Unraveling the gene regulatory network specifying urbilaterian photoreceptors - a comparative study between Branchiostoma (Chordata) and Platynereis (Annelida)

对老鼠和苍蝇眼睛发育的分子分析揭示了惊人的相似性（例如Pax6的保守作用），但也揭示了相关转录因子的重要差异（例如，Rx在脊椎动物中起关键作用，而在昆虫中不起作用）。我们假设这些差异反映了两种类型的光敏（光感受器）细胞，纤毛细胞和横纹肌细胞的不同部署。两者在许多双边动物群体中共存；然而，脊椎动物已经招募睫状体和无脊椎动物的横纹肌类型的视觉。因此，我们建议研究纤毛和/或横纹肌光受体特异性转录因子如Rx， Prox, Otx， Six 12和Lhx2在缓慢进化的海洋环节动物Platynereis dumerilii的眼睛和光受体细胞特异性中的功能。在这个新兴的分子模式物种中，两种类型的光感受器细胞都很早就分化了，眼睛的发育从幼虫早期就开始了。在与捷克共和国布拉格Zbynek Kozmik实验室的合作中，我们将通过染色质免疫沉淀结合第二代测序（ChiP-seq）确定这些转录因子直接调控的基因。对假定靶点的影响将通过最近为Platynereis建立的morpholino敲除技术和/或通过锌指核酸酶介导的基因敲除，然后进行qPCR和原位杂交分析来验证和表征。此外，我们将通过Wholemount In Silico表达谱来确定已验证的候选基因的细胞类型特异性表达，这是阿伦特实验室最近开发的另一种方案，允许在细胞分辨率下进行表达谱分析。随后，我们的数据将与脊索文昌鱼的类似数据进行比较，文昌鱼是另一种缓慢进化的海洋分子模型，具有纤毛和横纹肌感光细胞。通过这种方式，我们的目标是揭开双侧动物中光感受器特异性基因调控网络的保守核心，作为理解脊椎动物与无脊椎动物眼睛发育的异同的起点。

The molecular analysis of eye development in mice and fly has revealed stunning similarities (such as the conserved role of Pax6) but also uncovered important differences in the set of transcription factors involved (for example, Rx playing a crucial role in the vertebrates but not in insects). We hypothesize that these differences reflect the differential deployment of two types of light-sensitive (photoreceptor) cells, ciliary and rhabdomeric . Both co-exist in many bilaterian animal groups; however vertebrates have recruited the ciliary and invertebrates the rhabdomeric type for vision. Hence, we propose to investigate the function of ciliary and/or rhabdomeric photoreceptor-specific transcription factors such as Rx, Prox, Otx, Six 12 and Lhx2 in eye and photoreceptor cell specification in the slow-evolving marine annelid Platynereis dumerilii. In this emerging molecular model species, both types of photoreceptor cells differentiate early and eye development is fully accessible from early larval stages onwards. In a collaborative effort with the laboratory of Zbynek Kozmik in Prague, Czech Republic, we will determine the genes directly regulated by these transcription factors by chromatin immunoprecipitation combined with second generation sequencing (ChiP-seq). The effect on putative targets will be validated and characterized by the morpholino knockdown technique recently established for Platynereis and/or by Zinc finger nuclease-mediated gene knockout followed by qPCR and in situ hybridisation analysis. Also, we will determine the cell type-specific expression of validated candidate genes by Wholemount In Silico Expression Profiling, another protocol recently developed in the Arendt laboratory that allows expression profiling in cellular resolution. Subsequently, our data will be compared with similar data obtained for the chordate amphioxus, another slow evolving marine molecular model with both ciliary and rhabdomeric photoreceptor cells. This way we aim at unravelling the conserved core of the photoreceptor-specific gene regulatory network in Bilateria as a starting point to understand similarities and differences in vertebrate versus invertebrate eye development.