hedgehog
Lipid modifications of hedgehog Veratrum alkaloids and distal inhibitors of cholesterol biosynthesis have been studied for more than 30 years as potent
teratogens capable of inducing cyclopia and other birth defects. These compounds specifically block the
Sonic hedgehog (Shh) signaling pathway. These teratogens do not prevent the sterol modification of Shh during
autoprocessing but rather inhibit the response of target tissues to Shh, possibly acting through the sterol sensing domain
within the Patched protein regulator of Shh response (Cooper, 1998).
During hedgehog biosynthesis, autocatalytic processing produces a lipid-modified amino-terminal fragment (residues 24-197 in the
human Sonic hedgehog sequence) that is responsible for all known hedgehog signaling activity and that is highly conserved
evolutionarily. Published in vitro biochemical studies using Drosophila Hedgehog identified the membrane anchor as a cholesterol, and
localized the site of attachment to the COOH terminus of the fragment. Full-length human Sonic hedgehog has been expressed in insect
and in mammalian cells and it has been determined by mass spectrometry that, in addition to cholesterol, the human hedgehog protein is
palmitoylated. Peptide mapping and sequencing data indicate that the palmitoyl group is attached to the NH2 terminus of the protein on
the alpha-amino group of Cys-24. Cell-free palmitoylation studies demonstrate that radioactive palmitic acid is readily incorporated
into wild type Sonic hedgehog, but not into variant forms lacking the Cys-24 attachment site. The lipid-tethered forms of hedgehog
shows about a 30-fold increase in potency over unmodified soluble hedgehog in a cell- based (C3H10T1/2 alkaline phosphatase
induction) assay, suggesting that the lipid tether plays an important role in hedgehog function. The observation that an extracellular
protein such as Shh is palmitoylated is highly unusual and further adds to the complex nature of this protein (Pepinsky, 1998).
Sonic hedgehog (Shh) is a signaling molecule that is important for defining patterning in the developing vertebrate central nervous system.
After translation, Shh autoproteolyzes and covalently attaches cholesterol to the newly formed carboxyl terminus, a modification crucial
for normal Shh signaling. Acute severe sterol deprivation in cultured Chinese hamster ovary cells
expressing mouse Shh (mShh) inhibits autoprocessing of the protein. These conditions have allowed the first detailed kinetic analysis of mShh
autoprocessing and turnover rates, revealing that cells rapidly degrade both precursor and mature mShh, regardless of sterol content:
sterol deprivation increases the rate of precursor degradation. Inhibition of mShh autoprocessing also allows the determination of the
subcellular localization of mShh precursor that accumulates in a pre-medial Golgi intracellular compartment. Finally, the precursor form of mShh that results from
autoprocessing inhibition appears to accumulate as an amide rather than a stable thioester (Guy, 2000).
Sonic hedgehog (Shh) signaling from the posterior zone of polarizing activity (ZPA) is the primary determinant of anterior-posterior polarity in the vertebrate limb field. An active signal is produced by an autoprocessing reaction that covalently links cholesterol to the N-terminal signaling moiety (N-Shhp), tethering N-Shhp to the cell membrane. The role played by this lipophilic modification was examined in Shh-mediated patterning of mouse digits. Both the distribution and activity of N-Shhp indicate that N-Shhp acts directly over a few hundred microns. In contrast, N-Shh, a form that lacks cholesterol, retains similar biological activity to N-Shhp, but signaling is posteriorly restricted. Thus, cholesterol modification is essential for the normal range of signaling. It also appears to be necessary for appropriate modulation of signaling by the Shh receptor, Ptc1 (K. E. Lewis, 2001).
Thus, cholesterol modification, while dispensable for signaling over a limited range (several cell diameters), is essential for the long-range action (up to 30 cell diameters) of N-Shhp. N-Shh (produced by a recombinant allele
generates a truncated Shh protein differing from the autoproteolytically cleaved wild-type N-Shhp protein by the absence of the cholesterol modification) has comparable polarizing activity when compared to that of wild-type N-Shhp, but its range is confined to a more posteriorly restricted target field. These conclusions are based on several observations: (1) direct analysis of the distribution of N-Shhp detects protein over approximately 300 µm, which is most of the posterior half of the limb bud at 10.5 dpc; (2) expression of key Shh target genes correlates closely with the distribution of ligand; (3) cholesterol modification of N-Shh is not essential for formation of posterior digits (digits 5 and 4), but does appear to be required for formation of anterior digits (digits 3 and 2); (4) the loss of anterior digits correlates with the loss of expression of targets in the anterior half of the Shh signaling domain; (5) reducing N-Shhp production, and consequently the levels of active signal, leads to a preferential loss of the most posterior digit (digit 5) while more anterior digits remain; (6) N-Shh is effective in hair, whisker, tooth, and lung development where it seems less likely that N-Shhp partakes in long-range signaling (K. E. Lewis, 2001).
What role does cholesterol play in effecting the long-range action of N-Shh? In the fly, movement of N-Hhp requires the activity of Dispatched, a Ptc-related membrane protein, in cells synthesizing N-Hhp, and also, in the target field, the activity of Tout-velu, a member of the Ext family of glycosyl transferases. The latter result has led to the proposal that movement of N-Hhp through its target field may require the function of a membrane proteoglycan whose synthesis is regulated by Tout-velu. One possible role of cholesterol could be the membrane presentation of N-Shhp to an Ext-dependent process: both Ext1 and Ext2 are expressed in the limb bud at the appropriate times. The cholesterol linkage could also facilitate the interaction of low levels of N-Shhp protein with its receptor, Ptc1, by concentrating N-Shhp in the membrane so that small amounts of ligand present at some distance from the ZPA can interact with Ptc1 and elicit a signaling response. The cholesterol linkage may also influence membrane trafficking. N-Hhp is known to associate with cholesterol-rich membrane microdomains, and Ptc1 is predicted to have sterol-sensing properties. One intriguing possibility is that the sterol-sensing domain of Ptc1 and cholesterol modification on N-Shhp may facilitate the interaction of both proteins by targeting them to the same membrane domain. Finally, in vitro studies indicate that some fraction of N-Shhp is also palmitoyl modified and that cholesterol modification is essential for effective addition of this second lipophilic modification. If this modification occurs in the limb, then N-Shh will be largely nonpalmitoylated and this might also affect membrane interactions. Interestingly, palmitoylation is associated with high signaling activity, yet in the limb, N-Shh appears to be highly active. Clearly, further work will be necessary to explore the independent role of palmitoylation in N-Shhp action (K. E. Lewis, 2001 and references therein).
