Cellular localization of the cell cycle inhibitor Cdkn1c controls growth arrest of adult skeletal muscle stem cells

  1. Despoina Mademtzoglou
  2. Yoko Asakura
  3. Matthew J Borok
  4. Sonia Alonso-Martin
  5. Philippos Mourikis
  6. Yusaku Kodaka
  7. Amrudha Mohan
  8. Atsushi Asakura  Is a corresponding author
  9. Frederic Relaix  Is a corresponding author
  1. IMRB U955-E10, F-94010, France
  2. Faculté de medecine, F-94000, Université Paris-Est Creteil, France
  3. University of Minnesota Medical School, United States
  4. Etablissement Français du Sang, France
  5. DHU Pepsy & Centre de Référence des Maladies Neuromusculaires GNMH, France
7 figures, 2 tables and 1 additional file

Figures

Figure 1 with 1 supplement
Cdkn1c deficiency impairs normal muscle growth.

(A) Hematoxylin and Eosin (HE) and Sirius red staining of control (Ctrl) and Cdkn1c mutant (Cdkn1c-Mut) mouse Tibialis anterior (TA) muscles were performed to examine muscle histology, centrally …

https://doi.org/10.7554/eLife.33337.002
Figure 1—figure supplement 1
Cdkn1c mutant mice display smaller body weight.

(A) A few Cdkn1c mutant (Cdkn1cM/-) mice survived postnatally in a mixed CD1;B6 background. (B–C) Body weight average of control (Ctrl) and Cdkn1c mutant male (B) and female (C) mice. (D) Forelimb …

https://doi.org/10.7554/eLife.33337.003
Figure 2 with 1 supplement
CDKN1c deficiency delays muscle regeneration.

(A) Embryonic myosin (eMyHC)/LAMININ/DAPI, Hematoxylin and Eosin (HE), and Sirius red staining of twelve- to fifteen-week-old control (Ctrl) and Cdkn1c mutant mouse TA muscles were performed for …

https://doi.org/10.7554/eLife.33337.004
Figure 2—figure supplement 1
Myoblasts in Cdkn1c mutant mice display delayed cell cycle exit during muscle regeneration.

(A) EdU (green)/MYOD (red)/DAPI (blue) staining of TA muscle sections of control (Ctrl) and Cdkn1c mutant (Cdkn1c-Mut) mice at day 3 after CTX injection. Arrows indicate EdU-MYOD+ differentiating …

https://doi.org/10.7554/eLife.33337.005
Figure 3 with 2 supplements
Cdkn1c deficiency impairs myogenic differentiation.

(A) Time-course of tamoxifen (TMX) administration, muscle satellite cell harvest (FACS arrow) and culture (light gray bar for growth culture conditions, dark gray bar for differentiation culture …

https://doi.org/10.7554/eLife.33337.006
Figure 3—figure supplement 1
Myogenic marker expression in proliferating myoblasts.

(A) Time-course of tamoxifen administration, muscle satellite cell harvest (FACS arrow) and culture. Analyzed animals were Pax7CreERT2/+; Cdkn1cFlox(m)/+;RosamTmG (Cdkn1c cKO) and Pax7CreERT2/+; …

https://doi.org/10.7554/eLife.33337.007
Figure 3—figure supplement 2
Cdkn1c mutant myoblasts display increased proliferation and reduced differentiation.

(A) Control (Ctrl) and Cdkn1c mutant (Cdkn1c-Mut) primary myoblasts are positive for both PAX7 and MYOD. (B) Under growth conditions, EdU+ cells are significantly higher in Cdkn1c mutant primary …

https://doi.org/10.7554/eLife.33337.008
Figure 4 with 1 supplement
MuSC-specific Cdkn1c ablation hinders muscle regeneration.

(A) Time-course of tamoxifen administration, intramuscular injury of TA muscle (CTX arrow), and muscle harvest (D7 arrow). (B–G) Cryosections of TA muscle were stained for histological and satellite …

https://doi.org/10.7554/eLife.33337.009
Figure 4—figure supplement 1
In vivo MuSC-specific Cdkn1c ablation.
https://doi.org/10.7554/eLife.33337.010
Figure 5 with 1 supplement
Cdkn1c cytoplasmic expression after satellite cell activation.

