Tube formation is a basic process in the development of many organs, including the lungs, kidneys, heart, neural tube, and gut. This proposal uses Drosophila melanogaster egg chambers to investigate the mechanisms that control tube size and shape. In the fly ovary, certain follicle cells that surround the oocyte reorganize from a curved sheet into a pair of elongated tubes. These tubes produce eggshell structures called dorsal appendages (DAs), which facilitate air exchange in the developing embryo. DA formation provides a simple model system for investigating the highly conserved cell biological and molecular mechanisms that regulate tube formation. A genetic screen for DA mutants identified the female sterile mutation twin peaks (twk), which disrupts the zinc-finger transcription factor Tramtrack69 (TTK69;French et al. 2003). twk mutants produce short nubs of DAs. Studies with partial and null alleles demonstrated that TTK69 acts cell autonomously to promote expansion of apical surface area, thereby controlling tube lumen volume. A genetic interaction screen with twk identified four dominant suppressors of the twk nubs phenotype and suggested that trimeric G-proteins participate in the regulation of tube morphogenesis. Surprisingly, mosaic analyses showed that G213F acts in a subset of leading cells to regulate the rearrangement of cells within the tube and to control apical-basal dimensions. These factors affect overall tube length and width. These results suggest that two forces determine tube morphology: Trimeric G proteins act in leading cells to define the shape of the tube and TTK69 acts in all cells to control tube volume, within the bounds of the floor plan set by leading cells. This hypothesis makes several testable predictions that are the basis for this proposal. The plan is to: 1) Alter the balance between G-protein activity in leading cells and TTK69 activity in all cells to create tubes of various sizes and shapes. 2) Investigate potential cellular and molecular mechanisms for how G-protein function in leading cells influences the behavior of cells in distant regions of the primordium. 3) Analyze the apical expansion process and test promising candidates that likely mediate TTK69's role in regulating the size of the apical domain.
Dorsal appendage formation in the Drosophila egg chamber has emerged as an outstanding model for studying tubulogenesis. Although much is known about how individual cells sense signals, reorganize their cytoskeleton, and move in a directed fashion, less is known about how sheets of cells respond to environmental or genetic cues. This lab has developed the tools to tackle this important question. Synthesis of the dorsal respiratory appendages by the follicular epithelium resembles primary neurulation in vertebrates. The aberrant rearrangement of cells exhibited by G-protein mutants is similar to the tube-closure defects that produce spina bifida and other common birth defects. Identification of the genes involved in this process is an important first step in preventing these conditions in humans. TTK69 is conserved in animals and is a defining member of the BTB class of zinc-finger transcription factors. Defects in the human orthologue, Bcl6, promote the neoplastic transformation of B cells into lymphomas. These studies will reveal the normal mechanism of TTK69 action and give insight into how altered TTK69 function affects cell behavior. Such knowledge could lead to diagnostic advances. Organ failure is an increasing burden on our health care system. The development of replacement tissues is the goal of tissue engineers, who would benefit by a detailed understanding of the normal mechanisms that make and shape tubes.
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