Congenital retinal dystrophies are a major cause of unpreventable and incurable blindness worldwide. Mutations in CDHR1, a retina specific cadherin, are associated with cone-rod dystrophy. The ubiquitin proteasome system (UPS) is responsible for mediating orderly and precise targeting of protein degradation to maintain biological homeostasis and coordinate proper development, including retinal development. Recently, our lab uncovered that the seven in absentia (Siah) family of E3 ubiquitin ligases play a role in optic fissure fusion, and identified Cdhr1a as a potential target of Siah. Using two-color whole mount in situ hybridization and immunohistochemistry, we detected siah1 and cdhr1a co-expression as well as protein co-localization in the retinal outer nuclear layer (ONL), and more precisely in the connecting cilium of rods and cones between 3-5 days post fertilization (dpf). We confirmed that Siah1 targets Cdhr1a for proteasomal degradation by co-transfection and co-immunoprecipitation in cell culture. To analyze the functional importance of this interaction, we created two transgenic zebrafish lines that express siah1 or an inactive siah1 (siah1dRING) under the control of the heat shock promoter to modulate Siah activity during photoreceptor development. Overexpression of siah1, but not siah1dRING, resulted in a decrease in the number of rods and cones at 72 hours post fertilization (hpf). The number of retinal ganglion cells, amacrine and bipolar was not affected by Siah1 overexpression, and there was no significant reduction of proliferating cells in the Siah1 overexpressing retina. We did however detect increased cell death, confirmed by an increase in the number of TUNEL+ cells in the ONL, which was proteasome-dependent, as MG132 treatment rescued the cell death phenotype. Lastly, reduction in rods and cones resulting from increased Siah1 expression was rescued by injection of cdhr1 mRNA, and to an even greater extent by injection of a Siah1-insensitive cdhr1a variant mRNA. Taken together, our work provides the first evidence that Cdhr1a plays a role during early photoreceptor development and that Cdhr1a is regulated by Siah1 via the UPS. This work provides new avenues for investigation into the roles of CDHR1, and now also Siah1, in the predisposition and pathogenesis of inherited cone-rod dystrophy.

中文翻译：

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蛋白酶体介导的Siah1对Cdhr1a的调节可调节斑马鱼的感光细胞发育和存活。

先天性视网膜营养不良是世界范围内无法预防和无法治愈的失明的主要原因。CDHR1（一种视网膜特异性钙黏着蛋白）的突变与视杆营养不良有关。泛素蛋白酶体系统（UPS）负责介导蛋白质降解的有序和精确靶向，以维持生物稳态并协调包括视网膜发育在内的适当发育。最近，我们的实验室发现E3泛素连接酶的缺席（Siah）家族中的七个在视裂融合中发挥了作用，并确定Cdhr1a是Siah的潜在靶标。使用两种颜色的整体原位杂交和免疫组织化学，我们检测了siah1和cdhr1a的共表达以及在视网膜外核层（ONL）中的蛋白共定位，更确切地说，是在受精（dpf）后3-5天之间的棒和锥的连接纤毛中。我们证实，Siah1通过细胞培养中的共转染和免疫共沉淀作用将Cdhr1a靶向用于蛋白酶体降解。为了分析这种相互作用的功能重要性，我们创建了两个在热激启动子控制下表达siah1或非活性siah1（siah1dRING）的转基因斑马鱼品系，以调节感光细胞发育期间的Siah活性。siah1而不是siah1dRING的过表达导致受精（hpf）后72小时的杆和视锥细胞数量减少。视网膜神经节细胞，无长突和双极的数目不受Siah1过表达的影响，并且在Siah1过表达的视网膜中增殖细胞没有明显减少。但是，我们确实检测到了细胞死亡的增加，这是通过ONL中TUNEL +细胞数量的增加来证实的，这是蛋白酶体依赖性的，因为MG132治疗可以挽救细胞死亡的表型。最后，通过注射cdhr1 mRNA可以挽救因Siah1表达增加而引起的视杆和视锥细胞的减少，甚至可以通过注射Siah1不敏感的cdhr1a变体mRNA更大程度地挽救杆和视锥细胞。综上所述，我们的工作提供了第一个证据，证明Cdhr1a在早期感光体发育过程中发挥了作用，并且Cahr1a通过UPS通过Siah1对其进行调节。这项工作为调查CDHR1，现在还有Siah1，在遗传性锥状营养不良的易感性和发病机理中的作用提供了新的途径。MG132治疗挽救了细胞死亡表型。最后，通过注射cdhr1 mRNA挽救了因Siah1表达增加而引起的视杆和视锥细胞的减少，并且通过注射Siah1不敏感的cdhr1a变体mRNA更大程度地挽救了视杆和视锥细胞。综上所述，我们的工作提供了第一个证据，证明Cdhr1a在早期感光体发育过程中发挥了作用，并且Cahr1a通过UPS通过Siah1对其进行调节。这项工作为调查CDHR1，现在还有Siah1，在遗传性锥状营养不良的易感性和发病机理中的作用提供了新的途径。MG132治疗挽救了细胞死亡表型。最后，通过注射cdhr1 mRNA可以挽救因Siah1表达增加而引起的视杆和视锥细胞的减少，甚至可以通过注射Siah1不敏感的cdhr1a变体mRNA更大程度地挽救杆和视锥细胞。综上所述，我们的工作提供了第一个证据，证明Cdhr1a在早期感光体发育过程中发挥了作用，并且Cahr1a通过UPS通过Siah1对其进行调节。这项工作为调查CDHR1，现在还有Siah1，在遗传性锥状营养不良的易感性和发病机理中的作用提供了新的途径。我们的工作提供了第一个证据，证明Cdhr1a在早期感光体发育过程中发挥了作用，并且Cadhr1a由Siah1通过UPS调节。这项工作为调查CDHR1，现在还有Siah1，在遗传性锥状营养不良的易感性和发病机理中的作用提供了新的途径。我们的工作提供了第一个证据，证明Cdhr1a在早期感光体发育过程中发挥了作用，并且Cadhr1a由Siah1通过UPS调节。这项工作为调查CDHR1，现在还有Siah1，在遗传性锥状营养不良的易感性和发病机理中的作用提供了新的途径。