Carol A. Mason - US grants

The proposed research would investigate the maturation of growing axons, using mouse cerebellum as a model system, and would address three related issues: 1) the changes in form and cytology during the transition of growth cone to mature synaptic arbor; 2) to what extent the growing tip and mature synaptic bouton reflect the origin of the axon versus the interaction of the growing tip with its microenvironment; (3) to what extent the strategies taken by growing axons in formation of synaptic connection include error, and if so, what rearrangements they make to form proper connections. Once the steps in the formation of specific connections are delineated, the molecular basis for such specificity can in the future be explored. The form of immature axons and growing tips will be characterized during outgrowth towards target structures, during the period axons wait and during the formation of synaptic connections. Axons will be labeled with horseradish peroxidase (HRP) by injection into brain slices, followed by light and electron microscopic analysis. The three dimensional forms of axons and their growing tips will be correlated with their cytology, synaptic patterns and interactions with surrounding cells. The growing tips on axons during outgrowth will be contrasted with those during periods of waiting and synaptogenesis. The relations of waiting axons with target cells will be determined with HRP labeling and specific antisera to synapses and Purkinje cells. Climbing and mossy fibers will be identified by injection of their specific axon pathways or nucleus of origin, and the parameters of the rearrangement of transient connections of supernumerary climbing fiber branches and of axons with both climbing and mossy fiber features will be documented. The effect of deletion of the source of climbing fibers on the maturation of mossy fibers will be assessed. The interaction of axonal growing tips with target cells will be studied by co-culturing cerebellar afferents with purified granule or Purkinje cells.

The morphology and postnatal development of axonal terminal arbors will be studied. Once axons have reached their target, the sequence of structural events that occur to produce adult forms will be documented. Axons will be injected close to their terminations with horseradish peroxidase (HRP). The three-dimensional forms of terminal arbors and details of their appendages will be characterized in the light microscope and synaptic patterns of terminals will be identified in the electron microscope. Retino-geniculate axons in the kitten and cerebellar mossy and climbing fibers in postnatal mice will be studied with respect to their branching pattern as they expand and mature. The types of growing tips will be identified and their placement on the arbor, cytological features, and presence of specialized contacts will be described. Particular attention will be given to the refinement of mature shapes of terminal swellings and the corresponding development of synaptic relations. The structural changes accompanying potential withdrawal of supernumerary branches, as well as reduction of terminals will be documented. The development of shape of dendritic appendages of postsynaptic cells will also be followed in Golgi preparations to elucidate the relationship of axonal development to dendritic form. These studies should provide a morphological basis for development of axons in general and for future experiments utilizing surgical intervention, genetic mutants, or environmental manipulation.

The proposed research would investigate the maturation of growing axons, using mouse cerebellum as a model system, and would address three related issues: 1) the changes in form and cytology during the transition of growth cone to mature synaptic arbor; 2) to what extent the growing tip and mature synaptic bouton reflect the origin of the axon versus the interaction of the growing tip with its microenvironment; (3) to what extent the strategies taken by growing axons in formation of synaptic connection include error, and if so, what rearrangements they make to form proper connections. Once the steps in the formation of specific connections are delineated, the molecular basis for such specificity can in the future be explored. The form of immature axons and growing tips will be characterized during outgrowth towards target structures, during the period axons wait and during the formation of synaptic connections. Axons will be labeled with horseradish peroxidase (HRP) by injection into brain slices, followed by light and electron microscopic analysis. The three dimensional forms of axons and their growing tips will be correlated with their cytology, synaptic patterns and interactions with surrounding cells. The growing tips on axons during outgrowth will be contrasted with those during periods of waiting and synaptogenesis. The relations of waiting axons with target cells will be determined with HRP labeling and specific antisera to synapses and Purkinje cells. Climbing and mossy fibers will be identified by injection of their specific axon pathways or nucleus of origin, and the parameters of the rearrangement of transient connections of supernumerary climbing fiber branches and of axons with both climbing and mossy fiber features will be documented. The effect of deletion of the source of climbing fibers on the maturation of mossy fibers will be assessed. The interaction of axonal growing tips with target cells will be studied by co-culturing cerebellar afferents with purified granule or Purkinje cells.

