The mechanisms of Hedgehog signalling and its roles in development and disease.

The cloning of the founding member of the Hedgehog (HH) family of secreted proteins two decades ago inaugurated a field that has diversified to encompass embryonic development, stem cell biology and tissue homeostasis. Interest in HH signalling increased when the pathway was implicated in several cancers and congenital syndromes. The mechanism of HH signalling is complex and remains incompletely understood. Nevertheless, studies have revealed novel biological insights into this system, including the function of HH lipidation in the secretion and transport of this ligand and details of the signal transduction pathway, which involves Patched 1, Smoothened and GLI proteins (Cubitus interruptus in Drosophila melanogaster), as well as, in vertebrates, primary cilia.

The GTPase Rab8 differentially controls the long- and short-range activity of the Hedgehog morphogen gradient by regulating Hedgehog apico-basal distribution.

The Hedgehog (Hh) morphogen gradient is required for patterning during metazoan development, yet the mechanisms involved in Hh apical and basolateral release and how this influences short- and long-range target induction are poorly understood. We found that depletion of the GTPase Rab8 in Hh-producing cells induces an imbalance between the level of apically and laterally released Hh. This leads to non-cell-autonomous differential effects on the expression of Hh target genes, namely an increase in its short-range targets and a concomitant decrease in long-range targets. We further found that Rab8 regulates the endocytosis and apico-basal distribution of Ihog, a transmembrane protein known to bind to Hh and to be crucial for establishment of the Hh gradient. Our data provide new insights into morphogen gradient formation, whereby morphogen activity is functionally distributed between apically and basolaterally secreted pools.

TMED10 mediates the loading of neosynthesised Sonic Hedgehog in COPII vesicles for efficient secretion and signalling.

The morphogen Sonic Hedgehog (SHH) plays an important role in coordinating embryonic development. Short- and long-range SHH signalling occurs through a variety of membrane-associated and membrane-free forms. However, the molecular mechanisms that govern the early events of the trafficking of neosynthesised SHH in mammalian cells are still poorly understood. Here, we employed the retention using selective hooks (RUSH) system to show that newly-synthesised SHH is trafficked through the classical biosynthetic secretory pathway, using TMED10 as an endoplasmic reticulum (ER) cargo receptor for efficient ER-to-Golgi transport and Rab6 vesicles for Golgi-to-cell surface trafficking. TMED10 and SHH colocalized at ER exit sites (ERES), and TMED10 depletion significantly delays SHH loading onto ERES and subsequent exit leading to significant SHH release defects. Finally, we utilised the Drosophila wing imaginal disc model to demonstrate that the homologue of TMED10, Baiser (Bai), participates in Hedgehog (Hh) secretion and signalling in vivo. In conclusion, our work highlights the role of TMED10 in cargo-specific egress from the ER and sheds light on novel important partners of neosynthesised SHH secretion with potential impact on embryonic development.

Hherisomes, Hedgehog specialized recycling endosomes, are required for high level Hedgehog signaling and tissue growth.

In metazoans, tissue growth and patterning is partly controlled by the Hedgehog (Hh) morphogen. Using immuno-electron microscopy on Drosophila wing imaginal discs, we identified a cellular structure, the Hherisomes, which contain the majority of intracellular Hh. Hherisomes are recycling tubular endosomes, and their formation is specifically boosted by overexpression of Hh. Expression of Rab11, a small GTPase involved in recycling endosomes, boosts the size of Hherisomes and their Hh concentration. Conversely, increased expression of the transporter Dispatched, a regulator of Hh secretion, leads to their clearance. We show that increasing Hh density in Hherisomes through Rab11 overexpression enhances both the level of Hh signaling and disc pouch growth, whereas Dispatched overexpression decreases high-level Hh signaling and growth. We propose that, upon secretion, a pool of Hh triggers low-level signaling, whereas a second pool of Hh is endocytosed and recycled through Hherisomes to stimulate high-level signaling and disc pouch growth. Altogether, our data indicate that Hherisomes are required to sustain physiological Hh activity necessary for patterning and tissue growth in the wing disc.

