InteractiveFly: GeneBrief

interference Hedgehog: Biological Overview | References

BIOLOGICAL OVERVIEW

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).

Ihog and Boi elicit Hh signaling via Ptc but do not aid Ptc in sequestering the Hh ligand

Hedgehog (Hh) proteins are secreted molecules essential for tissue development in vertebrates and invertebrates. Hh reception via the 12-pass transmembrane protein Patched (Ptc) elicits intracellular signaling through Smoothened (Smo). Hh binding to Ptc is also proposed to sequester the ligand, limiting its spatial range of activity. In Drosophila, Interference hedgehog (Ihog) and Brother of ihog (Boi) are two conserved and redundant transmembrane proteins that are essential for Hh pathway activation. How Ihog and Boi activate signaling in response to Hh remains unknown; each can bind both Hh and Ptc and so it has been proposed that they are essential for both Hh reception and sequestration. Using genetic epistasis this study established that Ihog and Boi, and their orthologs in mice, act upstream or at the level of Ptc to allow Hh signal transduction. In the Drosophila developing wing model it was found that through Hh pathway activation Ihog and Boi maintain the boundary between the anterior and posterior compartments. The contributions of Ptc was dissociated from those of Ihog/Boi, and, surprisingly, it was found that cells expressing Ptc can retain and sequester the Hh ligand without Ihog and Boi, but that Ihog and Boi cannot do so without Ptc. Together, these results reinforce the central role for Ptc in Hh binding in vivo and demonstrate that, although Ihog and Boi are dispensable for Hh sequestration, they are essential for pathway activation because they allow Hh to inhibit Ptc and thereby relieve its repression of Smo (Camp, 2014).

Ihog and Boi have been shown to be absolutely essential within Hh-responding cells for activation of the Hh signaling pathway, acting upstream of Smo. The current experiments advance understanding of Ihog and Boi function by drawing three major conclusions: First, in genetic epistasis experiments it was found that Ihog and Boi also act upstream or at the level of Ptc, supporting the idea that they function through Ptc to relieve suppression of Smo. This epistatic relationship appears conserved in evolution, as it was found that Cdon and Boc also function upstream or at the level of Ptch1 for Hh signal transduction in mice. These genetic findings establish this relationship unequivocally, and so have profound implications for future studies to further clarify how these co-receptors participate in Hh reception and pathway activation (Camp, 2014).

Second, based on experiments to dissect the relative contributions of Ihog and Boi in processes involving Ptc, it is concluded that it is through their essential roles in Hh signal transduction that Ihog, Boi and Ptc contribute to anterior-posterior compartment segregation: once the pathway is activated, all three proteins are dispensable for maintenance of the compartment boundary, implicating other, yet unidentified, cell surface recognition molecules in compartment-specific cell affinity and adhesion (Camp, 2014).

Third, it is concluded that Ihog and Boi, unlike Ptc, are completely dispensable for the sequestration and retention of Hh. Cells lacking Ihog and Boi can sequester and retain the Hh signal if the pathway is activated and Ptc is upregulated. They do so via physiological levels of endogenous Ptc induced either by pathway activation in ^ptcS2 mutants or by expression of Smo^SD123. Incidentally, it was also found that Hh sequestration was rescued in boi;ihog double mutant clones overexpressing Ptc¹¹³⁰, a dominant-negative that fully activates the Hh pathway and upregulates endogenous, wild-type Ptc. This third conclusion is not consistent with the view that Ihog and Boi aid in addressing Ptc to the cell surface and that, once there, they are required for Ptc to bind and sequester Hh. This view is based primarily on Ptc and Ihog overexpression in cultured cells, and on an experiment that failed to restore Hh sequestration to boi;ihog double mutant cells with mutation of cAMP-dependent protein kinase 1 (Pka-C1), which upregulates Ptc and other target genes because loss of Pka-C1 disinhibits the activity of the transcription factor Ci. It is unclear why Ptc upregulation in Pka-C1 mutants was unable to rescue Hh sequestration in boi;ihog double mutants, whereas Ptc upregulation in the current experiments was able to do so. In cells lacking Ihog and Boi, perhaps the level to which Ptc is upregulated in Pka-C1 mutants is inadequate. Regardless, the current data indicate that Ptc has a central role in the binding and sequestration of Hh, whereas Ihog and Boi are dispensable, despite their requirement for Hh signal transduction (Camp, 2014).

The results are consistent with vertebrate systems, in which current models strongly favor direct contacts between Hh and Ptc, primarily because: (1) expression of the Ptc ortholog Ptch1 promotes binding of Shh to transfected cells, (2) radiolabeled Shh can be chemically cross-linked to Ptch1 expressed on the cell surface and (3) Ptch1 can reach the cell surface in the absence of the Ihog/Boi-related proteins Cdon and Boc. Whether Ptc is sufficient on its own to bind Hh remains an important question that awaits technically challenging studies using purified proteins. An alternative possibility is that Hh could have additional receptor(s), with candidates including the proteoglycans Dally and Dally-like (Dlp), and Shifted, a secreted protein of the Wnt inhibitory factor 1 (WIF1) family (Camp, 2014).

The results clearly distinguish a role for Ptc that relies on Ihog/Boi (Hh reception/signal transduction) from one that does not (Hh sequestration), and so they contribute to an emerging view of the function of Ihog, Boi and related proteins. In non-responding cells, others have shown that Ihog and Boi are involved in restricting the movement of Hh, and so may contribute to its overall distribution. Within Hh-responding cells, where Ptc is co-expressed with Ihog and Boi, it was found that Ihog and Boi are essential for Hh signal transduction, but not Hh sequestration and retention. As Ihog and Boi act upstream or at the level of Ptc, they must mediate a crucial, rate-limiting step in the inhibition of Ptc in response to Hh. However, as they are not essential for Ptc to bind Hh, how they affect Ptc function remains to be elucidated. Whereas the precise molecular mechanism remains elusive, several lines of evidence provide important clues. First, an Ihog variant lacking the cytoplasmic tail can rescue boi;ihog double mutants. Second, the second Fn3 domain of Ihog or Boi interacts physically with Ptc and is quite distinct from the first Fn3 domain that harbors the Hh-interacting surface. Third, the presence of Ihog or Boi potentiates co-immunoprecipitation of Hh and Ptc. Together, these results suggest that the primary role of Ihog and Boi in Hh signaling involves the ability of their ectodomains to form favorable protein complexes with Ptc or Hh, or with both simultaneously. Although the data indicate that Ptc does not need Ihog and Boi to bind Hh in vivo, it is surmised that it is through these multimolecular complexes that Ihog and Boi allow Hh to inhibit Ptc and thereby relieve its suppression of Smo and the Hh signaling cascade (Camp, 2014).

Dampening the signals transduced through Hedgehog via microRNA miR-7 facilitates Notch-induced tumourigenesis

Fine-tuned Notch and Hedgehog signalling pathways via attenuators and dampers have long been recognized as important mechanisms to ensure the proper size and differentiation of many organs and tissues. This notion is further supported by identification of mutations in these pathways in human cancer cells. However, although it is common that the Notch and Hedgehog pathways influence growth and patterning within the same organ through the establishment of organizing regions, the cross-talk between these two pathways and how the distinct organizing activities are integrated during growth is poorly understood. An unbiased genetic screen in the Drosophila melanogaster eye has found that tumour-like growth was provoked by cooperation between the microRNA miR-7 and the Notch pathway. Surprisingly, the molecular basis of this cooperation between miR-7 and Notch converged on the silencing of Hedgehog signalling. In mechanistic terms, miR-7 silenced the interference hedgehog (ihog) Hedgehog receptor, while Notch repressed expression of the brother of ihog (boi) Hedgehog receptor. Tumourigenesis was induced co-operatively following Notch activation and reduced Hedgehog signalling, either via overexpression of the microRNA or through specific down-regulation of ihog, hedgehog, smoothened, or cubitus interruptus or via overexpression of the cubitus interruptus repressor form. Conversely, increasing Hedgehog signalling prevented eye overgrowth induced by the microRNA and Notch pathway. Further, it was shown that blocking Hh signal transduction in clones of cells mutant for smoothened also enhance the organizing activity and growth by Delta-Notch signalling in the wing primordium. Together, these findings uncover a hitherto unsuspected tumour suppressor role for the Hedgehog signalling and reveal an unanticipated cooperative antagonism between two pathways extensively used in growth control and cancer (Da Ros, 2013).

A challenge to understand oncogenesis produced by pleiotropic signalling pathways, such as Notch, Hh, and Wnts, is to unveil the complex cross-talk, cooperation, and antagonism of these signalling pathways in the appropriate contexts. Studies in flies, mice, and in human cell cultures have provided critical insights into the contribution of Notch to tumourigenesis. These studies highlighted that Notch when acting as an oncogene needs additional mutations or genes to initiate tumourigenesis and for tumour progression, identifying several determinants for such co-operation. The identification of these co-operative events has often been knowledge-driven, although unbiased genetic screens also identified known unanticipated tumour-suppressor functions. In this sense, this study describes a conserved microRNA that cooperates with Notch-induced overproliferation and tumour-like overgrowth in the D. melanogaster eye, miR-7. Alterations in microRNAs have been implicated in the initiation or progression of human cancers, although such roles of microRNAs have rarely been demonstrated in vivo. In addition, by identifying and validating functionally relevant targets of miR-7 in tumourigenesis, this study also exposed a hitherto unsuspected tumour suppressor role for the Hh signalling pathway in the context of the oncogenic Notch pathway. Given the conservation of the Notch and Hh pathways, and the recurrent alteration of microRNAs in human cancers, it is speculated that the genetic configuration of miR-7, Notch, and Hh is likely to participate in the development of certain human tumours (Da Ros, 2013).

