ERK signaling is active at the leading fronts of tips. (A) pERK in a non-bifurcating branch (epithelial outline demarcated by white dotted line) detected by chromogenic antibody staining in a paraffin section of an E18.5 mammary gland. (B) Quantitation of nuclear pERK intensity within 0–50 µm (tip front) and 100–150 µm (trailing duct) range of distances of the leading edge (n = 10 branches from two embryos). (C) Distribution of pERK intensities per cell against cell distance to the leading edge (n = 10 branches from two embryos). (D) pERK in a bifurcating branch (epithelial border marked by white dotted line and branch point marked by black dotted line) detected by chromogenic antibody staining in a paraffin section of an E18.5 mammary gland. (E) Quantification of nuclear pERK intensity between the daughter tips and the branch point (n = 9 bifurcating branches of two embryos). (F) An illustration depicting the signaling domains that were identified and the cell behaviors that were associated with these domains in the present study (blue cells = leading front cells, red cells = branch point cells, purple cells = trailing duct cells). Data shown in B and E represents the median (line) with 25th and 75th percentiles (hinges) plus 1.5× interquartile ranges (whiskers) with individual paired data-points illustrated by connecting lines. Statistical significance was assessed with the two-tailed paired t test; ***, P ≤ 0.001; ****, P ≤ 0.0001.