www.sciencemag.org/cgi/content/full/317/5839/807/DC1

Supporting Online Material for

Increased Wnt Signaling During Aging Alters Muscle Stem Cell Fate

and Increases Fibrosis Andrew S. Brack, Michael J. Conboy, Sudeep Roy, Mark Lee, Calvin J. Kuo, Charles

Keller, Thomas A. Rando*

* To whom correspondence should be addressed. E-mail: rando@stanford.edu

Published 10 August 2007, Science 317, 807 (2007)

DOI: 10.1126/science.1144090

This PDF file includes

Materials and Methods Figs. S1 to S9 References

Brack et al.

SUPPORTING ONLINE MATERIAL

MATERIALS AND METHODS

Animals

C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME) and

NIA. Mice were used at 4-6 months of age (young) or 24-26 months of age (aged), unless

otherwise noted. TOPGAL mice were kindly provided by Elaine Fuchs (Rockefeller

University, NY). Pax7.Cre-ER.ROSA26 mice were generated by crossing a Pax7.Cre-ER

strain (Charles Keller, manuscript in preparation) with the ROSA26 strain (1). Animals

were housed and handled in accordance with the guidelines of Veterinary Medical Unit

of the VA Palo Alto Health Care System and the Administrative Panel on Laboratory

Animal Care of Stanford University.

Reagents

Antibodies to Pax7, embryonic Myosin Heavy Chain (eMyHC) and Myosin

Heavy Chain (MyHC; A4.1025) were obtained from DSHB; to GSK3βpY216 and Collagen

VI from Pharmingen/Beckton-Dickinson (San Jose, CA); to Desmin from Sigma (St.

Louis, MO); to MyoD from Vector Labs (Burlingame, CA) and Santa Cruz (Santa Cruz,

CA); to activated (non-phosphorylated) β-catenin from Upstate Biotechnology (Lake

Placid, NY); to β-gal from Cappel (Aurora, Ohio); to ER-TR7 from Novus Biologicals

(Littleton, Colorado). The chicken polyclonal Syndecan-4 (Syn-4) antibody was

generously provided by Brad Olwin (University of Colorado). Secondary conjugates used

for immunofluorescence detection and flow cytometry were goat anti-mouse Alexa546,

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goat anti-mouse APC, goat anti-rabbit Alexa488, goat anti-rat Alexa488 and donkey anti-

chicken Alexa488 (Molecular Probes).

Recombinant sFRP3, DKK1 and Wnt3A proteins were from R&D Systems

(Minneapolis, MN). Wnt3A was also kindly provided from Roel Nusse (Stanford

University). Tamoxifen and hydroxy-tamoxifen were from Sigma (St. Louis, MO).

Parabiosis

Parabiosis was established as previously described (2, 3). Pairs of mice were

anesthetized and prepared for surgery, and mirror-image incisions are made through the

skin down the side of each animal from behind the ear to before the tail. Shorter (~1 cm)

incisions were made through the abdominal wall. The abdominal openings were sutured

together, and the skin or each mouse was stapled (9 mm Autoclip, Clay Adams) to the

skin of its parabiont, thereby closing the incision. After closure, each mouse was injected

sub-cutaneously with Baytril antibiotic and Buprenex as directed for pain and monitored

during recovery.

Adenovirus and tail vein injections

Adenovirus containing DKK1 and the control expressing murine Fc IgG2a was

obtained as described previously (4). Briefly, DKK1 cDNA was isolated from embryonic

day 17.5 mouse cDNA, sequenced and cloned into the E1 region of E1-E3 Ad strain 5 by

homologues recombination. Mice received a single tail vein injection of 109 PFU of

either virus.

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Serum isolation

Whole blood was collected via partial decapitation of anaesthetized mice.

Collected blood was clotted at 37oC for 4 hrs. Serum was isolated by centrifugation

(9,000 rpm for 10 min). The top fraction was removed, the centrifugation was repeated,

and the top fraction was collected again.

