Salmon farms move to land Earlier dates for fish and their Browse the buffet of Gordon

for a cleaner future p. 530 ecosystems pp. 567 & 572 Research Conferences p. 592

$15
10 FEBRUARY 2023
science.org

CHANGING

COLOR
Regulation of carotenoid
pigment variation pp. 534 & 576
 “One of the
advantages of
being a scientist
is that you can
choose who you
work with.”
Shruti Naik, PhD
Assistant Professor at New York University’s
Grossman School of Medicine

Hear Shruti Naik, PhD and others

explore how human connections
drive science forward.

Find the podcasts here

abcam.me/together

0210Product.indd 514 2/2/23 7:43 AM

 CONTENTS
1 0 F E B RUA RY 2 0 2 3 • VO LU M E 3 7 9
ISSUE 6632

530
Growing Atlantic salmon
s entirely on land could help meet rising demand while lowering environmental impact.

528 Global alarm system watches 539 A window into prebiotic worlds?
NEWS for methane
Artificial intelligence–powered scans of
Zircons reveal the geochemistry of Earth’s
earliest hydrothermal systems
satellite data detect leaks of the greenhouse By L. E. Rodriguez
IN BRIEF gas By P. Voosen RESEARCH ARTICLE p. 582

520 News at a glance FEATURES POLICY FORUM

IN DEPTH 530 Oceans away 541 Public views on polygenic screening
Is raising salmon on land the next big thing of embryos
522 Should an also-ran in human in farming fish? By E. Stokstad
evolution get more respect? Understanding moral acceptability and
Oldest Oldowan tools—seen as a hallmark willingness to use is crucial for informing
policy By M. N. Meyer et al.
of our own genus—found with bones of an
unexpected hominin By A. Gibbons
RESEARCH ARTICLE p. 561
INSIGHTS BOOKS ET AL.
544 Lost and found in El Salvador
523 Autism researchers face off PERSPECTIVES Separated by civil war, families turn to genetic
over language
Terminology dispute underscores divide 534 Unusual suspects in flower color testing to locate missing children
evolution By V. Penchaszadeh
about what direction the field should take
By R. Zamzow The expression of phased small interfering
RNAs caused monkeyflower color evolution 545 Hacking and the social contract
524 Repurposed drug battles By M. Monniaux Although technical fixes can help, solutions
‘brain-eating’ amoeba RESEARCH ARTICLE p. 576
must focus on broader societal factors at play
A drug for urinary tract infections may also By V. Mayer-Schönberger
treat Balamuthia mandrillaris 536 Probing matter with nonlinear
By K. Kornei spectroscopy LETTERS
Ultrafast nonlinear spectroscopy can unravel 547 Soil microplastics pollution
526 Could a popular antiviral
the dynamics of highly excited electronic in agriculture
supercharge the pandemic?
Molnupiravir appears to be speeding states By S. R. Leone and D. M. Neumark By L. He et al.
SARS-CoV-2 evolution By R. F. Service
538 Exaggerated responses to a virus 548 Understanding the limits of AI coding
PHOTO: NARAYAN MAHON

527 Neotropical bird scientists call long gone By J. Hendler

for an end to bias Multisystem inflammatory syndrome in
Two manifestos offer recommendations for children is caused by abnormal cell activation 548 Effective implementation
shifting field’s flawed “northern lens” By P. Brodin of new biodiversity pact
By E. Rodríguez Mega RESEARCH ARTICLE p. 554 By R. Buckley

SCIENCE science.org 10 FEBRUARY 2023 • VOL 379 ISSUE 6632 515

0210TOC_16616775.indd 515 2/7/23 5:49 PM

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0210Product.indd 516 2/2/23 7:43 AM

 CONTE NTS

RESEARCH 567
IN BRIEF
549 From Science and other journals

REVIEW
552 Epigenetics
Epigenetics as a mediator of plasticity in
cancer A. P. Feinberg and A. Levchenko
REVIEW SUMMARY; FOR FULL TEXT:
DOI.ORG/10.1126/SCIENCE.AAW3835

RESEARCH ARTICLES
553 Development
Mitochondria metabolism sets the species-
specific tempo of neuronal development
R. Iwata et al.
RESEARCH ARTICLE SUMMARY; FOR FULL TEXT:
DOI.ORG/10.1126/SCIENCE.ABN4705

554 Coronavirus
Inborn errors of OAS–RNase L in
SARS-CoV-2–related multisystem
inflammatory syndrome in children
D. Lee et al.
RESEARCH ARTICLE SUMMARY; FOR FULL TEXT:
An artist’s reconstruction of the Guiyang Biota, which evolved 1.5 million years after the Permian-Triassic mass
DOI.ORG/10.1126/SCIENCE.ABO3627 extinction and is one of the earliest known modern-type marine ecosystems.
PERSPECTIVE p. 538
561 Hominin evolution DEPARTMENTS
Nanophotonics Expanded geographic distribution and dietary 519 Editorial
555 Negative refraction in hyperbolic hetero- strategies of the earliest Oldowan hominins
Students and postdocs deserve more
bicrystals A. J. Sternbach et al. and Paranthropus T. W. Plummer et al.
By S. M. Malcom and S. Parikh
558 Gate-tunable negative refraction of mid- NEWS STORY p. 522
infrared polaritons H. Hu et al.
567 Paleontology 614 Working Life
A Mesozoic fossil lagerstätte from 250.8 What no one told me
million years ago shows a modern-type By H. Mastro
555 marine ecosystem X. Dai et al.

572 Fish evolution ON THE COVER

Genome structures resolve the early A flower color locus that underlies pollinator-
diversification of teleost fishes E. Parey et al. mediated speciation in monkeyflowers
(Mimulus) produces regulatory small RNAs
576 Plant science that suppress the accumulation of yellow
Taxon-specific, phased siRNAs underlie a carotenoid pigments. Variation in the level
speciation locus in monkeyflowers of these small RNAs through either natural
M. Liang et al. mutations or transgenic
PERSPECTIVE p. 534 manipulation contributes
to a great diversity of
ILLUSTRATIONS: (LEFT TO RIGHT) ELLA MARU STUDIO; DINGHUA YANG

582 Geochemistry flower color in Mimulus,

Relatively oxidized fluids fed Earth’s earliest as shown in this image.
hydrothermal systems See pages 534 and
D. Trail and T. M. McCollom 576. Image: Photo: Peter
PERSPECTIVE p. 539; PODCAST Morenus/University of
Connecticut
586 Virology
Negative and positive refraction is observed Inhibition of cellular RNA methyltransferase
at the interface of two crystals, on the left and abrogates influenza virus capping and Gordon Research Conferences .................592
right, respectively. replication Y. Tsukamoto et al. Science Careers ..........................................611

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0210TOC_16616775.indd 517 2/7/23 5:49 PM

 RUTGERS CONGRATULATES
OUR NEWEST AAAS FELLOWS
Paul Copeland Robert Kopp
Professor of Biochemistry and Professor of Earth and Planetary
Molecular Biology, Robert Wood Sciences, School of Arts and Sciences;
Johnson Medical School Co-Director, University Office of
Advancements in understanding Climate Action
the molecular mechanism of Innovations in climate science,
mRNA translation and trace particularly using statistical process
element metabolism modeling to estimate sea level change

Shuchismita Dutta Malin Pinsky

Associate Research Professor, Institute Associate Professor of Ecology, Evolution,
for Quantitative Biomedicine and Natural Resources, School of
Innovations in molecular Environmental and Biological Sciences
visualization education for Advancements in marine biology,
teachers, undergraduates, particularly for predictions about
and the public the impacts of climate change and
population dynamics

Bonnie Firestein Jaideep Vaidya

Professor of Cell Biology and Distinguished Professor and Vice Chair
Neuroscience, School of Arts and Sciences of Management Science and Information
Contributions to molecular and Systems, Rutgers Business School
cellular neuroscience, particularly the Contributions to privacy protection
development of, and strategies to in data analytics, information
repair, damaged neural circuits sharing, and access control
management

William Hallman Rachael Winfree

Professor and Chair of Human Professor of Ecology, Evolution,
Ecology, School of Environmental and Natural Resources, School of
and Biological Sciences Environmental and Biological Sciences
Advancements in risk analysis, Advancements in ecosystem services,
assessment, and communication, particularly contributions to pollination
and educating the public on ecology and conservation
important scientific issues

Saurabh Jha
Professor of Physics and Astronomy, excellence is earned
School of Arts and Sciences
Contributions to the study of
Type Ia supernovae, including
insights on their intrinsic
properties and their use as
cosmological probes

0210Product.indd 518 2/2/23 7:43 AM

 EDITORIAL

Students and postdocs deserve more

A
slow-motion crisis is underway among gradu- the value they bring to the enterprise. As it is, the fed- Shirley M. Malcom
ate students and postdocs in the United States eral government does not pay the full cost of research. is a senior advisor
who comprise today’s indispensable research University presidents and provosts are rewarded for se- and director of
and teaching workforce and tomorrow’s sci- curing shiny new buildings and research centers while the STEM Equity
entific leaders. Low pay, lack of benefits, and squeezing the most work possible out of trainees and Achievement (SEA)
sometimes toxic research environments have contingent faculty. It is hard to expect money to come Change program
persisted for years. Frustrated graduate stu- from inside this broken system to supplement low sala- at the American
dents, postdocs, and nontenured faculty are protesting ries and stipends. Association for
and pursuing unionization to address worsening condi- Compounding the financial obstacles, graduate stu- the Advancement
tions. A few senior leaders are starting to recognize that dents and postdocs need more skills and knowledge
of Science (AAAS,
today’s research environment is much more challenging than ever, often leading to longer preparation times
the publisher
than that of their sepia-tinted memories. In response, before they are viewed as “independent” and capable
of Science),
some universities have offered salary and benefits relief. of striking out on their own. Their careers are in the
Unionization and overdue adjustments are incomplete hands of one or two senior scientists whose incentives Washington, DC,
and temporary responses to deeply generally are to produce the most USA. smalcom@
embedded problems—with long-term possible research rather than the aaas.org
implications that everyone may re- best experience and prospects for
gret. So where do solutions lie?
For the past 70 years, universities in
“…many must their students.
To survive this prolonged appren-
Sudip Parikh
is the chief executive
the United States have supported fun-
damental research, operating under the
leave academia ticeship, many students must take
out loans for living expenses, dispro-
officer of AAAS and
executive publisher
premise that research and education
should be integrated. How that inte-
for a salary portionately discouraging students
of color and those from low-income
of the Science
journals. sparikh@
gration is carried out must be reformed
because over the course of seven de-
sufficient to households. Without a safety net, many
must leave academia for a salary suf-
aaas.org

cades, the research ecosystem has been

optimized to flawed incentives.
support their ficient to support their own families.
Careers outside academia are valuable
In the current system, a student or
scholar will “apprentice” to a more own families.” and rewarding, but society also needs a
diverse and talented academy.
senior scientist, with training ex- Solutions will not come without
changed for low wages. But how low changing the incentives that led to
is too low? The model works when there are mentor- such deeply engrained problems. Only federal funding
ing, a supportive research environment, and growth agencies, including the National Institutes of Health,
opportunities that lead to rewarding, independent National Science Foundation, and Department of En-
careers. Unfortunately, each provision of this contract ergy, have the influence to align incentives for admin-
is strained. Training times are longer than necessary. istrators, researchers, and others toward a healthier
Mentoring is not rewarded. Tenure track positions are environment. Determining the metrics of a healthy
limited, and other career options are not spotlighted. research environment, and supporting and rewarding
Senior scientists and administrators who want to do scientists and institutions that optimize these metrics,
the right thing are constrained by the incentives of the would be a start. Many may not like the consequences,
system itself. Sadly, when some senior scientists take such as fewer but bigger grants or smaller labs, but do-
advantage of the huge differences in power, there may ing nothing is unsustainable. American writer James
be exploitation, harassment, or worse. Not enough safe- Baldwin famously said, “Not everything that is faced
guards are in place to prevent these abuses of power can be changed, but nothing can be changed until it is
that are built into the system itself. faced.” It is past time for those who lead the scientific
It is understandable that every actor in this training enterprise to face up to the fact that the student and
system seeks to advance their own position. Unfortu- postdoctoral experience is not how it might be remem-
nately, the disadvantages inevitably flow to the graduate bered. Otherwise, everyone will bear witness to a sys-
students and postdocs. Getting the maximum research tem that is crumbling under its own weight.
for the dollar too often devolves to more students and
postdocs receiving less than they need and less than –Shirley M. Malcom and Sudip Parikh

10.1126/science.adh0336

SCIENCE science.org 10 FEBRUARY 2023 • VOL 379 ISSUE 6632 519

0210Editorial_16616709.indd 519 2/7/23 5:51 PM

 NEWS Edited by
IN BRIEF Jeffrey Brainard

CLIMATE CHANGE

Melting glaciers threaten

millions downstream

A
bout 15 million people in 30 countries face injury outbursts have killed at least 12,000 people worldwide. The
or death from unstable glacial lakes, a study says. first global assessment of the threat, published this week
As Earth’s climate has warmed, glaciers have rap- in Nature Communications, estimates that half of the
idly melted in high mountain ranges 15 million people threatened live at high eleva-
across the globe, and the resulting wa- Peru’s 70-meter-deep tions in Asia. More than 1 million are at risk
Lake Palcacocha, fed by
ter accumulates in unstable gouges cut melting glaciers, threatens in one glacier basin, the Khyber Pakhtunkhwa
by the glaciers as they retreat. The lakes’ banks the nearby city of Huaraz, in Pakistan. Other high-risk areas identified in
can burst, flooding nearby communities; such population 100,000. the report are in Peru and China.

fund a new Sabatini lab for $5 million that would ban the entire PFAS category,
Sabatini to get lab, second chance per year for 5 years. Its location, struc- which includes more than 10,000 sub-
#METOO | Two donors have agreed to ture, and size haven’t been determined. stances. Used in thousands of consumer
fund a new laboratory for David Sabatini, “I called him and said, ‘We think it’s products such as stain-resistant fabrics and
the prominent biologist who was fired by important that you get back to work,’” nonstick cookware, PFAS have been linked
the Howard Hughes Medical Institute and Ackman told Science. Other Sabatini sup- to health problems. ECHA will begin a
resigned from the Whitehead Institute porters say the probe of the biologist was 6-month scientific evaluation of the health
for Biomedical Research in 2021. A probe unfair and his punishment disproportion- and socioeconomic impacts of a ban. Those
there had found he sexually harassed ate. Sabatini’s critics say the funding is a results will be forwarded to the European
a junior scientist, fostered a sexualized humiliating brush off of women and oth- Commission, which together with member
lab environment, threatened retaliation ers in science who are harassed by senior states will decide whether to restrict the
against lab members if they questioned scientists on whom their careers depend. substances. Other governments have taken
his conduct, and obstructed the investiga- more limited steps to restrict the chemi-

PHOTO: LUKA GONZALES/AFP/GETTY IMAGES

tion, which was conducted by an outside cals, and industry groups have generally
law firm. Sabatini has disputed the find- EU eyes ban on ‘forever’ chemicals opposed restricting the entire class of
ings and sued Whitehead and the junior | In one of the most sweep-
R E G U L AT I O N compounds, arguing they have many essen-
scientist, Kristin Knouse, for defamation ing proposals of its kind, the European tial uses.
and emotional distress. (She counter- Chemicals Agency (ECHA) is considering
sued.) Billionaire hedge fund manager Bill asking the European Union to ban per- and
Ackman said last week that his Pershing polyfluoroalkyl substances (PFAS), called Science boosted in U.K. shuffle
Square Foundation—for which Sabatini forever chemicals because they accumulate POLICY| Under a reorganization
has reviewed grant applications—and an and persist in the environment. On announced this week, the United
anonymous partner will, in equal parts, 7 February, ECHA published a proposal Kingdom will get a standalone ministry

520 10 FEBRUARY 2023 • VOL 379 ISSUE 6632 science.org SCIENCE

0210NewsInBrief_16621379.indd 520 2/7/23 5:56 PM

 for science, innovation, and technol- Progress by degrees degrees may be poised to increase, the
ogy and its head will have a seat in the The percentage of U.S. science, technology, agency noted a 56% increase from 2017 to
prime minister’s Cabinet. The ministry engineering, and math (STEM) degree recipients 2021 in the number of Hispanic students
will be led by Michelle Donelan, previ- who identify as an underrepresented racial or ethnic pursuing them.
ously the higher education minister. minority (URM) increased from 2011 to 2020, but
The shuffle—the latest of many under remains below URM’s share of the population ages 18
Conservative leadership since 2010—also to 34 for all degrees except associate. Inhaling soot is a bad chess move
creates separate ministries for energy and E N V I R O N M E N T | Chess players who
trade, which were previously combined 45 breathe relatively high levels of fine soot

Percent earned by underrepresented

with science. Commentators said the new 2011 2020 particles may be more likely to make
structure could bring greater influence mistakes, researchers say. Previous studies

racial or ethnic minorities

and funding to science, but cautioned that have linked fine particulate matter with
the separated ministries may now work 30 brain diseases and decreased cognitive
less cohesively and compete for resources. function; the new investigation used a
controlled method to focus on the pollut-
ants’ effects on high-pressure, rapid-fire
Indonesia head draws heat 15
decision-making. The authors examined
LEADERSHIP | The science committee of 609 chess matches across three different
Indonesia’s Parliament is calling on the tournaments between 2017 and 2019. They
nation’s president to fire the head of its 0 adjusted for the skill level of each player’s
embattled research agency. The demand Associate Bachelor’s Master’s Doctoral opponent and used artificial intelligence to
follows controversy over the government’s assess the quality of each side’s moves. (It
efforts to consolidate 38 research agen- defined as errors moves that put the player
cies under the National Research and Diversity gains in U.S. STEM in position to lose.) The research team
Innovation Agency (BRIN). After Laksana H I G H E R E D U CAT I O N | More people from also used sensors in the competition halls
Tri Handoko became the agency’s head in underrepresented racial and ethnic minor- to measure levels of PM2.5 particles—fine
2021, BRIN laid off hundreds of research ity groups are earning science, technology, particulate matter measuring 2.5 microns
assistants and downsized and renamed engineering, and math (STEM) degrees at or less. The group found that an increase
the prestigious Eijkman Institute for U.S. institutions than ever before, a report in PM2.5 particles of 10 micrograms per
Molecular Biology. Also fueling the com- indicates. But the many efforts to increase cubic meter raised the probability that
plaints was a recent audit that found more diversity have far to go. Only 16% of STEM players made a mistake by 26%. The
than 65% of BRIN’s $400 million budget Ph.D.s, 24% of master’s degrees, and scientists found no effect on performance
this year is not allocated to research proj- 26% of bachelor’s degrees were awarded from increased levels of carbon dioxide or
ect grants. Handoko has said the agency to Black, Hispanic, American Indian, or temperature. The range of PM2.5 levels
NATIONAL CENTER FOR SCIENCE AND ENGINEERING STATISTICS/NATIONAL SCIENCE FOUNDATION 2023; (PHOTO) JUST LIFE/SHUTTERSTOCK

should prioritize spending on research Alaska Native students in 2020, accord- measured was similar to those found in
equipment. His critics include members ing to the report released last week by U.S. cities. The findings highlight the need
CREDITS: (GRAPHIC) K. FRANKLIN/SCIENCE; (DATA) DIVERSITY AND STEM: WOMEN, MINORITIES, AND PERSONS WITH DISABILITIES,

of a coalition of opposition parties that the National Science Foundation. That is for installing air filters that can elimi-
aims to win the presidency in next still significantly lower than these groups’ nate the small particles, the authors say.
year’s elections. total share of the population ages 18 to 34. Their study was published last week in
In a sign that those numbers for graduate Management Science.
Big bridge, lower carbon
| Techniques to capture
C L I M AT E S C I E N C E
and store carbon dioxide (CO2) emitted
in manufacturing steel and concrete can
reduce global warming, but are expensive.
Relative to the total construction costs
of a long bridge, however, they can be
cost effective, a case study has found. Its
authors considered Louisiana’s
38-kilometer-long Lake Pontchartrain
Causeway, which opened in 1956. CO2
removal technologies available today
could have captured 51% of the gas emit-
ted by factories that made the bridge’s
steel and concrete while increasing its
cost by only 1%, according to a 2 February
paper in Environmental Science and
Technology. The authors say the find-
ings could inform similar cost-benefit
analyses for other construction projects.
The world’s cement and steel industries
together account for approximately 15% of
global CO2 emissions.

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 IN DEP TH

PALEOANTHROPOLOGY little about the beginnings of stone tools and

the emergence of early Homo,” says paleo-

Should an also-ran in human anthropologist Sileshi Semaw of Spain’s Na-

tional Research Center for Human Evolution
(CENIEH), who is not part of this study. This

evolution get more respect? is “why the Nyayanga discovery is important.”

It’s not the first time stone tools have
been found with fossils of Paranthropus, a
Oldest Oldowan tools—seen as a hallmark of our own

IMAGES: (TOP TO BOTTOM) ROMAN UCHYTEL/PREHISTORIC FAUNA STUDIO; T.W. PLUMMER/HOMA PENINSULA PALEOANTHROPOLOGY PROJECT
genus with several species that lived from
genus—found with bones of an unexpected hominin about 2.8 million to 1.2 million years ago
across Africa. In 1955, Louis and Mary
Leakey discovered the Nutcracker Man, a
By Ann Gibbons the Paranthropus molar, it got really, really skull with a robust jaw and teeth now classi-
exciting,” says Finestone, of the Cleveland fied as Paranthropus boisei, in the same 1.8-

A
s thunder boomed and dark rain Museum of Natural History. million-year-old layer of sediments as Old-
clouds gathered on the last day of the The tools, dated to about 2.8 million years owan tools. But Mary Leakey soon found a
field season in Kenya in 2017, paleo- ago, are the oldest known examples of the skull of Homo habilis (Latin for “handyman”)
anthropologist Emma Finestone was Oldowan toolkit. They also hint that Paran- in the same layer and thought that species, in
rushing to record the location of fos- thropus, often seen as an also-ran in the our own genus, was a better fit as the princi-
sils while excavators were hoisting an story of human evolution, might have made pal toolmaker. Paranthropus, with its power-
ancient hippo skeleton out of the ground. “I or at least used tools. “I have been skeptical ful jaws and teeth, was seen as not needing
was worried she would get struck by light- of Paranthropus using stone tools. … But tools to process tough food. “Homo was al-
ning because she was on top of a hill,” says maybe we do have multiple hominins using ways given credit for the tools,” says paleo-
Tom Plummer, a paleoanthropologist at the Oldowan,” Finestone says. “We know very anthropologist Bernard Wood of George
Queens College who led the excavation at Washington University.
Nyayanga, near Lake Victoria. As more Oldowan tools were discovered
Finestone received a shock of a differ- across Africa and beyond, most researchers
ent kind as the hippo was removed. Be- concluded that their appearance coincided
neath it, Blasto Onyango, head preparator with the earliest fossils of Homo, dating to
of the National Museums of Kenya, found 2.8 million years ago in Ethiopia. Many saw
a huge hominin molar. It lay intermingled the Oldowan as a key technological revolu-
with hammerstones and sharp flakes that tion that helped early Homo expand its diet,
Finestone recognized as early Oldowan tools, adapt to different habitats, and rapidly ex-
an ancient technological breakthrough long tend its range in Africa and beyond to Asia,
thought to be a defining hallmark of our ge- where some of the oldest Homo fossils are
nus, Homo. But the molar was from a very 2 cm found with Oldowan tools, also known as
different human relative: Paranthropus, Mode 1 tools. All of this, the theory goes,
known for its huge teeth and crested ape-size An Oldowan core and flakes were among the tools helped fuel Homo’s expanding brain.
skull, not toolmaking skills. “When we found found near a Paranthropus molar. But the 2011 discovery of crude stone tools

522 10 FEBRUARY 2023 • VOL 379 ISSUE 6632 science.org SCIENCE

0210NewsInDepth_16615111.indd 522 2/7/23 5:51 PM

 NE WS

With its powerful jaws and teeth, Paranthropus SCIENTIFIC COMMUNITY

(shown in a composite photo illustration) was thought
to have no need for stone tools to process food.

dating to 3.3 million years ago at Lomekwi in

Autism researchers
northern Kenya threw a wrench in that neat
view. The tools predated Homo and showed
that an earlier hominin, perhaps Australo-
face off over language
pithecus afarensis, already knew how to Terminology dispute underscores divide about
make flakes, albeit less sophisticated than
those of the Oldowan. Ever since, research-
what direction the field should take
ers have been eager to find fossils and tools
dating to the roughly 500,000-year gap in By Rachel Zamzow autistic people the study authors deemed
the fossil record between 3.3 million years dehumanizing, objectifying, or stigmatiz-

A
and 2.8 million years ago, says archaeologist long-smoldering debate among sci- ing. Some responses described autistic
Sonia Harmand of Stony Brook University, entists studying autism has erupted. people as “shut down from the outside
who reported the Lomekwi tools. At issue is language—for example, world” or “completely inexpressive and
The new tools and molars from Nyayanga whether researchers should describe apparently without emotions,” according
fall right in that gap. The ancient butchers autism as a “disorder,” “disability,” or to the November 2022 Frontiers in Psycho-
left two hippo carcasses, many large-animal “difference,” and whether its associ- logy study. “What is worse than I thought
bones bearing cutmarks from tools, and ated features should be called “symptoms” was how blatant a lot of the content was,
330 artifacts, including blades used to cut or simply “traits.” In scientific papers and which shows that, for [a] large proportion
meat and plants. Plummer’s team used multi- commentaries published in recent months, of participants, they did not consider the
ple methods to date the site to about 2.8 mil- some have decried ableist language among things they were saying to be problematic
lion years ago, with a range of 2.58 million to their colleagues whereas others have de- at all,” says lead author Monique Botha, a
3.03 million years. “They’ve made a solid case fended traditional terminology—with both psychologist at the University of Stirling.
with the evidence they have,” says geologist sides saying they have the best interests of Ableist language and the mindset that un-
Craig Feibel of Rutgers University, Piscataway. autistic people in mind. The vitriol is harm- derlies it also trickles down to study design,
By that time, toolmakers were al- ing the field and silencing researchers, some says Botha, who is autistic. In studies test-
ready skilled at knapping. “They are not fear, but others see it as a ing autism interventions,
newbies—they have bashed rocks together long-overdue reckoning. for example, researchers
before,” says Peter Ditchfield, a geologist at Since autism’s earli- “Specificity is always rarely track adverse re-
the University of Oxford who was part of
the dating team. “This hints at an earlier
est descriptions in the
academic literature as a
going to be more actions such as physical
harm or psychological dis-
stem to the Oldowan.” condition affecting social rigorous and accurate tress, found a 2021 study
The site of the discovery, more than interaction and communi- led by Kristen Bottema-
1300 kilometers from the next oldest Oldowan cation, researchers and cli- than generalization.” Beutel, who studies special
tools in Ethiopia, also shows the technology nicians have framed it as a Monique Botha, education at Boston Col-
spread faster and farther than was thought, medical disorder, with a set University of Stirling lege. This oversight sug-
says Mohamed Sahnouni, an archaeologist at of symptoms to be treated. gests many researchers see
CENIEH who has dated other Oldowan tools Historically, autistic children have been autistic people as less than human, Botha
to 2.4 million years ago at a site in Algeria. institutionalized and subjected to treat- says. “It’s one of the most pervasive prac-
The real “whodunnit” now, says co-author ments involving physical punishment, food tices that genuinely keeps me up at night.”
Rick Potts of the Smithsonian Institution’s restriction, and electric shocks. Even today, At the same time, others argue that
National Museum of Natural History, is: Who the most widely used autism therapy— making certain terms off-limits stifles
was the toolmaker? “We’re not claiming that applied behavior analysis—is seen by some the scientific process. “If you can’t use
Paranthropus made the tools, but I think it as a harmful tool of normalization. Many words like ‘challenging behaviors’ or ‘se-
could have used them,” he says. autistic people and their families have in- vere disorder’ or ‘symptoms’ or ‘comorbid
But there are other contenders as well. stead embraced the view that their difficul- disorder,’ then how are you supposed to
As many as a half-dozen species of Homo, ties lie not with their autism, but with a study those things?” asks Alison Singer,
Australopithecus, and Paranthropus lived in society that isn’t built to support them. president of the Autism Science Foun-
eastern Africa at that time. “We’re starting But according to some autism research- dation, who laid out these concerns in a
to see … lots of different species around that ers, the field still too often defaults to terms December 2022 commentary in Autism
could have been able to make stone tools,” with negative connotations. For example, in Research. Singer and others, includ-
Harmand says. “I like the idea that they addition to “symptom” and “disorder,” many ing her three co-authors, fear that us-
might have learned directly from each other.” scientists use the term “comorbid” rather ing neutral terminology—such as “traits”
The team will return to Nyayanga this than the more neutral “co-occurring” to de- or “features” in place of “symptoms”—
summer to look for answers. “This time pe- scribe conditions that tend to accompany downplays the experiences of autistic
riod keeps getting more and more interest- autism. Similarly, some argue the oft-used people who, like Singer’s daughter, have
ing,” Plummer says. “We have Oldowan tool phrase “people with autism,” as opposed to significant difficulty communicating, in-
use extended back, Paranthropus extended “autistic person,” can imply that autism is tellectual disabilities, or critical health
back, and early Homo extended back. It’s a necessarily an unwanted harmful condition. concerns. It “trivializes the severity of
time when there’s a lot of different lineages at In a recent survey of 195 autism research- autism,” says Singer, who is nonautistic.
critical points in their evolution.” j ers, 60% of responses included views about Botha, Bottema-Beutel, and 61 other re-

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searchers, clinicians, and advocates have in on the heated nature of the debate is to BIOMEDICINE
submitted a letter to the editor rebutting “entirely overlook what autistic people are
Singer’s commentary.
Singer and others also worry that mov-
ing toward neutral language could lead
saying or protesting.”
Some hold more moderate positions,
such as Zack Williams, an M.D.-Ph.D.
Repurposed
major funding agencies to shift support
from research exploring autism’s under-
lying biological causes and potential treat-
candidate in neuroscience at Vanderbilt
University. “If we have the appropriate lan-
guage to say something in a neutral and
drug battles
ments to other sectors, such as services
and supports for autistic people—though
whether this would be a positive or nega-
nonoffensive way, we should do it by de-
fault,” says Williams, who is autistic and
a co-author of the letter to the editor re-
‘brain-eating’
tive change is also a subject of debate. For
now, the bulk of funding for the field falls
sponding to Singer’s commentary. But, “If
someone goes and says ‘autism spectrum
amoeba
squarely on the side of biological research disorder,’ they shouldn’t be canceled,” he
in the United States, as well as in the says; the focus should be on whether and
A drug for urinary tract
United Kingdom and Australia. how the research is helping autistic people. infections may also treat
Amid the ongoing language dispute, re- Others say the field is too fractured to
searchers on all sides report coming un- even try to resolve the divide. “I think that
Balamuthia mandrillaris
der attack, in the form of vitriolic Twitter we don’t have a unified field anymore, and
exchanges, remarks at conferences, and I think the sooner we recognize that the By Katherine Kornei
less pain will be there,” says

I
Helen Tager-Flusberg, a n the summer of 2021, a 54-year-
psychologist at Boston Uni- old man was brought to a hospital
versity. Tager-Flusberg, who in Northern California after an un-
is nonautistic, and others explained seizure. When an MRI re-
say one source of the fric- vealed a mysterious mass in the left
tion is the use of a single set side of his brain, he was transferred to
of terms for an extremely the University of California, San Francisco
heterogeneous condition. (UCSF) Medical Center. A brain biopsy
She argues for splitting the and other tests revealed not a tumor, but
field and adopting sepa- an incredibly rare infection of the central
rate terminology. But the nervous system caused by the amoeba
autistic community largely Balamuthia mandrillaris. One of several
thinks dividing them into “brain-eating” amoebae that occasionally
groups such as “high-” or spark headlines, the pathogen kills more
“low-functioning” is harm- than 90% of people who contract it.
ful and unnecessarily seg- But despite initial setbacks, the patient
regating, according to a survived and has largely recovered after
December study exploring experimental treatment with a decades-old
the language preferences drug. As his UCSF medical team recounted
of 654 English-speaking in Emerging Infectious Diseases last month,
autistic adults across 30 a desperate hunt for a cure led them to a
countries. study published several years ago. In it,
Dehumanizing language is common among autism researchers, “I don’t get why we can’t researchers showed a drug originally de-
psychologist Monique Botha found in a recent study. have common ground,” says veloped in Europe to quell urinary tract in-
Bottema-Beutel, who is non- fections was effective against Balamuthia in
being shouted down during talks. This in- autistic. “I don’t think that anyone who is the laboratory. That discovery sent the med-
creasingly hostile environment threatens saying we should avoid dehumanizing lan- ical team rushing to obtain the drug, nitrox-
to drive some scientists out of the field guage is saying we should avoid accurately oline, from abroad so it could be given for
altogether—something Botha says they have describing people and what they need.” the first time to a Balamuthia patient.
seen firsthand among autistic researchers. “Why not just be specific?” Botha asks. Researchers not involved with the case
“People are getting reluctant to give pub- For example, if a study’s sample includes call the man’s recovery a breakthrough in
lic presentations or to be too vocal about autistic people who are nonspeaking, treating a brain infection that’s long been
what they’re finding,” says David Amaral, a have intellectual disability, and require presumed to be a death sentence. “It’s the
neuroscientist at the University of Califor- supported living, just say that. Phrasing best that I ever remember seeing with Bala-
nia, Davis, who is nonautistic. “Science is like this doesn’t minimize the very muthia,” says Dennis Kyle, a cell biologist
supposed to be about communication.” He real challenges some autistic people at the University of Georgia, Athens, who
wrote a December 2022 editorial in Autism experience—but it also doesn’t dehuman- studies amoebic diseases. The drug, which
Research, for which he is editor-in-chief, ize them, Botha says. “Specificity is always is not approved for regular use in the United
PHOTO: MONIQUE BOTHA

calling for civility across the board. going to be more rigorous and accurate States, has also been effective against other
But, Botha says, “Civility is only possi- than generalization.” j pathogenic amoebae in laboratory tests, ac-
ble when there is an equal playing field,” cording to the UCSF team.
which can’t exist, they say, as long as the Rachel Zamzow is a freelance journalist Balamuthia mandrillaris was first iden-
field marginalizes autistic people. Zeroing in Waco, Texas. tified in 1986—not in a hospital but at the

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 San Diego Wild Animal Park, where staff Spottiswoode and her colleagues started not approved by the Food and Drug Admin-
were eagerly anticipating the birth of a the patient on an aggressive suite of istration, which meant the medical team
mandrill, the largest species of monkey. antiparasitic, antibacterial, and antifungal had to file an Emergency Investigational
But one day, Nyani, the mother-to-be, be- drugs delivered via 47 daily pills and an in- New Drug application for permission to
gan dragging her right arm on the ground. travenous drip. But the drug cocktail was use it—and then find a source for the drug.
Within 48 hours she became lethargic, and somewhat ad hoc, Spottiswoode admits. Asieris Pharmaceuticals, a company devel-
she eventually stopped moving and died. A “It’s what’s recommended because it was oping nitroxoline to treat bladder cancer,
postmortem evaluation of Nyani’s brain tis- what happened to be used in patients who ultimately provided several hundred pills
sue revealed hemorrhaging and centimeter- survived,” she says. from its R&D facility in Shanghai and has
scale lesions. The culprits were plainly vis- It can also be highly toxic: The man’s continued to send resupply shipments, all
ible: Amoebae were eating Nyani’s brain. blood sugar levels and white blood cell pro bono.
Researchers later determined the organ- count both dipped dangerously low, and he After just 1 week of treatment with nitrox-
ism represented a new species of amoeba, suffered from kidney failure. All the while, oline, the patient’s lesions shrank. Follow-
constituting its own genus. Balamuthia lives the man’s lesions kept up MRIs have revealed con-
in soil, dust, and water, and it’s believed to growing. Desperate to save tinued improvement, and
enter the body via the lungs or via skin cuts. her patient, Spottiswoode the patient is now living
(Its full name is a nod to both Nyani’s spe- began poring over research at home. “I think he’s very
cies and the late William Balamuth, a zoo- about Balamuthia. much on his way to being
logist at UC Berkeley who studied amoebae.) One day, she found a one of the survivors of this
In the past few decades, only about 2018 study in the journal disease,” Spottiswoode told
100 human cases of Balamuthia have been mBio in which a team led by a group of her colleagues in
confirmed in the United States. “The vast Joseph DeRisi, a biochemist October 2022.
majority of doctors, even infectious dis- also at UCSF and president The UCSF team has been
ease doctors, have never seen a case,” says of the Chan Zuckerberg in touch with the Centers
UCSF infectious disease physician-scientist Biohub, had painstakingly for Disease Control and
Natasha Spottiswoode. screened more than 2100 Prevention about keep-
That was true for her as well until drugs in the laboratory to ing nitroxoline on hand
the California man came under her care identify compounds effec- Balamuthia mandrillaris for future use. It should
2 years ago. Even after his initial MRI results tive against Balamuthia. be available as an “emer-
PHOTOS: (TOP TO BOTTOM) K. YAMANOUCHI, H. ARIMA, Y. SAKAMOTO ET AL./WIKIMEDIA COMMONS; N. SPOTTISWOODE ET AL., EMERG. INFECT. DIS. (1) 197-201 (2023)

came back, the UCSF medical team, led by The effort, DeRisi recalls, “was inspired gency standby medication for 24-hour
Spottiswoode, had no reason to contemplate by our frustration” of watching an ulti- delivery anywhere in the United States,”
a Balamuthia infection because brain masses mately fatal progression of Balamuthia in a DeRisi says. But to save patients, doctors
can be caused by a slew of more common ail- 74-year-old woman being cared for at UCSF. also have to rapidly diagnose a Balamuthia
ments such as cancer, bacterial abscesses, One compound stood out in their screen- infection. Currently, a clinician must sus-
and tuberculosis. But after a biopsy of the ing: nitroxoline. But DeRisi and his collabo- pect an amoebic infection in the first place
brain mass revealed what appeared to be rators haven’t, until now, had a chance to and then order a specific PCR test.
amoebae, the team sent a piece of the man’s see it tested in an infected person. Balamu- “If you don’t think of what’s there, then
infected tissue to the University of Washing- thia cases are so rare, DeRisi says, and “by you’ll never find it,” says Michael Wilson, a
ton, Seattle, for a polymerase chain reaction the time you contact the treating clinical neurologist at UCSF and a member of the
(PCR) test that could confirm an amoebic team, the patients are dead.” research team.
infection and even identify the kind. The re- When Spottiswoode reached out to He notes that a technique called meta-
sults came back positive for Balamuthia. DeRisi, he urged her on. But nitroxoline is genomic next-generation sequencing, pio-
neered by DeRisi, could speed diagnosis.
The approach involves sequencing genetic
material from a patient sample and com-
paring it with enormous libraries of genetic
code from millions of known pathogens.
“You let the database match tell you what
organisms are in there,” Wilson says. “You’re
not going in with a preconceived notion.”
Spottiswoode and her colleagues are now
working with other doctors overseeing Bal-
amuthia cases. Just recently, a second pa-
tient was started on the drug, and the initial
results are already encouraging, she says.
Spottiswoode is hoping her frantic search
will have a lasting payoff for other Balamu-
thia patients. “For every patient who exists
with this disease, there’s a team of doctors
where I was, which is basically desperate to
find something that will help.” j

MRI scans of a man whose brain was infected with the amoeba Balamuthia mandrillaris show a dramatic Katherine Kornei is a journalist based
difference in inflammation (white) before and after (right) treatment with an experimental drug. in Portland, Oregon.

