Conservation of the Hedgehog Pathway

The Hedgehog (Hh) family of secreted proteins is involved in a number of developmental processes as well as in cancer. Genetic and biochemical data suggest that the Sonic hedgehog (Shh) receptor is composed of at least two proteins: the tumor suppressor protein Patched (Ptc) and the seven-transmembrane protein Smoothened (Smo). Using a biochemical assay for activation of the transcription factor Gli, a downstream component of the Hh pathway, it has been shown that Smo functions as the signaling component of the Shh receptor, and that this activity can be blocked by Ptc. The inhibition of Smo by Ptc can be relieved by the addition of Shh. Furthermore, oncogenic forms of Smo are insensitive to Ptc repression in this assay. Mapping of the Smo domains required for binding to Ptc and for signaling reveals that the Smo-Ptc interaction involves mainly the amino terminus of Smo, and that the third intracellular loop and the seventh transmembrane domain are required for signaling. This mapping was carried out by creating Smoothened-Frizzled chimeric proteins and assaying for Smo function. These data demonstrate that Smo is the signaling component of a multicomponent Hh receptor complex and that Ptc is a ligand-regulated inhibitor of Smo. Different domains of Smo are involved in Ptc binding and activation of a Gli reporter construct. The latter requires the third intracellular loop and the seventh transmembrane domain of Smo, regions often involved in coupling to G proteins. However, no changes in the levels of cyclic AMP or calcium associated with such pathways could be detected following receptor activation (Murone, 1999).

Sonic hedgehog (Shh) signaling from the posterior zone of polarizing activity (ZPA) is the primary determinant of anterior-posterior polarity in the vertebrate limb field. An active signal is produced by an autoprocessing reaction that covalently links cholesterol to the N-terminal signaling moiety (N-Shhp), tethering N-Shhp to the cell membrane. The role played by this lipophilic modification was examined in Shh-mediated patterning of mouse digits. Both the distribution and activity of N-Shhp indicate that N-Shhp acts directly over a few hundred microns. In contrast, N-Shh, a form that lacks cholesterol, retains similar biological activity to N-Shhp, but signaling is posteriorly restricted. Thus, cholesterol modification is essential for the normal range of signaling. It also appears to be necessary for appropriate modulation of signaling by the Shh receptor, Ptc1 (Lewis, 2001).

A significant difference between the fly wing disc and mouse limb studies is in the role that cholesterol modification plays in Ptc interactions. It has been proposed that the cholesterol modification on N-Hhp is not essential for N-Hh binding to Ptc, but is required for its Ptc-dependent sequestration, an interaction that attenuates Hh signaling. In the mouse limb, reducing Ptc1 dosage in the presence of a single N-Shhp allele has no detectable effect on digit patterning. However, reducing the dosage of active Ptc1 alleles in N-Shh/Shhn (Shhn is a null allele of Shh) embryos restores anterior digits, albeit with inappropriate identity for their position. This result suggests that reducing Ptc1 levels leads to an anterior extension of the N-Shh signaling domain, presumably due to decreased sequestration of N-Shh by Ptc1. Thus, cholesterol modification is not absolutely required for Ptc1-mediated sequestration of N-Shh, but higher levels of Ptc1 may be required than those that suffice for sequestration of N-Shhp. In summary, it is likely that addition of cholesterol to N-Shh is required for robust feedback control by Ptc1 (Lewis, 2001).

The fact that Ptc1 can sequester N-Shh might help to explain a paradoxical result. In N-Shh/+ limbs, Ptc1, Gli1, Gremlin, Fgf4, and Bmp2 are all ectopically expressed in more anterior positions and digit 1 is duplicated. This is indicative of an increased range of Shh signaling. It is postulated that the movement of N-Shhp through a field of target cells depends on the balance between Ptc1-mediated sequestration of ligand and a proposed Ext-dependent transport process. In N-Shh/Shhn embryos, only N-Shh is produced. N-Shh is sequestered by Ptc1 but, due to the absence of a cholesterol modification, is not transported normally by an Ext-dependent process and only has a limited range of activity. Both N-Shh and N-Shhp are present in N-Shh/+ embryos; this alters the balance between sequestration and transport. Because N-Shh can only be sequestered and not transported, one would expect that relatively less N-Shhp is sequestered. As a consequence, more N-Shhp may be available for transport, resulting in an anterior extension of N-Shhp-mediated signaling. Testing this model will require an approach that distinguishes N-Shh and N-Shhp protein in the developing limb bud (Lewis, 2001).

