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.     

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.     

Patched represses the Hedgehog signalling pathway by promoting modification of the Smoothened protein

Hedgehog (Hh) signalling plays a central role in many developmental processes in both vertebrates and invertebrates [1]. The multipass membrane-spanning proteins Patched (Ptc) [2-4] and Smoothened (Smo) [5-7] have been proposed to act as subunits of a putative Hh receptor complex. According to this view, Smo functions as the transducing subunit, the activity of which is blocked by a direct interaction with the ligand-binding subunit, Ptc [8]. Activation of the intracellular signalling pathway occurs when Hh binds to Ptc [8-11], an event assumed to release Smo from Ptc-mediated inhibition. Evidence for a physical interaction between Smo and Ptc is so far limited to studies of the vertebrate versions of these proteins when overexpressed in tissue culture systems [8,12]. To test this model, we have overexpressed the Drosophila Smo protein in vivo and found that increasing the levels of Smo protein per se was not sufficient for activation of the pathway. Immunohistochemical staining of wild-type and transgenic embryos revealed distinct patterns of Smo distribution, depending on which region of the protein was detected by the antibody. Our findings suggest that Smo is modified to yield a non-functional form and this modification is promoted by Ptc in a non-stoichiometric manner.     

The sterol-sensing domain of Patched protein seems to control Smoothened activity through Patched vesicular trafficking

The Hedgehog (Hh) family of signaling molecules function as organizers in many morphogenetic processes. Hh signaling requires cholesterol in both signal-generating and -receiving cells, and it requires the tumor suppressor Patched (Ptc) in receiving cells in which it plays a negative role. Ptc both blocks the Hh pathway and limits the spread of Hh. Sequence analysis suggests that it has 12 transmembrane segments, 5 of which are homologous to a conserved region that has been identified in several proteins involved in cholesterol homeostasis and has been designated the sterol-sensing domain (SSD). In the present study, we show that a Ptc mutant with a single amino acid substitution in the SSD induces target gene activation in a ligand-independent manner. This mutant Ptc(SSD) protein shows dominant-negative activity in blocking Hh signaling by preventing the downregulation of Smoothened (Smo), a positive effector of the Hh pathway. Despite its dominant-negative activity, the mutant Ptc protein functioned like the wild-type protein in sequestering and internalizing Hh. In addition, we show that Ptc(SSD) preferentially accumulates in endosomes of the endocytic compartment. All these results suggest a role of the SSD of Ptc in mediating the vesicular trafficking of Ptc to regulate Smo activity.     

Glypican-5 stimulates rhabdomyosarcoma cell proliferation by activating Hedgehog signaling

Glypican-5 (GPC5) is one of the six members of the glypican family. It has been previously reported that GPC5 stimulates the proliferation of rhabdomyosarcoma cells. In this study, we show that this stimulatory activity of GPC5 is a result of its ability to promote Hedgehog (Hh) signaling. We have previously shown that GPC3, another member of the glypican family, inhibits Hh signaling by competing with Patched 1 (Ptc1) for Hh binding. Furthermore, we showed that GPC3 binds to Hh through its core protein but not to Ptc1. In this paper, we demonstrate that GPC5 increases the binding of Sonic Hh to Ptc1. We also show that GPC5 binds to both Hh and Ptc1 through its glycosaminoglycan chains and that, unlike GPC3, GPC5 localizes to the primary cilia. Interestingly, we found that the heparan sulfate chains of GPC5 display a significantly higher degree of sulfation than those of GPC3. Based on these results, we propose that GPC5 stimulates Hh signaling by facilitating/stabilizing the interaction between Hh and Ptc1.     

Reduced white fat mass in adult mice bearing a truncated Patched 1

Hedgehog (Hh) signaling emerges as a potential pathway contributing to fat formation during postnatal development. In this report, we found that Patched 1 (Ptc1), a negative regulator of Hh signaling, was expressed in the epididymal fat pad of adult mice. Reduced total white fat mass and epididymal adipocyte cell size were observed in naturally occurring spontaneous mesenchymal dysplasia (mes) adult mice (Ptc1(mes/mes)), which carry a deletion of Ptc1 at the carboxyl-terminal cytoplasmic region. Increased expression of truncated Ptc1, Ptc2 and Gli1, the indicators of ectopic activation of Hh signaling, was observed in epididymal fat pads of adult Ptc1(mes/mes) mice. In contrast, expression of peroxisome proliferator-activated receptor gamma, CCAAT/enhancer binding protein alpha, adipocyte P2 and adipsin were reduced in epididymal fat pads of adult Ptc1(mes/mes) mice. Taken together, our results indicate that deletion of carboxyl-terminal tail of Ptc1 can lead to the reduction of white fat mass during postnatal development.     

