1. Developmental Biology

The glycosphingolipid MacCer promotes synaptic bouton formation in Drosophila by interacting with Wnt

  1. Yan Huang
  2. Sheng Huang
  3. Coralie Di Scala
  4. Qifu Wang
  5. Hans H Wandall
  6. Jacques Fantini
  7. Yong Q Zhang  Is a corresponding author
  1. CAS Center for Excellence in Brain Science and Intelligence Technology, China
  2. Sino-Danish Center for Education and Research, Chinese Academy of Sciences, China
  3. INSERM, France
  4. University of Copenhagen, Denmark
  5. Aix-Marseille Université, France
https://doi.org/10.7554/eLife.38183
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Cite this article
  1. Yan Huang
  2. Sheng Huang
  3. Coralie Di Scala
  4. Qifu Wang
  5. Hans H Wandall
  6. Jacques Fantini
  7. Yong Q Zhang
(2018)
The glycosphingolipid MacCer promotes synaptic bouton formation in Drosophila by interacting with Wnt
eLife 7:e38183.
https://doi.org/10.7554/eLife.38183
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9 figures, 1 table and 2 additional files

Figures

Figure 1
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NMJ growth depends on de novo synthesis of ceramides

(A) Simplified de novo biosynthesis pathway of sphingolipid in Drosophila is shown.(B–G) Representative images of NMJ4 co-stained with anti-HRP (green) and anti-CSP (magenta) in wild type (B), lace k…

https://doi.org/10.7554/eLife.38183.002
Figure 2 with 2 supplements
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GSL synthases Egh and Brn bi-directionally regulates NMJ growth presynaptically

(A) GSL synthesis pathway in Drosophila.(B–M) Images of NMJ4 co-stained with anti-HRP (green) and anti-CSP (magenta) in wild type (B), larvae treated with 0.5 mg/ml D, L-threo-PDMP (C), egh62d18/Y (D…

https://doi.org/10.7554/eLife.38183.003
Figure 2—source data 1

Numerical data for the statistical graphs.

https://doi.org/10.7554/eLife.38183.006
Figure 2—figure supplement 1
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Additional NMJ images and quantifications.

(A–D) Images of NMJ4 co-stained with anti-HRP (green) and anti-CSP (magenta) in wild type (A), egh62d18/+ (B), egh62d18/egh7 (C), brnfs107/Y (D). Scale bar: 10 μm. (E) Quantifications of muscle four …

https://doi.org/10.7554/eLife.38183.004
Figure 2—figure supplement 2
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Alteration of egh and brn level in glia or muscle does not affect NMJ growth.

(A–H) Images of NMJ4 co-stained with anti-HRP (green) and anti-CSP (magenta) in UAS-Egh/+; C57-Gal4/+ (A), egh62d18/Y; UAS-Egh/+; C57-Gal4/+ (B), UAS-brn-RNAi/+; C57-Gal4/+ (C), UAS-brn/C57-Gal4 (D),…

https://doi.org/10.7554/eLife.38183.005
Figure 3 with 1 supplement
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MacCer staining intensity at NMJs is bi-directionally regulated by Egh and Brn.

(A) Images of wild-type NMJ4 co-stained with anti-MacCer (green), anti-HRP (blue) and anti-DLG (red).MacCer puncta were apparent in presynaptic boutons. Scale bar: 10 μm. (B) Statistical results of …

https://doi.org/10.7554/eLife.38183.007
Figure 3—source data 1

Numerical data for the statistical graphs.

https://doi.org/10.7554/eLife.38183.009
Figure 3—figure supplement 1
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Images and quantifications of Fas II and Syx1A staining at NMJs of different genotypes.

(A–C) Images of NMJ4 co-stained with anti-Fas II (green) and anti-HRP (magenta) in wild type, egh62d18/Y, and nSyb-Gal4/UAS-brn. (D–F) Images of NMJ4 co-stained with anti-Syx1A (green) and anti-HRP …

https://doi.org/10.7554/eLife.38183.008
Figure 4 with 1 supplement
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Sterol-depletion inhibits NMJ growth in a common genetic pathway with MacCer.

