Alternative titles; symbols
HGNC Approved Gene Symbol: SHH
SNOMEDCT: 253159001, 38353004, 707609006; ICD10CM: Q04.6;
Cytogenetic location: 7q36.3 Genomic coordinates (GRCh38): 7:155,799,980-155,812,463 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 7q36.3 | Holoprosencephaly 3 | 142945 | Autosomal dominant | 3 |
| Microphthalmia with coloboma 5 | 611638 | Autosomal dominant | 3 | |
| Schizencephaly | 269160 | 3 | ||
| Single median maxillary central incisor | 147250 | Autosomal dominant | 3 |
The SHH gene encodes sonic hedgehog, a secreted protein that is involved in establishing cell fates at several points during development. SHH belongs to a family of vertebrate genes related to the Drosophila gene 'hedgehog' (hh) that encodes inductive signals during embryogenesis (Echelard et al., 1993; Roelink et al., 1994). These genes are involved in the organization and morphology of the developing embryo, which is established through a series of inductive interactions (Marigo et al., 1995).
Riddle et al. (1993) named the chicken homolog of the Drosophila gene 'Sonic hedgehog' after the Sega computer game cartoon character. Mammalian homologs of hh include Sonic hedgehog (Shh), Indian hedgehog (Ihh; see 600726), and desert hedgehog (Dhh; 605423) (Echelard et al., 1993).
Marigo et al. (1995) isolated human cDNA clones of the SHH and IHH genes. The SHH clone encodes a predicted protein 92.4% identical to its mouse homolog, while the IHH clone encodes a protein with 94.6% identity to its mouse homolog. IHH was expressed in adult kidney and liver. SHH expression was not detected in adult tissues examined; however, it was expressed in fetal intestine, liver, lung, and kidney.
Like its Drosophila cognate, Shh encodes a signal that is instrumental in patterning the early embryo. It is expressed in the Hensen node, the floorplate of the neural tube, the early gut endoderm, the posterior of the limb buds, and throughout the notochord. It has been implicated as the key inductive signal in patterning of the ventral neural tube (Echelard et al., 1993; Roelink et al., 1994), the anterior-posterior limb axis (Riddle et al., 1993), and the ventral somites (Johnson et al., 1994).
The mouse, chicken, and zebrafish Shh homologs are highly conserved (Marigo et al., 1995). Their functional properties appear to be conserved as well. Their probable importance in embryogenesis additionally suggests that alterations in the human hedgehog genes might be involved in congenital anomalies.
Porter et al. (1996) reviewed the molecular processing of hedgehog proteins. They noted that after signal sequence cleavage the hedgehog protein precursor of approximately 45 kD undergoes autocatalytic internal cleavage. This yields an approximately 20-kD N-terminal domain which has signaling activity and a 25-kD C-terminal domain which is active in precursor processing. Hedgehog protein autoprocessing includes peptide bond cleavage and the attachment of a lipophilic adduct to the C-terminal region. Porter et al. (1996) noted that the lipophilic modification is critical for the spatially restricted tissue localization of the hedgehog signal domain. Porter et al. (1996) demonstrated that cholesterol is the lipophilic moiety covalently attached to the N-terminal signaling domain during autoprocessing and that the C-terminal domain acts as an intramolecular cholesterol transferase. They postulated that some of the effects of perturbed cholesterol biosynthesis on animal development, such as those seen in Smith-Lemli-Opitz syndrome (SLO; 270400), may be due to the fact that cholesterol is used to modify embryonic signaling proteins.
Zeng et al. (2001) provided evidence for a freely diffusible form of Sonic hedgehog that is cholesterol modified, multimeric, and biologically potent. Zeng et al. (2001) further demonstrated that the availability of this form is regulated by 2 functional antagonists of the SHH pathway, Patched (Ptc; 601309) and hedgehog-interacting protein (HIP). Zeng et al. (2001) demonstrated a gradient of the freely diffusible form across the anterior-posterior axis of the chick limb, demonstrating the physiologic relevance of this form of SHH.
Drosophila Ihog, like its mammalian homolog Cdo (608707), contains multiple immunoglobulin and fibronectin type III (FNIII) repeats, and the first FNIII repeat binds the amino-terminal signaling domain of Hedgehog (HhN) in a heparin-dependent manner. Pull-down experiments had suggested that a mammalian Sonic hedgehog amino-terminal domain (ShhN) binds a nonorthologous FNIII repeat of Cdo. McLellan et al. (2008) reported biochemical, biophysical, and x-ray structural studies of a complex between ShhN and the third FNIII repeat of CDO. They showed that the ShhN-CDO interaction is completely unlike the HhN-Ihog reaction and requires calcium, which binds at a previously undetected site on ShhN. This site is conserved in nearly all Hedgehog proteins and is a hotspot for mediating interactions between ShhN and CDO, PTC, HIP (606178), and GAS1 (139185). Mutations in vertebrate Hedgehog proteins causing holoprosencephaly and brachydactyly type A1 (112500) map to this calcium-binding site and disrupt interactions with those partners.
Cryoelectron Microscopy
Using cryoelectron microscopy, Gong et al. (2018) determined the structures of human Patched1 (PTCH1; 601309) alone and in complex with the N-terminal domain of human SHH at resolutions of 3.9 and 3.6 angstroms, respectively. PTCH1 comprises 2 interacting extracellular domains, ECD1 and ECD2, and 12 transmembrane segments, with transmembrane segments 2 to 6 constituting the sterol-sensing domain. Two steroid-shaped densities are resolved in both structures, one enclosed by ECD1/2 and the other in the membrane-facing cavity of the sterol-sensing domain. Structure-guided mutational analysis showed that interaction between the N terminus of SHH and PTCH1 is steroid-dependent.
Qi et al. (2018) reported the cryoelectron microscopy structures of human PTCH1 alone and in complex with the N-terminal domain of 'native' SHH (SHH-N), which has both a C-terminal cholesterol and an N-terminal fatty acid modification, at resolutions of 3.5 and 3.8 angstroms, respectively. The structure of PTCH1 has internal 2-fold pseudosymmetry in the transmembrane core, which features a sterol-sensing domain and 2 homologous extracellular domains, resembling the architecture of Niemann-Pick C1 protein (NPC1; 607623). The palmitoylated N terminus of SHH-N inserts into a cavity between the extracellular domains of PTCH1 and dominates the PTCH1-SHH-N interface, which is distinct from that reported for SHH-N coreceptors. Qi et al. (2018) noted that their biochemical assays showed that SHH-N may use another interface, one that is required for its coreceptor binding, to recruit PTCH1 in the absence of a covalently attached palmitate.
The 1:1 PTCH1-HH complex structure reported by Qi et al. (2018) visualized a palmitate-mediated binding site on Hedgehog (HH), which was inconsistent with previous studies that implied a distinct, calcium-mediated binding site for PTCH1 and HH coreceptors. Qi et al. (2018) reported a 3.5-angstrom resolution cryoelectron microscopy structure of SHH-N in complex with PTCH1 at a physiologic calcium concentration that reconciled these disparate findings and demonstrated that 1 SHH-N molecule engages both epitopes to bind 2 PTCH1 receptors in an asymmetric manner. Functional assays using PTCH1 or SHH-N mutants that disrupted the individual interfaces illustrated that simultaneous engagement of both interfaces is required for efficient signaling in cells.
See Johnson and Tabin (1997) for a review of the role of the SHH gene in limb development.
Roessler and Muenke (2003) reviewed various aspects of hedgehog synthesis, secretion, distribution, and function in the context of holoprosencephaly (see 236100 and HPE3, 142945).
Ericson et al. (1996) analyzed the role of SHH signaling in the specification of vertebrate motor neuron identity using cultured explants of chick neural plate, neural tube and notochord tissue, and antibodies which block SHH signaling. They noted that the identity and pattern of cell types generated in the ventral neural tube is controlled by the notochord, an axial mesodermal organizing center. Previous studies revealed that the notochord secretes a locally acting factor that induces differentiation of the floorplate cells at the ventral midline of the neural tube and a diffusible factor that can initiate motor neuron differentiation (Placzek, 1995). Ericson et al. (1996) demonstrated that SHH function is required for the short-range induction of floorplate cells by the notochord. They also showed that SHH function is required independently for the induction of motor neurons by both the notochord and midline neural cells. Ericson et al. (1996) showed that motor neuron generation depends on 2 critical periods of SHH signaling: an early period, during which the neural plate cells are converted to ventralized progenitors, and a late period, during which SHH drives the differentiation of ventralized progenitors into motor neurons. They reported further that the ambient SHH concentration during the late period determines whether ventralized progenitors differentiate into motor neurons or interneurons, thus defining the pattern of neuronal cell types generated in the neural tube.
On the basis of their studies in Drosophila, Chen and Struhl (1996) presented evidence that Patched (Ptc; 601309) acts as a receptor for hedgehog (Hh) proteins. They suggested a novel signal transduction mechanism in which Hh proteins bind to Ptc or to a Ptc-Smo (SMOH; 601500) complex and thereby induce Smo activity. Their results showed further that Ptc limits the range of Hh action and that the high levels of Ptc induced by Hh serve to sequester any free Hh and thereby create a barrier to its further movement.
Marigo et al. (1996) reported that the Ptc gene product is the receptor for Sonic hedgehog. This was demonstrated by carrying out Shh binding studies on Xenopus laevis oocytes which had been injected with Ptc mRNA. Binding was shown to be dependent on glycosylation of Ptc and on the 2 large extracellular domains of Ptc. Independently and simultaneously, Stone et al. (1996) reported that epitope-tagged N-terminal Shh peptide binds specifically to mouse Ptc. They also demonstrated that Ptc and Smo form a complex, and that Shh binds the complex. Stone et al. (1996) noted that genetic mutations leading to a truncated or unstable Ptc protein are associated with familial or sporadic basal cell carcinoma (BCC; see 605462). They suggested that this finding, combined with the fact that Ptc is a high-affinity binding protein for Shh, suggests that the hedgehog system may provide mitogenic or differentiative signals to basal cells in the skin throughout life. Stone et al. (1996) raised the possibility that basal cell nevus syndrome (BCNS; 109400) and BCC might result from constitutive activation of Smo, which becomes oncogenic after its release from inhibition by Ptc.
Zuniga et al. (1999) reported that the secreted bone morphogenetic protein (BMP) antagonist gremlin (GREM1; 603054) relays the SHH signal from the polarizing region to the apical ectodermal ridge. Mesenchymal gremlin expression is lost in limb buds of mouse embryos homozygous for the 'limb deformity' (ld) mutation, which disrupts establishment of the Shh/Fgf4 (164980) feedback loop. Grafting gremlin-expressing cells into ld mutant limb buds rescued Fgf4 expression and restored the Shh/Fgf4 feedback loop. Analysis of Shh-null mutant embryos revealed that Shh signaling is required for maintenance of gremlin and formin (FMN1; 136535), the gene disrupted by the ld mutations. In contrast, formin, gremlin, and Fgf4 activation were independent of Shh signaling. Zuniga et al. (1999) concluded that the study uncovered the cascade by which the SHH signal is relayed from the posterior mesenchyme to the apical ectodermal ridge and established that formin-dependent activation of the BMP antagonist gremlin is sufficient to induce FGF4 and establish the SHH/FGF4 feedback loop.
