1. Developmental Biology

High hedgehog signaling is transduced by a multikinase-dependent switch controlling the apico-basal distribution of the GPCR smoothened

  1. Marina Gonçalves Antunes
  2. Matthieu Sanial
  3. Vincent Contremoulins
  4. Sandra Carvalho
  5. Anne Plessis  Is a corresponding author
  6. Isabelle Becam  Is a corresponding author
  1. Université Paris Cité, CNRS, Institut Jacques Monod, France
https://doi.org/10.7554/eLife.79843
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Cite this article
  1. Marina Gonçalves Antunes
  2. Matthieu Sanial
  3. Vincent Contremoulins
  4. Sandra Carvalho
  5. Anne Plessis
  6. Isabelle Becam
(2022)
High hedgehog signaling is transduced by a multikinase-dependent switch controlling the apico-basal distribution of the GPCR smoothened
eLife 11:e79843.
https://doi.org/10.7554/eLife.79843
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9 figures, 1 table and 2 additional files

Figures

Figure 1 with 3 supplements
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Cell surface Smoothened (SMO) is unevenly distributed along the apico-basal (Ap-Ba) axis and high levels of hedgehog (HH) promote its basolateral enrichment.

(A–A’) (A) Left: scheme of a wing imaginal disc (WID) with the posterior (P) compartment, where HH (in green) is produced, the anterior (A)/P boundary is represented by the dotted dark blue line and …

Figure 1—source data 1

Mean intensity of Surf SNAP-SMO along the apico-basal axis.

https://cdn.elifesciences.org/articles/79843/elife-79843-fig1-data1-v1.xlsx
Figure 1—source data 2

Relative intensity of Surf SNAP-SMO along the apico-basal axis.

https://cdn.elifesciences.org/articles/79843/elife-79843-fig1-data2-v1.xlsx
Figure 1—figure supplement 1
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SNAP-Smoothened (SNAP-SMO) activity.

(A) Wing of a smoD16/smoD16; BAC (CH322-98K24) SNAP-smo/BAC (CH322-98K24) SNAP-smo fly with a wild type phenotype. This indicates that BAC (CH322-98K24) SNAP-smo rescues the effects of smo loss of …

Figure 1—figure supplement 2
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Analysis of the apico-basal (Ap-Ba) distribution of SNAP-Smoothened (SNAP-SMO).

(A) Schematic representation of the method used for labeling SNAP-SMO. In brief, after dissection, the fraction of SNAP-SMO present at the cell surface (thereby called Surf SNAP-SMO) was …

Figure 1—figure supplement 2—source data 1

Mean intensity of Intra SNAP-SMO along the apico-basal axis.

https://cdn.elifesciences.org/articles/79843/elife-79843-fig1-figsupp2-data1-v1.xlsx
Figure 1—figure supplement 3
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Apico-basal (Ap-Ba) distribution of immunolabeled endogenous Smoothened (SMO) or Surf SNAP-SMO expressed at endogenous levels.

(A) XZ confocal images of wild-type wing imaginal discs (WIDs) immunolabeled for endogenous SMO (A, green in the merged image A”) and cubitus interruptus-F (CI-F) (A’, blue in merge image A”). (B) …

Figure 1—figure supplement 3—source data 1

Relative intensity of endogenous SMO (immunolabeling).

https://cdn.elifesciences.org/articles/79843/elife-79843-fig1-figsupp3-data1-v1.xlsx
Figure 2 with 1 supplement
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Blocking Smoothened (SMO) endocytosis favors its accumulation in the apical region independently of hedgehog (HH).

