Fig. 4: Peri-TEB fibroblasts represent a transient cell state and do not migrate with the branching epithelium.

a Schematic representation of the Acta2-CreERT2;R26-mTmG mouse model. b Lineage tracing strategy for the chase experiments. c, d Flow cytometry quantification of GFP+ cells within total stromal cells (c) and quantification of the proportion of GFP+ fibroblasts in the 3 fibroblast populations detected by FACS (d); n = 3 mice per time-point. Statistical analysis: Wilcoxon, two-sided (c) and chi-square test (d). e, f Projections of z-stack imaging of whole-mount mammary glands 24 h, 1 week or 3 weeks after tamoxifen induction. Red boxes in the whole organ overviews (e) indicate the regions presented in (f) Red boxes in detailed pictures of ductal or peri-TEB regions (f) indicate magnified regions with GFP+ fibroblasts (in insets i–iv). The GFP channel is shown as a “fire” lookup table. White arrowheads indicate GFP+ fibroblasts, orange arrowheads indicate GFP+ adipocytes, empty arrowheads indicate GFP+ mural cells. Scale bars: 1 cm in (e); 100 µm in (f), 50 µm in insets in (f); the images are representative of 3 mice per time-point. g Quantification of the distribution of GFP+ fibroblasts in TEBs or ductal regions after 24 h, 1 week or 3 weeks of chase; n = 3 mice per time-point, N = 38 z-stack for the 24 h chase, 22 z-stacks for 1 week and 51 z-stacks for the 3 weeks chase. Statistical analysis: Wilcoxon test, two-sided. h Quantification of the distance of GFP+ fibroblasts from TEBs. n = 3 mice per time-point, N = 343 fibroblasts for 24 h, 106 for 1 week and 144 for 3 week-chase. Statistical analysis: Wilcoxon test, two-sided.