The two distinct mechanisms of branching morphogenesis in the pubertal mouse mammary gland. The mouse mammary gland branches through two distinct mechanisms: bifurcation of TEBs and side branching. Bifurcation of TEBs to form primary and secondary branches occurs only from immature ducts. The branch point is formed through deposition of stroma at the cleft site, and the ducts extend directly into adipose tissue, without myoepithelial cells or stroma and with only a minimal basement membrane at their invasive front. In contrast, in side branching, a new branch forms from a mature duct. First, the region where the bud is to form must be defined. Then the emerging bud extrudes through and remodels a region containing layers of myoepithelial cells, basement membrane, and periductal stroma. Distinct molecules have been implicated in each type of branching. Factors involved in side branching include the progesterone receptor, Wnts, HSPGs, nuclear factor κB (NFκB), MMPs, TIMP-1, TGFβ and its receptor (TGFβIIR), gelsolin, P-cadherin, CCAAT/enhancer binding protein β (C/EBPβ), CSF-1, Stat5a, and Stat5b. Factors involved in TEB formation include β1 integrin, laminin-1, MMPs, discoidin domain receptor–1 (DDR-1), GH, IGF-I and its receptor IGF-IR, Ptc-1, inhibins and activins, and p27^Kip-1.

Stages of mouse mammary gland development. The mouse mammary gland is specified at embryonic day 10. The mammary epithelium invades the fat pad and forms a small, branched ductal network. After birth, the epithelium grows in concert with the mouse but does not begin to fill the fat pad until the release of ovarian hormones at puberty (around 3 weeks of age). With the onset of puberty, TEBs form and the ducts invade, branch, and eventually fill the fat pad by around 10 weeks of age. In the first stage of pregnancy, ducts branch laterally and form side branches with concomitant epithelial proliferation. Alveolar structures then form on the expanded ductal tree and differentiate into lobular alveoli. Finally, the lobular alveoli terminally differentiate and the epithelium becomes secretory, ready to provide milk for suckling pups upon parturition. At this stage, the epithelium has expanded to almost fill the mammary gland and the large fat cells have dedifferentiated into small pre-adipocytes. Upon involution, the secretory epithelium of the mammary gland dies by apoptosis, the fat cells redifferentiate, and the gland is remodeled back to a state resembling that of the adult nulliparous mouse.