InteractiveFly: GeneBrief

interference Hedgehog: Biological Overview | References

The iHog gene (interference hedgehog), identified by RNA interference in Drosophila cultured cells, encodes a type 1 membrane protein shown in this study to bind and to mediate response to the active Hedgehog (Hh) protein signal. ihog mutations produce defects characteristic of Hh signaling loss in embryos and imaginal discs, and epistasis analysis places ihog action at or upstream of the negatively acting receptor component, Patched (Ptc). The first of two extracellular fibronectin type III (FNIII) domains of the Ihog protein mediates a specific interaction with Hh protein in vitro, but the second FNIII domain is additionally required for in vivo signaling activity and for Ihog-enhanced binding of Hh protein to cells coexpressing Ptc. Other members of the Ihog family, including Drosophila Boi and mammalian CDO and BOC, also interact with Hh ligands via a specific FNIII domain, thus identifying an evolutionarily conserved family of membrane proteins that function in Hh signal response (Yao, 2006; full text of article).

Activity of the Hedgehog (Hh) signaling pathway is required for normal regulation of cell proliferation and differentiation during a diverse array of patterning events, ranging from embryonic segmentation in insects to neural tube differentiation in vertebrates. The Hh pathway also plays a homeostatic role in postembryonic tissues through its regulation of stem cell and precursor cell proliferation, and aberrant activity of the Hh pathway is associated with the initiation and growth of a variety of deadly human cancers. Despite the importance of this signaling pathway in development and disease, however, significant gaps remain in the understanding of Hh signal transduction (Yao, 2006).

The mature Hh ligand is derived from the Hh protein precursor by autoprocessing and lipid modification that generates an amino-terminal signaling peptide (HhN) dually modified by palmitoyl and cholesteryl adducts. Pathway activity is triggered by stoichiometric binding of this dually lipidated Hh ligand to Ptc, a 12-transmembrane transporter-like protein that in the absence of Hh acts catalytically to suppress activity of the seven-transmembrane protein Smoothened (Smo). The release of Smo inhibition by HhN binding to Ptc permits activation of an intracellular signal cascade that in turn activates latent cytoplasmic transcription factors, the zinc finger protein Ci (Cubitus interruptus) in Drosophila or the homologous Gli proteins in vertebrates, and these proteins subsequently stimulate transcriptional activation of pathway target genes (Yao, 2006).

The identification of Ptc as a Hh receptor is based on genetic epistasis studies demonstrating that Ptc functions downstream of Hh and upstream of Smo and other pathway components. Biochemical studies have further demonstrated that ShhN, the amino-terminal signaling domain of the mammalian Hh family member Sonic hedgehog, binds with high affinity to mammalian cells expressing murine Patched (mPtch). The signaling potencies of mutationally altered ShhN proteins in this cultured cell-based assay correlate with their apparent mPtch binding affinities. In addition to its cell-autonomous role in pathway activation, Ptc/HhN binding also results in sequestration and degradation of the HhN protein, thus preventing movement to distal cells and restricting the tissue range of Hh action in a cell nonautonomous fashion (Yao, 2006).

Despite the importance of Ptc/Hh binding in regulating both Hh tissue distribution and cell autonomous response to the Hh signal, it is curious that Ptc protein is mainly localized in vesicular structures within the cytoplasm, with barely detectable levels in the plasma membrane. The Hh receptor has not been biochemically defined and isolated, and the possibility remains that other pathway components may be involved in Hh binding to target cells. An RNAi-based genome-scale screen has recently identified several candidates for such action, including Dally-like (Dlp), a member of the glypican family of heparan sulfate proteoglycans (HSPGs) (Yao, 2006).