In contrast to results in the mammalian limb, ectopic expression of an N-Hh transgene in the Drosophila wing disc, in the absence of N-Hhp, results in an anterior expansion of Hh targets, suggesting that cholesterol modification normally restricts the range of N-Hh action. There are a number of possible explanations for the different results. The fly experiments used GAL4-mediated ectopic expression to drive high levels of N-Hh expression, whereas this study examined an N-Shh allele that is under the same transcriptional control as the wild-type Shh gene. Further, the cellular context and distances are quite different. Direct signaling in the wing disc occurs within an epithelium over a distance of only 8-10 cell diameters. In the vertebrate limb, signaling occurs within a mesenchymal target field over approximately 30 cell diameters (K. E. Lewis, 2001 and references therein).
A second significant difference between the fly wing disc and mouse limb studies is in the role that cholesterol modification plays in Ptc interactions. It has been proposed that the cholesterol modification on N-Hhp is not essential for N-Hh binding to Ptc, but is required for its Ptc-dependent sequestration, an interaction that attenuates Hh signaling. In the mouse limb, reducing Ptc1 dosage in the presence of a single N-Shhp allele has no detectable effect on digit patterning. However, reducing the dosage of active Ptc1 alleles in N-Shh/Shhn (Shhn is a null allele of Shh) embryos restores anterior digits, albeit with inappropriate identity for their position. This result suggests that reducing Ptc1 levels leads to an anterior extension of the N-Shh signaling domain, presumably due to decreased sequestration of N-Shh by Ptc1. Thus, cholesterol modification is not absolutely required for Ptc1-mediated sequestration of N-Shh, but higher levels of Ptc1 may be required than those that suffice for sequestration of N-Shhp. In summary, it is likely that addition of cholesterol to N-Shh is required for robust feedback control by Ptc1 (K. E. Lewis, 2001).
The fact that Ptc1 can sequester N-Shh might help to explain a paradoxical result. In N-Shh/+ limbs, Ptc1, Gli1, Gremlin, Fgf4, and Bmp2 are all ectopically expressed in more anterior positions and digit 1 is duplicated. This is indicative of an increased range of Shh signaling. It is postulated that the movement of N-Shhp through a field of target cells depends on the balance between Ptc1-mediated sequestration of ligand and a proposed Ext-dependent transport process. In N-Shh/Shhn embryos, only N-Shh is produced. N-Shh is sequestered by Ptc1 but, due to the absence of a cholesterol modification, is not transported normally by an Ext-dependent process and only has a limited range of activity. Both N-Shh and N-Shhp are present in N-Shh/+ embryos; this alters the balance between sequestration and transport. Because N-Shh can only be sequestered and not transported, one would expect that relatively less N-Shhp is sequestered. As a consequence, more N-Shhp may be available for transport, resulting in an anterior extension of N-Shhp-mediated signaling. Testing this model will require an approach that distinguishes N-Shh and N-Shhp protein in the developing limb bud (K. E. Lewis, 2001).
While these data provide evidence that cholesterol modification of N-Shh is required for its long-range action, they do not directly address whether N-Shhp acts as a morphogen. However, they do provide some interesting insights into the complex interactions that regulate digit pattern. One of these is the AER/ZPA feedback loop that maintains proximo-distal outgrowth of the limb bud. In this, Shh regulates Fgf4 expression in the posterior AER, while Fgf4 and, most likely, other Fgf family members, are required for the maintenance of Shh expression. Shh has been shown to regulate Fgf4 indirectly by activating expression of a Bmp antagonist, Gremlin, in the distal limb mesenchyme. In N-Shh/Shhn limb buds, Gremlin and Fgf4 expression are both more posteriorly restricted than in control embryos. Thus, it is likely that cholesterol modification of Shh plays a critical role in the long-range actions that maintain expression of these genes throughout the digit-forming region of the limb field. The loss of their anterior expression domains most likely explains the absence of digits 3 and 2. The corresponding reduction in AER signaling leads to a marked reduction in N-Shh protein. Yet, despite this, levels are sufficient for the specification of digit 5. Since N-Shh actually has lower bioactivity than N-Shhp in cell culture, it seems unlikely that N-Shh activity is increased in N-Shh/Shhn embryos. Interestingly, when production of wild-type N-Shhp is reduced to comparable levels, by reducing the number of Shh-expressing cells in the ZPA, Fgf4 and Gremlin expression are less severely affected. Thus, N-Shhp is better able to support this critical signaling relay (K. E. Lewis, 2001).
Surprisingly, Shh null mutants form a single hindlimb digit that closely resembles a biphalangeal digit 1. Thus, although the anterior-most digit can be induced in response to ectopic Shh, as observed in some polydactylous mouse models, there may not be a direct requirement for Shh in its specification. Indeed, in N-Shh/Shhn limbs, digit 1 most likely arises from cells that are not directly responding to N-Shh. In these mutant limbs, 'high' level signaling correlates with the specification of digits 5 and 4; whereas the abrupt transition to more anterior cells that show no response appears to be associated with the formation of digit 1, which is separated from digit 4 by a metacarpal rudiment. It is suggested that digit 1 is specified by Bmp2, a target of Shh signaling in the limb. In the chick wing bud, the anterior-most digit (digit 2) can be induced by Bmp2 implants, apparently without a requirement for Shh. In Shh null embryos, although most Bmp2 expression is lost, the small posterior patch that remains could be sufficient for specification of digit 1 (K. E. Lewis, 2001).
Studies in the chick wing bud also indicate that if anterior mesenchyme cells receive a prior exposure to Shh, the response to Bmp2 is altered, resulting in the specification of a more posterior digit identity. However, only Shh can induce the most posterior digit (digit 4). Interestingly, Bmp2 expression is posteriorly restricted in both the AER and mesenchyme of N-Shh/Shhn embryos. Therefore, N-Shh can activate or maintain Bmp2 expression, but only locally. Thus, it is likely that Bmp2 signaling is markedly reduced and Shh signaling entirely absent in some regions of the digit field that may ordinarily respond to both signals. In contrast, when levels of wild-type N-Shhp are severely reduced in Shhc/Shhn;MHoxCre embryos, no activation of Bmp2 is observed in the mesenchyme at 10.5 dpc, although extensive expression is maintained within the AER. This suggests that N-Shhp levels may be below a critical threshold for activation of mesenchymal Bmp2 expression, but nevertheless digits 4/3, 2, and 1 do form. Thus, if Bmp2 signaling is required for the specification of anterior digit identities in these embryos, Bmp2 production in the AER may suffice (K. E. Lewis, 2001).