In human cancer cells, miR-7 has been postulated to have an oncogene or a tumour suppressor functions that may reflect the participation of the microRNA in distinct pathways, due to the regulation of discrete target genes in different cell types, such as Fos, IRS-2, EGFR, Raf-1, CD98, IGFR1, bcl-2, PI3K/AKT, and YY1 in humans (Da Ros, 2013).

In Drosophila, multiple, cell-specific, targets for miR-7 have been previously validated via luciferase or in vivo eGFP-reporter sensors or less extensively via functional studiest. Although microRNAs are thought to regulate multiple target genes, when tested in vivo it is a subset or a given target that predominates in a given cellular context. Indeed, of the 39 predicted miR-7 target genes tested by direct RNAi, only downregulating ihog with several RNAi transgenes (UAS-ihog-IR) fully mimicked the effect of miR-7 overexpression in the transformation of Dl-induced mild overgrowth into severe overgrowth and even tumour-like growth. Moreover, it was confirmed that endogenous ihog is directly silenced by miR-7 and that this silencing involves direct binding of the microRNA to sequences in the 3'UTR of ihog both in vivo and in vitro (Da Ros, 2013).

Nevertheless, other miR-7 target genes may contribute to the cooperation with Dl-Notch pathway along with ihog, such as hairy and Tom. While miR-7 can directly silence hairy in the wing, this effect has been shown to be very modest, and thus, it is considered that while hairy may contribute to such effects, it is unlikely to be instrumental in this tumour model. Indeed, the loss of hairy is inconsequential in eye development, although retinal differentiation is accelerated by genetic mosaicism of loss of hairy and extramacrochaetae that negatively sets the pace of MF progression. It is unclear how Hairy might contribute to Dl-induced tumourigenesis (Da Ros, 2013).

The RNAi against Tom produced overgrowth with the gain of Dl albeit inconsistently and with weak penetrance, where one RNAi line did not modify the Dl-induced overgrowth and the other RNAi line caused tumours in less than 40% of the progeny. Tom is required to counteract the activity of the ubiquitin ligase Neuralized in regulating the Notch extracellular domain, and Dl in the signal emitting cells. These interactions are normally required to activate Notch signalling in the receiving cells through lateral inhibition and cell fate allocation. However, although it remains to be shown whether similar interactions are active during cell proliferation and growth, the moderate enhancement of Dl that is induced when Tom is downregulated by RNAi suggests that miR-7-mediated repression of Tom may contribute to the oncogenic effects of miR-7 in the context of Dl gain of function, along with other targets such as ihog (Da Ros, 2013).

Conversely, while the target genes of the Notch pathway, E(spl)m3 and E(spl)m4 as well as E(spl)mγ, Bob, E(spl)m5, and E(spl)mδ, have been identified as direct targets of miR-7 in the normal wing disc via analysis of 3'UTR sensors, there was no evidence that HLHm3, HLHm4, HLHm5, Bob, and HLHmγ are biological relevant targets of miR-7 in the Dl overexpression context. HLHmδ RNAi produced inconsistent phenotypes in the two RNAi transgenic lines available, causing tumour-like growth at very low frequency in only one of the lines. No evidence was obtained that miR-7 provoked overgrowth by targeting the ETS transcription factor in the EGFR pathway AOP/Yan, a functionally validated target of the microRNA miR-7 during retinal differentiation. Neither was any evidence obtained that RNAi of atonal provoked eye tumours with Dl overexpression, although a strong inhibition via expression of a fusion protein Atonal::EN that converts Atonal into a transcriptional repressor has been shown to be sufficient to trigger tumorigenesis together with Dl. Thus, it was reasoned that given that microRNA influenced target genes only subtly (even when using ectopic expression), it is possible that downregulation of atonal contributes to the phenotype along with the other targets (Da Ros, 2013).

In conclusion, this study has identified cooperation between the microRNA miR-7 and Notch in the D. melanogaster eye and identified and validated ihog as a direct target of the miR-7 in this context and have identified boi as a target of Notch-mediated activity at the DV eye organizer, although it remains whether this regulation is direct or indirect. A hitherto unanticipated tumour suppressor activity was uncovered of the endogenous Hh signalling pathway in the context of gain of Dl-Notch signalling that is also apparent during wing development (Da Ros, 2013).

Hh tumour suppressor role is revealed when components of the Hh pathway were lost in conjunction with a gain of Dl expression in both the eye and wing discs. Hh and Notch establish signalling centres along the AP and DV axes, respectively, of the disc to organize global growth and patterning. Where the organizer domains meet, the Hh and Notch conjoined activities specify the position of the MF in the eye disc and the proximodistal patterning in the wing disc. This study also unvailed that in addition antagonistic interaction between the Hh and Notch signalling might help to ensure correct disc growth. Thus, it was shown that Hh signalling limits the organizing activity of Dl-Notch signalling. Although it is often confounded whether Dl-Notch signalling instructs overgrowth by autonomous or nonautonomous (i.e., DV organizers) mechanisms, these findings uncover that loss of Hh signalling enhances a non-cell autonomous oncogenic role of Dl-Notch pathway (Da Ros, 2013).