In humans, dysfunctions of the Hedgehog receptors Patched and Smoothened are responsible for numerous pathologies. However, signaling mechanisms involving these receptors are less well characterized in mammals than in Drosophila. To obtain structure-function relationship information on human Patched and Smoothened, these human receptors were expressed in Drosophila Schneider 2 cells. As its Drosophila counterpart, human Patched is able to repress the signaling pathway in the absence of Hedgehog ligand. In response to Hedgehog, human Patched is able to release Drosophila Smoothened inhibition, suggesting that human Patched is expressed in a functional state in Drosophila cells. Human Smo, when expressed in Schneider cells, is able to bind the alkaloid cyclopamine, suggesting that it is expressed in a native conformational state. Furthermore, contrary to Drosophila Smoothened, human Smoothened does not interact with the kinesin Costal 2 and thus is unable to transduce the Hedgehog signal. Moreover, cell surface fluorescent labeling suggest that human Smoothened is enriched at the Schneider 2 plasma membrane in response to Hedgehog. These results suggest that human Smoothened is expressed in a functional state in Drosophila cells, where it undergoes a regulation of its localization comparable with its Drosophila homologue. Thus, it is proposed that the upstream part of the Hedgehog pathway involving Hedgehog interaction with Patched, regulation of Smoothened by Patched, and Smoothened enrichment at the plasma membrane is highly conserved between Drosophila and humans; in contrast, signaling downstream of Smoothened is different (De Rivoyre, 2006; full text of article).

Patched interaction with hedgehog proteins

Patched (Ptc) is the ligand-binding component of the Hedgehog (Hh) receptor complex. In the Drosophila embryo, Ptc and Hh colocalize in vesicular punctate structures. However, receptor-mediated endocytosis of Hh proteins has not been demonstrated. By using chick neural plate explants, it has been shown that Sonic hedgehog (Shh)-responsive neural precursor cells internalize recombinant and endogenous Shh and provide direct evidence for a gradient of endogenous Shh in the ventral neural tube. Shh internalization is blocked by a monoclonal antibody whose epitope overlaps the Ptc-binding site of Shh. These findings suggest that Shh internalization is mediated by Ptc-1 and may be linked to signaling. Concordantly, transfection of mammalian cell lines with a Ptc-1 cDNA confers the ability to internalize multiple forms of Shh, including transmembrane-anchored Shh, by a dynamin-dependent process (Incardona, 2000a).

Sonic hedgehog (Shh) signal transduction involves the ligand binding Patched1 (Ptc1) protein and a signaling component, Smoothened (Smo). Combined genetic and biochemical studies have indicated that Ptc inhibits a latent, tonic signaling activity of Smo, and that Hh binding to Ptc releases the inhibition of Smo. A select group of compounds inhibits both Shh signaling, regulated by Ptc1, and late endosomal lipid sorting, regulated by the Ptc-related Niemann-Pick C1 (NPC1) protein. NPC1 functions in the sorting and recycling of cholesterol and glycosphingolipids in the late endosomal/lysosomal system. It is suggested that Ptc1 regulates Smo activity through a common late endosomal sorting pathway also utilized by NPC1. During signaling, Ptc accumulates in endosomal compartments, but it is unclear if Smo follows Ptc into the endocytic pathway. The dynamic subcellular distributions of Ptc1, Smo, and activated Smo mutants has been characterized individually and in combination. Ptc1 and Smo colocalize extensively in the absence of ligand and are internalized together after ligand binding, but Smo becomes segregated from Ptc1/Shh complexes destined for lysosomal degradation. In contrast, activated Smo mutants do not colocalize with nor are they cotransported with Ptc1. Agents that block late endosomal transport and protein sorting inhibit the ligand-induced segregation of Ptc1 and Smo. Like NPC1-regulated lipid sorting, Shh signal transduction is blocked by antibodies that specifically disrupt the internal membranes of late endosomes, which provide a platform for protein and lipid sorting. These data support a model in which Ptc1 inhibits Smo only when in the same compartment. Ligand-induced segregation allows Smo to signal independent of Ptc1 after becoming sorted from Ptc1/Shh complexes in the late endocytic pathway (Incardona, 2002).

Loss-of-function mutations in glypican-3 (GPC3), one of the six mammalian glypicans, causes the Simpson-Golabi-Behmel overgrowth syndrome (SGBS), and GPC3 null mice display developmental overgrowth. Because the Hedgehog signaling pathway positively regulates body size, it was hypothesized that GPC3 acts as an inhibitor of Hedgehog activity during development. This study show that GPC3 null embryos display increased Hedgehog signaling and that GPC3 inhibits Hedgehog activity in cultured mouse embryonic fibroblasts. In addition, it is reportd that GPC3 interacts with high affinity with Hedgehog but not with its receptor, Patched, and that GPC3 competes with Patched for Hedgehog binding. Furthermore, GPC3 induces Hedgehog endocytosis and degradation. Surprisingly, the heparan sulfate chains of GPC3 are not required for its interaction with Hedgehog. It is concluded that GPC3 acts as a negative regulator of Hedgehog signaling during mammalian development and that the overgrowth observed in SGBS patients is, at least in part, the consequence of hyperactivation of the Hedgehog signaling pathway (Capurro, 2008).