(A) Satellite-cell-derived myoblasts (T24–T48) of single EDL myofibers stained with PAX7 (green) and CDKN1c (red). Arrowheads indicate PAX7+ cells. (B) Satellite cell-derived myoblasts of single EDL …

https://doi.org/10.7554/eLife.33337.011
Figure 5—figure supplement 1
Cdkn1c is not expressed in quiescent satellite cells.

(A) Muscle satellite cells (MuSCs; T0) stained with PAX7 (green) and Cdkn1c (red) in single myofiber cultures of EDL muscles. (B) Cdkn1c (red) presence in TA muscle section. MuSCs were marked with …

https://doi.org/10.7554/eLife.33337.012
Figure 6 with 2 supplements
Cdkn1c expression and subcellular localization during satellite cell activation and differentiation.

(A–D) Immunofluorescence for PAX7 (A; green), MYOD (B; green), MYOGENIN (C; green) or KI67 (D; green) and Cdkn1c (red) at T72 in single myofiber cultures of EDL muscles and quantification of PAX7+ (A

https://doi.org/10.7554/eLife.33337.013
Figure 6—figure supplement 1
Lack of CDKN1c binding in myogenic regulatory regions.

Chromatin immunoprecipitation followed by qPCR on C2C12 myogenic cells 4 days after differentiation induction. Enrichment was evaluated in myogenic regions that have previously been shown to be …

https://doi.org/10.7554/eLife.33337.014
Figure 6—figure supplement 2
Uncoupling of cell cycle exit and differentiation.

(A) Time-course of single myofiber culture, transduction of activated myoblasts with virus, and readouts. (B–D) Quantification of transduced (CFP+) myoblasts that were differentiating, as evaluated …

https://doi.org/10.7554/eLife.33337.015
Author response image 1
Quantification of M-cadherin+ MuSCs in regenerating muscle of wildtype littermate (Wt; Pax7+;Cdkn1c+), Cre control (Pax7CreERT2), and Cdkn1c cKO (Pax7CreERT2;Cdkn1cFlox) mice at D7 post-cardiotoxin injection.
https://doi.org/10.7554/eLife.33337.019