To date, Hh has not yet to be perceived as a tumour suppressor, although it is noteworthy that human homologs of ihog, CDO, and BOC were initially identified as tumour suppressors. Importantly, both CDO and BOC are downregulated by RAS oncogenes in transformed cells and their overexpression can inhibit tumour cell growth in vitro. Since human RAS regulates tumourigenesis in the lung by overexpressing miR-7 in an ERK-dependent manner, it is possible that RAS represses CDO and BOC via this microRNA. Indeed, the 3'UTR of both CDO and BOC like Drosophila ihog contains predicted binding sites for miR-7. There is additional clinical and experimental evidence connecting elements of the Hedgehog pathway with tumour-suppression. The function of Growth arrest specific gene 1 (GAS1), a Hh ligand-binding factor, overlaps that of CDO and BOC, while its overexpression inhibits tumour growth . More speculative is the association of some cancer cells with the absence of cilium, a structure absolutely required for Hh signal transduction in vertebrate cells (Da Ros, 2013).

Given the pleiotropic nature of Notch, Wnts, BMP/TGFβ, Ras, and Hh signalling pathways in normal development in vivo, it is speculated that competitive interplay as that described in this study between Notch and Hh may not be uncommon among core growth control and cancer pathways that act within the same cells at the same or different time to exert multiple outputs (such as growth and cell differentiation). Moreover, context-dependent tumour suppressor roles could explain the recurrent, unexplained, identification of somatic mutations in Hh pathway in human cancer samples. Indeed, the current findings stimulate a re-evaluation of the signalling pathways previously considered to be exclusively oncogenic, such as the Hh pathway (Da Ros, 2013).

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).

Dispatched mediates Hedgehog basolateral release to form the long-range morphogenetic gradient in the Drosophila wing disk epithelium

Hedgehog (Hh) moves from the producing cells to regulate the growth and development of distant cells in a variety of tissues. This study has investigated the mechanism of Hh release from the producing cells to form a morphogenetic gradient in the Drosophila wing imaginal disk epithelium. Hh reaches both apical and basolateral plasma membranes, but the apical Hh is subsequently internalized in the producing cells and routed to the basolateral surface, where Hh is released to form a long-range gradient. Functional analysis of the 12-transmembrane protein Dispatched, the glypican Dally-like (Dlp) protein, and the Ig-like and FNNIII domains of protein Interference Hh (Ihog) revealed that Dispatched could be involved in the regulation of vesicular trafficking necessary for basolateral release of Hh, Dlp, and Ihog. It was also shown that Dlp is needed in Hh-producing cells to allow for Hh release and that Ihog, which has been previously described as an Hh coreceptor, anchors Hh to the basolateral part of the disk epithelium (Callejo, 2011).

By using shi^ts1 mutant disks, it is possible to freeze Hh internalization and visualize on which side of the anterior compartment (A) wing disk epithelium, apical or basal, Hh gradient is being formed. Thus, an extended basolateral accumulation of Hh was observed in receiving cells, whereas, at the apical plane, Hh accumulation was only evident in the first row of A cells, indicating that the long-range Hh gradient is formed mainly basolaterally. Accordingly, Ptc accumulated in shi^ts1 disks equally apically and basolaterally but mainly colocalized with Hh at the basolateral sections, suggesting a specific mechanism to deliver Hh to Ptc in this surface of the disk epithelium. To analyze the mechanism of Hh release in the P cells, an apical accumulation of Hh was noticed in the producing cells in shi^ts1 mutant disks that was also observed in P shi^ts1 clones and in Rab5^DN ectopic clones, suggesting that the apically secreted Hh is also internalized in P cells and probably is recycling to other membranes. Accordingly, when blocking recycling endosomes (using the dominant-negative form of Rab8 or Rab4), Hh accumulation could be detected in producing cells. This Hh recycling in P cells is probably necessary to form a proper Hh gradient in the receiving cells. In agreement with the above, Hh signaling is compromised when endocytosis is blocked by expressing either Rab5^DN or Shi^DN in the P compartment. Interestingly, and in agreement with these results, Ayers (2011) suggested that apical Hh internalization in the Hh-producing cells is necessary to process or route Hh to activate the responses that require high levels of Hh. However, in contrast to the interpretation provided in this study, Ayers also proposed that the apical Hh pool is responsible for the long-range Hh gradient formation. It is not necessary to envision two Hh gradients, an apical long-range Hh and another basolateral short-range Hh, when all responses can be produced by means of a single gradient. Because only the recycled Hh is capable of activating the high-threshold Hh targets, there is no reason to believe that this processed Hh would not be efficient enough to induce the low-threshold targets basolaterally (Callejo, 2011).

