A rim of thin keratinocytes forms around the invaginating mammary rudiment. (A) Optical sections (planar and sagittal views; arrowheads indicate the section plane) of a confocal image from K14-Cre/mTmG mouse embryos at hillock (E12.5), bud (E13.0), and bulb (E13.5) stages. Epithelial tissue labeled with membrane-bound GFP and epithelial marker EpCAM (green). Mesenchymal tissue labeled with membrane-bound tdTomato (red). Yellow area in the planar and yellow line in the sagittal views show how epidermal contact area was defined. Dashed line marks epithelial-mesenchymal border. Scale bar, 50 µm. (B) Representative images showing the procedure determining the relative orientation of ring cells at the mammary hillock (E12.5) stage from R26-CreERT/tdTomato embryos. Upper left: Optical section (planar and sagittal views; arrowheads indicate the section plane) of the confocal image of the sparsely labeled tdTomato⁺ (red) cells with epithelial marker EpCAM (green), Golgi marker GM130 (white), and Hoechst (cyan). Dashed line marks epithelial-mesenchymal border. Scale bar, 50 µm. Upper middle: Planar view of the maximum intensity projection showing epithelial cells (EpCAM, green) and surface rendering of randomly selected tdTomato⁺ ring cells (red). Scale bar, 20 mm. Upper right: Planar view of surface rendering of the mammary primordium (green) and the staining for nuclei (Hoechst, cyan) and Golgi (GM130, magenta) in masked ring cells. Scale bar, 20 mm. Lower left: Planar view of the surface rendering of the mammary primordium (green) with reference points for nuclei (Hoechst, cyan) and Golgi marker (GM130, magenta) in masked tdTomato⁺ ring cells. Scale bar, 20 µm. Lower right: An example of the relative angles between cell vector (black arrow) and center of the bud’s top domain (red line toward orange dot).