(A) Schematic of experiment design whereby mice were administered VML injuries to the TA muscles, and the injured TAs were collected at 14 dpi for spatial transcriptomics analysis. (B) Representative image of H&E-stained section of VML defect at 14 dpi. (C) Representative tissue annotation into the 3 zones — a defect zone, a zone of intact muscle, and a transition zone between them. (D) Distribution of unique molecular identifiers shows higher read counts at the location of the defect. (E) Integration of spatial transcriptomics data sets with matched, cell type–annotated scRNA-Seq data sets using Seurat label transfer predicts the defect zone being predominantly inhabited by profibrotic mesenchymal-derived cells. Muscle stem cells are still largely absent from the defect zone but localize in the intact muscle zone. Scales indicate prediction scores. Representative of 2 replicates from 1 male and 1 female. (F) Heatmap of differentially expressed genes by zone and time point highlights loss of inflammatory transcripts within the defect zone by 14 dpi but continued expression of profibrotic genes. The transition zones at 7 dpi and 14 dpi were transcriptionally distinct, with more active regeneration occurring at 7 dpi. Color bar shows scaled expression (Z scores). (G) Volcano plots showing substantial differential gene expression across time points in both the defect (top) and transition (bottom) zones. Inflammatory genes are downregulated by 14 dpi in both zones, while myogenic terms are upregulated. Yellow indicates log₂ fold change greater than 0.0585 and adjusted P < 0.05, which was considered significant. Green indicates log₂ fold change greater than 0.0585 and P > 0.05. Magnification, 20×.