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Larval terminal cells of the Drosophila tracheal system generate extensive branched tubes, requiring a huge increase in apical membrane. We discovered that terminal cells compromised for apical membrane expansion – mTOR-vATPase axis and apical polarity mutants – were invaded by the neighboring stalk cell. The invading cell grows and branches, replacing the original single intercellular junction between stalk and terminal cell with multiple intercellular junctions. Here, we characterize disjointed, a mutation in the same phenotypic class. We find that disjointed encodes Drosophila Archease, which is required for the RNA ligase (RtcB) function that is essential for tRNA maturation and for endoplasmic reticulum stress-regulated nonconventional splicing of Xbp1 mRNA. We show that the steady-state subcellular localization of Archease is principally nuclear and dependent upon TOR-vATPase activity. In tracheal cells mutant for Rheb or vATPase loci, Archease localization shifted dramatically from nucleus to cytoplasm. Further, we found that blocking tRNA maturation by knockdown of tRNAseZ also induced compensatory branching. Taken together, these data suggest that the TOR-vATPase axis promotes apical membrane growth in part through nuclear localization of Archease, where Archease is required for tRNA maturation.

The terminal cells of the larval Drosophila tracheal system extend dozens of branched cellular processes, most of which become hollow intracellular tubes that support gas exchange with internal tissues. Previously, we undertook a forward genetic mosaic screen to uncover the pathways regulating terminal cell size, morphogenesis, and the generation and maintenance of new intracellular tubes. Our initial work identified several mutations affecting terminal cell size and branch number, and suggested that branch complexity and cell size are typically coupled but could be genetically separated. To deepen our understanding of these processes, we have further characterized and determined the molecular identities of mutations in the genes sprout, denuded and asthmatic, that had been implicated in our initial screen. Here we reveal the molecular identity of these genes and describe their function in the context of the TOR and Hippo pathways, which are widely appreciated to be key regulators of cell and organ size.

Embryos deficient for an essential gene may show complex phenotypes that reflect pleiotropic functions and non-cell-autonomous requirements for the encoded protein. The generation of mosaic animals, where most cells are wild type, but a few cells are mutant, is a powerful tool permitting the detailed analysis of the cell autonomous function of a gene, in a particular cell type, at cellular and subcellular resolutions. Here we apply this method to the analysis of the Cerebral Cavernous Malformations 3 (CCM3) pathway in Drosophila.The conserved CCM3 protein functions together with its binding partner, Germinal Center Kinase III (Wheezy/GckIII in Drosophila, MST3, STK24, and STK25 in human) in the regulation of tube morphogenesis (Bergametti et al. Am J Hum Genet. 76:42-51, 2005; Fidalgo et al. J Cell Sci. 123:1274-1284, 2010; Guclu et al. Neurosurgery. 57:1008-1013, 2005; Lant et al. Nat Commun. 6:6449, 2015; Song et al. Dev Cell. 25:507-519, 2013; Ceccarelli et al. J Biol Chem. 286:25056-25064, 2011; Rehain-Bell et al. Curr Biol. 27:860-867, 2017; Xu et al. Structure. 21:1059-1066, 2013; Zhang et al. Front Biosci. 17:2295-2305, 2012; Zhang et al. Dev Cell. 27:215-226, 2013; Zheng et al. J Clin Invest. 120:2795-2804, 2010). The Drosophila proteins play a role in the regulation of tube shape in the tracheal (respiratory) system, analogous to the role of the human proteins in the vascular system. To understand the cellular basis for tube dilation defects caused by loss of pathway function, we describe techniques for the generation and analysis of positively marked homozygous mutant GckIII tracheal cells, coupled with an "open book" preparation that can be subjected to immunofluorescent analysis. Dozens of mutant tracheal cells are generated per mosaic animal, and neighboring heterozygous cells in the same animal serve as ideal internal controls.

