The Drosophila ortholog of the human Wnt inhibitor factor Shifted controls the diffusion of lipid-modified Hedgehog

The Hedgehog (Hh) family of morphogenetic proteins has important instructional roles in metazoan development and human diseases. Lipid modified Hh is able to migrate to and program cells far away from its site of production despite being associated with membranes. To investigate the Hh spreading mechanism, we characterized Shifted (Shf) as a component in the Drosophila Hh pathway. We show that Shf is the ortholog of the human Wnt inhibitory factor (WIF), a secreted antagonist of the Wingless pathway. In contrast, Shf is required for Hh stability and for lipid-modified Hh diffusion. Shf colocalizes with Hh in the extracellular matrix and interacts with the heparan sulfate proteoglycans (HSPG), leading us to suggest that Shf could provide HSPG specificity for Hh. We also show that human WIF inhibits Wg signaling in Drosophila without affecting the Hh pathway, indicating that different WIF family members might have divergent functions in each pathway.     

Shifted, the Drosophila ortholog of Wnt inhibitory factor-1, controls the distribution and movement of Hedgehog

We here identify and characterize an extracellular modulator of Hedgehog signaling in Drosophila, Shifted. Shifted is required for high levels of long-range signaling in the developing wing imaginal disc. Surprisingly, shifted encodes the only Drosophila ortholog of the secreted vertebrate protein Wnt Inhibitory Factor-1 (WIF-1), whose known role is to bind to extracellular Wnts and inhibit their activity. However, Shifted does not regulate Hedgehog signaling by affecting Wingless or Wnt signaling. We show instead that Shifted is a secreted protein that acts over a long distance and is required for the normal accumulation of Hh protein and its movement in the wing. Our data further indicate that Shf interacts with Hh and the heparan sulfate proteoglycans. Therefore, we propose that Shf stabilizes the interaction between Hh and the proteoglycans, an unexpected role for a member of the WIF-1 family.     

Expression of a secreted form of Dally, a Drosophila glypican, induces overgrowth phenotype by affecting action range of Hedgehog

Glypicans, a family of heparan sulfate proteoglycans attached to the cell surface via a glycosylphosphatidylinositol (GPI)-anchor, play essential roles in morphogen signaling and distributions. A Drosophila glypican, Dally, regulates the gradient formation of Decapentaplegic (Dpp) in the developing wing. To gain insights into the function of glypicans in morphogen signaling, we examined the activities of two mutant forms of Dally: a transmembrane form (TM-Dally) and a secreted form (Sec-Dally). Misexpression of tm-dally in the wing disc had a similar yet weaker effect in enhancing Dpp signaling compared to that of wild-type dally. In contrast, Sec-Dally shows a weak dominant negative activity on Dpp signal transduction. Furthermore, sec-dally expression led to patterning defects as well as a substantial overgrowth of tissues and animals through the expansion of the action range of Hh. These findings support the recently proposed model that secreted glypicans have opposing and/or distinct effects on morphogen signaling from the membrane-tethered forms.     

Glypicans and cytonemes unite to distribute Wnt ligands

Hu et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202009082) show that Glypican 4 participates in filopodia-mediated Wnt transport from endoderm to mesoderm in zebrafish embryos to facilitate intercellular communication between germ layers.

