Fig. 6: Peri-TEB fibroblasts are recruited from preadipocytes.

a UMAP plot showing the expression of Fgf10 in the different fibroblast clusters from Fig. 1e. b Detection of Fgf10 expression in distal fat pad and peri-TEB areas using in situ hybridization. The dashed yellow line indicates the TEB border. The yellow boxes demarcate distal fat pad (A) and peri-TEB (B) regions shown in the magnified insets. White arrowheads point to Fgf10+ fat pad fibroblasts, empty arrowheads indicate Fgf10^neg peri-TEB fibroblasts. Scale bar: 100 µm and 20 µm in insets. The image is representative of 3 biological replicates. c Schematic representation of the Fgf10-CreERT2;R26-mTmG mouse model. d, e Flow cytometry quantification of GFP+ cells within total stromal cells (d) and quantification of the proportion of GFP+ fibroblasts among the fibroblast populations defined by FACS (e). n = 4 mice per time-point. Statistical analysis: Student’s t-test, two-sided in (d) and chi-square test in (e). f–j. Immunofluorescence analysis of mammary glands from tamoxifen-induced Fgf10-CreERT2;R26-mTmG females after 24 h, 3 days, 1 week or 3 weeks of chase. f Representative images of z-stack projections of mammary gland wholemounts. Scale bar: 100 µm. g, h Quantification of GFP+ fibroblasts in the TEBs and subtending duct regions (the regions are defined in schematics in g), as detected in wholemounts. In the box plots showing quantifications (h), each dot represents the number of GFP+ cells in one FOV. f–h n = 4 independent experiments, N = 15; 12 and 9 TEBs for 24 h, 3 days and 1 week time points, respectively. Statistical analysis: Wilcoxon test, two-sided. i, j Immunofluorescence analysis of mammary gland sections from tamoxifen-induced Fgf10-CreERT2;R26-mTmG females after 24 h, 3 days and 1 week of chase. i Representative images of tissue sections containing TEBs: dashed lines indicate GFP+ fibroblasts, empty arrowheads indicate GFP+ αSMA^neg cells and red arrowheads indicate GFP+ αSMA+ cells. Scale bars: 10 µm. j Quantification of the proportion of double positive GFP+ αSMA+ cells among all GFP+ cells, shown as box plots. Each dot indicates the proportion of double positive cells in one FOV; n = 3 independent experiments, N = 12; 3 and 13 FOVs for 24 h, 3 days and 1 week of chase, respectively. Statistical analysis: Wilcoxon test, two-sided.