Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified hedgehog from signaling cells

Members of the Hedgehog (Hh) family of secreted signaling proteins function as potent short-range organizers in animal development. Their range of action is limited by a C-terminal cholesterol tether and the upregulation of Patched (Ptc) receptor levels. Here we identify a novel segment-polarity gene in Drosophila, dispatched (disp), and demonstrate that its product is required in sending cells for normal Hh function. In the absence of Disp, cholesterol-modified but not cholesterol-free Hh is retained in these cells, indicating that Disp functions to release cholesterol-anchored Hh. Despite their opposite roles, Disp and Ptc share structural homology in the form of a sterol-sensing domain, suggesting that release and sequestration of cholesterol-modified Hh may be based on related molecular pathways.     

The zebrafish-secreted matrix protein you/scube2 is implicated in long-range regulation of hedgehog signaling

The Hedgehog (Hh) signal plays a pivotal role in induction of ventral neuronal and muscle cell types around the midline during vertebrate development [1]. We report that the gene disrupted in zebrafish you mutants, in which Hh signaling is impaired, encodes the secreted matrix protein Scube2. Consistently, epistasis analyses suggested that Scube2 functions upstream of Hh ligands or through a parallel pathway. In addition, overexpression analyses suggested that Scube2 is an essential, but a permissive, mediator of Hh signaling in zebrafish embryos. Surprisingly, the you gene is expressed in the dorsal neural tube, raising the possibility that Scube2 could indirectly act via a long-range regulator of Hh signaling. The dorsal Bmps have a long-range and opposing influence on Hh signaling [2-5]. We show that neural plate patterning is affected in you mutants in a way that is consistent with the aberrant long-range action of a Bmp-dependent signal. We further show that Bmp activity can be attenuated by the coexpression of Scube2. Our data support the idea that Scube2 can modulate the long-range action of Bmp-dependent signaling in the neural tube and somites.     

A genome-wide RNAi screen identifies regulators of cholesterol-modified hedgehog secretion in Drosophila

Hedgehog (Hh) proteins are secreted molecules that function as organizers in animal development. In addition to being palmitoylated, Hh is the only metazoan protein known to possess a covalently-linked cholesterol moiety. The absence of either modification severely disrupts the organization of numerous tissues during development. It is currently not known how lipid-modified Hh is secreted and released from producing cells. We have performed a genome-wide RNAi screen in Drosophila melanogaster cells to identify regulators of Hh secretion. We found that cholesterol-modified Hh secretion is strongly dependent on coat protein complex I (COPI) but not COPII vesicles, suggesting that cholesterol modification alters the movement of Hh through the early secretory pathway. We provide evidence that both proteolysis and cholesterol modification are necessary for the efficient trafficking of Hh through the ER and Golgi. Finally, we identified several putative regulators of protein secretion and demonstrate a role for some of these genes in Hh and Wingless (Wg) morphogen secretion in vivo. These data open new perspectives for studying how morphogen secretion is regulated, as well as provide insight into regulation of lipid-modified protein secretion.     

Mouse dispatched mutants fail to distribute hedgehog proteins and are defective in hedgehog signaling

Hedgehog (Hh) signaling plays a major role in multiple aspects of embryonic development, which involves both short- and long-range signaling from localized Hh sources. One unusual aspect of Hh signaling is the autoproteolytic processing of Hh followed by lipid modification. As a consequence, the N-terminal fragment of Hh becomes membrane anchored on the cell surface of Hh-producing cells. A key issue in Hh signaling is to understand the molecular mechanisms by which lipid-modified Hh protein is transported from its sites of synthesis and subsequently moves through the morphogenetic field. The dispatched gene, which encodes a putative multipass membrane protein, was initially identified in Drosophila and is required in Hh-producing cells, where it facilitates the transport of cholesterol-modified Hh. We report the identification of the mouse dispatched (Disp) gene and a phenotypic analysis of Disp mutant mice. Disp-null mice phenocopy mice deficient in the smoothened gene, an essential component for Hh reception, suggesting that Disp is essential for Hh signaling. This conclusion was further supported by a detailed molecular analysis of Disp knockout mice, which exhibit defects characteristic of loss of Hh signaling. We also provide evidence that Disp is not required for Hh protein synthesis or processing, but rather for the movement of Hh protein from its sites of synthesis in mice. Taken together, our results reveal a conserved mechanism of Hh protein movement in Hh-producing cells that is essential for proper Hh signaling.     