Signaling from one cell to another is fundamental for the development of multicellular organisms. Signaling is initiated by one cell releasing a ligand so that it can be recognized on the surface of another cell. To control cell signaling, then, it is imperative to control the ligand’s extracellular distribution. Two prominent mechanisms have been identified that regulate the distribution of extracellular ligands: first, restricted diffusion mediated by glypicans, and second, direct communication between cells mediated by cytonemes or signaling filopodia, which are actin-rich conduits that transport ligands directly from the signal-generating cell to the signal-receiving cell. In the report by Hu et al., these mechanisms are united, as they show that Glypican 4 (Gpc4) promotes the function of signaling filopodia to signal from one tissue to another (Fig. 1; 1).Open in a separate windowFigure 1.Endodermal Gpc4 aids in filopodia-mediated transport of endodermal Wnts to mesoderm to facilitate morphogenesis. Cell-surface Gpc4 is required for Wnt transport from endoderm to mesoderm in zebrafish embryos. Wnt transport occurs on actin-rich signaling filopodia, and Gpc4 is required for proper filopodial morphology to effectively transport Wnts.In a previous study, these authors found that Gpc4 was required for morphogenesis of the developing zebrafish embryo, specifically for convergence and extension (C&E) movements (2). In the current article, Hu et al. identify the mechanisms by which Gpc4 facilitates intercellular signaling, beginning with the interesting finding that supplying gpc4 transgenically only in the endoderm of gpc4 null animal rescues C&E not only in the endoderm but also in the other germ layers. This result indicates that Gpc4 acts both autonomously within the endoderm but also nonautonomously, somehow affecting the other germ layers of the transgenic animal even though it is not expressed in those layers (1).How does Gpc4 function nonautonomously? Typically, glypicans localize on the cell surface where they are attached via a glycosylphosphatidylinositol (GPI) anchor, and one possibility was that Gpc4 leaves the cells that synthesize it by getting cleaved at its GPI anchor. To address this question, the authors injected one cell of a 16-cell blastula with messenger RNAs encoding both GFP-Gpc4 and nuclear mCherry, then looked later in embryogenesis to see if GFP-Gpc4 was found only at the plasma membranes of cells with mCherry-labeled nuclei. Interestingly, GFP-Gpc4 was able to spread to unlabeled cells distant from expressing cells. The authors tested whether this nonautonomous spreading could account for the ability of Gpc4 to rescue C&E in all germ layers by reengineering the Gpc4 protein so that it could no longer spread: They replaced the GPI anchor with a transmembrane domain and showed that this modification effectively anchored Gpc4 to the cells that express it. To the authors’ surprise, however, when this transmembrane version of Gpc4 was expressed in the endoderm of transgenic embryos, it was still able to rescue C&E nonautonomously, in the mesoderm. Thus, the delivery of Gpc4 itself to other cells could not fully account for its nonautonomous function.Glypicans function by promoting the diffusion of secreted ligands, such as Wnts, in the extracellular space, ensuring proper ligand availability to the recipient cells (3, 4). Given the previous findings that Xenopus Gpc4 interacts with Wnt5, Wnt8, and Wnt11 to regulate C&E (5), the authors investigated if the Gpc4-mediated rescue of a C&E defect in zebrafish was also Wnt dependent. After finding that loss of these Wnts mimicked a gpc4 loss of function phenotype, they determined that Gpc4 physically interacted with functional Wnt5b and Wnt11f2 ligands in cultured cells, as they could be coimmunoprecipitated. Importantly, genetic interaction experiments showed that endodermal Gpc4 rescue of the C&E defect relied on endogenous Wnt5b and Wnt11f2 ligands. Based on these results, the authors tested a new model for the nonautonomous function of Gpc4—that the Wnts might spread to other cells.Some signaling ligands can be transported to other cells by signaling filopodia, actin-rich cellular protrusions that deliver ligands directly to the recipient cell. In the most significant and surprising results of this study, the authors discovered that Gpc4 was required for the functioning of endodermal signaling filopodia, as loss of gpc4 decreased their number and length. These filopodia were decorated with Wnt ligands Wnt5b and Wnt11f2, and live imaging showed labeled Wnt ligands being actively transported from cell to cell, released by and taken up by endodermal cells in embryos and even transported to mesodermal cells. Although it might have been expected that without Gpc4, these filopodia could not transport Wnt ligands, the authors found that both Wnt ligands were still available in the signaling filopodia, and instead the problem is that without Gpc4, the filopodia are too short and too few to effectively transport Wnts from cell to cell. This conclusion is supported by experiments that interfere genetically or pharmacologically with the actin polymerization necessary for filopodia formation: When Gpc4 was supplied only in the endoderm, embryos were especially susceptible to interference with filopodia, as these treatments abolished the ability of endodermal Gpc4 to rescue C&E in other germ layers.Thus, the conclusion of this work is that the nonautonomous effect of Gpc4 in rescuing C&E in all germ layers comes from the role of Gpc4 in promoting the function of signaling filopodia, likely delivering Wnt ligands from endodermal cells that express them to mesodermal and ectodermal cells that need them for morphogenesis to occur.Signaling filopodia or cytonemes, as they were first discovered and called in flies, emerged as a novel mechanism of ligand transport in vivo about two decades ago (6). Much of cytoneme biology, other than the general actin-based cellular machinery required for their formation, is largely unknown, and the discovery that glypicans contribute to their formation and function is exciting. Two prior observations in flies supported the idea that glypicans could be involved in cytoneme function. First, cytonemes cannot extend over a patch of cells mutant for fly glypicans (7). Second, glypicans localize on cytonemes (8), suggesting that they might contribute to cytoneme function. The advance by Hu et al. alters how we think about cytoneme-like actin protrusions: at least in some contexts, their ligand-delivery function is impaired in the absence of a glypican. This work also has the potential to revise how we think of the standard role of glypicans, understood to be exchange factors that passively distribute ligands randomly through the extracellular space by reversible binding. Coupling glypicans with the machinery of the actin cytoskeleton provides an active mechanism for the directed delivery of signaling ligands.There is still a lot to learn about how glypicans and actin protrusions work together. The big unanswered question is how glypicans can communicate with the actin cytoskeleton on a molecular level. Glypicans are GPI anchored and lack a transmembrane domain, so they cannot communicate directly with the inside of the cell. Therefore, there must exist a network of proteins that spans the membrane, connecting glypicans to the intracellular cytoskeletal machinery to facilitate changes in cell shape, which underlie cytoneme formation. The identity of these proteins, however, is not known. The lateral mobility afforded by GPI anchors could allow for focal accumulation of glypicans, initiating the underlying cytoskeletal machinery to form a protrusion. Another interesting possibility stems from the location of glypicans in the glycocalyx, a pericellular matrix made up of glycoproteins and glycolipids, which can alter cell membrane properties and shapes by altering physical forces experienced by the cell membrane (9). Perhaps glypicans and their unknown glycocalyx partners can promote actin protrusions at specific sites on the cell surface. Despite these black boxes, the discovery that glypicans can promote formation of filopodia is both significant and exciting because it opens avenues to investigate how specificities of signaling molecules and fine-tuning of development might be regulated.     

