Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

National Institutes of Health

&#x536B;&#x661F;&#x7EC6;&#x80DE; (SCs) &#x7EF4;&#x6301;&#x808C;&#x8089;&#x751F;&#x957F;&#x548C;&#x589E;&#x5F3A;&#x6210;&#x4EBA;&#x9AA8;&#x9ABC;&#x808C;&#x4E0E;&#x6709;&#x529B;&#x7684;&#x518D;&#x751F;&#x80FD;&#x529B;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x62A5;&#x544A; EZH2 Polycomb &#x9547;&#x538B;&#x590D;&#x6742; 2 (PRC2) &#x7684;&#x9176;&#x4E9A;&#x5355;&#x4F4D;&#x8868;&#x793A;&#x5728;&#x8FD9;&#x4E24;&#x4E2A; Pax7 + Myf5&#x207B; &#x5E72;&#x7EC6;&#x80DE;&#x4E0E; Pax7 + Myf5 + &#x72AF;&#x751F;&#x808C;&#x524D;&#x4F53;&#x548C;&#x9700;&#x8981;&#x7684;&#x6210;&#x4EBA;&#x7684; SC &#x6C60;&#x7684;&#x52A8;&#x6001;&#x5E73;&#x8861;&#x3002;&#x4E0E;&#x6761;&#x4EF6;&#x6D88;&#x878D;&#x672F;&#x5728;&#x516C;&#x52A1;&#x5458;&#x4E8B;&#x52A1;&#x5C40;&#x5C40;&#x957F; Ezh2 &#x8001;&#x9F20;&#x6709;&#x8F83;&#x5C11;&#x7684;&#x808C;&#x8089;&#x4EA7;&#x540E; Pax7 + &#x7EC6;&#x80DE;&#x548C;&#x51CF;&#x5C11;&#x7684;&#x808C;&#x8089;&#x8D28;&#x91CF;&#x548C;&#x672A;&#x80FD;&#x9002;&#x5F53;&#x5730;&#x91CD;&#x65B0;&#x751F;&#x6210;&#x3002;&#x8FD9;&#x4E9B;&#x7F3A;&#x9677;&#x662F;&#x4E0E;&#x53D7;&#x635F;&#x7684; SC &#x6269;&#x6563;&#x548C;&#x7CFB;&#x7EDF;&#x7684;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x5728;&#x975E;&#x808C;&#x7EC6;&#x80DE;&#x7EC6;&#x80DE;&#x8C31;&#x7CFB;&#x76F8;&#x5173;&#x8054;&#x3002;&#x56E0;&#x6B64;&#xFF0C;EZH2 &#x63A7;&#x5236;&#x81EA;&#x6211;&#x66F4;&#x65B0;&#x548C;&#x6269;&#x6563;&#xFF0C;&#x5E76;&#x4FDD;&#x6301;&#x9002;&#x5F53;&#x7684;&#x8F6C;&#x5F55;&#x7A0B;&#x5E8F;&#x5728;&#x516C;&#x52A1;&#x5458;&#x4E8B;&#x52A1;&#x5C40;&#x5C40;&#x957F;&#x3002;

Polycomb EZH2 &#x4FDD;&#x969C;&#x7684;&#x9AA8;&#x9ABC;&#x808C;&#x5E72;&#x7EC6;&#x80DE;&#x8F6C;&#x5F55;&#x6807;&#x8BC6;&#x548C;&#x63A7;&#x5236;&#x81EA;&#x6211;&#x66F4;&#x65B0;&#x3002;

Satellite cells (SCs) sustain muscle growth and empower adult skeletal muscle with vigorous regenerative abilities. Here, we report that EZH2, the enzymatic subunit of the Polycomb-repressive complex 2 (PRC2), is expressed in both Pax7+/Myf5â» stem cells and Pax7+/Myf5+ committed myogenic precursors and is required for homeostasis of the adult SC pool. Mice with conditional ablation of Ezh2 in SCs have fewer muscle postnatal Pax7+ cells and reduced muscle mass and fail to appropriately regenerate. These defects are associated with impaired SC proliferation and derepression of genes expressed in nonmuscle cell lineages. Thus, EZH2 controls self-renewal and proliferation, and maintains an appropriate transcriptional program in SCs.

