Abstract

Detailed knowledge of the Hedgehog signaling pathway is fundamental to an understanding of vertebrate development as well as several birth defects in humans. Here we review various aspects of Hedgehog synthesis, secretion, distribution and function in the context of the most common anomaly of the developing forebrain in humans, holoprosencephaly. Genetic studies in numerous model organisms are beginning to elucidate the factors that are likely candidates for the causes of early embryonic defects in humans, including holoprosencephaly.

INTRODUCTION

Each member of the Hedgehog (Hh) family of secreted proteins possesses remarkable morphogenic patterning activity (1). All of them can act as powerful mitogens, survival factors and inducers of distinct cell types in a dose-dependent manner (27) (Fig. 1A). In vertebrates, there are three Hedgehog factors (Sonic, Indian and Desert) of which Sonic Hedgehog (Shh) is the best characterized. All three factors are thought to activate a single signaling pathway. Shh is involved in numerous key developmental events at multiple times during embryogenesis. Shh participates in such diverse developmental steps as the establishment of the left–right axis (810) (which is essential for determining asymmetric organ positioning), beginning with the formation of the axial midline mesendoderm upon whose axis left and right are defined (1112). From this midline expression follows a crucial role in the specification of the floor plate in the spinal chord, as well as ventral identity of the brain along the entire rostral–caudal extent of the central nervous system (13,14). Later, during embryogenesis, Shh has a crucial role in defining the anterior–posterior axis of the limb (1517), and participating in the development of the pituitary gland (1822), neural crest cells (23), midbrain (24), cerebellum (2527), oligodendrocytes (28), eye (2933) and face (3438). We will not attempt to summarize all of these processes. Instead, our goal is to review the current understanding of the Shh signaling pathway in the context of a single disorder (39,40), holoprosencephaly (HPE).

HPE is the most common congenital anomaly of the brain during embryogenesis; however, only a fraction of affected embryos survive to term (39). Its pathogenesis can be best understood as a failure of the primordial single eye field and forebrain to divide into paired left and right eyes, or into separate cerebral hemispheres. Shh emanating from the most rostral extent of the axial mesendoderm [called the prechordal plate (PP); Fig. 1B] is thought to function as one of the midline signal(s) that defines a plane of division for cleavage. Precisely how this occurs is the subject of intense investigation.

THE SHH SIGNALING PATHWAY IN HPE

As medical geneticists, one of our strategies to better understand the tremendous genetic heterogeneity of HPE has been to systematically analyze the components of the Shh pathway through mutational analysis of HPE patients. In recent years, there has been an expanding list of potential HPE candidate genes derived from the Hh pathway. As shown in Table 1, it is very useful to classify components of the Hh pathway into factors that, when inactive (the most common effect of gene mutation or loss), would be predicted to lead to a decrease or increase in effective Shh target gene activation. Since several factors are known to exist in both loss or gain of function mutant forms, for clarity and consistency we will attempt to always describe the hypothetical effect that the loss of a gene would have on the expression of Hh target genes. Most of the known factors in the Hh pathway are best understood by their genetic and epistatic relationships worked out in the fruitfly, Drosophila, while a minority are seen only in vertebrates. This listing of candidate HPE genes is useful despite the fact that there remain substantial gaps in our understanding of Hh signal transduction and its regulation.

HEDGEHOG AND HUMAN DISEASE

HPE is by no means the only human disorder associated with changes in Shh signaling. As described in Figure 2, the majority of disorders of Hh signaling involve some form of ectopic or excessive activation of Hh target genes, such as occurs in gliomas, medulloblastomas or basal cell carcinoma (90). To a very large degree, the extensive characterization of mouse models has laid a solid foundation for a better understanding of these human diseases (13,49,50,55,60,66,8089,96). We are now beginning to appreciate how extensive the cell survival and proliferative effects of Hh function seen in cancer syndromesare. While these roles are essential during the explosive growth of the telencephalon during development of the brain (100,101), in the adult organism they must be strictly contained. When we consider the pathogenesis of HPE [and possibly VACTERL (102) syndrome] we are dealing with genetic lesions or teratogenic effects that are on the verge of lethality. As shown in the bottom right of Figure 2, there are at least five murine genotypes, which completely abrogate all Hh signaling, that lead to embryonic arrest at ∼e9.0–10.5 [i.e. Smo−/− (60), Shh/Ihh−/− (60), Sil−/−(96), Disp−/− (49,50), and Gli2/Gli3−/− (84)]. None of these genotypes are associated with any progression to a division of the brain, although the lethality may reflect failure to complete heart looping (60). Shh−/− mice die perinatally with cyclopia, absent floorplate or ventral brain types, limb reductions and laterality anomalies (13); apparently, Ihh is able to partially compensate for the absence of Shh function at earlier stages (60). One of the most useful advances over the past few years has been the appreciation that Hh target genes are effectively silenced by Hh-regulated repressor forms, such as vertebrate Gli3-R (76). Therefore, one of the most important actions of Hh is to relieve this repression. For example, much of the growth retardation (including brain size) and failure of ventral brain patterning is ameliorated by the concurrent loss of both the signaling factor, Shh, and the repressor, Gli3-R (42,89). This genotype prevents constitutive Shh target gene silencing. The concurrent reduction of Shh activity prevents the phenotypes associated with excessive Hh signaling and achieves a new balance between activators and repressors. Whether or not all of these activators are currently recognized mediators of Hh function, such as the ci/Gli/Zic family (106111), or parallel pathways is presently unknown.

COMPONENTS OF THE Hh PATHWAY ARE CANDIDATES FOR HPE

As Table 1 outlines, there is a growing list of factors that are necessary to produce a functional Hh protein, to distribute it within a tissue field, and for target cells to be able to bind and respond to graded Hh activity through the activation of target genes. Theoretically, any factor that interferes with the production of Hh protein could lead to cyclopia. Hh proteins are synthesized as a 45 kDa pre-protein that undergoes self-catalyzed cleavage to a 19 kDa amino terminal fragment, Shh-N, which is further modified at its carboxy terminus by the addition of cholesterol, Shh-Np (112). Recently, it has been demonstrated that the potency and diffusion of Hh is also influenced by a second lipid modification of the amino terminus of Shh-Np with palmitate, or related lipids (113117). In addition to these unusual lipid modifications, a new factor called Dispatched [structurally related to the Hh receptor Patched (55), SREBP (118,119) and NPC1 (120,121)] has been shown to be required for the release of Hh from the producing cell (Fig. 3A). Interestingly, DISP resides in a chromosomal position (HPE10) associated with HPE (49). Perhaps as a consequence of its lipid modifications, multimeric forms of Hh have been described that are extremely potent (122) (Figs 3A and 4A). Furthermore, the synthesis of specific heparin sulfate proteoglycans, mediated by the exostoses genes (EXTI-3), can influence the distribution of Hh activity through the target field, as does the tout veloux (Ttv) gene product (5154) in Drosophila. The Hh receptor, Patched (Ptc), binds the Hh signaling factor and targets it for intracellular degradation (123,124). Thus the putative HPE candidate genes in the secreting cells include those that add palmitate to SHH-Np, those that fulfill DISP function in secretion, or affect the transfer of SHH between cells.

As described in Figure 3B, loss-of-function and gain-of-function studies indicate that Smoothened (Smo) is necessary and sufficient to transduce the Hh signal (60,64). Genetic studies indicate that Ptc acts negatively to prevent Smo from signaling, although the biochemical mechanism is poorly understood. Cells exposed to Hh activity accumulate hyper-phosphorylated Smo protein in their plasma membranes (125), while most Ptch protein is detectable with Hh in intracellular vesicles. The functional significance of this Smo modification is presently unknown. Genetic and biochemical studies of Ptch indicate that the ability to inhibit Smo is separate and distinct from the binding of Hh (126). Ptch inhibition of Smo is catalytic (127) (as opposed to stoichiometric), involves vesicles (128131,148), and may be mediated by the transport of small molecules (132).