The adult basal ganglia arise from the medial and lateral ganglionic eminences, morphologically distinct structures found in the embryonic telencephalon. Temporal changes in sonic hedgehog responsiveness determine the sequential induction of embryonic neurons that populate the medial and lateral ganglionic eminences. LGE neurons do not express Nkx2.1. Shh-mediated differentiation of neurons that populate the lateral ganglionic eminence express different combinations of the homeobox-containing transcription factors Dlx, Mash1 and Islet 1/2. Dlx-expressing neurons are found in both the LGE and MGE, in both proliferating and differentiating zones. However, the numbers of Dlx-expressing progenitors were consistently greater in the LGE than the MGE. In addition there are more neurons co-expressing Dlx and Mash1 in the ventricular zone of LGE than the MGE. Dlx and Islet 1/2 are co-expressed in more differentiated neurons in both the LGE and MGE. Mash1 marks progenitors, whereas Islet 1/2 marks more differentiated neurons. These genes are present in distinct neurons in both the LGE and MGE. Individual Mash1- and Dlx2-expressing neurons incorporate BrdU (Kohtz, 2001).
N-terminal fatty-acylation of Shh significantly enhances its ability to induce the differentiation of rat E11 telencephalic neurons expressing Dlx, Islet 1/2 or Mash1. In utero injection of the E9.5 mouse forebrain with
retroviruses encoding wild-type Shh induces the ectopic expression of Dlx2 and severe deformities in the brain. Shh
containing a mutation at the site of acylation prevents either of these phenotypes. These results suggest that N-terminal fatty-acylation of Shh may
play an important role in Shh-dependent signaling during rodent ventral forebrain formation (Kohtz, 2001).
Although members of the Hedgehog (Hh) family were initially described as morphogens, many of these early conclusions were based on experiments that used non-physiologically relevant forms of Hh. Native Hh is modified by cholesterol (HhNp) and palmitate. These hydrophobic modifications are responsible for the ability of Hh to associate with cellular membranes, a property that initially appeared inconsistent with its ability to act far from its site of synthesis. Although it is now clear that Hh family members are capable of acting directly in long-range signaling, the form of Hh capable of this activity remains controversial. Evidence has been provided for a freely diffusible multimeric form of Sonic Hedgehog (Shh) termed s-ShhNp, which is capable of accumulating in a gradient fashion through a morphogenic field. Further evidence is now provided that s-ShhNp is the physiologically relevant form of Shh. The biological activity of freely diffusible ShhNp resides in its multimeric form, and this multimeric form is shown to be exceedingly stable, even at high concentrations of salt and detergent. Furthermore, the Shh-Shh interactions previously observed in the crystal structure of human Shh have now been validated; a highly conserved amino-terminal domain of Shh is important for the formation of s-ShhNp. Palmitoylation is required for s-ShhNp formation. Thus, these results identify both protein-protein and protein-lipid interactions that are required for s-ShhNp formation, and provide the first structural analyses supporting the existence of Shh multimers (Goetz, 2006).
Sonic hedgehog (Shh) secreted from the axial signaling centers of the notochord and prechordal plate functions as a morphogen in dorsoventral patterning of the neural tube. Active Shh is uniquely cholesterol-modified and the hydrophobic nature of cholesterol suggests that it might regulate Shh spreading in the neural tube. This study examined the capacity of Shh lacking the cholesterol moiety (ShhN) to pattern different cell types in the telencephalon and spinal cord. In mice expressing ShhN, low-level ShhN was detected in the prechordal plate and notochord, consistent with the notion that ShhN can rapidly spread from its site of synthesis. Surprisingly, it was found that low-level ShhN can elicit the generation of a full spectrum of ventral cell types in the spinal cord, whereas ventral neuronal specification and ganglionic eminence development in the ShhN/- telencephalon were severely impaired, suggesting that telencephalic patterning is more sensitive to alterations in local Shh concentration and spreading. In agreement, induction of Shh pathway activity and expression of ventral markers was observed at ectopic sites in the dorsal telencephalon indicative of long-range ShhN activity. These findings indicate an essential role for the cholesterol moiety in restricting Shh dilution and deregulated spread for patterning the telencephalon. It is proposed that the differential effect of ShhN in patterning the spinal cord versus telencephalon may be attributed to regional differences in the maintenance of Shh expression in the ventral neuroepithelium and differences in dorsal tissue responsiveness to deregulated Shh spreading behavior (Huang, 2007).
Genetic analyses in Drosophila have demonstrated that a
transmembrane protein Dispatched (Disp) is required for the release of
lipid-modified Hedgehog (Hh) protein from Hh secreting cells. Analysis of
Disp1 null mutant embryos has demonstrated that Disp1 plays
a key role in hedgehog signaling in the early mouse embryo. A hypomorphic allele in
Disp1(Disp1Delta2), was used to extend the
knowledge of Disp1 function in Hh-mediated patterning of the mammalian embryo.
Through genetic combinations with null alleles of patched 1 (Ptch1),
sonic hedgehog (Shh) and Indian hedgehog (Ihh), it has been demonstrated that Disp1 genetically interacts with Hh signaling components. Since
Disp1 activity is decreased a progressive increase in the
severity of hedgehog-dependent phenotypes is seen, that is further enhanced by
reducing hedgehog ligand levels. Analysis of neural tube patterning
demonstrates a progressive loss of ventral cell identities that most likely
reflects decreased Shh signaling, since Disp1 levels are attenuated. Conversely,
increasing available Shh ligand by decreasing Ptch1 dosage leads to
the restoration of ventral cell types in Disp1Delta2/Delta2 mutants.
Together, these studies suggest that Disp1 actively regulates the levels of
hedgehog ligand that are available to the hedgehog target field. Further, they
provide additional support for the dose-dependent action of Shh signaling in
patterning the embryo. Finally, in-vitro studies on Disp1 null mutant
fibroblasts indicate that Disp1 is not essential for membrane targeting or
release of lipid-modified Shh ligand (Tian, 2004).
