Additional Supporting Information may be found in the online version of this article.

STEM_775_sm_supplFigure1.pdf419KFigure S1. Notch signalling activity in muscle stem/progenitors. A) Cells isolated from adult Tg:Pax7-nGFP mice by FACS, cultured for 24h and immunostained for markers indicated. Arrow indicates a rare cell that does not express muscle markers and is not GFP positive. B) Comparison of Notch related genes from microarrays of Pax7-nGFP stem/progenitor cells at P8 (proliferating) and 4-5 weeks old adult (quiescent). Two independent probe sets for HeyL show upregulation; Dll1 and Hes6 are associated with the differentiated state and are downregulated. C) RT-qPCR of HeyL transcripts in Pax7- nGFP myogenic cells during prenatal and postnatal development. Data are represented as 2∧-δδCT ratios normalised to the E12.5 sample. D) Verification of Dll1-Fc and DAPT efficiency on activating and blocking Notch signalling, respectively. For activation, myogenic C2C12 cells were cultured for 24h in the presence of immobilised Dll1-Fc ligand, or the Fc fragment alone as control. For inhibition, samples were incubated in the presence of the γ-secretase inhibitor DAPT (20μM) or DMSO that is used as carrier. Relative mRNA levels of HeyL are presented, normalised to Fc DMSO control (basal activity). Note strong induction by Dll1-Fc is completely blocked by DAPT (third column). Data represent average values +/− s.e.m. E) Characterisation of Pax7-nGFP Hi, Intermediate and Low fractions by RT-qPCR for genes indicated. Scale bar: A, 50μm.
STEM_775_sm_supplFigure2.pdf619KFigure S2. Selective abrogation of Rbpj function results in loss of satellite cells. A) The Tg:Pax7-CT2 mouse was generated using a BAC containing the entire Pax7 gene (orange box indicates exons and introns) and approximately 100 kbp of flanking sequence (white boxes). The Cre recombinase fused to the simian virus poly(A) sequence (CreERT2::sv40pA) was cloned directly upstream of the starting ATG. Sequence of Exon I: in red is the 5′ UTR, in green the starting ATG and in blue two additional ATGs that were deleted to prevent the misuse of a starting codon (blue box highlights deleted segment upon recombination). Using the same targeting sequences, a loxP-nGFP-loxPnlacZ:: sv40pA transgene was knocked in the Pax7 locus. This knock in, here termed Pax7STOP-nlacZ, will be discussed in more detail in an independent report (R. Sambasivan and S. Tajbakhsh, in preparation). B) E11.5 Pax7nlacZ knock-in embryo (left) showing endogenous Pax7 expression. Tg:Pax7-CT2 :: ROSASTOP-lacZ embryo (right; Tmx-injected at E10.5, collected at E11.5. X-gal staining reveals nlacZ expression in the craniofacial region (neural crest cells) and somites; C) Targeting of quiescent satellite cells using Tg:Pax7-CT2::Rosa26mTomato-STOP-mGFP/+ mice; a, isolated EDL myofibre; b, cross-section of uninjured TA muscle; c, regenerated TA muscle post-injury ; mice were first injected with tamoxifen, injured the following day with notexin and muscle was collected 12 days later; Pax7+/GFP+ cells (arrows) are cells that have reassumed satellite cell, sub-laminal position. D) Whole hindlimb muscle cultures of dissociated skeletal muscles from control (Tg:Pax7-CT2:: Rbpjflox/+ :: Rosa26mTomato-STOP-mGFP/+) and cKO (Tg:Pax7-CT2:: Rbpjflox/- :: Rosa26mTomato-STOP-mGFP/+) mice at day 4 and 7 post-Tmx induction. Immunostaining for Rpbj shows heterogeneous expression in the targeted GFP+ cells on day 4 and no detectible protein on day 7, reflecting gradual loss of Rbpj; white arrows, high signal; arrowheads, low signal; blue arrow no signal. E) GFP+ cells purified by FACS from control and Rbpj cKO mice. Bars represent the average ratio ± s.d. of the percentage of GFP+ satellite cells of the total cell population of mutant relative to control mice (n = 3 mice/condition). F) (left) Whole muscle cultures from control and cKO animals at day 32 post-Tmx induction. The few surviving GFP+ cells in the cKO muscle preparation are Rbpj-positive (“recombination escapers”, data not shown). Dramatic increase in GFP+ myofibres in mutant diaphragm as a result of fusion of the differentiating GFP+ satellite cells to myofibres. Scale bars: C and D 10μm, F 100μm.
STEM_775_sm_supplFigure3.pdf387KFigure S3. The majority of satellite cells bypass S-phase and differentiate in absence of Rbpj. A) Cross-section of regenerating TA muscle 15 days post-injury stained for cleaved Caspase-3 to detect apoptotic cells. Note pycnotic nucleus of the apoptotic cell (blue dot, arrow). No apoptotic GFP+ satellite cells were detected in the Rbpj cKO mice at any time point (scored at days 7, 16 and 20 post-Tmx). B) Wild type TA muscle injured with notexin and exposed continuously to BrdU, then harvested and sectioned 7 days later. Immunostaining shows BrdU incorporation in all myonuclei within regenerated myofibres ensheathed in laminin using this protocol. C) Immunostaining on a regenerating TA muscle of a control mouse (Tg:Pax7-CT2:: Rbpjflox/+ :: Rosa26mTomato- STOP-mGFP/+; see Figure 2C scheme, collected 15 days post-injury). Left panel, low magnification of section; two right panels high magnification of designated area (dotted box). Note Ki67+/GFP+ double positive cell (arrow). D) Cells from control and cKO mice exposed continuously to BrdU ad libitum for 16 days. Cells were purified by FACS, sedimented by cytospin, and fixed immediately. Double immunostaining for BrdU and Myogenin showing a differentiating cKO cell that does not incorporate BrdU. Scale bars: A, 15μm; B, 20μm; C, 25μm, D, 10μm.
STEM_775_sm_supplInfo.pdf46KSupplementary Data

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