Wnt signaling is required for early development of zebrafish swimbladder

BACKGROUND: Wnt signaling plays critical roles in mammalian lung development. However, Wnt signaling in the development of the zebrafish swimbladder, which is considered as a counterpart of mammalian lungs, have not been explored. To investigate the potential conservation of signaling events in early development of the lung and swimbladder, we wish to address the question whether Wnt signaling plays a role in swimbladder development. METHODOLOGY/PRINCIPAL FINDINGS: For analysis of zebrafish swimbladder development, we first identified, by whole-mount in situ hybridization (WISH), has2 as a mesenchymal marker, sox2 as the earliest epithelial marker, as well as hprt1l and elovl1a as the earliest mesothelial markers. We also demonstrated that genes encoding Wnt signaling members Wnt5b, Fz2, Fz7b, Lef1, Tcf3 were expressed in different layers of swimbladder. Then we utilized the heat-shock inducible transgenic lines hs:Dkk1-GFP and hs:ΔTcf-GFP to temporarily block canonical Wnt signaling. Inhibition of canonical Wnt signaling at various time points disturbed precursor cells specification, organization, anterioposterior patterning, and smooth muscle differentiation in all three tissue layers of swimbladder. These observations were also confirmed by using a chemical inhibitor (IWR-1) of Wnt signaling. In addition, we found that Hedgehog (Hh) signaling was activated by canonical Wnt signaling and imposed a negative feedback on the latter. SIGNIFICANCE/CONCLUSION: We first provided a new set of gene markers for the three tissue layers of swimbladder in zebrafish and demonstrated the expression of several key genes of Wnt signaling pathway in developing swimbladder. Our functional analysis data indicated that Wnt/β-catenin signaling is required for swimbladder early development and we also provided evidence for the crosstalk between Wnt and Hh signaling in early swimbladder development.     

Mammalian Notum induces the release of glypicans and other GPI-anchored proteins from the cell surface

Glypicans are heparan sulfate proteoglycans that are attached to the cell surface by a GPI (glycosylphosphatidylinositol)anchor. Glypicans regulate the activity of Wnts, Hedgehogs,bone morphogenetic proteins and fibroblast growth factors. In the particular case of Wnts, it has been proposed that GPI-anchored glypicans stimulate Wnt signalling by facilitating and/or stabilizing the interaction between Wnts and their cell surface receptors. On the other hand, when glypicans are secreted to the extracellular environment, they can act as competitive inhibitors of Wnt. Genetic screens in Drosophila have recently identified a novel inhibitor of Wnt signalling named Notum. The Wnt inhibiting activity of Notum was associated with its ability to release Dlp [Dally (Division abnormally delayed)-like protein; a Drosophila glypican] from the cell surface by cleaving the GPI anchor. Because these studies showed that the other Drosophila glypican Dally was not released from the cell surface by Notum,it remains unclear whether this enzyme is able to cleave glypicans from mammalian cells. Furthermore, it is also not known whether Notum cleaves GPI-anchored proteins that are not members of the glypican family. Here, we show that mammalian Notum can cleave several mammalian glypicans. Moreover, we demonstrate that Notum is able to release GPI-anchored proteins other than glypicans. Another important finding of the present study is that,unlike GPI-phospholipase D, the other mammalian enzyme that cleaves GPI-anchored proteins, Notum is active in the extracellular environment. Finally, by using a cellular system in which GPC3 (glypican-3) stimulates Wnt signalling, we show that Notum can act as a negative regulator of this growth factor.     

