Monn Monn Myat

Epithelial Tube Morphology Is Determined by the Polarized Growth and Delivery of Apical Membrane

Formation of tubes of the correct size and shape is essential for viability of most organisms, yet little is understood of the mechanisms controlling tube morphology. We identified a new allele of hairy in a mutagenesis screen and showed that hairy mutations cause branching and bulging of the normally unbranched salivary tube, in part through prolonged expression of huckebein (hkb). HKB controls polarized cell shape change and apical membrane growth during salivary cell invagination via two downstream target genes, crumbs (crb), a determinant of the apical membrane, and klarsicht (klar), which mediates microtubule-dependent organelle transport. In invaginating salivary cells, crb and klar mediate growth and delivery of apical membrane, respectively, thus regulating the size and shape of the salivary tube.

Ribbon modulates apical membrane during tube elongation through Crumbs and Moesin

Although the formation and maintenance of epithelial tubes are essential for the viability of multicellular organisms, our understanding of the molecular and cellular events coordinating tubulogenesis is relatively limited. Here, we focus on the activities of Ribbon, a novel BTB-domain containing nuclear protein, in the elongation of two epithelial tubes: the Drosophila salivary gland and trachea. We show that Ribbon interacts with Lola Like, another BTB-domain containing protein required for robust nuclear localization of Ribbon, to upregulate crumbs expression and downregulate Moesin activity. Our ultrastructural analysis of ribbon null salivary glands by TEM reveals a diminished pool of subapical vesicles and an increase in microvillar structure, cellular changes consistent with the known role of Crumbs in apical membrane generation and of Moesin in the cross-linking of the apical membrane to the subapical cytoskeleton. Furthermore, the subapical localization of Rab11, a small GTPase associated with apical membrane delivery and rearrangement, is significantly diminished in ribbon mutant salivary glands and tracheae. These findings suggest that Ribbon and Lola Like function as a novel transcriptional cassette coordinating molecular changes at the apical membrane of epithelial cells to facilitate tube elongation.

Posterior migration of the salivary gland requires an intact visceral mesoderm and integrin function

The final overall shape of an organ and its position within the developing embryo arise as a consequence of both its intrinsic properties and its interactions with surrounding tissues. Here, we focus on the role of directed cell migration in shaping and positioning the Drosophila salivary gland. We demonstrate that the salivary gland turns and migrates along the visceral mesoderm to become properly oriented with respect to the overall embryo. We show that salivary gland posterior migration requires the activities of genes that position the visceral mesoderm precursors, such as heartless, thickveins, and tinman, but does not require a differentiated visceral mesoderm. We also demonstrate a role for integrin function in salivary gland migration. Although the mutations affecting salivary gland motility and directional migration cause defects in the final positioning of the salivary gland, most do not affect the length or diameter of the salivary gland tube. These findings suggest that salivary tube dimensions may be an intrinsic property of salivary gland cells.

Rho GTPase controls invagination and cohesive migration of the Drosophila salivary gland through Crumbs and Rho-kinase

Coordinated cell movements shape simple epithelia into functional tissues and organs during embryogenesis. Regulators and effectors of the small GTPase Rho have been shown to be essential for epithelial morphogenesis in cell culture; however, the mechanism by which Rho GTPase and its downstream effectors control coordinated movement of epithelia in a developing tissue or organ is largely unknown. Here, we show that Rho1 GTPase activity is required for the invagination of Drosophila embryonic salivary gland epithelia and for directed migration of the internalized gland. We demonstrate that the absence of zygotic function of Rho1 results in the selective loss of the apical proteins, Crumbs (Crb), Drosophila atypical PKC and Stardust during gland invagination and that this is partially due to reduced crb RNA levels and apical localization. In parallel to regulation of crb RNA and protein, Rho1 activity also signals through Rho-kinase (Rok) to induce apical constriction and cell shape change during invagination. After invagination, Rho-Rok signaling is required again for the coordinated contraction and dorsal migration of the proximal half of the gland. We also show that Rho1 activity is required for proper development of the circular visceral mesoderm upon which the gland migrates. Our genetic and live-imaging analyses provide novel evidence that the proximal gland cells play an essential and active role in salivary gland migration that propels the entire gland to turn and migrate posteriorly.

Making tubes in the Drosophila embryo

Epithelial and endothelial tubes come in various shapes and sizes and form the basic units of many tubular organs. During embryonic development, single unbranched tubes as well as highly branched networks of tubes form from simple sheets of cells by several morphogenic movements. Studies of tube formation in the Drosophila embryo have greatly advanced our understanding of the cellular and molecular mechanisms by which tubes are formed. This review highlights recent progress on formation of the hindgut, Malpighian tubules, proventriculus, salivary gland, and trachea of the Drosophila embryo, focusing on the cellular events that form each tube and their genetic requirements. Developmental Dynamics 232:617–632, 2005.

Pak1 control of E-cadherin endocytosis regulates salivary gland lumen size and shape

Generating and maintaining proper lumen size and shape in tubular organs is essential for organ function. Here, we demonstrate a novel role for p21-activated kinase 1 (Pak1) in defining the size and shape of the Drosophila embryonic salivary gland lumen by regulating the size and elongation of the apical domain of individual cells. Pak1 mediates these effects by decreasing and increasing E-cadherin levels at the adherens junctions and basolateral membrane, respectively, through Rab5- and Dynamin-dependent endocytosis. We also demonstrate that Cdc42 and Merlin act together with Pak1 to control lumen size. A role for Pak1 in E-cadherin endocytosis is supported by our studies of constitutively active Pak1, which induces the formation of multiple intercellular lumens in the salivary gland in a manner dependent on Rab5, Dynamin and Merlin. These studies demonstrate a novel and crucial role for Pak1 and E-cadherin endocytosis in determining lumen size and shape, and also identify a mechanism for multiple lumen formation, a poorly understood process that occurs in normal embryonic development and pathological conditions.