Patched in other insects

The origin of new morphological characters is a long-standing problem in evolutionary biology. Novelties arise through changes in development, but the nature of these changes is largely unknown. In butterflies, eyespots have evolved as new pattern elements that develop from special organizers called foci. Formation of these foci is associated with novel expression patterns of the Hedgehog signaling protein, its receptor Patched, the transcription factor Cubitus interruptus, and the engrailed target gene, all of which break the conserved compartmental restrictions on this regulatory circuit in insect wings. Redeployment of preexisting regulatory circuits may be a general mechanism underlying the evolution of novelties. hh is expressed in all cells of the posterior compartment of the butterfly wing disc, as it is in Drosophila, but hh transcript levels are increased in a striking pattern in cells just outside of the subdivision midlines at specific positions along the proximodistal axis of the wing. These domains of increased hh transcription flank cells that have the potential to form foci. Higher levels of hh transcripts accumulate specifically in cells that flank the developing foci. In the presence of high levels of Hh, Patched function is inhibited, resulting in the accumulation of the activator form of Ci. Because ptc is a direct target of Ci, cells that receive and transduce the Hh signal have increased levels of ptc transcription. Activation of ptc transcription, accompanied by the accumulation of Ci protein occurs in cells that are flanked by the domains of highest hh transcription and are destined to become eyespot foci. these results indicate that the Hh signal is received and transduced by cells that will differentiate as foci. These expression patterns break the A/P compartmental restrictions on gene expression known in Drosophila. During the course of eyespot evolution, there is a relaxation of the strict En-mediated repression of ci that occurs in the posterior compartment of Drosophila. During focal establishment, en and invected are targets, rather than inducers of Hh signaling. In most species of butterflies, eyespots are found only in the posterior compartment of the wing. But in those species in which eyespots are found in the anterior compartment, both En/Inv and Ci are coexpressed in eyespot foci, including the one in the anterior compartment. Thus the expression of the Hh signaling pathway and en/inv is associated with the development of all eyespot foci and has become independent of A/P compartmental restrictions. It is suggested that during eyespot evolution, the Hh-dependent regulatory circuit that establishes foci is recruited from the circuit that acts along the A/P boundary of the wing. This recruitment of an entire regulatory circuit through changes in the regulation of a subset of components increases the facility with which new developmental functions can evolve and may be a general theme in the evolution of novelties within extant structures (Keys, 1999).

The early embryo of the spider Achaearanea tepidariorum is emerging as a model for the simultaneous study of cell migration and pattern formation. A cell cluster internalized at the center of the radially symmetric germ disc expresses the evolutionarily conserved dorsal signal Decapentaplegic. This cell cluster migrates away from the germ disc center along the basal side of the epithelium to the germ disc rim. This cell migration is thought to be the symmetry-breaking event that establishes the orientation of the dorsoventral axis. In this study, knockdown of a patched homolog, At-ptc, that encodes a putative negative regulator of Hedgehog (Hh) signaling, prevents initiation of the symmetry-breaking cell migration (see The formation and migration of CM cells during early Achaearanea embryogenesis and cumulus-shift defects caused by At-ptc1 dsRNA injection). Knockdown of a smoothened homolog, At-smo, shows that Hh signaling inactivation also arrests the cells at the germ disc center, whereas moderate inactivation results in sporadic failure of cell migration termination at the germ disc rim. hh transcript expression patterns indicated that the rim and outside of the germ disc are the source of the Hh ligand. Analyses of patterning events suggests that in the germ disc, short-range Hh signal promotes anterior specification and long-range Hh signal represses caudal specification. Moreover, negative regulation of Hh signaling by At-ptc appears to be required for progressive derepression of caudal specification from the germ disc center. Cell migration defects caused by At-ptc and At-smo knockdown correlated with patterning defects in the germ disc epithelium. It is proposed that the cell migration crucial for dorsoventral axis orientation in Achaearanea is coordinated with anteroposterior patterning mediated by Hh signaling (Akiyama-Oda, 2010).

C. elegans patched homologs

Patched defines a class of multipass membrane proteins that control cell fate and cell proliferation. Biochemical studies in vertebrates indicate that the membrane proteins Ptc and Smoothened (Smo) form a receptor complex that binds Hedgehog (Hh) morphogens. Smo transduces the Hh signal to downstream effectors. The C. elegans genome encodes two Ptc homologs and one related pseudogene but does not encode obvious Hh or Smo homologs. ptc-1 was examined by RNAi and mutational deletion and it was found that ptc-1 is an essential gene, although the absence of ptc-1 has no detectable effect on body patterning or proliferation. Therefore, the C. elegans ptc-1 gene is functional despite the lack of Hh and Smo homologs, although a large number of predicted Hh relatives have been identified. Activity and expression of ptc-1 is essentially confined to the germ line and its progenitors. ptc-1 null mutants are sterile with multinucleate germ cells arising from a probable cytokinesis defect. A surprisingly large family of PTC-related proteins containing sterol-sensing domains, including homologs of Drosophila dispatched, has been found in C. elegans and other phyla (Kuwabara, 2000).

Despite the absence of Hh, the C. elegans genome encodes two homologs each of the Drosophila tout velu and disp proteins, which are postulated to facilitate the movement of Hh. In addition, the activity of the transcriptional regulator TRA-1, the single C. elegans homolog of Drosophila Ci and human GLI, appears to have been usurped by the sex determination pathway. The absence of ptc-1 activity has no detectable effect on sexual phenotype. Similarly, tra-1 mutations, either loss of function or gain of function, do not show phenotypes that resemble ptc-1 or ptc-3 mutants. Thus, PTC-1 and likely PTC-3 do not appear to mediate transcriptional regulation through TRA-1. Nonetheless, PTC proteins continue to have essential roles in C. elegans development, although they have no obvious role in somatic patterning (Kuwabara, 2000).