Neumann and Nuesslein-Volhard (2000) showed evidence that in zebrafish, SHH is also expressed in the first retinal neurons and that SHH drives a wave of neurogenesis across the retina, strikingly similar to the wave in Drosophila. The conservation of this patterning mechanism was unexpected, given the highly divergent structures of vertebrate and invertebrate eyes, and supports a common evolutionary origin of the animal visual system.
Agarwala et al. (2001) employed in vivo electroporation during midbrain development in chick embryos to create ectopic sources of Sonic hedgehog. Agarwala et al. (2001) provided direct evidence that a Sonic hedgehog source can control pattern at a distance in brain development and demonstrated that the size, shape, and orientation of the cell populations produced depend on the geometry of the morphogen source. Thus, a single regulatory molecule can coordinate tissue size and shape with cell-type identity in brain development.
By RT-PCR analysis, Bhardwaj et al. (2001) detected expression of SHH and its receptors PTCH and SMOH in primitive and differentiated (myeloid, B, and T) hematopoietic cells, as well as in stromal cells isolated from bone marrow and in endothelial cells. GLI transcription factors (see 165220), on the other hand, were only expressed in primitive hematopoietic cells, stromal cells, and endothelial cells. Cytokine-induced proliferation of primitive stem cells could be inhibited by antibodies to SHH or by the BMP4 (112262) inhibitor Noggin (NOG; 602991). Cytokine treatment induced an upregulation of BMP4; however, in the presence of SHH, BMP4 upregulation was markedly reduced, as was the upregulation of NOG. On the other hand, treatment with SHH induced the expansion of pluripotent hematopoietic cells in immunodeficient mice. In vitro, NOG could block SHH-induced proliferation of primitive hematopoietic cells. Bhardwaj et al. (2001) concluded that SHH is an important regulator of primitive hematopoietic cells that is dependent on downstream BMP signals.
Wang et al. (2002) demonstrated that Sonic hedgehog signaling from retinal ganglion cells is required for the normal laminar organization in the vertebrate retina.
In the embryonic spinal cord, the floor plate chemoattractant netrin-1 (601614) is required to guide commissural neuron axons to the midline. However, genetic evidence has suggested that other chemoattractant(s) are also involved. Charron et al. (2003) showed that mouse Shh could mimic the additional chemoattractant activity of the floor plate in vitro and could act directly as a chemoattractant on isolated axons. Cyclopamine-mediated inhibition of the Shh signaling mediator Smo or conditional inactivation of Smo in commissural neurons indicated that Smo activity was important for the additional chemoattractant activity of the floor plate in vitro and for the normal projection of commissural axons to the floor plate in vivo. These results provided evidence that SHH, acting via SMO, is a midline-derived chemoattractant for commissural axons and showed that a morphogen can also act as an axonal chemoattractant.
During early development in vertebrates, SHH is produced by the notochord and the floor plate. A ventrodorsal gradient of SHH directs ventrodorsal patterning of the neural tube. However, SHH is also required for the survival of neuroepithelial cells. Thibert et al. (2003) demonstrated that Patched (PTC; 601309) induces apoptotic cell death unless its ligand SHH is present to block the signal. Moreover, the blockade of Ptc-induced cell death partly rescues the chick spinal cord defect provoked by Shh deprivation. Thibert et al. (2003) concluded that the proapoptotic activity of unbound PTC and the positive effect of SHH-bound PTC on cell differentiation probably cooperate to achieve the appropriate spinal cord development.
Vertebrate limb outgrowth is driven by a positive feedback loop involving SHH, gremlin (GREM1; 603054), and FGF4 (164980). By overexpressing individual components of the loop at a time after these genes are normally downregulated in chicken embryos, Scherz et al. (2004) found that Shh no longer maintains gremlin in the posterior limb. Shh-expressing cells and their descendants cannot express gremlin. The proliferation of these descendants forms a barrier separating the Shh signal from gremlin-expressing cells, which breaks down the Shh-Fgf4 loop and thereby affects limb size and provides a mechanism explaining regulative properties of the limb bud.
Casali and Struhl (2004) demonstrated that a cell's measure of ambient Hh concentration is not determined solely by the number of active (unliganded) Ptc molecules. Instead, they found that Hh-bound Ptc can titrate the inhibitory action of unbound Ptc. Furthermore, this effect is sufficient to allow normal reading of the Hh gradient in the presence of a form of Ptc that cannot bind the ligand but retains its ability to inhibit Smo. Casali and Struhl (2004) concluded that their results supported a model in which the ratio of bound to unbound Ptc molecules determines the cellular response to Hh.
Chen et al. (2004) found that 2 molecules interact with mammalian Smoothened (SMO; 601500) in an activation-dependent manner: G protein-coupled receptor kinase-2 (GRK2; 109635) leads to phosphorylation of Smo, and beta-arrestin-2 (ARRB2; 107941) fused to green fluorescent protein interacts with Smo. These 2 processes promote endocytosis of Smo in clathrin-coated pits. Ptc inhibits association of Arrb2 with Smo, and this inhibition is relieved in cells treated with Shh. A Smo agonist stimulated and a Smo antagonist (cyclopamine) inhibited both phosphorylation of Smo by GRK2 and interaction of Arrb2 with Smo. Chen et al. (2004) suggested that Arrb2 and Grk2 are thus potential mediators of signaling by activated Smo.
Bourikas et al. (2005) found that Shh had a repulsive role in postcommissural axon guidance in chicken embryonic development. The effect of Shh was mediated by Hhip (606178) and not by Ptc or Smo.
The precise specification of left-right asymmetry is an essential process for patterning internal organs in vertebrates. In mouse embryonic development, the symmetry-breaking process in left-right determination is initiated by a leftward extraembryonic fluid flow on the surface of the ventral node. Tanaka et al. (2005) showed that FGF signaling triggers secretion of membrane-sheathed objects 0.3 to 5 microns in diameter, termed 'nodal vesicular parcels' (NVPs), which carry Sonic hedgehog and retinoic acid. These NVPs are transported leftward by the fluid flow and eventually fragment close to the left wall of the ventral node. The silencing effects of an FGF receptor (176943) inhibitor on NVP secretion and on a downstream rise in calcium were sufficiently reversed by exogenous Sonic hedgehog peptide or retinoic acid, suggesting that FGF-triggered surface accumulation of cargo morphogens may be essential for launching NVPs. Tanaka et al. (2005) proposed that NVP flow is a mode of extracellular transport that forms a left-right gradient of morphogens. Using time-lapse imaging, Tanaka et al. (2005) found that these NVPs were transported leftward once every 5 to 15 seconds.
Ahn and Joyner (2005) adopted an in vivo genetic fate-mapping strategy using Gli1 (165220) as a sensitive readout of Shh activity, to systematically mark and follow the fate of Shh-responding cells in the adult mouse forebrain. They showed that initially, only a small population of cells (including both quiescent neural stem cells and transit-amplifying cells) responds to Shh in regions undergoing neurogenesis. This population subsequently expands markedly to continuously provide new neurons in the forebrain. Ahn and Joyner (2005) concluded that their study of the behavior of quiescent neural stem cells provides in vivo evidence that they can self-renew for over a year and generate multiple cell types. Furthermore, Ahn and Joyner (2005) showed that the neural stem cell niches in the subventricular zone and dentate gyrus are established sequentially and not until late embryonic stages.
Working in Drosophila, Panakova et al. (2005) showed that Wingless (see WNT1, 164820), hedgehog, and glycophosphatidylinositol-linked proteins copurify with lipoprotein particles, and colocalize with them in the developing wing epithelium. In larvae with reduced glycoprotein levels, hedgehog accumulated near its site of production, and failed to signal over its normal range. Similarly, the range of Wingless signaling was narrowed. Panakova et al. (2005) proposed a novel function for lipoprotein particles, in which they act as vehicles for the movement of lipid-linked morphogens and glycophosphatidylinositol-linked proteins.
The anterior to posterior (A-P) polarity of the tetrapod limb is determined by the confined expression of Shh at the posterior margin of developing early limb buds, under the control of HOX proteins encoded by gene members of both the HoxA and HoxD clusters. Tarchini et al. (2006) used a set of partial deletions to show that only the last 4 Hox paralogy groups can elicit this response: i.e., precisely those genes whose expression is excluded from most anterior limb bud cells owing to their collinear transcriptional activation. Tarchini et al. (2006) proposed that the limb A-P polarity is produced as a collateral effect of Hox gene collinearity, a process highly constrained by its crucial importance during trunk development. In this view, the co-option of the trunk collinear mechanism, along with emergence of limbs, imposed an A-P polarity to these structures as the most parsimonious solution. This in turn further contributed to stabilize the architecture and operational mode of this genetic system. Deletion of Hoxd10 (142984), Hoxd11 (142986), Hoxd12 (142988), and Hoxd13 (142989) led to Hoxd9 (142982) upregulation in posterior cells; however, even a robust dose of Hoxd9 was unable to trigger Shh expression, demonstrating that HOXD10-HOXD13 expression is essential to elicit Shh expression.
Rohatgi et al. (2007) investigated the role of primary cilia in the regulation of PTCH1 (601309), the receptor for SHH. In mammalian cells, PTCH1 localized to cilia and inhibited Smoothened (SMO; 601500) by preventing its accumulation within cilia. When SHH bound to PTCH1, PTCH1 left the cilia, leading to accumulation of SMO and activation of signaling. Thus, Rohatgi et al. (2007) concluded that primary cilia sense SHH and transduce signals that play critical roles in development, carcinogenesis, and stem cell function.
Dessaud et al. (2007) provided evidence that changing the concentration or duration of SHH has an equivalent effect on intracellular signaling. They found that chick neural cells converted different concentrations of SHH into time-limited periods of signal transduction, such that signal duration was proportional to SHH concentration. This depended on the gradual desensitization of cells to ongoing SHH exposure, mediated by the SHH-dependent upregulation of PTC1, a ligand-binding inhibitor of SHH signaling. Thus, Dessaud et al. (2007) concluded that in addition to its role in shaping the SHH gradient, PTC1 participates cell autonomously in gradient sensing. Together, the data revealed a novel strategy for morphogen interpretation, in which the temporal adaptation of cells to a morphogen integrates the concentration and duration of a signal to control differential gene expression.
