Drosophila Epithelia Research Articles

The transmembrane protein Crumbs displays complex dynamics during follicular morphogenesis and is regulated competitively by Moesin and aPKC.

The transmembrane protein Crumbs (Crb) functions in apical polarity and epithelial integrity. To better understand its role in epithelial morphogenesis, we examined Crb localization and dynamics in the late follicular epithelium of Drosophila. Crb was unexpectedly dynamic during middle-to-late stages of egg chamber development, being lost from the marginal zone (MZ) in stage 9 before abruptly returning at the end of stage 10b, then undergoing a pulse of endocytosis in stage 12. The reappearance of MZ Crb is necessary to maintain an intact adherens junction and MZ. Although Crb has been proposed to interact through its juxtamembrane domain with Moesin (Moe), a FERM domain protein that regulates the cortical actin cytoskeleton, the functional significance of this interaction is poorly understood. We found that whereas the Crb juxtamembrane domain was not required for adherens junction integrity, it was necessary for MZ localization of Moe, aPKC and F-actin. Furthermore, Moe and aPKC functioned antagonistically, suggesting that Moe limits Crb levels by reducing its interactions with the apical Par network. Additionally, Moe mutant cells lost Crb from the apical membrane and accumulated excess Crb at the MZ, suggesting that Moe regulates Crb distribution at the membrane. Together, these studies reveal reciprocal interactions between Crb, Moe and aPKC during cellular morphogenesis.

The Spectrin cytoskeleton regulates the Hippo signalling pathway.

The Spectrin cytoskeleton is known to be polarised in epithelial cells, yet its role remains poorly understood. Here, we show that the Spectrin cytoskeleton controls Hippo signalling. In the developing Drosophila wing and eye, loss of apical Spectrins (alpha/beta-heavy dimers) produces tissue overgrowth and mis-regulation of Hippo target genes, similar to loss of Crumbs (Crb) or the FERM-domain protein Expanded (Ex). Apical beta-heavy Spectrin binds to Ex and co-localises with it at the apical membrane to antagonise Yki activity. Interestingly, in both the ovarian follicular epithelium and intestinal epithelium of Drosophila, apical Spectrins and Crb are dispensable for repression of Yki, while basolateral Spectrins (alpha/beta dimers) are essential. Finally, the Spectrin cytoskeleton is required to regulate the localisation of the Hippo pathway effector YAP in response to cell density human epithelial cells. Our findings identify both apical and basolateral Spectrins as regulators of Hippo signalling and suggest Spectrins as potential mechanosensors.

Bacterial uracil modulates Drosophila DUOX-dependent gut immunity via Hedgehog-induced signaling endosomes.

Genetic studies in Drosophila have demonstrated that generation of microbicidal reactive oxygen species (ROS) through the NADPH dual oxidase (DUOX) is a first line of defense in the gut epithelia. Bacterial uracil acts as DUOX-activating ligand through poorly understood mechanisms. Here, we show that the Hedgehog (Hh) signaling pathway modulates uracil-induced DUOX activation. Uracil-induced Hh signaling is required for intestinal expression of the calcium-dependent cell adhesion molecule Cadherin 99C (Cad99C) and subsequent Cad99C-dependent formation of endosomes. These endosomes play essential roles in uracil-induced ROS production by acting as signaling platforms for PLCβ/PKC/Ca2+-dependent DUOX activation. Animals with impaired Hh signaling exhibit abolished Cad99C-dependent endosome formation and reduced DUOX activity, resulting in high mortality during enteric infection. Importantly, endosome formation, DUOX activation, and normal host survival are restored by genetic reintroduction of Cad99C into enterocytes, demonstrating the important role for Hh signaling in host resistance to enteric infection.

The role of Drosophila antifungal immune response genes in intestinal homeostasis

Abstract The signaling pathways that control intestinal development, regeneration and disease show a high degree of conservation between Drosophila and mammals. The gut epithelia of Drosophila provide protection against invasion of microorganisms through production of reactive oxygen species (ROS) and antimicrobial peptides (AMPs). Although Drosophila gut immunity has been extensively studied, the specific responses to Gram-positive bacteria, fungi and toxic compounds are not fully understood. To identify the physiological role of genes involved in host defense we studied Drosophila mutants in antifungal genes identified previously and tested their survival upon feeding with various pathogens and toxic compounds. The results showed that several mutants displayed decreased viability compared with wild-type flies, and the lower survival rates were attributed to morphological change and excessive cell death in mutant guts. Thus, we identified several new Drosophila genes (spen, jumeau, inv, DDB1 and shg) required for intestinal homeostasis or stress responses.