Through a series of steps that are currently poorly understood, Smo activity leads to a shift from a constitutive ci/Gli repressor form to an uncleaved activator form. Those cells in the target field that have not seen Hh activity actively recruit histone deacetylase to ci/Gli binding sites and effectively shut off the transcription of Hh target genes (73). Most of the ci/Gli transcription factor protein is sequestered in a large multiprotein complex in the cytoplasm associated with microtubules (i.e. complexes consisting of costal2, fused, suppressor of fused, ci/Gli). A series of protein kinases (PKA, GSK, CKI) facilitate the cleavage of ci/Gli by proteosomes (utilizing slimb or related activity) to generate the p75 kDa repressor form (ci-R/Gli-R). In the nucleus, Su(Fu) serves as a molecular bridge between the DNA binding activity of ci/Gli and a multicomponent repressor complex (73). This repression continues throughout the life of the organism when Hh activity is no longer needed. Indeed, loss of SU(FU) function is now a recognized cause of medulloblastoma (71). Note that most of these factors (see Fig. 3B and Table 1) are important to constrain Hh signaling and therefore are not likely to be associated with HPE. Only the putative SMOH kinase and FUSED are likely candidates. Additional factors that initiate the signal downstream of SMO might also represent potential HPE candidate genes. SMOH itself was a promising candidate, however, no examples of mutations in SMOH have been described (133). Nor have we been able, thus far, to identify mutations in SIL (95) (which is genetically downstream of SMOH). In both cases, we suspect that these embryos lacked sufficient Hh function and thus do not survive gestation. Alternatively, heterozygous changes might simply be insufficient to perturb development.

HPE AND CHOLESTEROL

Cholesterol synthesis inhibitors are known teratogenic agents that can cause cylopia in animals (134,135). Furthermore, HPE can be seen in the Smith–Lemli–Opitz syndrome (SLO) (39) in humans caused by a defect in the pent-ultimate step in cholesterol synthesis mediated by the SSD-containing enzyme 7-dehydrocholesterol reductase (7-DHCR). Several inhibitors of this enzyme can cause cyclopia/HPE in animal models. Since Shh is modified by cholesterol, one of the first theories postulated that cholesterol depletion influences Hh processing; however, at least two groups have shown that this is incorrect (136,137). A subclass of cholesterol synthesis inhibitors (cyclopamine and jervine) appears to act on Hh-responding cells to inhibit the signaling of Smo in a Ptch-independent manner (138,139). Responding cells are exquisitely sensitive to these compounds, and these small molecules may be regulating Smo activity directly (132). Such modulation of Hh signaling might lead to therapeutic treatments for cancer. However, it is less likely that Hh agonists could be introduced in a spatial–temporal manner to counteract the defects of cyclopia/HPE.

Recent genetic studies have identified the Opb/Rab23 gene product as an essential factor in the inhibition of Hh signaling (66,67). The Rab family consist of an extremely large (at least 63 members) class of small GTP-ases that participate in the mobility and targeting of vesicles between intracellular compartments along the exocytic and endocytic pathways (140143). Mice lacking Opb/Rab23 are over-ventralized, suggesting hyperactivity of the Hh pathway. How could vesicle transport be important for Hh signaling?

One possible explanation for the role of cholesterol in Hh signaling is the recently described association of Ptch with intracellular cholesterol-rich vesicles containing caveolin (144146). The caveolin family of proteins are transcriptionally regulated by cholesterol and are associated with cholesterol-loaded vesicles (Fig. 4B). At the plasma membrane, flask-shaped membrane invaginations, called caveolae, form a unique membrane fluidity environment based on a distinct composition of sphingomyelin and cholesterol-rich components. Importantly, this microenvironment leads to the accumulation of subclasses of signaling molecules whose function can be modulated by this microenvironment (147,148). Similarly, membrane rafts contain a cholesterol-rich composition that also accumulates specific membranes proteins, especially signaling proteins.

We suggest that cholesterol synthesis inhibition leads to an indirect effect on the movement of Smo between intracellular compartments and the plasma membrane (Fig. 4B). This might be mediated by the SSD domain of Ptch, or an independent effect on the vesicles themselves, or of their cholesterol cargo. Hh leads to a stabilization and accumulation of Smo in the plasma membrane. A recent study suggests that Smo becomes segregated from the negative influence of Ptch in late endosomes (149) and that this sorting is the basis of Hh pathway activation (Fig. 4B). This model incorporates the similarity between the Ptch protein and NPC1, which also functions in late endosomes, and whose vesicle trafficking is controlled by Rab proteins. Genetic factors required for this sorting of Smo might also become candidate HPE genes. Finally, it would be interesting to know the effect of cholesterol depletion on the distribution of DISP and SHH (see hypothetical model outlined in Fig. 4A).

FUTURE DIRECTIONS

HPE is a subclass of diseases of the Hh signaling pathway associated with diminished activity. Multiple HPE candidate genes suggest themselves for mutational analysis based on our evolving understanding of the complexity and function of these components. The complexity of the Hh pathway mirrors the equally heterogeneous causation of HPE. Additional progress is anticipated through a continuing analysis of the Hh signaling pathway for mutations in HPE patients. There remain many gaps in our understanding of the Hh pathway and we can anticipate that it will remain a further source of mechanistic surprises.

A final challenge will be to begin to understand how various teratogens or HPE genes interact in the pathogenesis of HPE. The Hh pathway is merely the best understood pathogenetic mechanism that leads to HPE. Doubtless, there are parallel pathways that are currently unknown, and only a minority of patients have an identifiable genetic predisposition. What are the targets of Hh signaling? How do they execute their developmental program? If Hh specifies the midline of the brain, how is the cleavage of the eye field and brain performed? What genes are involved? Clearly, there are many gaps in our knowledge of HPE that remain to be identified and solved.

ACKNOWLEDGEMENTS

We are grateful to all of the families who have participated in our genetic studies of HPE, and the Don and Linda Carter Centers for Holoprosencephaly and related malformations. Research support for our HPE studies is from the Division of Intramural Research of the National Human Genome Research Institute, NIH.

*

To whom correspondence should be addressed. Tel: +1 3014028167; Fax: +1 3014807876; Email: muenke@nih.gov

Figure 1. (A) A transverse section of the spinal chord identifies two principal signaling centers: (in red) the notochord and floorplate are the source of Shh secretion that induces ventral patterning and form a gradient of activity (26) (red arrow; highest ventrally and decreasing towards the top); a similar gradient of BMP activity (gray arrow; highest in the roof plate) extends from the dorsal roof plate (41). Ventral motoneurons (MN) and interneurons (V0–V3) are specified by the combined actions of both gradients. The dorsal neural tube expresses the Gli3-repressor (Gli3-R), while the notochord is the source of BMP inhibitors (BMP-R). (B) A similar transverse section taken at the level of the presumptive forebrain reveals the proximity of the prechordal plate (PP) and the single eye field in the anterior neuroepithelium (adapted from 38). Both the PP and primitive foregut express Shh (red), although during pituitary development (18), Shh becomes excluded from Rathke's pouch (RP). We propose that in the immediate vicinity of Shh activity in the PP, GLI2 and GLI3 function as activators. ZIC2 (which is also associated with HPE) (see 39) may function as a co-factor with other GLI activators at more lateral positions.

Figure 1. (A) A transverse section of the spinal chord identifies two principal signaling centers: (in red) the notochord and floorplate are the source of Shh secretion that induces ventral patterning and form a gradient of activity (26) (red arrow; highest ventrally and decreasing towards the top); a similar gradient of BMP activity (gray arrow; highest in the roof plate) extends from the dorsal roof plate (41). Ventral motoneurons (MN) and interneurons (V0–V3) are specified by the combined actions of both gradients. The dorsal neural tube expresses the Gli3-repressor (Gli3-R), while the notochord is the source of BMP inhibitors (BMP-R). (B) A similar transverse section taken at the level of the presumptive forebrain reveals the proximity of the prechordal plate (PP) and the single eye field in the anterior neuroepithelium (adapted from 38). Both the PP and primitive foregut express Shh (red), although during pituitary development (18), Shh becomes excluded from Rathke's pouch (RP). We propose that in the immediate vicinity of Shh activity in the PP, GLI2 and GLI3 function as activators. ZIC2 (which is also associated with HPE) (see 39) may function as a co-factor with other GLI activators at more lateral positions.

Figure 2. Genotypes of humans and mice are predicted to directly affect the activity of Hh-regulated genes. These genotypes can be ranked from those associated with the least expression (bottom) to the most active expression (red arrowhead). Most human disorders such as cancer, Gorlin, Greig or Pallister–Hall syndrome (99,103) are associated with above normal SHH activity owing to inactivation of the tumor suppressor, PTCH, or the repressor and/or activator functions of GLI3 (104,105). In contrast, HPE (39) (and possibly VACTERL, see 102) are often lethal malformations associated with a substantial decrease in target gene expression. An extensive array of murine models has been generated by several laboratories whose analysis has allowed direct verification of these changes in gene expression. At least five genotypes are lethal, probably due to failure of cardiac development (60). Note the shift toward more normal brain development when both Shh−/− (or its transducer smo−/−) is genetically combined with a simultaneous inactivation of the Gli3 (repressor≫activator) function (42), or Shh−/− in combination with the obligatory repressor Opb/Rab23 (66, see Table 1). Loss of Gli1 leads to essentially normal mouse development (86), while inactivation of Gli2 is nearly normal with an absent floorplate and minor ventral anomalies (82,83). We now understand that a combined action of Gli2-A and Gli3-A, and factors acting in parallel, is the principal mediator of Shh function (see Fig. 1B) (42,89).