Towers et al. (2008) added an integral growth component to the known anteroposterior positional identity role of Sonic hedgehog in limb development. Towers et al. (2008) showed that SHH-dependent proliferation of prospective digit progenitor cells is essential for specifying the complete pattern of digits across the anteroposterior axis. Inhibiting Shh signaling in early chick wing buds reduced anteroposterior expansion, and posterior digits were lost because all prospective digit precursors formed anterior structures. Inhibiting proliferation also irreversibly reduced anteroposterior expansion, but instead posterior digits were lost because all prospective digit precursors formed posterior structures. When proliferation recovered in such wings, Shh transcription was maintained for longer than normal, suggesting that duration of Shh expression is controlled by a mechanism that measures proliferation. Rescue experiments confirmed that Shh-dependent proliferation controls digit number during a discrete time-window in which Shh-dependent specification normally occurs. Towers et al. (2008) concluded that their findings that Shh signaling has dual functions that can be temporally uncoupled have implications for understanding congenital and evolutionary digit reductions.
Capurro et al. (2008) found that glypican-3 (GPC3; 300037) inhibited soluble hedgehog activity in the medium of SHH-expressing mouse embryonic fibroblasts and IHH-expressing human embryonic kidney cells. GPC3 interacted with SHH, but not with Patched, and it competed with Patched for SHH binding. Furthermore, GPC3 induced SHH endocytosis and degradation.
Limb development is regulated by epithelial-mesenchymal feedback loops between SHH and fibroblast growth factor (FGF) signaling involving the bone morphogenetic protein (BMP) antagonist gremlin-1 (GREM1; 603054). By combining mouse molecular genetics with mathematical modeling, Benazet et al. (2009) showed that BMP4 (112262) first initiates and SHH then propagates epithelial-mesenchymal feedback signaling through differential transcriptional regulation of Grem1 to control digit specification. This switch occurs by linking a fast BMP4/GREM1 module to the slower SHH/GREM1/FGF epithelial-mesenchymal feedback loop. This self-regulatory signaling network results in robust regulation of distal limb development that is able to compensate for variations by interconnectivity among the 3 signaling pathways.
Martinelli and Fan (2007) found that Gas1 (139185) positively regulated Shh signaling in developing mouse and chicken, an effect particularly noticeable at regions where Shh acted at low concentrations. Combining studies of COS-7 cell-surface binding, in vitro activity, and mouse limb bud explants, Martinelli and Fan (2009) demonstrated that Gas1 positively regulates Shh signaling, and that murine Shh residues tyr81, glu90, asn116, and asp132 form part of a contiguous Gas1-Shh binding interface.
Huang et al. (2010) found that Shh signaling regulated proliferation and expansion of cerebellar radial glia and neuron progenitors derived from the ventricular zone in mice. The cerebellum itself was not the source of Shh. Shh was detected in circulating embryonic cerebrospinal fluid and was likely secreted by hindbrain choroid plexus epithelium.
In mice, Shin et al. (2011) showed that the proliferative response to bacterial infection or chemical injury within the bladder is regulated by signal feedback between basal cells of the urothelium and the stromal cells that underlie them. They demonstrated that these basal cells include stem cells capable of regenerating all cell types within the urothelium, and are marked by expression of the secreted protein signal Shh. On injury, Shh expression in these basal cells increases and elicits increased stromal expression of Wnt protein signals, which in turn stimulate the proliferation of both urothelial and stromal cells. The heightened activity of this signal feedback circuit and the associated increase in cell proliferation appear to be required for restoration of urothelial function and, in the case of bacterial injury, may help clear and prevent further spread of infection. Shin et al. (2011) concluded that their findings provided a conceptual framework for injury-induced epithelial regeneration in endodermal organs.
Alvarez et al. (2011) showed that astrocytes secrete Shh and that blood-brain barrier (BBB) endothelial cells express Hedgehog receptors, which together promote BBB formation and integrity during embryonic development and adulthood. Using pharmacologic inhibition and genetic inactivation of the hedgehog signaling pathway in endothelial cells, Alvarez et al. (2011) also demonstrated a critical role of the hedgehog pathway in promoting the immune quiescence of BBB endothelial cells by decreasing the expression of proinflammatory mediators and the adhesion and migration of leukocytes, in vivo and in vitro. Alvarez et al. (2011) concluded that the hedgehog pathway provides a barrier-promoting effect and an endogenous antiinflammatory balance to central nervous system-directed immune attacks.
Harjunmaa et al. (2012) reported that mouse tooth complexity can be increased substantially by adjusting multiple signaling pathways simultaneously. Harjunmaa et al. (2012) cultured teeth in vitro and adjusted ectodysplasin (EDA; 300451), activin A (see 147290), and sonic hedgehog (SHH) pathways, all of which are individually required for normal tooth development. The authors quantified tooth complexity using the number of cusps and a topographic measure of surface complexity, and found that whereas activation of EDA and activin A signaling and inhibition of SHH signaling individually cause subtle to moderate increases in complexity, cusp number is doubled when all 3 pathways are adjusted in unison. Furthermore, the increase in cusp number does not result from an increase in tooth size, but from an altered primary patterning phase of development. The combination of a lack of complex mutants, the paucity of natural variants with complex phenotypes, and their results of greatly increased dental complexity using multiple pathways, suggests that an increase may be inherently different from a decrease in phenotypic complexity.
To delineate the cellular mechanisms used by signaling proteins such as SHH that possess membrane-bound covalent lipid modifications to traverse long distances within the vertebrate limb bud in vivo, Sanders et al. (2013) directly imaged SHH ligand production under native regulatory control in chick embryos. They found that SHH is produced in the form of a particle that remains associated with the cell via long cytoplasmic extensions that span several cell diameters. Sanders et al. (2013) showed that these cellular extensions are a specialized class of actin-based filopodia with novel cytoskeletal features. Notably, particles containing SHH travel along these extensions with a net anterograde movement within the field of SHH cell signaling. Sanders et al. (2013) found that in SHH-responding cells, specific subsets of SHH coreceptors, including cell adhesion molecule downregulated by oncogenes (CDON; 608707) and brother of CDON (BOC; 608708), actively localize and substantially colocalize in specific microdomains within filopodial extensions, far from the cell body. Stabilized interactions are formed between filopodia containing SHH ligand and those containing coreceptors over a long range. Sanders et al. (2013) concluded that contact-mediated release propagated by specialized filopodia contributes to the delivery of SHH at a distance.
Peng et al. (2015) demonstrated that quiescence in the adult lung is an actively maintained state and is regulated by hedgehog signaling. Epithelial-specific deletion of sonic hedgehog during postnatal homeostasis in the murine lung results in a proliferative expansion of the adjacent lung mesenchyme. Hedgehog signaling is initially downregulated during the acute phase of epithelial injury as the mesenchyme proliferates in response, but returns to baseline during injury resolution as quiescence is restored. Activation of hedgehog during acute epithelial injury attenuates the proliferative expansion of the lung mesenchyme, whereas inactivation of hedgehog signaling prevents the restoration of quiescence during injury resolution. Finally, Peng et al. (2015) that hedgehog also regulates epithelial quiescence and regeneration in response to injury via a mesenchymal feedback mechanism. Peng et al. (2015) concluded that epithelial-mesenchymal interactions coordinated by hedgehog actively maintain postnatal tissue homeostasis, and deregulation of hedgehog during injury leads to aberrant repair and regeneration in the lung.
Nacu et al. (2016) clarified the molecular basis of the requirement for both anterior and posterior tissue during limb regeneration and supernumerary limb formation in axolotls. Nacu et al. (2016) showed that the 2 tissues provide complementary cross-inductive signals that are required for limb outgrowth. A blastema composed solely of anterior tissue normally regresses rather than forming a limb, but activation of hedgehog (HH) signaling was sufficient to drive regeneration of an anterior blastema to completion owing to its ability to maintain fibroblast growth factor (FGF) expression, the key signaling activity responsible for blastema outgrowth. In blastemas composed solely of posterior tissue, HH signaling was not sufficient to drive regeneration; however, ectopic expression of FGF8 (600483) together with endogenous HH signaling was sufficient. In axolotls, FGF8 is expressed only in the anterior mesenchyme and maintenance of its expression depends on SHH signaling from posterior tissue. Nacu et al. (2016) concluded that their data identified key anteriorly and posteriorly localized signals that promote limb regeneration.
Role in Cancer
Bale and Yu (2001) reviewed the hedgehog pathway and its disruption as a basis for basal cell carcinomas.
Berman et al. (2002) investigated the therapeutic efficacy of the hedgehog pathway antagonist cyclopamine in preclinical models of medulloblastoma (155255), the most common malignant brain tumor in children. Cyclopamine treatment of murine medulloblastoma cells blocked proliferation in vitro and induced changes in gene expression consistent with initiation of neuronal differentiation and loss of neuronal stem cell-like character. The compound also caused regression of murine tumor allografts in vivo and induced rapid death of cells from freshly resected human medulloblastomas, but not from other brain tumors, and thus established a specific role for hedgehog pathway activity in medulloblastoma growth.
Berman et al. (2003) demonstrated that a wide range of digestive tract tumors, including most of those originating in the esophagus, stomach, biliary tract, and pancreas, but not in the colon, display increased hedgehog pathway activity, which is suppressible by cyclopamine, a hedgehog pathway antagonist. Cyclopamine also suppresses cell growth in vitro and causes durable regression of xenograft tumor in vivo. Unlike tumors in Gorlin syndrome (109400), pathway activity and cell growth in these digestive tract tumors are driven by endogenous expression of hedgehog ligands, as indicated by the presence of Sonic hedgehog and Indian hedgehog transcripts, by the pathway- and growth-inhibitory activity of a hedgehog-neutralizing antibody, and by the dramatic growth-stimulatory activity of exogenously added hedgehog ligand. Berman et al. (2003) concluded that their results identified a group of common lethal malignancies in which hedgehog pathway activity, essential for tumor growth, is activated not by mutation but by ligand expression.
Watkins et al. (2003) investigated a role for the SHH pathway in regeneration and carcinogenesis of airway epithelium. They demonstrated extensive activation of the hedgehog pathway within the airway epithelium during repair of acute airway injury. This mode of hedgehog signaling is characterized by the elaboration and reception of the SHH signal within the epithelial compartment, and immediately precedes neuroendocrine differentiation. A similar pattern of hedgehog signaling in airway development during normal differentiation of pulmonary neuroendocrine precursor cells, and in a subset of small cell lung cancer (182280), was also observed. Small cell lung cancer tumors maintain their malignant phenotype in vitro and in vivo through ligand-dependent hedgehog pathway activation. Watkins et al. (2003) proposed that some types of small cell lung cancer might recapitulate a critical hedgehog-regulated event in airway epithelial differentiation. This requirement for hedgehog pathway activation identified a common lethal malignancy that may respond to pharmacologic blockade of the hedgehog signaling pathway.
