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

Abstract

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.

Fig. 1.

Hh processing in the wing imaginal disk. Hh protein distribution in a WT imaginal disk (A–A′′) and in a shi^ts1 mutant disk after 2.5 h at the restricted temperature (B–B′′) in apical, lateral, and basal confocal sections. Note that Hh is present at similar levels in the apical and basolateral sections in a WT disk (transversal section a) in the P compartment and is also present in punctuated structures in the A compartment (arrowheads in A). (B) Note also the accumulation of Hh in the apical part of P cells and in the first row of A cells abutting the A/P compartment border in a shi^ts1 disk. In lateral and basal sections, Hh levels decrease in P cells, whereas Hh accumulates in several rows of the A compartment (arrow in B′ and B′′ and orthogonal view b). hh-Gal4/UAS-HhGFP wing imaginal disk (C) and transversal section of the same disk (c). (D–D′′) In vivo labeling by internalized red fluorescent dextran (5-min pulse) in an hh-Gal4/UAS-HhGFP wing disk. Circles indicate the colocalization of HhGFP with the internalized red fluorescent dextran, and arrowheads indicate the HhGFP vesicles that do not colocalize with the red fluorescent dextran. (d–d′′) Transversal section of the same wing disk to show the colocalization between Hh puncta and red fluorescent dextran. (E and E′) Accumulation of Hh (red) in shi^ts1 clones (lack of green). (e and e′) Apical Hh accumulation in shi^ts1 clones is shown in a transversal section of the same disk. (F and F′) Extracellular staining using an anti-GFP antibody in shi^ts1 clones in an hh-Gal4/UAS-HhGFP wing disk. Observe the extracellular apical accumulation of GFP (red in F and gray in F′′) in shi^ts1 clones. (G and Insets g and g′) Ectopic HhGFP clone (marked in red by βGal staining) in the P compartment labeled by En expression (blue). Observe the basolateral and not the apical spreading of the HhGFP gradient outside the clone in the P (arrowheads). (H) Ectopic HhGFP clones in a shi^ts background stained with anti-βGal (red) and anti-En (blue) antibodies. Dynamin-mediated apical internalization of Hh is observed in the P ectopic clone cells only (Inset h′) and not in the adjacent A clone, where dynamin-mediated internalization takes place through the basolateral plasma membrane (Inset h, arrow). Note also that the basolateral spreading of HhGFP outside the clone in the P compartment, shown in Inset g, is not observed in Inset h, indicating that when there is “freezing” internalization, there is less Hh at the basolateral plane (arrowhead) and that apical internalization of Hh is needed for the formation of a basolateral Hh gradient. In all transversal sections, the apical part of disks is in the upper part of the panels.

Fig. 2.

Hh trafficking effects on gradient formation. (A–B′) Ectopic expression of Rab5^DN-YFP in clones (green) using the Tub-Gal80^ts system after 30 h at the restrictive temperature. Observe that Hh accumulates apically (red in A and gray in A′, arrowheads) and decreases in the basal part of the epithelium (red in B and gray in B′, arrowheads). (C–D′) Tub-Gal80^ts; hh-Gal4/Rab5^DN-YFP wing disk after 33 h at the restrictive temperature. Although Rab5^DN-YFP does not cause activation of the apoptotic marker Caspase 3 (blue in D), it does provoke a decrease in Hh signaling as reported by the expression of Ci¹⁵⁵ (blue in C and gray in C′), Ptc (red in C and gray in C′′), and Col (red in D and gray in D′). (E) Col expression in a WT disk. (F–F′′′) Tub-Gal80^ts; hh-Gal4/Shi^DN wing disk after 24 h at the restrictive temperature. Note that Ptc expression is reduced (F′) but Ci (F′′) and Dpp (F′′′) expression domains are expanded compared with WT expression of Ptc (G), Ci (H), and Dpp (I).

Fig. 3.

