Drosophila Src regulates anisotropic apical surface growth to control epithelial tube size.
ABSTRACT: Networks of epithelial and endothelial tubes are essential for the function of organs such as the lung, kidney and vascular system. The sizes and shapes of these tubes are highly regulated to match their individual functions. Defects in tube size can cause debilitating diseases such as polycystic kidney disease and ischaemia. It is therefore critical to understand how tube dimensions are regulated. Here we identify the tyrosine kinase Src as an instructive regulator of epithelial-tube length in the Drosophila tracheal system. Loss-of-function Src42 mutations shorten tracheal tubes, whereas Src42 overexpression elongates them. Surprisingly, Src42 acts distinctly from known tube-size pathways and regulates both the amount of apical surface growth and, with the conserved formin dDaam, the direction of growth. Quantitative three-dimensional image analysis reveals that Src42- and dDaam-mutant tracheal cells expand more in the circumferential than the axial dimension, resulting in tubes that are shorter in length-but larger in diameter-than wild-type tubes. Thus, Src42 and dDaam control tube dimensions by regulating the direction of anisotropic growth, a mechanism that has not previously been described.
Project description:Regulation of epithelial tube size is critical for organ function. However, the mechanisms of tube size control remain poorly understood. In the Drosophila trachea, tube dimensions are regulated by a luminal extracellular matrix (ECM). ECM organization requires apical (luminal) secretion of the protein Vermiform (Verm), which depends on the basolateral septate junction (SJ). Here, we show that apical and basolateral epithelial polarity proteins interact to control tracheal tube size independently of the Verm pathway. Mutations in yurt (yrt) and scribble (scrib), which encode SJ-associated polarity proteins, cause an expansion of tracheal tubes but do not disrupt Verm secretion. Reducing activity of the apical polarity protein Crumbs (Crb) suppresses the length defects in yrt but not scrib mutants, suggesting that Yrt acts by negatively regulating Crb. Conversely, Crb overexpression increases tracheal tube dimensions. Reducing crb dosage also rescues tracheal size defects caused by mutations in coracle (cora), which encodes an SJ-associated polarity protein. In addition, crb mutations suppress cora length defects without restoring Verm secretion. Together, these data indicate that Yrt, Cora, Crb, and Scrib operate independently of the Verm pathway. Our data support a model in which Cora and Yrt act through Crb to regulate epithelial tube size.
Project description:Many organs are composed of branched networks of epithelial tubes that transport vital fluids or gases. The proper size and shape of tubes are crucial for their transport function, but the molecular processes that govern tube size and shape are not well understood. Here we show that three genes required for tracheal tube morphogenesis in Drosophila melanogaster encode proteins involved in the synthesis and accumulation of chitin, a polymer of N-acetyl-beta-D-glucosamine that serves as a scaffold in the rigid extracellular matrix of insect cuticle. In all three mutants, developing tracheal tubes bud and extend normally, but the epithelial walls of the tubes do not expand uniformly, and the resultant tubes are grossly misshapen, with constricted and distended regions all along their lengths. The genes are expressed in tracheal cells during the expansion process, and chitin accumulates in the lumen of tubes, forming an expanding cylinder that we propose coordinates the behavior of the surrounding tracheal cells and stabilizes the expanding epithelium. These findings show that chitin regulates epithelial tube morphogenesis, in addition to its classical role protecting mature epithelia.
Project description:Most organs of multicellular organisms are built from epithelial tubes. To exert their functions, tubes rely on apico-basal polarity, on junctions, which form a barrier to separate the inside from the outside, and on a proper lumen, required for gas or liquid transport. Here we identify apnoia (apn), a novel Drosophila gene required for tracheal tube elongation and lumen stability at larval stages. Larvae lacking Apn show abnormal tracheal inflation and twisted airway tubes, but no obvious defects in early steps of tracheal maturation. apn encodes a transmembrane protein, primarily expressed in the tracheae, which exerts its function by controlling the localization of Crumbs (Crb), an evolutionarily conserved apical determinant. Apn physically interacts with Crb to control its localization and maintenance at the apical membrane of developing airways. In apn mutant tracheal cells, Crb fails to localize apically and is trapped in retromer-positive vesicles. Consistent with the role of Crb in apical membrane growth, RNAi-mediated knockdown of Crb results in decreased apical surface growth of tracheal cells and impaired axial elongation of the dorsal trunk. We conclude that Apn is a novel regulator of tracheal tube expansion in larval tracheae, the function of which is mediated by Crb.
Project description:Epithelial organ size and shape depend on cell shape changes, cell-matrix communication, and apical membrane growth. The <i>Drosophila melanogaster</i> embryonic tracheal network is an excellent model to study these processes. Here, we show that the transcriptional coactivator of the Hippo pathway, Yorkie (YAP/TAZ in vertebrates), plays distinct roles in the developing <i>Drosophila</i> airways. Yorkie exerts a cytoplasmic function by binding <i>Drosophila</i> Twinstar, the orthologue of the vertebrate actin-severing protein Cofilin, to regulate F-actin levels and apical cell membrane size, which are required for proper tracheal tube elongation. Second, Yorkie controls water tightness of tracheal tubes by transcriptional regulation of the <i>?-aminolevulinate synthase</i> gene (<i>Alas</i>). We conclude that Yorkie has a dual role in tracheal development to ensure proper tracheal growth and functionality.
