Monica Vetter - US grants

DESCRIPTION: The hypothesis in this application is that Xath regulates the differentiation of olfactory neurons in Xenopus and functions with another basic-helix-loop-helix (bHLH) gene, NeuroD, to regulate successive stages of olfactory neuron development. The first aim is to use in situ hybridization a immunohistochemistry to determine which cell types express Xath during development and regeneration of the olfactory epithelium and determine whether Xath expression coincides with specific stages in olfactory neurogenesis. The second aim is to overexpress Xath by RNA injection and to determine whether Xa expression is sufficient to promote differentiation of olfactory neuron precursors. The third aim is to perform double in situ hybridization and compare the expression of Xath and NeuroD to see if there is evidence for a functional relationship between these bHLH genes.

The development of neurons in the vertebrate retina is governed by the interplay between positive and negative regulators of differentiation. Several genes, including Notch, Delta, and HES1 have been shown to function as negative regulators of neurogenesis during retinal development. However, the positive factors controlling retinal neurogenesis have yet to be defined. We have identified a gene in Xenopus laevis called Xath5, that belongs to the basic helix-loop-helix (bHLH) family of transcription factors. Xath5 is a homologous to the Drosophila atonal gene, which is required for the determination and differentiation of photoreceptors during fly eye development. In Xenopus, Xath5 is relatively restricted in expression to the developing retina and appears to function as a differentiation factor for retinal neurons: it is expressed in differentiating retinal progenitor cells and appears to promote early differentiation of these cells when overexpressed. Xath5 may therefore function as an important positive regulator of neurogenesis in the vertebrate retina. The experiments is this proposal are directed towards understanding how Xath5 regulates retinal neuron differentiation during Xenopus eye development. First, we will use dominantly-interfering forms of Xath5 to disrupt its function and determine whether Xath5 is required for the differentiation of all or a subset of retinal neurons. Second, we will determine whether the negative regulators of retinal neurogenesis, Notch and Delta, block retinal cell differentiation by inhibiting the expression or function of Xath5. Third, we will assess the ability of Xath5 to heterodimerize with other bHLH proteins expressed in the developing retina, and determine the functional significance of this for retinal cell differentiation. Fourth, we will identify downstream target genes in the developing retina whose expression is directly regulated by Xath5. Ultimately the role of candidate target genes in regulating retinal cell differentiation will be determined. In summary, these experiments will lay the foundation for understanding the molecular mechanisms regulating retinal neuron development, and will underscore the importance of both positive and negative regulators in this process. In addition, these studies should provide more general insight into the role of bHLH proteins in the control of vertebrate nervous system development.

During development of the vertebrate neural retina, multiple neuronal cell
types are born and differentiate under the control of a complex set of
factors that are poorly understood. To gain a complete understanding of the
molecular mechanisms regulating retinal neurogenesis, it is essential to
define the hierarchy of genes involved in this process. The goal of this
project is to use recently developed transgenic methods in the model
organism Xenopus laevis to define which factors control expression of the
transcription factor Xath5, a key regulator of retinal neurogenesis. The
Xenopus transgenic method provides a unique opportunity to rapidly and
inexpensively map the regulatory control regions of developmentally
important genes, thus making it possible to define how genes come to be
expressed in precise spatial and temporal patterns during development.

These experiments will provide a more complete understanding of the steps
leading to normal retinal development. In addition, this work will advance
our understanding of nervous system development in general, since the
retina has proven to be a reliable model tissue for studying nervous system
development and function. Finally, the development and application of the
new transgenic method in Xenopus laevis will open up many possibilities for
future investigation and discovery in the field of developmental biology.

It is anticipated that this POWRE award will allow the principal investigator to
travel to learn a novel Xenopus transgenic method and to then establish it in her
laboratory.