Figure 2. Genotypes of humans and mice are predicted to directly affect the activity of Hh-regulated genes. These genotypes can be ranked from those associated with the least expression (bottom) to the most active expression (red arrowhead). Most human disorders such as cancer, Gorlin, Greig or Pallister–Hall syndrome (99,103) are associated with above normal SHH activity owing to inactivation of the tumor suppressor, PTCH, or the repressor and/or activator functions of GLI3 (104,105). In contrast, HPE (39) (and possibly VACTERL, see 102) are often lethal malformations associated with a substantial decrease in target gene expression. An extensive array of murine models has been generated by several laboratories whose analysis has allowed direct verification of these changes in gene expression. At least five genotypes are lethal, probably due to failure of cardiac development (60). Note the shift toward more normal brain development when both Shh−/− (or its transducer smo−/−) is genetically combined with a simultaneous inactivation of the Gli3 (repressor≫activator) function (42), or Shh−/− in combination with the obligatory repressor Opb/Rab23 (66, see Table 1). Loss of Gli1 leads to essentially normal mouse development (86), while inactivation of Gli2 is nearly normal with an absent floorplate and minor ventral anomalies (82,83). We now understand that a combined action of Gli2-A and Gli3-A, and factors acting in parallel, is the principal mediator of Shh function (see Fig. 1B) (42,89).

Figure 3. (A) A molecular symmetry is suggested between cells secreting SHH (red oval, top) and the responding cells (bottom) based on the homology between the SHH-interacting, sterol-sensing domain (SSD) proteins DISP (light blue) and PTCH (dark blue). While Ptch, as well as Shh, are known to associate with sphingolipid and cholesterol-rich (orange) microdomains (144), this is not yet demonstrated for DISP. However, examination of its primary sequence suggests that DISP has a putative caveolin binding site within its SSD that may function in influencing the vesicle trafficking of the SHH morphogen. Heparin sulfate proteoglycans are suggested to be required matrix components that act to facilitate the movement of the Shh morphogen between cells. Note that binding of SHH (monomer or multimer) by PTCH leads to rapid internalization which(1) removes SHH from further action(s), and (2) also removes PTCH from any influence on SMOH (turquoise, not shown). (B) Once SMOH is relieved from its inhibition, it accumulates in the cell membrane in a hyper-phosphorylated state. How SMOH is effectively inhibited or its immediate downstream signal is not well known. As shown by the red arrows, SMOH activation leads to the accumulation of full-length activator forms of GLI transcription factors. In the absence of Hh signaling, a series of sequential kinases (PKA, GSK, CK1) promotes the cleavage of GLI factors to a repressor form. SU(FU) is thought to serve as a molecular bridge between GLI-R and a recently described repressor complex which inactivates chromatin domains in the vicinity of GLI transcription factor binding sites (73). In the basal (unstimulated) state, most of the GLI factors are associated with a multicomponent complex associated with cytoplasmic microtubules.

Figure 3. (A) A molecular symmetry is suggested between cells secreting SHH (red oval, top) and the responding cells (bottom) based on the homology between the SHH-interacting, sterol-sensing domain (SSD) proteins DISP (light blue) and PTCH (dark blue). While Ptch, as well as Shh, are known to associate with sphingolipid and cholesterol-rich (orange) microdomains (144), this is not yet demonstrated for DISP. However, examination of its primary sequence suggests that DISP has a putative caveolin binding site within its SSD that may function in influencing the vesicle trafficking of the SHH morphogen. Heparin sulfate proteoglycans are suggested to be required matrix components that act to facilitate the movement of the Shh morphogen between cells. Note that binding of SHH (monomer or multimer) by PTCH leads to rapid internalization which(1) removes SHH from further action(s), and (2) also removes PTCH from any influence on SMOH (turquoise, not shown). (B) Once SMOH is relieved from its inhibition, it accumulates in the cell membrane in a hyper-phosphorylated state. How SMOH is effectively inhibited or its immediate downstream signal is not well known. As shown by the red arrows, SMOH activation leads to the accumulation of full-length activator forms of GLI transcription factors. In the absence of Hh signaling, a series of sequential kinases (PKA, GSK, CK1) promotes the cleavage of GLI factors to a repressor form. SU(FU) is thought to serve as a molecular bridge between GLI-R and a recently described repressor complex which inactivates chromatin domains in the vicinity of GLI transcription factor binding sites (73). In the basal (unstimulated) state, most of the GLI factors are associated with a multicomponent complex associated with cytoplasmic microtubules.

Figure 4. (A) A hypothetical model of SHH secretion postulates that adequate cholesterol (orange) can be a requirement for transport of SHH to the cell surface by influencing its association with the SSD-containing protein DISP (light blue). Cells secreting vertebrate Shh are known to accumulate immunoreative protein within the cell membrane, presumably due to the cholesterol moiety of Shh-Np. Cholesterol synthesis inhibitors do not affect the processing to the Shh-Np species, but might influence the trafficking to the cell membrane. (B) The mechanism(s) of inhibition of SMOH (turquoise) are poorly understood, but are likely to be multiple including: its translation, post-translational modification(s), vesicular trafficking between cellular compartments (influenced by Opb/Rab23 or cholesterol), its association with PTCH (inside and on the surface of cells), a balance between synthesis/degradation, as well as a hypothetical cholesterol requirement. In Drosophila, most of the Ptch protein in the steady state is associated with intracellular vesicles, and Hh signaling leads to a divergence in the distribution of Ptch and Smo. Similarly, in vertebrate cells, PTCH and SMOH appear to be co-localized in all compartments. Binding of the Hh ligand by PTCH leads to a sorting in late endosomes (149; see also 150), where PTCH/SHH complexes are degraded, and SMOH is diverted to the plasma membrane associated with signal transduction (red arrow).

Figure 4. (A) A hypothetical model of SHH secretion postulates that adequate cholesterol (orange) can be a requirement for transport of SHH to the cell surface by influencing its association with the SSD-containing protein DISP (light blue). Cells secreting vertebrate Shh are known to accumulate immunoreative protein within the cell membrane, presumably due to the cholesterol moiety of Shh-Np. Cholesterol synthesis inhibitors do not affect the processing to the Shh-Np species, but might influence the trafficking to the cell membrane. (B) The mechanism(s) of inhibition of SMOH (turquoise) are poorly understood, but are likely to be multiple including: its translation, post-translational modification(s), vesicular trafficking between cellular compartments (influenced by Opb/Rab23 or cholesterol), its association with PTCH (inside and on the surface of cells), a balance between synthesis/degradation, as well as a hypothetical cholesterol requirement. In Drosophila, most of the Ptch protein in the steady state is associated with intracellular vesicles, and Hh signaling leads to a divergence in the distribution of Ptch and Smo. Similarly, in vertebrate cells, PTCH and SMOH appear to be co-localized in all compartments. Binding of the Hh ligand by PTCH leads to a sorting in late endosomes (149; see also 150), where PTCH/SHH complexes are degraded, and SMOH is diverted to the plasma membrane associated with signal transduction (red arrow).

Table 1.