The absence of a C. elegans Hh homolog is interpreted to indicate that either Hh has been lost from the C. elegans genome or that the worm diverged from Drosophila and vertebrates before the acquisition of Hh. The closest C. elegans Hh relatives are those that share similarity to the carboxy-terminal autoprocessing domain; several of these proteins have the potential to be processed and may mediate cell signaling. Therefore, if the PTC proteins continue to function in signaling in C. elegans, it is possible that they function without a ligand or that they have acquired new ligands through coevolution with the worm Hh relatives and even new membrane protein partners. For example, although Smo is absent, the C. elegans genome encodes a number of frizzled homologs that are related by sequence to Smo. However, similar to Drosophila these proteins probably function as Wnt receptors (Kuwabara, 2000).

Alternatively, the discovery that mutations in the C. elegans ptc-1 gene disrupt germ-line cytokinesis suggests that a novel or perhaps an ancestral activity for PTC has been uncovered that is not dependent on Hh or Smo. Comparative studies suggest that the fundamental mechanisms of cytokinesis probably have been conserved among eukaryotes. These observations have led the authors to postulate that within the C. elegans germ-line syncytium, bipolar cytokinesis involves a contractile-process membrane fusion requiring vesicular transport, and stabilization of the incomplete membrane furrows within the syncytium. It is suggested that ptc-1 contributes to the process of syncytial cytokinesis by helping to establish or maintain the incomplete plasma membrane furrows separating individual nuclei within the syncytium. Furthermore, it may be found that one or more of the PTR proteins has a role in somatic cytokinesis similar to that of PTC-1 in the germ line (Kuwabara, 2000).

How might PTC-1 participate in cytokinesis? Three models are offered. (1) It is speculated that because the absence of ptc-1 disrupts either membrane deposition or stabilization during germ-line cytokinesis, PTC-1 may promote vesicle trafficking and membrane fusion events required to complete cytokinesis. It has been shown in C. elegans that membrane fusion during cytokinesis is dependent on the t-SNARE membrane fusion protein SYN-4. Furthermore, there is evidence that lipids and cholesterol have important roles in regulating the sorting of proteins and other lipids during intracellular trafficking. PTC has the ability to detect or be regulated by sterols because it carries a sterol sensing domain (SSD). In Drosophila, Hh carries a cholesterol moiety that may help to target it to the SSD of the PTC receptor, although this moiety is not essential for receptor recognition. It has also been suggested that the teratogenic effects of veratrum alkaloids, which block cholesterol biosynthesis and Sonic hedgehog (Shh) signaling, may be mediated through the SSD of Ptc and not by disrupting Hh processing. The possibility is raised that there is an additional connection between cholesterol homeostasis and Ce-PTC-1 mediated through the SSD, which may affect membrane deposition or stabilization during germ-line cytokinesis. (2) PTC-1 may have an adhesive function. It is not known how the membrane furrows separating individual germ nuclei within the germ-line syncytium retain their integrity; therefore, it is possible that the extracellular domains of PTC-1 interact with an extracellular protein or with each other to stabilize the newly formed furrows. (3) It remains possible that the cytoplasmic domains of PTC-1 interact with the cytoskeleton to promote cytokinesis (Kuwabara, 2000).

Sensory organs are often composed of neuronal sensory endings accommodated in a lumen formed by ensheathing epithelia or glia. Lumen formation in the C. elegans amphid sensory organ requires the gene daf-6. daf-6 encodes a Patched-related protein that localizes to the luminal surfaces of the amphid channel and other C. elegans tubes. While daf-6 mutants display only amphid lumen defects, animals defective for both daf-6 and the Dispatched gene che-14 exhibit defects in all tubular structures that express daf-6. Furthermore, DAF-6 protein is mislocalized, and lumen morphogenesis is abnormal, in mutants with defective sensory neuron endings. It is proposed that amphid lumen morphogenesis is coordinated by neuron-derived cues and a DAF-6/CHE-14 system that regulates vesicle dynamics during tubulogenesis (Perens, 2005).

Hedgehog signaling regulates gene expression in planarian glia

Hedgehog signaling is critical for vertebrate central nervous system (CNS) development, but its role in CNS biology in other organisms is poorly characterized. In the planarian Schmidtea mediterranea, hedgehog (hh; see Drosophila Hedgehog) is expressed in medial cephalic ganglia neurons, suggesting a possible role in CNS maintenance or regeneration. RNA sequencing of planarian brain tissue was performed following RNAi of hh and patched (ptc; see Drosophila Patched), which encodes the Hh receptor. Two misregulated genes, intermediate filament-1 (if-1) and calamari (cali), were expressed in a previously unidentified non-neural CNS cell type. These cells expressed orthologs of astrocyte-associated genes involved in neurotransmitter uptake and metabolism, and extended processes enveloping regions of high synapse concentration. It is proposed that these cells are planarian glia. Planarian glia were distributed broadly, but only expressed if-1 and cali in the neuropil near hh+ neurons. Planarian glia and their regulation by Hedgehog signaling present a novel tractable system for dissection of glia biology (Wang, 2016).

