Release and transportation of Hedgehog molecules

Secretion of the Hedgehog morphogen induces different cell fates over the short and long ranges during developmental patterning. Mature Hedgehog carries hydrophobic palmitic acid and cholesterol modifications essential for its correct spread. The long-range activity of Hedgehog raises questions about how a dually lipidated protein can spread in the hydrophilic environment of the extracellular space. There is compelling experimental evidence in favour of the existence of several different carriers for Hedgehog transportation, via very different routes. This suggests that different accessory proteins and cellular machineries may be involved in the specific release of Hedgehog. I suggest that Hh carriers may work in parallel within a given cell and that developmental context may condition the choice of Hh carrier in secreting cells.     

How does cholesterol affect the way Hedgehog works?

Members of the Hedgehog (Hh) family of proteins are conserved morphogens that spread and modulate cell fates in target tissue. Mature Hh carries two lipid adducts, a palmitoyl group at the N terminus and cholesterol at the C terminus. Recent findings have addressed how these lipid modifications affect the function and transport of Hh in Drosophila. In contrast to the palmitoyl adduct, cholesterol appears not to be essential for signalling. However, the absence of the cholesterol adduct affects the spread of Hh within tissues. As we discuss here, the exact nature of this effect is controversial.     

Hedgehog movement is regulated through tout velu-dependent synthesis of a heparan sulfate proteoglycan.

Hedgehog (Hh) molecules play critical roles during development as a morphogen, and therefore their distribution must be regulated. Hh proteins undergo several modifications that tether them to the membrane. We have previously identified tout velu (ttv), a homolog of the mammalian EXT tumor suppressor gene family, as a gene required for movement of Hh. In this paper, we present in vivo evidence that ttv is involved in heparan sulfate proteoglycan (HSPG) biosynthesis, suggesting that HSPGs control Hh distribution. In contrast to mutants in other HSPG biosynthesis genes, the activity of the HSPG-dependent FGF and Wingless signaling pathways are not affected in ttv mutants. This demonstrates an unexpected level of specificity in the regulation of the distribution of extracellular signals by HSPGs.     

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.     

Cholesterol modification is necessary for controlled planar long-range activity of Hedgehog in Drosophila epithelia

The Hedgehog morphogen is a major developmental regulator that acts at short and long range to direct cell fate decisions in invertebrate and vertebrate tissues. Hedgehog is the only known metazoan protein to possess a covalently linked cholesterol moiety. Although the role of the cholesterol group of Hedgehog remains unclear, it has been suggested to be dispensable for the its long-range activity in Drosophila. Here, we provide data in three different epithelia - ventral and dorsal embryonic ectoderm, and larval imaginal disc tissue - showing that cholesterol modification is in fact necessary for the controlled long-range activity of Drosophila Hedgehog. We provide an explanation for the discrepancy between our results and previous reports by showing that unmodified Hh can act at long range, albeit in an uncontrolled manner, only when expressed in squamous cells. Our data show that cholesterol modification controls long-range Hh activity at multiple levels. First, cholesterol increases the affinity of Hh for the plasma membrane, and consequently enhances its apparent intrinsic activity, both in vitro and in vivo. In addition, multimerisation of active Hh requires the presence of cholesterol. These multimers are correlated with the assembly of Hh into apically located, large punctate structures present in active Hh gradients in vivo. By comparing the activity of cholesterol-modified Hh in columnar epithelial cells and peripodial squamous cells, we show that epithelial cells provide the machinery necessary for the controlled planar movement of Hh, thereby preventing the unrestricted spreading of the protein within the three-dimensional space of the epithelium. We conclude that, as in vertebrates, cholesterol modification is essential for controlled long-range Hh signalling in Drosophila.     

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.     

The adventures of sonic hedgehog in development and repair. III. Hedgehog processing and biological activity

The Hedgehog (Hh) family of secreted proteins is necessary for aspects of the development and maintenance of the gastrointestinal tract. Hh is thought to function as a morphogen, a mitogen, a cell survival factor, and an axon guidance factor. Given its wide role in development, as well as in a variety of disease states, understanding the regulation of Hh function and activity is critically important. However, the study of Hh signaling has been impeded by its unusual biology. Hh is unique in that it is the only protein covalently modified by cholesterol, which in turn affects numerous aspects of its localization, release, movement, and activity. All are important factors when considering Hh's physiological role, and animals have developed an intricate system of regulators responsible for both promoting and inhibiting the activity of Hh. This review is intended to give a broad overview of how the biosynthesis and movement of Hh contributes to its biological activity.     

Differential range and activity of various forms of the Hedgehog protein

Background  

The Hedgehog (Hh) family of secreted proteins act as extra cellular messengers to control and coordinate growth and differentiation. The mechanism by which Hh protein travels across a field of cells, and results in a range of specific effects relating to the distance from the source, has been the subject of much debate. It has been suggested that the range and activity of the pathway can be linked to modifications of the Hh protein, specifically the addition of lipid groups at N- and C-terminal sites.     