A second membrane-associated Hh pathway component, previously known as CG9211 and here referred to as ihog (interference hedgehog), was identified in this RNAi screen. RNAi targeting of Ihog in cultured cells reveals its requirement for Hh response in cultured cells, and genetic analysis shows that loss of ihog function in embryos and imaginal discs leads to patterning defects associated with loss or reduction of Hh pathway activation. The ihog gene encodes a type I transmembrane protein with four immunoglobulin-like (Ig) domains and two fibronectin type III (FNIII) domains in its extracellular region. A biochemical interaction between HhN and the Ihog extracellular domain maps to the first FNIII domain (FN1), but signaling function in vivo requires the additional presence of the second FNIII domain (FN2). The binding of HhN to cultured cells is greatly enhanced by coexpression of Ptc and Ihog, and both FN1 and FN2 of Ihog are required to mediate this synergistic effect. A Drosophila homolog and two mammalian homologs can also interact directly with Hh ligands through a specific FNIII domain, suggesting that proteins in this family constitute conserved components of the Hh signal reception machinery (Yao, 2006).

The embryonic and imaginal phenotypes of ihog mutations are somewhat weaker than those of mutations affecting other pathway components such as hh and smo. Of possible relevance, the Drosophila genome contains a related gene, boi, (brother of ihog). The Ihog and Boi proteins, as well as the related mammalian proteins BOC and CDO (Kang, 2002; Aglyamova, 2007; Okada, 2006; Tenzen; 2006), contain amino-terminal clusters of four or five immunoglobulin (Ig) domains followed by two or three fibronectin type III (FNIII) domains, with a predicted transmembrane domain and cytoplasmic carboxy-terminal cytotail that displays no apparent homology to other family members or other proteins. The two FNIII domains of the Ihog and Boi proteins are particularly well conserved, with 54% amino acid identity as compared to 42% identity in the Ig domains or 45% identity between the two proteins as a whole, and these domains are most closely related to the second and third FNIII domains of BOC and CDO. Transfection of a Boi expression construct can rescue RNAi-mediated loss of Ihog in cl-8 cells; and Boi, like Ihog, appears to function at or upstream of the level of Ptc. Boi expression is also capable of rescuing RNAi-mediated loss of dlp function in cl-8 cells (Yao, 2006).

Ihog protein is detected as a single species of about 100KD in Western blots of lysates from Drosophila embryos and from various Drosophila cultured cell lines. Ihog is ubiquitously expressed throughout embryogenesis and in the wing imaginal disc. Confirming this distribution, ihog RNA was found by RT-PCR to be expressed in embryos, the wing imaginal disc, and in cl-8 and S2-R+ cultured cells. Two isoforms of boi cDNA were characterized, one full-length and the other lacking coding sequences for the signal sequence and the Ig domains. The full-length boi transcript, like the ihog transcript, is expressed in embryos, in the wing imaginal disc, and in S2 cells, but in cl-8 cells only the defective RNA is detected (Yao, 2006).

Given these nearly identical functional properties of Boi and Ihog proteins in cl-8 cultured cells, it seems likely that Boi expression may at least partially compensate for loss of ihog in embryos and imaginal discs and thus account for the intermediate phenotypes of ihog mutations. The relatively strong effect of ihog RNAi in cl-8 cells, where its pathway role was discovered, may be due to the absence of boi expression. Both ihog and boi are expressed in S2 cells, where hyperphosphorylation of Smo provides a rapid and direct readout of Hh pathway activation. Combined RNAi against both gene products indeed produced additive effects in this assay. A similar RNAi-based analysis in embryos was precluded by stability of the Ihog protein, and no boi mutation or nearby transposable element for imprecise excision is currently available (Yao, 2006).

Ihog function at or upstream of the level of Ptc suggests a possible role for Ihog as a central or accessory component of the Hh receptor. This possibility was further investigated by examining the subcellular localization of Ihog and by asking whether it can interact with the Hh protein. Consistent with its predicted structure, the Ihog protein appears to be localized mainly to the surface of embryonic cells and can be detected on the cell surface in both permeabilized and nonpermeabilized S2-R+ cultured cells (Yao, 2006).

The Ihog protein likely is modified by glycosylation, as it was enriched by Concanavalin A (Con A) Sepharose chromatography. Ihog also can be cross-linked to membrane-impermeable biotin, indicating that it is a cell surface glycoprotein. The levels of Ihog protein in cl-8 cells were not affected by Hh stimulation, consistent with its lack of spatial modulation in embryonic and imaginal disc expression (Yao, 2006).

