Figure 4. Betaglycan protein expression... | Rockefeller University Press

Figure 4.

$Betaglycan protein expression identifies self-renewing MuSCs that contribute to the in vivo stem cell pool in muscle regeneration. (A and B) Pairwise Pearson correlation coefficient (PCC) was calculated using (A) mRNA and (B) gene score of all the genes against that of Pax7 across all the single cells of MuSC/MPCs. The top-ranked genes with the most robust and significant PCC with Pax7 were plotted. (C) Violin plot showing Betaglycan mRNA expression across the six sub-clusters of MuSC/MPCs defined in Fig. 2 C. (D) Genome browser view of chromatin accessibility at the indicated gene loci of Betaglycan, Pax7, Myod1, and Myog within the five sub-clusters of MuSC/MPC defined by snATAC-seq shown in Fig. 2 A. The height of each tracks normalized against sequencing depth. (E) Acute muscle injury was induced in TA muscle by intramuscular injection of BaCl2. At 3 dpi (top) or 4.5 dpi (bottom), EdU solution was injected intraperitoneally to label the proliferating cells in vivo. TA muscle was collected at 4 h post EdU injection for cross-sections, followed by immuno-fluorescence staining. Arrowhead: the Pax7+/EdU+/Betaglycan+ cells, N = 3. (F) The lower hind limb muscle of Pax7/eGFP mice was used to prepare the single-cell suspensions for FACS analysis. Cells were prepared from uninjured muscle (top row), and the muscle at 3 d post injury (dpi; second row), 4.5 dpi (third row), 7 dpi (fourth row) and 14 dpi (fifth row), respectively. The cells were stained with APC-Cy7–conjugated Betaglycan antibody prior to FACS. Pink bar highlights the fraction of Betaglycan+ cells (right panels) within the eGFP+ populations (left panels). (G) The TA muscle of uninjured Pax7/eGFP reporter mice was collected for immunostaining analysis. N = 3. (H, I, and J) Following the same FACS condition described in F, using 3 dpi mice, the eGFP+/Betaglycan+ and the eGFP+/Betaglycan− cells were freshly sorted separately. An equal amount of eGFP+ cells were transplanted into TA muscle of recipient mice (10,000 cells per TA) that do not express eGFP transgene. The TA muscle of recipient mice was pre-injured 1 d prior to the transplantation. 30 d post-transplantation, the TA muscle was collected for analysis. (H and I) The immunostaining of TA muscle cross-sections. Arrowheads point to the eGFP+/Pax7+ mononuclear MuSCs surrounding newly formed eGFP+ myofibers. (H) A close-up view of the dash-line boxed region in (G, bottom row, Betaglycan+) for further visualization of PAX7 and Laminin staining on TA muscle cross-section. (I) Quantification of eGFP+ MuSCs on 20 cross-section slides of TA muscle collected from 6 mice (three for each genotype). Error bars represent the SD of the data. **: t test P < 0.01.$

Betaglycan protein expression identifies self-renewing MuSCs that contribute to the in vivo stem cell pool in muscle regeneration. (A and B) Pairwise Pearson correlation coefficient (PCC) was calculated using (A) mRNA and (B) gene score of all the genes against that of Pax7 across all the single cells of MuSC/MPCs. The top-ranked genes with the most robust and significant PCC with Pax7 were plotted. (C) Violin plot showing Betaglycan mRNA expression across the six sub-clusters of MuSC/MPCs defined in Fig. 2 C. (D) Genome browser view of chromatin accessibility at the indicated gene loci of Betaglycan, Pax7, Myod1, and Myog within the five sub-clusters of MuSC/MPC defined by snATAC-seq shown in Fig. 2 A. The height of each tracks normalized against sequencing depth. (E) Acute muscle injury was induced in TA muscle by intramuscular injection of BaCl₂. At 3 dpi (top) or 4.5 dpi (bottom), EdU solution was injected intraperitoneally to label the proliferating cells in vivo. TA muscle was collected at 4 h post EdU injection for cross-sections, followed by immuno-fluorescence staining. Arrowhead: the Pax7+/EdU+/Betaglycan+ cells, N = 3. (F) The lower hind limb muscle of Pax7/eGFP mice was used to prepare the single-cell suspensions for FACS analysis. Cells were prepared from uninjured muscle (top row), and the muscle at 3 d post injury (dpi; second row), 4.5 dpi (third row), 7 dpi (fourth row) and 14 dpi (fifth row), respectively. The cells were stained with APC-Cy7–conjugated Betaglycan antibody prior to FACS. Pink bar highlights the fraction of Betaglycan+ cells (right panels) within the eGFP+ populations (left panels). (G) The TA muscle of uninjured Pax7/eGFP reporter mice was collected for immunostaining analysis. N = 3. (H, I, and J) Following the same FACS condition described in F, using 3 dpi mice, the eGFP+/Betaglycan+ and the eGFP+/Betaglycan− cells were freshly sorted separately. An equal amount of eGFP+ cells were transplanted into TA muscle of recipient mice (10,000 cells per TA) that do not express eGFP transgene. The TA muscle of recipient mice was pre-injured 1 d prior to the transplantation. 30 d post-transplantation, the TA muscle was collected for analysis. (H and I) The immunostaining of TA muscle cross-sections. Arrowheads point to the eGFP+/Pax7+ mononuclear MuSCs surrounding newly formed eGFP+ myofibers. (H) A close-up view of the dash-line boxed region in (G, bottom row, Betaglycan+) for further visualization of PAX7 and Laminin staining on TA muscle cross-section. (I) Quantification of eGFP+ MuSCs on 20 cross-section slides of TA muscle collected from 6 mice (three for each genotype). Error bars represent the SD of the data. **: t test P < 0.01.

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