Polycomb EZH2 controls self-renewal and safeguards the transcriptional identity of skeletal muscle stem cells.

Cellules satellites (SCs) soutenir la croissance musculaire et l&#39;autonomisation des muscle squelettique adulte avec des capacit&eacute;s de r&eacute;g&eacute;n&eacute;ration vigoureuses. Nous rapportons ici que EZH2, la sous-unit&eacute; enzymatique de la Polycomb-r&eacute;pressif complexe 2 (PRC2), est exprim&eacute; dans les deux Pax7 + / Myf5&#x207B; cellules souches et Pax7 + / Myf5 + commis pr&eacute;curseurs myog&egrave;nes et est n&eacute;cessaire pour l&#39;hom&eacute;ostasie de la piscine de SC adulte. Souris avec ablation conditionnelle de Ezh2 dans SCs ont moins de muscle postnatals Pax7 + cellules et r&eacute;duit muscle masse et ne parviennent pas &agrave; se r&eacute;g&eacute;n&eacute;rer de fa&ccedil;on appropri&eacute;e. Ces d&eacute;fauts sont associ&eacute;s &agrave; la prolif&eacute;ration de SC avec facult&eacute;s affaiblies et d&eacute;r&eacute;pression de g&egrave;nes exprim&eacute;s dans des lign&eacute;es de cellules musculaires. Ainsi, EZH2 contr&ocirc;le l&#39;auto-renouvellement et prolif&eacute;ration et maintient un programme appropri&eacute; de transcriptional dans SCs.

Polycomb EZH2 contr&ocirc;le l&#39;auto-renouvellement et pr&eacute;serve l&#39;identit&eacute; de transcriptional des cellules souches musculaires squelettiques.

Cellule satellite (SCs) sostenere la crescita muscolare e potenziare il muscolo scheletrico adulto con vigorosa capacit&agrave; rigenerativa. Qui, segnaliamo che la EZH2, la subunit&agrave; enzimatica del Polycomb-repressivo complesso 2 (PRC2), si esprime in entrambi Pax7 + Myf5&#x207B; cellule staminali e Pax7 + Myf5 + commesso miogena precursori ed &egrave; richiesto per l&#39;omeostasi della piscina adulti SC. Topi con ablazione condizionale di Ezh2 in SCs hanno meno muscolo cellule Pax7 + postnatale e muscolo ridotto di massa e non riescono a rigenerarsi in modo appropriato. Questi difetti sono associati a ridotta proliferazione di SC e derepression di geni espressi in linee cellulari nonmuscle. Cos&igrave;, EZH2 controlli auto-rinnovamento e proliferazione e mantiene un adeguato programma transcriptional in SCs.

Polycomb EZH2 controlli auto-rinnovamento e salvaguarda l&#39;identit&agrave; trascrizionale di cellule staminali del muscolo scheletrico.

