JAG2-related muscular dystrophy and Notch signaling dysfunction in muscle stem cells

JAG2-related muscular dystrophy and Notch signaling dysfunction in muscle stem cells | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results JAG2 -related muscular dystrophy and Notch signaling dysfunction in muscle stem cells Nam Chul Kim , Minoru Tanaka , Isabelle Draper , Hannah R. Littel , Mekala Gunasekaran , Johnnie Turner , Natalya M. Wells , Qasim Mujteba , Yoko Asakura , View ORCID Profile Peter B. Kang , View ORCID Profile Atsushi Asakura doi: https://doi.org/10.1101/2025.07.23.665646 Nam Chul Kim 1 Department of Pharmacy Practice and Pharmaceutical Sciences, University of Minnesota College of Pharmacy , Duluth, Minnesota Find this author on Google Scholar Find this author on PubMed Search for this author on this site Minoru Tanaka 2 Stem Cell Institute, University of Minnesota Medical School , Minneapolis, Minnesota 3 Greg Marzolf Jr. Muscular Dystrophy Center and Department of Neurology, University of Minnesota Medical School , Minneapolis, Minnesota Find this author on Google Scholar Find this author on PubMed Search for this author on this site Isabelle Draper 4 Molecular Cardiology Research Institute, Tufts Medical Center , Boston, Massachusetts Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hannah R. Littel 3 Greg Marzolf Jr. Muscular Dystrophy Center and Department of Neurology, University of Minnesota Medical School , Minneapolis, Minnesota Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mekala Gunasekaran 3 Greg Marzolf Jr. Muscular Dystrophy Center and Department of Neurology, University of Minnesota Medical School , Minneapolis, Minnesota Find this author on Google Scholar Find this author on PubMed Search for this author on this site Johnnie Turner 3 Greg Marzolf Jr. Muscular Dystrophy Center and Department of Neurology, University of Minnesota Medical School , Minneapolis, Minnesota Find this author on Google Scholar Find this author on PubMed Search for this author on this site Natalya M. Wells 3 Greg Marzolf Jr. Muscular Dystrophy Center and Department of Neurology, University of Minnesota Medical School , Minneapolis, Minnesota Find this author on Google Scholar Find this author on PubMed Search for this author on this site Qasim Mujteba 1 Department of Pharmacy Practice and Pharmaceutical Sciences, University of Minnesota College of Pharmacy , Duluth, Minnesota Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yoko Asakura 2 Stem Cell Institute, University of Minnesota Medical School , Minneapolis, Minnesota 3 Greg Marzolf Jr. Muscular Dystrophy Center and Department of Neurology, University of Minnesota Medical School , Minneapolis, Minnesota Find this author on Google Scholar Find this author on PubMed Search for this author on this site Peter B. Kang 3 Greg Marzolf Jr. Muscular Dystrophy Center and Department of Neurology, University of Minnesota Medical School , Minneapolis, Minnesota 5 Institute for Translational Neuroscience, University of Minnesota Medical School , Minneapolis, Minnesota Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Peter B. Kang For correspondence: asakura{at}umn.edu pkang{at}umn.edu Atsushi Asakura 2 Stem Cell Institute, University of Minnesota Medical School , Minneapolis, Minnesota 3 Greg Marzolf Jr. Muscular Dystrophy Center and Department of Neurology, University of Minnesota Medical School , Minneapolis, Minnesota Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Atsushi Asakura For correspondence: asakura{at}umn.edu pkang{at}umn.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT We previously identified a muscular dystrophy caused by biallelic variants in JAG2 , whose protein product Jagged2 is a canonical Notch ligand. However, the disease mechanism remains unclear, particularly with respect to muscle stem cell (MuSC) function and muscle regeneration. We examined the consequences of JAG2 deficiency and modeled pathogenic JAG2 variants in vitro and in vivo , the latter in mouse and fly models and with particular attention to the MuSC-muscle endothelial cell (MuEC) niche. We found that both Jag2 deficiency and overexpression of pathogenic JAG2 variants impaired Notch signaling and myogenic self-renewal and differentiation. Hypomorphic Jag2 mutant ( Jag2 sm ) mice display depleted MuSCs, corresponding with impaired muscle regeneration in those mice. Co-culture experiments and the examination of cell-type-specific Jag2 conditional knockout mice demonstrated that MuEC-specific Jag2 knockout resulted in reduced MuSC self-renewal, while MuSC-specific Jag2 knockout resulted in reduced myogenic differentiation. Human reference JAG2 , but not human pathogenic variants of JAG2 , rescued the deficiency of Serrate (Ser) , the Drosophila ortholog of JAG2 . Therefore, pathogenic variants in JAG2 impair muscle development and regeneration through disrupted cell-autonomous cis- inhibition and non-autonomous trans- activation involving Notch signaling dysfunction. Our findings indicate that optimizing JAG2-mediated Notch signaling is a potential therapeutic approach for JAG2 -related muscular dystrophy. INTRODUCTION Muscle stem cells (MuSCs), also known as satellite cells, are normally quiescent cells located underneath the basal lamina of muscle fibers. In response to injury, MuSCs activate, proliferate, differentiate, and either form new myofibers or fuse with existing myofibers to repair the damaged muscle. A small proportion of activated MuSCs self-renew or escape activation in order to replenish the MuSC pool( 1 – 4 ). Defects in self-renewal lead to fewer MuSCs and diminished muscle regenerative capacity, particularly in aged and diseased muscle( 1 , 5 – 7 ). Prior studies have elucidated the molecular mechanisms regulating MuSCs through the Notch, Wnt, FGF, extracellular matrix signaling pathways, as well as juxtacrine interactions( 8 – 10 ). We and others have studied the role of muscle endothelial cells (MuECs) in regulating MuSCs( 11 – 14 ). We have shown that increasing vascular density can augment MuSC numbers( 15 – 17 ), which is mediated through the activation of Notch signaling in MuSCs by Dll4, an MuEC-derived Notch ligand( 14 ). In mammalian cells, Notch signaling involves the transmembrane Notch ligands Jag1, Jag2, Dll1, Dll3, and Dll4, which bind to Notch receptors 1-4( 18 , 19 ), and has been implicated in the homeostasis of stem cells, including MuSCs. Notch signaling regulates the maintenance of MuSC quiescence( 9 , 20 – 22 ) as well as the formation of the MuSC niche ( 23 ). However, the precise mechanisms of Notch ligand contributions to niche formation and Notch activation in MuSCs remain unclear. Endothelial cells express Notch ligands, which in turn regulate stem cells( 24 ), including hematopoietic stem cells (HSC)( 25 – 28 ), neural stem cells( 29 ), and MuSCs( 14 ). Disruption of Notch signaling is associated with several skeletal muscle diseases, particularly those associated with JAG2 ( 30 ), MEGF10 ( 31 , 32 ), POGLUT1 ( 33 ), and NOTCH2NLC ( 34 ). We discovered that biallelic pathogenic variants in JAG2 are associated with congenital muscular dystrophy (CMD) and limb-girdle muscular dystrophy (LGMD)( 30 ). Human JAG2 is a 1,238 amino acid membrane protein that interacts with Notch receptors via extracellular epidermal growth factor-like (EGF) domains( 35 ), triggering cell-cell interaction-mediated trans- activation of Notch signaling( 36 ). JAG2 binding to a Notch receptor leads to double cleavage, followed by migration of the Notch intracellular domain (NICD) to the nucleus, where it regulates transcription( 37 , 38 ). The orthologous Serrate gene in Drosophila was identified in 1990( 39 ), followed by Jag2 in rats( 40 ), Jag2 in mice( 41 ), and JAG2 in humans( 41 – 43 ). JAG2 is expressed in mammalian skeletal muscle( 44 ), brain( 45 , 46 ), gastrointestinal tract( 47 ), including the enteric nervous system( 48 ), immune system( 49 , 50 ), ovarian follicles( 51 , 52 ), and endothelial cells( 14 , 53 , 54 ). JAG2 is expressed in MuECs and MuSCs( 14 ). Notch ligands suppress or activate Notch signaling cell-autonomously, through cis- inhibition( 55 – 58 ) or cis- activation( 59 ), or via trans- activation of Notch receptors on neighboring cells. The exact ligand-receptor mechanisms that regulate Notch signaling in MuSCs remain unclear. For the current study, we examined the cis- and trans- regulatory activities of Jag2 for MuSC function and skeletal muscle regeneration in Jag2 hypomorphic ( Jag2 sm ) mice, as well as in conditional MuEC-specific and MuSC-specific Jag2 knockout mice. We utilized MuEC-MuSC coculture systems to examine Jagged2-mediated trans- activities of Notch for MuSCs. Lastly, we introduced reference and variant forms of human JAG2 into MuSC cultures and Drosophila . RESULTS Jag2 expression patterns in MuSCs and MuECs Jag2-Notch signaling is important for cell communication in skeletal muscle( 60 – 62 ). Previously, we performed a directional interactome analysis with MuECs as the sending cells and MuSCs as the receiving cells. Gene ontology (GO)-term-mediated interactome analysis identified Notch-signaling-mediated interactions between MuECs and MuSCs, including MuEC-derived Jag2 and Dll4 interacting with MuSC-derived Notch2 and Notch3( 14 ). We verified expression of Notch signaling pathway genes via RNA-seq ( Figure 1A ). Freshly isolated MuECs express the Notch ligands Dll1 , Dll4 , Jag1 , and Jag2 . Freshly isolated MuSCs express the Notch receptors Notch1 , Notch2 , and Notch3 , as well as the Notch ligands Dll1 and Jag1 , with lower expression levels of Dll4 and Jag2 . High expression of the Notch downstream genes Hes1 , Hey1 , and Heyl indicates that Notch signaling maintains MuSCs. We confirmed Jag2 expression in MuECs and MuSCs using a Jag2 LoxP/LoxP mouse line with a LacZ/Neo cassette that enabled us to detect Jag2 -expressing cells in muscle. X-gal staining of whole tibialis anterior (TA) muscle and purified MuSCs from LacZ/Neo - Jag2 LoxP/LoxP mice revealed high levels of β-galactosidase activity in CD31(+) capillaries, but low levels in Pax7(+) and MyoD(+) MuSCs ( Figure 1B ). qRT-PCR showed that Jag2 expression is low in quiescent MuSCs, is up-regulated during activation, then down-regulated during myogenic differentiation ( Figure 1C ). Jag2 is detected in the membranes of cultured MuECs and MuSCs ( Figure 1D ). We concluded that Jag2 is expressed in MuECs and variably expressed in MuSCs, depending on their stage in myogenesis. Download figure Open in new tab Figure 1. Jag2 is expressed in MuSCs and MuECs. (A) Heatmap for gene expression by RNA-seq reveals active Notch signaling in freshly isolated MuECs and MuSCs, but not in myofibers ( 14 ). (B) Upper panel: Schematic genomic structure of Jag2 LoxP/LoxP locus for conditional Jag2 mutant ( LacZ/Neo-Jag2 LoxP/LoxP ) mice before Flippase (Flp)-mediated recombination. Middle panels: On X-gal staining, capillaries (white arrow) in TA muscle from LacZ/Neo-Jag2 LoxP/LoxP mice are positive for LacZ(+) and CD31. Lower panels: MuSCs from LacZ/Neo-Jag2LoxP/LoxP mice are positive for LacZ(+) and Pax7 or MyoD. (C) qRT-PCR shows upregulation and downregulation of Dll1 and Jag2 following MuSC activation and differentiation, respectively. P0, P3, P5, Diff 1, and Diff 3 denote freshly isolated MuSCs, passage day 3, passage day 5, differentiation day 1, and differentiation day 5. (D) Anti-Jag2 antibody staining shows Jag2 expression at the membrane of MuECs and MuSCs. DAPI stained all nuclei (blue). Scale Downregulation of Notch signaling and myogenic differentiation genes in Jag2 deficiency To understand the influence of Jag2 on Notch signaling during myogenesis, we measured the gene expression levels of 93 Notch- and myogenesis-related genes (Table S1) via qPCR in C2C12 myoblasts treated with Jag2 shRNA compared to scrambled shRNA controls and cultured in differentiation media for 4 days. In Jag2 shRNA treated cells, 23 genes were significantly downregulated, notably Jag1 , Jag2 , Megf10 , Notch1 , Notch3 , and MyoG (Figure S1A, Table S1). No genes