Discontinuities in Rap1 activity determine epithelial cell morphology within the developing wing of Drosophila.
ABSTRACT: Mechanisms that govern cell-fate specification within developing epithelia have been intensely investigated, with many of the critical intercellular signaling pathways identified, and well characterized. Much less is known, however, about downstream events that drive the morphological differentiation of these cells, once their fate has been determined. In the Drosophila wing-blade epithelium, two cell types predominate: vein and intervein. After cell proliferation is complete and adhesive cell-cell contacts have been refined, the vast majority of intervein cells adopt a hexagonal morphology. Within vein territories, however, cell-shape refinement results in trapezoids. Signaling events that differentiate between vein and intervein cell fates are well understood, but the genetic pathways underlying vein/intervein cyto-architectural differences remain largely undescribed. We show here that the Rap1 GTPase plays a critical role in determining cell-type-specific morphologies within the developing wing epithelium. Rap1, together with its effector Canoe, promotes symmetric distribution of the adhesion molecule DE-cadherin about the apicolateral circumference of epithelial cells. We provide evidence that in presumptive vein tissue Rap1/Canoe activity is down-regulated, resulting in adhesive asymmetries and non-hexagonal cell morphologies. In particular Canoe levels are reduced in vein cells as they morphologically differentiate. We also demonstrate that over-expression of Rap1 disrupts vein formation both in the developing epithelium and the adult wing blade. Therefore, vein/intervein morphological differences result, at least in part, from the patterned regulation of Rap1 activity.
Project description:Egfr/Ras signaling promotes vein cell fate specification in the developing Drosophila wing. While the importance of Ras signaling in vein determination has been extensively documented, the mechanisms linking Ras activity to vein differentiation remain unclear. We found that Ras signaling regulates both the levels and subcellular localization of the cell adhesion molecule DE-cadherin/Shotgun (Shg) in the differentiating wing epithelium. High Ras activity in presumptive vein cells directs the apical localization of Shg containing adherens junctions, whereas low Ras activity in intervein cells allows Shg to relocalize basally. These alterations in Shg-mediated adhesion control cell shape changes that are essential for vein morphogenesis. While Decapentaplegic (Dpp) acts downstream of Ras to maintain vein cell identity in the pupal wing, our results indicate that Ras controls Shg localization via a Dpp-independent mechanism. Ras, therefore, regulates both the transcriptional responses necessary for vein cell identity, and the cell adhesive changes that determine vein and intervein cell morphology.
Project description:The small GTPase Rap1 affects cell adhesion and cell motility in numerous developmental contexts. Loss of Rap1 in the Drosophila wing epithelium disrupts adherens junction localization, causing mutant cells to disperse, and dramatically alters epithelial cell shape. While the adhesive consequences of Rap1 inactivation have been well described in this system, the effects on cell signaling, cell fate specification, and tissue differentiation are not known. Here we demonstrate that Egfr-dependent cell types are lost from Rap1 mutant tissue as an indirect consequence of DE-cadherin mislocalization. Cells lacking Rap1 in the developing wing and eye are capable of responding to an Egfr signal, indicating that Rap1 is not required for Egfr/Ras/MAPK signal transduction. Instead, Rap1 regulates adhesive contacts necessary for maintenance of Egfr signaling between cells, and differentiation of wing veins and photoreceptors. Rap1 is also necessary for planar cell polarity in these tissues. Wing hair alignment and ommatidial rotation, functional readouts of planar cell polarity in the wing and eye respectively, are both affected in Rap1 mutant tissue. Finally, we show that Rap1 acts through the effector Canoe to regulate these developmental processes.
Project description:We examined post-eclosion elimination of the Drosophila wing epithelium in vivo where collective "suicide waves" promote sudden, coordinated death of epithelial sheets without a final engulfment step. Like apoptosis in earlier developmental stages, this unique communal form of cell death is controlled through the apoptosome proteins, Dronc and Dark, together with the IAP antagonists, Reaper, Grim, and Hid. Genetic lesions in these pathways caused intervein epithelial cells to persist, prompting a characteristic late-onset blemishing phenotype throughout the wing blade. We leveraged this phenotype in mosaic animals to discover relevant genes and establish here that homeodomain interacting protein kinase (HIPK) is required for collective death of the wing epithelium. Extra cells also persisted in other tissues, establishing a more generalized requirement for HIPK in the regulation of cell death and cell numbers.