DESCRIPTION (provided by applicant): Retinal ganglion cell (RGC) axons from each eye grow towards the brain and meet at the midline of the ventral diencephalon, establishing an X-shaped decussation, the optic chiasm. The navigation of retinal axons as they diverge to ipsi- and contralateral targets at the optic chiasm is a model for axon guidance at the CNS midline. The optic chiasm also patterns binocular vision, and if RGC divergence is mis-apportioned in inherited defects such as albinism, reduced visual acuity and strabismus ensue. The proposed analyses build on work in the last fifteen years on the identification of RGC behavior and the cellular/molecular cues during chiasm formation, recently highlighting how the uncrossed projection is established. Our new work shows that guidance factor ephrin-B2 is expressed in chiasm midline radial gila, and is selectively inhibitory to ventrotemporal (VT) RGCs, source of the uncrossed projection in mouse. EphB1, a receptor for ephrin-B2, is uniquely expressed in VT RGCs during the formation of the uncrossed projection. In the absence of EphB1, the uncrossed RGC projection fails to develop. Two sets of analyses will investigate this guidance system during RGC divergence. In Aim 1, studies will determine whether EphB1 expression is conserved in other vertebrates and primates, and whether EphB1 directs the projection of all uncrossed axons in the mouse, especially the early uncrossed RGCs arising from dorsocentral retina. Misexpression of EphB1 will test whether it is sufficient to confer sensitivity to ephrin-B2 and to convert contralateral to ipsilateral projections. In Aim 2, EphB1 protein will be localized on retinal axons, the site of EphB1 synthesis established (soma vs. growth cone), and the fate of receptor and ligand tracked after growth cone interactions with midline gila. Other experiments will characterize intracellular events such as calcium dynamics as RGC growth cones respond to ephrin midline cues. Such studies are important for understanding how visual pathways form, key for aiding RGC regeneration after perturbation to the visual system.

This is a request for funds to purchase an electron microscope needed for studies of tissues, cells and molecules. A Primary User Group composed of 9 principal investigators and their laboratories, totaling nearly 30 individuals, is identified. The research of these investigators is supported by over 20 separate peer-reviewed grants from the National Institutes of Health and the National Science Foundation, and 8 peer-- reviewed grants from other sources (e.g., Muscular Dystrophy Association, etc.). The research of these investigators falls into two areas: developmental neurobiology, and cellular processes of neuronal and non-neuronal cells, both at the cellular and molecular levels. The equipment will form the basis of a shared ultrastructural facility in the Department of Pathology, shared by the primary user group who are faculty members of the Departments of Pathology, Anatomy and Cell Biology, Physiology, and Biochemistry and Molecular Biophysics. This equipment is needed to accommodate new investigators (all Pi's have assumed Columbia appointments in the last three years). The facility will also relieve overcrowding in existing facilities, namely a shared EM facility jointly run by the Departments of Anatomy and Cell Biology, and Physiology, used primarily by the designated Secondary User Group. Need for this equipment is justified by the numbers of investigators requiring access to high resolution ultrastructural tools. The equipment will be maintained by trained personnel and will be available 24 hours a day to the user group. Installation and start-up costs will be borne by Columbia University.

A long-standing question is how fiber pathways in the mammalian CNS diverge to both sides of the brain. Studies from our laboratory with single-fiber dye labeling have demonstrated that retinal axons from each eye distribute to both sides of the brain by diverging near the midline of the optic chiasm, a classic example of a region where fibers from two bilateral sources meet and redistribute. Immunocytochemistry with antisera to immature glia reveals a palisade of radial glia on either side of the chiasm midline. The palisade spans the region that appears to be permissive for the growth of crossed fibers, but inhibitory to the advance of uncrossed retinal fibers. The cellular structure of the chiasm midline during the period of axon outgrowth and its antigenic profile is remarkably similar to the floor plate of the spinal cord, strengthening the notion that the chiasm midline plays a role in axon guidance in the forebrain where the floor plate is absent. This proposal seeks to identify further the cellular components of the chiasm midline and their antigenic profiles, to describe the development of the chiasm and the underlying mesoderm, and to compare these features with those of the spinal cord floor plate. Finally, genes specific for the floor plate, discussed in Projects 1 and 2, will be localized in the developing chiasm. By studying the composition and development of the optic chiasm midline and the spinal cord floor plate in parallel, we will gain insight on cell patterning and the establishment of neural pathways, especially those that cross the neuraxis.