(A and B) Confocal images of single slice of NMJ4 boutons triple-labeled with anti-MacCer (red), anti-Syx1A (green) and anti-HRP (blue) in wild type with or without 20 mM MβCD treatment. Plot …

https://doi.org/10.7554/eLife.38183.010
Figure 4—source data 1

Numerical data for the statistical graphs.

https://doi.org/10.7554/eLife.38183.012
Figure 4—figure supplement 1
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Additional NMJ images and quantifications of bouton number and bouton size.

(A) Quantification of bouton number of NMJ4 from wild-type larvae fed with 0 mM (CT), 10 mM, 20 mM MβCD and 0 μg/ml (DMSO vehicle only), 25 μg/ml, 50 μg/ml filipin III. (B and C) Quantifications of …

https://doi.org/10.7554/eLife.38183.011
Figure 5 with 2 supplements
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MacCer is required for the NMJ growth-promoting effect of Wg signaling.

(A–J) Representative images of NMJ4 co-stained with anti-HRP (green) and anti-CSP (magenta) in egh62d18/+ (control, (A), UAS-Wg-HA/+; nSyb-Gal4/+ (B), egh62d18/+; UAS-Wg-HA/+; nSyb-Gal4/+ (C), egh62d…

https://doi.org/10.7554/eLife.38183.013
Figure 5—source data 1

Numerical data for the statistical graphs.

https://doi.org/10.7554/eLife.38183.016
Figure 5—figure supplement 1
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Additional NMJ images and quantifications.

Images of NMJ4 co-labeled for anti-HRP (green) and anti-CSP (magenta) in nSyb-Gal4/UAS-brn (A), UAS-Wg-HA/+; nSyb-Gal4/UAS-brn (B), wild type treated with 20 mM MβCD (C), UAS-Wg-HA/+; nSyb-Gal4/+ …

https://doi.org/10.7554/eLife.38183.014
Figure 5—figure supplement 2
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Additional NMJ images and quantifications.
https://doi.org/10.7554/eLife.38183.015
Figure 6 with 4 supplements
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MacCer is required for the local presynaptic Wg signaling at NMJs.

(A–D) Confocal images of single slice of NMJ4 boutons triple-labeled with anti-MacCer (red), anti-HRP (blue) and anti-Wg (green; A–B) or anti-Rab11 (green; C–D) in wild type and egh62d18 mutants. Mac…

https://doi.org/10.7554/eLife.38183.017
Figure 6—source data 1

Numerical data for the statistical graphs.

https://doi.org/10.7554/eLife.38183.022
Figure 6—figure supplement 1
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MacCer puncta showed few overlap with Rab5-YFP, Spinster-GFP and Mannose II-GFP.

Single sections of NMJ4 boutons triple-labeled with anti-MacCer (red), anti-HRP (blue), and specific organelle markers (green) in nSyb-Gal4/UAS-Spin-GFP (A), UAS-YFP-Rab5/+; nSyb-Gal4/+ (B), and nSyb…

https://doi.org/10.7554/eLife.38183.018
Figure 6—figure supplement 2
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The Wg level in egh brains by immunochemical analysis.

(A and B) Western blotting and quantification of Wg from 3rd instar larval brains of wild type and egh62d18 mutants, normalized to the level of Actin and Syntaxin 1A (Syx1A). n = 3; ns, no …

https://doi.org/10.7554/eLife.38183.019
Figure 6—figure supplement 3
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Additional images showing Wg and Futsch staining at NMJ of different genotypes.

(A–C) Representative images of NMJ4 co-labeled with anti-Wg (green) and anti-HRP (magenta) in wild type (A), nSyb-Gal4/UAS-brn (B) and UAS-brn-RNAi/+; nSyb-Gal4/+ (C). Scale bar: 5 μm. (D–F) …

https://doi.org/10.7554/eLife.38183.020
Figure 6—figure supplement 4
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Images and quantifications of MacCer, Rab11, and Fz2 staining at NMJ of different genotypes.

(A–C) Images of NMJ4 co-stained with anti-MacCer (green) and anti-HRP (magenta) in wild type (A), rab1193Bi (B) and wg1/wgCX4 (C). (D) Quantification of MacCer intensity normalized to HRP intensity …

https://doi.org/10.7554/eLife.38183.021
Figure 7
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Postsynaptic differentiation is normal in egh mutants.