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COVID-19 ing the dates and locations of the sequences

showed some of the mutated strains were

Could a popular antiviral spreading in the community. “Clearly some-

thing is happening here,” Peacock says.
Whether the changes will lead to variants

supercharge the pandemic? that are more pathogenic or transmissible

is unclear, the researchers say. “We are not
coming to a conclusion about risk,” says team
Molnupiravir appears to be speeding SARS-CoV-2 evolution member Theo Sanderson, a geneticist at the
Francis Crick Institute. Haseltine, however,
likens the danger to keeping a pet lion. “Just
By Robert F. Service SARS-CoV-2 genome sequences deposited because it didn’t bite you yesterday doesn’t
in the international GISAID database, look- mean it won’t bite you today,” he says.

A
widely used COVID-19 drug may be ing for the kinds of mutations expected to The Merck spokesperson says the link
driving the appearance of new SARS- be caused by molnupiravir. Rather than in- between the mutations and the drug is un-
CoV-2 variants, sparking concerns it ducing random changes in the virus’ RNA proved. “The authors … rely on circumstan-
could prolong the pandemic. The drug, genome, the drug is more likely to cause spe- tial associations between viral sequence
molnupiravir, produced by Merck & cific nucleic acid substitutions, with guanine origin and time frame of sequence collec-
Co., is designed to kill the virus by switching to adenine and cytosine to uracil. tion,” she says. But the new result comes on
inducing mutations in its genome. A survey One virus hunter, Ryan Hisner, a middle the heels of two others that could change the
of viral genomes reported in a new preprint, school science and math teacher in Monroe, risk-benefit calculus for the drug.
however, suggests some people treated with Indiana, identified dozens of sequences that In one, researchers in Australia found evi-
the drug generate novel viruses that not only showed clusters of those hallmark substitu- dence that molnupiravir treatment may be
remain viable, but spread. leading to new variants in immuno-
“It’s very clear that viable mutant compromised patients. Because these
viruses can survive [molnupiravir A drug’s fingerprint? patients’ immune systems have trou-
treatment] and compete [with exist- An analysis of SARS-CoV-2 genomes submitted to a global database ble clearing the virus, viral variants
ing variants],” says virologist William shows guanine-to-adenine mutations, often induced by molnupiravir, can accrue large numbers of muta-
Haseltine, chair of ACCESS Health are more common in countries where the drug is widely used, such tions, possibly causing big leaps in
International. “I think we are court- as Australia, the United Kingdom, and the United States, than in viral behavior. Repeatedly sequencing
ing disaster.” But a Merck spokes- countries where molnupiravir use is low, such as Canada and France. SARS-CoV-2 genomes from nine pa-
person disputes that the drug has tients, five of whom received the drug
led to the emergence of widely circu- Australia and four who did not, the researchers
100
lating variants, and some research- found that treated individuals har-
Guanine-to-adenine clusters*

United States

CREDITS: (GRAPHIC) K. FRANKLIN/SCIENCE; (DATA) T. SANDERSON ET AL., MEDRXIV, HTTPS://DOI.ORG/10.1101/2023.01.26.23284998 (2023)

ers downplayed the significance of bored an average of 30 new variants
molnupiravir-caused mutations. United Kingdom each within 10 days of the initial dose,
“Right now, it’s much ado about noth- Japan far more than the untreated patients.
ing,” says Raymond Schinazi, a medic- Germany “Our study demonstrates that this
10
inal chemist at the Emory University commonly used antiviral can ‘super-
School of Medicine, noting that the charge’ viral evolution in immuno-
virus is already mutating rapidly. compromised patients, potentially
Molnupiravir was authorized in generating new variants and prolong-
the United Kingdom and the United Canada
ing the pandemic,” the authors wrote
States in late 2021, and dozens of 1 France in a 22 December 2022 preprint.
0
other countries have followed. In 100 1000 10,000 100,000 1,000,000
A second report, on 28 January
2022, Merck estimated global sales at Total genomes submitted in 2022* in The Lancet, suggests that, at least
more than $5 billion. Though that is * Logarithmic scales were used for the axes. Countries with fewer than 100 total genomes
among people vaccinated against
well below the $18.9 billion in 2022 submitted in 2022 were excluded. COVID-19, molnupiravir offers limited
sales for Paxlovid, another oral SARS- benefits. The study tracked 26,411 vac-
CoV-2 antiviral, molnupiravir remains popu- tions. Hisner raised his concerns on Twit- cinated participants in the United Kingdom’s
lar in certain countries. ter and ultimately teamed up with Thomas PANORAMIC clinical trial, roughly half of
From the start, however, Haseltine and Peacock, a virologist from Imperial College whom were given the drug. It did reduce
others worried about the drug’s mechanism, London. With other colleagues, the pair sys- symptom severity and improve recovery
which involves introducing so many muta- tematically reviewed more than 13 million times, but it did not lower the frequency of
tions into the viral genome that it can no SARS-CoV-2 sequences in GISAID and ana- COVID-19–associated hospitalizations or
longer reproduce. One fear was that mutated lyzed those with clusters of more than 20 mu- deaths among high-risk adults.
virus would survive and propagate—and per- tations. In a preprint posted on 27 January, The new studies don’t prove molnupiravir
haps turn out to be more transmissible or they report that a large subset showed the is causing the emergence of dangerous new
virulent than before. Before the U.S. Food and hallmark substitutions; all dated from 2022, SARS-CoV-2 variants, says Ravindra Gupta,
Drug Administration authorized the drug, a after molnupiravir began to be widely used. a clinical microbiologist at the University of
Merck spokesperson called the worry “an in- These signature clusters were up to Cambridge. But he argues the drug’s limited
teresting hypothetical concern.” 100 times more common in countries where benefit suggests it’s no longer worth the risk.
Nevertheless, researchers and citizen sci- molnupiravir was widely used than in those “These results do call into question whether
entists from around the globe began to scan where it was not (see graphic, above). Track- molnupiravir should be used.” j

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 Ornithologist Francisca Helena Aguiar-Silva (right) tracks raptors in Brazil. She and others aim to improve work on neotropical birds such as the black-fronted piping guan (left).

ORNITHOLOGY

Neotropical bird scientists call for an end to bias

Two manifestos offer recommendations for shifting field’s flawed “northern lens”

By Emiliano Rodríguez Mega problem. Few people in neotropical nations ecology and evolution, they note, and much
are native English speakers, the authors note, of it comes from fieldwork in the neotropics.

T
wo years ago, some ornithologists were so journals often ask researchers from the But such work is often ineligible for funding
outraged by the publication of a paper region to have their manuscripts edited by a and can be hard to publish in key journals,
that highlighted how much scientists professional. But that can cost up to $600— which favor studies of ecology, biogeography,
still don’t know about birds from Latin more than many Latin American scientists or conservation.
America and the Caribbean. Many make in a month. To lower the language bar- The papers offer examples of how a lack of
criticized the authors—based at uni- rier, the authors recommend journals accept data from the neotropics has allowed flawed
versities in the United States and the United manuscripts in Spanish and Portuguese for ideas to linger. In the 1990s, North American
Kingdom—for citing few studies by scientists review, then translate them into English if researchers published an influential study
from the region and from journals that don’t accepted for publication—and also consider indicating neotropical birds thought to be
publish in English. Others said the paper, publishing a version in a second language. monogamous cheated on their partners less
published in Ornithological Advances, per- Language hegemony also hinders clear often than monogamous species in temper-
petuated an elitist, exclusionary, “northern” communication about bird names and im- ate regions. But Valentina Ferretti, an evolu-
approach that overlooked, for instance, the poses a “northern lens” on the field, the tionary ecologist at the Institute of Ecology,
knowledge of Indigenous people. authors say. Journals and meetings often Genetics, and Evolution in Argentina, noted
“It made me angry,” recalls bird ecologist require the use of English names, they note, the study’s sample included few neotropical
Ernesto Ruelas Inzunza of the University of “rather than the scientific (Latin) names that species. In 2019, after adding data that she
Veracruz in Xalapa, Mexico. “Deliberately are supposed to be a global standard.” Such and other researchers had gathered on addi-
or not,” he says, the article ignored “that to- rules not only require Latin American re- tional species, she found there was no clear
day’s neotropical ornithology is nurtured by searchers to learn the English names, which geographic pattern. “It’s a mistake to come
Latin American and Caribbean scientists.” He were often imposed by Europeans studying up with theories that are based on only a mi-
and others vowed to change that, by smash- museum specimens, but also discourage the nority of bird diversity,” Ferretti says.
ing barriers they say have disadvantaged use of names developed by Indigenous peo- The papers “will undoubtedly sensitize”
ornithologists from neotropical nations and ple, which can carry valuable information ornithologists from the north about the chal-
deprived the field of their contributions. This about how a bird sings or where it lives. lenges their colleagues face in the south, says
week, their resolve bore fruit in two papers “We continue to legitimize the idea that ornithologist Joseph Wunderle of the U.S.
PHOTOS: (LEFT TO RIGHT) DANIELA ZAFFIGNANI; OLIVIER JAUDOIN

published in Ornithological Applications. what’s important is European knowledge,” Forest Service, who is based in Puerto Rico.
In one, 124 authors from the region exam- says Kristina Cockle, a Canadian ornitho- The lead author of the 2020 study that ig-
ine numerous factors—including a shortage logist at the Institute of Subtropical Biology nited the discussion, ornithologist Alexander
of funding, few Latin American ornitho- in Argentina, where she has lived for nearly Lees of Manchester Metropolitan University,
logists in leadership roles, and a bias against 20 years. She and her co-authors urge funders says he hopes the new papers catalyze change.
citing papers in Spanish and Portuguese— and others to encourage collaboration with Initially, he felt stung by the criticism, par-
they say have often marginalized the region’s local communities, including by allowing ticularly because he has collaborated for de-
researchers. In the other, a smaller group of- nonacademics to help develop research ques- cades with local researchers in the Brazilian
fers 14 recommendations for how the field’s tions, co-lead projects, and author papers. Amazon. Now, he thinks his paper could pro-
major journals can revise their policies and Funders and others also need to do more duce “a bit of a funny legacy. … [I]f it means
practices to improve the flow of science from to encourage studies of the basic biology of that the field grows and becomes more inclu-
the region’s bird scientists. neotropical birds, the authors say. Descriptive sive, then, you know, I’ll take that.” j
Both papers identify “language hegemony,” information, such as a bird’s diet or behavior,
the use of English by major journals, as a is often foundational to broader insights into Emiliano Rodríguez Mega is a journalist in Mexico City.

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ATMOSPHERIC SCIENCE

Global alarm system watches for methane

Artificial intelligence–powered scans of satellite data detect leaks of the greenhouse gas

By Paul Voosen buy a bit more breathing room to achieve data, which made it hard to spot the plumes.
longer term CO2 cuts. Berend Schuit, a doctoral student at SRON,

M
ethane is a stealthy greenhouse In the past, researchers had to target loca- turned to AI for help. First he went through
gas, erupting unpredictably from tions already known to have methane releases years of TROPOMI data, ending in 2020,
sources such as pipelines and gas to find new ones, says Riley Duren, a remote- identifying some 800 scenes of confirmed
fields. Scientists have wanted to sensing scientist at the University of Arizona plumes, along with 2000 scenes without
catch these emitters in the act. In and CEO of Carbon Mapper, a $100 million them. Armed with these scenes, Schuit and
the past, watchdogs had to moni- project that later this year will launch a pair his colleagues trained their AI algorithm to
tor likely sites from the ground or by air- of satellites designed to spot plumes of meth- recognize the plumes. But it spat out too
plane. Now, massive, short-lived leaks can ane and CO2. The new technique, he says, many false positives, often caused by artifacts
be detected automatically, from space, “helps lay the foundation for future opera- such as the stray edge of a cloud. Eventually,
anywhere in the world—a first step toward tional monitoring with the expanding global they were able to produce a reliable filter by
plugging them and slowing climate change. ecosystem of methane satellites.” training a second AI to spot signs that a re-
The new technique, which sult might be spurious, such as
uses artificial intelligence (AI) to an increase in reflectivity from
scan through the 12 million daily clouds or an inconsistency be-
Pinpointing a source
observations collected by a Eu- A plume of methane over southern New Mexico was detected from orbit. A new tween the wind direction and the
ropean satellite, could aid future technique automates the search for leaks of the potent greenhouse gas. path of a “plume.”
efforts to spot plumes in data The team then tested the al-
collected by satellites, such as gorithms on TROPOMI’s mea-
the International Methane Emis- surements from 2021, finding
sions Observatory announced in 2974 unique methane plumes
2021 by the United Nations. Al- that they could confidently iden-
though so far the technique only tify from just one satellite pass.
captures the largest blowouts, More than 40% were associated
there’s no better place to begin, with oil and gas development,
says Ilse Aben, an atmospheric another one-third with landfills,
scientist at the Netherlands Insti- and 20% with coal mines.
tute for Space Research (SRON) TROPOMI’s roughly 5-kilome-
and co-author of the new work. ter resolution is not fine enough
“That’s the low-hanging fruit,” to spot the precise facility re-
she says. “If you’re putting in the sponsible for each plume. But
effort to stop leaks of methane, data from future satellites should
better to start with the big ones.” sharpen the pictures. This con-
The SRON team behind the 0 1 stellation will grow rapidly in the
work, which appeared last month Nor
North kkm next few years, including with
as a preprint in the journal Atmo- the launch of the Environmen-
spheric Chemistry and Physics, tal Defense Fund’s MethaneSAT
has now begun posting its weekly 0 200 400 600 800 1000 1200 1400 later this year, followed by Car-
results on its website. Last month Methane (parts per million/meter) bon Mapper and other satellites
alone, they detected 192 methane with even higher resolution.
plumes—some persistent, some intermittent— SRON’s automatic methane spotter relies Methane measurements from space have

GRAPHIC: NASA/JPL-CALTECH, ADAPTED BY C. BICKEL/SCIENCE

with an emissions rate greater than 10 tons on the Tropospheric Monitoring Instrument weaknesses, including difficulty distin-
per hour, concentrated in Asia but popping (TROPOMI) aboard the Sentinel-5 Precursor guishing fresh plumes from gas already in
up on every continent except Antarctica. satellite, launched in 2017 as part of Europe’s the air, says Xin Lan, a carbon cycle scientist
Although methane in the atmosphere Copernicus program of Earth observation. at the Earth System Research Laboratories
lasts for little more than a decade before Designed to monitor atmospheric pollution, of the National Oceanic and Atmospheric
decomposing, it is far more effective at trap- TROPOMI’s spectrometer has also proved Administration. And much of the recent
ping heat than carbon dioxide (CO2). Since capable of detecting the infrared glow of methane rise has come not from individ-
2006, atmospheric methane levels have methane. Investigators began to use the ual large emitters, but from more distrib-
grown some 7%, and the gas is responsible spectrometer to monitor regions already uted sources, including tropical wetlands,
for roughly one-third of the 1.2°C of warm- known to be large methane sources. But which could be emitting more methane in
ing since preindustrial times. As the world Aben says they realized the satellite’s frequent response to warming. But this warning sys-
approaches 1.5°C, the presumed threshold global coverage should enable it to sound the tem is still welcome, Lan says. “The ability
of “dangerous” warming, action to reduce alarm about any large methane releases. to detect emissions,” she says, “is the first
methane releases has been seen as a way to The problem was the sheer volume of step for action to reduce them.” j

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 AAAS® and Science® are registered trademarks of the American Association for the Advancement of Science, USA. Eppendorf® and the Eppendorf Brand Design are registered trademarks of Eppendorf SE, Germany.
2022 Winner
Ann Kennedy, Ph.D.
Northwestern University Feinberg
School of Medicine, Chicago, USA
For research on brain activity
underlying aggression

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0210Product.indd 529 2/6/23 3:11 PM

 FEATURES

OCEANS AWAY
Is raising salmon on land the next big thing in farming fish?

By Erik Stokstad,

W
hen drivers on Highway 94 cies. The challenges are driving vigorous
pass this tiny town, some are in Northfield, Wisconsin research efforts at universities and govern-
struck by a mysterious noc- ment laboratories.
turnal glow. Pink light ema- tors have pumped money into what many Here in rural Wisconsin, Summerfelt—
nates from the world’s largest see as the next big thing in farmed fish. Over who spent 26 years conducting aquaculture
aquaponic greenhouse, which the past decade, global sales of pink-fleshed research at the Freshwater Institute—has
can produce up to 2 million farmed salmon have nearly doubled to helped launch a salmon ranch that is putting
kilograms of salad greens each $12 billion, and demand is expected to keep innovative solutions into practice. These fish,
year. Less obvious, but also growing. Traditionally, that bounty has been unlike those at most salmon farms, never
unique at this scale, is the source of the nu- raised in large floating cages, called net pens, touch a drop of saltwater. The filters that pu-
trients used to fertilize the crops: wastewater located in coastal waters. But environmental rify the water use cheap sand instead of the
flowing from huge nearby tanks teeming with concerns and limited room for expansion costlier materials often found elsewhere. And
Atlantic salmon. The silvery fish grow in- have prompted companies to explore moving instead of simply dumping its waste, Supe-
doors, far from the ocean where wild salmon operations ashore. rior Fresh recycles it as fertilizer for the crops
normally spend the bulk of their lives. “Industry is realizing that this is a way to in that huge glowing greenhouse.
On a recent winter day, the surround- expand,” says Chris Good, head of research Summerfelt believes such approaches are
ing farmland blanketed in snow, Steve at the Conservation Fund’s Freshwater In- key to making land-based salmon farms both
Summerfelt opened the door to the fish stitute, a nonprofit research institute that economically viable and environmentally
house. Hundreds of meter-long fish swam supports sustainable aquaculture. Firms are sound. They’ve also made him something of
vigorously in each house-size tank, while fine-tuning technologies they hope will ulti- a maverick in the industry. Still, Summerfelt
an overhead crane delivered a 1-ton sack of mately enable them to raise huge numbers “has had a huge influence,” says Bendik Fyhn
feed into an automated dispenser. Rumbling of salmon in land-based facilities. These Terjesen, a fish physiologist with the Cer-
pumps and tanks filled with sand, separated cold-water fish can already be found in some maq Group in Norway, a leading producer of
from the fish tanks by a soundproof wall, improbable places, including semitropical farmed salmon. “All people in industry know
treated wastewater that had been stripped of Florida and the arid Gobi Desert. him and his work.”
fish poop. Nitrogen and phosphorus were di- For salmon farming to flourish on land—
verted to the vast greenhouse while cleansed and compete with existing sources—firms ALTHOUGH SALMON is considered seafood,
water recirculated to the salmon. “Sometimes will have to overcome some formidable wild salmon are no strangers to land.
the water is so clean it looks like the fish are challenges, including dealing with the fish The hatchlings begin their lives in coastal
swimming in air,” says Summerfelt, an en- waste and developing salmon variants that streams, where females lay thousands of
PHOTO: NARAYAN MAHON

gineer who is head of R&D at the company, thrive in tanks. Growing salmon, says Karl eggs each. The new generation heads down-
called Superior Fresh. Øystein Øyehaug, chief operating officer stream to the ocean as hand-size smolts.
The sprawling facility is just one of dozens of Atlantic Sapphire, another land-based Adult fish typically wander the high seas for
of indoor salmon farms that have sprung up farming firm, “is much, much more com- several years before returning to their home
around the world in recent years, as inves- plicated” than farming other seafood spe- streams to spawn.

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 NE WS

Life in a tank can help salmon grow faster, and with a at two farms, in Indiana and on Prince Ed- NOW 59, Summerfelt eats salmon at least
lower risk of disease, by providing optimal conditions. ward Island, and building a third in Ohio. 4 days a week. When he had his blood tested,
Over the past decade, investors have his levels of omega-3 fatty acids, healthful
In the 1730s, a German naturalist began poured billions of dollars into new land- compounds found in oily fish, were “off the
taking salmon eggs from streams and try- based salmon farms. Atlantic Sapphire has chart,” he says. He has spent decades think-
ing to hatch them. Less than a century some of the largest ambitions, aiming to grow ing about how to farm fish in a more sustain-
later, Europeans were building hatcheries 220,000 tons a year by 2031. The company’s able way. In the late 1980s, after studying
to mass raise fish to supplement declining Norwegian founders wanted to raise and chemical engineering, he was looking for a
wild stocks. These efforts didn’t have much sell salmon near North American markets doctoral project. His father, a fish biologist,
success, but in the 1960s they fueled the to avoid the need for expensive and carbon- suggested looking at the feasibility of con-
emergence of the salmon farming industry. intensive air freight from Norway. “We refer verting abandoned wastewater treatment
Norwegian farmers began to use cages float- to it as the insanity of the salmon market,” plants into aquaculture farms. “That sounds
ing in fjords to raise Atlantic salmon, fat- Øyehaug says. fun,” he remembers thinking. After wrapping
tened on special feed. The cold, clean water The company decided to set up shop in up the project, his first job was at the Fresh-
was just what the fish were used to, and they Florida, near the Everglades. It might seem water Institute.
were protected from predators. Today, fish like an unlikely location, but executives say Summerfelt eventually became director
raised in net pens, particularly off Norway the site has some hidden advantages: They of aquaculture research at the institute and
and Chile, account for 70% of the 3.7 million can pump freshwater for newborn salmon helped develop a demonstration unit, the
tons of salmon eaten worldwide each year. from one aquifer, pump saltwater from a largest at that time, for growing Atlantic
Even as the industry has boomed—and con- different aquifer for the smolts that nor- salmon commercially. In 2013, he was con-
sumer demand shows no signs of slackening— mally move to the ocean, and then inject the tacted by Brandon Gottsacker, a recent grad-
it has begun to face constraints. Prime near- wastewater into a separate cavern. “What uate of the University of Wisconsin, Stevens
shore areas are often already filled with pens, we’re doing involves micromanaging a huge Point, who was interested in setting up a
and some coastal communities have resisted number of different water quality param- fish farm. Gottsacker spent a year studying
new farms, concerned about their impact on eters that all need to be perfect,” Øyehaug at the Freshwater Institute, and then he and
views, recreational boating, and com-
mercial fishing. Researchers, regula-
tors, and environmental groups have
raised alarms about the pollution
produced by tightly packed fish, as
well as the parasitic lice and diseases
that can spread from farmed to wild
salmon. They also note that escapees
can breed with wild fish, potentially
weakening the genetic makeup of
native stocks. Such concerns have
led some areas, including British
Columbia and Washington state, to
consider barring or restricting new
coastal fish farms.
These developments have cata-
lyzed efforts to grow salmon entirely
on land in using what is known as
a recirculating aquaculture system.
The approach offers several advan-
tages, researchers say. Land-based Steve Summerfelt (left), now at Superior Fresh (right), has helped enable farming of Atlantic salmon entirely on land.
facilities can help managers control
environmental conditions and reduce dis- says. Getting it right can be tricky. In 2021, Summerfelt drew up the plans for Superior
ease risks, and they don’t face the same space about 500,000 salmon died at the Florida Fresh, which is backed by Karen Wanek and
limits as ocean farms. Production can also be facility after a clogged drain increased her husband Todd Wanek, owner and CEO
moved closer to markets, and in principle turbidity that may have generated deadly of Ashley Furniture, the world’s largest fur-
PHOTOS: (LEFT TO RIGHT) NARAYAN MAHON; SUPERIOR FRESH

growing salmon on land could contain the gases. The company’s output that year was niture manufacturer.
risks of pollution. just 2400 tons, far short of its long-range The firm began to raise salmon in a
AquaBounty, a firm with corporate head- target. (Superior Fresh sells about 680 tons pilot-scale plant in 2016. Two years later,
quarters in Maynard, Massachusetts, has of salmon per year.) Summerfelt left the Freshwater Institute to
long anticipated growing salmon in tanks. In Other companies have faced protests join Superior Fresh full time. “I finally said,
1991, the company began to commercialize over plans to build salmon farms in Maine ‘I want to jump in,’ because I couldn’t see
a strain of Atlantic salmon genetically engi- and California. Some communities fear the anyone else in the industry doing it right.”
neered for faster and more efficient growth. farms will deplete precious groundwater, The plant has since expanded 10-fold; now,
When U.S. and Canadian regulators finally or pollute aquifers or surface waters with a dozen tanks each hold up to 50,000 kilo-
approved the fish for sale years later, they re- their waste. In Maryland, one firm can- grams of fish at harvest.
quired that the salmon be raised entirely on celed plans to build a farm after scientists Designing Superior Fresh gave Summerfelt
land to prevent interbreeding with wild fish. said its wastewater could harm endan- a chance to realize some of his unorthodox
The company is now raising up to 1450 tons gered sturgeon. ideas. One involved dealing with waste, a

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 NEWS | F E AT U R E S

Salmon and salad

New land-based salmon farms have environmental advantages over marine production. But they face the challenge of dealing with the waste from the many
salmon in tanks. One company, Superior Fresh, recycles the water and converts fish waste into fertilizer for an adjacent greenhouse. The rest is added to fields.

1 Indoor facility 2 Fish tanks 3 Water treatment filters 4 Aquaponic greenhouse 5 Local efficiency
Tight biosecurity lowers the Optimal growing conditions Microbes convert toxic Salad greens grow in pallets Land farms use more power
risk of disease introduction. mean high growth rates and ammonia to nutrients for that float in water from than sea pens, but can be
Isolation from wild salmon year-round production. plants. Managers can adjust the fish tanks. Plants take closer to markets. Shipping fish
means farmed fish can’t Welfare is easier to monitor. conditions for fish health, for up nutrients and provide by truck rather than air boosts
spread parasites or viruses. example, by adding oxygen. extra revenue. overall energy efficiency.

1
3
Water
Solid waste
Liquid waste

6 2 4 5

6 Groundwater
Recycling water means 7
minimal well water is 7 Composting g
needed. Facility releases Fish waste and
almost no wastewater. plant matter aree
composted, then
applied to soil to
enrich crops and
native plants.

problem facing all factory farms with con- tower over a sump the size of a swimming Using sand as a filter can be problematic as
fined animals. In net-pen aquaculture, up to pool. Large pumps constantly push water well, researchers say. “When operating prop-
80% of the phosphorus and 60% of nitrogen up through the grains. “The whole technol- erly, it’s great,” says animal health specialist
in the feed is wasted as some drifts away un- ogy is based on injecting the water under- Deborah Bouchard, who directs the Aqua-
eaten and the fish excrete unused nutrients. neath it to lift it continuously, so the sand culture Research Institute at the University
The leftover nutrients can fuel harmful algal bed doesn’t ever collapse,” Summerfelt of Maine, Orono. But if a disease outbreak
blooms and acidification of coastal water. says. Superior Fresh keeps other aspects of strikes, sand can be much more difficult to
On land, salmon farmers have other op- the filtration system confidential and pro- disinfect. Terjesen adds another concern: “If
tions. Atlantic Sapphire, for example, land- hibits photographs. sand gets out of your biofilter then you are in
fills solid waste and pumps liquid waste Unlike at most other farms, the salmon big, big trouble with your pumps, which will
underground. Other companies convert their at Superior Fresh spend their entire lives in break down immediately.”
wastes to biogas, which is burned for energy. freshwater. Everything is simpler without
To extract as much value as possible from the salt, Summerfelt says. Freshwater means less TANKS FULL OF SALMON in another un-
nutrients, Superior Fresh uses them to grow corrosion of metal pumps and pipes, and likely location—a basement in downtown
greenhouse crops sold in about 2000 super- less risk in using ozone to purify the water; Baltimore—are a testing ground for other ap-
markets and convenience stores. They also in saltwater, ozone can cause the buildup of proaches to raising healthy fish efficiently on
restore soil nutrients in nearby fields. toxic bromine. The absence of salt also makes land. There, fish reproductive physiologist
Microbes in the indoor tanks can trans- dealing with waste easier, because biosolids Yonathan Zohar of the University of Mary-
form the toxic ammonia in wastewater into can be composted and liquids diverted to land’s Institute of Marine and Environmen-
nitrate, a form of nitrogen that can be used nearby farm fields—a nonstarter if they were tal Technology (IMET) recently tossed a
by plants. Most land-based salmon farms contaminated with salt. handful of brown pellets into a 4-meter-wide
rely on a technology called a moving bed Other firms don’t share Summerfelt’s en- tank. “Be careful, the fish—they splash,” he
bioreactor to purify the water. It uses count- thusiasm for either approach. They suggest said as several hundred large salmon lunged
less small plastic chips that swirl in bub- that although freshwater might be simpler, for the food. The scene is part of a $10 mil-
bling water, providing a substrate for the it’s not necessarily optimal for the fish. lion consortium, funded by the U.S. Depart-
microbial communities. Summerfelt, how- Young salmon get a growth spurt when they ment of Agriculture in September 2021. Led
ever, had long studied a different approach: first encounter salinity, and brackish water is by Zohar, who specializes in aquaculture, the GRAPHIC: V. ALTOUNIAN/SCIENCE

using biofilters containing sand grains, optimal for salmon’s regulation of water and partners are working on a range of biological
which provide a much greater surface area ions. In freshwater, they must expend more and engineering challenges.
for microbes to colonize. Sand is also cheap energy keeping the right balance, potentially One is water quality. The tank was sur-
and readily available. slowing their growth. Brackish water can rounded by a humming, bubbling, and
Inside Superior Fresh’s fish house, also inhibit bacterial diseases. And some frothing tangle of filtration pipes and
Summerfelt’s 6-meter-tall sand biofilters producers feel saltwater improves the taste. equipment. Zohar and his colleagues are

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 Pink lights help Superior Fresh’s leafy
greens grow through the night in the
world’s largest aquaponic greenhouse.

studying the microbes that live inside a pea, sit in refrigerated chambers. The eggs When it is time to harvest Superior Fresh’s
these filters, ultimately hoping to develop come from adult salmon in nearby tanks, salmon, workers in waterproof clothing use a
bacterial communities that are tailored which were bred by research partners in long-handled net to carefully lift the fish onto
to the specific fish strains, feed, and con- Maine. As part of ongoing trials, Zohar and a portable sluiceway. Within seconds, they
ditions used in land-based farms, which his IMET colleague Ten-Tsao Wong will im- land in a machine that stuns them. Within
could make it easier to maintain water merse some of the unfertilized eggs in a a day, the cleaned fish are being trucked
quality. “With optimal microbes the pro- bath containing antisense RNA, to disrupt to market, where they can fetch well over
cess becomes faster and more efficient,” the development of the gonads. $10 per kilogram.
says Keiko Saito, a microbiologist at IMET. The goal is to prevent a salmon’s sexual That’s about twice the price of the cheap-
“It’s balanced like an ecosystem in nature.” maturation, so that the fish instead continue est pen-raised salmon. And analysts say it
Nearby, Ph.D. student Matthew Stromberg, to invest their energy in muscle growth, en- will likely be difficult for land-based farms
an avid angler and tinkerer, fiddled with the suring large fillets. The researchers note to compete on cost. But Superior Fresh and
controls of a purification chamber, a shoul- that sexually immature fish are also less other firms have found buyers by offering
der-height blue metal tank. He is testing a sensitive to stress, which can make their a locally produced and sometimes higher
system that relies on ultraviolet (UV) light flesh less appealing. Ultimately, antisense quality fish. Superior Fresh, for example,
and a titanium catalyst to break down the might be used in commercial operations claims the feed it uses—which contains
water-borne chemicals that can give land- to prevent maturation without genetically more fish oil—gives the company’s salmon
reared salmon a muddy flavor. Bacteria that modifying the fish. twice as much of the healthful omega-3 fatty
grow in biofilms produce these metabolites, acids as other farmed salmon. The firm is
geosmin and 2-methylisoborneol, and they FOR OCEANGOING FISH confined to tanks, also betting that, by integrating its salmon
can be eliminated by purging salmon in sepa- stress is a constant risk, especially when farm with its greenhouses, it can become a
rate tanks with clean, untreated water. But they are handled. “When the fish don’t model for a profitable and environmentally
this step adds time and cost. Summerfelt is want to be moved, they can really hurt sensitive business.
also testing the UV system at Superior Fresh. themselves,” says Åsa Maria Espmark, an Superior Fresh already has plans to
Last year, he and others reported in Aquacul- aquaculture researcher with Nofima, the expand, with new farms on the East and
tural Engineering that the system, manufac- Norwegian food research institute. West coasts. And Summerfelt is once again
tured by Exciton Clean, efficiently reduces Summerfelt and his colleagues are keenly thinking about how to improve aqua-
the unwanted molecules. interested in keeping their fish calm. The culture practices. He envisions using even
PHOTO: SUPERIOR FRESH

In a separate room, Zohar and his col- biggest challenge comes when the growing bigger tanks, for example, with each hold-
leagues are working to optimize salmon fish must be moved to the largest tanks, ing 3 million liters of water—and some
for spending their entire lives in captivity. which requires a large centrifugal pump. 67,500 shimmering salmon. “It is about
Here, trays containing thousands of trans- The somersaulting journey can leave some economy of scale,” he says. And there is
lucent orange salmon eggs, each the size of fish bruised and unwilling to eat for days. plenty of room for fish on land. j

