Seth Blackshaw - US grants

[unreadable] DESCRIPTION (provided by applicant): Project summary: We aim to identify the molecular network required for development and survival of mammalian rod photoreceptors. We identified Protein Inhibitor of Activated STAT3 (PIAS3), a transcription co-regulator and site-specific SUMOylase as being strongly expressed in developing rod photoreceptors. We observed that Pias3 overexpression gives rise to an increase in rod photoreceptors, while PIAS3 knockdown produces excess Muller glia. We also identified two transcription factors, Crx and Nr2e3, as Pias3 interacting proteins. We thus hypothesize that PIAS3 plays a critical role in rod development and survival by interacting with photoreceptor-specific transcription factors. We propose to test this hypothesis with a series of experiments. [unreadable] [unreadable] First, we will use fluorescent in situ hybridization and immunocytochemistry to determine if Pias3 is expressed in developing rod photoreceptors and absent from developing cones and mitotic progenitors. [unreadable] [unreadable] Second, based on our preliminary data, we hypothesize that PIAS3 regulates both the fates of differentiating photoreceptors, the kinetics of rhodopsin expression, and the maintenance of newly differentiated photoreceptors. To address these questions, we will perform a detailed analysis of the effects of gain/loss of function of Pias3 using a panel of molecular markers. We will use BrdU-based birthdating to determine whether gain/loss of function of PIAS3 influences the kinetics of rhodopsin expression, and use rhodopsin-based expression constructs to determine whether gain/loss of Pias3 function has distinct effects in developing retina and differentiated photoreceptors. [unreadable] [unreadable] Finally, we will explore the mechanism by which Pias3 acts in retinal development. We hypothesize that the effects of PIAS3 are in part mediated by Crx, Nr2e3 and possibly Stat3. We also hypothesize that Pias3 directly regulates transcription of many rod, cone and possibly Muller glia-specific genes. We test whether PIAS3 interacts with Crx, Nr2e3 or Stat3 in vivo, and whether these factors mediate the effects of PIAS3 in developing retina. Following on from this, we will determine which domains of PIAS3 are required for its activity in the retina, and whether these are also required for interaction with Crx and Nr2e3. Finally, we will determine whether PIAS3 directly regulates expression of rod, cone and Muller-specific genes, and if this requires Crx and Nr2e3. [unreadable] [unreadable] Relevance: The molecular basis of cell specification in the central nervous system is poorly understood, and these studies will provide mechanistic insight into this process. Moreover, mutations in rod-enriched transcription factors very often lead to photoreceptor degeneration and blindness, and we anticipate that this may also hold for Pias3. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Hypothalamic tanycytes have been a cell type in search of a defined function for nearly sixty years. The prominent presence of tanycytes in the mediobasal hypothalamus, the key central regulator for a broad range of appetitive and emotional behaviors, has led to much speculation about their physiological function. A great deal of mostly indirect evidence has been accumulated to suggest their involvement in processes ranging from control of blood-brain barrier permeability, to regulation of neurohormone release and control of feeding, to adult neurogenesis. However, it has so far not been possible to effectively and selectively manipulate the function of tanycytes in vivo, and thus far these proposed functions of tanycytes have not been directed tested. We have identified a set of genes that are selectively expressed in hypothalamic tanycytes. We propose to use this data to generate transgenic mouse lines that will permit both selective and conditional ablation of tanycytes and selective and inducible Cre-based recombination in tanycytes. We will then selectively ablate hypothalamic tanycytes using these lines, and examine the contribution of these cells to aspects of hypothalamic physiology. PUBLIC HEALTH RELEVANCE: Hypothalamic tanycytes have been proposed to regulate many different physiological processes. However, it has thus far not been possible to effectively and selectively manipulate tanycyte function, and thus these proposed functions of tanycytes have not been directly tested. We propose to generate transgenic mice that will allow us to conditionally and selectively manipulate gene expression in hypothalamic tanycytes. We will use these lines to selectively ablate hypothalamic tanycytes and to study the behavior and physiology of mice lacking these cells.