(A and B) Representative images of NMJ4 from wild type and egh62d18 mutants co-stained with anti-HRP (magenta) and anti-GluRIIA (green). Scale bar: 2 μm. (C and D) Images of NMJ6/7 from wild type …

https://doi.org/10.7554/eLife.38183.023
Figure 7—source data 1

Numerical data for the statistical graphs.

https://doi.org/10.7554/eLife.38183.024
Figure 8 with 1 supplement
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Wg has a functional MacCer-binding domain

(A) Schematic representation of Wg protein and amino acid sequence of a putative GSL-binding motif (GBM) and mutated GBM with asterisks denoting the mutated amino acids.(B) A 3D model of Wg residues …

https://doi.org/10.7554/eLife.38183.025
Figure 8—figure supplement 1
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Additional monolayer data and GSL profiles of egh and brn mutants.

(A) Surface pressure increase (Δπ) by the addition of the Wg233-247 (GBM) peptide on a monolayer of LacCer. A maximal value was reached after 1 hr of incubation. (B) Surface pressure change (Δπ) by …

https://doi.org/10.7554/eLife.38183.026
Figure 9
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Wg GBM is required for Wg-MacCer colocalization and NMJ growth.

(A and B) Representative images of NMJ4 co-labeled with anti-Wg (green), anti-MacCer (red) and anti-HRP (blue) in wg1/+ (A) and wgGBM/wg1 mutants (B). (C) Quantification of Wg and MacCer intensities …

https://doi.org/10.7554/eLife.38183.027
Figure 9—source data 1

Numerical data for the statistical graphs.