Circulating Hedgehog: a fresh view of a classic morphogen.

Members of the Hedgehog family of morphogens mediate the intercellular communication necessary for the organisation and development of many animal tissues. They are modified by various lipid adducts, rendering them insoluble in hydrophilic environments and leading to the contentious question of how these molecules travel in the aqueous extracellular space. Seminal work carried out by Suzanne Eaton and her colleagues has shed light on how these morphogens can spread over long distances through their association with lipoprotein particles. In this Spotlight article, we discuss Suzanne's pioneering work and her contribution to our understanding of the transport and activity of morphogens, in particular Hedgehog. We also describe two other essential aspects of her work: the discovery and characterisation of endogenously present Hedgehog variants, as well as her proposition that, in addition to its role as a morphogen, Hedgehog acts as an endocrine hormone.

Functions of Wnt and Hedgehog-containing extracellular vesicles in development and disease.

Secreted morphogens play a major role in the intercellular communication necessary for animal development. It was initially thought that, in order to organize tissue morphogenesis and control cell fate and proliferation, morphogens diffused freely in the extracellular space. This view has since changed following the discovery that morphogens of the Wnt and Hedgehog (Hh) families are modified by various lipid adducts during their biosynthesis, providing them with high affinity for the membrane bilayer. Recent work performed in model organisms suggests that Wnt and Hh proteins are carried on extracellular vesicles. In this Review, we provide our perspectives on the mechanisms of formation of Wnt- and Hh-containing extracellular vesicles, and discuss their functions during animal development, as well as in various human physiopathologies.

The ESCRT machinery regulates the secretion and long-range activity of Hedgehog.

The conserved family of Hedgehog (Hh) proteins acts as short- and long-range secreted morphogens, controlling tissue patterning and differentiation during embryonic development. Mature Hh carries hydrophobic palmitic acid and cholesterol modifications essential for its extracellular spreading. Various extracellular transportation mechanisms for Hh have been suggested, but the pathways actually used for Hh secretion and transport in vivo remain unclear. Here we show that Hh secretion in Drosophila wing imaginal discs is dependent on the endosomal sorting complex required for transport (ESCRT). In vivo the reduction of ESCRT activity in cells producing Hh leads to a retention of Hh at the external cell surface. Furthermore, we show that ESCRT activity in Hh-producing cells is required for long-range signalling. We also provide evidence that pools of Hh and ESCRT proteins are secreted together into the extracellular space in vivo and can subsequently be detected together at the surface of receiving cells. These findings uncover a new function for ESCRT proteins in controlling morphogen activity and reveal a new mechanism for the transport of secreted Hh across the tissue by extracellular vesicles, which is necessary for long-range target induction.

Variations in Hedgehog signaling: divergence and perpetuation in Sufu regulation of Gli.

The Hedgehog (Hh) proteins play a universal role in metazoan development. Nevertheless, fundamental differences exist between Drosophila and vertebrates in the transduction of the Hh signal, notably regarding the role of primary cilia in mammalian cells. In this issue of Genes & Development, Chen and colleagues (pp. 1910-1928) demonstrate that mouse Suppressor of fused (Sufu) regulates the stability of the transcription factors Gli2 and Gli3 by antagonizing the conserved Gli degradation device mediated by Hib/Spop in a cilia-independent manner.

Dally and Notum regulate the switch between low and high level Hedgehog pathway signalling.

