hedgehog


EVOLUTIONARY HOMOLOGS


Table of contents

Effects of Hedgehog mutation

Holoprosencephaly (HPE) is a common developmental defect of the forebrain and frequently the midface in humans, with both genetic and environmental causes. HPE has a prevalence of 1:250 during embryogenesis and 1:16,000 newborn infants, and involves incomplete development and septation of midline structures in the central nervous system (CNS) with a broad spectrum of clinical severity. Alobar HPE, the most severe form which is usually incompatible with postnatal life, involves complete failure of division of the forebrain into right and left hemispheres and is characteristically associated with facial anomalies including cyclopia, a primitive nasal structure (proboscis) and/or midfacial clefting. At the mild end of the spectrum, findings may include microcephaly, mild hypotelorism, single maxillary central incisor and other defects. This phenotypic variability also occurs between affected members of the same family. The molecular basis underlying HPE is not known, although teratogens, non-random chromosomal anomalies and familial forms with autosomal dominant and recessive inheritance have been described. HPE3 on chromosome 7q36 is one of at least four different loci implicated in HPE. Human Sonic Hedgehog (SHH) is reported here as HPE3-the first known gene to cause HPE. Analyzing 30 autosomal dominant HPE (ADHPE) families, five families were found that segregate different heterozygous SHH mutations. Two of these mutations predict premature termination of the SHH protein, whereas the others alter highly conserved residues in the vicinity of the alpha-helix-1 motif or signal cleavage site (Roessler, 1996).

Holoprosencephaly (HPE) is a genetically and phenotypically heterogenous disorder involving the development of forebrain and midface, with an incidence of 1:16,000 live born and 1:250 induced abortions. This disorder is associated with several distinct facies and phenotypic variability: in the most extreme cases, anophthalmia or cyclopia is evident along with a congenital absence of the mature nose. The less severe form features facial dysmorphia characterized by ocular hypertelorism, defects of the upper lip and/or nose, and absence of the olfactory nerves or corpus callosum. Several intermediate phenotypes involving both the brain and face have been described. One of the gene loci, HPE3, maps to the terminal band of chromosome 7. Extensive physical mapping studies have been performed and a critical interval for HPE3 has been established. Subsequently the sonic hedgehog (SHH) gene has been identified as the prime candidate for the disorder. SHH lies within 15-250 kilobases (kb) of chromosomal rearrangements associated with HPE, suggesting that a 'position effect' has an important role in the aetiology of HPE. As detailed in the accompanying report, this role for SHH is confirmed by the detection of point mutations in hereditary HPE patients (Belloni, 1997).

Sonic hedgehog (Shh) is a secreted protein that is involved in the organization and patterning of several tissues in vertebrates. The zebrafish sonic-you (syu) gene, a member of a group of five genes required for somite patterning, encodes Shh. Embryos mutant for a deletion of syu display defects in the patterning of the somites, the lateral floor plate cells, the pectoral fins, the axons of motorneurons and the retinal ganglion cells. In contrast to mouse embryos lacking Shh activity, syu mutant embryos do form medial floor plate cells and motorneurons. Since ectopic overexpression of shh in zebrafish embryos does not induce ectopic medial floor plate cells, it is concluded that shh is neither required nor sufficient to induce this cell type in the zebrafish (Schauerte, 1998).

Targeted disruption of the mouse Sonic hedgehog gene shows that Shh plays a critical role in patterning of embryonic tissues, including the brain and spinal cord, the axial skeleton and limbs. The earliest detectable defect occurs in the future forbrain region at embryonic day 9.5. In Shh mutants, the midline is indistinct, the ventral lips of the cephalic folds are fused, and the normally separate optic vesicles appear instead as a continous single vesicle protruding at the ventral midline, with optic stalks deficient or absent. There is no invagination to form the characteristic double-layered optic cups, and the fused eye tissue at the midline forms a pigmented epithelium with no apparent differentiation of retinal tissue. The cephalic defects become even more apparent when the neural tube closes, with an overall reduction in size of the brain and spinal cord (Chiang, 1996).

By E15.5, mutants exhibit severe growth retardation throughout most of the embryo and lack the distinct forelimb and hindlimb structures. Relative growth defects in the forebrain and in craniofacial structures are so extreme that normal facial features such as the eyes, nose and oral structures are not identifiable. There are abnormalities of heart, lung, kidney and foregut development. There is absence of a morphologically distinct floorplate, but notochord tissue is present. Defects in cranio-facial, axial and appendicular skeleton are also evident. Brachyury (Drosophila homologs T-related gene) expression is transient, and the progressive rostral-to-caudal loss of notochord tissue concomitant with loss of Brachyury gene expression suggests that Shh is required for the maintenance, but not the formation, of the notochord. Although the initiation of HNF-3ß (Drosophila homolog: Forkhead) expression is normal, the failure to initiate HNF-3ß expression in mutant neural tube suggests that Shh protein is the notochord-derived signal required for HNF-3ß induction in the normal neural tube. In Shh mutants, LIM homeodomain protein Islet-1 (Drosophila homolog: Islet), normally expressed in motor neurons) is absent in the neural tube at all axial levels. Failure to induce Isl-1 suggests that Shh protein from notochord may required for differentiation of motor neurons. Expression limits of Pax-6, Pax-3 and Pax-2 in the neural tube expand beyond their normal limits (Chiang, 1996).

The mouse mutants of the hemimelia-luxate group (lx, lu, lst, Dh, Xt, and the more recently identified Hx, Xpl and Rim4) have in common preaxial polydactyly and longbone abnormalities. Associated with the duplication of digits are changes in the regulation of development of the anterior limb bud resulting in ectopic expression of signaling components such as Sonic hedgehog (Shh) and fibroblast growth factor-4 (Fgf4), but little is known about the molecular causes of this misregulation. A new member of this group of mutants, Sasquatch (Ssq) which disrupts aspects of both anteroposterior (AP) and dorsoventral (DV) patterning was generated by a transgene insertion event. The mutant displays preaxial polydactyly in the hindlimbs of heterozygous embryos, and in both hindlimbs and forelimbs of homozygotes. The Shh, Fgf4, Fgf8, Hoxd12 and Hoxd13 genes are all ectopically expressed in the anterior region of affected limb buds. The insertion site was found to lie close to the Shh locus. Furthermore, expression from the transgene reporter has come under the control of a regulatory element that directs a pattern mirroring the endogenous expression pattern of Shh in limbs. In abnormal limbs, both Shh and the reporter are ectopically induced in the anterior region, whereas in normal limbs the reporter and Shh are restricted to the zone of polarizing activity. These data strongly suggest that Ssq is caused by direct interference with the cis regulation of the Shh gene (Sharpe, 1999).

Considerable data suggest that sonic hedgehog (Shh) is both necessary and sufficient for the specification of ventral pattern throughout the nervous system, including the telencephalon. The regional markers induced by Shh in the E9.0 telencephalon are dependent on the dorsoventral and anteroposterior position of ectopic Shh expression. This suggests that by this point in development regional character in the telencephalon is established. To determine whether this prepattern is dependent on earlier Shh signaling, the telencephalon was examined in mice carrying either Shh- or Gli3-null mutant alleles. This analysis revealed that the expression of a subset of ventral telencephalic markers, including Dlx2 and Gsh2, although greatly diminished, persists in Shh-/- mutants, and that these same markers are expanded in Gli3-/- mutants. To understand further the genetic interaction between Shh and Gli3, Shh/Gli3 and Smoothened/Gli3 double homozygous mutants were examined. Notably, in animals carrying either of these genetic backgrounds, genes such as Gsh2 and Dlx2, which are expressed pan-ventrally, as well as Nkx2.1, which demarcates the ventral most aspect of the telencephalon, appear to be largely restored to their wild-type patterns of expression. These results suggest that normal patterning in the telencephalon depends on the ventral repression of Gli3 function by Shh and, conversely, on the dorsal repression of Shh signaling by Gli3. In addition, these results support the idea that, in addition to hedgehog signaling, a Shh-independent pathways must act during development to pattern the telencephalon (Rallu, 2002).

The essential roles of SHH in anteroposterior (AP) and apical ectodermal ridge AER-FGF signalling in proximodistal (PD) limb bud development are well understood. In addition, these morphoregulatory signals are key components of the self-regulatory SHH/GREM1/AER-FGF feedback signalling system that regulates distal progression of limb bud development. This study uncovers an additional signalling module required for coordinated progression of limb bud axis development. Transcriptome analysis using Shh-deficient mouse limb buds revealed that the expression of proximal genes was distally extended from early stages onwards, which pointed to a more prominent involvement of SHH in PD limb axis development. In particular, retinoic acid (RA) target genes were upregulated proximally, while the expression of the RA-inactivating Cyp26b1 enzyme was downregulated distally, pointing to increased RA activity in Shh-deficient mouse limb buds. Further genetic and molecular analysis established that Cyp26b1 expression is regulated by AER-FGF signalling. During initiation of limb bud outgrowth, the activation of Cyp26b1 expression creates a distal 'RA-free' domain, as indicated by complementary downregulation of a transcriptional sensor of RA activity. Subsequently, Cyp26b1 expression increases as a consequence of SHH-dependent upregulation of AER-FGF signalling. To better understand the underlying signalling interactions, computational simulations of the spatiotemporal expression patterns and interactions were generated. These simulations predicted the existence of an antagonistic AER-FGF/CYP26B1/RA signalling module, which was verified experimentally. In summary, SHH promotes distal progression of limb development by enhancing CYP26B1-mediated RA clearance as part of a signalling network linking the SHH/GREM1/AER-FGF feedback loop to the newly identified AER-FGF/CYP26B1/RA module (Probst, 2011).

Conservation of the Hedgehog pathway

A Sonic hedgehog (Shh) response element was identified in the chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) promoter (see Drosophila Seven-up). The Shh response element binds to a factor distinct from Gli, a gene known to mediate Shh signaling. Although this binding activity is specifically stimulated by Shh-N (amino-terminal signaling domain), it can also be unmasked with protein phosphatase treatment in the mouse cell line P19, and induction by Shh-N can be blocked by phosphatase inhibitors. Thus, Shh-N signaling may result in dephosphorylation of a target factor that is required for activation of COUP-TFII-, Islet1-, and Gli response element-dependent gene expression. This finding identifies another step in the Shh-N signaling pathway. The phosphatase that mediates this dephosphorylation in response to Shh-N treatment is PP2A or is like PP2A (see Drosophila Twins). This particular response is channeled through a protein with DNA binding activity apparently unrelated to that of the Ci/Gli family. A similar protein phosphatase activity is also required in the Ci/Gli-mediate branch of the Drosophila Hh signaling pathway (Krishnan, unpublished result). Thus, activation of specific protein phosphatase activity appears to be a general feature of Hh signal transduction (Krishnan, 1997).

