The mammary gland, or breast, distinguishes mammals by its ability to produce and secrete milk for nourishment of offspring. Throughout a female’s reproductive life, this remarkable organ retains the ability to generate milk-producing alveoli, undergoing prodigious proliferation and differentiation of mammary epithelial cells with each pregnancy and, on a minor scale, every estrous cycle. The mammary epithelium generates a tree-like, bi-layered ductal network, comprising an outer layer of myoepithelial basal cells that contract to expel milk, and an inner layer of luminal epithelial cells that either line the ducts – ductal epithelial cells – or generate milk during lactation – alveolar epithelial cells.

Lineage labeling studies have demonstrated that enduring, lineage-restricted progenitor cells play a crucial role in generating the large number of luminal and basal cells required to build a milk supply. Yet, molecular mechanisms governing the cyclical expansion, differentiation and renewal of such lineage-restricted progenitors are still largely unknown.

In this work, I provide evidence showing that Slit/Robo1 signaling governs the number and differentiation of alveolar progenitors. Loss of Robo1 in the basal compartment, leads to nuclear localization of CTNNB1, resulting in enhanced expression of the Notch ligand Jag1. This increased expression of JAG1 exerts paracrine control of Notch signaling in the luminal compartment and governs alveolar progenitor function. To date, there are limited tools to investigate specific lineage contributions of mammary epithelial cells. Here, I also describe a method for generating mosaic organoids using the two epithelial lineages of the mammary gland. This method also describes for the first time a process that results in alveolar cell differentiation and milk production ex vivo.