Components of Hedgehog signaling

Gene (invertebrate/vertebrate/human) Function LOF References 
Hedgehog synthesis/biogenesis    
Hh/Shh/SHH Secreted signaling molecule ↓↓↓ 13,40 
  ↑ (GOF) 43 
sit/ski/cm NH2-terminal palmitate ↓ 4447 
disp/Disp/DISPA Transport of Hh out of cell ↓↓↓ 4850 
 Contains a sterol sensing domain (SSD)   
Hedgehog transfer between cells    
ttv/Ttv/EXT Synthesis of heparin sulfate proteoglycans ↓ 5154 
Binding of Hh responsive cells    
ptc/Ptc/PTCH Membrane receptor, negative regulates target genes ↑↑↑ 5556 
 Sequesters Hh, contains a sterol sensing domain (SSD)   
 Dominant negative forms, putative constitutive activator ↑↓ (GOF) 57,58 
Hip/HIP Up-regulated by Hh, binds all family members and removes them from the cell surface; no known signaling role ↑ 59 
Biogenesis of a receptor signaling protein    
smo/Smo/SMO Initiates a signaling cascade to activate target genes ↓↓↓ 60,64 
  ↑↑ (GOF) 6163 
(phosphatase) (Associated with decreased activity) ↑ 65 
(kinase) (Associated with increased activity) ↓ 65 
rab23/Opb Involved in vesicle transport, required to inhibit smo ↑↑ 66,67 
Intracellular transduction of smo signals    
fused/Fused/FUSED Serine/threonine kinase, whose activity is Hh regulated, and acts as a positive effector of Hh signaling which disassociates ci from costal2 complex in microtubules and antagonizes su(fu) ↓ 68,69 
su(fu)/Su(Fu)/SU/(FU) Accompanies ci into the nucleus and bridges with SAP18 and mSin3-HDAC to form a repressor complex which inactivates genes containing a ci/GLI binding site ↑↑ 7073 
costal2 Kinesin-like protein sequesters ci complex [ci/su(fu)/fu] in the cytoplasm associated with microtubules ↑ 74 
ci/Gli1-3/GLI1-3 Transcription factor(s), exist in Hh-regulated activator or repressor forms ↓↓↓ 7588 
  ↑↑↑ 16,17 
   89 
pka/Pka/PKA cAMP dependent phosphorlyation of ci promotes cleavage to the repressor form, primes for gsk3 and ck1 phosphorylation ↑↑↑ 91,92 
gsk3 Also phosphorylates ci to promote the repressor form; also integrated into wnt signaling pathway ↑↑ 93,94 
ck1 Also phosphorylates ci to promote the repressor form ↑↑ 94 
Sil/SIL Genetically downstream of Smo, needed to activate target genes ↓↓↓ 95,96 
Slimb Proteosome protein involved in processing ci to a repressor ↑↑ 97,98 
Gene (invertebrate/vertebrate/human) Function LOF References 
Hedgehog synthesis/biogenesis    
Hh/Shh/SHH Secreted signaling molecule ↓↓↓ 13,40 
  ↑ (GOF) 43 
sit/ski/cm NH2-terminal palmitate ↓ 4447 
disp/Disp/DISPA Transport of Hh out of cell ↓↓↓ 4850 
 Contains a sterol sensing domain (SSD)   
Hedgehog transfer between cells    
ttv/Ttv/EXT Synthesis of heparin sulfate proteoglycans ↓ 5154 
Binding of Hh responsive cells    
ptc/Ptc/PTCH Membrane receptor, negative regulates target genes ↑↑↑ 5556 
 Sequesters Hh, contains a sterol sensing domain (SSD)   
 Dominant negative forms, putative constitutive activator ↑↓ (GOF) 57,58 
Hip/HIP Up-regulated by Hh, binds all family members and removes them from the cell surface; no known signaling role ↑ 59 
Biogenesis of a receptor signaling protein    
smo/Smo/SMO Initiates a signaling cascade to activate target genes ↓↓↓ 60,64 
  ↑↑ (GOF) 6163 
(phosphatase) (Associated with decreased activity) ↑ 65 
(kinase) (Associated with increased activity) ↓ 65 
rab23/Opb Involved in vesicle transport, required to inhibit smo ↑↑ 66,67 
Intracellular transduction of smo signals    
fused/Fused/FUSED Serine/threonine kinase, whose activity is Hh regulated, and acts as a positive effector of Hh signaling which disassociates ci from costal2 complex in microtubules and antagonizes su(fu) ↓ 68,69 
su(fu)/Su(Fu)/SU/(FU) Accompanies ci into the nucleus and bridges with SAP18 and mSin3-HDAC to form a repressor complex which inactivates genes containing a ci/GLI binding site ↑↑ 7073 
costal2 Kinesin-like protein sequesters ci complex [ci/su(fu)/fu] in the cytoplasm associated with microtubules ↑ 74 
ci/Gli1-3/GLI1-3 Transcription factor(s), exist in Hh-regulated activator or repressor forms ↓↓↓ 7588 
  ↑↑↑ 16,17 
   89 
pka/Pka/PKA cAMP dependent phosphorlyation of ci promotes cleavage to the repressor form, primes for gsk3 and ck1 phosphorylation ↑↑↑ 91,92 
gsk3 Also phosphorylates ci to promote the repressor form; also integrated into wnt signaling pathway ↑↑ 93,94 
ck1 Also phosphorylates ci to promote the repressor form ↑↑ 94 
Sil/SIL Genetically downstream of Smo, needed to activate target genes ↓↓↓ 95,96 
Slimb Proteosome protein involved in processing ci to a repressor ↑↑ 97,98 

LOF: loss-of-function; GOF: gain-of-function.

