| From the branched bronchi and bronchioles of the mammalian lung to the heart and circulatory system to the many organs contained within and attached to the digestive tract, humans are tubular organisms. Therefore, studying how tubes form is indispensable to our understanding of human embryogenesis and of the various diseases that arise due to defects in tube architecture and tube maintenance. The Drosophila salivary gland (SG), salivary duct (SD), and trachea, with their accessibility and availability of extensive genetic tools, provide relatively simple model systems to study tube morphogenesis. Although formation of these single layer epithelial tubes involves common cellular processes, morphogenesis of each tube type also involves distinct changes in cell behavior. For my thesis project, I have focused on processes affecting formation of one or more of these tubular organs.;As a major part of my thesis research, I collaborated with another graduate student in the lab to identify and characterize the cellular basis for defects caused by mutations in the ribbon (rib) gene. In rib mutants, the SGs stall at the point where wild-type SGs initiate posteriorly-directed migration. Also, migration of all tracheal branches is delayed with the dorsal trunk (DT), the major artery of the trachea, completely failing to form in rib mutants. We demonstrated that Rib, a BTB-domain-containing nuclear protein interacts with Lola like, another BTB-domain-containing nuclear protein to upregulate crumbs (crb) transcription and downregulate Moesin (Moe) activity. We showed by TEM analysis of rib SGs that there is an increase in microvillar structure and a diminished pool of subapical vesicles that are coincident with Rab11 staining by confocal microscopy. These cellular changes are consistent with the known roles of Crb in apical membrane generation and of Moe in the cross-linking of the apical membrane to the subapical cytoskeleton. Since all of the molecular and cellular changes in rib mutants relative to wild type localize to the apical surface and since increased Moe activity correlates with increased cortical stiffness and since perturbed membrane dynamics could also affect apical stiffness and viscosity, we hypothesized that rib mutants suffer from increased apical stiffness during tube elongation. To test this hypothesis, we analyzed the dynamics of SG and DT morphogenesis live. Consistently, both tissues exhibited slowed and incomplete lumenal morphogenesis. Using the live-imaging data, we constructed mechanical models of tube elongation, which suggested that rib mutant tubes exhibit three- to five-fold increased apical stiffness and two-fold increased effective apical viscosity. Our work on the cellular, molecular and mechanical basis for rib's defects in tube elongation was published in two manuscripts (Chapters 2 and 3 of my thesis).;As another major part of my thesis research, I also described the cell shape changes and cell movements that occur in the formation of the SD I demonstrated that this organ forms through mechanisms that include "cell wrapping" and "convergence and extension", which occur in the formation of many vertebrate organs, including the neural tube. I showed that these events can be relatively easily visualized and analyzed in the SD, making it a tractable model system for elucidating the events underlying the processes of wroapping and convergent extension (Appendix A of the thesis). To further analyze SD tube formation, I screened a collection of genes that affected tracheal development for mutations that also affected SD formation. I then carried out a detailed analysis of one of the genes I discovered to affect the SD, which I originally named anduril (aul). In aul mutants, frequent breaks in the DT were observed, the ventral tracheal branches were shorter, and individual SDs often had significant gaps or were completely missing. Through mapping the aul mutation, I discovered that aul is allelic to dalmation (dmt), a gene previously shown to be required for peripheral nervous system formation. I showed that dmt encodes a heterochromatin-associated nuclear protein that inhibits apoptosis by repressing expression of two proapoptotic genes, head involution defective (hid) and reaper ( rpr). I further demonstrated that inhibition of apoptosis can rescue and that over-expression of proapoptotic genes can mimic the dmt phenotypes, suggesting that keeping cells alive is the major function of Dmt during embryogenesis. Given Dmt heterochrmatin localization, transcriptional repression of hid and rpr, and timing of this repression, I propose that Dmt may limit cell death in committed cells through epigenetic mechanisms. This work has been submitted for publication and is included as Chapter 4 of the thesis. |
