1989 — 2005 |
Reh, Thomas A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Neuronal Proliferation and Differentiation @ University of Washington
The central nervous system (CNS) has many neuronal cells types, and within a given brain region, these different types of neurons are present in precise ratios. Such regular ratios of neurons are characteristic of all brain regions and are fundamental to the proper functioning of the nervous system. However, virtually nothing is known of the mechanisms that regulate the production of the various types of neurons during the development of the CNS. Several lines of evidence indicate that this process is in some way controlled by the microenvironment of the developing CNS. The experiments outlined in this proposal will test several specific predictions of a model of central neurogenesis in which age-dependent changes in the retinal microenvironment determine the types of neurons that are produced at any given time. The experiments can be divided into two parts: 1. Is the timing of production of the various retinal cell types important to their appropriate generations? 2. Do retinal cells express age- dependent cues that control the differentiation of germinal cells? An understanding of the factors that regulate CNS neuronal proliferation will undoubtedly prove invaluable in future attempts at reconstruction and regeneration of the damaged CNS as well as providing insight into the mechanisms of teratological agents that disrupt the normal development of the nervous system and neoplastic transformation of germinal neuroepithelial cells.
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1 |
1991 — 1997 |
Reh, Thomas |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Regulation of Retinal Regeneration @ University of Washington
The ability of the retina to regenerate in amphibians, teleosts and embryonic chicks is a particularly striking example of central nervous system repair following traumatic damage. In chick, the source of new retinal neural precursor cells in the regenerative process has been identified as the retinal pigmented epithelium. However, with increasing development the pigmented epithelium of the chick embryo loses its ability to generate neuronal cells. To study molecular mechanisms underlying this transdifferentiation and loss of regenerative capacity, the embryonic chick pigmented epithelium will be studied in a tissue culture system. The role of the extracellular matrix molecules, laminin and basic fibroblast growth factor, on transdifferentiation will be studied. Neuronal cells will be identified with antibodies to neuron specific markers. In addition, the polymerase chain reaction will be used to determine whether the GAP-43 or neurofilament genes are expressed. It also will be determined whether pigmented epithelium loses its regenerative capacity in the in vitro system. In addition, the expression of neuronal phenotype will be studied in relation to the expression of two chick genes that are homologous to the neural determination genes of the achaete scute complexes that play a role in neural determination in the fruit fly. These studies have the potential for identifying genes that play a role in the determination, as well as the regeneration, of retina.
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0.915 |
2000 — 2003 |
Reh, Thomas |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Factors That Control Eye Development @ University of Washington
0080197 REH
The Neurons and Photoreceptors in eyes are organized in regular arrays. The regular arrays of neurons in ten retina of the eye are like the pixels of a computer screen or video camera; they allow information to be arranged spatially in the nervous system, which is critical for the operation of the visual system. It is not known how these arrays arise during development. Imagining how difficult it would be for a video camera to assemble itself gives some idea of the fundamental mystery of this process. The purpose of the proposed experiments is to determine the cellular and molecular mechanisms that pattern the first neurons in vertebrate retina. These first neurons develop as a patterned array that spreads from the central to peripheral retina as a wave front of differentiation. It is proposed to further study the process of pattern formation in the retina by addressing the following questions: 1. Is the recently identified proneural Cath5 gene involved in the control of the spacing of the retinal ganglion cells at the front of differentiation? 2. Does the growth factor FGF regulate expression of the proneural gene Cath5? 3. Does FGF-15 control the onset of ganglion cell differentiation? 4. Does Cath5 co-ordinate ganglion cell differentiation? These studies will provide fundamental about the mechanisms by which patterned arrays of neurons are established in the vertebrate central nervous system.
It has been said that the human nervous system is the most complicated system in the Universe. The current understanding of this complex machine is still fragmentary; however, it has recently been recognized that those who study the nervous system are studying a "moving target"-the nervous system is a machine that has constructed itself, and is constantly changing. It would be as if a computer was constantly rewiring part of its circuitry every day to adapt to the changing world. The proposed research will help to understand how such a complex system arises during the development of every person, and how this complexity is maintained and refined throughout a lifetime.
