Fig. 4. Adult satellite cells derive from progenitors expressing Myf5 during prenatal development.

(A) An example of a YFP^+ve satellite cell (arrow) associated with an EDL fiber of an adult Myf5^CreER/wt; R26R^YFP/wt mouse treated with TMX at E10.5–E11.5. Staining with antibodies recognizing YFP and the satellite cell markers Pax7 and Syn4 is presented. Bar, 20 μm. (B) Xgal staining of an EDL fiber from an adult Myf5^CreER/wt; R26R^LacZ/wt mouse treated with TMX at E10.5–E11.5. A β-gal^+ve satellite cell is shown (arrow and insets). Bar, 30 μm. (C) An example of a satellite cell expressing the reporter gene YFP (arrowhead) after administration of TMX to E14.5–E15.5 is shown. Note that due to the relatively low efficiency of recombination, most of the satellite cells are YFP^−ve (asterisk). DAPI was used to stain the nuclei. Bar, 40 μm. (D) The fraction of the VCAM^+ve/CD45^−ve/CD31^−ve/Sca1^−ve satellite cells expressing the lineage marker YFP (FITC) was quantified by FACS on cells obtained from adult Myf5^CreER/wt; R26R^YFP/wt mice after TMX administration at different developmental stages, as indicated. Satellite cells from Pax7^CreER/wt; R26R^YFP/wt mice injected with TMX at adult stage were used as positive controls. Note that the analysis of the satellite cells obtained from Myf5^CreER/wt; R26R^YFP/wt not treated with TMX reveals a virtual absence of recombination. (E) Cells were isolated by enzymatic digestion from hindlimb muscles of Myf5^CreER/wt; R26R^YFP/wt mice treated with TMX at the indicated stages and allowed to adhere overnight in vitro. Cells were then stained with an antibody to YFP and with a cocktail of antibodies to Pax7, MyoD, and Mgn to reveal the total pool of myogenic cells. Bar, 25 μm. (F) Data from replicate studies shown in panel E were quantified to determine the percentage of myogenic cells that were marked by YFP. Results are expressed as mean ± standard deviation. The recombination obtained after TMX administration at E14.5–E15.5 or P7–P11 is significantly higher compared that detected at earlier stages (** P<0.01, * P<0.05). (G) Data from replicate studies shown in panel E were quantified to determine the percentage of myogenic cells that were marked by YFP and were normalized for the average efficiency of recombination observed after TMX treatment at different stages (see Fig. 3B). Each square represents the normalized value obtained in a single replicate (n=3 for each condition). Note that a minority of satellite cells traces its origin back to progenitors expressing Myf5 at the embryonic (E10.5–E11.5) or early fetal (E12.5–E13.5) stage, whereas >75% of the satellite cells derive from progenitors expressing Myf5 at the mid-fetal (E14.5–15.5) or neonatal (P7–11) stage.