Comparative analysis of neurogenesis in the branchiopod Daphnia magna and the malacostracan Orchestia cavimana

More than 80 % of the living animal species are arthropods, with over a million modern species described and a fossil record reaching back to the late proterozoic area. Arthropods are common throughout marine, freshwater, terrestrial, and even aerial environments. They include four groups: insects (e.g. flies and beetles), crustaceans (e.g. shrimps and lobsters), chelicerates (e.g. spiders and scorpions) and myriapods (e.g. millipedes and centipedes). There is a long-standing debate on the internal relationships of arthropods. Traditionally, myriapods were thought to be the closest relatives of insects. However, recent comparison of the sequences of similar genes in all arthropod groups resulted in a different hypothesis: insects are more closely related to crustaceans than to myriapods. Interestingly, data on the development of the nervous system in all four groups support this hypothesis and in addition even support a close relationship of chelicerates and myriapods. In contrast to insects and crustaceans, neural stem cells that divide and generate all the cells of the nervous system are absent in chelicerates and myriapods, rather, the area from which the nervous system develops consists of many more cells that directly develop into nerve cells. Despite these differences similar genes control the formation of the nervous system in insects, chelicerates and myriapods. However, their activity and function is adapted to the specific mode of nervous system formation. Crustaceans show the greatest diversity in shape and development among arthropods and therefore it is not clear if all crustacean groups share a common ancestor or if some groups, like the higher crustaceans (e.g. crabs, lobsters), are more closely related to insects while others are not. In addition, the similarities to insects have only been demonstrated in higher crustaceans. Except for two genes, neural development genes have not been identified in crustaceans yet. In the proposed project we will therefore analyse two representatives of crustaceans, the higher crustacean or malacostracan, Orchestia cavimana (a shrimp-like crustacean) and the basal branchiopod, Daphnia magna (a water flea). We will analyse the whole process of nervous system development from the generation of neural stem cells up to the formation of neural networks. On the one hand we will identify genes that are involved in the generation of neural stem cells and study their activity and function. On the other hand, we will verify if neural stem cells are present in the water flea and compare the number and positions of individual stem cells to Orchestia and to insects. Furthermore, we will analyse which nerve cells and support cells are generated by individual stem cells in both crustaceans by single cell labelling of neuroblasts that are adjacent to the ventral midline. In addition, this method will enable us to study the formation of neuronal networks, since the whole cell bodies including the long thin processes that connect individual nerve cells are labelled. The proposed project will (1) contribute substantially to our knowledge on crustacean neurogenesis, (2) contribute to the resolution of arthropod relationships and (3) contribute to the understanding in what way the developmental mechanisms leading to the formation of the nervous system have been modified during evolution in the individual arthropod groups.

Comparative analysis of neurogenesis in chelicerates, myriapods and insects has revealed that the genetic network controlling the recruitment of neural precursors is conserved in these three arthropod groups. However, the expression pattern and function of these genes is adapted to the distinct morphology of neural precursor formation in each group. Despite of having a similar function in the production of neural cells, insect and crustacean neuroblasts differ among other things in the way they are generated and in their position within the developing neuromeres. Until now, moleculargenetic analyses of neurogenesis in crustaceans are largely missing. In addition, morphological data on neuroblast lineages are only available for higher Crustacea and it is not clear if neuroblasts are present in other major crustacean groups, such as branchiopods. In the course of the proposed project we will analyse the generation of neuroblasts and their identity as well as the establishment of the axonal scaffold in two crustaceans, the branchiopod Daphnia magna and the malacostracan Orchestia cavimana, using both moleculargenetic and morphological approaches. The following techniques will be applied: PCR cloning, in situ hybridisation, cell labelling and functional analysis with RNA interference. We address two main questions: do crustaceans exhibit similar patterns of gene activity during differentiation of neuronal precursors despite apparent differences to insects, and is the situation within crustaceans the same despite differences in the generation of neuronal precursors in the various crustacean groups. We will compare our results to the data on neurogenesis in the remaining arthropod groups to reveal (1) in what way the developmental mechanisms have been modified in the individual arthropod groups and (2) which characters of neurogenesis can be considered as ancestral due to conservation in all groups and thus cannot be used for resolving euarthropod relationships.