Labeling satellite cells to assess contribution to myofibers

We assessed the contribution of satellite cells to myofibers during postnatal growth by evaluating the extent of satellite cell fusion into postnatal myofibers in mice of various ages up to 6 months old. We labeled satellite cells by recombination with an inducible Cre in the Pax7 locus [3] and a LSL-tdTomato reporter in the ROSA locus [8]. Recombination in satellite cells was induced with five consecutive daily I.P. tamoxifen injections (Fig. 1a), and 6 h post-injection, the tibialis anterior (TA) muscle was harvested and recombination efficiency determined by scoring the percentage of Pax7 immunoreactive cells located underneath the basal lamina (Fig. 1b, e) that were also positive for tdTomato fluorescence (Fig. 1d, e; Table 1). Myofibers were tdTomato negative (Fig. 1b, e), indicating that recombination in satellite cells during the 5 days of consecutive tamoxifen injections does not promote satellite cell activation and fusion into myofibers.

Fig. 1 Induction of tdTomato expression in satellite cells labels myofibers by satellite cell fusion. a Pax7Cre;tdTomato mice were injected with tamoxifen once a day for five consecutive days to induce tdTomato expression in satellite cells. Images shown in this figure are from 6-month-old mice. b–e Representative images of TA sections show that 96 % of Pax7-expressing satellite cells are also expressing tdTomato. Insert highlights a Pax7+, tdTomato+ sub-laminar satellite cell. f Pax7Cre;tdTomato mice were injected with tamoxifen once a day for five consecutive days and then tissue was collected 2 weeks following the last injection. g–j Representative images of TA sections show Pax7-expressing satellite cells as tdTomato+ but also unexpectedly show tdTomato+ myofibers. Insert highlights a tdTomato-expressing myofiber with an associated Pax7+, tdTomato+ satellite cell. k–n Representative images of TA sections from Pax7Cre;tdTomato mice that were injected with only with corn oil showing no basal tdTomato expression prior to tamoxifen injection. Images display DAPI (blue), Sdc4 (green), tdTomato (red), Pax7 (white). ^ marks indicate satellite cells. Scale bars are 50 μm Full size image

Table 1 Quantification of tdTomato recombination Full size table

When TA muscles were harvested 2 weeks post-final tamoxifen injection (Fig. 1f), recombination efficiencies in satellite cells were similar to those harvested 6 h post tamoxifen injection (Table 1) where nearly all Pax7+ immunoreacitve SCs were tdTomato+ (Fig. 1g, h, j). In contrast to muscle removed 6 h post tamoxifen treatment, we observed tdTomato+ myofibers in the TA muscle at 2 weeks post tamoxifen treatment (Fig. 1h, j), where ~15 % of the myofibers were tdTomato+ (Fig. 1h, j). Neither mononuclear tdTomato+ cells (Fig. 1k–n) nor tdTomato+ myofibers (Fig. 1k–n) were observed when Pax7Cre;tdTomato mice were injected with the vehicle (corn oil) demonstrating very low or nonexistent basal recombination.

We gave five daily tamoxifen injections to mice starting at various ages (4, 8, 12 weeks, and 6 months), isolated individual extensor digitorum longus (EDL) myofibers 2 weeks post final tamoxifen injection, and then visualized tdTomato, Pax7, and Syndecan-4 24 h post-isolation initially to assess recombination frequencies. Regardless of when the tamoxifen injections were started (4 weeks (Fig. 2a–e), 8 weeks (Fig. 2f–j), 12 weeks (Fig. 2k–o), or 27 weeks (Fig. 2p–t)), almost all Pax7 immunoreactive cells were tdTomato+, with recombination efficiencies from 90 to 99 % (Fig. 2u). We verified the recombination efficiencies with an independent satellite cell (SC) marker, Syndecan-4, where all myofiber-associated SCs were Syndecan-4+, Pax7+, and tdTomato+ in myofibers isolated from mice injected at 4 weeks (Fig. 2a–e), 8 weeks (Fig. 2f–j), 12 weeks (Fig. 2k–o), and 27 weeks (Fig. 2p–t) old. Recombination frequencies were indistinguishable between myofiber-associated Pax7+ SCs (Fig. 2u) and Syndecan-4+ SCs (Fig. 2v) recombined in vivo and fixed 24 h post-isolation.