were significantly upregulated. After 4 days of differentiation, Jag2 and MyoG expression levels were lower in Jag2 shRNA-treated cells (Figure S1B, C), with a lower myotube fusion index (Figure S1D). Phase contrast analysis showed reduced multinucleated myotube formation in Jag2 shRNA-treated cells (Figure S1E). These data suggest that Jag2 regulates myogenic differentiation by activating Notch signaling. Reduced MuSCs in homozygous Jag2 sm mice Jag2 sm mice harbor a naturally occurring p.Gly267Ser variant in the first EGF-repeat in the extracellular domain, which is important for Notch signaling( 63 ). Although the Jag2 sm mouse displays syndactyly and cleft palate( 64 ), a skeletal muscle phenotype has not been previously described. At 4 days after birth, the TA muscle of neonatal Jag2 sm mice had small myofiber diameters ( Figure 2A, B ). Pax7(+) MuSCs were reduced in Jag2 sm mice ( Figure 2A, C ). Adult Jag2 sm mice showed reduced body mass and muscle weight (Figure S2). The muscle defects in neonatal Jag2 sm mice persisted in adult TA muscle with reduced Feret’s fiber diameters and increased fibrosis ( Figure 2A, B, D ). Jag2 sm mice showed reduced forelimb muscle grip strength ( Figure 2E ), running durations and distances ( Figure 2F, G ), and rotarod running time ( Figure 2H ). Jag2 sm mice exhibit impaired MuSC development, leading to impaired skeletal muscle development and function. Download figure Open in new tab Figure 2. Muscle phenotypes in Jag2 sm mice. (A) Muscle from 4 day-old and 3-month-old Jag2 sm homozygous mice showed (B) reduced fiber diameters, (C) Pax7(+) MuSCs (arrows), and (D) increased Sirius red (+) fibrosis vs. WT mice. (E) Grip strength is reduced in Jag2 sm homozygous vs. WT mice. (F, G) Treadmill running time and distance are reduced in Jag2 sm homozygous vs. WT mice. (H) Motor coordination or balance on the rotarod was impaired in Jag2 sm homozygous vs. WT mice. DAPI stained all nuclei (blue). Scale bars, 100 μ m. An unpaired t-test showed *, p<0.05; **, p <0.01. ***, p <0.001. Error bars show SEM. We crossed homozygous Jag2 sm mice with Pax7 +/CreERT2 :ROSO26 +/Loxp-stop-Loxp-tdTomato ( Pax7 CreERT2 :R26R tdT or Pax7 tdT ) mice to generate Jag2 sm Pax7 tdT mice that mark MuSCs( 65 ). We confirmed that the tdTomato was specifically expressed in the cells of interest after tamoxifen (TMX) injection ( Figure 3A ). Reduced number of MuSCs were detected in TA muscle cross-sections from adult Jag2 sm mice ( Figure 3B ). Single myofiber analysis isolated from adult mice showed reduced Pax7(+) MuSC counts in Jag2 sm extensor digitorum longus (EDL) ( Figure 3C, D ). Download figure Open in new tab Figure 3. MuSCs are depleted in Jag2 sm homozygous mice. (A) WT: Pax7 tdT and Jag2 sm :Pax7 tdT mice were injected with TMX prior to sacrifice. (B, C, D) TA muscle sections and isolated single muscle fibers demonstrated reduced Pax7(+) MuSCs (arrows) in Jag2 sm homozygous vs. WT mice. Scale bars; 20 µ m (top panels), 50 µ m (fibers). (E, F) Freshly isolated MuSCs from homozygous Jag2 sm mice show reduced colony sizes and EdU(+) proliferating cells. (G) MuSCs isolated from homozygous Jag2 sm mice show (H) reduced EdU(+) proliferating cells (green), (I) MyHC(+) myogenic differentiation (green) and fusion in days 1 and 3 differentiation conditions, while (J) MyoD(+) cells (red) are not altered, compared with WT cells. DAPI stained all nuclei (blue). An unpaired t-test showed **, p <0.01, ***, p<0.001. Error bars show SEM. Reduced cell proliferation and myogenic differentiation of MuSCs isolated from homozygous Jag2 sm mice MuSCs were isolated from hindlimb muscles of adult Jag2 sm mice using antibody-mediated magnetic sorting. Freshly isolated MuSCs were cultured in growth medium to assess proliferation for 5 days. Jag2 sm MuSCs displayed reduced cell proliferation, as evidenced by smaller colony sizes ( Figure 3E, F ). A 5-ethynyl-2’-deoxyuridine (EdU) incorporation assay correspondingly revealed reduced proliferating cells. We assessed the cell proliferation of passaged MuSCs, then switched to differentiation medium for 1 or 3 days to evaluate myogenic differentiation ( Figure 3G ). There were reduced proliferating [EdU(+)] MuSCs in Jag2 sm ( Figure 3G, H ). Apoptosis of Jag2 sm MuSCs was slightly increased compared with WT MuSCs following thapsigargin or UV-treatment (Figure S3). These findings confirm Jag2’s essential role in niche-independent MuSC proliferation and survival. During myogenic differentiation, the number of MyoD(+)-committed myogenic progenitors remained unchanged in homozygous Jag2 sm mice ( Figure 3G ). However, immunostaining for myosin heavy chain (MyHC) after 1 or 3 days of differentiation showed diminished multinucleated myotubes in Jag2 sm MuSC cultures ( Figure 3G, I, J ). These findings suggest that while Jag2 deficiency does not affect the initial commitment of MuSCs to the myogenic lineage [MyoD(+) cells], it impairs their proliferative capacity and subsequent myogenic differentiation. Impaired muscle regeneration in homozygous Jag2 sm mice We assessed the regenerative capacity of TA muscles in adult Jag2 sm mice following intramuscular cardiotoxin (CTX)-induced injury ( Figure 4A ). At 7 days post-injury, Jag2 sm TA showed smaller regenerating myofibers ( Figure 4B ). Feret’s myofiber diameters were reduced in Jag2 sm TA ( Figure 4D, E ) at 7- and 21-days post-injury. Immunostaining showed that embryonic myosin heavy chain (eMyHC), a marker of newly formed fibers, persisted in Jag2 sm muscle at day 7 post-injury but was no longer detectable in regenerating WT muscle ( Figure 4B ), indicating delayed muscle regeneration in Jag2 deficiency. Oil Red O staining revealed increased lipid accumulation in regenerating Jag2 sm muscle ( Figure 4F ). At 28 days following sequential CTX injections in Jag2 sm mice, muscle regeneration was impaired ( Figure 4C, G ), indicating defects in MuSC self-renewal. No differences were observed in the number of CD31(+) capillaries between Jag2 sm and WT muscle ( Figure 4B ), indicating that the muscle regenerative defects are unlikely to be related to differences in muscle microvasculature. Download figure Open in new tab Figure 4. Jag2 sm hypomorphic mice have muscle regenerative defects. (A) Single or repeated CTX injections into the TA muscle were performed on WT and Jag2 sm mice. (B) TA histology [hematoxylin & eosin (H&E), Oil red-O] and immunostaining (eMyHC/Laminin/DAPI and CD31/Laminin) 7 days following CTX injection into the TA. Scale bars from left to right: 100, 25, 50 and 250μm. (C) H&E staining 21+7 days following sequential CTX injections into the TA. Scale bar, 100 μm. Feret’s diameters of TA fibers in WT and Jag2 sm homozygous mice at (D) 7 days and (E) following CTX injection. (F) Oil red-O (+) area was evaluated at 7 days following CTX injection. (G) Feret’s diameters of TA myofibers in WT and Jag2 sm homozygous mice 21+7 days following CTX injections. DAPI stained all nuclei (blue). An unpaired t-test showed **, p <0.01; ***, p<0.001. Error bars show the standard error of the mean (SEM). Transcriptome sequencing (RNA-seq) of homozygous Jag2 sm MuSCs To probe global gene expression changes in Jag2 deficiency, we performed whole transcriptome sequencing (RNA-seq) on MuSCs isolated from the hindlimb muscles of Jag2 sm and WT mice. MuSCs were isolated using antibody-mediated magnetic sorting. Total RNA was isolated, reverse-transcribed to cDNA, and sequenced using the Oxford Nanopore Technologies (ONT) long-read sequencing platform. Gene expression analysis revealed that 702 genes were significantly dysregulated (adjusted p-value < 0.05) in homozygous Jag2 sm compared to WT MuSCs. There were 186 upregulated genes and 516 downregulated genes ( Figure 5A, C , Tables S2, S3). Metascape Gene Ontology (GO) analysis indicated that 106 genes related to muscle structure development (GO:0061061), including 28 myogenic regulatory genes such as Myog , Myf6 , Mef2c , Mymk , and Igf2 , were downregulated in Jag2 sm MuSCs ( Figure 5B , Table S4). Negative regulators of cell proliferation, including Cxcl12 and Sox4 , were identified in the upregulated genes ( Figure 5C , Table S2). Among Notch receptor genes, Notch1 , Notch2 , and Notch3 were expressed in WT MuSCs, and Notch2 expression was upregulated in Jag2 sm MuSCs (Table S5). Dll1 , Jag1 and Jag2 were detected in both WT and Jag2 sm MuSCs (Table S5), suggesting cis- regulatory mechanisms for Notch signaling in MuSCs. Reduced myogenic differentiation and expression of Notch receptor and ligand genes was confirmed in Jag2 sm MuSCs via RT-qPCR ( Figure 5D ). These findings are consistent with observed defects in MuSC proliferation and myogenic differentiation in Jag2 deficiency. Increased expression of the Notch downstream effector genes Hes1 , Hey1 , and Heyl , was observed in Jag2 sm compared with WT MuSCs ( Figure 5D ). Download figure Open in new tab Figure 5. RNA-seq for gene expression profiles in Jag2 sm vs. WT MuSCs. (A) Gene ontology (GO) analysis reveals that numerous significantly downregulated genes in RNA samples from Jag2 sm vs. WT mouse MuSCs are muscle related. (B) Heatmap for down-regulated genes associated with myogenic regulatory genes, Notch receptor genes, and ligand genes. (C) GO analysis reveals that numerous significantly up-regulated genes in RNA samples from Jag2 sm vs. WT mouse MuSCs are involved in negative regulation of cell proliferation. (D) RT-qPCR was performed on WT and Jag2 sm MuSCs under growth, day 1, and day 3 differentiation conditions to detect the expression of myogenic and Notch pathway-related genes. Cis- inhibition of Notch signaling by JAG2 in MuSCs To determine whether human JAG2 suppresses Notch signaling in MuSCs via cis- inhibition, we co-transfected JAG2 with the Notch reporter gene pHes1-467-Luc , containing the 467 bp Hes1 gene upstream region, or with the pHes1-467-RBPJ ( - ) -Luc , which lacks a RBP-J binding site essential for assembly of the transcriptional complex with NICDs and other binding partners and subsequent Notch target gene activation ( Figure 6A ). Hes1-467-Luc activity was elevated in Jag2 sm MuSCs, then abolished when the reporter gene with the mutant RBP-J binding site was used ( Figure 6B ). Notch reporter activation was blunted by N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a global γ -secretase/Notch inhibitor( 66 ). These data indicate that Jag2 deficiency promotes Notch signaling in MuSCs. To determine which Notch receptors are targeted by Jag2, the Hes1-467-Luc vector was co-transfected with human JAG2 expression vector and Notch expression vectors. JAG2 suppressed Notch1, Notch2, and Notch3 but not Notch4-mediated Notch reporter gene activation ( Figure 6C ). Since Notch1, Notch2, and Notch3 are detected in quiescent and activated MuSCs, Jag2-mediated Notch signaling in MuSCs might be mediated through these Notch receptors. Since JAG2 suppresses Notch signaling in MuSCs while Jag2 sm MuSCs show increased Notch signaling, Jag2 may suppress Notch signaling via cis- inhibition. WT and Jag2 sm MuSCs transfected with reference JAG2 but not variant JAG2 ( p.Glu164Lys , p.Pro682Ser , or p.Phe977Ser ) show reduced Notch signaling inhibition, confirming that JAG2 pathogenic variants lack cis- inhibitory effects in MuSCs ( Figure 6D ). Download figure Open in new tab Figure 6. Human JAG2 suppresses Notch signaling and promotes myogenesis. (A) Hes1-467 -Luc vector contains Hes1 467bp upstream promoter-driving luciferase ( Luc ) gene with an RBPJ binding site and Hes1-467-Mut-Luc vector contains a mutated RBPJ binding site. (B) Homozygous Jag2 sm MuSCs show higher Hes1-467-Luc activity compared with WT MuSCs. Luc activities were diminished when the RBPJ-binding site was mutated or when treated with the Notch inhibitor DAPT. (C) Expression of Notch1-4 ( N1-4 ) increased Hes1-467-Luc activities that were suppressed by co-transfection of human JAG2 in WT and homozygous Jag2 sm MuSCs. (D) Human JAG2- mediated suppression was not observed when transfected with expression vectors carrying pathogenic JAG2 variants. (E) 