Fig 1: Gpr125+ cells have a MaSC/MRU profile and encompass distinct progenitor populations.a Representative FACS dot plots of total MECs (left) isolated from 6-week Adgra3lz/+ pubertal mice stained with antibodies against CD24 and CD49f. n = 4 mice/ experiment and repeated five times. The basal population is depleted in FDG? populations (center). Gpr125+/FDG+ cells gated within the CD24med/low/CD49f+/hi basal population (right). b Histograms showing expression of CD49f, CD29, Sca1 and CD61 in Gpr125+/FDG+ (red line), Gpr125?/FDG?(black line) and total MECs (gray line) n = 4 mice/experiment repeated three times. c Detection of reporters in the cap cell layer of TEBs: left panel: co-expression of s-SHIP-EGFP detected by immunohistochemistry (brown), on top of Gpr125-ß-gal detected by X-gal (dark blue) counterstained with hematoxylin (purple); and individual expression in center panel: Gpr125-ß-gal detected by X-gal (blue); right panel: s-SHIP-EGFP fluorescence (green). Scale bar = 50 µm (n = 2 mice/panel). d Adgra3 and Lgr5 mRNA expression in the basal cell cluster, visualized by t-SNE plots extracted from the Tabula Muris dataset42. e–g RNA-seq based transcriptomic comparison of FACS-sorted Gpr125+ with Gpr125? cells isolated from TEB-distal (purple) and nipple-proximal regions (yellow) of pubertal Adgra3lz/+ mammary glands. e Venn diagram shows 140 differentially expressed genes (gray) are shared by TEB-distal and nipple-proximal Gpr125+ cells (twofold change, FDR = < 0.1). Biological significance was evaluated by (f) gene ontology (GO) and (g) gene set enrichment analysis (GSEA) analysis of TEB-distal and nipple-proximal Gpr125+ cells compared to their Gpr-negative basal cell counterparts. GSEA shows a correlation between genes highly expressed in TEB-distal (blue) or nipple-proximal (yellow) Gpr125+ cells compared to the ‘Lim_mammary_stem_cell_UP’ gene signature. h Volcano plot comparing gene expression of nipple-proximal Gpr125+ cells and TEB-distal Gpr125+ including highlighted candidates in nipple Gpr125+(blue dots) and TEB cells (red dots) (FDR = 0.1). i GO analysis yielded gene enrichment in biological processes (red bars) and molecular function (green bars) that characterize Gpr125+ cells (FDR B&H). j Differently expressed genes between TEB-distal Gpr125+ and nipple-proximal Gpr125+ cells were confirmed and mapped to distinct basal cell clusters by scRNAseq analysis of the pubertal dataset of Pal et al.28. KNetL plots are shown for cell populations expressing Adgra3, Krt14, Tcf7, Sox11, Fbln1, Lrfn5, Lgr5, Slpi Wnt10a, Wnt5a, Wnt6. k Distal Sox11 expression in TEBs is detected in Tcf1+ cap and Tcf1? body cells and some adjacent mesenchymal cells. (n = 4 mice). Scale bar = 50 µm. Source data are provided as a Source Data file.