The long-term objective of this proposal is to understand the molecular events that lead to the determination, differentiation and maintenance of different retinal cell types. During mammalian retinogenesis, seven classes of cells are specified from multipotent progenitors by the action of various intrinsic and extrinsic factors. Recent molecular genetic studies involving loss-of-function and gain-of-function approaches have uncovered a number of transcription factors as pivotal intrinsic regulators of retinogenesis. These factors are found to act at different developmental processes to establish progenitor multipotency, define progenitor competence, determine cell fates, and/or specify cell types and subtypes. Therefore, transcription factors play essential roles in controlling cell specification ad differentiation during retinogenesis. Despite these important advances, however, the molecular machinery and regulatory gene networks of many transcription factors involved in retinal development still remain poorly understood. In this application, experiments are proposed that will focus on the molecular and developmental events regulated by the Ptf1a and Tcfap2 transcription factors that lead to amacrine and horizontal cell differentiation. Our preliminary data and previous work have led us to propose that Ptf1a may form a crucial protein complex with the Rbpj cofactor to activate the expression of Tcfap2a and 2b, which in turn may be redundantly required for the differentiation of amacrine and horizontal cells. This project primarily aims to test this hypothesis by pursuing three specific aims: i) to investigate the requirement for a Ptf1a-Rbpj complex in specifying amacrine and horizontal cell fates. By analyzing retinal phenotypes in several different Ptf1a and Rbpj loss- of-function mouse models, we aim to test the working hypothesis that Ptf1a may form a Notch-independent protein complex with Rbpj to specify amacrine and horizontal cells during retinogenesis;ii) to test Tcfap2a and 2b as Ptf1a targets in amacrine and horizontal cell development. By misimpression, chromatin immunoprecipitation and phenotype rescue experiments, we aim to investigate whether Ptf1a is be able to directly activate Tcfap2a and 2b expression which in turn may mediate the Ptf1a function in amacrine and horizontal cell development;and iii) to analyze the role of Tcfap2a and 2b in differentiation of amacrine and horizontal cells. By misexpressing Tcfap2a and 2b in retinal progenitors and by simultaneous knockdown of both genes in the developing retina, we aim to test the working hypothesis that Tcfap2a and 2b may function as Ptf1a effectors genes that are redundantly required for amacrine and horizontal cell differentiation. The proposed studies are expected to elucidate the Ptf1a-modulated molecular machinery that controls amacrine and horizontal cell specification from retinal progenitors, thereby providing important insights into te regulatory gene network underlying the differentiation of different retinal cell types as well as a framework for future improvement of stem cell-based therapy to treat retinal degeneration.
The proposed studies aim to understand the molecular basis of retinal development, in particular, to identify transcription factors and build regulatory gene networks leading to the specification and differentiation of different retinal cell types from progenitors. Because differentiation of stem cells normally recapitulates the events that occur during embryogenesis, a better understanding of retinal progenitors and their development will enable us to better manipulate, maintain and exploit retinal stem cells for therapeutic purposes. Thus, the proposed research on retinal development may provide a framework for future improvement of stem cell-based therapy to treat human retinal degeneration.
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