RAX Homeoprotein and NOTCH-HES Signaling Regulate Otx2 Expression in Embryonic Retinal Photoreceptor Cell Fate Determination

Abstract

Purpose: : The molecular mechanisms underlying cell fate determination from common progenitors in the vertebrate central nervous system remain elusive. We previously reported that the OTX2 homeoprotein regulates retinal photoreceptor cell fate determination. While Otx2 transactivation is a pivotal process for photoreceptor cell fate determination, its transactivation mechanism in the retina is unknown. We investigated the transactivation mechanism of Otx2 in retinal photoreceptor precursors.

Methods: : To identify the mouse Otx2 regulatory locus directing expression in the embryonic photoreceptor precursors, we first analyzed two transgenic mouse lines harboring bacterial artificial chromosomes (BAC), containing the mouse Otx2 gene inserted with a Beta-galactosidase (LacZ) reporter. We examined LacZ expression which was detected by X-gal staining, in the developing retina of transgenic lines at embryonic day 13.5 (EI3.5), when mouse Otx2 expression becomes obvious in the developing photoreceptors. We then narrowed down the critical region for the Otx2 regulatory activity by dividing the regulatory region identified in BAC transgenic mouse analysis.

Results: : We first found that OTX2 expression begins mainly in the final cell cycle of RPCs. We analyzed the regulatory region of the Otx2 gene during embryonic stages when Otx2 transcripts are distinctly expressed in the presumptive photoreceptor layer in contrast to postnatal stages when Otx2 expression shifts to the bipolar cell layer. We then identified an approximately 500 bp cis-regulatory region we called embryonic enhancer locus for photoreceptor Otx2 transcription (EELPOT) that can recapitulate initial Otx2 transcription in early developing photoreceptors. We found that the RAX homeoprotein interacts with EELPOT to transactivate Otx2, mainly in the final cell cycle of retinal progenitors. Conditional inactivation of Rax results in down-regulation of Otx2 expression in vivo. We also found that EELPOT is negatively regulated by the HES family of molecules, which are bHLH transcription repressors.

Conclusions: : Our results suggest that the integrated activity of cell intrinsic and extrinsic factors on EELPOT underlies the molecular basis of photoreceptor cell fate determination in the embryonic retina.

Keywords: differentiation • photoreceptors • transcription factors