Hh binding by Ihog raises the question of how Ihog may interact with Ptc, a previously characterized Hh receptor. Binding was measured of HhN enzymatically tagged by fusion to Renilla luciferase (HhN-Ren) to intact cells coexpressing Ihog and Ptc; tagging of HhN at this site is compatible with signaling activity, although the resulting HhN-Ren protein, like the HhN in conditioned medium used for signaling assays, is not cholesterol modified. COS1 monkey cells were selected for these experiments to avoid potential interference from endogenous Drosophila proteins that may function in binding. It was found that transfection for expression of Ptc only slightly increased HhN-Ren binding above endogenous levels and that transfection for expression of Ihog increased binding less than 2-fold. Cotransfection for expression of Ptc and Ihog in contrast produced a more than ten-fold increase in binding of HhN-Ren, far greater than the additive effects of Ptc and Ihog alone. Similar effects were noted for expression of Boi, alone and together with Ptc (Yao, 2006).

S2-R+ cells offer the possibility of reducing background binding by RNAi-mediated knockdown of specific endogenous proteins through bathing of cells in dsRNA. Significant binding of HhN-Ren to untransfected S2-R+ cells was noted, and RNAi targeting of ihog or ptc reduced this basal binding 2- to 5-fold, indicating that endogenous Ptc and Ihog indeed contribute to basal HhN-Ren binding activity. It was also found that coexpression of Ihog and Ptc synergistically increased HhN-Ren binding to levels many fold higher than those produced by expression of either protein alone. To more accurately compare synergistic binding to that of Ptc or Ihog individually, expression of one component with RNAi was reduced while transfecting for expression of the other. It was found that HhN-Ren binding with coexpression of Ihog and Ptc was 59-fold higher than that produced by combined Ihog expression and RNAi-mediated knockdown of ptc and 30-fold higher than that seen with combined expression of Ptc and RNAi-mediated knockdown of ihog (Yao, 2006).

The two closest vertebrate homologs of ihog are Cdo and Boc (Kang, 2002; Aglyamova, 2007; Okada, 2006; Tenzen; 2006). Cdo-/- mice display mild holoprosencephaly (HPE) (Cole, 2003), more severe forms of which are associated with embryonic loss of Hh signaling. Using an established reporter assay involving a Gli-luciferase reporter construct in NIH 3T3 cells, it was found that shRNA constructs targeting either Cdo or Boc, but not a control shRNA construct, inhibited cell response to ShhN, thus suggesting that HPE in Cdo-/- mice may be due to defective Hh signal response (Yao, 2006).

Upon testing of various human Fc fusions for a direct interaction with ShhN in vitro, it was found that only the third FNIII domain of CDO and BOC can precipitate ShhN from conditioned media. Thus, although CDO and BOC differ from their Drosophila counterparts in number of FNIII domains and, in the case of CDO, in the number of Ig domains, these mammalian proteins appear to retain the ability to interact with a Hh ligand via a specific FNIII domain (Yao, 2006).

Previous studies of ShhN binding utilized a series of altered proteins with structure-based alanine substitutions for four groups of conserved surface residues (A, B, C, and D). Using the Fc fusion to the FN3 domain of CDO, it was found that surface A and B mutants are as efficiently coprecipitated as wild-type ShhN but that the surface C mutant was not coprecipitated and the surface D mutant only poorly precipitated. Similar results were noted for FN3 of BOC. The ShhN determinants required for in vitro interaction with the FN3 domains of CDO and BOC thus are very similar to those required for binding of ShhN to cells expressing Ptch, consistent with the possibility of a cooperative role for CDO/BOC proteins with Ptch in binding and reception of the Hh signal in mammals (Yao, 2006).

Wing imaginal disc clones lacking Ihog function display a cell-autonomous loss or reduction in the expression of Hh pathway targets. Epistasis analysis in cultured cells revealed that Hh pathway activation by RNAi of cos2 or by expression of a constitutively activated form of Smo is not blocked by RNAi of ihog and that RNAi of ptc reverses the loss of Hh response caused by ihog RNAi. Furthermore, the ptc embryonic cuticle phenotype prevails in ihog ptc double mutant embryos, placing Ihog function at or upstream of the level of Ptc. Ihog protein is predominantly localized at the cell surface and binds specifically to Hh in vitro, as demonstrated biochemically and by crystallographic analysis of an Ihog:Hh complex (McLellan, 2006). Finally, Hh binding to cultured cells is synergistically augmented by coexpression of Ihog with Ptc. On the basis of its cell-autonomous role in mediating Hh signal response, its localization to the cell surface, and its binding to the Hh signal directly and in synergy with Ptc, it is concluded that Ihog functions as a component of the Hh signal reception machinery. This role appears to be conserved in mammals, since mutation of the mouse homolog Cdo produces holoprosencephaly (Cole, 2003), and CDO and its close relative BOC both bind to the Shh signal in vitro and contribute to Shh response in cultured cells (Yao, 2006).

