identification-skeletal-muscle-satellite-cells-immunofluorescence

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

We are interested in investigating the function of Hoxc8 in skeletogenesis during mouse development. Previous studies have shown that deregulation of Hoxc8 expression in the mouse leads to several skeletal defects, such as homeotic transformation in the thoracic vertebrae, abnormal development of the rib cage, and overproliferation of chondrocytes in the hypertrophic area. By deleting a crucial enhancer of Hoxc8 in vivo, we found that precise temporal expression of Hoxc8 is important for determining the correct identity of the vertebral column in early embryos. We also identified downstream targets of Hoxc8 relevant to osteoblast differentiation at later developmental stages.

Multiple roles of hoxc8 in skeletal development.

Polycomb group (PcG) proteins exert essential functions in the most disparate biological processes. The contribution of PcG proteins to cell commitment and differentiation relates to their ability to repress transcription of developmental regulators in embryonic stem (ES) cells and in committed cell lineages, including skeletal muscle cells (SMC). PcG proteins are preferentially removed from transcribed regions, but the underlying mechanisms remain unclear. Here, PcG proteins are found to occupy and repress transcription from an intronic region containing the microRNA miR-214 in undifferentiated SMC. Differentiation coincides with PcG disengagement, recruitment of the developmental regulators MyoD and myogenin, and activation of miR-214 transcription. Once transcribed, miR-214 negatively feeds back on PcG by targeting the Ezh2 3'UTR, the catalytic subunit of the PRC2 complex. miR-214-mediated Ezh2 protein reduction accelerates SMC differentiation and promotes unscheduled transcription of developmental regulators in ES cells. Thus, miR-214 and Ezh2 establish a regulatory loop controlling PcG-dependent gene expression during differentiation.

Mir-214-dependent regulation of the polycomb protein Ezh2 in skeletal muscle and embryonic stem cells.

MicroRNA-214 and polycomb group proteins: a regulatory circuit controlling differentiation and cell fate decisions.

Although β(2)-adrenergic receptors (β(2)AR) are expressed on most cell types, mechanisms that establish expression levels and regulate expression by chronic agonist remain unclear. The 3' UTR of ADRB2 has a conserved 8-nucleotide seed region that we hypothesized is targeted by the let-7 family of miRNAs leading to translational repression. In luciferase assays with transfected cells, luc-β(2)WT3'UTR had decreased expression when cotransfected with let-7f, but a mutated luc-β(2)3'UTR lacking the seed was unaffected by let-7f; a mutated let-7f also had no effect on luc-β(2)WT3'UTR expression. ADRB2 mRNA was in greater abundance in immunoprecipitates of Ago2, a core component of the miRNA-induced silencing complex, when cells were transfected with let-7f, but not with a mutated let-7f, indicating a direct interaction with the silencing mechanism. H292 cells transfected with let-7f caused ∼60% decrease in native β(2)AR expression, but transfection with let-7f-specific locked nucleic acid anti-miRNA increased β(2)AR expression by ∼twofold. We considered that an increase in let-7f leading to greater repression of translation contributes to agonist-promoted down-regulation. Paradoxically, in cells and in lungs from mice treated in vivo, an ∼50% decrease in let-7f occurs during long-term agonist exposure, indicating a counterregulatory event. Consistent with this notion, let-7f locked nucleic acid transfection caused depressed agonist-promoted down-regulation. Thus, let-7f miRNA regulates baseline β(2)AR expression and decreases in let-7f evoked by agonist attenuate down-regulation. This positive feedback loop has not previously been described for a G protein-coupled receptor and its miRNA. Methods to decrease let-7f expression in targeted cells may increase therapeutic responses to β-agonist by increasing β(2)AR expression or minimizing tachyphylaxis.

MicroRNA let-7 establishes expression of beta2-adrenergic receptors and dynamically down-regulates agonist-promoted down-regulation.

&#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.