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0.915 |
2002 — 2010 |
Reh, Thomas A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Stem Cells in the Retina @ University of Washington
DESCRIPTION (provided by applicant): The central nervous system (CNS) of people has only a limited capacity to regenerate after traumatic damage or degenerative diseases. However, in the past few years several lines of evidence have shown that new neurons are generated in the brains of higher vertebrates, and even humans. While the amount of neurogenesis that occurs in adults is limited, the capacity for stimulating this process for repair and regeneration is just beginning to be explored. We have found that the retina of the post hatch chick is capable of a limited amount of regeneration. In the past year we have also discovered a similar process in the rat and mouse retina. In this proposal we outline experiments that will lead to a better understanding of the molecular factors that regulate glial proliferation, and the mechanisms by which they maintain their phenotype. We also will attempt to define the basis for their plasticity and ability to serve as retinal progenitors and as a substrate for retinal regeneration. We use the retina, an easily accessible part of the central nervous system, as a model for the rest of the CNS.
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1 |
2007 — 2011 |
Reh, Thomas A |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Regulation of Human Embryonic Stem Cell Neuro-Retinal Differentation @ University of Washington
Over the past twenty years, there has been steady progress made in the development of methods for retinal repair in animal models. The best results have been obtained when fetal retina has been used as donor tissue, and results to date indicate that immature neurons survive the transplantation protocols much better than mature neurons. Due to ethical and practical concerns with the use of fetal human tissues, the translation of the efforts in animal models to the treatment of human disease will require a new source of developing retinal neurons. To this end, we have developed methods for directing human embryonic stem (hES) cells to the retinal progenitor fate. We have used two different lines of Federally approved human embryonic stem cells, and subjected them to a protocol based on previous molecular studies of eye development. We find that with our protocol up to 80% of the cells in the cultures show characteristic gene profiles of retinal progenitors after three weeks in Retinal Determination (RD) medium. Many of the cells also differentiate into functional retinal neurons in vitro, and preliminary experiments show that the retinal neurons and progenitors derived from hES cells will survive and differentiate following transplantation to degenerating mouse retinas. We now propose to extend these studies in the following three Aims. AIM 1. To determine whether the clock of changing competence is present in hES cell derived retinal progenitors. To determine whether Notch/delta signaling regulates differentiation of hES cell-derived retinal progenitors. AIM 2. To determine whether Notch/delta signaling regulates differentiation of hES cell-derived retinal progenitors. AIM 3. Develop methods to sort retinal neurons derived from human ES cells into types by expression of specific promoter-GFP constructs. AIM 4. To determine whether hES cell-derived retinal progenitors can differentiate into functional photoreceptors following transplantation to mouse models of Leber's congenital amaurosis and Retinitis Pigmentosa, using electrophysiological analyses. If we are successful in rescuing and/or restoring function (as assessed by ERG and behavioral analyses) to animal models of RP and LCA, the results will be a proof of principle that human embryonic stem cells can be used in cell replacement therapies for these retinal diseases.
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1 |
2010 — 2014 |
Ding, Sheng (co-PI) [⬀] Reh, Thomas A Zhang, Kang [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regeneration of Retinal Neurons by Chemically Induced Reprogramming of Muller Gli @ University of California San Diego
DESCRIPTION (provided by applicant): The retinal photoreceptors are the light-sensing cells that convert complex external visual stimuli to electrical and chemical signals. Degeneration of photoreceptors is the end point of the commonest causes of irreversible blindness including age-related macular degeneration and retinitis pigmentosa, affecting over 50 million people world-wide. In non-mammalian vertebrates such as fish, after retinal injury, resident Muller glia cells in the retina can proliferate, and differentiate into all retinal cell types including photoreceptors and restore visual functions. However, this regenerative potential is almost non-existent in mammals, with only very few new neurons generated after damage. We propose to develop and apply a high throughput screening to identify small molecules that will enhance Muller glia cells reprogramming and differentiation into retinal neurons in mammals, in vitro and in vivo. Identification, optimization, and characterizations of chemical tools for Muller cells reprogramming and differentiation will provide new avenues in developing cell-based therapy as well as conventional small molecule therapeutics for regenerative medicine, and facilitate new understanding of the transdifferentiation mechanisms. Our proposed research will facilitate the development of therapies to restore visual functions that have been lost in human patients with severe blindness.