In Vivo Imaging of Hedgehog Transport in Drosophila Epithelia.

The Hedgehog (Hh) signaling pathway is a regulator of patterning, cell migration and axon guidance during development as well as of homeostatic events in adult organs. It is highly conserved from Drosophila to humans. In many contexts during development, Hh appears to function as a morphogen; it spreads from producing cells to trigger concentration dependent responses in target cells, leading to their specification. During production, Hh undergoes two lipid modifications resulting in a highly hydrophobic molecule. The processes that create lipid-modified Hh for release from producing cells and that move it to target cells in a graded manner are complex. While most of the work done trying to explain Hh gradient formation is based on immunohistochemical studies in steady state, in vivo imaging in intact organisms is the finest technique to study gradient formation in real time. Both the wing imaginal disc epithelium and the adult abdominal epidermis of Drosophila are well suited for in vivo imaging. They allow us to observe the behavior of cells and fluorescently labeled proteins, without interfering with development. Here, we describe in vivo imaging methods for these two epithelia, which allowed us to study Hh transport along specialized cytoplasmic protrusions called cytonemes.

A dynamic intracellular distribution of Vangl2 accompanies cell polarization during zebrafish gastrulation.

During vertebrate gastrulation, convergence and extension movements elongate embryonic tissues anteroposteriorly and narrow them mediolaterally. Planar cell polarity (PCP) signaling is essential for mediolateral cell elongation underlying these movements, but how this polarity arises is poorly understood. We analyzed the elongation, orientation and migration behaviors of lateral mesodermal cells undergoing convergence and extension movements in wild-type zebrafish embryos and mutants for the Wnt/PCP core component Vangl2 (Trilobite). We demonstrate that Vangl2 function is required at the time when cells transition to a highly elongated and mediolaterally aligned body. vangl2 mutant cells fail to undergo this transition and to migrate along a straight path with high net speed towards the dorsal midline. Instead, vangl2 mutant cells exhibit an anterior/animal pole bias in cell body alignment and movement direction, suggesting that PCP signaling promotes effective dorsal migration in part by suppressing anterior/animalward cell polarity and movement. Endogenous Vangl2 protein accumulates at the plasma membrane of mesenchymal converging cells at the time its function is required for mediolaterally polarized cell behavior. Heterochronic cell transplantations demonstrated that Vangl2 cell membrane accumulation is stage dependent and regulated by both intrinsic factors and an extracellular signal, which is distinct from PCP signaling or other gastrulation regulators, including BMP and Nodals. Moreover, mosaic expression of fusion proteins revealed enrichment of Vangl2 at the anterior cell edges of highly mediolaterally elongated cells. These results demonstrate that the dynamic Vangl2 intracellular distribution is coordinated with and necessary for the changes in convergence and extension cell behaviors during gastrulation.

Vangl2 cooperates with Rab11 and Myosin V to regulate apical constriction during vertebrate gastrulation.

Core planar cell polarity (PCP) proteins are well known to regulate polarity in Drosophila and vertebrate epithelia; however, their functions in vertebrate morphogenesis remain poorly understood. In this study, we describe a role for PCP signaling in the process of apical constriction during Xenopus gastrulation. The core PCP protein Vangl2 is detected at the apical surfaces of cells at the blastopore lip, and it functions during blastopore formation and closure. Further experiments show that Vangl2, as well as Daam1 and Rho-associated kinase (Rock), regulate apical constriction of bottle cells at the blastopore and ectopic constriction of ectoderm cells triggered by the actin-binding protein Shroom3. At the blastopore lip, Vangl2 is required for the apical accumulation of the recycling endosome marker Rab11. We also show that Rab11 and the associated motor protein Myosin V play essential roles in both endogenous and ectopic apical constriction, and might be involved in Vangl2 trafficking to the cell surface. Overexpression of Rab11 RNA was sufficient to partly restore normal blastopore formation in Vangl2-deficient embryos. These observations suggest that Vangl2 affects Rab11 to regulate apical constriction during blastopore formation.

Planar Cell Polarity: The Importance of Getting It Backwards

The core and Fat-Dachsous signaling systems locally align planar cell polarities in Drosophila epithelia. Three recent papers address how coupling between these systems can be altered and reversed by the products of the gene prickle.

The roles and regulation of multicellular rosette structures during morphogenesis.