With the ability to study gene expression, trace axon pathways, and experimentally analyze axon guidance, cells in the paths of growing axons have been localized, but their identity and fates are not well understood. Our studies have outlined the paths of mouse retinal ganglion cell axons as they diverge to both sides of the brain within the optic chiasm. At this site we uncovered novel cellular specializations consisting of both early neurons and radial glia. In vitro analyses indicate that these cells present clues for axon divergence. In the first aim, in the period before retinal axon growth (E8-11), the early development of these cell populations (form, arrangement, and epitope expression) will be charted through immunocytochemistry, receptor- ligand binding, and in situ hybridization. The birthdates and lineage relationships of these cell will be determined by BrdU and retroviral labeling and their fates (transitions in form, and putative transience) investigated by thymidine labeling in combination with immunolabeling. The reciprocal cell-cell interactions between the early neurons and glia will be studied in dissociated cultures. In the second aim, the patterning of the terrain of the optic chiasm and ventral diencephalon, will be defined by combining in situ hybridization and immunocytochemistry. Analyses of mice deficient of these genes will address to what extent these genes regulate the patterning of the ventral diencephalon. The hypotheses of this work are: that early developing neurons and glia are organized in domains which are important for axon guidance, but are transient in their form, molecular expression and even existence; that expression patterns of regulatory genes in the ventral diencephalon correspond to these cellular domains; that these genes are critical to the development of these cells and ultimately to patterning of the brain. These analyses will interface with Dr. Dodd's studies of induction and early patterning of the anterior of the anterior neural tube, and relate to Dr. Jessell's studies of patterning in the spinal cord, in this poorly understood mid-gestational epoch of brain development. GRANT-P01NS305320003 Understanding the mechanisms that control the identity and diversity of cell types generated in the developing vertebrate nervous system is essential to a complete understanding of brain function and pathology. There is increasing evidence that secreted growth factors of the TGFbeta- like genes (GDF7, BMP6 and BMP7) that are expressed by roof plate cells, a specialized dorsal midline glial cell type, have an essential role in the specification of dorsal neural cell types. The function of GDF7, BMP6 and BMP7 will be assayed by analyzing the phenotype of mice in which each of these genes has been eliminated, singly and in combination, by gene targeting. A panel of markers of dorsal cell types will be used to assess developmental defects in CNS patterning in these mutant mice. These genetic studies will be complemented by in vitro assays of dorsal cell differentiation that are intended to define the potency and selectivity of action of these three proteins. Finally, the signaling properties of the roof plate will be assessed using a molecular genetic approach to the selective ablation of roof plate cells by expression of a toxin gene in transgenic mice. In the long term, these studies should provide important information on the generation and normal function of CNS neurons and the underlying causes of neuronal dysfunction.

This proposed research will study the interactions of axonal growth cones and their synaptic target neurons, in order to address the cellular mechanisms that direct and accompany synaptogenesis. Recent evidence from our laboratory suggests that a reciprocal relationship exists between afferent axon and target cell regulation of the growth of afferents by target cells, and the regulation of target cell differentiation by afferents. This relationship is the theme of this proposal. We will use the developing mouse cerebellum and analyze intact brain and tissue culture models, by static and dynamic microscopy. To address how the growth of afferents is influenced by target cells, we will examine the coordination of timing of afferent ingrowth with target cell availability in vivo. After dye labeling afferent axons, the cell contacts of mossy fibers will be analyzed in two situations where mossy fibers arrive before granule cells are present: vestibular mossy fiber ingrowth at E15, and in the neurological mutant meander tail, in which granule cells are missing in the anterior cerebellum. Second, the patterns of climbing fiber growth and growth cone behavior during their exuberant growth onto multiple Purkinje cells, and during focused arborization on individual Purkinje cells will be studied in slices of cerebellum, using time-lapse video microscopy. Third, in an in vitro model system based on purified Purkinje cells, the neural specificity of axon-target interactions will be investigated by analyzing the regulation of neurite growth and branching patterns of appropriate climbing and parallel fiber afferents and inappropriate afferents, with static and real time microscopy. Fourth, with the same tissue culture model, the cellular events associated with Purkinje and granule cell synaptogenesis, including afferent growth cone behavior, formation of synaptic junctions, and expression of synapse-associated proteins, will be examined. The regulation of survival and differentiation of Purkinje cells by afferent-target and other cell-cell interactions will be investigated, by co-culturing purified Purkinje cells with neurons and nonneuronal cells intrinsic and afferent to the cerebellum as well as other brain cell types. The extent to which these interactions regulate the construction of synaptic machinery in the target cell will also be examined. These studies will delineate temporal and spatial features of axon-target contacts, and the key cellular interactions that regulate afferent axonal growth and survival and differentiation of targets. The fundamental information obtained from the analysis of intact brain, coupled with the insight into cellular mechanisms obtained from in vitro experiments, will be essential to interpreting new molecular approaches to neuronal development.