Dose dependency of Disp1 and genetic interaction between Disp1 and other hedgehog signaling components in the mouse

Genetic analyses in Drosophila have demonstrated that a transmembrane protein Dispatched (Disp) is required for the release of lipid-modified Hedgehog (Hh) protein from Hh secreting cells. Analysis of Disp1 null mutant embryos has demonstrated that Disp1 plays a key role in hedgehog signaling in the early mouse embryo. Here we have used a hypomorphic allele in Disp1(Disp1(Delta)(2)), to extend our knowledge of Disp1 function in Hh-mediated patterning of the mammalian embryo. Through genetic combinations with null alleles of patched 1 (Ptch1), sonic hedgehog (Shh) and Indian hedgehog (Ihh), we demonstrate that Disp1 genetically interacts with Hh signaling components. As Disp1 activity is decreased we see a progressive increase in the severity of hedgehog-dependent phenotypes, which is further enhanced by reducing hedgehog ligand levels. Analysis of neural tube patterning demonstrates a progressive loss of ventral cell identities that most likely reflects decreased Shh signaling as Disp1 levels are attenuated. Conversely, increasing available Shh ligand by decreasing Ptch1 dosage leads to the restoration of ventral cell types in Disp1(Delta2/Delta2) mutants. Together, these studies suggest that Disp1 actively regulates the levels of hedgehog ligand that are available to the hedgehog target field. Further, they provide additional support for the dose-dependent action of Shh signaling in patterning the embryo. Finally, in-vitro studies on Disp1 null mutant fibroblasts indicate that Disp1 is not essential for membrane targeting or release of lipid-modified Shh ligand.     

Hedgehog secretion and signal transduction in vertebrates

Signaling by the Hedgehog (Hh) family of secreted proteins is essential for proper embryonic patterning and development. Dysregulation of Hh signaling is associated with a variety of human diseases ranging from developmental disorders such as holoprosencephaly to certain forms of cancer, including medulloblastoma and basal cell carcinoma. Genetic studies in flies and mice have shaped our understanding of Hh signaling and revealed that nearly all core components of the pathway are highly conserved. Although many aspects of the Drosophila Hh pathway are conserved in vertebrates, mechanistic differences between the two species have begun to emerge. Perhaps the most striking divergence in vertebrate Hh signaling is its dependence on the primary cilium, a vestigial organelle that is largely absent in flies. This minireview will provide an overview of Hh signaling and present recent insights into vertebrate Hh secretion, receptor binding, and signal transduction.     

Disp1 regulates growth of mammalian long bones through the control of Ihh distribution

Dispatched1 (Disp1) is required for the release of cholesterol modified hedgehog (Hh) proteins from producing cells. We investigated the role of Disp1 in Indian hedgehog (Ihh) signaling in the developing bone bypassing the lethality of the Disp1^C829F allele at early somite stages through the supply of non-cholesterol modified Sonic hedgehog (N-Shh). The long bones that develop in the absence of wild-type Disp1, while clearly shorter, have a juxtaposition of proliferating and non-proliferating hypertrophic chondrocytes that is markedly more normal in organization than those of ihh null mutants. Direct analysis of Ihh trafficking in the target field demonstrates that Ihh is distributed well beyond Ihh expressing cells though the range of movement and signaling action is more restricted than in wild-type long bones. Consequently, a PTHrP-Ihh feedback loop is established, but over a shorter distance, reflecting the reduced range of Ihh movement. These analyses of the Disp1^C829F mutation demonstrate that Disp1 is not absolutely required for the paracrine signaling role of Ihh in the skeleton. However, Disp1 is critical for the full extent of signaling within the chondrocyte target field and consequently the establishment of a normal skeletal growth plate.     

Inactivation of dispatched 1 by the chameleon mutation disrupts Hedgehog signalling in the zebrafish embryo

Searches of zebrafish EST and whole genome shotgun sequence databases for sequences encoding the sterol-sensing domain (SSD) protein motif identified two sets of DNA sequences with significant homology to the Drosophila dispatched gene required for release of secreted Hedgehog protein. Using morpholino antisense oligonucleotides, we found that inhibition of one of these genes, designated Disp1, results in a phenotype similar to that of the "you-type" mutants, previously implicated in signalling by Hedgehog proteins in the zebrafish embryo. Injection of disp1 mRNA into embryos homozygous for one such mutation, chameleon (con) results in rescue of the mutant phenotype. Radiation hybrid mapping localised disp1 to the same region of LG20 to which the con mutation was mapped by meiotic recombination analysis. Sequence analysis of disp1 cDNA derived from homozygous con mutant embryos revealed that both mutant alleles are associated with premature termination codons in the disp1 coding sequence. By analysing the expression of markers of specific cell types in the neural tube, pancreas and myotome of con mutant and Disp1 morphant embryos, we conclude that Disp1 activity is essential for the secretion of lipid-modified Hh proteins from midline structures.     

