An Essential Role for RAX Homeoprotein and NOTCH–HES Signaling inOtx2Expression in Embryonic Retinal Photoreceptor Cell Fate Determination

Abstract

The molecular mechanisms underlying cell fate determination from common progenitors in the vertebrate CNS 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. Here, we identified an evolutionarily conserved Otx2 enhancer of ϳ500 bp, named embryonic enhancer locus for photoreceptor Otx2 transcription (EELPOT), which can recapitulate initial Otx2 expression in the embryonic mouse retina. 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 downregulation of Otx2 expression in vivo. We also showed that NOTCH-HES signaling negatively regulates EELPOT to suppress Otx2 expression. These 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.

Prdm13Regulates Subtype Specification of Retinal Amacrine Interneurons and Modulates Visual Sensitivity

“…Immunohistochemistry was performed as described previously (Muranishi et al, 2011). Mouse embryos and eye cups were fixed by 4% PFA in PBS for 30 min at room temperature or on ice.…”

“…The S-phase labeling experiment was performed as described previously (Muranishi et al, 2011). Timed pregnant mice (ICR, E15.5) were injected intraperitoneally with BrdU (50 g/g body weight).…”

“…We subcloned an ϳ10 kb Prdm13 genomic fragment, inserted one loxP site into intron 1 and another loxP site into intron 3, cloned it into a modified pPNT vector to make a targeting construct, and transfected the linearized targeting construct into the JM8A3 ES cells (Pettitt et al, 2009). The culture, electroporation, and selection of ES cells were performed as described previously (Muranishi et al, 2011). ES cells that were heterozygous for the targeted gene disruption were microinjected into ICR (Charles River) blastocysts to obtain chimeric mice.…”

“…We hypothesized that transcripts from various genes important for amacrine cell development were relatively upregulated in the Otx2 CKO retina compared with those of the wild-type (WT) retina (Omori et al, 2011). We found that Prdm13 is a highly upregulated gene in Otx2 CKO retinas.…”

“…We previously reported that rod and cone cell fates were converted to those of amacrine-like cells in Otx2 conditional knockout (CKO) mouse retinas (Nishida et al, 2003;Sato et al, 2007). We hypothesized that transcripts from various genes important for amacrine cell development were relatively upregulated in the Otx2 CKO retina compared with those of the wild-type (WT) retina (Omori et al, 2011).…”