DEVELOPMENT OF THE LARVAL PERIPHERAL NERVOUS SYSTEM IN THE ASCIDIAN CIONA INTESTINALIS: ROLE OF THE RETINOIC ACID AND FGF/WNT SIGNALLING PATHWAYS AND OF THE POU TRANSCRIPTION FACTORS.

The research presented in this document focuses on the development of the larval peripheral nervous system of the Tunicate Ciona intestinalis with two main aims: a) to understand how the interactions between Retinoic Acid (RA) and the FGF/WNT pathways control its development; b) to study the role played by transcription factors of the POU family in its differentiation. Within chordates, tunicates represent the sister group of vertebrates and their larvae have a typical chordate body plan. Notably, larval nervous system is formed by few cells whose organization mirrors that of vertebrates. For these reasons the species C. intestinalis, whose genome is completely sequenced, is a good animal model often used to understand the basic mechanisms of Chordate development. POU genes are an important family of transcription factors with several members that regulate the neural patterning and differentiation in both vertebrate and invertebrate embryos. C. intestinalis has only three genes coding for POU transcription factors: Ci-POU-2 Ci-POU-IV and Ci-POU-like. The gene Ci-POU-IV is specifically expressed in all peripheral nervous system (PNS) territories and in some cells of the central nervous system (CNS) during development. Since the expression of the two other genes was not previously studied in detail, a part of this research consisted in their characterization. Several experiments of in situ hybridization showed that the transcripts of Ci-POU-2 are present early during cleavage stages while Ci-POU-like gene expression is restricted to the lateral mesenchime cells of the larva and to their precursors during embryonic development. Thus the research was focused on the Ci-POU-IV gene. Its expression had been formerly studied by the research team of Prof. De Bernardi that discovered the existence of two alternative transcripts. In order to study the role that they play in neural differentiation Morpholino oligos were designed to perform gene knock-down experiments for the two different isoforms. The results from these experiments revealed that the expression of the serotonin rate-limiting synthesis enzyme, tryptophane hydroxylase (TPH), and glutamate transporter (vGlut) in the PNS neurons could be regulated by the product of the long transcript. To verify if the alternative transcripts were expressed in different PNS neuron populations in situ hybridizations were performed with a probe selective for the short isoform. These in situ hybridizations, compared to the whole expression profile of Ci-POU-IV, showed a lack of expression of the short form in the sensory epidermal neurons of the trunk. Moreover experiments were performed to understand the relationship between Ci-POU-IV and the Delta/Notch pathway. The latter has an important role in determining the cell to cell interactions in a number of taxa and to affect the neural or the epidermal fate of the PNS precursors. The Delta/Notch pathway was inhibited both using DAPT that inhibits the –secretase, responsible of the correct functioning of the pathway and electroporing the pFOG::VeSu(H)DBM construct that blocks the pathway activity. Embryos treated showed an abnormal development of epidermic sensorial neurons and the following in situ hybridizations for Ci-POU-IV pointed out an ectopic expression of the gene. Furthermore the study looked for the Ci-POU-IV targets in order to identify the genes regulated by Ci-POU-IV during the PNS differentiation. A bioinformatic approach was used. The possible consensus sequences were obtained by bibliographic research of those known for the POU IV family in both invertebrates and vertebrates. These sequences have been used to build a matrix that was employed to perform a bioinformatic research in the whole C. intestinalis genome with a software elaborated by the Lemaire team of the IBDML of Marseille. The search identified 19 possible targets of Ci-POU-IV; 8 regions, corresponding to 6 genes including TPH, have been preliminary selected. The activity of the selected regions is being evaluated. The second part of the thesis has been developed during a period of research with Dr. A. Pasini at the Institut de Biologie du Développent of Marseille. The aim was to identify the mechanisms with which RA and the FGF/WNT pathways act on antero-posterior differentiation of PNS during C. intestinalis embryonic development. In vertebrates it was already known that during the antero-posterior extension of the body axis these pathways antagonize each other to coordinate mesoderm and nervous system differentiation. For this reason the hypothesis that an analogous mechanism could occur in other Chordates, including tunicates was tested. Thus in C. intestinalis I performed in situ hybridizations for different genes potentially involved in this mechanism. I employed also the electroporation technique with specific constructs. In particular results showed that the expression of the gene Ci-Raldh that codes for Retinaldehyde dehydrogenase, the enzyme responsible of RA synthesis, is confined to the anterior part of the tail in tailbud stage embryos. To this region is limited also the expression of some gene responsive to RA such as Ci-Hox-1 and Ci-Cyp26. On the contrary, at the posterior extremity of the tail it is predictable the existence of a source of FGF and WNT signals as shown by the expression of Ci-FGF-8 and Ci-WNT-5. Moreover, embryos treated with RA showed Ci-Hox-1 up-regulation at throughout tail epidermis and the inhibition of the posterior Ci-Hox-12 expression. On the contrary, embryos treated with FGF showed an opposite situation with Ci-Hox-12 activation and Ci-Hox-1 inhibition. Moreover, quantifications of differentiated caudal epidermal neurons and meticulous analysis of their position along the tail have been performed in late stage embryos treated with RA, FGF, their respective inhibitors and an inhibitor of the enzyme Ci-Cyp26, responsible for RA catabolism. This showed significant alterations in both the number of neurons and their position. In particular RA treatment increased the oveall number of caudal epidermal neurones but causes the loss of the most posterior ones; on the contrary FGF treatment induced a decrease in the number of neurons but maintained the posterior ones. Treatment with FGF and Ci-Cyp26 inhibitors mimicks the effects of RA while treatment with RA synthesis inhibitor mimicks the effect of FGF. On the whole a complex picture of antagonistic interactions, both direct and indirect has been revealed posing interesting questions from an evolutionary point of view.