Embryonic Dorsal Closure Research Articles

Rab11 negatively regulates wingless preventing JNK-mediated apoptosis in Drosophila epithelium during embryonic dorsal closure.

Rab11, a small Ras like GTPase marking the recycling endosomes, plays instrumental roles in Drosophila embryonic epithelial morphogenesis where an array of reports testify its importance in the maintenance of cyto-architectural as well as functional attributes of the concerned cells. Proper Rab11 functions ensure a precise regulation of developmentally active cell signaling pathways which in turn promote the expression of morphogens and other physico-chemical cues which finally forge an embryo out of a single layer of cells. Earlier reports have established that Rab11 functions are vital for fly embryonic development where amorphic mutants such as EP3017 homozygotes show a fair degree of epithelial defects along with incomplete dorsal closure. Here, we present a detailed account of the effects of Rab11 loss of function in the dorso-lateral epithelium which resulted in severe dorsal closure defects along with an elevated JNK-Dpp expression. We further observed that the dorso-lateral epithelial cells undergo epithelial to mesenchymal transition as well as apoptosis in Rab11 mutants with elevated expression levels of MMP1 and Caspase-3, where Caspase-3 contributes to the Rab11 knockout phenotype contrary to the knockdown mutants or hypomorphs. Interestingly, the elevated expressions of the core JNK-Dpp signaling could be rescued with a simultaneous knockdown of wingless in the Rab11 knockout mutants suggesting a genetic interaction of Rab11 with the Wingless pathway during dorsal closure, an ideal model of epithelial wound healing.

A Comparative Study of the Role of Formins in Drosophila Embryonic Dorsal Closure.

Dorsal closure is a late embryogenesis process required to seal the epidermal hole on the dorsal side of the Drosophila embryo. This process involves the coordination of several forces generated in the epidermal cell layer and in the amnioserosa cells, covering the hole. Ultimately, these forces arise due to cytoskeletal rearrangements that induce changes in cell shape and result in tissue movement. While a number of cytoskeleton regulatory proteins have already been linked to dorsal closure, here we expand this list by demonstrating that four of the six Drosophila formin type actin assembly factors are needed to bring about the proper fusion of the epithelia. An analysis of the morphological and dynamic properties of dorsal closure in formin mutants revealed a differential contribution for each formin, although we found evidence for functional redundancies as well. Therefore, we propose that the four formins promote the formation of several, and only partly identical, actin structures each with a specific role in the mechanics of dorsal closure.

The Tbx6 Transcription Factor Dorsocross Mediates Dpp Signaling to Regulate Drosophila Thorax Closure

Movement and fusion of separate cell populations are critical for several developmental processes, such as neural tube closure in vertebrates or embryonic dorsal closure and pupal thorax closure in Drosophila. Fusion failure results in an opening or groove on the body surface. Drosophila pupal thorax closure is an established model to investigate the mechanism of tissue closure. Here, we report the identification of T-box transcription factor genes Dorsocross (Doc) as Decapentaplegic (Dpp) targets in the leading edge cells of the notum in the late third instar larval and early pupal stages. Reduction of Doc in the notum region results in a thorax closure defect, similar to that in dpp loss-of-function flies. Nine genes are identified as potential downstream targets of Doc in regulating thorax closure by molecular and genetic screens. Our results reveal a novel function of Doc in Drosophila development. The candidate target genes provide new clues for unravelling the mechanism of collective cell movement.

Proximate larval epidermal cell layer generates forces for Pupal thorax closure in Drosophila.