4R-SV-Luc contains 4 x E-boxes for consensus binding sites for MyoD. (F) Expression of MyoD and human JAG2 activates 4R-SV-Luc in WT and homozygous Jag2 sm MuSCs . MyoD promoted myosin heavy chain (MyHC)(+) myogenic differentiation in WT and homozygous Jag2 sm MuSCs in growth (G) and differentiation conditions (days 1 and 3). (H) Anti-Hes1 antibody staining shows Hes1 is higher in homozygous Jag2 sm compared with WT MuSCs. Hes1 expression is down-regulated in MyHC(+) myocytes (arrows). Scale bars, 50μm. (I) The diagram shows a DuoLink PLA assay to examine a protein complex of Jag2 and Notch1 within MuSCs. Anti-Jag2 and anti-Notch1 antibodies were used followed by anti-rabbit and anti-mouse IgG with (+) and (–) strands of oligo DNAs. Red fluorescence tags were incorporated with successful ligation. (J) DuoLink labeling shows patchy red complexes around the cell membrane regions, with no positive complexes when antibodies were eliminated as a control. (K) Quantification of DuoLink(+) complexes/cell was performed. DAPI stained all nuclei (blue). Scale bars, 100μm. An unpaired t-test showed *, p <0.05; **, p<0.01; ***, p<0.001. Error bars show the standard error of the mean (SEM). Jag2 sm MuSCs display reduced myogenic differentiation but increased Notch activity, and consequently, reduced muscle regeneration after injury. To determine whether MyoD activity is down-regulated in Jag2 sm MuSCs, we tested the luciferase activity of the MyoD-binding site-driven 4R-SV-Luc reporter gene. The 4R-SV-Luc incorporates 4 x E-box elements and MyoD-binding motifs, which are sourced from the enhancer region of the muscle creatine kinase ( MCK ) gene ( Figure 6E )( 67 , 68 ). MyoD activity is lower in Jag2 sm MuSCs ( Figure 6F ). Co-transfection with MyoD promotes luciferase activity in Jag2 sm MuSCs. JAG2 promoted luciferase activities in both WT and Jag2 sm MuSCs. MyoD overexpression rescued myogenic differentiation of Jag2 sm MuSCs, as indicated by an increase in MyHC(+) myocytes ( Figures 6G , S4). Increased Hes1 protein was detected in Jag2 sm compared with WT MuSCs ( Figure 6H ). The Hes1 expression was abolished in MyHC(+) myocytes in WT MuSCs. These results indicate that Jag2 is a myogenic promoter that acts as a cis- inhibitory factor for Notch signaling. Using DuoLink technology, an in situ proximity ligation assay (PLA) that identifies two proteins in close proximity, we demonstrated the presence of a Jag2-Notch1 complex on the MuSC plasma membrane ( Figure 6I, J ). The quantity of DuoLink reaction products was measured, confirming the existence of Jag2-Notch1 complexes exclusively when specific antibodies were used ( Figure 6K ). These findings suggest that Jag2 is responsible for cis- inhibition of MuSC Notch signaling. In vitro rescue effects of human reference JAG2 versus variant JAG2 in MuSCs Following culturing in differentiation media for 4 days, overexpression of human reference JAG2 rescued myogenic differentiation defects in Jag2 sm MuSCs, while overexpression of three human JAG2 pathogenic variants ( p.Glu164Lys , p.Pro682Ser , and p.Phe977Ser )( 30 ) did not ( Figures 7A, B, C , S5). Similarly, overexpression of reference JAG2 but not variant JAG2 ( p.Glu164Lys , p.Pro682Ser , or p.Phe977Ser ) rescued myogenic cell fusion in Jag2 -deficient C2C12 myoblasts (Figure S6). These results demonstrate the essential roles of JAG2 in MuSC function, providing evidence for a loss of function (LOF) pathogenic mechanism of JAG2 variants. Download figure Open in new tab Figure 7. Overexpression of human JAG2 rescues differentiation defects in Jag2 sm MuSCs. MuSCs isolated from homozygous Jag2 mice were used for expression vector-mediated human JAG2 overexpression. (A) Overexpression of WT human JAG2 ( JAG2 WT ) but not human JAG2 pathogenic variants ( p.Glu164Lys , p.Pro682Ser , and p.Phe977Ser ) increased MyHC(+) myogenic differentiation (green) (B), and fusion in days 1 and 3 differentiation conditions (C), compared with control empty vector-transfected cells. DAPI stained all nuclei (blue). Scale bars, 100 μm. An unpaired t-test showed *, p <0.05; **, p<0.01. Error bars show the standard error of the mean (SEM). Jag2-mediated regulation of MuSC self-renewal via MuECs MuECs play an essential role in MuSC self-renewal during muscle regeneration ( 14 ), but the exact signaling mechanism between the two cell types is unclear. We examined Jag2-mediated effects of MuECs on MuSC self-renewal. We confirmed Jag2 expression in MuECs and MuSCs via RNA-seq ( Figure 1A ), histology using a Jag2 LoxP/LoxP mouse line with a LacZ/Neo cassette that enabled us to detect Jag2 -expressing cells ( Figure 1B ), RT-qPCR ( Figure 1C ) and immunostaining ( Figure 1D ) in muscle and dissociated cells. We performed co-culture experiments using MuECs and MuSCs isolated from WT mice. MuECs were transfected with either Jag2 or scrambled control siRNA, and their ability to support MuSC self-renewal was evaluated by quantifying Pax7(+)/MyoD(-) cells (i.e., self-renewing MuSCs or reserve cells) after 5 days of co-culture ( Figure 8A ). MuSCs co-cultured with Jag2 -depleted MuECs exhibited reduced Pax7(+)/MyoD(-) self-renewing reserve cells compared to those co-cultured with control MuECs ( Figure 8B, C, D ). This effect was replicated in co-cultures treated with DAPT, suggesting that Jag2-mediated Notch activation in MuSCs is required for self-renewal ( Figure 8C, D ). These results demonstrate that Jag2 in MuECs regulates MuSC self-renewal through trans- activation signaling, in addition to the cell-autonomous cis- inhibition effects of Jag2 ( Table 1 ). Download figure Open in new tab Figure 8. MuSC and MuEC co-culture experiments. (A) MuSCs were layered on top of the MuECs with scrambled or Jag2 siRNA, allowed to adhere, and then co-cultured in differentiation medium for 5 days. (B) MuECs transfected with Jag2 siRNA show a significant reduction of Jag2 expression vs. scrambled siRNA. (C, D) Pax7(+)MyoD(-) self-renewing MuSCs were reduced when Jag2 was co-cultured with Jag2-KD MuECs vs. control MuECs (arrows). Downregulation of Notch signaling through the pan-Notch inhibitor DAPT reduced the number of Pax7(+)MyoD(-) self-renewing MuSCs vs. PBS-treated cells in the co-cultures (arrows). (E) Diagram of the evaluation of MuSCs treated with Notch ligands. (F) Hes1-467-Luciferase activity was assessed in control and JAG2 -expressing MuSCs exposed to Notch ligand (Control-IgG-Fc, Dll1-Fc, Dll4-Fc, Jag1-Fc, and Jag2-Fc). Comparative mRNA expression levels of the Notch effector genes (G) Hes1 , (H) Hey1 , and (I) HeyL , in control and JAG2 -expressing MuSCs exposed to Notch ligand (Control-IgG-Fc, Dll1-Fc, Dll4-Fc, Jag1-Fc and Jag2-Fc). DAPI stained all nuclei (blue). An unpaired t-test showed **, p<0.01; and ***, p<0.001. Error bars show the standard error of the mean (SEM). Scale bars, 50 μm. View this table: View inline View popup Download powerpoint Table 1. Muscle phenotypes for Jag2 deficiency in various settings To determine whether extracellular Jag2 can trans- activate endogenous Notch activity and whether that activity is suppressed by intracellular Jag2 via cis- inhibition, WT MuSCs were plated onto dishes that were treated with Notch ligands linked to the Fc domain of IgG: Dll1-Fc, Dll4-Fc, Jag1-Fc, and Jag2-Fc ( Figure 8E ). Anti-goat IgG was a control. Dll1-Fc, Dll4-Fc, Jag1-Fc and Jag2-Fc increased Hes1-467-Luciferase activities, which were suppressed by JAG2 expression in WT MuSCs ( Figure 8F ), prompting elevated expression of the Notch effector genes Hes1 , Hey1 and HeyL , compared with control-Fc treatment ( Figure 8G-I ). These extracellular Notch-ligands mediated the up-regulation of Hes1 , Hey1 , and HeyL , which was suppressed by JAG2 . Therefore, Dll1, Dll4, Jag1, and Jag2 trans- activate Notch signaling and promote self-renewal in MuSC cultures, which are suppressed by intracellular Jag2-mediated cis- inhibition. MuECs-derived Jag2 is essential for MuSC self-renewal, while MuSC-derived Jag2 is essential for proper MuSC myogenic differentiation Our RNA-seq analysis and in vitro experiments demonstrated that Jag2 regulates MuSC proliferation, myogenic differentiation, and self-renewal via both cell-autonomous cis- activation and MuEC-MuSC-interaction-mediated trans- activation. To confirm these findings in vivo , we investigated the effects of MuEC- and MuSC-specific Jag2 deletions mediated by VE-cadherin CreERT2 and Pax7 CreERT2 , respectively, using the VEcad CreERT2 :Jag2 LoxP/LoxP :Pax7 tdT and Pax7 CreERT2 :Jag2 LoxP/LoxP :Pax7 tdT mice following TMX-injection ( Figures 1B , 9A ). TA muscles were harvested 7 days and 7+21days following CTX injections. Using qPCR, we verified efficient conditional Jag2 gene deletion ( Jag2 Δ/Δ ) in MuECs and MuSCs following Cre activation, in VEcad CreERT2 :Jag2 LoxP/LoxP :Pax7 tdT and Pax7 CreERT2 :Jag2 LoxP/LoxP :Pax7 tdT mice, respectively (compared to control Jag2 +/+ MuECs and MuSCs) (Figure S7). TA cross-sections in VEcad-mediated Jag2 conditional knockout (cKO) mice ( MuEC-Jag2 Δ/Δ ) displayed reduced muscle regeneration 7 days following CTX injection ( Figure 9B ). At 28 days following sequential CTX injections in MuEC-Jag2 Δ/Δ mice, muscle regeneration was impaired ( Figure 9B, C ), indicating defects in MuSC self-renewal. MuSCs were reduced in the regenerating TA at 3 weeks ( Figure 9E, F ), confirming that self-renewal of MuSCs is regulated by MuEC-derived Jag2 during muscle regeneration. The reduced MuSCs in fully repaired muscle suggest a failure of MuSC self-renewal in MuEC-Jag2 Δ/Δ mice. MuSC self-renewal was reduced in MuEC-specific Jag2 knockout mice, indicating that Jag2 is essential for MuSC self-renewal via MuEC-mediated trans- effects during muscle regeneration ( Table 1 ). Download figure Open in new tab Figure 9. Reduced self-renewal in Jag2 LoxP/LoxP :VEcad CreERT2 and reduced regeneration in Jag2 LoxP/LoxP :Pax7 mice. (A) Following tamoxifen (TMX) injection, single or repeated CTX injections into the TA were performed. (B) H&E staining of TA by 7 days following CTX injection or 28 days following sequential CTX injections in Jag2 LoxP/LoxP :VEcad CreERT2 and Jag2 LoxP/LoxP :Pax7 CreERT2 mice. Scale bars, 100 μm. (C, D) Feret’s diameters of TA muscle fibers in Jag2 LoxP/LoxP :VEcad CreERT2 and Jag2 LoxP/LoxP :Pax7 CreERT2 mice following CTX injections. (E, F) Single muscle fibers were isolated at 28 days following TMX treatment and CTX injection. Anti-Pax7 antibody staining shows a reduced number of self-renewing MuSCs in Jag2 Δ/Δ :VEcad CreERT2 but not in Jag2 Δ/Δ :Pax7 CreERT2 mice. (G) MuSCs isolated from homozygous Jag2 Δ/Δ :Pax7 CreERT2 mice with TMX treatment show (G, H) reduced EdU(+) proliferating cells (green) in growth, (G, I) MyHC(+) myogenic differentiation (green), and (G, J) fusion index in day 1 and 3 of differentiation conditions compared with control cells. DAPI stained all nuclei (blue). Scale bars, 50 μm for (E) and 100 μm for (G). An unpaired t-test showed *, p<0.05; **, p<0.01; and ***, p<0.001. Error bars show the standard error of the mean (SEM). TA cross-sections in Pax7-mediated Jag2 cKO ( MuSC-Jag2 Δ/Δ ) mice displayed reduced muscle regeneration by 1, 2, and 3 weeks following single CTX injections ( Figure 9B, D ). However, MuSC numbers were not altered in regenerating TA by 3 weeks ( Figure 9E, F ), indicating that self-renewal of MuSCs is not regulated by MuSC-derived Jag2 (i.e., cis- activation) during muscle regeneration. Isolated single muscle fibers from MuSC-Jag2 Δ/Δ mice also showed similar numbers of MuSCs compared with those in MuSC-Jag2 +/+ mice ( Figure 9E, F ). MuSCs cultured from Jag2 MuSC-specific knockout mice show reduced proliferation and differentiation ( Figure 9G, H-J ). We conclude that Jag2 is essential for MuSC differentiation via cell-autonomous cis- inhibition of Notch signaling during muscle regeneration ( Table 1 ). Human JAG2 rescues Serrate deficiency in Drosophila , but pathogenic variants do not Serrate ( Ser ), the Drosophila ortholog of human JAG1 and JAG2 , plays a critical role in wing development. In the