Hedgehog (Hh) moves from the producing cells to regulate the growth and development of distant cells in a variety of tissues. Here, we have investigated the mechanism of Hh release from the producing cells to form a morphogenetic gradient in the Drosophila wing imaginal disk epithelium. We describe that Hh reaches both apical and basolateral plasma membranes, but the apical Hh is subsequently internalized in the producing cells and routed to the basolateral surface, where Hh is released to form a longrange gradient. Functional analysis of the 12-transmembrane protein Dispatched, the glypican Dally-like (Dlp) protein, and the Iglike and FNNIII domains of protein Interference Hh (Ihog) revealed that Dispatched could be involved in the regulation of vesicular trafficking necessary for basolateral release of Hh, Dlp, and Ihog. We also show that Dlp is needed in Hh-producing cells to allow for Hh release and that Ihog, which has been previously described as an Hh coreceptor, anchors Hh to the basolateral part of the disk epithelium.

The Hedgehog signalling pathway is crucial for development, adult stem cell maintenance, cell migration and axon guidance in a wide range of organisms. During development, the Hh morphogen directs tissue patterning according to a concentration gradient. Lipid modifications on Hh are needed to achieve graded distribution, leading to debate about how Hh is transported to target cells despite being membrane-tethered. Cytonemes in the region of Hh signalling have been shown to be essential for gradient formation, but the carrier of the morphogen is yet to be defined. Here we show that Hh and its co-receptor Ihog are in exovesicles transported via cytonemes. These exovesicles present protein markers and other features of exosomes. Moreover, the cell machinery for exosome formation is necessary for normal Hh secretion and graded signalling. We propose Hh transport via exosomes along cytonemes as a significant mechanism for the restricted distribution of a lipid-modified morphogen.

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, lipidunmodified 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.

Hedgehog can signal both at a short and long-range, and acts as a morphogen during development in various systems. We studied the mechanisms of Hh release and spread using the Drosophila wing imaginal disc as a model system for polarized epithelium. We analyzed the cooperative role of the glypican Dally, the extracellular factor Shifted (Shf, also known as DmWif), and the Immunoglobulin-like (Ig-like) and Fibronectin III (FNNIII) domain-containing transmembrane proteins, Interference hedgehog (Ihog) and its related protein Brother of Ihog (Boi), in the stability, release and spread of Hh. We show that Dally and Boi are required to prevent apical dispersion of Hh; they also aid Hh recycling for its release along the basolateral part of the epithelium to form a long-range gradient. Shf/DmWif on the other hand facilitates Hh movement restrained by Ihog, Boi and Dally, establishing equilibrium between membrane attachment and release of Hh. Furthermore, this protein complex is part of thin filopodia-like structures or cytonemes, suggesting that the interaction between Dally, Ihog, Boi and Shf/DmWif is required for cytoneme-mediated Hh distribution during gradient formation.

The Hedgehog (Hh) family of morphogenetic proteins has important instructional roles in metazoan development and human diseases. Lipid modified Hh is able to migrate to and program cells far away from its site of production despite being associated with membranes. To investigate the Hh spreading mechanism, we characterized Shifted (Shf) as a component in the Drosophila Hh pathway. We show that Shf is the ortholog of the human Wnt inhibitory factor (WIF), a secreted antagonist of the Wingless pathway. In contrast, Shf is required for Hh stability and for lipid-modified Hh diffusion. Shf colocalizes with Hh in the extracellular matrix and interacts with the heparan sulfate proteoglycans (HSPG), leading us to suggest that Shf could provide HSPG specificity for Hh. We also show that human WIF inhibits Wg signaling in Drosophila without affecting the Hh pathway, indicating that different WIF family members might have divergent functions in each pathway.

The Hedgehog (Hh) family of secreted signaling proteins has a broad variety of functions during metazoan development and implications in human disease. Despite Hh being modified by two lipophilic adducts, Hh migrates far from its site of synthesis and programs cellular outcomes depending on its local concentrations. Recently, lipoproteins were suggested to act as carriers to mediate Hh transport in Drosophila. Here, we examine the role of lipophorins (Lp), the Drosophila lipoproteins, in Hh signaling in the wing imaginal disk, a tissue that does not express Lp but obtains it through the hemolymph. We use the up-regulation of the Lp receptor 2 (LpR2), the main Lp receptor expressed in the imaginal disk cells, to increase Lp endocytosis and locally reduce the amount of available free extracellular Lp in the wing disk epithelium. Under this condition, secreted Hh is not stabilized in the extracellular matrix. We obtain similar results after a generalized knock-down of hemolymph Lp levels. These data suggest that Hh must be packaged with Lp in the producing cells for proper spreading. Interestingly, we also show that Patched (Ptc), the Hh receptor, is a lipoprotein receptor; Ptc actively internalizes Lp into the endocytic compartment in a Hh-independent manner and physically interacts with Lp. Ptc, as a lipoprotein receptor, can affect intracellular lipid homeostasis in imaginal disk cells. However, by using different Ptc mutants, we show that Lp internalization does not play a major role in Hh signal transduction but does in Hh gradient formation.Drosophila ͉ LDLR ͉ morphogen

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