Single fiber cultures, explant cultures, and satellite cell isolation

Single fiber cultures were prepared as described previously (5), but with minor

but essential modifications. Briefly, extensor digitorum longus muscles were digested and

single fibers were carefully triturated. Approximately 100 single fibers were carefully

transferred into 10 ml of 5% horse serum (HS; Gibco BRL, Rockville, MD) in

Dulbecco's modified Eagle's medium (DMEM; Gibco BRL) and incubated at 37oC in 5%

CO2 for 15 min. This was repeated a minimum of five times to remove all loosely

associated cells. Single fibers were then cultured in plating medium (10% HS, 0.5% chick

embryo extract (CEE; US Biological, Swampscott, MA) in DMEM) for 1 day and then

switched to proliferation medium (20% fetal bovine serum (FBS; Mediatech, Herndon,

VA), 10% HS, 2% CEE in DMEM) or to medium containing young or old serum (5%

mouse serum in Optimem (Gibco BRL)) for a further 1.5 days. For modulation of Wnt

signaling in vitro, Wnt3A (100 ng/ml), sFRP3 (100 ng/ml), or DKK1 (100 ng/ml) was

incubated in fresh medium and added directly to cultures. Medium was replaced daily.

All cultures were done in 8-well permanox slides (Nunc, Rochester, NY) coated with

1/10 ECM gel (Sigma).

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Explant cultures of myofibers and associated satellite cells were prepared and

maintained exactly as previously described (6). Briefly, hindlimb muscles were incubated

in DMEM with 0.2% (w/v) collagenase II (Gibco BRL) for 90 min at 37oC. Digested

muscle was then washed in washing buffer (Ham’s F-10 nutrient mixture with 10% HS)

and dissociated into single myofibers by repeated triturating with a Pasteur pipette.

Myofiber fragments were rinsed and 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)) in flasks at 37oC

in 5% CO2. These cultures contain myofiber fragments and associated satellite cells.

Satellite cells were purified from bulk fibers as described (7). 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 20 ml of Hams F-10, 10% HS, 0.5 U/ml dispase

(Invitrogen, Carlsbad, CA), and 38 U/ml collagenase type II (US Biological) for 30 min

at 37oC with agitation. Satellite cells were liberated from the myofibers by trituration

with a 20-gauge syringe. The digests were then subjected to centrifugation at 500g for 1

min to pellet fiber debris, filtration through 50 micron mesh, and centrifugation at 1,000g

for 5 min to pellet satellite cells. The pellet was washed repeatedly with washing buffer.

The resulting preparation contained mononucleated cells that were almost all satellite

cells as described (7). Primary myoblast cultures were obtained as described (2).

Fibroblasts were obtained from muscle by enriching for cells that selectively attached to

non-coated dishes.

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Muscle injury

Focal injuries to tibialis anterior muscles were made by applying a metal probe, 4

mm in diameter, that had been cooled on dry ice directly to the exposed muscle surface

for 10 sec. To modulate Wnt signaling in vivo, 10 µl of recombinant Wnt3A (200 ng),

sFRP3 (500 ng), or DKK (500 ng) protein or control solution was introduced into the

muscle surrounding the injury site by direct intramuscular injection 1 day after the initial

injury. BrdU (50 mg/kg) was injected subcutaneous 2 days after injury.

Histology and immunofluorescence

Muscles were dissected and embedded for cryostat sectioning as previously

described (6). Gomori trichrome histological staining (Richard Allen Scientific) of tissue

sections was done according to manufacturer’s recommendations. Muscle wholemounts

for X-gal reactions were fixed in 2% PFA for 30 min at 4°C, permeabilized and

incubated in X-gal solution at 37°C. Sections were then cut and again incubated in X-gal

solution. Cells were fixed in 2% PFA for 5 min at 4°C, permeabilized for 10 min, and

incubated in X-gal solution.