Sonic hedgehog and Patched proteins are expressed in adjacent domains in the developing mouse retina. Treatment of cultures of perinatal mouse retinal cells with the amino-terminal fragment of Sonic hedgehog protein results in an increase in the proportion of cells that incorporate bromodeoxuridine, in total cell numbers, and in rod photoreceptors, amacrine cells and Müller glial cells, suggesting that Sonic hedgehog promotes the proliferation of retinal precursor cells. These finding suggest that Hedgehog and Patched are part of a conserved signaling pathway in the retinal development of both mammals and insects (Jensen, 1997).

Sonic hedgehog has been implicated in patterning of the developing chick limb. Such a role is suggested by the restricted expression of Shh along the posterior limb bud margin, and by the observation that heterologous cells expressing Shh have limb-polarizing activity resembling that of cells from the polarizing region of the posterior limb bud margin. It has not been demonstrated, however, that the Sonic hedgehog protein (SHH) alone is sufficient for limb patterning. SHH is proteolytically processed in developing chick limbs. Grafts of cells expressing SHH protein variants that correspond to individual cleavage products demonstrate that the ability to induce patterned gene expression and to impose morphological pattern upon the limb bud is limited to the amino-terminal product (SHH-N) of SHH proteolytic cleavage. The endogenous amino-terminal cleavage product is tightly localized to the posterior margin of the limb bud (Lopez-Martinez, 1995).

In vertebrate skin, sonic hedgehog is expressed specifically in the feather bud epithelia. Cyclic AMP, a protein kinase A (PKA) activator, suppresses the expression of Sonic hedgehog, (Shh) and continuous feather growth. The results suggest that Shh and PKA also have antagonistic action during vertebrate skin morphogenesis, similar to the interaction between Hedgehog and PKA in the fly (Noveen, 1996).

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, 1996d).

Branching morphogenesis of the embryonic murine lung requires interactions between the epithelium and the mesenchyme. Sonic hedgehog transcripts are present in the epithelium of the developing lung, with highest levels in the terminal buds. Transcripts of mouse patched, the putatitive Sonic hedgehog receptor, are expressed at high levels in the mesenchyme adjacent to the end buds. To investigate the function of SHH in lung development, Shh was overexpressed throughout the distal epithelium, using the surfactant protein-C (SP-C)-enhancer/promoter. Beginning around 16.5 dpc, when Shh and Ptc mRNA levels are normally both declining, this treatment causes an increase in the ratio of interstitial mesenchyme to epithelial tubules in transgenic compared to normal lungs. Transgenic newborn mice die soon after birth. Histological analysis of the lungs shows an abundance of mesenchyme and the absence of typical alveoli. Shh overexpression results in increased mesenchymal and epithelial cell proliferation at 16.5 and 17.5 dpc. However, there is no significant inhibition in the differentiation of proximal and distal epithelial cells. The expression of genes potentially regulated by SHH was also examined. No difference could be observed between transgenic and control lungs in either the level or distribution of Bmp4, Wnt2 and Fgf7 RNA. By contrast, Ptc is clearly upregulated in the transgenic lung. These results thus establish a role for SHH in lung morphogenesis, and suggest that SHH normally regulates lung mesenchymal cell proliferation in vivo (Bellusci, 1996).

Two members of the GLI family have been isolated from the chick, GLI and GLI3. Their expression patterns in a variety of tissues during embryogenesis suggest that these genes may be targets of Sonic hedgehog signals. The two GLI genes are differentially regulated by Sonic hedgehog during limb development. Sonic hedgehog up-regulates GLI transcription, while down-regulating GLI3 expression in the mesenchymal cells of the developing limb bud. An activated form of GLI can induce expression of Patched a known target of Sonic hedgehog, thus inplicating GLI as a key transcription factor in the vertebrate hedgehog signaling pathway. In conjunction with evidence from a mouse Gli3 mutant, these data suggest that GLI and GLI3 may have taken two different functions of their Drosophila homolog Cubitus interruptus. These two functions are the mediation of hedgehog signaling and the repression of hedgehog transcription. In Drosophila the same transcription factor can be utilized for both purposes because the cells expressing hedgehog and the cells reponsive to it are mutually exclusive populations. In the vertebrate limb, where the responsive cells overlap the cells producing Sonic hedgehog, the same factor cannot be used (Marigo, 1996c).

Expression of the vertebrate homolog of patched can be used as a marker for Sonic hedgehog function. Chicken PTC 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).

Zebrafish embryos injected with RNAs encoding Sonic hedgehog (Shh), Indian hedgehog (Ihh), or a dominant-negative regulatory subunit of PKA, PKI, have equivalent phenotypes. These include the expansion of proximal fates in the eye, ventral fates in the brain, and adaxial fates in somites and head mesenchyme. Moreover, ectopic expression of PKI partially rescues somite and optic stalk defects in no tail and cyclops mutants that lack midline structures that normally synthesize Shh. Conversely, all cell types promoted by ectopic expression of Shh and PKI are suppressed in embryos injected with RNA encoding a constitutively active catalytic subunit of PKA (PKA*). These results, together with epistasis studies on the block of ectopic Hh signaling by PKA*, indicate that PKA acts in target cells as a common negative regulator of Hedgehog signaling (Hammerschmidt, 1996).

The three mouse Gli genes are putative transcription factors that are the homologs of cubitus interruptus in Drosophila. Along with the gene patched, ci has been implicated in the Hedgehog (Hh) signal transduction pathway. To assess the role of Gli in embryogenesis, its expression was compared with that of Ptc and Hh family members in mouse. Gli and Ptc are expressed in similar domains in diverse regions of the developing mouse embryo and these regions are adjacent to Hh signals. Gli and different Hh isoforms show reciprocal relationships in the limb, digits, brain, gut, and whisker follicles. Gli is expressed ectopically along with Ptc and Shh in Strong's luxoid mutant mice. It is likely that Shh is expressed ectopically in the dominant Strong's Luxoid mutation. These results are consistent with conservation of the Hh signal transduction pathway in mice with Gli potentially mediating Hh signaling in multiple regions of the developing embryo (Platt, 1997).

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).

Apart from the gut, which expresses both Sonic hedgehog and Indian hedgehog, there is no overlap in the various Hh expression domains. Shh is predominantly expressed in epithelia at numerous sites of epithelial-mesenchymal interactions, including the tooth, hair, whisker, rugae, gut, bladder, urethra, vas deferens, and lung, Desert hedgehog in Schwann and Sertoli cell precursors, and Ihh in gut and cartilage. Thus, it is likely that Hh signaling plays a central role in a diverse array of morphogenetic processes. Hh expression was compared with that of a second family of signaling molecules, the Bone morphogenetic proteins (Bmps), vertebrate relatives of decapentaplegic, a target of the Drosophila Hh signaling pathway. The frequent expression of Bmp-2, -4, and -6 in similar or adjacent cell populations suggests a conserved role for Hh/Bmp interactions in vertebrate development (Bitgood, 1995).

Patched (Ptc) is a human tumor suppressor protein and a candidate receptor for Hedgehog (Hh) proteins, which regulate growth and patterning in embryos. Ptc represses expression of Hh target genes, such as Gli1 and ptc1 itself. Localized secretion of Hh appears to induce transcription of target genes in specific patterns by binding to Ptc and preventing it from functioning in recipient cells. People who are heterozygous for PTC1 exhibit a range of developmental defects, suggesting that some genes are inappropriately expressed when there is not enough Ptc protein. To test the idea that a balance between Hh and Ptc activities is essential for normal development, murine Ptc was overexpressed in the neural tube. Excess Ptc is sufficient to inhibit expression of Gli1 and ptc1, suggesting that Sonic hedgehog (Shh) cannot signal effectively. This leads to partial dorsalization of the neural tube and a wide spectrum of neural defects, ranging from embryonic lethality to hydrocephaly. This work further confirms the hypothesis that Shh and Ptc have opposing activities and provides evidence that an imbalance between these activities leads to patterning changes in the neural tube. In flies, ubiquitious expression of Hh induces the same target genes that are derepressed in a ptc mutant embryo. Conversely, excess Ptc in flies prevents expression of Hh target genes even in the presence of Hh signal. A similar relationship exists in mice. Increased levels of Ptc prevent transcription of Gli1 and ptc itself, two genes that are normally induced by Shh in the ventral tube (Goodrich, 1999).

The Cubitus interruptus (Ci) and Gli proteins are transcription factors that mediate responses to Hedgehog proteins (Hh) in flies and vertebrates, respectively. During development of the Drosophila wing, Ci transduces the Hh signal and regulates transcription of different target genes at different locations. In vertebrates, the three Gli proteins are expressed in overlapping domains and are partially redundant. To assess how the vertebrate Glis correlate with Drosophila Ci, each was expressed in Drosophila and their behaviors and activities were monitored. Each Gli has distinct activities that are equivalent to portions of the regulatory arsenal of Ci. Gli2 and Gli1 have activator functions that depend on Hh. Gli2 and Gli3 are proteolyzed to produce a repressor form able to inhibit hh expression. However, while Gli3 repressor activity is regulated by Hh, Gli2 repressor activity is not. These observations suggest that the separate activator and repressor functions of Ci are unevenly partitioned among the three Glis, yielding proteins with related yet distinct properties (Aza-Blanc, 2000).

Gli1 and Gli3 proteins can transduce Hh signals in Drosophila and both proteins are regulated by Hh -- Gli1, as an inducible activator, and Gli3 as a regulated repressor. Gli2, the Gli protein whose role and properties are least well understood, has also been characterized. Gli2 shares functional properties with Gli1 and Gli3. Gli2 contains a repressor activity able to inhibit hh expression in vivo, as well as an activator activity that is Hh sensitive. Recent studies have shown that Gli1 function is dispensable in mice if both copies of the Gli2 gene are present, suggesting that Gli2 can compensate for the absence of Gli1 if it is present in sufficient amounts. An explanation for this interaction is suggested, since Gli2 can mimic Gli1 as a regulated activator. Both Gli2 and Gli3 can be proteolyzed in Drosophila in a manner similar to Ci. In addition, these experiments have allowed for the detection of subtle, yet significant differences between Gli2 and Gli3 that might explain their distinct properties in vivo. (1) Gli2 accumulates less proteolytic product than Gli3, and this correlates with its lower repressor activity. This difference is thought to not be an artifact of the Drosophila system, since transfection of Gli2 and Gli3 constructs into 10T1/2 cells generates a similar profile of proteolysis (more Gli3 proteolytic product than Gli2). (2) Hh affects Gli2 and Gli3 differently. No change in the proportion of cleaved and uncleaved Gli2 is observed in these studies, indicating that the production of Gli2 repressor is not regulated by Hh. In contrast, Hh inhibits the accumulation of cleaved Gli3, reversing the proportion of full-length and processed forms. Consistent with this behavior in the fly system, production of Gli3 repressor is also regulated in the vertebrate limb bud, where it forms an anteroposterior gradient in response to Sonic Hh. Further studies on Gli2 in vertebrate systems will be required to validate these observations (Aza-Blanc, 2000).