Fig. 2 Induction of tdTomato expression does not vary with age of tamoxifen injection. a–t Images of EDL myofibers that were harvested 2 weeks after the final tamoxifen injection show that almost all satellite cells are expressing tdTomato. Images show DAPI (blue), Syndecan-4 (green), tdTomato (red), and Pax7 (white). ^ marks within micrographs indicate satellite cells. u–v Quantification showing the percentages of satellite cells on myofibers that are tdTomato+. w Quantification showing the percentages of myofibers that are tdTomato+. Scale bar is 50 μm. Three biological replicates were analyzed per time point Full size image

The majority of myofibers should be tdTomato+ when mice are injected with tamoxifen at 4 weeks old because satellite cells are actively contributing to growing muscle fibers. Mice injected at older ages when skeletal muscles have ceased growing should have fewer tdTomato+ myofibers. For the EDL muscle, 77 % of the isolated myofibers from mice injected at 4 weeks old were tdTomato+ (Fig. 2c, w), whereas only 12 % of EDL myofibers were tdTomato+ when isolated from mice injected at 27 weeks old (Fig. 2r, w), consistent with the cessation of postnatal skeletal muscle growth. The percentage of TdTomato+ myofibers decreased as the mouse age increased, but ~12 % of the isolated EDL myofibers from mice injected with tamoxifen at 27 weeks old were tdTomato+ when harvested 2 weeks post tamoxifen treatment.

To identify the extent of labeled SC incorporation into myofibers in vivo and to determine when cessation of postnatal growth occurs, we induced recombination as described earlier (see Fig. 1f); and 2 weeks post tamoxifen treatment, hindlimb muscles were harvested, sectioned, and immunostained for laminin to identify myofibers and visualized for tdTomato. Comparing tdTomato expression in mice injected with tamoxifen at 4 weeks of age (Fig. 3a, f) to mice injected at 27 weeks old (Fig. 3d, f) reveals a major decline in tdTomato+ myofibers in hindlimb muscles at the older age. The intensity of tdTomato was variable and thus, we utilized a “1–4” grading system, scoring the most tdTomato intense myofibers at 4 (+++) and background (no detectable tdTomato) scored 1. Myofibers clearly tdTomato+ but less intense than those at 4+ were scored a 3 (++). Myofibers above background fibers but much weaker than 3 were scored a 2 (+). Examples of representative myofibers in a single field of view were labeled (Fig. 3c) and when quantified reveal the greatest numbers of intensely labeled myofibers occur in mice injected with tamoxifen at 4 weeks of age and at 8 weeks of age (Fig. 3e). In mice injected at 4 weeks of age and at 8 weeks of age, ~78 and ~60 % of the myofibers were tdTomato+, respectively (Fig. 3f). However, with the 12- and 27-week-old tamoxifen injections, the tdTomato+ labeled myofibers declined to ~20 % and were maintained at 20 %, respectively (Fig. 3f), suggesting a cessation of postnatal muscle growth in the TA muscle between 8 and 12 weeks of age. No significant differences were observed in the percentages of tdTomato+ myofibers at either muscle end compared to the midbody (Fig. 3g). The high percentage (~20 %) of tdTomato+ myofibers in TA muscles of mice injected at 27 weeks old following a brief (2 weeks) chase suggests significant SC contribution to uninjured, resting TA muscle.

Fig. 3 Satellite cell contribution to tibialis anterior muscle plateaus between 8 and 12 weeks of age. a–d Images of TA muscle sections from mice that were harvested 2 weeks after the final tamoxifen injection show the percentages of tdTomato expressing myofibers decrease with age. Images display DAPI (blue), laminin (green), and tdTomato (red). Numbers in the 12-week image c illustrate tdTomato scoring metric. e Quantification of tdTomato-expressing myofibers in TA muscle sections by tdTomato expression level. +++ myofibers express high levels of tdTomato (1 in a), + myofibers clearly express tdTomato (2 in a), +/− myofibers have very low levels of tdTomato and are scored as negative (3 in a), and – myofibers do not express tdTomato at observable levels (4 in a). f Quantification showing the percentages of myofibers that express tdTomato (+ or +++) in TA muscle sections. g Quantification showing the percentages of tdTomato-expressing TA myofibers by location within the muscle. Scale bar is 50 μm. ****P < 0.0001. Mean ± standard error of the mean is plotted. Significance was determined by Student’s t test. Three to six biological replicates were analyzed per time point Full size image

