Tow (Target of Wingless), a novel repressor of the Hedgehog pathway in Drosophila

Hedgehog (Hh) signalling plays a crucial role in the development and patterning of many tissues in both vertebrates and invertebrates. Aberrations in this pathway lead to severe developmental defects and cancer. Hh signal transduction in receiving cells is a well studied phenomenon; however questions still remain concerning the mechanism of repression of the pathway activator Smoothened (Smo) in the absence of Hh. Here we describe a novel repressor of the Hh pathway, Target of Wingless (Tow). Tow represents the Drosophila homolog of a conserved uncharacterised protein family. We show that Tow acts in Hh receiving cells, where its overexpression represses all levels of Hh signalling, and that this repression occurs upstream or at the level of Smo and downstream of the Hh receptor Patched (Ptc). In addition, we find that like Ptc, overexpression of Tow causes an accumulation of lipophorin in the wing disc. We demonstrate that loss of tow enhances different ptc alleles in a similar manner to another pathway repressor, Suppressor of Fused (SuFu), possibly through mediating Ptc dependant lipophorin internalisation. Combined, these results demonstrate that Tow is an important novel regulator of the Hh pathway in the wing imaginal disc, and may shed light on the mechanism of Ptc repression of Smo.     

Patched controls the Hedgehog gradient by endocytosis in a dynamin-dependent manner, but this internalization does not play a major role in signal transduction

The Hedgehog (Hh) morphogenetic gradient controls multiple developmental patterning events in Drosophila and vertebrates. Patched (Ptc), the Hh receptor, restrains both Hh spreading and Hh signaling. We report how endocytosis regulates the concentration and activity of Hh in the wing imaginal disc. Our studies show that Ptc limits the Hh gradient by internalizing Hh through endosomes in a dynamin-dependent manner, and that both Hh and Ptc are targeted to lysosomal degradation. We also found that the ptc(14) mutant does not block Hh spreading, as it has a failure in endocytosis. However, this mutant protein is able to control the expression of Hh target genes as the wild-type protein, indicating that the internalization mediated by Ptc is not required for signal transduction. In addition, we noted that both in this mutant and in those not producing Ptc protein, Hh still occurred in the endocytic vesicles of Hh-receiving cells, suggesting the existence of a second, Ptc-independent, mechanism of Hh internalization.     

Establishing positional information through gradient dynamics: A lesson from the Hedgehog signaling pathway

A long standing question in developmental biology is how morphogen gradients establish positional information during development. Although the existence of gradients and their role in developmental patterning is no longer in doubt, the ability of cells to respond to different morphogen concentrations has been controversial. In the Drosophila wing disc, Hedgehog (Hh) forms a concentration gradient along the anterior-posterior axis and establishes at least three different gene expression patterns. In a recent study, we challenged the prevailing idea that Hh establishes positional information in a dose-dependent manner and proposed a model in which dynamics of the gradient, resulting from the Hh gene network architecture, determines pattern formation in the wing disc. In this Extra View, we discuss further the methodology used in this study, highlight differences between this and other models of developmental patterning, and also present some questions that remain to be answered in this system.Key words: Hedgehog, developmental patterning, morphogen, dynamics, mathematical modeling     

Temporal modulation of the Hedgehog morphogen gradient by a patched-dependent targeting to lysosomal compartment

Morphogenetic gradient of Hh is tightly regulated for correct patterning in Drosophila and vertebrates. The Patched (Ptc) receptor is required for restricting Hh long-range activity in the imaginal discs. In this study, we investigate the different types of Hh accretion that can be observed in the Drosophila embryonic epithelial cells. We found that, in receiving cells, large apical punctate structures of Hh (Hh-LPSs) are not depending on the Ptc receptor-dependent internalization of Hh but rather reflect Hh gradient. By analyzing the dynamic of the Hh-LPS gradient formation, we demonstrate that Hh distribution is strongly restricted during late embryonic stages compared to earlier stages. We demonstrate that the up-regulation of Ptc is required for the temporal regulation of the Hh gradient. We further show that dynamin-dependent internalization of Hh is not regulating Hh spreading but is involved in shaping Hh gradient. We found that Hh gradient modulation is directly related with the dynamic expression of the ventral Hh target gene serrate (ser) and with the Hh-dependent dorsal cell fate determination. Finally, our study shows that, in vivo, the Hh/Ptc complex is internalized in the Rab7-enriched lysosomal compartment in a Ptc-dependent manner without the co-receptor Smoothened (Smo). We propose that controlled degradation is an active mechanism important for Hh gradient formation.     

Membrane potential regulates Hedgehog signalling in the Drosophila wing imaginal disc

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.     

