Decreased number of satellite cells-derived myonuclei in both fast- and slow-twitch muscles in HeyL-KO mice during voluntary running exercise.

BACKGROUND: Skeletal muscles possess unique abilities known as adaptation or plasticity. When exposed to external stimuli, such as mechanical loading, both myofiber size and myonuclear number increase. Muscle stem cells, also known as muscle satellite cells (MuSCs), play vital roles in these changes. HeyL, a direct target of Notch signaling, is crucial for efficient muscle hypertrophy because it ensures MuSC proliferation in surgically overloaded muscles by inhibiting the premature differentiation. However, it remains unclear whether HeyL is essential for MuSC expansion in physiologically exercised muscles. Additionally, the influence of myofiber type on the requirement for HeyL in MuSCs within exercised muscles remains unclear. METHODS: We used a voluntary wheel running model and HeyL-knockout mice to investigate the impact of HeyL deficiency on MuSC-derived myonuclei, MuSC behavior, muscle weight, myofiber size, and myofiber type in the running mice. RESULTS: The number of new MuSC-derived myonuclei was significantly lower in both slow-twitch soleus and fast-twitch plantaris muscles from exercised HeyL-knockout mice than in control mice. However, expect for the frequency of Type IIb myofiber in plantaris muscle, exercised HeyL-knockout mice exhibited similar responses to control mice regarding myofiber size and type. CONCLUSIONS: HeyL expression is crucial for MuSC expansion during physiological exercise in both slow and fast muscles. The frequency of Type IIb myofiber in plantaris muscle of HeyL-knockout mice was not significantly reduced compared to that of control mice. However, the absence of HeyL did not affect the increased size and frequency of Type IIa myofiber in plantaris muscles. In this model, no detectable changes in myofiber size or type were observed in the soleus muscles of either control or HeyL-knockout mice. These findings imply that the requirement for MuSCs in the wheel-running model is difficult to observe due to the relatively low degree of hypertrophy compared to surgically overloaded models.

Flow Cytometer Analyses, Isolation, and Staining of Murine Muscle Satellite Cells.

Fluorescence-activated cell sorting (FACS) is a powerful and requisite tool for the analysis and purification of adult stem cells. However, it is difficult to separate adult stem cells from solid organs than from immune-related tissues/organs. This is because of the presence of large amounts of debris, which increases noise in the FACS profiles. In particular, it is extremely difficult for unfamiliar researchers to identify muscle stem cell (also known as muscle satellite cell: MuSC) fraction because all myofibers, which are mainly composed of skeletal muscle tissues, become debris during cell preparation. This chapter describes our FACS protocol, which we have used for more than a decade, to identify and purify MuSCs.

Chronology of embryonic and gonadal development in the Reeves' turtle, Mauremys reevesii.

Temperature-dependent sex determination (TSD) is a mechanism in which environmental temperature, rather than innate zygotic genotype, determines the fate of sexual differentiation during embryonic development. Reeves' turtle (also known as the Chinese three-keeled pond turtle, Mauremys reevesii) exhibits TSD and is the only species whose genome has been determined in Geoemydidae to date. Thus, M. reevesii occupy phylogenetically important position for the study of TSD and can be compared to other TSD species to elucidate the underlying molecular mechanism of this process. Nevertheless, neither embryogenesis nor gonadogenesis has been described in this species. Therefore, herein, we investigated the chronology of normal embryonic development and gonadal structures in M. reevesii under both female- and male-producing incubation temperatures (FPT 31 °C or MPT 26 °C, respectively). External morphology remains indistinct between the two temperature regimes throughout the studied embryonic stages. However, the gonadal ridges present on the mesonephros at stage 16 develop and sexually differentiate at FPT and MPT. Ovarian and testicular structures begin to develop at stages 18-19 at FPT and stages 20-21 at MPT, respectively, and thus, the sexual differentiation of gonadal structures began earlier in the embryos at FPT than at MPT. Our results suggest that temperature sensitive period, at which the gonadal structures remain sexually undifferentiated, spans from stage 16 (or earlier) to stages 18-19 at FPT and to stages 20-21 at MPT. Understanding the temperature-dependent differentiation in gonadal structures during embryonic development is a prerequisite for investigating molecular basis underlying TSD. Thus, the result of the present study will facilitate further developmental studies on TSD in M. reevesii.

Dlk1 regulates quiescence in calcitonin receptor-mutant muscle stem cells.

Muscle stem cells, also called muscle satellite cells (MuSCs), are responsible for skeletal muscle regeneration and are sustained in an undifferentiated and quiescent state under steady conditions. The calcitonin receptor (CalcR)-protein kinase A (PKA)-Yes-associated protein 1 (Yap1) axis is one pathway that maintains quiescence in MuSCs. Although CalcR signaling in MuSCs has been identified, the critical CalcR signaling targets are incompletely understood. Here, we show the relevance between the ectopic expression of delta-like non-canonical Notch ligand 1 (Dlk1) and the impaired quiescent state in CalcR-conditional knockout (cKO) MuSCs. Dlk1 expression was rarely detected in both quiescent and proliferating MuSCs in control mice, whereas Dlk1 expression was remarkably increased in CalcR-cKO MuSCs at both the mRNA and protein levels. It is noteworthy that all Ki67+ non-quiescent CalcR-cKO MuSCs express Dlk1, and non-quiescent CalcR-cKO MuSCs are enriched in the Dlk1+ fraction by cell sorting. Using mutant mice, we demonstrated that PKA-activation or Yap1-depletion suppressed Dlk1 expression in CalcR-cKO MuSCs, which suggests that the CalcR-PKA-Yap1 axis inhibits the expression of Dlk1 in quiescent MuSCs. Moreover, the loss of Dlk1 rescued the quiescent state in CalcR-cKO MuSCs, which indicates that the ectopic expression of Dlk1 disturbs quiescence in CalcR-cKO. Collectively, our results suggest that ectopically expressed Dlk1 is responsible for the impaired quiescence in CalcR-cKO MuSCs.