? DESCRIPTION (provided by applicant): Support is requested for training for 5 predoctoral and 1 postdoctoral trainee positions per year, in a university-wide training program in visual science, at the systems, cellular, and molecular levels. Training focuses on analysis of the visual pathways from eye to brain, and cellular, molecular and genetic aspects of the normal and diseased eye, in both basic science and disease-oriented research. Twenty-seven training faculty who are members of biomedical doctoral programs are distributed on two campuses of Columbia University: Twenty-two of these faculty are in basic and clinical science departments on the Health Sciences Campus, 168th Street and Broadway, and 5 are drawn from four departments from the main (Morningside) campus at 116th and Broadway. In late 2016, a majority of these faculty will move to the Greene building, 129th and Broadway in Manhattanville as part of the Zuckerman Mind Brain Behavior Institute. Section 1 (Systems/Computation) includes 14 faculty focused on the visual and oculomotor systems in humans and monkeys using neurophysiology, psychophysics, computational modeling, and imaging. These faculty are in the Departments of Psychology, Biomedical Engineering and Neuroscience, with 7 investigators in the Mahoney Center studying the primate visual cortex. Six faculty in Section 2 (Development and Plasticity) focus on cell specification, retinal axon guidance, eye development, and biophysics and plasticity of dendrites and cortical circuitry, and are in the departments of Genetics, Pathology and Cell Biology, Neuroscience, and Biological Sciences. Section 3 (Molecular/Genetic Approaches to the Normal and Diseased Eye) is composed of 7 faculty in the Department of Ophthalmology, Biochemistry, Medicine, and Pediatrics, who study retinal degeneration, retinoid processing, and the genetics, diagnostics and therapy of retinal disorders, with a focus on age-related macular degeneration and retinal edema. The research carried out by the mentors and trainees matches the goals in NEI's promotion of eye and vision research, including the Audacious Goals Initiative. Support is sought for up to three years for predoctoral students who have chosen their lab and mentor, and for one year for a postdoctoral trainee. Trainees will be recruited from selective graduate programs such as the Doctoral Program in Neurobiology and Behavior, and Integrated Program in Cellular, Molecular and Biomedical Sciences, both of which host MD-PhD students, and by advertisement. Through activities such as courses, thesis committees, symposia, seminars, and the Greater New York Vision Club (VisioNYC), it is expected that faculty and trainees will interact, collaborate, and produce a new generation of vision scientists who will elucidate information processing, development, and disease in the visual system.

DESCRIPTION (provided by applicant): The decussation of the retinal ganglion cell (RGC) projections in the optic chiasm is essential for normal mapping of visual information, and insures that each hemisphere receives information from both eyes to establish binocular vision. How the development of the binocular pathway is determined has been a longstanding enigma. Recent work in the spinal cord has shown that distinct subpopulations of neurons express combinations of homeodomain transcription factors and that such code determines projection to specific targets. We hypothesized that a similar code of regulatory gene expression might designate the uncrossed and crossed subpopulations of RGCs. Our laboratory has discovered that a zinc-finger containing transcription factor Zic2, is expressed in RGCs with an uncrossed trajectory. Zic2, but not other Zic family members, is postmitotically but dynamically expressed in RGCs in ventrotemporal (VT) retina in the period when the uncrossed RGC subpopulation projects axons from this location toward the chiasm. Gain- and loss-of-function experiments in vitro indicate that Zic2 is necessary and sufficient to switch the outgrowth behavior of retinal axons when co-cultured with chiasm cells, from crossed to uncrossed patterns, or vice versa. Moreover, Zic2 expression correlates with the degree of binocular vision across several species. Other work in our lab has identified EphB1 as a receptor system interacting with the inhibitory factor ephrinB2 at the chiasm midline in mouse. EphB1 is expressed coincident with Zic2, spatially and temporally. The proposed work will further analyze the hypothesis that Zic2 specifies the uncrossed retinal ganglion cell axon trajectory, by regulating expression of guidance receptors in the Eph family that mediate retinal axon divergence at the optic chiasm midline. Aims include verification that Zic2 is expressed in ganglion cells with an uncrossed trajectory and elucidation of its temporal expression (Aim1); confirmation that Zic2 is necessary and sufficient to establish an uncrossed trajectory, by using loss- and gain-of-function strategies in vivo (Aim 2); investigation of Zic2's influence on genes that are putative targets, especially with regard to the EphB1 receptor in the retina (Aim 3). This analysis should shed light on patterning the binocular pathway and should apply to the establishment of bilateral sensory pathways elsewhere in the nervous system.