Also in contrast to a previous report proposing a function for Disp in regulating the apical secretion of Hh in Drosophila epithelia, this study demonstrates that Disp is required for the basolateral release of Hh in the wing imaginal disk epithelium. The subcellular localization of Disp, and its function in the basolateral release of Hh, is in agreement with a recent report in vertebrates (Etheridge, 2010). The cellular phenotype of the loss of Disp function, such as the increase in the amount of Hh found in endocytic vesicles, which are supernumerary and disorganized, can be interpreted as a failure in Hh trafficking that subsequently affects its proper release. In this sorting process, Hh would interact with Disp either in the recycling endosome or in MVBs. Disp, a member of the RND family of proton-driven transporters, is likely to function only in compartments where a transmembrane proton gradient exists, such as in early and late endosomes, trans-Golgi, and MVBs. In support of this view, this study showed by confocal and EM studies that Disp protein is located not only at the basolateral plasma membrane but in vesicles and MVBs, where it colocalizes with Hh. Based on the disp^-/- phenotypes and the localization of Disp protein in MVBs, it is proposed that Disp might have a function in redirecting the apically internalized Hh toward the basal domain. Interestingly, and in agreement with the above, a form of Disp, mutant for the proton-driven transporter function (Disp^AAA), does not localize at the basolateral plasma membrane but in supernumerary cytoplasmic vesicles that do not colocalize with Hh puncta, implying that Disp^AAA may not participate in Hh vesicular trafficking to the basolateral plasma membrane (Callejo, 2011).

In noticeable contrast but also supporting the above, after freezing Hh internalization in shi^ts1 mutant disks, Hh accumulates apically in the first row of A compartment cells, suggesting that paracrine signaling could also occur through the apical plasma membrane. Interestingly, in P mutant cells for Dlp, Hh is able to signal to the abutting A cells but long-range signaling does not occur. A suggestive possibility is that the capacity for apical signaling is not affected in these mutant conditions; however, for long-range signaling to occur, a basolateral release implicating the coordinated actions of Dlp and Ihog together with Disp would be required. During Hh sorting in the producing cells, Disp may interact with Dlp and Ihog; in fact, the interaction of Disp with Dlp and Ihog may be important for the apical-to-basal transcytosis of these proteins, because the ectopic expression of Disp but not of mutant Disp^AAA increases Dlp and Ihog levels at the basolateral membranes. As in disp^-/- cells, dlp^-/- cells in the P compartment showed an accumulation of Hh at both the apical and basolateral plasma membranes, suggesting that Dlp might cooperate with Disp during Hh release. In agreement with the proposed mechanism, transcytosis of Dlp has previously been suggested to be important for Wingless (Wg) release and spreadin (Callejo, 2011).

Although the data cannot support that the total amount of synthesized Hh has to undergo this apical-to-basal transcytosis, an intriguing question is why Hh is placed and internalized apically and then shuttled to the basolateral part of the cell. One possibility is that the newly synthesized Hh protein, because of its unusual modifications with cholesterol and palmitate, uses the apical surface, which is enriched in cholesterol and glycosphingolipids, for primarily plasma membrane localization. Alternatively, it is also possible that the apical internalization of Hh allows its interaction with Disp, glypicans, and Ihog. Therefore, Hh that reaches the apical plasma membrane needs to be internalized to recycle to the basolateral plasma membrane, where the machinery for secretion and gradient formation is found. As has already been discussed, transcytosis of Dlp and Ihog together with Hh from the apical membrane to the basolateral membrane may also occur. Cholesterol and triglycerides also undergo apical-to-basolateral transcytosis across intestinal epithelial barriers to reach the blood. Cholesterol and palmitic acid modifications could attribute lipid-like properties to the Hh protein, such as the ability to be anchored to the plasma membrane, and could thus affect Hh intracellular trafficking. In agreement with the above, it has been described that lipid-unmodified Hh in the wing disk epithelium is not able to form a proper Hh gradien. As previously reported, it was observed that Hh mutant forms that lack lipid modifications are released and do not accumulate in disp^-/- clones. Interestingly, this study shows that lipid-unmodified Hh does not colocalize with Ihog-labeled basal cell extensions, indicating that lipid modifications are necessary to interact with Disp, Dlp, and Ihog, and therefore for proper Hh trafficking from the apical to basolateral plasma membrane regulated by Disp function. Reinforcing these findings, it has been reported that disp and ttv functions are not required for either release or transport of lipid-unmodified Hh, strongly suggesting that Disp and glypicans are needed for the appropriate basolateral release of Hh (Callejo, 2011).