Project summary: The vertebrate retina is a powerful model system for addressing mechanistic questions of how individual cell subtypes are specified during development. We have found that the homeodomain transcription factor Lhx2 regulates the development and function of retinal Muller glia. Loss of function of Lhx2 results in dramatic defects in Muller glial development. Furthermore, selective deletion of Lhx2 in mature Muller glia leads to altered expression of many genes known to be expressed in Muller glia. Furthermore, these cells becoming constitutively reactive following loss of Lhx2, mimicking the effect of injury. We propose to both identify the genes which are directly regulated by Lhx2 in mature Muller glia, and to examine the consequences of this constitutive reactivity in both uninjured and injured retina. We will determine whether mice that selectively lack Lhx2 expression in mature Muller glia demonstrate a gene expression profile that is fully characteristic of a reactive state, and determine whether Lhx2 directly represses expression of genes expressed in activated glia. Since glial reactivity has been proposed to regulate photoreceptor viability, we will conduct a detailed examination of cellular changes that occur throughout the lifespan of mutant animals following deletion of Lhx2 in mature Muller glia. Furthermore, we will determine the effects of mature glia- specific deletion of Lhx2 in models of photoreceptor injury. Finally, we will characterize any molecular signals released from Lhx2-deficient glia that regulate photoreceptor survival.

DESCRIPTION (provided by applicant): Project summary: Retinal cell types are generated during a series of discrete temporal intervals during the course of neurogenesis. The molecular mechanism by which retinal progenitors successively acquire and lose competence to generate specific cell types is largely unknown. Studies of ES cell differentiation, however, have provided insights into how this might occur. Prior to lineage commitment, the regulatory sequences of many cell-specific genes are marked by histone modifications characteristic of both transcriptionally active and repressed chromatin. These domains of bivalent chromatin are proposed to both facilitate recruitment of transcription factors that direct cell fate specificatio and to prime these genes for rapid activation following lineage commitment. We hypothesize that similar mechanisms may control the developmental competence of retinal progenitors. To address this question, we propose to use ChIP-Seq to map the genomic distribution of histone modifications associated with transcriptional regulation in both early and late-stage retinal progenitors. In parallel, we will determine whether changes in histone modifications directly correlate with changes in the genomic distribution of CHX10 and LHX2, two progenitor-specific transcription factors that regulate proliferation of early-stage progenitors and lineage commitment of late-born cell types. We anticipate that this work will provide a comprehensive map of active enhancers and promoters in retinal progenitor cells, and serve as the basis for future functional studies of the role of epigenetic processes in the regulation of retinal cell fat specification. These studies may also help inform research aimed at producing ccell-based therapies for blinding disorders, and help ensure the efficient generation of specific cell types that have been lost in disease.

(same as original) This is a proposal to create a production center capable of implementing a pipeline that includes acquisition and evaluation of human transcription factor immunogens, production and selection of monospecific Monoclonal antibodies (mMAbs), and biochemical characterization and validation of these protein capture (affinity) reagents. We emphasize the advantages of high throughput and scalability to be achieved within our production effort, and propose the development of process improvements, thereby improving cost and quality over the duration of the award. We will maintain a degree of scientific flexibility to develop approaches to producing affinity reagents to less tractable transcription factor protein targets as they are identified in the course of production. Finally we propose a viable distribution plan consistent with NIH policies, utilizing the existing Developmental Hybridoma Monoclonal Bank, which ensures broad availability and wide accessibility for future use of the reagents with minimal constraints, consistent with achieving the goals of this funding initiative.

