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. This study has investigated the mechanism of Hh release from the producing cells to form a morphogenetic gradient in the Drosophila wing imaginal disk epithelium. 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. It was also shown 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 (Callejo, 2011).

By using shits1 mutant disks, it is possible to freeze Hh internalization and visualize on which side of the anterior compartment (A) wing disk epithelium, apical or basal, Hh gradient is being formed. Thus, an extended basolateral accumulation of Hh was observed in receiving cells, whereas, at the apical plane, Hh accumulation was only evident in the first row of A cells, indicating that the long-range Hh gradient is formed mainly basolaterally. Accordingly, Ptc accumulated in shits1 disks equally apically and basolaterally but mainly colocalized with Hh at the basolateral sections, suggesting a specific mechanism to deliver Hh to Ptc in this surface of the disk epithelium. To analyze the mechanism of Hh release in the P cells, an apical accumulation of Hh was noticed in the producing cells in shits1 mutant disks that was also observed in P shits1 clones and in Rab5DN ectopic clones, suggesting that the apically secreted Hh is also internalized in P cells and probably is recycling to other membranes. Accordingly, when blocking recycling endosomes (using the dominant-negative form of Rab8 or Rab4), Hh accumulation could be detected in producing cells. This Hh recycling in P cells is probably necessary to form a proper Hh gradient in the receiving cells. In agreement with the above, Hh signaling is compromised when endocytosis is blocked by expressing either Rab5DN or ShiDN in the P compartment. Interestingly, and in agreement with these results, Ayers (2011) suggested that apical Hh internalization in the Hh-producing cells is necessary to process or route Hh to activate the responses that require high levels of Hh. However, in contrast to the interpretation provided in this study, Ayers also proposed that the apical Hh pool is responsible for the long-range Hh gradient formation. It is not necessary to envision two Hh gradients, an apical long-range Hh and another basolateral short-range Hh, when all responses can be produced by means of a single gradient. Because only the recycled Hh is capable of activating the high-threshold Hh targets, there is no reason to believe that this processed Hh would not be efficient enough to induce the low-threshold targets basolaterally (Callejo, 2011).

Also in contrast to a previous report proposing a function for Disp in regulating the apical secretion of Hh in Drosophila epithelia, this study demonstrates that Disp is required for the basolateral release of Hh in the wing imaginal disk epithelium. The subcellular localization of Disp, and its function in the basolateral release of Hh, is in agreement with a recent report in vertebrates (Etheridge, 2010). The cellular phenotype of the loss of Disp function, such as the increase in the amount of Hh found in endocytic vesicles, which are supernumerary and disorganized, can be interpreted as a failure in Hh trafficking that subsequently affects its proper release. In this sorting process, Hh would interact with Disp either in the recycling endosome or in MVBs. Disp, a member of the RND family of proton-driven transporters, is likely to function only in compartments where a transmembrane proton gradient exists, such as in early and late endosomes, trans-Golgi, and MVBs. In support of this view, this study showed by confocal and EM studies that Disp protein is located not only at the basolateral plasma membrane but in vesicles and MVBs, where it colocalizes with Hh. Based on the disp-/- phenotypes and the localization of Disp protein in MVBs, it is proposed that Disp might have a function in redirecting the apically internalized Hh toward the basal domain. Interestingly, and in agreement with the above, a form of Disp, mutant for the proton-driven transporter function (DispAAA), does not localize at the basolateral plasma membrane but in supernumerary cytoplasmic vesicles that do not colocalize with Hh puncta, implying that DispAAA may not participate in Hh vesicular trafficking to the basolateral plasma membrane (Callejo, 2011).

In noticeable contrast but also supporting the above, after freezing Hh internalization in shits1 mutant disks, Hh accumulates apically in the first row of A compartment cells, suggesting that paracrine signaling could also occur through the apical plasma membrane. Interestingly, in P mutant cells for Dlp, Hh is able to signal to the abutting A cells but long-range signaling does not occur. A suggestive possibility is that the capacity for apical signaling is not affected in these mutant conditions; however, for long-range signaling to occur, a basolateral release implicating the coordinated actions of Dlp and Ihog together with Disp would be required. During Hh sorting in the producing cells, Disp may interact with Dlp and Ihog; in fact, the interaction of Disp with Dlp and Ihog may be important for the apical-to-basal transcytosis of these proteins, because the ectopic expression of Disp but not of mutant DispAAA increases Dlp and Ihog levels at the basolateral membranes. As in disp-/- cells, dlp-/- cells in the P compartment showed an accumulation of Hh at both the apical and basolateral plasma membranes, suggesting that Dlp might cooperate with Disp during Hh release. In agreement with the proposed mechanism, transcytosis of Dlp has previously been suggested to be important for Wingless (Wg) release and spreadin (Callejo, 2011).

