Distinct Phases of Postnatal Skeletal Muscle Growth Govern the Progressive Establishment of Muscle Stem Cell Quiescence

Abstract

Muscle stem cells (or muscle satellite cells [MuSCs]) are required for postnatal growth. Yet, the detailed characterization of myogenic progression and establishment of quiescence during this process remains poorly documented. Here, we provide an overview of myogenic cells heterogeneity and dynamic from birth to adulthood using flow cytometry. We demonstrated that PAX7+ cells acquire an increasing ability to progress in the myogenic program from birth to adulthood. We then simultaneously analyzed the cycling state (KI67 expression) of the MuSCs and progenitors (PAX7+) and their progression into myogenic precursors (PAX7-MYOD+) and differentiating cells (MYOG+) in vivo. We identified two distinct peaks of myogenic differentiation between P7-P10 and P21-P28, and showed that the quiescent MuSC pool is established between 7 and 8 weeks of age. Overall our study provides a comprehensive in vivo characterization of myogenic heterogeneity and demonstrates the highly dynamic nature of skeletal muscle postnatal growth process.

Keywords: muscle satellite cells; muscle stem cells; myogenesis; myogenic precursors; myogenic progenitors; postnatal growth; quiescence.

Figure 1

Distinct Composition and In Vitro Behavior of Myogenic Cells of the CD34⁺ITGA7⁺ Fraction of Postnatal or Adult Muscles (A and B) Representative density scatterplots showing the gating strategy used to determine the proportion of PAX7+, MYOD+, and MYOG+ cells among the CD45⁻TER-119⁻CD31⁻SCA-1⁻CD34⁺ITGA7⁺ fraction (referred to as CD34⁺ITGA7⁺ fraction) from P7 mouse muscles. (A) Debris, doublets, CD45⁺TER-119⁺CD31⁺, and dead cells were excluded from the analysis (Figure S1; Table S1) and ITGA7⁺ cells were gated from the CD34⁺SCA-1⁻ fraction. (B) Gates used to determine the proportion of PAX7+, MYOD+, and MYOG+ cells. (C) Bar graph showing the proportion of PAX7+, MYOD+, and MYOG+ cells. Two-way ANOVA analysis, with ^∗∗∗p < 0.001, $p < 0.0001 relative to P0. (D) Purified CD34⁺ITGA7⁺ cells were plated at the same density and cultured in growth medium for 3, 4, and 5 days. (E–F) (E) Optical micrographs of cultured-myogenic cells. Scale bar, 100 μm. Quantification of (F) the total number of mononucleated cells, (G) their spontaneous fusion index, calculated as number of nuclei incorporated in myotubes on total number of nuclei per field. Values are represented as the mean ± SEM of three independent experiments (n = 3 mice/group). Two-way ANOVA analysis, with ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001.

Figure 2

Increasing Ability of Cultured-Myogenic Cells of the CD34⁺ITGA7⁺ Fraction to Progress in the Differentiation Program (A–F) (A–C) Representative density scatterplots showing the repartition of PAX7/MYOD (upper panel), MYOD/MYOG (lower panel) expression in cultured-myogenic cells. Debris and dead cells were excluded from the analysis. Gates were positioned based on FMO controls (Figures S2F and S2G). Graphs showing (D) the relative proportion of total PAX7+, MYOD+, and MYOG+ cells, and the repartition of (E) PAX7/MYOD and (F) MYOD/MYOG expression. Values are the mean ± SEM of three independent experiments (n = 3 mice/group). Statistical significance is represented relative to PAX7−MYOD+ cells (E) or MYOD+MYOG+ cells (F). Two-way ANOVA analysis, with ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001.

Figure 3

In Vivo Characterization of the Myogenic Populations from Birth to Adulthood (A) Experimental timeline. (B–G) (B) Strategy of analysis. Debris, doublets, and dead cells were excluded from the analysis. Representative density scatterplots showing the gates used to analyze myogenic cells in P7 mouse muscles. Joined gate PAX7+ or MYOD+ or MYOG+ cells was considered as the total myogenic cells (100%) referred to as TM. Representative density scatterplots showing, the repartition of (C) PAX7/MYOD and (D) MYOD/MYOG expression on TM (see also Figures S4A and S4B). Graphs showing (E) the proportion of total PAX7+, MYOD+, and MYOG+ cells, and the repartition of (F) PAX7/MYOD and (G) MYOD/MYOG expression on TM. (H) Analysis of the myogenic cells was refined based on their cycling state, monitored by KI67 expression. (I) Pie charts showing the distribution and the cycling state of the myogenic populations. Values represent the mean of data obtained from three to eight mice for each time point. Statistical significance is shown in Tables S5 and S6. Two-way ANOVA analysis, with ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, $ or ^∗∗∗∗p < 0.0001.

Figure 4

Immunohistological Analysis of Myogenic Cells Location during Postnatal Growth (A and B) Representative tibialis anterior (TA) muscle cross-sections immunostained for (A) PAX7 (green), MYOD (red), and laminin (yellow), (B) MYOG (green), MYOD (red), and laminin (yellow) (see also Figure S5). Nuclei were stained with DAPI. Scale bar, 20 μm. (C–E) (C) Quantification of the number of PAX7+, MYOD+, and MYOG+ cells normalized per 100 fibers. Quantification of the proportion of sub-laminal and interstitial (D) MuSCs and (E) progenitors. Yellow and white magnification inlets indicate sub-laminal and interstitial PAX7+MYOD+ progenitors, respectively. Values are the mean ± SEM of data obtained from minimum three different mice for each time point. Two-way ANOVA analysis, with ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, $ or ^∗∗∗∗p < 0.0001 versus P0.

Figure 5

Quiescent MuSC Pool Is Fully Established between P49 and P56 (A–C) (A) Representative TA muscle cross-sections immunostained for PAX7 (red), KI67 (green), and laminin (yellow). Nuclei were stained with DAPI. Scale bar, 20 μm. Quantification of (B) the total number of cycling (KI67+) and non-cycling (KI67−) PAX7+ cells per 100 fibers, and (C) the proportion of cycling and non-cycling PAX7+ cells determined on TA cross-sections immunostained as in (A). (D) Representative density scatterplots showing the gates used to determine the proportion of cycling PAX7+ cells by flow cytometry. (E) Proportion of cycling and non-cycling PAX7+ cells obtained by flow cytometry. (F) Pie charts showing the repartition of MYOD+ and KI67+ cells among total PAX7+ cells. Statistical significance is shown in Table S7. Values are the mean ± SEM of data obtained from three to eight mice for each time point. Two-way ANOVA analysis, with $p < 0.0001 versus P0.

Figure 6

Schematic Representation of Myogenic Cell Dynamics and Establishment of the Quiescent MuSC Pool during Muscle Postnatal Growth (A) Postnatal myogenesis is characterized by two successive phases of differentiation and progressive constitution of the quiescent MuSC pool. (B) Proposed model for myogenic cell progression in the differentiation program during postnatal growth. Dotted lines represent fates of myogenic cells that remain to be investigated.