? DESCRIPTION (provided by applicant): The mammalian central nervous system (CNS) contains many hundreds of molecularly and functionally distinct cell types, which comprise the basic building blocks of neural circuitry. Individual cell types can be labeled and manipulated using transgenic and knock-in animals, but this approach, is slow, expensive, and limited in scope. Furthermore, it cannot be applied to higher primates or humans. We propose to develop an approach that will allow the selective targeting of individual CNS cell types in wildtype individuals, from a range of mammalian species. This is a modification of a recently developed technology known as CRE-DOG that uses pairs of camelid nanobdies to scaffold assembly of functional split Cre recombinase in the presence of GFP. We propose to use this general approach to target endogenous cell subtype-specific transcription factors using Fn3-based recombinant monobodies, which can be rapidly produced and screened in vitro, and use these to induce assembly of split Cre and Dre recombinase. These reagents can then be used to induce cell-specific activation of expression of reporter and effector constructs delivered by electroporation or viral vector. As proof of principle for this approach, we will first use Fn3-basd pairs of anti- GFP monobodies to scaffold assembly of split Cre and Dre in vivo. We will next raise pairs of monobodies against cell-specific retinal transcription factors, and demonstrate that these can scaffold assembly of functional Cre recombinase, and develop expression constructs that allow Cre-dependent expression of these reagents to avoid potential disruptive effects of monobody expression. Following this, we will demonstrate that these reagents direct cell-specific Cre activation in neonatal retina. Finally, if proven successful, we will generate a toolbx of reagents that will enable selective activation of reporter and effector constructs in the major cell types of retina and cerebral cortex, in both mice and humans.

Project Summary: One potentially important approach to restore vision is the regeneration of lost retinal neurons from an endogenous population of retinal cells, the Müller glia. To explore the potential of ultimately stimulating the resident Müller glia in the damaged human retina, we will take a comparative approach using zebrafish (regeneration competent), chick (regeneration limited), and mouse (regeneration refractory). We will conduct a comprehensive and unbiased, comparative analysis of gene expression and chromatin conformation in isolated retinal progenitor cells and Müller glia in developing zebrafish, chick, and mouse retinas. We will also study changes Müller glia from all three model organisms as they are activated/reprogrammed in response to retinal injury (light damage, NMDA) or exposure to extrinsic factors that are capable of inducing their activation in the absence of retinal damage. Aims 1 and 2 will generate transcriptome and chromatin data of genes and chromatin structures that are associated with formation of Müller glia progenitor cells. In Aim 3, we will integrate this data using newly developed bioinformatic analysis to identify transcription factors and transcriptional networks that control neurogenic competence in Müller glia from each organism. In Aim 4, we will validate and test candidate genes in regulating the dedifferentiation of Müller glia in zebrafish, chick, and mice, using a combination of gain- and loss-of-function approaches. This work will begin to identify the transcription factors and miRNAs that regulate the extent of retinal regeneration in the three different model organisms. Understanding how to restore Müller glia to a youthful status will enable targeted regenerative retinal therapies.

? DESCRIPTION (provided by applicant): Obesity is the main risk factor for developing type II diabetes (T2D), a major and growing public health problem. Elevated adiposity leads to central leptin and insulin resistance, which in turn can trigger changes in homeostatic neural circuitry in the hypothalamus. This can ultimately lead to the development of metabolic syndrome and T2D. Neurogenesis in the adult hypothalamic parenchyma is disrupted by high fat diet (HFD) and leptin deficiency, leading to a reduction in the number of anorexigenic POMC- expressing neurons. We have recently identified hypothalamic tanycytes as a second source of newborn neurons in adult hypothalamus. Our preliminary data suggests that while HFD and leptin deficiency stimulate tanycyte-derived neurogenesis, tanycyte-derived neurons promote weight gain in wildtype animals but inhibit weight gain in leptin-deficient mice. The studies proposed here aim to improve our understanding of how tanycyte-derived neurons regulate body weight, and to determine how dietary signals regulate tanycyte-derived neurogenesis. First, using genetic approaches, we plan to investigate the physiological consequences of selectively disrupting and enhancing tanycyte-derived neurogenesis. Second, we plan to investigate the molecular mechanisms by which both HFD-induced cytokines such as CNTF and leptin regulate tanycyte-derived neurogenesis. Finally, we propose to determine the exact identity of tanycyte-derived neurons and to identify their post-synaptic targets. We anticipate that these studies will ultimately assist in the design of novel therapies for treatment of obesity and T2D.

