Supplementary Figure S1. Hoechst. cells/field. Myogenin. % of Myogenin+ cells. 0 h chase CONTROL. BrdU. 72 h chase. CONTROL BrdU CONTROL BrdU

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1 Hoechst Myogenin amyhc 24 h 48 h 72 h b cells/field h 48h 72h h after plating (4 days growth+ t) c e CONTROL 0 h chase % of Myogenin+ cells h after plating (4 days growth+ t) CONTROL CONTROL 24 h chase 48 h chase d % of Myogenin+ cells h 48h 72h 96h 120h 144h time after isolation from fibers CONTROL 72 h chase Supplementary Figure S1 1

2 1 cell clone 2 cell clone 3 cell clone 4 cell clone 5 cell clone 7 cell clone 8 cell clone 18 cell clone C2C12 Hoechst Figure S1 Growth kinetics and differentiation of satellite cell derived myoblasts. (a) Primary satellite cells were expanded for 4 days in culture, then replated at high density. Antibody staining for the transcription factor Myogenin which marks terminal differentiation, and the differentiation marker Myosin Heavy Chain (MyHC) shows differentiation appearing about 72h after replating; (b) myoblasts from satellite cells exhibit exponential growth kinetics after replating (c) by 72h, 40% of nuclei were Myogenin positive. For b and c, n = 3-5 fields counted with 10X objective; (d) single fibre derived satellite cells were grown in culture and differentiation was monitored by the appearance of Myogenin + cells by antibody staining; (e) the effect of on the expression of and in satellite cell derived myoblasts was assessed by antibody stainings. Single fibre derived satellite cells were grown for 4 days in the presence of, replated and assayed at 24h intervals after chase. Most cells coexpress and during this period (see Fig. 6b); (f) the sensitivity of anti- staining during the chase period was assayed in clones of different sizes obtained after 4 days growth in the presence of and several days of chase. + cells could be detected in satellite cell clones containing 18 cells and C2C12 clones of up to 41 cells (bottom row, right; a few cells in clone shown). -negative cells were detectable after 6-7 cell divisions, but + cells remained visible at this time; Scale bars: a, f 100 µm; e, 30 µm; C2C12, 20 µm. 2

3 a no chase 4 week chase % of /!-gal EDL double positive cells mean std.dev Soleus β-gal mean 58 6 std.dev 31 3 TA mean std.dev b β-gal % of -positive cells 5 days after injury (no chase) 4 weeks of chase injured TA 53±13 (n=3 mice) 9±1 (n=3 mice, 53 regenerating fibers counted) non-injured TA 0.5±0.9 (n=3 mice) not found (n=4 mice, 83 regenerating fibers counted) c d e Hoechst f EDL Soleus TA % of clones with single LRC 6.8±0.8 (n=165 clones, 3 experiments) 0.5±1 (n=414 clones, 3 experiments) 3.5±2 (n=192 clones, 3 experiments) Figure S2 A sub-population of label retaining cells in skeletal muscle. For growth (a) and injury (b) models, the number of + satellite cells were counted at the end of the last pulse. Counts were carried out on sections using anti- or anti-β-gal and anti- antibodies. Observed and normalised LRC percentages appear in Fig. 2 (adjusted to 100% theoretical saturation with taking into account% at end of last pulse assuming linear dependence). Individual mice indicated in Table. Assuming 7.5, 24 or 10 satellite cells per EDL, Soleus, or TA fibre respectively, using our observed LRC frequency values, we estimate that there is 1.7 (EDL), 1.4 (Soleus), or 1.1 (TA) LRC/fibre after 4 wk chase for these muscle groups; m, mean; s, standard deviation. For EDL (n=3; n=4), soleus (n=4; n=3), and TA (n=3; n=3) n values were for no chase and 4 weeks chase, respectively. Template DNA segregation was assessed in LRCs from growth model: (c) Anti- and anti- antibody staining of the first division of a recently divided satellite cell 4 wk of chase, 24h after single fibre isolation. Note template DNA segregation to only one daughter cell; both daughters express. (d) anti- and anti-desmin staining showing Desmin expression in an activated label retaining satellite cell 18h after single fibre isolation (4 wk chase). (e) clonogenic LRC containing a single + cell. Mice were pulse-chased (7 days and 2.5 weeks respectively), single satellite cell derived myoblasts were isolated by mouth pipette after 2-3 days of culture, and plated into individual wells of Terizaki plates. After 72h the clones were stained with anti- antibody (n = 2 clones); (f) single fibre derived satellite cells (4 wk chase in vivo) were plated as single cells and colonies containing a single + LRC were scored after 72h in culture (n, total number of colonies counted). Three mice were used for each muscle group (see Fig. 3d). Scale bars: a, 10 µm; b, 30 µm; e, 50 µm. 3

4 S U P P L E M E N TA R Y I N F O R M AT I O N a e f Numb Figure S3 Selective retention of -labelled template DNA, and preferential cosegregation with Numb, in one daughter cell in primary myogenic cells. (a) anti- and anti-phospho-met-histone3 staining showing selective segregation of + template DNA to one daughter cell during division of a satellite cell derived myoblast (see Fig. 5c); (b) pulsechase labelled cultures (see Fig. 5c) were incubated for 2.5 wk culture to allow differentiation, and stained with anti- and anti-myhc antibodies. + cells are MyHC-negative (n = 15 + cells); (c) control showing that the concentration used is compatible with differentiation. P11 satellite cells were isolated from single fibres, expanded inculture for 4 days, pulsed (4 days) with, chased (3 days) during differentiation. Cultures were stained with anti- and anti-myhc. (d) asymmetricaly dividing primary myogenic cells (18-22h chase) with Numb protein cosegregating with labelled template DNA (see Fig. 7a). Control anti-ki67 shows equivalent daughter cell staining; (e) scheme of labelling of non-template DNA with ; (f) asymmetricaly dividing myoblasts with Numb protein not cosegregating with labelled newly synthesised non-template DNA (top). A confocal section of a second example is shown (bottom). See Fig. 7β, ε. Σχαλε βαρσ: α, δ, φ: 5 µm; b: 100 µm; c: 50 µm. 4

5 Movies D video of asymmetric NumbGFP segregation in satellite cell derived myoblast from cultured single fibres, corresponding to frames in Fig. 1a. Confocal stacks (0.5 µm) were reconstructed for each frame (4 min/frame) and volume rendering was done in Velocity. At the end of the sequence, the intensity was increased to highlight the asymmetry. The cell in movie 2 was rotated to indicate the two daughter cells more readily. Movie 3 Movie of asymmetric NumbGFP segregation in cultured single fibre-derived satellite cells. For technical reasons, this sequence was captured on two movies and fused into one (movie3a: total time: 250 min; movie3b from 250min; total: 2min 12sec). The cell shifted in position during this time, between 224 and 250 min. Cell division occurs after 250min. Movie 4 Movie of symmetric NumbGFP segregation in cultured single fibre-derived satellite cells, taken with fluorescent microscope. Each frame 15 sec., total time min. Movie 5 Live imaging of template DNA segregation in label retaining cells. Mice were pulse-chased (3 days and 3 days respectively), satellite cells were plated, grown for 4 days and filmed for 13h. Cells were then stained with anti- antibody. Phase contrast video microscopy showing tracking of one cell before and after cell division. See Fig. 4b, c for still images and antibody staining. 5