Expression and Function of the Basic Helix-Loop-Helix Transcription Factor NeuroD in the Teleost Retina.

Abstract

NeuroD is a basic helix-loop-helix (bHLH) transcription factor critical for regulating withdrawal from the cell cycle and determining neuronal cell fate. In adult teleosts, neuroD is expressed in proliferating cells of rod and cone photoreceptor lineages and in differentiating cone photoreceptors, suggesting its potential role in photoreceptor genesis. The zebrafish has become a powerful tool for reverse genetic approaches to determine gene function. In the first part of my dissertation, I showed that, in the developing zebrafish retina, neuroD is expressed in proliferating cells of rod and cone photoreceptor lineages and transiently in nascent cone photoreceptors. In the second part of my dissertation, I used complementary gain and loss-of-function approaches to test the hypothesis that NeuroD promotes cell cycle withdrawal among rod and cone progenitor cells and promotes photoreceptor genesis. I created a line of zebrafish transgenic for Hsp70/4:nrd-EGFP, which allowed for temporal control of NeuroD induction following heat shock. I examined proliferation using a panel of cell cycle markers and investigated potential mechanisms utilizing probes for cell cycle regulatory proteins. Compared to controls, transgenic retinas show a significant decrease in the number of mitotically-active cells, indicating that NeuroD promotes cell cycle withdrawal. Induction of NeuroD results in the upregulation of p27 and p57, and downregulation of Cyclin B1, Cyclin D1, and Cyclin E2. Using a panel of cell type-specific antibodies, I showed that NeuroD promotes photoreceptor genesis and inhibits gliogenesis. I used morpholino oligonucleotides to block the translation of NeuroD in cells of the rod and cone photoreceptor lineages. In the absence of NeuroD, although cell type-specific markers show that retinal development is otherwise normal, cells of the rod and cone photoreceptor lineages continue to proliferate and fail to exit the cell cycle. In addition, the absence of NeuroD results in the upregulation of Cyclin D1. Taken together, the cellular pattern of neuroD expression and results from gain and loss-of-function experiments reveal that NeuroD promotes cell cycle withdrawal among photoreceptor progenitors. NeuroD may also function in early aspects of cone photoreceptor maturation. These experiments are the first in vivo demonstration of cell cycle regulation by NeuroD in the retina.