Disp subcellular distribution and trafficking effects. (A and A′) disp^−/− clones (lack of red) induced in an en-Gal4/UAS-HhGFP wing disk in a subapical view. Note the alteration in the subcellular distribution of apical Hh-GFP containing vesicles in disp mutant clones compared with twin clones (arrows in A and A′). (a–a′′) Transversal sections of a disp^−/− clone induced in an en-Gal4/UAS-HhGFP wing disk (lack of blue) and in vivo labeled with red fluorescent dextran. Note the increase in Hh levels within the clone and in endocytic vesicles which colocalized with red fluorescent dextran puncta. Lateral (B–B′′) and apical (C–C′′) sections of an hh-Gal4/UAS-DispYFP wing disk stained in vivo with the membrane dye FM4 (red). Note that Disp colocalizes with the FM4 at the lateral (B–B′′) but not the apical (C–C′′) plasma membranes. (D) Transversal section of an hh-Gal4/UAS-DispYFP wing disk stained with an anti-Cadherin antibody (red) to label the subapical cell junctions. Note that Disp is localized at basolateral plasma membranes. (E) Salivary gland expressing DispYFP, which localizes at the basolateral plasma membrane and is excluded from apical membranes (F and F′). Transversal section of an hh-Gal4/UAS-DispYFP wing disk is stained with fluorescent red fluorescent dextran at a 5-min pulse (F) and a 30-min pulse (F′). Note that DispYFP and red fluorescent dextran colocalize in apically located vesicles after a short incubation (early endosomes) (F) and also in laterally located vesicles after a longer incubation (F′). (G–G′′) Wing imaginal disk expressing DispYFP and HhCFP (artificially labeled in red). Note the colocalization of Hh and Disp in some apical vesicles (blue arrowheads). (H and I) Wing disks expressing either a Disp^AAA mutant construct in the dorsal compartment (Inset h) or random ectopic clones of the wild-type Disp protein (Inset i). Observe the distinct subcellular distribution of Disp^AAA (H and h) and WT Disp (I and i); Disp^AAA is less represented at the plasma membrane and is enriched in puncta (K′ and Inset h). (J) Immunoprecipitation (IP) of Hh using a specific anti-Disp antibody. IP was carried out in extracts from salivary glands overexpressing Disp and HhGFP and was tested with an anti-Disp antibody. IP control was made from salivary glands overexpressing both HhGFP and Disp and tested with the preimmune serum. IP^c, IP control. Anti-GFP antibody was used in the Western blot. Note that the 70- and 47-kDa bands (asterisks), not present in the control, correspond to unprocessed and processed Hh, respectively, and that immunoglobulins (50-kDa) are present both in the IP and in the control. (K and K′) Transversal section of wing imaginal disk expressing Disp^AAA-CFP (artificially labeled in green) with hhGal4 driver and stained with anti-Hh antibody. Note that there is not a significant colocalization between Hh and this Disp mutant form. (L–L′′) Transversal section of wing imaginal disk expressing DispYFP (artificially labeled in green) with hh-Gal4 driver and stained with anti-Hh antibody. Note the colocalization between Hh and DispYFP.

Fig. 4.

Hh accumulation in the P compartment of dlp mutants. (A) Hh levels in a WT wing disk. (B) Hh levels in a dlp^MH20 null mutant wing disk. (C) Graph representing Hh levels in WT wing disks and in dlp^MH20 homozygous wing disks (average of equivalent areas of 6 disks, error bars represent SDs). (D–E) Hh levels in dlp^−/− clones: an apical view is shown in D′, and a lateral view of another clone is shown in E. Hh accumulates at the cell membranes of dlp^MH20 mutant cells. (Inset e) Nonautonomous rescue in the mutant cells located near the clone border (arrowheads), which is also seen in D. (F) Ptc expression in a WT wing disk. (G) Ptc expression in an hh-Gal/UAS-Dlp^RNAi, Tub-Gal80^ts disk after 30 h at the restrictive temperature. (H) Graph representing Ptc levels in WT wing disks and in an hh-Gal4/UAS-Dlp^RNAi (average of expression levels of 6 disks). Observe the clear decrease of Ptc expression in hh-Gal4/UAS-Dlp^RNAi disks. (I) Disp and Dlp co-immunoprecipitation (co-IP) using a specific anti-Disp antibody. The IP was performed in extracts from salivary glands overexpressing Disp and Dlp-GFP and tested with an anti-Disp antibody. IP control was made in extracts from salivary glands overexpressing only DlpGFP and tested with the same anti-Disp antibody. IP^c, IP control. Anti-GFP antibody was used in the Western blot [Dlp^GFP, 112 kDa (asterisk), and immunoglobulins, 50 kDa]. (J and J′) Dlp expression (red) in ectopic UAS-DispYFP clones. (K and K′) Transversal section of an hh-Gal4/UAS-DispYFP wing imaginal disk. Note that Dlp is up-regulated in ectopic Disp clones. (L and L′) Dlp expression (red) in ectopic UAS-Disp^AAA using hh-Gal4 driver. (M and M′) Transversal section of the same disk. Observe that Disp^AAA-expressing cells do not accumulate Dlp.

Fig. 5.

Ihog attaches Hh basolaterally. (A and A′) Wing disk expressing IhogYFP for 24 h using the Gal80^ts system stained with an anti-Hh antibody. Note that Hh levels increase in the clones. (Inset a′) Basal view of the IhogYFP clone shown in A. Note that Hh accumulation extends several cell diameters at the most basal part of the epithelium. (a) Transversal section of an ectopic IhogYFP clone (yellow line in A). (B–C′) Extracellular Hh staining using an anti-Hh antibody in ectopic IhogYFP clones. Note the cell surface accumulation of Hh in lateral (B and B′) and basal (C and C′) confocal sections. (D–D′′) Ectopic Disp clones marked by anti-βGal staining (blue in D) and by the specific anti-Disp antibody (red in D and gray in D′) increase endogenous Ihog levels (green in D and gray in D′′). (E and E′) Wing disk expressing Disp^AAA in the dorsal compartment for 44 h using ap-Gal4; Tub-Gal80^ts driver marked by GFP and stained with anti-Disp (red in E) and anti-Ihog antibodies (blue in E and gray in E′). (E′) Note that the expression of Disp^AAA does not cause an increase of Ihog protein levels. (F–F′′) Basal confocal section of an ectopic clone coexpressing IhogRFP and HhC85SN-GFP, without cholesterol and palmitic acid adducts. Note that this unlipidated form of Hh, which cannot signal, is not able to decorate cellular extensions labeled with Ihog (arrowheads in F′ and F′′) as does the lipidated form (Inset a′).