David R. Hyde - US grants

The phototransduction cascade initiates with the photoexcitation of rhodopsin and terminated with the polarization of the cell by sodium ions in vertebrates and invertebrates. In Drosophila, an electroretinogram (ERG) measures the mass electrical graded potential across the retina, thus measuring the functional and structural integrity of the compound eye. Several ERG mutations have been isolated and have been shown to be involved in phototransduction. This proposal describes a neurogenic analysis of three of these mutations. Retinal degeneration B(rdgB) negatively interacts with phospholipase C or its enzymatic product, inositol 1,4,5-triphosphate, which is a second messenger in the cascade which activates the sodium channel to produce the graded potential. No on transient A (nonA) affects the transduction of the photoreceptor potential signal to the laminal interneurons, while slow receptor potential (slrp) exhibits a defect in returning to the ground state after the depolarization of the photoreceptor. 56 Drosophila visual system-specific cDNA clones were isolated, and two cDNAs mapped near rdgB and nonA or slrp. I will isolate the corresponding genomic clones and transform them into the Drosophila germline to examine if they are capable of rescuing the mutant ERG phenotype, demonstrating that the clone contains the wild-type gene. These genes will then be analyzed at a molecular level by intron/exon mapping, RNA 5' end analysis, and sequencing. The deduced amino acid sequence will be determined from the DNA sequence and will be used to search for homology to known functional protein domains and examined by hydrophobicity plots for transmembrane regions. These data should reveal potential roles of the molecules in the cascade. In vitro mutagenesis will be used to introduce point mutations within the deduced functional regions. The mutagenized gene will be introduced into flies and the phenotype will be observed. Failure to observe a wild-type ERG with the introduced gene will suggest that the mutagenized domain plays a vital role in the protein's function. Additionally, antibodies will be raised to examine the temporal and spatial localization of protein expression and immuno-electron microscopy will be used to localize the protein's function. It is the goal of this work to better understand the mechanism of signal transduction, particularly how the second messengers regulate the cellular response, such as photoreceptor depolarization, and how the second messengers are regulated, thereby also regulating the response.

We are interested in a deeper understanding of the components and regulation of the phototransduction cascade and learning how perturbations in photoreceptor cell physiology lead to various forms of retinal degeneration. We use the Drosophila visual system to examine these broad questions. Drosophila is amenable to molecular, electrophysiological, and genetic approaches to identify molecules and to study in vivo the protein relationships in biochemical pathways. Because many invertebrate visual transduction proteins have vertebrate homologs, both cascades may function in an analogous manner. Thus, the identification of molecules and mechanisms in Drosophila may clarify the vertebrate visual transduction pathway. Many visual transduction molecules exhibit retinal degeneration when altered in either invertebrates or vertebrates. Therefore, studying the role of these molecules in visual transduction will elucidate the mechanisms leading to degeneration. We will extend our analysis of Drosophila retinal degeneration-B (rdgB), which exhibits light-dependent degeneration of photoreceptors. The rdgB protein is a novel phosphatidylinositol transfer protein (PI-TP) required in the visual transduction cascade. One defined role for PI-TPs is regulation of vesicular transport. Immunolocalization of the rdgB protein near the cytosolic face of the rhabdomeres is consistent with the protein functioning in vesicular transport from a trans-Golgi compartment to the rhabdomeres. The rdgB polyclonal antiserum stains vertebrate rod inner segments, suggesting that a vertebrate "photoreceptor" homolog exists. Detailed analysis of the Drosophila molecule and identification of the vertebrate homolog could elucidate aspects of rod outer disc assembly and possibly a novel form of retinal degeneration. The dgq gene encodes two Drosophila photoreceptor-specific G-protein a subunits, DGq1 and DGq2. We demonstrated genetically and biochemically that DGq1 is involved in the light-activated pathway. Further analyses will reveal what is DGq1 's effector molecule and the role of DGq2 in the phototransduction cascade. In addition, an analogous dominant mutation in both DGq1 and transducin leads to an abnormal adaptation response in Drosophila and mouse, respectively. Therefore, further genetic and molecular analyses of DGq1 may elucidate a common adaptation mechanism in both vertebrates and invertebrates.