Differential satellite cell contribution among individual hindlimb muscles

To determine the age when satellite cell contribution plateaus among various hindlimb muscles, tdTomato expression was analyzed in the EDL, soleus, and gastrocnemius 2 weeks after tamoxifen injection of Pax7 Cre;LSL-tdTomato mice. In contrast to the TA muscle, the percentage of tdTomato-expressing myofibers significantly decreased in the EDL (Fig. 4a), soleus (Fig. 4b), and gastrocnemius (Fig. 4c) between the 4- and 8-week-old injections. Similar to the TA (Fig. 3f), there was a further decrease in tdTomato-expressing myofibers in the soleus (Fig. 4a) and gastrocnemius (Fig. 4c) muscles between the injections given at 8 and 12 weeks of age. The EDL was unique among the hindlimb muscles as no significant decreases in tdTomato expressing myofibers was observed in mice injected older than 8 weeks (Fig. 4a). The percentage of tdTomato+ myofibers did not significantly change in mice injected at 12 versus 27 weeks, demonstrating that all assayed hindlimb muscles reach a steady state SC fusion by 12 weeks of age. A minimum of 15 % of the labeled SCs fuse into myofibers during the 2-week chase in non-exercised, uninjured hindlimb muscle. The only muscle in which satellite cell fusion plateaus prior to 12 weeks of age is the EDL, which reaches steady state satellite cell fusion (~20 %) at 8 weeks of age. The data do not appear to be biased by the longitudinal section selected for scoring as the extent of SC incorporation at either end or at the midbody of the EDL (Fig. 4d), soleus (Fig. 4e), or gastrocnemius (Fig. 4f) was not significantly different from each other.

Fig. 4 Satellite cell contribution is similar in all hindlimb muscles as a function of age. a–c Quantification of tdTomato-expressing myofibers among the EDL (a), soleus (b), and gastroc (c) muscles 2 weeks after the final tamoxifen injection. d–f Quantification of tdTomato-expressing myofibers by location within the EDL (d), soleus (e), and gastroc (f). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. For all graphs, mean ± standard error of the mean is plotted. Significance was determined by Student’s t test. Three biological replicates were analyzed per time point Full size image

Differential satellite contribution to fast and slow-twitch muscle myofibers is age-dependent

The extent of SC fusion into adult myofibers and the age at which SC fusion plateaus was similar for all hindlimb muscles was analyzed. We selected the soleus muscle, which is comprised of ~50 % slow twitch myofibers to determine if differences were observed for SC incorporation into slow twitch (type 2) vs. fast twitch (type 1) myofibers. Pax7 Cre ;LSL-tdTomato mice were injected with tamoxifen at the indicated ages and the soleus muscles collected 2 weeks post final tamoxifen injection. Muscle sections were then stained with an antibody that recognizes all fast twitch muscle fibers types (Fig. 5a–c; IIa/b/x) (Havenith et al. [9]). Unlabeled myofibers were assumed to be slow twitch (Fig. 5a–c), and the percentages of fast and slow myofibers were quantified for each age (Fig. 5f). When tamoxifen was injected into 27-week-old mice, 2 weeks later, 25 % of the soleus myofibers were tdTomato+ (Fig. 4b). Further analysis of the muscle from mice injected at 27 weeks of age identified 60 % of the tdTomato+ soleus myofibers as fast twitch and 40 % of the tdTomato+ myofibers as slow twitch, (Fig. 5a–c, g), the same ratio of fast twitch to slow twitch muscle among the entire 27-week myofiber population (Fig. 5f). The contribution of satellite cells to type 1 fast twitch myofibers (Fig. 5d) plateaus at an earlier age than for type II slow twitch (Fig. 5e), indicating that satellite cell fusion rates differ for slow and fast twitch myofibers during postnatal muscle growth.