https://doi.org/10.7554/eLife.38183.028

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or
reference		IdentifiersAdditional information
Genetic reagent (D. melanogaster)wgGBMthis paperNAThis allele carries the point mutation of K234E and M238R in Wg
Genetic reagent (D. melanogaster)wg1doi: 10.1523/Jneurosci.3714–13.2014RRID: BDSC_2978
Genetic reagent (D. melanogaster)wgCX4,wgl-17doi: 10.1016/S0092-8674 (02)01047–4RRID: BDSC_2980
Genetic reagent (D. melanogaster)UAS-Wg-HA(6C)doi: 10.1016/S0092-8674 (02)01047–4RRID: BDSC_5918
Genetic reagent (D. melanogaster)UAS-Wg-HA(3C)doi: 10.1016/S0092-8674 (02)01047–4RRID: DGGR_108488
Genetic reagent (D. melanogaster)egh7PMID: 9012507RRID: BDSC_3902
Genetic reagent (D. melanogaster)egh62d18PMID: 9012507NADr. Stephen M. Cohen (National University of Singapore)
Genetic reagent (D. melanogaster)UAS-Egh-Mycdoi: 10.1074/jbc.C400571200NA
Genetic reagent (D. melanogaster)brnfs107PMID: 1483386RRID: BDSC_4303
Genetic reagent (D. melanogaster)brn1.6P6PMID: 1483386RRID: BDSC_50762
Genetic reagent (D. melanogaster)UAS-Brndoi: 10.1016/j.ydbio.2007.04.013NA
Genetic reagent (D. melanogaster)UAS-brn-RNAidoi: 10.1038/nmethRRID: BDSC_55386
Genetic reagent (D. melanogaster)arrk08131doi: 10.1534/genetics.104.026427RRID: BDSC_665
Genetic reagent (D. melanogaster)arr2doi: 10.1038/35035110RRID: BDSC_3087
Genetic reagent (D. melanogaster)evi2doi: 10.1074/jbc.M112.342667NADr. Vivian Budnik (University Massachusetts Medical School)
Genetic reagent (D. melanogaster)lace2doi: 10.1128/MCB.19.10.7276RRID: BDSC_3159
Genetic reagent (D. melanogaster)lacek05305doi: 10.1128/MCB.19.10.7276RRID: BDSC_12176
Genetic reagent (D. melanogaster)Df(2L)Exel7063NARRID:BDSC_7831
Genetic reagent (D. melanogaster)schlankG0489doi:10.1038/emboj.2009.305RRID: DGGR_111936
Genetic reagent (D. melanogaster)schlankG0061doi:10.1038/emboj.2009.305RRID: BDSC_11665
Genetic reagent (D. melanogaster)Sk2KG05894doi: 10.1194/jlr.M300005-JLR200RRID: BDSC_14133
Genetic reagent (D. melanogaster)Df(3L)BSC671NARRID: BDSC_26523
Genetic reagent (D. melanogaster)UAS-ManII-GFPdoi:10.1016/j.cell.2007.06.032RRID: BDSC_65248Dr. Yuh Nung Jan (University of California, San Francisco)
Genetic reagent (D. melanogaster)UAS-Spin-GFPdoi: S0896627302010140RRID:BDSC_39668Dr. Graeme Davis (University of California, San Francisco)
Genetic reagent (D. melanogaster)UAS-YFP-Rab5doi: 10.1534/genetics.106.066761RRID: BDSC_24616
Genetic reagent (D. melanogaster)nSyb-Gal4NARRID: BDSC_51635
Genetic reagent (D. melanogaster)OK6-Gal4NARRID: BDSC_64199
Genetic reagent (D. melanogaster)C57-Gal4NARRID: BDSC_32556
Genetic reagent (D. melanogaster)Repo-Gal4NARRID: BDSC_7415
Genetic reagent (D. melanogaster)act-Cas9NARRID: BDSC_54590
AntibodyMouse IgM anti-MacCerdoi: 10.1074/jbc.C400571200NAIHC (1:1)
AntibodyMouse anti-CSPDSHB Cat. #: 6D6RRID:AB_528183IHC (1:300)
AntibodyMouse anti-WgDSHB Cat. #: 4D4RRID:AB_528512IHC (1:10), WB (1:50)
AntibodyMouse anti-FutschDSHB Cat. #: 22C10RRID:AB_528403IHC (1:50)
AntibodyMouse anti-DLGDSHB Cat. #: 4F3RRID:AB_528203IHC (1:500)
AntibodyMouse anti-GluRIIADSHB Cat. #: 8B4D2RRID:AB_528269IHC (1:200)
AntibodyMouse anti-Fas IIDSHB Cat. #: 1D4RRID:AB_528235IHC (1:50)
AntibodyMouse anti-Syx1ADSHB Cat. #: 8C3RRID:AB_528484IHC (1:20), WB (1:1000)
AntibodyMouse anti-dFz2DSHB Cat. #:12A7RRID:AB_528257IHC (1:5)
AntibodyMouse anti-Rab11BD Biosciences Cat. #: 610656RRID:AB_397983IHC (1:50)
AntibodyMouse anti-HAMBL International Cat. #: M180RRID:AB_10951811IHC (1:1000)
AntibodyMouse anti-MycCWBIO Cat. #: CW0259IHC (1:300), WB (1:1000)
AntibodyMouse anti-MycMBL International Cat. #: M192RRID:AB_11160947IHC (1:300), WB (1:1000)
AntibodyRat anti-GFPMBL International Cat. #: D153RRID:AB_591820IHC (1:200)
Antibodyfluorescence-conjugated anti-HRPJackson ImmunoResearchRRID:AB_2314647IHC (1:200)
AntibodyMouse anti-β-actinMillipore Bioscience ResearchRRID:AB_2223041WB (1:50000)
Chemical compound, drugD,L-threo-PDMPMatreyaCat.#: 17190.5 mg/ml
Chemical compound, drugfilipin IIICayman7044050 μg/ml
Chemical compound, drugMβCDSigma-AldrichC455520 mM
Chemical compound, drugLactosylceramideMatreyaCat.#: 1500
Peptide, recombinant proteinGBMthis paperCKCHGMSGSCTVKTC
Peptide, recombinant proteinGBMmutthis paperCECHGRSGSCTVKTC
Recombinant DNA reagentpcDNA3.1-Myc-WgInvitrogenV790-20
Recombinant DNA reagentU6b-sgRNAAddgene65956
Recombinant DNA reagentpBluescript SK (-)Stratagene212206
Commercial assay or kitTNT T6 Quick Coupled
Transcription/Translation System		PromegaCat.#: L1171
Commercial assay or kitLacCer-coated beadsEchelonCat.#: P-B0LC
Commercial assay or kitControl beadsEchelonCat.#: P-B000
Commercial assay or kitHPTLC Silica Gel (aluminium plates)Merck-MilliporeCat.#: 105547
Software, algorithmHyperchem softwareHypercube

Additional files

Supplementary File 1

Genetic screen for NMJ morphology by manipulating selected genes involved in synthesis and turnover of membrane lipids.

https://doi.org/10.7554/eLife.38183.029
Transparent reporting form
https://doi.org/10.7554/eLife.38183.030

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