Asymmetric distribution of Spalt in Drosophila wing squamous and columnar epithelia ensures correct cell morphogenesis.
ABSTRACT: The Drosophila wing imaginal disc is a sac-like structure that is composed of two opposing cell layers: peripodial epithelium (PE, also known as squamous epithelia) and disc proper (DP, also known as pseudostratified columnar epithelia). The molecular mechanism of cell morphogenesis has been well studied in the DP but not in the PE. Although proper Dpp signalling activity is required for proper PE formation, the detailed regulation mechanism is poorly understood. Here, we found that the Dpp target gene sal is only expressed in DP cells, not in PE cells, although pMad is present in the PE. Increasing Dpp signalling activity cannot activate Sal in PE cells. The absence of Sal in the PE is essential for PE formation. The ectopic expression of sal in PE cells is sufficient to increase the PE cell height. Down-regulation of sal in the DP reduced DP cell height. We further demonstrated that the known PE cell height regulator Lines, which can convert PE into a DP cell fate, is mediated by sal mis-activation in PE because sal-RNAi and lines co-expression largely restores PE cell morphology. By revealing the microtubule distribution, we demonstrated that Lines- and Sal-heightened PE cells are morphologically similar to the intermediate cell with cuboidal morphology.
Project description:The Drosophila BMP, decapentaplegic (dpp), controls morphogenesis of the ventral adult head through expression limited to the lateral peripodial epithelium of the eye-antennal disc by a 3.5 kb enhancer in the 5' end of the gene. We recovered a 15 bp deletion mutation within this enhancer that identified a homeotic (Hox) response element that is a direct target of labial and the homeotic cofactors homothorax and extradenticle. Expression of labial and homothorax are required for dpp expression in the peripodial epithelium, while the Hox gene Deformed represses labial in this location, thus limiting its expression and indirectly that of dpp to the lateral side of the disc. The expression of these homeodomain genes is in turn regulated by the dpp pathway, as dpp signalling is required for labial expression but represses homothorax. This Hox-BMP regulatory network is limited to the peripodial epithelium of the eye-antennal disc, yet is crucial to the morphogenesis of the head, which fate maps suggest arises primarily from the disc proper, not the peripodial epithelium. Thus Hox/BMP interactions in the peripodial epithelium of the eye-antennal disc contribute inductively to the shape of the external form of the adult Drosophila head.
Project description:Central to embryonic development is the generation of molecular asymmetries across fields of undifferentiated cells. The Drosophila wing imaginal disc provides a powerful system with which to understand how such asymmetries are generated and how they contribute to formation of a complex structure. Early in development, the wing primordium is subdivided into a thin layer of peripodial epithelium (PE) and an apposing thickened layer of pseudostratified columnar epithelium (CE), known as the disc proper (DP). The DP gives rise to the wing blade, hinge and dorsal mesothorax, whereas the PE makes only a minor contribution to the ventral hinge and pleura. The mechanisms that generate this major asymmetry and its contribution to wing development are poorly understood. The Lines protein destabilizes the nuclear protein Bowl in ectodermal structures. Here, we show that Bowl accumulates in the PE from early stages of wing development and is absent from the DP. Broad inhibition of Bowl in the PE resulted in the replacement of the PE with a mirror image duplication of the DP. The failure to generate the PE severely compromised wing growth and the formation of the notum. Conversely, the activation of bowl in the DP (by removal or inhibition of lines function) resulted in the transformation of the DP into PE. Thus, we provide evidence that bowl and lines act as a binary switch to subdivide the wing primordium into PE and DP, and assign crucial roles for this asymmetry in wing growth and patterning.
