Normal mammary morphogenesis involves transitions between simple and multilayered epithelial organization. We used electron microscopy and molecular markers to determine whether intercellular junctions and apico-basal polarity were maintained in the multilayer. We found that multilayered elongating ducts had polarized apical and basal tissue surfaces both in 3D culture and in vivo. However, individual cells were only polarized on surfaces in contact with the lumen or extracellular matrix. The basolateral marker Scribble and the apical marker atypical protein kinase C zeta localized to all interior cell membranes, while Par-3 displayed cytoplasmic localization, suggesting incomplete apico-basal polarity. Despite membrane localization of E-cadherin and ß-catenin, we did not observe a defined zonula adherens connecting interior cells. Instead, interior cells were connected through desmosomes and exhibited complex, interdigitating membrane protrusions. Single cell labeling revealed that individual cells were both protrusive and migratory within the epithelial multilayer. Inhibition of Rho kinase (ROCK) further reduced intercellular adhesion on apical and lateral surfaces, but did not disrupt basal tissue organization. Following morphogenesis, segregated membrane domains were re-established and junctional complexes reformed. We observed similar epithelial organization during mammary morphogenesis in organotypic culture and in vivo. We conclude that mammary epithelial morphogenesis involves a reversible, spatially limited, reduction in polarity and intercellular junctions and active, individualistic cell migration. Our data suggest that reductions in polarity and adhesion during breast cancer progression may reflect partial recapitulation of a normal developmental program.

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