Embryos deficient for an essential gene may show complex phenotypes that reflect pleiotropic functions and non-cell-autonomous requirements for the encoded protein. The generation of mosaic animals, where most cells are wild type, but a few cells are mutant, is a powerful tool permitting the detailed analysis of the cell autonomous function of a gene, in a particular cell type, at cellular and subcellular resolutions. Here we apply this method to the analysis of the Cerebral Cavernous Malformations 3 (CCM3) pathway in Drosophila.The conserved CCM3 protein functions together with its binding partner, Germinal Center Kinase III (Wheezy/GckIII in Drosophila, MST3, STK24, and STK25 in human) in the regulation of tube morphogenesis (Bergametti et al. Am J Hum Genet. 76:42-51, 2005; Fidalgo et al. J Cell Sci. 123:1274-1284, 2010; Guclu et al. Neurosurgery. 57:1008-1013, 2005; Lant et al. Nat Commun. 6:6449, 2015; Song et al. Dev Cell. 25:507-519, 2013; Ceccarelli et al. J Biol Chem. 286:25056-25064, 2011; Rehain-Bell et al. Curr Biol. 27:860-867, 2017; Xu et al. Structure. 21:1059-1066, 2013; Zhang et al. Front Biosci. 17:2295-2305, 2012; Zhang et al. Dev Cell. 27:215-226, 2013; Zheng et al. J Clin Invest. 120:2795-2804, 2010). The Drosophila proteins play a role in the regulation of tube shape in the tracheal (respiratory) system, analogous to the role of the human proteins in the vascular system. To understand the cellular basis for tube dilation defects caused by loss of pathway function, we describe techniques for the generation and analysis of positively marked homozygous mutant GckIII tracheal cells, coupled with an "open book" preparation that can be subjected to immunofluorescent analysis. Dozens of mutant tracheal cells are generated per mosaic animal, and neighboring heterozygous cells in the same animal serve as ideal internal controls.

Hippo-like pathways are ancient signalling modules first identified in yeasts. The best-defined metazoan module forms the core of the Hippo pathway, which regulates organ size and cell fate. Hippo-like kinase modules consist of a Sterile 20-like kinase, an NDR kinase and non-catalytic protein scaffolds. In the Hippo pathway, the upstream kinase Hippo can be activated by another kinase, Tao-1. Here, we delineate a related Hippo-like signalling module that Tao-1 regulates to control tracheal morphogenesis in Drosophila melanogaster. Tao-1 activates the Sterile 20-like kinase GckIII by phosphorylating its activation loop, a mode of regulation that is conserved in humans. Tao-1 and GckIII act upstream of the NDR kinase Tricornered to ensure proper tube formation in trachea. Our study reveals that Tao-1 activates two related kinase modules to control both growth and morphogenesis. The Hippo-like signalling pathway we have delineated has a potential role in the human vascular disease cerebral cavernous malformation.

The last decade has seen an explosion of research on exosomes, small (30 to 100 nm) vesicles that are trafficked to the extracellular environment by the fusion of the multivesicular body to the plasma membrane (1, 2). Exosomes arise from endosomal microdomains that, through the agency of ESCRT proteins and Alix, curve inward and bud as intraluminal vesicles (50 to 150 nm) into the lumen of the multivesicular body. The small GTPases, Ral1, Rab27a, and Rab27b, mediate the transport and fusion of multivesicular bodies to plasma membrane, resulting in the exocytic release of the exosomes into the extracellular space (3, 4). Once thought of primarily as disposal devices, tiny garbage bags, exosomes are now appreciated to act as hormone-like signals. Cells may use exosomes to exert systemic influence through the activity of exosomal microRNAs (miRNAs), messenger … [↵][1]1Email: asg2236{at}cumc.columbia.edu. [1]: #xref-corresp-1-1

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.

Genetic mosaic analysis in Drosophila melanogaster shows that there are two classes of cells in the budding tracheal branches: those that migrate to the tip, and those that follow these lead cells and form a tube. The position of the cell results from competition within the population, with those expressing the highest level of FGF taking the lead positions at the tip, and the others forming the branch stalk. Many organs are composed of tubular networks that arise by branching morphogenesis in which cells bud from an epithelium and organize into a tube1,2,3. Fibroblast growth factors (FGFs) and other signalling molecules have been shown to guide branch budding and outgrowth4,5,6,7, but it is not known how epithelial cells coordinate their movements and morphogenesis. Here we use genetic mosaic analysis in Drosophila melanogaster to show that there are two functionally distinct classes of cells in budding tracheal branches: cells at the tip that respond directly to Branchless FGF and lead branch outgrowth, and trailing cells that receive a secondary signal to follow the lead cells and form a tube. These roles are not pre-specified; rather, there is competition between cells such that those with the highest FGF receptor activity take the lead positions, whereas those with less FGF receptor activity assume subsidiary positions and form the branch stalk. Competition appears to involve Notch-mediated lateral inhibition that prevents extra cells from assuming the lead. There may also be cooperation between budding cells, because in a mosaic epithelium, cells that cannot respond to the chemoattractant, or respond only poorly, allow other cells in the epithelium to move ahead of them.