Project description:The pattern of the Drosophila melanogaster adult wing is heavily influenced by the expression of proteins that dictate cell fate decisions between intervein and vein during development. dSRF (Blistered) expression in specific regions of the larval wing disc promotes intervein cell fate, whereas EGFR activity promotes vein cell fate. Here, we report that the chromatin-organizing protein CAP-D3 acts to dampen dSRF levels at the anterior/posterior boundary in the larval wing disc, promoting differentiation of cells into the anterior crossvein. CAP-D3 represses KNOT expression in cells immediately adjacent to the anterior/posterior boundary, thus blocking KNOT-mediated repression of EGFR activity and preventing cell death. Maintenance of EGFR activity in these cells depresses dSRF levels in the neighboring anterior crossvein progenitor cells, allowing them to differentiate into vein cells. These findings uncover a novel transcriptional regulatory network influencing Drosophila wing vein development, and are the first to identify a Condensin II subunit as an important regulator of EGFR activity and cell fate determination in vivo.
Project description:The establishment and maintenance of apical-basal cell polarity is critical for assembling epithelia and maintaining organ architecture. Drosophila embryos provide a superb model. In the current view, apically positioned Bazooka/Par3 is the initial polarity cue as cells form during cellularization. Bazooka then helps to position both adherens junctions and atypical protein kinase C (aPKC). Although a polarized cytoskeleton is critical for Bazooka positioning, proteins mediating this remained unknown. We found that the small GTPase Rap1 and the actin-junctional linker Canoe/afadin are essential for polarity establishment, as both adherens junctions and Bazooka are mispositioned in their absence. Rap1 and Canoe do not simply organize the cytoskeleton, as actin and microtubules become properly polarized in their absence. Canoe can recruit Bazooka when ectopically expressed, but they do not obligatorily colocalize. Rap1 and Canoe play continuing roles in Bazooka localization during gastrulation, but other polarity cues partially restore apical Bazooka in the absence of Rap1 or Canoe. We next tested the current linear model for polarity establishment. Both Bazooka and aPKC regulate Canoe localization despite being "downstream" of Canoe. Further, Rap1, Bazooka, and aPKC, but not Canoe, regulate columnar cell shape. These data reshape our view, suggesting that polarity establishment is regulated by a protein network rather than a linear pathway.
Project description:The expression of the spalt genes is regulated by the Decapentaplegic signalling pathway in the Drosophila wing. These genes participate in the patterning of the longitudinal wing veins by regulating the expression of vein-specific genes, and in the establishment of cellular affinities in the central region of the wing blade epithelium. The Spalt proteins act as transcription factors, most likely regulating gene expression by repression, but the identity of their target genes in the wing is still unknown. As a preliminary step to unravel the genetic hierarchy controlled by the Spalt proteins, we have analysed their requirements during wing development, and addressed to what extent they mediate all the functions of the Decapentaplegic pathway in this developmental system. We identify additional functions for Spalt in cell division, survival, and maintenance of epithelial integrity. Thus, Spalt activity is required to promote cell proliferation, acting in the G2/M transition of the cell cycle. The contribution of Spalt to cell division is limited to the central region of the wing blade, as they do not mediate the extra growth triggered by Decapentaplegic signalling in the peripheral regions of the wing disc. In addition, Spalt function is required to maintain cell viability in cells exposed to high levels of Decapentaplegic signalling. This aspect of Spalt function is related to the repression of JNK signalling in the spalt domain of expression. Finally, we further characterise the requirements of Spalt to maintain epithelial integrity by regulating cellular affinities between cells located in the central wing region. Our results indicate that Spalt function mediates most of the requirements identified for Decapentaplegic signalling, contributing to establish the cellular qualities that differentiate central versus peripheral territories in the wing blade.