wing disc, Serrate is expressed in the epithelium adjacent to adult muscle progenitor cells (AMPs), where it may promote the proliferation of muscle progenitors via trans- activation of Notch( 69 ). Ser is also expressed in a distinct subset of AMPs( 70 ). We generated transgenic fly lines carrying UAS-human JAG2 constructs, allowing for tissue-specific expression of JAG2 using the Gal4/UAS system. When reference human JAG2 ( JAG2 Ref ) was overexpressed using the Serrate-Gal4 driver mimicking the endogenous expression pattern of Serrate , flies developed normally. However, the wings in the corresponding adult transgenic flies showed a characteristic "delta" wing vein phenotype( 71 ), indicating a genetic interaction between human JAG2 and the Drosophila Delta-Notch pathway ( Figure 10A ). Expression of either human pathogenic JAG2 variant ( p.Glu164Lys or p.Pro682Ser ) associated with muscular dystrophy in humans( 30 ) led to an attenuated wing vein delta phenotype, suggesting a LOF effect ( Figure 10A ). Download figure Open in new tab Figure 10. Reference human JAG2 ( JAG2 Ref ) rescues Serrate deficiency in Drosophila , while human variant JAG2 E164K does not. (A) Human JAG2 Ref induced delta-shaped wing veins,but variants showed marginal effects. (B) Serrate deficiency generated progressive melanotic spots on the legs. Expression of JAG2 Ref rescued manifestations of serrate deficiency, whereas expression of JAG2 E164K did not, on measures of (C) flight and (D, E) negative geotaxis, along with (F) melanotic spots. n=5 replicates, ***, p<0.001 (two-sided t-test). Likewise, our results in mice showed that the pathogenic JAG2 variants result in LOF ( Figures 2 - 4 , 9 , and Table 1 ). RNA interference (RNAi) suppressed endogenous Serrate expression in Serrate -expressing cells with Serrate-Gal4 . RNAi-mediated Ser knockdown did not induce developmental abnormalities, such as pupal lethality or eclosion defects. Right after eclosion, the Ser RNAi adult flies displayed normal walking and flight behavior. However, a rapid decline in locomotor activity occurred within a week post-eclosion, including both flight and gait impairments ( Figure 10B-E ). Flies with reduced Serrate exhibited progressive development of dark melanotic spots, indicating tissue necrosis and a hemocyte-mediated inflammatory response. This is consistent with the need for Notch signaling in the leg imaginal discs to promote leg segment formation( 72 ). The degenerative phenotypes observed with Serrate LOF were useful to assess whether human JAG2 could rescue the defects. Expression of reference human JAG2 , but not JAG2 p.Glu164Lys , in Serrate -deficient flies rescued the locomotor deficits and necrotic legs ( Figure 10C-F ). These findings demonstrate the functional conservation of JAG2 across species and provide in vivo evidence for the LOF mechanism of pathogenic JAG2 variants ( Table 1 ). DISCUSSION Biallelic pathogenic variants in the canonical Notch ligand JAG2 cause a form of muscular dystrophy( 30 , 73 , 74 ). Pathogenic variants in the paralogous gene JAG1 are associated with Alagille syndrome( 75 , 76 ), which does not prominently involve skeletal muscles, yet JAG1 augmentation shows promise as a therapeutic target for muscular dystrophy( 77 , 78 ). Notch signaling affects several biological functions associated with skeletal muscle, including MuSC self-renewal, maintenance, and muscle regeneration. Jag2 is expressed in both MuSCs and MuECs, but the Significance of Jag2 for skeletal muscle development and health was not clear. Our data indicate that Jag2 deficiency in MuSCs impairs their myogenic differentiation potential via failed cis- inhibition effects for Notch activity, while Jag2 deficiency in MuECs impairs MuSC self-renewal via failed trans- activation effects for Notch activity as niche cells, suggesting that Jag2-related cell-autonomous ( cis ) and cell-nonautonomous ( trans ) Notch signaling affects skeletal muscle development, regeneration and health in different ways ( Figure 11 ). In Drosophila , Serrate -expressing niche cells in the wing epithelium regulate the proliferation and maintenance of adult muscle precursors (AMPs)( 69 ). We demonstrated that in vivo knockdown of Serrate (the fly ortholog of JAG2 ) in Serrate -expressing cells resulted in motor function and morphological defects. These phenotypes are postulated to result from reduced trans- activation. It is unclear if cis- inhibitory activity is also involved in Drosophila adult muscle development ( Figure 11 ). Download figure Open in new tab Figure 11. A diagram illustrating the role of Jag2 expression in mammal (left) and Drosophila (right) muscles. In mammals, neighboring capillary MuECs trans -activate Notch signaling in MuSCs via Jag2 for MuSC self-renewal. MuSCs, which do not receive Jag2 -mediated trans -activation by MuECs suppress Notch signaling via cis- inhibition by cell-autonomous Jag2 expression, stimulating myogenic differentiation. In Drosophila wing discs, the ortholog Serrate is expressed in epithelial cells, which activates Notch signaling in adjacent adult muscle precursors (AMPs), which are MuSC-like cells, to maintain the progenitor pool. AMPs express Serrate , but it is unclear whether cis- inhibition by Serrate occurs in AMPs. Homozygous Jag2 hypomorphic ( Jag2 sm ) mice display digit and craniofacial developmental defects( 79 , 80 ). We showed that homozygous Jag2 sm mice display impaired muscle regeneration due to a reduced Pax7-positive MuSC population during muscle development. The surviving homozygous Jag2 sm MuSCs showed reduced proliferation and decreased myogenic differentiation. To reveal whether MuSC defects seen in Jag2 sm mice are due to the trans- effects via neighboring niche cells or cell-autonomous cis- effects, we utilized MuSC-MuEC co-culture experiments, Notch ligand-Fc treatment cultures, and Cre-recombinase-mediated conditional Jag2 gene KO mice. We demonstrated that MuEC-specific Jag2 knockout resulted in reduced MuSC self-renewal. Our MuEC and MuSC co-culture experiments demonstrated that MuEC-derived Jag2 is required for sufficient MuSC self-renewal, underscoring the significance of direct cellular interaction between MuECs and MuSCs for the activation of Notch signaling in vitro and in vivo . Trans- activation of Notch signaling by Jag2 Multiple investigations have implicated MuECs in the functionality of MuSCs( 11 – 13 ). We and others have demonstrated that vascular network enhancement augments MuSC populations in mouse models of Duchenne muscular dystrophy (DMD)( 15 – 17 , 81 ). Recent works, including our studies, reveal a molecular mechanism that connects MuECs and MuSCs, along with the functional outcomes of this signaling. We demonstrated that the proximity of MuSCs to capillaries is actively orchestrated by VEGFA secreted by MuSCs, attracting capillaries to create a juxtavascular environment for MuSCs( 14 ). In addition, MuSC self-renewal is induced by Notch activation, which is stimulated by the adjacent capillary MuECs through Dll4 as a trans- activator. By contrast, the MuEC-derived secreted form of Dll4 regulates muscle fiber atrophy( 82 ). Our in vitro co-culture experiments and in vivo conditional gene KO mice showed that MuEC-derived Jag2 is essential for MuSC self-renewal. Therefore, MuEC-mediated MuSC self-renewal requires at least two Notch ligands, Dll4 and Jag2. Recent findings underscore the necessity of Notch receptors for MuSCs to revert to quiescence and establish stem cell populations( 83 , 84 ). Regarding the neighboring cell origin for Notch activation, Dll4, derived from mature myofibers, activates Notch3 expression in MuSCs, facilitating their return to quiescence( 84 ). Mature myofiber-derived Dll4 is important for the maintenance of quiescent MuSCs on myofibers in myofiber-specific Dll4 KO mice( 85 ). Moreover, Dll4 and Jag2 from muscle fibers regulate Notch signaling in the proximal MuSCs to enhance their regenerative potential via increased self-renewal( 86 ). We found that Dll4 and Jag2 levels were reduced in myofibers compared to MuECs, suggesting that MuECs play a crucial role as Notch ligand-synthesizing cells that support MuSC self-renewal in skeletal muscle. Serrate-Notch -mediated Drosophila myogenic progenitor maintenance Using Serrate-GAL4 to knock-down Serrate in Serrate -expressing cells, we observed previously unreported adult Drosophila phenotypes, which we believe are due to reduced trans- activation. Whether Drosophila Serrate is also involved in cis- inhibition is unknown. Recent single-nucleus sequencing( 87 ) suggests that a discrete population of Serrate -expressing cells is present in the adult muscle system, although these are fewer than Delta -expressing cells and do not co-express Delta , and thus remains the main cis- inhibitory signal in fruit flies. Cis- inhibition We demonstrated that MuSC-specific Jag2 knockout resulted in reduced myogenic differentiation without affecting MuSC self-renewal capacity. These results are consistent with our RNA-seq and gene knockdown data. Notch signaling relies on families of ligands and receptors that relay messages to adjacent cells in various combinations across distinct cell types, as seen in MuEC-MuSC interactions (in trans ), and within the same MuSCs (in cis ). Notch ligands and their corresponding receptors that are present within the same cell display cis- inhibition of Notch signaling( 88 – 90 ). This cis- inhibition plays a crucial role in various developmental processes, such as wing disc formation in Drosophila , maintenance of epidermal stem cells, neurogenesis, pancreatic cell differentiation, and hematopoiesis ( 58 , 88 , 91 ). It has been proposed that Notch ligands are capable of binding to Notch receptors and of cis- activation of Notch signaling within the same cells( 59 , 92 ). The interaction between the Notch receptor and its ligand arises from trans- activation and cis- activation of the Notch receptor mediated by the ligand as a monomer, while cis- inhibition is induced by the ligand as a dimer( 90 ). Therefore, cis- activation indicates innovative methods through which cells can integrate various Notch interactions. However, more recent work demonstrated that with competition from trans- activating ligands from neighboring cells, Dll1 and Dll4 can cis- inhibit Notch1 but cis- activate Notch2 signaling, while both Jag1 and Jag2 only cis- inhibit both Notch receptors( 93 ). A systematic examination of cis- and trans- interactions between Jag2 and different Notch receptors could yield more profound insights into cell-cell communication-mediated MuSC functions. Notch activity modifiers Activation of Notch signaling induces MuSC proliferation, self-renewal, and maintenance, and suppresses terminal myogenic differentiation via the trans -activation of Notch effector genes belonging to the negative bHLH family, including Hes1 , Hes5 , Hey1 and HeyL , by antagonizing MyoD activity( 94 , 95 ). Our data indicated that Jag2 mutant MuSCs isolated from homozygous Jag2 sm and MuSC-specific Jag2 KO mice display myogenic differentiation defects. The Jag2 deficiency-mediated reduced myogenic differentiation is due to the failed cis- inhibition of Notch activity, since overexpression of reference JAG2 but not pathogenic variants suppresses Notch activity and promotes myogenic differentiation. Several Notch modifying factors have been identified. The Notch-controlled ankyrin repeat molecule (NRARP) functions as a counteracting regulator of Notch activity in many cell types in a negative feedback loop( 96 , 97 ). Notch3 KO mice display increased quiescent MuSCs and muscle hypertrophy due to hyperplasia of MuSC-derived myogenic precursor cells during muscle regeneration, potentially by upregulation of NRARP to suppress Notch1 activity( 98 ). Numb regulates asymmetrical cell division, with one daughter cell inheriting Numb and the other inheriting Notch via antagonizing Notch activity( 99 ). Reduced Notch signaling through elevated Numb expression in MuSCs resulted in myogenic differentiation by suppressing