Figure 4.
A rim of thin keratinocytes encircles the invaginating mammary bud. (A) Optical sections (planar and sagittal views; arrowheads show the section plane) of confocal whole-mount microscopy images from K14-Cre/mTmG mouse mammary primordia at placode (E11.25), hillock (E12.25 and E12.5), bud (E13.0), and bulb (E13.5) stages; epithelium in green (cell-membrane localized GFP and EpCAM staining) and mesenchyme in red. Dashed line marks the epithelial-mesenchymal border. Lower panel: inserts (i) are close-ups of planar views marked in orange boxes in the upper panel. Arrowheads indicate ring cells. Scale bar, 50 µm. (B) Quantification of epidermal contact area of the mammary rudiment. One-way ANOVA with Šidák’s post hoc comparison; ***, P ≤ 0.001; ****, P ≤ 0.0001; nE12.5 = 8; nE13.0 = 9; nE13.5 = 9. Data are shown as mean ± SD. (C) Planar views of maximum intensity projections showing epithelial tissue (cell-membrane localized GFP) at E12.5 and E13.5. Surface rendering of the keratinocytes (ring cells, red) surrounding the mammary bud at E12.5 and cells in the neck at E13.5. Scale bar, 20 µm. See also Video 2. (D) Quantification of the volume and sphericity of mammary epithelial, ring, neck, and epidermal cells (EC) before (E12.5) and after (E13.5) invagination; six biological replicates for both stages. nE12.5 ring = 86 ring cells, 142 MECs, and 136 ECs; nE13.5 = 38 neck cells, 175 MECs, and 175 ECs. Data are shown as mean ± SD. Statistical significance was calculated by Student’s t test to compare ring and neck cells or one-way ANOVA with Šidák’s post hoc comparison or Kruskal-Wallis test to compare ring cells, MECs, and ECs at the same developmental stage; **, P ≤ 0.01; ****, P ≤ 0.0001. (E) Planar views of surface rendering of ring cells (red) and mammary bud (green) at the hillock (E12.5) stage. Cell vectors (white arrowheads) show the polarity of ring cells as defined by the nucleus (cyan) to Golgi (magenta) vector. Orange spot marks the center of the bud’s top surface. Scale bar, 20 µm. (F) Coordinate system showing ring cells’ vectors (arrows) and their opposing directionality at E12.5. Red dot marks the center of the hillock’s top domain. A, anterior; D, dorsal; P, posterior; V, central (nE12.5 = 55 ring cells from four biological replicates). (G) Rose plot representing the angles between the cell vector and the center of mammary bud’s top domain. Rayleigh test for nonuniformity, H0 = random.

A rim of thin keratinocytes encircles the invaginating mammary bud. (A) Optical sections (planar and sagittal views; arrowheads show the section plane) of confocal whole-mount microscopy images from K14-Cre/mTmG mouse mammary primordia at placode (E11.25), hillock (E12.25 and E12.5), bud (E13.0), and bulb (E13.5) stages; epithelium in green (cell-membrane localized GFP and EpCAM staining) and mesenchyme in red. Dashed line marks the epithelial-mesenchymal border. Lower panel: inserts (i) are close-ups of planar views marked in orange boxes in the upper panel. Arrowheads indicate ring cells. Scale bar, 50 µm. (B) Quantification of epidermal contact area of the mammary rudiment. One-way ANOVA with Šidák’s post hoc comparison; ***, P ≤ 0.001; ****, P ≤ 0.0001; nE12.5 = 8; nE13.0 = 9; nE13.5 = 9. Data are shown as mean ± SD. (C) Planar views of maximum intensity projections showing epithelial tissue (cell-membrane localized GFP) at E12.5 and E13.5. Surface rendering of the keratinocytes (ring cells, red) surrounding the mammary bud at E12.5 and cells in the neck at E13.5. Scale bar, 20 µm. See also Video 2. (D) Quantification of the volume and sphericity of mammary epithelial, ring, neck, and epidermal cells (EC) before (E12.5) and after (E13.5) invagination; six biological replicates for both stages. nE12.5 ring = 86 ring cells, 142 MECs, and 136 ECs; nE13.5 = 38 neck cells, 175 MECs, and 175 ECs. Data are shown as mean ± SD. Statistical significance was calculated by Student’s t test to compare ring and neck cells or one-way ANOVA with Šidák’s post hoc comparison or Kruskal-Wallis test to compare ring cells, MECs, and ECs at the same developmental stage; **, P ≤ 0.01; ****, P ≤ 0.0001. (E) Planar views of surface rendering of ring cells (red) and mammary bud (green) at the hillock (E12.5) stage. Cell vectors (white arrowheads) show the polarity of ring cells as defined by the nucleus (cyan) to Golgi (magenta) vector. Orange spot marks the center of the bud’s top surface. Scale bar, 20 µm. (F) Coordinate system showing ring cells’ vectors (arrows) and their opposing directionality at E12.5. Red dot marks the center of the hillock’s top domain. A, anterior; D, dorsal; P, posterior; V, central (nE12.5 = 55 ring cells from four biological replicates). (G) Rose plot representing the angles between the cell vector and the center of mammary bud’s top domain. Rayleigh test for nonuniformity, H0 = random.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal