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The ESCRT machinery regulates the secretion and long-range activity of Hedgehog

Abstract

The conserved family of Hedgehog (Hh) proteins acts as short- and long-range secreted morphogens, controlling tissue patterning and differentiation during embryonic development1. Mature Hh carries hydrophobic palmitic acid and cholesterol modifications essential for its extracellular spreading2. 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)3. 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.

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Figure 1: ESCRT proteins are required for the long-range activity of Hh.
Figure 2: Lack of ESCRT function in posterior cells leads to defects in Hh secretion and target gene induction.
Figure 3: Hh is secreted on extracellular vesicles.
Figure 4: ESCRT-positive exovesicles transport Hh in the extracellular space of Drosophila wing discs and haemolymph.

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Acknowledgements

This work was supported by the French Government (National Research Agency, ANR) through the “Investments for the Future” LABEX SIGNALIFE (program reference number ANR-11-LABX-0028-01) and by the Fondation pour la Recherche Médicale (reference number DEQ20110421324). T.M. and F.W. were supported by the Fondation Association pour la Recherche Contre le Cancer (ARC) and Ligue Nationale Contre le Cancer, respectively. M.F. was supported by ATIP/Avenir, ARC and the Human Frontier Science Program. The authors acknowledge S. Pagnotta for technical support. We thank K. Haglund for sharing the anti-AliX antibody before publication. We thank L. Staccini-Lavenant and M.-A. Derieppe for technical help.

Author information

Authors and Affiliations

Authors

Contributions

P.P.T. conceived the project and supervised the study. P.P.T. and T.M. designed the genetic studies. T.M. performed all the genetic tests and most of the immunohistochemistry on WIDs. F.W. and P.P.T. designed the biochemical studies. F.W. performed the biochemistry/molecular biology experiments. M.F. designed and performed the live imaging experiments and generated the Chmp1b antibody. M.F. and S.Po. analysed the localization of Hh, Chmp1 and Lpp in wild-type animals and in discs expressing Vps4DN, Ptc(1130X) and Lpp RNAi. G.D’A. produced reagents and contributed to the experimental design of extracellular labelling of Hh. S.Pi. performed the electron microscopy and immunoelectron microscopy analysis, and performed the counting on the electron microscopy data. P.P.T., M.F., F.W., S.Pi. and T.M. analysed the individual collected data sets. P.P.T. and T.M. assembled and edited the figures. P.P.T. wrote the manuscript. All authors commented on the manuscript versions. S.Pi. and S.Po. contributed equally to this work.

Corresponding author

Correspondence to Pascal P. Thérond.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 ESCRT depletion in posterior cells rescues the Hh gain-of-function phenotype in adult Drosophila wings.

ad, Morphology and dpp-lacZ expression pattern of wild-type (WT; a, b) and Hh-overexpressing WIDs (c, d; tester line). Scale bar, 100 μm. Dorsal is towards the top and anterior is to the left. The white arrow in c indicates anterior outgrowth, whereas in d it points to cells expressing ectopic dpp-lacZ. e, Quantification (mean of the ratios of absolute values) of the v3–v4 intervein space (shorter blue line on f) relative to the whole wing span (longer blue line on f) normalized to the tester line. Sample numbers (indicating the number of wings examined) are shown at the top of each column. fk, Representative examples of controls (fi) and wings of tester line plus ESCRT RNAi (j, k) escapers. The amount of eclosed escaper flies in the control w;tubGal80ts/+;hhGal4<UAS-Hh/+ genotype is below 1%. l, hhGal4/hhts2 mutant wing from animals kept at restrictive temperature for 4 days from L1 larval stage. m, Representative western blot of WID lysates from rescued discs (ESCRT RNAi, UAS-Hh; hhGal4). n, Quantification of band density from m. HhNp values were normalized to tubulin values in each individual case.

Extended Data Figure 2 Mild ESCRT RNAi knockdown does not lead to apoptosis or cell polarity changes.

ag, Control (a, b) and ESCRT RNAi (cg) discs stained with antibodies against the apoptotic marker Caspase III (green). Peripodial nuclei are labelled with DAPI (red). Rescued discs with wild-type appearance did not display any sign of apoptosis or cell-architecture defects with the exception of Vps22, investigation of which was not further pursued. Scale bar, 100 μm. hk, XZ section of WIDs expressing ESCRT RNAi in the dorsal compartment (D) with apGal4 driver and labelled for the polarity markers DE-cadherin (DCAD2; green) and Disc-large (Dlg; red). The ventral domain (V) is wild type. Scale bar, 20 μm. lo, Secretion of Dlp is not affected in discs expressing ESCRT RNAi in the dorsal compartment for 12–30 h. ECDlp, for extracellular Dlp protein. ps, DispHA subcellular localization is not affected in Hh-producing cells simultaneously expressing ESCRT RNAi for 30 h. In all XZ images, apical is towards the top and dorsal is to the left. The dorsal–ventral boundary is indicated by a dashed white line. White arrowheads in ps indicate the apical side of the columnar cells.