Notch activity( 100 , 101 ). Prox1-expressing MuSCs undergo myogenic differentiation through reciprocal suppression of Notch1( 102 ). The Fringe homologs Lunatic fringe (Lfng), Manic fringe (Mfng), and Radical fringe (Rfng) are β3-N-acetylglucosaminyltransferases that influence Notch function by altering O-fucose modifications on EGF repeats in Notch receptors. Lfng enhances Notch2 activation via Dll1 and Dll4, whereas Mfng suppresses Notch2 activation through Jag1 and Jag2( 103 ). Lfng enhances Dll1/4-driven trans- and cis- activation for Notch receptors( 93 ). Thus, cellular environments with different expression levels of Notch receptors and Notch modifiers may influence Jag2-mediated Notch signaling. Notch for muscular dystrophies Reduced MuSC self-renewal, maintenance, and differentiation contribute to the disease mechanism of muscular dystrophies. Therapies targeting Notch signaling could selectively enhance MuSC replication, potentially alleviating the symptoms of muscular dystrophy. Two other inherited muscle disease genes aside from JAG2 have been linked to the Notch signaling pathway: MEGF10 ( 104 , 105 ) and POGLUT1 ( 106 ). We determined that Megf10 and Notch1 interact at their intracellular domains and that this interaction is impaired by pathogenic variants( 107 ). Loss of Notch signaling in POGLUT1 deficiency has been demonstrated in skeletal muscle tissue from affected individuals and in Drosophila ( 106 ). Jag1 -mediated augmentation of Notch signaling ameliorated DMD in canine and zebrafish models( 77 ). Conclusions We have demonstrated that Jag2-mediated trans- activation and cis- inhibition of Notch signaling regulate muscle stem cell function during muscle regeneration. JAG2 shows promise as a therapeutic target for muscular dystrophy, and our findings will help fine-tune interventions to focus on specific desirable downstream effects of JAG2-related interventions. METHODS Mice C57BL/6N- A tm1Brd Jag2 tm1a(KOMP)Wtsi /HMmucd ( Jag2 LoxP/LoxP ; MMRRC stock # 048257-UCD) were obtained from the Mutant Mouse Resource & Research Centers (MMRRC). B6.Cg-Pax7 tm1(cre/ERT2)Gaka/J [ Pax7 +/CreERT2 ; JAX stock# 017763 ( 65 )], B6.Cg-Gt(ROSA) 26Sortm9(CAG- tdTomato)Hze/J [ Ai9 ; JAX stock# 007909 ( 108 )], and STOCK Jag2 sm /J [ Jag2 sm ; JAX stock# 000239;( 80 )], and B6.129S4-Gt(ROSA)26Sor tm2(FLP*Sor /J ( FLP ; JAX stock# 012930) were obtained from Jackson Laboratory. Kdr tm2.1Jrt/J ( Flk1 +/GFP ) were obtained from Masatsugu Ema ( 109 ). Cdh5 +/CreERT2 mice were obtained from Dr. Yoshiaki Kubota ( 110 ). B6.Cg-Pax7 tm1(cre/ERT2)Gaka/J ( Pax7 +/CreERT2 ) mice were crossed with B6.Cg-Gt(ROSA) 26Sortm9(CAG-tdTomato)Hze/J ( Ai9 ) to yield the Pax7 +/CreERT2 :R26R tdT ( Pax7 tdT ) mice. Pax7 tdT mice were bred with Jag2 LoxP/LoxP and Flk1 +/GFP to yield Jag2 LoxP/LoxP :Pax7 +/tdT : Flk1 +/GFP mice. Cdh5 +/CreERT2 mice were crossed with B6.Cg-Gt(ROSA) 26Sortm9(CAG-tdTomato)Hze/J ( Ai9 ) to yield Cdh5 +/CreERT2 :R26R tdT ( Cdh5 tdT ) mice. Cdh5 tdT mice were bred with Jag2 LoxP/LoxP and Flk1 +/GFP to yield Jag2 LoxP/LoxP :Cdh5 +/tdT : Flk1 +/GFP mice. Jag2 sm mice( 80 ) were crossed with Pax7 tdT to yield the Jag2 sm :Pax7 tdT mice. All mouse colonies were established ( Table 1 ) and genotyped (Table S6) in the laboratory. Cre recombination was induced using tamoxifen (TMX; T5648, MilliporeSigma) 75mg/kg body weight x 3 over 1 week at 3–6 weeks of age. CreERT2 mice were used as controls. TA muscle regeneration was induced by intramuscular injection of 20µl of 10µM cardiotoxin (CTX) (V9125, MilliporeSigma). The animals were housed in an SPF environment and were monitored by Research Animal Resources (RAR) of the University of Minnesota. All protocols (2204–39969A) were approved by the Institutional Animal Care and Usage Committee (IACUC) of the University of Minnesota and complied with NIH guidelines for the use of animals in research. Cell culture and immunostaining C2C12 myoblasts (CRL-1772) were obtained from American Type Culture Collection (ATCC) and cultured in DMEM medium with 10% FBS, 100units/ml of penicillin, and 100μg of streptomycin at 37°C in 5% O 2 and 5% CO 2 . C2C12 cells were STR profiled to confirm their identity and tested negative for mycoplasma. MuSCs were isolated from adult mice( 111 ). After collagenase type II (CLS-2, Worthington) treatment, dissociated cells from mouse hindlimb muscles were incubated with anti-CD31-PE (12-0311-82, eBiosciences), anti-CD45-PE (12-0451-81, eBiosciences), anti-Sca1-PE (A18486, eBiosciences), and anti-Integrin α7 (ABIN487462, MBL International), followed by anti-PE microbeads (130-048-801, Miltenyi Biotec), then underwent LD column (130-042-901, Miltenyi Biotec) separation. Negative cell populations were incubated with anti-Mouse IgG beads (130-048-402, Miltenyi Biotec), and then MS column (130-042-201, Miltenyi Biotec) separation was performed to isolate Integrin α7(+) MuSCs. MuSCs were maintained in culture on collagen-coated plates in myoblast growth medium containing 20% FBS, 20ng/ml bFGF (PHG0263, Invitrogen), 100units/ml of penicillin, and 100μg of streptomycin in HAM’s-F10 medium. MuECs were isolated from adult mice( 14 ). Dissociated muscle cells were obtained as described above. Dissociated cells were incubated with CD45 MicroBeads (130-052-301, eBiosciences) and anti-CD45-PE (12-0451-81, eBiosciences), and then underwent LD column (130-042-901, Miltenyi Biotec) separation. Negative cell populations were incubated with CD31 MicroBeads (130-097-418, Miltenyi Biotec), and then MS column (130-042-201, Miltenyi Biotec) separation was performed to isolate CD45(-)CD31(+) MuECs. MuECs were maintained in culture on fibronectin-coated plates in EGM-2 Endothelial Cell Growth Medium-2 Bullet Kit (CC-3162, Lonza). All antibody-related materials are listed in Table S7. All cell cultures were maintained in a humidified incubator at 37°C with 5% CO 2 and 5% O 2 . 4-Hydroxy tamoxifen (4-OHT, H6278, MilliporeSigma) treatment (1µM in EtOH) was used to induce Jag2 deletion in MuSCs isolated from Jag2 LoxP/LoxP :Pax7 CreERT2 mice. For the cell proliferation assay, cells were exposed to 1µM EdU for 3 hours before being fixed and stained by the Click-iT EdU Alexa Fluor 488 or 647 Imaging Kit (C10337 or C10340, Fisher Scientific). To induce differentiation of MuSCs, the myoblast growth medium was replaced with differentiation medium that contained DMEM supplemented with 5% horse serum for 1 to 5 days. Following cell cultures, anti-MyHC (MF20, DSHB) and anti-MyoD (C-20, Santa Cruz Biotechnology) antibodies followed by secondary anti-mouse-Alexa-488 (A11001, Thermofisher Scientific) and anti-rabbit-Alexa-647 (A32795, Thermofisher Scientific), anti-MyHC (MF20, DSHB) and anti-Hes1 (D6P2U, Cell Signaling Technology) antibodies followed by secondary anti-mouse-Alexa-568 (A11004, Thermofisher Scientific) and anti-rabbit-Alexa-488 (A11008, Thermofisher Scientific), or anti-Jag2 antibody (C23D2, Cell Signaling Technology) followed by secondary anti-rabbit-Alexa-488 (A11008, Thermofisher Scientific) were used (Table S7). LacZ expression in MuSCs obtained from LacZ/Neo-Jag2 LoxP/LoxP mice was detected by X-gal staining overnight as described previously ( 15 ). Following X-gal staining, anti-Pax7 (DSHB) or anti-MyoD (C-20, Santa Cruz Biotechnology) antibodies followed by secondary anti-mouse-Alexa-488 (#A-11001, Thermofisher Scientific) or anti-rabbit-Alexa-488 (#A-11008, Thermofisher Scientific) were used. The fusion index (containing two or more nuclei in MyHC-positive myotubes) was measured. For induction of apoptotic cell death in MuSCs, Thapsigargin-mediated apoptosis was induced by 1µM of thapsigargin (T9033, MilliporeSigma) dissolved in EtOH for 24 hours. UV light-mediated apoptosis was induced by exposing the cells to UV light in a cell culture hood for 1 min without medium. After UV exposure, cell survival was assessed 24 hours following culture in 0.1% FBS in HAM’s F10 medium using the Annexin V Assay Kit (ab232855, Abcam). DAPI was used for nuclei staining. shRNA knockdown of Jag2 in C2C12 mouse myoblasts C2C12 cells were transfected with a cocktail of shRNA plasmids (Genecopoeia) against mouse Jag2 using Lipofectamine 3000 (L3000001, ThermoFischer Scientific). The Jag2 and scrambled shRNA sequences are shown in Table S6. shRNA transfection and positive clone selection were performed as described( 30 ). Notch plate array and myotube-fusion index of differentiated C2C12 cells Scrambled and Jag2 shRNA cells were switched at 80% confluence from normal growth medium containing 10% FBS to differentiation medium containing 2% horse serum and differentiated for 4 days. Total RNA was isolated using an RNA isolation kit (Zymo Research). Reverse transcription of mRNA was performed using a high-capacity RNA to cDNA kit (Applied Biosystems). qPCR-based gene expression analysis was conducted using the TaqMan Fast Advanced Master Mix in the QuantStudio 3 Real-Time PCR System ( ThermoFisher Scientific ) . The Taqman probes used were: mouse Jag2 (Mm01325629_m1); mouse MyoG (Mm00446194_m1); human JAG2 (Hs99999198_m1), and mouse Gapdh (Mm99999915_g1) from ThermoFisher Scientific. The cDNA samples were also examined via the TaqMan Array Mouse Notch Signaling Pathway, Fast 96-well plate (ThermoFisher Scientific) containing a set of Notch signaling pathway-associated genes and endogenous control genes as reported( 30 ). For myotube fusion index analysis, the cells were fixed in 4% paraformaldehyde (PFA) for 15 mins and blocked in serum medium for 1 hour. The cells were stained with MHC [MF20, Developmental Study Hybridoma Bank (DSHB)] primary antibody for 1 hour. Cells were then stained with anti-rabbit or anti-mouse Alexa Fluor-568 secondary antibody for 1 hour. Nuclei were stained using DAPI (4’,6-diamidino-2-phenylindole dihydrochloride, D1306, Thermofisher Scientific), and the coverslips were mounted using Fluoromount Aqueous Mounting Medium (MilliporeSigma). The slides were imaged using a DM6000B or DM5500B epifluorescent microscope (Leica). The myotube-fusion index was determined by counting the number of nuclei within MHC-positive myotubes divided by the total number of nuclei in the field of view using ImageJ (National Institutes of Health). Site-directed Mutagenesis and overexpression of JAG2 variants in C2C12 myoblasts Site-directed mutagenesis was performed on the JAG2 coding sequence in the pCDNA3.1 backbone to generate the variants of interest using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs). pCDNA3.1JAG2 was a gift from Sandra Coppens. Mutagenic primers harboring the desired variants were used in a PCR reaction with wild-type JAG2 sequence as the template. PCR was performed using the primers for the indicated JAG2 variants ( p.Glu164Lys , p.Pro682Ser , and p.Phe977Ser ) (Table S6). Generation and the stable overexpression of the JAG2 human variants in scrambled and Jag2 shRNA cells was performed. RNA and genomic DNA isolation and qPCR Cultured cells were washed with ice-cold PBS and lysed in place with Trizol. RNA was isolated using the DirectZol RNA Microprep Kit (R2062, Zymo Research) with on-column DNase digestion followed by cDNA synthesis using the Transcriptor First Strand cDNA synthesis kit (04379012001, Roche Molecular Diagnostics) with random primers. Genomic DNA for genotyping was isolated from mouse tail snips with lysis buffer containing Proteinase K (P2308, MilliporeSigma). qPCR was performed using GoTaq qPCR Master Mix (A6002, Promega). The input RNA amount was normalized across all samples, and 18S rRNA was used for normalization of qPCR across samples. All primers were synthesized as custom DNA oligos from Integrated DNA Technologies (IDT) (Table S6). Single-muscle fiber isolation and staining Extensor digitorum longus (EDL) muscle was dissected from uninjured or CTX-injected lower hindlimb muscle and digested with 0.2% collagenase type I (C0130, MilliporeSigma) for single muscle fiber isolation( 15 ). Single muscle fibers were fixed with 2% PFA/PBS, permeabilized with 0.2% Triton-X100. Anti-Pax7 antibody (DSHB), followed by secondary anti-mouse-Alexa-488 (#A-11029, Thermofisher Scientific), was used for immunostaining (Table S7). DAPI was used for nuclear staining. Co-culture Co-culture was performed by plating a monolayer of MuECs overlaid with MuSCs and culturing them in low-serum media to induce myogenic differentiation and reserve cell induction for 5 days ( 14 ). Jag2 siRNAs (sc-39673, Santa Cruz Biotechnology) and control scramble siRNA-A (sc-37007, Santa Cruz Biotechnology) were transfected in ECs by Polyjet (11668019, ThermoFisher Scientific) before co-culture. A γ-secretase inhibitor, DAPT 10 μM, (D5942, Sigma-Aldrich), was used to block Notch signaling. Following co-cultures, anti-Pax7 (DSHB) and anti-MyoD (C-20, Santa Cruz Biotechnology) antibodies followed by secondary anti-mouse-Alexa-488 (#A-11029, Thermofisher Scientific) and anti-rabbit-Alexa-594 (#A-21207, Thermofisher Scientific) were used to detect Pax7(+)MyoD(-) self-renewing reserve cell population (Table S7). DAPI was used for nuclear staining. Luciferase reporter assays The firefly luciferase reporter genes Hes1-467-luciferase [ pHes1 ( 467 ) -Luc ] and Hes1-467-Mut-luciferase [ pHes1(467 RBPj(-)-Luc ] were obtained from Addgene [41723 and 43805, Addgene; ( 112 )]. 