Drosophila melanogaster has a tubular heart called the dorsal vessel, which is composed of contractile cardiomyocytes and hemolymph filtering pericardial cells. During larval development the dorsal vessel (heart) grows in size, and the luminal space inside the heart expands, however it has not been clear which cells are responsible for laying the extracellular matrix (ECM) during this expansion. Hemocytes (white blood cells), pericardial cells and cells of the fat body are candidate cell types that may secrete ECM for assembly during the growth of the heart lumen. With gene knock-down techniques we are exploring whether hemocytes participate in assembly of the heart ECM at this location. Additionally, studies of fluorescently tagged hemocytes in intact larvae reveal that hemocytes aggregate around pericardial cells of the dorsal vessel in 3rd instars. Confocal studies of dissected larval hearts indicate that hemocytes aggregate within infoldings of basement membrane associated with pericardial cells. Hemocyte-pericardial cell association could indicate that hemocytes take up proteins that are produced by pericardial cells and deliver them to other locations or that there might be a previously unidentified hematopoietic site at the Drosophila larval heart. / Thesis / Master of Science (MSc)

This dissertation characterizes the geometry, kinematics, and physical properties of insect internal structures that make up the respiratory and circulatory systems. This characterization is necessary to better understand how these systems function to transport fluids at the microscale, and ultimately, how we might computationally model this flow. Chapter 2 describes the geometry of the insect tracheal system, specifically testing if Murray's law applies to this system using three-dimensional imaging of tracheal tubes. Chapter 3 begins to characterize the physical properties of insect hemolymph, specifically the viscosity and density of hemolymph, using experimental measurements. Because insects are strongly affected by environmental temperature, this chapter also explores how hemolymph viscosity may be affected by temperature. Chapter 4 builds on the results of Chapter 3, exploring the effects of developmental responses to temperature on hemolymph viscosity and properties, as well as performance of the insect using experimental measurements. Finally, Chapter 5 presents a kinematic and structural characterization of the insect heart using a variety of imaging techniques and analyses. / Doctor of Philosophy / Insect physiology and morphology has long been studied by biologists and entomologists, with many of the basic features understood and characterized. The insect circulatory and respiratory systems differ greatly from those of many other organisms. Physically, these systems transport fluids through microscale environments which include a variety of pumps, networks, and other structures that facilitate flow. Functionally, the circulatory and respiratory systems are largely decoupled, unlike in vertebrates. The respiratory system transports air directly to deliver oxygen to tissues, whereas the circulatory system transports various nutrients and other chemicals via hemolymph. With these unique differences, investigation of these major biological transport systems in insects is essential to fully understand their structure and function. This dissertation addresses many of the basic structural and physical properties of the insect respiratory and circulatory systems that are still unknown, despite growing engineering analysis. First, I measured specific geometric features of the insect tracheal network and determined if Murray’s law applies to this system. Second, I quantified the viscosity of insect hemolymph, including in response to temperature. To expand upon this relationship further, I measured hemolymph viscosity, hemolymph composition, and insect performance after temperature acclimation during development. Last, I investigated the morphology and kinematics of the insect heart, using many methods of imaging and analysis to measure structural features of the heart wall, including during function. Hemolymph properties and heart morphology provide the physical basis of flow production within the circulatory system. Understanding flow production within the circulatory system, as well as design features of the respiratory system, are crucial in the construction of mathematical models of both hemolymph and air flow within the insect.

The cardiovascular system of Drosophila melanogaster consists of a cardiac tube composed of myogenic cardiomyocytes and associating non-contractile pericardial cells, pumping hemolymph into the open circulatory system. The cardiac tube, known as the dorsal vessel, is embedded in a highly regulated extracellular matrix environment, required to maintain cellular integrity and cardiac function. After embryogenesis, the dorsal vessel undergoes extensive physiological changes, relying on the extracellular matrix to adapt and remodel accordingly. Three extracellular matrix proteins are investigated throughout this thesis: Type IV Collagen, Laminin and Pericardin. Due to their localization, morphology, and role in early development, the pericardial cells are candidate cells responsible for dorsal vessel extracellular matrix deposition and regulation throughout post-embryonic growth. Using immunofluorescence techniques in combination with confocal microscopy, I characterize the association between pericardial cells and extracellular matrix proteins, and quantify extracellular matrix protein deposition at the dorsal vessel throughout post-embryonic development. Gene knock-down experiments assess pericardial cell contribution to extracellular matrix synthesis and deposition at the dorsal vessel in third instar larva. Moreover, I quantify extracellular matrix protein deposition at the dorsal vessel in the absence of pericardial cells. These data suggests that pericardial cells regulate extracellular matrix protein deposition, localization and contribute to proper cardiac morphology in post-embryonic development. / Thesis / Master of Science (MSc)