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 INSIGHTS

PERSPECTIVES

PLANT BIOLOGY

Unusual suspects in flower color evolution

The expression of phased small interfering RNAs caused monkeyflower color evolution

By Marie Monniaux The finding that flower color change was looked for the molecular determinant for
caused by an inverted repeat that produces carotenoid deposition in petals of monkey-

P
igmentation in flower petals mainly phased siRNAs constitutes an unexpected flowers. Out of the many different color-
relies on anthocyanins and carot- molecular mechanism for flower morpho- ation phenotypes found in this genus (see
enoids, two classes of pigments whose logical evolution. the photo), Liang et al. focused their study
biosynthetic and regulatory pathways Flowers display a stunning diversity of on two of them: flowers with pale pink pet-
are well characterized (1) and consti- shapes, sizes, and colors, which makes them als (low levels of anthocyanins and no carot-
tute theoretically ideal targets for evo- amenable to studying the mechanisms that enoids) and yellow nectar guides (high local
lution to create new color patterns. However, underlie morphological evolution. Flower amounts of carotenoids) and flowers with
evolution of flower color sometimes works color is a deceivingly simple trait that has bright red petals (a combination of high lev-
in mysterious ways. On page 576 of this is- been extremely labile during evolution: els of both anthocyanins and carotenoids).
sue, Liang et al. (2) show that a major spe- Sister species from several genera can show A single locus called YELLOW UPPER

PHOTO: PETER MORENUS/UNIVERSITY OF CONNECTICUT

ciation locus in Mimulus (monkeyflowers) substantially different pigmentation pat- (YUP) was found to govern carotenoid depo-
that causes change in flower color contains terns, and repeated shifts in flower color sition in monkeyflowers but was never finely
a noncoding inverted repeat region. This re- are observed within a single genus. In most mapped (4). YUP is a classical example of a
peat produces a long noncoding RNA that is cases, changes in flower color during evo- speciation locus because exchanging YUP al-
cleaved into small interfering RNAs (siRNAs) lution are simply caused by regulatory or leles between species is sufficient to directly
in a regularly spaced (“phased”) pattern, and coding mutations in the genes that control influence pollinator visits and cause repro-
some of these siRNAs regulate the transla- pigment biosynthesis. For example, the evo- ductive isolation of emerging species (5). By
tion of a carotenoid biosynthesis regulator. lution of red floral color in Petunia involved crossing two monkeyflower species that had
changes in multiple players of the anthocy- not been used in previous studies, Liang et al.
anin biosynthesis pathway (3). narrowed down YUP to an interval contain-
Laboratoire de Reproduction et Développement des
Plantes, ENS de Lyon, CNRS, INRAE, UCBL, Lyon, France. However, this is not always the case, ac- ing eight protein-coding genes. However, they
Email: [email protected] cording to the findings of Liang et al., who found that reducing the expression of these

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0210Perspectives_16562412.indd 534 2/3/23 4:13 PM

 bright red–colored petals, mutations in the reduction in anthocyanin accumulation in
YUP sequence have disrupted the phased pale pink petals was found to be caused by
pattern of phasiRNA production, and, as a an alternative start codon in the 59 untrans-
result, the major siRNA that targets RCP2 is lated region of PELAN, which causes reduced
not produced and carotenoid accumulation translation (12). This was unforeseen because
is not repressed. most of the cis-regulatory changes that have
Liang et al. also found that the se- been found to shape phenotypic variation
quence of YUP strongly matches that of a affect transcription (10). In Capsella, the
CYTOCHROME P450 (CYP450) gene found reduction of petal size has recently evolved
a small distance away, suggesting that YUP through mutations in the CYP724A1 gene, a
originated from the duplication and inver- member of the CYP450 gene family that is
sion of part of this gene. CYP450 enzymes involved in the synthesis of brassinosteroids,
take part in a large range of biosynthetic a plant hormone that controls cell prolifera-
reactions during plant development (for ex- tion (13). The mutations were found in the
ample, they participate in the production of exons of CYP724A1 and increased splicing ef-
membrane sterols, plant hormones, or de- ficiency and transcription (13). Excessive lev-
fense compounds) but were unlikely to be els of brassinosteroids limit cell proliferation,
specific players in flower color evolution, and which results in smaller petals. That exonic
they share no phylogenetic relationship with mutations affect splicing efficiency is rarely
RCP2. Pinpointing the unexpected origin and reported because they are more likely to af-
mode of action of the YUP locus would have fect protein function, and this effect could
been impossible without a well-established have been completely overlooked.
model genus such as Mimulus, which comes Therefore, it appears that each step of
with genomic information and efficient the regulation of gene expression, in all its
The petal pigmentation of transgenesis protocols. complexity (including recruitment of tran-
Mimulus (monkeyflowers) Liang et al. also found YUP to be physi- scription factors, initiation of transcrip-
involves a long noncoding cally linked to two regulators of anthocy- tion, splicing, mRNA stability, and transla-
RNA that generates small anin production in petals—PETAL LOBE tion efficiency), might be a possible target
interfering RNAs. ANTHOCYANIN (PELAN) (8) and SISTER for morphological evolution. In particular,
OF LIGHT AREAS1 (SOLAR) (2)—in a small mutations that affect protein translation or
region that they propose acts as a superlocus stability have been largely overlooked (14),
that controls petal pigmentation. A superlo- but researchers of evolution now have to
ight genes did not affect carotenoid deposi- cus (or supergene) is a group of genes that address these types of challenges for the
tion. The authors instead identified a long participate in a related function and are phys- field to move forward.
inverted repeat that was being actively tran- ically close on a chromosome; these genes are Is there something special about flower
scribed in this region; this repeat produced therefore inherited as a single locus. This al- evolution that explains why it involves un-
abundant secondary siRNAs in a phased pat- lows a favorable combination of alleles to be common molecular mechanisms more fre-
tern, called phasiRNAs (6). PhasiRNAs can maintained through generations, which is quently than the evolution of other traits in
originate from a long noncoding RNA that particularly important to avoid the dissolu- plants? It is probably more likely to be the
is cleaved into secondary siRNAs. Cleavage tion of pollination syndromes (the suite of case that flowers are visually attractive [and
sites in the RNA that contains phasiRNAs are traits jointly recognized by pollinators) over scientists are as sensitive to this as insects
regularly spaced, which generates a phased generations. A superlocus in Petunia con- can be (15)] and their morphology is ex-
accumulation of siRNAs. But of the pool of tains genes that control petal visible color, tremely diverse and directly affects reproduc-
phasiRNAs that accumulates, one siRNA ultraviolet absorption, floral scent produc- tive isolation and speciation; therefore, they
is more abundant, and, in the case of YUP, tion, pistil length, and stamen length (9). constitute an excellent system to explore the
the major siRNA matched the sequence of In monkeyflowers, the YUP-PELAN-SOLAR molecular determinants of morphological
REDUCED CAROTENOID PIGMENTATION 2 superlocus controls most of the carotenoid evolution. This has revealed the unforeseen
(RCP2), which encodes a major activator of and anthocyanin accumulation in the petals, but likely widespread ways that diversity can
carotenoid biosynthesis (7). which could maintain the two distinct petal be generated through evolution. j
Liang et al. found that a functional YUP pigmentation phenotypes that are recognized
REF ERENCES AND NOTES
locus would produce abundant siRNAs that by different pollinators.
1. E. Grotewold, Annu. Rev. Plant Biol. 57, 761 (2006).
target RCP2 and thereby reduce carotenoid That noncoding regions of the genome, 2. M. Liang et al., Science 379, 576 (2023).
accumulation in the petals of the pale pink such as the YUP locus, underlie morphologi- 3. A. E. Berardi et al., Plant Cell 33, 2273 (2021).
4. H. D. Bradshaw Jr. et al., Nature 376, 762 (1995).
flowers. The authors demonstrated that the cal evolution is not conceptually new, but 5. H. D. Bradshaw Jr., D. W. Schemske, Nature 426, 176
siRNA targets RCP2 mainly by reducing its evidence for such mechanisms is rare (10). (2003).
translation. Moreover, they provided a sat- Intriguingly, recent studies have identified 6. Y. Liu et al., Plant Cell 32, 3059 (2020).
7. L. E. Stanley et al., Plant Cell 32, 1536 (2020).
isfactory explanation for why carotenoids other unusual molecular mechanisms that 8. Y.-W. Yuan et al.,New Phytol. 204, 1013 (2014).
accumulate in the yellow nectar guides, are involved in floral evolution. For example, 9. K. Hermann et al., Curr. Biol. 23, 873 (2013).
10. A. Martin, V. Orgogozo, Evolution 67, 1235 (2013).
which serve as guiding signals for bumble similar to YUP, the recent appearance of 11. D. Bradley et al., Science 358, 925 (2017).
bee visitation: They found that another ac- a yellow pigmentation pattern in the pet- 12. M. Liang, C. E. Foster, Y.-W. Yuan, Sci. Adv. 8, eabo1113
tivator of carotenoid biosynthesis, RCP1, is als of Antirrhinum (snapdragon) is caused (2022).
13. U. Fujikura et al., Dev. Cell 44, 192 (2018).
present in nectar guides and that RCP1 re- by the SULF locus, an inverted duplication 14. A. E. Berardi, Sci. Adv. 8, eade2347 (2022).
presses YUP transcription so that the pha- that generates siRNAs that target a pigment 15. M. Adamo et al., Nat. Plants 7, 574 (2021).
siRNAs are not produced. In species with biosynthesis gene (11). In monkeyflowers, a 10.1126/science.adg2774

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 INSIGHTS | P E R S P E C T I V E S

CHEMICAL PHYSICS

Probing matter with nonlinear spectroscopy

Ultrafast nonlinear spectroscopy can unravel the dynamics of highly excited electronic states

By Stephen R. Leone1,2,3 and photons typically excite core electrons into lower bound to the shortest achievable pulse
Daniel M. Neumark1,2 unoccupied valence orbitals. Because core at 800 nm. Therefore, higher photon ener-
electrons are localized on specific atoms, the gies are needed for which the period is cor-

I
n the Bohr model of the atom, the energies of core-to-valence transitions are respondingly shorter; for example, at 100 eV
nominal orbiting period for a 1s elec- highly element specific and thus allow moni- (12.5 nm) the optical period is 41 as. Hence,
tron in the hydrogen atom is 150 as toring of the evolving electronic environment in many of the attosecond experiments per-
(1 as = 10 –18 s). Attosecond light pulses on specific atoms in photoexcited molecules. formed to date, the attosecond pulses are
are thus in principle short enough to As an example, one can use attosecond XUV formed through the process of high harmonic
follow electron dynamics in real time pulses to monitor core-to-valence transitions generation (HHG) by using a tabletop laser
(1). Moreover, with such pulses, the time- around 47 eV on the iodine atom in photo- system; HHG provides a relatively straight-
resolved measurements can be extended to excited iodomethane (CH3I). This experiment forward means of upconverting infrared and
subfemtosecond lifetimes, probe electronic captures the instant of passage through a visible electromagnetic radiation into XUV
and vibrational coherences in atoms and conical intersection (CI), a multidimensional and SXR wavelengths.
molecules, and by means of attosecond pho- crossing between electronic states in a poly- In HHG (3), an intense femtosecond la-
tochemistry, follow the earliest dynamics of atomic molecule in which the outcome of a ser pulse interacts with a nonlinear me-
photoexcited molecules as they dissociate photodissociation event is determined (2). dium—typically, inert gases at low pressure.
or isomerize. Attosecond light pulses have More generally, CIs mitigate photodamage The rare gas atoms undergo tunnel or field
recently been incorporated into four-wave of molecules and regulate many processes in ionization driven by the oscillating electric
mixing (FWM) experiments in gases and living systems. field of the laser. As the sign of the oscil-
solids, by combining one attosecond pulse To understand the technical challenges in- lating electric field reverses, the electron is
with two visible optical pulses or multiple volved in generating attosecond light pulses, driven back toward the ion and recombines,
attosecond pulses themselves. This now al- it must be understood that light with a wave- converting the gained kinetic energy into
lows more accurate determination of quan- length l represents an electromagnetic field an attosecond burst of XUV or SXR light
tum pathways in transient electronic states. with period l/c, where c is the speed of light, upon recollision. Methods have been devel-
Attosecond pulses generally lie in the ex- and that the minimum duration of a light oped that select a single burst from this pro-
treme ultraviolet (XUV; from 15 to 100 eV) pulse is approximately one optical period. For cess, a so-called isolated attosecond pulse.
and soft x-ray (SXR; from 100 to 2000 eV) near-infrared (NIR) light at 800 nm, which It is now possible to obtain 50 isolated
regions of the electromagnetic spectrum. is the center wavelength of the titanium- pulses whose photon energies extend well
XUV light below 30 eV generally excites va- sapphire lasers that are most commonly used into the SXR region (4). This allows the in-
lence electrons in atoms and molecules into in ultrafast applications, the optical period is vestigation of core-to-valence transitions in
unoccupied orbitals, whereas higher-energy 2.6 fs. This value represents an approximate an expanding number of elements because

Nonlinear spectroscopy with extreme-ultraviolet attosecond laser pulses

Attosecond four-wave mixing experiment Rydberg series Core-exciton wave function
Extreme ultraviolet (XUV) pulses and two near-infrared (NIR) XUV excitation of the 3s Rydberg state of O2 at 22 XUV excitation of a core-exciton in solid
beams pass through a sample. The off-axis emission is spatially eV decays by autoionization or dissociation. NaCl at 20 to 45 eV excites a 2p core
separated from the incident XUV beam and dispersed by a electron on the Na ion to a vacant 3s
grating onto a charge-coupled device (CCD) camera. [O2+ c4Su–] 3s
26
Oxygen Core-exciton 2.8 Å
Off-axis emission Sample NIR

XUV Na+ Cl–

24
Grating 3s
Potential energy (eV)

v=1 e–
CCD camera 22
3p

Quantum pathway Autoionization 2p

This reveals a possible quantum pathway involving one XUV 20 or
(+kXUV) and two NIR photons (–kNIR and +kNIR) that leads to v=1
GRAPHIC: A. MASTIN/SCIENCE
off-axis emission (kEMIT). Na+ core-exciton wave function
+kXUV 18 Dissociation Wave function Na Cl
XUV
–kNIR
kEMIT +kNIR
16
2 3 4 5
(g) Energy (d) (b)
Internuclear distance (Bohr)

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 the energies of these transitions generally states created by the attosecond pulse with experiment (11) provides an exquisite probe
increase with atomic number (5). Isolated respect to, for example, processes such as au- of the competition between these two chan-
attosecond pulses can also be generated toionization, dissociation, and Auger-Meitner nels, which is of fundamental interest in un-
with free-electron lasers (FELs) (6). These decay (in which one electron fills a vacancy derstanding the decay dynamics of highly
accelerator-based light sources are much and a different electron is ejected). excited molecular electronic states.
more expensive and complex than tabletop However, because multiple quantum path- Attosecond FWM can also be applied to
systems, but the reported attosecond pulse ways contribute to transient absorption in solid samples, such as the dynamics of core
intensities and photon energies are consid- this configuration, the resulting signals can excitons in solid sodium chloride (NaCl)
erably higher. be challenging to interpret. To address this (12). NaCl exhibits strong absorption bands
One of the most powerful experimental challenge, nonlinear spectroscopic tech- around 33.5 eV that are attributed to excita-
methods in attosecond science is attosec- niques are being developed, one of which is tion of Na(2p) core electrons into localized
ond transient absorption (ATAS) (7). This attosecond FWM (see the figure). These ex- electronic states of unoccupied Na(3s) or-
approach explores ultrafast dynamics in periments are based on ideas developed in bitals, essentially creating a localized hydro-
both gas-phase and solid targets. In its most infrared and visible FWM experiments, in genic-like Rydberg state within the solid. A
straightforward implementation, a NIR, which a third-order polarization created by combination of bright- and dark-state scans
visible, or UV pulse photoexcites the sam- three light fields interacting with a sample shows evidence for at least five distinct
ple, and the resulting dynamics are probed yields an emission signal at a new angle that states underlying this absorption band, all
with a time-delayed, broadband attosecond is determined by phase matching consider- of which exhibit decoherence lifetimes of
pulse that passes through the sample and is ations dictated by momentum conservation less than 10 fs. These short lifetimes are
then spectrally dispersed by a grating onto of the light field (10). attributed mainly to electronic effects, in-
an x-ray camera. An appealing feature of In tabletop experiments, an attosecond cluding configuration interactions from
this method is that even though the atto- XUV or SXR beam is incident on a target electron-electron correlation that couple
second pulse has a large range of possible and crossed by two NIR beams at a slight the multiple electronic states that compose
energies (10 to 40 eV), the spectral resolu- angle. The two NIR pulses can be indepen- the excitonic bands in this energy range.
tion is determined by the grating-camera dently delayed. However, the experiments These attosecond FWM experiments
combination and is typically 0.02 to 0.2 eV. are often run in either a “bright-state” pave the way for more ambitious nonlinear
A distinctive example of ATAS is its ap- mode, in which the two NIR pulses are co- attosecond and x-ray spectroscopy experi-
plication to the electronic nonadiabatic incident and their time delay relative to the ments, some of which are already being
(state-switching) dynamics that occur upon XUV pulse is varied, or a “dark-state” mode, carried out at FELs (13). Moreover, such
ionization of ethylene (C2H4) (8). Gas-phase in which the XUV and one NIR pulse are experiments provide the foundation for
C2H4 is ionized through its interaction with coincident and the delay between those two nonlinear spectroscopy experiments with
a 10-fs pulse at a wavelength of 1.6 mm. The pulses and the second NIR pulse is varied. multiple attosecond XUV and/or SXR light
dynamics of the resulting C2H4+ cation are In a bright-state scan, the FWM persists pulses (14). Current tabletop laser systems
followed by transient absorption of a broad- only over the lifetime of the bright state, do not provide sufficient flux at high pho-
band attosecond pulse spanning 250 to 450 yielding the lifetime of this state. Similarly, ton energies to conduct such multiple-at-
eV (28 to 50 nm). The probe pulse covers a dark-state scan probes states that are ac- tosecond-pulse experiments, but it will be
the region of carbon core-to-valence transi- cessible only by a two-photon (XUV+NIR) possible to perform them at FELs in the
tions around 280 to 290 eV in which a C(1s) transition. The noncollinear geometry future. For example, one proposed experi-
electron is excited to an unoccupied valence of the beams provides angular phase- ment that combines x-ray diffraction and
orbital. The energies of these transitions de- matching separation to distinguish the vari- stimulated x-ray Raman spectroscopy has
pend sensitively on the electronic state of the ous photon interaction pathways. the capability to directly probe the changes
cation. In this work (8), the cation is formed Attosecond FWM has been used to probe in electron density associated with passage
initially in its first excited state (the D1 state) the decay dynamics of very short-lived elec- through a CI (15). j
but then undergoes a nonadiabatic transition tronic states in both gas-phase and solid
REF ERENCES AND NOTES
to its ground state (the D0 state) in 6.8 fs. The samples. As an example, attosecond FWM
1. M. Drescher et al., Science 291, 1923 (2001).
high time resolution in this experiment thus was applied to the 3s Rydberg state of oxygen 2. K. F. Chang et al., J. Chem. Phys. 154, 234301 (2021).
shows that electronic state switching—of (O2) that lies at 21 eV (11). This state is a mem- 3. P. B. Corkum, Phys. Rev. Lett. 71, 1994 (1993).
which passage through a CI—occurs almost ber of a Rydberg series that converges to the 4. J. Li et al., Nat. Commun. 8, 186 (2017).
5. T. Popmintchev et al., Science 336, 1287 (2012).
immediately after photoexcitation. c4S–u state of O2+. It comprises a very shal- 6. J. Duris et al., Nat. Photonics 14, 30 (2020).
These experiments can also be carried out low well that supports two vibrational 7. E. Goulielmakis et al., Nature 466, 739 (2010).
in the reverse order, in which the attosecond levels. These vibrational states can decay 8. K. S. Zinchenko et al., Science 371, 489 (2021).
9. H. Wang et al., Phys. Rev. Lett. 105, 143002 (2010).
pulse interacts with the sample first, creat- through predissociation to form two O at- 10. P. Hamm, M. Zanni, Concepts and Methods of 2D Infrared
ing a coherent superposition of highly ex- oms, or through autoionization to O2+ + e¯. Spectroscopy (Cambridge Univ. Press, 2011).
cited electronic and vibrational states. In this Bright-state scans were carried out on the 11. Y.-C. Lin et al., Faraday Discuss. 228, 537 (2021).
12. J. D. Gaynor et al., Phys. Rev. B 103, 245140 (2021).
case, transmission of the attosecond pulse is valence (v) = 0 and v = 1 levels supported 13. F. Bencivenga et al., Nature 520, 205 (2015).
affected by the subsequent NIR pulse, pro- by the potential energy curve, yielding life- 14. M. Kowaleski et al., Chem. Rev. 117, 12165 (2017).
vided that the second pulse arrives before times of 5.8 and 4.5 fs, respectively. Analysis 15. Y. Nam et al., J. Phys. Chem. Lett. 12, 12300 (2021).
the highly excited states decay (9). In this of these results shows that autoionization
ACKNOWL EDGMENTS
way, lifetimes can be obtained for the excited is the dominant decay channel for both The authors are primarily supported by the director, Office of
vibrational states. Moreover, although the Basic Energy Science, Chemical Sciences Division of the US
1
Department of Chemistry, University of California, v = 1 level predissociates more rapidly than Department of Energy, under contract DE-AC02-05CH11231.
Berkeley, CA, USA. 2Chemical Sciences Division, Lawrence Additional support was provided by the Air Force Office of
the v = 0 level, its autoionization rate is Scientific Research under grant FA9550-20-1-0334 (to S.R.L.).
Berkeley National Laboratory, Berkeley, CA, USA.
3
Department of Physics, University of California, Berkeley, higher too, and both channels need to be
CA, USA. Email: [email protected]; [email protected] accounted for to interpret the results. This 10.1126/science.add4509

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 INSIGHTS | P E R S P E C T I V E S

CORONAVIRUS

Exaggerated responses to a virus long gone

Multisystem inflammatory syndrome in children is caused by abnormal cell activation

By Petter Brodin1,2 is apparent only through a symptomatic mediated T cell activation. Additionally, Lee
parent or sibling. The finding by Lee et al. et al. show that some of the MIS-C patients

T
he clearest determinant of COVID-19 that variants causing exuberant antiviral in- with OAS–RNAse L pathway mutations have
severity is age, with the majority of flammatory responses are overrepresented disproportionate expansion of T cells ex-
children experiencing mild or asymp- in 558 children with MIS-C elucidates a pre- pressing the Vb21.3 chain, suggesting com-
tomatic infections (1). But one disease viously unknown aspect of disease patho- bined or even synergistic effects of these phe-
presentation observed during the genesis but also presents a paradox. It is un- nomena underlying MIS-C (see the figure).
peak of the pandemic shows an oppo- clear why children carrying these variants Analyses of plasma cytokines indicate
site pattern. The rare but sometimes life- do not present exuberant inflammatory re- broad T cell activation in children with
threatening multisystem inflammatory syn- sponses upon initial infection. MIS-C and autoantibody repertoires, sug-
drome in children (MIS-C) has been a major Another important observation, reported gesting that such T cells can also lead to wide-
cause of pediatric morbidity and mortality by multiple groups investigating children spread B cell activation and loss of tolerance
during the pandemic. Symptoms of MIS-C with MIS-C (5), presents a similar paradox. (7–12). This could explain additional aspects
overlap partially with Kawasaki disease, a Given overlapping clinical presentations of the disease presentation and responses to
postinfectious vasculitis, and immunomodulatory therapies,
with toxic shock syndrome, such as intravenous immuno-
a bacterial toxin–mediated Exuberant inflammatory responses in MIS-C globulins. Similar to exuber-
disease caused by nonspe- Persistence of SARS-CoV-2 in the intestine of children with MIS-C could drive a self- ant myeloid cell responses in
cific activation of T lympho- propagating hyperactivation loop of tissue-resident myeloid cells and T cells. Variants in patients with deficiencies in
cytes by so-called superan- the OAS–RNase L viral RNA sensing pathway could drive inflammatory responses while the OAS–RNase L pathway,
tigens. On page 554 of this a SARS-CoV-2–derived superantigen nonspecifically activates T cells expressing the why would a superantigen-
issue, Lee et al. (2) report that Vß21.3 chain of the TCR. mediated disease present 1
variants in genes encoding Myeloid T cell to 2 months after the initial
Acute phase MIS-C cell
the 29-59-oligoadenylate syn- Mild respiratory 1 to 2 Persistent virus infection? These questions re-
thetase (OAS)–ribonuclease L infection months activates tissue- main to be answered and pre-
(RNase L) viral RNA sensing Virus persists resident myeloid sent intriguing possibilities for
pathway lead to exuberant in- in the intestine cells and T cells future research.
Hyperinflammation T cell
flammatory responses in my- SARS-CoV-2 Viral RNA activation SARS-CoV-2 vaccination or
eloid cells in individuals with Superantigen prior infection reduce the
OAS
MIS-C. But why does MIS-C RNase L risk of developing MIS-C,
develop 1 month after the ini- +++ – although there are some
Vß21.3
tial infection? Innflamma
Inflammatory
ammato rryy ×
ttory
oory reports of rare vaccine-
The strength and early cytokines triggered MIS (MIS-V) (13).
activation of the type I in- MIS-C, multisystem inflammatory syndrome in children; OAS, 2′-5′-oligoadenylate synthetase; RNase L, ribonuclease
However, it is difficult to be
terferon (IFN-I) pathway L; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TCR, T cell receptor; Vß21.3, variable-ß21.3. sure of a causal role for SARS-
at the site of initial severe CoV-2 vaccination in trigger-
acute respiratory syndrome coronavirus 2 among children with MIS-C and patients ing MIS-V in populations with extensive
(SARS-CoV-2) infection—the airways and with toxic shock syndrome—a disease SARS-CoV-2 circulation and possible simul-
mouth—is the most important determinant caused by bacterial toxins that nonspecif- taneous exposure to virus and vaccine. It
of disease severity; individuals who fail to ically activate T lymphocytes through the would be interesting to investigate the ge-
mount such responses are at higher risk of expression of superantigens—a search for netic variants reported by Lee et al. in the
severe COVID-19 and death (3). Children on superantigens in SARS-CoV-2 infections was context of suspected MIS-V because mRNA
average seem to have a primed and more initiated. Superantigens are bacterial or viral encoding the Spike protein from vaccines
efficient IFN-I system in the airway mucosa proteins that bind T cell receptors (TCRs) in would also be expected to trigger exuberant
and consequently respond efficiently, lead- nonspecific ways, in contrast to the peptide responses in individuals with OAS–RNase
ing to mostly mild and even asymptomatic complexed with a major histocompatibility L deficiencies. Interestingly, with increas-
disease (4). Such efficient IFN-I responses complex (MHC) molecule that only trig- ing population immunity and/or changing
also likely occur in children who subse- gers a subset of T cells that recognize such properties of SARS-CoV-2 variants, decreas-
quently develop MIS-C because most such complexes through their TCR. Structural ing rates of MIS-C have been seen during
children report mild and sometimes unno- modeling revealed one potential superan- 2022. Long Covid, another postinfectious GRAPHIC: N. CARY/SCIENCE

ticeable COVID-19, and often the infection tigen motif within the Spike protein (6). A disorder that is triggered by SARS-CoV-2 in-
disproportionate increase in T cells carrying fection, also has multisystem involvement
the variable-b21.3 (Vb21.3) chain of the TCR that partially overlaps with the clinical
1
Department of Women’s and Children’s Health, Karolinska paired with various Va chains in children features of MIS-C, including vasculopathy,
2
Institutet, Solna, Sweden. Department of Immunology and
Inflammation, Imperial College London, London, UK. with MIS-C has been reported (5), which in- coagulopathy, and immune cell activation.
Email: [email protected] directly suggests SARS-CoV-2 superantigen– Variants in the OAS–RNase L pathway

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 would also be of interest in children who GEOCHEMISTRY
develop Long Covid (14).
Overall, the findings of Lee et al. together
with reports of superantigen-mediated T
cell activation, the rapid response to im-
A window into prebiotic worlds?
munomodulatory therapies, and the rare Zircons reveal the geochemistry of Earth’s earliest
reoccurrence of inflammation after a few
days of treatment suggest that MIS-C is
hydrothermal systems
driven by a dysregulated and possibly self-
propagating loop of activated myeloid cells By Laura E. Rodriguez riod, a broad understanding of the early
and T cells. The main question to be re- Earth has been informed by 3.3- to 4.4-Ga-

T
solved relates to the delayed presentation o search for life on other worlds, old zircon (ZrSiO4) specimens from Jack
after acute infection and the role of SARS- understanding the prebiotic chem- Hills, Australia.
CoV-2 in specifically driving MIS-C disease. istry of those worlds is necessary Zircons are extremely resilient miner-
Most children with MIS-C have negative (1). However, to know which organic als and can survive beyond the weathering
SARS-CoV-2 polymerase chain reaction compounds form in the absence of away of their host rock. Upon crystalliza-
(PCR) tests in the respiratory tract, but life, and the conditions conducive for tion, zircons record the isotopic and el-
the virus may persist elsewhere and drive life’s emergence, requires an understand- emental signature of their surroundings.
MIS-C. SARS-CoV-2 can persist for a longer ing of the geochemical environment (e.g., Hence, studies of zircons formed in an-
period in the intestines, particularly in chil- fluid salinity, metal availability, etc.) in cient mantle melts have revealed much of
dren, and most children with MIS-C present which those compounds existed. As Earth what is known about early Earth, includ-
with intestinal symptoms, including pain, is the only example for life, origins-of-life ing that liquid water was present by ~4.3
vomiting, or loose stools (15). studies simulating early Earth conditions Ga ago (5). They also indicate that ~4.4 Ga
One possible explanation for the delayed can inform this endeavor. Unfortunately, ago, Earth’s mantle was near its current
presentation is viral persistence in the in- the geological record of early Earth is redox state (the balance of oxidants and
testine and the need for repeated stimula- sparse, and the constituents of ancient reductants) (6).
tion to pathogenically activate myeloid and rock specimens have been heavily altered The redox state of the early Earth
T cells. A potential explanation for the de- over time. Consequently, has been at the center
layed presentation in MIS-C is viral persis-
tence and repeated stimulation of intestinal
the fluid geochemistry of
prebiotic environments
“…contrary to popular of origins-of-life discus-
sions since the field took
myeloid cells and T cells. It is also possible on early Earth remains opinion, relatively off, with many suggest-
that the reason for MIS-C not occurring dur- uncertain. On page 582 ing that a reducing at-
ing acute infection in the airways but only of this issue, Trail and oxidizing systems…can mosphere—that is, one
during the postinfectious phase is a result
of distinct aspects of tissue-resident cells in
McCollom (2) report that
zircons—natural sili- create exceptionally dominated by gases that
donate electrons (H2,
the intestine. The dependency on repeated
stimulation of tissue-resident cells could
cate minerals in Earth’s
crust—can constrain the
favorable conditions NH3, CH4, and H2S)—was
required for life to arise
explain the delayed presentation of disease, fluid geochemistry of an- for prebiotic synthesis.” (7). Earth’s early atmo-
despite the systemic expression of predis- cient hydrothermal sys- sphere was composed of
posing OAS–RNase L variants. Support for tems. This provides a potential path to mantle gases expelled during volcanism.
this idea comes from the observation that unraveling the fluid composition of early The speciation of mantle gases was con-
SARS-CoV-2 persists in the stool of MIS-C Earth and discerning life’s origins. trolled by the mantle redox state, mea-
patients, and increased intestinal permea- The origins of life on Earth are gener- sured by the amount of oxygen present
bility and inflammation lead to detectable ally thought to have occurred sometime (oxygen fugacity, fO2). Within the mantle,
Spike protein in circulation (15). Thus, the between 4.5 and 3.5 billion years (Ga) ago. the fO2 is buffered by the surrounding min-
rarity of MIS-C could be explained by the This range is partially constrained by the eral assemblage. For present-day Earth,
frequencies of predisposing variants, such oldest accepted evidence for life—3.48- the mineral buffer is fayalite, magnetite,
as those reported by Lee et al., and possi- Ga-old fossilized stromatolites. These are and quartz (FMQ), which favors outgas-
ble variants associated with superantigen- carbonate structures produced by photo- sing of neutral and weakly oxidized gases
mediated T cell activation (12). j synthetic microorganisms that were discov- (N2, H2O, CO2, and SO2) (8). Given that
REFERENCES AND NOTES
ered in the Dresser Formation, Australia, Earth’s mantle ~4.4 to 3.8 Ga ago had an
1. C. Smith et al., Nat. Med. 28, 185 (2022). an area containing some of Earth’s oldest estimated FMQ value similar to that of the
2. D. Lee et al., Science 379, eabo3627 (2023). rocks (3). Additionally, formation of the present day, the prebiotic atmosphere was
3. Q. Zhang et al., Nature 603, 587 (2022).
4. P. Brodin, Immunity 55, 201 (2022).
Moon constrained how early life could likely redox-neutral (6).
5. M. N. Rivas et al., Front. Immunol. 13, 941009 (2022). have arisen. It is generally thought that the Zircons (>650°C, ~5 to 10 kbar) have been
6. M. H. Cheng et al., Proc. Natl. Acad. Sci. U.S.A. 117, 25254 Moon formed during a collision between used to deduce the redox state of the mantle
(2020).
7. C. R. Consiglio et al., Cell 183, 968 (2020). Earth and another small planet. This im- and, in turn, inform the atmospheric com-
8. C. N. Gruber et al., Cell 183, 982 (2020). pact event likely would have wiped out any position of the early Earth. What is less
9. K. Sacco et al., Nat. Med. 28, 1050 (2022).
10. A. Ramaswamy et al., Immunity 54, 1083 (2021).
life predating 4.5 Ga ago (4). Despite there known is the redox state and geochemis-
11. M. Moreews et al., Sci. Immunol. 6, eabh1516 (2021). being scarce geologic evidence of this pe- try of near-surface aqueous environments
12. R. A. Porritt et al., J. Clin. Invest. 131, e151520 (2021). where prebiotic organic chemistry relevant
13. N. Ouldali et al., Lancet Reg. Health Eur. 17, 100393 (2022).
14. P. Brodin et al., Nat. Med. 28, 879 (2022). to the origins of life would have been most
Lunar and Planetary Institute, Universities Space Research
15. L. M. Yonker et al., J. Clin. Invest. 131, e149633 (2021). Association (USRA), 3600 Bay Area Boulevard, Houston, prevalent. Notably, zircons formed in man-
10.1126/science.adg2776 TX 77058, USA. Email: [email protected] tle melts can be transported upward in the

