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.

Regulation of the collagen IV network by the basement membrane protein perlecan is crucial for squamous epithelial cell morphogenesis and organ architecture.

All epithelia have their basal side in contact with a specialized extracellular matrix, the basement membrane (BM). During development, the BM contributes to the shaping of epithelial organs via its mechanical properties. These properties rely on two core components of the BM, collagen type IV and perlecan/HSPG2, which both interact with another core component, laminin, the initiator of BM assembly. While collagen type IV supplies the BM with rigidity to constrain the tissue, perlecan antagonizes this effect. Nevertheless, the number of organs that has been studied is still scarce, and given that epithelial tissues exhibit a wide array of shapes, their forms are bound to be regulated by distinct mechanisms. This is underscored by mounting evidence that BM composition and assembly/biogenesis is tissue-specific. Moreover, previous reports have essentially focused on the mechanical role of the BM in morphogenesis at the tissue scale, but not the cell scale. Here, we took advantage of the robust conservation of core BM proteins and the limited genetic redundancy of the Drosophila model system to address how this matrix shapes the wing imaginal disc, a complex organ comprising a squamous, a cuboidal and a columnar epithelium. With the use of a hypomorphic allele, we show that the depletion of Trol (Drosophila perlecan) affects the morphogenesis of the three epithelia, but particularly that of the squamous one. The planar surface of the squamous epithelium (SE) becomes extremely narrow, due to a function for Trol in the control of the squamous shape of its cells. Furthermore, we find that the lack of Trol impairs the biogenesis of the BM of the SE by modifying the structure of the collagen type IV lattice. Through atomic force microscopy and laser surgery, we demonstrate that Trol provides elasticity to the SE's BM, thereby regulating the mechanical properties of the SE. Moreover, we show that Trol acts via collagen type IV, since the global reduction in the trol mutant context of collagen type IV or the enzyme that cross-links its 7S -but not the enzyme that cross-links its NC1- domain substantially restores the morphogenesis of the SE. In addition, a stronger decrease in collagen type IV achieved by the overexpression of the matrix metalloprotease 2 exclusively in the BM of the SE, significantly rescues the organization of the two other epithelia. Our data thus sustain a model in which Trol counters the rigidity conveyed by collagen type IV to the BM of the SE, via the regulation of the NC1-dependant assembly of its scaffold, allowing the spreading of the squamous cells, spreading which is compulsory for the architecture of the whole organ.

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.

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.

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.

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.

Two different sources of Perlecan cooperate for its function in the basement membrane of the Drosophila wing imaginal disc.

BACKGROUND: The basement membrane (BM) provides mechanical shaping of tissues during morphogenesis. The Drosophila BM proteoglycan Perlecan is vital for this process in the wing imaginal disc. This function is thought to be fostered by the heparan sulfate chains attached to the domain I of vertebrate Perlecan. However, this domain is not present in Drosophila, and the source of Perlecan for the wing imaginal disc BM remains unclear. Here, we tackle these two issues. RESULTS: In silico analysis shows that Drosophila Perlecan holds a domain I. Moreover, by combining in situ hybridization of Perlecan mRNA and protein staining, together with tissue-specific Perlecan depletion, we find that there is an autonomous and a non-autonomous source for Perlecan deposition in the wing imaginal disc BM. We further show that both sources cooperate for correct distribution of Perlecan in the wing imaginal disc and morphogenesis of this tissue. CONCLUSIONS: These results show that Perlecan is fully conserved in Drosophila, providing a valuable in vivo model system to study its role in BM function. The existence of two different sources for Perlecan incorporation in the wing imaginal disc BM raises the possibility that inter-organ communication mediated at the level of the BM is involved in organogenesis.

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.

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.

Functional Analysis of ESCRT-Positive Extracellular Vesicles in the Drosophila Wing Imaginal Disc.

A large number of studies have shown that proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) can trigger the biogenesis of different types of Extracellular Vesicles (EV). The functions that these vesicular carriers exert in vivo remain, however, poorly understood. In this chapter, we describe a series of experimental approaches that we established in the Drosophila wing imaginal disc to study the importance of ESCRT-positive EVs for the extracellular transport of signaling molecules, as exemplified by a functional analysis of the mechanism of secretion and propagation of the major developmental morphogen Hedgehog (Hh).Through the combined use of genetic, cell biological, and imaging approaches, we investigate four important aspects of exovesicle biology: (1) The genetic identification of ESCRT proteins that are specifically required for Hh secretion. (2) The imaging of ESCRT and Hh-positive EVs in the lumenal space of both living and fixed wing imaginal discs. (3) The receptor-mediated capture of Hh-containing EVs on the surface of Hh-receiving cells. (4) The effect of manipulations of ESCRT function on the extracellular pool of Hh ligands.

Protein association changes in the Hedgehog signaling complex mediate differential signaling strength.

Hedgehog (Hh) is a conserved morphogen that controls cell differentiation and tissue patterning in metazoans. In Drosophila, the Hh signal is transduced from the G protein-coupled receptor Smoothened (Smo) to the cytoplasmic Hh signaling complex (HSC). How activated Smo is translated into a graded activation of the downstream pathway is still not well understood. In this study, we show that the last amino acids of the cytoplasmic tail of Smo, in combination with G protein-coupled receptor kinase 2 (Gprk2), bind to the regulatory domain of Fused (Fu) and highly activate its kinase activity. We further show that this binding induces changes in the association of Fu protein with the HSC and increases the proximity of the Fu catalytic domain to its substrate, the Costal2 kinesin. We propose a new model in which, depending on the magnitude of Hh signaling, Smo and Gprk2 modulate protein association and conformational changes in the HSC, which are responsible for the differential activation of the pathway.

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.

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.

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.

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.

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.

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.

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.

Distinct phosphorylations on kinesin costal-2 mediate differential hedgehog signaling strength.

The graded Hedgehog (Hh) signal is transduced by the transmembrane Smoothened (Smo) proteins in both vertebrates and invertebrates. In Drosophila, associations between Smo and the Fused (Fu)/Costal-2 (Cos2)/Cubitus Interruptus (Ci) cytoplasmic complex lead to pathway activation, but it remains unclear how the cytoplasmic complex responds to and transduces different levels of Hh signaling. We show here that, within the Hh gradient field, low- and high-magnitude Smo activations control differentially the phosphorylation of Cos2 on two distinct serines. We also provide evidence that these phosphorylations depend on the Fu kinase activity and lead to a shift of Cos2 distribution from the cytoplasm to the plasma membrane. Moreover, the distinct Cos2 phosphorylation states mediate differential Hh signaling magnitude, suggesting that phosphorylation and relocation of Cos2 to the plasma membrane facilitate high-level Hh signaling through the control of Ci nuclear translocation and transcriptional activity.