(A–B) XZ confocal images of wing imaginal discs (WID) from shi+; apGal4; UAS SNAP-smo (A, called ctr for control in C and C’) or shits; apGal4; UAS SNAP-smo (B, called shits in C and C’) male flies …

Figure 2—source data 1

Mean intensity of Surf SNAP-SMO for control and shits flies.

https://cdn.elifesciences.org/articles/79843/elife-79843-fig2-data1-v1.xlsx
Figure 2—source data 2

Relative intensity of Surf SNAP-SMO for control and shits flies.

https://cdn.elifesciences.org/articles/79843/elife-79843-fig2-data2-v1.xlsx
Figure 2—source data 3

Relative intensity of Surf SNAP-SMO for control and RAB5CA flies.

https://cdn.elifesciences.org/articles/79843/elife-79843-fig2-data3-v1.xlsx
Figure 2—source data 4

Relative intensity of Surf SNAP-SMO for control and smurf RNAi flies.

https://cdn.elifesciences.org/articles/79843/elife-79843-fig2-data4-v1.xlsx
Figure 2—figure supplement 1
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Effect of shits and rab5CA on Surf SNAP-Smoothened (SNAP-SMO).

(A–B) The false color representation of XY confocal lateral section of a wing imaginal disc (WID) (dorsal part of pouch) of shi+; apGal4; UAS SNAP-smo (A) or shits; apGal4; UAS SNAP-smo (B) male …

Figure 2—figure supplement 1—source data 1

Relative intensity of Surf SNAP-SMO in shibirets flies at 18°C or 30°C.

https://cdn.elifesciences.org/articles/79843/elife-79843-fig2-figsupp1-data1-v1.xlsx
Figure 2—figure supplement 1—source data 2

Relative intensity of Surf SNAP-SMO in control and shibirets flies at 18°C.

https://cdn.elifesciences.org/articles/79843/elife-79843-fig2-figsupp1-data2-v1.xlsx
Figure 3 with 1 supplement
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Hedgehod (HH) controls the fate of endocytosed Smoothened (SMO), favoring its recycling and leading to its basolateral accumulation.

XZ confocal images of dissected wing imaginal discs expressing UAS SNAP-smo fixed immediately (0 min) (A) or 15 min (B) after labeling of Surf SNAP-SMO. Quantification of the mean intensity (C) and …

Figure 3—source data 1

Mean intensity of Surf SNAP-SMO at T0 and at T15'.

https://cdn.elifesciences.org/articles/79843/elife-79843-fig3-data1-v1.xlsx
Figure 3—source data 2

Relative intensity of Surf SNAP-SMO at T0 and T15'.

https://cdn.elifesciences.org/articles/79843/elife-79843-fig3-data2-v1.xlsx
Figure 3—figure supplement 1
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The fate of endocytosed SNAP-Smoothened (SNAP-SMO).

(A–A”) ( B–B”) Colocalization of endocytosed Surf SNAP-SMO with RAB7. XY (A–A”) and XZ (B–B”) confocal images of apGal4; UAS SNAP-smo wing imaginal discs labeled for Surf SNAP-SMO A, B, green in A”, …

Figure 3—figure supplement 1—source data 1

Percentage of decrease in Surf SNAP-SMO between T0 and T15.

https://cdn.elifesciences.org/articles/79843/elife-79843-fig3-figsupp1-data1-v1.xlsx
Figure 4 with 1 supplement
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The apico-basal (Ap-Ba) distribution of Smoothened (SMO) is controlled by phosphorylation by the protein kinase A (PKA)/ casein kinase I (CKI) and Fused (FU) kinases.

(A–D) Confocal images of wing imaginal discs labeled for Surf SNAP-SMOWT (A), SNAP-SMOPKA-SD (B), SNAP-SMOPKA-SD FU-SA (C), and SNAP-SMOPKA-SD FU-SD (D). Note that for better visualization of SMO, …

Figure 4—source data 1

Relative intensity of Surf SNAP-SMO for different SMO mutants.

https://cdn.elifesciences.org/articles/79843/elife-79843-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
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Effect of the phosphorylation of Smoothened (SMO) by the protein kinase A (PKA) and Fused (FU) kinases on its accumulation and signaling activity.