Project description:Insulin-like growth factors (IGFs) control cell and organism growth through evolutionarily conserved signaling pathways. The mammalian acid-labile subunit (ALS) is a secreted protein that complexes with IGFs to modulate their activity. Recent work has shown that a Drosophila homolog of ALS, dALS, can also complex with and modulate the activity of a Drosophila IGF. Here we report the first mutations in the gene encoding dALS. Unexpectedly, we find that these mutations are allelic to a previously described mutation in convoluted (conv), a gene required for epithelial morphogenesis. In conv mutants, the tubes of the Drosophila tracheal system become abnormally elongated without altering tracheal cell number. conv null mutations cause larval lethality, but do not disrupt several processes required for tracheal tube size control, including septate junction formation, deposition of a lumenal/apical extracellular matrix, and lumenal secretion of Vermiform and Serpentine, two putative matrix-modifying proteins. Clearance of lumenal matrix and subcellular localization of clathrin also appear normal in conv mutants. However, we show that Conv/dALS is required for the dynamic organization of the transient lumenal matrix and normal structure of the cuticle that lines the tracheal lumen. These and other data suggest that the Conv/dALS-dependent tube size control mechanism is distinct from other known processes involved in tracheal tube size regulation. Moreover, we present evidence indicating that Conv/dALS has a novel, IGF-signaling independent function in tracheal morphogenesis.
Project description:Tubes with distinct shapes and sizes are critical for the proper function of many tubular organs. Here we describe a unique phenotype caused by the loss of a novel, evolutionarily-conserved, Drosophila Smad-like protein, Expansion. In expansion mutants, unicellular and intracellular tracheal branches develop bubble-like cysts with enlarged apical membranes. Cysts in unicellular tubes are enlargements of the apical lumen, whereas cysts in intracellular tubes are cytoplasmic vacuole-like compartments. The cyst phenotype in expansion mutants is similar to, but weaker than, that observed in double mutants of Drosophila type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. Ptp4E and Ptp10D negatively regulate the receptor tyrosine kinase (RTK) pathways, especially epithelial growth factor receptor (EGFR) and fibroblast growth factor receptor/breathless (FGFR, Btl) signaling to maintain the proper size of unicellular and intracellular tubes. We show Exp genetically interacts with RTK signaling, the downstream targets of RPTPs. Cyst size and number in expansion mutants is enhanced by increased RTK signaling and suppressed by reduced RTK signaling. Genetic interaction studies strongly suggest that Exp negatively regulates RTK (EGFR, Btl) signaling to ensure proper tube sizes. Smad proteins generally function as intermediate components of the transforming growth factor-? (TGF-?, DPP) signaling pathway. However, no obvious genetic interaction between expansion and TGF-? (DPP) signaling was observed. Therefore, Expansion does not function as a typical Smad protein. The expansion phenotype demonstrates a novel role for Smad-like proteins in epithelial tube formation.
Project description:Skeletal muscle provides inspiration on how to achieve reversible, macroscopic, anisotropic motion in soft materials. Here we report on the bottom-up design of macroscopic tubes that exhibit anisotropic actuation driven by a thermal stimulus. The tube is built from a hydrogel in which extremely long supramolecular nanofibers are aligned using weak shear forces, followed by radial growth of thermoresponsive polymers from their surfaces. The hierarchically ordered tube exhibits reversible anisotropic actuation with changes in temperature, with much greater contraction perpendicular to the direction of nanofiber alignment. We identify two critical factors for the anisotropic actuation, macroscopic alignment of the supramolecular scaffold and its covalent bonding to polymer chains. Using finite element analysis and molecular calculations, we conclude polymer chain confinement and mechanical reinforcement by rigid supramolecular nanofibers are responsible for the anisotropic actuation. The work reported suggests strategies to create soft active matter with molecularly encoded capacity to perform complex tasks.
Project description:How do flat sheets of cells form gut and neural tubes? Across systems, several mechanisms are at play: cells wedge, form actomyosin cables, or intercalate. As a result, the cell sheet bends, and the tube elongates. It is unclear to what extent each mechanism can drive tube formation on its own. To address this question, we computationally probe if one mechanism, either cell wedging or intercalation, may suffice for the entire sheet-to-tube transition. Using a physical model with epithelial cells represented by polarized point particles, we show that either cell intercalation or wedging alone can be sufficient and that each can both bend the sheet and extend the tube. When working in parallel, the two mechanisms increase the robustness of the tube formation. The successful simulations of the key features in Drosophila salivary gland budding, sea urchin gastrulation, and mammalian neurulation support the generality of our results.
Project description:The control of the size of internal tubular organs, such as the lungs or vascular system, is critical for proper physiological activity and to prevent disease or malformations. This control incorporates the intrinsic physical anisotropy of tubes to generate proportionate organs that match their function. The exact mechanisms underlying tube size control and how tubular anisotropy is translated at the cellular level are still not fully understood. Here we investigate these mechanisms using the Drosophila tracheal system. We show that the apical polarity protein Crumbs transiently accumulates anisotropically at longitudinal cell junctions during tube elongation. We provide evidence indicating that the accumulation of Crumbs in specific apical domains correlates with apical surface expansion, suggesting a link between the anisotropic accumulation of Crumbs at the cellular level and membrane expansion. We find that Src42A is required for the anisotropic accumulation of Crumbs, thereby identifying the first polarised cell behaviour downstream of Src42A. Our results indicate that Src42A regulates a mechanism that increases the fraction of Crb protein at longitudinal junctions, and genetic interaction experiments are consistent with Crb acting downstream of Src42A in controlling tube size. Collectively, our results suggest a model in which Src42A would sense the inherent anisotropic mechanical tension of the tube and translate it into a polarised Crumbs accumulation, which may promote a bias towards longitudinal membrane expansion, orienting cell elongation and, as a consequence, longitudinal growth at the tissue level. This work provides new insights into the key question of how organ growth is controlled and polarised and unveils the function of two conserved proteins, Crumbs and Src42A, with important roles in development and homeostasis as well as in disease, in this biological process.
Project description: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.