Membrane potential regulates Hedgehog signalling in the Drosophila wing imaginal disc.
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ABSTRACT: While the membrane potential of cells has been shown to be patterned in some tissues, specific roles for membrane potential in regulating signalling pathways that function during development are still being established. In the Drosophila wing imaginal disc, Hedgehog (Hh) from posterior cells activates a signalling pathway in anterior cells near the boundary which is necessary for boundary maintenance. Here, we show that membrane potential is patterned in the wing disc. Anterior cells near the boundary, where Hh signalling is most active, are more depolarized than posterior cells across the boundary. Elevated expression of the ENaC channel Ripped Pocket (Rpk), observed in these anterior cells, requires Hh. Antagonizing Rpk reduces depolarization and Hh signal transduction. Using genetic and optogenetic manipulations, in both the wing disc and the salivary gland, we show that membrane depolarization promotes membrane localization of Smoothened and augments Hh signalling, independently of Patched. Thus, membrane depolarization and Hh-dependent signalling mutually reinforce each other in cells immediately anterior to the compartment boundary.
Project description:Hedgehog (Hh) is an evolutionarily conserved signaling protein that has essential roles in animal development and homeostasis. We investigated Hh signaling in the region of the Drosophila wing imaginal disc that produces Hh and is near the tracheal air sac primordium (ASP) and myoblasts. Hh distributes in concentration gradients in the anterior compartment of the wing disc, ASP and myoblasts, and activates genes in each tissue. Some targets of Hh signal transduction are common to the disc, ASP and myoblasts, whereas others are tissue-specific. Signaling in the three tissues is cytoneme-mediated and cytoneme-dependent. Some ASP cells project cytonemes that receive both Hh and Branchless (Bnl), and some targets regulated by Hh signaling in the ASP are also dependent on Bnl signal transduction. We conclude that the single source of Hh in the wing disc regulates cell type-specific responses in three discreet target tissues.
Project description:Growth and patterning are coordinated during development to define organ size and shape. The growth, proliferation and differentiation of Drosophila wings are regulated by several conserved signaling pathways. Here, we show that the Salvador-Warts-Hippo (SWH) and Notch pathways converge on an enhancer in the expanded (ex) gene, which also responds to levels of the bHLH transcription factor Daughterless (Da). Separate cis-regulatory elements respond to Salvador-Warts-Hippo (SWH) and Notch pathways, to bHLH proteins, and to unidentified factors that repress ex transcription in the wing pouch and in the proneural region at the anterior wing margin. Senseless, a zinc-finger transcription factor acting in proneural regions, had a negative impact on ex transcription in the proneural region, but the transcriptional repressor Hairy had no effect. Our study suggests that a complex pattern of ex transcription results from integration of a uniform SWH signal with multiple other inputs, rather than from a pattern of SWH signaling.
Project description:Signaling between cells in the anterior (A) and posterior (P) compartments directs Drosophila wing disc development and is dependent on expression of the homeodomain transcription factor Engrailed (En) in P cells. Downstream of en, posteriorly expressed Hedgehog (Hh) protein signals across the A/P border to establish a developmental organizer that directs pattern formation and growth throughout the wing primordium. Here we extend investigations of the processes downstream of en by using expression array analysis to compare A and P cells. A total of 102 candidate genes were identified that express differentially in the A and P compartments; four were characterized: Stubble (Sb) expression is restricted to A cells due to repression by en. CG15905, CG16884; CG10200/hase und igel (hui) are expressed in A cells downstream of Hh signaling; and RNA interference for hui, Stubble, and CG16884 revealed that each is essential to wing development.
Project description:Organ growth and size are finely tuned by intrinsic and extrinsic signaling molecules. In Drosophila, the BMP family member Dpp is produced in a limited set of imaginal disc cells and functions as a classic morphogen to regulate pattern and growth by diffusing throughout imaginal discs. However, the role of TGF?/Activin-like ligands in disc growth control remains ill-defined. Here, we demonstrate that Myoglianin (Myo), an Activin family member, and a close homolog of mammalian Myostatin (Mstn), is a muscle-derived extrinsic factor that uses canonical dSmad2-mediated signaling to regulate wing size. We propose that Myo is a myokine that helps mediate an allometric relationship between muscles and their associated appendages.
Project description:The apical and basolateral membranes of epithelia are insulated from each other, preventing the transfer of extracellular proteins from one side to the other. Thus, a signalling protein produced apically is not expected to reach basolateral receptors. Evidence suggests that Wingless, the main Drosophila Wnt, is secreted apically in the embryonic epidermis. However, in the wing imaginal disc epithelium, Wingless is mostly seen on the basolateral membrane where it spreads from secreting to receiving cells. Here we examine the apico-basal movement of Wingless in Wingless-producing cells of wing imaginal discs. We find that it is presented first on the apical surface before making its way to the basolateral surface, where it is released and allowed to interact with signalling receptors. We show that Wingless transcytosis involves dynamin-dependent endocytosis from the apical surface. Subsequent trafficking from early apical endosomes to the basolateral surface requires Godzilla, a member of the RNF family of membrane-anchored E3 ubiquitin ligases. Without such transport, Wingless signalling is strongly reduced in this tissue.
