Fig. 3: Bcl11b labels a group of bipotent mammary stem cells.

a Schematic diagram of doxycycline-inducible Bcl11b-rtTA/TetO-Cre/R26-tdTomato model. Bcl11b-rtTA mice were crossed with TetO-Cre mice and R26-tdTomato reporter mice to generate doxycycline inducible reporter mouse model for Bcl11b⁺ cell lineage tracing. b Schematic strategy for lineage tracing Bcl11b⁺ cells from puberty stage. Bcl11b-rtTA/TetO-Cre/tdTomato mice were administered with 2 mg/mouse doxycycline (100 µL, 20 mg/mL in PBS) intraperitoneally for three consecutive days at 4 weeks of age. The mammary glands were harvested for tdTomato labeling analysis 2 days after pulsing. c Representative FACS plots showing the percentage of tdTomato-positive cells in epithelial population (left panel) and the proportion of basal or luminal subpopulation in tdTomato⁺ epithelial cells (right panel), 2 days after doxycycline induction at puberty. d Representative immunofluorescence images showing the co-staining of Bcl11b, tdTomato and basal cell marker αSMA in nipple and duct in mammary glands 2 days after doxycycline induction at puberty (left panel). Yellow, Bcl11b; red, tdTomato; green, αSMA; blue, DAPI. Scale bars, 50 μm. And bar chart showing the labeling efficiency of Bcl11b⁺ cells by tdTomato in nipple and duct of mouse mammary glands (right panel). Statistical analysis was performed using two-tailed unpaired t test. Data were presented as means ± SEM, n = 3. *P < 0.05. e Representative FACS plots showing the tdTomato-labeling percentage in basal cells (Lin⁻Epcam^low CD49f⁺) of TEB, duct or nipple 2 days after doxycycline induction from puberty stage. n = 3. f Bar graph showing the statistical analysis of the percentage of tdTomato⁺ cells in basal or luminal compartments from TEB, duct and nipple 2 days after doxycycline induction from 4-week-old mice by FACS as in e. Bcl11b-rtTA/TetO-Cre/ tdTomato mice with PBS treatment were control group (CTR), while that with doxycycline treatment were experimented group (EXP). Statistical analysis was performed using two-tailed unpaired t-test. Data were presented as means ± SEM, n = 3−5. g Schematic diagram showing the lineage tracing strategy of Bcl11b⁺ cells from embryonic day 15.5 in h−j. h Representative whole-mount images of tdTomato-labeled mammary glands in 8-week-old mice traced from embryonic stage. Scale bars, 5 mm. i Representative FACS plots display the percentage of tdTomato-positive cells in epithelial population (middle panel), and the proportion of basal or luminal subpopulation in tdToamto⁺ epithelial cells (right panel), from 8-week-old mice traced from embryonic stage. j Bcl11b-lineage tracing from the embryonic stage (E15.5 d) to adulthood (P8w). Bcl11b⁺ epithelial cells were traced from embryonic day 15.5 (E15.5 d). When these offspring reached 8 weeks of age (P8w), their mammary glands were harvested for tdTomato-labeling analysis. j1, j2 Bar graphs showing the percentage of tdTomato-labeled cells in luminal or basal population (j1) and the percentage of luminal or basal cells among the total tdTomato-labeled epithelial cells (j2). Statistical analysis was performed using two-tailed unpaired t-test. Data were presented as means ± SEM, n = 5. k Schematic diagram showing the lineage-tracing strategy of Bcl11b⁺ cells for induction in pubertal mice at 4 weeks of age, with various chase periods as indicated (2 days, 8 weeks, 20 weeks, 1 year) in l−p. l Representative FACS plots depict the distribution of tdTomato-traced cells in epithelial gate, 8 weeks after induction at puberty. m, n Bar charts showing the composition of tdTomato-labeled epithelial cells (m), the percentage of tdTomato-labeled epithelial cells in total epithelial cells (n) in Bcl11b-lineage tracing at designated tracing times after pulsing at puberty. Statistical analysis was performed using two-tailed unpaired t-test. Data were presented as means ± SEM, n = 14 (2-day chase), n = 5 (8-week chase), n = 5 (20-week chase) and n = 6 (1-year chase). *P < 0.05, **P < 0.01 and ***P < 0.001. o FACS plots showing the contribution of Bcl11b-traced cells to various mammary populations 20 weeks after pulsing at puberty. Sca-1 was used as a marker of the ESR1⁺ luminal cells. p Representative immunofluorescence image showing tdTomato-labeled clone containing basal cell (magenta arrowhead), ESR1⁺ luminal cell (white arrowhead) and ESR1⁻ luminal cell (green arrowhead) 20 weeks after pulsing at puberty. Scale bar, 10μm.