A large number of retinal degeneration mutants have been generated in Drosophila. Analyzing these mutants have identified several key components of the visual transduction cascade and other critical cell biological components for the photoreceptor cell, which has led to models for the underlying degeneration mechanisms. This project will employ molecular, genetic, and cell biological approaches to examine the degeneration mechanisms associated with two different mutants. Dominant rhodopsin mutations are a major cause of one form of autosomal dominant retinitis pigmentosa in humans. Several mechanisms have been proposed for the retinal degeneration process. One class of dominant rhodopsin mutants was previously characterized in Drosophila and shown to undergo degeneration by blocking the maturation of the wild-type rhodopsin protein. We isolated two additional dominant rhodopsin mutations (ninaEpp36 and ninaEpp100 that exhibit retinal degeneration, but do not block rhodopsin maturation. Additionally, several lines of data strongly suggest that the ninaEpp36 and ninaEpp100 mutations utilize different mechanisms to produce the retinal degeneration phenotype. We will examine these underlying degeneration mechanisms to elucidate additional models for autosomal dominant retinitis pigementosa. The retinal degeneration G (rdgG) mutation exhibits a light-independent, temperature-sensitive retinal degeneration phenotype. The electrophysiological light response (measured by the electroretinogram) of the rdgG mutant is wild-type, except for the inability to dark recover after an extended saturating light stimulus. This suggests that the rdgG mutant may exhibit "run-down" of a critical component in the visual transduction process.

[unreadable] DESCRIPTION (provided by applicant): Dark-adapted albino zebrafish undergo loss of rods and cones when placed in constant intense light. The photoreceptors are regenerated from a population of adult stem cells in the inner nuclear layer. The Muller glia may also proliferate and either dedifferentiate into neuronal precursors or trans-differentiate into photoreceptors during regeneration. To examine the role of the Muller glia in regeneration, it is necessary to develop techniques to regulate the temporal-spatial expression of transgenes, such as the Cre-lox site- specific recombination and the Tet-On systems. While both of these systems are effectively used in mouse genetics, neither has been fully demonstrated to function in transgenic zebrafish. We will develop the Cre-lox system to express a reporter (EGFP) in a cell-specific manner to examine cell lineages. The Cre enzyme, which will be expressed in Muller glia using the GFAP or glutamine synthetase promoter, will catalyze the recombination between two lox sites in a second transgene. This will excise transcriptional and translational termination signals and express EGFP from a ubiquitous promoter. Thus, EGFP will only be expressed in Muller glia and any cells derived from these glia. Using immunohistochemical methods to examine retinas at different time points during regeneration, we will unambiguously identity the EGFP-positive cells and the lineage of the Muller glia during regeneration. We will also determine if the Tet-On system functions in zebrafish. We will express the Tet trans-activator (rtTA) using either an ubiquitous or cell-specific promoter. Addition of doxycycline will permit the rtTA to bind the TRE sequence and activate transcription of the EGFP reporter. Demonstrating that the Tet-On system is a tight inducible system will allow us to express a variety of transgenes to examine the roles of the inner nuclear layer stem cells and Muller glia in the regeneration response. [unreadable] Relevance: Regulating the temporal-spatial expression of transgenes in zebrafish will allow us to examine the function of either normal or mutant forms of a gene throughout development. We will initially use these techniques to examine the role of radial glia in the regeneration of photoreceptors. Elucidating the role of Muller glia in zebrafish photoreceptor regeneration may reveal inductive cues that could be used to induce Muller glia to regenerate photoreceptors in inherited human retinal degenerative diseases. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The retinas of most adult vertebrates cannot regenerate neurons. In contrast, adult zebrafish regenerate all classes of retinal neurons including the photoreceptors. For example, light-induced photoreceptor cell damage of the adult zebrafish retina induces the Muller glia to reenter the cell cycle and generate neuronal progenitor cells. These Muller glial-derived progenitors migrate to the outer nuclear layer and differentiate into new rods and cones. In contrast to zebrafish, human retinal Muller cells cannot regenerate any retinal cell class. By understanding the genetic and biochemical basis of Muller glia-based neuronal regeneration in the light-damaged zebrafish retina, we may identify approaches to stimulate a similar regeneration response in the human retina. This could yield a strategy to restore vision to individuals with genetic and environmental forms of blindness. We determined Pax6 is expressed in the new neuronal progenitor cells shortly after being generated from the Muller glial cells. To determine the role of the Pax6 protein in these progenitors during regeneration, we need to disrupt Pax6 expression. Unfortunately, Pax6 is required for retinal development and disruption of Pax6 expression during early zebrafish development will prevent eye formation. Thus, we developed an electroporation technique to deliver antisense morpholinos into the progenitor cells of the regenerating adult retina and conditionally block Pax6 protein expression. Our preliminary results demonstrate we can block Pax6 expression in the neuronal progenitor cells, which prevents their proliferation. We will demonstrate the utility of this new technique and determine the role of Pax6 in progenitor cell proliferation and photoreceptor regeneration. We will also begin to identify Pax6 transcriptional targets that may play roles during the retinal regeneration response. Relevance: While several candidate genes were identified that may play important roles for retinal regeneration in zebrafish, a method must be developed to test the function of these candidate genes/proteins. Standard forward genetic approaches will not be amenable for this analysis because most of these genes are also required for retinal development. We describe a morpholino electroporation technique to conditionally knockdown the expression of the desired protein in the adult retina. To demonstrate the power of this method, we will examine the speculative function of the Pax6 protein for zebrafish retinal regeneration.Many vision diseases cause loss of human photoreceptors that cannot be regenerated. Robust photoreceptor regeneration in zebrafish involves expressing the Pax6 protein as the Muller glial cells divide and produce neuronal progenitor cells, which then renew the lost photoreceptors. Understanding the role of Pax6 in zebrafish retinal regeneration may provide clues as to why the human Muller glial cells cannot regenerate photoreceptors, and ultimately lead to the ability to induce endogenous Muller glial cells to regenerate photoreceptors in the human retina.