During tissue closures, such as embryonic dorsal closure in Drosophila melanogaster, a proximate extra-embryonic layer, amnioserosa, generates forces that drive migration of the flanking lateral embryonic epidermis, thereby zip-shutting the embryo. Arguably, this paradigm of tissue closure is also recapitulated in mammalian wound healing wherein proximate fibroblasts transform into contractile myofibroblasts, develop cell junctions, and form a tissue layer de novo: contraction of the latter then aids in wound closure. Given this parallelism between disparate exemplars, we posit a general principle of tissue closure via proximate cell layer-generated forces. Here, we have tested this hypothesis in pupal thorax closure wherein 2 halves of the presumptive adult thorax of Drosophila, the contralateral heminotal epithelia, migrate over an underlying larval epidermal cell layer. We show that the proximate larval epidermal cell layer promotes thorax closure by its active contraction, orchestrated by its elaborate actomyosin network-driven epithelial cell dynamics, cell delamination, and death-the latter being prefigured by the activation of caspases. Larval epidermal cell dynamics generate contraction forces, which when relayed to the flanking heminota-via their mutual integrin-based adhesions-mediate thorax closure. Compromising any of these contraction force-generating mechanisms in the larval epidermal cell layer slows down heminotal migration, while loss of its relay to the flanking heminota abrogates the thorax closure altogether. Mathematical modeling further reconciles the biophysical underpinning of this emergent mechanism of thorax closure. Revealing mechanism of thorax closure apart, these findings show conservation of an essential principle of a proximate cell layer-driven tissue closure.

Evolutionary conserved role of eukaryotic translation factor eIF5A in the regulation of actin-nucleating formins

Elongation factor eIF5A is required for the translation of consecutive prolines, and was shown in yeast to translate polyproline-containing Bni1, an actin-nucleating formin required for polarized growth during mating. Here we show that Drosophila eIF5A can functionally replace yeast eIF5A and is required for actin-rich cable assembly during embryonic dorsal closure (DC). Furthermore, Diaphanous, the formin involved in actin dynamics during DC, is regulated by and mediates eIF5A effects. Finally, eIF5A controls cell migration and regulates Diaphanous levels also in mammalian cells. Our results uncover an evolutionary conserved role of eIF5A regulating cytoskeleton-dependent processes through translation of formins in eukaryotes.

α-Catenin stabilises Cadherin-Catenin complexes and modulates actomyosin dynamics to allow pulsatile apical contraction.

We have investigated how cell contractility and adhesion are functionally integrated during epithelial morphogenesis. To this end, we have analysed the role of α-Catenin, a key molecule linking E-Cadherin-based adhesion and the actomyosin cytoskeleton, during Drosophila embryonic dorsal closure, by studying a newly developed allelic series. We find that α-Catenin regulates pulsatile apical contraction in the amnioserosa, the main force-generating tissue driving closure of the embryonic epidermis. α-Catenin controls actomyosin dynamics by stabilising and promoting the formation of actomyosin foci, and also stabilises DE-Cadherin (Drosophila E-Cadherin, also known as Shotgun) at the cell membrane, suggesting that medioapical actomyosin contractility regulates junction stability. Furthermore, we uncover a genetic interaction between α-Catenin and Vinculin, and a tension-dependent recruitment of Vinculin to amniosersoa apical cell membranes, suggesting the existence of a mechano-sensitive module operating in this tissue.

The receptor tyrosine kinase Pvr promotes tissue closure by coordinating corpse removal and epidermal zippering.

A leading cause of human birth defects is the incomplete fusion of tissues, often manifested in the palate, heart or neural tube. To investigate the molecular control of tissue fusion, embryonic dorsal closure and pupal thorax closure in Drosophila are useful experimental models. We find that Pvr mutants have defects in dorsal midline closure with incomplete amnioserosa internalization and epidermal zippering, as well as cardia bifida. These defects are relatively mild in comparison to those seen with other signaling mutants, such as in the JNK pathway, and we demonstrate that JNK signaling is not perturbed by altering Pvr receptor tyrosine kinase activity. Rather, modulation of Pvr levels in the ectoderm has an impact on PIP3 membrane accumulation, consistent with a link to PI3K signal transduction. Polarized PI3K activity influences protrusive activity from the epidermal leading edge and the protrusion area changes in accord with Pvr signaling intensity, providing a possible mechanism to explain Pvr mutant phenotypes. Tissue-specific rescue experiments indicate a partial requirement in epithelial tissue, but confirm the essential role of Pvr in hemocytes for embryonic survival. Taken together, we argue that inefficient removal of the internalizing amnioserosa tissue by mutant hemocytes coupled with impaired midline zippering of mutant epithelium creates a situation in some embryos whereby dorsal midline closure is incomplete. Based on these observations, we suggest that efferocytosis (corpse clearance) could contribute to proper tissue closure and thus might underlie some congenital birth defects.

Regulating cell morphogenesis: The drosophila jun N‐terminal kinase pathway

The Jun-N-terminal Kinase pathway (JNK), known also as stress activated protein kinase pathway (SAPK), is an eukaryotic evolutionarily conserved signaling pathway. From a purported evolutionarily "ancient" function as stress mediator, it evolved in multicellular eukaryotes to permanent roles in development, without leaving its original function. In Drosophila melanogaster, it is required for follicle cell morphogenesis, embryonic dorsal closure, pupal thoracic closure and genital disc rotation closure, all processes with requisite cell shape changes. Besides, it is activated during wound healing and in response to stress (UV irradiation, oxidative stress) where it may signal cell death or proliferation. Despite these varied roles, it has a conserved core of molecules that follow the MAPKKK/MAPKK/MAPK logic of mitogen activated protein kinases pathways. Regulation of the JNK pathway appears majorly negative, with phosphatases, transcription factors and proteins of novel structure "holding back" on JNK activation in different tissues. This particular mode of regulation may hark back to the pathway's origin as stress detector and responder, implying readiness to respond, from which the developmental roles may have evolved as conditions demanding obligate and predicted stress responses (i.e., embryonic dorsal closure viewed as a "wound of development").

Planar Cell Polarity Signaling in Collective Cell Movements During Morphogenesis and Disease

Collective and directed cell movements are crucial for diverse developmental processes in the animal kingdom, but they are also involved in wound repair and disease. During these processes groups of cells are oriented within the tissue plane, which is referred to as planar cell polarity (PCP). This requires a tight regulation that is in part conducted by the PCP pathway. Although this pathway was initially characterized in flies, subsequent studies in vertebrates revealed a set of conserved core factors but also effector molecules and signal modulators, which build the fundamental PCP machinery. The PCP pathway in Drosophila regulates several developmental processes involving collective cell movements such as border cell migration during oogenesis, ommatidial rotation during eye development, and embryonic dorsal closure. During vertebrate embryogenesis, PCP signaling also controls collective and directed cell movements including convergent extension during gastrulation, neural tube closure, neural crest cell migration, or heart morphogenesis. Similarly, PCP signaling is linked to processes such as wound repair, and cancer invasion and metastasis in adults. As a consequence, disruption of PCP signaling leads to pathological conditions. In this review, we will summarize recent findings about the role of PCP signaling in collective cell movements in flies and vertebrates. In addition, we will focus on how studies in Drosophila have been relevant to our understanding of the PCP molecular machinery and will describe several developmental defects and human disorders in which PCP signaling is compromised. Therefore, new discoveries about the contribution of this pathway to collective cell movements could provide new potential diagnostic and therapeutic targets for these disorders.

Transcriptional Activity and Nuclear Localization of Cabut, the Drosophila Ortholog of Vertebrate TGF-β-Inducible Early-Response Gene (TIEG) Proteins

BackgroundCabut (Cbt) is a C2H2-class zinc finger transcription factor involved in embryonic dorsal closure, epithelial regeneration and other developmental processes in Drosophila melanogaster. Cbt orthologs have been identified in other Drosophila species and insects as well as in vertebrates. Indeed, Cbt is the Drosophila ortholog of the group of vertebrate proteins encoded by the TGF-ß-inducible early-response genes (TIEGs), which belong to Sp1-like/Krüppel-like family of transcription factors. Several functional domains involved in transcriptional control and subcellular localization have been identified in the vertebrate TIEGs. However, little is known of whether these domains and functions are also conserved in the Cbt protein.Methodology/Principal FindingsTo determine the transcriptional regulatory activity of the Drosophila Cbt protein, we performed Gal4-based luciferase assays in S2 cells and showed that Cbt is a transcriptional repressor and able to regulate its own expression. Truncated forms of Cbt were then generated to identify its functional domains. This analysis revealed a sequence similar to the mSin3A-interacting repressor domain found in vertebrate TIEGs, although located in a different part of the Cbt protein. Using β-Galactosidase and eGFP fusion proteins, we also showed that Cbt contains the bipartite nuclear localization signal (NLS) previously identified in TIEG proteins, although it is non-functional in insect cells. Instead, a monopartite NLS, located at the amino terminus of the protein and conserved across insects, is functional in Drosophila S2 and Spodoptera exigua Sec301 cells. Last but not least, genetic interaction and immunohistochemical assays suggested that Cbt nuclear import is mediated by Importin-α2.Conclusions/SignificanceOur results constitute the first characterization of the molecular mechanisms of Cbt-mediated transcriptional control as well as of Cbt nuclear import, and demonstrate the existence of similarities and differences in both aspects of Cbt function between the insect and the vertebrate TIEG proteins.