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain background
(M. musculus)
Cdkn1ctm1SjeThe Jackson Laboratory;
PMID: 9144284
MGI: J40203,
RRID:IMSR_JAX:003336
Strain, strain background
(M. musculus)
p57floxPMID: 28196404Mouse line generated by the
group of F.Relaix and
characterized in
Mademtzoglou et al. (2017);
Genesis 55(4) doi:
10.1002/dvg.23025
Strain, strain background
(M. musculus)
Pax7CreERT2/+The Jackson Laboratory;
PMID: 19554048
MGI: J:150962;
RRID:IMSR_JAX:012476
Mouse line obtained
from C.M. Fan
Strain, strain background
(M. musculus)
RosamTmGThe Jackson Laboratory;
PMID: 17868096
MGI: J:124702;
RRID:IMSR_JAX:007576
Genetic reagent (synthetic)pGEMT-Easy vectorPromegaA1360
Cell line (Homo sapiens)293TDSMZACC635;
RRID:CVCL_0063
https://www.dsmz.de/catalogues/details/culture/ACC-635.html?tx_dsmzresources_pi5%5BreturnPid%5D=192
Cell line (M. musculus)C2C12American Type Culture
Collection (ATCC);
PMID: 28966089
CRL-1772;
RRID: CVCL_0188
Cell line maintained
in E. Gomes lab
Antibodyanti-CD31-PE (monoclonal)eBiosciences12-0311-81;
RRID:AB_465631
Antibodyanti-CD45-PE (monoclonal)eBiosciences12-0451-81;
RRID:AB_465667
Antibodyanti-embryonic MyHC
(mouse monoclonal)
DSHBF1.652;
RRID:AB_528358
Antibodyanti-embryonic MyHC
(mouse monoclonal)
Santa Cruzsc53091;
RRID:AB_670121
Antibodyanti-GFP (chicken
polyclonal)
Abcamab13970;
RRID:AB_300798
Antibodyanti-integrin a-biotin
(mouse)
Miltenyi Biotec130-101-979;
RRID:AB_2652472
Antibodyanti-IgG (rabbit)DiagenodeC15410206
Antibodyanti-KI67 (mouse
monoclonal)
BD Pharmingen556003;
RRID:AB_396287
Antibodyanti-Laminin (rabbit
polyclonal)
Sigma-AldrichL9393;
RRID:AB_477163
Antibodyanti-Laminin (rat
monoclonal)
Sigma-Aldrich4H8-2;
RRID:AB_784266
Antibodyanti-Laminin (rabbit
polyclonal)
Novus BiologicalNB300-144AF647
Antibodyanti-MyHC (mouse
monoclonal)
DSHBmf20-c;
RRID:AB_2147781
Antibodyanti-MyoD (mouse
monoclonal)
DAKOM3512;
RRID:AB_2148874
Antibodyanti-MyoD (rabbit
polyclonal)
Santa Cruzsc-760;
RRID:AB_2148870
Antibodyanti-Myogenin
(mouse monoclonal)
DSHBF5D;
RRID:AB_2146602
Antibodyanti-p57 (goat polyclonal)Santa Cruzsc1039;
RRID:AB_2078158
Antibodyanti-p57 (mouse
monoclonal)
Santa Cruzsc56431;
RRID:AB_2298043
Antibodyanti-p57 (rabbit
polyclonal)
Santa Cruzsc8298;
RRID:AB_2078155
Antibodyanti-Pax7 (mouse
monoclonal)
DSHBPAX7-c;
RRID:AB_528428
Antibodyanti-Sca-1-PE (mouse)eBiosciences12-5981-81;
RRID:AB_466085
Antibodyfab fragment affinity-
purified antibody (goat)
Jackson ImmunoResearch115-007-003
Sequence-based reagentAGGGCATATCC
AACAACAAACTT
EurogentecN/AqPCR HPRT (Forward primer)
Sequence-based reagentGTTAAGCAGTA
CAGCCCCAAA
EurogentecN/AqPCR HPRT (Reverse primer)
Sequence-based reagentCTGAAGGACCA
GCCTCTCTC
EurogentecN/AqPCR p57 (Forward primer)
Sequence-based reagentAAGAAGTCGTT
CGCATTGGC
EurogentecN/AqPCR p57 (Reverse primer)
Sequence-based reagentATCTGAGGTCA
GCCATTTGGT
EurogentecN/AChIP qPCR Mef2a
(Forward primer)
Sequence-based reagentGCTAAGGACAG
CTGTGACCTG
EurogentecN/AChIP qPCR Mef2a
(Reverse primer)
Sequence-based reagentTTAAAGACATGTG
GCAACAGACTAC
EurogentecN/AChIP qPCR Lmn2b
(Forward primer)
Sequence-based reagentTGCTCTTTCTGTA
CTGTGTGGTG
EurogentecN/AChIP qPCR Lmn2b
(Reverse primer)
Sequence-based reagentGGAGTGATTGA
GGTGGACAGA
EurogentecN/AChIP qPCR Lincmd1
(Forward primer)
Sequence-based reagentCTCTCCCACCTG
TTTGTGTCTT
EurogentecN/AChIP qPCR Lincmd1
(Reverse primer)
Sequence-based reagentAATTACAGCCG
ACGGCCTCC
EurogentecN/AChIP qPCR Myogenin
(Forward primer)
Sequence-based reagentCCAACGCCACA
GAAACCTGA
EurogentecN/AChIP qPCR Myogenin
(Reverse primer)
Sequence-based reagentCAGCTCCTTG
CCCTGTGAAA
EurogentecN/AChIP qPCR Desmin-proximal
(Forward primer)
Sequence-based reagentTGTAGCCCTCC
TGACATCAC
EurogentecN/AChIP qPCR Desmin proximal
