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


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.

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Available from: Amin S Ghabrial, Jul 07, 2014
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    • "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. "
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    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.
    PLoS ONE 07/2014; 9(7):e101609. DOI:10.1371/journal.pone.0101609 · 3.23 Impact Factor
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    • "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. "
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    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.
    Development 07/2014; 141(14):2750-2759. DOI:10.1242/dev.104794 · 6.46 Impact Factor
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    • "Its dynamic expression is determined by the interplay between Hh and Wg [44] and is required for the proper migration of myotubes and tracheal branches [43], [45], [46]. In addition, sr is expressed by bnl-expressing cells, and sr null mutants display tracheal migration phenotypes (Fig. 7 G, H and [46], [47]). Therefore, we examined whether sr could be the effector of Hh signalling responsible for modulating bnl levels during tracheal migration. "
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    ABSTRACT: Cell migration is a widespread and complex process that is crucial for morphogenesis and for the underlying invasion and metastasis of human cancers. During migration, cells are steered toward target sites by guidance molecules that induce cell direction and movement through complex intracellular mechanisms. The spatio-temporal regulation of the expression of these guidance molecules is of extreme importance for both normal morphogenesis and human disease. One way to achieve this precise regulation is by combinatorial inputs of different transcription factors. Here we used Drosophila melanogaster mutants with migration defects in the ganglionic branches of the tracheal system to further clarify guidance regulation during cell migration. By studying the cellular consequences of overactivated Hh signalling, using ptc mutants, we found that Hh positively regulates Bnl/FGF levels during embryonic stages. Our results show that Hh modulates cell migration non-autonomously in the tissues surrounding the action of its activity. We further demonstrate that the Hh signalling pathway regulates bnl expression via Stripe (Sr), a zinc-finger transcription factor with homology to the Early Growth Response (EGR) family of vertebrate transcription factors. We propose that Hh modulates embryonic cell migration by participating in the spatio-temporal regulation of bnl expression in a permissive mode. By doing so, we provide a molecular link between the activation of Hh signalling and increased chemotactic responses during cell migration.
    PLoS ONE 03/2014; 9(3):e92682. DOI:10.1371/journal.pone.0092682 · 3.23 Impact Factor
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