Hedgehogs, diffusion, and the extracellular matrix

Patterning of the vertebrate neural tube depends on intercellular signals emanating from sources such as the notochord and the floor plate. The secreted protein Sonic hedgehog and the extracellular matrix protein Vitronectin are both expressed in these signaling centers and have both been implicated in the generation of ventral neurons. The proteolytic processing of Sonic hedgehog is fundamental for its signaling properties. This processing generates two secreted peptides with all the inducing activity of Shh residing in the highly conserved 19 kDa amino-terminal peptide (N-Shh). Vitronectin is also proteolitically processed in the embryonic chick notochord, floor plate and ventral neural tube and this processing is spatiotemporally correlated with the generation of motor neurons. The processing of Vitronectin produces two fragments of 54 kDa and 45 kDa. The 45 kDa fragment lacks the heparin-binding domain and the integrin-binding domain, RGD, present in the non-processed Vitronectin glycoprotein. N-Shh binds to the three forms of Vitronectin (70, 54 and 45 kDa) isolated from embryonic tissue, although it is preferentially associated with the 45 kDa form. In cultures of dissociated neuroepithelial cells, the combined addition of N-Shh and Vitronectin significantly increases the extent of motor neuron differentiation, as compared to the low or absent inducing capabilities of either N-Shh or Vitronectin alone. Thus, it is concluded that the differentiation of motor neurons is enhanced by the synergistic action of N-Shh and Vitronectin, and that Vitronectin may be necessary for the proper presentation of the morphogen N-Shh to one of its target cells, the differentiating motor neurons (Pons, 2000).

Hh proteins undergo an autocatalytic cleavage to yield an N-terminal and a C-terminal peptide, with the signaling capacities confined to the N peptide. Drosophila Hh-N has been shown to act via both short- and long-range signaling. In vertebrates, however, attempts to directly demonstrate Shh (SHH) or Ihh (IHH) proteins at a distance from producing cells have been largely unsuccessful. Furthermore, the fact that the Hh N peptides occur in a cholesterol-modified, membrane-tethered form is not easily reconciled with long-range signaling. This study used optimized immunohistochemistry combined with tissue separation and biochemical analyses in vivo and in vitro to determine the range of action of SHH and IHH in the mouse embryo. In all embryonic structures studied, signaling peptides were detected in producing cells, but ligands move over considerable distances depending on the tissue. These data provide direct evidence for the presence of Hedgehog signaling peptides in target compartments, suggesting a direct long-range action without a need for secondary mediators. Visualization of Hedgehog proteins in target tissues was achieved only under conditions that allowed proteoglycan/glycosaminoglycan (PG/GAG) preservation. Furthermore, induced changes of the composition of PG/GAG in the tooth alter SHH signaling. These data suggest a crucial role for PG/GAGs in Hedgehog movement (Gritli-Linde, 2001).

During odontogenesis, from dental lamina formation to predentin secretion by odontoblasts, a stage-specific dental basement membrane (BM) separates the dental epithelium and mesenchyme. The dental BM undergoes a progressive breakdown during terminal differentiation of ameloblasts. At this stage, preameloblasts establish cellular contact with odontoblasts through localized interruptions of the BM. During early tooth development, SHH protein is able to move across the BM to reach dental mesenchyme cells. Movement of growth factors during epithelial-mesenchymal interactions across a BM is not a novelty. However, compared to other growth factors, SHH is characterized by the presence of a cholesterol modification making it membrane-tethered. Recent evidence suggests that cholesterol modification may not be necessary for long-range signaling of SHH in tissues involving epithelial-mesenchymal interactions, including the tooth. However, whether another lipid modification, namely palmitoylation, is involved is not yet known. The PG/GAG-rich BM may act as a vehicle to concentrate and deliver the signaling peptide during early stages of odontogenesis. After early predentin secretion, SHH may be transported from young ameloblasts to odontoblasts via cell processes, since predentin is permissive for heterotypic cell contacts. This would be in agreement with the suggested mode of Hh transport involving cytonemes. Alternatively, since early predentin is not completely impermeable to growth factors, SHH may still be able to cross it to reach the dental papilla. In effect, both modes of SHH trafficking may be involved. In cartilage, because chondrocytes are matrix-encased cells, it is likely that IHH is transported through the PG/GAG-rich extracellular matrix (Gritli-Linde, 2001).

At the neural plate/early neural fold stages and soon after neural tube closure, given the absence of a continuous external limiting membrane (ELM, neuroepithelium basement membrane) at the midline, notochordal SHH may be transported to the basal plate largely via cell-cell contacts. The early neural tube is a pseudostratified epithelium, with the ELM serving as its base and the boundary of the central canal serving as its apex. The ventral aspect of the ELM, the notochordal basement membrane, as well as the interstitial matrix of the ventral mesenchyme are known for their relatively high amounts of PG/GAGs. Given the pattern of distribution of SHH in the basal plate, it is likely that the PG/GAG-rich ELM plays a role in SHH movement at the basal surface of neuroepithelium. In the ventricular layer, protein movement might involve cell-to-cell transfer and/or incriminate cell surface proteoglycans (Gritli-Linde, 2001).

The secreted protein Sonic hedgehog exerts many of its patterning effects through a combination of short- and long-range signaling. Three distinct mechanisms, which are not necessarily mutually exclusive, have been proposed to account for the long-range effects of Shh: simple diffusion of Shh, a relay mechanism in which Shh activates secondary signals, and direct delivery of Shh through cytoplasmic extensions, termed cytonemes. Although there is much data (using soluble recombinant Shh [ShhN]) to support the simple diffusion model of long-range Shh signaling, there has been little evidence to date for a native form of Shh that is freely diffusible and not membrane-associated. Evidence is provided for a freely diffusible form of Shh (s-ShhNp) that is cholesterol modified, multimeric and biologically potent. The availability of s-ShhNp is regulated by two functional antagonists of the Shh pathway: Patched (Ptc) and Hedgehog-interacting protein (Hip). A gradient of s-ShhNp across the anterior-posterior axis of the chick limb is shown, demonstrating the physiological relevance of s-ShhNp (Zeng, 2001).

To address how s-ShhNp, a protein with two lipophilic modifications, is soluble in an aqueous environment it is proposed that Shh might bind additional proteins to bury its hydrophobic moieties. Conditioned media was isolated from cells transfected with full-length Shh or ShhN and subjected to analysis by gel filtration chromatography. These conditioned media were isolated under serum-free conditions in which they are still biologically active to verify that s-ShhNp is not just binding to proteins present in serum. s-ShhNp migrates at about six times its native molecular weight. In contrast, ShhN migrates through the column close to its predicted molecular weight. To determine whether Shh can multimerize with itself to form the large Shh complex, s-ShhNp was immunoprecipitated from the conditioned media of cells co-transfected with Shh and a Flag-tagged Shh construct. Antibodies to the Flag epitope are able to co-immunoprecipitate untagged Shh, suggesting that Shh multimerizes with itself (Zeng, 2001).

It is speculated that mammalian homologs of the Drosophila protein Dispatched (Disp) might be involved in either the packaging of s-ShhNp or the targeting of ShhNp to lipid rafts, given that amorphic disp mutants are insensitive to HhNp. Additionally, Tout velu (TTV), an enzyme involved in heparin sulphate biosynthesis, has been implicated as a necessary component in Hh-receiving cells. Although the function that mammalian TTV homologs have in Shh target cells has not been analyzed, biochemical data is presented consistent with the proposed function of TTV in Drosophila. That is, ttv mutations are insensitive to HhNp but responsive to HhN, suggesting that Hh's lipid modifications are necessary for TTV to exert its normal biological effects. There is a central difference between ShhN and ShhNp: ShhNp can form large, stable multimers whereas ShhN cannot. Therefore, TTV homologs might regulate the biosynthesis of a molecule that is necessary to recognize s-ShhNp but is not necessary for signaling by ShhN (Zeng, 2001).

Heparan sulphate proteoglycans have been implicated in the binding and presentation of several growth factors to their receptors, thereby regulating cellular growth and differentiation. To investigate the role of heparan sulphate proteoglycans in mouse spinal neurulation, chlorate (a competitive inhibitor of glycosaminoglycan sulphation) was administered to cultured E8.5 embryos. Treated embryos exhibit accelerated posterior neuropore closure, accompanied by suppression of neuroepithelial bending at the median hinge point and accentuated bending at the paired dorsolateral hinge points of the posterior neuropore. These effects appear specific, as they can be prevented by addition of heparan sulphate to the culture medium, whereas heparitinase-treated heparan sulphate and chondroitin sulphate are ineffective. Both N- and O-sulphate groups appear to be necessary for the action of heparan sulphate. In situ hybridization analysis demonstrates a normal distribution of sonic hedgehog mRNA in chlorate-treated embryos. By contrast, patched 1 transcripts are abnormally abundant in the notochord, and diminished in the overlying neuroepithelium, suggesting that sonic hedgehog signaling from the notochord may be perturbed by inhibition of heparan sulphation. Together, these results demonstrate a regulatory role for heparan sulphate in mouse spinal neurulation (Yip, 2002).

Sonic hedgehog promotes proliferation of developing cerebellar granule cells. Since sonic hedgehog is expressed in the cerebellum throughout life it is not clear why proliferation occurs only in the early postnatal period and only in the external granule cell layer. It was asked whether heparan sulfate proteoglycans might regulate sonic hedgehog-induced proliferation and thereby contribute to the specialized proliferative environment of the external granule cell layer. A conserved sequence within sonic hedgehog was identified that is essential for binding to heparan sulfate proteoglycans, but not for binding to the receptor Patched. Sonic hedgehog interactions with heparan sulfate proteoglycans promote maximal proliferation of postnatal day 6 granule cells. By contrast, proliferation of less mature granule cells is not affected by sonic hedgehog-proteoglycan interactions. The importance of proteoglycans for proliferation increases during development in parallel with increasing expression of the glycosyltransferase genes, exostosin 1 and exostosin 2. These data suggest that heparan sulfate proteoglycans, synthesized by exostosins, may be critical determinants of granule cell proliferation (Rubin, 2002).