Karhadkar et al. (2004) found that activity of the hedgehog signaling pathway, which has essential roles in developmental patterning, was required for regeneration of prostate epithelium, and that continuous pathway activation transformed prostate progenitor cells and rendered them tumorigenic. Elevated pathway activity furthermore distinguished metastatic from localized prostate cancer (176807), and pathway manipulation modulated invasiveness and metastasis. Pathway activity was triggered in response to endogenous expression of hedgehog ligands, and was dependent upon the expression of Smoothened, which is not expressed in benign prostate epithelial cells. Karhadkar et al. (2004) concluded that monitoring and manipulating hedgehog pathway activity may offer significant improvements in diagnosis and treatment of prostate cancers with metastatic potential.
Sims-Mourtada et al. (2007) showed that inhibition of SHH signaling increased the response of human cancer cell lines to multiple structurally unrelated chemotherapies. SHH activation induced chemoresistance in part by increasing drug efflux in an ABC transporter-dependent manner. SHH signaling regulated expression of the ABC transporters ABCB1 (171050) and ABCG2 (603756), and targeted knockdown of ABCB1 and ABCG2 expression by small interfering RNA partially reversed SHH-induced chemoresistance.
Although a cell-autonomous role for hedgehog signaling in tumors has been described (Berman et al., 2003; Thayer et al., 2003; Karhadkar et al., 2004), Yauch et al. (2008) found that hedgehog ligands failed to activate signaling in tumor epithelial cells. In contrast, their data supported ligand-dependent activation of the hedgehog pathway in the stromal microenvironment. Specific inhibition of hedgehog signaling using small molecule inhibitors, a neutralizing anti-hedgehog antibody, or genetic deletion of Smo in the mouse stroma resulted in growth inhibition in xenograft tumor models. Yauch et al. (2008) concluded that their studies demonstrated a paracrine requirement for hedgehog ligand signaling in tumorigenesis of hedgehog-expressing cancers and have important implications for the development of hedgehog pathway antagonists in cancer.
By PCR analysis of DNA from a panel of rodent/human somatic cell hybrids, Marigo et al. (1995) assigned the SHH gene to 7q and the IHH gene to chromosome 2. SHH was more precisely localized by linkage studies using a CA repeat sequence tagged site identified in a P1 genomic clone of SHH in members of a family with polysyndactyly, or triphalangeal thumb-polysyndactyly syndrome (TPT; see 174500), previously reported by Tsukurov et al. (1994). SHH was found to be closely linked to but distinct from the TPT1 locus at 7q36; maximum lod score = 4.82 at theta = 0.05. It was tightly linked to En2, the engrailed-2 locus (131310). Marigo et al. (1995) mapped the mouse homologs Shh, Ihh, and Dhh by linkage analysis of an interspecific backcross. Shh mapped to a position 0.6 cM distal to En2 and 1.9 cM distal to Il6, or interleukin-6 (147620), on mouse chromosome 5. This location is closely linked to but distinct from the murine limb mutation Hx and is in an area with homology of synteny to human 7q36.
Holoprosencephaly 3
Belloni et al. (1996) identified SHH as a candidate gene for autosomal dominant holoprosencephaly-3 (HPE3; 142945) by detailed characterization of HPE3 patient chromosome rearrangements and contigs of the HPE3 region. Further analysis revealed that SHH mapped approximately 250 and 15 kb centromeric of T1 and T2, respectively (T1 and T2 represent the translocation breakpoints in 2 unrelated patients with a mild form of HPE3). Belloni et al. (1996) proposed that the chromosomal rearrangements remove distal cis-acting regulatory elements or exert long-term position effects causing aberrant expression of the gene.
Roessler et al. (1996) defined the intron-exon boundaries of SHH by direct sequencing and then designed primers for exon amplification and SSCP analysis in 30 families with HPE3. The authors then identified mutations in SHH which caused HPE3 in these families. Two families that showed chromosome 7q36 linkage demonstrated band shifts on SSCP of exon 1. The mutation in one family was a gly31-to-arg substitution (G31R; 600725.0001). In the second family the mutation occurred at gln100, resulting in a stop codon (600725.0002) and leading to synthesis of a truncated protein. In exon 2, a nonsense mutation leading to a stop codon (600725.0003) and 2 missense mutations (600725.0004 and 600725.0005) were identified. Roessler et al. (1996) noted that loss of one SHH allele was sufficient to cause HPE in humans, whereas both Shh alleles need to be lost to produce a similar phenotype in mice (Chiang et al., 1996).
Roessler et al. (1997) identified a total of 5 different mutations in the processing domain encoded by exon 3 of the SHH gene in familial and sporadic HPE. This was the initial report in humans of SHH mutations in the domain responsible for autocatalytic cleavage and cholesterol modification of the N-terminal signaling domain of the protein.
Schell-Apacik et al. (2003) assessed the biologic significance of 2 SHH mutations identified in HPE patients: W117G (600725.0004) and W117R (600725.0005). The studies were initiated in the chick spinal cord and demonstrated in vivo that these mutations perturbed the normal patterning activity of SHH. In addition, these mutations altered the immunoreactivity of the SHH protein, suggesting that the conformation of the protein had been disrupted.
Nanni et al. (1999) performed mutation analysis on the complete coding region and intron-exon junctions of the SHH gene in 344 unrelated individuals with holoprosencephaly. They identified 13 unrelated affected individuals with novel SHH mutations, including nonsense and missense mutations, deletions, and an insertion. These mutations occurred throughout the gene. No specific genotype-phenotype association was evident based on the correlation of the type or position of the mutations. In conjunction with their previous studies (Roessler et al., 1996; Roessler et al., 1997), Nanni et al. (1999) identified a total of 23 mutations in 344 unrelated cases of HPE. These mutations accounted for 14 cases of familial HPE and 9 cases of sporadic HPE. Mutations in the SHH gene were detected in 10 of 27 (37%) families showing autosomal dominant transmission of the HPE spectrum, based on structural anomalies. Three patients with SHH mutations also had abnormalities in another gene that is expressed during forebrain development. Nanni et al. (1999) speculated that, given the great intrafamilial clinical variability in kindreds carrying an SHH mutation, other genes acting in the same or different developmental pathways might act as modifiers for expression of the HPE spectrum. They identified a gly290-to-asp mutation of the SHH gene (600725.0011) that was associated with a mutation predicting an expansion of an ala repeat in exon 2 of the ZIC2 gene (603073), the site of mutations causing holoprosencephaly-5. In a second patient, a pro424-to-ala mutation in the SHH gene (600725.0012) was identified both in a child who was deleted for 18pter and TGIF, the site of mutations causing holoprosencephaly-4 (142946), and in her mother, who carried a balanced translocation involving chromosome 18. A third example was a 9-bp deletion in the SHH gene (600725.0013) in a child with HPE who also had a thr151-to-ala mutation in the TGIF gene (602630.0003).
Nanni et al. (1999) presented a panel of 12 photographs illustrating the range of severity in holoprosencephaly resulting from mutation in the SHH gene.
Nanni et al. (2001) studied 13 patients with solitary median maxillary central incisor (SMMCI; 147250), also known as single central incisor, which is often associated with holoprosencephaly. Although these patients did not have holoprosencephaly, in 1 SMMCI family the authors identified a new missense mutation, ile111 to phe (I111F; 600725.0014), which they suggested may be specific for the SMMCI phenotype since it had not been found in cases of holoprosencephaly or in normal controls.
Heussler et al. (2002) reported a large family ascertained following the identification of HPE in the index case by antenatal ultrasound. Six members of the family over 2 generations carried an asp88-to-val mutation in the SHH gene (D88V; 600725.0015). The family highlighted the intrafamilial variability of expression of an identical mutation.
In a family previously reported by Nanni et al. (1999), Verlinsky et al. (2003) performed preimplantation diagnosis for a Sonic hedgehog mutation causing familial holoprosencephaly. The parents had had 2 children with holoprosencephaly. Their second child, a girl with severe holoprosencephaly and cleft lip and palate, died shortly after birth. The girl was found to have a glu256-to-stop mutation in the SHH gene (E256X; 600725.0017). The same mutation was found in the couple's 5-year-old son, who had less severe facial dysmorphism than his sister, including microcephaly, Rathke pouch cyst, single central incisor, and choanal stenosis (the latter was dilated surgically after birth). He also had clinodactyly of the fifth fingers and incurved fourth toes bilaterally. His growth was slow during the first 2 years but subsequently improved, and his social and cognitive development were apparently normal. The mutation was not found in either parent, although paternity testing showed that the father was the biologic father of both affected children, suggesting a new gonadal mutation in one of the parents. The use of preimplantation genetic diagnosis, followed by confirmation of mutation-prestatus by amniocentesis, resulted in the birth of a healthy girl.
Traiffort et al. (2004) developed 3-dimensional models of human N-terminal (SHH-N) and C-terminal (SHH-C) hedgehog proteins and characterized the functional consequences linked to various mutations in the SHH gene, dividing them into 3 classes. In the first group, the production of the active SHH-N fragment was dramatically impaired in transfected HEK293 cells, and supernatants from those cell cultures showed no significant SHH signaling activity in a reporter cell-based assay. The second group showed lower production of SHH-N and signaling activity, and the third displayed an activity comparable to that of the wildtype protein. Traiffort et al. (2004) concluded that most of the holoprosencephaly-associated SHH mutations analyzed have a deleterious effect on the availability of SHH-N and its biologic activity, but that the lack of genotype/phenotype correlations suggested that other factors intervene in the development of the spectrum of holoprosencephalic anomalies.
Maity et al. (2005) analyzed sequence alterations in the N-terminal signaling domain of mouse Shh corresponding to human missense mutations associated with holoprosencephaly. Five of the mutations, including G31R (600725.0001), W117G (600725.0004), and W117R (600725.0005), affected normal processing, Ptc binding, and signaling to varying degrees.
Singh et al. (2009) analyzed a panel of human HPE-associated SHH missense mutations that encode changes in the amino terminus by studying their expression in human embryonic kidney cells. Several mutant proteins (e.g., D88V, 600725.0015; I111F, 600725.0014; G31R, 600725.0001) showed defective processing, impaired secretion into the lysate, and/or compromised stability, consistent with overall reduced SHH activity compared to wildtype. In addition, some variants, including G31R, appeared to have a dominant-negative effect on SHH activity when coexpressed with the wildtype protein. Singh et al. (2009) concluded that the range of phenotypes associated with heterozygous SHH mutations may reflect distinct pathogenic mechanisms resulting from different mutations that interfere with SHH biogenesis and signaling at multiple steps.
