Ann C. Morris - US grants

DESCRIPTION (provided by applicant): Retinitis pigmentosa (RP) and allied inherited retinal dystrophies are a leading cause of blindness for which there is currently no cure. One potential treatment for retinal degenerative diseases is cell-based transplantation therapy, whereby progenitor cells are transplanted into the diseased eye to replace the lost photoreceptors. While this is an exciting possibility, several challenges to the implementation of transplantation therapies must be overcome, including the inefficient integration of retinal progenitor cells (RPCs) into the recipient retina, and the difficulty of obtaining sufficient numbers of photoreceptor precursors for clinical application. Protocols for the in vitro culture of RPCs must be developed that will produce large numbers of photoreceptor precursors and will promote their survival and differentiation once transplanted. Defining the transcriptional networks that promote specification of photoreceptor precursors is a necessary next step for the evolution of therapeutic strategies. One of the long-term goals of our laboratory is to contribute to these efforts by studying photoreceptor development and regeneration in the zebrafish. The zebrafish is especially useful for studying photoreceptor biology, because its retina contains numerous cone subtypes in addition to rods. Furthermore, unlike mammals, the zebrafish retina is able to regenerate neurons in response to experimental damage. We have recently identified the transcriptional repressor Insm1a as a candidate regulator of photoreceptor differentiation and regeneration. The experiments described in this proposal will define the role of Insm1a during retinal neurogenesis through the application of genetic and molecular genetics approaches. Our specific aims are as follows: Specific Aim I: Determine the role of Insm1a in regulating photoreceptor differentiation during retinal development at the cellular level, using both loss- and gain-of-function experiments to place Insm1 within the genetic hierarchy of known photoreceptor development genes and to determine whether it is required for retinal progenitor cell cycle exit; Specific Aim II: Identify the molecular targets of Insm1 regulation in the retina, using a combination of gene expression profiling, digital gene expression analysis, in vitro reporter assays and in vivo chromatin immunoprecipitation. Completion of our proposal will bridge important gaps in our understanding of the molecular mechanisms of vertebrate photoreceptor differentiation, and reveal underlying principles relevant to the development of approaches for the effective treatment of human retinal disease. PUBLIC HEALTH RELEVANCE: The development of therapies to treat visual impairment resulting from photoreceptor degeneration will directly benefit from studies of animal models such as the zebrafish, which develops ex utero, remains optically transparent throughout development, and which displays the capacity for injury-induced neural regeneration. The goal of our proposal is to define the role of a transcriptional regulator, Insm1, in the development of photoreceptor cells, and to identify the genes that are controlled by Insm1 in the developing retina. This information will facilitate efforts to guide photoreceptor differentiation from stem clls in vitro and in vivo for therapeutic purposes.

? DESCRIPTION (provided by applicant): This proposal requests funding for the purchase of a Zeiss Z.1 Lightsheet Microscope at the University of Kentucky, which will be housed in a new Imaging Center operated by the College of Arts and Sciences Biology Department. This instrument would be the first of its kind in the state of Kentucky. The lightsheet microscope incorporates state-of-the-art technology that will allow users to conduct long-term time-lapse imaging of live specimens, as well as to image whole fixed, cleared tissues at subcellular resolution. Lightsheet microscopy has several advantages over conventional fluorescence microscopy techniques, including greater sample penetration depth, faster image acquisition times, superior signal-to-noise ratio, and significantly reduced exposure, which minimizes photobleaching and toxicity. This equipment will serve a growing group of NIH-funded investigators in the Biology department studying developmental and regenerative processes in a variety of model organisms (mouse, zebrafish, salamander, lamprey, and fly) whose research will be greatly enhanced by the ability to conduct live cell and tissue imaging. It will also serve scientists located in the neighboring Colleges of Medicine, Agriculture, and Pharmacy. Research areas that will be enhanced through the acquisition of this lightsheet microscope include studies of germ cell and organ system development, tissue regeneration, circadian rhythms, the role of scaffolding proteins in hematopoiesis, and the regulation of signaling pathways during development.

PROJECT SUMMARY Inherited retinal dystrophies such as retinitis pigmentosa (RP) are a leading cause of blindness for which there is currently no cure. One potential treatment for retinal degenerative diseases is cell- based transplantation therapy, whereby precursor cells are transplanted into the diseased eye to replace the lost photoreceptors. While this is an exciting possibility, several challenges to the implementation of transplantation therapies must be overcome, including the inefficient integration of photoreceptor precursors into the recipient retina, and the difficulty of obtaining sufficient numbers of photoreceptor precursors for clinical application. To improve protocols for the in vitro culture of such cells, we must have a better understanding of the transcriptional networks that promote specification of photoreceptor precursors. One of the long-term goals of our laboratory is to contribute to these efforts by studying photoreceptor development and regeneration in the zebrafish. The zebrafish is especially useful for studying photoreceptor biology, because its retina contains numerous cone subtypes in addition to rods. Furthermore, unlike mammals, the zebrafish retina is able to regenerate neurons in response to experimental damage. The experiments described in this proposal will define the role of three transcription factors -- Sox, Sox11, and Her9 -- during retinal neurogenesis through the application of gene targeting and molecular genetic approaches. Our specific aims are as follows: Specific Aim I: Determine the role of Sox4 and Sox11 in photoreceptor differentiation. Using newly generated loss-of-function mutants, in this aim, we will 1) determine whether there is a dosage effect of Sox4/11 activity on rod photoreceptor number; 2) use state-of-the-art time-lapse imaging to determine when and where Sox4 expression is required to achieve proper rod photoreceptor development; 3) determine precisely how Sox4/11 regulate Hh and Bmp signaling; and 4) identify the molecular targets of Sox4/11. Specific Aim II: Determine the cause and extent of photoreceptor defects in the zebrafish her9 mutant. In this aim, we will 1) determine the mechanism for photoreceptor defects in her9 mutants; 2) determine whether loss of Her9 impairs vision; 3) identify the upstream signaling pathway(s) that regulate her9 expression in the retina; and 4) determine whether Her9-mediated VEGF expression in the avascular zebrafish retina regulates retinal progenitor cell proliferation and differentiation. Completion of our proposal will bridge important gaps in our understanding of the molecular mechanisms of vertebrate photoreceptor differentiation, and reveal underlying principles relevant to the development of approaches for the treatment of human retinal degenerative disease.