Zebrafish Patched

The signaling molecule encoded by Sonic hedgehog participates in the patterning of several embryonic structures including limbs. During early fin development in zebrafish, a subset of cells in the posterior margin of pectoral fin buds express shh. Regulation of shh in pectoral fin buds is consistent with a role in mediating the activity of a structure analogous to the zone of polarizing activity (ZPA). During growth of the bony rays of both paired and unpaired fins, and during fin regeneration, there does not seem to be a region equivalent to the ZPA and one would predict that shh would play a different role, if any, during these processes specific to fish fins. The expression of shh was examined in the developing fins of 4-week old larvae and in regenerating fins of adults. A subset of cells in the basal layer of the epidermis in close proximity to the newly formed dermal bone structures of the fin rays, the lepidotrichia, express both shh and ptc1 (which is thought to encode the receptor of the SHH signal). The expression domain of ptc1 is broader than that of shh; adjacent blastemal cells releasing the dermal bone matrix also express ptc1. Further observations indicate that the bmp2 gene, in addition to being expressed in the same cells of the basal layer of the epidermis as shh, is also expressed in a subset of the ptc1-expressing cells of the blastema. Amputations of caudal fins immediately after the first branching point of the lepidotrichia, and global administration of all-trans-retinoic acid, two procedures known to cause fusion of adjacent rays, result in a transient decrease in the expression of shh, ptc1 and bmp2. The effects of retinoic acid on shh expression occur within minutes after the onset of treatment, suggesting direct regulation of shh by retinoic acid. These observations suggest a role for shh, ptc1 and bmp2 in the patterning of the dermoskeleton of developing and regenerating teleost fins (Laforest, 1998).

Hedgehog signaling has been implicated in a variety of processes in vertebrate development, and in each case, the activity of Hh proteins is thought to be mediated by their interaction with a large multipass transmembrane protein encoded by the patched (ptc) gene. The full-length coding sequence is presented and the wild-type expression pattern is described of a second ptc gene in zebrafish, Zf-ptc2. At the sequence level Zf-ptc2 is more closely related than Zf-ptc1 to the ptc genes initially characterized in other vertebrate species. Transcription of Zf-ptc2, like Zf-ptc1, is dependent upon Hh signalling and evidence is presented that Zf-ptc2 is activated in response to lower levels of Hh activity than is Zf-ptc1. No evidence is found for any specificity in the regulatory interactions between the various Hh proteins and the two ptc genes in the zebrafish (Lewis, 1999a).

The specification of different muscle cell types in the zebrafish embryo requires signals that emanate from the axial mesoderm. Overexpression of different members of the Hedgehog protein family can induce the differentiation of two types of slow-twitch muscles: the superficially located slow-twitch fibers and the medially located muscle pioneer (MP) cells. The requirement for Hedgehog signalling in the specification of these distinct muscle cell types has been investigated in two ways: (1) by characterising the effects on target gene expression and muscle cell differentiation of the u-type (you; you-too; sonic you; chameleon; u-boot). mutants, members of a phenotypic group previously implicated in Hedgehog signaling, and (2) by analyzing the effects of overexpression of the Patched1 protein, a negative regulator of Hedgehog signaling. Embryos mutant for u-type genes all have normal notochords, leading to the suggestion that they may directly disrupt the signaling pathway required for MP induction. Two members of this class map to genes encoding components of the Hh signaling pathway. The syu mutations map to the shh gene itself, while mutations in the gene encoding the transcription factor Gli2, a homolog of the Drosophila Ci protein, are responsible for the yot mutant phenotype. The results support the idea that most u-type genes are required for Hedgehog signaling. The analysis of ptc1 expression has confirmed a role for two other members of the u-type class, con and you, in the propagation or transduction of the Hh signals between the notochord and the paraxial mesoderm. It is striking that the effects of both these mutants are like those of syu, initially weak and increasing in severity with developmental time. Whether this reflects a hypomorphism of the you and con alleles or a specificity in the function of the you and con gene products remains to be elucidated. While hedgehog signalling is essential for slow myocyte differentiation, the loss of activity of one signal, Sonic hedgehog, can be partially compensated for by other Hedgehog family proteins (Lewis, 1999c).

