Photoreceptor Cell Development Requires CDHR1a Protein Stability Controlled by Siah E3 Ubiquitin Ligase During Zebrafish Eye Development

Abstract

E3 ubiquitin ligases mediate orderly and precise targeting of protein degradation to maintain biological homeostasis and coordinate proper development. Our lab recently discovered that Siah family of E3 ligases plays a role in early ocular morphogenesis, in particular fusion of the optic fissure. Interestingly, Siah ligases are also expressed during photoreceptor development and are predicted to target CDHR1a, a cadherin superfamily of calcium‐dependent cell adhesion molecules and photoreceptor‐specific cadherin. Mutations in this cadherin are associated with inherited retinal dystrophies, such as cone‐rod dystrophy. Using whole mount in situ hybridization and Immuno‐histochemistry we detected Siah gene expression and protein localization in the outer nuclear layer (ONL) and in the retinal ganglion cell layer starting at 3 days‐post fertilization (dpf) until 7dpf. CDHR1a expression and protein localization overlaps with both Siah genes in the outer segment of the retina from 3 until 7dpf. We therefore hypothesized that Siah regulates CDHR1a during photoreceptor development. To test our hypothesis, I first confirmed siah‐mediated targeting of CDHR1a for degradation using a mammalian cell culture, co‐immunoprecipitation followed by western blotting analysis. Second, I created two transgenic zebrafish lines that express Siah1 or a dominant negative (Siah1DRING) under the control of the heat shock promoter. Using the heat shock line to overexpress Siah1 at 2dpf and 2.5dpf, we observed a decrease in the number of developing rods and cones in the retina at 3dpf. The number of retinal ganglion cells, amacrine, bipolar and horizontal cells, however, did not change. In addition, there was a significant reduction of proliferating (EdU+ and PH3+ cells in ONL) cells in the retina. Taken together, our results suggest that Siah ubiquitin ligases may control CDHR1a stability and therefore regulate the photoreceptor cell proliferation and differentiation.Support or Funding InformationThis work was supported by the Brazilian National Council for Scientific and Technological Development (CNPq) under grant number 202970/2014‐0.