Regulation of epithelial morphogenesis by Drosophila Rho GTPase and Abl kinase

The goal of embryogenesis is to convert a fertilized egg into an adult. To ensure that this process occurs normally, regulatory proteins act on the structural elements of a cell to change its position or shape. The resulting changes at a cellular level are coordinated within tissues and constitute a process termed morphogenesis. To date, many morphogenesis regulators have been identified in many model systems. The current aim is to understand how each regulator interacts with the others and with the structural elements of the cell such as the cell adhesion machinery and the cytoskeleton. In my thesis work, I have utilized epithelial development in Drosophila embryos as a model to investigate functions of the morphogenesis regulators Rho1 GTPase and Abelson (Abl) kinase. Rho GTPases have been linked to both regulation of cell-cell adhesion at adherens junctions (AJs) and the actin cytoskeleton. With respect to adhesion, conflicting evidence exists concerning how Rho interacts with core AJ components and the accessory AJ protein p120-catenin (p120). As part of my thesis work, I examined the role of Drosophila Rho1 during epithelial morphogenesis. I found that Rho1 function is not dependent on p120, but that Rho1 regulates core AJ components. Further, my work suggests a mechanistic role for Rho1 in trafficking of the AJ protein cadherin. The second major area of my work examined the role of the non-receptor tyrosine kinase Abl during Drosophila morphogenesis. I identified a novel role for Abl in a specific type of apical cell shape change. Abl is required for apical constriction of mesodermal cells during gastrulation. Abl's regulation of its target Enabled and, consequently, the apical actin cytoskeleton is crucial for this function. This observation led me to test the roles of other ventral furrow regulators in apical actin regulation. I found that RhoGEF2 but not Concertina (G-alpha 12/13) also regulates apical actin during ventral furrow formation, possibly clarifying the difference in phenotypes of mutants of these two morphogenesis regulators. Finally, I developed and characterized new tools for studying Abl localization and activation. Overall, these studies will aid in efforts in understanding how to build an animal

Balancing different types of actin polymerization at distinct sites: roles for Abelson kinase and Enabled

The proto-oncogenic kinase Abelson (Abl) regulates actin in response to cell signaling. Drosophila Abl is required in the nervous system, and also in epithelial cells, where it regulates adherens junction stability and actin organization. Abl acts at least in part via the actin regulator Enabled (Ena), but the mechanism by which Abl regulates Ena is unknown. We describe a novel role for Abl in early Drosophila development, where it regulates the site and type of actin structures produced. In Abl's absence, excess actin is polymerized in apical microvilli, whereas too little actin is assembled into pseudocleavage and cellularization furrows. These effects involve Ena misregulation. In abl mutants, Ena accumulates ectopically at the apical cortex where excess actin is observed, suggesting that Abl regulates Ena's subcellular localization. We also examined other actin regulators. Loss of Abl leads to changes in the localization of the Arp2/3 complex and the formin Diaphanous, and mutations in diaphanous or capping protein β enhance abl phenotypes

Abelson kinase (Abl) and RhoGEF2 regulate actin organization during cell constriction in Drosophila

Morphogenesis involves the interplay of different cytoskeletal regulators. Investigating how they interact during a given morphogenetic event will help us understand animal development. Studies of ventral furrow formation, a morphogenetic event durin

Endoreplication and polyploidy: insights into development and disease

Polyploid cells have genomes that contain multiples of the typical diploid chromosome number and are found in many different organisms. Studies in a variety of animal and plant developmental systems have revealed evolutionarily conserved mechanisms that control the generation of polyploidy and have recently begun to provide clues to its physiological function. These studies demonstrate that cellular polyploidy plays important roles during normal development and also contributes to human disease, particularly cancer

Physiology, development, and disease modeling in the Drosophila excretory system

The insect excretory system contains two organ systems acting in concert: the Malpighian tubules and the hindgut perform essential roles in excretion and ionic and osmotic homeostasis. For over 350 years, these two organs have fascinated biologists as a model of organ structure and function. As part of a recent surge in interest, research on the Malpighian tubules and hindgut of Drosophila have uncovered important paradigms of organ physiology and development. Further, many human disease processes can be modeled in these organs. Here, focusing on discoveries in the past 10 years, we provide an overview of the anatomy and physiology of the Drosophila excretory system. We describe the major developmental events that build these organs during embryogenesis, remodel them during metamorphosis, and repair them following injury. Finally, we highlight the use of the Malpighian tubules and hindgut as accessible models of human disease biology. The Malpighian tubule is a particularly excellent model to study rapid fluid transport, neuroendocrine control of renal function, and modeling of numerous human renal conditions such as kidney stones, while the hindgut provides an outstanding model for processes such as the role of cell chirality in development, nonstem cell–based injury repair, cancer-promoting processes, and communication between the intestine and nervous system

Drosophila p120catenin plays a supporting role in cell adhesion but is not an essential adherens junction component

Cadherin–catenin complexes, localized to adherens junctions, are essential for cell–cell adhesion. One means of regulating adhesion is through the juxtamembrane domain of the cadherin cytoplasmic tail. This region is the binding site for p120, leading to the hypothesis that p120 is a key regulator of cell adhesion. p120 has also been suggested to regulate the GTPase Rho and to regulate transcription via its binding partner Kaiso. To test these hypothesized functions, we turned to Drosophila, which has only a single p120 family member. It localizes to adherens junctions and binds the juxtamembrane region of DE-cadherin (DE-cad). We generated null alleles of p120 and found that mutants are viable and fertile and have no substantial changes in junction structure or function. However, p120 mutations strongly enhance mutations in the genes encoding DE-cadherin or Armadillo, the β-catenin homologue. Finally, we examined the localization of p120 during embryogenesis. p120 localizes to adherens junctions, but its localization there is less universal than that of core adherens junction proteins. Together, these data suggest that p120 is an important positive modulator of adhesion but that it is not an essential core component of adherens junctions

Assessing Differential Drug Effect

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Rho1 regulates Drosophila adherens junctions independently of p120ctn

During animal development, adherens junctions (AJs) maintain epithelial cell adhesion and coordinate changes in cell shape by linking the actin cytoskeletons of adjacent cells. Identifying AJ regulators and their mechanisms of action are key to understanding the cellular basis of morphogenesis. Previous studies linked both p120catenin and the small GTPase Rho to AJ regulation and revealed that p120 may negatively regulate Rho. Here we examine the roles of these candidate AJ regulators durin