In zebrafish, Hedgehog (Hh) signalling from ventral midline structures is necessary and sufficient to specify posterior otic identity. Loss of Hh signalling gives rise to mirror symmetric ears with double anterior character, whereas severe upregulation of Hh signalling leads to double posterior ears. By contrast, in mouse and chick, Hh is predominantly required for dorsoventral otic patterning. Whereas a loss of Hh function in zebrafish does not affect dorsoventral and mediolateral otic patterning, this study shows that a gain of Hh signalling activity causes ventromedial otic territories to expand at the expense of dorsolateral domains. In a panel of lines carrying mutations in Hh inhibitor genes, Hh pathway activity is increased throughout the embryo, and dorsolateral otic structures are lost or reduced. Even a modest increase in Hh signalling has consequences for patterning the ear. In ptc1-/- and ptc2-/- mutant embryos, in which Hh signalling is maximal throughout the embryo, the inner ear is severely ventralised and medialised, in addition to displaying the previously reported double posterior character. Transplantation experiments suggest that the effects of the loss of Hh pathway inhibition on the ear are mediated directly. These new data suggest that Hh signalling must be kept tightly repressed for the correct acquisition of dorsolateral cell fates in the zebrafish otic vesicle, revealing distinct similarities between the roles of Hh signalling in zebrafish and amniote inner ear patterning (Hammond, 2010).

Shh promotes direct interactions between epidermal cells and osteoblast progenitors to shape regenerated zebrafish bone

Zebrafish innately regenerate amputated fins by mechanisms that expand and precisely position injury-induced progenitor cells to re-form tissue of the original size and pattern. For example, cell signaling networks direct osteoblast progenitors (pObs) to rebuild thin cylindrical bony rays with a stereotypical branched morphology. Hedgehog/Smoothened (Hh/Smo) signaling has been variably proposed to stimulate overall fin regenerative outgrowth or promote ray branching. Using a photoconvertible patched2 (see Drosophila Patched) reporter, active Hh/Smo output to a narrow distal regenerate zone comprising pObs and adjacent motile basal epidermal cells was identified. This Hh/Smo activity is driven by epidermal Sonic hedgehog a (Shha) rather than Ob-derived Indian hedgehog a (Ihha), which nevertheless functions atypically to support bone maturation. Using BMS-833923, a uniquely effective Smo inhibitor, and high-resolution imaging, it was shown that Shha/Smo is functionally dedicated to ray branching during fin regeneration. Hh/Smo activation enables transiently divided clusters of Shha-expressing epidermis to escort pObs into similarly split groups. This co-movement likely depends on epidermal cellular protrusions that directly contact pObs only where an otherwise occluding basement membrane remains incompletely assembled. Progressively separated pObs pools then continue regenerating independently to collectively re-form a now branched skeletal structure (Armstrong, 2017).

Xenopus Patched

Patched (Ptc) is a putative twelve transmembrane domain protein that is both a Hedgehog (Hh) receptor and transcriptional target of Hh. Xenopus Ptc cDNAs, Ptc-1 and Ptc-2, have been isolated and comparative analyses on their expression patterns has been carried out. The putative Ptc-2 protein has a long C-terminal extension that has similarities in both length and sequence to those of Ptc-1 proteins in mouse, chick and human. In both early embryogenesis and hindlimb development, Ptc-2 expression is restricted to cells that receive a Hh signal, a pattern similar to that of Gli-1. Ptc-1, however, shows a broader distribution, mainly non-overlapping with that of Ptc-2. Despite the difference in their expression patterns, both are induced synergistically in animal cap explants by Shh and Noggin, showing a conserved regulation in their activation mechanisms (Takabatake, 2000).

The open reading frames of Xenopus Ptc-1 (Xptc-1) and Ptc-2 (Xptc-2) are predicted to encode 1258 and 1413 amino acids, respectively. The overall amino acid identity between Xptc-1 and Xptc-2 is 63%. Like all the other Ptc proteins, both Xenopus Ptc proteins are predicted to contain 12 hydrophobic membrane-spanning domains with two large extracellular loops. Both Xenopus proteins have eight cysteine residues, conserved in all the other Ptc proteins and exhibit extensive similarity (36% identical in Xptc-1 and 34% identical in Xptc-2) in a region containing five predicted transmembrane domains (domains 2 to 6) homologous to a potential sterol-sensing domain in Niemann-Pick type C protein, that has been implicated in intracellular trafficking of cholesterol. The most obvious structural difference between the two Xenopus proteins is the C-terminal extension present in Ptc-2. Whereas Ptc-1, but not Ptc-2, has a C-terminal extension in mouse, chick and human, phylogenic analysis clearly indicates close relationships of Xptc-1 to Ptc-1 proteins in mouse, chick and human. Like zebrafish Ptc-2 (ZFptc-2), Xptc-1 lacks the C-terminal extension. Xptc-2 shows the highest similarity (80% identical) to zebrafish Ptc1 (ZFptc-1). Although Xptc-2 has a long C terminal cytoplasmic domain similar in both length and sequence to those of Ptc-1 proteins in mouse, chick and human, the overall amino acid sequence of Xptc-2 is less similar to Ptc-1 (about 60% identical) than to Ptc-2 (about 70% identical) proteins in mouse and human. In addition, Xptc-2 has only two putative glycosylation sites, like ZFptc-1 and Ptc-2 proteins in mouse and human, while Xptc-1 has an additional three sites that are conserved among ZFptc-2 and Ptc-1 proteins in mouse, chick and human. Judging from these characteristics, Xptc-1 appears to be the ortholog of ZFptc-2 and Ptc-1 in mouse, chick and human, and Xptc-2 would belong to the same or closely related class of Ptc-2 in mouse and human. These data suggest that a common ancestral form of the two types of Ptc proteins might have had a long C-terminal extension that was eliminated from some Ptc proteins after gene duplication during vertebrate evolution (Takabatake, 2000).