Project Summary New tools are urgently needed to selectively target constructs that monitor and manipulate the activity of individual cell types without having to rely on genetic manipulation. This proposal aims to develop viral tools that use cell type-specific alternative splicing events to drive cell type-specific gene expression in the nervous system independent of genetic manipulation, an approach we term splicing-linked expression design (SLED). SLED-based vectors use evolutionarily conserved, highly cell type-specific exons, identified using the ASCOT database developed by our group, to drive expression of reporter and effector constructs. We have demonstrated feasibility of this approach using constructs that selectively target retinal photoreceptors, muscle cells, and cortical neurons. We propose to extend this by combining cell-specific alternative exon/intron sequences with appropriate promoter sequences to generate a toolbox of AAV and lentiviral SLED vectors that selectively target multiple cell types of interest to the neuroscience research community. We will first generate SLED vectors that target primary sensory and motor neurons, as well as multiple subtypes of cortical neurons and glia, and validate the specificity of these reagents in mice. We will next test the cell specificity of SLED reagents that are validated in mice in rats, ferrets, as well as human cortical organoids and rat-human chimeras. Finally, highly specific SLED fluorescent reporter constructs will be converted to drive expression of calcium indicators, as well as optogenetic and chemogenetic constructs. We anticipate that SLED-based reagents will allow highly cell type-specific expression of a broad range of molecular tools useful for analysis of neural circuitry in multiple mammalian species.

Project Summary Müller glia of cold-blooded vertebrates can re-enter the cell cycle and rise to photoreceptors following retinal injury, while mammals have lost this ability. As part of the NEI Audacious Goals Initiative, we have conducted a ü comprehensive analysis of injury-induced changes in gene expression and chromatin accessibility in zebrafish, chick and mouse M ller glia, allowing us to identify both evolutionarily consüerved and species-specific gene regulatory networks that regulate glial reprogramming. This has identified a set of dedicated gene regulatory networks in mice tühat restrict proliferative and neurogenic competence in M ller glia. We aim to use these findings to gain a more complete insight into the molecular mechanisms that regulate neurogenic competence in mammalian M ller glia, and to develop treatments that can maximize generation of glial-derived photoreceptors while simultaneously not depleting the number of existing glia. To do this, we propose to generate individual loss of function mutants of the top candidate negative regulators of proliferative and ü neurogenic competence using AAV-mediated CRISPR/Cas9 gene disruption. We will first validate efficacy of sgRNAs targeting individual TFs, comprehensively profile chanüges in geüne expression in reactive M ller glia following loss of function of these genes, and characterize the fate of M ller glia-derived cells. We will then conduct combinatorial loss of function of negative regulators of M ller glia reprogramming to enhance generation ü of glial-derived retinal progenitor cells in wildtype and Nfia/b/x-deficient mice. Finally, we will combine CRISPR- mediated loss of function analysis with overexpression of Ascl1, Crx and Nrl to enhance generation of M ller glia-derived rod photoreceptors in both wildtype and dystrophic retina. We predict that these studies may substantially advance cell-based regenerative treatments aimed at restoring retinal photoreceptors lost due to blinding diseases.