DESCRIPTION (provided by applicant): In most vertebrates, the adult retina is unable to regenerate lost neurons, which results in loss of vision. In contrast, the zebrafish retina possesses the ability to regenerate any class of retinal neurons that are lost due to a variety of insults. For example, constant intense light causes rod and cone photoreceptor death. Photoreceptor regeneration originates from the Muller glia that reenter the cell cycle and divide to produce neuronal progenitors, which continue to proliferate and then differentiate into the regenerated neurons. The human retina also possesses Muller glia, but fails to regenerate any retinal cell class. We are interested in identifying the processes that regulate retinal regeneration of photoreceptors in the light-damaged zebrafish retina. In this proposal, we will investigate the molecular mechanisms that generate and maintain the neuronal progenitor cell population in an undifferentiated and proliferating state during regeneration, which will identify approaches to induce a full regeneration response in the damaged mammalian retina. This could provide a strategy to restore vision to individuals who suffer from genetic forms of blindness, such as retinitis pigmentosa or macular degeneration. We will explore the roles of the Pax6a, Pax6b, and Olig2 proteins during proliferation of neuronal progenitors in the light-damaged zebrafish retina. We will use a technique that we developed to electroporate antisense morpholinos into the regenerating retina. This technique gives us the powerful ability to conditionally block the translation of specific proteins during regeneration of the light-damaged retina. We will test the hypothesis that the Pax6b protein is required for the initiation of neuronal progenitor cell proliferation, but is not required for the subsequent expression of progenitor cell transcription factors, and ultimately, photoreceptor cell opsins (S. A. 1). We will then determine if Pax6a is required for the continued proliferation of the neuronal progenitors and the transcription of the olig2 gene (S. A. 2). Finally, we will determine if Olig2 is also required for the continued proliferation of the neuronal progenitor cells and, if, in the absence of Olig2, the neuronal progenitors prematurely differentiate into photoreceptors at the expense of Muller glial cells (S. A. 3). PUBLIC HEALTH RELEVANCE: This proposal explores key genetic checkpoints that are required to regenerate retinal neurons in the adult eye. Uncovering the molecular processes that generate neuronal progenitor cells from the Muller glia and stimulating these progenitors to proliferate until they must differentiate into the missing neurons may reveal approaches to induce neuronal regeneration from the Muller glia in the damaged human retina. Ultimately, these results may lead to therapies for different forms of human blindness.

An award was made to the University of Notre Dame, Department of Biological Sciences, to provide research training for 10 weeks for 12 students during the summers of 2010-2012. The program, "Integrative Cell and Molecular Biology", will provide students with independent research projects within an interdisciplinary research community. Participants will formulate their research project and proposal with their mentor, speak about their work at the beginning of the summer and present their results at the end of the summer at the Regional Summer Symposium. A weekly seminar program will be included to develop students' intellectual and professional skills, as well as to educate them about various career paths in biological sciences. In addition, students will attend interactive workshops on integrative research, ethics and the responsible conduct of research, the publication process, scientific writing, and graduate school applications. The underlying aim of the program is to give students strong incentives for pursuing careers in research. Students will be recruited through general and targeted approaches with partner institutions. Underrepresented minority students and students who do not have access to research at their home institutions will be encouraged to apply. Participant tracking data will be obtained using a background, pre-program, post-program, and alumni survey each year. In addition, the BIO REU common assessment tool will be used. For more information, students may visit http://nd.edu/~biosreu/ or contact the PI, Dr Michelle A Whaley, at (574) 631-9343 or Michelle.A.Whaley.3@nd.edu.