Vertebrate inner ear development is initiated by the specification of the otic placode, an ectodermal structure induced by signals from neighboring tissue. Although several signaling molecules have been identified as candidate otic inducers, many details of the process of inner ear induction remain elusive. Both gain- and loss-of-function approaches reveal that otic induction is responsive to the level of Hedgehog (Hh) signaling activity in Xenopus. Ectopic activation of Hedgehog signaling results in the development of ectopic vesicular structures expressing the otic marker genes XPax-2, Xdll-3, and Xwnt-3A, thus revealing otic identity. Induction of ectopic otic vesicles is also achieved by misexpression of two different inhibitors of Hh signaling: the putative Hh antagonist mHIP and XPtc1DeltaLoop2, a dominant-negative form of the Hh receptor Patched. In addition, misexpression of XPtc1DeltaLoop2 as well as treatment of Xenopus embryos with the specific Hh signaling antagonist cyclopamine results in the formation of enlarged otic vesicles. In summary, these observations suggest that a defined level of Hh signaling provides a restrictive environment for otic fate in Xenopus embryos (Koerbernick, 2003).

Chicken Patched

Chicken PTC, a homolog of Drosophila patched is regulated by Sonic Hedgehog in the developing neural tube. PTC is expressed in neural and somite development in all regions of these tissues known to be responsive to Sonic Hedgehog signal. PTC expression is found in neural tissue, from the caudal end of the neural tube through the diencephalon. In the developing hindbrain, PTC is expressed in the rhombomeres in a gradient that is higher ventrally and lower dorsally. PTC is also expressed in a variety of non-neural tissues, including posterior mesoderm of the first and second branchial arches, in the caudal intestinal portal and in the paraxial mesoderm, as well as the developing limb, the tongue and buccal region, and in the feather germs, in addition to the brain. As in the limb bud, ectopic expression of Sonic hedgehog leads to ectopic induction of PTC in the neural tube and paraxial mesoderm. The pattern of PTC expression suggests that Sonic hedgehog may play an inductive role in more dorsal regions of the neural tube than had been previously demonstrated. Examination of the pattern of PTC expression also suggests that PTC may act in a negative feedback loop to attenuate hedgehog signaling (Marigo, 1996a).

talpid3 is an embryonic-lethal chicken mutation in a molecularly un-characterised autosomal gene. The recessive, pleiotropic phenotype includes polydactylous limbs with morphologically similar digits. Previous analysis established that hox-D and bmp genes [normally expressed posteriorly in the limb bud in response to a localized, posterior source of Sonic Hedgehog (Shh)] are expressed symmetrically across the entire anteroposterior axis in talpid3 limb buds. In contrast, Shh expression itself is unaffected. Expression of patched (ptc), which encodes a component of the Shh receptor and is probably itself a direct target of Shh signaling, was examined to establish whether talpid3 acts in the Shh pathway. ptc expression has been found to be significantly reduced in talpid3 embryos. talpid3 function is not required for Shh signal production but is required for normal response to Shh signals, implicating talpid3 in transduction of Shh signals in responding cells. Analysis of expression of putative components of the Shh pathway (gli1, gli3 and coupTFII) shows that genes regulated by Shh are either ectopically expressed or no longer responsive to Shh signals in talpid3 limbs, suggesting possible bifurcation in the Shh pathway. Genetic mapping of gli1, ptc, shh and smoothened in chickens is described. Co-segregation analysis confirms that none of these genes correspond to talpid3 (Lewis, 1999b).

A chicken Patched homolog is strongly expressed adjacent to all tissues where members of the hedgehog family are expressed. As in Drosophila, ectopic expression of Sonic hedgehog leads to ectopic induction of chicken Patched. Based on this regulatory conservation, vertebrate Patched is likely to be directly downstream of Sonic hedgehog signaling. An important role for Sonic hedgehog is the regulation of anterior/posterior pattern in the developing limb bud. Since Patched is directly downstream of the Hedgehog signal, the extent of high level Patched expression provides a measure of the distance that Sonic hedgehog diffuses and directly acts. On this basis, Sonic hedgehog is found to directly act as a signal over only the posterior third of the limb bud. During limb patterning, secondary signals are secreted in both the mesoderm (e.g. Bone Morphogenetic Protein-2) and apical ectodermal ridge (e.g. Fibroblast Growth Factor-4) in response to Sonic hedgehog. Thus knowledge of which is the direct target tissue is essential for unraveling the molecular patterning of the limb. The expression of Patched provides a strong indication that the mesoderm and not the ectoderm is the direct target of Sonic hedgehog signaling in the limb bud. Induction of Patched requires Sonic hedgehog but, unlike Bone Morphogenetic Protein-2 and Hox genes, does not require Fibroblast Growth Factor as a co-inducer. It is therefore a more direct target of Sonic hedgehog than other patterning genes (Marigo, 1996c).