Project description:Morphogens are classically defined as molecules that control patterning by acting at a distance to regulate gene expression in a concentration-dependent manner. In the Drosophila wing imaginal disc, secreted Hedgehog (Hh) forms an extracellular gradient that organizes patterning along the anterior-posterior axis and specifies at least three different domains of gene expression. Although the prevailing view is that Hh functions in the Drosophila wing disc as a classical morphogen, a direct correspondence between the borders of these patterns and Hh concentration thresholds has not been demonstrated. Here, we provide evidence that the interpretation of Hh signaling depends on the history of exposure to Hh and propose that a single concentration threshold is sufficient to support multiple outputs. Using mathematical modeling, we predict that at steady state, only two domains can be defined in response to Hh, suggesting that the boundaries of two or more gene expression patterns cannot be specified by a static Hh gradient. Computer simulations suggest that a spatial "overshoot" of the Hh gradient occurs, i.e., a transient state in which the Hh profile is expanded compared to the Hh steady-state gradient. Through a temporal examination of Hh target gene expression, we observe that the patterns initially expand anteriorly and then refine, providing in vivo evidence for the overshoot. The Hh gene network architecture suggests this overshoot results from the Hh-dependent up-regulation of the receptor, Patched (Ptc). In fact, when the network structure was altered such that the ptc gene is no longer up-regulated in response to Hh-signaling activation, we found that the patterns of gene expression, which have distinct borders in wild-type discs, now overlap. Our results support a model in which Hh gradient dynamics, resulting from Ptc up-regulation, play an instructional role in the establishment of patterns of gene expression.
Project description:Changes in cell metabolism and plasma membrane potential have been linked to shifts between tissue growth and differentiation, and to developmental patterning. How such changes mediate these effects is poorly understood. Here, we use the developing wing of Drosophila to investigate the interplay between cell metabolism and a key developmental regulator-the Hedgehog (Hh) signalling pathway. We show that reducing glycolysis both lowers steady-state levels of ATP and stabilizes Smoothened (Smo), the 7-pass transmembrane protein that transduces the Hh signal. As a result, the transcription factor Cubitus interruptus accumulates in its full-length, transcription activating form. We show that glycolysis is required to maintain the plasma membrane potential and that plasma membrane depolarization blocks cellular uptake of N-acylethanolamides-lipoprotein-borne Hh pathway inhibitors required for Smo destabilization. Similarly, pharmacological inhibition of glycolysis in mammalian cells induces ciliary translocation of Smo-a key step in pathway activation-in the absence of Hh. Thus, changes in cell metabolism alter Hh signalling through their effects on plasma membrane potential.
Project description:Regeneration is the ability that allows organisms to replace missing organs or lost tissue after injuries. This ability requires the coordinated activity of different cellular processes, including programmed cell death. Apoptosis plays a key role as a source of signals necessary for regeneration in different organisms. The imaginal discs of Drosophila melanogaster provide a particularly well-characterised model system for studying the cellular and molecular mechanisms underlying regeneration. Although it has been shown that signals produced by apoptotic cells are needed for homeostasis and regeneration of some tissues of this organism, such as the adult midgut, the contribution of apoptosis to disc regeneration remains unclear. Using a new method for studying disc regeneration in physiological conditions, we have defined the pattern of cell death in regenerating discs. Our data indicate that during disc regeneration, cell death increases first at the wound edge, but as regeneration progresses dead cells can be observed in regions far away from the site of damage. This result indicates that apoptotic signals initiated in the wound spread throughout the disc. We also present results which suggest that the partial inhibition of apoptosis does not have a major effect on disc regeneration. Finally, our results suggest that during disc regeneration distinct apoptotic signals might be acting simultaneously.
Project description:Wing imaginal discs were dissected to generate body wall and wing/hinge fragments. Targets from three biological replicates of each were generated and the expression profiles were determined using Affymetrix Drosophila Genechip 1 arrays. Comparisons between the sample groups allow the identification of genes with localized expression patterns. Keywords = Drosophila, wing disc, spatially restricted transcripts Keywords: repeat sample
Project description:The Drosophila Hedgehog receptor functions to regulate the essential downstream pathway component, Smoothened, and to limit the range of signaling by sequestering Hedgehog protein signal within imaginal disc epithelium. Hedgehog receptor function requires both Patched and Ihog activity, the latter interchangeably encoded by interference hedgehog (ihog) or brother of ihog (boi). Here we show that Patched and Ihog activity are mutually required for receptor endocytosis and degradation, triggered by Hedgehog protein binding, and causing reduced levels of Ihog/Boi proteins in a stripe of cells at the anterior/posterior compartment boundary of the wing imaginal disc. This Ihog spatial discontinuity may contribute to classically defined cell segregation and lineage restriction at the anterior/posterior wing disc compartment boundary, as suggested by our observations that Ihog activity mediates aggregation of otherwise non-adherent cultured cells and that loss of Ihog activity disrupts wing disc cell segregation, even with downstream genetic rescue of Hedgehog signal response.