References

1
Ingham, P.W. and McMahon, A.P. (
2001
) Hedgehog signaling in animal development: paradigms and principles.
Genes Dev.
 ,
15
,
3059
–3087.
2
Ericson, J., Rashbass, P., Schedl, A., Brenner-Morton, S., Kawakami, A., van Heyningen, V., Jessell, T.M. and Briscoe, J. (
1997
) Pax6 controls progenitor cell identity and neuronal fate in response to graded Shh signaling.
Cell
 ,
90
,
169
–180.
3
Briscoe, J. and Ericson, J. (
1999
) The specification of neuronal identity by graded Sonic hedgehog signalling.
Semin. Cell Dev. Biol.
 ,
10
,
353
–362.
4
Patten, I. and Placzek, M. (
2000
) The role of Sonic hedgehog in neural tube patterning.
Cell Mol. Life Sci.
 ,
57
,
1695
–1708.
5
Briscoe, J., Pierani, A., Jessell, T.M. and Ericson, J. (
2000
) A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube.
Cell
 ,
101
,
435
–445.
6
Briscoe, J. and Ericson, J. (
2001
) Specification of neuronal fates in the ventral neural tube.
Curr. Opin. Neurobiol.
 ,
11
,
43
–49.
7
Kohtz, J.D., Baker, D.P., Corte, G. and Fishell, G. (
1998
) Regionalization within the mammalian telencephalon is mediated by changes in responsiveness to Sonic hedgehog.
Development
 ,
125
,
5079
–5089.
8
Meyers, E.N. and Martin, G.R. (
1999
) Differences in left–right axis pathways in mouse and chick: functions of FGF8 and Shh.
Science
 ,
285
,
403
–406.
9
Tsukui, T., Capdevila, J., Tamura, K., Ruiz-Lozano, P., Rodreguez-Esteban C., Yonei-Tamura, S., Magallon, J., Chandraratna, R.A., Chien, K., Blumburg, B., Evans, R.M. and Belmonte, J.C.I. (
1999
) Multiple left–right asymmetry defects in Shh(−/−) mutant mice unveil a convergence of the shh and retinoic acid pathways in the control of Lefty-1.
Proc. Natl Acad. Sci. USA
 ,
96
,
11376
–11381.
10
Purandare, S.M., Ware, S.M., Kwan, K.M., Gebbia, M., Bassi, M.T., Deng J.M., Vogel, H., Behringer, R.R., Belmont, J.W. and Casey, B. (
2002
) A complex syndrome of left–right axis, central nervous system and axial skeletal defects in Zic3 mutant mice.
Development
 ,
129
,
2293
–2303.
11
Marlow, F., Zwarikruis, F., Malicki, J., Neuhauss, S.C.F., Abbas, L., Weaver, M., Driever, W. and Solnica-Krezel, L. (
1998
) Functional interactions of genes mediating convergent-extension, knypck and trilobite, during the partitioning of the eye primordium in zebrafish.
Dev. Biol.
 ,
203
,
382
–399.
12
Roessler, E. and Muenke, M. (
2001
) Midline and laterality defects: left and right meet in the middle.
Bioessays
 ,
23
,
888
–900.
13
Chiang, C., Litingtung, Y., Lee, E., Young, K.E., Corden, J.L., Westphal, H. and Beachy, P.A. (
1996
) Cyclopia and defective axial patterning in mice lacking sonic hedgehog gene function.
Nature
 ,
383
,
407
–413.
14
Ho, K.S. and Scott, M.P. (
2002
) Sonic hedgehog in the nervous system: functions, modifications and mechanisms.
Curr. Opin. Neurobiol.
 ,
12
,
57
–63.
15
Chiang, C., Littingtung, Y., Harris, M.P., Simaudl, B.K., Beachy, P.A. and Fallon, J.F. (
2000
) Manifestation of the limb prepattern: limb development in the absence of sonic hedgehog function.
Dev. Biol.
 ,
236
,
421
–425.
16
Welscher, P., Zuniga, A., Kuljper, S., Drenth, T., Goedemans, H.J., Meijlink, F. and Zeller, R. (
2002
) Progression of vertebrate limb development through Shh-mediated counteraction of Gli3.
Science
 ,
298
,
827
–830.
17
Littingtung, Y., Dahn, R.D., Fallon, J.F. and Chiang, C. (
2002
) Shh and Gli3 are dispensable for limb skeleton formation but regulate digit number and identity.
Nature
 ,
418
,
979
–983.
18
Sheng, H. and Westphal, H. (
1999
) Early steps in pituitary organogenesis.
Trends Gene.
 ,
15
,
236
–240.
19
Dasen, J.S. and Rosenfeld, M.G. (
1999
) Combinatorial codes in signaling and synergy: lessons from pituitary development.
Curr. Opin. Genet. Dev.
 ,
9
,
566
–574.
20
Trier, M., O'Connell, S., Gleiberman, A., Price, J., Szeto, D.P., Burgess, R., Chuang, P.-T., McMahon, A.P. and Rosenfeld, M.G. (
2001
) Hedgehog signaling is required for pituitary gland development.
Development
 ,
128
,
377
–386.
21
Karlstrom, R.O., Talbot, W.S. and Schier, A.F. (
1999
) Comparative synteny cloning of zebrafish you-too: mutations in the hedgehog target gli2 affect ventral forebrain patterning.
Genes Dev.
 ,
13
,
388
–393.
22
Kondoh, H., Uchikawa, M., Yoda, H., Furutani-Seiki, M. and Karlstrom, R.O. (
2000
) Zebrafish mutations in Gli-mediated hedgehog signaling lead to lens transdifferentiation from the adenohypophysis anlage.
Mech. Dev.
 ,
96
,
165
–174.
23
Ahlgren, S.C. and Bronner-Fraser, M. (
1999
) Inhibition of Sonic hedgehog signaling in vivo results in craniofacial neural crest cell death.
Curr. Biol.
 ,
9
,
1304
–1314.
24
Agarwala, S., Sanders, T.A. and Ragsdale, C.W. (
2001
) Sonic hedgehog control of size and shape in midbrain pattern formation.
Science
 ,
291
,
2147
–2150.
25
Dahmane, N. and Ruiz-i-Altaba, A. (
1999
) Sonic hedgehog regulates the growth and patterning of the cerebellum.
Development
 ,
126
,
3089
–3100.
26
Rowitch, D.H., St.-Jacques, B., Lee, S.M.K., Flax, J.D., Snyder, E.Y. and McMahon, A.P. (
1999
) Sonic hedgehog regulates proliferation and inhibits differentiation of CNS precursor cells.
J. Neurosci.
 ,
19
,
8954
–9658.
27
Wechsler-Reya, R.J. and Scott, M.P. (
1999
) Control of neuronal precursor proliferation in the cerebellum by Sonic hedgehog.
Neuron
 ,
22
,
103
–114.
28
Davies, J.E. and Miller, R.H. (
2001
) Local Sonic hedgehog signaling regulates oligodendrocyte precursor appearance in multiple ventricular zone domains in the chick metencephalon.
Dev. Biol.
 ,
233
,
513
–525.
29
Dominguez, M. and Hafen, E. (
1997
) Hedgehog directly controls initiation and propagation of retinal differentiation in the Drosophila eye.
Genes Dev.
 ,
11
,
3254
–3264.
30
Huang, Z. and Kunes, S. (
1998
) Signals transmitted along retinal axons in Drosophila: Hedgehog signal reception and the cell circuitry of lamina cartridge assembly.
Development
 ,
125
,
3753
–3764.
31
Stenkamp, D.L., Frey, R.A., Prabhudesai, S.N. and Raymond, P.A. (
2000
) Function for Hedgehog genes in zebrafish retinal development.
Dev. Biol.
 ,
220
,
238
–252.
32
Huang, Z. and Kunes, S. (
1996
) Hedgehog, transmitted along retinal axons, triggers neurogenesis in the developing visual centers of the Drosophila brain.
Cell
 ,
86
,
411
–422.
33
Jarman, A.P. (
2000
) Developmental genetics: vertebrates and insects see eye to eye.
Curr. Biol.
 ,
10
,
R857
–859.
34
Helms, J.A., Kim, C.H., Hu, D., Minkoff, R., Thaller, C. and Eichele, G. (
1997
) Sonic hedgehog participates in craniofacial morphogenesis and is down-regulated by teratogenic doses of retinoic acid.
Dev. Biol.
 ,
187
,
25
–35.
35
Golden, J.A., Bracilovic, A., McFadden, K.A., Beesley, J.S., Rubenstein, J.L.R. and Grinspan, J.B. (
1999
) Ectopic bone morphogenetic proteins 5 and 4 in the chicken forebrain lead to cyclopia and holoprosencephaly.
Proc. Natl Acad. Sci. USA
 ,
96
,
2439
–2444.
36
Nasrallah, I. and Golden, J.A. (
2001
) Brain, eye, and face defects as a result of ectopic localization of sonic hedgehog protein in the developing rostral neural tube.
Teratology
 ,
64
,
107
–113.
37
Schneider, R.A., Hu, D., Rubenstein, J.L., Maden, M. and Helms, J.A. (
2001
) Local retinoid signaling coordinates forebrain and facial morphogenesis by maintaining FGF8 and SHH.
Development
 ,
128
,
2755
–2767.
38
Ohkubo, Y., Chiang, C. and Rubenstein, J.L. (
2002
) Coordinate regulation and synergistic actions of BMP4, SHH, and FGF8 in the rostral prosencephalon regulate morphogenesis of the telencephalic and optic vesicles.
Neuroscience
 ,
111
,
1
–17.
39
Muenke, M. and Beachy, P.A. (
2001
) Holoprosencephaly. In Scriver, C.R. et al. (ed.)