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0.946 |
2011 — 2021 |
Reh, Thomas A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Stimulation of Retinal Regeneration @ University of Washington
Abstract ! Like other areas of the nervous system, the retina is subject to many acquired and inherited neuronal degenerative diseases. Since the retina provides the input for all visual sensory information to the brain, the loss of cells results in visual impairment and potentially complete blindness. Many retinal degenerative diseases affect only a subset of the retinal cells, although, frequently in more advanced disease, loss and reorganization of the entire retina can occur. In mammals, there is very limited regeneration of the degenerated cells; however, in fish, new neurons of all types regenerate from Müller glia (MG) following retinal damage and they are functionally integrated into the existing circuitry. Although mammals, including people, lack this ability, MG, the cellular source for regeneration, are present in all vertebrate retinas. We hypothesize that regeneration from mammalian MG is limited because they fail to express the proneural program of gene expression after injury. We have found that viral over-expression of a proneural transcription factor can partly reprogram mammalian MG to a neurogenic state in vitro. For in vivo confirmation, we generated a transgenic mouse to express Ascl1 in MG. When we induce Ascl1 expression in adult MG, the combination of Ascl1 and histone deacetylase (HDAC) inhibition can stimulate new neuron production from MG in adult mice after NMDA induced damage. The MG-derived neurons primarily resemble bipolar or amacrine cells, and form connections with the existing retinal circuitry. These results show for the first time that functional neurons can be regenerated in an adult mammalian retina and properly integrate within the existing host circuit, but raise a key question: Why do the MG only produce bipolar cells and amacrine cells? In this proposal we outline studies to better understand, and potentially overcome, the barriers to regeneration in mammalian retina. !
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1 |
2013 — 2017 |
Reh, Thomas A |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Regulation of Human Embryonic Stem Cell Neuro-Retinal Differentiation @ University of Washington
One of the most important potential uses of embryonic stem cells and induced pluripotent stem cell research is in the development of disease models. Although many diseases can be modeled in mice, others are difficult or impossible to adequately model in non-human organisms for a variety of reasons. In the retina, for example, there are mouse models for many inherited degenerations, but these have been more difficult to generate for diseases like macular degeneration and many forms of Usher's disease. Therefore, disease models from human patient iPS cells would be of great utility in retinal research. In the past six years, our group, and others, have developed protocols for efficient production of retinal cells from human ESCs and iPSCs. The early stages of retinal development are extremely well recapitulated by our protocol of directed differentiation of hESC cells; however, the cells fail to attain the level of expression of markers that we observe in the late stages of human fetal or postnatal development, even with prolonged culture periods. Therefore, a significant challenge for creation of disease models from iPSCs will be to better understand and promote the progression of the cultured cells to mature stages of retina. The mechanisms that control the developmental timing of retinal progenitor cells are not clear; however, recent evidence from our lab shows a key role for miRNAs. Specifically, we found that the loss of Dicer in retinal progenitor cells leads to their failure to progress from the early state to the late state. We therefore hypothesize (1) that miRNAs regulate developmental timing in retinal progenitor cells and (2) that misregulation of the heterochronic pathway in ESC-derived retinal cells accounts for their failure to progress in vitro at the same rate as in vivo. We propose to test these hypotheses with the following specific aims. Aim 1: Determine whether the expression of specific miRNAs (and the genes they regulate) correlates with the progression of retinal progenitors from the early to late stage. Aim 2.Determine whether the developmental progression of mouse ESC/iPSC-derived retinal progenitors is controlled by stage-specific progenitor miRNAs. Aim 3. Determine whether the developmental progression of human ESC derived retinal progenitors can be accelerated by stage-specific miRNAs. The results of these studies will enable us to better control the development of hESC-derived retinal cells and produce more appropriate models of retinal disease.
