Abstract
Branching morphogenesis requires dramatic changes in cell polarity, proliferation, migration, and actin dynamics to elaborate tubular networks. However, little is known about how microtubules support these cell behaviors in 3D tissues. Using organotypic cultures, we first examined the organization of the microtubule cytoskeleton. Simple luminal epithelial cells exhibited non-centrosomal, apico-basally oriented microtubule arrays, while stratified luminal cells had centrosomally radiating microtubules. During collective migration, luminal cells adopted an ameboid-like organization with a rear-facing nuclear-centrosomal axis. Multiple staining approaches suggest that cells in the basal-most luminal cell layer had more stable microtubules than cells deeper within the stratified layer. Finally, we tested the requirement for microtubules using pharmacologic inhibitors. Both microtubule stabilization and destabilization prevented bud formation and arrested duct elongation. Cell tracking analysis demonstrated that microtubules coordinated luminal cell migration within elongating buds. Destabilizing microtubules reduced cell directionality, while stabilizing microtubules did not affect directionality but reduced cell motility. Our data reveal that microtubules are essential for collective migration of luminal cells and for mammary branching morphogenesis.