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.
Tesi di Dottorato
Data di Pubblicazione:
2010
Citazione:
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 / R. Manenti ; Tutors: Roberta Pennati, Andrea Pasini. Università degli Studi di Milano, 2010 Dec 02. 23. ciclo, Anno Accademico 2010. [10.13130/manenti-raoul_phd2010-12-02].
Abstract:
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 e
Tipologia IRIS:
Tesi di dottorato
Keywords:
development ; ascidian ; tunicates ; evolution ; chordates ; biology ; EST
Elenco autori:
R. Manenti
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