Rho GTPase controls Drosophila salivary gland lumen size through regulation of the actin cytoskeleton and Moesin

Generation and maintenance of proper lumen size is important for tubular organ function. We report on a novel role for the Drosophila Rho1 GTPase in control of salivary gland lumen size through regulation of cell rearrangement, apical domain elongation and cell shape change. We show that Rho1 controls cell rearrangement and apical domain elongation by promoting actin polymerization and regulating F-actin distribution at the apical and basolateral membranes through Rho kinase. Loss of Rho1 resulted in reduction of F-actin at the basolateral membrane and enrichment of apical F-actin, the latter accompanied by enrichment of apical phosphorylated Moesin. Reducing cofilin levels in Rho1 mutant salivary gland cells restored proper distribution of F-actin and phosphorylated Moesin and rescued the cell rearrangement and apical domain elongation defects of Rho1 mutant glands. In support of a role for Rho1-dependent actin polymerization in regulation of gland lumen size, loss of profilin phenocopied the Rho1 lumen size defects to a large extent. We also show that Ribbon, a BTB domain-containing transcription factor functions with Rho1 in limiting apical phosphorylated Moesin for apical domain elongation. Our studies reveal a novel mechanism for controlling salivary gland lumen size, namely through Rho1-dependent actin polymerization and distribution and downregulation of apical phosphorylated Moesin.

Rac1 GTPase acts downstream of αPS1βPS integrin to control collective migration and lumen size in the Drosophila salivary gland

During collective migration of the Drosophila embryonic salivary gland, the distal gland cells mediate integrin-based contacts with surrounding tissues while proximal gland cells change shape and rearrange. Here, we show that αPS1βPS integrin controls salivary gland migration through Rac1 GTPase which downregulates E-cadherin in proximal and distal gland cells, and promotes extension of actin-rich basal membrane protrusions in the distal cells. In embryos mutant for multiple edematous wings (mew), which encodes the αPS1 subunit of the αPS1βPS integrin heterodimer, or rac1 and rac2 GTPases, salivary gland cells failed to migrate, to downregulate E-cadherin and to extend basal membrane protrusions. Selective inhibition of Rac1 in just the proximal or distal gland cells demonstrate that proximal gland cells play an active role in the collective migration of the whole gland and that continued migration of the distal cells depends on the proximal cells. Loss of rac1rac2 also affected gland lumen length and width whereas, loss of mew affected lumen length only. Activation of rac1 in mew mutant embryos significantly rescued the gland migration, lumen length and basal membrane protrusion defects and partially rescued the E-cadherin defects. Independent of mew, Rac regulates cell shape change and rearrangement in the proximal gland, which is important for migration and lumen width. Our studies shed novel insight into a Rac1-mediated link between integrin and cadherin adhesion proteins in vivo, control of lumen length and width and how activities of proximal and distal gland cells are coordinated to result in the collective migration of the entire salivary gland.

Genetic Regulation of Salivary Gland Development in Drosophila melanogaster

Studies of salivary gland development in the Drosophila embryo have revealed the morphogenic events by which the salivary gland tubes are formed, and identified the genes and genetic networks that regulate these events. Invagination of the salivary gland primordium occurs by an apical constriction mechanism regulated by the tyrosine kinase, Tec29, the transcription factors, Fork head and Huckebein, and Rho GTPase-mediated actomyosin contraction. After invagination is complete, transcriptional control of the apical membrane protein, Crumbs, by the transcription factors, Hairy, Hkb and Ribbon, and downregulation of Moesin-dependent apical stiffness promotes elongation of the salivary gland lumen. Integrin-mediated adhesion between the gland and surrounding mesoderm, coupled with Rho GTPase-mediated contraction of the proximal gland cells and downregulation of E-cadherin-based cell-cell adhesion by the Rac GTPases, allow turning and posterior migration of the salivary gland. Further posterior migration of the salivary gland is dependent on axon guidance cues, such as Robo and Slit, and close association with surrounding tissues. Many of the genes identified as regulators of salivary gland invagination and migration also control invagination of other epithelial tissues and migration of diverse cell types. Thus, studies of Drosophila salivary gland morphogenesis continue to illuminate the conserved mechanisms by which cells give rise to three-dimensional tissues and organs during embryogenesis.

Deficiency screen identifies a novel role for beta 2 tubulin in salivary gland and myoblast migration in the Drosophila embryo.

The Drosophila embryonic salivary gland is an epithelial organ formed by the coordinated invagination and migration of primordial cells. To identify genes that regulate gland migration we performed a deficiency screen of the third chromosome. Here, we report on the analysis of the beta 2 tubulin isoform (β2t) that maps at 85D15. We show that, in β2t mutant embryos, salivary glands did not complete their posterior migration and that migration of fusion competent myoblasts and longitudinal visceral muscle founder cells between the gland and circular visceral mesoderm was delayed. We also demonstrate that gland migration defects correlate with reduced βPS and αPS2 integrin expression in the surrounding mesoderm and that β2t genetically interacts with genes encoding integrin αPS1 and αPS2 subunits. Our studies reveal for the first time that β2t is expressed in embryogenesis and that β2t plays an important role in salivary gland and myoblast migration, possibly through proper regulation of integrin adhesion proteins. Developmental Dynamics 238:853–863, 2009.