A positive role for Patched in Hedgehog signaling revealed by the intracellular trafficking of Sex-lethal, the Drosophila sex determination master switch

The sex determination master switch, Sex-lethal (Sxl), controls sexual development as a splicing and translational regulator. Hedgehog (Hh) is a secreted protein that specifies cell fate during development. We show that Sxl is in a complex that contains all of the known Hh cytoplasmic components, including Cubitus interruptus (Ci) the only known target of Hh signaling. Hh promotes the entry of Sxl into the nucleus in the wing disc. In the anterior compartment, the Hh receptor Patched (Ptc) is required for this effect, revealing Ptc as a positive effector of Hh. Some of the downstream components of the Hh signaling pathway also alter the rate of Sxl nuclear entry. Mutations in Suppressor of Fused or Fused with altered ability to anchor Ci are also impaired in anchoring Sxl in the cytoplasm. The levels, and consequently, the ability of Sxl to translationally repress downstream targets in the sex determination pathway, can also be adversely affected by mutations in Hh signaling genes. Conversely, overexpression of Sxl in the domain that Hh patterns negatively affects wing patterning. These data suggest that the Hh pathway impacts on the sex determination process and vice versa and that the pathway may serve more functions than the regulation of Ci.     

Ptc1 and Ptc2 transcripts provide distinct readouts of Hedgehog signaling activity during chick embryogenesis.

Hedgehog (Hh) signaling in vertebrates controls patterning and differentiation of a broad range of tissues during development. The Hh receptor Patched (Ptc) is a critical regulator of signaling, maintaining active repression of the pathway in the absence of stimulation, limiting excess diffusion of ligand, and providing an efficient negative feedback mechanism for fine-tuning the responsiveness of receiving cells. Two distinct Ptc genes have been isolated from several vertebrates. Here, we describe the cloning of a second Ptc gene from chick (Ptc2). We show that Ptc1 and Ptc2 are both upregulated at sites of active Hh signaling but that the expression patterns of these genes only partially overlap, thus providing distinct readouts of Hh pathway stimulation. We also show that chick Ptc2 is expressed in the posterior apical ectodermal ridge (AER) of the limb bud in a pattern similar to Fgf4 and that the induction of Ptc2 within the AER, like that of Fgf4, is mediated via antagonism of BMP signaling. The differential responsiveness of cells to Hh pathway stimulation (as marked by the differential induction of Ptc genes) suggests heterogeneity in the mechanisms by which Hh signals are transduced within different populations of receiving cells.     

Multiple roles of the gene zinc finger homeodomain-2 in the development of the Drosophila wing

The gene zfh2 and its human homolog Atbf1 encode huge molecules with several homeo- and zinc finger domains. It has been reported that they play important roles in neural differentiation and promotion of apoptosis in several tissues of both humans and flies. In the Drosophila wing imaginal disc, Zfh2 is expressed in a dynamic pattern and previous results suggest that it is involved is proximal–distal patterning. In this report we go further in the analysis of the function of this gene in wing development, performing ectopic expression experiments and studying its effects in genes involved in wing development. Our results suggest that Zfh2 plays an important role controlling the expression of several wing genes and in the specification of those cellular properties that define the differences in cell proliferation between proximal and distal domains of the wing disc.     

DSulfatase-1 fine-tunes Hedgehog patterning activity through a novel regulatory feedback loop

Sulfs are secreted sulfatases that catalyse removal of sulfate from Heparan Sulfate Proteoglycans (HSPGs) in the extracellular space. These enzymes are well known to regulate a number of crucial signalling pathways during development. In this study, we report that DSulfatase-1 (DSulf1), the unique Drosophila Sulf protein, is a regulator of Hedgehog (Hh) signalling during wing development. DSulf1 activity is required in both Hh source and Hh receiving cells for proper positioning of Hh target gene expression boundaries. As assessed by loss- and gain-of-function experiments in specific compartments, DSulf1 displays dual functions with respect to Hh signalling, acting as a positive regulator in Hh producing cells and a negative regulator in Hh receiving cells. In either domain, DSulf1 modulates Hh distribution by locally lowering the concentration of the morphogen at the apical pole of wing disc cells. Thus, we propose that DSulf1, by its desulfation catalytic activity, lowers Hh/HSPG interaction in both Hh source and target fields, thereby enhancing Hh release from its source of production and reducing Hh signalling activity in responding cells. Finally, we show that Dsulf1 pattern of expression is temporally regulated and depends on EGFR signalling, a Hh-dependent secondary signal in this tissue. Our data reveal a novel Hh regulatory feedback loop, involving DSulf1, which contributes to maintain and stabilise expression domains of Hh target genes during wing disc development.     

BMP morphogen gradients in flies

Bone morphogenetic proteins (BMPs) act as morphogens to control patterning and growth in a variety of developing tissues in different species. How BMP morphogen gradients are established and interpreted in the target tissues has been extensively studied in Drosophila melanogaster. In Drosophila, Decapentaplegic (Dpp), a homologue of vertebrate BMP2/4, acts as a morphogen to control dorsal–ventral patterning of the early embryo and anterior–posterior patterning and growth of the wing imaginal disc. Despite intensive efforts over the last twenty years, how the Dpp morphogen gradient in the wing imaginal disc forms remains controversial, while gradient formation in the early embryo is well understood. In this review, we first focus on the current models of Dpp morphogen gradient formation in these two tissues, and then discuss new strategies using genome engineering and nanobodies to tackle open questions.     

