Figure 3.
Cell influx drives mammary placode formation. (A) Optical section (planar and sagittal views; arrowhead shows section plane) of a mammary placode (left) and nonmammary epidermis (right) from E11.5 Fucci, mKO2;TCF/Lef1:H2B-GFP embryos. Cells in G1/G0 express nuclear mKO2 (red), while Wnt reporter active cells express nuclear GFP (green); Hoechst staining for nuclei (cyan) and EpCAM for epithelial cells (white). Dashed line marks the epithelial-mesenchymal border. Scale bar, 50 µm. (B) Quantification of nuclear sphericity of MECs and epidermal cells at E11.5. Student’s t test; ****, P ≤ 0.0001; n = 652 MECs and 882 epidermal cells from 14 biological replicates. Data shown represent median (line) with 25th and 75th percentiles (hinges) plus min to max ranges (whiskers). (C) Cells in E11.5 R26-CreERT/tdTomato embryos were sparsely labeled by low dosage of tamoxifen 24 h earlier and stained with EpCAM (white), GM130 (magenta), and Hoechst (cyan) to mark epithelial cells, Golgi, and nuclei, respectively. Figure shows planar views of a representative masked single cell (1–6) to define cell polarity. The surface-rendered cell (red; 1), nucleus (cyan; 2), Golgi (magenta; 3), overlay of both nucleus and Golgi (4), overlay of nucleus (cyan) and Golgi (magenta) with reference points for both (5), and cell vector (white arrow) from nuclear reference point (cyan) to Golgi reference point (magenta) (6). Scale bar, 5 µm. (D) Cell vectors (white arrows) show the polarity of MECs and epidermal cells at E11.5. Center of the placode or the control epidermal region is indicated by the orange spot. Scale bar, 20 µm. (E) Schematic image showing two examples of the angle between the cell vector and center of the placode (orange spot). (F) Rose plots showing the angles between cell vectors and the center of a mammary placode or image center of a corresponding epidermal region. Rayleigh test for nonuniformity, H0 = random. Watson’s U2 test revealed a significant difference between MEC and epidermal cell plots. n = 172 MECs and 170 epidermal cells from six biological replicates. (G) Confocal time-lapse 3D imaging of a forming mammary placode. Images show tracks of representative individual MECs and epidermal cells expressing Fucci, mKO transgene (nuclear red) on the left and track direction (arrows) on the right. Dashed line demarcates the mammary placode. Scale bar, 50 µm. See also Video 1. (H) Quantification of track straightness, length, and net displacement of MECs (n = 30) and epidermal cells (EC, n = 30) from three biological replicates. Statistical significance was tested with Mann-Whitney U test and Student’s t test; ***, P ≤ 0.001; ****, P ≤ 0.0001. (I) Rose plots of the escape angles (the angle between cell trajectories at the beginning and end of the time lapse with respect to the center of the placode or a corresponding epidermal region). Rayleigh test for nonuniformity, H0 = random. Watson’s U2 test revealed a significant difference between mammary placode cell and epidermal cell plots.

Cell influx drives mammary placode formation. (A) Optical section (planar and sagittal views; arrowhead shows section plane) of a mammary placode (left) and nonmammary epidermis (right) from E11.5 Fucci, mKO2;TCF/Lef1:H2B-GFP embryos. Cells in G1/G0 express nuclear mKO2 (red), while Wnt reporter active cells express nuclear GFP (green); Hoechst staining for nuclei (cyan) and EpCAM for epithelial cells (white). Dashed line marks the epithelial-mesenchymal border. Scale bar, 50 µm. (B) Quantification of nuclear sphericity of MECs and epidermal cells at E11.5. Student’s t test; ****, P ≤ 0.0001; n = 652 MECs and 882 epidermal cells from 14 biological replicates. Data shown represent median (line) with 25th and 75th percentiles (hinges) plus min to max ranges (whiskers). (C) Cells in E11.5 R26-CreERT/tdTomato embryos were sparsely labeled by low dosage of tamoxifen 24 h earlier and stained with EpCAM (white), GM130 (magenta), and Hoechst (cyan) to mark epithelial cells, Golgi, and nuclei, respectively. Figure shows planar views of a representative masked single cell (1–6) to define cell polarity. The surface-rendered cell (red; 1), nucleus (cyan; 2), Golgi (magenta; 3), overlay of both nucleus and Golgi (4), overlay of nucleus (cyan) and Golgi (magenta) with reference points for both (5), and cell vector (white arrow) from nuclear reference point (cyan) to Golgi reference point (magenta) (6). Scale bar, 5 µm. (D) Cell vectors (white arrows) show the polarity of MECs and epidermal cells at E11.5. Center of the placode or the control epidermal region is indicated by the orange spot. Scale bar, 20 µm. (E) Schematic image showing two examples of the angle between the cell vector and center of the placode (orange spot). (F) Rose plots showing the angles between cell vectors and the center of a mammary placode or image center of a corresponding epidermal region. Rayleigh test for nonuniformity, H0 = random. Watson’s U2 test revealed a significant difference between MEC and epidermal cell plots. n = 172 MECs and 170 epidermal cells from six biological replicates. (G) Confocal time-lapse 3D imaging of a forming mammary placode. Images show tracks of representative individual MECs and epidermal cells expressing Fucci, mKO transgene (nuclear red) on the left and track direction (arrows) on the right. Dashed line demarcates the mammary placode. Scale bar, 50 µm. See also Video 1. (H) Quantification of track straightness, length, and net displacement of MECs (n = 30) and epidermal cells (EC, n = 30) from three biological replicates. Statistical significance was tested with Mann-Whitney U test and Student’s t test; ***, P ≤ 0.001; ****, P ≤ 0.0001. (I) Rose plots of the escape angles (the angle between cell trajectories at the beginning and end of the time lapse with respect to the center of the placode or a corresponding epidermal region). Rayleigh test for nonuniformity, H0 = random. Watson’s U2 test revealed a significant difference between mammary placode cell and epidermal cell plots.

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