Drosophila as a model of wound healing and tissue regeneration in vertebrates

Understanding the molecular basis of wound healing and regeneration in vertebrates is one of the main challenges in biology and medicine. This understanding will lead to medical advances allowing accelerated tissue repair after wounding, rebuilding new tissues/organs and restoring homeostasis. Drosophila has emerged as a valuable model for studying these processes because the genetic networks and cytoskeletal machinery involved in epithelial movements occurring during embryonic dorsal closure, larval imaginal disc fusion/regeneration, and epithelial repair are similar to those acting during wound healing and regeneration in vertebrates. Recent studies have also focused on the use of Drosophila adult stem cells to maintain tissue homeostasis. Here, we review how Drosophila has contributed to our understanding of these processes, primarily through live-imaging and genetic tools that are impractical in mammals. Furthermore, we highlight future research areas where this insect may provide novel insights and potential therapeutic strategies for wound healing and regeneration.

The ATAC Acetyltransferase Complex Coordinates MAP Kinases to Regulate JNK Target Genes

In response to extracellular cues, signal transduction activates downstream transcription factors like c-Jun to induce expression of target genes. We demonstrate that the ATAC (Ada two A containing) histone acetyltransferase (HAT) complex serves as a transcriptional cofactor for c-Jun at the Jun N-terminal kinase (JNK) target genes Jra and chickadee. ATAC subunits are required for c-Jun occupancy of these genes and for H4K16 acetylation at the Jra enhancer, promoter, and transcribed sequences. Under conditions of osmotic stress, ATAC colocalizes with c-Jun, recruits the upstream kinases Misshapen, MKK4, and JNK, and suppresses further activation of JNK. Relocalization of these MAPKs and suppression of JNK activation by ATAC are dependent on the CG10238 subunit of ATAC. Thus, ATAC governs the transcriptional response to MAP kinase signaling by serving as both a coactivator of transcription and as a suppressor of upstream signaling.

The Drosophila serine protease homologue Scarface regulates JNK signalling in a negative-feedback loop during epithelial morphogenesis

In Drosophila melanogaster, dorsal closure is a model of tissue morphogenesis leading to the dorsal migration and sealing of the embryonic ectoderm. The activation of the JNK signal transduction pathway, specifically in the leading edge cells, is essential to this process. In a genome-wide microarray screen, we identified new JNK target genes during dorsal closure. One of them is the gene scarface (scaf), which belongs to the large family of trypsin-like serine proteases. Some proteins of this family, like Scaf, bear an inactive catalytic site, representing a subgroup of serine protease homologues (SPH) whose functions are poorly understood. Here, we show that scaf is a general transcriptional target of the JNK pathway coding for a secreted SPH. scaf loss-of-function induces defects in JNK-controlled morphogenetic events such as embryonic dorsal closure and adult male terminalia rotation. Live imaging of the latter process reveals that, like for dorsal closure, JNK directs the dorsal fusion of two epithelial layers in the pupal genital disc. Genetic data show that scaf loss-of-function mimics JNK over-activity. Moreover, scaf ectopic expression aggravates the effect of the JNK negative regulator puc on male genitalia rotation. We finally demonstrate that scaf acts as an antagonist by negatively regulating JNK activity. Overall, our results identify the SPH-encoding gene scaf as a new transcriptional target of JNK signalling and reveal the first secreted regulator of the JNK pathway acting in a negative-feedback loop during epithelial morphogenesis.