This work shows that Hh has a more complicated mechanism for release than has been previously anticipated. The finding of a basolateral route for Hh release and gradient formation will help to understand Hh interaction with different Hh pathway components, such as Disp, Dally, Dlp, Ihog, Boi, and Ptc, during the process of Hh gradient formation. Related to this issue, it is quite intriguing to find Disp, Dlp, Ihog, and Hh decorating long basal cellular extensions in disk cells expressing Ihog ectopically. Some of the long filaments labeled with IhogYFP extend up to several cell diameters and are reminiscent of the 'cytonemes', with a function in the transport of morphogens. In contrast to the previously described apical cytonemes, the extensions this study visualize are mainly found at the basal part of the disk epithelium. Interestingly, in the context of Notch signaling, basal actin-based filopodia are important for lateral inhibition between nonneighboring cells. However, further investigation will be necessary to demonstrate the implication of cytonemes in Hh gradient formation (Callejo, 2011).

Balancing Hedgehog, a retention and release equilibrium given by Dally, Ihog, Boi and shifted/DmWif

Hedgehog can signal both at a short and long-range, and acts as a morphogen during development in various systems. The mechanisms of Hh release and spread were studied using the Drosophila wing imaginal disc as a model system for polarized epithelium. The cooperative role of the glypican Dally, the extracellular factor Shifted (Shf, also known as DmWif), and the Immunoglobulin-like (Ig-like) and Fibronectin III (FNNIII) domain-containing transmembrane proteins, Interference hedgehog (Ihog) and its related protein Brother of Ihog (Boi), was analyzed in the stability, release and spread of Hh. Dally and Boi were shown to be required to prevent apical dispersion of Hh; they also aid Hh recycling for its release along the basolateral part of the epithelium to form a long-range gradient. Shf/DmWif on the other hand facilitates Hh movement restrained by Ihog, Boi and Dally, establishing equilibrium between membrane attachment and release of Hh. Furthermore, this protein complex is part of thin filopodia-like structures or cytonemes, suggesting that the interaction between Dally, Ihog, Boi and Shf/DmWif is required for cytoneme-mediated Hh distribution during gradient formation (Bilioni, 2013).

This study has approached the functional interaction of the ECM components Shf/dWif and Dally, and of the Hh coreceptors Ihog and Boi in Hh release and/or spreading to form a gradient. Two major findings are described: one is an unpredicted role of Dally and Boi in the apical retention and subsequent internalization of Hh in producing cells, and the other the interaction of Dally and Ihog/ Boi with Shf/dWif, facilitating Hh movement in the basolateral part of the disc epithelium. Interactions between these components allow retention at the apical plasma membranes of producing cells necessary to prevent apical Hh spreading and facilitate subsequent recycling to basolateral side, as well as Hh release and movement in this side of the epithelium (Bilioni, 2013).

Apical Hh levels in the Hh producing cells are affected in both dally and boi (but not in ihog) null mutant conditions. Moreover, ectopic Dally or Boi (but not Ihog) cause an increase in Hh retention at the apical plane of the disc. Accordingly, Dally and Boi have an Ihog-independent function in maintaining Hh concentration at the apical part of the disc epithelium. As has been previously demonstrated, apically externalized Hh does not form a gradient (Callejo, 2011); thus, this might be accounted as a mechanism preventing the spread of apically externalized Hh. In agreement with an Ihog-independent role of Boi in apical Hh retention, a recently published study (Hartman, 2010) demonstrates that Boi is expressed in apical cells of the ovary and suppresses follicular stem cells (FSC) proliferation by binding to and sequestering Hh on the apical cell surface, thereby inhibiting Hh long range distribution (Bilioni, 2013).

In addition, it has been observed that the apically externalized Hh is subsequently internalized and recycled to the basolateral membranes of the wing disc epithelium (Callejo, 2011). In strong support of this Hh recycling scheme, overexpression of Dally in the P compartment was shown to enhance Hh apical retention, decreasing Hh levels in the most basal side and reducing Hh target activation in the A compartment. Therefore, it is likely that Dally and Boi not only prevent apical Hh spreading but also mediate the apical Hh internalization in the Hh producing cells. The observation that Hh, Dally and Boi accumulate in the apical surface when internalization is blocked by a dynamin mutation reinforces this possibility (Bilioni, 2013).

The increment of apical spreading of Hh in the A compartment cells caused by overexpression of a secretable form of Dally in the P compartment also supports Dally's function in Hh apical retention in the P compartment cells. Given that the enhanced apical spreading of Hh correlates with a reduced basolateral Hh gradient, it is proposed that in normal conditions recycling of the apical Hh pool results in the formation of a basolateral Hh gradient. In contrast, it has been propose that the hydrolase, Notum, is implicated in the release of Dally and the abnormal increase in the apical spreading of Hh in the A compartment cells by overexpression of a DallySec has been interpreted as a direct evidence of a long-range apical Hh gradient. However, notum mutants show a Wg (not a Hh) signaling phenotype, which argues against this hypothesis. Furthermore, the cell-autonomous requirement of wild type Dally for keeping Hh in the ECM suggests that Dally may not be released from its GPI anchor for this function. In addition, no non-autonomous effects of Dally were observed on Shf/dWif stability, which implies that Dally remains membrane-anchored (Bilioni, 2013).

