Isabel Guerrero

The sterol-sensing domain of Patched protein seems to control Smoothened activity through Patched vesicular trafficking.

The Hedgehog (Hh) family of signaling molecules function as organizers in many morphogenetic processes. Hh signaling requires cholesterol in both signal-generating and -receiving cells, and it requires the tumor suppressor Patched (Ptc) in receiving cells in which it plays a negative role. Ptc both blocks the Hh pathway and limits the spread of Hh. Sequence analysis suggests that it has 12 transmembrane segments, 5 of which are homologous to a conserved region that has been identified in several proteins involved in cholesterol homeostasis and has been designated the sterol-sensing domain (SSD). In the present study, we show that a Ptc mutant with a single amino acid substitution in the SSD induces target gene activation in a ligand-independent manner. This mutant Ptc(SSD) protein shows dominant-negative activity in blocking Hh signaling by preventing the downregulation of Smoothened (Smo), a positive effector of the Hh pathway. Despite its dominant-negative activity, the mutant Ptc protein functioned like the wild-type protein in sequestering and internalizing Hh. In addition, we show that Ptc(SSD) preferentially accumulates in endosomes of the endocytic compartment. All these results suggest a role of the SSD of Ptc in mediating the vesicular trafficking of Ptc to regulate Smo activity.

Patched controls the Hedgehog gradient by endocytosis in a dynamin-dependent manner, but this internalization does not play a major role in signal transduction

The Hedgehog (Hh) morphogenetic gradient controls multiple developmental patterning events in Drosophila and vertebrates. Patched (Ptc), the Hh receptor, restrains both Hh spreading and Hh signaling. We report how endocytosis regulates the concentration and activity of Hh in the wing imaginal disc. Our studies show that Ptc limits the Hh gradient by internalizing Hh through endosomes in a dynamin-dependent manner, and that both Hh and Ptc are targeted to lysosomal degradation. We also found that the ptc14 mutant does not block Hh spreading, as it has a failure in endocytosis. However, this mutant protein is able to control the expression of Hh target genes as the wild-type protein, indicating that the internalization mediated by Ptc is not required for signal transduction. In addition, we noted that both in this mutant and in those not producing Ptc protein, Hh still occurred in the endocytic vesicles of Hh-receiving cells, suggesting the existence of a second, Ptc-independent, mechanism of Hh internalization.

The function of engrailed and the specification of Drosophila wing pattern.

The adult Drosophila wing (as the other appendages) is subdivided into anterior and posterior compartments that exhibit characteristic patterns. The engrailed (en) gene has been proposed to be paramount in the specification of the posterior compartment identity. Here, we explore the adult en function by targeting its expression in different regions of the wing disc. In the anterior compartment, ectopic en expression gives rise to the substitution of anterior structures by posterior ones, thus demonstrating its role in specification of posterior patterns. The en-expressing cells in the anterior compartment also induce high levels of the hedgehog (hh) and decapentaplegic (dpp) gene products, which results in local duplications of anterior patterns. Besides, hh is able to activate en and the engrailed-related gene invected (inv) in this compartment. In the posterior compartment we find that elevated levels of en product result in partial inactivation of the endogenous en and inv genes, indicating the existence of a negative autoregulatory mechanism. We propose that en has a dual role: a general one for patterning of the appendage, achieved through the activation of secreted proteins like hh and dpp, and a more specific one, determining posterior identity, in which the inv gene may be implicated.

Cytoneme-Mediated Delivery of Hedgehog Regulates the Expression of Bone Morphogenetic Proteins to Maintain Germline Stem Cells in Drosophila

Genetic manipulation of the germline stem cell niche in Drosophila ovaries reveals that support cells ensure the maintenance of stem cells by modulating the spread of Hedgehog within the niche.

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.

Dispatched mediates Hedgehog basolateral release to form the long-range morphogenetic gradient in the Drosophila wing disk epithelium

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 long-range gradient. Functional analysis of the 12-transmembrane protein Dispatched, the glypican Dally-like (Dlp) protein, and the Ig-like 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 fu gene discriminates between pathways to control dpp expression in Drosophila imaginal discs

The genes decapentaplegic (dpp) and wingless (wg), which encode secreted factors of the TGF-beta and Wnt families, respectively, are required for the proper development of the imaginal discs. The expression of these genes must be finely regulated since their ectopic expression induces overgrowth and pattern alterations in wings and legs. Genes like patched (ptc) and costal-2 (cos-2), and the gene encoding the catalytic subunit of the protein kinase A gene (pkA) are required to restrict dpp and wg expression in their proper positions. We show here that some mutations in the cubitus interruptus (ci) gene also show ectopic dpp expression in the wing disc. We have also analyzed the functional hierarchy between these genes and the gene fused (fu), in the activation of dpp by the hedgehog (hh) signal. fu is required to transmit the hh signal in imaginal discs, since fu mutations rescue the phenotype due to the ectopic hh expression or to the lack of ptc activity. fu is also required for the activation of engrailed (en) caused when hh is ectopically activated in the wing disc. By contrast, fu mutations do not rescue the phenotypic consequences of the abnormal ci, cos-2 or pkA activity. Although fu, cos-2 and ci probably form part of the same pathway that controls dpp expression, pkA probably controls dpp transcription by a different pathway.

Exosomes as Hedgehog carriers in cytoneme-mediated transport and secretion

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.

SFRPs act as negative modulators of ADAM10 to regulate retinal neurogenesis

It is well established that retinal neurogenesis in mouse embryos requires the activation of Notch signaling, but is independent of the Wnt signaling pathway. We found that genetic inactivation of Sfrp1 and Sfrp2, two postulated Wnt antagonists, perturbs retinal neurogenesis. In retinas from Sfrp1−/−; Sfrp2−/− embryos, Notch signaling was transiently upregulated because Sfrps bind ADAM10 metalloprotease and downregulate its activity, an important step in Notch activation. The proteolysis of other ADAM10 substrates, including APP, was consistently altered in Sfrp mutants, whereas pharmacological inhibition of ADAM10 partially rescued the Sfrp1−/−; Sfrp2−/− retinal phenotype. Conversely, ectopic Sfrp1 expression in the Drosophila wing imaginal disc prevented the expression of Notch targets, and this was restored by the coexpression of Kuzbanian, the Drosophila ADAM10 homolog. Together, these data indicate that Sfrps inhibit the ADAM10 metalloprotease, which might have important implications in pathological events, including cancer and Alzheimers disease.

Patched, the receptor of Hedgehog, is a lipoprotein receptor

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.