The sonic hedgehog (SHH) sequence was examined for identifiable motifs that might bind to heparin/HSPGs. A highly conserved Cardin-Weintraub consensus sequence for heparin binding was discovered at the N terminus of the biologically active fragment of SHH. This motif, XBBBXXBX, is characterized by a cluster of basic amino acids (B) that allows for electrostatic interaction between the positive charges on the protein and the negatively charged sulfates of HSPGs. All hedgehog proteins contain the sequence with slight variations in amino acid composition. Two basic amino acid positions within the potential heparin-binding motif are absolutely conserved between Drosophila and the family of vertebrate hedgehogs. In order to assess the role of this sequence in heparin/HSPG binding, these two basic amino acids, Arg33 and Lys37, were mutated to alanine or glutamine. The mutation to both alanine and glutamine allowed for an assessment of the contribution of hydrogen bonding to interactions between heparin and this domain. In addition, an alternative mutation was created in which an extra arginine was added in position 31 as a tool for evaluating whether total positive charge influenced SHH-HSPG interactions. All mutations were introduced into the biologically active conjugate protein comprised of the N-terminal fragment of mouse SHH and human placental alkaline phosphatase. The data indicate that the Cardin-Weintraub sequence is the domain that mediates high affinity interactions between SHH and heparin (Rubin, 2002).

The evolutionary conservation of the heparin-binding domain among the family of hedgehog proteins suggests that it plays an important role in hedgehog biology. Genetic analyses in Drosophila support this notion. Mutation of Tout-velu (Ttv), a glycosyltransferase involved in HSPG synthesis, has been demonstrated to phenocopy the Hh mutation. The expression of the closest vertebrate homologs of Ttv, Ext1 and Ext2 in developing mouse brain was examined in order to identify developmental systems in which to evaluate interactions of SHH and HSPGs. In the neonatal mouse brain Ext1 and Ext2 exhibit overlapping patterns of expression, with the highest levels of mRNA evident in the cerebellum. Expression was also detectable in the hippocampus as well as the olfactory and neo-cortices. Within the cerebellum, Ext1 and Ext2 are expressed by granule cells of both the internal (IGL) and external (EGL) granule cell layers as well as by Purkinje cells. Northern blot analysis has indicated that expression of Ext 2 is developmentally regulated in the cerebellum. No Ext2 mRNA was evident in total RNA samples from P0 mice. However at P2 and P4, equivalent, low levels of mRNA were detected and expression of Ext2 increased 4.5-fold from P4 to P9. Ext1 expression was similarly regulated during cerebellar development, with a comparable fourfold increase in expression between P4 and P9. The increase in Ext expression parallels the increase in granule cell proliferation observed in vivo during the first postnatal week. This early proliferation requires SHH. Together, these findings suggest that HSPGs, synthesized by the gycosyltransferases Ext1 and Ext2, might be present at an appropriate time and place to regulate SHH-induced proliferation during cerebellar development (Rubin, 2002).

There is an age-dependent change in the effect of HSPGs on cerebellar granule cell proliferation in response to SHH. Primary cultures from P3 mice display a sigmoidal dose response curve to SHH, that is not affected by mutation of the Cardin-Weintraub sequence, nor by treatment of cultures with heparinase or sodium perchlorate. At this stage, expression of Ext1 and Ext2 are low, and SHH:AP binds at low levels to HSPGs in cerebellar slices. These correlations suggest that HSPGs that participate in SHH responses may not be synthesized during the early neonatal period (Rubin, 2002).

By contrast, proliferation in cultures derived from P6 mice was modulated by SHH-HSPG interactions. Primary cultures derived from P6 mice display a bell-shaped dose-response curve to SHH. Mutation of the Cardin-Weintraub sequence, or treatment of cultures with heparinase or sodium perchlorate, reduces the peak proliferative response to SHH. This developmentally regulated dependence on HSPG interactions is accompanied by increased expression of Ext1 and Ext2, and the synthesis of HSPGs capable of binding to SHH. Thus, at P6, the developmental stage when granule cell proliferation is maximal, HSPGs contribute to SHH-induced proliferation. Furthermore, SHH binds at highest levels to HSPGs in the EGL, the location of proliferating granule cell precursors. Thus, the regulated synthesis of HSPGs may allow optimal proliferation to occur at both the right time and place (Rubin, 2002).

Precise patterning of cell types along the dorsal-ventral axis of the spinal cord is essential to establish functional neural circuits. In order to prove the feasibility of studying a single biological process through random mutagenesis in the mouse, recessive ENU-induced mutations were identified in six genes that prevent normal specification of ventral cell types in the spinal cord. The genes responsible for two of the mutant phenotypes, smoothened and dispatched, which are homologs of Drosophila Hh pathway components, were identified and cloned. The Dispatched homolog1 (Disp1) mutation causes lethality at midgestation and prevents specification of ventral cell types in the neural tube, a phenotype identical to the Smoothened (Smo) null phenotype. As in Drosophila, mouse Disp1 is required to move Shh away from the site of synthesis. Despite the existence of a second mouse disp homolog, Disp1 is essential for long-range signaling by both Shh and Ihh ligands. These data indicate that Shh signaling is required within the notochord to maintain Shh expression and to prevent notochord degeneration. Disp1, unlike Smo, is not required for this juxtacrine signaling by Shh (Caspary, 2002).

Drosophila disp is required in Hh-producing cells to allow release of active Hh, and clones of homozygous disp mutant cells in imaginal discs appear to accumulate high levels of Hh protein. The distribution of Shh in embryos at e9.5 was examined by confocal microscopy. In wild-type embryos, Shh protein made in the notochord spreads to the ventral neural tube and then activates Shh expression in the floor plate. High levels of Shh protein are present in the notochord cells of Disp1icb embryos; however, there is no detectable Shh protein in the ventral neural tube. Thus, the Shh made in the Disp1icb notochord fails to induce Shh expression in the ventral neural tube. Given that Disp1icb acts upstream of Patched, the results suggest that mouse Disp1, like Drosophila disp, is required for the spread of Shh ligand from the cells where it is synthesized. Further, the resemblance of the Shh Ihh, Smo, and Disp1icb phenotypes suggests that Disp1 is required for the spread of both Shh and Ihh ligands (Caspary, 2002).

The Shh expression pattern revealed an interesting difference between the Disp1icb and the Smobnb mutant phenotypes: the notochord in Disp1icb is intact and expresses high levels of Shh protein, whereas the notochord in Smobnb is small and expresses lower levels of Shh. The notochord begins to degenerate at e9.0 in Shh mutants, similar to what is observed in Smobnb embryos. Thus, Shh signaling is required, directly or indirectly, for maintenance of Shh expression in the notochord. The robust notochord of e9.0 Disp1icb mutants suggests that Disp1 is not required for Shh activity within the notochord. Similarly, Drosophila Hh produced in posterior compartment wing cells can activate the signaling pathway locally, but not at a distance, in the absence of disp. Together, the results suggest that Disp1 is not required for juxtacrine signaling by Shh and is specifically required for release of Shh to an extracellular compartment from which Shh can move to more distant cells. Long-range signaling by Shh is important for patterning the mouse neural tube, somites, and limb bud. In contrast to the case in Drosophila, where disp is expressed ubiquitously, localized expression of Disp1 in the mouse embryo could play a decisive role in determining where Shh can act at a distance (Caspary, 2002).

Hedgehog (Hh) signaling plays a major role in multiple aspects of embryonic development, which involves both short- and long-range signaling from localized Hh sources. One unusual aspect of Hh signaling is the autoproteolytic processing of Hh followed by lipid modification. As a consequence, the N-terminal fragment of Hh becomes membrane anchored on the cell surface of Hh-producing cells. A key issue in Hh signaling is to understand the molecular mechanisms by which lipid-modified Hh protein is transported from its sites of synthesis and subsequently moves through the morphogenetic field. The dispatched gene, which encodes a putative multipass membrane protein, was initially identified in Drosophila and is required in Hh-producing cells, where it facilitates the transport of cholesterol-modified Hh. The mouse dispatched (Disp) gene has now been identified. The complete Disp cDNA (4721bp) encodes a predicted protein of 1521 amino acids with a relative molecular mass of 170,047. Both Disp and a second gene, Disp-related, encode proteins with twelve predicted membrane-spanning domains as well as stretches of sequences similar to a conserved domain known as the sterol sensing domain (SSD). Proteins containing the SSD include several classes of proteins that are involved in different aspects of cholesterol homeostasis or cholesterol-linked signaling. Notably, Ptch, the Hh receptor, also contains an SSD. Disp-null mice phenocopy mice deficient in the smoothened gene, an essential component for Hh reception, suggesting that Disp is essential for Hh signaling. This conclusion is further supported by a detailed molecular analysis of Disp knockout mice, which exhibit defects characteristic of loss of Hh signaling. Evidence is provided that Disp is not required for Hh protein synthesis or processing, but rather for the movement of Hh protein from its sites of synthesis in mice. Taken together, these results reveal a conserved mechanism of Hh protein movement in Hh-producing cells that is essential for proper Hh signaling (Kawakami, 2002).

The dispatched (disp) gene is required for long-range Hedgehog (Hh) signaling in Drosophila. One of two murine homologs, mDispA, can rescue disp function in Drosophila and is essential for all Hh patterning activities examined in the early mouse embryo. Embryonic fibroblasts lacking mDispA respond normally to exogenously provided Sonic hedgehog (Shh) signal, but are impaired in stimulation of other responding cells when expressing Shh. A biochemical assay has been developed that directly measures the activity of Disp proteins in release of soluble Hh proteins. This activity is disrupted by alteration of residues functionally conserved in Patched and in a related family of bacterial transmembrane transporters, thus suggesting similar mechanisms of action for all of these proteins (Ma, 2002).

Perhaps the most striking and unexpected aspect of these results is the extreme nature of the pattern disruptions in mDispA-/- embryos. The mDispA-/- mutant phenotype is more severe than that of mutations in any single gene encoding a Hh protein. In contrast, the Drosophila disp mutant phenotype is less severe than the hh phenotype. This discrepancy appears in part due to disruption of signaling by multiple Hh proteins, because the mDispA-/- phenotype resembles that of Smo-/- and that of the Shh-/-; Ihh-/- double mutant. However, the phenotype also appears to owe its severity, at least in part, to a distinct balance in the relative importance of long-range and short-range signaling in Drosophila and in the mouse. In Drosophila disp mutants, short-range Hh signaling is intact and contributes to maintenance of target gene expression and to patterning. In mDispA-/- embryos, some signal response is retained in cells that express Shh (Ptch-lacZ is expressed in the notochord), but this response appears not to contribute to morphological pattern. The null function phenotype for the mDispA gene thus permits dissection of the relative importance of long- and short-range Hh signaling in mouse embryos and reveals a near absolute dependence on long-range signaling in patterning of the mouse embryo (Ma, 2002).