Most organs are composed of tubes of differing cellular architectures, including intracellular 'seamless' tubes. Two studies examining the morphogenesis of the seamless tubes formed by the excretory canal cell in Caenorhabditis elegans reveal a previously unappreciated role for osmoregulation of tubulogenesis: hyperosmotic shock recruits canalicular vesicles to the lumenal membrane to promote seamless tube growth.

▪ Abstract Many organs including the mammalian lung and vascular system consist of branched tubular networks that transport essential gases or fluids, but the genetic programs that control the development of these complex three-dimensional structures are not well understood. The Drosophila melanogaster tracheal (respiratory) system is a network of interconnected epithelial tubes that transports oxygen and other gases in the body and provides a paradigm of branching morphogenesis. It develops by sequential sprouting of primary, secondary, and terminal branches from an epithelial sac of ∼80 cells in each body segment of the embryo. Mapping of the cell movements and shape changes during the sprouting process has revealed that distinct mechanisms of epithelial migration and tube formation are used at each stage of branching. Genetic dissection of the process has identified a general program in which a fibroblast growth factor (FGF) and fibroblast growth factor receptor (FGFR) are used repeatedly to control branch budding and outgrowth. At each stage of branching, the mechanisms controlling FGF expression and the downstream signal transduction pathway change, altering the pattern and structure of the branches that form. During terminal branching, FGF expression is regulated by hypoxia, ensuring that tracheal structure matches cellular oxygen need. A branch diversification program operates in parallel to the general budding program: Regional signals locally modify the general program, conferring specific structural features and other properties on individual branches, such as their substrate outgrowth preferences, differences in tube size and shape, and the ability to fuse to other branches to interconnect the network.

Small open-reading frames are difficult to detect both computationally and by mutagenesis. An mRNA previously thought to be non-coding has now been found to produce four tiny peptides that function non-cell autonomously to organize epithelial actin during Drosophila development.

The Drosophila gene torpedo/Egfr (top/Egfr) encodes a homolog of the vertebrate Epidermal Growth Factor receptor. This receptor is required several times during the life cycle of the fly for the transmission of developmental cues. During oogenesis, Top/Egfr activation is required for the establishment of the dorsal/ventral axis of the egg and the embryo. To examine how ectopic Top/Egfr activation affects cell fate determination, we constructed an activated version of the protein. Expression of this activated form (λtop) in the follicle cells of the ovary induces dorsal cell fates in both the follicular epithelium and the embryo. Different levels of expression resulted in different dorsal follicle cell fates. These dorsal cell fates were expanded in the anterior, but not the posterior, of the egg, even in cases where all the follicle cells covering the oocyte expressed λtop. The expression of genes known to respond to top/Egfr activation, argos (aos), kekkon1 (kek 1) and rhomboid (rho), was also expanded in the presence of the λtop construct. When λtop was expressed in all the follicle cells covering the oocyte, kek 1 and argos expression was induced in follicle cells all along the anterior/posterior axis of the egg chamber. In contrast, rho RNA expression was only activated in the anterior of the egg chamber. These data indicate that the response to Top/Egfr signaling is regulated by an anterior/posterior prepattern in the follicle cells. Expression of λtop in the entire follicular epithelium resulted in an embryo dorsalized along the entire anterior/posterior axis. Expression of λtop in anterior or posterior subpopulations of follicle cells resulted in regionally autonomous dorsalization of the embryos. This result indicates that subpopulations of follicle cells along the anterior/posterior axis can respond to Top/Egfr activation independently of one another.

Summary Tubes of differing cellular architecture connect into networks. In the Drosophila tracheal system, two tube types connect within single cells (terminal cells); however, the genes that mediate this interconnection are unknown. Here we characterize two genes that are essential for this process: lotus , required for maintaining a connection between the tubes, and wheezy , required to prevent local tube dilation. We find that lotus encodes N-ethylmaleimide sensitive factor 2 (NSF2), whereas wheezy encodes Germinal center kinase III (GCKIII). GCKIIIs are effectors of Cerebral cavernous malformation 3 (CCM3), a protein mutated in vascular disease. Depletion of Ccm3 by RNA interference phenocopies wheezy ; thus, CCM3 and GCKIII, which prevent capillary dilation in humans, prevent tube dilation in Drosophila trachea. Ectopic junctional and apical proteins are present in wheezy terminal cells, and we show that tube dilation is suppressed by reduction of NSF2, of the apical determinant Crumbs, or of septate junction protein Varicose.