Mutations in the sterol-sensing domain of Patched suggest a role for vesicular trafficking in Smoothened regulation

The tumor suppressor gene patched (ptc) encodes an approximately 140 kDa polytopic transmembrane protein [1-3] [corrected] that binds members of the Hedgehog (Hh) family of signaling proteins [4-6] [corrected] and regulates the activity of Smoothened (Smo), a G protein-coupled receptor-like protein essential for Hh signal transduction [7-9] [corrected]. Ptc contains a sterol-sensing domain (SSD) [10, 11] [corrected], a motif found in proteins implicated in the intracellular trafficking of cholesterol [12] [corrected], and/or other cargoes [13-15] [corrected]. Cholesterol plays a critical role in Hedgehog (Hh) signaling by facilitating the regulated secretion and sequestration of the Hh protein [16] [corrected], to which it is covalently coupled. In addition, cholesterol synthesis inhibitors block the ability of cells to respond to Hh [18, 19] [corrected], and this finding points to an additional requirement for the lipid in regulating downstream components of the Hh signaling pathway. Although the SSD of Ptc has been linked to both the sequestration of, and the cellular response to Hh [16, 20, 21] [corrected], definitive evidence for its function has so far been lacking. Here we describe the identification and characterization of two missense mutations in the SSD of Drosophila Ptc; strikingly, while both mutations abolish Smo repression, neither affects the ability of Ptc to interact with Hh. We speculate that Ptc may control Smo activity by regulating an intracellular trafficking process dependent upon the integrity of the SSD.     

A role for Sonic hedgehog in axon-to-astrocyte signalling in the rodent optic nerve.

Retinal ganglion cell (RGC) axons have been shown to stimulate the proliferation of astrocytes in the developing rodent optic nerve, but the signals that mediate this effect have not been identified. The following findings suggest that Sonic hedgehog (Shh) is one of the signals. (1) RGCs express both Shh mRNA and protein, whereas the optic nerve contains the protein but not the mRNA. (2) Astrocytes and their precursors in the developing optic nerve express the Hedgehog (Hh) receptor gene Patched (Ptc), suggesting that they are being signalled by an Hh protein. (3) Ptc expression in the nerve is greatly decreased by either nerve transection or by treatment with neutralizing anti-Shh antibodies, suggesting that it depends on axon-derived Shh. (4) Astrocyte proliferation in the developing nerve is reduced by treatment with anti-Shh antibodies, suggesting that Shh normally helps stimulate this proliferation.     

Dynamic Interpretation of Hedgehog Signaling in the Drosophila Wing Disc

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.     

A hedgehog-insensitive form of patched provides evidence for direct long-range morphogen activity of sonic hedgehog in the neural tube.

Cell pattern in the ventral neural tube is organized by Sonic hedgehog (Shh) secreted by floor plate cells. To assay the range of direct Shh action, we developed a general method for blocking transduction of Hedgehog (Hh) signals through ectopic expression of a deleted form of the Hh receptor Patched (Ptc), termed Ptc(Deltaloop2). We validated this method in Drosophila and used mouse Ptc1(Deltaloop2) (mPtc1(Deltaloop2)) to block Shh transduction in the chick neural tube. mPtc1(Deltaloop2) expression caused cell-autonomous ventral-to-dorsal switches in progenitor identity and neuronal fate throughout the ventral neural tube, supporting a gradient mechanism whereby Shh acts directly and at long range. mPtc1(Deltaloop2) expression also caused the abnormal spread of Shh to more dorsal cells, indicating that Shh in the neural tube, like Hh in Drosophila, induces a feedback mechanism that limits its range of action.     