Dispatched 1 (Disp1) encodes a twelve transmembrane domain protein that is required for long-range sonic hedgehog (Shh) signaling. Inhibition of Disp1 function, both by RNAi or dominant-negative constructs, prevents secretion and results in the accumulation of Shh in source cells. Measuring the Shh response in neuralized embryoid bodies (EBs) derived from embryonic stem (ES) cells, with or without Disp1 function, demonstrates an additional role for Disp1 in cells transporting Shh. Co-cultures with Shh-expressing cells revealed a significant reduction in the range of the contact-dependent Shh response in Disp1-/- neuralized EBs. These observations support a dual role for Disp1, not only in the secretion of Shh from the source cells, but also in the subsequent transport of Shh through tissue (Etheridge, 2010).
These results suggest that Ptch1 and Disp1 act in concert to mediate the transport of Shh through tissues. The similarities between Ptch1 and Disp1, such as their ability to trimerize and their putative proton channel, indicates that their function might be conserved with that of the resistance-nodulation-cell division (RND) family of proton-driven transporters in bacteria. In general, the role of Disp1 is in the secretion of Shh, whereas Ptch1 is involved in the uptake of Shh, and the function of both is necessary for long-range Shh signaling. These observations are consistent with a model in which reiterated secretion (by Disp1) and uptake (by Ptch1) are involved in the long-range transport of Shh. The non-directionality of this process, combined with the incomplete secretion of all internalized Shh, would sufficiently distribute Shh in a gradient away from the source (Etheridge, 2010).
Based on these results the following model is proposed. Disp1 is active in MVBs and mediates the loading of Shh onto exosome/lipoprotein-like particles, which are then secreted. These particles are specifically recognized by Ptch1 at the surface of adjacent cells, which traffics them into early/late endosomes, where the particles are disassembled. Shh can either be degraded, trafficked to the apical surface or trafficked into MVBs, where it would be loaded onto exosomes again for re-secretion. This model accounts for the putative function of Disp1 as a proton-driven transporter and explains the high molecular weight complex that Shh is found in outside of cells, the pH-dependent action of Disp1 and Ptch1 in the intracellular trafficking of Shh and the role of Disp1 in the re-secretion of Shh (Etheridge, 2010).
Intraflagellar transport (IFT) proteins were first identified as essential factors for the growth and maintenance of flagella in the single-celled alga Chlamydomonas reinhardtii. In a screen for embryonic patterning mutations induced by ethylnitrosourea, two mouse mutants, wimple (wim) and flexo (fxo) were identified that lack ventral neural cell types and show other phenotypes characteristic of defects in Sonic hedgehog signalling. Both mutations disrupt IFT proteins: the wim mutation is an allele of the previously uncharacterized mouse homolog of IFT172; and fxo is a new hypomorphic allele of polaris, the mouse homolog of IFT88. Genetic analysis shows that Wim, Polaris and the IFT motor protein Kif3a (see Drosophila Kinesin associated protein 3) are required for Hedgehog signalling at a step downstream of Patched1 (the Hedgehog receptor) and upstream of direct targets of Hedgehog signalling. These data show that IFT machinery has an essential and vertebrate-specific role in Hedgehog signal transduction (Huangfu, 2003).
Hip: a Hedgehog interacting protein The Hedgehog signaling pathway is essential for the development of diverse tissues during embryogenesis. Signaling is activated by binding of Hedgehog protein to the multipass membrane
protein Patched (Ptc). A novel component in the vertebrate signaling pathway,
name Hip (for Hedgehog-interacting protein) because of its ability to bind Hedgehog proteins, has been identifed. Hip encodes a membrane glycoprotein that binds to all three mammalian Hedgehog proteins
with an affinity comparable to that of Ptc-1. Hip-expressing cells are located next to cells that express each Hedgehog gene. Hip expression is induced by ectopic Hedgehog signaling and is lost in
Hedgehog mutants. Thus, Hip, like Ptc-1, is a general transcriptional target of Hedgehog signaling. Overexpression of Hip in cartilage, where Indian hedgehog (Ihh) controls growth, leads to a shortened skeleton, which resembles that seen when Ihh function is lost. These findings support a model in which Hip attenuates Hedgehog signaling as a result of binding to Hedgehog proteins: a negative regulatory feedback loop established in this way could thus modulate the responses to any Hedgehog signal (Chuang, 1999).
The integration of multiple signaling pathways is a key issue in several aspects of embryonic development. In this context, extracellular inhibitors of secreted growth factors play an important role, which is to antagonize specifically the activity of the corresponding signaling molecule. The Hedgehog-interacting protein (Hip) from Xenopus, previously described as a Hedgehog-specific antagonist in the mouse, interferes with Wnt-8 and eFgf/Fgf-8 signaling pathways as well. To address the function of Hip during early embryonic development, gain- and loss-of-function studies were performed in the frog. Overexpression of Xhip or mHip1 resulted in a dramatic increase of retinal structures and larger olfactory placodes primarily at the expense of other brain tissues. Furthermore, loss of Xhip function results in a suppression of olfactory and lens placode formation. Therefore, the localized expression of Xhip may counteract certain overlapping signaling activities, which inhibit the induction of distinct sensory placodes (Cornesse, 2005).
Overall amino acid identities are 76% and 75% in a comparison of Xhip with human Hip and mouse Hip1, respectively. The mammalian Hip proteins, the predicted Xenopus HIP harbors an N-terminal signal peptide and a hydrophobic domain at the C-terminus, which is necessary to anchor the mature protein to the cell membrane, four potential glycosylation sites and two EGF-like modules. A computer-aided search for additional structural motifs within the HIP protein revealed the existence of a 'six 4-stranded beta-sheet motif'. This motif was first described in soluble quinoprotein glucose dehydrogenase, but has been recently identified also in members of the low-density lipoprotein (LDL) receptor superfamily, such as Megalin and LRP6, and in the Wnt-inhibitory protein (WIF). WIF and LRP6 have been shown to interact with Wnt molecules, whereas Megalin binds to the N-terminal fragment of Sonic Hedgehog with high affinity (Cornesse, 2005).
