Supplemental Experimental Procedures

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1 Cell Stem Cell, Volume 2 Supplemental Data A Temporal Switch from Notch to Wnt Signaling in Muscle Stem Cells Is Necessary for Normal Adult Myogenesis Andrew S. Brack, Irina M. Conboy, Michael J. Conboy, Jeanne Shen, Thomas A. Rando Supplemental Experimental Procedures Single fiber cultures, Explant Cultures, and Satellite Cell Isolation Single fiber cultures were prepared as described previously (Chargé et al., 2002). Briefly, extensor digitorum longus muscles were digested and single fibers were carefully triturated and cultured in plating medium (10% horse serum (HS); Gibco BRL, Rockville, MD), 0.5% chick embryo extract (CEE; US Biological, Swampscott, MA) in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL)) for 1 day and then switched to proliferation medium (20% fetal bovine serum (FBS); Mediatech, Herndon, VA), 10% HS, 2% CEE in DMEM). Proliferation medium was replaced daily. All cultures were done in 8-well permanox slides (Nunc, Rochester, NY) coated with 1/10 ECM gel (Sigma) at 37 o C in 5% CO 2. Explant cultures of myofibers and associated satellite cells were prepared and maintained, with some modifications, as previously described (Conboy and Rando, 2002). Briefly, hindlimb muscles were incubated in DMEM with 0.2% (w/v) collagenase II (Gibco BRL) for 90 min at 37 o C. Digested muscle was then rinsed in washing buffer

2 (Ham s F-10 nutrient mixture with 10% HS, 10 mm HEPES and 1% penicillin/streptomycin (Gibco BRL)) and dissociated into single myofibers by repeated triturating with a Pasteur pipette. Myofiber fragments were rinsed in washing buffer and centrifuged at 1,000 rpm for 30 seconds. The supernatant was removed, leaving just single myofibers. This was repeated 3 times leaving a pure pellet of myofibers and their associated mononucleated cells. The fibers were then resuspended in growth medium (GM) (Ham s F-10 nutrient mixture with 20% FBS, 5 ng/ml basic fibroblast growth factor (Atlanta Biological, Atlanta, GA) and 1% penicillin/streptomycin (Gibco BRL)) and cultured in flasks coated with ECM gel (Sigma) at 37 o C in 5% CO 2. These cultures contain myofiber fragments and associated satellite cells. Satellite cells were purified from bulk fibers as described (Conboy et al., 2003), with minor modifications. Muscles were subjected to the same procedure described above for bulk myofiber explants, but then rinsed more extensively with washing buffer. Satellite cells were then liberated by further digesting the myofiber fragments in 10 volumes of digesting solution (DS) (washing buffer and 0.5 U/ml dispase (Invitrogen, Carlsbad, CA), and 38 U/ml collagenase type II (US Biological)) for 20 min at 37 o C with agitation. Digests were then dissociated with a 20-guage syringe and further incubated in DS for 10 min. The digest was then subjected to centrifugation at 500g for 1 min to pellet fiber debris, filtration of supernatant through 50 micron mesh, and centrifugation at 1,000g for 5 min to pellet satellite cells. A second wash and spin in washing buffer was done to further purify for myogenic cells.

3 REFERENCES Chargé, S.B., Brack, A.S., and Hughes, S.M. (2002). Aging-related satellite cell differentiation defect occurs prematurely after Ski-induced muscle hypertrophy. Am. J Physiol Cell Physiol 283, C1228-C1241. Conboy, I.M., Conboy, M.J., Smythe, G.M., and Rando, T.A. (2003). Notch-mediated restoration of regenerative potential to aged muscle. Science 302, Conboy, I.M. and Rando, T.A. (2002). The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis. Dev. Cell 3,

4 Figure S1. Wnt signaling and expression of Wnts and Wnt receptors during satellite cell activation and myogenic lineage progression. A) Real-time qrt-pcr analysis of Axin2 a direct downstream target of Wnt signaling, from uninjured and injured TA muscle collected after 3 days of regeneration. Transcript levels were normalized to GAPDH. B) Myofiber explants from TOPGAL mice were plated in GM and myogenic progenitors were stripped off myofibers 1, 2, and 4 days later. Real-time qrt-pcr was performed with the primers specific to LacZ transcript as an index of Wnt signaling activity. Transcript levels were normalized to GAPDH, and changes are presented relative to the levels on day 1 (** p < 0.01). C) Real-time qrt-pcr analysis of expression of canonical Wnts (Wnt1, Wnt3A, and Wnt7A), Wnt receptors (Frizzled-1 and Frizzled-2), and Axin2 from myofiber explants collected after 4 days in culture and compared to values obtained after 1 day in culture. Transcript levels were normalized to GAPDH (* p < 0.05). D) Real-time qrt-pcr analysis of expression of Wnt3A and Frizzled-1 from myogenic progenitors stripped off myofibers at 1, 2, and 4 days in culture. Transcript levels were normalized to GAPDH, and changes are presented relative to the levels on day 1 (* p < 0.05).