Combining cell surface binding, in vitro activity, and mouse limb bud explants, Martinelli and Fan (2009) demonstrated that Gas1 (139185) positively regulates Shh signaling, and that murine Shh residues tyr81, glu90, asn116, and asp132 form part of a contiguous Gas1-Shh interface. A constructed murine Shh N116K mutant, which corresponds to the HPE3-associated N115K mutation (600725.0020), caused markedly decreased binding to Gas1, resulting in decreased Shh signaling. These findings indicated that HPE due to the N115K mutation results from an inability of mutant SHH to bind to GAS1 normally, thus interrupting positive effect of GAS1.
Schell-Apacik et al. (2009) identified a heterozygous G290D mutation (600725.0011) in a boy with schizencephaly (269160) and developmental delay. Brain MRI at age 5 months showed a complex brain malformation with partial absence of the corpus callosum, bilateral parietotemporal closed-lip schizencephaly, polymicrogyria, and optic atrophy. Dysmorphic features included microbrachycephaly, hypotelorism, broad nasal root, short philtrum, and a thin upper lip. The patient's unaffected mother was also heterozygous for the mutation. The findings expanded the phenotypic spectrum resulting from SHH mutations.
Isolated Microphthalmia with Coloboma
In a boy with bilateral colobomatous microphthalmia (MCOPCB5; 611638), Schimmenti et al. (2003) identified heterozygosity for a 24-bp deletion in the SHH gene (600725.0016). His mother, who had unilateral iris and uveoretinal coloboma, and 3 unaffected family members carried the same deletion. The authors noted that incomplete expression of SHH mutations had also been observed in several holoprosencephaly pedigrees (see Nanni et al., 1999).
In a cohort of 236 individuals with developmental eye anomalies, primarily microphthalmia, clinical anophthalmia, and coloboma, Bakrania et al. (2010) identified 2 patients with heterozygous SHH variants (600725.0011 and 600725.0016, respectively) and a patient with a de novo 152.23- to 156.20-Mb deletion on chromosome 7q36.2-q36.3 encompassing SHH and 23 other genes. The latter patient had right microphthalmia, chorioretinal coloboma, and funnel retinal detachment with subretinal opacities, and left optic nerve and chorioretinal coloboma. In addition to her ocular phenotype, she had plagiocephaly, microcephaly, delayed motor development, and mild choanal atresia, suggesting a mild form of holoprosencephaly spectrum. Citing the incomplete penetrance and relatively mild ocular phenotype seen in these patients, Bakrania et al. (2010) suggested that genetic modifiers and/or environmental influences might be important.
Cleft Lip and/or Palate
Cleft lip and/or palate can occur in pedigrees with autosomal dominant holoprosencephaly due to mutations in SHH. In addition, animal models have shown that SHH is involved in face development. Orioli et al. (2002) examined the SHH gene in 220 newborn infants with nonsyndromic oral clefts registered in the Latin American Collaborative Study of Congenital Malformations (ECLAMC). They found 15 sequence changes in 13 patients with oral clefts, all of which were found by sequencing to represent silent polymorphisms. Four occurred in introns. No clearly disease-causing mutation was found. The authors concluded that SHH mutations are not a frequent cause of isolated oral clefts.
SHH Regulatory Element-Associated Syndromes
Lettice et al. (2003) showed that chromosome 7q36-associated preaxial polydactyly II (PPD2; 174500) results from point mutations in an SHH regulatory element. SHH, normally expressed in the zone of polarizing activity (ZPA) posteriorly in the limb bud, is expressed in an additional ectopic site at the anterior margin in mouse models of PPD. Lettice et al. (2003) identified an enhancer element that drives normal SHH expression in the ZPA. The regulator, designated ZPA regulatory sequence (ZRS; 620738), lies within intron 5 of the LMBR1 gene (605522), 1 Mb from the target gene SHH. The ZRS contained point mutations (620738.0001-620738.0004) that segregated with polydactyly in 4 unrelated families with PPD2 as well as in the Hx mouse mutant.
Other limb anomalies caused by mutation in the ZRS region include triphalangeal thumb-polysyndactyly syndrome (190605), type IV syndactyly (SDTY4; 186200), tibial hypoplasia or aplasia with polydactyly (THYP; 188740), and Laurin-Sandrow syndrome (LSS; 135750).
Gomez-Ospina et al. (2012) reported a 41-year-old man with a germline translocation t(7;Y) in which the middle of the SHH promoter was fused with Y-chromosome sequences, leaving intact 140 kb of regulatory sequences upstream of the SHH transcriptional start site. The patient had microcephaly, hypotelorism, flat nasal bridge, and T-shaped incisors, suggestive of mild holoprosencephaly. He also had several advanced basal cell carcinomas (see 605462) on his head, trunk, and all 4 extremities. The onset of skin tumors occurred around age 9 years. Gomez-Ospina et al. (2012) suggested that the translocation resulted in partial loss of SHH during development, causing the mild holoprosencephaly, and that the mutant promoter resulted in overexpression of SHH in the skin. Tumors from the patient showed higher levels of SHH protein and mRNA compared to control.
Human evolution is characterized by a dramatic increase in brain size and complexity. To probe its genetic basis, Dorus et al. (2004) examined the evolution of genes involved in diverse aspects of nervous system biology. These genes, including SHH, displayed significantly higher rates of protein evolution in primates than in rodents. This trend was most pronounced for the subset of genes implicated in nervous system development. Moreover, within primates, the acceleration of protein evolution was most prominent in the lineage leading from ancestral primates to humans. Dorus et al. (2004) concluded that the phenotypic evolution of the human nervous system has a salient molecular correlate, i.e., accelerated evolution of the underlying genes, particularly those linked to nervous system development.
Lopez-Rios et al. (2014) analyzed bovine embryos to establish that polarized gene expression is progressively lost during limb development in comparison to the mouse. Notably, the transcriptional upregulation of the Ptch1 gene (601309), which encodes an SHH receptor, is disrupted specifically in the bovine limb bud mesenchyme. This is due to evolutionary alteration of a Ptch1 cis-regulatory module, which no longer responds to graded Shh signaling during bovine handplate development. Lopez-Rios et al. (2014) concluded that their study provided a molecular explanation for the loss of digit asymmetry in bovine limb buds, and suggested that modifications affecting the Ptch1 cis-regulatory landscape have contributed to evolutionary diversification of artiodactyl limbs.
Olson and Srivastava (1996) reviewed the role of Sonic hedgehog in cardiac morphogenesis in the chick and mouse, particularly in the initiation of rightward looping of the heart tube in early embryogenesis. Before looping, Sonic hedgehog is expressed on the left side and Acvr2, the activin A receptor type II (102581), is expressed on the right side. On the right side of the embryo, activin (147290) or an activin-like molecule induces Acvr2a and suppresses expression of Shh, thereby creating left to right asymmetry. On the left side, the nodal-related morphogen (601265) is induced by Shh. Olson and Srivastava (1996) stated that evidence for the role of these morphogens in the control of looping direction is indicated by the finding that looping is randomized when Shh is expressed on the right side or when Acvr2 is expressed on the left side.
Chiang et al. (1996) generated mice that were homozygous for a disrupted Sonic hedgehog gene by using homologous recombination in embryonic stem cells. Morphologic studies in these mice revealed defects in the establishment of maintenance of midline structures such as the notochord and floorplate. Other defects observed included absence of distal limb structures, cyclopia, absence of ventral cell types within the neural tube, and absence of the spinal column and most of the ribs. Chiang et al. (1996) reported that defects in all tissues extend beyond the normal sites of Shh transcription, and that this observation confirmed the proposed role of Shh protein as an extracellular signal required for the tissue organizing properties of several vertebrate patterning centers.
Oro et al. (1997) showed that transgenic mice overexpressing SHH in the skin developed many features of the basal cell nevus syndrome, demonstrating that SHH is sufficient to induce basal cell carcinomas (BCCs) in mice. The data suggested that SHH may have a role in human tumorigenesis. Activating mutations of SHH or another 'hedgehog' gene may be an alternative pathway for BCC formation in humans. The human mutation his133 to tyr (his134 to tyr in mouse) is a candidate. It is distinct from loss-of-function mutations reported for individuals with holoprosencephaly. His133 lies adjacent in the catalytic site to his134 (mouse his135), one of the conserved residues thought to be necessary for catalysis. Oro et al. (1997) suggested that SHH may be a dominant oncogene in multiple human tumors, a mirror of the tumor suppressor activity of the opposing 'Patched' (PTCH) gene. The rapid and frequent appearance of Shh-induced tumors in the mice suggested that disruption of the SHH-PTC pathway is sufficient to create BCCs. The mouse BCCs appeared within the first 4 days of skin development, unlike mouse squamous neoplasia where tumors arise 1 to 12 months after oncogene expression. The kinetics of the tumors in these mice were consistent with previous clinical and epidemiologic data, which suggested that BCCs, in contrast to melanomas and squamous carcinomas, lack precursor or intermediate cellular phenotypes. The gene PTCH joins APC (611731) in a class of genes instrumental for controlling early epithelial proliferation. Mutations in APC cause familial adenomatous polyposis, a condition that predisposes individuals to many benign polyps, akin to the hundreds of nodular BCCs that can occur in patients with the basal cell nevus syndrome. Nodular BCCs are reminiscent of polyps in colonic epithelium, as both lack aneuploidy and are locally invasive.
Litingtung et al. (1998) found that mice with a targeted deletion of Shh have foregut defects that are apparent as early as embryonic day 9.5, when the tracheal diverticulum begins to outgrow. Homozygous Shh-null mutant mice showed esophageal atresia/stenosis, tracheoesophageal fistula, and tracheal and lung anomalies, features similar to those observed in humans with foregut defects. The lung mesenchyme showed enhanced cell death, decreased cell proliferation, and downregulation of Shh target genes. These results indicated that Shh is required for the growth and differentiation of the esophagus, trachea, and lung, and suggested that mutations in SHH and its signaling components may be involved in foregut defects in humans. Of relevance is the demonstration of Motoyama et al. (1998) that Gli2 (165230) and Gli3 (165240), which are involved in the transduction of Shh signal, are essential to the formation of lung, trachea, and esophagus.
Hair follicles form in prenatal skin and mature in the postnatal period, establishing a growth cycle in 3 phases: telogen (resting), anagen (growth), and catagen (regression). Based on the knowledge that Shh expression is necessary for the embryonic development of hair follicles, and that anagen in the postnatal cycling follicle has morphologic similarities to the epithelial invagination process in embryonic skin, Sato et al. (1999) hypothesized that localized, but transient, enhanced expression of the Shh gene in postnatal skin would accelerate initiation of anagen in the hair follicle cycle, with concomitant accelerated hair growth. To assess this concept, an adenovirus vector (AdShh) was used to transfer the murine Shh cDNA to skin of postnatal day 19 C57BL/6 mice. The treated skin showed increased mRNA expression of Shh, the Shh receptor Patched, and Gli1 (165220), a transcription factor in the Shh pathway. In mice receiving the treatment, but not in controls, acceleration into anagen was evident, since hair follicle size and melanogenesis increased and the hair-specific keratin Hb1 (KRT81; 602153) and melanin synthesis-related tyrosinase (see 606933) mRNAs accumulated. Finally, C57BL/6 mice showed marked acceleration of the onset of new hair growth in the region of AdShh administration to skin 2 weeks after treatment, but not in control vector-treated or untreated areas. After 6 months, AdShh-treated skin showed normal hair and normal skin morphology. Together, these observations were considered consistent with the concept that upregulation of Shh activity in postnatal skin functions as a biologic switch that induces resting hair follicles to enter anagen with consequent hair growth.
