The transgenic model for tracing the formation of skeletal muscle fibers
To better define the development pattern of skeletal muscle fiber number, we used a doxycycline-inducible tracing system that can permanently label all pre-existing myofibers during the desired time period. Two transgenic strains were employed: Tg-Cre and LacZ reporter (Fig. 1a). Tg-Cre line contains two transgenic constructs: cre recombinase under the control of the TRE and rtTA under the control of the human ACTA1 (actin, alpha 1) skeletal muscle promoter (Fig. 1a). The LacZ reporter line contains a targeted mutation that the DNA sequences encoding LacZ with a loxP-flanked STOP cassette upstream were knocked in the Rosa26 site (Fig. 1a). By crossing these two transgenic lines, the triple transgenic offspring called ActaLabel mice are generated (Fig. 1a). In the ActaLabel line exposed to Dox, the STOP sequence is removed and LacZ is expressed only in skeletal muscle myocytes (Fig. 1a). Consequently, myofibers expressing LacZ can be stained blue in the β-galactosidase (β-gal) assay.
It has been well documented that efficient induction can be achieved in various tissues in this triple transgenic strain of various ages, including embryonic period and postnatal growth [21, 22]. In order to verify our model, 8-week-old ActaLabel mice were treated with doxycycline for 5 days before being analyzed. β-gal staining showed that all of the myofibers (TA, EDL, LD, GA, RF) were labeled blue (Fig. 1b), indicating nearly 100 % induction efficiency. As expected, LacZ expression was rarely detected in the counterparts of no Dox-treated ActaLabel mice (Fig. 1b). Cre and LacZ mRNA expressions were also detected in skeletal muscle of Dox-treated ActaLabel mice, but not in heart, liver, lung and kidney (Fig. 1c). Thus, these transgene expressions were doxycycline-dependent and skeletal myocytes-specific.
To test the washout period of doxycycline in mice, ActaLabel mice were fed with doxycycline diet and drinking water for one week and then switched to control diet and water without Dox for three time periods (12 h, 24 h and 96 h). Quantitative PCR (qPCR) analysis showed that Cre expression was slight after 12 h of Dox withdrawal and negligible after 24 h or 96 h Dox removal (Fig. 1d), which was consistent with the previous report [21]. In contrast, LacZ was expressed at the similar level in all groups (Fig. 1d), indicating that upon the stop cassette is removed, LacZ could be expressed continually even in the absence of Dox. Collectively, these observations suggest that our transgenic model is desirable and suitable for labeling myofibers both spatially and temporally.
According to the donor’s description, expression mosaicism between muscle fibers and muscles was detected when the adult mice were treated with Dox in drinking water for 3 days [22]. In our study, we also observed slight expression mosaicism when mice were fed with Dox for 5 days. However, expression mosaicism almost could not be detected when the mice were fed with Dox both in diet and drinking water for 7 days. Therefore, we speculated that expression mosaicism may be caused by the inadequate Dox exposure. We added Dox both into the diet and drinking water and simultaneously extended induction time to ensure more successive and sufficient induction. Moreover, the induction efficiency of Dox in the embryo may be lower by the mother transmission. For these reasons, we treated the pregnant mice with Dox from E0.5 to ensure more adequate and robust induction to avoid expression mosaicism phenomenon. In addition, induction performed at the beginning of embryonic development ensured that all myofibres formed during embryogenesis could be labelled by LacZ. Remarkably, there was no negative effect of the prolonged administration with Dox observed by us and previous reports [21, 24].
The myofiber number of LD, GA and RF is determined prenatally
To determine the precise timing of myogenesis during development, ActaLabel mice were continuously exposed to Dox during two periods (E0.5–19.5 and E0.5-P7) respectively and analyzed at postnatal day 60 (P60). All the myofibers pre-formed during the induction period would express LacZ permanently, even after doxycycline withdraw. If the myofibers newly emerge after doxycycline removal, no LacZ signals could be detected in them. In the β-gal assay, it was showed that nearly 100 % LD, GA and RF myofibers were LacZ positive in both female and male adult ActaLabel mice which were administrated with Dox for whole pregnancy (Fig. 2a). Similar results were also observed in adult ActaLabel mice on Dox during E0.5-P7 (Fig. 2b). These observations demonstrate that all of the LD, GA and RF myofibers in mice are formed during embryogenesis, which is gender-independent. Thus, we conclude that the total myofiber number of LD, GA and RF has been determined prenatally in mice, without postnatal increase.
The myofiber number of TA and EDL is determined within postnatal one week
Intriguingly, unlike LD, GA and RF, TA and EDL displayed a significantly distinctive development pattern. In the 60-day-old mice treated with Dox throughout the whole pregnancy, only around 76 % of the EDL myofibers were LacZ-positive (Fig. 3), whereas nearly 100 % positive β-gal staining was observed after prolonging Dox administration (E0.5-P7) (Fig. 3). These results imply that the LacZ-negative EDL fibers were formed in the postnatal first week and no extra addition of myofibers occurred afterwards. As for TA, a similar pattern was observed in 60-day-old mice which were induced during embryogenesis, with approximately 31 % LacZ-negative fibers were present (Fig. 4). These data indicate that part of the total myofibers in TA emerge after birth. In contrast, when ActaLabel mice were exposed to Dox during the period E0.5-P7, all myofibers in TA were labeled blue at P60 (Fig. 4), demonstrating that there was no increase in the fiber number afterwards. According to these results, we conclude that the total myofiber number of TA and EDL is determined within postnatal one week in both genders.
By using this elegant genetic system, we demonstrate that not all the number of skeletal muscle fibers is determined before birth. TA and EDL continue to add fibers up through the first week of postnatal development. These newly-emerging myofibers were formed from the individual myoblasts instead of the existing myotubes. Thus, it is quite likely that the de novo forming occurs for TA and EDL within the first week of postnatal growth. It has been well documented that postnatal skeletal muscle growth occurs by proliferation, differentiation and fusion of the committed muscle progenitors (mainly satellite cell) [1, 25]. Therefore, we speculated that the newly-formed myofibers within the first postnatal week may be due to the differentiation of muscle progenitors and myocytes fusion.
Remarkably, the visible blue labeling makes the monitor for myofiber number changes more credible and accurate than the traditional counting. Taken together, our data present an overview that anatomically distinct skeletal muscles have different development time windows. The elucidation of kinetics of myofiber number development would provide theoretical basis for meat production and muscle-associated diseases therapy.