Conditional Deletion of the Rx Gene in the Developing Neural Retina Leads to Cell Cycle Exit and Biased Retinal Cell Fate

Abstract

Purpose:: The purpose of these studies is to understand the mechanisms controlling neural retinal cell fate determination and to analyze the role of the Rx homeobox gene is regulating cell fate decisions.

Methods:: We have used a conditional deletion allele of the Rx gene along with the Pax6 alpha-Cre transgenic line to delete Rx activity during neural retinal development. We have also used Cre-activated lineage tracing alleles to determine which cells have undergone Rx deletion within the proximal retina. Mutant and control retinas have been analyzed by immunohistochemistry and in situ hybridization using cell-specific markers for each mature retinal cell type. Proliferation assays involved BrdU-incorporation followed by immunoreactivity to BrdU.

Results:: Alpha Cre-mediated deletion of Rx activity within the distal optic cup leads to reduced clones of lineage-marked cells in this region. BrdU incorporation is severely reduced in the neonatal retina and modestly reduced at E14.5, suggesting a depletion of retinal progenitor cells upon Rx deletion. The fate of these Rx-deleted cells is altered from that of control retinas, with a bias toward early born cell fates, including retinal ganglion cells, amacrine cells, and horizontal cells. Surprisingly, cones are not present in the mutant retinas.

Conclusions:: Rx is known to play a critical role in specifying the early retina and promoting proliferation of retinal stem cells. These current studies demonstrate that the Rx gene continues to function during retinogenesis. Similar to the roles of Pax6 and Notch, Rx appears to promote retinal progenitor proliferation and helps to maintain these progenitors in a multipotent state. In addition, these studies suggest that Rx may be crucial for cone cell specification.

Keywords: retinal development • gene/expression • transgenics/knock-outs