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 INSIGHTS | P E R S P E C T I V E S

crust where they are exposed and altered cient hydrothermal fluids would have thus characterize the geochemistry of other hy-
by hydrothermal (>100°C) fluids (9). Upon been ripe for prebiotic organic synthesis. In drothermal systems captured within ancient
recrystallization, the zircons capture the sum, Trail and McCollom have shown that, zircons, including those found on other
geochemistry of these fluids (see the figure). contrary to popular opinion, relatively oxi- worlds. Mars is predicted to have a pre-
Leveraging synthetic zircons, Trail and dizing systems—which are thought to not ponderance of zircons (13) from rocks that
McCollom devised an approach to deter- be conducive for origins-of-life chemistry had been altered by ancient fluids. Indeed,
mine the geochemistry of the hydrothermal (7)—can create exceptionally favorable con- zircons that recrystallized within hydrother-
fluids (~580°C) from which eight of the Jack ditions for prebiotic synthesis. mal fluids have been identified in martian
Hills zircons had recrystallized ~3.9 Ga ago The calibration and approach developed meteorites (14). The NASA–European Space
(10). The synthetic zircons were crystallized by Trail and McCollom could be applied to Agency’s Mars Sample Return campaign
in hydrothermal fluids (1200° will return samples collected
to 900°C, 10 kbar) of varying re- from within the Jezero crater
dox states to formulate an equa- Zircons capture the geochemistry of ancient Earth which, having been subjected
tion that calculated fO2 from The habitability of aqueous environments on early Earth can be discerned to impact-induced hydrother-
redox-sensitive cerium (Ce3+, from chemical information captured in zircons, resilient silicate minerals found in mal alteration ~3.8 Ga ago (15),
Ce4+) and redox-insensitive ele- the mantle and crust. Fluid geochemistry deduced from zircons can serve as could host recrystallized zircons
ments lanthanum (La) and pra- input for models and simulations to explore the synthesis of organic compounds that captured the geochemistry
seodymium (Pr). The partition in ancient fluids. of potentially prebiotic fluids.
coefficient—a ratio of total Ce in Applying the approach from
the fluid versus in zircon—was Zircon exposure to hydrothermal fluids Trail and McCollom to these
Natural zircons from igneous rocks are present in mantle melts. These zircons can
determined and normalized to transport upward into the crust where they can be exposed to—and altered samples may not only inform
the partition coefficients of La by—hydrothermal fluids. Fluid sources include rainfall or seawater that circulate fluids the habitability of Mars but
and Pr. Notably, Ce4+ is more into and out of the crust. could also provide geochemical
readily captured in crystallizing constraints for prebiotic studies.
zircons than Ce3+. Thus, nor- Such constraints, in turn, could
malizing to La and Pr reveals elucidate the origins of martian
if there is excess Ce in zircon, Hydrothermal pool organics detected within these
which would indicate that the Seawater
samples and the probability
zircon formed in oxidized flu- of their having an abiotic or bi-
ids enriched in Ce4+. Using this otic origin. j
calibration, Trail and McCollom
REF ERE NCES AND NOTES
determined that the redox state Crust 1. L. M. Barge, L. E. Rodriguez, J. M. Weber,
of the hydrothermal fluids from B. P. Theiling, Astrobiology 22, 481
which the eight Jack Hills zir- (2022).
cons formed was slightly more 2. D. Trail, T. M. McCollom, Science 379,
oxidized than the upper mantle 582 (2023).
3. R. J. Baumgartner et al., Geology 47,
from that time period (11). Mantle 1039 (2019).
Once equipped with the fO2, 4. G. R. Osinski et al., Astrobiology 20,
temperature (10), and salinity 1121 (2020).
5. S. J. Mojzsis, T. M. Harrison, R. T.
(10) of the hydrothermal fluids, Zircon crystallization Pidgeon, Nature 409, 178 (2001).
Trail and McCollom simulated A zircon crystal present in the crust can dissolve into its elemental constituents in a 6. D. J. Smythe, J. M. Brenan, Earth Planet.
the geochemical evolution of hydrothermal fluid. Consequently, its age, as determined by uranium (U) decay into lead Sci. Lett. 453, 260 (2016).
(Pb), is lost. However, zircons can recrystallize, trapping certain constituents of the 7. G. Schlesinger, S. L. Miller, J. Mol. Evol.
the fluids as they progressed environment. This process “records” the geochemistry of the fluid and resets its age. 19, 383 (1983).
upward through Earth’s crust. 8. J. F. Kasting, Science 259, 920 (1993).
Although the fluids were rela- O2– Pb 9. M. J. Bojanowski, B. Bagiński,
Pb Pb E. Clarkson, R. Macdonald, L.
tively more oxidized than the O2–
Marynowski, Contrib. Mineral. Petrol.
Pb U
modern mantle, the litho- Pb 164, 245 (2012).
spheric fluids at equilibrium Dissolve
Dis Recrystallize 10. H. Tang, D. Trail, E. A. Bell, T. M. Harrison,
Geochem. Perspect. Lett. 9, 49 (2019).
(580°C, 5 kbar) were predicted 11. L. Gao et al., Nat. Commun. 13, 3283
O2– Zn4+ U
to have been enriched in H2S U (2022).
(versus SO4) and transition U Si4+ O2–
12. G. D. Cody et al., Geochim. Cosmochim.
metals (Cu, Mn, and Zn) that Pb Acta 68, 2185 (2004).
U 13. M. M. Costa et al., Proc. Natl. Acad. Sci.
together could have driven U.S.A. 117, 30973 (2020).
abiotic carbon fixation—the 14. M. Guitreau, J. Flahaut, Nat. Commun.
A snapshot of ancient hydrothermal fluids 10, 2457 (2019).
conversion of inorganic carbon As zircon recrystallizes, certain ions (Ce3+, Ce4+, Cl–, Ti4+) are incorporated, depending on 15. K. A. Farley et al., Science 377, 1512
into organic compounds (12). the redox state, salinity, and temperature of the fluid environment. For example, in a (2022).
The models suggested that by high-salinity fluid, the crystal traps more Cl–; at higher temperatures, it traps more Ti4+.
the time the fluids breached ACKNOWLE DGMENTS GRAPHIC: A. FISHER/SCIENCE
More reduced More oxidized Higher salinity Higher temperature
the surface, they would have This article is Lunar and Planetary Institute
Ce3+ Ce4+ Cl– (LPI) contribution no. 2874. LPI is operated
been enriched in ions with Ce3+
Ti4+
by Universities Space Research Association
2+ Ce3+ Ce 4+
– Ti4+
known catalytic potential (Fe , Cl under a cooperative agreement with the
Fe3+, Ni2+, Mn2+, and Ca2+) and Ce 3+
Ce 3+
Ce 4+ Science Mission Directorate of NASA.
reduced gases (CH4 and H2S).
Ce3+ Cl–
A pool sourced from these an- 10.1126/science.adg2630

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 P OLICY FORUM
HUMAN GENETICS

Public views on polygenic screening of embryos

Understanding moral acceptability and willingness to use is crucial for informing policy

By Michelle N. Meyer1,2, Tammy Tan3, for biological sex and conditions such as be distinct from whether they themselves
Daniel J. Benjamin3,4,5, David Laibson3,6, deafness. In contrast to those, a PGI—also would use the technology. To our knowl-
Patrick Turley3,7,8 called a polygenic risk score—is based on edge, this paper is the first to measure PGT-
the estimated associations (calculated from P use intentions—and the effects of social

F
or decades, people have used genetic a large-scale genetic study) between com- norming on these intentions.
information to exercise control over mon genetic variants and a particular phe-
the kinds of children they will have. notype. This gene-based index can then be ACCEPTABILITY AND WILLINGNESS
These technologies have largely tar- used to make phenotypic predictions—not In January 2022, we conducted a preregis-
geted chromosomal and monogenic only to avoid serious disease but also to try tered, nationally representative US survey-
disorders and traits; but most hu- to select for phenotypes such as greater cog- based experiment on the attitudes of 6823
man phenotypes are highly polygenic (and nitive ability or educational attainment. people towards three services: PGT-P, gene
influenced by the environment). One tech- Another technology that targets the entire editing, and—as a nongenetic benchmark
nology that targets the entire genome— genome and could, in principle, vastly ex- for attitudes toward interventions targeted
preimplantation genetic testing for poly- pand our ability to select for or against any at college admissions—courses to prepare
genic risk (PGT-P)—uses polygenic indexes heritable phenotype is germline genome ed- for the SAT test (effective N after applying
(PGIs) to predict the expected value of the iting (hereafter “gene editing”)—for instance, weights, 3805; see table S1 for sample char-
phenotype(s) that would arise for each em- acteristics). We randomized participants to
bryo if successfully transferred; parents can
use these predictions to select an embryo
“…public views should influence answer two questions, in randomized order,
about one of these three services. One ques-
for in vitro fertilization (IVF). Seeing gaps
in evidence and analysis relevant for po-
policy-making but alone do not tion asked whether the respondent views
the service as morally acceptable, morally
tential policy discussions around PGT-P, we determine appropriate policy.” wrong, or not a moral issue; participants
conducted a survey of public attitudes. Our could also indicate whether they were un-
data suggest that it would be unwise to as- with clustered regularly interspaced short sure. For this question, both PGT-P and gene
sume that use of PGT-P—even for controver- palindromic repeats (CRISPR). This might editing were described as being potentially
sial traits—will be limited to idiosyncratic someday be used to try to influence offspring used for “medical and nonmedical traits.”
individuals, or that it has little potential to characteristics by making thousands of DNA The other question measured willing-
cause or contribute to society-wide changes edits (or more) to a gamete or embryo. ness to use each service by asking partici-
and inequities. However—with the notable exception of pants how likely it was—on a scale from 0
Historically, technologies to enable con- three Chinese children whose C-C chemo- to 100%—that they would use the service to
trol over offspring have included carrier kine receptor type 5 (CCR5) genes were il- increase the odds that their offspring will
screening, ultrasound, preimplantation licitly edited while they were embryos in attend a top-100 college by selecting for
genetic diagnosis, amniocentesis, chori- a misguided attempt to provide them with genetic variants, or enrolling their child in
onic villus sampling, noninvasive prenatal AIDS resistance (1, 2)—gene editing has courses, associated with higher educational
screening, and selective abortion. Using not been used. Indeed, it is not permitted attainment. We asked participants to as-
them, people have selected against diseases in some 70 countries (1), and experts have sume that each service was free. We also
such as Huntington’s, Down syndrome and called for a global moratorium (2). asked them to assume a realistic effect size:
other trisomies, and alleles [such as patho- PGT-P, by contrast, is already offered by We told them that about 3% of high school
genic breast cancer gene (BRCA) variants] at least one US company whose embryo seniors attend a top-100 ranked college, and
that increase an individual’s lifetime risk screening business operates in several that each service would raise their likeli-
of certain diseases. They have also selected countries and US states (3). Yet it has re- hood of having such a child by two percent-
ceived far less academic, policy, and regula- age points (from 3 to 5%). In the cases of
1
Department of Bioethics and Decision Sciences, Geisinger tory analysis than gene editing, leading to gene editing and PGT-P, we asked them to
Health System, Danville, PA 17822, USA. 2Geisinger calls for urgent research about public at- assume that they were already using IVF
Commonwealth School of Medicine, Scranton, PA 18510, titudes towards PGT-P (4). Recent surveys and that the add-on service was safe. Fi-
USA. 3National Bureau of Economic Research, Cambridge,
MA 02138, USA. 4Anderson School of Management, have measured acceptance of gene editing nally, we further randomized participants
University of California Los Angeles, Los Angeles, CA (5, 6), intentions to use gene editing (7), and within each “service condition” to be told
90095, USA. 5David Geffen School of Medicine, University views about whether certain forms of em- that it was used on average by either “1 out
of California Los Angeles, Los Angeles, CA 90095, USA.
6
Department of Economics, Harvard University, Cambridge, bryo selection should be legally permitted of every 10” or “9 out of every 10” similarly
MA 02138, USA. 7Center for Economic and Social Research, (8). Someone’s view about whether the law situated people (for the PGT-P and gene
University of Southern California, Los Angeles, CA 90089, should prohibit a technology may be dis- editing arms, “people currently having ba-
USA. 8Department of Economics, University of Southern
California, Los Angeles, CA 90089, USA. tinct from their view of whether the tech- bies”; for the SAT prep arm, “people who
Email: [email protected] nology is morally acceptable, and both may currently have high-school–age children”).

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 INSIGHTS | P O L I C Y F O RU M

SOCIAL NORM, AGE, AND EDUCATION (65% versus 54%, P = 1.7 × 10-4), and SAT ing as a future technology; people’s views
A minority of participants (41%) said they prep (86% versus 71%, P = 3.9 × 10-10) are of gene editing might become more posi-
had no moral objection to gene editing for morally acceptable or not a moral issue (see tive if it becomes available. To isolate par-
“certain medical and nonmedical traits” the figure and table S5). They also reported ticipants’ attitudes about shaping offspring
(i.e., they reported it was morally accept- a greater likelihood that they themselves characteristics, in assessing willingness to
able or not a moral issue), and a majority would use gene editing (38% versus 32%, use, we also asked them to assume that
of participants reported no moral objection P = 0.008), PGT-P (48% versus 40%, P = 1.6 each service was free—and, for PGT-P and
to PGT-P (58%) or SAT prep (76%) (fig. S1 × 10-4), and SAT prep (78% versus 67%, P = gene editing, that they were already using
and table S2). On average, participants said 4.7 × 10-17) to increase the odds that their IVF. Our data thus do not measure willing-
they were 34% likely to use gene editing, child attends a top-100 college (see the fig- ness to use PGT-P or gene editing among
43% likely to use PGT-P, and 69% likely to ure and table S5). These results might re- those who would not otherwise already be
use SAT prep to increase the odds of their flect parents’ tendency to try to mirror their using IVF. However, the number of babies
child attending a top-100 college (see the own educational outcomes in the outcomes born through assisted reproductive tech-
figure and table S2). Furthermore, a ma- of their offspring, which would have impli- nologies has more than tripled between
terial fraction of participants reported a cations for other phenotypes. 1996 and 2017 (9). Moreover, developments
>50% likelihood of using each service (28% in stem cell research are expected to make
gene editing, 38% PGT-P, 68% SAT prep; DISCUSSION IVF much less financially and physically
table S2). As predicted, those who were told In the US, there appears to be both greater costly (10).
that 90% of relevant people use each service moral acceptance of, and greater willing- Other aspects of the scenario we pre-
were more likely to say that they, too, would ness under certain circumstances to use, sented suggest that our results may repre-
use it, compared to those who were told PGT-P versus gene editing—and the more sent an underestimate of acceptability and
that 10% of people were using it. The mean people use PGT-P, the more likely others potential uptake. For one, in measuring
willingness to use gene editing, PGT-P, and say they would, too. Those circumstances, potential uptake, we asked about an espe-
SAT prep was 4 (P = 0.020), 5 (P = 0.007), of course, matter. We asked participants to cially controversial use of these technolo-
and 4 (P = 0.022) percentage points higher, assume that each service is safe; because gies: not to avoid serious disease, but to
respectively, for those in the 90% condition CRISPR currently carries considerable increase the odds that the resulting child
(table S3). These effect sizes are typical of risks to offspring (2), our results may over- will be admitted to a top-100 college. Prior
those reported for behavioral intentions estimate acceptability of and willingness to surveys measured attitudes toward gene
from other social norm manipulations. use CRISPR. Additionally, we (accurately) editing and PGT-P without quantifying
A recent study of 2233 UK residents portrayed PGT-P as available but gene edit- how effective the technology would be,
between the ages of 16 and 75 found that
those under 35 were more likely than older
participants to “support” (rather than “op- Moral acceptability and willingness to use, by age and education
pose”) both sex selection by IVF patients Left: Mean likelihood of using gene editing, preimplantation genetic testing for polygenic risk (PGT-P), and
and embryo gene editing for “preferred courses to prepare for the SAT college admissions test, to increase participants’ chances of having a child who
characteristics” such as “eye color, height, attends a top-100 college by 2 percentage points (from 3% to 5%). Error bars are 95% confidence intervals.
hair color, etc.” (5). In the first of two ex- Low educational attainment reflects associate degree or below, high reflects bachelor’s degree or above.
ploratory analyses that we conducted, al- See supplementary materials for P values and standard errors. Right: Degree of moral acceptability of each
though the moral attitudes of those under service. Some bars do not sum to 100% owing to rounding.
35 years of age toward all three services
were statistically indistinguishable when Willingness to use each service Moral acceptability of each service
compared to the full sample (all P values >
Gene editing PGT-P SAT prep Morally wrong Not Sure
0.59; fig. S1 and table S4), those under 35 100 Not a moral issue Morally acceptable
reported a higher willingness than the full
Mean likelihood of use

sample to use gene editing (41% versus 34%, 75 Gene editing

P = 4.0 × 10-4), PGT-P (48% versus 43%, P
= 0.013), and SAT prep (72% versus 69%, 50 Low 29 33 22 17
P = 0.039) for educational attainment (see 69 72
the figure and table S4). (All of these esti- 25 48 High 31 23 22 24
34 41 43
mates account for sample overlap between
Educational attainment

the two groups; the statistically equivalent 0

All Under 35 All Under 35 All Under 35 PGT-P
tests comparing those under 35 and those
Age
35 and older are in table S4.) Approximately Low 17 29 30 24
equal moral acceptance among age groups Gene editing PGT-P SAT prep
but differential willingness to use these ser- 100 High 17 19 31 34
Mean likelihood of use

vices might reflect younger people being

the natural use population for reproductive 75
technologies and (as recent students rather
SAT prep GRAPHIC: K. FRANKLIN/SCIENCE

than parents) SAT prep. 50

78 Low 8 21 44 27
In the second exploratory analysis, com- 64
pared to those with less educational attain- 25
40 48
32 38 High 6 9 59 27
ment, those who had at least a bachelor’s
0
degree were more likely to say that gene Low High Low High Low High 0 25 50 75 100
editing (46% versus 39%, P = 0.012), PGT-P Educational attainment Percent of participants

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 whereas we stipulated a realistic effect size US, found that only 18% “agreed with the genetic ancestries, we must consider the ex-
(see supplementary materials). In prior re- use of ” gene editing for intelligence, and a tent to which a free market for PGT-P might
search, we expressed concern that an unre- 2017 survey of 11 countries, again includ- exacerbate or create new health or social
alistic lay view of what PGT-P can deliver ing the US, found very little intention to inequities, and ask whether society should
might drive appetite for the service. We use gene editing to “enhance” offspring tolerate that result. How can we ensure that
recommended (inter alia) that advertised “memory and learning capacities” and lit- those with traits that others select against
effect sizes focus on absolute gains rather tle variation across countries (7). In light remain fully welcomed members of our so-
than relative (proportional) gains, which of our recent findings of much higher gene ciety? Should PGT-P be limited to certain
seem large in part because they are calcu- editing acceptance (41%) and substantial traits, and if so, who would draw that line,
lated from a small base (11). Others have gene editing and PGT-P use intentions in and how? Finally, in the wake of the 2022
similarly warned that PGT-P is of “limited the US to increase educational attainment, decision by the US Supreme Court in Dobbs
utility” for phenotypes such as height and it is plausible that considerable gene edit- v. Jackson Women’s Health Organization,
cognitive ability (12). Yet, although we can- ing and PGT-P use intention now also char- the US is experiencing uncertainty about
not know whether the realistic effect size acterizes attitudes in other countries. the legal status not only of abortion but also
that we stipulated, appropriately commu- Public views of technology should in- of other reproductive decisions that are un-
nicated in absolute terms, was perceived fluence policy-making in a democratic precedented in the past half century. Could
by participants as large or small, it still society. Experts who are critical of a tech- choices about PGT-P be regulated without
resulted in a substantial share of people nology should not assume that the public further threatening other reproductive
expressing interest in using PGT-P. shares their knowledge or viewpoint. Un- choices?
As important as how many people are derstanding which members of the public Aside from urging the Federal Trade
interested in PGT-P is which people are are most likely to use a technology can Commission to ensure proper communica-
interested. That potential uptake is higher inform predictions about societal impact— tion of PGT-P and its expected gains (10),
among younger generations compared to for example, the extent to which it is likely we do not prejudge what additional regu-
the full sample is notable, because younger to enlarge existing or create new dispari- lation, if any, is warranted. But we call on
generations are the ones who are in a posi- ties. Much more should be learned about any policy choice—including not to further
tion to use PGT-P. Some critics of PGT-P the public’s reflexive as well as considered intervene—to be made deliberately, after in-
argue that the technology’s effectiveness in judgments. For instance, which other traits put by experts and the public. j
selecting for offspring phenotype is trivial. do people want to select for or against?
REF ERENCES AND NOTES
But PGT-P has been estimated by multiple How does relaxing the assumptions of free
1. F. Baylis, M. Darnovsky, K. Hasson, T. M. Krahn, CRISPR J.
independent researchers to have some im- access and safety affect the rates, and dis- 3, 365 (2020).
pact, even if the expected gains are not tribution across groups, of moral accept- 2. E. S. Lander et al., Nature 567, 165 (2019).
what some consumers might imagine and ability and willingness to use? We should 3. LifeView, https://www.lifeview.com/tests_pgtp.html.
some companies might suggest (11–13). learn the extent to which people who would 4. G. Lázaro-Muñoz, S. Pereira, S. Carmi, T. Lencz, Genet.
Med. 23, 432 (2021).
That those who themselves have higher ed- not otherwise use IVF would do so to use 5. Progress Educational Trust, “Fertility, Genomics
ucational attainment are more interested PGT-P. More subtly, legal frameworks for and Embryo Research: Public Attitudes and
in using PGT-P for this phenotype raises reproductive technologies vary widely (1); Understanding,” June 2022; https://www.progress.
the risk that PGT-P will exacerbate exist- the current US path is one of continuing to org.uk/wp-content/uploads/2022/06/pet_
fertilitygenomicsembryoresearch.pdf.
ing inequalities. Over several generations, treat offspring-influencing technologies as
6. T. McCaughey et al., Cell Stem Cell 18, 569 (2016).
the gains from PGT-P could build on one a matter of individual, private choice, de- 7. G. Gaskell et al., Nat. Biotechnol. 35, 1021 (2017).
another, resulting in familial transfers of spite their potential societal impact. Peo- 8. S. Zhang, R. A. Johnson, J. Novembre, E. Freeland, D.
socially-favored phenotypes that mirror ple might have preferences against societal Conley, Soc. Sci. Med. 274, 113796 (2021).
9. S. Sunderam et al., MMWR Surveill. Summ. 69, 1 (2020).
and—given the costs of IVF—amplify un- inequality that coexist with the preference
10. H. T. Greely, The End of Sex and the Future of Human
equal familial transfers of wealth. to improve outcomes for their own family Reproduction (Harvard Univ. Press, 2018).
members. Specifically, someone might pre- 11. P. Turley et al., N. Engl. J. Med. 385, 78 (2021).
CONCLUSION fer that inequality be reduced at the aggre- 12. E. Karavani et al., Cell 179, 1424 (2019).
Media reports of early adopters of PGT-P gate level, but still choose an inequality- 13. T. Lencz et al., eLife 10, e64716 (2021).
14. C. Goldberg, “The Pandora’s box of embryo testing
might suggest that it is a fringe issue un- amplifying technology for themselves. Our is officially open,” Bloomberg, 26 May 2022; https://
worthy of policy attention (14). But the study does not explore these issues, and www.bloomberg.com/news/features/2022-05-26/
sharp turn in public opinion about IVF future work should. dna-testing-for-embryos-promises-to-predict-genetic-
itself shows that innovations that are ini- But despite their relevance, public at- diseases#xj4y7vzkg.
15. A. V. Bell, in Misconception: Social Class and Infertility in
tially met with limited uptake and even titudes alone do not determine appropri- America (Rutgers Univ. Press, 2014), p. 6.
active resistance can quickly become nor- ate policy. Additional research and diverse
malized and widely adopted. A 1969 Harris expert input should inform several policy ACKNOWL EDGMENTS
poll found that most Americans objected to questions: Given our limited knowledge of The authors are supported by Open Philanthropy grant
IVF, and the American Medical Association the unintended effects of selecting for and 010623-00001 (M.N.M., P.T., D.J.B.); National Institute
on Aging grant R24-AG065184 (T.T., D.J.B.); National
called for a moratorium on IVF research. against particular phenotypes, does wide- Institute on Aging grant R01-AG042568 (T.T., D.J.B.) and
In 1978, 1 month after the well-publicized spread use of PGT-P pose acceptable popu- R00-AG062787-04 (P.T.); and Pershing Square Fund for
birth of the first IVF baby, the same poll lation risks? How can the complexities of Research on the Foundations of Human Behavior (D.L.). Sur-
found that over 60% supported IVF and PGT-P—e.g., pleiotropy, relative risk reduc- vey materials and code are available at https://osf.io/krj8t/.
All Understanding America Study (UAS) data are available at
would consider using it themselves (15). In tion—be conveyed to achieve appropriate https://uasdata.usc.edu.
our survey, 78% said they view IVF as mor- consumer literacy? Given the costs of PGT-P
ally acceptable or not a moral issue; only and those of IVF and that PGIs developed SUPPL EMENTARY MATE RIALS
6% said it was morally wrong (table S6). A with participants of European genetic an- science.org/doi/10.1126/science.ade1083
2016 survey of 185 countries, including the cestries are less predictive for those of other 10.1126/science. ade1083

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 INSIGHTS

Children gather in the Mesa Grande refugee camp

in Honduras, 40 km from the Salvadoran border.

The book itself is a collection of mov-

ing narratives that document a number of
encounters and interviews conducted by
the author over many years with relatives
of disappeared children as well as with
“jóvenes encontrados” (found youth) them-
selves. Working with Pro-Búsqueda, the
author followed up on dozens of very sen-
sitive cases of forced separation and docu-
mented the efforts of parents and family
members to find their missing loved ones.
Some of the found youth Barnert meets
were discovered still living in orphanages,
whereas many others had been adopted il-
legally by American or European families
during the war. Most of the latter were
aware that they had been adopted from
El Salvador and were very keen to learn
B O OKS et al . more about their biological families.
Barnert’s ability to describe the diverse
feelings of the book’s protagonists—be
ANTHROPOLOGY they searching relatives or found youth—
is admirable, and many of the stories she

Lost and found in El Salvador recounts, particularly those described by

mothers of disappeared children, are truly
harrowing. One mother describes having
Separated by civil war, families turn to genetic lost a child during the crossing of a river in
the middle of a battle, for example, while
testing to locate missing children another recounts being forced to give up
her newborn for adoption because she
By Victor Penchaszadeh a database of relatives searching for miss- could not care for her. Among the found
ing family members built with the support youth interviewed, Barnert documents

B
etween 1979 and 1992, the people of a group of forensic geneticists from the psychological troubles attributable to iden-
of El Salvador were subjected to a United States. tity ambivalence as well as pervasive feel-
civil war that claimed the lives of In 2005, more than a decade after the ings of abandonment that have led some to
at least 75,000, left thousands dis- end of the war, Elizabeth Barnert, a Cuban join armed gangs.
abled, and resulted in more than American of Jewish descent, who was then The most emotional intensity takes
1 million displaced and innumerable a young medical student in California, place during family reunions after identifi-
disappeared persons. The disappearance volunteered to go to El Salvador to re- cation. Although each “case” is distinctive,
of children, specifically, numbered in the search and write about the psy- a common denominator among
thousands and occurred under a variety chosocial and human rights searching relatives and found
of circumstances. Some were separated issues surrounding the disap- youth is psychological pain and
from their families as they fled attacks or peared children. Her new book, suffering. Barnert’s compassion-
were abducted by the military for various Reunion, documents her expe- ate approach to her interviews
reasons; others were forcibly placed in or- rience over the next 17 years, helps bring to the surface many
phanages or smuggled from the country as she completed her medical complex feelings for her subjects
and illegally adopted abroad. and public health studies at the and, hopefully, contributes to
In 1994, in the absence of any govern- University of California, con- their healing.
Reunion
mental support, Jon Cortina, a Spanish ducted research in El Salvador, This book, beautifully writ-
Elizabeth Barnert
Jesuit priest who lived and preached in and worked with Pro-Búsqueda University of California ten from the heart, is an essen-
El Salvador, founded the nongovernmen- to document the reunification of Press, 2023. 370 pp. tial tool for anyone interested
tal organization Pro-Búsqueda (“For the long-separated families. in recent Latin America history,
Search”), which was dedicated to finding Reunion opens with a foreword by which has been plagued by authoritarian
the missing children. DNA testing of youth Philippe Bourgois, an American anthropol- regimes and military dictatorships that
suspected of having been disappeared be- ogist who worked with indigent communi- have violated fundamental human rights. PHOTO: PHILIPPE BOURGOIS

came a crucial tool used by the group, as ties in El Salvador during the war and who Among an endless list of atrocities, the
the organization worked to find matches in provides a very useful historical account of book rightly focuses on the right to identity
the country. This section emphasizes the as a fundamental human right and a basis
prevailing social injustice, authoritarian- for dignity, freedom, and truth. j
The reviewer is at the Department of Genetics and Human
Rights, Universidad Nacional de Tres de Febrero, Buenos ism, and political repression during the
Aires, Argentina. Email: [email protected] decades that preceded the civil war. 10.1126/science.adg2895

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 SCIENCE AND SOCIETY A Hacker’s Mind:
How the Powerful Bend

Hacking and the social contract Society’s Rules, and How

to Bend Them Back
Bruce Schneier
Norton, 2023. 304 pp.
Although technical fixes can help, solutions must focus
on broader societal factors at play
By Viktor Mayer-Schönberger using their avatars’ body positions to com- shift focus to social systems that provide
municate letters. Seen from this perspective, boundaries but are themselves mutable.

T
he concept of “hacking” is not an in- hackers can be social innovators—probing, A Hacker’s Mind is highly recommended
vention of the digital age. Nor is it a reinterpreting, circumventing, and even reading, precisely because of the broader
purely technical process, although to- breaking established norms. context it provides on this point, without
day it often requires some technical ex- Schneier is at his best when he details the which remedies remain elusive.
pertise. Humans have always tried to balancing act in society between our social Perhaps the only limitation of the book
find loopholes in the systems of rules systems that ensure rules are followed and is that Schneier does not go far enough in
we find ourselves beholden to. When we the importance of keeping an open mind to- his analysis. For instance, he repeatedly
reach a wall, we try to find a way around it. ward norm innovation–causing hacking. We embarks on examinations of the social and
Bruce Schneier’s A Hacker’s Mind is a ought to conceive of hacking as a human be- technical interplay of a particular case—
collection of fairly short, often insightful havior that is neither intrinsically dangerous how Boeing got away with the problematic
commentaries about hacking. Schneier is nor inherently laudable, he argues. To judge Maneuvering Characteristics Augmentation
one of the nation’s most well- System (MCAS) hack in its 737
known cybersecurity experts, aircraft, or how banks success-
and his prose is clear, jargon- fully hacked the Dodd-Frank
free, and a pleasure to read. A Act that aimed to regulate de-
reader might pick up this book rivatives—only to reduce the
for the numerous instructive problem to simple questions of
cases and vignettes it offers, but power and wealth. The rich and
conceptually, A Hacker’s Mind powerful hack unconstrained, he
advances an important point; argues, while the rest of us are
and it is hugely revealing that forced to follow the rules. As so-
Schneier—a computer scientist— cial science research has shown
is making it. time and again, that is too meek
People have long sought to an explanation (1).
hack society’s conventions and In other instances, Schneier
legal norms—from taking advan- offers thoughtful descriptions of
tage of tax loopholes to creatively the social elements of a hack but
evading military drafts—and then advocates a technical fix. He
they do so most often by bending makes clear that such fixes are
social rules. Hence, Schneier sees only one part of a comprehensive
hacking as a social rather than a solution, but one cannot help but
technical phenomenon. see the computer scientist at-
This might sound like a sim- tempting to fix bugs at such mo-
ple reframing, but it has huge ments. As Schneier seems to have
consequences. It effectively dis- discovered himself, understand-
penses with the naïve hope that ing social systems is hard and re-
hacking can be “solved” through verting to trotted paths enticing.
technical fixes, although technology can, of its value, we need to evaluate a hack along That Schneier has pushed himself be-
course, be a part of the solution. Effective multiple dimensions and in context. The yond his own comfort zone, confronting
responses, argues Schneier, need to be so- result often highlights complicated trade- hacking as something bigger and more
cially anchored as well. offs. We can be impressed, for example, by multifaceted than simply sand in the ma-
When we talk about hacks, we often think a hack’s effectiveness but loathe its unethi- chinery of digital systems, is what makes A
of their negative consequences, or what econ- cal nature, as with the “Double Irish with a Hacker’s Mind unique and valuable. If his
omists call “externalities”: taxes go unpaid, Dutch Sandwich,” an intricate tax hack that message is received, our social systems will
ILLUSTRATION: PM IMAGES/GETTY IMAGES

cars designed to cheat emissions inspections reduced US corporations’ tax liability by bil- soon begin to evolve to interact with hack-
pollute the environment. But some hackers lions, or find it disruptive, but eventually ing with greater agility, nuance, and even—
repurpose a social system for their own ends. come to accept it as the new normal, as we in some instances—appreciation. j
For instance, a group of kids hacked the pro- did with dunking in basketball, a practice
REF ERENCES AND NOTES
hibition of free-form text communication in that leagues initially tried to ban but was
1. For example, Lawrence Lessig argues in Republic, Lost
Disney’s online kids game Club Penguin by eventually accepted, as fans liked it. (Twelve, 2011) that even lobbying Congress is not only
By giving hacking a social meaning, we about money but also about information.
The reviewer is at the Oxford Internet Institute, University of link it to the human desire to influence the
Oxford, Oxford OX1 3JS, UK. Email: [email protected] decisions and behaviors of others and we 10.1126/science.adg3002

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The Vetlesen Prize 2023
Achievement in the Earth Sciences
The Lamont-Doherty Earth Observatory and The G. Unger Vetlesen
Foundation are pleased to congratulate the 2023 Vetlesen Prize Laureate
David Kohlstedt for his pioneering work showing how processes
occurring at inaccessible depths of the Earth drive what happens on
our planet’s surface. The Vetlesen Prize honors
scientific achievement
Columbia University will present David Kohlstedt with the 2023 that results in a clearer
Vetlesen Prize at a formal dinner in April 2023. understanding of the
Earth, its history, or its
lamont.columbia.edu/about/vetlesen-prize relation to the universe.

Subscribe to News from Science for unlimited

access to authoritative, up-to-the-minute news
on research and science policy.

bit.ly/NewsFromScience

0210Product.indd 546 2/2/23 7:43 AM

 INSI GHTS

Ducipsap erspelit ut faccat as nobit vitiunt et

magniam volorro rercili quost, sandit is quasint

LET TERS
Plastics used in agriculture can lead to soil contamination.

Edited by Jennifer Sills found in human intestines, lungs, blood, industry. Farmers should receive incentives
brain, and breast milk (9). These foreign to explore nature-based farm equipment,
Soil microplastics substances can cause tissue rejection and
inflammation, similar to the impact of par-
phase out single-use plastic film, and pri-
oritize reusable plastic products. Scientists
pollution in agriculture ticulate matter of less than 2.5 mm on the
human respiratory system (10). Moreover,
should work to create affordable biode-
gradable agricultural plastic options and
Soil microplastics contaminate the soil most compounds added to plastics, such as technology that can remove microplastics
when macroplastics used in farming plasticizers, stabilizers, and pigments, are from the soil. Finally, legislation must be
decompose, threatening both agriculture harmful to the human endocrine system (11). passed to regulate the use of agricultural
and human health (1). Demand for agri- Mitigating the damages of soil micro- plastics and the level of microplastics pol-
cultural plastics to regulate field climate is plastics in farmland will require compre- lution in soil.
estimated to increase by 50% by 2030 (2). hensive action, including research, produc- Liuyue He1, Zhongbin Li1, Qian Jia2, Zhenci Xu3*
1
Emergency action is required to mitigate tion, administration, and legislation. The School of Remote Sensing and Information
Engineering, Wuhan University, Wuhan 430079,
soil microplastics pollution and to help global soil microplastics content in farm-
China. 2College of Environmental Science and
guide sustainable agricultural production. land caused by agricultural plastics must Engineering, Tongji University, Shanghai 200092,
Frequent extreme weather and growing be quantified by combining long-term China. 3Department of Geography, The University
food demand have exacerbated reliance field monitoring, satellite remote sensing, of Hong Kong, Hong Kong 999077, China.
*Corresponding author. Email: [email protected]
on plastics to increase grain yield (3). and theoretical modeling. The potential
Plastics used in agriculture include green- damages of soil microplastics to land pro- REF ERENCES AND NOTES
houses and plastic films for temperature ductivity, soil and field biodiversity, and 1. United Nations Environment Programme, “Plastics
control, irrigation pipes, and seed plastic human health must be determined. The in agriculture–an environmental challenge” (2022);
https://www.unep.org/resources/emerging-issues/
coating to prevent pests and diseases. site-specific maximum threshold for agri- plastics-agriculture-environmental-challenge.
When these plastics are left to decompose cultural plastics use must be determined, 2. Food and Agriculture Organization, “Assessment of
in the fields, microplastics enter the soil especially in key agricultural regions with agricultural plastics and their sustainability: A call for
action” (2021); https://www.fao.org/documents/card/
(4), decreasing the number, diversity, high microplastics content. Coordinated en/c/cb7856en.
PHOTO: DENG HEPING/VCG VIA GETTY IMAGES

movement, and reproduction rate of soil action plans such as consistent monitoring 3. J. Yates et al., Nat. Food 2, 80 (2021).
biota (5). Microplastics can also change and assessment, global and cross-sectoral 4. C. M. Rochman, Science 360, 28 (2018).
5. F. Zhu et al., Bull. Environ. Contam. Toxicol. 102, 741
the physiochemical properties of soil, cooperation, and open data sharing are (2019).
such as its structure, water-holding capac- urgently required. Governments should 6. M. C. Rillig, A. Lehmann, Science 368, 1430 (2020).
ity, and density (6), which could restrict encourage the transition to affordable bio- 7. M. C. Rillig et al., New Phytol. 223, 1066 (2019).
8. Y. S. Jung et al., Environ. Pollut. 315, 120442 (2022).
root growth, nutrient uptake, and yield of degradable plastics through policy incen- 9. A. D. Vethaak, J. Legler, Science 371, 672 (2021).
future crops (7). tives, technological innovation, agricultural 10. X. Z. Lim, Nature 593, 22 (2021).
Soil microplastics can be transferred to subsidies, public outreach, and collabora- 11. M. Sendra et al., J. Hazard. Mater. 409, 124975 (2021).
humans through the food chain or water tion with other stakeholders such as non-
cycle (8). Microplastic particles have been governmental organizations, scientists, and 10.1126/science.adf6098

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 INSIGHTS | L E T T E R S

REF ERENCES AND NOTES countries’ natural resources.