&#x885B;&#x661F;&#x7D30;&#x80DE; &#xFF08;SCs&#xFF09; &#x306F;&#x3001;&#x7B4B;&#x8089;&#x306E;&#x6210;&#x9577;&#x3092;&#x7DAD;&#x6301;&#x3057;&#x3001;&#x7A4D;&#x6975;&#x7684;&#x306A;&#x518D;&#x751F;&#x80FD;&#x529B;&#x3092;&#x6301;&#x3064;&#x5927;&#x4EBA;&#x306E;&#x9AA8;&#x683C;&#x7B4B;&#x306B;&#x529B;&#x3092;&#x4E0E;&#x3048;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x6211;&#x3005; &#x306E;&#x6291;&#x5727;&#x7684;&#x3001;&#x30DD;&#x30EA;&#x30B3;&#x30FC;&#x30E0;-&#x306A;&#x8907;&#x96D1;&#x306A; 2 &#xFF08;PRC2 &#x306E;&#xFF09;&#x3001;&#x9175;&#x7D20;&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8; ezh2 &#x306F;&#x4E21;&#x65B9; Pax7 &#x3067;&#x8868;&#x3055;&#x308C;&#x308B;&#x3053;&#x3068;&#x5831;&#x544A; + Myf5&#x207B; &#x5E79;&#x7D30;&#x80DE;&#x30FB; Pax7 + Myf5 + &#x30B3;&#x30DF;&#x30C3;&#x30C8;&#x7B4B;&#x524D;&#x99C6;&#x4F53;&#x3068;&#x5927;&#x4EBA;&#x7528;&#x306E; SC &#x30D7;&#x30FC;&#x30EB;&#x306E;&#x6052;&#x5E38;&#x6027;&#x306B;&#x5FC5;&#x8981;&#x3067;&#x3059;&#x3002;&#x6761;&#x4EF6;&#x4ED8;&#x304D;&#x306E;&#x30A2;&#x30D6;&#x30EC;&#x30FC;&#x30B7;&#x30E7;&#x30F3; ezh2 &#x306F; SCs &#x3067;&#x306E;&#x30DE;&#x30A6;&#x30B9;&#x304C;&#x3042;&#x308B;&#x5C11;&#x306A;&#x3044;&#x7B4B;&#x8089;&#x7523;&#x5F8C; Pax7 + &#x7D30;&#x80DE;&#x3068;&#x7B4B;&#x8CEA;&#x91CF;&#x3057;&#x3001;&#x9069;&#x5207;&#x306B;&#x518D;&#x751F;&#x6210;&#x306B;&#x5931;&#x6557;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x6B20;&#x9665;&#x969C;&#x5BB3;&#x306E; SC &#x5897;&#x6B96;&#x3068;&#x975E;&#x7D30;&#x80DE;&#x7CFB;&#x8B5C;&#x306B;&#x767A;&#x73FE;&#x3059;&#x308B;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x6291;&#x5236;&#x304C;&#x89E3;&#x9664;&#x306B;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;ezh2 &#x306F;&#x81EA;&#x5DF1;&#x8907;&#x88FD;&#x3068;&#x5897;&#x6B96;&#x3092;&#x5236;&#x5FA1;&#x3057;&#x3001;&#x9069;&#x5207;&#x306A;&#x8EE2;&#x5199;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30E0; SCs &#x3067;&#x7DAD;&#x6301;&#x3057;&#x307E;&#x3059;&#x3002;

&#x30DD;&#x30EA;&#x30B3;&#x30FC;&#x30E0; ezh2 &#x306F;&#x81EA;&#x5DF1;&#x8907;&#x88FD;&#x3092;&#x5236;&#x5FA1;&#x3057;&#x3001;&#x9AA8;&#x683C;&#x7B4B;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x8EE2;&#x5199;&#x306E; id &#x3092;&#x4FDD;&#x8B77;&#x3057;&#x307E;&#x3059;&#x3002;

&#xC704;&#xC131; &#xC138;&#xD3EC; (SCs) &#xADFC;&#xC721; &#xC131;&#xC7A5;&#xC744; &#xC720;&#xC9C0; &#xD558; &#xACE0; &#xD65C;&#xBC1C; &#xD55C; &#xC7AC;&#xC0DD; &#xB2A5;&#xB825;&#xC744; &#xAC00;&#xC9C4; &#xC131;&#xC778; &#xACE8;&#xACA9; &#xADFC;&#xC721;&#xC758; &#xB2A5;&#xB825;&#xC744; &#xC8FC;&#xC18C; &#xC11C;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC;&#xB294; &#xBAA8;&#xB450; Pax7 ezh2&#xB294; Polycomb-&#xC5B5;&#xC555; &#xBCF5;&#xC7A1; &#xD55C; 2 (PRC2)&#xC758; &#xD6A8;&#xC18C; &#xC18C; &#xB2E8;&#xC704; &#xD45C;&#xD604; &#xB41C; &#xBCF4;&#xACE0;&#xC11C; + Myf5&#x207B; &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC640; Pax7 + Myf5 + &#xC870;&#xC9C1;&#xC801; &#xC120;&#xAD6C;&#xC790; &#xD22C;&#xC785; &#xBC0F; &#xC131;&#xC778; &#xC0AC;&#xC6B0;&#xC2A4; &#xCE90;&#xB864;&#xB77C;&#xC774;&#xB098; &#xC218;&#xC601;&#xC7A5;&#xC758; &#xD56D;&#xC0C1; &#xC131;&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC694; &#xD569;&#xB2C8;&#xB2E4;. Scs&#xC5D0;&#xC11C; ezh2&#xC758; &#xC870;&#xAC74;&#xBD80; &#xC808;&#xC81C;&#xC640; &#xC950;&#xAC00; &#xC801;&#xC740; &#xADFC;&#xC721; &#xCD9C;&#xC0DD; &#xD6C4; Pax7 + &#xC140; &#xBC0F; &#xAC10;&#xC18C; &#xADFC;&#xC721; &#xC9C8;&#xB7C9; &#xBC0F; &#xC801;&#xC808; &#xD558; &#xAC8C; &#xB2E4;&#xC2DC; &#xC0DD;&#xC131; &#xD558;&#xB824;&#xBA74; &#xC2E4;&#xD328; &#xD569;&#xB2C8;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xACB0;&#xD568; &#xC7A5;&#xC560;&#xC778;&#xB41C; SC &#xD655;&#xC0B0; &#xBC0F; nonmuscle &#xC138;&#xD3EC; &#xACC4;&#xBCF4;&#xC5D0;&#xC11C; &#xD45C;&#xD604; &#xD558;&#xB294; &#xC720;&#xC804;&#xC790;&#xC758; derepression&#xC640; &#xC5F0;&#xACB0; &#xB429;&#xB2C8;&#xB2E4;. &#xB530;&#xB77C;&#xC11C;, EZH2 &#xC790;&#xAE30; &#xAC31;&#xC2E0; &#xBC0F; &#xD655;&#xC0B0;, &#xC81C;&#xC5B4; &#xD558; &#xACE0; Scs&#xC5D0; &#xC801;&#xC808; &#xD55C; transcriptional &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC744; &#xC720;&#xC9C0; &#xD569;&#xB2C8;&#xB2E4;.