Upregulation of Patched (Ptc), the Drosophila Hedgehog (Hh) receptor in response to Hh signaling, limits the range of signaling within a target field by sequestering Hh. In vertebrates, Ptch1 also exhibits ligand-dependent transcriptional activation, but mutants lacking this response show surprisingly normal early development. The identification of Hh-interacting protein 1 (Hhip1), a vertebrate-specific feedback antagonist of Hh signaling, raises the possibility of overlapping feedback controls. The significance of feedback systems in sonic hedgehog (Shh)-dependent spinal cord patterning was addressed. Mouse embryos lacking both Ptch1 and Hhip1 feedback activities exhibit severe patterning defects consistent with an increased magnitude and range of Hh signaling, and disrupted growth control. Thus, Ptc/Ptch1-dependent feedback control of Hh morphogens is conserved between flies and mice, but this role is shared in vertebrates with Hhip1. Furthermore, this feedback mechanism is crucial in generating a neural tube that contains appropriate numbers of all ventral and intermediate neuronal cell types (Jeong, 2005).
Unlike Drosophila, vertebrates have several Hh-binding proteins
that are transcriptionally regulated by Hh signaling; patched 2
(Ptch2) and Hh-interacting protein 1 (Hhip1) are positively
regulated, whereas growth arrest specific gene 1 (Gas1) is negatively
regulated. The role of Ptch2 or Gas1 in Hh-mediated
patterning processes during normal development has yet to be established.
Overexpression and loss-of-function studies in the mouse indicate that Hhip1,
a cell-surface glycoprotein, is an antagonist of Hh signaling;
Hhip1-/- embryos die soon after birth, owing to lung
defects indicative of overactive Hh signaling. However, other parts of the body where Hh signaling plays important roles, e.g. the limb, face and spinal cord, develop normally in Hhip1 mutants. Taken together, the mild phenotypes of both MtPtch1;Ptch1-/- and Hhip1-/- embryos suggest that Ptch1 and Hhip1 may be functionally redundant in providing feedback ligand dependent antagonism (LDA) to Hh ligands. Consistent with this view, removing one copy of Ptch1 allele in Hhip1-/- embryos (Hhip1-/-;Ptch1+/-) causes earlier lethality (around E12.5) and more severe lung and pancreas defects than those observed in Hhip1-/- embryos (Jeong, 2005).
Although the previous studies point to a role for Ptch1 and Hhip1 in attenuation of paracrine Hh signaling, they did not address the issue of how LDA might contribute to controlling the magnitude (pathway activity at a given position in the tissue) or range (total distance over which the pathway is activated) of a morphogen signaling gradient to generate a specific pattern. The best evidence for Shh acting as a morphogen comes from studies in the vertebrate spinal cord. Here, Shh is first produced from the notochord that underlies the neural tube, and directs the formation of floor plate which in turn expresses Shh. Shh from these two ventral midline sources forms a concentration gradient along the dorsoventral (DV) axis of the neural tube, and represses (Class I proteins) or induces (Class II proteins) expression of several homeodomain and basic helix-loop-helix transcription factors at different thresholds. Cross-repression between the transcription factors sharing a border further sharpens the boundaries of their territories to define five neural progenitor domains in the ventral half of the spinal cord (from ventral to dorsal, p3, pMN, p2, p1, p0). Finally, cells in each domain differentiate into specific types of neurons (from ventral to dorsal, V3, motoneuron [MN], V2, V1, V0) based on the combinations of transcription factors they express. Since Hh signaling controls the specification of individual progenitor domains by a direct and dose-dependent mechanism, changes in the expression of progenitor domain-associated transcription factors provide sensitive readouts for any perturbations in the Shh morphogen gradient (Jeong, 2005).
The role of negative feedback regulation on Hh signaling was investigated in vertebrates by analyzing mouse embryos that lack both Ptch1 and Hhip1 feedback mechanisms (MtPtch1;Ptch1-/-;Hhip1-/-). The findings indicate that the LDA mediated by these components plays a crucial role in controlling the magnitude and most likely the range of Shh morphogen signaling (Jeong, 2005).
Cdo and Boc (homologs of Drosophila ihog) encode cell surface Ig/fibronectin superfamily members linked to muscle differentiation. They are also targets and signaling components of the Sonic hedgehog (Shh) pathway. Although Cdo and Boc are generally negatively regulated by Hedgehog (HH) signaling, in the neural tube Cdo is expressed within the Shh-dependent floor plate while Boc expression lies within the dorsal limit of Shh signaling. Loss of Cdo results in a Shh dosage-dependent reduction of the floor plate. In contrast, ectopic expression of Boc or Cdo results in a Shh-dependent, cell autonomous promotion of ventral cell fates and a non-cell-autonomous ventral expansion of dorsal cell identities consistent with Shh sequestration. Cdo and Boc bind Shh through a high-affinity interaction with a specific fibronectin repeat that is essential for activity. A model is proposed where Cdo and Boc enhance Shh signaling within its target field (Tenzen, 2006).
Holoprosencephaly (HPE), a common defect of human forebrain development, is associated with haploinsufficiency for genes encoding Sonic Hedgehog (SHH) pathway components. Clinical expression of HPE is extremely variable, but it is rarely associated with defects in other SHH-dependent structures, such as limbs. This study reports that mice lacking the transmembrane protein Cdo, previously implicated in myogenesis, display HPE with strain-specific severity and without limb defects, modeling human HPE and implicating modifier genes as a cause of variability. Shh target gene expression is reduced in the developing forebrains of Cdo-/- mice, and Cdo positively regulates Shh signaling in vitro. These data suggest that Cdo enhances pathway activity in multiple ways, including at signal reception and via a parallel mechanism required at the level of Gli transcription factors. Specific Cdo domains required for its promyogenic effect are dispensable for its Shh signaling role, suggesting that Cdo has multiple, independent functions (Zhang, 2006).
Transcriptional targets of the hedgehog pathway Drosophila transcription factor cubitus interruptus (Ci) and its co-activator CRE (cAMP response
element)-binding protein (CBP) activate a group of target genes on the anterior-posterior border in response
to Hedgehog protein (Hh) signaling. In contrast, in the anterior region, the carboxyl-truncated form of Ci
generated by protein processing represses Hh expression. In vertebrates, three Ci-related transcription
factors (glioblastoma gene products [GLIs] 1, 2, and 3) have been identified, but their functional difference in Hh
signal transduction is unknown. Distinct roles are reported for GLI1 and GLI3 in Sonic hedgehog (Shh)
signaling. GLI3, which contains both repression and activation domains, acts both as an activator and a repressor,
as does Ci, whereas GLI1 contains only the activation domain. Consistent with this, GLI3, but not GLI1, is
processed to generate the repressor form. Transcriptional co-activator CBP binds to GLI3, but not to GLI1.