Rho-family small GTPases are required for cell polarization and directional sensing in Drosophila wound healing

A wound induces cell polarization, in which myosin II is localized at the rear end of individual cells in a migrating epithelial sheet of the Drosophila larval epidermis. Here, we use myosin localization to demonstrate that Rac1, Cdc42, and Rho1 are each required for cell polarization and directional sensing of the wound. The three GTPases are also required for actin cable formation at the wound leading edge. Rac1, Cdc42, and Rho1 act upstream of c-Jun N-terminal kinase (JNK) to organize actin assembly. These results highlight the similarities between the molecular mechanism of Drosophila wound healing and those of Drosophila embryonic dorsal closure and the chemotactic response of Dictyostelium and leukocytes.

POSH misexpression induces caspase‐dependent cell death inDrosophila

POSH (Plenty of SH3 domains) is a scaffold for signaling proteins regulating cell survival. Specifically, POSH promotes assembly of a complex including Rac GTPase, mixed lineage kinase (MLK), MKK7, and Jun kinase (JNK). In Drosophila, genetic analysis implicated POSH in Tak1-dependent innate immune response, in part through regulation of JNK signaling. Homologs of the POSH signaling complex components, MLK and MKK7, are essential in Drosophila embryonic dorsal closure. Using a gain-of-function approach, we tested whether POSH plays a role in this process. Ectopic expression of POSH in the embryo causes dorsal closure defects due to apoptosis of the amnioserosa, but ectodermal JNK signaling is normal. Phenotypic consequences of POSH expression were found to be dependent on Drosophila Nc, the caspase-9 homolog, but only partially on Tak1 and not at all on Slpr and Hep. These results suggest that POSH may use different signaling complexes to promote cell death in distinct contexts.

Interaction betweenDrosophilabZIP proteins Atf3 and Jun prevents replacement of epithelial cells during metamorphosis

Epithelial sheet spreading and fusion underlie important developmental processes. Well-characterized examples of such epithelial morphogenetic events have been provided by studies in Drosophila, and include embryonic dorsal closure, formation of the adult thorax and wound healing. All of these processes require the basic region-leucine zipper (bZIP) transcription factors Jun and Fos. Much less is known about morphogenesis of the fly abdomen, which involves replacement of larval epidermal cells (LECs) with adult histoblasts that divide, migrate and finally fuse to form the adult epidermis during metamorphosis. Here, we implicate Drosophila Activating transcription factor 3 (Atf3), the single ortholog of human ATF3 and JDP2 bZIP proteins, in abdominal morphogenesis. During the process of the epithelial cell replacement, transcription of the atf3 gene declines. When this downregulation is experimentally prevented, the affected LECs accumulate cell-adhesion proteins and their extrusion and replacement with histoblasts are blocked. The abnormally adhering LECs consequently obstruct the closure of the adult abdominal epithelium. This closure defect can be either mimicked and further enhanced by knockdown of the small GTPase Rho1 or, conversely, alleviated by stimulating ecdysone steroid hormone signaling. Both Rho and ecdysone pathways have been previously identified as effectors of the LEC replacement. To elicit the gain-of-function effect, Atf3 specifically requires its binding partner Jun. Our data thus identify Atf3 as a new functional partner of Drosophila Jun during development.

Developmental expression of Rab11, a small GTP‐binding protein in Drosophila epithelia

Intracellular membrane trafficking regulates a wide variety of developmental processes, including cell and tissue morphogenesis. Here we report developmental expression of Drosophila Rab11, a small GTP-binding protein, required for both endocytic recycling and exocytosis. Rab11 is expressed in the epithelial cell types of diverse lineages at all developmental stages, beginning from the cellular blastoderm in early embryos to adult primordia and adult tissues, like the columnar epithelia lining male ejaculatory bulb. A robust expression of Rab11 is seen both in the amnioserosa and in the lateral epidermis during embryonic dorsal closure, a morphogenetic event that involves spreading and fusion of the contra-lateral sides of epidermis. Rab11 mutant embryos fail to display the characteristic morphological changes in these two epithelial tissues during dorsal closure, providing a strong basis to dissect the role of Rab11 in coordinated epithelial sheet movements. genesis 47:32-39, 2009. (c) 2008 Wiley-Liss, Inc.