DESCRIPTION (provided by applicant): This grant is aimed at understanding how transcription factor codes pattern the retina and control guidance receptor expression to establish the crossed and uncrossed visual projections. Our previous work defined such a program for the ipsilateral projection through the mouse optic chiasm: The transcription factor Zic2 and the guidance receptor EphB1 are expressed in ventrotemporal (VT) retinal ganglion cells (RGCs), which give rise to the ipsilateral projection. EphrinB2 is expressed on radial glia at the optic chiasm midline, and the repulsive EphB1-ephrinB2 interaction produces the ipsilateral projection. Zic2 and EphB1 expression correlate with the degree of binocularity across species and in genetic models with a reduced ipsilateral projection, such as the albino. In the last funding period, we determined that EphB1 expression is downregulated in Zic2 mutants and upregulated after ectopic expression of Zic2;thus, Zic2 controls EphB1 expression. Further, Zic2 is necessary and sufficient to drive an ipsilateral projection in vivo and in vitro. We also found that Foxd1 is expressed in the VT quadrant and is required for Zic2 and EphB1 expression, placing Foxd1 upstream of this transcriptional program for the ipsilateral projection. In other studies, we found that the Ig-CAM NrCAM and the Semaphorin receptor PlexinA1 are expressed by RGCs in non-VT and late-forming VT retina, both regions giving rise to the contralateral projection. NrCAM appears to modulate an inhibitory response by RGCs to semaphorins, as found for midline crossing in other systems. In Aim 1, we will continue a focus on Zic2 and identify additional genes regulated by Zic2. The transcription factor Islet2 has an identical expression pattern to NrCAM and PlexinA1, but its role is restricted to the late-born crossed projection from VT, similar to NrCAM. Therefore, we will focus on the late VT retina and determine whether Islet2 controls NrCAM and/or PlexinA1 expression to encode the contralateral retinal projection from this region. We will also determine if Islet2 interacts functionally with Zic2. In Aim 2, we will search for additional genes that may specify the retinal sectors giving rise to the crossed and uncrossed projection, and investigate whether Foxg1 (the nasal counterpart to Foxd1) is upstream of the contralateral program. In Aim 3, we will apply information gained from Aims 1 and 2 to understand how the the albino retina is (mis)specified to produce a diminished ipsilateral projection. Aim 4 will examine whether the genes important for RGC and retinal specification implement midline guidance and/or targeting of eye- specific zones in the dorsal lateral geniculate nucleus (dLGN). These studies use innovative methods for gene delivery (in utero and ex vivo electroporation), in vitro assays, and circuit tracing to define the molecular control of the formation of binocular visual projections. PUBLIC HEALTH RELEVANCE: This research aims to understand how retinal ganglion cells grow out from each eye, meet at the X-shaped optic chiasm, then diverge toward targets on both sides of the brain. Proper binocular vision is dependent on a normal distribution of retinal axons crossing at the optic chiasm, and if altered, reduced visual acuity and depth perception ensue. This work investigates the genes that pattern the retina into sectors giving rise to crossed and uncrossed projections and that drive expression of guidance receptors to enable retinal axons to take the appropriate route.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We will test the hypothesis that binge alcohol consumption will cause the reactivation of latent TB disease in primates. Towards this end, we will use previously developed protocol to generate binge alcohol consumption in rhesus macaques and infect them with latent Mtb via the aerosol route. We will ask if latent infection reactivates, relative to historic controls, which received a similar amount of the same Mtb strain.