Although the data cannot support that the total amount of synthesized Hh has to undergo this apical-to-basal transcytosis, an intriguing question is why Hh is placed and internalized apically and then shuttled to the basolateral part of the cell. One possibility is that the newly synthesized Hh protein, because of its unusual modifications with cholesterol and palmitate, uses the apical surface, which is enriched in cholesterol and glycosphingolipids, for primarily plasma membrane localization. Alternatively, it is also possible that the apical internalization of Hh allows its interaction with Disp, glypicans, and Ihog. Therefore, Hh that reaches the apical plasma membrane needs to be internalized to recycle to the basolateral plasma membrane, where the machinery for secretion and gradient formation is found. As has already been discussed, transcytosis of Dlp and Ihog together with Hh from the apical membrane to the basolateral membrane may also occur. Cholesterol and triglycerides also undergo apical-to-basolateral transcytosis across intestinal epithelial barriers to reach the blood. Cholesterol and palmitic acid modifications could attribute lipid-like properties to the Hh protein, such as the ability to be anchored to the plasma membrane, and could thus affect Hh intracellular trafficking. In agreement with the above, it has been described that lipid-unmodified Hh in the wing disk epithelium is not able to form a proper Hh gradien. As previously reported, it was observed that Hh mutant forms that lack lipid modifications are released and do not accumulate in disp-/- clones. Interestingly, this study shows that lipid-unmodified Hh does not colocalize with Ihog-labeled basal cell extensions, indicating that lipid modifications are necessary to interact with Disp, Dlp, and Ihog, and therefore for proper Hh trafficking from the apical to basolateral plasma membrane regulated by Disp function. Reinforcing these findings, it has been reported that disp and ttv functions are not required for either release or transport of lipid-unmodified Hh, strongly suggesting that Disp and glypicans are needed for the appropriate basolateral release of Hh (Callejo, 2011).

This work shows that Hh has a more complicated mechanism for release than has been previously anticipated. The finding of a basolateral route for Hh release and gradient formation will help to understand Hh interaction with different Hh pathway components, such as Disp, Dally, Dlp, Ihog, Boi, and Ptc, during the process of Hh gradient formation. Related to this issue, it is quite intriguing to find Disp, Dlp, Ihog, and Hh decorating long basal cellular extensions in disk cells expressing Ihog ectopically. Some of the long filaments labeled with IhogYFP extend up to several cell diameters and are reminiscent of the 'cytonemes', with a function in the transport of morphogens. In contrast to the previously described apical cytonemes, the extensions this study visualize are mainly found at the basal part of the disk epithelium. Interestingly, in the context of Notch signaling, basal actin-based filopodia are important for lateral inhibition between nonneighboring cells. However, further investigation will be necessary to demonstrate the implication of cytonemes in Hh gradient formation (Callejo, 2011).


To determine the expression pattern of disp, wild-type embryos and pupal imaginal discs were probed with anti-sense DISP RNA. Ubiquitous disp expression is observed throughout the embryo and imaginal discs. Thus, based on its expression pattern, disp is neither a transcriptional target nor a spatial determinant of Hh signaling (Burke, 1999).

Effects of Mutation or Deletion

To determine if disp is required specifically for either the Hh or Wg pathway, large disp minus clones were generated in the adult wing, a tissue in which the two pathways function independently of one another in distinct subpopulations of cells. Hh plays a role in the functioning of the anterior-posterior boundary in the wing, while Wg functions at the wing margin. A reduction in Hh activity causes a strong narrowing of the intervein region between longitudinal veins L3 and L4, a reflection of Hh's role at the anterior-posterior boundary, while loss of Wg signaling in the wing primordium results in loss of wing margin. Although disp minus clones can encompass large regions of Wg-sending and Wg-receiving cells, such clones contribute to wild-type wing margin structures, which indicates that disp function is not required for the Wg signaling pathway. However, when located in the posterior compartment, large clones cause a significant reduction in the distance between veins L3 and L4, a phenotype typical for the reduction of Hh signaling at the A/P boundary. Thus, it is concluded that disp is acting in the Hh signaling pathway (Burke, 1999).


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dispatched: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 30 December 2012

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