The embryonic and imaginal disc phenotypes of ihog mutations are intermediate in severity, likely due to overlapping expression of boi, a closely related member of the Ihog family whose Hh pathway function in cultured cells can substitute for that of ihog. Boi interacts with HhN in vitro and synergistically with Ptc in vivo. Residual pathway function in embryos and imaginal disc cells lacking ihog may well be due to expression of boi in these tissues, and more severe phenotypes thus would be predicted to result from the combined absence of Boi and Ihog function, once boi mutations become available. Consistent with this prediction, RNAi-mediated targeting in S2-R+ cells of both ihog and boi produced a stronger effect on Smo accumulation and phosphorylation than targeting of either gene alone (Yao, 2006).

Although the binding interactions between Ihog and HhN are mediated by FN1 in vitro (McLellan, 2006), in vivo signaling function depends upon the additional presence of FN2. This requirement for FN2 likely relates to the finding that combined expression of Ihog and Ptc in cells produces 30- to 60-fold higher levels of HhN binding than either protein alone; this synergistic binding of Hh, like signaling, requires the presence of FN1 and FN2. Much, though not all, Ptc protein is localized in intracellular vesicles, and neither Ihog nor Ptc proteins detectably change localization when coexpressed. Despite this predominant intracellular localization for Ptc, HhN-Ren binding by Ptc/Ihog nevertheless appears to occur on the surface, since binding studies were carried out at 4°C, preventing endocytosis, and bound HhN-Ren is sensitive to treatment with trypsin. The basis of synergistic binding seems likely to be an increased affinity for Hh, as Scatchard analysis of HhN-Ren binding to cells expressing Ihog and Ptc yields an estimate of HhN affinity as much as two orders of magnitude stronger than that for Ihog alone (McLellan, 2006). This increase in affinity could be based on simultaneous interaction of Hh with Ptc and Ihog, since the Ptch/ShhN interface defined by mutational analysis is adjacent to and partially overlapping with the Ihog/Hh interface identified crystallographically (McLellan, 2006) (Yao, 2006).

The Ihog family of proteins has previously been studied primarily in mammals, where CDO and BOC were identified as members of a distinctive subgroup of the Ig/FNIII family. One of the distinguishing features of the Ihog/CDO subgroup is a higher degree of conservation within their membrane-proximal FNIII domains as compared to the Ig domains, which contrasts with higher conservation for the Ig domains in the Robo receptors and other subgroups of the larger Ig/FNIII family. Interestingly, these FNIII domains are critically important for Hh signaling function in functional dissection of the Drosophila proteins (Yao, 2006).

The CDO/BOC proteins were initially linked to myogenesis based on their overexpression in C2C12 and 10T1/2 cells, which promoted increased levels of myogenic transcription factors and myotube differentiation. The myogenesis-promoting effects of CDO and BOC were attributed to a promyogenic interaction of these proteins with cadherins (Kang, 2003) and with neogenin, a netrin receptor (Kang, 2004). Consistent with a role in myogenesis, loss of Cdo function in mouse embryos caused a reduction or delay in expression of promyogenic transcription factors Myf-5, MyoD, and myogenin and a delay in muscle development (Cole, 2004). It is interesting to note, however, that expression of Myf-5 and MyoD is also reduced in Shh-/- embryos, particularly in the epaxial domain of the newly forming somite and later in the epaxial dermomyotome. An effect on embryonic Shh signal response thus might account, at least in part, for the effect of Cdo loss on embryonic myogenesis (Yao, 2006).