Metabolic Molecular Basis of Inherited Disease
 . 8th edn., (Chap. 250). McGraw Hill, New York.
40
Roessler, E., Belloni, E., Gaudenz, K., Jay, P., Berta, P., Scherer, S.W., Tsui, L.C. and Muenke, M. (
1996
) Mutations in the human sonic Hedgehog gene cause holoprosencephaly.
Nat. Genet.
 ,
14
,
357
–360.
41
Anderson, R.M., Lawrence, A.R., Stottmann, R.W., Bachiller, D. and Klingensmith, J. (
2002
) Chordin and noggin promote organizing centers of forebrain development in the mouse.
Development
 ,
129
,
4975
–4987.
42
Railu, M., Machold, R., Galano, N., Corbin, J.G., McMahon, A.P. and Fischell, G. (
2002
) Dorsoventral patterning is established in the telencephalon of mutants lacking both Gli3 and Hedgehog signaling.
Development
 ,
129
,
4963
–4974.
43
Oro, A.E., Higgins, K.M., Hu, Z., Bonifas, J.M., Epstein, E.H. and Scott, M.P. (
1997
) Basal cell carcinoma in mice overexpressing sonic Hedgehog.
Science
 ,
276
,
817
–821.
44
Amanai, K. and Jiang, J. (
2001
) Distinct roles of central missing and dispatched in sending the hedgehog signal.
Development
 ,
128
,
5119
–5127.
45
Micchelli, C.A., The, I., Selva, E., Mogila, V. and Perrimon, N. (
2002
) rasp, a putative transmembrane acyltransferase, is required for Hedgehog signaling.
Development
 ,
129
,
843
–851.
46
Chamoun, Z., Mann, R.K., Nellen, D., von Kessler, D.P., Bellotto, M., Beachy, P.A. and Basler, K. (
2001
) Skinny Hedgehog, an acyltransferase required for palmitoylation and activity of the Hedgehog signal.
Science
 ,
293
,
2080
–2084.
47
Lee, J.D. and Treisman, J.E. (
2001
) Sightless has homology to transmembrane acyltransferases and is required to generate active Hedgehog protein.
Curr. Biol.
 ,
11
,
1147
–1152.
48
Burke, R., Nellen, D., Bellotto, M., Hafen, E., Senti, K.A., Dickson, B.J. and Basler, K. (
1999
) Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified Hedgehog from signaling cells.
Cell
 ,
99
,
803
–815.
49
Ma, Y., Erkner, A., Gong, R., Yao, S., Taipale, J., Basler, K. and Beachy, P.A. (
2002
) Hedgehog-mediated patterning of the mammalian embryo requires transporter-like function of dispatched.
Cell
 ,
111
,
63
–75.
50
Kawakami, T., Kawcak, T., Li, Y-J, Zhang, W., Hu, Y. and Chuang, P-T. (
2002
) Mouse dispatched mutants fail to distribute hedgehog proteins and are defective in hedgehog signaling.
Development
 ,
129
,
5753
–5765.
51
Bellaiche, Y., The, I. and Perrimon, N. (
1998
) Tout-velu is a Drosophila homologue of the putative tumour suppressor EXT-1 and is needed for Hh diffusion.
Nature
 ,
394
,
85
–88
52
The, I., Bellaiche, Y. and Perrimon, N. (
1999
) Hedgehog movement is regulated through tout velu-dependent synthesis of a heparan sulfate proteoglycan.
Mol. Cell
 ,
4
,
633
–639.
53
Pons, S. and Marti, E. (
2000
) Sonic hedgehog synergizes with the extracellular matrix protein vitronectin to induce spinal motor neuron differentiation.
Development
 ,
127
,
333
–342.
54
Baeg, G.H. and Perrimon, N. (
2000
) Functional binding of secreted molecules to heparan sulfate proteoglycans in Drosophila.
Curr. Opin. Cell Biol.
 ,
12
,
575
–580.
55
Goodrich, L.V., Milenkovic, L., Higgins, K.M. and Scott, M.P. (
1997
) Altered neural cell fates and medulloblastoma in mouse Patched mutants.
Science
 ,
277
,
1109
–1113.
56
Milenkovic, L., Goodrich, L.V., Higgins, K.M. and Scott, M.P. (
1999
) Mouse patched 1 controls body size determination and limb patterning.
Development
 ,
126
,
4431
–4440.
57
Briscoe, J., Chen, Y., Jessell, T.M. and Struhl, G. (
2001
) A Hedgehog-insensitive form of Patched provides evidence for direct long-range morphogen activity of Sonic hedgehog in the neural tube.
Mol. Cell.
 ,
7
,
1279
–1291.
58
Ming, J.E., Kaupas, M.E., Roessler, E., Brunner, H.G., Golabi, M., Tekin, M., Stratton, R.F., Sujansky, E., Bale, S.J. and Muenke, M. (
2002
) Mutations in PATCHED-1, the receptor for SONIC HEDGEHOG, are associated with holoprosencephaly.
Hum. Genet.
 ,
110
,
297
–301.
59
Huo, L., Roessler, E., Dutra, A., Chuang, P.-T., McMahon, A.P. and Muenke, M. (
2000
) Determination of the chromosomal location and genomic structure of the Hedgehog-interacting protein gene, and analysis of its role in holoprosencephaly.
Gene Funct Dis.
 ,
3–1
,
119
–127.
60
Zhang, X.M., Ramalho-Santos, M. and McMahon, A.P. (
2001
) Smoothened mutants reveal redundant roles for Shh and Ihh regulation of L/R symmetry by the mouse node.
Cell
 ,
106
,
781
–792.
61
Reifenberger, J., Wolter, M., Weber, R.G., Megahed, M., Ruzicka, T., Lichter, P. and Reifenberger, G. (
1998
) Missense mutations in SMOH in sporadic basal cell carcinoma of the skin and primitive neuroectodermal tumors of the central nervous system.
Cancer Res.
 ,
58
,
1798
–1803.
62
Lam, C.-W., Xie, J., To, K.-F., Ng, H.-K., Lee, K.-C., Yuen, N.W.-F., Lim, P.-L., Chan, Y.-S., Tong, S.-F. and McCormick, F. (
1999
). A frequent activated smoothened mutation in sporadic vassal cell carcinomas.
Oncogene
 ,
18
,
833
–836.
63
Xie, J., Murone, M., Luoh, S.-M., Ryan, A., Gu, Q., Zhang, C., Bonifas, J.M., Lam, C.W., Hynes, M., Goddard, A. et al. (
1998
) Activating smoothened mutations in sporadic basal cell carcinoma.
Nature
 ,
391
,
90
–92.
64
Hynes, M., Ye, W., Stone, D., Murone, M., de Sauvage, F. and Rosenthal, A. (
2000
) The seven transmembrane receptor Smoothened cell-autonomously induces multiple ventral cell types.
Nat. Neurosci.
 ,
3
,
41
–46.
65
Kalderon, D. (
2000
) Transducing the Hedgehog signal.
Cell
 ,
103
,
371
–374.
66
Eggenschwiler, J.T., Espinoza, E. and Anderson, K.V. (
2001
) Rab23 is an essential negative regulator of the mouse Sonic hedgehog signaling pathway.
Nature
 ,
412
,
194
–198.
67
Eggenschwiler, J.T. and Anderson, K.V. (
2000
) Dorsal and lateral fates in the mouse neural tube require the cell-autonomous activity of the open brain gene.
Dev. Biol.
 ,
227
,
648
–660.
68
Nybakken, K.E., Turck, C.W., Robbins, D.J. and Bishop, J.M. (
2002
) Hedgehog-stimulated phosphorylation of the kinesin-related protein costal2 is mediated by the serine/threonine kinase fused.
J. Biol. Chem.
 ,
277
,
24638
–24647.
69
Fukumoto, T., Watanabe-Fukunaga, R., Fugisawa, K., Nagata, S. and Fukunaga, R. (
2001
) The fused protein kinase regulates hedgehog-stimulated transcriptional activation in Drosophila Schneider 2 cells.
J. Biol. Chem.
 ,
276
,
38441
–38448.
70
Murone, M., Luoh, S.-M., Stone, D., Li, W., Gumey, A., Armanini, M., Grey, C., Rosenthal, A. and de Sauvage, F.J. (
2000
) Gli regulation by the opposing activities of fused and suppressor of fused.
Nat. Cell Biol.
 ,
2
,
310
–312.
71
Taylor, M.D., Liu, L, Raffel, C., Hui, C., Mainprize, T.G., Zhang, X., Agatep, R., Chiappa, S., Gao, L. et al. (
2002
) Mutations in SUFU predispose to medulloblasoma.
Nat. Genet.
 ,
31
,
306
–310.
72
Pearse, R.V., Collier, L.S., Scott, M.P. and Tabin. C.J. (
1999
) Vertebrate homologs of Drosophila suppressor of fused interact with the Gli family of transcriptional regulators.
Dev. Biol.
 ,
212
,
323
–336.
73
Cheng, S.Y. and Bishop, J.M. (
2002
) Suppressor of fused represses Gli-mediated transcription by recruiting the SAP18-mSin3 corepressor complex.
Proc. Natl Acad. Sci. USA
 ,
99
,
5442
–5447.
74
Monnier, V., Ho, K.S., Sanial, M., Scott, M.P. and Plessis, A. (
2002
) Hedgehog signal transduction proteins: contacts of the fused kinase and ci transcription factor with the kineisin-related protein costal2.
BMC Dev. Biol.
 ,
2
(4),
1
–9.
75
Aza-Blanc, P. and Kornberg, T.B. (
1999
) Ci: a complex transducer of the hedgehog signal.
Trends Genet.
 ,
15
,
458
–462.
76
Ruiz i Altaba, A., Palma, V. and Dahmane, N. (
2002
) Hedgehog-Gli signaling and the growth of the brain.
