Requirement of Multiple bHLH Genes for Retinal Cell Fate Specification

Abstract

Abstract: : Purpose: The basic helix-loop-helix(bHLH) genes Math3, Mash1, NeuroD and Ngn2 are expressed by developing retinal precursors. Previous studies have demonstrated that Mash1-Math3 double-mutant retina exhibits a fate switch from bipolar cells to Müller glial cells while Math3-NeuroD double-mutant retina shows a fate switch from amacrine cells to both ganglion cells and Müller glial cells. However it is still unknown well which bHLH genes regulate generation of horizontal cells, rods or cones. In this study, to clarify the role of bHLH genes in retinal development, we examined the retina mutant for these bHLH genes. Methods: Retinal explants were prepared from E17.5 mutant embryos. After 2 weeks of culture, we analyzed each retinal cell type by immunohistochemistry. Results: Development of horizontal cells and rods was not detected in Math3-Ngn2, Mash1-NeuroD, or NeuroD-Ngn2 double-mutant retina. In contrast, in Math3-NeuroD-Ngn2 triple-mutant retina, the inner nuclear layer became thinner than the wild type. In the mutant retina, bipolar cells were completely missing and the number of horizontal cells was significantly reduced while that of Müller glial cells was increased. However, photoreceptor development was not significantly affected in the triple-mutant retina. Conclusions: Because the horizontal cell number is severely reduced in Math3-NeuroD-Ngn2 triple-mutant retina but not in Math3-NeuroD, NeuroD-Ngn2, or Math3-Ngn2 double-mutant retina, the three bHLH genes are likely to compensate each other for horizontal cell development. Since photoreceptor development is not severely affected in this triple-mutant retina, it remains to be determined which bHLH genes regulated rod and cone genesis.

Keywords: retinal development • transcription factors • transgenics/knock-outs