On the role of glypicans in the process of morphogen gradient formation

Glypicans are cell surface molecules that influence signaling and gradient formation of secreted morphogens and growth factors. Several distinct functions have been ascribed to glypicans including acting as co-receptors for signaling proteins. Recent data show that glypicans are also necessary for morphogen propagation in the tissue. In the present study, a model describing the interaction of a morphogen with glypicans is formulated, analyzed and compared with measurements of the effect of glypican Dally-like (Dlp) overexpression on Wingless (Wg) morphogen signaling in Drosophila melanogaster wing imaginal discs. The model explains the opposing effect that Dlp overexpression has on Wg signaling in the distal and proximal regions of the disc and makes a number of quantitative predictions for further experiments. In particular, our model suggests that Dlp acts by allowing Wg to diffuse on cell surface while protecting it from loss and degradation, and that Dlp rather than acting as Wg co-receptor competes with receptors for morphogen binding.     

Drosophila glypicans control the cell-to-cell movement of Hedgehog by a dynamin-independent process

The signalling molecule Hedgehog (Hh) functions as a morphogen to pattern a field of cells in animal development. Previous studies in Drosophila have demonstrated that Tout-velu (Ttv), a heparan sulphate polymerase, is required for Hh movement across receiving cells. However, the molecular mechanism of Ttv- mediated Hh movement is poorly defined. We show that Dally and Dally-like (Dly), two Drosophila glypican members of the heparan sulphate proteoglycan (HSPG) family, are the substrates of Ttv and are essential for Hh movement. We show that embryos lacking dly activity exhibit defects in Hh distribution and its subsequent signalling. However, both Dally and Dly are involved and are functionally redundant in Hh movement during wing development. We further demonstrate that Hh movement in its receiving cells is regulated by a cell-to-cell mechanism that is independent of dynamin-mediated endocytosis. We propose that glypicans transfer Hh along the cell membrane to pattern a field of cells.     

Wnt inhibitory factor (WIF)‐1 promotes melanogenesis in normal human melanocytes

Wnt signaling plays a role in the differentiation as well as the development of melanocytes. Using a microarray analysis, hyperpigmentary skin of melasma expressed high levels of Wnt inhibitory factor‐1 (WIF‐1) compared with perilesional normal skin. In this study, the expression and functional roles of WIF‐1 on melanocytes were investigated. WIF‐1 was expressed both in the melanocytes of normal human skin and in cultured melanocytes. The upregulation of WIF‐1 on cultured normal human melanocytes significantly induced expressions of MITF and tyrosinase, which were associated with increased melanin content and tyrosinase activity. Consistent with the stimulatory effect of WIF‐1, WIF‐1 siRNA reduced melanogenesis in the cells. Moreover, WIF‐1 increases pigmentation in melanocytes co‐cultured with WIF‐1‐overexpressed fibroblasts and of organ‐cultured human skin. These findings suggest that melanocytes express WIF‐1 constitutively in vivo and in vitro and that WIF‐1 promotes melanogenesis in normal human melanocytes.     

NMR structure of the WIF domain of the human Wnt-inhibitory factor-1

The human Wnt-binding protein Wnt-inhibitory factor-1 (WIF-1) comprises an N-terminal WIF module followed by five EGF-like repeats. Here we report the three-dimensional structure of the WIF domain of WIF-1 determined by NMR spectroscopy. The fold consists of an eight-stranded beta-sandwich reminiscent of the immunoglobulin fold. Residual detergent (Brij-35) used in the refolding protocol was found to bind tightly to the WIF domain. The binding site was identified by intermolecular nuclear Overhauser effects observed between the WIF domain and the alkyl chain of the detergent. The results point to a possible role of WIF domains as a recognition motif of Wnt and Drosophila Hedgehog proteins that are activated by palmitoylation.     

Identification and Expression Analysis of Zebrafish Glypicans during Embryonic Development

Heparan sulfate Proteoglycans (HSPG) are ubiquitous molecules with indispensable functions in various biological processes. Glypicans are a family of HSPG’s, characterized by a Gpi-anchor which directs them to the cell surface and/or extracellular matrix where they regulate growth factor signaling during development and disease. We report the identification and expression pattern of glypican genes from zebrafish. The zebrafish genome contains 10 glypican homologs, as opposed to six in mammals, which are highly conserved and are phylogenetically related to the mammalian genes. Some of the fish glypicans like Gpc1a, Gpc3, Gpc4, Gpc6a and Gpc6b show conserved synteny with their mammalian cognate genes. Many glypicans are expressed during the gastrulation stage, but their expression becomes more tissue specific and defined during somitogenesis stages, particularly in the developing central nervous system. Existence of multiple glypican orthologs in fish with diverse expression pattern suggests highly specialized and/or redundant function of these genes during embryonic development.     