Nat. Rev. Neurosci.
 ,
3
,
24
–33.
77
Aza-Blanc, P., Lin, H.Y., Ruiz i Altaba, A. and Komberg, T.B. (
2000
) Expression of the vertebrate Gli proteins in Drosophila reveals a distribution of activator and repressor activities.
Development
 ,
127
,
4293
–4301.
78
Muller, B. and Basler, K. (
2000
) The repressor and activator forms of cubitus interruptus control hedgehog target genes through common generic gli-binding sites.
Development
 ,
127
,
2999
–3007.
79
Pavletich, N.P. and Pabo, C.O. (
1993
) Crystal structure of a five-finger GLI-DNA complex: new perspectives on zinc fingers.
Science
 ,
261
,
1701
–1707.
80
Hui, C.-c., Slusarski, D., Platt, K.A., Holmgren, R. and Joyner, A.L. (
1994
) Expression of three mouse homologs of the Drosophila segment polarity gene cubitus interruptus, Gli, Gli-2, and Gli-3, in endoderm and mesoderm-derived tissues suggests multiple roles during postimplantation development.
Dev. Biol.
 ,
162
,
402
–413.
81
Mo, R., Freer, A.M., Zinyk, D.L., Crackower, M.A., Michaud, J., Heng, H.H.-Q., Chik, K.W., Shi, X.-M., Tsui, L.-C., Cheng, S.H., Joyner, A.L. and Hui, C.-c. (
1997
) Specific and redundant functions of Gli2 and Gli3 zinc finger genes in skeletal patterning and development.
Development
 ,
124
,
113
–123.
82
Matise, M.P., Epstein, D.J., Park, H.L., Platt, K.A. and Joyner, A.L. (
1998
) Gli2 is required for induction of floor plate and adjacent cells, but not most ventral neurons in the mouse central nervous system.
Development
 ,
125
,
2759
–2770
83
Ding, Q., Motoyama, J., Gasca, S., Mo, R., Sasaki, H., Rossant, J. and Hui, C.-c. (
1998
) Diminished Sonic hedgehog signaling and lack of floor plate differentiation in Glii2 mutant mice.
Development
 ,
125
,
2533
–2543.
84
Motoyama, J., Liu, J., Mo, R., Ding, Q., Post, M. and Hui, C.-c. (
1998
) Essential function of Gli2 and Gli3 in the formation of lung, trachea and oesophagus.
Nature Genet.
 ,
20
,
54
–57.
85
Hardcastle, Z., Mo, R., Hui, C.-c. and Sharpe, P.T. (
1998
) The Shh signaling pathway in tooth development: defects in Gli2 and Gli3 mutants.
Development
 ,
125
,
2803
–2811.
86
Park, H.L., Bai, C., Platt, K.A., Matise, M.P., Beeghly, A., Hui, C.-c., Nakashima, M. and Joyner, A.L. (
2000
) Mouse Gli 1 mutants are viable but have defects in SHH signaling in combination with a Gli2 mutation.
Development
 ,
127
,
1593
–1605.
87
Bai, C.B. and Joyner, A.L. (
2001
) Gli1 can rescue the in vivo function of Gli2.
Development
 ,
128
,
5161
–5172.
88
Bai, C.B., Auerbach, W., Lee, J.S., Stephen, D. and Joyner, A.L. (
2002
) Gli2, but not Gli1, is required for initial Shh signaling and ectopic activation of the Shh pathway.
Development
 ,
129
,
4753
–4761.
89
Littingtung, Y. and Chiang, C. (
2000
) Specification of ventral neuron types is mediated by an antagonistic interaction between Shh and Gli3.
Nat. Nerosci.
 ,
3
,
979
–985.
90
Ruiz I Altaba, A., Sanchez, P. and Dahmane, N. (
2002
) Gli and hedgehog in cancer: tumors, embryos and stem cells.
Nat. Rev. Cancer
 ,
2
,
361
–372.
91
Ungar, A.R. and Moon, R. (
1996
) Inhibition of protein kinase A phenocopies ectopic expression of hedgehog in the CNS of wild-type and cyclops mutant embryos.
Dev. Biol.
 ,
178
,
186
–191.
92
Hammerschmidt, M., Bitgood, M.J. and McMahon, A.P. (
1996
) Protein kinase A is common negative regulator of hedgehog signaling in the vertebrate embryo.
Genes Dev.
 ,
10
,
647
–658.
93
Jia, J., Amanai, K., Wang, G., Tang, J., Wang, B. and Jiang, J. (
2002
) Shaggy/GSK3 antagonizes hedgehog signaling by regulating cubitus interruptus.
Nature
 ,
416
,
548
–552.
94
Price M.A. and Kalderon, D. (
2002
) Proteolysis of the hedgehog signaling effector cubitus in interruptus requires phophorylation by glycogen synthase kinase 3 and casein kinase 1.
Cell
 ,
108
,
823
–835.
95
Karkera, J.D., Izraeli, S., Roessler, E., Dutra, A., Kirsch, I. and Muenke, M. (
2002
) The genomic structure, chromosomal localization, and analysis of SIL as a candidate gene for holoprosencephaly.
Cytogenet. Genome Res.
 ,
97
,
62
–67.
96
Izraeli, S., Lowe, L.A., Bertness, V.L., Campaner, S., Hahn, H., Kirsch, I.R. and Kuehn, M.R. (
2001
) Genetic evidence that Sil is required for the sonic hedgehog response pathway.
Genesis
 ,
31
,
72
–77.
97
Theodosiou, N.A., Zhang, S., Wang, W.-Y. and Xu, T. (
1998
) slimb coordinates wg and dpp expression in the dorsal-ventral and anterior-posterior axes during limb development.
Development
 ,
125
,
3411
–3416.
98
Jiang, J. and Struhl, G. (
1998
) Regulation of the hedgehog and wingless signaling pathways by the F-box/WD40-repeat protein Slimb.
Nature
 ,
391
,
493
–498.
99
Bale, A.E. (
2002
) Hedgehog signaling and human disease.
A. Rev. Genomics Hum. Genet.
 ,
3
,
47
–65.
100
Britto, J.M., Tannahill, D. and Keynes, R.J. (
2000
) Life, death and Sonic hedgehog.
Bioessays
 ,
22
,
499
–502.
101
Britto, J., Tannahill, D. and Keynes, R. (
2002
) A critical role for sonic hedgehog signaling in the early expansion of the developing brain.
Nat. Neurosci.
 ,
5
,
103
–110.
102
Kim, J., Kim, P. and Hui, C.-c. (
2001
) The Vacterl association: lessons from the Sonic Hedgehog pathway.
Clin. Genet.
 ,
59
,
306
–315.
103
Mullor, J.L., Sanchez, P. and Ruiz i Altaba, A. (
2002
) Pathways and consequences: hedgehog in human disease.
Trends Cell Biol.
 ,
12
,
562
–569.
104
Shin, S.H., Kogerman, P., Lindstrom, E., Toftgard, R. and Biesecker, L.G. (
1999
) Gli3 mutations in human disorder mimic Drosophila cubitus interruptus protein functions and localization.
Proc. Natl Acad. Sci. USA
 ,
96
,
2880
–2884.
105
Radhakrishna, U., Bomholdt, D., Scott, H.S., Patel, U.C., Rossier, C., Engel, H., Bottani, A., Chandal, D., Blouin, J.-L., Solanki, J.V., Grzeschik, K.H. and Antonarakis, A.E. (
1999
) The phenotypic spectrum of GLI3 morphopathies includes autosomal dominant preaxial polydactyly Type-IV and postaxial polydactyly type A/B; no phenotype prediction from the position of GLI3 mutations.
Am. J. Hum. Genet.
 ,
65
,
645
–655.
106
Aruga, J., Inoue, T., Hoshino, J. and Mikoshiba, K. (
2002
) Zic2 controls cerebellar development in cooperation with Zic1.
J. Neurosci.
 ,
22
,
218
–225.
107
Nagai, T., Aruga, J., Minowa, O., Sugimoto, T., Ohno, Y., Noda, T. and Mikoshiba, K. (
2000
) Zic2 regulates the kinetics of neurulation.
Proc. Natl Acad. Sci. USA
 ,
97
,
1618
–1623.
108
Aruga, J., Mizugishi., K., Koseki., H., Imai, K., Balling, R., Noda, T. and Mikoshiba, K. (
1999
) Zicl regulates the patteming of vertebral arches in cooperation with Gli3.
Mech. Dev.
 ,
89
,
141
–150.
109
Nagai, T., Aruga, J., Takada, S., Gunther, T., Sporle, R., Schughart, K. and Mikoshiba, K. (
1997
) The expression of the mouse Zic1, Zic2, and Zic3 gene suggests an essential role for Zic genes in body pattern formation.
Dev. Biol.
 ,
182
,
299
–313.
110
Mizugishi, K., Aruga, J., Nakata, K. and Mikoshiba, K. (
2001
) Molecular properties of Zic proteins as transcriptional regulators and their relationship to GL1 proteins.
J. Biol. Chem.
 ,
276
,
2180
–2188.
111
Koyabu, Y., Nakata, K., Mizugishi, K., Aruga, J. and Mikoshiba, K. (
2001
) Physical and functional interactions between Zic and Gli proteins.
J. Biol. Chem.
 ,
278
,
6889
–6892.
112
Porter, J.A., Ekker, S.C., Park, W.J., von Kessler, D.P., Young, K.E., Chen, C.H., Ma, Y., Woods, A.S., Cotter, R.J., Koonin, E.V. and Beachy, P.A. (
1996
) Hedgehog patteming activity: role of a lipophilic modification mediated by the carboxyterrninal autoprocessing domain.
Cell
 ,
86
,
21
–34.
113
Pepinsky, R.B., Zeng, C., Wen, D., Rayhom, P., Baker, D.P., Williams, K.P., Bixler, S.A., Ambrose, C.M., Garber, E.A., Miatkowski, K., Taylor, F.R., Wang, E.A. and Galdes, A. (
