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Establishing Drosophila as a model to study the functional relevance of conserved heart genesCatterson, James Harold January 2013 (has links)
Background/Aims. Understanding the fundamental mechanisms underlying the development of congenital heart disease and cardiomyopathies is a goal of researchers worldwide, with the ultimate goal being the establishment of effective therapeutics for the amelioration of cardiac dysfunction. Unfortunately these disorders are often polygenic in aetiology, making it difficult for researchers to probe complex interactions that may contribute to the severity of the disease. Over the last decade, the adult fruit fly (Drosophila melanogaster) has emerged as an invaluable tool with which to study the genetic and molecular mechanisms underlying heart function. The aim of my thesis research was to establish the adult fruit fly as a model of human heart function, and to exploit this powerful genetic system to screen for conserved genes affecting the development and function of its cardiac syncytium. Methodology/Results Baseline measures of heart function and other factors contributing to variability in heart function (i.e. age, temperature, and the time of day) were assessed to establish the adult Drosophila heart model. I then performed an a priori RNAi screen, knocking down expression of individual conserved genes via cardiomyocyte-specific overexpression utilising the yeast GAL4/UAS system. Heart-specific ablation of Fermitin 1 and Fermitin 2 (Fit1, Fit2), the two Drosophila orthologs of Kindlin 2 (Kind2, a gene thought to be important for cardiomyocyte-cardiomyocyte junction integrity in human myocardium), caused severe cardiomyopathy characterised by the failure of cardiomyocytes to develop as a functional syncytium and loss of synchrony between cardiomyocytes. I generated a null allele of Fit1 via P-element mobilisation, but this had no impact on heart development or function. Similarly, the silencing of Fit2 failed to affect heart development or function. In contrast, the silencing of Fit2 in the cardiomyocytes of Fit1-null flies disrupted syncytium development, leading to severe cardiomyopathy. Temperature-sensitive cardiac-specific GAL4/GAL80ts lines were also generated, and knockdown of Fit (Fit1 and Fit2) function at different developmental stages was assessed. I observed the strongest effects of Fit knockdown on adult cardiac morphology during stages of heart development and remodelling, with significant cardiomyocyte decoupling. After 3-weeks of Fit knockdown during adulthood, cardiomyocytes were significantly decoupled, and these hearts were significantly arrhythmic compared to control animals. Conclusions/Discussion. My data provide clarity about the role of Kind2 by demonstrating a cell autonomous role for this family in the development of a functional cardiac syncytium in Drosophila. My findings also show that the Fermitins can functionally compensate for each other in order to control syncytium development. Therefore, my thesis demonstrates the power of the fruit fly as a model of human cardiac physiology, and supports the concept that abnormalities in cardiomyocyte KIND2 expression or function may contribute to cardiomyopathies in humans.
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The Rab5 GTPase is required for lumen formation in the embryonic Drosophila heartPerry, Katie L. January 2019 (has links)
Tube formation, or tubulogenesis, is an elaborate form of epithelial morphogenesis that includes processes such as cell migration and cell shape changes. The embryonic Drosophila heart, or dorsal vessel, is an excellent model of tubulogenesis and more specifically the signaling mechanisms required for cell migration and lumen formation. Similar to vertebrate heart formation, Drosophila heart tubulogenesis begins with the collective migration of cardioblasts that meet at the midline and adhere at specialised junctions, enclosing a lumen between them. Roundabout, and its ligand Slit, are required to restrict cell-to-cell adhesions to the junctional domains of contralateral cardioblasts, as well as maintain the integrity of the lumen. The localisation patterns of Robo, and other luminal cell surface receptors important for lumen formation are significantly modified throughout heart formation. Initial receptor expression is broadly distributed over the cardioblast surface. Receptors are then relocalised to specific cell surface domains by late embryonic development. The mechanisms by which Robo and other cell surface receptors are localised have yet to be determined. Endocytosis is a promising mechanism by which cell surface receptors are targeted and trafficked to cell surface domains. Specifically, vesicular trafficking proteins, such as Rab GTPases, are molecular switches that regulate endocytic events. Here, we investigated the roles of Rab5, Rab11, and Sec6 during heart formation. Of these, only Rab5, a regulator of the early endosome, was required for lumen formation. Particularly, gain of function, loss of function, and overexpression of rab5 resulted in reduced lumen phenotype, characterised by lumen pockets rather than a continuous lumen along the anterior-posterior axis. Perturbed Rab5 function also resulted in the mislocalisation of Robo at the basal domain. Live imaging showed that expression of rab5 dominant negative, constitutively active, and overexpression constructs did not perturb apical membrane motility of migrating cardioblasts in the developing heart. / Thesis / Master of Science (MSc)
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