Towards the vertebrate eye - Exploring photoreceptors of basal deuterostomes

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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.