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Identifier 000390584
Title Study of the development of the trachea system of Drosophila melanogaster.
Alternative Title Μελέτη της ανάπτυξης του τραχειακού συστήματος της Drosophila melanogaster
Author Τσικαλά, Γεωργία
Thesis advisor Καραγωγέως, Δόμνα
Reviewer Δελιδάκης, Χρήστος
Ζέρβας, Χρήστος
Μαυροθαλασσίτης, Γιώργος
Ταβερναράκης, Νεκτάριος
Αλεξανδράκη, Δέσποινα
Βόντας, Ιωάννης
Abstract Epithelia are key building blocks of many organs. They play important roles from both a structural and functional point of view. Epithelia line most if not all organs and they can play important roles in the interactions with the surrounding environment and nearby tissues. Transport properties of epithelia are key to many physiological functions in a multicellular organism, like nutrient uptake and blood filtering. Epithelia also protect the body from the invasion of external pathogens. Finally, they work as permeability barriers and limit the free diffusion of water and solutes, allowing the establishment and maintenance of specific intra-organ and luminal milieu across the two sides of the epithelium. The respiratory system of insects is a vital organ primarily consisting of a complex network of epithelial tubes (the trachea and spiracles) that deliver air-borne oxygen to all organs. It develops in the embryo and becomes functional as the embryo hatches to become a crawling larva. In particular the airways and spiracles of Drosophila melanogaster have long been studied as a tractable model to understand the development, maturation and function of epithelial tubular organs. A genetic screen had been conducted in our laboratory to identify new candidate genes involved in the development and maturation of the fly respiratory system, and in particular the establishment of the permeability barrier of the tracheal epithelium. This screen led to the identification of Btk29A as a gene essential for spiracle morphogenesis, tracheal barrier function and airways maturation. Btk29A encodes for a non-receptor tyrosine kinase, a member of the Tec family of tyrosine kinases, conserved across vertebrates and invertebrates. The aim of this study was to unravel the role of Btk29A in the formation of the respiratory system. Analysis of Btk29A expression pattern with in situ hybridization and antibody staining (using a newly developed polyclonal Btk29A antibody) enabled the documentation of the expression in the trachea and posterior spiracles. Btk29A expression is detected in both tissues from embryonic stage 11 till late embryogenesis. Interestingly, in the embryo only one of the two Btk29A isoforms, the Short (or Type 1) isoform, is expressed by the cells of the respiratory system, while the Long (or Type 2) isoform appears solely in the cells of the central nervous system. The posterior spiracles of Drosophila connect the tracheal tubes to the environment. They consist internally of the spiracular chamber, an invaginated tube with filtering properties that is continuous to the trachea and externally of the stigmatophore, an extensible epidermal structure that covers the spiracular chamber. The tissue develops from a two-dimensional group of ectoderm- 4 derived epithelial cells and involves multiple morphogenetic steps. In the end of embryogenesis the cells have rearranged their positions to form three-dimensional tubular structures. Both of the posterior spiracle compartments express Btk29A. The spiracles of Btk29A mutant embryos are shorter and display abnormal shape. In vivo imaging of the posterior spiracle morphogenesis in Btk29A mutants revealed that although the invagination process is not completely abolished, the most distal of the spiracular chamber cells fail to invaginate. In order to explain the phenotype, several parameters known to affect the fine-tuning of spiracular chamber invagination were examined: apical constriction, cell shape changes, organization of actin-myosin network and activation of the Rho1 GTPase. None of these parameters seems impaired in Btk29A mutants. In parallel, phenotypic rescue experiments, using different tissue specific GAL4 drivers, showed that recovery of Btk29A expression by the spiracular chamber cells is not sufficient to rescue the invagination phenotype. Interestingly the recovery of the kinase in the stigmatophore cells led to normally shaped posterior spiracles. The above suggests that Btk29A might regulate spiracular chamber cell invagination through stigmatophore cells in a non-cell autonomous manner. In vivo imaging of the morphogenetic movements of the stigmatophore cells further showed that Btk29A is indeed important for the regulation of the spatial rearrangements of the stigmatophore cells. Additionally, it revealed that the positioning of the spiracular chamber cells and the execution of their invagination program is highly dependent on the extent of stigmatophore cell rearrangements. Overall, it seems that Btk29A, acting downstream of the spiracle master regulator Abdominal-B, affects spiracle morphogenesis in two distinct ways: first it cell-autonomously controls the convergent extension of stigmatophore cells; second, Btk29A ensures, non-cell autonomously, the efficient invagination of spiracular chamber cells. The respiratory system of Btk29A mutant embryos is additionally characterised by a leaky tracheal permeability barrier and a failure to fill with air at the end of embryogenesis. The overall trachea morphology is not affected: the airways have normal size, branch patterning and chitin deposition in the tracheal lumen. In order to understand the cause of the permeability barrier defect, specific intercellular junctions, the Septate Junctions (SJs) were examined. These junctions are crucial for the establishment of the epithelial permeability barriers in Drosophila. The ultrastructure of the SJs, as well as the subcellular localization of the SJ proteins, is not impaired in the tracheal cells of Btk29A mutants. It is thus likely that the permeability phenotype is not due to gross impairment in SJ formation or maintenance. 5 In conclusion the work undertaken during my doctoral studies offers insights into the role of Btk29A kinase in the development and maturation of the Drosophila respiratory system. In particular my work has highlighted the complex physical interactions that take place among organ components during morphogenesis, which contribute to their final form and function.
Language English
Subject BTK29A tyrosine kinase
BTK29A κινάση τυροσίνης
Drosophila melanogaster
Permeability barrier
Respiratory system
Tissue morphogenesis
Αναπνευστικό σύστημα
Φραγμός διαπερατότητας
Issue date 2015-03-31
Collection   Faculty/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
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