Tissue sections for BrdU detection were fixed in ethanol, washed in PBS and

permeabilzed with 2M HCl for 20 min. Sections were washed in PBS for 30 min and

blocked in 5% goat serum (GS)/PBT then incubated in rat anti-BrdU antibody (1/400) for

2 hr at room temperature. Sections were then washed and incubated in donkey anti-rat

Alexa488 secondary antibody (1/1500) with DAPI for 1 hr at room temperature. Tissue

sections for immunofluorescence of Collagen VI and eMyHC were fixed in 2% PFA,

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permeabilized in 0.2% PBT, blocked in 5% GS/PBT, and then incubated in rabbit anti-

collagen antibody (1/50) and mouse anti-eMyHC (1/20) overnight at 4°C. Sections were

washed and blocked in 5% GS/PBT and incubated in goat anti-rabbit Alexa488 and goat

anti-mouse Alexa546 with DAPI for 1 hr at room temperature.

Immunofluorescence was performed on fixed cells (4% PFA, 10 min) after

permeabilization (0.2% PBT; 10 min) and block (5% GS in PBT). Cells were incubated

in primary antibodies overnight at 4°C at the following dilutions: Pax7 (1/2), Desmin

(1/100), MyoD (1/100), Fibronectin (1/1000), β-gal (1/3000), ER-TR7 (1/10). Cells were

washed and blocked in 5% GS/PBS then incubated in fluorophore-conjugated antibody

(goat anti-mouse Alexa546, donkey anti-rat Alexa488, and goat anti-rabbit Alexa488 at

1/1500) and DAPI to visualize nuclei for 1 hr at room temperature.

Quantification of “Fibrotic Index”

From tissue sections, the total injured area was measured. Fiber sizes of all

eMyHC+ fibers were measured. The “Fibrotic Index” was calculated as: (1 – ((total fiber

number x fiber cross-sectional area)/ total injury area)) x 100%.

Quantification of β-galactosidase activity in TOPGAL mice

Cells were isolated in lysis buffer (Tropix) and heat-inactivated to inactivate any

endogenous β-gal activity. β-gal activity was determined (Galacto-light; Tropix). DNA

was quantified as optical density at 260 nm using a UV spectrophotometer (Pharmacia).

β-gal activity was normalized to DNA content.

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Wnt depletion studies

Chimeric Frizled1-Fc, Frizzled7-Fc, and human IgG-Fc control (50 µl; 200

ng/mL) (R&D Systems) were incubated in the presence or absence of anti-human IgG-

conjugated agarose beads (0.001µl) (Sigma), for 1 hr at 4°C. Beads were washed three

times in 0.1% BSA interspersed with spins at 5,000 rpm for 1 min. Serum from young or

aged mice (70 µl) or Wnt (40 ng/mL) was diluted in 5% HS in Optimem and added to

beads for 1 hr at 4°C. The mixture was spun and the supernatant was collected. The

supernatant was then added to LSL cells to quantify Wnt signaling as previously

described (8).

Real-time RT-PCR

RNA was isolated from muscle using TRIZOL reagent (Invitrogen) and cDNA

was synthesized using Superscript First Strand Synthesis System for RT-PCR

(Invitrogen) according to manufacturer’s instructions. Relative quantitation by RT-PCR

was carried out using SYBR-green detection of PCR products in real time using the

MyiQ single-color detection system (Biorad). In each experiment, GAPDH was

amplified as the reference standard. Each RT-PCR reaction (25 µl) contained 2 µl of

cDNA, 10 µl of 2X SYBR Green Master Mix (Applied Biosystems Inc., Foster City,

CA), including Amplitaq polymerase (Perkin-Elmer), and primers (available on request)

at a final concentration of 20 nM.

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Gene expression was quantitated using an ABI SYBR® green PCR detection

system (Applied Biosystem). All reactions were performed using the following thermal

cycler conditions: 95°C for 15 min followed by 45 cycles of a 3 step reaction;

denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec and extension and data

collection at 72°C for 30 sec.