? DESCRIPTION (provided by applicant): Vision loss is among the top ten disabilities in the United States, which results in a heavy financial burden on society. To remedy this significant problem, the National Eye Institute recently announced the Audacious Goal to regenerate neurons and neural connections in the eye and visual system (://www.nei.nih.gov/audacious/). To accomplish this Audacious Goal, it is necessary to identify the molecular signals needed to activate latent endogenous cells to replace lost host neurons. To identify these potential regulators, we are studying zebrafish, where retinal damage stimulates Müller glia to proliferate and produce neuronal progenitors that regenerate the missing zebrafish neurons. While the human retina also possesses Müller glia, they are unable to regenerate retinal neurons. Identifying the molecular switches that induce the zebrafish Müller glia to initiate the regeneration response may reveal approaches to induce a similar retinal regeneration response in humans. We recently identified tumor necrosis factor alpha (TNFa) and Notch signaling as positive and negative regulators of Müller glia proliferation, respectively. However, the signalin pathways of TNFa and Notch are less clear. Elucidating these pathways could significantly advance the NEI's Audacious Goal by yielding a strategy to regenerate retinal neurons in individuals who suffer from a variety of forms of blindness. Our long-term goal is to identify and characterize the molecular and cellular events required to regenerate the damaged zebrafish retina. We recently found that tumor necrosis factor-alpha (TNFa) is produced in the dying zebrafish photoreceptors and is necessary and sufficient for Müller glia proliferation. We also observed that repressing Notch signaling is sufficient to induce Müller glia to reenter the cell cycle, suggesting that Notch is a negative regulator of initiating the regeneration response. Our central hypothesis is that dying photoreceptors produce TNFa, which binds receptors on the Müller glia and activates Stat3. Stat3 then regulates the expression of Ascl1a to induce Müller glia proliferation. Additionally, retinal damage represses Notch signaling to increase expression of Ascl1a and Müller glia proliferation, likely through the decreased expression of his/her genes. Aim 1 will explore the components of the TNFa signaling pathway that initiates Müller glia proliferation and in what cells they act, including the ability of TNFa to induce expression of Stat3 and Ascl1a, the potential role of Stat3 in inducing Ascl1a expression, if Stat3 must be activated in Müller glia or another retinal cell type for Müller glia proliferation, and if TNFa activates Stat3 directly or through an intermediate such as NF-?B (NF-kappaB), JNK, or p38. Aim 2 will examine the role of the Notch signaling pathway to maintain Müller glia in a quiescent (non-proliferating) state in undamaged retinas. We will determine if Notch activity must be in the Müller glia to maintain quiescence and determine the identity of the Notch receptor and ligand that are required to keep the Müller glia from reentering the cell cycle in undamaged retinas. Thus, the expected outcomes of this project will reveal the relationships of TNFa and Notch as positive and negative regulators of Müller glia proliferation and how Stat3 and Ascl1a are regulated in the damaged zebrafish retina. We anticipate that the impact of this work will lead to a better understanding of what regulates Müller glia reentry into the cell cycle in the damaged retina. This work will also assist in the development of potential therapeutic approaches that use endogenous Müller glia to regenerate lost retinal neurons in individuals suffering from vision loss.

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.

This REU site award to the University of Notre Dame, located in South Bend, IN, will support the training of 12 students for 10 weeks during summers 2017-2019. The REU program will provide students with independent research projects within an interdisciplinary research community. Participants will formulate their research project and proposal with their mentor, speak about their work at the beginning of the summer, and present their results at the end of summer at the Summer REU Symposium. A weekly seminar program will be included to develop students' intellectual and professional skills, as well as to educate them about various career paths in biological sciences. In addition, students will attend interactive workshops on integrative research, ethics and responsible conduct of research, the publication process, scientific writing, and graduate school applications. The underlying aim of the program is to give students strong incentives for pursuing careers in research.

It is anticipated that a total of 36 students, primarily from schools with limited research opportunities, will be trained in the program. They will be recruited through general and targeted approaches with partner institutions. Students will be selected based on academic record, the quality of the personal statement/cover letter, recommendation letters, a sincere desire to pursue a research career, and potential for outstanding research in cell/molecular biology. Students will learn how research is conducted, and many will present the results of their work at scientific conferences.

Participant tracking data will be obtained using a background, pre-program, post-program, and alumni survey. In addition, a common web-based assessment tool used by all REU Site programs funded by the Division of Biological Infrastructure will be used to determine the effectiveness of the training program. Continued contact with each participant will take place through planning for conference presentations, encouraging students to pursue additional research, and preparation for graduate school. More information and application materials are available at http://nd.edu/~biosreu/, or by contacting the PI (Dr. Michelle Whaley, (574) 631-9343, Michelle.A.Whaley.3@nd.edu) or the co-PI (Dr. David Hyde, (574) 631-8054, David.R.Hyde.1@nd.edu).