A Junction-Dependent Mechanism Drives Mammary Cell Intercalation for Ductal Elongation

Abstract

Mammary glands contain branched networks of ducts and alveoli that function to produce milk for offspring. While the murine luminal epithelium is organized as a cellular monolayer, it originates from multilayered structures called terminal end buds (TEB). The TEBs generate ducts of monolayered epithelial cells as they invade the fat pad, but little is known about underlying mechanisms. While apoptosis provides a plausible mechanism for cavitation of the ductal lumen, it does not account for elongation of ducts behind the TEBs. Our spatial calculations suggest that most cells in TEBs need to intercalate into the outermost luminal layer and that this migration of cells is the primary driver of cavitation and ductal elongation. To study the progression of multilayered to monolayered epithelium, we developed a quantitative cell culture assay that determines the efficiency of intercalation into an epithelial monolayer. Using this tool, we verified that loss of adherens junctions prevents stable integration of cells into monolayers, consistent with previous data in cultured cells and in primary tissue. Interestingly, tight junction (TJ) proteins also play a key role in this integration process. Although loss of the ZO-1 TJ protein in intercalating cells suppresses intercalation, loss of ZO-1 in the monolayer has the reverse effect, promoting intercalation - even though ZO-1 is not necessary for establishment of TJs. ZO-1-positive puncta form between cells and the monolayer, which then resolves into a new intercellular boundary as intercalation proceeds. ZO-1 loss also reduces engraftment when cells are transplanted into the mammary gland via intraductal injection. We further show that intercalation is dependent on dynamic cytoskeletal rearrangements in both the existing monolayer and intercalating cells. These data identify luminal cell rearrangements necessary for mammary gland development and suggest a molecular mechanism for integration of cells into an existing monolayer.