Multicellular rosettes have recently been appreciated as important cellular intermediates that are observed during the formation of diverse organ systems. These rosettes are polarized, transient epithelial structures that sometimes recapitulate the form of the adult organ. Rosette formation has been studied in various developmental contexts, such as in the zebrafish lateral line primordium, the vertebrate pancreas, the Drosophila epithelium and retina, as well as in the adult neural stem cell niche. These studies have revealed that the cytoskeletal rearrangements responsible for rosette formation appear to be conserved. By contrast, the extracellular cues that trigger these rearrangements in vivo are less well understood and are more diverse. Here, we review recent studies of the genetic regulation and cellular transitions involved in rosette formation. We discuss and compare specific models for rosette formation and highlight outstanding questions in the field.

C-Src drives intestinal regeneration and transformation.

The non-receptor tyrosine kinase c-Src, hereafter referred to as Src, is overexpressed or activated in multiple human malignancies. There has been much speculation about the functional role of Src in colorectal cancer (CRC), with Src amplification and potential activating mutations in up to 20%of the human tumours, although this has never been addressed due to multiple redundant family members. Here, we have used the adult Drosophila and mouse intestinal epithelium as paradigms to define a role for Src during tissue homeostasis, damage-induced regeneration and hyperplasia. Through genetic gain and loss of function experiments, we demonstrate that Src is necessary and sufficient to drive intestinal stem cell (ISC) proliferation during tissue self-renewal, regeneration and tumourigenesis. Surprisingly, Src plays a non-redundant role in the mouse intestine, which cannot be substituted by the other family kinases Fyn and Yes. Mechanistically, we show that Src drives ISC proliferation through upregulation of EGFR and activation of Ras/MAPK and Stat3 signalling. Therefore, we demonstrate a novel essential role for Src in intestinal stem/progenitor cell proliferation and tumourigenesis initiation in vivo.

ESCRT-0 Is Not Required for Ectopic Notch Activation and Tumor Suppression in Drosophila

Multivesicular endosome (MVE) sorting depends on proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) family. These are organized in four complexes (ESCRT-0, -I, -II, -III) that act in a sequential fashion to deliver ubiquitylated cargoes into the internal luminal vesicles (ILVs) of the MVE. Drosophila genes encoding ESCRT-I, -II, -III components function in sorting signaling receptors, including Notch and the JAK/STAT signaling receptor Domeless. Loss of ESCRT-I, -II, -III in Drosophila epithelia causes altered signaling and cell polarity, suggesting that ESCRTs genes are tumor suppressors. However, the nature of the tumor suppressive function of ESCRTs, and whether tumor suppression is linked to receptor sorting is unclear. Unexpectedly, a null mutant in Hrs, encoding one of the components of the ESCRT-0 complex, which acts upstream of ESCRT-I, -II, -III in MVE sorting is dispensable for tumor suppression. Here, we report that two Drosophila epithelia lacking activity of Stam, the other known components of the ESCRT-0 complex, or of both Hrs and Stam, accumulate the signaling receptors Notch and Dome in endosomes. However, mutant tissue surprisingly maintains normal apico-basal polarity and proliferation control and does not display ectopic Notch signaling activation, unlike cells that lack ESCRT-I, -II, -III activity. Overall, our in vivo data confirm previous evidence indicating that the ESCRT-0 complex plays no crucial role in regulation of tumor suppression, and suggest re-evaluation of the relationship of signaling modulation in endosomes and tumorigenesis.

Methods for studying planar cell polarity

Planar cell polarity (PCP) is the polarity of epithelial cells in the plane orthogonal to the apical–basal axis, and is controlled by a partially defined signaling system [54,76]. PCP related signaling also plays roles in cell migration, tissue re-organization and stem cell differentiation during embryonic development, and later, in regeneration and repair [29,58]. Aberrant signaling has been linked to a broad range of pathophysiologies including cancer, developmental defects, and neurological disorders [29,58,89]. The deepest mechanistic insights have come from studies of PCP in Drosophila[53,76]. In this chapter we review tools and methods to study PCP signaling in Drosophila epithelia, where it was found to involve asymmetric protein localization that is coordinated between adjacent cells. Such signaling has been most extensively studied in wing, eye, and abdomen, but also in other tissues such as leg and notum [1,45]. In the adult fly, PCP is manifested in the coordinated direction of hairs and bristles, as well as the organization of ommatidia in the eye. The polarity of these structures is preceded by asymmetric localization of PCP signaling proteins at the apical junctions of epithelial cells. Based on genetic and molecular criteria, the proteins that govern PCP can be divided into distinct modules, including the core module, the Fat/Dachsous/Four-jointed (Fat/Ds/Fj) module (often referred to as the ‘global’ module) as well as tissue specific effector modules [36,54,91]. Different tissues and tissue regions differ in their sensitivity to disturbances in the various modules of the PCP signaling system, leading to controversies about the interactions among the modules, and emphasizing the value of studying PCP in multiple contexts [45,62]. Here, we review methods including those generally applicable, as well as some that are selectively useful for analyses of PCP in eye (including eye discs), wing (including wing discs), pupal and adult abdomen, and the cuticle of larvae and embryos.