Conserved expression control and shared activity between cognate T-box genes Tbx2 and Tbx3 in connection with Sonic hedgehog signaling during Xenopus eye development

Tbx2 and Tbx3 are considered to be cognate genes within a Tbx2/3/4/5 subfamily of T-box genes and are expressed in closely overlapping areas in a variety of tissues, including the eye. Herein, we show that misexpression of Tbx2 and Tbx3 in Xenopus embryos gave rise to defective eye morphogenesis, which was reminiscent of the defect caused by attenuated Sonic hedgehog (Shh) signaling. Indeed, Tbx2/3 misexpression suppressed Gli1, Gli2, Ptc2 and Pax2, mediators or targets of Hedgehog (Hh) signals. From these data, Tbx2/3 may have a shared function in inhibiting Gli-dependent Shh signaling during eye development. Conversely, the expression of Tbx2/3 was severely affected by both Shh and a putative dominant negative form of Hh, as well as by both transactivator and transrepressor forms of Gli-fusion proteins, suggesting that the expression of Tbx2/3 may be regulated by a Gli-dependent Hh signal transduction pathway. Because the Shh signal has been considered to play crucial roles in the formation of the proximal-distal and dorsal-ventral axes in the eyes, these findings about the mutual regulatory mechanism between Tbx2/3 and Gli-dependent Hh signaling provide valuable insight into the cause of the localized expression of Tbx2/3 and their role during the formation of these axes. In addition, our findings also imply the conserved regulation and shared activity between the cognate genes of Tbx2 and Tbx3.     

Regulation of Smoothened by Drosophila G-protein-coupled receptor kinases

The Hedgehog (Hh) signaling pathway plays a conserved and essential role in regulating development and homeostasis of numerous tissues. Cytoplasmic signaling is initiated by Smoothened (Smo), a G-protein-coupled receptor (GPCR) family member, whose levels and activity are regulated by the Hh receptor Patched (Ptc). In response to Hh binding to Ptc, Ptc-mediated repression of Smo is relieved, leading to Smo activation, surface accumulation, and downstream signaling. We find that downregulation of Drosophila Smo protein in Hh-responding imaginal disc cells is dependent on the activity of G-protein-coupled receptor kinase 2 (Gprk2). By analyzing gain- and null loss-of-function phenotypes, we provide evidence that Gprk2 promotes Smo internalization subsequent to its activation, most likely by direct phosphorylation. Ptc-dependent regulation of Smo accumulation is normal in gprk2 mutants, indicating that Gprk2 and Ptc downregulate Smo by different mechanisms. Finally, we show that both Drosophila G-protein-coupled receptor kinase orthologues, Gprk1 and Gprk2, act in a partially redundant manner to promote Hh signaling. Our results suggest that Smo is regulated by distinct Ptc-dependent and Gprk2-dependent trafficking mechanisms in vivo, analogous to constitutive and activity-dependent regulation of GPCRs. G-protein-coupled receptor kinase activity is also important for efficient downstream signaling.     

The zebrafish mutants dre, uki, and lep encode negative regulators of the hedgehog signaling pathway

Proliferation is one of the basic processes that control embryogenesis. To identify factors involved in the regulation of proliferation, we performed a zebrafish genetic screen in which we used proliferating cell nuclear antigen (PCNA) expression as a readout. Two mutants, hu418B and hu540A, show increased PCNA expression. Morphologically both mutants resembled the dre (dreumes), uki (ukkie), and lep (leprechaun) mutant class and both are shown to be additional uki alleles. Surprisingly, although an increased size is detected of multiple structures in these mutant embryos, adults become dwarfs. We show that these mutations disrupt repressors of the Hedgehog (Hh) signaling pathway. The dre, uki, and lep loci encode Su(fu) (suppressor of fused), Hip (Hedgehog interacting protein), and Ptc2 (Patched2) proteins, respectively. This class of mutants is therefore unique compared to previously described Hh mutants from zebrafish genetic screens, which mainly show loss of Hh signaling. Furthermore, su(fu) and ptc2 mutants have not been described in vertebrate model systems before. Inhibiting Hh activity by cyclopamine rescues uki and lep mutants and confirms the overactivation of the Hh signaling pathway in these mutants. Triple uki/dre/lep mutants show neither an additive increase in PCNA expression nor enhanced embryonic phenotypes, suggesting that other negative regulators, possibly Ptc1, prevent further activation of the Hh signaling pathway. The effects of increased Hh signaling resulting from the genetic alterations in the uki, dre, and lep mutants differ from phenotypes described as a result of Hh overexpression and therefore provide additional insight into the role of Hh signaling during vertebrate development.