Polycomb EZH2 &#xC790;&#xAE30; &#xAC31;&#xC2E0;&#xC744; &#xC81C;&#xC5B4; &#xD558; &#xACE0; &#xACE8;&#xACA9; &#xADFC;&#xC721; &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC758; transcriptional &#xC2E0;&#xC6D0; &#xBCF4;&#xD638;.

Las c&eacute;lulas sat&eacute;lite (SCs) sostener crecimiento muscular y la autonom&iacute;a de m&uacute;sculo esquel&eacute;tico adulto con vigorosa capacidad regenerativa. Aqu&iacute;, Divulgamos que EZH2, la subunidad enzim&aacute;tica de la Polycomb-represivas complejo 2 (PRC2), se expresa en ambos Pax7 + / Myf5&#x207B; c&eacute;lulas madre y Pax7 + / Myf5 + cometidos mi&oacute;genas precursores y es necesaria para la homeostasis de la piscina para adultos de SC. Ratones con ablaci&oacute;n condicional de Ezh2 en SCs tienen menos m&uacute;sculo postnatales Pax7 + c&eacute;lulas y muscular reducida masa y no regenerar adecuadamente. Estos defectos se asocian con deterioro proliferaci&oacute;n de SC y derepression de genes expresados en linajes celulares de valvas. As&iacute;, EZH2 controla auto renovaci&oacute;n y proliferaci&oacute;n y mantiene un adecuado programa transcripcional en SCs.

Polycomb EZH2 controla la auto-renovaci&oacute;n y protege la identidad transcripcional de c&eacute;lulas de m&uacute;sculo esquel&eacute;tico.

Transcription factors and DNA regulatory binding motifs are fundamental components of the gene regulatory network. Here, by using genome-wide binding profiling, we show extensive occupancy of transcription factors of myogenesis (MyoD and Myogenin) at extragenic enhancer regions coinciding with RNA synthesis (i.e., eRNA). In particular, multiple regions were transcribed to eRNA within the regulatory region of MYOD1, including previously characterized distal regulatory regions (DRR) and core enhancer (CE). While (CE)RNA enhanced RNA polymerase II (Pol II) occupancy and transcription at MYOD1, (DRR)RNA acted to activate the downstream myogenic genes. The deployment of transcriptional machinery to appropriate loci is contingent on chromatin accessibility, a rate-limiting step preceding Pol II assembly. By nuclease sensitivity assay, we found that eRNAs regulate genomic access of the transcriptional complex to defined regulatory regions. In conclusion, our data suggest that eRNAs contribute to establishing a cell-type-specific transcriptional circuitry by directing chromatin-remodeling events. 

eRNAs promote transcription by establishing chromatin accessibility at defined genomic loci.

