Spatially coordinated cell cycle activity and motility govern mammary ductal growth and tip bifurcation

Branching morphogenesis is the common evolutionary solution of multiple organs to combine maximal epithelial function with compact organ size. It involves successive rounds of branch elongation and branch point generation to generate a branched epithelial network. Branch points form most commonly at the tips of branches as they split into two. However, it is unclear how epithelial cells in tips drive both elongation and branching. In this study, we used ex vivo live imaging to identify these fundamental cellular mechanisms in the embryonic mammary gland. Our 4D analyses show that tips of branches are driven forward by directional cell migration, while elongation of the subtending duct is supported by cell proliferation that feeds a retrograde flow of lagging cells into the duct, established upon differential cell motility. Tip bifurcation involved localized repression of both cell cycle and cell motility at the branch point. Cells in the nascent daughter tips remained proliferative, but changed the direction of their movement to elongate new branches. Our study also reports the fundamental importance of the contractile actin cytoskeleton in regulating branch point generation in the mammary epithelium.