An Emerging Role of Sonic Hedgehog Shedding as a Modulator of Heparan Sulfate Interactions

Major developmental morphogens of the Hedgehog (Hh) family act at short range and long range to direct cell fate decisions in vertebrate and invertebrate tissues. To this end, Hhs are released from local sources and act at a distance on target cells that express the Hh receptor Patched. However, morphogen secretion and spreading are not passive processes because all Hhs are synthesized as dually (N- and C-terminal) lipidated proteins that firmly tether to the surface of producing cells. On the cell surface, Hhs associate with each other and with heparan sulfate (HS) proteoglycans. This raises the question of how Hh solubilization and spreading is achieved. We recently discovered that Sonic hedgehog (Shh) is solubilized by proteolytic processing (shedding) of lipidated peptide termini in vitro. Because unprocessed N termini block Patched receptor binding sites in the cluster, we further suggested that their proteolytic removal is required for simultaneous Shh activation. In this work we confirm inactivity of unprocessed protein clusters and demonstrate restored biological Shh function upon distortion or removal of N-terminal amino acids and peptides. We further show that N-terminal Shh processing targets and inactivates the HS binding Cardin-Weintraub (CW) motif, resulting in soluble Shh clusters with their HS binding capacities strongly reduced. This may explain the ability of Shh to diffuse through the HS-containing extracellular matrix, whereas other HS-binding proteins are quickly immobilized. Our in vitro findings are supported by the presence of CW-processed Shh in murine brain samples, providing the first in vivo evidence for Shh shedding and subsequent solubilization of N-terminal-truncated proteins.     

Oligomerization and endocytosis of Hedgehog is necessary for its efficient exovesicular secretion

Hedgehog (Hh) is a secreted morphogen involved in both short- and long-range signaling necessary for tissue patterning during development. It is unclear how this dually lipidated protein is transported over a long range in the aqueous milieu of interstitial spaces. We previously showed that the long-range signaling of Hh requires its oligomerization. Here we show that Hh is secreted in the form of exovesicles. These are derived by the endocytic delivery of cell surface Hh to multivesicular bodies (MVBs) via an endosomal sorting complex required for transport (ECSRT)–dependent process. Perturbations of ESCRT proteins have a selective effect on long-range Hh signaling in Drosophila wing imaginal discs. Of importance, oligomerization-defective Hh is inefficiently incorporated into exovesicles due to its poor endocytic delivery to MVBs. These results provide evidence that nanoscale organization of Hh regulates the secretion of Hh on ESCRT-derived exovesicles, which in turn act as a vehicle for long-range signaling.     

Improving the understanding of cytoneme-mediated morphogen gradients by in silico modeling

Morphogen gradients are crucial for the development of organisms. The biochemical properties of many morphogens prevent their extracellular free diffusion, indicating the need of an active mechanism for transport. The involvement of filopodial structures (cytonemes) has been proposed for morphogen signaling. Here, we describe an in silico model based on the main general features of cytoneme-meditated gradient formation and its implementation into Cytomorph, an open software tool. We have tested the spatial and temporal adaptability of our model quantifying Hedgehog (Hh) gradient formation in two Drosophila tissues. Cytomorph is able to reproduce the gradient and explain the different scaling between the two epithelia. After experimental validation, we studied the predicted impact of a range of features such as length, size, density, dynamics and contact behavior of cytonemes on Hh morphogen distribution. Our results illustrate Cytomorph as an adaptive tool to test different morphogen gradients and to generate hypotheses that are difficult to study experimentally.     

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.     

Three Drosophila EXT genes shape morphogen gradients through synthesis of heparan sulfate proteoglycans

The signaling molecules Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg) function as morphogens and organize wing patterning in Drosophila. In the screen for mutations that alter the morphogen activity, we identified novel mutants of two Drosophila genes, sister of tout-velu (sotv) and brother of tout-velu (botv), and new alleles of tout-velu (ttv). The encoded proteins of these genes belong to an EXT family of proteins that have or are closely related to glycosyltransferase activities required for biosynthesis of heparan sulfate proteoglycans (HSPGs). Mutation in any of these genes impaired biosynthesis of HSPGs in vivo, indicating that, despite their structural similarity, they are not redundant in the HSPG biosynthesis. Protein levels and signaling activities of Hh, Dpp and Wg were reduced in the cells mutant for any of these EXT genes to a various degree, Wg signaling being the least sensitive. Moreover, all three morphogens were accumulated in the front of EXT mutant cells, suggesting that these morphogens require HSPGs to move efficiently. In contrast to previous reports that ttv is involved exclusively in Hh signaling, we found that ttv mutations also affected Dpp and Wg. These data led us to conclude that each of three EXT genes studied contribute to Hh, Dpp and Wg morphogen signaling. We propose that HSPGs facilitate the spreading of morphogens and therefore, function to generate morphogen concentration gradients.     