Mill et al. (2003) found that Gli2-null mice showed grossly normal epidermal differentiation, but like Shh-null mice, they exhibited arrested hair follicle development with reduced cell proliferation and Shh-responsive gene expression. A constitutively active form of Gli2, but not wildtype Gli2, activated Shh-responsive gene expression and promoted cell proliferation in Shh-null skin.
Using the Cre/loxP system, Sun et al. (2000) found that Shh expression is maintained and limb formation is normal when Fgf4 (164980) is inactivated in mouse limbs, contradicting another model which suggested that Fgf4 expression is not maintained in Shh -/- mouse limbs. Sun et al. (2000) also found that maintenance of Fgf9 (600921) and Fgf17 (603725) expression is dependent on Shh, whereas Fgf8 (600483) expression is not. Sun et al. (2000) developed a model in which no individual Fgf expressed in the apical ectodermal ridge is solely necessary to maintain Shh expression, but instead the combined activity of 2 or more apical ectodermal ridge Fgfs function in a positive feedback loop with Shh to control limb development.
To gain insight into the role of SMO in hedgehog signaling in vertebrates, Zhang et al. (2001) generated a null allele of Smo by gene targeting in mouse embryonic stem (ES) cells. They showed that Smo acts epistatic to Ptc1 to mediate Shh and Ihh signaling in the early mouse embryo. Smo and Shh/Ihh compound mutants had identical phenotypes: embryos failed to turn, arresting at somite stages with a small, linear heart tube, an open gut, and cyclopia. The absence of visible left/right (L/R) asymmetry led the authors to examine the pathways controlling L/R situs. Zhang et al. (2001) presented evidence consistent with a model in which hedgehog signaling within the node is required for activation of GDF1 (602880) and induction of left-side determinants. Further, they demonstrated an absolute requirement for hedgehog signaling in sclerotomal development and a role in cardiac morphogenesis.
Murdoch et al. (2001) cloned the causative gene for craniorachischisis (a severe neural tube defect) in 'loop-tail' (Lp) mice, which they named Lpp1 (see VANGL2; 600533). Lpp1 is expressed in the ventral part of the developing neural tube, but is excluded from the floorplate where Shh is expressed. Embryos lacking Shh express Lpp1 throughout the ventral neural tube, suggesting negative regulation of Lpp1 by Shh. The authors suggested that the mutual interaction between Lpp1 and Shh may define the lateral boundary of floorplate differentiation. They hypothesized that loss of Lpp1 function may disrupt neurulation by permitting more extensive floorplate induction by Shh, thereby inhibiting midline bending of the neural plate during initiation of neurulation.
By in situ hybridization, Treier et al. (2001) detected Shh expression in the ventral diencephalon and oral ectoderm during development of the pituitary gland in mouse embryos. Using loss- and gain-of-function studies in transgenic mice, they investigated the role of Shh in outgrowth and differentiation of the pituitary gland. They targeted overexpression of Hip (HHIP; 606178) in transgenic mice to specifically block hedgehog signaling in the oral ectoderm and Rathke pouch within the head region and observed a failure of pituitary organogenesis. Using in situ hybridization, Treier et al. (2001) observed an absence of ventral cell-type markers in Rathke pouch. Using a transgenic approach in gain-of-function studies, they targeted overexpression of Shh to Rathke pouch, resulting in an expansion of ventral cell types. Treier et al. (2001) concluded that Shh exerts effects on both proliferation and cell-type determination in pituitary gland development.
Inactivation of the Shh gene in mice leads to annular pancreas (167750) in certain genetic backgrounds (Ramalho-Santos et al., 2000). Gut malrotations and imperforate anus (301800), often associated with annular pancreas in humans, are also found in Shh mutant mice.
Litingtung et al. (2002) reported genetic analyses in mice showing that Shh and Gli3 are dispensable for formation of limb skeletal elements. The limbs of double-knockout Shh/Gli mice are distally complete and polydactylous, but completely lack wildtype digit identities. Litingtung et al. (2002) showed that the effects of Shh signaling on skeletal patterning and ridge maintenance are necessarily mediated through Gli3. The authors proposed that the function of Shh and Gli3 in limb skeletal patterning is limited to refining autopodial morphology, imposing pentadactyl constraint on the limb's polydactyl potential, and organizing digit identity specification, by regulating the relative balance of Gli3 transcriptional activator and repressor activities.
Alcohol is a teratogen that induces a variety of abnormalities including brain and facial defects (Jones and Smith, 1973), referred to as the fetal alcohol syndrome, with the exact nature of the defect depending on the time and magnitude of the dose of ethanol to which the developing fetus is exposed. In addition to abnormal facial structures, ethanol-treated embryos exhibit a highly characteristic pattern of cell death. Dying cells are observed in the premigratory and migratory neural crest cells that normally populate most facial structures. The observation that blocking Shh signaling results in similar craniofacial abnormalities prompted Ahlgren et al. (2002) to examine whether there is a link between this aspect of fetal alcohol syndrome and loss of Shh. They demonstrated that administration of ethanol to chick embryos resulted in a dramatic loss of Shh, as well as a loss of transcripts involved in Shh signaling pathways. In contrast, other signaling molecules examined did not demonstrate such dramatic changes. Furthermore, they demonstrated that both ethanol-induced cranial neural crest cell death and the associated craniofacial growth defect can be rescued by application of Shh. These data suggested that craniofacial abnormalities resulting from fetal alcohol exposure are caused at least partially by loss of Shh and subsequent neural crest cell death.
Te Welscher et al. (2002) reported that the polydactyly of Gli3 (165240)-deficient mice arises independently of Shh signaling. Disruption of one or both Gli3 alleles in mouse embryos lacking Shh progressively restored limb distal development and digit formation. Te Welscher et al. (2002) concluded that SHH signaling counteracts GLI3-mediated repression of key regulator genes, cell survival, and distal progression of limb bud development. The limbs of Gli3-deficient embryos were polydactylous, whereas 1 fused forearm bone and no digit arch formed in limbs of Shh-deficient embryos. Disruption of 1 Gli3 allele on an Shh-knockout background resulted in embryos with 2 forearm bones and rudimentary digits. The limbs of double homozygous mouse embryos were grossly morphologically indistinguishable from the limbs of Gli3 homozygous embryos. Te Welscher et al. (2002) showed that, whereas the polydactyly of Gli3-deficient mice is Shh-independent, the polydactyly of Alx4 (605420) mutant mice depends on Shh signaling,
Lai et al. (2003) found high expression of the Shh receptor Patched (Ptc; 601309) in both adult rat hippocampus and neural progenitor cells isolated from this region. In vitro, Shh promoted neural progenitor cell proliferation, and in vivo, adeno-associated viral vector delivery of Shh cDNA to the rat hippocampus elicited a 3.3-fold increase in cell proliferation. Injection of cyclopamine, an inhibitor of Shh signaling, reduced hippocampal neural progenitor proliferation in vivo. Lai et al. (2003) concluded that Shh is an important regulator of adult hippocampal neural stem cells.
Cooper et al. (2003) identified a defective response to hedgehog (Hh) signaling in the disorders of cholesterol biosynthesis Smith-Lemli-Opitz syndrome (SLOS; 270400) and lathosterolosis (607330). Many of the developmental malformations in these syndromes occur in tissues and structures whose embryonic patterning depends on signaling by the Hh family of secreted proteins. Cooper et al. (2003) reported that response to the Hh signal is compromised in mutant cells from mouse models of SLOS and lathosterolosis and in normal cells pharmacologically depleted of sterols. They showed that decreasing levels of cellular sterols correlated with diminishing responsiveness to the Hh signal. This diminished response occurred at sterol levels sufficient for normal autoprocessing of Hh protein, which requires cholesterol as cofactor and covalent adduct. They also found that sterol depletion affects the activity of Smoothened (Smo; 601500), an essential component of the Hh signal transduction apparatus.
Riccomagno et al. (2002) found that the morphogenesis of the inner ear of Shh-null mouse embryos was greatly perturbed by midgestation, whereas otic induction proceeded normally. Ventral otic derivatives including the cochlear duct and cochleovestibular ganglia failed to develop in the absence of Shh. The inner ear defects were due to alterations in the expression of a number of genes involved in cell fate specification including Pax2 (167409), Otx1 (600036), Otx2 (600037), Tbx1 (602054), and Ngn1 (601726).
In studies in transgenic mice, Riccomagno et al. (2005) demonstrated that Wnt3a (606359) and Wnt1 (164820) signaling in dorsal regions of the otic vesicle regulates expression of genes (i.e., Dlx5/6, 600029, 600030; Gbx2, 601135) necessary for vestibular morphogenesis. In addition, they found that restriction of the Wnt target genes to the dorsal otocyst is also influenced by Shh. Riccomagno et al. (2005) suggested that a balance between Wnt and Shh signaling activities is key in distinguishing between vestibular and auditory cell types.
Thayer et al. (2003) reported that Sonic hedgehog is abnormally expressed in pancreatic adenocarcinoma and its precursor lesions, pancreatic intraepithelial neoplasia. The pancreata of Pdx1- (600733) Shh mice (in which Sonic hedgehog is misexpressed in the pancreatic endoderm) developed abnormal tubular structures, a phenocopy of human pancreatic intraepithelial neoplasia-1 and -2. Moreover, these pancreatic intraepithelial neoplasia-like lesions also contained mutations in Kras (190070) and overexpressed Erbb2 (164870), which are genetic mutations found early in the progression of human pancreatic cancer. Furthermore, hedgehog signaling remained active in cell lines established from primary and metastatic pancreatic adenocarcinomas. Notably, inhibition of hedgehog signaling by cyclopamine induced apoptosis and blocked proliferation in a subset of the pancreatic cancer cell lines both in vitro and in vivo. Thayer et al. (2003) concluded that their data suggested that the hedgehog pathway may have an early and critical role in the genesis of pancreatic cancer, and that maintenance of hedgehog signaling is important for aberrant proliferation and tumorigenesis.