Tumor suppressor roles of CENP-E and Nsl1 in Drosophila epithelial tissues

Depletion of spindle assembly checkpoint (SAC) genes in Drosophila epithelial tissues leads to JNK-dependent programmed cell death and additional blockade of the apoptotic program drives tumorigenesis. A recent report proposes that chromosomal instability (CIN) is not the driving force in the tumorigenic response of the SAC-deficient tissue, and that checkpoint proteins exert a SAC-independent tumor suppressor role. This notion is based on observations that the depletion of CENP-E levels or prevention of Bub3 from binding to the kinetochore in Drosophila tissues unable to activate the apoptotic program induces CIN but does not cause hyperproliferation. Here we re-examined this proposal. In contrast to the previous report, we observed that depletion of CENP-E or Nsl1—the latter mediating kinetochore targeting of Bub3—in epithelial tissues unable to activate the apoptotic program induces significant levels of aneuploidy and drives tumor-like growth. The induction of the JNK transcriptional targets Wingless, a mitogenic molecule, and MMP1, a matrix metaloproteinase 1 involved in basement membrane degradation was also observed in these tumors. An identical response of the tissue was previously detected upon depletion of several SAC genes or genes involved in spindle assembly, chromatin condensation, and cytokinesis, all of which have been described to cause CIN. All together, these results reinforce the role of CIN in driving tumorigenesis in Drosophila epithelial tissues and question the proposed SAC-independent roles of checkpoint proteins in suppressing tumorigenesis. Differences in aneuploidy rates might explain the discrepancy between the previous report and our results.

2P135 アポトーシスに伴う上皮恒常性維持への機械的力の寄与(09. 発生・分化,ポスター,第52回日本生物物理学会年会(2014年度))

2P135 アポトーシスに伴う上皮恒常性維持への機械的力の寄与(09. 発生・分化,ポスター,第52回日本生物物理学会年会(2014年度))

Polyploidization and Cell Fusion Contribute to Wound Healing in the Adult Drosophila Epithelium

Reestablishing epithelial integrity and biosynthetic capacity is critically important following tissue damage. The adult Drosophila abdominal epithelium provides an attractive new system to address how postmitotic diploid cells contribute to repair. Puncture wounds to the adult Drosophila epidermis close initially by forming a melanized scab. We found that epithelial cells near the wound site fuse to form a giant syncytium, which sends lamellae under the scab to re-epithelialize the damaged site. Other large cells arise more peripherally by initiating endocycles and becoming polyploid, or by cell fusion. Rac GTPase activity is needed for syncytium formation, while the Hippo signaling effector Yorkie modulates both polyploidization and cell fusion. Large cell formation is functionally important because when both polyploidization and fusion are blocked, wounds do not re-epithelialize. Our observations indicate that cell mass lost upon wounding can be replaced by polyploidization instead of mitotic proliferation. We propose that large cells generated by polyploidization or cell fusion are essential because they are better able than diploid cells to mechanically stabilize wounds, especially those containing permanent acellular structures, such as scar tissue.

Cytonemes are required for the establishment of a normal Hedgehog morphogen gradient in Drosophila epithelia

SummaryHedgehog (Hh) signalling is important in development, stem cell biology and disease. In a variety of tissues, Hh acts as a morphogen to regulate growth and cell fate specification. Several hypotheses have been proposed to explain morphogen movement, one of which is transport via filopodia-like protrusions called cytonemes. Here, we analyse the mechanism underlying Hh movement in the wing disc and the abdominal epidermis of Drosophila. We show that, in both epithelia, cells generate cytonemes in regions of Hh signalling. These protrusions are actin-based and span several cell diameters. Various Hh signalling components localise to cytonemes, as well as to punctate structures that move along cytonemes and are probably exovesicles. Using in vivo imaging, we show that cytonemes are dynamic structures and that Hh gradient establishment correlates with cytoneme formation in space and time. Indeed, mutant conditions that affect cytoneme formation reduce both cytoneme length and Hh gradient length. Our results suggest that cytoneme-mediated Hh transport is the mechanistic basis for Hh gradient formation.

