Characterization of Genes Expressed During Early Retinal Development: Role of Foxn4 and Its Functional Domains in Retinal Differentiation

Abstract

Abstract: : Purpose: During development, retinal progenitor cells (RPC) give birth to the different subtypes of neurons and glial cells of the neural retina. Cell division and differentiation of these progenitors are under the control of endogenous factors produced within the retina. Suppressive Subtractive Hybridization (SSH) and DNA chips have been used to isolate genes involved in retinal differentiation. Methods: We have used the SSH technique and microarray hybridization to isolate genes which expression is higher at embryonic day 16 (E16) when neural retina is mostly composed of RPC, than in Post Natal day 0 (P0). The functions of these genes were investigated by electroporation of naked DNA plasmid in cultured explants at E16 or P0 and subsequent analysis of the fate of transfected cells and total cell population by immunohistochemistry Results: Microarray hybridization demonstrated that 14 genes of the forkhead/winged helix transcription factor (Fox) familly were expressed in retina and 4 of them were downregulated during retinal differentiation. Among them, Foxn4 a recently characterized mouse transcription factor involved in amacrine and horizontal cell differentiation was the most downregulated gene during development and was also present in our substractive library. Overexpression of Foxn4 cDNA in combination with eGFP in rat retinal explants at E16 and P0 dramatically reduced the number of photoreceptor cells and increased the number of amacrine cells. Conversely, no modifications were observed in the fate of the RPC when Foxn4 lacking the putative activation domain was overexpressed. The deletion of three putative phosphorylation sites didn’t affect the functions of the protein. Conclusions: The role of Foxn4 in amacrine differentiation and its inhibitory effect on photoreceptor differentiation was further confirmed in rats. Our experiments also demonstrate that the presence of a putative activation domain (aa407 to 455) is required to achieve the functions of Foxn4.

Keywords: photoreceptors • retinal development • transcription factors