Gofflot et al. (2003) developed an in vivo rat model of cholesterol deficiency. Treatment with triparanol, a distal inhibitor of cholesterol biosynthesis, induced patterning defects of the autopod at high frequency, including preaxial syndactyly and postaxial polydactyly, thus reproducing limb anomalies frequently observed in humans. In situ hybridization showed that these malformations originated from a modification of SHH signaling in the limb bud at 13 days postcoitum, leading to a deficiency of the anterior part of the limb. This deficiency resulted in an imbalance of IHH expression in the forming cartilage, ultimately leading to reduced interdigital apoptosis and syndactyly.
By using an inversion of and a large deficiency in the mouse HoxD cluster, Zakany et al. (2004) found that a perturbation in the early collinear expression of Hoxd11 (142986), Hoxd12 (142988), and Hoxd13 (142989) in limb buds led to a loss of asymmetry. Ectopic Hox gene expression triggered abnormal Shh transcription, which in turn induced symmetrical expression of Hox genes in digits, thereby generating double posterior limbs. Zakany et al. (2004) concluded that early posterior restriction of Hox gene products sets up an anterior-posterior prepattern, which determines the localized activation of Shh. This signal is subsequently translated into digit morphologic asymmetry by promoting the late expression of Hoxd genes, 2 collinear processes relying on opposite genomic topographies, upstream and downstream Shh signaling.
In Fgf10 (602115) -/-, Fgfr2b (see FGFR2, 176943) -/-, and Shh -/- mice, which all exhibit cleft palate, Rice et al. (2004) showed that Shh is a downstream target of Fgf10/Fgfr2b signaling. Using BrdU staining, they demonstrated that mesenchymal Fgf10 regulated the epithelial expression of Shh, which in turn signaled back to the mesenchyme. This was confirmed by the finding that cell proliferation was decreased not only in the palatal epithelium but also in the mesenchyme of Fgfr2b -/- mice. Rice et al. (2004) concluded that coordinated epithelial-mesenchymal interactions are essential during the initial stages of palate development and require an FGF-SHH signaling network.
Wilbanks et al. (2004) showed that the functional knockdown of beta-arrestin-2 (107941) in zebrafish embryos recapitulates the many phenotypes of hedgehog pathway mutants. Expression of wildtype beta-arrestin-2, or constitutive activation of the hedgehog pathway downstream of Smoothened (SMO; 601500), rescues the phenotypes caused by beta-arrestin-2 deficiency. These results suggested to Wilbanks et al. (2004) that a functional interaction between beta-arrestin-2 and Smo may be critical to regulate hedgehog signaling in zebrafish development.
Yamamoto et al. (2004) used the teleost Astyanax mexicanus, a single species with an eyed surface-dwelling form (surface fish) and many blind cave forms (cavefish), to study the evolution of eye degeneration. Small eye primordia are formed during cavefish embryogenesis, which later arrest in development, degenerate, and sink into the orbits. Eye degeneration is caused by apoptosis of the embryonic lens, and transplanting a surface fish embryonic lens into a cavefish optic cup can restore a complete eye. Yamamoto et al. (2004) showed that Sonic hedgehog and tiggy-winkle hedgehog (Twhh) gene expression is expanded along the anterior embryonic midline in several different cavefish populations. The expansion of hedgehog signaling results in hyperactivation of downstream genes, lens apoptosis, and arrested eye growth and development. These features can be mimicked in surface fish by Twhh and/or Shh overexpression, supporting the role of hedgehog signaling in the evolution of cavefish eye regression.
Niedermaier et al. (2005) mapped the radiation-induced short digits (Dsh) mouse phenotype to a region of chromosome 5 that contains Shh. Using a positional cloning approach, they demonstrated an 11.7-Mb inversion with a distal breakpoint 13.298 kb upstream of Shh, separating the coding sequence from several putative regulatory elements. The inversion results in temporal and spatial dysregulation of Shh expression with almost complete downregulation during embryonic day 9.5 (E9.5) to E12.5 and upregulation at E13.5 and E14.5, the latter occurring in the phalangeal anlagen of Dsh +/- mice, at a time point and in a region where wildtype Shh is never expressed. Niedermaier et al. (2005) concluded that the formation of phalangeal elements and joints mutually depend on each other and that Dsh is a model for abnormal joint formation.
Roper et al. (2006) found that a deficit in cerebellar granule cell neurons in a mouse model of Down syndrome (190685) was associated with reduced mitogenic response of granule cell precursors to hedgehog protein signaling in early postnatal development. Systemic treatment of newborn trisomic mice with a small molecule agonist of the hedgehog signaling pathway increased mitosis and restored the granule cell precursor population in vivo.
Yamagishi et al. (2006) found that Shh-null mice had hypoplasia and midline fusion of the first pharyngeal arch due to defective epithelial-mesenchymal signaling.
Huang et al. (2007) stated that a mutant mouse Shh allele encoding an Shh protein that could not be modified by cholesterol (ShhN) elicited ectopic Shh signaling in limb bud. They found that mice with 1 ShhN allele (ShhN/+ mice) exhibited a spectrum of features, including partial division of cerebral hemispheres, hydrocephalus, and cleft palate, with apparently normal external craniofacial features. These features were similar to those of a human patient with milder HPE due to an N-terminal truncation mutation in 1 SHH allele (Nanni et al., 1999). Persistent ectopic ShhN signaling in the dorsal telencephalon of ShhN/+ mice altered Bmp and Wnt signaling from dorsal patterning centers, resulting in altered behavior of roof plate cells and impaired roof plate invagination. Huang et al. (2007) proposed that elevated ectopic Shh signaling can impair dorsal telencephalic midline morphogenesis.
By removing a conditional Shh allele at defined times during mouse limb development, Zhu et al. (2008) determined the temporal dependence of digit specification on Shh. Shh was required only very early and transiently for digit patterning, but it was required continuously to ensure sufficient cell numbers to produce the normal complement of digits. Zhu et al. (2008) concluded that Shh plays dual and distinct roles in regulating digit identity and number.
Limb bud outgrowth is driven by signals in a positive feedback loop involving fibroblast growth factor (Fgf) genes, Sonic hedgehog, and gremlin-1 (GREM1; 603054). Precise termination of these signals is essential to restrict limb bud size. That the sequence in mouse limb buds is different from that in chick limb buds drove Verheyden and Sun (2008) to explore alternative mechanisms. By analyzing compound mouse mutants defective in genes comprising the positive loop, Verheyden and Sun (2008) provided genetic evidence that Fgf signaling can repress Grem1 expression, revealing a novel Fgf/Grem1 inhibitory loop. The repression occurs in both mouse and chick limb buds and is dependent on high Fgf activity. These data supported a mechanism where the positive Fgf/Shh loop drives outgrowth and an increase in Fgf signaling, which triggers the Fgf/Grem1 inhibitory loop. The inhibitory loop then operates to terminate outgrowth signals in the order observed in either mouse or chick limb buds. Verheyden and Sun (2008) concluded that their study unveils the concept of a self-promoting and self-terminating circuit that may be used to attain proper tissue size in a broad spectrum of developmental and regenerative settings. Verheyden and Sun (2008) demonstrated that Fgf8 (600483) repression of Fgf4 (164980) expression is dependent on Grem1 but not Sonic hedgehog.
Rink et al. (2009) characterized the hedgehog pathway in planarians. Hedgehog signaling is essential for establishing the anterior/posterior axis during regeneration by modulating wnt expression. Moreover, RNA interference methods to reduce signal transduction proteins Cos2/Kif27/Kif7, Fused, or Iguana do not result in detectable hedgehog signaling defects; however, these proteins are essential for planarian ciliogenesis. Rink et al. (2009) concluded that their study expanded the understanding of hedgehog signaling in the animal kingdom and suggests an ancestral mechanistic link between hedgehog signaling and the function of cilia.
Zhao et al. (2009) analyzed a mouse preaxial polydactyly model with a T-to-A point mutation in a conserved locus about 1 Mb upstream of the Shh coding region. A core element of mutation (CEM) with putative enhancer activity was identified by promoter activity assay and shown to contain a matrix attachment region. HnRNPU (602869) preferentially bound to the mutant but not the wildtype CEM. HnRNPU also bound to the 5-prime UTR of the Shh gene, which was not located in the nuclear matrix in wildtype embryonic cells. The authors proposed that the 5-prime UTR of Shh was pulled into the nuclear matrix by HnRNPU when the CEM was mutated, and consequently affected Shh expression. Therefore, distant cis-elements may modulate gene expression by altering the affinity of HNRNPU for certain mediator proteins and nuclear relocation.
Ezratty et al. (2011) measured Shh signaling following disruption of ciliogenesis in embryonic mouse keratinocytes in utero and in culture. They found that Shh was not required for epidermal ciliogenesis, Notch signaling, or differentiation, but was required later for development of hair follicles.
In zebrafish, Wang et al. (2015) found that genetic depletion of the epicardium after myocardial loss inhibits cardiomyocyte proliferation and delays muscle regeneration. The epicardium vigorously regenerates after its ablation, through proliferation and migration of spared epicardial cells as a sheet to cover the exposed ventricular surface in a wave from the chamber base toward its apex. By reconstituting epicardial regeneration ex vivo, Wang et al. (2015) showed that extirpation of the bulbous arteriosus, a distinct, smooth muscle-rich tissue structure that distributes outflow from the ventricle, prevents epicardial regeneration. Conversely, experimental repositioning of the bulbous arteriosus by tissue recombination initiated epicardial regeneration and could govern its direction. Hedgehog (Hh) ligand is expressed in the bulbous arteriosus, and treatment with a Hh signaling antagonist arrested epicardial regeneration and blunted the epicardial response to muscle injury. Transplantation of Sonic hedgehog (Shh)-soaked beads at the ventricular base stimulated epicardial regeneration after bulbous arteriosus removal, indicating that Hh signaling can substitute for the influence of the outflow tract. Thus, Wang et al. (2015) concluded that the ventricular epicardium has pronounced regenerative capacity, regulated by the neighboring cardiac outflow tract and Hh signaling.
Cohen (2006) pointed out the difficulty in using in the clinic the whimsical names that have been given to the homologous genes in Drosophila or mice, such as 'Sonic hedgehog' for the gene involved in one form of holoprosencephaly. The 'lunatic fringe' gene (LFNG; 602576), which is mutant in spondylocostal dysostosis (609813), is another case in point. Cohen (2006) suggested that if it is necessary to refer to the gene in which the mutation has been found in affected families, perhaps the gene symbol can be used.
Roessler et al. (1996) identified a GGG-to-AGG transition resulting in a gly31-to-arg (G31R) substitution of the SHH gene in a family with HPE3 (142945). This exon 1 residue is conserved in hedgehog proteins and is adjacent to the putative signal cleavage site.
Maity et al. (2005) found that the G31R mutation in mouse Shh introduced a cleavage site for a furin-like protease, resulting in abnormal protein processing. Cleavage at this site removed 11 amino acids from the N-terminal domain and reduced affinity of Shh for Ptc (PTCH1; 601309) and Shh signaling potency in assays using chicken embryo neural plate explants and mouse C3H10T1/2 stem cells.