Loss of Cdo function also produced a mild form of HPE in mice (Cole, 2003). The role of Hh signaling in HPE is clearly established from genetic analysis in mice and humans (Muenke, 2001), and the HPE phenotype of Cdo-/- mice thus may well be accounted for by a partial reduction of Hh pathway activation. Morpholino oligonucleotide-based disruption of boc expression in zebrafish embryos also has implicated BOC function in axonal growth guidance for ventrally projecting forebrain neurons (Connor, 2005). This defect could be due to an effect on Hh pathway activity, given the role of Hh signal response in ventrally directed axonal guidance of commissural neurons in the developing spinal cord (Yao, 2006).

The primary importance of individual Ihog FN1 and FN2 domains in Hh ligand binding and response suggests that other parts of the Ihog protein, which also are evolutionarily conserved, may play functional roles in other signaling pathways. Further genetic and biochemical analysis of variant forms of Ihog family proteins will be required to identify such roles and to learn how such pathways may be integrated with Hh signaling through use of a common receptor (Yao, 2006).

Genetic and biochemical definition of the Hedgehog receptor

Although the transporter-like protein Patched (Ptc) is genetically implicated in reception of the extracellular Hedgehog (Hh) protein signal, a clear definition of the Hh receptor is complicated by the existence of additional Hh-binding proteins and, in Drosophila, by the lack of physical evidence for direct binding of Hh to Ptc. This study shows that activity of Ihog (Interference hedgehog), or of its close relative Boi (Brother of Ihog), is absolutely required for Hh biological response and for sequestration of the Hh protein to limit long-range signaling. This study shows that Ihog interacts directly with Ptc, is required for presentation of Ptc on the cell surface, and that Ihog and Ptc are both required for high-affinity Hh binding. On the basis of their joint roles in ligand binding, signal transduction, and receptor trafficking, it is concluded that Ihog and Ptc together constitute the Drosophila Hh receptor (Zheng, 2010).

Using the targeted alleles of ihog and boi developed in this study, evidence is provided that Ihog proteins are an essential component required for all biological responses to the Hh signal, including target gene induction and patterning in the embryonic segment and in the wing imaginal disc. The central role of Ihog proteins in Hh response was not noted previously because of the functionally overlapping expression of Ihog and Boi in embryos and imaginal discs, which complicates genetic screens and analysis and accounts for the observation that neither the ihog nor boi targeted alleles are lethal in homozygous form. The cl-8 cells used in the genome-scale RNAi screen, in contrast, do not express Boi, and this exposed a critical role for Ihog and facilitated initial discovery of this essential component. In addition to functional overlap, analysis of these functions has been complicated by the required removal of all maternal function for fully penetrant expression of embryonic phenotypes, although maternal expression is neither necessary nor sufficient for Hh response (Zheng, 2010).

The interaction of Ihog Fn2 (the second FNIII domain) with Ptc is essential for presentation of wild-type Ptc on the cell surface. It is not possible, at present, to distinguish between the possibilities that Ihog-mediated surface presentation of Ptc is due to an increased rate of transport to the surface or to an increased duration of residence on the surface. Whatever the mechanism, Fn2 can interact with Ptc in vitro and promote surface presentation of Ptc in cells, even in the absence of the first FNIII domain (Fn1). Similarly, Fn1 alone can interact with the cleavage and cholesterol modified Hh protein HhN in vitro, and Fn1 and Fn2 thus have demonstrably independent functions. Neither domain alone, however, suffices for formation of a high-affinity complex, and the presence of both domains is required for Hh signal reception and transduction and participation in signaling in vivo (Zheng, 2010).

In addition to surface presentation of Ptcour evidence indicates that Ihog proteins also play a direct role in binding the Hh ligand in a multimolecular receptor complex that is critical for transduction. It was thus found that Hh ligand is bound to the surface of cultured cells expressing a variant of Ptc (Ptc1130) with increased localization on the surface. It was also found, with the use of quantitative assays, that endogenous Ihog expressed in these cultured cells contributes critically to binding, and that additional Ihog expression can dramatically enhance binding. In addition, expression of Ptc1130 in the wing imaginal disc clearly produces visible accumulation of the Hh protein on what appears to be the surface of anterior cells at the compartment boundary; this accumulation depends critically on the expression of Ihog/Boi (Zheng, 2010).

