Analysis of nuclear shape and cell polarity in forming mammary placodes. (A) Representative images showing surface rendering of randomly selected nuclei based on the expression of nuclear mKO2 (G1/G0 marker of the Fucci cell cycle reporter, red) and nuclear GFP (TCF/Lef:H2B-GFP Wnt reporter, green) in a mammary placode (left) and epidermis (right) at E11.5; double positive mKO2 and GFP nuclei in yellow. Epithelial cells were stained with EpCAM (white). Gray area in the upper picture delineates the placode. Insets are close-ups of the indicated areas. Scale bar, 20 µm. (B) Optical sections (planar and sagittal views; arrowheads indicate the section plane) of confocal microscopy images from E11.5 R26-CreERT/tdTomato mouse embryos. Cells were sparsely labeled with low dosage of tamoxifen 24 h before to induce the expression of cytoplasmic tdTomato (red). The cells were stained with EpCAM (white), GM130 (green), and Hoechst (cyan) to mark epithelial cells, Golgi, and nuclei, respectively. Dashed line marks the epithelial-mesenchymal border. Scale bar, 50 µm. (C) Planar views of representative images showing the process of creating reference points using nuclear marker (Hoechst, cyan) and Golgi marker (GM130, green) of mammary placode (upper panel) and epidermis (lower panel) at mammary placode stage (E11.5) that are required for cell angle analysis shown in Fig. 3, C–F. Epithelial cells are further stained with EpCAM (white). First column shows overlay of maximum intensity projection of confocal image and surface rendering of randomly selected cells expressing tdTomato (from R26-CreERT/tdTomato, red). Second column shows surface rendering (white) of mammary placode or epidermis together with surface rendering of randomly selected tdTomato⁺ epithelial cells (red). Third column shows the nucleus (cyan) and GM130 (Golgi marker, magenta) staining in masked tdTomato⁺ epithelial cells from the mammary placode and epidermis. Fourth column shows surface rendering of the mammary placode and epidermis with reference points for nuclei (Hoechst, cyan; reference point, cyan) and Golgi marker (GM130, magenta; reference point, magenta) in masked tdTomato⁺ epithelial cells from the mammary placode and epidermis. Scale bar, 20 µm. (D) Summary of all the relative cell orientations in 2D of MECs (left, n = 172 from six biological replicates) and epidermal cells (right, n = 170 from six biological replicates) at E11.5. Each arrow represents one cell. Red star marks the mammary placode center (left) or image center of the epidermis (right).