Extended Data Figure 3 The anterior expression of Engrailed and Patched is not affected by ESCRT RNAi in conditions of Hh gain of function.

al, In all images, dorsal is towards the top and anterior is to the left. En (af) and Ptc (gl) expression in control discs (ac, gi) and discs with tester line plus ESCRT RNAi (df, jl) in the posterior compartment. White triangles indicate the anterior–posterior border. Scale bar, 50 μm.

Extended Data Figure 4 Ptc production is maintained in conditions in which iro-lacZ and dpp-lacZ expression is reduced by ESCRT RNAi.

ad, The long-range target iro-lacZ expression is reduced in discs expressing ESCRT RNAi in the posterior compartment. The red dashed lines indicate the areas used for the quantification in e, f. e, f, Quantification of the expression area (ratio of the area of iro-expressing cells to the whole WID pouch) (e) and intensity (where absolute average intensity values of iro-lacZ-expressing cells in the WID pouch were normalized to the background) (f) of iro-lacZ expression in ESCRT RNAi discs compared with wild type. gj, Ptc (g, i) and En (h, j) expression in wild type and conditions of Chmp1 RNAi in posterior cells. White triangles indicate the anterior–posterior (A/P) border. k, Quantification of Ptc protein expression in discs expressing ESCRT RNAi in the posterior compartment. lo, Representative examples of the decrease in dpp-lacZ expression in hhts2 discs with RNAi against ESCRT in posterior cells (ESCRT RNAi;hhts2) grown at restrictive temperature for 4 days from L1 onwards. At restrictive temperature, the hhts2 allele is semi-lethal, with up to 60% of the flies dying as pharate adults (Methods). The surviving flies displayed a 50% decrease in the v3–v4 intervein space (Extended Data Fig. 1e, l). dpp-lacZ expression, which was mildly affected in the hhts2 WID (l, p), became strongly reduced in all ESCRT RNAi discs in the hhts2 background analysed (mp). A magnification of the dpp-lacZ stripe is shown for each genotype below the panels. p, Quantification of dpp-lacZ expression in wild-type, hhts2 and ESCRT RNAi;hhts2 discs. qt, Ptc expression in conditions of ESCRT RNAi;hhts2 discs. ESCRT RNAis had no effect on the decrease of Ptc expression observed in hhts2 WIDs, suggesting that Hh long-range activity is sensitive to ESCRT function, but not short-range Hh signalling (qt). On all fluorescence images, dorsal is shown to the top and anterior is to the left. Scale bar, 50 μm. bd, lo, Numbers on bottom right indicate number of discs displaying reduced iro or dpp activity per whole population analysed.

Extended Data Figure 5 Specificity controls for ESCRT RNAi.

ae, Expression of Vps4DN (b) or ESCRT RNAi (ce, and see also in np) in the posterior compartment results in the accumulation of the poly-Ubi epitope compared with wild type (a). fh, Chmp1 RNAi in the Hh-producing cells leads to the depletion of Chmp1 epitopes (f), whereas overexpression of UAS-Chmp1 in the Ptc domain results in the accumulation of the Chmp1 signal (g, h). i, AliX RNAi in the Hh-producing cells leads to the depletion of AliX epitopes. j, k, AliX RNAi in Hh-producing cells does not result in the accumulation of either poly-Ubi or Lpp epitopes. This disc is the same as the one in Fig. 2b. lp, Panels show four examples of discs expressing RNAi against Vps32 in the posterior compartment stained for Lpp (l), poly-Ubi (mp, top row) and dpp-lacZ (mp, bottom row). Note that dpp-lacZ expression can be reduced (m, bottom panel) even in a disc with no change in poly-Ubi (m, top panel) and Lpp distribution (l). Increased poly-Ubi accumulation is not correlated with a further decrease in dpp-lacZ expression. l, m, The disc shown is the same as the one in Fig. 2c. qt, Expression of an Lpp RNAi construct in the fat body using the CgGal4 (CgG4) driver decreases Lpp protein levels in the WID (r, t), but does not cause Hh accumulation at the apical cell surface (q, s). Shown is a close-up of the central disc quadrant overlying the anterior-posterior compartment boundary. In all images dorsal is at the top and anterior is to the left. s, t, Note that because of general depletion of Lpp the size of the disc is considerably reduced. a, q, s, Scale bars, 30 μm. In all images dorsal is at the top and anterior is to the left.

