Papers by James D Lauderdale
Light-sheet microscopy is an ideal imaging modality for long-term live imaging in model organisms... more Light-sheet microscopy is an ideal imaging modality for long-term live imaging in model organisms. However, significant optical aberrations can be present when imaging into an organism that is hundreds of microns or greater in size. To measure and correct optical aberrations, an adaptive optics system must be incorporated into the microscope. Many biological samples lack point sources that can be used as guide stars with conventional Shack-Hartmann wavefront sensors. We have developed a scene-based Shack-Hartmann wavefront sensor for measuring the optical aberrations in a light-sheet microscopy system that does not require a point-source and can measure the aberrations for different parts of the image. The sensor has 280 lenslets inside the pupil, creates an image from each lenslet with a 500 micron field of view and a resolution of 8 microns, and has a resolution for the wavefront gradient of 75 milliradians per lenslet. We demonstrate the system on both fluorescent bead samples and zebrafish embryos.
Eukaryotic chromatin consists of extensive, ordered nucleosome arrays along the DNA chain. The di... more Eukaryotic chromatin consists of extensive, ordered nucleosome arrays along the DNA chain. The distance between nucleosome centers, known as the nucleosome spacing periodicity or repeat length, varies in a tissue- and species-specific manner. In nature repeat lengths ranging from 160 to 250 base pairs have been observed; however, the mechanisms by which nucleosome arrays are generated, the precision of the spacing, and the factors which might influence nucleosome alignment are not understood. We have achieved physiological nucleosome spacing on cloned DNA using a fully defined in vitro chromatin assembly system. This is the first time anyone has achieved physiological nucleosome spacing using highly purified DNA and histones, and clearly demonstrates that all the information for nucleosome alignment is contained in the histones and the DNA. Histone H1- or H5-mediated nucleosome alignment has been found to nucleate from certain regions of DNA, which we call chromatin organizing regions or CORs. A COR was initially identified in pBR327 by Shin Wu Jeong; sequences with COR-like activity have now been identified in the chicken ovalbumin gene. Nucleosome alignment in our chromatin assembly system appears to be essentially an all-or-none mechanism. A necessary requirement for histone H5-mediated nucleosome alignment is that the total DNA length must be close to an integral multiple of the repeat length generated for small $(\u3c$4 kb) plasmids, a type of boundary effect. Only plasmids with DNA lengths close to integral multiples of 210 $\pm$ 3 bp permitted nucleosome alignment. This suggests that nucleosome arrays can be quasi-crystalline, and are capable of transmitting information over a distance of more than 2 kb. Taken together, these data suggest a possible mechanism through which chromatin can regulate eukaryotic gene expression. It is possible that nucleosome spacing periodicities might in some way be specified by different CORs. Different cell types might use different CORs to alter chromatin structure and function in a manner analogous to the way different cell types might use different replication origins and chromosomal scaffold attachment regions. Changes in the boundary conditions, by binding of a regulatory protein for example, might be sufficient to affect the chromatin structure within that particular domain thereby altering the expression of genes within the affected region
Nature Protocols, 2013
Multipotent neural crest stem cells (NCSCs) have the potential to generate a wide range of cell t... more Multipotent neural crest stem cells (NCSCs) have the potential to generate a wide range of cell types including melanocytes; peripheral neurons; and smooth muscle, bone, cartilage and fat cells. This protocol describes in detail how to perform a highly efficient, lineage-specific differentiation of human pluripotent cells to a NCSC fate. The approach uses chemically defined media under feeder-free conditions, and it uses two small-molecule compounds to achieve efficient conversion of human pluripotent cells to NCSCs in ~15 d. After completion of this protocol, NCSCs can be used for numerous applications, including the generation of sufficient cell numbers to perform drug screens, for the development of cell therapeutics on an industrial scale and to provide a robust model for human disease. This protocol can be also be applied to patient-derived induced pluripotent stem cells and thus used to further the knowledge of human disease associated with neural crest development, for example, Treacher-Collins Syndrome.