Mutations in the sterol-sensing domain of Patched suggest a role for vesicular trafficking in Smoothened regulation

The tumor suppressor gene patched (ptc) encodes an approximately 140 kDa polytopic transmembrane protein [1-3] [corrected] that binds members of the Hedgehog (Hh) family of signaling proteins [4-6] [corrected] and regulates the activity of Smoothened (Smo), a G protein-coupled receptor-like protein essential for Hh signal transduction [7-9] [corrected]. Ptc contains a sterol-sensing domain (SSD) [10, 11] [corrected], a motif found in proteins implicated in the intracellular trafficking of cholesterol [12] [corrected], and/or other cargoes [13-15] [corrected]. Cholesterol plays a critical role in Hedgehog (Hh) signaling by facilitating the regulated secretion and sequestration of the Hh protein [16] [corrected], to which it is covalently coupled. In addition, cholesterol synthesis inhibitors block the ability of cells to respond to Hh [18, 19] [corrected], and this finding points to an additional requirement for the lipid in regulating downstream components of the Hh signaling pathway. Although the SSD of Ptc has been linked to both the sequestration of, and the cellular response to Hh [16, 20, 21] [corrected], definitive evidence for its function has so far been lacking. Here we describe the identification and characterization of two missense mutations in the SSD of Drosophila Ptc; strikingly, while both mutations abolish Smo repression, neither affects the ability of Ptc to interact with Hh. We speculate that Ptc may control Smo activity by regulating an intracellular trafficking process dependent upon the integrity of the SSD.     

Loss of Function Mutation in the Palmitoyl-Transferase HHAT Leads to Syndromic 46,XY Disorder of Sex Development by Impeding Hedgehog Protein Palmitoylation and Signaling

The Hedgehog (Hh) family of secreted proteins act as morphogens to control embryonic patterning and development in a variety of organ systems. Post-translational covalent attachment of cholesterol and palmitate to Hh proteins are critical for multimerization and long range signaling potency. However, the biological impact of lipid modifications on Hh ligand distribution and signal reception in humans remains unclear. In the present study, we report a unique case of autosomal recessive syndromic 46,XY Disorder of Sex Development (DSD) with testicular dysgenesis and chondrodysplasia resulting from a homozygous G287V missense mutation in the hedgehog acyl-transferase (HHAT) gene. This mutation occurred in the conserved membrane bound O-acyltransferase (MBOAT) domain and experimentally disrupted the ability of HHAT to palmitoylate Hh proteins such as DHH and SHH. Consistent with the patient phenotype, HHAT was found to be expressed in the somatic cells of both XX and XY gonads at the time of sex determination, and Hhat loss of function in mice recapitulates most of the testicular, skeletal, neuronal and growth defects observed in humans. In the developing testis, HHAT is not required for Sertoli cell commitment but plays a role in proper testis cord formation and the differentiation of fetal Leydig cells. Altogether, these results shed new light on the mechanisms of action of Hh proteins. Furthermore, they provide the first clinical evidence of the essential role played by lipid modification of Hh proteins in human testicular organogenesis and embryonic development.     

Hedgehog-mediated patterning of the mammalian embryo requires transporter-like function of dispatched

The dispatched (disp) gene is required for long-range Hedgehog (Hh) signaling in Drosophila. Here, we demonstrate that one of two murine homologs, mDispA, can rescue disp function in Drosophila and is essential for all Hh patterning activities examined in the early mouse embryo. Embryonic fibroblasts lacking mDispA respond normally to exogenously provided Sonic hedgehog (Shh) signal, but are impaired in stimulation of other responding cells when expressing Shh. We have developed a biochemical assay that directly measures the activity of Disp proteins in release of soluble Hh proteins. This activity is disrupted by alteration of residues functionally conserved in Patched and in a related family of bacterial transmembrane transporters, thus suggesting similar mechanisms of action for all of these proteins.     