Untapped resources for gene discovery include an explosion of genome sequence information, high-throughput methods for transcript profiling and high-throughput, functional genomics technologies. Experiments are proposed whose long-term goals are to refine and validate new genomics- and functional genomics-based techniques for the identification and analysis of genes that acts in neural development or function. The approaches developed here are generally applicable for gene discovery and expression analysis in the nervous system. The specific experiments proposed, analysis of CREB-dependent pathways of neural plasticity, should serve as a paradigm to guide future studies. A computational screen of a Drosophila genome database for CREB-induced genes involved in neural plasticity will be performed and validated. The screen will establish the viability of promoter sequence-based approaches to studying gene regulation in metazoa; it will also provide: i) a test panel of activity-induced Drosophila genes, and ii) the opportunity to implement new software designed for more mathematically rigorous microarray analysis. A commercially available mammalian microarray will be screened to select mammalian genes strongly induced in human SHSY5Y cultured cells by depolarization and cAMP agonists, treatments known to activate genes involved in long-term plasticity. Once downstream CREB-responsive genes are identified, reporters based on their promoter elements will be engineered in order to embark on a fluorescence-based functional genomics screen for critical regulators of the CREB pathway. First, GFP fusions will be created in which GFP coding sequences are fused downstream of 5' regulatory elements from the genes whose expression behavior correlates with the CREB-response. By a series of genetic engineering steps and FACS selections, cell lines will be generated in which fluorescence serves as a surrogate phenotype for activation of the CREB pathway. A set of such lines, each of which coopts a particular CREB-induced gene, will be developed and used in transdominant genetic experiments to identify modulators of the CREB pathway. Experiments to study the functions of these modulators in vivo will be initiated in rodents and in Drosophila using resources developed as part of this proposal.

DESCRIPTION (provided by applicant): An essential event in the development of the neural retina is the transition of proliferating progenitors to postmitotic differentiated retinal neurons. Inhibition of this process alters the complement of cell types required for normal retinal organization and function. Proneural basic helix-loop-helix (bHLH) transcription factors play a central role in regulating the commitment of retinal progenitor cells to a differentiated retinal neuron fate. The Ath5 gene is required for the genesis of retinal ganglion cells (RGCs). The long-term goal of this proposal is to define the molecular pathway by which Ath5 regulates retinal neurogenesis. Ath5 expression is tightly coupled with the onset of retinal neurogenesis, and the timing of its expression is important for its ability to regulate RGC differentiation. The first aim of the proposal will focus on characterizing the transcriptional regulatory mechanisms that provide spatial and temporal control of Ath5 expression. Next, to understand how Ath5 drives retinal differentiation it is important to investigate the function of genes that are regulated by Ath5 during retinal development. One of these Ath5 target genes, SBT1 (shared bHLH target 1), is a novel gene that functions as an essential effector for proneural bHLH factors in the developing retina. To understand the function of this novel gene, Aim 2 will focus on testing a predicted interaction between SBT1 and histone deactylase, while Aim 3 will be directed towards defining the molecular pathways that are dependent upon SBT1 during eye development. This work will advance our understanding of the mechanisms controlling retinal neuron differentiation, and will reveal the function of an important new gene in the proneural regulatory pathway, which could provide insight into developmental disorders affecting the retina. Ultimately, a detailed understanding of the factors determining retinal neuron fate in vivo will impact future efforts at replacing neurons lost to disease. PUBLIC HEALTH RELEVANCE: An important goal of our research is to understand how cells in the retinal are generated during early eye development, since we may gain a better understanding of how these processes are disrupted in pathological situations, such as congenital disorders affecting vision. A long-term hope is that we may ultimately be able to manipulate the differentiation of retinal stem cells or progenitors to treat retinal degenerative diseases. Our work will contribute to this effort by characterizing the mechanisms underlying retinal neuron development.