4R-SV-luciferase ( 4R-SV-Lux ) was obtained from Andrew Lassar ( 67 ). pRL-TK (E1910, Promega) was used as an internal control. WT and homozygous MuSCs were transfected with expression vectors for human JAG2 ( pcDNA3-JAG2 ), mouse MyoD ( pcDNA3-MyoD ), mouse Notch1 ( pCS2-Notch1 ), mouse Notch2 ( pPB[Exp]-Notch2 , VectorBuilder), human NOTCH3 ( pPB[Exp]-NOTCH3 , VectorBuilder), mouse Notch4 ( pHyTc-Notch4 , Addgene), or empty vectors, and the luciferase reporter genes using PolyJet™ In Vitro DNA Transfection Reagent (SL100688, SignaGen Laboratories). Cells were harvested 48 hours after transfection. Luciferase activity was measured with a plate reader (LD400; Beckman Coulter) using a dual luciferase reporter assay system (E1910, Promega). Ligand-coating Notch signaling assay A total of 5×10 4 WT MuSCs, Jag2 sm homozygous MuSCs, WT MuSCs carrying JAG2 , and Jag2 sm homozygous MuSCs carrying JAG2 were placed in a 48-well tissue culture plate pretreated with 5 μg/ml of DLL1-Fc, DLL4-Fc, JAG1-Fc, or JAG2-Fc (5026-DL, 10089-D4, 10969-JG, and 4748-JG, R&D) and allowed to settle at room temperature for 1 hour. After 16–18 hours, the cells were transfected with pHes1 ( 467 ) -Luc using PolyJet™ In Vitro DNA Transfection Reagent (SL100688, SignaGen Laboratories). After 3 hours, the medium was changed to growth medium for an additional 48 hours. Cells were harvested for luciferase assays and RNA isolation. Histology and immunostaining for sections and cell cultures The mouse tibialis anterior (TA) muscle was used for all histological analyses. Tissues were snap-frozen using LiN 2 chilled isopentane and stored at –80°C. Eight-μm thick transverse cryosections were used for histological analysis. Hematoxylin & Eosin (H&E) staining was performed( 15 ). Sirius red (Direct Red 80, 365548, MilliporeSigma) staining was performed on muscle sections to detect fibrosis( 75 ). Muscle sections were stained in Oil Red O solution (O1391-250ML, MilliporeSigma)( 76 ). LacZ expression in whole muscle and MuSCs obtained from LacZ/Neo-Jag2 LoxP/LoxP mice was detected by X-gal staining overnight as described previously ( 15 ). Muscle sections obtained from X-gal-stained muscle were used for anti-CD31 antibody staining followed by anti-rat Alexa-488 (A11006, ThermoFisher Scientific). Anti-eMyHC (F1.652, DSHB) and anti-Laminin (L0663, MilliporeSigma) antibodies, followed by anti-mouse Alexa-488 (A11001, ThermoFisher Scientific) and anti-rat Alexa-568 antibodies (A11077, ThermoFisher Scientific) were used to detect regenerating muscle fibers. For capillary density measurement, anti-CD31 antibody (550274, BD Biosciences) and anti-Laminin antibody (L9393, MilliporeSigma) were used for TA sections, followed by anti-rat Alexa-488 (A11006, ThermoFisher Scientific) and anti-rabbit Alexa-568 (A11011, ThermoFisher Scientific). Immunostaining was performed on 35mm tissue culture plates or 8-well Permanox® Chamber slides (C7182-1PAK, MilliporeSigma). Cells were fixed with 2% PFA for 5 min and immunostained( 15 ). Cells were permeabilized with 0.2% Triton-X in PBS, blocked with 1% BSA in PBS, and incubated with primary antibodies followed by secondary antibodies. PBS with 0.01% Triton-X was used for washing cells. Nuclei were counterstained with DAPI. The antibodies are listed in Table S7. Microscopic images were captured by a DP-1 digital camera attached to a BX51 fluorescence microscope with 10×, 20×, or 40×UPlanFLN objectives with cellSens Entry 1.11 (Olympus). Photoshop (Adobe) and Fiji (NIH) were used for image processing and enumerating Feret’s diameters( 77 ). Over-expression experiments The pcDNA3 expression vectors for WT human JAG2 , human JAG2 pathogenic variants ( p.Glu164Lys , p.Pro682Ser , and p.Phe977Ser ), and mouse MyoD were transfected to low-passaged (2–3 passages) WT or homozygous Jag2 sm MuSCs using PolyJet™ In Vitro DNA Transfection Reagent (SL100688, SignaGen Laboratories) for 3 hours. For the generation of the stable cell lines, the medium was replaced with myoblast growth medium, and G418 (Geneticin, 300 μg/ml; 10131035, Gibco) was added for the selection of transformant cells, which were used for cell growth and differentiation assays. Proximity ligation assay (PLA) Cells were inoculated into 3-cm culture dishes. The following day, cells were fixed with 4% PFA, followed by permeabilization for 5 minutes using PBS + 0.2% Triton X-100. After rinsing, the PLA was carried out using a Duolink in situ PLA kit (DUO92101, MilliporeSigma). Following incubation with primary antibodies (anti-Jag2 and anti-Notch1 antibodies; Table S7), two distinct PLA probes (anti-rabbit minus and anti-mouse plus) were combined and incubated on the dishes for 1 hour. Microscopic images were captured as described above. Grip strength test A forelimb grip strength test was performed( 113 ). Mice were gently pulled by the tail after forelimb-grasping a metal bar attached to a force transducer (Grip Strength Meter, 1027CSM-D52, Columbus Instruments). Grip strength tests were performed by the same blinded examiner. Five consecutive grip strength tests were recorded, and then the mice were returned to the cage for a resting period of 20 min. Then, three series of pulls were performed, each followed by 20-min resting period. The average of the three highest values out of the 15 values collected was normalized to the body weight for comparison. Treadmill running Exer-3/6 Treadmill (Columbus Instruments) was used for treadmill running tests( 114 ). For acclimation, mice in each lane ran on a treadmill for 5 minutes at 10 m/min on a 0% uphill grade daily for 3 days. Then, the mice ran on a treadmill with a 10% uphill grade, starting at 10 m/min for 5 minutes. Then, every 2 minutes, the speed was increased by 2 m/min until exhaustion, defined as the mice’s inability to remain on the treadmill. The running time and distance were recorded. Rotarod test Mice were trained on the rotarod (0890M-D54 Rotamex-5, Columbus Instruments) for 2 days before data collection( 115 ). During each trial, mice were placed on the rod at 10 rpm for 60 seconds, and the rod accelerated from 10 to 30 rpm at 30-second intervals. The total maximum testing time was 240 seconds. Each trial was performed twice daily at 2-hour intervals for three consecutive days. The latency to fall was recorded, and the most prolonged latency was used for analysis. Transcriptome (RNA-seq) analysis Three WT and 4 Jag2 sm homozygous RNA samples were extracted from MuSCs isolated from hindlimb muscles. The RNA samples underwent Tapestation analysis (Agilent) to ensure RNA quality for long-read sequencing. 1μg of each RNA sample was used as input for cDNA synthesis library prep following the Ligation sequencing V14 - Direct cDNA sequencing (SQK-LSK114) protocol from Oxford Nanopore Technologies (ONT) until elution at the cDNA repair and end-prep step. The cDNA was eluted in 23.5μl water. Following cDNA end prep, barcodes were ligated so that the cDNA could be pooled. 22.5μl end-prepped cDNA, 2.5μl Native Barcode (NB05-11 from SQK-NBD114.24), and 25µl Blunt/TA Ligase Master Mix were combined, following the Direct cDNA sequencing - native barcoding (SQK-DCS109 with EXP-NBD104 and EXP-NBD114) protocol. 5μl EDTA was used to inactivate the ligation reaction after 20 min of incubation. Half of each barcoded cDNA sample was pooled into one library, and the remaining half was pooled into a second library. The rest of the library prep and loading followed the protocol for Ligation sequencing amplicons - Native Barcoding Kit 24 V14 (SQK-NBD114.24) . The barcoded pooled libraries were loaded on two different PromethION flow cells (FLO-PRO114M). Sequencing was performed on a P2-solo (ONT) for ∼23 hours, after data acquisition had plateaued. The raw data was base called using dorado/0.5.3 with the --min-qscore 7 --kit-name ’SQK-NBD114-24’ --no-trim flags, and the data were assembled to the GRCm39 mouse genome (GRCm39). The resulting .bam file was demultiplexed by barcode using dorado demux using the --emit-fastq flag. The resulting .fastq files from corresponding barcodes of the two different libraries were concatenated together, and then transcripts from each sample were counted using minimap2/2.17 htseq. The transcript counts were input into DESeq2 using R 4.3.0 using default analysis parameters, and significantly differentially expressed genes (padj < 0.05) were recorded. Genes with low expression (baseMean < 10) were filtered out of gene enrichment analysis, but the results from the whole transcriptome were saved. GO analysis was performed using Metascape v3.5.20250101( 116 ). Gene information was obtained from NCBI database ( https://www.ncbi.nlm.nih.gov/gene ).= Transgenic Drosophila generation Human reference JAG2 and variants were cloned from the pcDNA3 constructs into the pUASTattB vector using the EcoRI and XbaI sites for Drosophila expression via the in-fusion cloning method (Takara). The resulting plasmids were sequenced and verified (Eurofins Genomics). Transgenic animals were generated by Bestgene through φC31 integrase-mediated transgenesis on the second chromosome landing site, attP40. Drosophila husbandry All Drosophila lines were maintained with standard Bloomington food at 25°C with 70% humidity and a 12-hour light:12-hour dark cycle. All experiments were conducted at 27°C to enhance transgene expression efficiency. Drosophila behavioral assays For a flight assay, 25 flies from each group were collected and aged for four days. They were funneled into a 500-ml glass cylinder. The distribution of flies in the cylinder was recorded by a document camera (IPEVO), and the average scores from five independent experiments were calculated. For a negative geotaxis assay, 25 flies from each group were collected and aged for four days. Flies were tapped down by gently striking the vials on the surface of a table. Their climbing behaviors were recorded by a document camera (IPEVO) for 30 seconds. Movie clips were exported and analyzed by ImageJ. Statistical significance was calculated by a two-sided t-test. Statistical analysis Statistical analysis was performed using Prism 10 (Graphpad). For comparison between two groups, an unpaired t-test was used. For comparison between multiple groups, a one-way ANOVA was used with multiple comparisons to the control. Distributions were compared using a chi-squared test. Graphing of the data was performed using Prism 10. All values are means ± SEM unless noted otherwise. *, p<0.05; **, p<0.01; ***, p<0.001. Acknowledgments We thank the Minnesota Supercomputing Institute (MSI) and the University of Minnesota Imaging Center (UIC). We thank Dr. Yoshiaki Kubota for kindly providing Cdh5 +/CreERT2 mice. This work was supported by the Department of Defense (DoD) Award (HT9425-23-1-0461), Muscular Dystrophy Association (MDA) Research Grant (1297954) and Greg Marzolf Jr. Research Foundation to AA. Funder Information Declared Department of Defense (DoD) Award Muscular Dystrophy Association (MDA) Research Grant , 1297954 References 1. ↵ Chakkalakal JV , et al. The aged niche disrupts muscle stem cell quiescence . Nature . 