Glycobiology, Jun 17, 2023
Previous in vitro studies demonstrated that Fringe glycosylation of the NOTCH1 extracellular doma... more Previous in vitro studies demonstrated that Fringe glycosylation of the NOTCH1 extracellular domain at O-fucose residues in Epidermal Growth Factor-like Repeats (EGFs) 6 and 8 is a significant contributor to suppression of NOTCH1 activation by JAG1 or enhancement of NOTCH1 activation by DLL1, respectively. In this study, we sought to evaluate the significance of these glycosylation sites in a mammalian model by generating 2 C57BL/6J mouse lines carrying NOTCH1 point mutations, which eliminate O-fucosylation and Fringe activity at EGFs 6 (T232V) or 8 (T311V). We assessed changes to morphology during retinal angiogenesis, a process in which expression of Notch1, Jag1, Dll4, Lfng, Mfng, and Rfng genes coordinate cell-fate decisions to grow vessel networks. In the EGF6 O-fucose mutant (6f/6f ) retinas, we observed reduced vessel density and branching, suggesting that this mutant is a Notch1 hypermorph. This finding agrees with prior cell-based studies showing that the 6f mutation increased JAG1 activation of NOTCH1 during co-expression with inhibitory Fringes. Although we predicted that the EGF8 O-fucose mutant (8f/8f ) would not complete embryonic development due to the direct involvement of the O-fucose in engaging ligand, the 8f/8f mice were viable and fertile. In the 8f/8f retina, we measured increased vessel density consistent with established Notch1 hypomorphs. Overall, our data support the importance of NOTCH1 O-fucose residues for pathway function and confirms that single O-glycan sites are rich in signaling instructions for mammalian development.
Glycobiology
Previous in vitro studies demonstrated that Fringe glycosylation of the NOTCH1 extracellular doma... more Previous in vitro studies demonstrated that Fringe glycosylation of the NOTCH1 extracellular domain at O-fucose residues in Epidermal Growth Factor-like Repeats (EGFs) 6 and 8 is a significant contributor to suppression of NOTCH1 activation by JAG1 or enhancement of NOTCH1 activation by DLL1, respectively. In this study, we sought to evaluate the significance of these glycosylation sites in a mammalian model by generating 2 C57BL/6J mouse lines carrying NOTCH1 point mutations, which eliminate O-fucosylation and Fringe activity at EGFs 6 (T232V) or 8 (T311V). We assessed changes to morphology during retinal angiogenesis, a process in which expression of Notch1, Jag1, Dll4, Lfng, Mfng, and Rfng genes coordinate cell-fate decisions to grow vessel networks. In the EGF6 O-fucose mutant (6f/6f) retinas, we observed reduced vessel density and branching, suggesting that this mutant is a Notch1 hypermorph. This finding agrees with prior cell-based studies showing that the 6f mutation increas...
Adaptive Optics and Wavefront Control for Biological Systems IX, Mar 16, 2023
Biomedical Optics Express
In primates, the fovea is a pit in the center of the macula, which is a region of the retina with... more In primates, the fovea is a pit in the center of the macula, which is a region of the retina with a high concentration of cone photoreceptor cells that accounts for a large degree of visual acuity in primates. The maturation of this primate visual acuity area is characterized by the shallowing and widening of the foveal pit, a decrease in the diameter of the rod-free zone, and packing of photoreceptor cells more densely into this area of the retina, which occurs sometime after birth. Maturation occurs concurrently with progressing age and development, which further correlates with increasing eye size, retinal length, and retinal area. These observations have led to the hypothesis that the maturation of the fovea might be a function of mechanical variables that remodel the retina. However, this has never been explored outside of primates. Here, we take advantage of the Anolis sagrei lizard, which has a bifoveated retina, to study maturation of the fovea and macula. Eyes were collecte...
Investigative Ophthalmology & Visual Science, Jun 10, 2020
Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXV, 2018
Zebrafish are a promising vertebrate model for elucidating how neural circuits generate behavior ... more Zebrafish are a promising vertebrate model for elucidating how neural circuits generate behavior under normal and pathological conditions. The Baraban group first demonstrated that zebrafish larvae are valuable for investigating seizure events and can be used as a model for epilepsy in humans. Because of their small size and transparency, zebrafish embryos are ideal for imaging seizure activity using calcium indicators. Light-sheet microscopy is well suited to capturing neural activity in zebrafish because it is capable of optical sectioning, high frame rates, and low excitation intensities. We describe work in our lab to use light-sheet microscopy for high-speed long-time imaging of neural activity in wildtype and mutant zebrafish to better understand the connectivity and activity of inhibitory neural networks when GABAergic signaling is altered in vivo. We show that, with light-sheet microscopy, neural activity can be recorded at 23 frames per second in twocolors for over 10 minutes allowing us to capture rare seizure events in mutants. We have further implemented structured illumination to increase resolution and contrast in the vertical and axial directions during high-speed imaging at an effective frame rate of over 7 frames per second.