[unreadable] DESCRIPTION (provided by applicant): A number of congenital eye disorders stem from alterations in early development, including proliferation and differentiation of progenitors within the neural retina, although in many cases the mechanisms underlying these disorders is not understood. By defining the mechanisms controlling normal retinal neurogenesis, we may gain a better understanding of how these processes are disrupted in pathological situations. bHLH transcription factors play a pivotal role in retinal neurogenesis, but remarkably little is known about how they control the process of retinal neuron differentiation. We have identified a new gene, sbt1, which encodes a novel protein that is expressed in the developing Xenopus nervous system and which is regulated by proneural bHLH activity. Our preliminary results indicate that sbt1 is an essential regulator of retinal neurogenesis in Xenopus. We have identified a mouse ortholog of this gene, and showed that it is also expressed in the nervous system and has a conserved ability to regulate retinal neuron differentiation. Here we propose to develop two new directions for the analysis of sbt1 function. In Specific Aim 1 we will initiate a study of sbt1 during mouse retinal development by performing a detailed analysis of sbt1 expression, and determining whether its expression depends upon proneural bHLH factors. In Specific Aim 2 we will analyze subcellular localization of sbt1 and define protein partners for this novel protein. The experiments in this proposal will provide the conceptual framework for an in-depth genetic study of sbt1, a novel and exciting gene that appears to be a key regulator of neuronal differentiation in the developing retina. A long-term hope is that we may ultimately be able to manipulate the differentiation of retinal stem cells or progenitors to treat retinal degenerative diseases. Our work will contribute to this effort by characterizing the molecular mechanisms underlying retinal neuron differentiation and cell fate specification. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The hyaloid vascular system (HVS) is a transient vascular network that nourishes the immature lens and retina before formation of the retinal vasculature. Defects in HVS development can lead to serious congenital eye diseases, including persistent hyperplastic primary vitreous (PHPV). PHPV is characterized by the presence of fibrovascular retrolental mass tissue in the vitreous, microphthalmia and leukokoria, and is associated with cataract, retinal folding and detachment. A better understanding of how development of the HVS is governed should provide insights into the pathogenesis of such congenital eye diseases. We found that selective knockout of the gene for the Wnt receptor Frizzled-5 (Fzd5) in the developing mouse retina and optic stalk causes early hyperplasia of the vitreous hyaloid vasculature and persistence at later stages, resembling the human disease PHPV. Since Fzd5 was selectively disrupted in the retina and not the vasculature itself, the effect is non-autonomous. We hypothesize that loss of Fzd5 in the retina alters signaling between the retina and components of the developing HVS, resulting in hyperplasia and subsequent persistence of this tissue. The goal of this proposal is to identify candidate retina-derived factors and reveal the mechanisms through which the development of the HVS may be regulated by signals from retina. This may lead to novel strategies to treat congenital eye disease such as PHPV. PUBLIC HEALTH RELEVANCE: The hyaloid vascular system (HVS) is a transient arterial network that nourishes the immature lens as well as the retinal neuroepithelium before formation of the retinal vasculature. The mechanisms governing the normal development of the HVS have not been well defined. Defects in HVS development can lead to serious congenital eye diseases, including persistent hyperplastic primary vitreous (PHPV). A better understanding of this process may lead to strategies to potentially prevent or treat congenital eye diseases such as PHPV.

The challenge to understand the molecular basis of the evolutionary origin and development of the eye and visual system has intrigued scientists for centuries. The eye and visual system has long been recognized as a fascinating and uniquely informative model in which to explore how sensory structures develop. Vision is a highly specialized sensory function that demands an exquisitely organized and highly structured neural system, and it is this precision that creates an ideal experimental system to uncover principles of development and evolution. The community of scientists who study development of the eye and visual system have come to rely on the Visual System Development GRC as a 'must attend' event that provides a high level of information and a valuable forum for exchange.
The aim of the Gordon Research Conference on Visual System Development is to bring together investigators studying the development, disease, and the evolution of the visual system. The meeting provides an environment that stimulates discussion and interaction across disciplines and among students, early-career investigators and leaders in the field. The goals of this biennial conference are to generate an understanding of common principles that regulate visual system development in diverse species, to share the latest and most exciting new results, and to stimulate new ideas and collaborations that impact our understanding of eye development. In addition, through this meeting there is an effort to apply our growing knowledge of eye development and maturation to the fields of regeneration and stem cell biology. Support from the NSF will help enable outstanding young scientists to attend this high-profile international meeting, to present their research findings to the international scientific community, and to interact with the leaders in the field. In particular, the funds requested from NSF will be used to facilitate participation by U.S. students and postdocs from a variety of backgrounds.