Hedgehog lipid modifications are required for Hedgehog stabilization in the extracellular matrix

The Hedgehog (Hh) family of morphogenetic proteins has important instructional roles in metazoan development. Despite Hh being modified by Ct-cholesterol and Nt-palmitate adducts, Hh migrates far from its site of synthesis and programs cellular outcomes, depending on its local concentrations. We show that in the receiving cells of the Drosophila wing imaginal disc, lipid-unmodified Hh spreads across many more cell diameters than the wild type and this spreading leads to the activation of low but not high threshold responses. Unlipidated Hh forms become internalized through the apical plasma membrane, while wild-type Hh enters through the basolateral cell surface - in all cases via a dynamin-dependent mechanism. Full activation of the Hh pathway and the spread of Hh throughout the extracellular matrix depend on the ability of lipid-modified Hh to interact with heparan sulfate proteoglycans (HSPG). However, neither Hh-lipid modifications nor HSPG function are required to activate the targets that respond to low levels of Hh. All these data show that the interaction of lipid-modified Hh with HSPG is important both for precise Hh spreading through the epithelium surface and for correct Hh reception.     

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.     

Distinct and collaborative roles of Drosophila EXT family proteins in morphogen signalling and gradient formation

Heparan sulfate proteoglycans (HSPG) have been implicated in regulating the signalling activities of secreted morphogen molecules including Wingless (Wg), Hedgehog (Hh) and Decapentaplegic (Dpp). HSPG consists of a protein core to which heparan sulfate (HS) glycosaminoglycan (GAG) chains are attached. The formation of HS GAG chains is catalyzed by glycosyltransferases encoded by members of the EXT family of putative tumor suppressors linked to hereditary multiple exostoses. Previous studies in Drosophila demonstrated that tout-velu (ttv), the Drosophila EXT1, is required for Hh movement. However, the functions of other EXT family members are unknown. We have identified and isolated the other two members of the Drosophila EXT family genes, which are named sister of tout-velu (sotv) and brother of tout-velu (botv), and encode Drosophila homologues of vertebrate EXT2 and EXT-like 3 (EXTL3), respectively. We show that both Hh and Dpp signalling activities, as well as their morphogen distributions, are defective in cells mutant for ttv, sotv or botv in the wing disc. Surprisingly, although Wg morphogen distribution is abnormal in ttv, sotv and botv, Wg signalling is only defective in botv mutants or ttv-sotv double mutants, and not in ttv nor sotv alone, suggesting that Ttv and Sotv are redundant in Wg signalling. We demonstrate further that Ttv and Sotv form a complex and are co-localized in vivo. Our results, along with previous studies on Ttv, provide evidence that all three Drosophila EXT proteins are required for the biosynthesis of HSPGs, and for the gradient formation of the Wg, Hh and Dpp morphogens. Our results also suggest that HSPGs have two distinct roles in Wg morphogen distribution and signalling.     

Cellular trafficking of the glypican Dally-like is required for full-strength Hedgehog signaling and wingless transcytosis

Hedgehog (Hh) and Wingless (Wg) morphogens specify cell fate in a concentration-dependent manner in the Drosophila wing imaginal disc. Proteoglycans, components of the extracellular matrix, are involved in Hh and Wg stability, spreading, and reception. In this study, we demonstrate that the glycosyl-phosphatidyl-inositol (GPI) anchor of the glypican Dally-like (Dlp) is required for its apical internalization and its subsequent targeting to the basolateral compartment of the epithelium. Dlp endocytosis from the apical surface of Hh-receiving cells catalyzes the internalization of Hh bound to its receptor Patched (Ptc). The cointernalization of Dlp with the Hh/Ptc complex is dynamin dependent and necessary for full-strength Hh signaling. We also demonstrate that Wg is secreted apically in the disc epithelium and that apicobasal trafficking of Dlp allows Wg transcytosis to favor Wg spreading along the basolateral compartment. Thus, Dlp endocytosis is a common regulatory mechanism of both Hh and Wg morphogen action.     

Sonic Hedgehog as a mediator of long-range signaling

The ability of Hedgehog (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 many cell diameters. Accordingly, the loss of gene products required for these processes abrogates the Hh proteins' abilities to exert their effects, which can be long range, short range, or both. We review here recent evidence demonstrating that Hh proteins are directly responsible for their long-range biological effects. Additionally, we integrate both genetic and biochemical data to delineate a model illustrating how the unusual biochemistry of Hh family members may allow them to act as morphogens, signaling over both short and long distances.