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6 Figure S2. Activation of Wnt pathway promotes downstream Wnt reporter activity in myogenic cells. A) Myofibers from TOPGAL mice were maintained in growth conditions for 2 days. Myogenic progenitors were stripped off fibers and plated overnight in GM containing either Wnt3A or the GSK3 inhibitor, each at two different concentrations as indicated. Cells were collected and β-gal activity was measured and normalized to total protein. Activities are presented relative to control levels (** p < 0.01) B) Exogenous Wnt3A (100 ng/ml) or control solution (0.1% BSA) was added overnight to single fiber cultures 2 days after isolation. Cells were fixed and then stained for active (non-phosphorylated) β-catenin (β-catenin*). The percentage of myoblasts expressing β-catenin* increased in cultures exposed to Wnt 3A compared to controls. C) Primary myoblasts in proliferation medium were treated with Wnt3A (60 ng/ml) or control (0.1% BSA) for 8 hrs, fixed and stained for β-catenin. Nuclear localization of β-catenin was observed only in Wnt-treated cultures.

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9 Figure S3. Activation of canonical Wnt pathway promotes myogenic lineage progression and differentiation in vitro. A) Myogenic cells from single fibers were cultured in the presence or absence of GSK inhibitor (BIO, 0.35 mg/ml) overnight, and the cells were then fixed and stained with an antibody against Desmin (green) and with DAPI (blue). Desmin expression increased with GSK inhibitor treatment, phenocopying the effect of exogenous Wnt3A (Fig. 2A). B) After 2 days in culture, myogenic cells from single fiber cultures were treated overnight with either Wnt3A (100 ng/ml) or GSK3 inhibitor (0.35 mg/ml), and the expression of Myf5 (green) was analyzed immunohistochemically and compared to control cultures. DAPI (blue) labels all nuclei. (Left) Representative images. (Right) quantitative analysis. Both Wnt3A and GSK inhibitor treatment promoted myogenic lineage progression, leading to a decrease in the fraction of cells expressing the early lineage marker Myf5 (* p < 0.05). C) Analysis of proliferation of myogenic progenitors in single fiber cultures two days after isolation, with or without exogenous Wnt3A (100 ng/ml) added to the medium. Proliferation was assessed as the percentage of BrdU + cells in cultures in which BrdU was added to the medium for the final 12 hrs (left panel) after a 36 hr exposure to Wnt3A or the percentage of phospho-h3 + cells (right panel) after an overnight exposure to Wnt3A (** p < 0.01).

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11 Figure S4. CD45 + cells present in resting or injured muscle are almost exclusively infiltrating leukocytes and do not reveal active Wnt signaling. A. FACS analysis of TCF-4 expression in CD45 + and CD45 - cells from resting muscle and from regenerating muscle 1 and 4 days after focal injury. In uninjured muscle, almost no cells expressed the TCF-4, a read out of active Wnt signaling. After injury, there was an increase in the percentage of cells expressing TCF-4, but nearly 100% were CD45 -. B. FACS analysis of co-expression of CD45 and leukocyte lineage markers (for T cells, B cells, granulocytes, macrophages, or erythrocytes, using the hematopoietic lineage antibody cocktail from Pharmingen/Beckton-Dickinson (San Jose, CA)) was performed using mononucleated cells from resting (non-injured) and regenerating muscle at different times after injury, as indicated. Nearly all CD45 + cells were positive for lineage markers at every time point identifying them as components of the inflammatory response to muscle injury. Splenocytes were used as a positive control for CD45 and lineage markers (not shown). C. Mononucleated cells present in resting and injured muscle were analyzed for the expression of PCNA by FACS. In injured muscle a clear subset of M-cadherin + activated satellite cells were proliferating based on PCNA expression, but virtually none of the CD45 + cells were proliferative.

12 SOM Figure 4 A Resting Day 1 Day 4 TCF CD45 B Resting Day 1 Day 2 Day 4 Lin CD45 C Resting Injured Resting Injured PCNA PCNA M-cadherin CD45

13 Figure S5. Direct activation of the Notch signaling pathway leads to an increase in active GSK3β in myoblasts. Myoblasts were treated with Notch activating antibody or hamster isotype control antibody for 1 hour and plated in 5% horse serum/dmem for 1 hour. Cells were fixed and analyzed for active Notch (Notch1*), GSK3β py216 (GSK3β*) and total GSK3β (GSK3β). The histogram represents the increased levels of staining after Notch activation compared to control expressed as a percentage. Myoblasts treated with Notch activating antibody had increased levels of Notch1* and GSK3β* compared to control treated myoblasts. Experiments were done in triplicate (* p < 0.05).

14 Figure S6. Model of interaction and temporal switch from Notch to Wnt signaling regulating myogenic lineage progression during postnatal myogenesis. We present a model of the inductive signaling during satellite cell activation and myogenic lineage progression. During satellite cell activation Notch signaling predominates early, when Wnt signaling is relatively low. This leads to satellite cell activation and an expansion of the intermediate progenitor cell population. For myogenic progenitor lineage to progress, there is a necessary increase in Wnt signaling, and a concomitant decrease in Notch signaling leading to further myogenic lineage progression and terminal differentiation. Both pathways alter GSK3β activity (Notch increasing and Wnt decreasing GSK3β activity) making this a potential nodal point for regulating the temporal downstream effects of these two pathways.

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