Project description:Netrin receptors of the DCC/NEO/UNC-40/Frazzled family have well established roles in cell migration and axon guidance but can also regulate epithelial features such as adhesion, polarity and adherens junction (AJ) stability. Previously, we have shown that overexpression of Drosophila Frazzled (Fra) in the peripodial epithelium (PE) inhibits wing disc eversion and also generates cellular protrusions typical of motile cells. Here, we tested whether the molecular pathways by which Fra inhibits eversion are distinct from those driving motility. We show that in disc proper (DP) epithelial cells Fra, in addition to inducing F-Actin rich protrusions, can affect localization of AJ components and columnar cell shape. We then show that these phenotypes have different requirements for the three conserved Fra cytoplasmic P-motifs and for downstream genes. The formation of protrusions required the P3 motif of Fra, as well as integrins (mys and mew), the Rac pathway (Rac1, wave and, arpc3) and myosin regulatory light chain (Sqh). In contrast, apico-basal cell shape change, which was accompanied by increased myosin phosphorylation, was critically dependent upon the P1 motif and was promoted by RhoGef2 but inhibited by Rac1. Fra also caused a loss of AJ proteins (DE-Cad and Arm) from basolateral regions of epithelial cells. This phenotype required all 3 P-motifs, and was dependent upon the polarity factor par6. par6 was not required for protrusions or cell shape change, but was required to block eversion suggesting that control of AJ components may underlie the ability of Fra to promote epithelial stability. The results imply that multiple molecular pathways act downstream of Fra in epithelial cells.
Project description:Tissue mechanics play a crucial role in organ development. They rely on the properties of cells and the extracellular matrix (ECM), but the relative physical contribution of cells and ECM to morphogenesis is poorly understood. Here, we have analyzed the behavior of the peripodial epithelium (PE) of the Drosophila leg disc in the light of the dynamics of its cellular and ECM components. The PE undergoes successive changes during leg development, including elongation, opening and removal to free the leg. During elongation, we found that the ECM and cell layer are progressively uncoupled. Concomitantly, the tension, mainly borne by the ECM at first, builds up in the cell monolayer. Then, each layer of the peripodial epithelium is removed by an independent mechanism: while the ECM layer withdraws following local proteolysis, cellular monolayer withdrawal is independent of ECM degradation and is driven by myosin II-dependent contraction. These results reveal a surprising physical and functional cell-matrix uncoupling in a monolayer epithelium under tension during development.This article has an associated 'The people behind the papers' interview.
Project description:Locusta has strong fly wings to ensure its long distance migration, but the molecular mechanism that regulates the Locusta wing development is poorly understood. To address the developmental mechanism of the Locusta flying wing, we cloned the Dpp target gene spalt (sal) and analyzed its function in wing growth in the Locusta. The Locusta wing size is apparently reduced with vein defects when sal is interfered by injection of dsRNA, indicating that sal is required for locust wing growth and vein formation. This function is conserved during the Drosophila wing development. To better understand sal's function in wing growth, we then used Drosophila wing disc as a model for further study. We found that sal promotes cell proliferation in the whole wing disc via positive regulation of a microRNA bantam. Our results firstly unravel sal's function in the Locusta wing growth and confirm a highly conserved function of sal in Locusta and Drosophila.
Project description:The transcription factor Pax6 is considered the master control gene for eye formation because (1) it is present within the genomes and retina/lens of all animals with a visual system; (2) severe retinal defects accompany its loss; (3) Pax6 genes have the ability to substitute for one another across the animal kingdom; and (4) Pax6 genes are capable of inducing ectopic eye/lens in flies and mammals. Many roles of Pax6 were first elucidated in Drosophila through studies of the gene eyeless (ey), which controls both growth of the entire eye-antennal imaginal disc and fate specification of the eye. We show that Ey also plays a surprising role within cells of the peripodial epithelium to control pattern formation. It regulates the expression of decapentaplegic (dpp), which is required for initiation of the morphogenetic furrow in the eye itself. Loss of Ey within the peripodial epithelium leads to the loss of dpp expression within the eye, failure of the furrow to initiate, and abrogation of retinal development. These findings reveal an unexpected mechanism for how Pax6 controls eye development in Drosophila.