Understanding the limits 1. J. Wei et al., arXiv 10.48550/arXiv.2206.07682 (2022).
2. K. Valmeekam, A. Olmo, S. Sreedharan, S. Kambhampati,
The framework provides for an addi-
tional US$170 billion per year for biodi-
arXiv 10.48550/arXiv.2206.10498 (2022).
of AI coding 3. ACM Technology Policy Council, “Statement on prin-
ciples for responsible algorithmic systems” (2022);
versity funding within each country’s own
borders (4), including mechanisms such as
In the 9 December 2022 issue, the https://www.acm.org/binaries/content/assets/public- ecosystem services payments and the pur-
policy/final-joint-ai-statement-update.pdf.
Research Article “Competition-level chase and protection of unprotected land of
code generation with AlphaCode” (Y. high conservation value to compensate for
10.1126/science.adg4246
Li et al., p. 1092) and the accompany- biodiversity loss elsewhere. However, such
ing Perspective, “AlphaCode and ‘data- purchases, known as biodiversity offsets, are
driven’ programming” (J. Z. Kolter, p.
1056) describe an artificial intelligence
Effective implementation subject to political manipulation and largely
unsuccessful (8). Offsets have even been
(AI)–based system for generating code.
The authors explain that the system can
of new biodiversity pact claimed within an existing national park
(9), already fully protected by law.
be used for small coding problems, such In his Science Insider piece “New biodi- The US$30 billion per year the GBF
as tests for computing students, and that versity pact sets ambitious goals, but will international fund will be distributed
they are far from being useful in comput- nations deliver?” (22 December 2022, through the Global Environment Facility
ing applications that include millions of https://scim.ag/1jG). E. Stokstad explains (4). This new GBF sum is more than 80
lines of code, such as word processing. that the Kunming-Montréal Global times Global Environment Facility’s 2022
As we enter an era of AI where tools like Biodiversity Framework (GBF) of the to 2026 biodiversity allocation (10). Most
AlphaCode and chatGPT will change how Convention on Biological Diversity (CBD) Global Environment Facility funds go to
tasks are performed, it is important to is scientifically strong but legally weak. newly industrialized rather than developing
understand the boundaries of what they The GBF lacks hallmarks of effective inter- nations, and there is a risk that the organi-
can and cannot do. national agreements, such as reciprocal zation will disseminate GBF funds similarly.
To make sure that code can be main- responsibilities, dispute resolution systems, To strengthen the GBF, all financial
tained and managed by other program- uncompromisable goals, and noncompli- transfers during its implementation need
mers, human developers use mnemonic ance penalties. For the GBF to be effective, auditable accounting. Every new protected
variable names and embed explanatory these shortfalls must be addressed. area needs internationally transparent
comments. Understanding, debugging, As Stokstad reports, the GBF aims to boundaries, budgets, standards, and moni-
and extending code written by other protect 30% of all lands and oceans, but toring; the World Heritage model (11, 12)
humans remains a formidable chal- the goal is global rather than national, could serve as an example. Penalties are
lenge—perhaps even more difficult than which makes accountability difficult. needed for noncompliance. For example,
producing the code in the first place. In Countries such as Brazil, Indonesia, and countries that declare reserves but don’t
addition, many techniques are used for the Democratic Republic of Congo, which protect them could be charged a levy on
validation and verification, and code used have more than 30% rainforest cover, are exports, used to fund new reserves else-
in mission-critical applications, such likely to continue current rapid deforesta- where. CBD could require these as condi-
as airline flight systems, goes through tion (1). Moreover, because conservation tions of GBF funds, with additional moni-
substantial quality assurance testing. AI goals pertain to all ecosystems and do toring by nongovernmental organizations
models have yet to address the challenges not prioritize biodiverse areas, a country and research scientists.
of maintaining code, ensuring that users could declare rangelands as protected Ralf Buckley
can decipher it, and subjecting programs areas while continuing to allow logging Griffith University, Gold Coast, QLD 4222,
Australia. Email: [email protected]
to safety protocols. in rainforests. Countries could also allow
Understanding and evaluating the lim- areas to be logged or cleared before declar- REF ERENCES AND NOTES
its of these techniques is crucial before ing them as national parks, as has hap- 1. D. M. Lapola et al., Science 379, eabp8622 (2023).
they are put into real-world use. Some pened in Australia (2). The wording of the 2. J. Ward, “The relative net economic benefits of logging
and tourism in native Australian forests,” PhD Thesis,
testing of capabilities has been applied GBF (3) would allow countries to count
Griffith University, Gold Coast (1999).
to language generation tools (1, 2), but the national park toward the target for 3. UN Environment Programme (UNEP), Kunming-
AI coding remains a nascent field. The protected land and then restore the area Montreal Global Biodiversity Framework: CBD/
Technology Policy Committee of the and count it again, this time toward GBF’s COP/15/L.25 (2022).
4. UNEP, Financial Mechanism: CBD/COP/15/L.33 (2022).
Association for Computing Machinery separate target for restoration. 5. R. Buckley et al., PLOS ONE 7, e44134 (2012).
recommends more investment in trans- The GBF multilateral fund of US$30 6. “Global trade set to hit record $32 trillion in 2022, but
parency and accountability for AI algo- billion per year (4) is more than 10 times outlook increasingly gloomy for 2023,” UNCTAD (2022).
7. International Monetary Fund, World Datasets (2022);
rithms (3). The promise of systems like the aggregate budgets of all developing- https://www.imf.org/external/datamapper/profile/
AlphaCode must be carefully balanced country parks agencies worldwide (5), and WEOWORLD.
against the risks of their use. The inter- it could enable them to fund new land pur- 8. S. zu Ermgassen et al., Conserv. Lett. 12, e12664 (2019).
9. L. Cox, “NSW government seeks to use national park
action between AI code-generation sys- chases, protection, and anti-poaching mea- management to offset Warragamba Dam wall project,”
tems and human programmers must be sures. However, these funds represent a The Guardian (2022).
resolved before such systems can become transfer of just 0.1% of international trade 10. Global Environment Facility, “Initial GEF-8 star country
allocations: GEF/C.63/Inf.05” (2022).
an integral part of the future of computing. (6) and just 0.03% of global gross national 11. UN Educational, Scientific, and Cultural Organization
James Hendler product (7) from taxpayers in developed (UNESCO), State of Conservation Information System
Future of Computing Institute, Rensselaer nations to governments in developing (2023); https://whc.unesco.org/en/soc/.
Polytechnic Institute, Troy, NY, USA and Technology 12. UNESCO, World Heritage Convention: Periodic
nations. Meanwhile, multinational cor-
Policy Council, Association for Computing Reporting (2023); https://whc.unesco.org/en/
Machinery, New York, NY, USA. porations that primarily serve wealthy periodicreporting/.
Email: [email protected] countries continue to exploit developing 10.1126/science.adg5018

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 RESEARCH
IN S CIENCE JOURNAL S
Edited by Michael Funk

PALEONTOLOGY

Mass extinction rebellion

T
he Triassic recovery of life
from the devastating end-Permian
mass extinction was an amaz-
ing period of evolution. Whether
biodiversity had to rebuild from
near annihilation or from refugia is a mat-
ter of conjecture, but recovery heralded
the development of recognizably modern
ecosystems. Dai et al. present examples of
diverse fishes, ammonoids, bivalves, protists,
sts,
and malacostracan arthropods from a fossil sil
site dated approximately 1 million years after
ter
the crisis. These fossil assemblages, particularly
icularly
the presence of vertebrate predators, reveal al a
surprisingly early diversity of animals afterr the end-
Permian mass extinction. —CA and SNV
Science, adf1622, this issue p. 567

New fossils from a site in China, including the ammonoid shell pictured
here, reveal a surprisingly diverse ecosystem in the early Triassic.

NANOPHOTONICS hexagonal boron nitride bicrys- enable their own replication in COMPLEMENT
tals to show that negative a process called cap snatching.
Nanoscale negative refraction of mid-IR polaritons Before they can enact a snatch,
Lung protection by local
refraction occurs for propagation normal influenza viruses specifically complement
Refraction is a familiar effect in to the interface. Polaritonic require host cap maturation by Complement proteins are innate
which a light beam alters direc- negative refraction in the mid- a host methyltransferase called immune defense molecules
tion as it propagates from one IR provides opportunities for MTr1. Tsukamoto et al. screened that protect the host against
medium to another. Negative optical and thermal applications a compound library and found pathogenic microorganisms. The
refraction is a nonintuitive but such as IR super-resolution that trifluoromethyl-tubercidin activities of the C3 component
well-established effect in which imaging, nanoscale thermal (TFMT) inhibits host Mtr1 and of complement are mostly attrib-
the light beam is bent in the manipulation, and chemical suppresses virus replication. uted to circulating C3 primarily
“wrong” direction. Two groups sensing devices with enhanced TFMT inhibits host cap RNA synthesized by hepatocytes.
now independently demon- sensitivity. —ISO maturation and impedes bind- Sahu et al. used mouse models
strate negative refraction at the Science, adf1251, adf1065, this issue ing of host cap RNAs with the of conditional gene deletion
interface of two-dimensional pp. 558 and 555 viral polymerase, thus disabling to investigate the contribution
van der Waal materials. Hu et al. viral replication. TFMT was not of local C3 protein production
used molybdenum trioxide with only effective in inhibiting viral in the lung to protecting the
a graphene overlayer to show VIROLOGY replication in human lung cells, pulmonary mucosal surface
that in-plane negative refraction but was also effective in mice, from cell damage caused by
Catching out
PHOTO: DAI ET AL.

of mid-infrared (mid-IR) polari- displayed little toxicity, and Pseudomonas aeruginosa infec-
tons occurs at the interface and cap snatching acted in synergy with approved tion. Mice lacking C3 only in lung
is gate tunable. Sternbach et Soome virus families hijack anti-influenza drugs. —CA epithelial cells exhibited more
al. used molybdenum trioxide/ part of their hosts’ RNA to Science, add0875, this issue p. 586 pronounced acute lung injury

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 RESEARCH | I N S C I E N C E J O U R NA L S

than control mice even when evolution. Using new genomes

circulating C3 levels were main- from fishes in the basal group
tained at near-normal levels by that includes tarpons and eels, as IN OTHER JOURNALS
maintenance of liver C3 produc- well as approaches that integrate
tion. These findings point to lung patterns of genome structure not Edited by Caroline Ash
epithelial cell–intrinsic protec- just sequence, Parey et al. show and Jesse Smith
tive functions provided by C3 as that this group forms a mono-
being an essential contributor phyletic lineage within another
to the first-line defense against group that includes the arapaima
bacterial pneumonia. —IRW and elephant nose fishes. This
Sci. Immunol. 8, eabp9547 (2023). single lineage is resolved as sister
to all the other teleosts, settling
a debate about patterns of early
HOMININ EVOLUTION
fish evolution. —SNV
Earlier Oldowan Science, abq4257, this issue p. 572
Oldowan tools, consisting of
stones with one to a few flakes
MALARIA
removed, are the oldest wide-
spread and temporally persistent A different antibody angle
hominin tools. The oldest of Natural killer (NK) cells have
these were previously known been shown to mediate immune
from around 2.6 million years responses against the malaria
ago in Ethiopia, and by 2 million parasite Plasmodium falciparum
years ago, they were found to be using multiple effector functions.
quite widespread. Plummer et Odera et al. now show that P.
al. report on an older fossil site falciparum merozoites marked
from around 3 to 2.6 million years by antibodies can induce NK cell
ago in Kenya, where Oldowan degranulation and interferon-g
tools were not only present, but production. This response was
were also being used to process not strain specific and reduced ICE SHEETS
a variety of foods, including invasion of merozoites into
hippopotamus. Thus, it appears uninfected red blood cells. Using In winter like in summer
that these tools were widespread a controlled human malaria infec-

W
ater from melting of the surface of the Greenland
much earlier than previous esti- tion study in adults, the authors ice sheet can flow through channels in the ice to the
mates and were widely used for observed that antibody-depen- base of the ice sheet, where it may reduce the fric-
food processing. Which hominins dent NK (ab-NK) cell activity tion between the ice sheet and the underlying bed
were using these tools remains correlated directly with the con- and thereby accelerate the flow of the overlying ice.
uncertain, but Paranthropus fos- trol of parasitemia. In addition, This process normally occurs in the summer, but Maier et al.
sils occur at the site. —SNV in a cohort of children living in an show that meltwater-induced ice flow also can occur during
Science, abo7452, this issue p. 561 endemic malaria setting, ab-NK the winter. These wintertime events, which are caused by the
cell frequency increased with age, cascading release of water stored in supraglacial lakes, are
was boosted during P. falciparum likely to have a larger impact on ice dynamics than previously
FISH EVOLUTION
infection, and was linked to a realized. —HJS Geophys. Res. Lett. 10.1029/2022GL102251 (2023).
Debate resolved? reduced risk of clinical malaria.
Teleost (or “ray-finned”) fishes These findings highlight a key role Summertime melting of surface ice can be stored in supraglacial lakes
make up half of all vertebrates, for antibody-mediated NK cell and released to the bottom of the ice sheet during winter.

PHOTOS: (LEFT TO RIGHT) CINOBY /ISTOCKPHOTO; WANDERLUSTER/ISTOCKPHOTO

but there has been long-stand- responses during malaria. —CNF
ing debate about their early Sci. Transl. Med. 15, eabn5993 (2023).

QUANTUM SIMULATION atoms, the right half of which were

subjected to a disorder potential.
Spotting quantum After a quench, the clean left
avalanches half of the chain quickly thermal-
Disorder can prevent interacting ized. The disordered right half
quantum systems from thermal- initially resisted thermalization,
izing, a phenomenon known but at longer times, characteristic
as many-body localization. If, anticorrelations appeared that
however, a section of the system were strongest near the interface
is free of disorder, then that between the two halves. The
subsystem has been predicted dynamics of the anticorrelation
to thermalize and grow in an spread were consistent with a
Analysis of fish genomes reveals the evolutionary relationships between teleost avalanche-like process. To test quantum avalanche. —JS
fishes, a diverse group that includes eels (such as the giant moray eel, pictured this prediction, Léonard et al. Nat. Phys. 10.1038/
here), zebrafish, and the giant arapaima. used a chain of 12 interacting cold s41567-022-01887-3 (2023).

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 PHOTONICS 88% of patients, but 70.8% of fluid of muscle or fat from mice. against pathogens, but it is a
patients had a partial response The authors then applied question of getting the dose right.
Optically addressing or better with a median duration mass spectrometry to identify Compared with other insects,
3D laser arrays of 8.3 months. Therefore, ALLO- proteins whose abundance bees lack the genetic capacity
Coping with the insatiable need 715 treatment appears to be changed in response to cold to fully metabolize xenobiot-
for the transfer and processing of feasible and could be beneficial exposure or exercise. Some ics, so Motta et al. investigated
vast amounts of data is expected for treating multiple myeloma. unexpected myokines and adi- whether the bee microbiome
to see a shift from mainly elec- —GKA pokines emerged, which could could supply the missing func-
tronics-based platforms to either Nat. Med. 10.1038/ potentially mediate signaling tions. Honeybees feeding on
hybrid opto-electronic or solely s41591-022-02182-7 (2023). involved in some of the well- almond blossom ingest the
optical platforms. Such a shift to known beneficial physiological cyanogenic glycoside amygdalin
an integrated photonics technol- effects of exercise. —LBR when foraging. The authors found
ogy requires the ability to use and PHYSIOLOGY Cell Metab. 10.1016/ that microbiota-depleted bees
address arrays of optical sources. j.cmet.2022.12.014 (2023). metabolized amygdalin to the
Song et al. demonstrate the abil-
Searching muscle and more toxic prunasin, which accu-
ity to fabricate three-dimensional fat regulators mulated in the insect’s gut. Bees
MICROBIOTA
(3D) arrays of photonic crystal Extracellular fluid contains with a full complement of gut
nanolasers by printing them along proteins that are difficult to Honeybee self-medication microbiota, including one called
the length of an optic fiber. They detect or are absent in serum. Plants generate a range of toxic Bifidobacterium wkB204, can
then show that each nanolaser Such proteins include secreted metabolites, many of which are fully degrade amygdalin by means
can be addressed individually regulatory factors that control intended to deter insect pests. of a glycoside hydrolase 3. Host
through an interference effect by tissue physiology. Mittenbühler Pollinators are also exposed and microbiota thus join forces
simply adjusting the pulse width et al. adapted a technique in to these so-called xenobiotic to maintain levels of a potential
and polarization of the input light which low-speed centrifugation compounds in nectar and pollen. toxin at tolerable levels to ward off
into the optic fiber. The abil- allowed the rapid isolation of Some of these chemicals can parasites. —CA
ity to program such a 3D array proteins from the extracellular confer benefits to pollinators eLife 11, e82595 (2022).
offers possibilities for developing
high-density optical circuits and
may have applications in sensing.
—ISO
Optica 9, 1424 (2022).

IMMUNOTHERAPY
Off-the-shelf T cell
therapy
Multiple myeloma is character-
ized by the accumulation of
malignant plasma cells in the
bone marrow. Various B cell
maturation antigen (BCMA)–
targeted drugs are approved
for the treatment of relapsed/
refractory multiple myeloma,
including an engineered chime-
ric antigen receptor (CAR) T cell
therapy. However, extracting T
cells from individual patients to PLANT SCIENCE
produce autologous CAR T cells
presents numerous challenges. Nematodes hijack root development
ALLO-715 is an allogeneic, “off-

C
yst nematode parasites cause substantial damage to crop plants. These parasites invade
the-shelf” BCMA-targeted CAR plant roots, destroying internal root tissue structure and inducing root branching to provide
T cell therapy. Mailankody et al. additional feeding sites. Guarneri et al. found that cyst nematode infection triggers the accu-
report interim results from the mulation of the plant hormone jasmonic acid and up-regulates a key transcription factor for
PHOTO: NIGEL CATTLIN/SCIENCE SOURCE

ongoing first-in-human phase 1 root induction. These events lead to increased local biosynthesis of auxin that initiates de
trial of ALLO-715 in 43 patients novo root formation in a similar manner to wound-associated root induction. Although infected
with relapsed/refractory plants maintained their overall root system size, increased secondary root growth was balanced
multiple myeloma. CAR T cell by reduced primary root lengths. Therefore, pathogens can manipulate the dynamic responses of
regimens are associated with root systems for their own benefit. —MRS New Phytol. 237, 807 (2023).
adverse events that are treat-
Cyst nematodes (Heterodera rostochiensis is shown) are important crop plant pests because of the damage
able if managed early. ALLO-715
they cause by stimulating secondary root growth.
treatment resulted in grade
3 or higher adverse events in

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 RESEARCH

ALSO IN SCIENCE JOURNALS Edited by Michael Funk

CHEMICAL PHYSICS cancer cells drive oncogenic genes are components of a sig- that regulate floral carotenoid
phenotypes. Such analyses can naling pathway that suppresses pigmentation. Revealing the
Unraveling excited also reveal pathogenic signaling inflammation in double-stranded opportunistic nature of evolu-
electronic states and therapeutic targets. —GKA RNA-stimulated mononuclear tion, the locus evolved from a
For more than 100 years, linear Science, aaw3835, this issue p. 552 phagocytes. Thus, single-gene fragment of a gene encoding a
spectroscopy has served as the recessive inborn errors of the cytochrome protein not involved
primary means of exploring and OAS–RNase L pathway can in floral pigment production.
validating quantum mechanical DEVELOPMENT result in uncontrolled inflam- —PJH
energy levels of atoms and mol- matory cytokine production by Science, adf1323, this issue p. 576;
ecules. With strong light fields,
Metabolism sets neuronal mononuclear phagocytes after see also adg2774, p. 534
nonlinear spectroscopic meth- development pace SARS-CoV-2 infection, poten-
ods using multiple pulses yield The pace of neuronal develop- tially explaining the origins of
sensitive molecular information ment varies between species, MIS-C in some children. —STS CALCIUM SIGNALING
and provide powerful tools, and the relatively slow devel- Science, abo3627, this issue p. 554;
such as magnetic resonance opment of the human brain see also adg2776, p. 538
Restoring a quiescent
imaging. A recent revolution may help allow its exceptional state for STIM1
uses multiple x-ray pulses and complexity. Iwata et al. propose Gain-of-function mutations
GEOCHEMISTRY
newly developed nonlinear x-ray that the pace of neuronal devel- in the endoplasmic reticulum
spectroscopy methods to derive opment depends on the rate of Testing Earth’s water Ca2+ sensor STIM1 result in
time-resolved atomic, molecular, metabolic activity in mitochon- The chemistry of aqueous constitutive activity of the
and solid-state dynamics. In a dria. Human and mouse neurons systems early in Earth’s history plasma membrane Ca2+ channel
Perspective, Leone and Neumark exhibited distinct paces of devel- is important for the eventual Orai1 and underlie a disease
discuss nonlinear attosecond opment in culture correlated development of life on the called Stormorken syndrome.
four-wave mixing in which an with the tricarboxylic acid (TCA) planet. However, the relative lack Gamage et al. found that dele-
x-ray pulse is merged with two cycle and oxidative activity in of rocks from billions of years tion of a glutamate residue in
optical pulses in molecules and mitochondria. Manipulations ago presents a great challenge STIM1 reversed the effects of a
materials to unveil ultrashort to increase oxygen consump- for solving this puzzle. Trail and common Stormorken syn-
processes such as quantum tion rates and TCA cycle activity McCollom used the chemistry drome–associated mutation.
pathways in transient electronic in human cells increased the of four-billion-year-old zircons Mice expressing STIM1 with both
states. —GKA rate of neuronal development. to estimate the fluid chemistry modifications were phenotypi-
Science, add4509, this issue p. 536 Slowing metabolic rates in and temperature for mineral cally similar to wild-type mice.
mouse neurons slowed neuronal formation (see the Perspective The combined modifications
maturation. Thus, metabolic by Rodriguez). They found more restored the protein domain
EPIGENETICS rates in mitochondria appear to oxidizing fluids feeding hydro- interactions that prevent STIM1
somehow help set the speed of thermal pools than previously from associating with and
Plasticity of cancer cell neuronal development. —LBR expected, which has implications activating Orai1 in the absence of
phenotypes Science, abn4705, this issue p. 553 for how minerals and organics stimuli. —WW
During differentiation, cells may have formed in the time just Sci. Signal. 16, eadd0509 (2023).
adopt phenotypic states of pro- after Earth’s accretion. —BG
gressive specificity. Cancer cells CORONAVIRUS Science, adc8751, this issue p. 582;
violate this property, instead see also adg2630, p. 539
adopting increased plastic-
OAS–RNase L pathway the
ity of structure and function. “MIS-C”ing link?
Epigenetic change has been Multisystem inflammatory PLANT SCIENCE
considered a developmental syndrome in children (MIS-C) is
landscape that can channel a severe complication of severe
Noncoding gene drives
specific differentiation events acute respiratory syndrome floral speciation
and define and constrain distinct coronavirus 2 (SARS-CoV-2) In monkeyflowers (Mimulus
phenotypic and gene expression that affects one in 10,000 spp.), variation in the YELLOW
states. In a Review, Feinberg and infected children, is reminiscent UPPER (YUP) genetic locus
Levchenko discuss how cancer of Kawasaki disease, and its causes changes in flower color
epigenetic landscapes can be etiology remains unknown. Lee patterns that in turn drive
defined quantitatively, borrowing et al. performed whole-exome speciation with shifts in whether
from theory used in physical sci- and whole-genome sequencing the flower is pollinated by bees
ences to define potential energy on a cohort of MIS-C patients or by hummingbirds. Liang et
and its relationship to physical or and uncovered autosomal- al. now show that the locus
chemical states. This strat- recessive deficiencies of OAS1, does not encode a protein (see
egy has yielded new insights OAS2, or RNase L in around the Perspective by Monniaux).
whereby stochastic changes 1% of the cohort (see the Instead, YUP produces phased
in the epigenetic landscape of Perspective by Brodin). These small interfering RNAs (siRNAs)

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RES EARCH

◥ uncertainty. These measures, which we define

REVIEW SUMMARY in detail, enable a precise and quantitative def-
inition of the underlying epigenetic landscapes,
EPIGENETICS coordinately reflected by gene expression land-
scapes. Cancer-related genetic and epigenetic
Epigenetics as a mediator of plasticity in cancer alterations can increase the entropy of the
landscape as a whole and result in higher
Andrew P. Feinberg* and Andre Levchenko* variability and occupancy of otherwise cryp-
tic attractors. An increase in entropy and thus
heterogeneity of the responses—rather than
BACKGROUND: During differentiation, living Such approaches are particularly appealing alteration of the average response—is emerg-
cells within complex organisms adopt pheno- in describing the cancer epigenetic landscape ing as a key and often overlooked feature of
typic states of progressive specificity. Cancer- given that the plasticity of cell states realized the landscape deformation in cancer patho-
ous cells and tissues violate this property, on such a landscape lies at the functional core genesis. Changes in entropy can also accom-
adopting increased plasticity of cell states, of the disease. pany cell differentiation and aging in ways
tissue structure, and function during their that further inform cancer etiology. They also
progression. The information about the rep- ADVANCES: Recent developments in experimen- permit distinguishing phenotypic plasticity
ertoire of normal differentiation outcomes is tal technologies, including single cell–resolution from phenotypic heterogeneity. Using recent
genetically encoded, but the information about analysis of mRNA and protein expression as observations and landscape conceptualization,
the particular realization of this potential and well as molecular assays of epigenetic mod- we outline several scenarios that can occur dur-
cell regulation in response to the environment ifications of DNA and histones, have enriched ing precancerous and cancerous progression.
is encoded epigenetically in DNA methyla- our understanding of the diversity of pheno- We also discuss the molecular mechanisms
tion and biochemical modification of chro- typic states defined by genomic information enabling these scenarios, relating them to spe-
matin. Dating back to Conrad Waddington’s and epigenetic control. In this work, we expand cific landscape transformations. We suggest
prescient work, epigenetic change has been on the emerging view that there is consider- how the relationship between the epigenetic
viewed schematically as a developmental land- able variability in the expression of biological landscape alterations and corresponding pheno-
scape that can channel specific differentia- molecules even within presumably isogenic typic changes can be quantitatively assessed
tion events and define and constrain distinct cells in normal homeostatic tissues or in well- and used to further understand the informa-
phenotypic and gene expression states. More defined cell lines in cell culture. This revela- tion transfer in signaling pathways and to
recently, cancer onset and progression have tion suggests that the biological processes may develop new therapeutic interventions. This
been viewed as a reversal or deformation of be essentially stochastic and that biological approach can also incorporate recently intro-
this landscape. In the physical sciences, po- variability on the cellular level can be indica- duced ideas of the archetypical states of cells
tential energy landscapes and their relation- tive of—or even drive—important aspects of within normal and cancerous tissues.
ships to the probability distribution of physical biological function. This analysis has also en-
or chemical states have been developed and abled assessment of the probability distributions OUTLOOK: New integrated theoretical and ex-
refined for decades, but they have only re- of different cellular states, or quasipotential perimental methods in quantitative analyses
cently been applied to more quantitatively energy, and the use of these to determine the of the cancer epigenetic landscape provide the
realize Waddington’s classical landscape idea. associated entropy, a measure of informational tools to understand the connections between
genetic and environmental drivers of cancer
evolution and the relationships between epi-
genetic regulatory networks that mediate the
Epigenetic landscapes and Environmental inputs landscape. Continued advances in single-cell
phenotypic plasticity in cancer. Aging Mutagenesis measurements, including assessment of DNA
Regulatory networks can define methylation, genomic sequencing, and chro-
the number and probabilities matin analysis, will allow further understand-
of stable cellular states ing of the dynamics of the landscapes having
adopted by a cell population, Landscape 1 Landscape 2 progressively increasing complexity and ac-
representing attractors in the counting for tumor evolution, progression to
epigenetic landscape. Diverse invasive and metastatic spread, and associated
inputs can promote transitions alterations in anatomical organization and
(and corresponding phenotypic Plasticity structure. Moreover, a greater understanding
plasticity) between cellular of biological stochasticity, defined mathemat-
states within landscapes ically as epigenetic and gene expression entropy,
corresponding to the normal can uncover the cellular actors and mechanisms
tissue (fewer attractors) and by which cancer plasticity enables escape from
cancerous tumors (emergence
of new attractors), as defined
P2 P2 natural defenses and therapeutic interventions.
▪
by parameters P1 and P2 that The list of author affiliations is available in the full article online.
correspond to effective *Corresponding author. Email: [email protected] (A.P.F.);
concentrations of landscape- Normal [email protected] (A.L.)
Cite this article as A. P. Feinberg and A. Levchenko, Science
defining molecules. 379, eaaw3835 (2023). DOI: 10.1126/science.aaw3835
Cancer
READ THE FULL ARTICLE AT
P1 P1 https://doi.org/10.1126/science.aaw3835

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◥ for nondeterministic, many-fate behaviors of the

REVIEW system. Although the Waddington landscape
is frequently interpreted as the epigenetic land-
EPIGENETICS scape, these concepts may no longer be fully
synonymous. Whereas the term epigenetic pre-
Epigenetics as a mediator of plasticity in cancer viously referred to any mechanism outside tra-
ditional gene expression control, today it more
Andrew P. Feinberg1* and Andre Levchenko2* commonly refers to a specific set of regulatory
processes affecting in particular DNA methyla-
The concept of an epigenetic landscape describing potential cellular fates arising from pluripotent cells, tion and chromatin modifications.
first advanced by Conrad Waddington, has evolved in light of experiments showing nondeterministic The probabilistic nature of biochemical pro-
outcomes of regulatory processes and mathematical methods for quantifying stochasticity. In this cesses has been historically addressed in sev-
Review, we discuss modern approaches to epigenetic and gene regulation landscapes and the associated eral ways. Most simply, added noise might be
ideas of entropy and attractor states, illustrating how their definitions are both more precise described by extending the ODE description
and relevant to understanding cancer etiology and the plasticity of cancerous states. We address the using the Langevin approach (Box 1, Eq. 1).
interplay between different types of regulatory landscapes and how their changes underlie cancer However, this approach can break down if
progression. We also consider the roles of cellular aging and intrinsic and extrinsic stimuli in modulating the number of molecules becomes very small,
cellular states and how landscape alterations can be quantitatively mapped onto phenotypic outcomes making the functions defining the ODE sys-
and thereby used in therapy development. tem behavior discontinuous. This considera-
tion drove a conceptual quantum leap, akin to

T
the transition from classical to quantum me-
umor cell heterogeneity and phenotypic capabilities—e.g., by recruiting DNA methylases chanics in physics. In this view, instead of
plasticity have long been known to be and histone acetyltransferases—it is not clear describing the biological variables, such as
principal drivers of invasion and metas- whether epigenetic control might be viewed as molecular concentrations, one operates with
tasis (1). Complex and diverse behaviors a constituent part of transcriptional regulation the probabilities that system-defining vari-
displayed by cancer cells frequently in- or whether it has independent and essential ables take on certain values. This probabilistic
volve coordinated expression of multiple genes, roles not fully predictable by the analysis of dynamics is captured by the master equation,
constituting programs that may be very dis- gene networks alone. based on the Markovian assumption about the
tinct from those defining the phenotypes of the In this Review, we argue that epigenetic system dynamics (Box 1, Eq. 2). However, al-
tissue of origin. Because oncogenic transforma- mechanisms can have several key functions though appearing superficially simple, such
tions are frequently accompanied by extensive that are frequently not evident from tradi- equations require advanced computational
mutagenesis, analysis of phenotypic hetero- tional quantitative approaches involving the resources to solve or estimate, though some
geneity displayed by cancer cell populations analysis of transcriptional regulation or RNA success has been achieved for simpler cases
has typically been ascribed to the emergence and protein stability. In particular, epigenetic (13–15). A fundamental challenge facing both
of multiple genetically distinct clonal cell sub- regulation may have a critical role in regulat- the Langevin and master equation approaches
populations within the same tumor (2). This ing variability of gene expression, accounting is the lack of complete knowledge of the mech-
genetic heterogeneity should be distinguished for variance in current cellular states and the anisms that govern interactions between sys-
from phenotypic heterogeneity (i.e., the emer- emergence of new ones. Recent methodolog- tem variables. To date, relatively few biological
gence of stable phenotypic states in an iso- ical advances allow us to explicitly define and networks have been fully characterized, and
genic population) and phenotypic plasticity measure epigenetic landscapes that, along with frequently this mechanistic characterization is
(i.e., the ability of cells to adopt different pheno- gene regulation landscapes, can be used to only semiquantitative. It is therefore difficult
typic states transiently). Phenotypic plastic- much more precisely understand the etiology to theoretically or computationally predict the
ity is thought to underlie complex phenotypic of cancer and to better understand the mech- probability distributions of different molecu-
changes, such as epithelial-mesenchymal tran- anisms underlying phenotypic plasticity and lar and cellular states. However, one can now
sition (EMT), drug resistance, and increased heterogeneity. directly experimentally estimate these proba-
cell proliferation (3–6). Therefore, although bilities (16–18) and thus attempt to solve an
genetic mutations may increase the proba- Theoretical approaches to describe genetic inverse problem of reconstructing the mecha-
bility of new phenotypic states, they may not and epigenetic landscapes nisms underlying gene expression regulation
completely define them and may not be strict- Regulatory networks impose complex inter- and the corresponding genetic and epigenetic
ly necessary for their emergence. dependencies on gene expression, which are landscapes from experimental data.
Elucidation of the interplay between epige- commonly modeled using ordinary differential The inferences based on experimentally ob-
netic states and regulation of gene expression equations (ODEs) (Box 1, Eq. 1). A related concept tained probability distributions can be put on
is crucial for our understanding of phenotypic of landscape introduced by Waddington and a solid theoretical foundation that provides
plasticity. Multiple questions remain regarding further elaborated mathematically (3, 7–9) has specific meanings to the notions of epigenetic
coupling of these regulatory mechanisms. For been instructive for simpler and well-characterized and genetic landscapes, distinct from those im-
example, because many regulators of gene ex- systems but has faced important challenges. plied by Waddington. Critical to this approach
pression may also have epigenetic modification In particular, molecular interactions constitut- is the fundamental connection of potential en-
ing regulatory networks are frequently un- ergy and probability distributions provided by
1 known and may be restricted by epigenetic the Gibbs-Boltzmann approach (Box 1, Eqs. 3
Center for Epigenetics, Johns Hopkins University Schools of
Medicine, Biomedical Engineering, and Public Health, mechanisms, such as DNA methylation. Fur- and 4), which are particularly useful for def-
Baltimore, MD 21205, USA. 2Yale Systems Biology Institute thermore, these interactions and networks inition and analysis of landscape attractors
and Department of Biomedical Engineering, Yale University, are subject to biomolecular noise or variabil- corresponding to specific coherent molecular,
West Haven, CT 06516, USA.
*Corresponding author. Email: [email protected] (A.P.F.); ity of molecular concentrations or alterations cellular, and phenotypic states. Specifically,
[email protected] (A.L.) of molecular states (10–12), which are crucial this approach associates the probability of

