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.