Extended Data Fig. 2: Notch signalling regulates Col5 and Col6 expression in vivo.
From: Reciprocal signalling by Notch–Collagen V–CALCR retains muscle stem cells in their niche
a, Satellite cells isolated by FACS at day 10 after tamoxifen injections, from resting tibialis anterior muscle from control (Tg:Pax7-CT2;Rbpj+/−;R26mTmG/+) and Rbpj-null (Tg:Pax7-CT2;Rbpjflox/−;R26mTmG/+) mice immunostained for RBPJ. b, RT–qPCR of collagen genes in Rbpj cKO and control satellite cells. Hey1 used as control for Notch signalling (n = 3 mice per genotype). c, Induction of collagen genes in E17.5 control (Myf5Cre/+;R26mTmG/+) and Myf5Cre-NICD (Myf5Cre/+;R26stop-NICD-nGFP/+) cells isolated by FACS. RT–qPCR was normalized to Gapdh, n = 3 fetuses per genotype. HeyL reports Notch activity. d, FACS plots showing fractionation of GFP+ cells from E17.5 Tg:Pax7-nGFP fetuses into Pax7high (20% of population), Pax7mid (40%), and Pax7low (20%). The intensity of the GFP signal reflects the activity of the Pax7 promoter. e, Transcript levels of GFP+ cells isolated by FACS show a tight correlation between lineage progression, Notch signalling activity and collagen gene expression (n = 3 fetuses per genotype). f, Specificity of α3-COLV antibody assessed by immunostaining of tibialis anterior muscle transverse section from wild-type and Col5a3 cKO P14 postnatal pups (n = 3 mice per genotype). g, Time course of gene expression performed by RT–qPCR on freshly isolated satellite cells (Quiescent), 48 h or 60 h after cardiotoxin injury of tibialis anterior muscle (48 hours post injury (hpi), 60 hpi), and isolated single myofibres from extensor digitorum longus muscle of Tg:Pax7-nGFP mice. Col5a1 and Col5a3 were strongly downregulated in activated and differentiated cells. Quiescence (Pax7, Calcr) and differentiation (Myog) markers are indicated. Col4a2, a major component of the basement membrane, is expressed mainly by myofibres (n = 3 mice per condition). Data are mean ± s.d.; one-sided unpaired t-test. Scale bars, 50 μm.
