Distribution of cell cycle indicators and cell fate markers in embryonic mammary glands. (A) Pipeline for spatial analysis of cell cycle activity based on the Fucci2a reporter. (B) Distribution of cell cycle status (% M/G2/S cells) and cell density (n cells) by the distance to the surface of the gland from seven glands of four embryos. (C and D) Gray values of keratin 14 (K14) and keratin 8 (K8) per cell (C) and the ratio of K14-to-K8 gray values by the distance of the cell to the gland surface (D) derived from confocal 3D images of five E18.5 mammary glands of three embryos (n = 77,921 cells, color-coding indicates the density of cell neighbors; turbo LUT, blue→red = low→high). (E) The average % of cells with more K14 than K8 expression per gland (n = 5 glands of three embryos) by the distance from the gland surface. (F) Image of a paraffin section of a E18.5 mammary gland (top) and a close-up of a terminal branch (bottom), stained for p63 (cyan) and K8 (magenta). (G) Distribution of cell cycle status (% M/G2/S cells) in the basal (≤6 µm) and inner (>6 µm) compartment of ducts (sampled within ≥50 µm radius of the estimated center point) by the distance to the closest tip (n = 216 ducts from seven glands of four embryos). (H) Distribution of cell cycle status (% M/G2/S cells) in the “basal” and inner compartment of branch points (sampled within ≥50 µm radius of the estimated center point) by the distance to the closest tip (n = 94 branch points from seven glands of four embryos). (I) Distribution of basal and inner cell cycle status (% M/G2/S cells) along terminal branches, from the terminal edge to the branch point (n = 74 terminal branches ≥150 µm in length of seven glands, four embryos). Correlation between variables in G and H was assessed with the Pearson coefficient (R) and P value for the linear correlation given.