Cellular and Molecular Foundations of Mammary Branching Morphogenesis

Epithelial tubes provide the compartmentalization required for processes such as fluid and gas exchange, nutrient absorption, secretion, and waste elimination. Decades of study have provided insight into the genetic and molecular regulators of mammalian tube formation, but internal development left us with an incomplete understanding of how cells respond to these signals to build epithelial organs. Using organotypic culture of primary mouse mammary epithelium and time-lapse microscopy, we were able to study mammalian epithelial morphogenesis in real-time at subcellular resolution. This allowed us to identify the cellular and molecular mechanisms that underlie epithelial stratification and elongation. Mammary ducts transition from a simple polarized state to low-polarity stratified architecture at the onset of development. The low-polarity stratified epithelium then functions as the elongation front during mammary branch elongation. We determined that stratification was initiated by a novel vertical cell division of apically localized epithelial cells in culture and in vivo. The vertical divisions also directly resulted in the polarity-loss that is associated with the stratified epithelium. We then showed that this developmental mechanism of stratification could be hijacked in response to acute oncogene activation during the initial steps of tumor formation. To determine the cellular mechanism of mammary duct elongation we tracked the migration of individual cells within the epithelium. We found that cells at the elongation front displayed a significant increase in cell motility. When we decreased cell motility by inhibition of Rac we found that branches stopped elongating. The high motility cells were specifically enriched for high levels of mitogen-activated protein kinase (MAPK) signaling and MAPK signaling was required for branching. Mosaic expression of an activated mitogen-activated protein kinase kinase (MEK), a member of the MAPK cascade, was sufficient to induce branch elongation in the absence of external stimulation. We concluded that mammary branches are elongated by a subset of actively migrating epithelial cells and that the increase in cell motility is driven by MAPK signaling. Finally it is worth noting that collective cell migration through aberrant MAPK signaling is a key feature of tumor progression and here we show that MAPK signaling also drives collective cell migration in normal epithelium