UC Santa Cruz

The goal of my thesis work was to resolve the molecular mechanisms governing mammary gland alveologenesis during pregnancy. Alveologenesis is the essential process by which alveolar progenitor cells undergo rapid expansion during early pregnancy and subsequent differentiation into mature milk-producing alveolar cells during late pregnancy, generating a functional mammary gland capable of providing nourishment for offspring. How the mammary gland maintains a balance between massive proliferation and functional differentiation, however, remains unclear. Here, we show that the mammary gland couples proliferation with differentiation by means of endoreplication, the cellular process of replicating DNA in the absence of cell division to generate a polyploid cell. Through pharmacological inhibition and transgenic loss-of-function studies, we show that the DNA damage response to replication stress is activated during the rapid proliferation of early pregnancy. This, in turn, results in early mitotic arrest through WEE1-mediated CDK1 inhibition and subsequent endoreplication during early lactation, generating a population of terminally differentiated polyploid alveolar cells that are essential for efficient milk production. Furthermore, Notch signaling has long been known to be essential for maintenance of the alveolar progenitor population, and it must be attenuated to achieve alveologenesis. Despite the importance of Notch signaling, however, the mechanisms that regulate Notch activity to ensure proper alveolar proliferation and differentiation during pregnancy are not well understood. Through transgenic loss-of-function and 3D organoid studies, our preliminary data suggest ROBO2 functions from the luminal epithelial compartment to promote expression of the Notch ligand JAG1 in the basal epithelial compartment, potentially through the direct inhibition of ROBO1 in basal cells. This, in turn, induces the Notch activity of alveolar progenitor cells in a juxtacrine manner, maintaining them in the progenitor state to inhibit their differentiation. Moreover, our preliminary data also suggest NOTCH1 activation inhibits the DNA damage response to replication stress and alveolar endoreplication through the interaction of its intracellular domain with the DNA damage response kinase ATR. Taken together, the results of my thesis work elucidate multiple molecular mechanisms by which the mammary gland achieves a balance between the rapid proliferation of alveolar progenitor cells and their functional differentiation into mature milk-producing alveolar cells during late pregnancy and lactation.