Feinberg and Levchenko, Science 379, eaaw3835 (2023) 10 February 2023 1 of 11

RES EARCH | R E V I E W

any outcome—e.g., gene expression or DNA

Box 1. Mathematical analysis of stochastic biological systems. methylation pattern and the corresponding
phenotypic outcome—with a generically de-
Biological systems are governed by underlying interactions that could be described mathematically. fined quasipotential energy, a term adapted
These interactions can be stochastic because of the variability of the participating elements (molecular from physics to describe the origin of prob-
modifications, molecular and cellular numbers, etc.). The mathematical approach frequently used to ability distributions of the possible cellular
capture stochastic interactions is the Langevin approximation based on the ODEs describing temporal states (states with lower energy are occupied
dynamics of variables xi, using the function f(x) of the vector x of all variables xj, with the added noise with higher probability; see Fig. 1A and the
terms h(xi) definition in Box 1, Eq. 3). This association
dxi allows one to define the corresponding genetic
¼ fi ðxÞ þ hi ðxi Þ ð1Þ (gene expression) or epigenetic (e.g., DNA meth-
dt
ylation markers) landscapes as the quasipo-
An alternative but equivalent description that is particularly useful for systems with very small values of tential energy distributions. This landscape
xi that are expressed as whole numbers is to consider the dynamics of probabilities of xi rather than definition is consistent with the one implied
the dynamics of xi themselves. This is done using the so-called master equation based on the as- by the Waddingtonian ODE approach, afford-
sumption that the underlying processes are Markovian (memoryless) ing easy interpretation and visualization. The
dPðxi Þ X
attractors within this landscape representa-
¼ k P xj
j ij
ð2Þ tion would be seen as the values of biological
dt
variables for which the local and global en-
Unfortunately, other than for a few relatively simple systems, the mechanistic information that would define ergy minima would be reached, thus consti-
the functions f(x) and kij is largely incomplete for most biological systems. Therefore, it is frequently hard to tuting states with high relative probability
use these models for real biological systems to characterize their dynamic behavior and thus values (Fig. 1A). The stability of the attractor
landscapes. Instead, it has proven easier to experimentally measure the distributions P(xi). Therefore, is further defined by such landscape charac-
the problem can be inverted—i.e., reconstructing the underlying regulatory landscape from the knowl- teristics as the depth and width of the cor-
edge of P(xi). The recipe for doing so is provided by statistical mechanics (Boltzmann-Gibbs distribution) responding energy wells and the proximity,
and in particular the relationship between the energy U(xi) associated with a specific xi and the probability and thus accessibility, of other attractors (19).
of this value of the variable (the energy here is quasipotential, corresponding to measuring the energy in Increased variability or noise makes energy
units of kBT—i.e., the product of the Boltzmann constant kB and temperature T) wells effectively more shallow, thereby widen-
ing the distribution of biological variables
hX i
Uðxi Þ ¼ ln Pðxi Þ ln j
e Uðxj Þ ð3Þ (20). This allows cellular networks to explore
multiple states across different attractors. The
local energy (landscape) minima may be re-
If the distribution of different values that can be taken by variables xi is constant (i.e., the landscape alized in different cells within a population or
does not change), then the second term in Eq. 3 is constant and, because the energy U(xi) is only could only be accessible in specific develop-
determined up to a constant, it can be omitted for the analysis of a constant landscape mental, physiological, or pathological situa-
Uðxi Þ ’ ln Pðxi Þ ð4Þ tions. Below, we illustrate and discuss these
and other important ideas, such as the en-
tropy associated with the whole landscape and
The relationship (Eq. 3) can be used to directly define the energy (quasipotential) landscape corre-
specific attractor states and the information
sponding to the gene expression and epigenetic landscapes and to relate it to the entropy of the
associated with changes of this entropy (Box 1),
system, again up to a constant
particularly in the context of emergence and
X X
HðxÞ ¼ Pðxi Þlog Pðxi Þ ’ Pðxi ÞlnPðxi Þ ’ hUðxi Þi ð5Þ progression of cancer.
i i
Landscape models of cancer
This relationship, linking the entropy and average energyhUi for a system, emphasizes the global nature of the
Gene regulatory landscapes can be evaluated
entropy measure, involving summation over multiple states. Thus, as illustrated in Fig. 1, different landscapes
from experimentally determined gene expres-
may have the same entropy and thus cannot be uniquely distinguished using this function of the state.
sion probability distributions, particularly those
Conversely, the distributions captured by P(xi) and U(xi), can be more informative.
yielded by single-cell RNA sequencing (scRNA-
Finally, the mutual information is defined as the change of entropy (and thus the underlying landscape) seq) data or from the analysis of multiple tissue
of a state DH(x) given a regulatory event or a signal. If the entropy decreases, the mutual information is samples (e.g., cancerous tissues from multiple
positive, which provides a measure of comparison between two distributions. Another important measure of patients) (Fig. 1A). The landscape attractors in
dissimilarity between two distributions P(xi) and Q(xi) is the frequently used Kullback-Leibler (KL) these distributions correspond to groups of co-
divergence ordinately expressed genes and gene products,
X   whose abundance and activity may be regulated
Pðxi Þ
DKL ðPkQÞ ¼ P ðxi Þlog ð6Þ by self-sustaining feedback interactions, en-
i Qðxi Þ
dowing cells with specific phenotypic prop-
erties. Cancerous states may represent the
Mutual information is thus the KL divergence between the joint distribution of the variable values
genetic landscape attractors that are inac-
before and after the event (or signal) and the product of their marginal distributions. A symmetrized
cessible in untransformed tissues but can be
version of the KL divergence of particular value in comparing epigenetic landscapes is the Jensen-
unmasked by oncogenic mutations (19, 21).
Shannon distance, defined as
This is exemplified by the emergence of the
1 1 1 mesenchymal state in EMT (3). The Langevin
JSDðPkQÞ ¼ DKL ðPkMÞ þ DKL ðQkMÞ where M ¼ ðP þ QÞ ð7Þ
2 2 2 ODE approach has been used to analyze a tri-
stable regulatory circuit assumed to control

Feinberg and Levchenko, Science 379, eaaw3835 (2023) 10 February 2023 2 of 11

RES EARCH | R E V I E W

A B B B

A A
H3 H5

H1
Attractor

B H2 H4

B B
C
A

Cancer attractor

u
Dev ctors
elop ra
t
m en t a l at

A A

Fig. 1. Gene expression and epigenetic landscapes control normal and attractors with the overall entropy H2 greater than the original entropy H1
cancer cell functions. (A) Gene regulatory networks and availability of genes for (H2 > H1), generating phenotypic heterogeneity (input 1′) or, alternatively,
expression can define the probabilistic distributions of proteins expressed within enlarging the existing attractor with the new entropy H3 > H1, generating a more
a cell population. In this example, the network of interacting proteins that plastic state (phenotypic plasticity), with cells capable of stochastically and
includes the molecules A and B (top) and the underlying epigenetic control dynamically exploring this attractor and thus transiently adopting different
determining the availability of the corresponding genes for expression define the phenotypes. In both cases, entropy increases versus H1 and it is possible
distribution of the expression of molecules A and B (middle). This probability that H2 = H3, thus making entropy less discriminating than the full landscape
distribution can be experimentally measured and converted into a gene picture in the analysis of cell states. These new landscapes can be further
expression landscape by calculating the corresponding quasipotential distribution altered by oncogenic and environmental inputs, so that one of the attractors
(bottom) (see Box 1). The epigenetic landscape can be similarly determined becomes dominant (input 2′), associated with a lower entropy value (H4 < H2)
by experimentally measuring the probabilistic distributions of epialleles, or, alternatively, with the narrowing of the wider (and more plastic) attractor
measuring DNA methylation marks at specific loci, or by performing other (input 2; H5 < H3). Again, it is possible that H4 = H5, requiring the landscape
measurements of epigenetic regulation across populations of cells and tissues analysis rather than entropy analysis alone for full characterization. The
and then also converting these probability distributions into corresponding narrowing of the wide attractor because of either environmental or intrinsic
underlying quasipotential landscapes. The landscape analysis allows conceptual inputs (input 2) is frequently reversible and context dependent, further
accounting for abundance and dynamics of molecular species, shown here as a elaborating the more plastic overall state (transient nature of input 2 described
trajectory of a particle inside a quasipotential well, with the particle position by a bidirectional arrow). The transiently occupied attractors can be
defined by the current concentrations of A and B that can change probabilisti- simultaneously occupied by distinct cells in the population. Small arrows
cally in time, with the quasipotential wells interpreted as the landscape correspond to stochastic fluctuations of molecular concentrations within
attractors. (B) Various scenarios of landscape alterations and the corresponding individual attractors. (C) Gene regulation and epigenetic landscapes of cancer
changes in the molecular distributions, shown as joint distributions of the cells can be complex and have multiple attractors, corresponding to distinct and
molecules A and B and the corresponding entropies H1 to H5. Implementation of stable cell states and phenotypes, which may be reshaped by oncogenic
these scenarios in the context of carcinogens is extensively illustrated and mutations, cell aging, environmental inputs, and other perturbations, leading to
discussed in the text. Oncogenic mutations of epigenetic modifiers and mutual accessibility of the attractors, more plastic cell states, and an increase
modulators or environmental inputs can lead to the formation of new stable in the phenotypic plasticity.

epithelial-mesenchymal plasticity (22). Addi- gene regulation landscapes. However, they A key consequence of both the emergence of
tionally, four attractor states have been pro- were based primarily on theoretical modeling new attractors and elevated variability within
posed to control EMT, including metastatic, constructs that require experimental valida- the same attractors is the increase in the en-
antimetastatic, and two intermediate states tion, as we discuss below. Alternatively, the tropy of the underlying regulatory landscapes
(23). These studies have illustrated putative properties of experimentally measured reg- (inputs 1 and 1′ in Fig. 1B; see Box 1). Indeed, it
feedback-based mechanisms of emergence of ulatory landscapes can be inferred without is consistently observed that the entropy of
well-defined attractors in cancer-associated imposing hypothetical mechanisms. gene expression increases in cancer versus the

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corresponding noncancerous tissue of origin, phenotypic characteristics, such as EMT, cell the Ising model enabled a more sophisticated
as revealed, for example, by examining gene proliferation, and drug resistance (input 2 in analysis, such as the calculation of the Jensen-
expression data alone (24) or by integrating Fig. 1B). These phenotypes may be related to Shannon distance to evaluate the mutual in-
these data with protein interaction networks those occurring separately in normal develop- formation between two epigenomes and thus
(to account for molecular interactions stabiliz- ment and tissue homeostasis but might be- to rank genomic features by their relative con-
ing the attractors) (25). A similar approach come mutually accessible in the context of a tribution to epigenetic landscape change (38).
using protein-interaction networks and RNA- larger underlying attractor in the phenotypi- This more-rigorous treatment revealed substan-
seq for multiple tissue samples rather than cally plastic cancerous states (Fig. 1C). Thus, a tial entropy increases in the epigenetic land-
single cells within the same sample has sug- more detailed landscape analysis is required scapes of a variety of cancer types compared
gested that tumor progression is associated to understand the mechanisms underlying the with matched normal tissues of origin (37).
with increasing entropy (26). This approach emergence and dynamics of cancerous cell Notably, it was found that an increase in dis-
was further used to link the entropy of gene populations. Furthermore, these studies leave ordered methylation patterns predicts poor
expression to the pluripotency of the under- the following questions open: What controls clinical outcomes in chronic lymphocytic leu-
lying cell population (27) and to demonstrate the availability of various transcriptional pro- kemia (39) and acute myelogenous leukemia
that specific cell differentiation, in contrast to grams that enable diverse landscape attractors (AML) (40). This recent progress has been con-
cancer emergence, could be represented by and what mechanisms regulate the transitions spicuous because cancer epigenetics was thought
emergent attractors associated with progres- between different attractor states and thus the to be computationally intractable owing to
sively lower variability and entropy. Consistent degree of plasticity of various phenotypes? being derived from hundreds of landscapes of
with this analysis, it was shown that cancer In contrast to gene regulation landscapes, in normal tissue (41).
stemness may be inferred from increased en- which variables, such as molecular concen- The increase in epigenetic landscape en-
tropy of the attractor defining a specific cell trations, can be viewed as continuously adopt- tropy observed in various cancers is accompa-
subpopulation (27). Notably, this analysis re- ing multiple values, epigenetic landscapes are nied by a greater variability in gene expression
vealed that decreased entropy was associated essentially binary or digital in nature—e.g., the (42), thus linking epigenetic and gene regu-
not only with cell fate–specifying molecular methylation patterns of specific CpG sites or lation landscapes. A possible explanation for
circuits, but also with specific cancer-related the presence or absence of specific histone mod- this linkage is that genetic programs account-
phenotypic states, such as increased cell pro- ifications. Nevertheless, the landscape analysis ing for new phenotypic properties can become
liferation (21), implying correlated gene ex- framework can also be used for these types of accessible because of increased epigenetic var-
pression and molecular interaction of the experimental data. For instance, epigenetic var- iability, leading to the emergence of new gene
components of the cell cycle, representing iation in the patterns of sets of four consecu- regulation attractors. This hypothesis is sup-
another example of a well-defined attractor tive CpG sites display increasing stochasticity ported by the observation that the increased
in the gene expression landscape. However, in aging fibroblasts and in cancer cells within variation in gene methylation and expression
this high proliferation attractor may not be particular regions of the genome (34). The pat- is often enriched in genomic regions that are
fully stable, with recent studies suggesting terns of consecutive CpG sites, also known as larger than a single gene (42, 43)—e.g., regions
that cells may stochastically transition into epialleles, can thus be treated probabilistically, associated with EMT (42) or smaller domains
and out of a cell proliferation state (6, 28). with their distributions within the same tumor observed in pancreatic cancer (44), perhaps
Other studies have pointed to a transient rather estimated using a Bayesian model (35). The related to hypomethylated regions inferred for
than stable nature of other key cancer-associated probability analysis lends itself to examination hematopoietic stem cells (45). Thus, epigenetic
phenotypes—e.g., drug resistance and EMT— of entropy and information metrics (defined control of multiple genes needed to enable a
which indicates that they may be a consequence in Box 1), which was applied to several brain specific phenotype could loosen, and the cor-
of phenotypic plasticity within a broader at- tumor and normal samples, revealing an in- responding genetic attractor may emerge and
tractor state, potentially allowing these pheno- creased methylation entropy of Alu repeat se- enable variable onset of a new phenotypic
types to be transiently achieved (29–32). This quences (36). The epigenetic patterns of epialleles state in a cell population. It has in fact been
broader attractor may be associated with a may display certain correlations, interpreted found that the transition to the mesenchymal
higher entropy stemness state, whereas each as specific attractor states in the underlying state depends on altered chromatin accessibility
of the transiently visited phenotypic states landscapes. Mechanistically, these correlated of the DNA regions incorporating EMT-related
may have much lower associated entropy—i.e., patterns may arise because DNA methylation genes (46). Furthermore, large heterochromatic
they can be considered subattractors that can at heritable (i.e., CpG) dinucleotides is affected domains related to developmentally regulated
be occupied by the cells either stochastically or by the state of the cell or location in the ge- EMT (47) are topologically altered in colon can-
in response to environmental inputs (33) (in- nome as well as the nearest CpG neighbors, cer (48). These data suggest that a key cancer-
puts 1 and 2 in Fig. 1B). because DNA methyltransferase I, the enzyme related phenotype is enabled by changes in both
These studies reveal that reasons for higher that copies CpG from the parent to the daughter gene expression landscape (actualization of a
entropy in gene expression networks in cancer strand during DNA replication, resides at the cryptic attractor state) and epigenetic land-
cells can be complex, because the same increase replication fork, conferring a nonrandom meth- scape (changes in DNA and histone markers
in entropy could be due to the emergence of ylation pattern to nearby CpG dinucleotides. that enable accessibility to the genetic infor-
new stable attractors in the cell population The underlying landscapes may be more mation encoding components of this newly
(phenotypic diversity; input 1′ in Fig. 1B) or formally defined recognizing the binary na- accessible genetic landscape attractor).
increased noisiness of the individual attrac- ture of the experimental data by exploiting Together, these results suggest that a key con-
tors that may already be present in the nor- the Ising model, introduced in the 1930s for dition for the emergence of a new attractor in
mal, precancerous tissue or emerge as a result one-dimensional magnetic dipoles with bi- the genetic landscape is the epigenetic reorga-
of cell dedifferentiation (phenotypic plasticity; nary states that are also influenced by external nization of the corresponding genetic material,
input 1 in Fig. 1B). Furthermore, phenotypic forces and the nearest dipole neighbors (37). It leading to increasing stochastic variability with-
plasticity can enable the emergence of tran- also allowed a highly accurate computation of in the current attractor and increased proba-
siently stable, stochastically visited attractors the Shannon normalized methylation entro- bility of transition to a new state (20) (input 1 in
that can nevertheless have important functional py (NME) for DNA methylation. Furthermore, Fig. 1B). It is therefore of interest to explore the

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triggers of epigenetic and gene regulation outcomes, whereas broader distributions were broader epiallele distribution, which may result
landscape reorganization that occur during generally associated with poor clinical prog- in broader, more noisy attractors; phenotypic
carcinogenesis, a task that is complicated by noses. Furthermore, higher epiallele diversity plasticity; and poorer prognosis (input 1 in Fig.
interdependencies of epigenetic and gene reg- was associated with gene expression hyper- 1B and Fig. 2). A greater phenotypic plasticity
ulation mechanisms. Indeed, epigenetic mod- variability and, thus, likely higher phenotypic enabled by a more global epigenetic dysregu-
ification, for example, DNA methylation of and developmental plasticity. This result was lation may enhance the escape of cancer cells
gene regulatory sequences, can directly influ- consistent with an independent analysis of from various treatments and other pressures,
ence gene expression, and many transcription AML involving genetic rearrangement of the including immune surveillance.
factors can recruit epigenetic regulators, which the histone methyltransferase gene KMT2A Whereas mutational alterations of some
suggests substantial epigenetic and genetic (also known as MLL), which, when compared modulators can lead to the emergence of spe-
landscape interdependence (Fig. 2). To provide with untransformed controls, also demonstrated cific attractor states, mutations of other mod-
more detailed insight into oncogenic processes a greatly increased epigenetic entropy (more ulator proteins may exert their effect indirectly
that shape and are shaped by epigenetic and specifically, NME) and high expression varia- by regulating the broad specificity epigenetic
gene regulation landscapes, it will be instruc- bility of genes implicated in AML etiology (53). modifiers, thereby also leading to extensive
tive to examine the genetic and environmental Notably, animal models of AML revealed that epigenetic alterations and broader, more plas-
inputs that can lead to precancerous and can- epigenetic variability was a precursor of trans- tic attractor states. For example, inhibition of
cerous states. formation to AML, pointing to the causal rather DNA methylation was highly effective in AML
than correlative nature of the greater entropy patients harboring TP53 mutations (encoding
Interplay between epigenetic states (52). Overall, these data suggest that the tumor suppressor p53) but had much lower
and genetic landscapes mutations affecting particular regulatory path- effects in tumors with wild-type (WT) TP53
How can the large number of recurrent so- ways may result in adoption of specific ge- (54). These results were supported by lung can-
matic mutations that drive carcinogenesis be netic and epigenetic states (narrow and stable cer xenograft studies and in vitro experiments
connected to the pathways and genes respon- landscape attractors). Conversely, dysregula- (55), which together suggest that p53 is an epi-
sible for cancer phenotypes (49)? Traditionally, tion of epigenetic modifiers can lead to a much genetic modulator that is critically important
this question has been addressed by analyzing
the effects of various mutations on the magni-
tude of phenotypic changes, such as increased
proliferation, in response to overexpression of
diverse mitogenic regulators. What is currently
less understood is how the increased variabil-
ity (and thus entropy) of epigenetic and gene
expression regulation is linked to cancer pro-
gression. Such analyses are now progressively
enabled by the rank ordering of oncogenic
mutations using metrics such as the Jensen-
Shannon distance (Box 1, Eq. 7), which es-
tablish the effect size and its importance in
epigenetic control of cancer etiology (38).
The roles of mutations affecting molecules
that directly (so-called epigenetic modifiers)
and indirectly (epigenetic modulators) control
epigenetic landscapes through their enzymatic
functions (50) can be elucidated in cancerous
states (Fig. 2). For example, in AML, driver
mutations can affect both epigenetic modu-
lator molecules [such as transcription factors Narrow to wide Single to multiple
and signaling pathways that can recruit epi- attractor attractors
genetic modifiers to specific gene targets (51)]
and modifier molecules [such as the DNA
demethylation-related gene TET2, which is
directly mutated in a subset of AML patients
(52)]. Analysis of epiallele distribution across
AML patients with different driver mutations Fig. 2. Interplay between epigenetic and gene expression landscapes. Developmental and environmental
has indicated an increase in the entropy of factors and genetic mutations can affect diverse modulators of epigenetic control and gene expression, such
epiallele distribution, consistent with other as signaling and cell communication networks, frequently leading to diversification of cell states. These
cancers. Notably, this analysis revealed a more modulators may directly affect mediators of epigenetic states, such as DNA demethylases, and gene
fine-grained epigenetic landscape structure expression, such as transcription factors, which also can directly interact with each other. Examples of these
underlying entropy increase. In particular, molecular regulators discussed in the text are shown here. The result is alterations of the epigenetic and gene
epialleles commonly showed specific patterns regulation landscapes that are tightly coupled, for example through the action of mediators of epigenetic
for a subset of modulator mutations, whereas control, influencing accessibility of genes for regulation, and the magnitude and variability of gene expression.
the distribution was much broader for mod- Certain additional inputs may be more specific to each of the landscapes, such as the epigenetic drift
ifier mutations. This study further indicated with cell aging primarily leading to a widening of the landscape attractors, higher plasticity and higher entropy
that tighter and more specific epiallele dis- of the state, or protein-protein interaction and gene regulatory networks, stabilizing various attractors
tributions were correlated with better clinical and serving to decrease the plasticity and entropy.

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for epigenetic stability. In embryonic stem potent stem cell–like states and increased plas- attractors more likely, maintaining a plastic
cells (ESCs), p53 restricts the expression of ticity of cancer cells. state and metaplastic tissue composition. A
modifiers DNMT3A and DNMT3B, thus inhib- particularly interesting example of this is
iting methylation, and regulates TET1 and TET2, Stabilization of attractor states found in kidney differentiation. WT1, a tissue-
making the loss of TRP53 a major driver of by endogenous and exogenous inputs specifying transcription factor that is critical
epigenetic heterogeneity (55, 56). Other genes Exogenous inputs for embryonic kidney development, can be
commonly mutated in AML, including WT1, Enhanced variability of both gene expression seen as a molecule stabilizing the correspond-
IDH1, and IDH2, can also have more global and epiallele abundance indicative of broad ing differentiation program attractor. Loss-of-
effects on epigenetic landscapes through in- genetic and epigenetic attractors may permit function mutation of WT1 leading to a loss of
ducing loss of TET2 function (57). These results cells to transiently achieve multiple alternative DNA binding in the germline causes Denys-
suggest that although epigenetic modifiers may states that could be potentially further stabi- Drash syndrome with renal dysplasia (67). The
not be mutated in specific tumors, in many lized by additional inputs. For example, ge- resultant absence of WT1 in somatic cells leads
cases, the effect of modulator mutations can netic perturbations of DNMT3A and DNMT3B to metaplastic transdifferentiation of early pro-
lead to changes in the methylation status of a markedly increase genetic and epigenetic en- nephric cells into other mesodermal elements,
large number of genes, similar to the effects tropy in ESCs, whereas the effects on mean such as skeletal muscle, fat, and cartilage, and
of modifier mutations. The more specific, tar- methylation are much more modest (59). Fur- generation of preneoplastic intralobar nephro-
geting effects of modifiers can be revealed thermore, several genes that are differentially genic rests relatively early in the developmen-
through comparative gene expression anal- controlled by this perturbation can promote tal axis, increasing the risk of Wilms tumor
ysis, for example by searching for enrichment cellular differentiation. Notably, genetic per- development (68). WT1 also controls EMT, likely
of binding sites for specific transcription fac- turbations of DNMT and TET genes revealed through interaction with the YAP transcrip-
tors in the control regions of epigenetically var- that the differentiated state is a separate at- tional coregulator in controlling E-cadherin
iable genes. Enrichment of specific binding tractor in the genetic landscape and that this expression (69), which may modulate the tis-
sites would suggest that a particular tran- attractor exists dynamically, with cells reach- sue organization and further influence the
scription factor can both recruit an epigenetic ing and leaving it according to the relative metaplastic state. Timed experimental acti-
modifier to a particular gene and more di- activities of DNMT and TET enzymes (60). vation of stem cell reprogramming factors in
rectly control the expression of this gene once These results are consistent with the current the developing mouse kidney can induce Wilms
it is epigenetically modified. This type of anal- understanding of the importance of epige- tumor in the absence of other perturbations,
ysis yielded a number of putative modulator netic regulation of induced pluripotency by including absence of any WT1 mutations (70).
proteins, including GATA2, coupled to epi- the Yamanaka factors, particularly the increased This same effect arises from loss of imprinting
allele variation in many AML subtypes driven expression of TET enzymes to stabilize this state (LOI) of IGF2 in Beckwith-Wiedemann syn-
by distinct mutation events (52). Similar studies (61). Critically, this differentiation attractor can drome, or sporadically in the general population,
have further suggested specific roles of NFKB1 also be regulated by modulators in the form of with hyperproliferation of developing nephrons
and MYBL1 as modulator proteins control- signaling pathways, particularly dual inhibi- also creating predisposition for Wilms tumor
ling the drug resistance phenotype in chronic tion of mitogen-activated protein kinase (MAPK) development. These results are consistent with
lymphocytic leukemia (58). signaling and glycogen synthase kinase-3b losses of stabilizing control leading to a broad,
Overall, these studies suggest the following (GSK3b) (62–64), which suggests that exter- variable, and plastic landscape attractor, which
refinement of our understanding of the in- nal signaling inputs can stabilize the differ- can also decrease the probability of occupying
terplay between genetic and epigenetic land- entiation state. the attractor corresponding to a stably differ-
scapes in the emergence of genetically driven The insights from epigenetic regulation of entiated nephron state (Fig. 1B and Fig. 3).
cancers (Fig. 2). The effect of mutations in stemness can elucidate how conditional attrac- Metaplastic states do not need to involve
mediator and a subset of modulator genes may tors can emerge and be stabilized, for example processes that occur early in development or
be broad, encompassing multiple genes across by exogenous signaling inputs in cancer (input 2 depend on reprogramming cues. Rather they
the genome and generating a high degree of in Fig. 1B). As an example of signaling attrac- may arise from stabilization of one of the mu-
epiallelic and genetic heterogeneity, which cor- tor stabilization, it was found in two indepen- tually accessible attractors even in the mature
relates with poor clinical outcome. The effect dent studies that paracrine signaling involving adult tissue (input 2′ in Fig. 1B). Acinar-ductal
of mutations in another subset of modulators insulin-like growth factor 1 (IGF-1) receptor metaplasia (ADM) (i.e., the process in which
may be more specific, leading to the emer- activation or NOTCH pathway signaling can acinar cells in the pancreas differentiate into
gence of less variable epiallele distributions lead to an altered chromatin structure that is ductal cells) can occur in the untransformed
and coordinated gene expression patterns, cor- mediated by the histone demethylases KDM5 adult pancreas as a part of the tissue repair
responding to a limited number of coupled and KDM6, respectively, which in turn control process (71) but may also lead to preneoplas-
attractors in genetic and epigenetic landscapes. the drug resistance phenotype (5, 65). The ac- tic lesions and, ultimately, progression to pan-
In both cases, the entropy of epigenetic and quisition of this phenotype was, in both cases, creatic ductal adenocarcinoma (PDAC) (72).
genetic landscapes goes up but for different transient and stochastic, suggesting continuous In addition to ADM, which may occur as a
reasons: In the former case, a more plastic transitions between distinct states associated normal adaptive process, destabilization of
state implies a noisy and relatively unstable with two or more attractors in the underly- the attractor corresponding to the acinar cell
attractor state, whereas in the latter case, in- ing landscape. The dynamic acquisition can be type can lead to the emergence of pancreatic
creased entropy may correspond to the emer- analyzed in substantial detail in vivo, as was intraepithelial neoplasms (PanINs). Further-
gence and coexistence of new stable attractors the case for AML in a genetically engineered more, other cell types may also emerge in this
in the genetic and epigenetic spaces (Fig. 1B). mouse model (66). process, for example tuft cells. This attractor
This view implies that there may be different destabilization may involve mutations of mod-
landscape paths to cancer, but the emergence Metaplasia ulators KRAS and DPC4 (also known as SMAD4)
of a more plastic, less defined attractor state The landscape-based analysis implies that a and up-regulation of KLF4 during inflam-
may be particularly interesting to consider, loss of stabilizing inputs, exogenous or endoge- mation or injury (73, 74) as well as changes
because it may be related to both more pluri- nous, may make transitions to other available in the epigenetic control of key regulators,

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A B C

Fig. 3. Connection between an epigenetic landscape and variable pheno- example, the mapping can be visualized as WT and LOI cell distributions mapped
typic outcomes. (A) An epigenetic alteration—LOI of the IGF2 gene, implicated with respect to the areas of cell survival and death on the (BAX, Bcl-2)
in Wilms tumor, doubling the signaling input—can lead to rewiring of the signaling phenotypic plane, which suggests how treatments targeting LOI cells may be
network activated by the IGF2 receptor IGF1R (depicted as IGF1Rp) through developed to spare the WT cells. Arrows in the lower panel represent the effect of
altered receptor trafficking (IGF1Rint), degradation (f), and altered balance of drugs, such as IGF1R inhibitors, shifting the landscape and phenotypic
activation of the downstream signaling pathways activating Erk (Erkpp) and Akt distribution toward the boundary separating survival and death, with the red
(Aktpp) kinases. Rebalancing of Erk and Akt activities translates into areas depicting the effect on the WT and LOI cell populations. (C) A more
transcriptional up-regulation of IGF1R and a higher proliferation rate but also general view of landscape mapping onto the apoptosis phenotypic plane. By
rebalancing of pro- and antiapoptotic protein abundances (BAX versus Bcl-2, analogy with Fig. 1B, one can contrast mapping of a large attractor versus two
respectively), leading to an increased propensity for cell death (108). The integral more limited attractors, representing the difference between a plastic and
signs represent integration over time of signaling activities. (B) The landscape stochastic state (phenotypic plasticity) versus a state with two alternative
alterations that correspond to a change in phenotype (top) are the altered stable attractors (phenotypic diversity). The more plastic state can allow cells to
expression and activity of signaling pathway molecules (and thus gene regulatory escape from the death area to the survival area even in the presence of a
landscapes in the bottom panel) in response to alteration of epigenetic treatment [such as in (B)], by stochastically exploring the available attractor,
landscapes (IGF2 LOI). This leads to emergence of a new attractor in addition whereas a combination of more stable attractors (with the same overall entropy
to the WT attractor, resulting in a mosaic WT-LOI cell distribution in the tissue. as the more plastic state) can allow for selective targeting of one but not
This landscape alteration can be mapped onto, for example, the apoptosis the other attractor. Therefore, the treatment strategy suggested in (B) may
phenotype-defining network by a quantitative analysis of the dependence of the benefit from the initial intervention, stabilizing smaller attractors within a larger
BCL family protein distributions on the signaling inputs, thus enabling a direct one and thus decreasing the plasticity of the state, particularly through
translation of the landscape alterations into phenotype distributions. In this epigenetic perturbations.

such as AATK, as revealed by studies of a mouse Together, these observations suggest that viously dominant attractors may become less
model of ADM and pancreatic cancer (75). metaplasia or alternative differentiation can likely, whereas preexisting alternative attrac-
Another example of an environmentally in- be an important characteristic of premalignant tors can be occupied with higher probabilities,
duced metaplastic process occurs in Barrett’s tissue states. Consequently, the new states ex- leading to further metaplasia and potentially
esophagus. In this case, the environmental plored by the cells may not be random but fre- cancerous states (Fig. 1B). In another scenario,
stimulus (modulator) is acid reflux and inflam- quently belong to closely related lineages (79), if epigenetic modulators are directly affected
matory cytokine enrichment, which promote in agreement with the expectation that cells by genetic mutations or environmental inputs,
transdifferentiation of normal squamous epi- explore adjacent attractors along the under- even tissues with low initial plasticity can be-
thelium of the esophagus into intestine-like lying epigenetic and genetic landscapes be- come more plastic and permit occupancy of
columnar epithelium (76). Animal and human cause of an increased stochasticity and thus previously inaccessible, cryptic attractors (in-
data suggest that in this setting, the attractor entropy of the regulatory networks (Fig. 1C). put 1′ in Fig. 1B). These attractors can become
corresponding to the esophageal cell type is com- Notably, up-regulation of epigenetic modifiers, stable as a result of either internal feedback or
promised as a result of induction of intestinal- such as ARID1A, is commonly observed both additional stabilizing mutations, or they can
specific factors CDX1 and CDX2, which might in the metaplastic states and multiple cancers remain transient and stochastically visited de-
be induced by inflammatory inputs and can (80–82). Tissue disorganization related to meta- pending on the presence of an external cue.
self-stabilize through autoregulation (77). This plasia as well as the common dysregulation of The higher degree of epigenetic plasticity and
process is associated with a bimodality in the the EMT-MET program can further perturb conditional, rather than stable, attractors can
DNA methylation distribution reflective of co- local cell-cell interactions, leading to hyperpla- increase the corresponding phenotypic plas-
existence of esophageal and intestinal attrac- sia and the onset of neoplastic growth (83–85). ticity, which can result in poor prognosis be-
tors, characteristic of this type of metaplasia Therefore, if these genetic or environmental cause of increased metastatic propensity, drug
(78). Transdifferentiation in this setting is again inputs occur in tissues with a high potential resistance, or immune escape. In this sense,
a major risk factor of ensuing development of for metaplasticity, the occupancy of the attrac- stabilization of particular attractors, by reduc-
esophageal adenocarcinoma. tors may change. The occupancy of the pre- ing the plasticity of cell regulation, can make