Fluorescent activated cell sorting (FACS)

To enrich for cells that expressed Syn-4, cells were isolated from myofibers as

described above. Cells were incubated in Sorting Medium (5% FBS, 10% BlokHen in

Hams F10) for 10 min then incubated in anti-Syn-4 (1/300) or chicken isotype antibody

for 1 hr on ice. Cells were washed and incubated in Sorting Medium for 5 min.

Secondary antibody (1/2000 goat anti-chicken Alexa488) was applied for 30 min in the

dark. Cells were washed and resuspended in Sorting Medium. Cells were sorted using

FACS Vantage SE (BD Biosciences) equipped with a 488 nm wavelength laser. Cells

were sorted with a gating hierarchy of forward and side scatter and gated to include cells

with fluorescence staining above isotype control. A minimum of 30,000 cells were

collected per sample. After collection the cells were washed, spun and prepared for RNA

isolation as described above.

Cells were fixed with 4% PFA. For detection using single antibodies, cells were

permeabilized with 5% FBS in 0.1% Triton-X 100. Fixed cells were then stained with

primary antibodies or with isotype-matched control antibodies (2 µg in 100 µl) for 1 hr at

room temperature followed by incubation with fluorochrome-labeled secondary

antibodies (1:1000) for 1 hr at room temperature. For double antibody labeling

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experiments, the primary antibodies were sequentially applied. Cells were blocked in

10% BlokHen (Aves Labs, Tigard, OR) for 30 min and stained with the Syn-4 antibody

(1/2000) for 1 hr. Cells were washed and blocked in 5% GS in 0.1% PBT for 15 min.

Secondary antibody (1/1500; donkey anti-chick Alexa488 (Molecular Probes)) was

applied for 1 hr. Cells were then washed and fixed in 4% PFA for 5 min. The second

antibody was then applied using the same protocol as single antibody labeling. Cells were

analyzed by FACScaliber (Beckton-Dickinson).

Statistical analysis

A minimum of 3 and up to 5 replicates was done for experiments presented. Data

are presented as means and standard deviations. Comparisons between groups were done

using Student’s t-test assuming two-tailed distribution and unequal variances. For

multiple comparisons, ANOVA was used when data were normally distributed. When

data were not normally distributed, the Kruskal Wallis test was used for multiple

comparisons. Differences were considered statistically significant at the p < 0.05 level.

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SOM FIGURE LEGENDS

Fig. S1. Age-related impairment of muscle regeneration.

A) Ten days after focal injury to muscles of mice aged 4, 24, and 32 months, muscles

were analyzed by cryosectioning and histochemical and immunohistochemical

staining. The top panels are Gomori-stained sections in which darkly stained muscle

fibers are surrounded by lightly stained connective tissue. The middle panels show

sections immunostained for MyHC (red) and nuclei stained with DAPI (blue). The

lower panel shows sections immunostained for MyHC (red) and Collagen VI (green).

During aging there is a progressive impairment of muscle regeneration, as evidenced

by the appearance of fewer and smaller muscle fibers and by a greater deposition of

connective tissue.

B) After twenty days of regeneration after focal injury, there is more connective tissue

surrounding smaller myofibers in the aged muscle. The top panels show sections

stained with H&E, and the bottoms panels show sections immunostained for MyHC

(red) and Collagen VI (green), with DAPI (blue) labeling all nuclei.

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Fig. S2. Age-related decline in proliferation rate of non-myogenic cells derived from

skeletal muscle.

A) Mononucleated cells from muscles of young and aged mice were isolated without any

purification and plated in growth medium for 3 days. BrdU was added to the medium

for the final 8 hrs. The graph shows the percentage of non-myogenic cells that were

also positive for BrdU. (* p < 0.05)

B) Culture derived exactly as in panel A were immunostained for Ki67. The graph shows

the percentage of non-myogenic cells that were also positive for Ki67. (* p < 0.05)

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Fig. S3. Loss of myogenic fate of young myogenic progenitors maintained in serum

from aged mice.