In contrast to the functional role of heparan sulfate proteoglycans (HSPGs), the importance of chondroitin sulfate proteoglycans (CSPGs) in modulating signaling pathways involving hedgehog proteins, wingless-related proteins and fibroblast growth factors remains unclear. To elucidate the importance of sulfated CSPGs in signaling paradigms required for endochondral bone formation, the brachymorphic (bm) mouse was used as a model for undersulfated CSPGs. The bm mouse exhibits a postnatal chondrodysplasia caused by a mutation in the phosphoadenosine phosphosulfate (PAPS) synthetase (Papss2) gene, leading to reduced levels of PAPS and undersulfated proteoglycans. Biochemical analysis of the glycosaminoglycan (GAG) content in bm cartilage via sulfate labeling and fluorophore-assisted carbohydrate electrophoresis revealed preferential undersulfation of chondroitin chains (CS) and normal sulfation of heparan sulfate chains. In situ hybridization and immunohistochemical analysis of bm limb growth plates showed diminished Indian hedgehog (Ihh) signaling and abnormal Ihh protein distribution in the extracellular matrix. Consistent with the decrease in hedgehog signaling, BrdU incorporation exhibited a significant reduction in chondrocyte proliferation. Direct measurements of Ihh binding to defined GAG chains demonstrated that Ihh interacts with CS, particularly chondroitin-4-sulfate. Furthermore, co-immunoprecipitation experiments showed that Ihh binds to the major cartilage CSPG aggrecan via its CS chains. Overall, this study demonstrates an important function for CSPGs in modulating Ihh signaling in the developing growth plate, and highlights the importance of carbohydrate sulfation in regulating growth factor signaling (2009).

Hedgehogs interactions with their receptors

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).

Patched1 regulates hedgehog signaling at the primary cilium

Primary cilia are essential for transduction of the Hedgehog (Hh) signal in mammals. This study investigated the role of primary cilia in regulation of Patched1 (Ptc1), the receptor for Sonic Hedgehog (Shh). Ptc1 localizes to cilia and inhibites Smoothened (Smo) by preventing its accumulation within cilia. When Shh binds to Ptc1, Ptc1 leaves the cilia, leading to accumulation of Smo and activation of signaling. Thus, primary cilia sense Shh and transduce signals that play critical roles in development, carcinogenesis, and stem cell function (Rohatgi, 2007).

In Drosophila, Ptc inhibits the movement of Smo to the plasma membrane. Binding of Hh causes the internalization of Ptc from the plasma membrane to vesicles, allowing Smo to translocate to the plasma membrane and activate downstream signaling. The discovery that protein components of primary cilia are required for Hh signaling suggested that subcellular localization has an important role in mammalian Hh signaling. Primary cilia are cell surface projections found on most vertebrate cells that function as sensory 'antennae' for signals. Several components of the Hh pathway, including Smo and the Gli proteins, accumulate in primary cilia, and Smo is enriched in cilia upon stimulation with Shh (Rohatgi, 2007).

The dynamic subcellular localization of Ptc1 and Smo in mammalian cells was studied with the use of novel antibodies to the two proteins. These antibodies allowed detection of endogenous Ptc1 and Smo in cultured mouse fibroblasts (NIH 3T3 cells) and mouse embryonic fibroblasts (MEFs), two Hh-responsive cell types. Hh signaling was activated in NIH 3T3 cells by treatment with either Shh or SAG (Shh-agonist), a small molecule that directly binds and activates Smo. Because ptc1 is itself a transcriptional target of Hh signaling, increases in Ptc1 protein levels can serve as a metric for pathway activation. Ptc1 protein levels began to rise by 4 hours and continued to increase until 24 hours after addition of Shh. After stimulation of cells with Shh or SAG, endogenous Smo was enriched in primary cilia. The mean fluorescence intensity of Smo in cilia began to increase as early as 1 hour after stimulation of cells with Shh or SAG. This likely represented relocalization from a cytoplasmic pool, because the total amount of Smo protein did not increase at this time point (Rohatgi, 2007).

To determine whether Ptc1 regulates the localization of Smo, Smo localization was examined in MEFs from ptc1-/- mice. These cells showed constitutive activation of Hh target gene transcription. Consistent with a role of Ptc1 in regulating Smo trafficking, Smo was constitutively localized to primary cilia in these cells even in the absence of Shh or SAG. Reintroduction of Ptc1 into these cells by means of a retrovirus suppressed Hh-pathway activity and prevented Smo accumulation in primary cilia. Thus, the regulation of Smo localization by Ptc1 is conserved from flies to mammals (Rohatgi, 2007).

To understand how Ptc1 may regulate entry of Smo into the cilium, the localization of Ptc1 was examined in MEFs and mouse embryos. Endogenous Ptc1 was present in small amounts in MEFs, near the limit of detection by immunofluorescence. Therefore the amounts of Ptc1 protein was increased by stimulating cells with SAG. Under these conditions, Ptc1 was highly enriched in primary cilia. The ciliary localization of Ptc1 was confirmed in three additional ways. First, Ptc1 fused to yellow fluorescent protein (Ptc1-YFP) was found around the base and in the shaft of cilia in unstimulated ptc1-/- cells infected with a retrovirus encoding Ptc1-YFP. Second, Ptc1-YFP overproduced in ptc1-/- cells by transfection showed clear ciliary localization in both live and fixed cells. Third and most important, endogenous Ptc1 was found in the cilia of mouse embryo mesoderm cells responsive to Shh (Rohatgi, 2007).

Ptc1 staining in cross sections of embryonic day 9.5 (E9.5) embryos was detected in cells of the ventral neural tube, notochord, splanchnic mesoderm, and paraxial mesoderm, precisely the regions where Hh signaling is known to be active and Shh target genes such as ptc1 are highly expressed. Focus was placed on mesoderm cells because they are likely the cells that gave rise to the MEFs that were analyzed in culture. Endogenous Ptc1 showed asymmetric localization to a domain surrounding the base of the cilium and in particles along the shaft of the cilium. This localization pattern around the base and in a particulate pattern along the shaft of the cilium is similar to that seen in cultured fibroblasts. In embryo cells, there was more variability in the amount of Ptc1 in the shaft of cilia, a finding likely related to differences in the amount of Shh signal received by cells in the complex milieu of embryonic tissue. The concentration of Ptc1 at the base of primary cilia suggests a mechanism for how it may inhibit Smo activation. Transport of proteins in and out of primary cilia is thought to be regulated at their base, and Ptc1 could function at this location to inhibit a protein-trafficking step critical for Smo activation (Rohatgi, 2007).

Shh could inactivate Ptc1 by binding to it at the cilium and inducing its internalization, degradation, or movement to other regions of the plasma membrane. To determine whether Ptc1 at the cilium can bind to Shh, a fluorescently labeled version of the N-terminal signaling fragment of Shh (ShhN-A594) was produced. Minute amounts of ShhN-A594, one-hundredth of those required to activate signaling, were added to live ptc1-/- cells transfected with Ptc1-YFP and a marker for cilia, inversin fused to cyan fluorescent protein (inversin-CFP). Live cells were used because the interaction between Shh and Ptc1 does not survive fixation. ShhN-A594 concentrated at cilia containing Ptc1-YFP and colocalized with puncta of Ptc1-YFP. Ptc1-/- cells expressing inversin-CFP alone did not bind ShhN-A594, and an excess of unlabeled ShhN prevented binding of ShhN-A594 (Rohatgi, 2007).

It was next asked whether the interaction of Shh with Ptc1 influences the localization of Ptc1. Ptc1 was concentrated at cilia after treatment of cells with SAG alone but not after treatment with Shh or a combination of Shh and SAG. This suggested that Shh binding might trigger the removal of the Ptc1-Shh complex from the cilium, or that new Ptc1 produced in response to Shh was not localized in the cilium. To distinguish these possibilities, the production of large amounts of Ptc1 was induced in the cilia of NIH 3T3 cells with SAG treatment and then the cells were switched to control medium or medium containing Shh. Ptc1 levels in the cilium remained stable in the control, but Shh treatment caused a time-dependent disappearance of Ptc1 from the primary cilium. The loss of Ptc1 from cilia was not associated with a decrease in total Ptc1 protein levels and thus implied movement of Ptc1 from cilia to another location in the cell. This delocalization was only evident with the endogenous protein and not upon examination of transfected Ptc1-YFP, a far more abundant protein (Rohatgi, 2007).

Ptc1 and Smo localization were examined in the same experiment. Because the localization changes for Ptc1 and Smo described above were each seen in >80% of the cilia visualized, the levels of Ptc1 and Smo in cilia were inversely correlated. The reciprocal time courses of Ptc1 disappearance and Smo appearance at cilia after Shh addition support a model in which Shh triggers the removal of Ptc1 from the cilium, allowing Smo to enter and activate signaling. Consistent with this idea, cells of the ventral neural tube and floor plate, which receive large amounts of Shh, showed high levels of Smo and low levels of Ptc1 in cilia. The movement of Ptc1 and Smo at the cilium is analogous to the situation in Drosophila, where pathway activation is associated with Smo movement to the plasma membrane and movement of Ptc away (Rohatgi, 2007).

Ptc1 may regulate Smo localization through a small molecule. Because Smo translocation to the primary cilium appears to be a critical step in its activation, a regulatory small molecule would be predicted to control this step. Naturally occurring oxysterols are good candidates for endogenous small molecules that regulate Smo function. Cellular sterol concentrations are important determinants of a cell's responsiveness to Shh, and oxysterols can activate Hh signaling. When NIH 3T3 cells were treated with activating concentrations of the oxysterol 20alpha-hydroxycholesterol, Smo rapidly translocated to the primary cilium with kinetics that were identical to those seen in cells treated with SAG or Shh. Treatment with 7alpha-hydroxycholesterol, an oxysterol that does not activate the Hh pathway, did not induce translocation of Smo. This result provides a specific molecular mechanism -- Smo translocation to cilia -- to explain how oxysterols regulate Hh signaling (Rohatgi, 2007).