Das Dorsalgefäß von Drosophila ist ein wertvolles Modellsystem für die Untersuchung der genetischen und molekularen Mechanismen der Kardiogenese. Ein Schlüsselereignis der Kardiogenese ist die Bildung eines Herzlumens, durch das die Hämolymphe gepumpt wird, um Nährstoffe und Zellen des angeborenen Immunsystems zu zirkulieren. Ein Schwerpunkt meiner Arbeit umfasste die Identifizierung neuer Gene, die im embryonalen Dorsalgefäß von Drosophila exprimiert sind. Dafür habe ich die Expression von 101 Genen, deren Orthologe spezifisch im Herzen von Zebrafisch exprimiert sind, in Drosophila untersucht. Ich identifizierte ein Gen, das für das Cbl-assoziierte Protein (CAP) kodiert. Durch Herstellung eines anti-CAP Antikörpers konnte ich erstmals eine detaillierte Lokalisation des CAP-Proteins im Dorsalgefäß beschreiben. Interessanterweise stellte sich dabei heraus, dass CAP ähnlich wie die homologen Vertebraten Proteine embryonal an den fokalen Adhäsionskontakten der Kardioblasten und im adulten Dorsalgefäß auch an den Z-Scheiben und den Zell-Zell-Kontaktstellen der Kardiomyozyten lokalisiert ist. Des Weiteren untersuchte ich, welche Auswirkungen der Verlust der CAP Funktion auf die Herzentwicklung hat. Für die Analyse der CAP-Mutanten nutzte ich neben Immunhistochemischen Methoden auch ultrastrukturelle Analysen mittels TEM-Mikroskopie. So konnte ich zeigen, dass embryonale Dorsalgefäße von CAP-Mutanten eine fehlerhafte Anzahl sowie Anordnung der Kardioblasten und Lumendefekte aufweisen. Ein genetischer Interaktionstest untermauerte meine Vermutung, dass CAP mit dem Integrinsignalweg während der embryonalen Dorsalgefäßentwicklung interagiert. Live-Aufnahmen des pumpenden Dorsalgefäßes von Drosophila L3-Larven und Injektionstests an späten Puppen zeigten zudem, dass der Verlust der CAP Funktion auch zu starken Defekten in der Funktionalität des larvalen und adulten Dorsalgefäßes führt. / The heart of Drosophila provides a valuable model system for the examination of the genetic and molecular mechanisms that guide cardiogenesis. A key event of cardiogenesis is the formation of a heart lumen through which the hemolymph is pumped to circulate nutrients and cells of the innate immune system. A main focus of my work was the identification of new genes that are expressed in the embryonic heart of Drosophila. Therefore I studied the expression of 101 genes, whose orthologues are expressed specifically in the heart of zebrafish. I identified a gene that encodes for the Cbl-associated protein (CAP). By generating an anti-CAP antibody I could describe the localization of the CAP protein in the heart for the first time in detail. Interestingly, it turned out that CAP is located similar to the homologous vertebrate proteins at the focal adhesion contacts of cardioblasts in the embryo and at the Z-discs and the cell-cell contact sites of cardiomyocytes in the adult heart. I also examined the consequences of the loss of CAP function on heart development. For the analysis of the CAP mutants I used immunohistochemical and ultrastructural analysis by TEM microscopy. So I was able to demonstrate that embryonic hearts of CAP mutants show a defective number and arrangement of cardioblasts and lumen defects. A genetic interaction test substantiated my guess that CAP interacts with the Integrin signaling pathway during embryonic heart development. Live recordings of the pumping heart of Drosophila L3 larvae and injection tests of late pupae also showed that the loss of CAP function leads to severe defects in the functionality of the larval and adult heart.