Consistent with the role of Ihog in binding, a striking contribution was noted of Ihog to binding in membrane vesicle preparations when present in combination with Ptc. In addition, purified, immobilized HhN and detergent-solubilized extracts containing Ptc and Ihog could be used to demonstrate Ihog-dependent, enhanced precipitation of Ptc. In these biochemical experiments, it was observed that immobilized HhN fails to precipitate detergent-solubilized Ptc alone, but does so in the presence of detergent-solubilized Ihog, and that Ihog alone precipitates Ptc much less efficiently than when HhN is present. This enhancement of Ptc precipitation was dependent on the presence of both the HhN-binding Fn1 domain and the Ptc-binding Fn2 domain of Ihog, consistent with the formation of a multimolecular complex involving HhN, Ptc, and Ihog. Similar results were noted for (Zheng, 2010).

It is interesting to note that little interaction between HhN and Ptc was observed in the absence of Ihog. Formally, it is possible that the interaction of Ptc with HhN is indirect and mediated through enhanced Ihog interaction due to Ptc-induced multimerization or allosteric effects on Ihog. This is thought to be unlikely, however, because Ihog is capable of dimerization in the absence of Ptc, and because the HhN-interacting surface of Ihog is located on the Fn1 domain, which folds independently and is quite distinct from the Ptc-interacting Fn2 domain, thus making allostery unlikely. Thus the interpretation is favored that favorable energetic contributions in the multimolecular receptor/ligand complex derive from Ptc-HhN contacts as well as contacts between Ihog-Ptc and Ihog-HhN (Zheng, 2010).

It is important to note that, despite a direct physical interaction of Ihog and Ptc and their mutual contributions to formation and surface presentation of receptor, and to ligand binding, these two pathway components have opposing roles in pathway regulation. Ihog proteins are thus absolutely required for pathway activation in response to Hh ligand, whereas Ptc alone suffices for suppression of Smo activity in the absence of ligands (Zheng, 2010).

Functional genetic analyses of the mammalian Ihog proteins Cdo and Boc have revealed roles in Hh signaling. Cdo mutant mice thus display mild to intermediate forms of holoprosencephaly, a classic manifestation of Hh signaling deficiency, with the severity of the effect depending on strain background and subject to modifying effects of mutations in other Hh pathway components. Boc mutant mice also show defects in Hh signal-dependent axonal pathfinding by dorsal neurons with ventral commissural projections in the developing neural tube. Neither of these mutants displays phenotypes as severe as those seen in the Shh mutant mouse, or in the Smo mutant, which affects all aspects of Hh signaling. It is possible, however, that a systematic analysis of the double mutant Cdo; Boc animals might reveal more severe phenotypes, as is noted in this study for ihog; boi in Drosophila. In addition, phenotypic characterization of ihog and boi mutants was not designed to reveal defects in axonal pathfinding functions like that of murine Boc, and the possibility of such a function in Drosophila remains to be explored (Zheng, 2010).

The cell-surface proteins Dally-like and Ihog differentially regulate Hedgehog signaling strength and range during development

Hedgehog (Hh) acts as a morphogen in various developmental contexts to specify distinct cell fates in a concentration-dependent manner. Hh signaling is regulated by two conserved cell-surface proteins: Ig/fibronectin superfamily member Interference hedgehog (Ihog) and Dally-like (Dlp), a glypican that comprises a core protein and heparan sulfate glycosaminoglycan (GAG) chains. Dlp core protein can interact with Hh and is essential for its function in Hh signaling. In wing discs, overexpression of Dlp increases short-range Hh signaling while reducing long-range signaling. By contrast, Ihog has biphasic activity in Hh signaling in cultured cells: low levels of Ihog increase Hh signaling, whereas high levels decrease it. In wing discs, overexpression of Ihog represses high-threshold targets, while extending the range of low-threshold targets, thus showing opposite effects to Dlp. It was further shown that Ihog and its family member Boi are required to maintain Hh on the cell surface. Finally, Ihog and Dlp have complementary expression patterns in discs. These data have led to a proposal that Dlp acts as a signaling co-receptor. However, Ihog might not act as a classic co-receptor; rather, it may act as an exchange factor by retaining Hh on the cell surface, but also compete with the receptor for Hh binding (Yan, 2010).