DESCRIPTION (provided by applicant): Glaucoma is an age-related neurodegenerative disease that affects up to 60 million people worldwide. Visual field loss and blindness result from progressive deterioration of the optic axons and selective death of retinal ganglion cells (RGCs). Intraocular pressure (IOP) elevation constitutes a risk factor in glaucoma, but is not sufficient to cause disease, suggesting that other conspiring events lead to progressive decline of optic axons, loss of RGC viability and final apoptosis. The exact mechanism responsible for RGC degeneration in glaucoma is not known. In this proposal we investigate the role of microglia and complement pathways in retinal ganglion cell decline in the DBA/2J model of glaucoma. Microglia, the resident immune surveillance cells of the CNS, are exquisitely sensitive to neuronal stress and injury, and have been implicated in multiple neurodegenerative diseases. There is evidence that microglia become activated in human glaucoma as well as in various animal models, however the consequences of microglial activation in glaucoma remains unknown. In this proposal we will document the relationship between microglia activation and RGC decline. We will then inducibly deplete microglia from the retina, or inhibit complement pathways in the retina, and assess the effects on RGC degeneration. This study will provide the first detailed investigation of the functional consequences of microglia activation and complement expression in an animal model of glaucoma. The findings from this work may form the basis for testing therapeutic interventions targeting microglia or complement pathways in human glaucoma. PUBLIC HEALTH RELEVANCE: Glaucoma is a devastating neurodegenerative disease of the eye, and is a leading cause of blindness worldwide. The cellular and molecular players involved in disease progression are only partially understood, limiting the ability to develop new innovative treatments for the disease. The experiments in this proposal will extend the knowledge about the role of microglia and innate immunity pathways in glaucoma. This may reveal potential candidate biomarkers for the disease as well as novel targets for therapeutic intervention.

DESCRIPTION (provided by applicant): As retinal axons grow along the retinotectal pathway from their origins in the retina to their final targets in the tectum, they are guided by a complex array of signals in the extracellular matrix, and by interactions with other retinal axons. This process is not only critical for the development of visual function, but serves as an accessible model for axon guidance in general. This proposal will use genetic, embryological, and imaging approaches in the zebrafish embryo to analyze retinal axon guidance in vivo, studying how Slit- Robo signaling and axon-axon interactions act to shape the retinotectal projection. We will address three main questions about Slit-Robo signaling. First, can Slits change from repulsive to attractive in different contexts as growth cones pass from one part of their pathway to another? Expression studies, time lapse analysis, in vitro assays, and a novel in vivo method for targeted misexpression will address this question definitively. Second, do different cytoplasmic domains of the Robo2 receptor have different required functions? And third, is the alternative splicing that we have seen in the robo2 gene functionally significant? We will address these two questions by employing splice-blocking morpholino oligonucleotides for a powerful new strategy, targeted exon deletion. Within the retina, our preliminary data show that early-born central RGCs are required for later-born peripheral axons to exit the eye. We will first comprehensively analyze retinal axon pathfinding within the retina, then use time lapse and transplant experiments to ask how early RGCs act to guide later axons. Finally, we will test which cell-adhesion molecules mediate retinal axon fasciculation, and what role they play in guiding retinal axons within the retina, in the chiasm, and in the optic tract. Relevance to public health. Visual system anatomy and genetic control mechanisms are both highly conserved across the vertebrates from fish to humans. Understanding the molecular mechanisms that work together to assemble the zebrafish visual system is thus directly relevant to human development. Furthermore, our data showing the importance of axon-axon interactions during development highlight the necessity of considering such interactions during axon regeneration in response to injury or disease.

DESCRIPTION (provided by applicant): Glaucoma is an age-related neurodegenerative disease that causes blindness due to selective deterioration and death of retinal ganglion cells (RGCs). While multiple risk factors, including elevated intraocular pressure (IOP) can contribute to glaucoma, the molecular and cellular mechanisms responsible for RGC degeneration are not known. Microglia have been implicated in multiple neurodegenerative diseases, including human glaucoma as well as various animal models of the disease. Here we investigate the fractalkine signaling pathway since it regulates communication between neurons and microglia in the CNS. We propose that disruption of this pathway contributes to enhanced microglia activation and increased degeneration of RGCs in glaucoma. Using two different animal models of glaucoma, we will first test whether disruption of fractalkine signaling increases microglia activation and/or RGC degeneration. Next, we will use adeno-associated viral delivery to increase fractalkine expression and test whether this limits microglia activation and/or is reduces RGC degeneration. Finally, to elucidate the molecular signature of microglia responses in glaucoma, we will generate a comprehensive molecular profile of microglia activation, and regulation by fractalkine signaling. The findings from this work will provide significant insight ito the molecular pathways involved in glaucomatous pathology, and advance efforts to develop therapeutic interventions for slowing or preventing vision loss in glaucoma.