(Reverse primer)
Sequence-based reagentCCAAAAGGG
CCGATGAGGAA
EurogentecN/AChIP qPCR Desmin distal
(Forward primer)
Sequence-based reagentTAGAGACAGA
CCAGTGGCGG
EurogentecN/AChIP qPCR Desmin distal
(Reverse primer)
Commercial assay or kitLightCycler 480 SYBR
Green I Master
Roche-Sigma-Aldrich04887352001
Commercial assay or kitiDeal ChIP-seq kitDiagenodeC01010051
Commercial assay or kitRNasy Micro KitQIAGEN74004
Commercial assay or kitTranscriptor First Strand
cDNA Synthesis Kit
Roche-Sigma-Aldrich4379012001
Chemical compound, drugbFGFPeprotech450–3320 ng/ml
Chemical compound, drugbFGFThermo Fisher ScientificPHG026320 ng/ml
Chemical compound, drugBovine serum
albumin (BSA)
Jackson ImmunoResearch100016200.2%
Chemical compound, drugCardiotoxinLatoxanL810210 µM
Chemical compound, drugCardiotoxinSigma-Aldrich217503–1 mg10 µM
Chemical compound, drugChicken embryo extractMP-Biomedical928501450.5%
Chemical compound, drugChicken embryo extractSeralabCE-650-J1%
Chemical compound, drugcollagenBD Biosciences354236culture dish coating
Chemical compound, drugCollagenase type ISigma-AldrichC01300.2%
Chemical compound, drugCollagenase type IWorthington Biochemical Corp9001-12-1
Chemical compound, drugCollagenase ARoche-Sigma-Aldrich110887930010.2% w/v
Chemical compound, drugDAPI (4’,6-diamidino-2-
phenylindole
dihydrochloride)
Thermo Fisher ScientificD1306
Chemical compound, drugDispase IIRoche-Sigma-Aldrich49420780012.4 U/ml
Chemical compound, drugDNaseIRoche-Sigma-Aldrich1128493200110 ng/mL
Chemical compound, drugDulbecco’s Modified
Eagle’s Medium (DMEM)
Thermo Fisher Scientific41966single myofiber culture
Chemical compound, drugDMEM with GlutaMAXThermo Fisher Scientific61965myoblast culture
Chemical compound, drugEdUThermo Fisher ScientificC103402 μM
Chemical compound, drugF-10 Ham's mediaSigma-AldrichN6635N/A
Chemical compound, drugFetal bovine serum (FBS)Thermo Fisher Scientific1027020%
Chemical compound, drugFetal calf serum (FCS)EurobioCVFSVF00-0110% (prol/tion medium),
2% (diff/tion medium)
Chemical compound, drugFluoromount-GSouthern Biotech0100–01
Chemical compound, drugHanks' Balanced
Salt Solution (HBSS)
Thermo Fisher Scientific14025
Chemical compound, drugHepesThermo Fisher Scientific156300.1M
Chemical compound, drugHorse serumThermo Fisher Scientific260500885% (coating), 10% (culture)
Chemical compound, drugL-glutamineThermo Fisher Scientific2503020 mM
Chemical compound, drugmatrigelCorning Life Sciences3542301:20 in DMEM
Chemical compound, drugPenicillin/streptomycinLife Technologies151401X
Chemical compound, drugPyruvateThermo Fisher Scientific1136010 mM
Software, algorithmPhotoshop CS5https://www.adobe.com/products/photoshop.htmlRRID:SCR_014199
Other (anti-biotin beads)anti-biotin beadsMiltenyi Biotec130-090-485;
RRID:AB_244365
MACS
Other (anti-PE beads)anti-PE beadsMiltenyi Biotec130-048-801;
RRID:AB_244373
MACS
Other (chamber slides)chamber slideNalge Nunc International177445myoblast culture
Other (culture plates)petri dishSigma-AldrichZ692301single myofiber culture
Other (LD column)LD columnMiltenyi Biotec130-042-901MACS
Other (MS column)MS columnMiltenyi Biotec130-042-201MACS
Other (grip strength meter)grip strength meterColumbus Instruments1027CSM-D54
Table 1
Sequences of primers used for the ChIP-qPCR.
https://doi.org/10.7554/eLife.33337.016
Regulated gene/regionForward primerReverse primer
Mef2aATCTGAGGTCAGCCATTTGGTGCTAAGGACAGCTGTGACCTG
Lmn2bTTAAAGACATGTGGCAACAGACTACTGCTCTTTCTGTACTGTGTGGTG
Lincmd1GGAGTGATTGAGGTGGACAGACTCTCCCACCTGTTTGTGTCTT
MyogeninAATTACAGCCGACGGCCTCCCCAACGCCACAGAAACCTGA
Desmin (proximal)CAGCTCCTTGCCCTGTGAAATGTAGCCCTCCTGACATCAC
Desmin (distal)CCAAAAGGGCCGATGAGGAATAGAGACAGACCAGTGGCGG

Additional files

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