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 key roles in controlling cell determination and differentiation during retinogenesis. Despite these important advances, however, the molecular targets and signaling events downstream from 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 controlled by the Brn3b POU domain transcription factor, a crucial regulator of retinal ganglion cell (RGC) differentiation and survival, and its downstream molecules.
Four specific aims will be pursued: i) to understand Brn3b as a negative regulator of retinal cell differentiation. Utilizing molecular, cellular and overexpression approaches, the proposed studies aim to verify in Brn3b-/- retinas the upregulation of multiple genes involved in non-RGC cell development, confirm the inhibitory effect of Brn3b on non-RGC cell differentiation, and identify downstream genes that mediate its repressive effects;ii) to investigate the role of Ebf factors during retinal development by overexpression analysis.
We aim to employ an overexpression approach to investigate the effect of Ebf factors and their dominant-negative forms or their knockdown on retinal cell differentiation and RGC axon pathfinding;iii) to study the biological function of Ebf factors during retinal development using targeted mutants. The goal is to use targeted Ebf mouse mutants to complement the overexpression studies to uncover the role of Ebf factors and establish a functional relationship between them and Brn3b during mammalian retinogenesis;and iv) to analyze the role of Cxcr4 during retinal development. Both loss-of-function and gain-of-function approaches will be used in this aim to define the role of the Cxcl12-Cxcr4 chemokine signaling pathway during mouse retinal cell specification and RGC axon projection and to explore Brn3b-mediated downstream signaling events. The proposed studies together are expected to provide important insights into the genetic regulatory networks involved in retinal cell diversification and differentiation.
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