In the avian embryo, previous work has demonstrated that the notochord provides inductive signals to activate myoD and pax1 regulatory genes, which are expressed in the dorsal and ventral somite cells that give rise to myotomal and sclerotomal lineages. Bead implantation and antisense inhibition experiments have been carried out that show that Sonic hedgehog is both a sufficient and essential notochord signal molecule for myoD and pax1 activation in somites. Genes of the Sonic hedgehog signal response pathway [specifically patched (the Sonic hedgehog receptor) and gli and gli2/4, (two zinc-finger transcription factors)] are activated in coordination with somite formation, establishing that Sonic hedgehog response genes play a regulatory role in coordinating the response of somites to the constitutive notochord Sonic hedgehog signal. The expression of patched, gli and gli2/4 is differentially patterned in the somite, providing mechanisms for differentially transducing the Sonic hedgehog signal to the myotomal and sclerotomal lineages. The activation of gli2/4 is controlled by the process of somite formation and signals from the surface ectoderm, whereas upregulation of patched and activation of gli is controlled by the process of somite formation and a Sonic hedgehog signal. Therefore, the Sonic hedgehog signal response genes carry out important functions in regulating the initiation of the Sonic hedgehog response in newly forming somites and in regulating the patterned expression of myoD and pax1 in the myotomal and sclerotomal lineages following somite formation (Borycki, 1998).

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. This paper describes the cloning of a second Ptc gene from chick (Ptc2). Ptc1 and Ptc2 are both upregulated at sites of active Hh signaling but the expression patterns of these genes only partially overlap, thus providing distinct readouts of Hh pathway stimulation. chick Ptc2 is expressed in the posterior apical ectodermal ridge (AER) of the limb bud in a pattern similar to Fgf4, and 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 (Pearse, 2001).

WT1 regulates the development of the posterior taste field

Despite the importance of taste in determining nutrient intake, understanding of the processes that control the development of the peripheral taste system is lacking. Several early regulators of taste development have been identified, including sonic hedgehog, bone morphogenetic protein 4 and multiple members of the Wnt/β-catenin signaling pathway. However, the regulation of these factors, including their induction, remains poorly understood. This study identified a crucial role for the Wilms' tumor 1 protein (WT1) in circumvallate (CV) papillae development. WT1 is a transcription factor that is important in the normal development of multiple tissues, including both the olfactory and visual systems. In mice, WT1 expression is detectable by E12.5, when the CV taste placode begins to form. In mice lacking WT1, the CV fails to develop normally and markers of early taste development are dysregulated compared with wild type. Expression of the WT1 target genes Lef1, Ptch1 and Bmp4 is significantly reduced in developing tongue tissue derived from Wt1 knockout mice, and, in normal tongue, WT1 was shown to be bound to the promoter regions of these genes. Moreover, siRNA knockdown of WT1 in cultured taste cells leads to a reduction in the expression of Lef1 and Ptch1. These data identify WT1 as a crucial transcription factor in the development of the CV through the regulation of multiple signaling pathways that have established roles in the formation and patterning of taste placodes (Gao, 2014).

Hedgehog, Patched and axolotl tail regeneration

Tail regeneration in urodeles requires the coordinated growth and patterning of the regenerating tissues types, including the spinal cord, cartilage and muscle. The dorsoventral (DV) orientation of the spinal cord at the amputation plane determines the DV patterning of the regenerating spinal cord as well as the patterning of surrounding tissues such as cartilage. This phenomenon was investigated on a molecular level. Both the mature and regenerating axolotl spinal cord express molecular markers of DV progenitor cell domains found during embryonic neural tube development, including Pax6, Pax7 and Msx1. Furthermore, the expression of Sonic hedgehog (Shh) is localized to the ventral floor plate domain in both mature and regenerating spinal cord. Patched1 receptor expression indicates that hedgehog signaling occurs not only within the spinal cord but is also transmitted to the surrounding blastema. Cyclopamine treatment revealed that hedgehog signaling is not only required for DV patterning of the regenerating spinal cord but also has profound effects on the regeneration of surrounding, mesodermal tissues. Proliferation of tail blastema cells is severely impaired, resulting in an overall cessation of tail regeneration, and blastema cells no longer expressed the early cartilage marker Sox9. Spinal cord removal experiments reveal that hedgehog signaling, while required for blastema growth is not sufficient for tail regeneration in the absence of the spinal cord. By contrast to the cyclopamine effect on tail regeneration, cyclopamine-treated regenerating limbs achieve a normal length and contain cartilage. This study represents the first molecular localization of DV patterning information in mature tissue that controls regeneration. Interestingly, although tail regeneration does not occur through the formation of somites, the Shh-dependent pathways that control embryonic somite patterning and proliferation may be utilized within the blastema, albeit with a different topography to mediate growth and patterning of tail tissues during regeneration (Schnapp, 2005).

Mammalian Patched Homologs

Patched Evolutionary homologs part 2/2

patched: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.