Fig. 5 Differential contribution of satellite cells to fast and slow twitch myofibers. a–c Images shows soleus muscle sections from mice given tamoxifen injections at 27 weeks old and collected 2 weeks post injection. tdTomato-expressing myofibers are shown in red, and myofibers labeled with an antibody to identify fast twitch myofibers are shown in green. d Quantification of tdTomato expression in type I myofibers. e Quantification of tdTomato expression in type II myofibers. f Soleus fiber type distribution by age. g Fiber type distribution of tdTomato-expressing myofibers by age. Pseudocolored images display DAPI (blue), laminin (white), tdTomato (red), and fast myosin heavy chain (green, IIa, IIb, and IIx). Scale bars are 50 μm. *P < 0.05, ***P < 0.001. Mean ± standard error of the mean is plotted. Significance was determined by Student’s t test. Three biological replicates were analyzed per time point Full size image

As shown in the previous figure, the percentage of tdTomato expressing myofibers in the soleus muscle decreases from 60 to 30 % in mice injected at 4 versus 8 weeks of age (Fig. 4b), yet the percentage of fast twitch tdTomato+ myofibers remains constant at approximately 40 % (Fig. 5e). Therefore, between 4 and 8 weeks of age, satellite cells contribute predominately to fast twitch myofibers (Fig. 5d). Collectively, satellite cells in the soleus preferentially contribute to fast twitch myofibers during adolescent growth and then satellite cells contribute equally to fast and slow twitch muscle in adult, once postnatal growth has ceased.

Satellite cell contribution to the extraocular and diaphragm muscle differs from hindlimb muscles

The diaphragm and extraocular (EOM) muscles were selected for analysis as the diaphragm is severely affected in dystrophic mice and the EOM is spared in dystrophic mice, respectively [10]. To analyze the EOM and diaphragm muscles, mice were given tamoxifen injections at the same time points that were used in the hindlimb analysis (4, 8, 12, and 27 weeks of age). EOM and diaphragm muscles were collected 2 weeks later and scored for tdTomato+ myofibers. Satellite cell incorporation into EOM was much greater than that observed in hindlimb muscles, where 80 % of the EOM myofibers were tdTomato+ in mice injected at 8 weeks of age (Fig. 6e). Distinct from the hindlimb muscles, SC fusion into the diaphragm and EOM did not plateau, where tdTomato+ myofibers were at 64 (Fig. 6a, e) and 48 % (Fig. 6b, e) when injected at 12 and 27 weeks of age, respectively. We did not analyze older mice and do not know whether SC fusion reaches a steady state in EOM.

Fig. 6 Satellite cell contribution to EOM and diaphragm muscles does not plateau with age. a–d Images show tdTomato expressing myofibers in the EOM and diaphragm 2 weeks after the final tamoxifen injection. e–f Quantification showing the percentages of tdTomato-expressing myofibers in sections of the e EOM and f diaphragm. Images display DAPI (blue), laminin (green), and tdTomato (red). Scale bar is 50 μm. *P < 0.05, **P < 0.01. For all graphs, mean ± standard error of the mean is plotted. Significance was determined by Student’s t test. Three biological replicates were analyzed per time point Full size image

Satellite cell fusion into the diaphragm, similar to the EOM, does not plateau by 27 weeks of age yet is very high, where 95 (Fig. 6f), 90 (Fig. 6c, f), and 80 % (Fig. 6d, f) of the diaphragm myofibers were tdTomato+ following in mice injected at 8, 12, and 27 weeks old, respectively. Satellite contribution in adult EOM and diaphragm is significantly higher than satellite cell contributions in hindlimb muscle of mice injected at 27 weeks old.

A comparison of the muscles analyzed illustrates overall similarities between the hindlimb muscles, where hindlimb muscle SC fusion plateaus by 12 weeks of age (Fig. 7b). However, the number of tdTomato+ myofibers differs among the hindlimb muscles when tamoxifen is given prior to 12 weeks of age (Fig. 7b), where the TA muscle is the last to stabilize SC fusion at 15 % at 12 weeks of age (Fig. 7b). In contrast, the diaphragm and EOM decline more linearly over time (Fig. 7a) and do not achieve steady state SC fusion with high levels of tdTomato+ myofibers present when tamoxifen is injected at 27 weeks of age. Plotting the relative intensity of tdTomato fluorescence for each muscle between the 8-, 12-, and 27-week-old injections reveals a decline in the tdTomato fluorescence intensity with increasing age until a steady state is reached in the hindlimb muscles (Fig. 7c).