Previous studies have shown that Dlp is specifically required for Hh signaling in cell-based assays and in embryos. However, the molecular basis of this specificity was unknown. This study shows that overexpression of Hh can restore naked cuticle in sugarless (sgl) and sulfateless (sfl) embryos, but not in dlp embryos. It was further demonstrated that the specificity of Dlp in Hh signaling results from its core protein. The Dlp core protein can restore Hh signaling autonomously in dlp embryos and in dlp-RNAi cells. In addition, the Dlp core protein interacts with Hh and promotes Hh signaling in the disc. Overexpression of Dlp increases Hh signaling strength, but reduces signaling range as well as Hh gradient range. These data suggest that the Dlp core protein could act as a classic co-receptor in Hh signaling by facilitating Hh-Ptc interaction (Yan, 2010).

Recent studies have shown that the vertebrate glypican-3 core protein directly promotes Wnt signaling in cancer cells, but inhibits sonic hedghog signaling during development. That the same glypican has opposite effects on Wnt and Hh is interesting because Dlp can inhibit high-threshold Wg signaling when overexpressed in discs. In addition to the essential role of the core protein, the attached GAG chains are important for the non-cell-autonomous functions of Dlp. The current results demonstrated that wild-type Dlp can rescue non-cell-autonomously in dlp embryos, whereas the core protein mainly acts in its expression domains. Interestingly, the CD2 forms of Dlp also lose the non-autonomous activity in dlp embryos. Several studies suggest that the GPI anchor of Dlp can be cleaved by the hydrolase Notum and that the GAG chains of Dlp can recruit lipoprotein particles. Thus, it will be interesting to determine the mechanism of Dlp non-autonomous activity (Yan, 2010).

This study suggests that the GPI anchor of Dlp is not essential for its activity in Hh signaling. Most importantly, two CD2 forms of Dlp, Dlp(-HS)-CD2 and GFP-Dlp-CD2, can effectively rescue Hh signaling in dlp embryos. In addition, CD2 forms of Dlp can also signal in cultured cells and discs. It is important to note that although the GPI, but not the CD2, form of Dlp is colocalized with Ptc in intracellular vesicles, both forms have similar activities in promoting Hh signaling in the disc. These data argue that colocalization of Dlp with Ptc in endocytic vesicles is not essential for Dlp function in Hh signaling. Consistent with this view, several studies have shown that endocytosis is not essential for Hh signaling in Drosophila wing discs. The current conclusion differs from a recent publication arguing that the GPI anchor of Dlp is required for its function in Hh signaling (Gallet, 2008). In that study, it was shown that overexpression of GFP-Dlp-CD2 can reduce Hh signaling in the wing discs. However, this study did not observe any dominant-negative effect of GFP-Dlp-CD2, which can rescue dlp mutant embryos and enhance Hh signaling strength in cells and discs. A possible explanation for the discrepancy is that expression of Dlp enhances signaling strength but also reduces signaling range. ap-Gal4 was always used, allowing use of the ventral disc as an internal control. The observed dominant-negative effect might reflect the reduced signaling range rather than signaling strength (Yan, 2010).