The hexosamine biosynthetic pathway, whose end product is UDP-N acetylglucosamine (UDP-GlcNAc), lies at the base of cellular glycosylation pathways, including glycosylation of lipids, formation of heparin sulfated proteoglycans, and N- and O-linked glycosylation of proteins. Forward genetic studies in Drosophila have revealed that mutations in genes encoding different enzymes of the hexosamine biosynthetic pathway result in reduction of UDP-GlcNAc to different extents, with a consequent disruption of distinct glycosylation pathways and developmental processes. A maternal and zygotic loss-of-function screen has identified mutations in nesthocker ( nst ), which encodes an enzyme in the hexosamine biosynthetic pathway. Embryos lacking maternal and zygotic nst gene products show defective O-GlcNAcylation of a fibroblast growth factor receptor (FGFR)–specific adaptor protein, which impairs FGFR-dependent migration of mesodermal and tracheal cells.

Seamless tubes form intracellularly without cell–cell or autocellular junctions. Such tubes have been described across phyla, but remain mysterious despite their simple architecture. In Drosophila, seamless tubes are found within tracheal terminal cells, which have dozens of branched protrusions extending hundreds of micrometres. We find that mutations in multiple components of the dynein motor complex block seamless tube growth, raising the possibility that the lumenal membrane forms through minus-end-directed transport of apical membrane components along microtubules. Growth of seamless tubes is polarized along the proximodistal axis by Rab35 and its apical membrane-localized GAP, Whacked. Strikingly, loss of whacked (or constitutive activation of Rab35) leads to tube overgrowth at terminal cell branch tips, whereas overexpression of Whacked (or dominant-negative Rab35) causes formation of ectopic tubes surrounding the terminal cell nucleus. Thus, vesicle trafficking has key roles in making and shaping seamless tubes.

The Drosophila respiratory organ (tracheal system) consists of epithelial tubes, the morphogenesis of which is controlled by distinct sets of signaling pathways and transcription factors. The downstream events controlling tube formation and shape are only now beginning to be identified. Here we review recent insight into the communication between neighboring tracheal cells, their interactions with the surrounding matrix, and the impact of these processes on tube morphogenesis. We focus on cell-cell interactions that drive rearrangement of cells within the epithelium and that are essential for maintenance of epithelial integrity, and also on cell-matrix interactions that play key roles in determining and maintaining the size and shape of tube lumens.

Epithelial tubes that compose many organs are typically long lasting,except under specific developmental and physiological conditions when network remodeling occurs. Although there has been progress elucidating mechanisms of tube formation, little is known of the mechanisms that maintain tubes and destabilize them during network remodeling. Here, we describe Drosophila tendrils mutations that compromise maintenance of tracheal terminal branches, fine gauge tubes formed by tracheal terminal cells that ramify on and adhere tightly to tissues in order to supply them with oxygen. Homozygous tendrils terminal cell clones have fewer terminal branches than normal but individual branches contain multiple convoluted lumens. The phenotype arises late in development: terminal branches bud and form lumens normally early in development, but during larval life lumens become convoluted and mature branches degenerate. Their lumens, however, are retained in the remaining branches, resulting in the distinctive multi-lumen phenotype. Mapping and molecular studies demonstrate that tendrils is allelic to rhea, which encodes Drosophila talin, a large cytoskeletal protein that links integrins to the cytoskeleton. Terminal cells mutant for myospheroid, the major Drosophila β-integrin, or doubly mutant for multiple edematous wings and inflatedα-integrins, also show the tendrils phenotype, and localization of myospheroid β-integrin protein is disrupted in tendrils mutant terminal cells. The results provide evidence that integrin-talin adhesion complexes are necessary to maintain tracheal terminal branches and luminal organization. Similar complexes may stabilize other tubular networks and may be targeted for inactivation during network remodeling events.