Fig. 7 Satellite cell contribution to myofibers is age dependent and varies in different muscles. a–b Non-linear regression trend lines for percentages of tdTomato-expressing myofibers through experimental time points from the EOM and diaphragm (a) and EDL, soleus, and gastrocnemius (b). c–e Quantification of myofiber tdTomato-expression of hindlimb, EOM, and diaphragm muscles with recombination induced at 4 (c), 12 (d), or 27 weeks (e). Quantification of tdTomato was evaluated at four relative levels of expression: +++ very high tdTomato intensity (see example myofiber “1” in Fig. 3c, 12 weeks), + intermediate tdTomato intensity (see example myofiber “2” in Fig. 3c, 12 weeks), +/− very low tdTomato intensity myofibers and scored as negative (see example myofiber “3” in Fig. 3c, 12 weeks), and – fibers do tdTomato intensity indistinguishable from background (see example myofiber “4” in Fig. 3c, 12 weeks) Full size image

Satellite cell fusion into uninjured myofibers can occur with either an intervening cell division or without cell division

EdU was added to the drinking water of 5-month-old Pax7Cre;tdTomato mice concomitant with the initiation of tamoxifen injections. Muscle tissue was collected 2 weeks following the final tamoxifen injection and individual EDL myofibers fixed immediately after isolation. Essentially, all Pax7 expressing SCs were tdTomato+, while ~12 % of the EDL myofibers were tdTomato+. Approximately 10 % of the SCs were labeled with EdU following the chase period (Fig. 8a, b, d), with ~35 % of the myofibers possessing one or more EdU+ -associated SC (Fig. 8e). Similar numbers of EdU+ SCs were present on tdTomato+ myofibers and tdTomato− myofibers (Fig. 8d, e) with about 25 % possessing one or more EdU+ myonucleus (Fig 8a, c, f). No more than two EdU+ nuclei were found associated or within any single myofiber where the percentage of myofibers possessing at least one EdU+ myonucleus did not significantly differ between tdTomato+ myofibers (23 %) and tdTomato– myofibers (27 %) (Fig. 8f). Since the majority of tdTomato+ myofibers did not possess an EdU+ nucleus (77 %) the majority of SC fusion occurs without an intervening cell division. Furthermore, EdU+ myonuclei in tdTomato− myofibers suggests that multiple SCs must fuse for visualization of tdTomato expression in myofibers. This conclusion is consistent with fewer SCs on tdTomato+ myofibers compared to the SC number on tdTomato– myofibers (Fig. 8g).

Fig. 8 Satellite cells fuse into uninjured myofibers either with our without an intervening cell division. a–c Representative images show EdU+ satellite cells and EdU-labeled nuclei within myofibers. a Myofiber showing an EdU+ satellite cell (arrow) and an EdU+ differentiated myonuclei (arrowhead). b Images show a myofiber with two satellite cells that are EdU+. c A myofiber with two EdU+ myonuclei within the myofiber. For a–c, DAPI (blue), tdTomato (red), EdU (green), and Pax7 (white). Scale bar is 20 μm. d Quantification showing the percentages of satellite cells on dtTomato+ (Tom pos) myofibers versus dtTomato− myofibers (Tom neg) that were labeled with EdU. e Bar graph showing the percentages of myofibers that have at least one EdU+ associated satellite cell. f Quantification showing the percentages of myofibers that have at least one EdU+ nuclei within the myofiber. g Quantification of the satellite cell number present on dtTomato+ myofibers versus dtTomato− myofibers (*p ≤ 0.01). h Images of TA muscle sections show an EdU+, tomato+ SC. tdTomato (red), EdU (green), laminin (white), and DAPI (blue). Scale bar is 40 μm. For bar graphs, ns denotes not significant. Significance was determined by Student’s t test. N = 3. For all graphs, the mean with the standard error of the mean is plotted Full size image

TA muscle, EOM, and diaphragm were collected from EdU-exposed recombined Pax7CreER;LSL-tdTomato 5 months old mice where EdU+ cells are clearly visible in the muscle tissue sections (Fig. 8h). Attempting to reliably quantify EdU+ SCs proved impractical due to the EdU uptake by other cells and the difficulty in identifying EdU+ cells following antigen retrieval necessary for Pax7 detection.