Figure 2.
Mammary rudiments are characterized by low level of cell proliferation. (A) Optical sections (planar and sagittal views; arrowhead indicates section plane) of confocal microscopy images from R26-CreERT/tdTomato mouse embryos at placode (E11.5), hillock (E12.5), and bulb (E13.5) stages stained with EpCAM (white). Cells were sparsely labeled (cytoplasmic tdTomato, red) with low dosage of tamoxifen 24 h earlier. Scale bar, 50 µm. Dashed line marks the epithelial-mesenchymal border. (B) Planar views of maximum intensity projections of tdTomato-expressing epithelial cells. Cell surface rendering was performed on randomly selected cells (white); dashed line marks the border of the mammary rudiment. Scale bar, 20 µm. (C and D) Quantification of cell sphericity (C) and volume (D) at E11.5, E12.5, and E13.5 (six biological replicates for each stage). nE11.5 = 172 MECs and 170 epidermal cells; nE12.5 = 142 MECs and 136 epidermal cells; nE13.5 = 175 MECs and 175 epidermal cells. (E) Quantification of the total cell number in the mammary rudiment (nE11.5 = 7; nE12.5 = 6; nE13.5 = 6). (F) Optical sections (planar and sagittal views; arrowhead indicates section plane) of confocal images of mammary rudiments from Fucci embryos stained with EpCAM (white); nuclei in G1/G0 in red and in S/G2/M in green. Scale bar, 50 µm. Dashed line marks the epithelial-mesenchymal border. (G) Quantification of the proportions of cells in G1/G0 and S/G2/M in MECs and epidermis (nE11.5 = 7; nE12.5 = 6; nE13.5 = 6). Statistical significances were calculated by Student’s t test to compare mammary rudiment and epidermis at the same developmental stage or one-way ANOVA with Šidák’s post hoc comparison to compare mammary rudiments from three developmental stages. *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001. Data are shown as mean ± SD.

Mammary rudiments are characterized by low level of cell proliferation. (A) Optical sections (planar and sagittal views; arrowhead indicates section plane) of confocal microscopy images from R26-CreERT/tdTomato mouse embryos at placode (E11.5), hillock (E12.5), and bulb (E13.5) stages stained with EpCAM (white). Cells were sparsely labeled (cytoplasmic tdTomato, red) with low dosage of tamoxifen 24 h earlier. Scale bar, 50 µm. Dashed line marks the epithelial-mesenchymal border. (B) Planar views of maximum intensity projections of tdTomato-expressing epithelial cells. Cell surface rendering was performed on randomly selected cells (white); dashed line marks the border of the mammary rudiment. Scale bar, 20 µm. (C and D) Quantification of cell sphericity (C) and volume (D) at E11.5, E12.5, and E13.5 (six biological replicates for each stage). nE11.5 = 172 MECs and 170 epidermal cells; nE12.5 = 142 MECs and 136 epidermal cells; nE13.5 = 175 MECs and 175 epidermal cells. (E) Quantification of the total cell number in the mammary rudiment (nE11.5 = 7; nE12.5 = 6; nE13.5 = 6). (F) Optical sections (planar and sagittal views; arrowhead indicates section plane) of confocal images of mammary rudiments from Fucci embryos stained with EpCAM (white); nuclei in G1/G0 in red and in S/G2/M in green. Scale bar, 50 µm. Dashed line marks the epithelial-mesenchymal border. (G) Quantification of the proportions of cells in G1/G0 and S/G2/M in MECs and epidermis (nE11.5 = 7; nE12.5 = 6; nE13.5 = 6). Statistical significances were calculated by Student’s t test to compare mammary rudiment and epidermis at the same developmental stage or one-way ANOVA with Šidák’s post hoc comparison to compare mammary rudiments from three developmental stages. *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001. Data are shown as mean ± SD.

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