Article

A Systematic Screen for Tube Morphogenesis and Branching Genes in the Drosophila Tracheal System

Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA.
PLoS Genetics (Impact Factor: 7.53). 07/2011; 7(7):e1002087. DOI: 10.1371/journal.pgen.1002087
Source: PubMed

ABSTRACT

Many signaling proteins and transcription factors that induce and pattern organs have been identified, but relatively few of the downstream effectors that execute morphogenesis programs. Because such morphogenesis genes may function in many organs and developmental processes, mutations in them are expected to be pleiotropic and hence ignored or discarded in most standard genetic screens. Here we describe a systematic screen designed to identify all Drosophila third chromosome genes (∼40% of the genome) that function in development of the tracheal system, a tubular respiratory organ that provides a paradigm for branching morphogenesis. To identify potentially pleiotropic morphogenesis genes, the screen included analysis of marked clones of homozygous mutant tracheal cells in heterozygous animals, plus a secondary screen to exclude mutations in general "house-keeping" genes. From a collection including more than 5,000 lethal mutations, we identified 133 mutations representing ∼70 or more genes that subdivide the tracheal terminal branching program into six genetically separable steps, a previously established cell specification step plus five major morphogenesis and maturation steps: branching, growth, tubulogenesis, gas-filling, and maintenance. Molecular identification of 14 of the 70 genes demonstrates that they include six previously known tracheal genes, each with a novel function revealed by clonal analysis, and two well-known growth suppressors that establish an integral role for cell growth control in branching morphogenesis. The rest are new tracheal genes that function in morphogenesis and maturation, many through cytoskeletal and secretory pathways. The results suggest systematic genetic screens that include clonal analysis can elucidate the full organogenesis program and that over 200 patterning and morphogenesis genes are required to build even a relatively simple organ such as the Drosophila tracheal system.

Download full-text

Full-text

Available from: Amin S Ghabrial, Jul 07, 2014
    • "Compensatory stalk cell branching also occurs upon physiological challenge, not just in genetically compromised animals: we find that injured terminal cells are rapidly invaded by their neighboring stalk cell. RESULTS Ectopic branched autocellular tubes extend into okg and cnj terminal cells We characterized the role of okg and cnj (Ghabrial et al., 2011) in tube architecture and connectivity in mosaic animals, with a focus on the connection between autocellular and seamless tubes. In wildtype larvae, a gas-filled autocellular tube connects the stalk cell to its terminal cell neighbor. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Tubes are essential for nutrient transport and gas exchange in multicellular eukaryotes, but how connections between different tube types are maintained over time is unknown. In the Drosophila tracheal system, mutations in oak gall (okg) and conjoined (cnj) confer identical defects, including late onset blockage near the terminal cell-stalk cell junction and the ectopic extension of autocellular, seamed tubes into the terminal cell. We determined that okg and cnj encode the E and G subunits of the vacuolar ATPase (vATPase) and showed that both the V0 and V1 domains are required for terminal cell morphogenesis. Remarkably, the ectopic seamed tubes running along vATPase-deficient terminal cells belonged to the neighboring stalk cells. All vATPase-deficient tracheal cells had reduced apical domains and terminal cells displayed mislocalized apical proteins. Consistent with recent reports that the mTOR and vATPase pathways intersect, we found that mTOR pathway mutants phenocopied okg and cnj. Furthermore, terminal cells depleted for the apical determinants Par6 or aPKC had identical ectopic seamed tube defects. We thus identify a novel mechanism of compensatory branching in which stalk cells extend autocellular tubes into neighboring terminal cells with undersized apical domains. This compensatory branching also occurs in response to injury, with damaged terminal cells being rapidly invaded by their stalk cell neighbor. © 2015. Published by The Company of Biologists Ltd.
    No preview · Article · Jun 2015 · Development
  • Source
    • "One puzzling aspect of our results is that previous work by Ghabrial et al. showed that single cell clones of a Wts mutation in the larval dorsal trunk caused what they described as a “general overgrowth” phenotype, and what appears to be a roughly isotropic increase in tracheal cell apical surface [64]. This increase is in marked contrast to the reduction in apical surface area we observed in the shortened dorsal trunks of homozygous wts338/1489 embryos. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Precise control of epithelial tube size is critical for organ function, yet the molecular mechanisms remain poorly understood. Here, we examine the roles of cell growth and a highly conserved organ growth regulatory pathway in controlling the dimensions of the Drosophila tracheal (airway) system, a well-characterized system for investigating epithelial tube morphogenesis. We find that tracheal tube-size is regulated in unexpected ways by the transcription factor Yorkie (Yki, homolog of mammalian YAP and TAZ) and the Salvador/Warts/Hippo (SWH) kinase pathway. Yki activity typically promotes cell division, inhibits apoptosis, and can promote cell growth. However, reducing Yki activity in developing embryos increases rather than decreases the length of the major tracheal tubes, the dorsal trunks (DTs). Similarly, reduction of Hippo pathway activity, which antagonizes Yki, shortens tracheal DTs. yki mutations do not alter DT cell volume or cell number, indicating that Yki and the Hippo pathway regulate cell shape and apical surface area, but not volume. Yki does not appear to act through known tracheal pathways of apical extracellular matrix, septate junctions (SJs), basolateral or tubular polarity. Instead, the Hippo pathway and Yki appear to act downstream or in parallel to SJs because a double mutant combination of an upstream Hippo pathway activator, kibra, and the SJ component sinu have the short tracheal phenotype of a kibra mutant. We demonstrate that the critical target of Yki in tube size control is Drosophila Inhibitor of Apoptosis 1 (DIAP1), which in turn antagonizes the Drosophila effector caspase, Ice. Strikingly, there is no change in tracheal cell number in DIAP1 or Ice mutants, thus epithelial tube size regulation defines new non-apoptotic roles for Yki, DIAP1 and Ice.
    Full-text · Article · Jul 2014 · PLoS ONE
  • Source
    • "E-cadherin is actively reduced in the stalk cells via endocytosis (Shaye et al., 2008), which facilitates intercalation, as disrupting endocytic trafficking of E-cadherin blocks stalk cell intercalation and tracheal extension. This is consistent with the finding that genes involved in vesicle trafficking are required for tracheal morphogenesis (Ghabrial et al., 2011). Laser ablation experiments have also revealed that pulling forces from the tip cell are required to induce stalk cell intercalation (Caussinus et al., 2008): the stalk cells are subjected to tensile forces by the tip cell and ablating their connection with the tip cell not only prevents subsequent intercalation, but also causes the stalk cells to retract. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Branching morphogenesis is the developmental program that builds the ramified epithelial trees of various organs, including the airways of the lung, the collecting ducts of the kidney, and the ducts of the mammary and salivary glands. Even though the final geometries of epithelial trees are distinct, the molecular signaling pathways that control branching morphogenesis appear to be conserved across organs and species. However, despite this molecular homology, recent advances in cell lineage analysis and real-time imaging have uncovered surprising differences in the mechanisms that build these diverse tissues. Here, we review these studies and discuss the cellular and physical mechanisms that can contribute to branching morphogenesis.
    Preview · Article · Jul 2014 · Development
Show more