Negative regulation of Hedgehog signaling by the cholesterogenic enzyme 7-dehydrocholesterol reductase

Cholesterol regulates Hedgehog (Hh) signaling during early vertebrate development. Smith-Lemli-Opitz syndrome (SLOS) is caused by defects in 7-dehydrocholesterol reductase (DHCR7), an enzyme catalyzing the final step of cholesterol biosynthesis. Many developmental malformations attributed to SLOS occur in tissues and organs where Hh signaling is required for development, but the precise role of DHCR7 deficiency in this disease remains murky. We report that DHCR7 and Sonic Hedgehog (Shh) are co-expressed during midline development in Xenopus embryos. DHCR7 has previously been implicated to function as a positive regulator of Hh signaling that acts to regulate the cholesterol adduction of Hh ligand or to affect Hh signaling in the responding cell. We present gain- and loss-of-function analyses suggesting that DHCR7 functions as a negative regulator of Hh signaling at the level or downstream of Smoothened (Smo) and affects intracellular Hh signaling. Our analysis also raises the possibility that the human condition SLOS is caused not only by disruption of the enzymatic role of DHCR7 as a reductase in cholesterol biosynthesis, but may also involve defects in DHCR7 resulting in derepression of Shh signaling.     

Greased hedgehogs: new links between hedgehog signaling and cholesterol metabolism

The close link between signaling by the developmental regulators of the Hedgehog family and cholesterol biochemistry has been known for some time. The morphogen is covalently attached to cholesterol in a peculiar autocatalytic reaction and embryonal disruption of cholesterol synthesis leads to malformations that mimic Hh signaling defects. Recently, it was furthermore shown that secreted Hh could hitchhike on lipoprotein particles to establish its morphogenic gradient in the developing embryo. Additionally, there is new evidence that the Hh-receptor Patched transmits the Hh signal by modulating the secretion of an inhibitory sterol molecule from the receiving cells. Here we present some of the most recent discoveries on the Hh-sterol link and discuss their implications from a systems design perspective. We predict that a robust functioning of the Hh pathway will require the involvement of more sterol metabolites, and these should be the subject of future research.     

On the range of hedgehog signaling

Hedgehog (Hh) is a secreted signaling protein that regulates the development of many organ systems. It can travel from its site of synthesis, a process that involves covalent attachment of cholesterol to its carboxyl terminus, proteins with putative sterol sensing domains in both sending and receiving cells, and glycosaminoglycans. Understanding how the movement of Hh is controlled and propelled will be key to understanding how it carries out its essential roles.     

Lipid Modification of Secreted Signaling Proteins

Proteins of the Hedgehog, Wnt and Epidermal Growth Factor Receptor (EGFR) ligand families are secreted signals that induce concentration-dependent responses in surrounding cells. Although these proteins must diffuse through the aqueous extracellular environment, recent work has shown that hydrophobic lipid modifications are essential for their functions. All three classes of ligands are palmitoylated in the secretory pathway by related enzymes, and Hedgehog also carries a C-terminal cholesterol modification as a result of its autocatalytic cleavage. Palmitoylation is required for Wingless secretion and contributes to the signaling activity of Hedgehog and Wnt3a, but is not required for secretion or receptor activation by the EGFR ligand Spitz. While lipid modifications enhance the long-range activity of Sonic hedgehog, they restrict the range and increase the local concentration of Spitz. We discuss the diverse functions and the possible extent of palmitoylation of secreted ligands.     

Boc and Gas1 each form distinct Shh receptor complexes with Ptch1 and are required for Shh-mediated cell proliferation

Hedgehog (Hh) proteins regulate important developmental processes, including cell proliferation and differentiation. Although Patched acts as the main Hh receptor in Drosophila, Hh signaling absolutely requires the additional Hh-binding proteins Ihog and Boi. Here we show that, unexpectedly, cerebellar granule neuron progenitors (CGNPs) lacking Boc and Cdon, the vertebrate orthologs of Ihog and Boi, still proliferate in response to Hh. This is because in their absence, Gas1, an Hh-binding protein not present in Drosophila, mediates Hh signaling. Consistently, only CGNPs lacking all three molecules-Boc, Cdon, and Gas1-have a complete loss of Hh-dependent proliferation. In a complementary manner, we find that a mutated Hh ligand that binds Patched1 but not Boc, Cdon, or Gas1 cannot activate Hh signaling. Together, this demonstrates an absolute requirement for Boc, Cdon, and Gas1 in Hh signaling and reveals a distinct requirement for ligand-binding components that distinguishes the vertebrate and invertebrate Hh receptor systems.     

Recent progress in the study of Hedgehog signaling

The Hedgehog (Hh) family of secreted signaling proteins plays a critical role in regulating the development of several tissues and organ systems.The ability of Hh proteins to exert their biological effects is regulated by a series of post-translational processes.These processes include an intramolecular cleavage,covalent addition of cholesterol and/or palmitate,and conversion into a multimeric freely diffusible form.The processing of Hh proteins affects their trafficking,potency,and ability to signal over several cell diameters.Here we review the current understanding of the Hh signaling mechanisms that govern the establishment of the Hh gradient and the transduction of the Hh signal in the light of recent data.