Project Summary The proper distribution of retinal ganglion cell (RGC) axons at the optic chiasm to the same (ipsilateral) and opposite (contralateral) sides of the brain is crucial for proper binocular vision. Our work has defined axon guidance receptors and transcription factors directing the ipsilateral and contralateral pathways in pigmented animals. We have shown that a molecular program of transcription and guidance factors, including Foxd1, Zic2, and EphB1, is essential for directing the ipsilateral retinal projection, and a complex of factors including guidance molecules Nr-CAM, Plexin-A1, and Semaphorin6D regulate the contralateral projection. These genes control RGC axon growth from the eye as RGC axons cross or avoid the chiasm midline, and subsequently innervate the thalamus and superior colliculus. Albinism is characterized by perturbed melanogenesis in the skin and the eye and results in fewer RGCs that project ipsilaterally and, consequently, impaired stereovision. The molecular mechanisms that link altered melanin biogenesis in the RPE with the reduction in ipsilaterally-projecting (i) RGCs are not understood. However, our studies have indicated that in the albino, neurogenesis in the ventrotemporal (VT) retina, where iRGCs originate, is temporally shifted, and that fewer RGCs express genes such as Zic2 that specify an ipsilateral trajectory. These findings provide new inroads on how to approach the albino mystery. We hypothesize that factors related to melanin production in the normally pigmented RPE influence the development of RGCs in the neural retina, namely their genesis and fate specification. Based on our new RPE studies and those of other labs, it is likely that such factors could affect RGC development via junctions at the RPE-retina interface, where we have identified alterations in the albino. In addition, some RPE factors, missing or upregulated in the albino, are likely to be important for RPE cell health. In the proposed studies, Aim 1 will identify genes that are differentially expressed in pigmented and albino RPE during the period of iRGC genesis, by conducting candidate-based and unbiased gene profiling analyses, followed by validation of these genes by immunostaining, in situ hybridization and qPCR. In Aim 2, we will test a role for select factors on RGC subtype specification through in vitro assays of RGCs alone, RPE alone, and in organotypic slice cultures. By addition of proteins or exogenous gene expression, we will determine if candidate RPE factors can rescue or induce albino retinal and RPE phenotypes. Identification of RPE-derived factors that influence RGC subtype specification will reveal ocular tissue interactions during vision system development, that relate to human disease. These studies will also illuminate how neuronal differentiation programs govern axon projection and connectivity in the nervous system.

DESCRIPTION (provided by applicant): In the mouse visual system, axons of retinal ganglion cells (RGCs) exit from each eye and converge at the ventral midline of the brain to form the optic chiasm (OC), then grow to visual centers in the thalamus, where they synapse onto second-order neurons that project to the cerebral cortex. In animals with frontally located eyes, some RGC axons do not cross the midline and instead project to the ipsilateral, or same-side, visual centers. This circuit mediates binocular vision and we have long focused on the formation of this circuit. The present proposal addresses how ipsilateral (ipsi) and contralateral (contra) RGCs axons extend together in common tracts through the developing brain, converging and diverging throughout their journey to the target, and whether such cohort integrity in paths is relevant to connectivity within target regions. In this proposal, we seek to more fully characterize eye-specific axonal cohort maneuvers in the pathway from eye through the OC to first target. In Aim 1, we will investigate the organization of the ipsi and contra axons, viewed with different tracers in the same preparation, focusing on where the ipsi and contra RGC axons from the same and opposite eyes interleave and where they segregate. We will use classical and novel morphological analysis at the LM and EM levels to chart fasciculation of ipsi and contra RGCs along the path and their relationships with glial cells. We will also devise explant assays to dissect the cellular and molecular mechanisms for fiber-fiber interactions during eye-specific axon pathway formation. In Aim 2, we will investigate the effects of mutations that have known effects on decussation and targeting on RGC eye- specific fiber organization. Throughout, we will compare and contrast the cell and molecular differences of ipsi and contra RGCs as they diverge amongst radial glia at the optic chiasm midline, associate with their own and opposite eye cohorts, exit the chiasm and grow in the tract where astroglia are organized to potentially influence their trajectory, and enter and terminate in the dorsal lateral geniculate nuclei (dLGN).. Approaches developed over the last few decades in our lab will facilitate analysis of axons at the single fiber and cohort levels, aided by a newly developed clearing technique and special microscopy, and electroporation in utero for delivery of GFP-constructs into the retina. We will address the relationship between decussation, eye-specific organization within the path, and early target innervation, linking phases of axon guidance that were previously addressed as discrete steps and restricted to focus on a single cohort. Understanding how tracts are laid down is essential for unraveling the phenotypes of neurological disorders, in which defects in fiber pathways and synapse formation are implicated but their relationship is not fully understood. Moreover, implementing axon regeneration depends on knowledge of how the entire pathway is organized and to what extent each phase of axon organization prepares axons for the next phase.

