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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.

Key Points The Hedgehog signalling pathway has diverse functions in animal development and tissue homeostasis. Dysregulation of the pathway has been implicated in several developmental syndromes and cancers. The production of Hedgehog (HH) ligands involves autoproteolytic cleavage and the addition of lipid moieties such as cholesterol and palmitic acid. Several dedicated proteins are required for the secretion of the ligand. Novel mechanisms involving ligand multimerization, complex formation with additional soluble proteins and association with components of the extracellular matrix, such as the heparan sulphate proteoglycans, have been proposed to explain the dispersion of lipidated HH proteins through tissues. Signal transduction requires a number of transmembrane proteins, most notably Patched 1 (PTC) and Smoothened (SMO). The mechanism by which the signal is transduced from PTC to SMO is unclear. Strikingly, in vertebrates, but not Drosophila melanogaster , signal transduction requires the primary cilia of cells, identifying a function for this previously enigmatic organelle. Intracellularly, signalling involves Suppressor of Fu (SUFU), the kinesin proteins Costal 2 (Cos2) in D. melanogaster and KIF7 in vertebrates and several kinases. Signalling culminates with the regulation of the D. melanogaster Cubitus interruptus (Ci) and vertebrate GLI families of transcription factors. HH signalling converts the Ci and GLI proteins from transcriptional repressors to activators by inhibiting their proteolytic cleavage. The various functions of HH signalling have predominately been ascribed to its regulation of gene expression, and the relevant transcriptional networks are beginning to be dissected. Recently, however, non-canonical branches to the signalling pathway have been found, and these might contribute to some HH functions. HH signalling regulates the survival and proliferation of tissue progenitor and stem populations. This function is linked to its role in tumour formation. Several small-molecule antagonists and agonists that target SMO have been identified, and these show promise in the treatment of HH-related pathologies. In 2012, the approval of the Food and Drug Administration (FDA) was obtained for the first drug that targets HH signalling in the skin cancer basal cell carcinoma. The founding member of the Hedgehog (HH) family of secreted proteins was cloned two decades ago. The mechanism of HH signalling is incomplete, but insight has been gained into the function of lipidation in ligand secretion and transport, as well as into key components of the signalling pathway. 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.

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. In Drosophila , protein kinase A (PKA) phosphorylates the transcription factor Cubitus interruptus (Ci) in the absence of Hedgehog signalling and the transducer Smoothened in its presence. Here, the authors investigate the mechanism underlying this switch and propose that Smoothened outcompetes Ci for association with PKA on Hedgehog signalling activation.

A new role for the endosomal sorting complex required for transport (ESCRT) is identified in fly larvae, where it is shown to be essential for the secretion and long-range signalling of the embryonic development morphogen Hedgehog. ESCRT involved in Hedgehog signalling ESCRT, the endosomal sorting complex required for transport, is best known for its roles in vesicular trafficking, cell division and mediating viral escape from cells. Pascal Thérond and co-workers now report an unexpected role for this complex. During embryonic development, Hedgehog proteins control tissue patterning and differentiation over short and long distances. The authors find that ESCRT activity is essential for Hedgehog secretion in fly larvae. Pools of Hedgehog and ESCRT are secreted together into the extracellular space and both can subsequently be detected together at the surface of receiving cells, where ESCRT activity seems to be required for long-range Hedgehog signalling. The conserved family of Hedgehog (Hh) proteins acts as short- and long-range secreted morphogens, controlling tissue patterning and differentiation during embryonic development 1 . Mature Hh carries hydrophobic palmitic acid and cholesterol modifications essential for its extracellular spreading 2 . 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) 3 . 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.

The Hedgehog (Hh) signalling pathway has a crucial role in several developmental processes and is aberrantly activated in a variety of cancers. In Drosophila , many of the canonical Hh pathway components are phosphorylated, yet the precise role of these phosphorylation events in the regulation of Hh signal transduction is unclear. Furthermore, the Hh pathway receives input from several kinases that have well‐described roles in other cellular functions, some of which have both positive and negative effects on Hh signalling. Several recent studies have characterized the role of specific phosphorylation events in the Hh pathway, and have begun to shed light on how phosphorylation of Hh signalling components affects their subcellular location, stability and activity to mediate the transcriptional response to the Hh gradient.

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 β-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/β-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.

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.

ABSTRACT The regulation and coordination of developmental processes involves the secretion of morphogens and membrane carriers, including extracellular vesicles, which facilitate their transport over long distance. The long-range activity of the Hedgehog morphogen is conveyed by extracellular vesicles. However, the site and the molecular basis of their biogenesis remains unknown. By combining fluorescence and electron microscopy combined with genetics and cell biology approaches, we investigated the origin and the cellular mechanisms underlying extracellular vesicle biogenesis, and their contribution to Drosophila wing disc development, exploiting Hedgehog as a long-range morphogen. We show that microvilli of Drosophila wing disc epithelium are the site of generation of small extracellular vesicles that transport Hedgehog across the tissue. This process requires the Prominin-like protein, whose activity, together with interacting cytoskeleton components and lipids, is critical for maintaining microvilli integrity and function in secretion. Our results provide the first evidence that microvilli-derived extracellular vesicles contribute to Hedgehog long-range signaling activity highlighting their physiological significance in tissue development in vivo. Competing Interest Statement The authors have declared no competing interest. Footnotes * ↵* gisela.dangelo{at}curie.fr

The hedgehog gene (hh) of Drosophila melanogaster exerts both short- and long-range effects on cell patterning during development. The product of hedgehog is a secreted protein that apparently acts by triggering an intracellular signaling pathway, but little is known about the details of that pathway. The Drosophila gene fused (fu) encodes a serine/threonine-protein kinase that genetic experiments have implicated in signaling initiated by hedgehog. Here we report that the fused protein is phosphorylated during the course of Drosophila embryogenesis, as a result of hedgehog activity. In cell culture, phosphorylation of fused protein occurs in response to the biologically active form of hedgehog and cannot be blocked by activation of protein kinase A, which is thought to be an antagonist of signaling from hedgehog. These results suggest that fused and protein kinase A function downstream of hedgehog but in parallel pathways that eventually converge distal to fused. The reconstruction of signaling from hedgehog in cell culture should provide further access to the mechanisms by which hedgehog acts.