2012 ; 490 ( 7420 ): 355 – 360 . OpenUrl CrossRef PubMed Web of Science 2. Chakkalakal JV , et al. Early forming label-retaining muscle stem cells require p27kip1 for maintenance of the primitive state . Development . 2014 ; 141 ( 8 ): 1649 – 1659 . OpenUrl Abstract / FREE Full Text 3. Schultz E . Satellite cell proliferative compartments in growing skeletal muscles . Dev Biol . 1996 ; 175 ( 1 ): 84 – 94 . OpenUrl CrossRef PubMed Web of Science 4. ↵ Shea KL , et al. Sprouty1 regulates reversible quiescence of a self-renewing adult muscle stem cell pool during regeneration . Cell Stem Cell . 2010 ; 6 ( 2 ): 117 – 129 . OpenUrl CrossRef PubMed Web of Science 5. ↵ Dumont NA , et al. Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division . Nat Med . 2015 ; 21 ( 12 ): 1455 – 1463 . OpenUrl CrossRef PubMed 6. Rhoads RP , et al. Satellite cell-mediated angiogenesis in vitro coincides with a functional hypoxia-inducible factor pathway . Am J Physiol Cell Physiol . 2009 ; 296 ( 6 ): C1321 – 1328 . OpenUrl CrossRef PubMed Web of Science 7. ↵ Urciuolo A , et al. Collagen VI regulates satellite cell self-renewal and muscle regeneration . Nat Commun . 2013 ; 4 : 1964 . OpenUrl CrossRef PubMed 8. ↵ Bentzinger CF , et al. Fibronectin regulates Wnt7a signaling and satellite cell expansion . Cell Stem Cell . 2013 ; 12 ( 1 ): 75 – 87 . OpenUrl CrossRef PubMed Web of Science 9. ↵ Bjornson CRR , et al. Notch signaling is necessary to maintain quiescence in adult muscle stem cells . Stem Cells . 2012 ; 30 ( 2 ): 232 – 242 . OpenUrl CrossRef PubMed Web of Science 10. ↵ Goel AJ , et al. Niche Cadherins Control the Quiescence-to-Activation Transition in Muscle Stem Cells . Cell Rep . 2017 ; 21 ( 8 ): 2236 – 2250 . OpenUrl CrossRef PubMed 11. ↵ Abou-Khalil R , et al. Autocrine and paracrine angiopoietin 1/Tie-2 signaling promotes muscle satellite cell self-renewal . Cell Stem Cell . 2009 ; 5 ( 3 ): 298 – 309 . OpenUrl CrossRef PubMed Web of Science 12. Christov C , et al. Muscle satellite cells and endothelial cells: close neighbors and privileged partners . Mol Biol Cell . 2007 ; 18 ( 4 ): 1397 – 1409 . OpenUrl Abstract / FREE Full Text 13. ↵ Kostallari E , et al. Pericytes in the myovascular niche promote post-natal myofiber growth and satellite cell quiescence . Development . 2015 ; 142 ( 7 ): 1242 – 1253 . OpenUrl Abstract / FREE Full Text 14. ↵ Verma M , et al. Muscle Satellite Cell Cross-Talk with a Vascular Niche Maintains Quiescence via VEGF and Notch Signaling . Cell Stem Cell . 2018 ; 23 ( 4 ): 530 – 543.e9 . OpenUrl CrossRef PubMed 15. ↵ Verma M , et al. Flt-1 haploinsufficiency ameliorates muscular dystrophy phenotype by developmentally increased vasculature in mdx mice . Hum Mol Genet . 2010 ; 19 ( 21 ): 4145 – 4159 . OpenUrl CrossRef PubMed Web of Science 16. Verma M , et al. Inhibition of FLT1 ameliorates muscular dystrophy phenotype by increased vasculature in a mouse model of Duchenne muscular dystrophy . PLoS Genet . 2019 ; 15 ( 12 ): e1008468 . OpenUrl CrossRef PubMed 17. ↵ Verma M , et al. Endothelial cell signature in muscle stem cells validated by VEGFA-FLT1-AKT1 axis promoting survival of muscle stem cell . Elife . 2024 ; 13 : e73592 . OpenUrl PubMed 18. ↵ Chillakuri CR , et al. Notch receptor-ligand binding and activation: insights from molecular studies . Semin Cell Dev Biol . 2012 ; 23 ( 4 ): 421 – 428 . OpenUrl CrossRef PubMed 19. ↵ Vargas-Franco D , et al. The Notch signaling pathway in skeletal muscle health and disease . Muscle Nerve . 2022 ; 66 ( 5 ): 530 – 544 . OpenUrl CrossRef PubMed 20. ↵ Seandel M , et al. Generation of a functional and durable vascular niche by the adenoviral E4ORF1 gene . Proc Natl Acad Sci U S A . 2008 ; 105 ( 49 ): 19288 – 19293 . OpenUrl Abstract / FREE Full Text 21. Rios AC , et al. Neural crest regulates myogenesis through the transient activation of NOTCH . Nature . 2011 ; 473 ( 7348 ): 532 – 535 . OpenUrl CrossRef PubMed 22. ↵ Mourikis P , et al. A critical requirement for notch signaling in maintenance of the quiescent skeletal muscle stem cell state . Stem Cells . 2012 ; 30 ( 2 ): 243 – 252 . OpenUrl CrossRef PubMed Web of Science 23. ↵ Bröhl D , et al. Colonization of the satellite cell niche by skeletal muscle progenitor cells depends on Notch signals . Dev Cell . 2012 ; 23 ( 3 ): 469 – 481 . OpenUrl CrossRef PubMed Web of Science 24. ↵ Morrison SJ , Spradling AC . Stem cells and niches: mechanisms that promote stem cell maintenance throughout life . Cell . 2008 ; 132 ( 4 ): 598 – 611 . OpenUrl CrossRef PubMed Web of Science 25. ↵ Butler JM , et al. Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells . Cell Stem Cell . 2010 ; 6 ( 3 ): 251 – 264 . OpenUrl CrossRef PubMed Web of Science 26. Hadland BK , et al. Endothelium and NOTCH specify and amplify aorta-gonad-mesonephros-derived hematopoietic stem cells . J Clin Invest . 2015 ; 125 ( 5 ): 2032 – 2045 . OpenUrl CrossRef PubMed 27. Kobayashi H , et al. Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells . Nat Cell Biol . 2010 ; 12 ( 11 ): 1046 – 1056 . OpenUrl CrossRef PubMed Web of Science 28. ↵ Rafii S , et al. Human ESC-derived hemogenic endothelial cells undergo distinct waves of endothelial to hematopoietic transition . Blood . 2013 ; 121 ( 5 ): 770 – 780 . OpenUrl Abstract / FREE Full Text 29. ↵ Ottone C , et al. Direct cell-cell contact with the vascular niche maintains quiescent neural stem cells . Nat Cell Biol . 2014 ; 16 ( 11 ): 1045 – 1056 . OpenUrl CrossRef PubMed Web of Science 30. ↵ Coppens S , et al. A form of muscular dystrophy associated with pathogenic variants in JAG2 . Am J Hum Genet . 2021 ; 108 ( 5 ): 840 – 856 . OpenUrl CrossRef PubMed 31. ↵ Logan CV , et al. Mutations in MEGF10, a regulator of satellite cell myogenesis, cause early onset myopathy, areflexia, respiratory distress and dysphagia (EMARDD) . Nat Genet . 2011 ; 43 ( 12 ): 1189 – 1192 . OpenUrl CrossRef PubMed 32. ↵ Boyden SE , et al. Mutations in the satellite cell gene MEGF10 cause a recessive congenital myopathy with minicores . Neurogenetics . 2012 ; 13 ( 2 ): 115 – 124 . OpenUrl CrossRef PubMed Web of Science 33. ↵ Servián-Morilla E , et al. A POGLUT1 mutation causes a muscular dystrophy with reduced Notch signaling and satellite cell loss . EMBO Mol Med . 2016 ; 8 ( 11 ): 1289 – 1309 . OpenUrl Abstract / FREE Full Text 34. ↵ Ogasawara M , et al. CGG expansion in NOTCH2NLC is associated with oculopharyngodistal myopathy with neurological manifestations . Acta Neuropathol Commun . 2020 ; 8 ( 1 ): 204 . OpenUrl PubMed 35. ↵ Appella E , Weber IT , Blasi F . Structure and function of epidermal growth factor-like regions in proteins . FEBS Lett . 1988 ; 231 ( 1 ): 1 – 4 . OpenUrl CrossRef PubMed Web of Science 36. ↵ Vargas-Franco D , et al. The Notch signaling pathway in skeletal muscle health and disease . Muscle Nerve . [published online ahead of print: August 15, 2022 ]. doi: 10.1002/mus.27684 . OpenUrl CrossRef PubMed 37. ↵ Shimizu K , et al. Binding of Delta1, Jagged1, and Jagged2 to Notch2 rapidly induces cleavage, nuclear translocation, and hyperphosphorylation of Notch2 . Mol Cell Biol . 2000 ; 20 ( 18 ): 6913 – 6922 . OpenUrl Abstract / FREE Full Text 38. ↵ Shimizu K , et al. Physical interaction of Delta1, Jagged1, and Jagged2 with Notch1 and Notch3 receptors . Biochem Biophys Res Commun . 2000 ; 276 ( 1 ): 385 – 389 . OpenUrl CrossRef PubMed 39. ↵ Fleming RJ , et al. The gene Serrate encodes a putative EGF-like transmembrane protein essential for proper ectodermal development in Drosophila melanogaster . Genes Dev . 1990 ; 4 ( 12A ): 2188 – 2201 . OpenUrl Abstract / FREE Full Text 40. ↵ Shawber C , et al. Jagged2: a serrate-like gene expressed during rat embryogenesis . Dev Biol . 1996 ; 180 ( 1 ): 370 – 376 . OpenUrl CrossRef PubMed Web of Science 41. ↵ Valsecchi C , et al. JAGGED2: a putative Notch ligand expressed in the apical ectodermal ridge and in sites of epithelial-mesenchymal interactions . Mech Dev . 1997 ; 69 ( 1–2 ): 203 – 207 . OpenUrl CrossRef PubMed Web of Science 42. Luo B , et al. Isolation and functional analysis of a cDNA for human Jagged2, a gene encoding a ligand for the Notch1 receptor . Mol Cell Biol . 1997 ; 17 ( 10 ): 6057 – 6067 . OpenUrl Abstract / FREE Full Text 43. ↵ Deng Y , et al. Characterization, chromosomal localization, and the complete 30-kb DNA sequence of the human Jagged2 (JAG2) gene . Genomics . 2000 ; 63 ( 1 ): 133 – 138 . OpenUrl CrossRef PubMed 44. ↵ Gray GE , et al. Human ligands of the Notch receptor . Am J Pathol . 1999 ; 154 ( 3 ): 785 – 794 . OpenUrl CrossRef PubMed Web of Science 45. ↵ Irvin DK , et al. Patterns of Jagged1, Jagged2, Delta-like 1 and Delta-like 3 expression during late embryonic and postnatal brain development suggest multiple functional roles in progenitors and differentiated cells . J Neurosci Res . 2004 ; 75 ( 3 ): 330 – 343 . OpenUrl CrossRef PubMed Web of Science 46. ↵ Stump G , et al. Notch1 and its ligands Delta-like and Jagged are expressed and active in distinct cell populations in the postnatal mouse brain . Mech Dev . 2002 ; 114 ( 1–2 ): 153 – 159 . OpenUrl CrossRef PubMed Web of Science 47. ↵ Sander GR , Powell BC . Expression of notch receptors and ligands in the adult gut . J Histochem Cytochem . 2004 ; 52 ( 4 ): 509 – 516 . OpenUrl CrossRef PubMed Web of Science 48. ↵ Sander GR , Brookes SJH , Powell BC . Expression of Notch1 and Jagged2 in the enteric nervous system . J Histochem Cytochem . 2003 ; 51 ( 7 ): 969 – 972 . OpenUrl CrossRef PubMed Web of Science 49. ↵ DeHart SL , Heikens MJ , Tsai S . Jagged2 promotes the development of natural killer cells and the establishment of functional natural killer cell lines . Blood . 2005 ; 105 ( 9 ): 3521 – 3527 . OpenUrl Abstract / FREE Full Text 50. ↵ Kared H , et al. Jagged2-expressing hematopoietic progenitors promote regulatory T cell expansion in the periphery through notch signaling . Immunity . 2006 ; 25 ( 5 ): 823 – 834 . OpenUrl CrossRef PubMed Web of Science 51. ↵ Johnson J , et al. Notch pathway genes are expressed in mammalian ovarian follicles . Mech Dev . 2001 ; 109 ( 2 ): 355 – 361 . OpenUrl CrossRef PubMed 52. ↵ Vanorny DA , et al. Notch signaling regulates ovarian follicle formation and coordinates follicular growth . Mol Endocrinol . 2014 ; 28 ( 4 ): 499 – 511 . OpenUrl CrossRef PubMed 53. ↵ Guo P , et al. Endothelial jagged-2 sustains hematopoietic stem and progenitor reconstitution after myelosuppression . J Clin Invest . 2017 ; 127 ( 12 ): 4242 – 4256 . OpenUrl CrossRef PubMed 54. ↵ Palano MT , et al. Jagged Ligands Enhance the Pro-Angiogenic Activity of Multiple Myeloma Cells . Cancers (Basel ) . 2020 ; 12 ( 9 ): E2600 . OpenUrl 55. ↵ Becam I , et al. A role of receptor Notch in ligand cis - inhibition in Drosophila . Curr Biol . 2010 ; 20 ( 6 ): 554 – 560 . OpenUrl CrossRef PubMed 56. Fiuza U-M , et al. Mechanisms of ligand-mediated inhibition in Notch signaling activity in Drosophila . Dev Dyn . 2010 ; 239 ( 3 ): 798 – 805 . OpenUrl CrossRef PubMed Web of Science 57. Sprinzak D , et al. Cis - interactions between Notch and Delta generate mutually exclusive signalling states . Nature . 2010 ; 465 ( 7294 ): 86 – 90 . OpenUrl CrossRef PubMed Web of Science 58. ↵ Thambyrajah R , et al. Cis inhibition of NOTCH1 through JAGGED1 sustains embryonic hematopoietic stem cell fate . Nat Commun . 2024 ; 15 ( 1 ): 1604 . OpenUrl CrossRef PubMed 59. ↵ Nandagopal N , Santat LA , Elowitz MB . Cis - activation in the Notch signaling pathway . Elife . 2019 ; 8 : e37880 . OpenUrl CrossRef PubMed 60. ↵ Gu JM , et al. An NF-κB - EphrinA5-Dependent Communication between NG2+ Interstitial Cells and Myoblasts Promotes Muscle Growth in Neonates . Developmental Cell . 2016 ; 36 ( 2 ): 215 – 224 . OpenUrl CrossRef PubMed 61. Vaz R , et al. Fibronectin promotes migration, alignment and fusion in an in vitro myoblast cell model . Cell and tissue research . 