Towards the vertebrate eye - Exploring photoreceptors of basal deuterostomes

150多年来，眼睛器官的进化一直是科学争论的问题。研究棘皮动物和肠口动物的感光器官和感光细胞类型将阐明特定的感光细胞类型和系统的进化，最终导致脊椎动物眼睛的发育。这将通过部署一套广泛的分子、结构、行为和功能分子方法的综合方法来实现。针对各种视蛋白感光色素的抗体染色可以在棘皮动物和肠囊虫物种的广泛分类群样本中识别迄今未知的原癌细胞。随后通过原位杂交对已鉴定的PRC类型进行的基因表达谱(分子指纹)将有助于阐明所提出的推动(光感受器)细胞类型进化的机制(例如，多功能细胞类型分离为功能降低的姊妹细胞类型，独立的基因共选)。原癌基因与神经系统的联系(以及刺激整合的方式)对于我们理解PRC的功能是必不可少的，我们将用免疫组织化学方法和透射电子显微镜进行研究。由于棘皮动物不同的亚类已被证明将骨骼元素作为一种屏蔽或聚焦装置，棘皮动物骨骼成分的功能作用将在原始组织环境下通过非侵入性X射线显微断层扫描进行研究。所获得的综合数据集将允许在非脊椎动物和其他(后口)门之间进行系统发育比较。此外，这些数据还构成棘皮动物和肠结节PRC系统光学性质的生物物理模型的基础，该系统正在与DAN-E合作建造。尼尔森来自瑞典隆德大学愿景小组。功能分子分析，集中在紫海胆的两种不同的视蛋白上，最终将使我们能够收集到棘皮动物PRC系统功能的第一个直接证据。这些实验将与那不勒斯Stazione Zoologica的Maria I.Arone密切合作进行，并将通过注射吗啡实现海胆基因敲除，以及通过Sigma定制锌指技术实现基因敲除。对所获得数据的总结将使我们能够得出关于后口生物基础上的原生生物进化的重要结论，以及关于中华人民共和国在进化的“古代”海洋生态背景下发挥作用的特性的重要结论。

The evolution of eye organs is a matter of scientific debate since more than 150 years. Investigating light sensitive organs and photoreceptor cell (PRC) types in Ambulacraria (echinoderms + enteropneusts) - the most basal recent deuterostome phyla - will elucidate the evolution of specific PRC types and systems eventually leading to the development of the vertebrate eye. This will be achieved by deployment of an integrative approach with a broad set of molecular, structural, behavioural and functional molecular methods. Antibody stainings against various opsin photopigments will allow identification of hitherto unknown PRCs in a broad taxon sampling of echinoderm and enteropneust species. Subsequent gene expression profiling (´molecular fingerprinting´) of the identified PRC types via in situ-hybridization will assist in elucidating proposed mechanisms driving (photoreceptor) cell type evolution (e.g. segregation of multifunctional cell types into sister cell types with reduced functions, independend gene co-option). The connection of the PRCs to the nervous system (and thus the mode of stimulus integration) is essential for our understanding of PRC function and will be investigated with immunohistochemical methods as well as with transmission electron microscopy. As different echinoderm subgroups have been demonstrated to deploy skeletal elements either as a shielding- or a focussing device, the functional role of echinoderm skeletal components will be studied by non-invasive X-ray microtomography in the original tissue context. The integrative dataset obtained will allow for phylogenetic comparison between ambulacrarian PRCs and those of other (deuterostome) phyla. Furthermore these data comprise the basis for the biophysical modelling of the optical properties of echinoderm and enteropneust PRC systems which is under construction in collaboration with Dan-E. Nilsson from The Vision Group of Lund University, Sweden. Functional molecular analysis, focussed on two different opsin proteins of the purple sea urchin Strongylocentrotus purpuratus will finally allow us to gather first direct evidence for the function of an echinoderm PRC system. Those experiments will be performed in close collaboration with Maria I. Arnone from Stazione Zoologica in Naples, and will deploy sea urchin 'conventional' gene knock down via morpholino injection as well gene knock out via Sigma customized Zinc Finger Technology. The summary of obtained data will allow us to draw important conclusions about the evolution of PRCs at the base of deuterostomes as well as about PRC properties functioning in an evolutionary 'ancient' marine ecological context.