DESCRIPTION (provided by applicant): Normal visual function depends upon the generation of a balanced complement of retinal cell types during development. Although much has been learned about retinal progenitor proliferation and differentiation, little is known about how this s modulated by microglia, which is the resident neuroimmune cell of the central nervous system (CNS). Microglia infiltrates the mouse retina at early stages of eye development and is present by e11.5 coincident with the onset of retinal neurogenesis. However, despite the prevalence of microglia at the earliest stages of neurogenesis and suggestive evidence for a role in cortical neurogenesis, we lack a detailed understanding of whether these cells regulate progenitor cell survival, proliferation or differentiation, particularly in the developing retina. Here we propose o define the pattern of microglia distribution, activation and gene expression changes during retinal development, first by imaging GFP labeled microglia at multiple stages, and then by generating a comprehensive molecular profile of microglia over developmental time. Next we will test whether inhibiting or eliminating microglia alters retinal progenitor proliferation, survval or differentiation. This study will provide a detailed investigation of microglia function during embryonic eye development, and will shed light on the role of neuro-immune interactions on visual system development.

ABSTRACT During development of the retina, it is critical to precisely coordinate the balance between proliferation and differentiation of progenitors to ensure that the appropriate complement of each cell type is generated. While much has been learned about transcription factors and signaling pathways that regulate this process, there is evidence that epigenetic mechanisms, including histone modifications, play a key role. The polycomb repressive complex 2 (PRC2), catalyzes and maintains methylation of lysine 27 on histone H3, which promotes gene repression. The goal of this proposal is to address the role of PRC2 in the developing retina. We previously found that the PRC2 catalytic subunit EZH2 is critical during postnatal retinal development for maintaining retinal progenitor proliferation, ensuring transcriptional integrity, and coordinating the timing and balance of late retinal cell differentiation. However, it is unclear whether PRC2 plays a role during early retinal neurogenesis, and whether it promotes the maintenance of retinal ganglion cells (RGCs), which are born early during retinal development. The first aim of the proposal will focus on addressing how PRC2 regulates the differentiation and the maintenance of early born retinal cell types, particularly RGCs. The second aim will determine how PRC2 activity and target gene selection is regulated by a key accessory protein, JARID2. This work will advance our understanding of how histone modification governs gene expression during retinal neurogenesis. In particular, addressing how a key repressive modification is regulated as RGCs are generated and maintained will be a major step forward. This will help inform strategies for cell replacement in diseases where RGCs degenerate and are lost. In addition, by determining how changes in histone modification contribute to neurodegeneration, we may gain insight into fundamental mechanisms of blinding eye diseases such as glaucoma and other diseases impacting RGCs.

PROJECT SUMMARY Microglia, the resident neuroimmune cells of the CNS, play important developmental roles. While brain microglia have been extensively studied, less is known about the molecular properties and function of microglia in the developing retina. The goal of this proposal is to address how retinal microglia change over the course of development, the molecular pathways involved and how this relates to their function. The first aim will determine whether there are molecularly distinct subpopulations of microglia in embryonic and early postnatal retina. The second aim will address the function of microglia in the early postnatal retina by utilizing knowledge about their gene expression signature and targeting them for depletion. Finally, in the last aim we will test how developmental events govern retinal microglia phenotype and function, and the molecular pathways involved. By determining how changes in microglial properties are regulated and contribute to development of the retina we will gain insight into fundamental mechanisms of retina development and microglial function. This may ultimately inform our understanding of how microglia are modulated and participate in degenerative disease processes resulting in loss of vision.

PROJECT SUMMARY Microglia, the resident neuroimmune cells of the CNS, play important developmental roles. During embryonic development we showed that retinal microglia target a subset of viable newborn retinal ganglion cells (RGCs) for phagocytic elimination through a process that requires complement signaling. The embryonic retina is a simple system to address how microglia target non-apoptotic cells for phagocytosis, with important implications for the role of microglia in cell loss during injury and disease. To understand the mechanisms of retinal cell elimination, the first aim will address the role of complement expression and stress pathways in RGCs. The second aim will test specific microglial recognition pathways required for phagocytic retinal cell elimination, including RGCs and astrocytes. This study will shed light on how microglia influence developmental remodeling in the retina, and may ultimately inform our understanding of how microglia participate in retinal disease processes resulting in loss of vision.