Visualization of PAX7 protein dynamics in muscle satellite cells in a YFP knock-in-mouse line

Figures

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  Fig. 1. Generation of Pax7-YFP knock-in mice. a Schematic diagrams showing the knock-in construct and knock-in allele. A targeting vector for generating a Pax7-YFP knock-in mouse line was constructed by inserting an EYFP sequence downstream of exon 9 of the Pax7 gene locus. b Genotype of Pax7-YFP knock-in mice was verified by a two-primer PCR strategy (wild-type = 134 bp, Pax7-EYFP = 2441 bp). Pax7+/+, wild-type; Pax7YFP/+, heterozygous Pax7YFP/+; Pax7YFP/YFP, homozygous Pax7YFP/YFP. c Immunohistochemistry of YFP in the neural tube (NT) and somite region of a Pax7YFP/YFP mouse embryo at E10.5, scale bar 50 μm. d YFP fluorescence signals in the dermomyotome (DM) region of a Pax7YFP/YFP mouse embryo at E10.5, scale bar 50 μm. e Representative fluorescence images of YFP+ cells in soleus (Sol) and extensor digitorum longus (EDL) muscle tissues freshly isolated from Pax7YFP/YFP mice, scale bar 50 μm. f Immunohistochemistry of YFP and laminin α2 in cryosections of TA muscle from adult Pax7YFP/YFP mice. Arrows indicate YFP+ cells, scale bar 50 μm. g Immunohistochemistry showed that YFP+ cells were co-localized with PAX7+ satellite cells in transverse sections of TA muscle in Pax7YFP/YFP mice.…

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  Fig. 2. Satellite cell population is sortable from Pax7-YFP mice. a–c FACS profiles show a CD31−CD45−Sca1−Vcam1+ cell fraction of mononuclear cells isolated from limb muscles of wild-type mice (a). CD31−CD45−Sca1−Vcam1+ cells were sorted by FACS and stained with a PAX7 antibody (b) [quantified in (c)], scale bar 50 μm. Data represent the mean ± s.e.m. (n = 3). d A CD31−CD45−Sca1−Vcam1+ cell fraction was almost completely positive for YFP fluorescence. Top panel: FACS profiles of mononuclear cells derived from limb muscles of wild-type (Pax7+/+) mice. Bottom panel: FACS profiles of mononuclear cells derived from limb muscles of Pax7YFP/+ mice. e FACS plots demonstrated that YFP+ cells isolated from limb muscle of Pax7YFP/+ mice were positive for Vcam1 and negative for CD31, CD45, and Sca1. YFP+ cells were stained with (lower panel) or without (upper panel) a Vcam1 antibody. f–j A YFP+ cell population was isolated from limb muscles of Pax7YFP/+ mice by FACS without antibody staining. FACS profiles of mononuclear cells derived from Pax7+/+ or Pax7YFP/+ mice (f). The red circles indicate YFP+ cells. A representative image of isolated YFP+ cells cultured in growth medium (GM) for 3 days (g),…

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  Fig. 3. YFP expression levels mirror the endogenous dynamics of PAX7. a–h Regeneration of the TA muscle was induced by injection of cardiotoxin (CTX). H&E staining of cross-sections of TA muscle in Pax7+/+ and Pax7YFP/+ mice, 0, 3, 7, and 14 days following CTX injection, scale bar 50 μm (a). Q-PCR analysis of gene expression dynamics of myogenic regulatory factors (Pax7, Myf5, MyoD, and Myogenin) and YFP during muscle regeneration (b–f). Data represent means ± s.e.m. (ND, not detected; n = 3–5, per group). g Immunohistochemistry for YFP and Laminin α2 proteins in cross-sections of TA muscle isolated from Pax7+/+ or Pax7YFP/+ mice at day 3 after CTX injection, scale bar 50 μm. h Immunohistochemistry for YFP and PAX7 proteins in cross-sections of TA muscle isolated from Pax7YFP/+ mice at day 3 after CTX injection, scale bar 10 μm. i–o Satellite cells were isolated from limb muscles of Pax7YFP/+ mice and cultured in growth medium (GM). Myogenic differentiation was induced by differentiation medium (DM) for 3 days. Representative images are shown (i), scale bar 100 μm. j–o Q-PCR analysis shows a positive correlation between Pax7 and YFP mRNA levels during myogenic differentiation [quantified…

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  Fig. 4. Homozygous Pax7-YFP mouse-derived satellite cells exhibit a wild-type stem cell function. a–c Regeneration was induced in the TA muscle of Pax7+/+, Pax7YFP/+, and Pax7YFP/YFP mice by injection of CTX as shown in Fig. 3. Regenerating muscles were isolated at day 14 following CTX injection. Representative immunohistochemical images for Laminin α2 in cross-sections of TA muscle (a). Cross-sectional area (CSA) of centrally nucleated regenerating myofibers was quantified (b), scale bar 50 μm. (n = 4). Muscle weight of TA muscles was measured (c) (n = 3–7). Data represent means ± s.e.m. One-way ANOVA followed by the Bonferroni post hoc test: NS non-significant. d–j Satellite cell populations were isolated from limb muscles of Pax7+/+ or Pax7YFP/YFP mice by FACS and cultured as shown in Fig. 2. Time course for analysis (d). e Representative images of EdU staining of plated satellite cells in GM conditions [quantified in (f)]. Nuclei were stained with DAPI. > 300 cells per mouse were counted (n = 3 mice). Data represent means ± s.e.m. (NS non-significant), scale bar 50 μm. g Myogenic differentiation was induced under differentiation medium (DM) conditions for 4 days and cells…