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Human Molecular Genetics, 2003, Vol. 12, Review Issue 1 R15-R25
DOI: 10.1093/hmg/ddg058
© 2003 Oxford University Press

How a Hedgehog might see holoprosencephaly

Erich Roessler and Maximilian Muenke*

Medical Genetics Branch, National Human Genome Research Institute, Bldg 10, 10C103, National Institutes of Health, Bethesda, MD 20892-1852, USA

Received December 2, 2002; Accepted January 6, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 THE SHH SIGNALING PATHWAY...
 HEDGEHOG AND HUMAN DISEASE
 COMPONENTS OF THE Hh...
 HPE AND CHOLESTEROL
 FUTURE DIRECTIONS
 REFERENCES
 
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
TOP
 ABSTRACT
 INTRODUCTION
THE SHH SIGNALING PATHWAY...
 HEDGEHOG AND HUMAN DISEASE
 COMPONENTS OF THE Hh...
 HPE AND CHOLESTEROL
 FUTURE DIRECTIONS
 REFERENCES
 
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 (2–7) (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 (8–10) (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 (11–12). 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 (15–17), and participating in the development of the pituitary gland (18–22), neural crest cells (23), midbrain (24), cerebellum (25–27), oligodendrocytes (28), eye (29–33) and face (34–38). 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).




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  		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 (2–6) (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.

 
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
TOP
 ABSTRACT
 INTRODUCTION
 THE SHH SIGNALING PATHWAY...
HEDGEHOG AND HUMAN DISEASE
 COMPONENTS OF THE Hh...
 HPE AND CHOLESTEROL
 FUTURE DIRECTIONS
 REFERENCES
 
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.


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  		Table 1. Components of Hedgehog signaling
 

   HEDGEHOG AND HUMAN DISEASE
TOP
 ABSTRACT
 INTRODUCTION
 THE SHH SIGNALING PATHWAY...
 HEDGEHOG AND HUMAN DISEASE
COMPONENTS OF THE Hh...
 HPE AND CHOLESTEROL
 FUTURE DIRECTIONS
 REFERENCES
 
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 (13,49,50,55,60,66,80–89,96). 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 (106–111), or parallel pathways is presently unknown.



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  		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).

 

   COMPONENTS OF THE Hh PATHWAY ARE CANDIDATES FOR HPE
TOP
 ABSTRACT
 INTRODUCTION
 THE SHH SIGNALING PATHWAY...
 HEDGEHOG AND HUMAN DISEASE
 COMPONENTS OF THE Hh...
HPE AND CHOLESTEROL
 FUTURE DIRECTIONS
 REFERENCES
 
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 (113–117). 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 (51–54) 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.




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  		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.

 



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  		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).

 
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 (128–131,148), 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
TOP
 ABSTRACT
 INTRODUCTION
 THE SHH SIGNALING PATHWAY...
 HEDGEHOG AND HUMAN DISEASE
 COMPONENTS OF THE Hh...
 HPE AND CHOLESTEROL
FUTURE DIRECTIONS
 REFERENCES
 
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 (140–143). 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 (144–146). 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
TOP
 ABSTRACT
 INTRODUCTION
 THE SHH SIGNALING PATHWAY...
 HEDGEHOG AND HUMAN DISEASE
 COMPONENTS OF THE Hh...
 HPE AND CHOLESTEROL
 FUTURE DIRECTIONS
REFERENCES
 
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.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +1 3014028167; Fax: +1 3014807876; Email: muenke{at}nih.gov Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 THE SHH SIGNALING PATHWAY...
 HEDGEHOG AND HUMAN DISEASE
 COMPONENTS OF THE Hh...
 HPE AND CHOLESTEROL
 FUTURE DIRECTIONS
 REFERENCES
 

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