Oligomerization and endocytosis of Hedgehog is necessary for its efficient exovesicular secretion

Hedgehog (Hh) is a secreted morphogen involved in both short- and long-range signaling necessary for tissue patterning during development. It is unclear how this dually lipidated protein is transported over a long range in the aqueous milieu of interstitial spaces. We previously showed that the long-range signaling of Hh requires its oligomerization. Here we show that Hh is secreted in the form of exovesicles. These are derived by the endocytic delivery of cell surface Hh to multivesicular bodies (MVBs) via an endosomal sorting complex required for transport (ECSRT)–dependent process. Perturbations of ESCRT proteins have a selective effect on long-range Hh signaling in Drosophila wing imaginal discs. Of importance, oligomerization-defective Hh is inefficiently incorporated into exovesicles due to its poor endocytic delivery to MVBs. These results provide evidence that nanoscale organization of Hh regulates the secretion of Hh on ESCRT-derived exovesicles, which in turn act as a vehicle for long-range signaling.     

The wing imaginal disc

The Drosophila wing imaginal disc is a tissue of undifferentiated cells that are precursors of the wing and most of the notum of the adult fly. The wing disc first forms during embryogenesis from a cluster of ∼30 cells located in the second thoracic segment, which invaginate to form a sac-like structure. They undergo extensive proliferation during larval stages to form a mature larval wing disc of ∼35,000 cells. During this time, distinct cell fates are assigned to different regions, and the wing disc develops a complex morphology. Finally, during pupal stages the wing disc undergoes morphogenetic processes and then differentiates to form the adult wing and notum. While the bulk of the wing disc comprises epithelial cells, it also includes neurons and glia, and is associated with tracheal cells and muscle precursor cells. The relative simplicity and accessibility of the wing disc, combined with the wealth of genetic tools available in Drosophila, have combined to make it a premier system for identifying genes and deciphering systems that play crucial roles in animal development. Studies in wing imaginal discs have made key contributions to many areas of biology, including tissue patterning, signal transduction, growth control, regeneration, planar cell polarity, morphogenesis, and tissue mechanics.     

Notch and Wingless modulate the response of cells to Hedgehog signalling in the Drosophila wing

During Drosophila wing development, Hedgehog (Hh) signalling is required to pattern the imaginal disc epithelium along the anterior-posterior (AP) axis. The Notch (N) and Wingless (Wg) signalling pathways organise the dorsal-ventral (DV) axis, including patterning along the presumptive wing margin. Here, we describe a functional hierarchy of these signalling pathways that highlights the importance of competing influences of Hh, N, and Wg in establishing gene expression domains. Investigation of the modulation of Hh target gene expression along the DV axis of the wing disc revealed that collier/knot (col/kn), patched (ptc), and decapentaplegic (dpp) are repressed at the DV boundary by N signalling. Attenuation of Hh signalling activity caused by loss of fused function results in a striking down-regulation of col, ptc, and engrailed (en) symmetrically about the DV boundary. We show that this down-regulation depends on activity of the canonical Wg signalling pathway. We propose that modulation of the response of cells to Hh along the future proximodistal (PD) axis is necessary for generation of the correctly patterned three-dimensional adult wing. Our findings suggest a paradigm of repression of the Hh response by N and/or Wnt signalling that may be applicable to signal integration in vertebrate appendages.     

Differential Involvement of Hedgehog Signaling in Butterfly Wing and Eyespot Development

Butterfly eyespots may have evolved from the recruitment of pre-existent gene circuits or regulatory networks into novel locations on the wing. Gene expression data suggests one such circuit, the Hedgehog (Hh) signaling pathway and its target gene engrailed (en), was recruited from a role in patterning the anterior-posterior insect wing axis to a role patterning butterfly eyespots. However, while Junonia coenia expresses hh and en both in the posterior compartment of the wing and in eyespot centers, Bicyclus anynana lacks hh eyespot-specific expression. This suggests that Hh signaling may not be functioning in eyespot development in either species or that it functions in J. coenia but not in B. anynana. In order to test these hypotheses, we performed functional tests of Hh signaling in these species. We investigated the effects of Hh protein sequestration during the larval stage on en expression levels, and on wing size and eyespot size in adults. Hh sequestration led to significantly reduced en expression and to significantly smaller wings and eyespots in both species. But while eyespot size in B. anynana was reduced proportionately to wing size, in J. coenia, eyespots were reduced disproportionately, indicating an independent role of Hh signaling in eyespot development in J. coenia. We conclude that while Hh signaling retains a conserved role in promoting wing growth across nymphalid butterflies, it plays an additional role in eyespot development in some, but not all, lineages of nymphalid butterflies. We discuss our findings in the context of alternative evolutionary scenarios that led to the differential expression of hh and other Hh pathway signaling members across nymphalid species.