During development, secreted morphogens, such as Hedgehog (Hh), control cell fate and proliferation. Precise sensing of morphogen levels and dynamic cellular responses are required for morphogen-directed morphogenesis, yet the molecular mechanisms responsible are poorly understood. Several recent studies have suggested the involvement of a multi-protein Hh reception complex, and have hinted at an understated complexity in Hh sensing at the cell surface. We show here that the expression of the proteoglycan Dally in Hh-receiving cells in Drosophila is necessary for high but not low level pathway activity, independent of its requirement in Hh-producing cells. We demonstrate that Dally is necessary to sequester Hh at the cell surface and to promote Hh internalisation with its receptor. This internalisation depends on both the activity of the hydrolase Notum and the glycosyl-phosphatidyl-inositol (GPI) moiety of Dally, and indicates a departure from the role of the second glypican Dally-like in Hh signalling. Our data suggest that hydrolysis of the Dally-GPI by Notum provides a switch from low to high level signalling by promoting internalisation of the Hh-Patched ligand-receptor complex.

Microvilli-derived extracellular vesicles carry Hedgehog morphogenic signals for Drosophila wing imaginal disc development.

Morphogens are secreted molecules that regulate and coordinate major developmental processes, such as cell differentiation and tissue morphogenesis. Depending on the mechanisms of secretion and the nature of their carriers, morphogens act at short and long range. We investigated the paradigmatic long-range activity of Hedgehog (Hh), a well-known morphogen, and its contribution to the growth and patterning of the Drosophila wing imaginal disc. Extracellular vesicles (EVs) contribute to Hh long-range activity; however, the nature, the site, and the mechanisms underlying the biogenesis of these vesicular carriers remain unknown. Here, through the analysis of mutants and a series of Drosophila RNAi-depleted wing imaginal discs using fluorescence and live-imaging electron microscopy, including tomography and 3D reconstruction, we demonstrate that microvilli of the wing imaginal disc epithelium are the site of generation of small EVs that transport Hh across the tissue. Further, we show that the Prominin-like (PromL) protein is critical for microvilli integrity. Together with actin cytoskeleton and membrane phospholipids, PromL maintains microvilli architecture that is essential to promote its secretory function. Importantly, the distribution of Hh to microvilli and its release via these EVs contribute to the proper morphogenesis of the wing imaginal disc. Our results demonstrate that microvilli-derived EVs are carriers for Hh long-range signaling in vivo. By establishing that members of the Prominin protein family are key determinants of microvilli formation and integrity, our findings support the view that microvilli-derived EVs conveying Hh may provide a means for exchanging signaling cues of high significance in tissue development and cancer.

The full-length unprocessed hedgehog protein is an active signaling molecule.

The hedgehog (HH) family of ligands plays an important instructional role in metazoan development. HH proteins are initially produced as approximately 45-kDa full-length proteins, which undergo an intramolecular cleavage to generate an amino-terminal product that subsequently becomes cholesterol-modified (HH-Np). It is well accepted that this cholesterol-modified amino-terminal cleavage product is responsible for all HH-dependent signaling events. Contrary to this model we show here that full-length forms of HH proteins are able to traffic to the plasma membrane and participate directly in cell-cell signaling, both in vitro and in vivo. We were also able to rescue a Drosophila eye-specific hh loss of function phenotype by expressing a full-length form of hh that cannot be processed into HH-Np. These results suggest that in some physiological contexts full-length HH proteins may participate directly in HH signaling and that this novel activity of full-length HH may be evolutionarily conserved.

Stability and association of Smoothened, Costal2 and Fused with Cubitus interruptus are regulated by Hedgehog.

The mechanisms involved in transduction of the Hedgehog (Hh) signal are of considerable interest to developmental and cancer biologists. Stabilization of the integral membrane protein Smoothened (Smo) at the plasma membrane is a crucial step in Hh signalling but the molecular events immediately downstream of Smo remain to be elucidated. We have shown previously that the transcriptional mediator Cubitus interruptus (Ci) is associated in a protein complex with at least two other proteins, the kinesin-like Costal2 (Cos2) and the serine-threonine kinase Fused (Fu). This protein complex governs the access of Ci to the nucleus. Here we show that, consequent on the stabilization of Smo, Cos2 and Fu are destabilized. Moreover, we find that the Cos2-Fu-Ci protein complex is associated with Smo in membrane fractions both in vitro and in vivo. We also show that Cos2 binding on Smo is necessary for the Hh-dependent dissociation of Ci from this complex. We propose that the association of the Cos2 protein complex with Smo at the plasma membrane controls the stability of the complex and allows Ci activation, eliciting its nuclear translocation.

