Browsing by Subject "Dorsal closure"

Cell sheet morphogenesis is essential for metazoan development and homeostasis, contributing to key developmental stages such as neural tube closure as well as tissue maintenance through wound healing. Dorsal closure, a well-characterized stage in Drosophila embryogenesis, has emerged as a model for cell sheet morphogenesis. Closure is a remarkably robust process where coordination of conserved gene expression and signaling cascades regulate cellular movements that drive closure. While well-characterized, new ‘dorsal closure genes’ continue to be discovered due to advances in microscopy and genetics. Here, we use live imaging and a set of large deletions, deficiencies (Dfs), that together remove 98.9% of the genes on 2L in order to identify regions of the genome required for normal closure. We successfully screened 96.1% of the genes on 2L and identified diverse dorsal closure defects in embryos homozygous for 47 Dfs, 26 of which have no known dorsal closure gene located within the Df region. We have already identified pimples, odd-skipped, paired, and sloppy-paired 1 as dorsal closure genes on the 2L affecting lateral epidermal cell shapes, and anticipate we will continue to identify novel ‘dorsal closure genes’ with further analysis. We also investigate the changes in dorsal closure dynamics and forces in the even-skipped (eve) mutant, which has aberrant cell shapes and behaviors as well as reduced actin and myosin at the purse string, but completes closure. We find that loss of wg/wnt-1 signaling in eve causes the observed defects in closure and that crumbs, a regulator of actin and myosin, is mis-expressed. Additionally, laser microsurgery demonstrates that the eve or wg mutant embryos are under a global tension in the anterior-posterior direction. Lastly, we identify a lesion in echinoid that is responsible for the jagged purse string and ectopic zipping dorsal closure phenotype previously thought to be due to a lesion in Zasp52.

Cell sheet morphogenesis characterizes key developmental transitions throughout phylogeny. As such, it plays a crucial role in developmental milestones in vertebrate morphogenesis including gastrulation, neural tube formation, and palate formation. It also plays important roles in wound healing. Dorsal closure, a process during Drosophila embryogenesis, has emerged as a model for cell sheet morphogenesis due to the ability to image embryos in-vivo the genetic tractability of Drosophila. While 140 genes are currently published to affect dorsal closure, new genes are identified each year. In addition our understanding of dorsal closure is far from complete with many questions remaining regarding the molecular mechanisms involved in this complex process. To identify a more complete list of genes involved in dorsal closure, we used a set of large deletions (deficiencies), which collectively remove 98.5% of the genes on the right arm of the 2nd chromosome. Through two crosses, we unambiguously identified homozygous deficiencies and imaged them for the duration of dorsal closure. Images were then analyzed for defects in the cell shapes and morphogenesis. 48 deficiencies were identified to have notable defects on dorsal closure. We anticipate these deficiencies will lead to the identification of at least 31 novel dorsal closure genes. We expect the large number of novel dorsal closure will identify links to pathways already known to coordinate various aspects of closure in addition to new processes and pathways that are currently unidentified as involved in closure.

Cell-cell adhesions and intercellular ion channels, such as gap junctions regulate the synchronization of cells in an epithelium and promote proper embryonic epithelial organization and morphogenesis. Gap junctions regulate the exchange of small molecules and ions, such as calcium (Ca2+) between apposed cells. The strict maintenance of Ca2+ ion concentration across a cell membrane allows Ca2+ to function as an effective intracellular signal in epithelial sheet morphogenesis, including development and wound repair, where it coordinates cell behavior throughout the tissue. Here I used live imaging to observe Drosophila dorsal closure (DC), a model of epithelial sheet morphogenesis. I quantified endogenous calcium flashes in the lateral epidermis, the spread of Ca2+ following single cell wounds, and perturbed extracellular Ca2+ concentrations to demonstrate the necessity of calcium in maintaining the coordinated movements of epithelial sheets in DC. I also genetically and pharmacologically reduced gap junction subunit functionality. I have observed that the knockdown of single ion channel subunits results in the partial penetrance of morphological abnormalities during DC. The intercellular calcium dynamics in the lateral epidermis are modified in gap junction mutants. Lastly, pan-innexin inhibition using gap junction antagonists completely halts DC, resulting in a large coordinated release of intracellular Ca2+ and disruption of cell junction adhesion in the amnioserosa.

Physical forces play a key role in the morphogenesis of embryos. As cells and tissues change shape, grow, and migrate, they exert and respond to forces via mechanosensitive proteins and protein complexes. How the response to force is regulated is not completely understood.

Dorsal closure in Drosophila is a model system for studying cell sheet forces during morphogenesis. We demonstrate a role for mechanically gated ion channels (MGCs) in dorsal closure. Microinjection of GsMTx4 or GdCl₃, inhibitors of MGCs, blocks closure in a dose-dependent manner. UV-mediated uncaging of intracellular Ca2+ causes cell contraction whereas the reduction of extra- and intracellular Ca2+ slows closure. Pharmacologically blocking MGCs leads to defects in force generation via failure of actomyosin structures during closure, and impairs the ability of tissues to regulate forces in response to laser microsurgery.

We identify three genes which encode candidate MGC subunits that play a role in dorsal closure, ripped pocket, dtrpA1, and nompC. We find that knockdown of these channels either singly or in combination leads to defects in force generation and cell shapes during closure.

Our results reveal a key role for MGCs in closure, and suggest a mechanism for the coordination of force producing cell behaviors across the embryo.

Epithelial sheet morphogenesis is characterized by dynamic tissue movements, resulting in the recognition and adhesion of cells to generate a seamless epithelium. Each step is mediated by carefully organized, cellular actin structures, including contractile purse strings, cellular protrusions, and dynamic medioapical arrays. I used live, 4D imaging to observe Drosophila dorsal closure, a model of epithelial sheet morphogenesis. I compared four fluorescently tagged F-actin probes widely used by Drosophila researchers to determine which was optimal for imaging dorsal closure. I observed differences in the intensity of the probes and the viability of the stocks that carry them. I quantified the rate of closure and the oscillatory behavior of amnioserosa cells when embryos expressed each F-actin probe. My findings demonstrated that each probe can be used to image F-actin during dorsal closure, and that the effects of probe expression make one probe more or less suitable than another for answering specific questions. I investigated the structure, kinematics and location of medioapical, actomyosin arrays during dorsal closure. I resolved medioapical arrays in vivo at the level of individual cytoskeletal components using total internal reflection structured illumination microscopy (TIRF-SIM). In concert with lattice light-sheet images, I show that when amnioserosa cells are relaxed, actin and myosin form a loose, domed meshwork that protrudes apically from the cellular junctions to which they are anchored. As the amnioserosa cells contract, this meshwork condenses, rearranges and is drawn basally towards the plane of the junctional belts. As the cells relax, so too does the actin and myosin meshwork in a new configuration. The medioapical arrays are juxtaposed to the plasma membrane and continuous with the extending lamellipodia and filopodia. Thus, medioapical arrays are modified cell cortex.