The trans-activating capacity of GLI3 is positively and negatively regulated by Shh and cAMP-dependent
protein kinase, respectively, through a specific region of GLI3, which contains the CBP-binding domain and
the phosphorylation sites of cAMP-dependent protein kinase. GLI3 directly binds to the Gli1 promoter and
induces Gli1 transcription in response to Shh. Thus, GLI3 may act as a mediator of Shh signaling in the
activation of the target gene Gli1 (Dai, 1999).
The regulation of the Gli genes during somite formation has
been investigated in quail embryos. The Gli genes are a
family encoding three related zinc finger transcription
factors, Gli1, Gli2 and Gli3, which are effectors of Shh
signaling in responding cells. A quail Gli3 cDNA has been
cloned and its expression compared with Gli1 and Gli2.
These studies show that Gli1, Gli2 and Gli3 are co-activated
at the time of somite formation, thus providing a
mechanism for regulating the initiation of Shh signaling in
somites. Embryo surgery and paraxial mesoderm explant
experiments show that each of the Gli genes is regulated by
distinct signaling mechanisms. Gli1 is activated in response
to Shh produced by the notochord, which also controls the
dorsalization of Gli2 and Gli3 following their activation by
Wnt signaling from the surface ectoderm and neural tube.
This surface ectoderm/neural tube Wnt signaling has both
negative and positive functions in Gli2 and Gli3 regulation:
these signals repress Gli3 in segmental plate mesoderm
prior to somite formation and then promote somite
formation and the somite-specific activation of Gli2 and
Gli3. These studies, therefore, establish a role for Wnt
signaling in the control of Shh signal transduction through
the regulation of Gli2 and Gli3, and provide a mechanistic
basis for the known synergistic actions of surface
ectoderm/neural tube and notochord signaling in somite
cell specification (Borycki, 2000).
A model is presented for Wnt and Shh
signaling in the control of Gli gene
activation during somite formation.
In this model, in the segmental plate mesoderm, Gli3
is maintained in a repressed state by
Wnt signaling through beta-catenin.
When anteriormost segmental plate
mesoderm initiates somite
formation, Wnt/beta-catenin signaling
undergoes a negative to positive
switch, leading to derepression of
Gli3, to the initiation of somite
formation, and to activation of the
somite-specific expression of Gli2
and Gli3. It is suggested that this switch in Wnt/beta-catenin
function might be mediated by transcription cofactors such as
Groucho, NLK and CtBP, factors that are known to control the transcription activities of beta-catenin/LEF1/tcf complexes in segmental plate mesoderm. The process of somite formation and the regulated expression of beta-catenin cofactors might be be under the control of the segmentation genes. Quantitative changes in Wnt signaling at the time of somite formation, resulting from the
activation of Wnt expression in the neural tube and loss of Wnt inhibitors in newly forming somites, would then mediate increased levels of beta-catenin. This high level of beta-catenin would participate in both the cytoplasmic
cell adhesion processes to initiate somite formation as well as in new beta-catenin/LEF1/tcf transcription complexes for Gli2 and Gli3 activation.
The Gli2 and Gli3 proteins produced in newly formed somites would then become activated as nuclear transcription factors in response to the Shh
that is produced by the notochord, leading to their participation in the activation of Shh response genes, including Gli1 and Ptc1 (Borycki, 2000).
A novel chick WD-protein, cSWiP-1, is described that is expressed in somitic mesoderm and developing limb buds as well as in other
embryonic structures where Hedgehog signaling has been shown to play a role. Using embryonic manipulations it has been shown that
in somites cSWiP-1 expression integrates two signals originating from structures adjacent to the segmental mesoderm: a positive
signal from the notochord and a negative signal from intermediate and/or lateral mesoderm. In explant cultures of somitic
mesoderm, Shh protein induces cSWiP-1, while a blocking antibody to Shh inhibits the induction of cSWiP-1 by the notochord.
These results show that the positive signal from the notochord is mediated by Shh. In limb buds cSWiP-1 is
upregulated by ectopic Shh. This occurs in about the same time period as upregulation of BMP2, placing cSWiP-1 among the
earliest markers for the change of limb pattern caused by ectopic Shh. A human homolog of cSWiP-1 is described as well as a
mouse gene, mSWiP-2, which is more distantly related to SWiP-1. This suggests that SWiP-1 belongs to a novel subfamily of
WD-proteins (Vasiliauskas, 1999).
Dorsoventral polarity of the somitic mesoderm is
established by competitive signals originating from
adjacent tissues. The ventrally located notochord provides
the ventralizing signals to specify the sclerotome, while the
dorsally located surface ectoderm and dorsal neural
tube provide the dorsalizing signals to specify the
dermomyotome. Noggin and SHH-N (the amino-terminal cleavage product of Sonic Hedgehog) have been implicated
as the ventralizing signals produced by the notochord. However, the
members of the WNT family of proteins
have been implicated as the dorsalizing signals derived
from the ectoderm and dorsal neural tube. When
presomitic explants are confronted with cells secreting
SHH-N and WNT1 simultaneously, competition to specify
the sclerotome and dermomyotome domains within the
naive mesoderm can be observed. Using these explant
cultures, evidence is provided that SHH-N competes with
WNT1, not only by upregulating its own receptor Ptc1, but
also by upregulating Sfrp2 (Secreted frizzled-related protein
2), which encodes a potential WNT antagonist. Among the
four known Sfrps, Sfrp2 is the only member expressed in
the sclerotome and upregulated by SHH-N recombinant
protein. SFRP2-expressing cells can
reduce the dermomyotome-inducing activity of WNT1 and
WNT4, but not that of WNT3a. Together, these results
support the model that SHH-N at least in part employs
SFRP2 to reduce WNT1/4 activity in the somitic mesoderm (Lee, 2000).