Investigative Ophthalmology & Visual Science, Jun 21, 2021
Imaging and Applied Optics 2018 (3D, AO, AIO, COSI, DH, IS, LACSEA, LS&C, MATH, pcAOP), 2018
Light-sheet microscopy is an ideal imaging modality for long-term live imaging in model organisms... more Light-sheet microscopy is an ideal imaging modality for long-term live imaging in model organisms. However, significant optical aberrations can be present when imaging into an organism that is hundreds of microns or greater in size. To measure and correct optical aberrations, an adaptive optics system must be incorporated into the microscope. Many biological samples lack point sources that can be used as guide stars with conventional Shack-Hartmann wavefront sensors. We have developed a scene-based Shack-Hartmann wavefront sensor for measuring the optical aberrations in a light-sheet microscopy system that does not require a point-source and can measure the aberrations for different parts of the image. The sensor has 280 lenslets inside the pupil, creates an image from each lenslet with a 500 micron field of view and a resolution of 8 microns, and has a resolution for the wavefront gradient of 75 milliradians per lenslet. We demonstrate the system on both fluorescent bead samples and zebrafish embryos.
Brain Research, 2021
The paired-box 6 (PAX6) gene encodes a highly conserved transcription factor essential for the pr... more The paired-box 6 (PAX6) gene encodes a highly conserved transcription factor essential for the proper development of the eye and brain. Heterozygous loss-of-function mutations in PAX6 are causal for a condition known as aniridia in humans and the Small eye phenotype in mice. Aniridia is characterized by iris hypoplasia and other ocular abnormalities, but recent evidence of neuroanatomical, sensory, and cognitive impairments in this population has emerged, indicating brain-related phenotypes as a prevalent feature of the disorder. Determining the neurophysiological origins of brain-related phenotypes in this disorder presents a substantial challenge, as the majority of extra-ocular traits in aniridia demonstrate a high degree of heterogeneity. Here, we summarize and integrate findings from human and rodent model studies, which have focused on neuroanatomical and functional consequences of PAX6 mutations. We highlight novel findings from PAX6 central nervous system studies in adult mammals, and integrate these findings into what we know about PAX6's role in development of the central nervous system. This review presents the current literature in the field in order to inform clinical application, discusses what is needed in future studies, and highlights PAX6 as a lens through which to understand genetic disorders affecting the human nervous system.
Investigative Ophthalmology & Visual Science, 2013
Light Sheet Microscopy has developed rapidly over the past decade and is the ideal approach for i... more Light Sheet Microscopy has developed rapidly over the past decade and is the ideal approach for imaging model organisms such as zebrafish and other thick tissue specimens. Despite the superior optical sectioning capability, high imaging speed, and large field of view, the performance of light sheet microscopy still suffers from optical aberrations. We have implemented a scene-based Shack-Hartmann wavefront sensor for directly measuring the optical aberrations on the emission side of the light-sheet microscope. In this work, we show that our system is capable of AO correction using sensor based and sensorless based approaches. We demonstrate correction up-to one hundred microns deep in zebrafish and fruitfly embryos.
Investigative Ophthalmology & Visual Science, 2013
Investigative Ophthalmology & Visual Science, 2015
Background. The fovea, a pit in the retina, is believed to be important for high-acuity vision an... more Background. The fovea, a pit in the retina, is believed to be important for high-acuity vision and is a feature found in the eyes of humans and a limited number of vertebrate species that include certain primates, birds, lizards, and fish. At present, model systems currently used for ocular research lack a foveated retina and studies investigating fovea development have largely been limited to histological and molecular studies in primates. As a result, progress towards understanding the mechanisms involved in regulating fovea development in humans is limited and is completely lacking in other, non-primate, vertebrates. To address this knowledge gap, we provide here a detailed histological atlas of retina and fovea development in the bifoveated Anolis sagrei lizard, a novel reptile model for fovea research. We also further test the hypothesis that retinal remodeling, which leads to fovea formation and photoreceptor cell packing, is related to asymmetric changes in eye shape. Results...
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Papers by James D Lauderdale