Identification and functional analysis of new factors that mediate tramtrack's function during Drosophila tracheal system development / Identificación y análisis funcional de nuevos factores que median la función de tramtrack durante el desarrollo del sistema traqueal de Drosophila

摘要

A stereotyped tubular epithelial network forms many of our bodies’ organs and, defects in the formation of these tubules often leads to organ failure. My Thesis project aims to further understand the mechanisms underling the formation of tubular networks using the tracheal system of Drosophila melanogaster as a model system. Tramtrack (Ttk) is a widely expressed transcription factor (TF) whose function has been analysed in different adult and embryonic tissues in Drosophila. Interestingly, previous work from our lab showed Ttk as a key tracheal regulator. Ttk is involved in a wide range of developmental decisions, ranging from early embryonic patterning to differentiation processes in organogenesis (Araújo et al., 2007). Given the wide spectrum of functions and pleiotropic effects that hinder a comprehensive characterisation, many of the tissue specific functions of this transcription factor are poorly understood. We profiled gene expression experiments after Ttk loss- and gain-of-function in whole embryos and cell populations enriched for tracheal cells in order to identified some of the underlying genetic components that are responsible for the tracheal phenotypes of Ttk mutants. Our transcriptomes analysis revealed widespread changes in gene expression. Interestingly, one of the most prominent gene classes that showed significant opposing responses to loss- and gain-of-function was annotated with functions in chitin metabolism, along with additional genes that are linked to cellular responses, which are impaired in ttk mutants. The expression changes of these genes were validated by quantitative real-time PCR and further functional analysis of these candidate genes and other genes also expected to control tracheal tube size revealed at least a partial explanation of Ttk’s role in tube size regulation (Rotstein et al., 2011). In addition, from all the target genes found in this study, we identified and selected one of them expressed in the tracheal system, CG13188, for further functional analysis. After validating CG13188 differential expression by quantitative PCR, we corroborated and refined CG13188 expression pattern by generating an anti-CG13188 antibody. This protein accumulation analysis recapitulated the predicted CG13188 mRNA expression, but also indicated that CG13188 has a dynamic sub-cellular accumulation during the development of the tracheal system. We have confirmed that CG13188 protein expression levels are, as expected, dependent on the general regulator ttk. Interestingly, we saw that ttk also controls CG13188 protein localization, as in ttk mutant embryos, CG13188 protein did not accumulate apically in the tracheal cells at late embryonic stages, as compared to wild-type. These results suggested on one hand, that CG13188 in order to perform its function might need to be transported from the cytoplasm to the apical membrane of the tracheal cells. On the other hand, the CG13188 protein accumulation differences observed could be just a consequence of CG13188 protein levels between the conditions analysed. We have also observed that CG13188 expression is also controlled by the branch specific regulator spalt. CG13188-RNAi down-regulation in the tracheal system gave tracheal morphogenesis defects during mid and late embryonic stages. In particular, we observed that from embryonic stage 15, CG13188-RNAi embryos can not accumulate chitin into the luminal space of the most dorsal and ventral tracheal branches. These experiments indicated that CG13188 gene is required to form the chitin luminal cable in a branch specific manner. Its down-regulation in the tracheal system produced also physiological maturation phenotypes at late embryonic stages, as CG13188-RNAi embryos did not gas filled completely. Together our results suggested on one hand, the possibility of coexistence of more than one ECM assembly mechanism involved in tracheal tube size control. This novel chitin organisation mechanism might be dependent on branch type. On the other hand, our results might provide a first evidence of a link between chitin cable formation and gas filling tube morphogenesis processes.