The hierarchical relationships between stem cells and progenitors that guide mammary gland morphogenesis are still poorly defined. While multipotent basal stem cells have been found within the myoepithelial compartment, the in vivo lineage potential of luminal progenitors is unclear. Here we used the expression of the Notch1 receptor, previously implicated in mammary gland development and tumorigenesis, to elucidate the hierarchical organization of mammary stem/progenitor cells by lineage tracing. We found that Notch1 expression identifies multipotent stem cells in the embryonic mammary bud, which progressively restrict their lineage potential during mammary ductal morphogenesis to exclusively generate an ERαneg luminal lineage postnatally. Importantly, our results show that Notch1-labelled cells represent the alveolar progenitors that expand during pregnancy and survive multiple successive involutions. This study reveals that postnatal luminal epithelial cells derive from distinct self-sustained lineages that may represent the cells of origin of different breast cancer subtypes.

How pluripotent stem cells differentiate into the main germ layers is a key question of developmental biology. Here we show that the chromatin-related factor Whsc1 has a dual role in pluripotency exit and germ layer specification of embryonic stem cells (ESCs). Upon induction of differentiation, a proportion of Whsc1-depleted ESCs remain entrapped in a pluripotent state and fail to form mesoderm and endoderm, although they are still capable of generating neuroectoderm. These functions of Whsc1 are independent of its methyltransferase activity. Whsc1 binds to enhancers of the mesendodermal regulators Gata4, Brachyury, Gata6 and Foxa2 together with Brd4, and activates the genes' expression. Depleting each of these regulators also delays pluripotency exit, suggesting that they mediate the effects observed with Whsc1. Hence, up-regulation of mesendoderm instructive transcription factors is required for the timely egress from pluripotency. Our data suggest that the silencing of the pluripotency regulatory network and the activation of lineage-restricted networks are tightly interconnected.