Cells treated with 20alpha-hydroxycholesterol also retained Ptc1 in cilia in a pattern similar to that seen in cells treated with SAG. Thus, oxysterols appear to function not like Shh, by causing the removal of Ptc1 from cilia, but at a more downstream step to make Smo insensitive to the inhibitory effects of Ptc1. However, oxysterols function differently from SAG because they likely do not directly bind to Smo (Rohatgi, 2007).

These results suggest that Ptc1 localization to primary cilia inhibits the Hh pathway by excluding Smo and also allows cilia to function as chemosensors for the detection of extracellular Shh. Binding of Shh to Ptc1 at primary cilia is coupled to pathway activation by the reciprocal movement of Ptc1 out of the cilia and Smo into the cilia, a process that may be mediated by oxysterols. Elucidating the molecular machinery that controls Ptc1 and Smo trafficking at primary cilia will likely provide new targets for modulation of this important pathway (Rohatgi, 2007).

Hedgehog signaling and cilia

Several studies have linked cilia and Hedgehog signaling, but the precise roles of ciliary proteins in signal transduction remain enigmatic. A mouse mutation, hennin (hnn), causes coupled defects in cilia structure and Sonic hedgehog (Shh) signaling. The hnn mutant cilia are short with a specific defect in the structure of the ciliary axoneme, and the hnn neural tube shows a Shh-independent expansion of the domain of motor neuron progenitors. The hnn mutation is a null allele of Arl13b, a small GTPase of the Arf/Arl family, and the Arl13b protein is localized to cilia. Double mutant analysis indicates that Gli3 repressor activity is normal in hnn embryos, but Gli activators are constitutively active at low levels. Thus, normal structure of the ciliary axoneme is required for the cell to translate different levels of Shh ligand into differential regulation of the Gli transcription factors that implement Hedgehog signals (Caspary, 2007).

In mutants that lack cilia altogether, such as Ift172 and Ift88 mutants, no Gli activator is produced. In contrast, in hnn mutants, it appears there is a constitutive low level of Gli activator in all cells in the neural tube. It is therefore proposed that there are two cilia-dependent steps in the formation of Gli activators. It is proposed that full-length Gli proteins are modified within the cilium in a Hh-independent process to have a low level of activator function, and the low-level activator is normally tethered in the cilium in the absence of ligand. The idea that there are ligand-independent steps in Hh signaling that depend on cilia is not novel: processing of Gli3 to make Gli3 repressor is also cilia dependent and occurs in the absence of ligand. In response to Hh, full-length Gli proteins can be further modified to create high-level activator, which is then released from the cilium to the nucleus. In the abnormal cilia that lack Arl13b, the modification that produces full-length Gli protein with low-level activity takes place, but this low-level activator is not effectively tethered in the cilium and is released inappropriately to the nucleus in the absence of Hh ligand. This step retains a small amount of sensitivity to upstream signals in hnn mutant, as the phenotype of Ptch1 hnn double mutants is not identical to the hnn phenotype (Caspary, 2007).

Two alternative views of the relationship between cilia and Hh signaling have been proposed. In the simpler view, cilia represent a site where Hh pathway components are enriched, and the high local concentration of the proteins allows efficient signaling transduction. Alternatively, dynamic trafficking within the cilium may allow a sequence of protein interactions that promote the activity of the pathway. The hnn phenotype supports the latter model, as it reveals a complexity of events that occur within the cilium (Caspary, 2007).

Cilia are essential for mammalian embryonic development as well as for the physiological activity of various adult organ systems. Despite the multiple crucial roles that cilia play, the mechanisms underlying ciliogenesis in mammals remain poorly understood. A forward genetic approach identified Hearty (Hty), a recessive lethal mouse mutant with multiple defects, including neural tube defects, abnormal dorsal-ventral patterning of the spinal cord, a defect in left-right axis determination and severe polydactyly (extra digits). By genetic mapping, sequence analysis of candidate genes and characterization of a second mutant allele, Hty was identified as C2cd3, a novel gene encoding a vertebrate-specific C2 domain-containing protein. Target gene expression and double-mutant analyses suggest that C2cd3 is an essential regulator of intracellular transduction of the Hedgehog signal. Furthering a link between Hedgehog signaling and cilia function, it was found that cilia formation and proteolytic processing of Gli3 are disrupted in C2cd3 mutants. Finally, C2cd3 protein was identified at the basal body, consistent with its essential function in ciliogenesis. Interestingly, the human ortholog for this gene lies in proximity to the critical regions of Meckel-Gruber syndrome 2 (MKS2) and Joubert syndrome 2 (JBTS2), making it a potential candidate for these two human genetic disorders (Hoover, 2008).

Cilia have been implicated in Hedgehog (Hh) and Wnt signaling in mouse but not in Drosophila. To determine whether the role of cilia is conserved in zebrafish, maternal-zygotic (MZ) oval (ovl; ift88) mutants were generated that lack all cilia. MZovl mutants display normal canonical and non-canonical Wnt signaling but show defects in Hh signaling. As in mouse, zebrafish cilia are required to mediate the activities of Hh, Ptc, Smo and PKA. However, in contrast to mouse Ift88 mutants, which show a dramatic reduction in Hh signaling, zebrafish MZovl mutants display dampened, but expanded, Hh pathway activity. This activity is largely due to gli1, the expression of which is fully dependent on Hh signaling in mouse but not in zebrafish. These results reveal a conserved requirement for cilia in transducing the activity of upstream regulators of Hh signaling but distinct phenotypic effects due to differential regulation and differing roles of transcriptional mediators (Huang, 2009).

Zfp423 regulates Sonic Hedgehog signaling via primary cilium function

Zfp423 (see Drosophila Oaz) encodes a 30-zinc finger transcription factor that intersects several canonical signaling pathways. Zfp423 mutations result in ciliopathy-related phenotypes, including agenesis of the cerebellar vermis in mice and Joubert syndrome (JBTS19) and nephronophthisis (NPHP14) in humans. Unlike most ciliopathy genes, Zfp423 encodes a nuclear protein and its developmental expression is complex, leading to alternative proposals for cellular mechanisms. This study shows that Zfp423 is expressed by cerebellar granule cell precursors, that loss of Zfp423 in these precursors leads to cell-intrinsic reduction in proliferation, loss of response to Shh (see Drosophila hh), and primary cilia abnormalities that include diminished frequency of both Smoothened (see Drosophila smo) and IFT88 (see Drosophila nompB) localization. Loss of Zfp423 alters expression of several genes encoding key cilium components, including increased expression of Tulp3 (see Drosophila ktub). Tulp3 is a direct binding target of Zfp423 and reducing the overexpression of Tulp3 in Zfp423-deficient cells suppresses Smoothened translocation defects. These results define Zfp423 deficiency as a bona fide ciliopathy, acting upstream of Shh signaling, and indicate a mechanism intrinsic to granule cell precursors for the resulting cerebellar hypoplasia.

Transcriptional regulation of Hedgehogs

In developing limbs, numerous signaling molecules have been identified but less is known about the mechanisms by which such signals direct patterning. Signal transduction pathways in the chicken limb bud have been explored. A cDNA encoding RACK1, a protein that binds and stabilizes activated protein kinase C (PKC), was isolated in a screen for genes induced by retinoic acid (RA) in the chick wing bud. Fibroblast growth factor (FGF) also induces RACK1 and such induction of RACK1 expression is accompanied by a significant augmentation in the number of active PKC molecules and an elevation of PKC enzymatic activity. This suggests that PKCs mediate signal transduction in the limb bud. Application of chelerythrine, a potent PKC inhibitor, to the presumptive wing region results in buds that do not express sonic hedgehog (Shh) and develop into wings that are severely truncated. This observation suggests that the expression of Shh depends on PKCs. Providing ectopic SHH protein, RA or ZPA grafts overcomes the effects of blocking PKC with chelerythrine and results in a rescue of the wing morphology. Taken together, these findings suggest that the responsiveness of Shh to FGF is mediated, at least in part, by PKCs (Lu, 2001).

The establishment of the floor plate at the ventral midline of the CNS is dependent on an inductive signaling process mediated by the secreted protein Sonic hedgehog (Shh). To understand molecularly how floor plate induction proceeds a Shh-responsive regulatory element was identified that directs transgene reporter expression to the ventral midline of the CNS and notochord in a Shh-like manner and critical cis-acting sequences regulating this element were characterized. Cross-species comparisons narrowed the activity of the Shh floor plate enhancer to an 88-bp sequence within intron 2 of Shh that included highly conserved binding sites matching the consensus for homeodomain, Tbx and Foxa transcription factors. Mutational analysis revealed that the homeodomain and Foxa binding sites are each required for activation of the Shh floor plate enhancer, whereas the Tbx site was required for repression in regions of the CNS where Shh is not normally expressed. Shh enhancer activity is detected in the mouse node from where the floor plate and notochord precursors derive. Shh reporter expression was restricted to the ventral (mesodermal) layer of the node in a pattern similar to endogenous Shh. X-gal-positive cells emerging from the node were only detected in the notochord lineage, suggesting that the floor plate and notochord arise from distinct precursors in the mouse node (Jeong, 2003).

The Shh-dependent pathway resulting in floor plate formation relies on triggering a transcription factor cascade culminating in the stable expression of Shh in the ventral midline of the neural tube. Shh signaling from the notochord activates Gli2, a zinc-finger transcriptional regulator, in the overlying neural plate. Gli2, which is required for floor plate development, is responsible for initiating the transcription of Foxa2 (formerly Hnf3b). Although, the misexpression of Foxa2 in the CNS can under certain conditions result in the ectopic activation of Shh, it remains unclear whether Foxa2 is required to regulate Shh transcription within sites of endogenous expression including the floor plate. Attempts at addressing this question through conventional loss-of-function studies is confounded by the requirement for Foxa2 in node formation, resulting in Foxa2-/- embryos that lack both the notochord and floor plate (Jeong, 2003 and references therein).

Given that vertebrate species show similar patterns of Shh expression in the CNS and that regulatory sequences directing floor plate expression have been localized to intron 2 in mouse, chicken and zebrafish, the premise that conservation of sequence underscores conservation of function was explored. Comparative sequence analysis of the 746-bp fragment of mouse DNA previously attributed with Shh floor plate enhancer 2 (Sfpe2) activity was undertaken with comparable regions from human, chicken and zebrafish using ClustalW algorithms. As expected, alignment of mouse and human sequence showed the highest degree of overall homology at 67%. Conservation of chicken and zebrafish sequences was found on average to be lower when compared to that of mouse, with homologies of 44% and 36%, respectively. On closer inspection however, the 4-way alignment revealed higher homology scores over short stretches of sequence compared to the overall average. Of these short stretches of sequence, three homologous regions corresponding to HR-a (nucleotide position: 60-139), HR-b (184-233) and HR-c (221-308) were remarkable given that all of the 2-by-2 comparisons between mouse and the individual species in question displayed higher homologies than the overall average for that species (Jeong, 2003).