Previous studies in both Drosophila and vertebrates have demonstrated positive roles of the Ihog family proteins in Hh signaling. Ihog and Ptc synergize in mediating Hh binding to cells. These observations suggest that Ihog functions as a co-receptor for Hh. However, the new data argue that Ihog does not simply act as a classic co-receptor that only increases the binding of ligand to the signaling receptor. By altering Ihog levels in vivo and in vitro, it was shown that Ihog has biphasic activity in Hh signaling, with too much or too little Ihog leading to reduced signaling. Overexpression of Ihog leads to the accumulation of Hh, and knockdown of Ihog results in reduced Hh levels, suggesting that one major activity of Ihog is to retain Hh on the cell surface. Moreover, knockdown of both Ihog and Boi dramatically reduces Hh levels and signaling. This reduction of Hh signaling activity is likely to be due to an absence of Hh on the cell surface of the double-mutant tissue. However, a high level of Ihog causes a large amount of Hh to accumulate on the cell surface, but also reduces signaling strength. One explanation for this result is that Ihog can compete with Ptc for Hh binding. Thus, a low level of Ihog is required to maintain Hh on the cell surface, whereas a high level of Ihog can sequester Hh from its receptor. In other words, depending on the context, Ihog can either provide Hh for the receptor by retaining Hh on the cell surface, or compete with the receptor for Hh binding. This activity of Ihog is very similar to the recently proposed 'exchange factor' model, which allows the exchange of Hh between Ptc and Ihog. A recent study has demonstrated that Ihog also interacts with Ptc and that Ihog, Ptc and Hh form a triple complex (Zheng, 2010). The close association between the Ihog and Ptc receptors may thus allow them to exchange Hh ligand. It will be important to determine whether the triple complex has a greatly reduced ability to signal, a prediction from the mathematical exchange factor model (Yan, 2010).

Although this is the first demonstration of a biphasic co-factor in Hh signaling, similar biphasic co-factors have been reported. Drosophila Cv-2 enhances BMP signaling at low concentrations, but inhibits signaling at high concentrations. Syndecan-1 shows a similar concentration-dependent activation or inhibition of BMP signaling in Xenopus. Interestingly, Dlp has biphasic activity in Wg signaling, depending on its protein levels. All these co-factors are likely to act by a similar mechanism, suggesting that the biphasic co-factor is a recurring motif in different morphogen systems (Yan, 2010).

This study has shown that Dlp and Ihog play distinct roles in Hh signaling. Expression of Dlp enhances Hh signaling strength, but reduces signaling range. By contrast, expression of Ihog reduces Hh signaling strength, but extends signaling range. In addition, the Dlp level is elevated in the high Hh signaling area, whereas the Ihog level is reduced in that region. It is important to consider from a system point of view what these two co-factors provide for the Hh morphogen. For morphogens to work, they should be able to generate sharp boundaries between target genes with different thresholds. They also need to diffuse over a certain range without being lost in the extracellular space. The positive feedback of Dlp expression in the high Hh signaling areas helps to sharpen the boundaries between high- and low-threshold target genes. The negative-feedback regulation of Ihog might ensure that a strong Hh signal is attained in the areas close to the Hh source and also allow the Hh gradient to diffuse to those areas distant from the source (Yan, 2010).

Search PubMed for articles about Drosophila iHog

Aglyamova, G. V. and Agarwala, S. (2007). Gene expression analysis of the hedgehog signaling cascade in the chick midbrain and spinal cord. Dev. Dyn. 236(5): 1363-73. Medline abstract: 17436280

Cole, F. and Krauss, R. S. (2003). Microform holoprosencephaly in mice that lack the Ig superfamily member Cdon. Curr. Biol. 13: 411-415. Medline abstract: 12620190

Cole, F., Zhang, W., Geyra, A., Kang, J. S. and Krauss, R. S. (2004). Positive regulation of myogenic bHLH factors and skeletal muscle development by the cell surface receptor CDO. Dev. Cell 7: 843-854. Medline abstract: 15572127

Connor, R. M., et al. (2005). BOC, brother of CDO, is a dorsoventral axon-guidance molecule in the embryonic vertebrate brain. J. Comp. Neurol. 485: 32-42. Medline abstract: 15776441

Gallet, A., Staccini-Lavenant, L. and Therond, P. P. (2008). Cellular trafficking of the glypican Dally-like is required for full-strength Hedgehog signaling and wingless transcytosis. Dev. Cell 14: 712-725. PubMed Citation: 18477454

Kang, J. S., et al. (2002). BOC, an Ig superfamily member, associates with CDO to positively regulate myogenic differentiation. EMBO J. 21: 114-124. Medline abstract: 11782431

Kang, J. S., et al. (2003). Promyogenic members of the Ig and cadherin families associate to positively regulate differentiation. Proc. Natl. Acad. Sci. 100: 3989-3994. Medline abstract: 12634428

Kang, J. S., et al. (2004). Netrins and neogenin promote myotube formation. J. Cell Biol. 167: 493-504. Medline abstract: 15520228

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date revised: 1 November 2010

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