Roessler et al. (1996) identified a CAG-to-TAG transition resulting in a gln100-to-ter nonsense mutation of the SHH gene in a family with HPE3 (142945).
Roessler et al. (1996) identified a AAG-to-TAG transversion resulting in a lys105-to-ter nonsense mutation of the SHH gene in a large multigenerational family with HPE3 (142945).
Roessler et al. (1996) identified a TGG-to-GGG transversion resulting in a trp117-to-gly (W117G) substitution of the SHH gene in a family with HPE3 (142945). The W117 residue is invariant in all hedgehog protein sequences and occurs immediately following the first alpha-helix of the murine Shh N fragment.
Maity et al. (2005) found that the W117G mutation in mouse Shh caused failure of Shh processing, leading to retention of the immature glycosylated protein within the ER of transfected cells. In vitro binding assays using recombinant proteins showed that the mutation caused a temperature-dependent conformational change that allowed Shh to bind Ptc (PTCH1; 601309) at 4 or 32 degrees C, but not at 37 degrees C. The W117G mutation drastically reduced signaling potency in chicken embryo neural plate explant assays.
Roessler et al. (1996) identified a TGG-to-CGG transversion resulting in a trp117-to-arg (W117R) substitution in the SHH gene in a family with HPE3 (142945). The W117 residue is invariant in all of the hedgehog protein sequences and occurs immediately following the first alpha-helix of the murine Shh N fragment.
Maity et al. (2005) found that the W117R mutation in mouse Shh caused failure of Shh processing, leading to retention of the immature glycosylated protein within the ER of transfected cells. In vitro binding assays using recombinant proteins showed that the mutation caused a temperature-dependent conformational change that allowed Shh to bind Ptc (PTCH1; 601309) at 4 or 32 degrees C, but not at 37 degrees C. The W117R mutation drastically reduced signaling potency in chicken embryo neural plate explant assays.
In a large multigeneration family with autosomal dominant holoprosencephaly (HPE3; 142945), Roessler et al. (1997) identified a T-to-A transversion in the SHH gene resulting in a val-to-glu substitution at codon 224 (V224E). The mutation was present in all affected individuals but in none of the unaffected family members. The mutation created a novel AluI restriction site, and the site of the mutation is 28 amino acids from the cleavage site between SHH-N and SHH-C.
In a family with autosomal dominant holoprosencephaly (HPE3; 142945), Roessler et al. (1997) identified a G-to-A transition in the SHH gene resulting in an ala-to-thr substitution at codon 226 (A226T), an invariant amino acid in all of the vertebrate hedgehog proteins. This mutation occurred 2 codons 3-prime of the val224-to-glu mutation (600725.0006), implicating this region of the protein in processing. This mutation resulted in the loss of a FnuIVH restriction site. The clinically unaffected father was also a mutation carrier.
In a family segregating autosomal dominant holoprosencephaly (HPE3; 142945), Roessler et al. (1997) found that 21 basepairs of the SHH gene were deleted, resulting in deletion of 7 amino acids (RLLLTAA) between and including codons 263 and 269. These 7 amino acids immediately precede a key histidine residue that is thought to be involved in the processing of Drosophila hedgehog protein.
In a family with autosomal dominant holoprosencephaly (HPE3; 142945), Roessler et al. (1997) identified a G-to-T transversion in the SHH gene, resulting in a glu-to-ter change at codon 284 (E284X).
In a child with holoprosencephaly (HPE3; 142945), Roessler et al. (1997) identified a G-to-A transition in the SHH gene that resulted in an ala-to-thr substitution at codon 384 (A384T). In a screen of 184 sporadic HPE cases, this was the only sequence variation found that predicted a change in the primary coding sequence of the SHH protein. The parents of this child were unavailable for analysis.
In a 20-year-old woman with holoprosencephaly (HPE3; 142945), Nanni et al. (1999) identified a G-to-A transition at codon 290 in exon 3 of the SHH gene, resulting in a gly290-to-asp (G290D) substitution. The patient also had a mutation predicting an expansion of an ala repeat in exon 2 of the ZIC2 gene (603073), the site of mutations causing holoprosencephaly-5. DNA samples from her parents were not available for genetic analysis. The mutation was not found in over 200 control chromosomes from Caucasian individuals or in over 200 control chromosomes from Hispanic individuals.
Schell-Apacik et al. (2009) identified a heterozygous G290D mutation in a boy with schizencephaly (269160) and developmental delay. Brain MRI at age 5 months showed a complex brain malformation with partial absence of the corpus callosum, bilateral parietotemporal closed-lip schizencephaly, polymicrogyria, and optic atrophy. Dysmorphic features included microbrachycephaly, hypotelorism, broad nasal root, short philtrum, and a thin upper lip. The patient's unaffected mother was also heterozygous for the mutation. The findings expanded the phenotypic spectrum resulting from SHH mutations.
In an Asian Indian girl with isolated left microphthalmia and her unaffected father, Bakrania et al. (2010) identified heterozygosity for the G290D variant in the SHH gene. The authors did not detect the mutation in 182 controls, but noted that Garcia-Barcelo et al. (2008) found the G290D variant at an approximately 4% frequency in 88 Chinese patients with anorectal malformations and in 96 controls; thus Bakrania et al. (2010) suggested that the variant might represent a rare polymorphism rather than a true disease-causing mutation.
In a child with holoprosencephaly (HPE3; 142945) and in her clinically unaffected mother, Nanni et al. (1999) identified a C-to-G transversion at codon 424 of the SHH gene, resulting in a pro-to-ala substitution. The affected child also showed loss of 18pter and TGIF (602630), the site of mutations causing holoprosencephaly-4 (HPE4; 142946), that was derived from a maternal balanced translocation involving 18p. The mutation was not found in over 200 control chromosomes from Caucasian individuals or in over 200 control chromosomes from Hispanic individuals.
In a child with holoprosencephaly (HPE3; 142945) and in her clinically unaffected mother, Nanni et al. (1999) identified a 9-bp deletion encompassing nucleotides 1283-1291 of the SHH gene, resulting in the loss of 3 amino acids (ala378, pro379, and phe380). The patient also carried a thr151-to-ala mutation in her TGIF gene (602630.0003), the site of mutations causing holoprosencephaly-4 (HPE4; 142946). The mutation was not found in over 200 control chromosomes from Caucasian individuals or in over 200 control chromosomes from Hispanic individuals.
In 8 individuals in 3 generations of a family with SMMCI (147250), Nanni et al. (2001) identified an ile111-to-phe (I111F) mutation in the SHH gene. The dental anomaly was present in a girl, her mother, and her mother's sister. The affected daughter also had choanal stenosis. Garavelli et al. (2004) stated that 2 members of this family who carried the mutation had an entirely normal phenotype.
Heussler et al. (2002) reported a large family ascertained for HPE (142945) in which the proband presented in utero with alobar holoprosencephaly and at post mortem was noted to have microcephaly, hypotelorism, and premaxillary agenesis. Analysis of the SHH gene in fetal DNA demonstrated a novel missense mutation which resulted from an A-to-T transition at nucleotide position 263. This was predicted to result in an asp88-to-val (D88V) amino acid substitution. This residue is located in the N-terminal signaling domain at an invariant position in the hedgehog family of proteins and is conserved in human, mouse, chicken, and zebrafish SHH and in Drosophila hedgehog. Although the functional effects of the V88D change were not studied, the authors suggested that this mutation probably caused an alteration in the biologic activity of SHH. The mutation was identified in other family members, and was associated with striking phenotypic variation. Two mutation carriers had attention difficulties, with one of them clinically diagnosed with attention deficit-hyperactivity disorder (143465). The authors suggested that the combination of microcephaly, hypotelorism, subtle midline facial anomalies, and attention deficit-hyperactivity disorder within a sibship should alert the physician to the possible diagnosis of HPE.
Maity et al. (2005) found that the D88V mutation in mouse Shh moderately reduced Ptc (PTCH1; 601309) binding in vitro and signaling potency in chicken embryo neural plate explant assays compared with wildtype Shh.
Schimmenti et al. (2003) described an 8-month-old boy with bilateral microphthalmia and iris, uveal, and retinal colobomas (MCOPCB5; 611638) whose development was normal. On subsequent examinations, the mother was found to have a history of refractive corrections since late childhood and an incomplete uveoretinal coloboma of the right eye. Both she and her son were heterozygous for a 24-bp deletion in the 3-prime end of the SHH gene coding region, which was predicted to lead to deletion of 8 amino acids near the C terminus of the autocatalytic region of SHH. Three unaffected family members carried the same deletion; Schimmenti et al. (2003) noted that incomplete expression of SHH mutations had also been observed in several holoprosencephaly pedigrees (see Nanni et al., 1999).
In a male patient with right microphthalmia and microcornea, iris coloboma, and small optic nerve, who also had plagiocephaly and developed type 1 diabetes at 6 months of age, Bakrania et al. (2010) identified a 24-bp deletion in the SHH gene that was identical at the protein level to that previously detected by Schimmenti et al. (2003). Bakrania et al. (2010) noted that the deletion was also present in their patient's unaffected brother, mother, and maternal grandfather.
In a female fetus with holoprosencephaly (142945) and her brother, who was less severely affected, Nanni et al. (1999) identified a G-to-T transversion in the SHH gene, resulting in a glu256-to-ter (E256X) mutation. Neither parent carried the mutation.
In a patient with solitary median maxillary central incisor (147250), Garavelli et al. (2004) identified a de novo val332-to-ala (V332A) mutation in the SHH gene. The mutation was absent in the parents. The patient had microcephaly, a flat face, hypotelorism, horizontal palpebral fissures, flattened nose, low nasal bridge, recessed premaxillary region, abnormal and hypoplastic columella, and a long philtrum.
Marini et al. (2003) identified a 383G-A transition in the SHH gene, resulting in a trp128-to-ter nonsense mutation, in a family in which the mother had a single central maxillary incisor (147250) and a daughter and 2 male fetuses had HPE3 (142945).
In a child with HPE3 (142945), Nanni et al. (1999) identified a heterozygous C-to-A transversion in the SHH gene, resulting in an asn115-to-lys (N115K) substitution in an invariant position in the hedgehog proteins in the N-terminal domain. The patient's unaffected mother also carried the N115K mutation. The mutation was not found in over 200 control chromosomes from Caucasian individuals or in over 200 control chromosomes from Hispanic individuals.
Martinelli and Fan (2009) demonstrated that a constructed mouse Shh N116K mutant, which corresponds to the HPE3-associated N115K mutation, caused markedly decreased binding to Gas1, resulting in decreased Shh signaling. These findings indicated that HPE due to the N115K mutation results from an inability of mutant SHH to bind to GAS1 normally, thus interrupting the positive effect of GAS1.
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