Fig 3. Head-derived MuSCs adopt a limb-like transcriptome upon transplantation in limb muscles.

(A) Experimental scheme. EOM and TA-derived MuSCs were engrafted into pre-injured recipient TA muscles. After regeneration, engrafted MuSCs were re-isolated and processed through RNA- and BS-sequencing. ‘Post-graft’ MuSCs were compared to their ‘Pre-graft’ counterparts. N = 10 donor mice and N = 10 recipient mice. For each donor mouse, equal number of EOM and TA MuSCs (in the range of 10,000 cells) were transplanted to TA muscles of the same recipient mouse. For each donor muscle, 500 pre-graft cells and 60 re-isolated post-graft cells (mean value) were analysed (S1 Table). (B) Expression analysis of EOM post-graft and EOM pre-graft. Each dot represents a gene. Genes are colour-coded according to their fold change in pre-graft TA vs EOM MuSCs (from Fig 1, without transplantation). Note that genes highly expressed in pre-graft TA MuSCs (from Fig 1, coloured in pink) were mostly upregulated in EOM MuSCs following grafting, while genes highly expressed in pre-graft EOM MuSCs (from Fig 1, coloured in blue) were mostly downregulated in EOM MuSCs following grafting. (C) PCA analysis of TA pre-graft, EOM pre-graft and EOM post-graft MuSCs using the expression values of genes differentially expressed between EOM and TA pre-graft MuSCs. PC1 separates EOM and TA MuSCs and shows that after engrafting EOM MuSCs into the TA muscle, they resemble transcriptionally TA MuSCs more than EOM MuSCs. (D) (left) Classification of EOM and TA-specific genes as resistant, responsive, or intermediate upon heterotopic transplantation of EOM MuSCs. Determining the responsiveness of each gene to transplantation was carried out in two steps. First, expression values for TA-specific genes (resp EOM-specific) in pre-graft MuSCs were rescaled to 0–100, where 0 represented the mean expression in EOM (resp TA) MuSCs and 100 represented the mean expression in TA (resp EOM) MuSCs. Next, the expression of EOM and TA-specific genes were rescaled similarly in post-graft EOM MuSCs. Each dark blue dot represents a gene with corresponding rescaled value in post-graft EOM MuSCs. EOM-specific genes with rescaled values in post-graft EOM MuSCs less than 25 were classified as responsive, between 25 and 75 as intermediate and above 75 as resistant. TA-specific genes with rescaled values in post-graft EOM MuSCs less than 25 were classified as resistant, between 25 and 75 as intermediate and above 75 as responsive. (right) Distribution of responses of EOM and TA-specific genes in post-graft EOM MuSCs. (E) Expression of selected markers between TA pre-graft, EOM pre-graft and EOM post-graft MuSCs. Many TA marker genes were upregulated to TA-like levels when EOM MuSCs were grafted into TA muscle. In addition, some EOM marker genes such as Lmx1a and Mobp were downregulated in this scenario. *** p < 0.001, ** p < 0.01, * p < 0.05 by Welch’s t test. (F) Gene expression analysis throughout the HoxA, HoxB, HoxC and HoxD clusters in post-graft EOM (heterologous graft, left panel) and TA (homologous graft, middle panel) MuSCs. Genes were colour-coded according to their antero-posterior expression domain in mouse at embryonic day 12.5 (right panel, adapted from [57]). All TA-specific Hox genes were upregulated in post-graft EOM MuSCs. See Fig 1E for pre-graft expression levels in EOM and TA MuSCs.