The distribution of Dishevelled in convergently extending mesoderm

Convergent extension (CE) is a conserved morphogenetic movement that drives axial lengthening of the primary body axis and depends on the planar cell polarity (PCP) pathway. In Drosophila epithelia, a polarised subcellular accumulation of PCP core components, such as Dishevelled (Dvl) protein, is associated with PCP function. Dvl has long been thought to accumulate in the mediolateral protrusions in Xenopus chordamesoderm cells undergoing CE. Here we present a quantitative analysis of Dvl intracellular localisation in Xenopus chordamesoderm cells. We find that, surprisingly, accumulations previously observed at mediolateral protrusions of chordamesodermal cells are not protrusion-specific but reflect yolk-free cytoplasm and are quantitatively matched by the distribution of the cytoplasm-filling lineage marker dextran. However, separating cell cortex-associated from bulk Dvl signal reveals a statistical enrichment of Dvl in notochord–somite boundary-(NSB)-directed protrusions, which is dependent upon NSB proximity. Dvl puncta were also observed, but only upon elevated overexpression. These puncta showed no statistically significant spatial bias, in contrast to the strongly posteriorly-enriched GFP-Dvl puncta previously reported in zebrafish. We propose that Dvl distribution is more subtle and dynamic than previously appreciated and that in vertebrate mesoderm it reflects processes other than protrusion as such.

Protein trafficking abnormalities inDrosophilatissues with impaired activity of the ZIP7 zinc transporter Catsup

Developmental patterning requires the precise interplay of numerous intercellular signaling pathways to ensure that cells are properly specified during tissue formation and organogenesis. The spatiotemporal function of the Notch signaling pathway is strongly influenced by the biosynthesis and intracellular trafficking of signaling components. Receptors and ligands must be trafficked to the cell surface where they interact, and their subsequent endocytic internalization and endosomal trafficking is crucial for both signal propagation and its down-modulation. In a forward genetic screen for mutations that alter intracellular Notch receptor trafficking in Drosophila epithelial tissues, we recovered mutations that disrupt the Catsup gene, which encodes the Drosophila ortholog of the mammalian ZIP7 zinc transporter. Loss of Catsup function causes Notch to accumulate abnormally in the endoplasmic reticulum (ER) and Golgi compartments, resulting in impaired Notch signaling. In addition, Catsup mutant cells exhibit elevated ER stress, suggesting that impaired zinc homeostasis causes increased levels of misfolded proteins within the secretory compartment.

Activation of the proton pump, V-ATPase, triggers JNK-dependent cell invasion and overgrowth in a Drosophila epithelium

Activation of the proton pump, V-ATPase, triggers JNK-dependent cell invasion and overgrowth in a <i>Drosophila</i> epithelium

Parvin Overexpression Uncovers Tissue-Specific Genetic Pathways and Disrupts F-Actin to Induce Apoptosis in the Developing Epithelia in Drosophila

Parvin is a putative F-actin binding protein important for integrin-mediated cell adhesion. Here we used overexpression of Drosophila Parvin to uncover its functions in different tissues in vivo. Parvin overexpression caused major defects reminiscent of metastatic cancer cells in developing epithelia, including apoptosis, alterations in cell shape, basal extrusion and invasion. These defects were closely correlated with abnormalities in the organization of F-actin at the basal epithelial surface and of integrin-matrix adhesion sites. In wing epithelium, overexpressed Parvin triggered increased Rho1 protein levels, predominantly at the basal side, whereas in the developing eye it caused a rough eye phenotype and severely disrupted F-actin filaments at the retina floor of pigment cells. We identified genes that suppressed these Parvin-induced dominant effects, depending on the cell type. Co-expression of both ILK and the apoptosis inhibitor DIAP1 blocked Parvin-induced lethality and apoptosis and partially ameliorated cell delamination in epithelia, but did not rescue the elevated Rho1 levels, the abnormal organization of F-actin in the wing and the assembly of integrin-matrix adhesion sites. The rough eye phenotype was suppressed by coexpression of either PTEN or Wech, or by knock-down of Xrp1. Two main conclusions can be drawn from our studies: (1), high levels of cytoplasmic Parvin are toxic in epithelial cells; (2) Parvin in a dose dependent manner affects the organization of actin cytoskeleton in both wing and eye epithelia, independently of its role as a structural component of the ILK-PINCH-Parvin complex that mediates the integrin-actin link. Thus, distinct genetic interactions of Parvin occur in different cell types and second site modifier screens are required to uncover such genetic circuits.