LSM 4232
Advanced Cell Biology
Lecture I-October 3, 2011
Reshma TANEJA
Associate Professor
Dept of Physiology
NUS Graduate School of Integrative Sciences and
Engineering
E-mail:phsrt@[Link]
"Overview of Lectures
Lecture I: Making Muscle in the Embryo
"
How is skeletal muscle formed?
"
Transcription factors in muscle differentiation
Lecture II: Skeletal muscle differentiation
"
Regulation of myogenic transcription factors
"
Lecture III: Making Muscle in the Adult
" Muscle stem cells & regeneration
Lecture IV: The Aging Muscle & Muscular dystrophies
" Stem cell numbers or stem cell function?
" Factors underlying muscle degeneration
Why study muscle development?
Making muscle
Muscle pathologies
LEARNING OBJECTIVES FOR LECTURES I & II
1. FORMATION OF SKELETAL MUSCLE
"EMBRYONIC AND ADULT MYOGENESIS
2. REGULATORS OF MYOGENESIS
"MYOD AND MEF2 FAMILIES
What is myogenesis?
Literally muscle formation
Two types:
(a) Embryonic (formation of skeletal muscle before birth)
(b) Adult (required for regeneration)
Skeletal muscle development
Embryonic myogenesis
Current Opinion in Genetics & Development, 2003, 13- 413-422
Adult myogenesis
Stages of skeletal muscle development
Signals affect somite determination
Transcription factors regulate gene
expression
Embryonic
mesodermal cells
somite
Determination- a change in a cells
that commits to a particular cell
fate
1. Determination
myoblasts
Proliferation - increase in cell number
2. Proliferation & migration
Premuscle masses
3. Differentiation and fusion
Differentiation - process in which a
precursor cells become a specialized cell
Myotube (muscle cell)
Embyronic Myogenesis
Where does muscle come from?
Mesoderm
Formation of somites from paraxial mesoderm
neural tube
somite
Thick bands of mesodermal
cells alongside neural tube
unsegmented mesoderm
separates into blocks of cells
(somites)
paraxial mesoderm
Somite formation
Block of mesodermal cells
Transient structure formed from paraxial mesoderm
Paraxial mesoderm somite
What role do somites play?
Help define the body plan
Somites
Somites
give rise
givetorise
myotome
to myotome
and muscle
Skeletal cartilage
muscle
dermis
Cell-cell signals induce changes in somites
Cell fates within the somite
Sclerotome
Gives rise to cartilage/rib
Dermamyotome
epaxial
hypaxial
Forms:
1.
2.
Dermatome - gives rise to dermis
Myotome which gives rise to:
Epaxial myotome back muscle precursors and
Hypaxial myotome body wall, limb, tongue muscle precursors
Epaxial myotome induction
Closest to the neural tube
Induction of epaxial myotome by neural
tube products
Wnt1
Wnt3a
Low sonic hedgehog (Shh) levels
Hypaxial myotome induction
Farthest from the neural tube
Induction of hypaxial myotome by epidermis
Wnt proteins
Induction of hypaxial myotome by lateral plate
mesoderm
BMP4
FGF5
Brief review
noggin
Stages of skeletal muscle development
Kollias, H. D. et al. J Appl Physiol 104: 579-587 2008;
doi:10.1152/japplphysiol.01091.2007
Copyright 2008 American Physiological Society
Where are we
Fast & slow muscle fibers identified by myosin
staining
Slow twitch
fibers
Fast twitch
fibers
Transcription factors in development of skeletal muscle
Dermamyotome
(gives rise to dermis of skin and muscle
precursors)
mesodermal cells
somite
Determined cells - committed but
not yet differentiated
1. Determination
MyoD and Myf5
Myoblasts (precursor cells)
2. Proliferation & migration
Premuscle masses
3. Differentiation and fusion
Myogenin and MEF2
Differentiation - cells align, stop
dividing, differentiate and fuse to form a
multinuncleated cell
Myotube (muscle cell)
Transcription factors that regulate muscle
differentiation
Myogenic regulatory factors (MRFs)
MyoD, Myf5, myogenin, Mrf4
Basic helix loop helix transcription factors
Activate muscle specific gene expression
Myocyte enhancer family 2 (Mef2)
Transcription factors
Cooperate with MRFs
Two classes of transcription factors regulate
myogenesis
MRFs
MyoD
Myogenin
Myf5
MRF4
MADS family
Muscle
specific
Muscle and
other tissues
Muscle differentiation genes
MyoD
Transfection of a single gene - MyoD- is SUFFICIENT to convert
fibroblasts to myoblasts!
-MyoD is a basic helix-loop-helix (bHLH) transcription factor
A master regulator of muscle differentiation
The Myogenic regulatory factor (MRF) family
Muscle regulatory factors (MRF)
Vector
alone
MyoD
Myosin
heavy
chain
staining
(green)
Conversion of C3H10T1/2 cells (fibroblasts) to muscle
All MRFs when expressed in fibroblast cells can convert
fibroblasts to muscle (UNIQUE to MRF family)
Role of MRFs in myogenesis
nucleus
MHC staining
myotube
MRF functions
MRFs have partially overlapping, but distinct, functions
MyoD and Myf5 are expressed early
Myogenin and MRF4 are late factors
MyoD
Myf5
Cell in somite
Myogenin
MRF4
myoblast
Differentiated muscle
Sabourin and Rudnicki (2000)
MRF genetics
MyoD-/- mice
Hypaxial development is delayed
Myf5-/- mutants
Epaxial development is delayed
MyoD and Myf5 mark two different myoblast
populations
Lack of Myotome Formation in Homozygous Myf-5
embryos at E9.75.
Myogenin
MHC
MRF genetics
Sabourin and Rudnicki (2000)
MyoD/Myf5 double mutants lack myoblasts and myotubes
Wild type
MyoD/Myf5 mutants
-actin
Stains muscle
fibers
-Desmin
stains myoblasts
& myotubes
MRF genetics
Myogenin mutants
Early events normal, but show severe
reduction in multinucleate muscle fibers
Suggests a late role for myogenin
MRF4 mutants
variable results, but some muscle fibers are
formed
Suggests a late role for MRF4
Muscle deficiency in myogenin mutants
Wild type muscle
Arrow indicates muscle fibers
Myogenin -/- muscle
Very few fibers in mutant
muscle
Muscle differentiation genes
MEF2 gene family
MEF2 in muscle development
Expressed in cardiac, skeletal and smooth muscle
-Unlike MRFs, cannot convert fibroblast cells to muscle
phenotype but cooperate with MRFs to increase efficiency of
conversion
Synergize with MRFs to regulate muscle gene expression
Expression during skeletal myogenesis
-low levels in myoblasts induced at differentiation
-activity coincident with expression of muscle specific markers
Mutation of MEF2 in mice
-MEF2C: Embryos die very early due to cardiac development
defects
-MEF2B: No phenotype
Regulatory interaction between MRF & MEF
Control of myogenesis - summary
Extracellular signals in the
developing embryo control
proliferation (cell number) and
migration (location) of determined
yet undifferentiated myoblasts
somite
Determination
MyoD
or Myf5
Myoblasts
Arrest in G1 of the
cell cycle, cyclins
and cyclin
dependent kinases
Differentiation
MyoD or Myf5 required for
determination of myoblasts
Myogenin and MEF2
required for
differentiation into
myotube
Myogenin
and MEF2
Activators - and inhibitory protein
regulate MRF and MEF2 activity
Myotube
Next time.
Regulation of MyoD and MEF2 activity