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cancer progression more predictable and po- organisms (94–97). In the context of hyperpro- teins, such as Bcl-2) (106, 107) (Fig. 3A). The
tentially increase the probability of success of liferation associated with cancer, epigenetic equally matched abundance of pro- and anti-
personalized therapeutic interventions. drift is because of epimutations accumulating apoptotic proteins defines the boundary be-
with the increasing number of cell divisions, tween these states within a phenotypic space
Attractor stability and aging which can happen at a high frequency (98). (life versus death) onto which the epigenetic and
Stabilization of conditional attractors by an This increasing entropy of the epigenetic land- genetic landscapes can be mapped (Fig. 3B).
environmental cue enabled by dysregulated scape is distinct from the recurrent changes This mapping is based on biochemical input-
epigenetic control can occur through a variety in DNA methylation serving as a basis for the output relatonships between the phenotype-
of mechanisms but particularly through the epigenetic clocks (99). Unlike the age-related defining molecules and the upstream genetic
activation of intracellular signaling networks. DNA hyper- or hypomethylation signatures at and epigenetic mediators, and it is a natural
Indeed, new attractors, such as the mesenchy- specific CpG sites, epigenetic drift is a reflec- dimensionality reduction mechanism that al-
mal cell state emerging in EMT, can be trig- tion of increased DNA methylation variability, lows the evaluation of effects of the underlying
gered by biochemical and biomechanical inputs which frequently occurs at loci related to ma- molecular network variability and regulation.
from the microenvironment, and increased cell lignancy and can be predictive of cancer emer- In many circumstances—e.g., in the turnover
proliferation can be controlled by a progres- gence (100). One example of this site specificity of multiple epithelial tissues—the entropy asso-
sive accumulation of growth factors and other is methylation at the locus encoding polycomb ciated with the underlying landscape and thus
ligands in the cellular milieu (69). Attractor sta- repressive complex-2 (PRC2) (itself responsi- variability in the expression of apoptosis pro-
bilization may result in a decrease in entropy ble for trimethylation of H3K27) (98). The en- teins can result in a subset of cells undergoing
of gene expression and epiallele occurrence, tropy increase associated with epigenetic drift cell death even under homeostatic conditions.
which can be precisely quantified experimen- may also explain the emergence of a rare 5% The distribution of cells across the epigenetic
tally and used to evaluate the information car- subpopulation of bipotent-like breast cancer landscape can therefore define the fractions
ried by the signaling input (86) (see Box 1; progenitor cells (101). Intratumor epigenetic of cells displaying different phenotypes.
information is defined as the decrease in en- drift may be dependent on the replication his- The mapping of signaling network activity
tropy after an input). The entropy decrease is tory of the individual clones, leading to a con- onto this apoptosis-regulating circuit (Fig. 3B)
frequently modest, with only 1 bit of informa- tinually expanding spectrum of EMT identities can be established experimentally and visual-
tion about the signal amplitude commonly trans- in colorectal tumors (102). Overall, the landscape ized in two dimensions as the joint single-cell
ferred in such events on a cell-by-cell basis entropy is not static and can decrease and distributions of Bcl-2–Bax pairs. The regula-
(12, 86–92). Coordination of signaling between increase in a quantifiable fashion, even with- tory landscapes upstream of the phenotype-
multiple cells can allow for an increase in this out genetic alterations, creating a more com- defining molecular circuit control the relative
information transfer and thus further entropy plex context for normal tissue function and abundances of the proteins in these pairs, which
decrease; however, because cellular communi- progression to cancer. can alter in response to an epigenetic change,
cation is noisy, the effect may also not be very such as LOI of IGF2 (108). The misregulation of
substantial (93). A further entropy decrease Mapping regulatory landscapes onto the signaling inputs resulting from epigenetic
can occur if cells are engaged in response to phenotypic responses alteration [particularly the rebalancing of
the same input but are not necessarily com- Can quantitatively assessed changes in epige- signaling through growth factor–responsive
municating (92). netic and genetic landscapes inform the se- extracellular signal–regulated kinase (ERK)–
One bit of information is sufficient to define lection of appropriate treatments? Because MAPK and AKT pathways] is the key aspect of
a binary phenotypic outcome, such as cell treatments are driven by disease-specific pheno- the newly emerging attractor state that trans-
death/survival, differentiation/self-renewal, types and alterations of landscapes occur at lates into a new Bcl-2–Bax distribution, resulting
or proliferation/migration. This binary outcome the level of DNA modifications and molecular in a higher propensity for cell death (Fig. 3C).
frequently reflects occupancy of alternative networks, it is important to consider in more Therefore, an epigenetic landscape change, in
attractor states modulated by the signaling detail how the specific phenotypes are con- addition to promoting higher cell proliferation
input (inputs 2 and 2′ in Fig. 1B). In the con- trolled. In many cases, a phenotypic state is and tissue hyperplasia, creates a vulnerability
text of cancer progression, multiple external defined by a specific biomolecular circuit. For (an increased propensity to undergo apoptosis)
cues can trigger signaling events, leading to instance, cell migration phenotypes are con- that can be exploited to selectively eliminate
diversification of cell responses through con- trolled by cytoskeleton reorganization, which the cell population undergoing IGF2 LOI. These
ditional occupancy of different attractors as in turn is frequently controlled by a subset of landscape perturbations cause the LOI cells
a function of the combinations of different Rho-family small guanosine triphosphatases but not the WT cells to cross the death bound-
cues (and their persistence) in a specific tu- (GTPases) (103, 104). Similarly, cell prolifera- ary (Fig. 3C), thus predicting the therapeutic
mor location or the cues received by cancer tion is phenotypically driven by the circuit in- window relevant to this precancerous state for
cells during metastatic spread. Similar quan- volving regulation of cyclin-dependent kinases specific drugs, such as receptor tyrosine kinase
titative analysis can be used to gauge the effects (105). It is therefore important to understand inhibitors (108). This analysis highlights the
of different mutations and drug treatments how the function of these specific, phenotype- direct translation of landscape concepts to the
that can perturb the entropy of different at- defining circuits may be influenced by the more development of more-targeted therapeutic ap-
tractors more specifically or the entropy asso- global changes in cellular regulatory networks, proaches, particularly if an epigenetic altera-
ciated with the overall epigenetic or genetic as discussed below for the case of apoptosis. tion is considered an important therapeutic
state more generally (12). Although the molecular circuits controlling target (109).
In contrast to the entropy decrease because apoptosis are complex, one can use the relative This phenotypic mapping analysis can be
of signaling inputs, entropy of a particular at- abundances of pro- and antiapoptotic members further extended within the recently intro-
tractor and thus the associated phenotypic of the BCL protein family controlling the pheno- duced framework of cellular archetypes in
plasticity can also naturally increase because typic outcomes of either regulated cell death tissues (31, 32). Archetypes are defined as ex-
of epigenetic drift (input 1 in Fig. 1B). Epi- (relatively higher abundance of proapoptotic treme phenotypic states spanning the avail-
genetic drift occurs both in tissue culture proteins, such as Bax) or cytoprotection (rela- able optimized phenotypic space and thus
and in vivo with increasing age of cells and tively higher abundance of antiapoptotic pro- reflecting the available degrees of freedom of

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the phenotypic plasticity. The analysis of Wilms the ability of cells (and their molecular net- In this Review, we primarily focused on DNA
tumor patient samples revealed three archetypal works) to dynamically explore diverse states methylation as a key epigenetic mechanism for
phenotypic states, suggesting a more complex and corresponding phenotypes—and pheno- which landscape and entropic analysis has
landscape distribution in vivo. More-advanced typic diversity—i.e., stable occupancy of different already been performed in diverse biological
stages of the disease, accompanied by emer- attractors by different cells in the population systems. However, epigenetic control can oc-
gence of IGF2 LOI, lead to a shift of cell popu- (Fig. 3C). In both cases, the entropy of the cur on other levels, including through histone
lation from a relatively uniform distribution tumor tissue may be higher compared with modifications. As rich data accounting for fur-
across three archtypes—an epithelial and two the parental WT tissue state, but the landscapes ther levels of epigenetic control are gathered,
mesenchymal states (one of which is also char- would be distinct, which can be a crucial level of an important challenge will be to understand
acterized by higher cell proliferation)—toward resolution to determine prognosis and possi- whether extensive, landscape-based description
a greater abundance of mesenchymal states ble interventions. Nevertheless, entropy can of all levels of epigenetic and gene regulatory
versus the epithelial one (30). This result sug- still be a convenient metric, particularly in as- controls is necessary or whether this complexity
gested a specific landscape change, potentially sessing the effects of signaling inputs, muta- and data dimensionality can be reduced to a
resulting from an increased expression of IGF2 tional and epigenetic perturbations, or medical simpler measurement set. In these efforts, a key
promoting more aggressive cell behaviors re- interventions. In particular, changes in entropy role may be played by various machine learning
lated to cell proliferation and EMT. Despite this [assessed for example through different in- techniques to reveal mutual interdependencies
more complex landscape structure, we suggest formation theory–based measures (Box 1)] can of various molecular control mechanisms and
that phenotypic mapping analysis can still be quantitatively characterize the effects of these their influences on phenotypic plasticity.
used to target specific cell states. diverse inputs or evaluate how long-term pro-
REFERENCES AND NOTES
In general, measuring and mapping genetic cesses, such as cellular aging, may affect cel-
1. I. J. Fidler, Tumor heterogeneity and the biology of cancer
and epigenetic landscapes onto phenotypic lular landscapes. invasion and metastasis. Cancer Res. 38, 2651–2660 (1978).
spaces can be an effective strategy to both New experimental approaches may also al- pmid: 354778
infer the corresponding phenotypic diversity low further analysis of causality versus corre- 2. P. C. Nowell, The clonal evolution of tumor cell populations.
Science 194, 23–28 (1976). doi: 10.1126/science.959840;
and target specific subpopulations defined lation in the relationship between landscape pmid: 959840
by landscape attractors. Notably, it can also alterations and the emergence of cancer states. 3. D. Jia, M. K. Jolly, P. Kulkarni, H. Levine, Phenotypic Plasticity
elucidate the effects of phenotypic plasticity For instance, new CRISPR methods can allow and Cell Fate Decisions in Cancer: Insights from Dynamical
Systems Theory. Cancers 9, 70 (2017). doi: 10.3390/
and guide corresponding treatment strat- recruitment of specific epigenetic modifiers to
cancers9070070; pmid: 28640191
egies. If mutations, epigenetic changes, or en- a targeted locus within the genome and direct 4. A. Sacchetti et al., Phenotypic plasticity underlies local
vironmental inputs leading to the emergence control of epigenetic and gene expression var- invasion and distant metastasis in colon cancer. eLife 10,
of precancerous and cancerous states also iability and attractor properties (29). Studies e61461 (2021). doi: 10.7554/eLife.61461; pmid: 34036938
5. S. V. Sharma et al., A chromatin-mediated reversible
increase the plasticity within emerging attrac- such as this combined with further analysis of drug-tolerant state in cancer cell subpopulations. Cell 141,
tors, cell populations may be substantially less precancerous states in patients and animal 69–80 (2010). doi: 10.1016/j.cell.2010.02.027; pmid: 20371346
targetable owing to a larger overlap with WT models involving specific perturbations of 6. C. A. Chang et al., Ontogeny and Vulnerabilities of Drug-
Tolerant Persisters in HER2+ Breast Cancer. Cancer Discov.
attractors and faster escape from a vulnerable genetic and epigenetic mechanisms can pro- 12, 1022–1045 (2022). doi: 10.1158/2159-8290.CD-20-1265;
area of the attractor (Fig. 3C). This consider- vide further data to refine our understanding pmid: 34911733
ation both provides further interpretation for of cancer-specific lanscapes. 7. J. X. Zhou, M. D. Aliyu, E. Aurell, S. Huang, Quasi-potential
landscape in complex multi-stable systems. J. R. Soc.
poor prognostic correlates of the more plastic Within oncology applications, key advan- Interface 9, 3539–3553 (2012). doi: 10.1098/rsif.2012.0434;
tumor attractors and suggests that cancer tages of the landscape and entropy concepts pmid: 22933187
treatment should reduce plasticity. More spe- lie in an improvement of our understanding 8. J. Wang, K. Zhang, L. Xu, E. Wang, Quantifying the Waddington
landscape and biological paths for development and
cifically, the clinical intervention would be of cancer etiology and development of ap- differentiation. Proc. Natl. Acad. Sci. U.S.A. 108, 8257–8262
aimed at reshaping the underlying landscape proaches for targeted and personalized can- (2011). doi: 10.1073/pnas.1017017108; pmid: 21536909
to first stabilize the cancer-specific attractors cer treatments. More plastic attractors can 9. C. Li, J. Wang, Landscape and flux reveal a new global
view and physical quantification of mammalian cell cycle.
by perturbing the attractor-defining pathways allow cells to potentially rapidly adjust to both
Proc. Natl. Acad. Sci. U.S.A. 111, 14130–14135 (2014).
and molecular circuits and to then eliminate pharmacological and immune therapeutic in- doi: 10.1073/pnas.1408628111; pmid: 25228772
cells occupying these attractors while mini- terventions as well as enable cells to dynam- 10. G. Balázsi, A. van Oudenaarden, J. J. Collins, Cellular decision
mizing the effect on untransformed cells. ically switch between diverse phenotypic states making and biological noise: From microbes to mammals.
Cell 144, 910–925 (2011). doi: 10.1016/j.cell.2011.01.030;
required, for example, for metastatic spread. pmid: 21414483
Conclusions and future directions Therefore, arguably, further approaches may 11. N. Eling, M. D. Morgan, J. C. Marioni, Challenges in measuring
The studies reviewed here support the utility focus on a combination of treatments that and understanding biological noise. Nat. Rev. Genet. 20,
536–548 (2019). doi: 10.1038/s41576-019-0130-6;
of the genetic and epigenetic landscape ap- would not only attempt to eliminate specific pmid: 31114032
proaches in our understanding of cancer cell subpopulations but also reduce the pheno- 12. A. Levchenko, I. Nemenman, Cellular noise and information
etiology, ultimately allowing us to arrive at a typic plasticity of tumor cells. In developing transmission. Curr. Opin. Biotechnol. 28, 156–164 (2014).
doi: 10.1016/j.copbio.2014.05.002; pmid: 24922112
more general and conceptual understanding of these approaches, characterization of the un- 13. A. Golightly, C. S. Gillespie, in In Silico Systems Biology,
carcinogenesis. Notably, this approach presents derlying landscapes will be of particular im- M. V. Schneider, Ed. (Springer, 2013), pp. 169–187.
a detailed view of the system, which may focus portance. In these efforts, it will be important 14. D. T. Gillespie, A. Hellander, L. R. Petzold, Perspective:
Stochastic algorithms for chemical kinetics. J. Chem. Phys.
on a specific attractor but may also focus on a to refine our understanding of how the land- 138, 170901 (2013). doi: 10.1063/1.4801941
combination of multiple attractors and dy- scapes determined on the epigenetic and gene 15. M. Ullah, O. Wolkenhauer, Stochastic approaches in systems
namic transitions between them. Conversely, regulation levels can be quantitatively trans- biology. Wiley Interdiscip. Rev. Syst. Biol. Med. 2, 385–397
(2010). doi: 10.1002/wsbm.78; pmid: 20836037
entropy is frequently a less-resolved and more lated into the corresponding descriptions of
16. D. Grün, A. van Oudenaarden, Design and analysis of single-
global metric of the overall diversity of cell states. the activity of the molecular circuits controlling cell sequencing experiments. Cell 163, 799–810 (2015).
Both approaches are useful and frequently com- specific phenotypes. Such phenotypic mapping doi: 10.1016/j.cell.2015.10.039; pmid: 26544934
plementary. For example, the landscape view can direct more-specific drug development ef- 17. G. Kelsey, O. Stegle, W. Reik, Single-cell epigenomics:
Recording the past and predicting the future. Science 358,
may be better at capturing the difference be- forts and quantitatively establish the thera- 69–75 (2017). doi: 10.1126/science.aan6826;
tween an increase in phenotypic plasticity—i.e., peutic windows of specific compounds. pmid: 28983045

Feinberg and Levchenko, Science 379, eaaw3835 (2023) 10 February 2023 9 of 11

RES EARCH | R E V I E W

18. E. Shema, B. E. Bernstein, J. D. Buenrostro, Single-cell and 40. S. Li et al., Distinct evolution and dynamics of epigenetic and 62. C. A. Doege et al., Early-stage epigenetic modification during
single-molecule epigenomics to uncover genome regulation genetic heterogeneity in acute myeloid leukemia. Nat. Med. somatic cell reprogramming by Parp1 and Tet2. Nature 488,
at unprecedented resolution. Nat. Genet. 51, 19–25 22, 792–799 (2016). doi: 10.1038/nm.4125; pmid: 27322744 652–655 (2012). doi: 10.1038/nature11333; pmid: 22902501
(2019). doi: 10.1038/s41588-018-0290-x; pmid: 30559489 41. The American Association for Cancer Research Human 63. Y. Gao et al., Replacement of Oct4 by Tet1 during iPSC
19. Q. Li et al., Dynamics inside the cancer cell attractor reveal Epigenome Task Force, European Union, Network of induction reveals an important role of DNA methylation and
cell heterogeneity, limits of stability, and escape. Proc. Natl. Excellence, Scientific Advisory Board, Moving AHEAD with an hydroxymethylation in reprogramming. Cell Stem Cell 12,
Acad. Sci. U.S.A. 113, 2672–2677 (2016). doi: 10.1073/ international human epigenome project. Nature 454, 711–715 453–469 (2013). doi: 10.1016/j.stem.2013.02.005;
pnas.1519210113; pmid: 26929366 (2008). doi: 10.1038/454711a; pmid: 18685699 pmid: 23499384
20. E. Pujadas, A. P. Feinberg, Regulated noise in the epigenetic 42. K. D. Hansen et al., Increased methylation variation in 64. J. Silva et al., Nanog is the gateway to the pluripotent
landscape of development and disease. Cell 148, 1123–1131 epigenetic domains across cancer types. Nat. Genet. 43, ground state. Cell 138, 722–737 (2009). doi: 10.1016/
(2012). doi: 10.1016/j.cell.2012.02.045; pmid: 22424224 768–775 (2011). doi: 10.1038/ng.865; pmid: 21706001 j.cell.2009.07.039; pmid: 19703398
21. A. Taherian Fard, M. A. Ragan, Modeling the Attractor 43. B. P. Berman et al., Regions of focal DNA hypermethylation 65. B. B. Liau et al., Adaptive Chromatin Remodeling Drives
Landscape of Disease Progression: A Network-Based Approach. and long-range hypomethylation in colorectal cancer coincide Glioblastoma Stem Cell Plasticity and Drug Tolerance.
Front. Genet. 8, 48 (2017). doi: 10.3389/fgene.2017.00048; with nuclear lamina–associated domains. Nat. Genet. 44, Cell Stem Cell 20, 233–246.e7 (2017). doi: 10.1016/
pmid: 28458684 40–46 (2012). doi: 10.1038/ng.969; pmid: 22120008 j.stem.2016.11.003; pmid: 27989769
22. S. Tripathi, P. Chakraborty, H. Levine, M. K. Jolly, A mechanism 44. O. G. McDonald et al., Epigenomic reprogramming during 66. R. C. Rockne et al., State-Transition Analysis of Time-
for epithelial-mesenchymal heterogeneity in a population of pancreatic cancer progression links anabolic glucose Sequential Gene Expression Identifies Critical Points
cancer cells. PLOS Comput. Biol. 16, e1007619 (2020). metabolism to distant metastasis. Nat. Genet. 49, 367–376 That Predict Development of Acute Myeloid Leukemia.
doi: 10.1371/journal.pcbi.1007619; pmid: 32040502 (2017). doi: 10.1038/ng.3753; pmid: 28092686 Cancer Res. 80, 3157–3169 (2020). doi: 10.1158/
23. Y. Cheng et al., Role of metastasis-associated lung 45. M. Jeong et al., Large conserved domains of low DNA 0008-5472.CAN-20-0354; pmid: 32414754
adenocarcinoma transcript-1 (MALAT-1) in pancreatic cancer. methylation maintained by Dnmt3a. Nat. Genet. 46, 17–23 67. J. Pelletier et al., Germline mutations in the Wilms’ tumor
PLOS ONE 13, e0192264 (2018). doi: 10.1371/journal. (2014). doi: 10.1038/ng.2836; pmid: 24270360 suppressor gene are associated with abnormal urogenital
pone.0192264; pmid: 29389953 46. K. S. Johnson et al., Gene expression and chromatin development in Denys-Drash syndrome. Cell 67, 437–447
24. W. N. van Wieringen, A. W. van der Vaart, Statistical analysis accessibility during progressive EMT and MET linked to (1991). doi: 10.1016/0092-8674(91)90194-4; pmid: 1655284
of the cancer cell’s molecular entropy using high-throughput dynamic CTCF engagement. bioRxiv2020.05.11.089110 68. A. Schedl, N. Hastie, Multiple roles for the Wilms’ tumour
data. Bioinformatics 27, 556–563 (2011). doi: 10.1093/ [Preprint] (2020); doi: 10.1101/2020.05.11.089110 suppressor gene, WT1 in genitourinary development.
bioinformatics/btq704; pmid: 21172912 47. O. G. McDonald, H. Wu, W. Timp, A. Doi, A. P. Feinberg, Mol. Cell. Endocrinol. 140, 65–69 (1998). doi: 10.1016/
25. J. West, G. Bianconi, S. Severini, A. E. Teschendorff, Genome-scale epigenetic reprogramming during epithelial-to- S0303-7207(98)00031-8; pmid: 9722170
Differential network entropy reveals cancer system mesenchymal transition. Nat. Struct. Mol. Biol. 18, 867–874 69. J. Park et al., Switch-like enhancement of epithelial-
hallmarks. Sci. Rep. 2, 802 (2012). doi: 10.1038/srep00802; (2011). doi: 10.1038/nsmb.2084; pmid: 21725293 mesenchymal transition by YAP through feedback regulation
pmid: 23150773 48. S. E. Johnstone et al., Large-Scale Topological Changes of WT1 and Rho-family GTPases. Nat. Commun. 10, 2797
26. F. Cheng, C. Liu, B. Shen, Z. Zhao, Investigating cellular Restrain Malignant Progression in Colorectal Cancer. Cell 182, (2019). doi: 10.1038/s41467-019-10729-5; pmid: 31243273
network heterogeneity and modularity in cancer: A network 1474–1489.e23 (2020). doi: 10.1016/j.cell.2020.07.030; 70. K. Ohnishi et al., Premature termination of reprogramming in
entropy and unbalanced motif approach. BMC Syst. Biol. 10, 65 pmid: 32841603 vivo leads to cancer development through altered epigenetic
(2016). doi: 10.1186/s12918-016-0309-9; pmid: 27585651 49. L. A. Garraway, E. S. Lander, Lessons from the cancer genome. regulation. Cell 156, 663–677 (2014). doi: 10.1016/
27. A. E. Teschendorff, T. Enver, Single-cell entropy for accurate Cell 153, 17–37 (2013). doi: 10.1016/j.cell.2013.03.002; j.cell.2014.01.005; pmid: 24529372
estimation of differentiation potency from a cell’s pmid: 23540688 71. M. Reichert, A. K. Rustgi, Pancreatic ductal cells in
transcriptome. Nat. Commun. 8, 15599 (2017). doi: 10.1038/ 50. A. P. Feinberg, M. A. Koldobskiy, A. Göndör, Epigenetic development, regeneration, and neoplasia. J. Clin. Invest. 121,
ncomms15599; pmid: 28569836 modulators, modifiers and mediators in cancer aetiology and 4572–4578 (2011). doi: 10.1172/JCI57131; pmid: 22133881
28. R. Chaligne et al., Epigenetic encoding, heritability and progression. Nat. Rev. Genet. 17, 284–299 (2016). 72. P. Storz, Acinar cell plasticity and development of
plasticity of glioma transcriptional cell states. Nat. Genet. 53, doi: 10.1038/nrg.2016.13; pmid: 26972587 pancreatic ductal adenocarcinoma. Nat. Rev. Gastroenterol.
1469–1479 (2021). doi: 10.1038/s41588-021-00927-7; 51. T. Zhen et al., RUNX1 and CBFb-SMMHC transactivate target Hepatol. 14, 296–304 (2017). doi: 10.1038/nrgastro.2017.12;
pmid: 34594037 genes together in abnormal myeloid progenitors for leukemia pmid: 28270694
29. A. Lu et al., Reprogrammable CRISPR/dCas9-based development. Blood 136, 2373–2385 (2020). doi: 10.1182/ 73. D. Wei et al., KLF4 is essential for induction of cellular
recruitment of DNMT1 for site-specific DNA demethylation blood.2020007747; pmid: 32929473 identity change and acinar-to-ductal reprogramming during
and gene regulation. Cell Discov. 5, 22 (2019). doi: 10.1038/ 52. S. Li et al., Somatic Mutations Drive Specific, but Reversible, early pancreatic carcinogenesis. Cancer Cell 29, 324–338
s41421-019-0090-1; pmid: 31016028 Epigenetic Heterogeneity States in AML. Cancer Discov. 10, (2016). doi: 10.1016/j.ccell.2016.02.005; pmid: 26977883
30. A. Trink et al., Geometry of Gene Expression Space of Wilms’ 1934–1949 (2020). doi: 10.1158/2159-8290.CD-19-0897; 74. V. Giroux, A. K. Rustgi, Metaplasia: Tissue injury adaptation
Tumors From Human Patients. Neoplasia 20, 871–881 pmid: 32938585 and a precursor to the dysplasia-cancer sequence. Nat. Rev.
(2018). doi: 10.1016/j.neo.2018.06.006; pmid: 30029183 53. M. A. Koldobskiy et al., A Dysregulated DNA Methylation Cancer 17, 594–604 (2017). doi: 10.1038/nrc.2017.68;
31. J. Hausser, U. Alon, Tumour heterogeneity and the Landscape Linked to Gene Expression in MLL-Rearranged pmid: 28860646
evolutionary trade-offs of cancer. Nat. Rev. Cancer 20, AML. Epigenetics 15, 841–858 (2020). doi: 10.1080/ 75. L. Y. Ding et al., Epigenetic silencing of AATK in acinar
247–257 (2020). doi: 10.1038/s41568-020-0241-6; 15592294.2020.1734149; pmid: 32114880 to ductal metaplasia in murine model of pancreatic cancer.
pmid: 32094544 54. A. J. Levine, The p53 protein plays a central role in the Clin. Epigenetics 12, 87 (2020). doi: 10.1186/
32. J. Hausser et al., Tumor diversity and the trade-off between mechanism of action of epigentic drugs that alter s13148-020-00878-6; pmid: 32552862
universal cancer tasks. Nat. Commun. 10, 5423 (2019). the methylation of cytosine residues in DNA. Oncotarget 8, 76. S. J. Spechler, R. F. Souza, Barrett’s esophagus. N. Engl.
doi: 10.1038/s41467-019-13195-1; pmid: 31780652 7228–7230 (2017). doi: 10.18632/oncotarget.14805; J. Med. 371, 836–845 (2014). doi: 10.1056/NEJMra1314704;
33. P. B. Gupta et al., Stochastic state transitions give rise to pmid: 28129641 pmid: 25162890
phenotypic equilibrium in populations of cancer cells. 55. P. T. Filipczak et al., p53-Suppressed Oncogene TET1 Prevents 77. R. Barros et al., CDX2 autoregulation in human intestinal
Cell 146, 633–644 (2011). doi: 10.1016/j.cell.2011.07.026; Cellular Aging in Lung Cancer. Cancer Res. 79, 1758–1768 metaplasia of the stomach: Impact on the stability of the
pmid: 21854987 (2019). doi: 10.1158/0008-5472.CAN-18-1234; pmid: 30622117 phenotype. Gut 60, 290–298 (2011). doi: 10.1136/
34. G. Landan et al., Epigenetic polymorphism and the stochastic 56. A. Tovy et al., p53 is essential for DNA methylation gut.2010.222323; pmid: 21148572
formation of differentially methylated regions in normal homeostasis in naïve embryonic stem cells, and its loss 78. E. G. Luebeck et al., Identification of a key role of widespread
and cancerous tissues. Nat. Genet. 44, 1207–1214 (2012). promotes clonal heterogeneity. Genes Dev. 31, 959–972 epigenetic drift in Barrett’s esophagus and esophageal
doi: 10.1038/ng.2442; pmid: 23064413 (2017). doi: 10.1101/gad.299198.117; pmid: 28607180 adenocarcinoma. Clin. Epigenetics 9, 113 (2017). doi: 10.1186/
35. J. E. Barrett et al., Quantification of tumour evolution and 57. R. Rampal et al., DNA hydroxymethylation profiling reveals s13148-017-0409-4; pmid: 29046735
heterogeneity via Bayesian epiallele detection. BMC Bioinformatics that WT1 mutations result in loss of TET2 function 79. J. Wu et al., A single-cell survey of cellular hierarchy in acute
18, 354 (2017). doi: 10.1186/s12859-017-1753-2; pmid: 28743252 in acute myeloid leukemia. Cell Rep. 9, 1841–1855 myeloid leukemia. J. Hematol. Oncol. 13, 128 (2020).
36. H. Xie et al., Genome-wide quantitative assessment of (2014). doi: 10.1016/j.celrep.2014.11.004; pmid: 25482556 doi: 10.1186/s13045-020-00941-y; pmid: 32977829
variation in DNA methylation patterns. Nucleic Acids Res. 39, 58. A. Marusyk, M. Janiszewska, K. Polyak, Intratumor 80. M. M. Streppel et al., Next-generation sequencing of
4099–4108 (2011). doi: 10.1093/nar/gkr017; pmid: 21278160 heterogeneity: The rosetta stone of therapy resistance. endoscopic biopsies identifies ARID1A as a tumor-suppressor
37. G. Jenkinson, E. Pujadas, J. Goutsias, A. P. Feinberg, Cancer Cell 37, 471–484 (2020). doi: 10.1016/ gene in Barrett’s esophagus. Oncogene 33, 347–357 (2014).
Potential energy landscapes identify the information- j.ccell.2020.03.007; pmid: 32289271 doi: 10.1038/onc.2012.586; pmid: 23318448
theoretic nature of the epigenome. Nat. Genet. 49, 719–729 59. A. M. Tsankov et al., Loss of DNA methyltransferase activity in 81. G. Livshits et al., Arid1a restrains Kras-dependent changes in
(2017). doi: 10.1038/ng.3811; pmid: 28346445 primed human ES cells triggers increased cell-cell variability and acinar cell identity. eLife 7, e35216 (2018). doi: 10.7554/
38. G. Jenkinson, J. Abante, M. A. Koldobskiy, A. P. Feinberg, transcriptional repression. Development 146, dev174722 (2019). eLife.35216; pmid: 30014851
J. Goutsias, Ranking genomic features using an information- doi: 10.1242/dev.174722; pmid: 31515224 82. T. D. Treger, T. Chowdhury, K. Pritchard-Jones, S. Behjati,
theoretic measure of epigenetic discordance. BMC Bioinformatics 60. Z. S. Singer et al., Dynamic heterogeneity and DNA The genetic changes of Wilms tumour. Nat. Rev. Nephrol. 15,
20, 175 (2019). doi: 10.1186/s12859-019-2777-6; pmid: 30961526 methylation in embryonic stem cells. Mol. Cell 55, 319–331 240–251 (2019). doi: 10.1038/s41581-019-0112-0;
39. D. A. Landau et al., Locally disordered methylation forms the (2014). doi: 10.1016/j.molcel.2014.06.029; pmid: 25038413 pmid: 30705419
basis of intratumor methylome variation in chronic 61. Y. Costa et al., NANOG-dependent function of TET1 and TET2 83. V. Conteduca et al., Barrett’s esophagus and esophageal
lymphocytic leukemia. Cancer Cell 26, 813–825 (2014). in establishment of pluripotency. Nature 495, 370–374 cancer: An overview. Int. J. Oncol. 41, 414–424 (2012).
doi: 10.1016/j.ccell.2014.10.012; pmid: 25490447 (2013). doi: 10.1038/nature11925; pmid: 23395962 doi: 10.3892/ijo.2012.1481; pmid: 22615011

Feinberg and Levchenko, Science 379, eaaw3835 (2023) 10 February 2023 10 of 11

RES EARCH | R E V I E W

84. J. H. Taube et al., Core epithelial-to-mesenchymal transition 94. A. R. Mendelsohn, J. W. Larrick, Epigenetic drift is a determinant Comput. Biol. 13, e1005524 (2017). doi: 10.1371/
interactome gene-expression signature is associated of mammalian lifespan. Rejuvenation Res. 20, 430–436 (2017). journal.pcbi.1005524; pmid: 28472054
with claudin-low and metaplastic breast cancer subtypes. doi: 10.1089/rej.2017.2024; pmid: 28942711 105. O. Kapuy et al., System-level feedbacks control cell cycle
Proc. Natl. Acad. Sci. U.S.A. 107, 15449–15454 (2010). 95. S. Maegawa et al., Widespread and tissue specific age-related progression. FEBS Lett. 583, 3992–3998 (2009).
doi: 10.1073/pnas.1004900107; pmid: 20713713 DNA methylation changes in mice. Genome Res. 20, 332–340 doi: 10.1016/j.febslet.2009.08.023; pmid: 19703449
85. A. D. Rhim et al., EMT and dissemination precede pancreatic (2010). doi: 10.1101/gr.096826.109; pmid: 20107151 106. L. D. Walensky, BCL-2 in the crosshairs: Tipping the balance
tumor formation. Cell 148, 349–361 (2012). doi: 10.1016/ 96. A. E. Teschendorff, J. West, S. Beck, Age-associated of life and death. Cell Death Differ. 13, 1339–1350 (2006).
j.cell.2011.11.025; pmid: 22265420 epigenetic drift: Implications, and a case of epigenetic thrift? doi: 10.1038/sj.cdd.4401992; pmid: 16763614
86. R. Cheong, A. Rhee, C. J. Wang, I. Nemenman, A. Levchenko, Hum. Mol. Genet. 22, R7–R15 (2013). doi: 10.1093/hmg/ 107. R. J. Youle, A. Strasser, The BCL-2 protein family: Opposing
Information transduction capacity of noisy biochemical ddt375; pmid: 23918660 activities that mediate cell death. Nat. Rev. Mol. Cell Biol. 9,
signaling networks. Science 334, 354–358 (2011). 97. H. Takeshima, T. Ushijima, Accumulation of genetic and 47–59 (2008). doi: 10.1038/nrm2308; pmid: 18097445
doi: 10.1126/science.1204553; pmid: 21921160 epigenetic alterations in normal cells and cancer risk. NPJ 108. K. G. Vanaja, W. Timp, A. P. Feinberg, A. Levchenko, A loss of
87. A. Rhee, R. Cheong, A. Levchenko, The application of information Precis. Oncol. 3, 7 (2019). doi: 10.1038/s41698-019-0079-0; epigenetic control can promote cell death through reversing
theory to biochemical signaling systems. Phys. Biol. 9, 045011 pmid: 30854468 the balance of pathways in a signaling network. Mol. Cell 72,
(2012). doi: 10.1088/1478-3975/9/4/045011; pmid: 22872091 98. S. C. Zheng, M. Widschwendter, A. E. Teschendorff, Epigenetic 60–70.e3 (2018). doi: 10.1016/j.molcel.2018.08.025;
88. M. D. Brennan, R. Cheong, A. Levchenko, How information drift, epigenetic clocks and cancer risk. Epigenomics 8, 705–719 pmid: 30244832
theory handles cell signaling and uncertainty. Science 338, (2016). doi: 10.2217/epi-2015-0017; pmid: 27104983 109. H. Zhong et al., Overproduction of IGF-2 drives a subset of
334–335 (2012). doi: 10.1126/science.1227946; 99. S. Horvath, DNA methylation age of human tissues and cell colorectal cancer cells, which specifically respond to an anti-
pmid: 23087235 types. Genome Biol. 14, 3156 (2013). doi: 10.1186/gb-2013- IGF therapeutic antibody and combination therapies.
89. S. Uda et al., Robustness and compensation of information 14-10-r115; pmid: 24138928 Oncogene 36, 797–806 (2017). doi: 10.1038/onc.2016.248;
transmission of signaling pathways. Science 341, 558–561 100. A. E. Teschendorff, A. Jones, M. Widschwendter, Stochastic pmid: 27399333
(2013). doi: 10.1126/science.1234511; pmid: 23908238 epigenetic outliers can define field defects in cancer. BMC
90. J. Selimkhanov et al., Accurate information transmission Bioinformatics 17, 178 (2016). doi: 10.1186/s12859-016-1056-z; AC KNOWLED GME NTS
through dynamic biochemical signaling networks. Science 346, pmid: 27103033 We thank O. Kilic and J. Fairman for illustration preparation.
1370–1373 (2014). doi: 10.1126/science.1254933; pmid: 25504722 101. W. Chen et al., Single-cell landscape in mammary epithelium Funding: This study was supported by NIH grants R01CA54358 to
91. A. S. Hansen, E. K. O’Shea, Limits on information reveals bipotent-like cells associated with breast cancer risk A.P.F. and U54CA209992 to A.L. Competing interests: A.P.F.
transduction through amplitude and frequency regulation and outcome. Commun. Biol. 2, 306 (2019). doi: 10.1038/ is an inventor on a pending US patent related to this work
of transcription factor activity. eLife 4, e06559 (2015). s42003-019-0554-8; pmid: 31428694 (“Detecting cancer through generalized loss of stability of
doi: 10.7554/eLife.06559; pmid: 25985085 102. Z. Meir, Z. Mukamel, E. Chomsky, A. Lifshitz, A. Tanay, Single- epigenetic domains,” US20210071262A1, EP2707506B1). The
92. R. Suderman, J. A. Bachman, A. Smith, P. K. Sorger, cell analysis of clonal maintenance of transcriptional and authors declare no other competing interests. License
E. J. Deeds, Fundamental trade-offs between information flow epigenetic states in cancer cells. Nat. Genet. 52, 709–718 information: Copyright © 2023 the authors, some rights reserved;
in single cells and cellular populations. Proc. Natl. Acad. (2020). doi: 10.1038/s41588-020-0645-y; pmid: 32601473 exclusive licensee American Association for the Advancement of
Sci. U.S.A. 114, 5755–5760 (2017). doi: 10.1073/ 103. C. D. Lawson, A. J. Ridley, Rho GTPase signaling complexes in Science. No claim to original US government works. https://www.
pnas.1615660114; pmid: 28500273 cell migration and invasion. J. Cell Biol. 217, 447–457 (2018). science.org/about/science-licenses-journal-article-reuse
93. D. Ellison et al., Cell-cell communication enhances the doi: 10.1083/jcb.201612069; pmid: 29233866
capacity of cell ensembles to sense shallow gradients during 104. W. R. Holmes, J. Park, A. Levchenko, L. Edelstein-Keshet,
morphogenesis. Proc. Natl. Acad. Sci. U.S.A. 113, E679–E688 A mathematical model coupling polarity signaling to Submitted 24 December 2020; accepted 22 December 2022
(2016). doi: 10.1073/pnas.1516503113; pmid: 26792522 cell adhesion explains diverse cell migration patterns. PLOS 10.1126/science.aaw3835

Feinberg and Levchenko, Science 379, eaaw3835 (2023) 10 February 2023 11 of 11

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 RESEAR CH

◥ mouse cortical neurons over time. We thus

RESEARCH ARTICLE SUMMARY profiled, across time and species, mitochondria
morphology, gene expression, oxygen consump-
DEVELOPMENT tion, and glucose metabolism. Next, we used
pharmacological or genetic manipulation of
Mitochondria metabolism sets the species-specific human or mouse neurons to enhance or de-
crease their mitochondria function, and de-
tempo of neuronal development termined the consequences on the speed of
neuronal development.
Ryohei Iwata†, Pierre Casimir†, Emir Erkol, Leïla Boubakar, Mélanie Planque, Isabel M. Gallego López,
Martyna Ditkowska, Vaiva Gaspariunaite, Sofie Beckers, Daan Remans, Katlijn Vints, Anke Vandekeere, RESULTS: We found that mitochondria are ini-
Suresh Poovathingal, Matthew Bird, Ine Vlaeminck, Eline Creemers, Keimpe Wierda, Nikky Corthout, tally low in size and quantity in newborn neurons,
Pieter Vermeersch, Sébastien Carpentier, Kristofer Davie, Massimiliano Mazzone, Natalia V. Gounko, and then grow gradually as neurons undergo
Stein Aerts, Bart Ghesquière, Sarah-Maria Fendt, Pierre Vanderhaeghen* maturation following a species-specific time-
line. Whereas in mouse neurons, mitochon-
dria reach mature patterns in 3 to 4 weeks,
INTRODUCTION: During embryonic development, their species-specific timeline. This suggests they only do so after several months in human
the temporal sequence of events is usually con- that species-specific developmental timing is neurons.
served throughout evolution, but it can occur controlled by cell-intrinsic mechanisms, but We next measured mitochondria oxidative
at very different time scales depending on the these remain essentially unknown. activity and glucose metabolism in human
species or cell type considered. The human ce- and mouse developing cortical neurons. This
rebral cortex is characterized by a considerably RATIONALE: Metabolism and mitochondria are revealed a species-specific timeline of func-
prolonged timing of neuronal development key drivers of cell fate transitions in many tional maturation of mitochondria, with mouse
compared with other species, taking months systems, including the developing brain. Here, neurons displaying a much faster increase in
to years to reach maturity compared with only we tested whether they could be involved in mitochondria-dependent oxidative activity than
a few weeks in the mouse. The resulting neo- the species-specific tempo of cortical neuron human neurons. We also found that human
teny is thought to be a key mechanism en- development and human brain neoteny. cortical neurons displayed lower levels of
abling enhanced function and plasticity of the We developed a system of genetic birth- mitochondria-driven glucose metabolism than
human brain. Human and nonhuman corti- dating to label newly born neurons with high did mouse neurons at the same age.
cal neurons cultured in vitro or xenotrans- temporal and cellular resolution, and direct- Finally, we tested whether mitochondria func-
planted into the mouse brain develop along ly compared the development of human and tion affects neuronal developmental timing. We
performed pharmacological or genetic manipu-
lation of human developing cortical neurons to
enhance mitochondria oxidative metabolism.
This led to accelerated neuronal maturation,
with neurons exhibiting more mature features
weeks ahead of time, including complex mor-
phology, increased electrical excitability, and
functional synapse formation. Similar treat-
ments on mouse neurons also led to faster
maturation, whereas inhibition of mitochon-
dria metabolism in mouse neurons led to a
decrease in developmental rates.