To test the functional relevance of the conserved sequences, each of the homologous regions was assayed for its ability to direct transgene expression to the floor plate of the hindbrain and spinal cord either independently or in combination. A series of reporter constructs (Rc1-8) were generated that contained a lacZ reporter cassette cloned upstream of fragments of the 746-bp region mediating Sfpe2 activity. Transgenic embryos were generated with each of the reporter constructs and assayed for Sfpe2 activity at 9.5 dpc by X-gal staining. An 88-bp fragment constitutes an essential component of Sfpe2 and in cooperation with Shh brain enhancer 1, is sufficient to direct lacZ expression to the floor plate in a Shh-like manner (Jeong, 2003).

Foxa2 is not sufficient to mediate Sfpe2 function; cooperative interactions with a homeodomain transcription factor are required to direct reporter expression to the floor plate in a Shh-like manner. These results are seemingly inconsistent with previous reports documenting that forced expression of Foxa2 is sufficient to activate Shh transcription. However, additional observations are supportive of this conclusion: Foxa2 is expressed along the length of the floor plate yet the activities of the enhancers regulating Shh are regionalized along the anteroposterior axis of the neural tube; moreover, the sequences mediating Sbe1 and Sfpe2 activity, although both possessing Foxa binding sites, cannot independently direct reporter expression to the floor plate even when multimerized. Therefore, additional transcription factors must be acting in concert with Foxa2 to regulate Shh expression in the floor plate. To reconcile differences between these results and the Foxa2 gain-of-function studies, it is speculated that: (1) Foxa2 may be inducing the expression of the cooperating transcription factor(s); (2) Foxa2 may only be capable of activating Shh transcription where the cooperating transcription factor(s) is/are expressed. Restrictions in where Foxa2 can activate Shh within the neural tube have been described; and (3) ectopic expression of Foxa2 may be activating Shh transcription through enhancers other than Sfpe2 (Jeong, 2003).

The data implicating homeodomain binding-sites in the regulation of Shh floor plate enhancer activity leaves open the identity of the trans-acting factor binding to these sites. A survey of genes encoding homeodomain proteins expressed in the ventral midline of the mouse CNS has identified members of the Arx, Hox, Lmx and Nkx families. Despite this large number of factors, members of the Nkx family stand out as candidate regulators of Sfpe2 based on their temporal and spatial overlap with Shh. At least four Nkx genes including Nkx2.2, 2.9, 6.1 and 6.2 are expressed in the ventral midline of the CNS prior to the onset of Shh transcription. The list of candidates can be further narrowed based on the divergent DNA-binding properties exhibited by Nkx2 [T(T/C)AAGT(A/G)(C/G)TT] and Nkx6 (TTAATTAC) class family members. Because the homeodomain binding-site in HR-c better matches the consensus for Nkx6 versus Nkx2 family members -- confirmed by DNA binding studies showing that Nkx6.1 but not Nkx2.2 could bind to the homeodomain site in HR-c -- Nkx6.1 and Nkx6.2 are favored as candidate regulators of Sfpe2 activity (Jeong, 2003).

Notwithstanding the agreement of this data with a role for Nkx6 family members in regulating Shh expression in the floor plate, genetic studies supporting the requirement of Nkx6 genes in this process have not been forthcoming. Thus, homeodomain proteins other than Nkx6.1 or Nkx6.2 may regulate Sfpe2. Furthermore, detecting a down-regulation in Shh transcription in Nkx6 mutants may be confounded by the presence of another floor plate enhancer (Sfpe1), located upstream of the Shh gene, which may compensate in the absence of Sfpe2. It is interesting to note that the regulation of Shh expression in more rostral regions of the CNS is also dependent on an Nkx gene. In embryos lacking Nkx2.1, Shh expression in the ventral telencephalon is completely absent. Not surprisingly Nkx2.1 is the only Nkx family member expressed in this region of the CNS (Jeong, 2003).

How might Foxa2 and the cooperating homeodomain factor interact to regulate Shh transcription? Previous studies have shown that the binding of Foxa proteins to their recognition sites on active enhancers can result in the stabilization of nucleosome position, thus facilitating the binding of additional transcription factors to the enhancer complex. Foxa2 may be functioning in a similar capacity on Sfpe2 by promoting the stable binding of homeodomain proteins such as Nkx6 family members (Jeong, 2003).

Using Sfpe2 reporter activity to trace the lineage of Shh-expressing cells in the node it has been shown that X-gal staining was restricted to the ventral layer, as was endogenous Shh. Consequently, only the notochordal plate, a mesodermal derivative emerging from the ventral layer of the node was positive for X-gal staining. Because the floor plate precursors residing in the dorsal layer of the node showed no X-gal staining, it is concluded that floor plate and notochord progenitors in the mouse node do not derive from a common origin. These results are consistent with previous dye I labeling studies of the mouse node but are in disagreement with data from the chick that support a common origin for floor plate and notochord precursors. In the chick, floor plate precursors in the node segregate from a common population of progenitors and subsequently insert into the medial position of the overlying neural plate. Because mixing between X-gal-positive ventral cells and X-gal-negative dorsal cells in or around the mouse node was not observed, it is concluded that floor plate precursors in the mouse are not generated by the same mechanism as in chick. Instead, these results agree with the prevailing model that the mouse floor plate forms by inductive Shh signaling (Jeong, 2003).

The observation that the Shh target genes Ptc and Foxa2 are expressed in the dorsal layer of the node offers further support that the process of floor plate induction begins in the mouse node at early somite stages and doesn't terminate until Shh transcription is activated in the ventral midline of the CNS between 8 to 12 somite stages. In this homeogenetic model of floor plate induction, Shh secreted from the axial mesoderm signals to the overlying neural plate to activate effectors of the Shh signal transduction cascade. A consequence of this vertical signaling step is the initiation of Shh transcription, through the direct binding of Foxa2 and a homeodomain protein to specific enhancer sequences. Given that sequences mediating Sbe1 activity are also required for floor plate expression, it is speculated that additional transcriptional activators are participating in the regulation of Shh expression. Identifying the critical sequences mediating Sbe1 activity and the factors binding to these sites should further elucidate how Shh expression is activated in the floor plate of the mouse spinal cord (Jeong, 2003).

The cerebellum provides an excellent system for understanding how afferent and target neurons coordinate sequential intercellular signals and cell-autonomous genetic programs in development. Mutations in the orphan nuclear receptor RORalpha block Purkinje cell differentiation with a secondary loss of afferent granule cells. Early transcriptional targets of RORalpha include both mitogenic signals for afferent progenitors and signal transduction genes required to process their subsequent synaptic input. RORalpha acts through recruitment of gene-specific sets of transcriptional cofactors, including ß-catenin, p300, and Tip60, but appears independent of CBP. One target promoter is Sonic hedgehog, and recombinant Sonic hedgehog restores granule precursor proliferation in RORalpha-deficient cerebellum. These results suggest a link between RORalpha and ß-catenin pathways, confirm that a nuclear receptor employs distinct coactivator complexes at different target genes, and provide a logic for early RORalpha expression in coordinating expression of genes required for reciprocal signals in cerebellar development (Gold, 2003).

Differentiation of mesenchymal cells into chondrocytes and chondrocyte proliferation and maturation are fundamental steps in skeletal development. Runx2 is essential for osteoblast differentiation and is involved in chondrocyte maturation. Although chondrocyte maturation is delayed in Runx2-deficient (Runx2–/–) mice, terminal differentiation of chondrocytes does occur, indicating that additional factors are involved in chondrocyte maturation. The involvement of Runx3 in chondrocyte differentiation was investigated by generating Runx2-and-Runx3-deficient (Runx2–/–3–/–) mice. Chondrocyte differentiation is inhibited depending on the dosages of Runx2 and Runx3, and Runx2–/–3–/– mice show a complete absence of chondrocyte maturation. Further, the length of the limbs is reduced depending on the dosages of Runx2 and Runx3, due to reduced and disorganized chondrocyte proliferation and reduced cell size in the diaphyses. Runx2–/–3–/– mice do not express Ihh, which regulates chondrocyte proliferation and maturation. Adenoviral introduction of Runx2 in Runx2–/– chondrocyte cultures strongly induces Ihh expression. Moreover, Runx2 directly binds to the promoter region of the Ihh gene and strongly induces expression of the reporter gene driven by the Ihh promoter. These findings demonstrate that Runx2 and Runx3 are essential for chondrocyte maturation and that Runx2 regulates limb growth by organizing chondrocyte maturation and proliferation through the induction of Ihh expression (Yoshida, 2004).

Mutations in a conserved non-coding region in intron 5 of the Lmbr1 locus, which is 1 Mb away from the sonic hedgehog (Shh) coding sequence, are responsible for mouse and human preaxial polydactyly with mirror-image digit duplications. In the mouse mutants, ectopic Shh expression is observed in the anterior mesenchyme of limb buds. Furthermore, a transgenic reporter gene flanked with this conserved non-coding region shows normal polarized expression in mouse limb buds. This conserved sequence has therefore been proposed to act as a long-range, cis-acting regulator of limb-specific Shh expression. Previous phylogenetic studies have also shown that this sequence is highly conserved among tetrapods, and even in teleost fishes; the core sequence (160 bp in length) identity exceeds 60%-75% identity between mouse and medaka. Paired fins of teleost fishes and tetrapod limbs have evolved from common ancestral appendages, and polarized Shh expression is commonly observed in fins. In this study, it is first shown that this conserved sequence motif is also physically linked to the Shh coding sequence in a teleost fish, the medaka, by homology search of a newly available genomic sequence database. Next, it is shown that deletion of this conserved intronic sequence by targeted mutation in the mouse results in a complete loss of Shh expression in the limb bud and degeneration of skeletal elements distal to the stylopod/zygopod junction. This sequence contains a major limb-specific Shh enhancer that is necessary for distal limb development. These results suggest that the conserved intronic sequence evolved in a common ancestor of fishes and tetrapods to control fin and limb development (Sagai, 2005).