After young myogenic progenitors had been isolated and plated in plating medium for 1.5

days, they were maintained in aged serum for another 1.5 days and then subsequently

incubated in low serum medium (2% HS, DMEM) for 3 days to induce myogenic

differentiation. The panel on the left shows a phase contrast image of an area of the

culture showing a high percentage of non-fusing fibroblastic cells. The same field is

shown on the right stained for MyHC (red), demonstrating a MyHC+ nascent myotube

adjacent to several MyHC-negative mononucleated cells. All nuclei were identified by

DAPI staining (blue).

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Fig. S4. Specificity of anti-fibroblast antibody.

(A) Primary myoblasts and fibroblasts were immunostained for ER-TR7 (green) and

MyoD (red); DAPI (blue) stains all nuclei.

(B) Fibroblast cultures were stained for ER-TR7; DAPI (blue) stains all nuclei.

Heterogeneity of the intensity and pattern of staining can be observed. Cells can be

seen with extensive network of staining (white arrows), whereas other cells had more

discrete, punctuate staining (white arrow heads).

(C) Histogram showing the percentage of cells positive for ER-TR7 in both myoblast and

fibroblast cultures.

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Fig. S5. Pax7.Cre-ER.ROSA26 can be used to monitor cells of myogenic origin.

Mice were injected with tamoxifen two weeks prior to sacrifice. Myoblasts were prepared

and treated with hydroxy-tamoxifen (1 µM) for two days in proliferation media. Cells

were stained with (upper panels) X-gal (blue) and MyoD (red) or (center panels) X-gal

(blue), Pax7 (red) and MyoD (green). Fibroblasts were prepared and treated with

hydroxy-tamoxifen for two days in proliferation medium. Cells were then incubated in

serum from aged mice for 3 days. Cells were stained with X-gal (blue), ER-TR7 (green),

and DAPI (blue) (bottom panel).

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Fig. S6. FACS sorting for Syndecan-4 to isolate pure myogenic cells

Fiber-associated cells were isolated from muscles of young or aged mice. Cells were

stained for Syn-4 or chicken isotype control and analyzed by FACS (top panels). Cells

above the fluorescence background (inside the gate) were collected from young muscle

(center panel) and aged muscle (right panel). Cells were then placed into culture in GM

for 1 day, and then stained for Pax7 (red) and MyoD (green) (bottom panels). A

representative image of the fluorescent staining of sorted cells from young muscle is

shown on the left, with DAPI staining (blue) is shown on the right. 96% of sorted cells

from young muscle and 97% of sorted cells from aged muscle were positive for Pax7 or

MyoD.

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Fig. S7. Enhanced Wnt signaling in aged muscle and myogenic progenitors

A) RNA was isolated from whole, uninjured muscle of young and aged mice. Axin2

transcrip levels were assessed by real-time RT-PCR, normalized to glyceraldehyde

phosphate dehydrogenase (GAPDH) levels, and plotted relative to values in muscle

from young animals. (* p < 0.05)

B) Analysis of active β-catenin in myogenic progenitors from aged mice five days after

tail vein injection of either a control adenovirus or an adenovirus expressing the Wnt

inhibitor DKK1 (4). Myogenic progenitors were isolated and analyzed as by FACS

for the percentage with β-catenin*. (* p < 0.05)

C) Analysis of active β-catenin in myogenic progenitors isolated from muscle of young

and aged mice that had been injured 2 days previously. The populations were

analyzed by FACS for all myogenic progenitors (Syn-4+) that were also positive for

β-catenin*. Isotype controls (left panel) were used to define cells negative for Syn-4

and β-catenin*. (* p < 0.05)

D) Analysis of Wnt signaling activity in myogenic progenitors from TOPGAL mice at

different ages (6, 14 and 24 months). Cells were isolated 1 and 2 days after injury; β-

gal activity was quantified and normalized to DNA content. Letters in parenthesis (Y,

A, O) refer to young, adult and old mice. (* p < 0.05)

E) A wholemount X-gal reaction of muscle 4 days after focal injury to muscles of young

and aged TOPGAL mice.