Project description:Development requires coordination between cell proliferation and cell growth to pattern the proper size of tissues, organs, and whole organisms. The Drosophila wing has landmark features, such as the location of veins patterned by cell groups and trichome structures produced by individual cells, that are useful to examine the genetic contributions to both tissue and cell size. Wing size and trichome density have been measured manually, which is tedious and error prone, and although image processing and pattern-recognition software can quantify features in micrographs, this approach has not been applied to insect wings. Here we present FijiWings, a set of macros designed to perform semiautomated morphophometric analysis of a wing photomicrograph. FijiWings uses plug-ins installed in the Fiji version of ImageJ to detect and count trichomes and measure wing area either to calculate trichome density of a defined region selected by the user or generate a heat map of overall trichome densities. For high-throughput screens we have developed a macro that directs a trainable segmentation plug-in to detect wing vein locations either to measure trichome density in specific intervein regions or produce a heat map of relative intervein areas. We use wing GAL4 drivers and UAS-regulated transgenes to confirm the ability of these tools to detect changes in overall tissue growth and individual cell size. FijiWings is freely available and will be of interest to a broad community of fly geneticists studying both the effect of gene function on wing patterning and the evolution of wing morphology.
Project description:Epithelial apical-basal polarity drives assembly and function of most animal tissues. Polarity initiation requires cell-cell adherens junction assembly at the apical-basolateral boundary. Defining the mechanisms underlying polarity establishment remains a key issue. Drosophila embryos provide an ideal model, as 6000 polarized cells assemble simultaneously. Current data place the actin-junctional linker Canoe (fly homolog of Afadin) at the top of the polarity hierarchy, where it directs Bazooka/Par3 and adherens junction positioning. Here we define mechanisms regulating Canoe localization/function. Spatial organization of Canoe is multifaceted, involving membrane localization, recruitment to nascent junctions and macromolecular assembly at tricellular junctions. Our data suggest apical activation of the small GTPase Rap1 regulates all three events, but support multiple modes of regulation. The Rap1GEF Dizzy (PDZ-GEF) is crucial for Canoe tricellular junction enrichment but not apical retention. The Rap1-interacting RA domains of Canoe mediate adherens junction and tricellular junction recruitment but are dispensable for membrane localization. Our data also support a role for Canoe multimerization. These multifactorial inputs shape Canoe localization, correct Bazooka and adherens junction positioning, and thus apical-basal polarity. We integrate the existing data into a new polarity establishment model.
Project description:The Spalt-like family of zinc finger transcription factors is conserved throughout evolution and is involved in fundamental processes during development and during embryonic stem cell maintenance. Although human SALL1 is modified by SUMO-1 in vitro, it is not known whether this post-translational modification plays a role in regulating the activity of this family of transcription factors. Here, we show that the Drosophila Spalt transcription factors are modified by sumoylation. This modification influences their nuclear localization and capacity to induce vein formation through the regulation of target genes during wing development. Furthermore, spalt genes interact genetically with the sumoylation machinery to repress vein formation in intervein regions and to attain the wing final size. Our results suggest a new level of regulation of Sall activity in vivo during animal development through post-translational modification by sumoylation. The evolutionary conservation of this family of transcription factors suggests a functional role for sumoylation in vertebrate Sall members.
Project description:In the vertebrates, the BMP/Smad1 and TGF-beta/Smad2 signaling pathways execute antagonistic functions in different contexts of development. The differentiation of specific structures results from the balance between these two pathways. For example, the gastrula organizer/node of the vertebrates requires a region of low Smad1 and high Smad2 signaling. In Drosophila, Mad regulates tissue determination and growth in the wing, but the function of dSmad2 in wing patterning is largely unknown. In this study, we used an RNAi loss-of-function approach to investigate dSmad2 signaling during wing development. RNAi-mediated knockdown of dSmad2 caused formation of extra vein tissue, with phenotypes similar to those seen in Dpp/Mad gain-of-function. Clonal analyses revealed that the normal function of dSmad2 is to inhibit the response of wing intervein cells to the extracellular Dpp morphogen gradient that specifies vein formation, as measured by expression of the activated phospho-Mad protein. The effect of dSmad2 depletion in promoting vein differentiation was dependent on Medea, the co-factor shared by Mad and dSmad2. Furthermore, double RNAi experiments showed that Mad is epistatic to dSmad2. In other words, depletion of Smad2 had no effect in Mad-deficient wings. Our results demonstrate a novel role for dSmad2 in opposing Mad-mediated vein formation in the wing. We propose that the main function of dActivin/dSmad2 in Drosophila wing development is to antagonize Dpp/Mad signaling. Possible molecular mechanisms for the opposition between dSmad2 and Mad signaling are discussed.