(A, A’-D, D’) False color representation of the accumulation at the cell surface of wild type and mutant forms of SNAP-SMO (as indicated and expressed in the dorsal part of the wing discs using the …

Figure 4—figure supplement 1—source data 1

Relative intensity of Surf SNAP-SMO in WT1 and WT2.

https://cdn.elifesciences.org/articles/79843/elife-79843-fig4-figsupp1-data1-v1.xlsx
Figure 5 with 2 supplements
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Trapping the Fused (FU) kinase to the apical region promotes the stabilization of Smoothened (SMO) at the cell surface.

Confocal images of wing imaginal discs coexpressing (using the apGal4 driver) GFP-fu and T48 or NVR1 fused to the mcherry (mche). XY sections are shown in (A–A”’ and C–C”’), anterior YZ sections in …

Figure 5—figure supplement 1
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Fused (FU) and Smoothened (SMO) colocalization.

(A-A’) Confocal images of wild type discs immunolabeled for FU. XY section in (A) and XZ section in (A’). (B–B”) XY confocal images of endogenous SMO (left panels) and FU (center panels) …

Figure 5—figure supplement 2
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Effect on the subcellular localization of Smoothened (SMO) of trapping the Fused (FU) kinase to the apical (ap) or basolateral region.

(A–A’’’, B–B’’’) YZ confocal images of wing imaginal discs (WIDs) coexpressing (using the apGal4 driver), GFP-fu with either mche-T48 or mche-NVR1. mcherry (A and B, respectively, red in the merged …

Figure 5—figure supplement 2—source data 1

Relative and mean intensity of endogenous SMO in T48 GFP-FU flies.

https://cdn.elifesciences.org/articles/79843/elife-79843-fig5-figsupp2-data1-v1.xlsx
Figure 6 with 1 supplement
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Trapping the Fused (FU) kinase to the apical region is sufficient to promote medium but not high hedgehog (HH)/ Smoothened (SMO) signaling.

Confocal XY images of discs expressing GFP-fused with mche-T48 in fuWT (A–B) or knockout (fuKO) context (E–F), with mche-NVR1 in fuWT (C–D) or alone in fuKO context (G–H) and immunolabeled for …

Figure 6—figure supplement 1
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Effect of trapping the Fused (FU) kinase on decapentaplegic (dpp) expression.

(A, B) Confocal images of discs expressing GFP-fu with mche-T48 (A) or with mche-NVR1 (B) immunolabeled for the β-galactosidase (dpp-Z, to report dpp expression). (C, D) Construction of the fuKO …

Figure 7
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Model: hedgehog (HH) controls the fate of endocytosed Smoothened (SMO) via a multikinase phosphorylation barcode.

SMO is initially targeted (curved arrow) to the apical plasma membrane (presented in pink), where it is endocytosed (vesicle on the top, with a pink membrane). Endocytosed SMO can then be sent to …