PROJECT SUMMARY The 2020 Gordon Research Conference (GRC) and Gordon Research Seminar (GRS) on Visual System Development are a paired set of biennial meetings that bring together investigators studying development, disease and evolution of the visual system. Over the years, these meetings have provided an exciting and unique forum in which to explore the similarities and differences underlying visual system development and function across a broad range of species. The goal of these meetings is to foster an appreciation of common principles that mediate the construction and function of the visual system in diverse organisms, and to share the latest exciting new ideas and findings on this topic. By including sessions that highlight emerging topics with translational impact, such as ?Retinal regeneration? and ?Development of the primate retina?, the meeting will also expand its scope and stimulate cross-talk between developmental biologists and investigators focused on translational aspects of vision science. The GRS provides a unique platform for students and postdoctoral research fellows in the visual research field to share current, unpublished research amongst their peers. The format of the GRC and GRS meetings provides a highly interactive and stimulating venue for cross- fertilization of ideas and developing new collaboration. The Visual System Development GRC has established a reputation as the leading conference in its field, and is the only meeting on the topic that brings together vision researchers working on the full range of experimental systems in the field, ranging from Drosophila to human. The current proposal requests funds to help defray conference fees for attendees at both meetings. The Visual System GRC and GRS will feature scientists at the cutting edge of the field, with careful attention taken to ensure gender, ethnic, and geographic diversity, and to include scientists at all stages of their careers.

Project Summary GABAergic neurons of the dorsolateral hypothalamus play an essential role in both the regulating the sleep/wake cycle, but little is known about the molecular mechanisms that control their development. We have recently found that the LIM homeodomain transcription factor Lhx6 is necessary for development of a population of sleep-promoting GABAergic neurons in the zona incerta in the hypothalamus. Lhx6 is essential for development and migration of most telencephalic interneurons, in the hypothalamus Lhx6 is expressed in a much more restricted subset of neurons that do not express markers of telencephalic Lhx6-positive interneurons. The role of Lhx6 in development of telencephalic and hypothalamic neurons differs in several important respects. Lhx6 expression is regulated by different transcription factors in the two regions. Preliminary gene expression analysis has identified a number of genes that are strong candidates for mediating these differences. In this proposal, we aim to identify the molecular mechanisms that guide the specification and survival of hypothalamic Lhx6 neurons. First, we will use a combination of genetic approaches to identify transcriptional regulatory networks that are required for initiation and maintenance of hypothalamic expression of Lhx6, and that control development of distinct subtypes of Lhx6-positive neurons. Next, we will determine whether tangential cell migration plays a critical role in the localization of sleep-promoting Lhx6-positive neurons, and identify the molecular mechanisms that control this process. Finally, we will identify molecular subtypes of sleep-activated Lhx6-positive hypothalamic neurons, and investigate how mutants that disrupt the development of these neurons result in altered regulation of the sleep/wake cycle. This will provide insight into molecular pathways that control the assembly of key components of hypothalamic neural circuitry, and may identify therapeutic targets for treatment of sleep disorders.

Project Summary While degeneration of cone photoreceptors is the ultimate cause of blindness in photoreceptor dystrophies, mouse and human retinas are rod photoreceptor-dominant, a fact that has hindered progress in understanding how cones are specified. New insight into this problem can potentially come from studying the molecular mechanisms of photoreceptor development in mammalian species with naturally cone-dominant retinas. One such species is the 13-lined ground squirrel (13-LGS) Ictidomys tridecemlineatus, which is endemic to the American Midwest. The genome of 13-LGS is fully sequenced, and their developmental staging is well- characterized and readily comparable to mouse. We propose to comprehensively profile gene expression and chromatin accessibility during retinal neurogenesis in 13-LGS using single-cell RNA- and ATAC-Seq. We will then use computational approaches to identify gene regulatory networks predicted to control cone photoreceptor development in 13-LGS, and compare these with results obtained from mouse and human retinas to identify species-specific differences in gene expression and regulation that underlie differences in the rod:cone ratio. We will also test whether the 13-LGS orthologue of the Nrl gene, a master regulator of photoreceptor specification, shows reduced ability to promote rod, and repress cone, development relative to its mouse counterpart. We expect these studies will identify multiple previously unidentified genes that are strong candidate positive and negative regulators of cone development, and can be functionally tested in future studies. Ultimately, this may help improve protocols for directed differentiation of cone photoreceptors from stem and progenitor cells for use in therapeutic transplantation.