CONCLUSION: Our work identifies a species-

specific temporal pattern of mitochondria
and metabolic development that sets the
tempo of neuronal maturation. Accelerated
human neuronal maturation using metabolic
manipulation might benefit pluripotent stem
cell–based modeling of neural diseases, which
remains greatly hindered by protracted neu-
ron development. Tools to accelerate or decel-
erate neuronal development could allow testing
of the impact of neuronal neoteny on brain
function, plasticity, and disease.
▪
The list of author affiliations is available in the full article online.
Mitochondria metabolism sets the tempo of neuronal development. Mitochondria dynamics and *Corresponding author. Email: [email protected]
These authors contributed equally to this work.
metabolism display species-specific timelines during cortical neuron development. In newborn neurons,
Cite this article as R. Iwata et al., Science 379, eabn4705
mitochondria are small in number and metabolic activity, and then increase gradually during neuronal (2023). DOI: 10.1126/science.abn4705
maturation. Enhanced mitochondria metabolism in human neurons leads to accelerated maturation, including
increased neurite complexity, excitability, and synaptic function. Decreased mitochondria metabolism in READ THE FULL ARTICLE AT
mouse neurons leads to decelerated neuronal maturation. https://doi.org/10.1126/science.abn4705

SCIENCE science.org 10 FEBRUARY 2023 • VOL 379 ISSUE 6632 553

RES EARCH

◥ purification of the labeled cells (Fig. 1A and fig.

RESEARCH ARTICLE S1B). Because the NeuroD1 promoter is turned
on transiently at the time of neuron genera-
DEVELOPMENT tion, a pulse of 4-hydroxy-tamoxifen (4-OHT)
allowed the selective labeling of a cohort of
Mitochondria metabolism sets the species-specific neurons born at precise time points, as as-
sessed by 5-ethynyl-2′-deoxyuridine nuclear
tempo of neuronal development labeling (fig. S1, C to E), which revealed a timely
progression in growth and complexity (Fig.
Ryohei Iwata1,2,3†, Pierre Casimir1,2,3†, Emir Erkol1,2, Leïla Boubakar1,2,3, Mélanie Planque4,5, 1B). To maximize the number of neuronal
Isabel M. Gallego López1,2, Martyna Ditkowska1,2, Vaiva Gaspariunaite1,2, Sofie Beckers1,2, cells born at similar time points, the cultures
Daan Remans1,2, Katlijn Vints2,6, Anke Vandekeere4,5, Suresh Poovathingal1, Matthew Bird7, were treated simultaneously with the gamma-
Ine Vlaeminck1,8, Eline Creemers1,8, Keimpe Wierda1,8, Nikky Corthout1,9, Pieter Vermeersch10, secretase inhibitor N-[N-(3,5-difluorophenacetyl)-
Sébastien Carpentier11, Kristofer Davie1, Massimiliano Mazzone12,13, Natalia V. Gounko2,6, L-alanyl]-S-phenylglycine t-butyl ester (DAPT),
Stein Aerts1,2, Bart Ghesquière14, Sarah-Maria Fendt4,5, Pierre Vanderhaeghen1,2,3* which inhibits Notch signaling and thereby
increases cortical neurogenesis, and cells were
Neuronal development in the human cerebral cortex is considerably prolonged compared with that of purified by CD8+ magnetic-activated cell sort-
other mammals. We explored whether mitochondria influence the species-specific timing of cortical ing (MACS) purification. The purified NNN-
neuron maturation. By comparing human and mouse cortical neuronal maturation at high temporal and labeled neurons thus displayed homogeneous
cell resolution, we found a slower mitochondria development in human cortical neurons compared maturation patterns and identity (Fig. 1C and
with that in the mouse, together with lower mitochondria metabolic activity, particularly that of oxidative fig. S1, F and G).
phosphorylation. Stimulation of mitochondria metabolism in human neurons resulted in accelerated To examine mitochondrial dynamics during
development in vitro and in vivo, leading to maturation of cells weeks ahead of time, whereas its cortical neuron development, we combined
inhibition in mouse neurons led to decreased rates of maturation. Mitochondria are thus important NNN labeling with mitochondria tagging with
regulators of the pace of neuronal development underlying human-specific brain neoteny. emerald-green fluorescent protein fused to
mitochondrial targeting sequence of cyto-

D
chrome c oxidase subunit 8A (COX8A) (Fig. 1,
evelopmental processes display species- (6–8), pointing to cell-intrinsic develop- D to F). We observed that mitochondria were
specific differences in timeline, or heter- mental timing mechanisms. Metabolism and initially small and sparse in newly born mouse
ochrony, which can lead to divergence in mitochondria are drivers of cell fate transi- neurons (20) and gradually grew in size and
size, cell composition, or organ function tions (9–12) and maturation (13–15) in many quantity over 3 weeks during neuronal matu-
(1–3). Human brain development is char- systems, including the brain (16–21). Species ration, and the same was observed in mouse
acterized by a prolonged timing of maturation differences in metabolism at the organism PSC–derived cortical neurons (Fig. 1, D and F,
of cortical neurons compared with that of other level are linked to developmental growth and fig. S2). However a similar examination of
species (4). Such neoteny may underlie the en- (22, 23), but whether metabolism influences PSC-derived human cortical neurons revealed
hanced performance of the human brain (5). species-specific developmental tempo remains a more prolonged timeline of mitochondria
Human and nonhuman primate cortical neu- unclear. development, spanning several months (Fig. 1,
rons derived from pluripotent stem cells (PSCs) E and F). We monitored mitochondria dynam-
and xenotransplanted into the mouse brain Mitochondria development during ics in developing cortical neurons in vivo. We
develop along their species-specific timeline cortical neuronal maturation follows labeled mouse cortical neurons with eGFP by
a species-specific timeline in utero electroporation at embryonic day 14.5
1
VIB-KU Leuven Center for Brain & Disease Research, 3000 To test whether metabolism influences the (E14.5), followed by a time course of correl-
Leuven, Belgium. 2KU Leuven, Department of Neurosciences
species-specific timing of neuronal develop- ative light and electron microscopy (CLEM)
& Leuven Brain Institute, 3000 Leuven, Belgium. 3Université
Libre de Bruxelles (ULB), Institut de Recherches en Biologie ment, we used in vitro cultures of cortical py- analysis (Fig. 1G and fig. S3A). This confirmed
Humaine et Moléculaire (IRIBHM), and ULB Neuroscience ramidal neurons derived from PSCs (human that mitochondria gradually reach maximal
Institute (UNI), 1070 Brussels, Belgium. 4Laboratory of and mouse) or from embryonic brain (mouse), levels of growth and size in 3 weeks in mouse
Cellular Metabolism and Metabolic Regulation, VIB-KU
Leuven Center for Cancer Biology, VIB, 3000 Leuven, which recapitulate species-specific timelines cortical neurons in vivo (Fig. 1F). We also
Belgium. 5Laboratory of Cellular Metabolism and Metabolic of corticogenesis (6, 24) (fig. S1A). Given that xenotransplanted DAPT-treated PSC-derived
Regulation, Department of Oncology, KU Leuven and Leuven neurogenesis is not synchronous, it is chal- human cortical neurons transduced with eGFP.
Cancer Institute (LKI), 3000 Leuven, Belgium. 6VIB-KU
Leuven Center for Brain & Disease Research, Electron lenging to study neuronal maturation because In this system, xenotransplanted neurons dis-
Microscopy Platform & VIB-Bioimaging Core, 3000 Leuven, populations of neurons born at different time play a months-long protracted pattern of mat-
Belgium. 7Clinical Department of Laboratory Medicine, points coexist at the same stage of brain de- uration (7). CLEM performed on such neurons
University Hospitals Leuven, 3000 Leuven, Belgium.
8
Electrophysiology Unit, VIB-KU Leuven Center for Brain &
velopment. To study neuronal development revealed slower mitochondria growth than in
Disease Research, 3000 Leuven, Belgium. 9VIB Bio Imaging with optimal temporal resolution, we devel- their mouse counterparts, taking months to
Core, 3000 Leuven, Belgium. 10Department of Cardiovascular oped a neuronal birth-dating system called reach similar mitochondrial development (Fig.
Sciences, KU Leuven, Leuven, Belgium, and Department of
Laboratory Medicine, University Hospitals Leuven, 3000
neuroD1-dependent newborn neuron (NNN) 1H and fig. S3B). We used transmission electron
Leuven, Belgium. 11SYBIOMA, KU Leuven Center for SYstems labeling. NNN combines the expression of microscopy to visualize mitochondria ultra-
BIOlogy based MAss spectrometry, 3000 Leuven, Belgium.
12
tamoxifen-inducible CreERT2 (Cre recombinase structure in greater detail. In newborn neu-
Laboratory of Tumor Inflammation and Angiogenesis, Center
fused to a mutant estrogen receptor ligand- rons, mitochondria were mostly devoid of
for Cancer Biology, VIB, 3000 Leuven, Belgium. 13Laboratory of
Tumor Inflammation and Angiogenesis, Center for Cancer binding domain) under the control of the pro- cristae, whereas there were more, better de-
Biology, Department of Oncology, KU Leuven, 3000 Leuven, moter of Neuronal Differentiation 1 (NeuroD1), fined cristae as neurons matured, following a
Belgium. 14Metabolomics Expertise Center, Center for Cancer together with the Cre-dependent reporters en- species-specific timeline that was more pro-
Biology, VIB, KU Leuven, 3000 Leuven, Belgium.
*Corresponding author. Email: [email protected] hanced green fluorescent protein (eGFP) and tracted in human neurons (fig. S3, C and D).
These authors contributed equally to this work. truncated CD8, enabling the identification or Thus, mitochondria morphological development

Iwata et al., Science 379, eabn4705 (2023) 10 February 2023 1 of 9

RES EARCH | R E S E A R C H A R T I C L E

A NeuroD1-dependent Newborn Neuron (NNN) B 18d post NNN-labeling 38d post NNN-labeling 58d post NNN-labeling
labeling system

MATURATION

GFP
NNN-labeled
Newborn
Neuron
100 µm

Progenitor
C 9d post NNN-labeling
24d post NNN-labeling SEMA3C

TIME 39d post NNN-labeling NEUROD1

FOXP4
NEUROD2
NeuroD1 pr.-CreERT2 LV vector NeuroD1+ cell NeuroD1+
SOX4
(Newborn neuron) Marker+ cell
NeuroD1 pr. CreERT2 WPRE DCX
LTR LTR Marker GRIA2
CreERT2
MAPT
Cre-dependent reporter LV vector SYT1 Max.

Expression
Marker SNAP25
CAG pr. WPRE
CAMK2B
LTR lox2272 loxP LTR
MEF2C 0

D 2 dp NNN 4 dp NNN 8 dp NNN 16 dp NNN F Mouse

Mouse (PSC-derived)
tdTomato / mito-EmGFP

4 Human
****

Average mitochondrial
3

length (µm)
Mouse

††††

2 ****
****
**** *** ****
1 **

10 µm 0
0 10 20 30 40 50 60 70
E

Mitochondrial volume (µm3)

13 dp NNN 31 dp NNN 45 dp NNN 66 dp NNN 600

****
GFP / Mitochondria

400
****
††††
Human

200 **** ****

****
****
0
0 10 20 30 40 50 60 70
10 µm TIME
Days post NNN-labeling (Days post 4-OHT induction)

G Mouse cell labeling/preparation

H Human cell labeling/preparation
E16.5 Ms Progenitor E16.5 Ms Neuron
IUE at E14.5 +X days Near-infrared hPSC-derived Neonatal +X days Near-infrared
Dissection branding cortical cells Xenotransplantation Dissection branding

CLEM CLEM

GFP+ cell GFP+ cell GFP+ cell

P0 Ms Neuron P7 Ms Neuron P14 Ms Neuron P30 Ms Neuron 5 weeks Hu Neuron 6 months Hu Neuron 15 months Hu Neuron

Fig. 1. Interspecies differences in mitochondria biogenesis and dynamics derived mouse: 4 and 18 dp NNN labeling: length: n = 9 and 9, respectively;
during neuronal development. (A) Schematic of the NNN-labeling system. volume: n = 9 and 9, respectively. Human: 13, 31, 45, and 66 dp NNN labeling:
(B) Representative images after NNN labeling in human PSC-derived cortical length: n = 20, 15, 17, and 18, respectively; volume: n = 9, 11, 22, and 20,
neurons. (C) Left: Uniform manifold approximation and projection (UMAP) respectively. (G and H) Schematic of mouse (Ms) or human (Hu) cortical neuron
of MACS-purified NNN-labeled cells. Right: Heatmap of gene expression after birth-dating followed by CLEM, and representative images. E, embryonic day;
pseudotime analysis. (D and E) Representative images of mitochondrial P, postnatal day. Yellow indicates mitochondria. Bottom images are 20 × 20 mm.
morphology in NNN-labeled mouse (D) and human (E) neurons. (F) Quantifica- (F) Data are shown as mean ± SEM. Dunnett’s multiple-comparisons test
tion of mitochondrial length (top) and mitochondria volume (bottom) per cell. or unpaired t test were used to compare with first time point. Unpaired t test
Mouse: 2, 4, 8, 16, and 32 days post (dp) NNN labeling: length: n = 10, 11, 10, or Mann-Whitney test was used to compare mouse and human. **P < 0.01,
10, and 5 cells, respectively; volume: n = 6, 8, 10, 9, and 5, respectively. PSC- ***P < 0.001, ****P < 0.0001, and ††††P < 0.0001.

Iwata et al., Science 379, eabn4705 (2023) 10 February 2023 2 of 9

RES EARCH | R E S E A R C H A R T I C L E

follows a species-specific timeline that is highly mouse and human cortical neurons at simi- measurement using tetramethylrhodamine
correlated with neuronal maturation. lar time points after birth (fig. S4, A and B). methyl ester (fig. S5).
From early stages, mitochondrial OCR was We next examined glucose metabolism using
Mitochondria metabolic activity is lower higher in mouse than in human neurons, and mass spectrometryÐbased 13C tracer analysis
in human than in mouse developing neurons also increased more rapidly, which is con- (26) of enriched preparations of human and
We examined the functional properties of sistent with their morphological development mouse developing cortical neurons at similar
mitochondria during neuronal development, (Fig. 2, A to C, and fig. S4, C to E). As a result, ages (19 days after neuronal birth) (Fig. 2, D
focusing on mitochondrial oxidative phos- stimulated OCR was >10 times higher in mouse to F, and fig. S6A). Although this led to sim-
phorylation (OXPHOS) and electron trans- than in human counterparts after 2 weeks of ilar labeling of glycolytic metabolites in the
port chain (ETC) capacity (Fig. 2, A to C). We differentiation (Fig. 2, B and C, and fig. S4). two species, it revealed a higher enrichment
measured the mitochondrial oxygen consump- The increased mitochondria ETC activity dur- of lactate from 13C6-glucose and a higher
tion rate (OCR) using oxygraphy (25) on high- ing mouse neuronal development was con- secretion of lactate in human than in mouse
ly enriched preparations (>95% neurons) of firmed by mitochondria membrane potential neurons (Fig. 2, D to F, and figs. S6 and S7).

A Resting B Maximum OXPHOS C Maximum ETC

120 *** 800 1500
(pmols O2/mL per million cells)

(pmols O2/mL per million cells)

(pmols O2/mL per million cells)

****
100 **** 1200
600
80 900 ****
60 **** 400 ****
* 600
40
OCR

OCR

OCR
†† 200 300 †††
*** ††† **
20 80 120 ****
*
15 60 **** 80
10 40 ††
†††
40
5 20
0 0 0
0 10 20 30 40 0 10 20 30 40 0 10 20 30 40
TIME TIME TIME
(days post 4-OHT/DAPT induction) (days post 4-OHT/DAPT induction) (days post 4-OHT/DAPT induction)
Mouse Mouse (PSC-derived) Human

D 30h after U-13C glucose addition E Mouse (n=9)

13 12
(Normalized by FL of pyruvate)
Mouse: Fraction of C Fraction of C Human (n=9)
Fractional labeling at 30h

Human: Fraction of 13C 1.2

***
1.0 ** ** *** ****
*
2-Phosphoglycerate
0.8
19d post 19d post ** ***
DAPT induced NNN-labeled 0.6 ****
Mouse neurons Human neurons 0.4
n=9 n=9
0.2
PEP 0

ate

e
te

te
te
te

te

te
ine

te
lat
ra

ma
cta
va

tra

ina

rta
ar
an

Ma
ma
ru

t
Ci

pa
La

uta
cc
glu
Al
Py

Fu

As
Su
n=9 n=9
to

Gl
Ke
α-

Alanine Pyruvate Lactate F

400 Mouse (n=9)
C lactate secretion rate

Human (n=9)
to media (AU/mg/h)

**
n=9 n=9 n=9 n=9 n=9 n=9
300

200
Malate α-ketoglutarate
TCA 100
cycle
0
13

n=9 n=9 n=9 n=9

Fig. 2. Interspecies differences in mitochondria metabolism during neuro- PEP, phosphoenolpyruvate. (E) Fractional labeling of metabolites normalized by
nal development. (A to C) Quantification of OCR during neuronal development the labeled fraction of pyruvate at 30 hours after tracer addition. (F) 13C-labeled
from at least two biological replicates. (A) Resting OCR. (B) Maximum OXPHOS lactate secretion rate. AU, arbitrary units. All data are shown as mean ± SEM.
capacity under coupled condition. (C) Maximum ETC capacity under uncoupled For (A) to (C), Dunnett’s or Dunn’s multiple-comparisons test (mouse) and
condition. Mouse: 4, 13, and 19 dp DAPT induction: n = 6, 8, and 4, respectively. unpaired t test or Mann-Whitney test (PSC mouse and human) were used to
PSC-derived mouse: 4 and 18 dp DAPT induction: n = 14 and 16, respectively. compare with first time point. For (E) and (F), unpaired t test or Mann-Whitney
Human: 9 and 37 to 41 dp NNN labeling, n = 6 and 7, respectively. (D) 13C test was used to compare mouse and human. *P < 0.05, **P < 0.01 or ††P <
enrichment patterns of the metabolites in mouse (orange) and human (blue). 0.01, ***P < 0.001 or †††P < 0.001, and ****P < 0.0001.

Iwata et al., Science 379, eabn4705 (2023) 10 February 2023 3 of 9

RES EARCH | R E S E A R C H A R T I C L E

Conversely, the labeling of tricarboxylic acid lactate to pyruvate (29) (Fig. 4A). To enhance distinguish the effects on neuronal develop-
(TCA) cycle metabolites was comparatively mitochondria activity in developing neurons, ment from an acute impact on mitochondria
lower in human than in mouse neurons, in- we therefore targeted LDHA. Treatment with function (Fig. 4, D and E, and fig. S15). Few
dicating lower mitochondria metabolic activ- a chemical inhibitor of LDHA, GSK-2837808A untreated neurons responded to KCl treat-
ity (Fig. 2E). Whole-cell oxidized nicotinamide (hereafter referred to as GSK) (30), resulted ment, but inhibition of LDHA increased the
adenine dinucleotide (NAD+) and reduced in increased mitochondria OCR in human proportion of neurons displaying NPAS4 re-
NAD (NADH) measurements revealed that the neurons (Fig. 4B and fig. S13), consistent with sponses (Fig. 4D), indicating acceleration of
amounts of NAD+ and NADH were both higher earlier reports (31). Similarly, exposure of cells neuronal maturation in response to increased
in human neurons, whereas the NAD+/NADH to free fatty acids (AlbuMAX), a fuel in addi- mitochondrial respiration (Fig. 4B). Increased
ratio was similar in both species (fig. S6H). This tion to glucose for mitochondrial TCA cycle neuronal maturation was also observed with
may suggest a difference in NAD recovery and activity, led to increased OCR, as did its com- other treatments that enhance mitochondrial
synthesis between the two species. We also bination with GSK (referred to as AlbuMAX- metabolism, including AlbuMAX and PS10, a
found a higher ratio of reduced glutathione GSK) (Fig. 4B and fig. S13). These treatments, pyruvate dehydrogenase (PDH) kinase (PDK1-4)
to oxidized glutathione in human neurons alone or in combination, led to increased OCR, inhibitor (33) that increases the conversion of
(fig. S6D), indicating a lower oxidative stress but not to the level found in mouse neurons pyruvate into acetyl-CoA. The effects of AlbuMAX
in human cells consistent with a lower activity (compare Fig. 2, A to C, with Fig. 4B), even with and GSK were additive (Fig. 4, D and E), fur-
of the mitochondrial ETC (Fig. 2C). Adenosine higher concentrations of GSK (fig. S14). ther linking mitochondrial metabolic activ-
triphosphate (ATP) and adenosine diphos- We examined the impact of AlbuMAX-GSK ity to the speed of neuronal maturation. These
phate (ADP) levels and ATP/ADP ratios were treatment on mitochondria morphology and experiments were performed on human corti-
higher in human than in mouse neurons, protein composition. Morphology analyses did cal neurons cultured on mouse astrocytes, but
whereas their fractional labeling from glucose not reveal significant effects on mitochondria a similar acceleration was observed after the
was lower (fig. S6, E and F), suggesting more length and volume (fig. S16, F to H). Western same treatment on pure neuronal populations
de novo biosynthesis in mouse neurons. blot analyses did not reveal changes in most (figs. S13, H to J, S14, C to E, and S15, C to E).
These data indicate that human developing tested mitochondria proteins, but some key Increased mitochondrial activity appears to
cortical neurons have lower mitochondria-driven components were increased in abundance lead to accelerated neuronal differentiation.
TCA cycle and oxidative activity than do their after the treatment, specifically COXII, a com- We tested this further by performing patch-
mouse counterparts at a similar age. ponent of complex IV (fig. S17). The effects of clamp recordings of human cortical neurons
Oxygraphy data point to temporal differences AlbuMAX-GSK treatments on OCR could still after AlbuMAX-GSK treatment. This resulted
in mitochondrial metabolism during neuro- be detected at similar levels of magnitude in an increased frequency and amplitude of
nal maturation. To gain insights into what 1 to 5 days after stopping the treatment (fig. synaptic currents (Fig. 4, H and I), together
might cause such differences and their in vivo S15). Thus, there appears to be selective mo- with a decrease of membrane potential (fig.
relevance, we examined our single-cell RNA- lecular and metabolic effects of LDH inhibi- S18), consistent with a global increase in the
sequencing data from in vitro birth-dated tion leading to increased OXPHOS, rather than functional maturation of the treated neurons.
human neurons with those from human and global effects on mitochondria biogenesis or We also found an increased number of puncta
mouse fetal cortex in vivo (27, 28) (Fig. 3 and dynamics. This could contribute to the ob- containing the presynaptic marker synapsin I
figs. S8 to S11). The in vitro and in vivo pat- served limits of the effects obtained after manip- on dendrites of treated neurons (Fig. 4, F and
terns of expression of mitochondrial or meta- ulations of mitochondria function in human G). The density of synapsin I puncta in 24-day-
bolic genes were similar and correlated (Fig. 3, neurons, which never reached the levels found old treated human neurons was still lower than
E and F), providing in vivo validation of our in mouse neurons. None of the treatments en- that in 13-day-old mouse neurons (fig. S16, J
in vitro observations. Human and mouse cells hancing mitochondria appeared to cause and K).
had similar temporal patterning of mitochon- changes in neuronal identity or survival, be- We examined the consequences of AlbuMAX-
drial and metabolic genes, with an overall cause the amounts of apoptosis remained GSK treatment on neuronal morphogenesis,
increase in genes related to oxidative phos- low and identity marker expression was un- a crucial parameter of neuronal maturation.
phorylation and a decrease in genes related changed after AlbuMAX-GSK treatments (figs. AlbuMAX-GSK–treated human cortical neu-
to glycolysis, which occurred earlier in mouse S13G and S16, A to E). rons had larger neuronal size and increased
than in human neurons (Fig. 3, F and J, and We tested the impact of these treatments on dendritic length and complexity (Fig. 5, A and
data S2). neuronal developmental rates. To determine B). The morphology of 24-day-old AlbuMAX-
the speed of maturation, we assessed neuro- GSK–treated neurons reached the same mat-
Increasing mitochondria activity nal excitability by measuring the response of uration level as for 58-day-old control neurons
in human neurons leads to accelerated neurons to membrane depolarization induced (Figs. 1B and 5A), indicating that enhanced
neuronal maturation by the addition of KCl using as a readout the mitochondrial metabolism could accelerate
We tested whether the species differences ob- expression of the activity-dependent immediate- neuronal morphological development sev-
served in mitochondria metabolic functions early gene Neuronal PAS Domain Protein 4 eral weeks ahead of time.
could set the speed of neuronal maturation. (NPAS4) (32). Few NNN-labeled human corti- To explore the relevance of these observa-
Glucose tracer experiments revealed an inter- cal neurons responded to KCl shortly after tions across species, we tested the influence of
species difference in the conversion of pyruvate their birth (9 to 24 days), but most of them mitochondria activity on rates of mouse neu-
to lactate (Fig. 2, D to F), which is catalyzed by became responsive weeks later (39 days) (Fig. ron maturation. Treatment of mouse neurons
lactate dehydrogenase (LDH). LDH is com- 4C and fig. S13E). We then examined the im- with AlbuMAX-GSK resulted in increased den-
posed of A and B subunits, both of which were pact of LDHA inhibitors on human cortical dritic growth, although to a lesser extent than
expressed in both mouse and human cortical neuron maturation by treating the cells from in human neurons (Fig. 5, C and D). Reduction
neurons, with higher amounts in human than day 9 to 23, when the neurons were still poor- of mitochondria activity using either 2-cyano-
in mouse (fig. S12). ly responsive to KCl (Fig. 4D and fig. S13F). 3-(1-phenyl-1H-indol-e-yl)-2-peopenoic acid
LDHA favors the conversion of pyruvate to LDHA inhibitor treatment was stopped for (UK-5099) as a mitochondrial pyruvate car-
lactate, and LDHB favors the conversion of 1 to 5 days before assessing maturation to rier inhibitor or rotenone as an ETC complex I

Iwata et al., Science 379, eabn4705 (2023) 10 February 2023 4 of 9

RES EARCH | R E S E A R C H A R T I C L E

A C D PAX6 EOMES
Human
Hu CS22
2: Developing neuron
(Bhaduri et al., 2020) 8

Expression

Expression
0: Newborn neuron
9d post NNN-labeling 5
4: Newborn neuron & IPC-1
24d post NNN-labeling 4 0
7: IPC-2
39d post NNN-labeling
1 1: RGC-1
7 NEUROD2 MEF2C
3: RGC-2
2
6 6: RGC-3

Expression

Expression
3 5: Low quality cell
8: Unknown

B Principal component E NNN-labeled F

correlation heatmap cell Pseudo-time_hPSC-derived NNN-labeled cell
7 6 1 3 5 8 4 0 9d 39d 2 24d 9d 24d39d
24d 0 1

Normalized
expression
2
39d 1

CS22_Bhaduri et al., 2020

9d
GAPDH Glycolysis
0 2 PDHX PDH complex
4
8 3 0 SDHA
5 TCA cycle
3 4 0
Pseudo-time 1 SUCLA2
1
5 NDUFA5
6 Pseudo-time_Cluster 0 & 2 from CS22_Bhaduri et al., 2020 OXPHOS
7 NDUFA9
6
VDAC1 Outer
-1 0 1 mitochondrial
7 1

Normalized
expression
VDAC3 membrane
8
Correlation
0
0.75 1.0
0 1
Pseudo-time
G H Mouse E13.5 mouse cortex
I
Cross species comparison Pax6 Eomes
(Di Bella et al., 2021)
3 5
0.8

Expression

Expression
Bhaduri et al., 2020

2: Developing neuron
Human CS22

6
1 6 2
3 7 0: Newborn neuron
7
4 0 4: Newborn neuron & IPC-1
0.4
0 7: IPC-2
2 4
1: RGC-1 Neurod2 Mef2c
5
8 3: RGC-2
0 1

Expression

Expression
AP IP MN ImN PC EC MG 6: RGC-3
Mouse E13.5 5: Low quality cell
Di Bella et al., 2021 8: Unknown

J Glycolysis PDH complex TCA OXPHOS 2: Developing neuron

pathway pathway pathway pathway 0: Newborn neuron
1
4: Newborn neuron & IPC-1
Bhaduri et al., 2020

0.3
Human CS22

0.2 0.45 7: IPC-2

1: RGC-1
0
0 0.3
Expression score

0
-0.3
0 1 0 1 0 1 0 1

0.8
Di Bella et al., 2021

0.4 0.9
Mouse E13.5

0.25

0
0
0.6
0 -0.4

0 1 0 1 0 1 0 1
Pseudo-time

Fig. 3. Temporal pattern of expression of metabolic genes during neuronal complex, TCA, and OXPHOS; table S1). (F) Expression trends of representative
development. (A to C) UMAP of NNN-labeled human cortical neurons and metabolic pathway genes plotted along the pseudotime axis for NNN-labeled
human cortical cells from Carnegie stage (CS) 22 (27). (B) Principal component neurons (top) and CS22 neurons (bottom) from cluster 0 and 2. (G) Heatmap
correlation heatmap. (C) Unsupervised Leiden clustering and classification of cell type alignment scores representing the cross-species comparison of
of cells after integration with Harmony algorithm. (D) Normalized expressions E13.5 mouse and CS22 human using SAMap. (H) UMAP of mouse cortical
of PAX6, EOMES, NEUROD2, and MEF2C are shown as radial glial cells cells showing transferred annotations from merged human cells to the
(RGCs), intermediate progenitor cells (IPCs), excitatory neurons, and developing mouse dataset. (I) Normalized expressions of Pax6, Eomes, Neurod2, and
neuron markers, respectively. (E) Heatmap of Pearson correlation coefficients Mef2c. (J) Temporal pattern of metabolic pathway score in CS22 human (top)
per time point of the mean scores of metabolic pathways (glycolysis, PDH and E13.5 mouse (bottom).

Iwata et al., Science 379, eabn4705 (2023) 10 February 2023 5 of 9

RES EARCH | R E S E A R C H A R T I C L E

A B 28-31 dp NNN-labeling
Glucose
400 DMSO (n=13)
****
GSK-2837808A Cytoplasm GSK (n=17)

(pmols O2/mL/million cells)

****
AlbuMAX-DMSO (n=10) ****
300
LDHA AlbuMAX-GSK (n=19)
Lactate Pyruvate

OCR
****
LDHB 200 * E
Mitochondrion ***

% of NPAS4+ cell among HN+

AlbuMAX
A Pyruvate PDP *** 100 ††††
Inactive 100
P P
** ††††
Fatty acid PDH PDH P
** 80 ****

(after KCl treatment)

****
Acyl-CoA 0
Acetyl-CoA PDK 60
Resting Maximum Maximum
OXPHOS ETC **** ****
PS10
TCA
cycle H + C 9 dp NNN 24 dp NNN 39 dp NNN
40 **
ADP ATP
20

NPAS4 / MAP2
ETC I II III IV V
Complex
OXPHOS 0

SO

10
Al X-D SK

Al AX-P O
MA S10

K
GS
M MS
PS
MA G
DM

X-
bu
50 μm

bu
bu
Al
D 24 dp NNN
DMSO PS10 GSK AlbuMAX-DMSO AlbuMAX-PS10 AlbuMAX-GSK
NPAS4
Human Nuclei (HN)

50 µm

F 9 dp NNN 24 dp NNN G DMSO (n=15)

H
DMSO AlbuMAX-GSK AlbuMAX-GSK (n=16) DMSO
DAPI / MAP2

25
per 100µm dendrite
Synapsin I

***
# of SynI puncta

20
AlbuMAX-GSK
15
10
5 pA 10 pA
5 200 ms 2.5 ms
0
Synapsin I

I
sEPSC Frequency (Hz)

DMSO (n=35)
sEPSC Amplitude (pA)

3 20
*