In conclusion, these data show that Dally has a cell-autonomous role in Hh attachment to the ECM, with a double purpose: in the producing cells Dally facilitates Hh retention necessary to prevent Hh spreading, and in receiving cells it supports Hh presentation to the receptor. An autonomous Dally requirement for Hh signaling has been recently proposed. This cell-autonomous requirement for Dally in maintaining extracellular Hh concentration is in agreement with the previously described role of Dally in Wg and Dpp signaling (Bilioni, 2013).

It has been suggested that Shf/dWif mediates the function of the HSPGs in Hh stability in the ECM. This study finds that Shf/dWif stabilization also depends on Dally, Ihog and Boi because Shf/dWif levels vary accordingly in both loss and gain of function of these genes. In these mutants, Shf/dWif levels are reduced at the basolateral side of the disc epithelium and, as a consequence, Hh levels also decrease. Thus, Dally together with Shf/dWif, Boi and Ihog is implicated in Hh stability in the ECM. On the other hand, despite an increment in Hh levels when overexpressing Dally in a shf mutant background, the target expression remains severely impaired. Thus, an excess of Dally or Ihog/Boi can offset the effects of Shf/dWif mutation in terms of Hh concentration but not in terms of Hh movement. Taken together, these results lead to the conclusion that Shf/dWif is an ECM factor that counteracts the impact of Dally and Ihog/Boi on Hh attachment at the membranes. Interestingly, ectopic expression of Ihog, Boi or Dally stabilizes Shf/dWif mainly in the basolateral domain where most of Shf/dWif protein is located (Bilioni, 2013).

Shf/dWif is then required for Hh movement even when overexpressing Hh. Counteracting this effect, Ihog and Boi mediate the attachment of extracellular Hh to plasma membranes in Hh producing cells. In ihog and boi mutant cells Hh levels, mainly at the basolateral plane, are very low, and overexpression of Ihog or Boi not only causes Hh accumulation at the plasma membrane but also a restriction in Hh movement. Moreover, Shf/dWif can rescue the phenotype of restricted Hh movement imposed by ectopic Ihog or Boi, and is necessary to allow the increment of Hh spreading when knocking down Boi and Ihog in the P compartment, demonstrating that proper gradient formation requires equilibrium between these proteins. This is further confirmed when Ihog overexpression in the P compartment restricts Hh movement and decreases Hh signaling in the region anterior to a smo⁻ clone located at the A/P compartment border; and then again simultaneous overexpression of Shf/dWif reestablishes the equilibrium so Hh can now reach the wild type territory and signal across the clone (Bilioni, 2013).

It has been proposed that Boi and Ihog are not required in P compartment cells because double boi and ihog mutant clones had no effect on Hh signaling. However, this conclusion did not take account of Hh non-autonomy. Indeed, it has been reported that wings develop normally even with P compartments that have large hh mutant clones. This non-autonomy of Hh is also supported by the lack of an effect on Hh signaling of disp^−/− clones or by long-range spreading of Hh through large smo mutant clones. Because of Hh non-autonomy, the function of Boi and Ihog in Hh-producing cells was only reveled when Boi and Ihog were knocked down in the whole P compartment. Interestingly, despite of the low Hh levels in the P compartment in the absence of Boi and Ihog functio, an increase of long-range Hh gradient was observed. It is thought is that a low Hh retention at the plasma membrane of P compartment cells causes an increase of Hh release, so the bulk of Hh that reaches the A compartment is higher than in the wild type condition. Supporting this hypothesis, it was shown that Ihog and Boi from A cells have indeed the capacity to 'capture' Hh from P compartment cells (Bilioni, 2013).

In addition, the ectopic expression of Ihog increases not only the endogenous levels of Hh but also Shf/dWif, Dally along cytonemes located at the basolateral side of the disc epithelium, as previously described for Dlp (Callejo, 2011). Some of these long filaments extend up to several cell diameters and are reminiscent of 'cytonemes'. In the context of Notch signaling, basal actin-based filopodia are important for lateral inhibition between non-neighboring cells. In Hh signaling, it was have observed that cytonemes act as vectors for Hh movement in the ECM, contributing to Hh gradient formation. Since Boi and Ihog are absolutely required for reception, Ptc, Ihog- and Boi-labeled cytonemes emanating from A compartment cells are probably essential for sequestering Hh from P cells. Although this work does not provide the molecular mechanism by which Shf/dWif, Dally, Ihog and Boi proteins affect cytoneme-mediated Hh transport, it is suggested that Shf/dWif might be responsible for maintaining the equilibrium between Hh attachment to cytonemes -- mediated by Dally, Ihog and Boi -- and Hh release or movement (Bilioni, 2013).