Glypican-mediated endocytosis of Hedgehog has opposite effects in flies and mice

Glypicans are glycosylphosphatidylinositol-linked heparan sulfate proteoglycans that play an essential part in the regulation of morphogen signalling. Two new reports using Drosophila and mice have highlighted the importance of glypican endocytosis in the regulation of Hedgehog (Hh) signalling and in Wingless gradient formation. One Drosophila glypican, Dally-like, acts positively in Hh signalling, whereas mouse Glypican-3 is a negative regulator. This difference seems to be dependent on whether glypicans promote the internalization of Hh alone or as a complex with its receptor, Patched.     

Wnt11 controls cell contact persistence by local accumulation of Frizzled 7 at the plasma membrane

Wnt11 is a key signal, determining cell polarization and migration during vertebrate gastrulation. It is known that Wnt11 functionally interacts with several signaling components, the homologues of which control planar cell polarity in Drosophila melanogaster. Although in D. melanogaster these components are thought to polarize cells by asymmetrically localizing at the plasma membrane, it is not yet clear whether their subcellular localization plays a similarly important role in vertebrates. We show that in zebrafish embryonic cells, Wnt11 locally functions at the plasma membrane by accumulating its receptor, Frizzled 7, on adjacent sites of cell contacts. Wnt11-induced Frizzled 7 accumulations recruit the intracellular Wnt signaling mediator Dishevelled, as well as Wnt11 itself, and locally increase cell contact persistence. This increase in cell contact persistence is mediated by the local interaction of Wnt11, Frizzled 7, and the atypical cadherin Flamingo at the plasma membrane, and it does not require the activity of further downstream effectors of Wnt11 signaling, such as RhoA and Rok2. We propose that Wnt11, by interacting with Frizzled 7 and Flamingo, modulates local cell contact persistence to coordinate cell movements during gastrulation.     

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.     

Hedgehog and Wnt coordinate signaling in myogenic progenitors and regulate limb regeneration

Amphibians have a remarkable capacity for limb regeneration. Following a severe injury, there is complete regeneration with restoration of the patterning and cellular architecture of the amputated limb. While studies have focused on the structural anatomical changes during amphibian limb regeneration, the signaling mechanisms that govern cellular dedifferentiation and blastemal progenitors are unknown. Here, we demonstrate the temporal and spatial requirement for hedgehog (Hh) signaling and its hierarchical correlation with respect to Wnt signaling during newt limb regeneration. While the dedifferentiation process of mature lineages does not depend on Hh signaling, the proliferation and the migration of the dedifferentiated cells are dependent on Hh signaling. Temporally controlled chemical inactivation of the Hh pathway indicates that Hh-mediated antero-posterior (AP) specification occurs early during limb regeneration and that Hh is subsequently required for expansion of the blastemal progenitors. Inhibition of Hh signaling results in G0/G1 arrest with a concomitant reduction in S-phase and G2/M population in myogenic progenitors. Furthermore, Hh inhibition leads to reduced Pax7-positive cells and fewer regenerating fibers relative to control tissue. We demonstrate that activation of Wnt signaling rescues the inhibition of Hh pathway mainly by enhancing proliferative signals, possibly mediated through TCF4 activity. Collectively, our results demonstrate coordinated signaling of Hh and Wnt activities in regulating blastemal progenitors and their hierarchical positioning during limb regeneration.     

Heparan sulfate proteoglycans are critical for the organization of the extracellular distribution of Wingless

Recent studies in Drosophila have shown that heparan sulfate proteoglycans (HSPGs) are required for Wingless (Wg/Wnt) signaling. In addition, genetic and phenotypic analyses have implicated the glypican gene dally in this process. Here, we report the identification of another Drosophila glypican gene, dally-like (dly) and show that it is also involved in Wg signaling. Inhibition of dly gene activity implicates a function for DLY in Wg reception and we show that overexpression of DLY leads to an accumulation of extracellular Wg. We propose that DLY plays a role in the extracellular distribution of Wg. Consistent with this model, a dramatic decrease of extracellular Wg was detected in clones of cells that are deficient in proper glycosaminoglycan biosynthesis. We conclude that HSPGs play an important role in organizing the extracellular distribution of Wg.