Extended Data Figure 6 Control experiments for exovesicle characterization.

a, The S10 fraction was run at 160,000g for 24 h. Hh protein split equally between the 160,000g supernatant and the 160,000g pellet. These two fractions induced Fu phosphorylation (P-Fused) with the same intensity. b, The relative amount of S120-Hh and P120-Hh before and after incubation of cells in Fig. 3c. c, Immunogold labelling of P120 fraction isolated from non-Hh-expressing Cl8 cells. In this experimental condition the Hh epitope is not detectable on the surface of exovesicles. This is a control for Fig. 3e.

Extended Data Figure 7 Visualization of ESCRT-positive exovesicles in the luminal space of Drosophila WIDs.

a, b, Live imaging of Vps32–GFP in a WID. Cell membranes are stained with FM4-64 (red). Scale bar, 20 μm. a, XY section through the WID pouch showing the posterior Vps32–GFP-producing cells on the right (strong green signal) and the anterior luminal space on the left. The white square indicates the region shown at a higher magnification in Fig. 4b. b, XZ section showing the Vps32–GFP-expressing columnar cells on the right (bright green) and a Vps32–GFP-positive vesicle in the anterior lumen. cf, Colocalization of Hh–GFP and endogenous Chmp1 protein in the WID lumen. ce, Transverse section through the ventral part of the wing pouch parallel to the dorsal–ventral axis. Scale bars, 20 μm. d, e, Higher magnification views of the region indicated by the white box in c. Note the presence of Hh- and Chmp1-positive exovesicles in the luminal space in cf. Cell outlines are visualized by the staining of cortical filamentous actin (phalloidin, blue). f, Transverse section through the WID across the dorsal–ventral axis. The white square indicates the region shown at a higher magnification in Fig. 4f–h. Scale bar, 50 μm. Schematic representations of the positions of the optical sections (dashed yellow lines) used in c and f are shown in the bottom-right corner of the corresponding images.

Extended Data Figure 8 Controls and additional examples for the extracellular colocalization of Hh and Vps32–GFP.

ai, Partial colocalization of extracellular Hh and Vps32–GFP on the apical side of both the producing (ac) and recipient cells (di). Scale bar, 10 μm. Pearson coefficients: 0.426 (df) and 0.542 (gi). M1M2 coefficients: 0.156 for Hh and 0.146 for Vps32–GFP (df); 0.284 for Hh and 0.382 for Vps32–GFP (gi). jl, GAP43–GFP, an inner plasma membrane marker, was expressed in the posterior compartment. No colocalization with Hh was observed in the anterior compartment (Pearson coefficient: 0.009; M1M2 coefficients: 0.001for Hh and 0.142 for GAP43–GFP). Note that GAP43–GFP-labelled peripodial membrane above the columnar cells. Scale bar, 20 μm. Apical is towards the top and anterior is to the left. The anterior–posterior border is marked with a dashed white line. Magnification of Hh punctae is shown in the bottom-right corner.

Extended Data Figure 9 Endogeneous Chmp1 is secreted into the luminal space of WID.

ad, Transverse section through the ventral part of the wing pouch across the anterior–posterior axis. Scale bars, 20 μm. bd, Higher magnification views of the region indicated by the white box in a. Note the presence of Chmp1-positive exovesicles in the luminal space. Cell outlines are visualized by the staining of cortical filamentous actin (phalloidin, red). Schematic representation of the position of the optical section (dashed yellow line) used in ad is shown in the bottom-right corner of a.

Extended Data Figure 10 Specificity controls for Drosophila haemolymph staining.

ad, Endogenous Hh colocalizes with AliX on non-cellular (DAPI-negative) particles in the Drosophila haemolymph. White arrows indicate examples of colocalization. eh, Most of the Hh epitope is depleted from the circulating particles in the hhts2 homozygous mutant, which was kept at a restrictive temperature for 4 days. il, GAP43–GFP expressed in all posterior cells is not secreted into the Drosophila circulation. mp, The ESCRT II member Vps36–GFP expressed in all posterior cells does not colocalize with endogeneous Hh in the fly blood, but is secreted as different particles instead. Scale bar, 20 μm. White circles mark DAPI-positive blood cells.

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Matusek, T., Wendler, F., Polès, S. et al. The ESCRT machinery regulates the secretion and long-range activity of Hedgehog. Nature 516, 99–103 (2014). https://doi.org/10.1038/nature13847

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