Hedgehog proteins have been implicated in the control of myogenesis in the
medial vertebrate somite. In the mouse, normal epaxial expression of the myogenic transcription factor gene myf5 is dependent on Sonic hedgehog. The interaction between Hedgehog signals, the expression of myoD family genes, including the newly cloned zebrafish myf5, and slow myogenesis have been examined in zebrafish. Sonic hedgehog is necessary for normal expression of both myf5 and myoD in adaxial slow muscle precursors, but not in lateral paraxial mesoderm. Expression of both genes is initiated normally in rostral presomitic mesoderm in sonic you mutants, which lack all Sonic hedgehog. Similar initiation continues during tailbud outgrowth when the cells forming caudal somites are generated. However, adaxial cells in sonic you embryos are delayed in terminal differentiation and caudal adaxial cells fail to maintain myogenic regulatory factor expression. Despite these defects, other signals are able to maintain, or reinitiate, some slow muscle development in sonic you mutants. In the cyclops mutant, the absence of floorplate-derived Tiggywinkle hedgehog and Sonic hedgehog has no discernible effect on slow adaxial myogenesis. Similarly, the absence of notochord-derived Sonic hedgehog and Echidna hedgehog in mutants lacking notochord delays, but does not prevent, adaxial slow muscle development. In contrast, removal of both Sonic hedgehog and a floorplate signal, probably Tiggywinkle hedgehog, from the embryonic midline in cyclops;sonic you double mutants essentially abolishes slow myogenesis. It is concluded that several midline signals, likely to be various Hedgehogs, collaborate to maintain adaxial slow myogenesis in the zebrafish embryo. Moreover, the data demonstrate that, in the absence of this required Hedgehog signaling, expression of myf5 and myoD is insufficient to commit cells to adaxial myogenesis (Coutelle, 2001).
Cerebellar granule cells are the most abundant neurons in the brain, and granule cell precursors (GCPs) are a common target of transformation in the pediatric brain tumor medulloblastoma. Proliferation of GCPs is regulated by the secreted signaling molecule Sonic hedgehog (Shh), but the mechanisms by which Shh controls proliferation of GCPs remain inadequately understood. DNA microarrays have been used to identify targets of Shh in these cells; Shh was found to activate a program of transcription that promotes cell cycle entry and DNA replication. Among the genes most robustly induced by Shh are cyclin D1 and N-myc. N-myc transcription is induced in the presence of the protein synthesis inhibitor cycloheximide, so it appears to be a direct target of Shh. Retroviral transduction of N-myc into GCPs induces expression of cyclin D1, E2F1, and E2F2, and promotes proliferation. Moreover, dominant-negative N-myc substantially reduces Shh-induced proliferation, indicating that N-myc is required for the Shh response. Finally, cyclin D1 and N-myc are overexpressed in murine medulloblastoma. These findings suggest that cyclin D1 and N-myc are important mediators of Shh-induced proliferation and tumorigenesis (Oliver, 2003).
Sonic hedgehog (Shh) acts as a morphogen to mediate the specification of distinct cell identities in the ventral neural tube through a Gli-mediated (Gli1-3) transcriptional network. Identifying Gli targets in a systematic fashion is central to the understanding of the action of Shh. This issue was examined in differentiating neural progenitors in mouse. An epitope-tagged Gli-activator protein was used to directly isolate cis-regulatory sequences by chromatin immunoprecipitation (ChIP). ChIP products were then used to screen custom genomic tiling arrays of putative Hedgehog (Hh) targets predicted from transcriptional profiling studies, surveying 50-150 kb of non-transcribed sequence for each candidate. In addition to identifying expected Gli-target sites, the data predicted a number of unreported direct targets of Shh action. Transgenic analysis of binding regions in Nkx2.2, Nkx2.1 (Titf1) and Rab34 established these as direct Hh targets. These data also facilitated the generation of an algorithm that improved in silico predictions of Hh target genes. Together, these approaches provide significant new insights into both tissue-specific and general transcriptional targets in a crucial Shh-mediated patterning process (Volkes, 2007).
Hedgehog and butterfly eyespot evolution The origin of new morphological characters is a long-standing problem in evolutionary biology. Novelties
arise through changes in development, but the nature of these changes is largely unknown. In butterflies,
eyespots have evolved as new pattern elements that develop from special organizers called foci. Formation
of these foci is associated with novel expression patterns of the Hedgehog signaling protein, its receptor
Patched, the transcription factor Cubitus interruptus, and the engrailed target gene, all of which break the conserved
compartmental restrictions on this regulatory circuit in insect wings. Redeployment of preexisting
regulatory circuits may be a general mechanism underlying the evolution of novelties. hh is expressed in all cells of the posterior compartment of the butterfly wing disc, as it is in Drosophila, but hh transcript levels are increased in a striking pattern in cells just outside of the subdivision midlines at specific positions along the proximodistal axis of the wing. These domains of increased hh transcription flank cells that have the potential to form foci. Higher levels of hh transcripts accumulate specifically in cells that flank the developing foci. In the presence of high levels of Hh, Patched function is inhibited, resulting in the accumulation of the activator form of Ci. Because ptc is a direct target of Ci, cells that receive and transduce the Hh signal have increased levels of ptc transcription. Activation of ptc transcription, accompanied by the accumulation of Ci protein occurs in cells that are flanked by the domains of highest hh transcription and are destined to become eyespot foci. these results indicate that the Hh signal is received and transduced by cells that will differentiate as foci. These expression patterns break the A/P compartmental restrictions on gene expression known in Drosophila. During the course of eyespot evolution, there is a relaxation of the strict En-mediated repression of ci that occurs in the posterior compartment of Drosophila. During focal establishment, en and invected are targets, rather than inducers of Hh signaling. In most species of butterflies, eyespots are found only in the posterior compartment of the wing. But in those species in which eyespots are found in the anterior compartment, both En/Inv and Ci are coexpressed in eyespot foci, including the one in the anterior compartment. Thus the expression of the Hh signaling pathway and en/inv is associated with the development of all eyespot foci and has become independent of A/P compartmental restrictions. It is suggested that during eyespot evolution, the Hh-dependent regulatory circuit that establishes foci is recruited from the circuit that acts along the A/P boundary of the wing. This recruitment of an entire regulatory circuit through changes in the regulation of a subset of components increases the facility with which new developmental functions can evolve and may be a general theme in the evolution of novelties within extant structures (Keys, 1999).
The Hedgehog (Hh) pathway plays important roles during embryogenesis and carcinogenesis. This study shows that ablation of the mouse Suppressor of fused (Sufu), an intracellular pathway component, leads to embryonic lethality at ~E9.5 with cephalic and neural tube defects. Fibroblasts derived from Sufu−/− embryos showed high Gli-mediated Hh pathway activity that could not be modulated at the level of Smoothened and could only partially be blocked by PKA activation. Despite the robust constitutive pathway activation in the Sufu−/− fibroblasts, the GLI1 steady-state localization remained largely cytoplasmic, implying the presence of an effective nuclear export mechanism. Sufu+/− mice develop a skin phenotype with basaloid changes and jaw keratocysts, characteristic features of Gorlin syndrome, a human genetic disease linked to enhanced Hh signaling. These data demonstrate that, in striking contrast to Drosophila, in mammals, Sufu has a central role, and its loss of function leads to potent ligand-independent activation of the Hh pathway (Svärd, 2006).