2012 ; 348 ( 3 ): 569 – 78 . OpenUrl CrossRef PubMed 62. ↵ Bentzinger CF , et al. Article Fibronectin Regulates Wnt7a Signaling and Satellite Cell Expansion . Stem Cell . 2013 ; 12 ( 1 ): 75 – 87 . OpenUrl 63. ↵ Sidow A , et al. Serrate2 is disrupted in the mouse limb-development mutant syndactylism . Nature . 1997 ; 389 ( 6652 ): 722 – 725 . OpenUrl CrossRef PubMed Web of Science 64. ↵ Jiang R , et al. Defects in limb, craniofacial, and thymic development in Jagged2 mutant mice . Genes Dev . 1998 ; 12 ( 7 ): 1046 – 1057 . OpenUrl Abstract / FREE Full Text 65. ↵ Murphy MM , et al. Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration . Development . 2011 ; 138 ( 17 ): 3625 – 3637 . OpenUrl Abstract / FREE Full Text 66. ↵ Dong Z , et al. Gamma-Secretase Inhibitor (DAPT), a potential therapeutic target drug, caused neurotoxicity in planarian regeneration by inhibiting Notch signaling pathway . Sci Total Environ . 2021 ; 781 : 146735 . OpenUrl CrossRef PubMed 67. ↵ Weintraub H , et al. MyoD binds cooperatively to two sites in a target enhancer sequence: occupancy of two sites is required for activation . Proc Natl Acad Sci U S A . 1990 ; 87 ( 15 ): 5623 – 5627 . OpenUrl Abstract / FREE Full Text 68. ↵ Watanabe S , et al. MyoD gene suppression by Oct4 is required for reprogramming in myoblasts to produce induced pluripotent stem cells . Stem Cells . 2011 ; 29 ( 3 ): 505 – 516 . OpenUrl CrossRef PubMed Web of Science 69. ↵ Gunage RD , Reichert H , VijayRaghavan K . Identification of a new stem cell population that generates Drosophila flight muscles . eLife . 2014 ; 3 : e03126 . OpenUrl CrossRef PubMed 70. ↵ Deng M , et al. Single cell transcriptomic landscapes of pattern formation, proliferation and growth in Drosophila wing imaginal discs . Development . 2019 ; 146 ( 18 ): dev179754 . OpenUrl Abstract / FREE Full Text 71. ↵ Zeng C , et al. Delta and Serrate are redundant Notch ligands required for asymmetric cell divisions within the Drosophila sensory organ lineage . Genes Dev . 1998 ; 12 ( 8 ): 1086 – 1091 . OpenUrl Abstract / FREE Full Text 72. ↵ de Celis JF , et al. Notch signalling mediates segmentation of the Drosophila leg . Development . 1998 ; 125 ( 23 ): 4617 – 4626 . OpenUrl Abstract 73. ↵ Dofash L , et al. Three novel missense variants in two families with JAG2-associated limb-girdle muscular dystrophy . Neuromuscul Disord . 2024 ; 42 : 36 – 42 . OpenUrl PubMed 74. ↵ Nikitin S , et al. Case Report: Exploring the clinical spectrum of LGMD R27: insights from a case study with homozygous pathogenic variant in the JAG2 gene . Front Pediatr . 2024 ; 12 : 1414465 . OpenUrl PubMed 75. ↵ Oda T , et al. Mutations in the human Jagged1 gene are responsible for Alagille syndrome . Nat Genet . 1997 ; 16 ( 3 ): 235 – 242 . OpenUrl CrossRef PubMed Web of Science 76. ↵ Li L , et al. Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1 . Nat Genet . 1997 ; 16 ( 3 ): 243 – 251 . OpenUrl CrossRef PubMed Web of Science 77. ↵ Vieira NM , et al. Jagged 1 Rescues the Duchenne Muscular Dystrophy Phenotype . Cell . 2015 ; 163 ( 5 ): 1204 – 1213 . OpenUrl CrossRef PubMed 78. ↵ De Souza Leite F , et al. Muscle-specific increased expression of JAG1 improves skeletal muscle phenotype in dystrophin-deficient mice [preprint] . 2025 . doi: 10.1101/2025.03.12.642857 . OpenUrl Abstract / FREE Full Text 79. ↵ Lan Y , et al. The Jagged2 gene maps to chromosome 12 and is a candidate for the lgl and sm mutations . Mamm Genome . 1997 ; 8 ( 11 ): 875 – 876 . OpenUrl PubMed 80. ↵ Casey LM , et al. Jag2-Notch1 signaling regulates oral epithelial differentiation and palate development . Dev Dyn . 2006 ; 235 ( 7 ): 1830 – 1844 . OpenUrl CrossRef PubMed Web of Science 81. ↵ Matsakas A , et al. Muscle ERRγ mitigates Duchenne muscular dystrophy via metabolic and angiogenic reprogramming . FASEB J . 2013 ; 27 ( 10 ): 4004 – 4016 . OpenUrl CrossRef PubMed Web of Science 82. ↵ Fujimaki S , et al. The endothelial Dll4–muscular Notch2 axis regulates skeletal muscle mass . Nat Metab . 2022 ; 4 ( 2 ): 180 – 189 . OpenUrl PubMed 83. ↵ Fujimaki S , et al. Notch1 and Notch2 Coordinately Regulate Stem Cell Function in the Quiescent and Activated States of Muscle Satellite Cells . Stem Cells . 2018 ; 36 ( 2 ): 278 – 285 . OpenUrl CrossRef PubMed 84. ↵ Low S , et al. Delta-Like 4 Activates Notch 3 to Regulate Self-Renewal in Skeletal Muscle Stem Cells . Stem Cells . 2018 ; 36 ( 3 ): 458 – 466 . OpenUrl CrossRef PubMed 85. ↵ Eliazer S , et al. Heterogeneous levels of delta-like 4 within a multinucleated niche cell maintains muscle stem cell diversity . Elife . 2022 ; 11 : e68180 . OpenUrl CrossRef PubMed 86. ↵ Bi P , et al. Stage-specific effects of Notch activation during skeletal myogenesis . Elife . 2016 ; 5 : e17355 . OpenUrl CrossRef PubMed 87. ↵ Li H , et al. Fly Cell Atlas: A single-nucleus transcriptomic atlas of the adult fruit fly . Science . 2022 ; 375 ( 6584 ): eabk2432 . OpenUrl CrossRef PubMed 88. ↵ del Álamo D , Rouault H , Schweisguth F . Mechanism and significance of cis-inhibition in Notch signalling . Curr Biol . 2011 ; 21 ( 1 ): R40 – 47 . OpenUrl CrossRef PubMed 89. Yaron A , Sprinzak D . The cis side of juxtacrine signaling: a new role in the development of the nervous system . Trends Neurosci . 2012 ; 35 ( 4 ): 230 – 239 . OpenUrl CrossRef PubMed Web of Science 90. ↵ Chen D , et al. A new model of Notch signalling: Control of Notch receptor cis-inhibition via Notch ligand dimers . PLoS Comput Biol . 2023 ; 19 ( 1 ): e1010169 . OpenUrl PubMed 91. ↵ Xu X , et al. Jag1-Notch cis - interaction determines cell fate segregation in pancreatic development . Nat Commun . 2023 ; 14 ( 1 ): 348 . OpenUrl CrossRef PubMed 92. ↵ Formosa-Jordan P , Ibañes M . Competition in notch signaling with cis enriches cell fate decisions . PLoS One . 2014 ; 9 ( 4 ): e95744 . OpenUrl CrossRef PubMed 93. ↵ Kuintzle R , Santat LA , Elowitz MB . Diversity in Notch ligand-receptor signaling interactions . Elife . 2025 ; 12 : RP91422 . OpenUrl PubMed 94. ↵ Fukada S , et al. Molecular signature of quiescent satellite cells in adult skeletal muscle . Stem Cells . 2007 ; 25 ( 10 ): 2448 – 2459 . OpenUrl CrossRef PubMed Web of Science 95. ↵ Den Hartog L , Asakura A . Implications of notch signaling in duchenne muscular dystrophy . Front Physiol . 2022 ; 13 : 984373 . OpenUrl PubMed 96. ↵ Lamar E , et al. Nrarp is a novel intracellular component of the Notch signaling pathway . Genes Dev . 2001 ; 15 ( 15 ): 1885 – 1899 . OpenUrl Abstract / FREE Full Text 97. ↵ Jarrett SM , et al. Extension of the Notch intracellular domain ankyrin repeat stack by NRARP promotes feedback inhibition of Notch signaling . Sci Signal . 2019 ; 12 ( 606 ): eaay2369 . OpenUrl Abstract / FREE Full Text 98. ↵ Kitamoto T , Hanaoka K . Notch3 Null Mutation in Mice Causes Muscle Hyperplasia by Repetitive Muscle Regeneration . Stem Cells . 2010 ; 28 ( 12 ): 2205 – 2216 . OpenUrl CrossRef PubMed Web of Science 99. ↵ Roegiers F , Jan YN . Asymmetric cell division . Curr Opin Cell Biol . 2004 ; 16 ( 2 ): 195 – 205 . OpenUrl CrossRef PubMed Web of Science 100. ↵ Conboy IM , Rando TA . The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis . Dev Cell . 2002 ; 3 ( 3 ): 397 – 409 . OpenUrl CrossRef PubMed Web of Science 101. ↵ Shinin V , et al. Asymmetric division and cosegregation of template DNA strands in adult muscle satellite cells . Nat Cell Biol . 2006 ; 8 ( 7 ): 677 – 687 . OpenUrl CrossRef PubMed Web of Science 102. ↵ Kivelä R , et al. The transcription factor Prox1 is essential for satellite cell differentiation and muscle fibre-type regulation . Nat Commun . 2016 ; 7 : 13124 . OpenUrl CrossRef PubMed 103. ↵ Kakuda S , et al. Canonical Notch ligands and Fringes have distinct effects on NOTCH1 and NOTCH2 . J Biol Chem . 2020 ; 295 ( 43 ): 14710 – 14722 . OpenUrl Abstract / FREE Full Text 104. ↵ Logan CV , et al. Mutations in MEGF10, a regulator of satellite cell myogenesis, cause early onset myopathy, areflexia, respiratory distress and dysphagia (EMARDD) . Nat Genet . 2011 ; 43 ( 12 ): 1189 – 1192 . OpenUrl CrossRef PubMed 105. ↵ Boyden SE , et al. Mutations in the satellite cell gene MEGF10 cause a recessive congenital myopathy with minicores . Neurogenetics . 2012 ; 13 ( 2 ): 115 – 124 . OpenUrl CrossRef PubMed Web of Science 106. ↵ Servián-Morilla E , et al. A POGLUT1 mutation causes a muscular dystrophy with reduced Notch signaling and satellite cell loss . EMBO Mol Med . 2016 ; 8 ( 11 ): 1289 – 1309 . OpenUrl Abstract / FREE Full Text 107. ↵ Saha M , et al. Consequences of MEGF10 deficiency on myoblast function and Notch1 interactions . Hum Mol Genet . 2017 ; 26 ( 15 ): 2984 – 3000 . OpenUrl CrossRef PubMed 108. ↵ Madisen L , et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain . Nat Neurosci . 2010 ; 13 ( 1 ): 133 – 140 . OpenUrl CrossRef PubMed Web of Science 109. ↵ Ema M , Takahashi S , Rossant J . Deletion of the selection cassette, but not cis - acting elements, in targeted Flk1-lacZ allele reveals Flk1 expression in multipotent mesodermal progenitors . Blood . 2006 ; 107 ( 1 ): 111 – 117 . OpenUrl Abstract / FREE Full Text 110. ↵ Okabe K , et al. Neurons limit angiogenesis by titrating VEGF in retina . Cell . 2014 ; 159 ( 3 ): 584 – 596 . OpenUrl CrossRef PubMed Web of Science 111. ↵ Motohashi N , Asakura Y , Asakura A . Isolation, culture, and transplantation of muscle satellite cells . J Vis Exp . [published online ahead of print: April 8, 2014 ];( 86 ). doi: 10.3791/50846 . OpenUrl CrossRef 112. ↵ Nishimura M , et al. Structure, chromosomal locus, and promoter of mouse Hes2 gene, a homologue of Drosophila hairy and Enhancer of split . Genomics . 1998 ; 49 ( 1 ): 69 – 75 . OpenUrl CrossRef PubMed Web of Science 113. ↵ Aartsma-Rus A , van Putten M . Assessing functional performance in the mdx mouse model . J Vis Exp . 2014 ;( 85 ): 51303 . OpenUrl CrossRef 114. ↵ Fukada S , et al. Genetic Background Affects Properties of Satellite Cells and mdx Phenotypes . The American Journal of Pathology . 2010 ; 176 ( 5 ): 2414 – 2424 . OpenUrl CrossRef PubMed Web of Science 115. ↵ Jin Q , et al. A GDF11/myostatin inhibitor, GDF11 propeptide-Fc, increases skeletal muscle mass and improves muscle strength in dystrophic mdx mice . Skeletal Muscle . 2019 ; 9 ( 1 ): 16 . OpenUrl PubMed 116. ↵ Zhou Y , et al. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets . Nat Commun . 2019 ; 10 ( 1 ): 1523 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted July 27, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following JAG2-related muscular dystrophy and Notch signaling dysfunction in muscle stem cells Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share JAG2 -related muscular dystrophy and Notch signaling dysfunction in muscle stem cells Nam Chul Kim , Minoru Tanaka , Isabelle Draper , Hannah R. Littel , Mekala Gunasekaran , Johnnie Turner , Natalya M. Wells , Qasim Mujteba , Yoko Asakura , Peter B. Kang , Atsushi Asakura bioRxiv 2025.07.23.665646; doi: https://doi.org/10.1101/2025.07.23.665646 Share This Article: Copy Citation Tools JAG2 -related muscular dystrophy and Notch signaling dysfunction in muscle stem cells Nam Chul Kim , Minoru Tanaka , Isabelle Draper , Hannah R. Littel , Mekala Gunasekaran , Johnnie Turner , Natalya M. Wells , Qasim Mujteba , Yoko Asakura , Peter B. Kang , Atsushi Asakura bioRxiv 2025.07.23.665646; doi: https://doi.org/10.1101/2025.07.23.665646 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Developmental Biology Subject Areas All Articles Animal Behavior and Cognition (7618) Biochemistry (17636) Bioengineering (13860) Bioinformatics (41847) Biophysics (21401) Cancer Biology (18536) Cell Biology (25424) Clinical Trials (138) Developmental Biology (13353) Ecology (19860) Epidemiology (2067) Evolutionary Biology (24287) Genetics (15583) Genomics (22463) Immunology (17701) Microbiology (40300) Molecular Biology (17141) Neuroscience (88434) Paleontology (666) Pathology (2825) Pharmacology and Toxicology (4813) Physiology (7633) Plant Biology (15107) Scientific Communication and Education (2042) Synthetic Biology (4285) Systems Biology (9808) Zoology (2268)