1998
) Identification of a palmitic acid-modified form of human Sonic hedgehog.
J. Biol. Chem.
 ,
273
,
14037
–14045.
114
Lee, J.D., Kraus, P., Gaiano, N., Nery, S., Kohtz, J., Fishell, G., Loomis, C.A. and Treisman, J.E. (
2001
) An acylatable residue of Hedgehog is differentially required in Drosophila and mouse limb development.
Dev. Biol.
 ,
233
,
122
–136.
115
Kohtz, J.D., Lee, H.Y., Gaiano, N., Segal, J., Ng, E., Larson, T., Baker, D.P., Garber, E.A., Williams, K.P. and Fishell, G. (
2001
) N-terminal fatty-acylation of Sonic hedgehog enhances the induction of rodent ventral forebrain neurons.
Development
 ,
128
,
2351
–2363.
116
Taylor, F.R., Wen, D., Garber, E.A., Carmillo, A.N., Baker, D.P., Arduini, R.M., Williams, K.P., Weinreb, P.H., Rayhom, P., Hronowski, X. et al. (
2001
) Enhanced potency of human Sonic hedgehog by hydrophobic modification.
Biochemistry
 ,
40
,
4359
–4371.
117
Lewis, P.M., Dunn, M.P., McMahon, J.A., Logan, M., Martin, J.F., St-Jacques, B. and McMahon, A.P. (
2001
) Cholesterol modification of Sonic hedgehog is required for long-range signaling activity and effective modulation of signaling by Ptcl.
Cell
 ,
105
,
599
–612.
118
Brown, A.J., Sun, L., Feramisco, J.D., Brown, M.S. and Goldstein, J.L. (
2002
) Cholesterol addition to ER membranes alters conformation of SCAP, the SREBP escort protein that regulates cholesterol metabolism.
Mol. Cell
 ,
10
,
237
–245.
119
Brown, M.S. and Goldstein, J.L. (
1997
) The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor.
Cell
 ,
89
,
331
–340.
120
Carstea, E.D., Morris, J.A., Coleman, K.G., Loftus, S.K., Zhang, D., Cummings, C., Gu, S.K., Rosenfeld, M.A., Pavan, W.J., Krizman, D.B. et al. (
1997
) Niemann-Pick Cl disease gene: homology to mediators of cholesterol homeostatsis.
Science
 ,
277
,
228
–231.
121
Loftus, S.K., Morris, J.A., Carstea, E.D., Gu, J.Z., Cummings, C., Brown, A., Ellison, J., Ohno, K., Rosenfeld, M.A., Tagle, D.A., Pentchev, P.G. and Pavan, W.J. (
1997
) Murine model of Niemann–Pick disease: mutation in a cholesterol homeostasis gene.
Science
 ,
277
,
232
–235.
122
Zeng, X., Goetz, J.A., Suber, L.M., Scott, W.J. Jr, Schreiner, C.M. and Robbins, D.J. (
2001
) A freely diffusible form of Sonic hedgehog mediates long-range signalling.
Nature
 ,
411
,
716
–720.
123
Chen, Y. and Struhl, G. (
1996
) Dual roles for Patched in sequestering and transducing Hedgehog.
Cell
 ,
87
,
553
–563.
124
Incardona, J.P., Lee, J.H., Robertson, C.P., Enga, K., Kapur, R.P. and Roelink, H. (
2000
) Receptor-mediated endocytosis of soluble and membrane tethered Sonic Hedgehog by Patched-1.
Proc. Natl Acad. Sci. USA
 ,
97
,
12044
–12049.
125
Denef, N., Neubuser, D., Perez, L. and Cohen, S.M. (
2000
) Hedgehog induces opposite changes in turnover and subcellular localization of Patched and Smoothened.
Cell
 ,
102
,
521
–531.
126
Johnson, R.L., Milenkovic, L. and Scott, M.P. (
2000
) In vivo functions of the patched protein: requirement of the c terminus for target gene inactivation but not Hedgehog sequestration.
Mol. Cell
 ,
6
,
467
–478.
127
Taiplae, J., Cooper, M.K., Marti, T. and Beachy, P.A. (
2002
) Patched acts catalytically to suppress the activity of smoothened.
Nature
 ,
418
,
892
–896.
128
Strutt, H., Thomas, C., Nakano, Y., Stark, D., Neave, B., Taylor, A.M. and Ingham, P.W. (
2001
) Mutations in the sterol-sensing domain of Patched suggest a role for vesicular trafficking in Smoothened regulation.
Curr. Biol.
 ,
11
,
608
–613.
129
Ingham, P.W., Nystedt, S., Nakano, Y., Brown, W., Stark, D., van den Heuvel, M. and Taylor, A.M. (
2000
) Patched represses the Hedgehog signaling pathway by promoting modification of the smoothened protein.
Curr. Biol.
 ,
10
,
1315
–1318.
130
Alcedo, J., Zou, Y. and Noll, M. (
2000
) Posttranscriptional regulation of smoothened is part of a self-correcting mechanism in the Hedgehog signaling system.
Mol. Cell.
 ,
6
,
457
–465.
131
Martin, V., Carrillo, G., Torroja, C. and Guerrero, I. (
2001
) The sterol-sensing domain of Patched protein seems to control Smoothened activity through Patched vesicular trafficking.
Curr. Biol.
 ,
11
,
601
–607.
132
Berman, D.M., Karhadkar, S.S., Hallahan, A.R., Pritchard, J.I., Eberhart, C.G., Watkins, D.N., Chen, J.K., Cooper, M.K., Taiplae, J., Olson, J.M. and Beachy, P.A. (
2002
) Medulloblastoma growth inhibition by hedgehog pathway blockade.
Science
 ,
297
,
1559
–1561.
133
Ming, J.E., Jeng, B., de Sauvage, F.J. and Muenke, M. (
2002
) Analysis of Smoothened as a candidate gene for human holoprosencephaly.
Gene Funct Dis.
  (in press).
134
Gofflot, F., Kolf-Clauw, M., Roux, C. and Picard, J.J. (
1999
) Absence of ventral cell populations in the developing brain in a rat model of the Smith–Lemli–Optiz syndrome.
Am. J. Med. Genet.
 ,
87
,
207
–216.
135
Incardona, J.P. and Roelink, H. (
2000
) The role of cholesterol in Shh signaling and teratogen-induced holoprosencephaly.
Cell. Mol. Life Sci.
 ,
57
,
1709
–1719.
136
Cooper, M.K., Porter, J.A., Young, K.E. and Beachy, P.A. (
1998
) Teratogen-mediated inhibition of target tissue response to Shh signaling.
Science
 ,
280
,
1603
–1607.
137
Incardona, J.P., Gaffield, W., Kapur, R.P. and Roelink, H. (
1998
) The teratogenic Veratrum alkaloid cyclopamine inhibits Sonic Hedgehog signal transduction.
Development
 ,
125
,
3553
–3562.
138
Incardona, J.P., Gaffield, W., Lange, Y., Cooney, A., Pentchev, P.G., Liu, S., Watson, J.A., Kapur, R.P. and Roelink, H. (
2000
) Cyclopamine inhibition of Sonic hedgehog signal transduction is not mediated through effects on cholesterol transport.
Dev. Biol.
 ,
224
,
440
–452.
139
Taipale, J., Chen, J.K., Cooper, M.K., Wang, B., Mann, R.K., Milenkovic, L., Scott, M.P. and Beachy, P.A. (
2000
) Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine.
Nature
 ,
406
,
1005
–1009.
140
Chavrier, P. and Goud, B. (
1999
) The role of ARF and Rob GTPases in membrane transport.
Curr. Opin. Cell Biol.
 ,
11
,
466
–475.
141
Jeong, J. and McMahon, A.P. (
2001
) Developmental biology. Vesicles and the spinal cord.
Nature
 ,
412
,
136
–137.
142
Choudhury, A., Dominguez, M., Puri, V., Sharma, D.K., Narita, K., Wheatley, C.L., Marks, D.L. and Pagano, R.E. (
2002
) Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann–Pick C cells.
J. Clin. Invest.
 ,
109
,
1541
–1550.
143
Zerial, M. and McBride, H. (
2002
) Rab proteins as membrane organizers.
Nat. Mol. Cell Biol.
 ,
2
,
107
–119.
144
Karpen, H.E., Bukowski, J.T., Hughes, T., Gratton, J.-P., Sessa, W.C. and Gailani, M.R. (
2001
) The Sonic hedgehog receptor Patched associates with caveolin-l in cholesterol-rich microdomains of the plasma membrane.
J. Biol. Chem.
 ,
276
,
19503
–19511.
145
Parton, R. (
1998
) Caveolae and caveolins.
Curr. Opin. Cell Biol.
 ,
8
,
542
–548.
146
Kurzchalia, T.V. and Parton, R.G. (
1999
) Membrane microdomains and caveolae.
Curr. Opin. Cell Biol.
 ,
11
,
424
–431.
147
Roy, S., Luetterforst, R., Harding, A., Apolloni, A., Etheridge, M, Stang, E., Rolls, B., Hancock, J.F. and Parton, R.G. (
1999
) Dominant-negative caveolin inhibits II-Pas function by disrupting cholesterol-rich plasma membrane domains.
Nat. Cell Biol.
 ,
1
,
98
–105.
148
Oh, P. and Schnitzer, J.E. (
2001
) Segregation of heterotrimeric G proteins in cell surface microdomains.
Mol. Biol. Cell
 ,
12
,
685
–698.
149
Incardona, J.P., Gruenberg, J. and Roelink, II. (
2002
) Sonic hedgehog induces the segregation of patched and smoothened in endosomes.
Curr. Biol.
 ,
12
,
983
–995.
150
Kuwabara, P.E. and Labouesse, M. (
2002
) The sterol-sensing domain: multiple families, a unique role?
Trends Genet.
 ,
18
,
193
–201.