Project description:The epicardium, the outer mesothelial layer enclosing the myocardium, plays key roles in heart development and regeneration. During embryogenesis, the epicardium arises from the proepicardium (PE), a cell cluster that appears in the dorsal pericardium (DP) close to the venous pole of the heart. Little is known about how the PE emerges from the pericardial mesothelium. Using a zebrafish model and a combination of genetic tools, pharmacological agents and quantitative in vivo imaging, we reveal that a coordinated collective movement of DP cells drives PE formation. We found that Bmp signaling and the actomyosin cytoskeleton promote constriction of the DP, which enables PE cells to extrude apically. We provide evidence that cell extrusion, which has been described in the elimination of unfit cells from epithelia and the emergence of hematopoietic stem cells, is also a mechanism for PE cells to exit an organized mesothelium and fulfil their developmental fate to form a new tissue layer, the epicardium.
Project description:The motor protein non-muscle myosin II is a major driver of the movements that sculpt three-dimensional organs from two-dimensional epithelia. The machinery of morphogenesis is well established but the logic of its control remains unclear in complex organs. Here we use live imaging and ex vivo culture to report a dual role of myosin II in regulating the development of the Drosophila wing. First, myosin II drives the contraction of a ring of cells that surround the squamous peripodial epithelium, providing the force to fold the whole disc through about 90°. Second, myosin II is needed to allow the squamous cells to expand and then retract at the end of eversion. The combination of genetics and live imaging allows us to describe and understand the tissue dynamics, and the logic of force generation needed to transform a relatively simple imaginal disc into a more complex and three-dimensional adult wing.
Project description:All organisms have developed mechanisms to respond to organ or tissue damage that may appear during development or during the adult life. This process of regeneration is a major long-standing problem in Developmental Biology. We are using the Drosophila melanogaster wing imaginal disc to study the response to major damage inflicted during development. Using the Gal4/UAS/Gal80(TS) conditional system, we have induced massive cell killing by forcing activity of the pro-apoptotic gene hid in two major regions of the disc as defined by Gal4 inserts in the genes rotund (rn) and spalt (sal). The procedure ensures that at the end of a 40-48 hrs of ablation period the great majority of the cells of the original Rn or Sal domains have been eliminated. The results indicate that the damage provokes an immediate response aimed to keep the integrity of the epithelium and to repair the region under ablation. This includes an increase in cell proliferation to compensate for the cell loss and the replacement of the dead cells by others from outside of the damaged area. The response is almost contemporaneous with the damage, so that at the end of the ablation period the targeted region is already reconstructed. We find that the proliferative response is largely systemic, as the number of cells in division increases all over the disc. Furthermore, our results indicate that the Dpp and Wg pathways are not specifically involved in the regenerative response, but that activity of the JNK pathway is necessary both inside and outside the ablated domain for its reconstruction.
Project description:Quantitative data from the Drosophila wing imaginal disc reveals that the amplitude of the Decapentaplegic (Dpp) morphogen gradient increases continuously. It is an open question how cells can determine their relative position within a domain based on a continuously increasing gradient. Here we show that pre-steady state diffusion-based dispersal of morphogens results in a zone within the growing domain where the concentration remains constant over the patterning period. The position of the zone that is predicted based on quantitative data for the Dpp morphogen corresponds to where the Dpp-dependent gene expression boundaries of spalt (sal) and daughters against dpp (dad) emerge. The model also suggests that genes that are scaling and are expressed at lateral positions are either under the control of a different read-out mechanism or under the control of a different morphogen. The patterning mechanism explains the extraordinary robustness that is observed for variations in Dpp production, and offers an explanation for the dual role of Dpp in controlling patterning and growth. Pre-steady-state dynamics are pervasive in morphogen-controlled systems, thus making this a probable general mechanism for the scaled read-out of morphogen gradients in growing developmental systems.