ERα^neg luminal cells represent self-sustained mammary progenitors that clonally expand and self-renew in vivo.

<p><b>(A–B)</b> N1Cre^ERT2R26^mTmG females induced with a low dose of tamoxifen (1µg/g of mouse body weight) at 4 wk of age and analyzed at different time points: 24 h (pulse), 2 wk (2 wk chase), 6 wk (6 wk chase), or 10 wk later (10 wk chase). <b>(A)</b> Representative images of whole mount digestion of mammary ducts labeling single cells (one cell), small clones (two to five cells), and large clones (six or more cells). Notch1-labelled cells are in green and Tomato fluorescence labels non-targeted cells. <i>n</i> = 11 mice. <b>(B)</b> Quantification of the in vivo clonal expansion of GFP-marked cells, at the indicated time points; <i>n</i> = 11. <b>(C–E)</b> N1Cre^ERT2R26^mTmG females were induced with a single dose of tamoxifen (0.1mg/g of mouse body weight) at 4 wk of age and analyzed 10 wk later. The clonal progeny of Notch1-expressing cells (marked in green by GFP) is still luminal (K5^neg in red in C), and ERα^neg (in red in D) and PR^neg (in red in E), as the cells from which it derives. DAPI stains DNA in blue, <i>n</i> = 4. <b>(F)</b> FACS plots of N1Cre^ERT2R26^mTmG females induced at puberty and analyzed after three cycles of pregnancy, lactation, and involution. Mammary epithelial cells (MEC) from females in the third week of the last involution were dissociated and analyzed by flow cytometry. GFP^pos cells represent 28.9 ±1.4% of the luminal population (CD24⁺and CD29^low), <i>n</i> = 2. (<b>G–H</b>) Representative sections of mammary ducts from N1Cre^ERT2R26^mTmG females induced at 4 wk of age and analyzed after three serial pregnancies. GFP-expressing cells (in green) remain invariably negative for ERα (in red in G) and PR expression (in red in H). DAPI stains DNA in blue, <i>n</i> = 2. Scale bars correspond to 40 µm in A; 20 µm in C–E, G and H; and 10 µm in the insets in C–E and G–H.