Neurogenesis in the compound eyes of Drosophila and the camera eyes of vertebrates spreads in a wave-like fashion. In both phyla, waves of hedgehog expression are known to drive the wave of neuronal differentiation. The mechanism controlling the propagation of hedgehog expression during retinogenesis of the vertebrate eye is poorly understood. The Iroquois homeobox genes play important roles in Drosophila eye development; they are required for the up-regulation of hedgehog expression during propagation of the morphogenetic furrow. This study shows that the zebrafish Iroquois homolog irx1a is expressed during retinogenesis and knockdown of irx1a results in a retinal phenotype strikingly similar to those of sonic hedgehog mutants. Analysis of shh-GFP transgene expression in irx1a knockdown retinas revealed that irx1a is required for the propagation of shh expression through the retina. Transplantation experiments illustrated that the effects of irx1a on shh expression are both cell-autonomous and non-cell-autonomous. These results reveal a role for Iroquois genes in controlling hedgehog expression during vertebrate retinogenesis (Cheng, 2006).

Increasing evidence reveals a striking conservation of genetic pathways regulating morphogenesis of the Drosophila and fish eyes. One example is between R8 photoreceptor differentiation in the Drosophila eye discs and retinal ganglion cell (RGC) specification in the vertebrate retinas. In both systems, the wave of differentiation is controlled in part by hedgehog signaling. In the Drosophila eye discs, a border of Irx+/Irx cells is required and sufficient to trigger an up-regulation of hh expression in the posterior most region, which drives the propagation of the morphogenetic furrow (Cavodeassi, 1999). Intriguingly, Iroquois genes act non-cell-autonomously in controlling hh propagation in the eye discs although the underlying molecular mechanism remains unknown. This study shows that irx1a also regulates shh propagation in the zebrafish retina in a non-cell-autonomous manner adding another conserved genetic component between Drosophila and vertebrate eye morphogenesis. However, there is significant divergence in the expression and function of the Iroquois genes in vertebrate eye development. While all six mouse Irx genes are expressed in the GCL, irx1a, but not irx1b and irx7, is expressed in the zebrafish retina. Moreover, to date mutation of neither mouse Irx gene has been shown to have such a dramatic effect on retinal neurogenesis as the knock-down of irx1a in the zebrafish. Analysis of Irx2, Irx4 and Irx5 mutant mice revealed a subtle phenotype in the differentiation of a subset of bipolar interneurons only in the Irx5 mutant retinas. In contrast, the current results illustrate that irx1a is pivotal for retinogenesis in the zebrafish and suggest that irx1a acts in a critical step after the specification of retinal progenitor cells. Furthermore, this study demonstrates the importance of irx1a in regulating the propagation of neurogenic waves in the retina (Cheng, 2006).

Holoprosencephaly (HPE), the most common forebrain malformation, is characterized by an incomplete separation of the cerebral hemispheres. Mutations in the homeobox gene SIX3 account for 1.3% of all cases of human HPE. Using zebrafish-based assays, it has been determined that HPE-associated Six3 mutant proteins function as hypomorphs. Haploinsufficiency of Six3 caused by deletion of one allele of Six3 or by replacement of wild-type Six3 with HPE-associated Six3 mutant alleles was sufficient to recapitulate in mouse models most of the phenotypic features of human HPE. Shh is a direct target of Six3 in the rostral diencephalon ventral midline (RDVM). Reduced amounts of functional Six3 protein fail to activate Shh expression in the mutant RDVM and ultimately lead to HPE. These results identify Six3 as a direct regulator of Shh expression and reveal a crossregulatory loop between Shh and Six3 in the ventral forebrain (Geng, 2008).

The expression of Sonic hedgehog (Shh) in mouse limb buds is regulated by a long-range enhancer 1Mb upstream of the Shh promoter. 3D-FISH and chromosome conformation capture assays were used to track changes at the Shh locus; long-range promoter-enhancer interactions were found to be specific to limb bud tissues competent to express Shh. However, the Shh locus loops out from its chromosome territory only in the posterior limb bud (zone of polarizing activity or ZPA), where Shh expression is active. Notably, while Shh mRNA is detected throughout the ZPA, enhancer-promoter interactions and looping out were observed only in small fractions of ZPA cells. In situ detection of nascent Shh transcripts and unstable EGFP reporters revealed that active Shh transcription is likewise only seen in a small fraction of ZPA cells. These results suggest that chromosome conformation dynamics at the Shh locus allow transient pulses of Shh transcription (Amano, 2009).

The sonic hedgehog (Shh) pathway plays indispensable roles in the morphogenesis of mouse epithelial linings of the oral cavity and respiratory and digestive tubes. However, no enhancers that regulate regional Shh expression within the epithelial linings have been identified so far. In this study, comparison of genomic sequences across mammalian species and teleost fishes revealed three novel conserved non-coding sequences (CNCSs) that cluster in a region 600 to 900 kb upstream of the transcriptional start site of the mouse Shh gene. These CNCSs drive regional transgenic lacZ reporter expression in the epithelial lining of the oral cavity, pharynx, lung and gut. Together, these enhancers recapitulate the endogenous Shh expression domain within the major epithelial linings. Notably, genomic arrangement of the three CNCSs shows co-linearity that mirrors the order of the epithelial expression domains along the anteroposterior body axis. The results suggest that the three CNCSs are epithelial lining-specific long-range Shh enhancers, and that their actions partition the continuous epithelial linings into three domains: ectoderm-derived oral cavity, endoderm-derived pharynx, and respiratory and digestive tubes of the mouse. Targeted deletion of the pharyngeal epithelium specific CNCS results in loss of endogenous Shh expression in the pharynx and postnatal lethality owing to hypoplasia of the soft palate, epiglottis and arytenoid. Thus, this long-range enhancer is indispensable for morphogenesis of the pharyngeal apparatus (Sagai, 2009).

The Shh topological domain facilitates the action of remote enhancers by reducing the effects of genomic distances

Gene expression often requires interaction between promoters and distant enhancers, which occur within the context of highly organized topologically associating domains (TADs) (see Drosophila chromatin organization). Using a series of engineered chromosomal rearrangements at the Shh locus, this study carried out an extensive fine-scale characterization of the factors that govern the long-range regulatory interactions controlling Shh expression. It was shown that Shh enhancers act pervasively, yet not uniformly, throughout the TAD. Importantly, changing intra-TAD distances have no impact on Shh expression. In contrast, inversions disrupting the TAD alter global folding of the region and prevent regulatory contacts in a distance-dependent manner. Data indicate that the Shh TAD promotes distance-independent contacts between distant regions that would otherwise interact only sporadically, enabling functional communication between them. In large genomes where genomic distances per se can limit regulatory interactions, this function of TADs could be as essential for gene expression as the formation of insulated neighborhoods (Symmons, 2016).

Mapping the Shh long-range regulatory domain

Coordinated gene expression controlled by long-distance enhancers is orchestrated by DNA regulatory sequences involving transcription factors and layers of control mechanisms. The Shh gene and well-established regulators are an example of genomic composition in which enhancers reside in a large desert extending into neighbouring genes to control the spatiotemporal pattern of expression. Exploiting the local hopping activity of the Sleeping Beauty transposon, the lacZ reporter gene was dispersed throughout the Shh region to systematically map the genomic features responsible for expression activity. Enhancer activities were found to be retained inside a genomic region that corresponds to the topological associated domain (TAD) defined by Hi-C. This domain of approximately 900 kb is in an open conformation over its length and is generally susceptible to all Shh enhancers. Similar to the distal enhancers, an enhancer residing within the Shh second intron activates the reporter gene located at distances of hundreds of kilobases away, suggesting that both proximal and distal enhancers have the capacity to survey the Shh topological domain to recognise potential promoters. The widely expressed Rnf32 gene lying within the Shh domain evades enhancer activities by a process that may be common among other housekeeping genes that reside in large regulatory domains. Finally, the boundaries of the Shh TAD do not represent the absolute expression limits of enhancer activity, as expression activity is lost stepwise at a number of genomic positions at the verges of these domains (Anderson, 2014).

Comparative biological responses to human Sonic, Indian, and Desert hedgehog

A comprehensive comparison of Sonic (Shh), Indian (Ihh), and Desert (Dhh) hedgehog biological activities has not previously been undertaken. To test whether the three higher vertebrate Hh proteins have distinct biological properties, recombinant forms of the N-terminal domains of human Shh, Ihh, and Dhh were examined in a variety of cell-based and tissue explant assays in which their activities could be assessed at a range of concentrations. While the proteins are similar in their affinities for the Hh-binding proteins [Patched (Ptc) and Hedgehog-interacting protein (Hip)], and are equipotent in their ability to induce Islet-1 in chick neural plate explant,there are dramatic differences in their potencies in several other assays. Most dramatic are the Hh-dependent responses of C3H10T1/2 cells, where relative potencies range from 80 nM for Shh, to 500 nM for Ihh, to >.5 microM for Dhh. Similar trends in potency are seen in the ability of the three Hh proteins to induce differentiation of chondrocytes in embryonic mouse limbs, and to induce the expression of nodal in the lateral plate mesoderm of early chick embryos. However, in a chick embryo digit duplication assay used to measure polarizing activity, Ihh was the least active, and Dhh was almost as potent as Shh. These findings suggest that a mechanism for fine-tuning the biological actions of Shh, Ihh, and Dhh, exists beyond the simple temporal and spatial control of their expression domains within the developing and adult organism (Pathi, 2001).

The Eda pathway functions upstream of Sonic Hedgehog

Ectodermal organogenesis is regulated by inductive and reciprocal signalling cascades that involve multiple signal molecules in several conserved families. Ectodysplasin-A (Eda), a tumour necrosis factor-like signalling molecule, and its receptor Edar are required for the development of a number of ectodermal organs in vertebrates. In mice, lack of Eda leads to failure in primary hair placode formation and missing or abnormally shaped teeth, whereas mice overexpressing Eda are characterized by enlarged hair placodes and supernumerary teeth and mammary glands. Two signalling outcomes of the Eda pathway are reported in this study: suppression of bone morphogenetic protein (Bmp) activity and upregulation of sonic hedgehog (Shh) signalling. Recombinant Eda counteracts Bmp4 activity in developing teeth and, importantly, inhibition of BMP activity by exogenous noggin partially restores primary hair placode formation in Eda-deficient skin in vitro, indicating that suppression of Bmp activity is compromised in the absence of Eda. The downstream effects of the Eda pathway are likely to be mediated by transcription factor NF-kappaB, but the transcriptional targets of Edar have remained unknown. Using a quantitative approach, it is shown, in cultured embryonic skin, that Eda induces the expression of two Bmp inhibitors, Ccn2/Ctgf (CCN family protein 2/connective tissue growth factor) and follistatin. Moreover, the data indicate that Shh is a likely transcriptional target of Edar, but, unlike noggin, recombinant Shh is unable to rescue primary hair placode formation in Eda-deficient skin explants (Pummila, 2007).

Table of contents


hedgehog continued: Biological Overview | Regulation | Targets of Activity | 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.