Author response image 1
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Author response image 2
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Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (D. melanogaster)apGal4Weihe et al., 2001FLYB: FBtp0084979FlyBase symbol: P{ap-GAL4.U}
Genetic reagent (D. melanogaster)UAS SNAP-smoWTSanial et al., 2017
Genetic reagent (D. melanogaster)UAS SNAP-smoPKA-SDThis paperGenerated by BestGene Inc
using the PhiC31
integration system
Genetic reagent (D. melanogaster)UAS SNAP-smoPKA-SD FU-SAThis paperGenerated by BestGene Inc
using the PhiC31
integration system
Genetic reagent (D. melanogaster)UAS SNAP-smoPKA-SD FU-SDThis paperGenerated by BestGene
Inc using the PhiC31
integration system
Genetic reagent (D. melanogaster)hhtsHeemskerk and DiNardo, 1994FLYB: FBal0031490
Genetic reagent (D. melanogaster)shibiretsvan der Bliek and Meyerowitz, 1991FLYB: FBal0015610
Genetic reagent (D. melanogaster)apGal4 Gal80tsGift from F Schweisguth
Genetic reagent (D. melanogaster)UAS-YFP-rab5CAHugo J. Bellen,
Baylor College of Medicine		RRID:BDSC_9773
Genetic reagent (D. melanogaster)UAS smurf RNAiPerkins et al., 2015RRID:BDSC_40905
Genetic reagent (D. melanogaster)UAS mCherry-NVR1-GFP nanobodyHarmansa et al., 2017RRID:BDSC_68175
Genetic reagent (D. melanogaster)UAS mCherry-T48-GFP nanobodyHarmansa et al., 2017RRID:BDSC_68178
Genetic reagent (D. melanogaster)UAS GFP-fusedRuel et al., 2003
Genetic reagent (D. melanogaster)dpp-LacZ, apGal4Gift from R Holmgrendpp-LacZ RRID:BDSC_12379
Genetic reagent (D. melanogaster)w1118Hazelrigg et al., 1984FLYB: FBal0018186FlyBase symbol: Dmel\w1118
Genetic reagent (D. melanogaster)BAC SNAP- smoThis papersnap insertion via
recombineering mediated
gap-repair. Fly generated
by BestGene Inc using
the PhiC31 integration system.
Genetic reagent (D. melanogaster)fuKO/FM0BarThis paperfu knockout (KO) mutant
fly generated via CRISPR/
Cas9 by inDROSO
Antibodyanti-PTC (mouse monoclonal)DSHB, 
Martín et al., 2001		DSHB Cat# Drosophila Ptc (Apa 1), RRID:AB_528441IF(1:50)
Antibodyanti-EN (mouse monoclonal)DSHB, University of Iowa, USA, 
Patel et al., 1989		DSHB Cat# 4D9 anti-engrailed/invected, RRID:AB_528224IF(1:50)
Antibodyanti-DLG (mouse monoclonal)DSHB, Parnas et al., 2001DSHB Cat# 4F3 anti-discs large, RRID:AB_528203IF(1:50)
Antibodyanti-SMO (mouse monoclonal)DSHB, Lum et al., 2003DSHB Cat# Smoothened (20C6), RRID:AB_528472IF(1:100)
Antibodyanti-Rab7 (mouse monoclonal)DSHB, University of Iowa, USA,DSHB Cat# Rab7, RRID:AB_2722471IF(1:25)
Antibodyanti-FU (rabbit polyclonal)Gift from Ruel et al., 2003IF(1:100)
Antibodyanti-β-Galactosidase (rabbit polyclonal)MP Biomedicals085597-CFIF(1:100)
Antibodyanti-CI (rat monoclonal 2A1)Gift from Motzny and Holmgren, 1995IF(1:5)
Antibodyanti-mouse IgG Alexa Fluor Plus 555 (goat polyclonal)Thermo Fisher ScientificThermo Fisher Scientific Cat# A32727, RRID:AB_2633276IF(1:200)
Antibodyanti-rat IgG Alexa Fluor Plus 647 (goat polyclonal)Thermo Fisher ScientificThermo Fisher Scientific, Cat# A-21247, RRID: AB_141778IF(1:200)
Antibodyanti-rabbit IgG Alexa Fluor Plus 555 (goat polyclonal)Thermo Fisher ScientificThermo Fisher Scientific, Cat# A32732, RRID: AB_2633281IF(1:200)