Satellite cells (SCs) are the main source of adult skeletal muscle stem cells responsible for muscle growth and regeneration. By interpreting extracellular cues, developmental regulators control quiescence, proliferation, and differentiation of SCs by influencing coordinate gene expression. The scope of this review is limited to the description and discussion of protein complexes that introduce and decode heritable histone and chromatin modifications and how these modifications are relevant for SC biology.

Sculpting chromatin beyond the double helix: epigenetic control of skeletal myogenesis.

MicroRNAs (miRNAs) are small non-coding RNAs, which regulate gene expression by inhibiting translation or promoting degradation of specific target messenger RNAs (mRNAs). Alteration of the levels of a number of miRNAs is common in solid and hematological tumors. We have shown previously that miR-214 regulates Ezh2 in skeletal muscle and embryonic stem cells. The current study was aimed at examining the role of miR-214 in breast cancer where miR-214 levels are reduced but whether this phenomenon bears a functional relevance is unknown. MiR-214 expression was inversely correlated with Ezh2 mRNA and protein levels in breast cancer cell lines and at least one copy of the miR-214 alleles was found to be deleted in 24% (6/25) of primary breast tumors. Experimental increase of miR-214 in breast cancer cell lines correlated with reduction of Ezh2 protein levels, a known marker of invasion and aggressive breast cancer behavior. Supporting a direct targeting mechanism, miR-214 decreased luciferase activity from a construct containing the Ezh2 3' untranslated region. Expression of miR-214 specifically reduced cell proliferation of breast cancer cells and inhibited the invasive potential of a highly metastatic breast cancer cell line. These findings indicate that reduced miR-214 levels may contribute to breast tumorigenesis by allowing abnormally elevated Ezh2 accumulation and subsequent unchecked cell proliferation and invasion.

Decreased microRNA-214 levels in breast cancer cells coincides with increased cell proliferation, invasion and accumulation of the Polycomb Ezh2 methyltransferase.

Although the genetic interactions between signaling pathways and transcription factors have been largely decoded, much remains to be learned about the epigenetic regulation of cerebellar development. Here, we report that cerebellar deletion of Ezh2, the methyltransferase subunit of the PRC2 complex, results in reduced H3K27me3 and profound transcriptional dysregulation, including that of a set of transcription factors directly involved in cerebellar neuronal cell-type specification and differentiation. Such transcriptional changes lead to increased GABAergic interneurons and decreased Purkinje cells. Transcriptional changes also inhibit the proliferation of granule precursor cells derived from the rhombic lip. The loss of both cell types ultimately results in cerebellar hypoplasia. These findings indicate Ezh2/PRC2 plays crucial roles in regulating neurogenesis from both cerebellar germinal zones.

Polycomb Ezh2 controls the fate of GABAergic neurons in the embryonic cerebellum.

Signal-directed chromatin recruitment of mammalian Polycomb complexes is a fundamental component of epigenetic regulation. In this issue, Yi et al. (2016) reveal how mTORC1 activation deploys the ribosomal serine/threonine kinase S6K1 and Polycomb proteins at genomic regulatory regions to repress expression of anti-adipogenic developmental regulators.

S6K1ing to ResTOR Adipogenesis with Polycomb.

The polycomb repressive complex 2 (PRC2) methylates lysine 27 of histone H3 (H3K27) through its catalytic subunit Ezh2. PRC2-mediated di- and tri-methylation (H3K27me2/H3K27me3) have been interchangeably associated with gene repression. However, it remains unclear whether these two degrees of H3K27 methylation have different functions. In this study, we have generated isogenic mouse embryonic stem cells (ESCs) with a modified H3K27me2/H3K27me3 ratio. Our findings document dynamic developmental control in the genomic distribution of H3K27me2 and H3K27me3 at regulatory regions in ESCs. They also reveal that modifying the ratio of H3K27me2 and H3K27me3 is sufficient for the acquisition and repression of defined cell lineage transcriptional programs and phenotypes and influences induction of the ESC ground state.

Aster H. Juan

Laboratory of Muscle Stem Cells and Gene Regulation

National Institutes of Health

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