Stem cells undergo a shift in metabolic substrate utilization during specification and/or differentiation, a process that has been termed metabolic reprogramming. Here, we report that during the transition from quiescence to proliferation, skeletal muscle stem cells experience a metabolic switch from fatty acid oxidation to glycolysis. This reprogramming of cellular metabolism decreases intracellular NAD(+) levels and the activity of the histone deacetylase SIRT1, leading to elevated H4K16 acetylation and activation of muscle gene transcription. Selective genetic ablation of the SIRT1 deacetylase domain in skeletal muscle results in increased H4K16 acetylation and deregulated activation of the myogenic program in SCs. Moreover, mice with muscle-specific inactivation of the SIRT1 deacetylase domain display reduced myofiber size, impaired muscle regeneration, and derepression of muscle developmental genes. Overall, these findings reveal how metabolic cues can be mechanistically translated into epigenetic modifications that regulate skeletal muscle stem cell biology. 

The NAD(+)-dependent SIRT1 deacetylase translates a metabolic switch into regulatory epigenetics in skeletal muscle stem cells.

Histone variants complement and integrate histone post-translational modifications in regulating transcription. The histone variant macroH2A1 (mH2A1) is almost three times the size of its canonical H2A counterpart, due to the presence of an ∼25 kDa evolutionarily conserved non-histone macro domain. Strikingly, mH2A1 can mediate both gene repression and activation. However, the molecular determinants conferring these alternative functions remain elusive. Here, we report that mH2A1.2 is required for the activation of the myogenic gene regulatory network and muscle cell differentiation. H3K27 acetylation at prospective enhancers is exquisitely sensitive to mH2A1.2, indicating a role of mH2A1.2 in imparting enhancer activation. Both H3K27 acetylation and recruitment of the transcription factor Pbx1 at prospective enhancers are regulated by mH2A1.2. Overall, our findings indicate a role of mH2A1.2 in marking regulatory regions for activation.

The Histone Variant MacroH2A1.2 Is Necessary for the Activation of Muscle Enhancers and Recruitment of the Transcription Factor Pbx1.

The nuclear envelope protein lamin A is encoded by thelamin A/C(LMNA) gene, which can contain missense mutations that cause Emery-Dreifuss muscular dystrophy (EDMD) (p.R453W). We fused mutated forms of the lamin A protein to bacterial DNA adenine methyltransferase (Dam) to define euchromatic-heterochromatin (epigenomic) transitions at the nuclear envelope during myogenesis (using DamID-seq). Lamin A missense mutations disrupted appropriate formation of lamin A-associated heterochromatin domains in an allele-specific manner-findings that were confirmed by chromatin immunoprecipitation-DNA sequencing (ChIP-seq) in murine H2K cells and DNA methylation studies in fibroblasts from muscular dystrophy patient who carried a distinctLMNAmutation (p.H222P). Observed perturbations of the epigenomic transitions included exit from pluripotency and cell cycle programs [euchromatin (open, transcribed) to heterochromatin (closed, silent)], as well as induction of myogenic loci (heterochromatin to euchromatin). In muscle biopsies from patients with either a gain- or change-of-functionLMNAgene mutation or a loss-of-function mutation in theemeringene, both of which cause EDMD, we observed inappropriate loss of heterochromatin formation at theSox2pluripotency locus, which was associated with persistent mRNA expression ofSox2 Overexpression ofSox2inhibited myogenic differentiation in human immortalized myoblasts. Our findings suggest that nuclear envelopathies are disorders of developmental epigenetic programming that result from altered formation of lamina-associated domains.

Stefania Dell'Orso

Office of Science and Technology,
National Institute of Arthritis,
Musculoskeletal and Skin Diseases (NIAMS),
Office of Science and Technology, National Institute of Arthritis, Musculoskeletal and Skin Diseases (NIAMS)

National Institutes of Health

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Stefania Dell'Orso

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Office of Science and Technology,National Institute of Arthritis,Musculoskeletal and Skin Diseases (NIAMS),Office of Science and Technology, National Institute of Arthritis, Musculoskeletal and Skin Diseases (NIAMS)

National Institutes of Health

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Office of Science and Technology,
National Institute of Arthritis,
Musculoskeletal and Skin Diseases (NIAMS),
Office of Science and Technology, National Institute of Arthritis, Musculoskeletal and Skin Diseases (NIAMS)