Arf6 is necessary for senseless expression in response to wingless signalling during Drosophila wing development.

Wnt signalling is a core pathway involved in a wide range of developmental processes throughout the metazoa. In vitro studies have suggested that the small GTP binding protein Arf6 regulates upstream steps of Wnt transduction, by promoting the phosphorylation of the Wnt co-receptor, LRP6, and the release of ß-catenin from the adherens junctions. To assess the relevance of these previous findings in vivo, we analysed the consequence of the absence of Arf6 activity on Drosophila wing patterning, a developmental model of Wnt/Wingless signalling. We observed a dominant loss of wing margin bristles and Senseless expression in Arf6 mutant flies, phenotypes characteristic of a defect in high level Wingless signalling. In contrast to previous findings, we show that Arf6 is required downstream of Armadillo/ß-catenin stabilisation in Wingless signal transduction. Our data suggest that Arf6 modulates the activity of a downstream nuclear regulator of Pangolin activity in order to control the induction of high level Wingless signalling. Our findings represent a novel regulatory role for Arf6 in Wingless signalling.

Hedgehog Signaling Modulates Glial Proteostasis and Lifespan.

The conserved Hedgehog signaling pathway has well-established roles in development. However, its function during adulthood remains largely unknown. Here, we investigated whether the Hedgehog signaling pathway is active during adult life in Drosophila melanogaster, and we uncovered a protective function for Hedgehog signaling in coordinating correct proteostasis in glial cells. Adult-specific depletion of Hedgehog reduces lifespan, locomotor activity, and dopaminergic neuron integrity. Conversely, increased expression of Hedgehog extends lifespan and improves fitness. Moreover, Hedgehog pathway activation in glia rescues the lifespan and age-associated defects of hedgehog mutants. The Hedgehog pathway regulates downstream chaperones, whose overexpression in glial cells was sufficient to rescue the shortened lifespan and proteostasis defects of hedgehog mutants. Finally, we demonstrate the protective ability of Hedgehog signaling in a Drosophila Alzheimer's disease model expressing human amyloid beta in the glia. Overall, we propose that Hedgehog signaling is requisite for lifespan determination and correct proteostasis in glial cells.

Cholesterol modification of hedgehog is required for trafficking and movement, revealing an asymmetric cellular response to hedgehog.

Hedgehog family members are secreted proteins involved in numerous patterning mechanisms. Different posttranslational modifications have been shown to modulate Hedgehog biological activity. We investigated the role of these modifications in regulating subcellular localization of Hedgehog in the Drosophila embryonic epithelium. We demonstrate that cholesterol modification of Hedgehog is responsible for its assembly in large punctate structures and apical sorting through the activity of the sterol-sensing domain-containing Dispatched protein. We further show that movement of these specialized structures through the cellular field is contingent upon the activity of proteoglycans synthesized by the heparan sulfate polymerase Tout-Velu. Finally, we show that the Hedgehog large punctate structures are necessary only for a subset of Hedgehog target genes across the parasegmental boundary, suggesting that presentation of Hedgehog from different membrane compartments is responsible for Hedgehog functional diversity in epithelial cells.

Confocal analysis of Hedgehog morphogenetic gradient coupled with fluorescent in situ hybridization of hedgehog target genes.

Hedgehog (Hh) family members are secreted proteins that can act at short and long range to direct cell fate decisions during developmental processes. In both Drosophila and vertebrates, the morphogenetic gradient of Hh must be tightly regulated for correct patterning. The posttranslational modification of Hh by a cholesterol adduct participates in such regulation. We have shown that cholesterol modification is necessary for the controlled long-range activity of Drosophila Hh, as observed for its vertebrate counterpart Sonic Hh. The presence of cholesterol on Hh allows the observation of large apical punctuate structures of Hh (Hh-LPSs) at a distance from the Hh source both in embryos and in imaginal discs. The Hh-LPSs apical distribution reflects the Hh gradient and is temporally regulated. Hh gradient modulation is directly related to the dynamic expression of the Hh target gene serrate (ser), shown by immunofluorescent detection of Hh coupled with fluorescent in situ hybridization of ser.