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1 |
2016 — 2017 |
Bermingham-Mcdonogh, Olivia Mary [⬀] Reh, Thomas A |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Cis Regulatory Elements in the Inner Ear @ University of Washington
? DESCRIPTION (provided by applicant): Hearing loss is a large health issue in the US with approximately 36 million adults reporting some loss. Much of this deafness is caused by loss of the sensory hair cells in the inner ear either from noise or drug damage or just aging. Much research has aimed at replacing these lost cells. Another approach is to restart the developmental program by which these cells develop to begin with. The inner ear is a highly complex tissue, with many different cell types. Understanding the development of this complex organ will ultimately require characterization of gene expression and its regulation in developing and mature inner ear cell types. DNase I hypersensitivity mapping (DNase I-seq) has recently been developed to map enhancers of genes in particular tissues at particular stages of development. In this proposed research we aim to map the enhancers that are used in the inner ear during development. Knowledge of the way genes are regulated in the inner ear may provide us with additional targets for manipulation in our attempts to restore hearing and balance function. This research proposes to completely characterize the enhancers using DNase I hypersensitivity mapping, RNA-seq and using mutant mice that lack sensory domains to determine which are the critical genes. This will be accomplished with the following three specific aims: Aim 1. Mapping the accessible chromatin in the inner ear at three developmental ages. Aim 2. Mapping DNase I hypersensitive regions in a mutant lacking the sensory domain. Aim 3. Characterization of enhancer activity in explant culture.
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1 |
2017 |
Reh, Thomas A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Supplement to Ey021482 to Carry Out a Screen For Retinal Regeneration Using Crispr-Cas9 Gene Activation. @ University of Washington
DESCRIPTION (provided by applicant): Like other areas of the nervous system, the retina is subject to many acquired and inherited neuronal degenerative diseases. Since the retina provides the input for all visual sensory information to the brain, the loss of cells results in viual impairment and potentially complete blindness. Many retinal degenerative diseases affect only a subset of the retinal cells, although, frequently in more advanced disease, loss and reorganization of the entire retina can occur. In mammals, there is very limited regeneration of the degenerated cells; however, in fish, new neurons of all types regenerate from M¿ller glia following retinal damage and they are functionally integrated into the existing circuitry. Nevertheless, M¿ller glia, the cellular source for regeneration, is present in all vertebrate retins. In the proposal we submitted three years ago, we hypothesized that regeneration from mammalian M¿ller glia was limited because they fail to express a key proneural transcription factor, Ascl1, after injury. We proposed to test this hypothesis by virally-mediated expression of Ascl1 in mouse M¿ller glia. In the two years of funding, we have tested the hypothesis, and found that viral expression of Ascl1 is sufficient to activate a neurogenic program in mouse M¿ller glia, both in dissociated cultures and in the intact retina. The reprogrammed M¿ller glia generates cells that resemble neurons in morphology, gene expression and their responses to neurotransmitters. In the next funding period, we propose to further optimize this reprogramming process, using other transcription factors and epigenetic modifiers, and then to test whether Ascl1-reprogrammed Muller glia can provide a source for regeneration in vivo in a newly developed line of transgenic mice.
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1 |
2019 — 2021 |
Calkins, David J. (co-PI) [⬀] Goldberg, Jeffrey L [⬀] Reh, Thomas A Zack, Donald J. (co-PI) [⬀] |
U24Activity Code Description: To support research projects contributing to improvement of the capability of resources to serve biomedical research. |
Retinal Ganglion Cell Replacement in Optic Neuropathies
PROJECT SUMMARY Glaucoma is a leading causes of blindness and along with other optic neuropathies is characterized by the loss of retinal ganglion cells (RGCs). Increased intraocular pressure (IOP) management is the current standard of care for glaucoma patients, but fails to stop the irreversible loss of RGCs and progressive visual dysfunction. Vision restoration through RGC replacement therapy, one of the NEI?s Audacious Goals program, could be a potential solution, and considerable progress has been made in understanding the molecular signals that regulate RGC specification from human stem cells, as well as in RGC transplant and integration in rodents. However, when considering translation of laboratory advances to human testing, rodent models are limited by critical differences in retinal physiology, and proof-of-concept in non-human primates would greatly increase confidence and aid in therapeutic development before moving to human testing. Thus there is a considerable need for a tractable non-human primate model. Here we will establish a squirrel monkey-induced glaucoma model and the parameters to study human stem cell-derived RGC integration and potential vision restoration in a retina and visual system closer to those of human. Through this 5-year proposal we will achieve critical milestones, including validating the monkey glaucoma model, studying key structural and functional measures using innovative new modalities that should be portable between monkey and humans, and demonstrating the model?s ability to move across institutions. All of this will be accomplished in the setting of studying RGC transplant: differentiation, migration, local integration and synapse formation, growth down the optic nerve, and targeting to distal brain nuclei, with the goal of vision restoration.
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0.954 |