Previous analysis on Hh release in the wing imaginal disc epithelium indicates that although Hh is initially externalized through all plasma membranes, the apical Hh pool is internalized and recycled to basolateral plasma membranes where the long-range Hh gradient is formed (Callejo, 2011). This article has provided a compressive genetic analysis that confirms the hypothesis. A novel role is described of the glypican Dally and of the transmembrane protein Boi in the process of the apical internalization of Hh in P compartment cells, which is essential to guarantee that the bulk of Hh protein produced in the P compartment cells is redirected towards the basolateral domain. A role is also described of the Hh coreceptors Ihog and Boi, and the diffusible Shf/dWif factor at the basolateral plane of the epithelium. These proteins interact physically and together with Dally act to establish a balance between Hh attachment to membranes and movement across the ECM to promote gradient formation and signaling. Moreover, all these proteins associate to cytonemes in the basolateral part of the disc epithelium. Thus, the interplay of all these proteins creates an environment supporting Hh transport along cytonemes to shape a proper gradient (Bilioni, 2013).

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

Ayers, K. L., Gallet, A., Staccini-Lavenant, L. and Thérond, P. P. (2010). The long-range activity of Hedgehog is regulated in the apical extracellular space by the glypican Dally and the hydrolase Notum. Dev Cell 18: 605-620. PubMed ID: 20412775

Bilioni, A., Sanchez-Hernandez, D., Callejo, A., Gradilla, A. C., Ibanez, C., Mollica, E., Carmen Rodriguez-Navas, M., Simon, E. and Guerrero, I. (2013). Balancing Hedgehog, a retention and release equilibrium given by Dally, Ihog, Boi and shifted/DmWif. Dev Biol 376: 198-212. PubMed ID: 23276604

Callejo, A., Bilioni, A., Mollica, E., Gorfinkiel, N., Andres, G., Ibanez, C., Torroja, C., Doglio, L., Sierra, J. and Guerrero, I. (2011). Dispatched mediates Hedgehog basolateral release to form the long-range morphogenetic gradient in the Drosophila wing disk epithelium. Proc Natl Acad Sci U S A 108: 12591-12598. PubMed ID: 21690386

Camp, D., He, B. H., Li, S., Althaus, I. W., Holtz, A. M., Allen, B. L., Charron, F. and van Meyel, D. J. (2014). Ihog and Boi elicit Hh signaling via Ptc but do not aid Ptc in sequestering the Hh ligand. Development 141(20):3879-88. PubMed ID: 25231763

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

Da Ros, V. G., Gutierrez-Perez, I., Ferres-Marco, D. and Dominguez, M. (2013). Dampening the signals transduced through Hedgehog via microRNA miR-7 facilitates Notch-induced tumourigenesis. PLoS Biol 11(5): e1001554. PubMed ID: 23667323

Etheridge, L. A., Crawford, T. Q., Zhang, S. and Roelink, H. (2010). Evidence for a role of vertebrate Disp1 in long-range Shh signaling. Development 137: 133-140. PubMed ID: 20023168

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 ID: 18477454

Hartman, T. R., Zinshteyn, D., Schofield, H. K., Nicolas, E., Okada, A. and O'Reilly, A. M. (2010). Drosophila Boi limits Hedgehog levels to suppress follicle stem cell proliferation. J Cell Biol 191: 943-952. PubMed ID: 21098113

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

McLellan, J. S., et al. (2006). Structure of a heparin-dependent complex of Hedgehog and Ihog. Proc. Natl. Acad. Sci. 103(46): 17208-13. Medline abstract: 17077139

Muenke, M. and Beachy, P. A. (2001). Holoprosencephaly. In: C. Scriver, A. Beaudet, W. Sly and D. Valle, Editors, The Metabolic and Molecular Bases of Inherited Disease, McGraw-Hill, New York pp. 6203-6230.

Okada, A., et al. (2006). Boc is a receptor for sonic hedgehog in the guidance of commissural axons. Nature 444(7117): 369-73. Medline abstract: 17086203

Tenzen, T., et al. (2006). The cell surface membrane proteins Cdo and Boc are components and targets of the Hedgehog signaling pathway and feedback network in mice. Dev. Cell 10(5): 647-56. Medline abstract: 16647304

Yao, S., Lum. L. and Beachy, P. (2006). The ihog cell-surface proteins bind Hedgehog and mediate pathway activation. Cell 125: 343-357. Medline abstract: 16630821

Yan, D., et al. (2010). The cell-surface proteins Dally-like and Ihog differentially regulate Hedgehog signaling strength and range during development. Development 137(12): 2033-44. PubMed ID: 20501592

Zheng, X., Mann R. K., Sever N. and Beachy P. A. (2010). Genetic and biochemical definition of the Hedgehog receptor. Genes Dev. 24: 57-71. PubMed ID: 20048000

date revised: 23 June 2023

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