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Fig. S8. Testing of Wnt-signaling activity in mouse serum

A) Dose response of Wnt reporter activity in LSL cells with overnight incubation in

commercially available Wnt3A (R&D Systems) or Wnt3A provided from Roel

Nusse, Stanford University (Wnt3A*). Data were normalized to control.

B) Anti-human IgG conjugated agarose beads were incubated with Frizzled1-Fc,

Frizzled7-Fc or Human IgG-Fc control. After washing the beads, Wnt was added to

the bead mixture. The mixture was isolated and incubated on LSL cells for 24 hrs and

Wnt signaling quantified. Data are expressed as luciferase activity normalized to β-

gal activity. (* p < 0.05)

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Fig. S9. The effects of the aged environment on muscle regeneration are mediated

by the Wnt signaling pathway

A) Effects of exogenous Wnt on cell proliferation. Muscles of young mice were injured,

and either Wnt3A (200 ng /10 µl) or control solution (10 µl of 0.1% BSA) was

injected into regenerating tissues 1 day after injury. BrdU was injected

subcutaneously 2 days after injury, and muscles were analyzed 3 days later.

Cryosections were immunostained for BrdU and quantified for the percentage of

BrdU+ mononucleated cells in control and Wnt-treated muscles. (* p < 0.05)

B) Muscles of young or aged mice were injured, and either sFRP3 (500 ng/10 µl) or

control solution (10 µl of 0.1% BSA) was injected into the regenerating tissues 1 day

after injury. BrdU was injected subcutaneously 2 days after injury, and muscles were

analyzed 3 days later. The graph represents the percentage of mononucleated cells

that were BrdU+ in the control and sFRP-treated aged muscles. BrdU incorporation in

regenerating muscles of young mice is shown for comparison. (* p < 0.05)

C) Muscles of young or aged mice were injured, and either sFRP3 (500 ng/10 µl) or

control solution (10 µl of 0.1% BSA) was injected into the regenerating tissues 1 day

after injury. BrdU was injected subcutaneously 2 days after injury, and bulk cultures

of myogenic progenitors were prepared 3 days later. The cells were immunostained

for Desmin to identify myoblasts and for BrdU to identify proliferating cells. The

“Myogenic progenitor proliferation Index” refers to the percentage of Desmin+ cells

that were also BrdU+. (* p < 0.05)

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REFERENCES FOR SOM

1. P. Soriano, Nat. Genet. 21, 70 (1999).

2. C. M. McCay, F. Pope, W. Lunsford, G. Sperling, P. Sambhavaphol, Gerontologia. 1, 7 (1957).

3. I. M. Conboy et al., Nature 433, 760 (2005).

4. F. Kuhnert et al., Proc. Natl. Acad. Sci U. S. A. 101, 266 (2004).

5. S. B. Chargé, A. S. Brack, S. M. Hughes, Am. J Physiol Cell Physiol 283, C1228 (2002).

6. I. M. Conboy, T. A. Rando, Dev. Cell 3, 397 (2002).

7. I. M. Conboy, M. J. Conboy, G. M. Smythe, T. A. Rando, Science 302, 1575 (2003).

8. J. T. Blitzer, R. Nusse, BMC. Cell Biol. 7, 28 (2006).

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Figure S1A

10d Regeneration4 month 24 month 32 month

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Figure S5

X-gal MyoD

X-gal Pax7 MyoD

X-gal ER-TR7 DAPI

Figure S6

Chicken Syn4 Syn4

Young Aged

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Figure S7

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