Sufu knockouts are embryonic lethal and show strong similarities with Ptch1 knockouts, and Hh signaling is strongly activated in a ligand-independent manner in Sufu−/− cells. Moreover, Sufu+/− mice develop a skin phenotype with many features found in Gorlin syndrome. Furthermore, no support for a direct role of Sufu in the Wnt pathway was found (Svärd, 2006).
There is a remarkable agreement between the Sufu−/− and Ptch1−/− embryos in terms of the spatiotemporal expression pattern of all the markers examined both in the whole-mount embryo and neural tube analysis. High-level expression of the Ptch1 and Gli1 target genes in the Sufu−/− embryos suggests that Sufu is endowed with a strong repressor activity, removal of which causes ectopic activation of the Hh pathway. It should be emphasized that in the ventralized spinal cord from the Sufu−/− embryos, prominent ectopic expression of target genes such as FoxA2 and Nkx2.2, which require the highest levels of Hh signaling, is evident. Evidently, this high Hh activity is dominant in patterning the neural tube over the dorsalizing signals mediated by the Bmp proteins, which normally act in an antagonizing manner to Shh in the neural tube. Taken together, this indicates that mammalian Sufu, in striking contrast to the situation in Drosophila, is an equally strong repressor of the Hh pathway, as Ptch1 and removal of either one is sufficient to induce a similar high level of cell autonomous Hh signaling (Svärd, 2006).
Based on studies of the effects of activators and inhibitors (SAG and cyclopamine, respectively) of Smo activity in the Sufu−/− MEFs, it appears that these cells have uncoupled the upstream ligand-dependent activation of Smo from the downstream Gli activity. This observation further suggests that constitutive activation of Hh signaling in cells lacking Sufu is determined by their intrinsic competence for Hh response and not actual exposure to Hh ligands. This independence of Hh ligand is further demonstrated in mouse Sufu−/−; Shh−/− double mutant embryos, which appear morphologically similar to the Sufu−/− mutants. It has similarly been shown that downregulation of Sufu levels by RNAi in NIH-3T3 or Smo−/− cells results in activation of Hh signaling (Svärd, 2006).
The severe functional consequences and the high level of Hh signaling caused by deleting the Sufu gene in the mouse was surprising given the overall conservation of the Hh pathway during evolution and the lack of an altered phenotype in the corresponding Drosophila mutants. Recently, another unanticipated result was revealed when a possible mouse ortholog of Drosophila Fu was targeted and no apparent Hh-dependent phenotypes during embryonic development were seen. In contrast, Drosophila fu mutants are embryonic lethal, and, in zebrafish, MO knockdown of Fu abrogates Hh-dependent specification of myotome cell types. Moreover, genetic inactivation or MO knockdown of Sufu in zebrafish results in a detectable Hh-related phenotype, but the effect is much weaker than that caused by elimination of Ptc. In contrast, the data presented here indicate that, in the mouse, null mutations of Sufu and Ptch1 produce equally strong perturbations of the Hh pathway. This suggests that not only has the Hh pathway evolved differently in vertebrates compared to invertebrates, but also within vertebrates, since mammals and teleosts show divergence in the pathway. Another such example of differences in the Hh pathway is illustrated by the recent finding that intraflagellar transport (IFT) proteins are required for transduction of the Hh signal in the mouse, whereas analysis of IFT mutants in zebrafish and Drosophila did not reveal any Hh-dependent phenotypes. This fundamental divergence in regulatory mechanisms has several important implications. In the fly, it is presently believed that Su(fu) together with the atypical kinesin Cos2 represent intracellular negative regulators of Hh signaling and that Cos2 plays a major role by tethering the transcriptional effector Ci to microtubules and, in the absence of ligand, promotes processing of Ci to its truncated repressor form. Upon ligand stimulation, a direct interaction between the C-terminal tail of Smo and Cos2 is enhanced, leading to inhibition of Ci processing and nuclear availability of activated full-length Ci. Su(fu), on the other hand, resides mostly in a separate intracellular complex also containing Ci and functions both to tether Ci in the cytoplasm and to inhibit activated full-length Ci. However, even in the absence of Su(fu), Cos2 is able to sequester Ci in the cytoplasm and maintain normal regulation of Hh signaling, whereas, in the absence of Cos2, constitutive activation of Ci ensues. In the mouse, Sufu loss alone leads to complete activation of the Hh signaling pathway, and mice therefore may not utilize a Cos2-like activity that in Drosophila is sufficient to maintain Hh pathway regulation in the absence of Sufu. Thus, when Sufu is removed, unrestrained Gli-mediated transcriptional activation is allowed by eliminating repression mechanisms normally inhibiting Gli activity. In mammalian cells, Gli1 and Gli2 serve mainly as positive transcriptional regulators, whereas Gli3 is processed and, for the most part, acts as a repressor. Since Sufu can interact with the N-terminal part of Gli proteins and the Sufu binding motif is retained in the Gli3 repressor form, it appeared likely that Sufu also exerts a negative influence on the repressor activity of Gli3. Since a strong activation of Hh signaling was found in cells lacking Sufu in vivo and in vitro, it is proposed that Gli-mediated transcriptional activation is dominant over Gli-mediated repression. Alternatively, this result may be due to the fact that Sufu, in analogy to the situation with Gli1, must interact with two different domains on Gli3, one of which is missing in the Gli3 repressor form, to achieve effective repression. This result may also be due to differential expression domains for the activating and repressive Gli isoforms (Svärd, 2006).
The results presented in this study highlight an important evolutionary divergence in the basic regulatory circuits controlling Hh signaling, a pathway having a key role in development and human disease, including cancer, thereby illustrating the need to understand in detail the function of the pathway in mammalian systems. The unanticipated role of Sufu as a specific and potent repressor of Hh signaling also opens new therapeutic avenues to control deregulated Hh pathway activation, for example, by development of Sufu mimetics (Svärd, 2006).
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continued:
Biological Overview
| Regulation
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| Protein Interactions
| Developmental Biology
| Effects of Mutation
| References
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