Analysis of the hypoxia-sensing pathway in Drosophila melanogaster.

The mechanism by which hypoxia induces gene transcription involves the inhibition of HIF-1alpha (hypoxia-inducible factor-1 alpha subunit) PHD (prolyl hydroxylase) activity, which prevents the VHL (von Hippel-Lindau)-dependent targeting of HIF-1alpha to the ubiquitin/proteasome pathway. HIF-1alpha thus accumulates and promotes gene transcription. In the present study, first we provide direct biochemical evidence for the presence of a conserved hypoxic signalling pathway in Drosophila melanogaster. An assay for 2-oxoglutarate-dependent dioxygenases was developed using Drosophila embryonic and larval homogenates as a source of enzyme. Drosophila PHD has a low substrate specificity and hydroxylates key proline residues in the ODD (oxygen-dependent degradation) domains of human HIF-1alpha and Similar, the Drosophila homologue of HIF-1alpha. The enzyme promotes human and Drosophila [(35)S]VHL binding to GST (glutathione S-transferase)-ODD-domain fusion protein. Hydroxylation is enhanced by proteasomal inhibitors and was ascertained using an anti-hydroxyproline antibody. Secondly, by using transgenic flies expressing a fusion protein that combined an ODD domain and the green fluorescent protein (ODD-GFP), we analysed the hypoxic cascade in different embryonic and larval tissues. Hypoxic accumulation of the reporter protein was observed in the whole tracheal tree, but not in the ectoderm. Hypoxic stabilization of ODD-GFP in the ectoderm was restored by inducing VHL expression in these cells. These results show that Drosophila tissues exhibit different sensitivities to hypoxia.

Endocytosis of Hedgehog through dispatched regulates long-range signaling.

The proteins of the Hedgehog (Hh) family are secreted proteins exerting short- and long-range control over various cell fates in developmental patterning. The Hh gradient in Drosophila wing imaginal discs consists of apical and basolateral secreted pools, but the mechanisms governing the overall establishment of the gradient remain unclear. We investigated the relative contributions of endocytosis and recycling to control the Hh gradient. We show that, upon its initial apical secretion, Hh is re-internalized. We examined the effect of the resistance-nodulation-division transporter Dispatched (Disp) on long-range Hh signaling and unexpectedly found that Disp is specifically required for apical endocytosis of Hh. Re-internalized Hh is then regulated in a Rab5- and Rab4-dependent manner to ensure its long-range activity. We propose that Hh-producing cells integrate endocytosis and recycling as two instrumental mechanisms contributing to regulate the long-range activity of Hh.

Switch of PKA substrates from Cubitus interruptus to Smoothened in the Hedgehog signalosome complex.

Hedgehog (Hh) signalling is crucial for developmental patterning and tissue homeostasis. In Drosophila, Hh signalling is mediated by a bifunctional transcriptional mediator, called Cubitus interruptus (Ci). Protein Kinase A (PKA)-dependent phosphorylation of the serpentine protein Smoothened (Smo) leads to Ci activation, whereas PKA-dependent phosphorylation of Ci leads to the formation of Ci repressor form. The mechanism that switches PKA from an activator to a repressor is not known. Here we show that Hh signalling activation causes PKA to switch its substrates from Ci to Smo within the Hh signalling complex (HSC). In particular, Hh signalling increases the level of Smo, which then outcompetes Ci for association with PKA and causes a switch in PKA substrate recognition. We propose a new model in which the PKA is constitutively present and active within the HSC, and in which the relative levels of Ci and Smo within the HSC determine differential activation and cellular response to Hh signalling.

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.