The genes okra and spindle-B act during meiosis in Drosophila to repair double-stranded DNA breaks (DSBs) associated with meiotic recombination. Unexpectedly, mutations in these genes cause dorsoventral patterning defects during oogenesis. These defects result from a failure to accumulate Gurken protein, which is required to initiate dorsoventral patterning during oogenesis. Here we find that the block in Gurken accumulation in the oocyte cytoplasm reflects activation of a meiotic checkpoint in response to the persistence of DSBs in the nucleus. We also show that Vasa is a target of this meiotic checkpoint, and so may mediate the checkpoint-dependent translational regulation of Gurken.

okra (okr), spindle-B (spnB), andspindle-D (spnD) are three members of a group of female sterile loci that produce defects in oocyte and egg morphology, including variable dorsal–ventral defects in the eggshell and embryo, anterior–posterior defects in the follicle cell epithelium and in the oocyte, and abnormalities in oocyte nuclear morphology. Many of these phenotypes reflect defects in grk-Egfr signaling processes, and can be accounted for by a failure to accumulate wild-type levels of Gurken and Fs(1)K10. We have cloned okr and spnB, and show that okr encodes the Drosophila homolog of the yeast DNA-repair protein Rad54, and spnB encodes a Rad51-like protein related to the meiosis-specific DMC1 gene. In functional tests of their role in DNA repair, we find that okrbehaves like its yeast homolog in that it is required in both mitotic and meiotic cells. In contrast, spnB and spnD appear to be required only in meiosis. The fact that genes involved in meiotic DNA metabolism have specific effects on oocyte patterning implies that the progression of the meiotic cell cycle is coordinated with the regulation of certain developmental events during oogenesis.

Summary Most tubes have seams (intercellular or autocellular junctions that seal membranes together into a tube), but "seamless" tubes also exist [1–3]. In Drosophila , stellate-shaped tracheal terminal cells make seamless tubes, with single branches running through each of dozens of cellular extensions. We find that mutations in braided impair terminal cell branching and cause formation of seamless tube cysts. We show that braided encodes Syntaxin7 and that cysts also form in cells deficient for other genes required either for membrane scission ( shibire ) or for early endosome formation ( Rab5 , Vps45 , and Rabenosyn-5 ). These data define a requirement for early endocytosis in shaping seamless tube lumens. Importantly, apical proteins Crumbs and phospho-Moesin accumulate to aberrantly high levels in braided terminal cells. Overexpression of either Crumbs or phosphomimetic Moesin induced lumenal cysts and decreased terminal branching. Conversely, the braided seamless tube cyst phenotype was suppressed by mutations in crumbs or Moesin . Indeed, mutations in Moesin dominantly suppressed seamless tube cyst formation and restored terminal branching. We propose that early endocytosis maintains normal steady-state levels of Crumbs, which recruits apical phosphorylated (active) Moe, which in turn regulates seamless tube shape through modulation of cortical actin filaments.

Sequence data from this article have been deposited with the Gen-Bank data libraries under accession no. AY257540., In Drosophila, mutations in double-strand DNA break (DSB) repair enzymes, such as spn-B, activate a meiotic checkpoint leading to dorsal-ventral patterning defects in the egg and an abnormal appearance of the oocyte nucleus. Mutations in spn-D cause an array of ovarian phenotypes similar to spn-B. We have cloned the spn-D locus and found that it encodes a protein of 271 amino acids that shows significant homology to the human RAD51C protein. In mammals the spn-B and spn-D homologs, XRCC3 and RAD51C, play a role in genomic stability in somatic cells. To test for a similar role for spn-B and spn-D in double-strand DNA repair in mitotic cells, we analyzed the sensitivity of single and double mutants to DSBs induced by exposure to X rays and MMS. We found that neither singly mutant nor doubly mutant animals were significantly sensitized to MMS or X rays. These results suggest that spn-B and spn-D act in meiotic recombination but not in repair of DSBs in somatic cells. As there is no apparent ortholog of the meiosis-specific DMC1 gene in the Drosophila genome, and given their meiosis-specific requirement, we suggest that spn-B and spn-D may have a function comparable to DMC1., This work was supported by the PHS Grant PO1CA41086 and by the Howard Hughes Medical Institute. A.G.-R. acknowledges the financial support of the Spanish Ministerio de Ciencia y Tecnología (grant nos. PM99-0107 and BMC2001-4822-E), the Junta de Andalucía (CVI 724), and the EMBO Young Investigator Programme.