Racioppi, Claudia (2013) Gene regulatory Network controlling anterior-posterior patterning of Central Nervous System and melanosomal logistics in Ciona intestinalis: a transcriptomic approach. [Tesi di dottorato]

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Abstract

Fibroblast growth factor (FGF) signaling plays an important role in neural induction and subsequent development throughout the vertebrates. Likewise, the FGF signaling pathway also functions in neural development in ascidians, that are considered as basal chordate sharing conserved molecular developmental mechanisms with vertebrates. FGF and Wnt signaling are involved in development of the pigment cells in the sensory organs of C. intestinalis. Nevertheless the FGF downstream targets, involved in pigment cell precursors formation, are not yet characterized and less is known about the patterning mechanisms in specification of the anteriormost nervous system in Ciona. Here, I employed a cell sorting, microarrays, and targeted manipulations to analyse the FGF signaling gene regulatory network controlling Central Nervous System (CNS) differentiation and pigmented cell formation in C. intestinalis. Microarray data analysis revealed that FGF signaling is required for setting the anterior-posterior CNS patterning in Ciona. My results indicate that when FGF signaling is blocked in Pigment Cell Precursors (PCPs), the most anterior cells change their fate, behaving like cells of the anterior CNS. Microarray data analysis produced hundreds genes differentially expressed when FGF signaling is pertubated in pigment cell lineage. A 'reverse engineered' approach has been employed to create a pigment cell lineage-specific transcriptional network from my microarray data set and neighbors analysis permitted to identify new potential candidates involved in FGF signaling regulatory architecture. Clustering algorithms permitted to group genes according to similar expression pattern; a bioinformatic tool was used to identify statistically the over-representation of conserved transcription factor binding sites on co-expressed gene promoter regions. This analysis allowed to computationally recover putative transcription factors involved in the FGF signaling regulatory network. Furthermore from microarray data analysis, I identified new specific markers of Ciona pigmented cell lineage and several genes that may function as key factors to prime the melanosomal logistic, including biogenesis and transport. Among these, I focused on Ci-Rab38/32/rab-rp1/ltd, the only orthologue of two highly conserved small Rab GTPase, RAB38 nad RAB32. These proteins play a crucial role in the transport of melanogenic enzymes during melanosome maturation in vertebrate melanocytes. Targeted expression of the double mutant in the GTP binding pocket of Ci-Rab38/32/rab-rp1/ltd protein in Ciona pigment cell lineage, leads to the formation of larvae with defects or completely lacking of pigment cells in the sensory vesicle.

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