PROJECT SUMMARY Retinal ganglion cells (RGCs) from each eye extend their axons ipsi- and contralaterally to the brain to establish the circuit for binocular vision. The lack of appropriate sidedness of RGC projections is debilitating in genetic disorders such as albinism, in which hypopigmentation of the retinal pigment epithelium (RPE) is linked to optic nerve misrouting and therefore altered stereo vision. Progress on this grant includes the identification of transcriptional regulators of the specification and differentiation of ipsi and contra RGCs; demonstration of the ventral ciliary margin zone (CMZ) as a source of ipsi RGCs; expression of the cell cycle regulator Cyclin D2 in the ventral CMZ; and dependence of the ipsi RGC projection on Cyclin D2. In the albino retina, these processes and the cellular integrity of the RPE, are disrupted, making the albino an excellent comparative model for the proposed studies. Here we explore a novel role for the CMZ in neural retinal development and RGC specification, and seek to uncover potential signaling networks for establishing proper RGC connections. We propose to combine studies of neurogenesis and fate mapping with transcriptomics of the CMZ, RPE, and neural retina to gain mechanistic insight into how loss of melanin in the RPE of albino animals causes a shift in cell fate from ipsi to contra during the establishment of the binocular circuit. To this end, we will establish a role for Cyclin D2 in the pace of the cell cycle, plane of cell division, and migration from the CMZ to the neural retina (Aim 1); study signaling between the RPE and CMZ, starting with the Wnt pathway, as a route to regulate RGC fate (Aim 2); identify the transcriptional networks that regulate RGC cell fate by performing single-cell RNA-Seq of the CMZ, neural retina, and RPE (Aim 3). Throughout, we will compare albino and pigmented retina. We hypothesize that events in the CMZ control timing of neurogenesis, which specifies RGC projection fate and that factors in the RPE provide directives to the CMZ and neural retina for ipsi/contra RGC fate acquisition. Significance: Probing early neural retinal development and parsing regulators of neurogenesis and cell fate emanating from the CMZ and RPE offer a novel mechanistic entry point to the long-standing enigma of how pigment and the RPE influences cell fate and RGC axon segregation at the optic chiasm. Identifying gene programs on how ipsi/contra RGC diversity arises reveals how decussating systems such as the binocular circuit are established. Such information is critical for driving stem cells into RGCs for replacement therapy and directing axon regeneration in injured and degenerating visual pathways.

Project Summary/Abstract Sensory neurons in the dorsal spinal cord (dINs) represent the first site in the CNS to receive somatosensory information from the periphery and are a crucial component in sensorimotor circuits that control posture and behavior. Defining the developmental principles of somatosensory circuit formation and the molecular identity of neurons that serve specific sensory functions is key to our understanding of the logic of sensorimotor integration and for strategic interventions following injury or disease. All dINs arise from a small array of molecularly defined embryonic cell populations, but markers are expressed only transiently and how these early neurons diversify and integrate into sensory circuits serving distinct sensory modalities in the mature animal remains largely unknown. There is an urgent need for genetic access to express tracers and to manipulate individual classes of developing dINs as they emerge during the embryonic period but establish circuits postnatally. Here, we will generate for the first time, mice that bridge the gap between early identity and mature function by providing access selectively to one population of dINs, called dI1s, throughout development. We will create mouse lines to introduce anterograde and retrograde viral tracers to reveal and map the synaptic targets of dI1s in brain and spinal cord, and to identify input neurons to dI1s, in DRG, brain and spinal cord. These lines will extend temporal genetic access selectively to developing dI1s into postnatal stages, by, first, a Cre-dependent Cre approach, permitting access by Cre-dependent axonal and synaptic tracers, and second, mice that express the proteins required for transsynaptic tracing using rabies virus. We will also develop platforms to analyze efficiently the anatomy and connectivity of the circuitry. Furthermore, we will create a widely needed 3D spinal cord atlas for assessment of neuronal identity and mapping circuitry. To understand the programs that regulate dI1 development, we will also identify and follow dynamic changes of transcriptional signatures in dI1s throughout embryonic and postnatal ontogeny by single cell RNA sequencing. The tools established in the proposed experiments and analyses will be applicable to all dIN subsets and will contribute both resources and information to the field, and provide a template for manipulation and functional analysis of somatosensory pathways in health and in disease.