Raw Mediates Antagonism of AP-1 Activity in Drosophila

High baselines of transcription factor activities represent fundamental obstacles to regulated signaling. Here we show that in Drosophila, quenching of basal activator protein 1 (AP-1) transcription factor activity serves as a prerequisite to its tight spatial and temporal control by the JNK (Jun N-terminal kinase) signaling cascade. Our studies indicate that the novel raw gene product is required to limit AP-1 activity to leading edge epidermal cells during embryonic dorsal closure. In addition, we provide the first evidence that the epidermis has a Basket JNK-independent capacity to activate AP-1 targets and that raw function is required broadly throughout the epidermis to antagonize this activity. Finally, our mechanistic studies of the three dorsal-open group genes [raw, ribbon (rib), and puckered (puc)] indicate that these gene products provide at least two tiers of JNK/AP-1 regulation. In addition to Puckered phosphatase function in leading edge epidermal cells as a negative-feedback regulator of JNK signaling, the three dorsal-open group gene products (Raw, Ribbon, and Puckered) are required more broadly in the dorsolateral epidermis to quench a basal, signaling-independent activity of the AP-1 transcription factor.

Genetic Analysis of Slipper/Mixed Lineage Kinase Reveals Requirements in Multiple Jun-N-Terminal Kinase-Dependent Morphogenetic Events During Drosophila Development

Mixed lineage kinases (MLKs) function as Jun-N-terminal kinase (JNK) kinase kinases to transduce extracellular signals during development and homeostasis in adults. slipper (slpr), which encodes the Drosophila homolog of mammalian MLKs, has previously been implicated in activation of the JNK pathway during embryonic dorsal epidermal closure. To further define the specific functions of SLPR, we analyzed the phenotypic consequences of slpr loss and gain of function throughout development, using a semiviable maternal-effect allele and wild-type or dominant-negative transgenes. From these analyses we confirm that failure of dorsal closure is the null phenotype in slpr germline clones. In addition, there is a functional maternal contribution, which can suffice for embryogenesis in the zygotic null mutant, but rarely suffices for pupal metamorphosis, revealing later functions for slpr as the maternal contribution is depleted. Zygotic null mutants that eclose as adults display an array of morphological defects, many of which are shared by hep mutant animals, deficient in the JNK kinase (JNKK/MKK7) substrate for SLPR, suggesting that the defects observed in slpr mutants primarily reflect loss of hep-dependent JNK activation. Consistent with this, the maternal slpr contribution is sensitive to the dosage of positive and negative JNK pathway regulators, which attenuate or potentiate SLPR-dependent signaling in development. Although SLPR and TAK1, another JNKKK family member, are differentially used in dorsal closure and TNF/Eiger-stimulated apoptosis, respectively, a Tak1 mutant shows dominant genetic interactions with slpr, suggesting potential redundant or combinatorial functions. Finally, we demonstrate that SLPR overexpression can induce ectopic JNK signaling and that the SLPR protein is enriched at the epithelial cell cortex.

Anthrax lethal factor and edema factor act on conserved targets in Drosophila

Many bacterial toxins act on conserved components of essential host-signaling pathways. One consequence of this conservation is that genetic model organisms such as Drosophila melanogaster can be used for analyzing the mechanism of toxin action. In this study, we characterize the activities of two anthrax virulence factors, lethal factor (LF) and edema factor, in transgenic Drosophila. LF is a zinc metalloprotease that cleaves and inactivates most human mitogen-activated protein kinase (MAPK) kinases (MAPKKs). We found that LF similarly cleaves the Drosophila MAPK kinases Hemipterous (Hep) and Licorne in vitro. Consistent with these observations, expression of LF in Drosophila inhibited the Hep/c-Jun N-terminal kinase pathway during embryonic dorsal closure and the related process of adult thoracic closure. Epistasis experiments confirmed that LF acts at the level of Hep. We also found that LF inhibits Ras/MAPK signaling during wing development and that LF acts upstream of MAPK and downstream of Raf, consistent with LF acting at the level of Dsor. In addition, we found that edema factor, a potent adenylate cyclase, inhibits the hh pathway during wing development, consistent with the known role of cAMP-dependent PKA in suppressing the Hedgehog response. These results demonstrate that anthrax toxins function in Drosophila as they do in mammalian cells and open the way to using Drosophila as a multicellular host system for studying the in vivo function of diverse toxins and virulence factors.