Antibodyanti-rabbit IgG Alexa Fluor Plus 488 (Goat polyclonal)Thermo Fisher ScientificThermo Fisher Scientific, Cat# A32731, RRID: AB_2633280IF(1:200)
Antibodyanti-rabbit IgG Alexa Fluor Plus 647 (goat polyclonal)Thermo Fisher ScientificThermo Fisher Scientific, Cat# A-21245, RRID: AB_253581IF(1:200)
Antibodyanti-mouse IgG cross adsorbed 488 (chicken polyclonal)Thermo Fisher ScientificThermo Fisher Scientific, Cat# A-21200, RRID: AB_2535786IF(1:200)
Antibodyanti-mouse IgG Dylight 649 (donkey polyclonal)Jackson Immuno715-495-151IF(1:200)
Recombinant DNA reagent (D. melanogaster)BAC smoBAC Resources PACCH322-98K24https://bacpacresources.org/home.htm
Recombinant DNA reagentBAC snap-smoThis papersnap cDNA introduced in
CH322-98K24 after
Serine 33 codon of smo
Recombinant DNA reagentpENTR/D-TOPO-snap smoSanial et al., 2017Template for snap amplification
Recombinant DNA reagentpENTR/D-TOPO-snap smoPKA-SDSanial et al., 2017
Recombinant DNA reagentpENTR/D-TOPO-snap smoPKA-SD FU-SASanial et al., 2017
Recombinant DNA reagentpENTR/D-TOPO-snap smoPKA-SD FU-SDSanial et al., 2017
Recombinant DNA reagentpUASt-GW-attBBrigui et al., 2015
Sequence-based reagentrpsL-neo/smo mRNA 360 /FEurofins GenomicsPCR primersAGGTTGCGATCTTATGCCTGTGGGTGGTCGCAGACGCATCGGCCAGTTCGggcctggtgatgatggcgggatcg
Sequence-based reagentrpsL-neo/smo mRNA 462 /REurofins GenomicsPCR primersAGTTCCACATCCGACTGCTGCGCACTTGCGGGCGTTGTGCTGCCGAACTTGGCtcagaagaactcgtcaagaaggcg
Sequence-based reagentpEnSnapSmo/smo mRNA 360 /FEurofins GenomicsPCR primersAGGTTGCGATCTTATGCCTGTGGGTGGTCGCAGACGCATCGGCCAGTTCG
Sequence-based reagentpEnSnapSmo/smo mRNA 462 /REurofins GenomicsPCR primersAGTTCCACATCCGACTGCTGCGCACTTGCGGGCGTTGTGCTGCCGAACTTGGC
Sequence-based reagentsmo mRNA 289/Seq/FEurofins GenomicsPCR primersGACTCGCCTCTGGCAAATGG
Sequence-based reagentsmo mRNA 512/Seq/REurofins GenomicsPCR primersTGCCCTTCTTGGCGTACAGTCGG
Sequence-based reagentRpsl/neo/273/Seq/FEurofins GenomicsPCR primersGAACTCCGCGCTGCGTAAAGTATG
Sequence-based reagentsnap/203/Seq/FEurofins GenomicsPCR primersCCTACTTTCACCAGCCTGAG
Chemical compound, drugSNAP-Surface Alexa Fluor 488New England BiolabsCat#S9124S3.3 µM
Chemical compound, drugSNAP-Surface Alexa Fluor 546New England BiolabsCat#S9132S3.3 µM
Chemical compound, drugSNAP-Surface BlockNew England BiolabsCat#S9143S2 µM
Chemical compound, drugSNAP-Surface Alexa Fluor 647New England BiolabsCat#S9136S3.3 µM
Software, algorithmFijiSchindelin et al., 2012Fiji, RRID:SCR_002285http://fiji.sc/
Software, algorithmAdobe PhotoshopAdobeRRID: SCR_014199https://www.adobe.com/products/photoshop.html
Software, algorithmAdobe IllustratorAdobeRRID:SCR_010279http://www.adobe.com/products/illustrator.html
Software, algorithmAdobe InDesignAdobeRRID:SCR_021799https://www.adobe.com/fr/products/indesign.html
Software, algorithmPrism – GraphPad 9Graph Pad SoftwareRRID:SCR_002798https://www.graphpad.com/scientific-software/prism/

Additional files

MDAR checklist
https://cdn.elifesciences.org/articles/79843/elife-79843-mdarchecklist1-v1.pdf
Source code 1

Fiji Macro for the quantification.

https://cdn.elifesciences.org/articles/79843/elife-79843-code1-v1.zip

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