Changes in Shh Signaling in the Absence of Chx10 During Retinal Development

Abstract

Purpose:: Proliferative expansion of retinal progenitor cells (RPCs) is crucial to ensure generation of the appropriate number and types of differentiated cells during retinal histogenesis. The homeobox transcription factor, Chx10, is expressed in RPCs and required for such proliferation to occur. The mitogen Sonic hedgehog (Shh) is also required for sufficient RPC proliferation. Shh is produced by retinal ganglion cells (RGCs) and functions as a signaling molecule, activating the Hedgehog (Hh) signaling pathway. The present study was undertaken to investigate whether coordination of these two genes’ functions occurs in the regulation of proliferative expansion in the vertebrate retina.

Methods:: Shh expression and Hh signaling was compared between wild type and ocular retardation J (Chx10^orj) mice, which carry a null mutation in the Chx10 gene. Specifically, Shh expression was determined by immunohistochemistry, western blots and RT-PCR. Hh signaling was analyzed by determining expression of Hh target genes using in situ hybridization and RT-PCR and assessing the proliferative responsiveness of RPCs to Shh-N. Responses to endogenous Shh were examined using cyclopamine, an inhibitor of Hh signaling.

Results:: Several changes in Shh signaling were identified in the Chx10^orj eye. Delayed onset of Shh and Hh target gene expression in the Chx10^orj retina correlate with delayed RGC differentiation during embryonic retinal development. Although differentiation and gene expression patterns extend appropriately to the peripheral retina by birth, reduced expression of Hh target genes was observed. Furthermore, RPCs at this age failed to exhibit a proliferative response to Shh-N treatment in dissociated cell culture; however, a response was observed in explant culture. The reduced proliferation and Hh target gene expression persisting in the Chx10^orj retina was inhibited by cyclopamine treatment.

Conclusions:: Early in retinal development, Chx10 indirectly controls the onset of Shh signaling by regulating differentiation of RGCs, the retinal source of Shh. Chx10 may further control RPC proliferation by regulating the responsiveness of RPCs to Shh.

Keywords: retinal development • proliferation • growth factors/growth factor receptors