Mammary epithelium can functionally regenerate upon transplantation. This renewal capacity has been classically ascribed to the function of a multipotent mammary gland stem cell population, which has been hypothesized to be a primary target in the etiology of breast cancer. Several complementary approaches were employed in this study to identify and enrich mammary epithelial cells that retain stem cell characteristics. Using long-term BrdU labeling, a population of label retaining cells (LRCs) that lack expression of differentiation markers has been identified. LRCs isolated from mammary primary cultures were enriched for stem cell antigen-1 (Sca-1) and Hoechst dye-effluxing "side population" properties. Sca-1(pos) cells in the mammary gland were localized to the luminal epithelia by using Sca-1(+/GFP) mice, were progesterone receptor-negative, and did not bind peanut lectin. Finally, the Sca-1(pos) population is enriched for functional stem/progenitor cells, as demonstrated by its increased regenerative potential compared with Sca-1(neg) cells when transplanted into the cleared mammary fat pads of host mice.

Research Article 1138 development has not been evaluated. However, a role for Wnt-4 has been suggested by rescue of Wnt-4-null mammary epithelial cells (MECs) by transplantation into the cleared fat pads of wild-type recipients (Brisken et al., 2000). In these studies, a delay in lobuloalveolar development was observed at mid-pregnancy, but by day 1 of lactation no differences were observed between the outgrowths of wild-type and Wnt-4 null MECs, presumably because of compensation by other family members. This problem of Wnt redundancy has been addressed in the current study by examining the requirement for β-catenin-mediated signalling as a convergence point for the canonical Wnt signal transduction pathway.β-Catenin, the vertebrate orthologue of Drosophila Armadillo (McCrea et al., 1991;Peifer et al., 1991), is a multifunctional protein, characterized by a stretch of arm repeats that are the sites of multiple protein-protein interactions (Huber et al., 1997;Peifer et al., 1994). β-Catenin binds to Ecadherin at the adherens junctions, modulates cadherindependent cell-cell adhesion (Barth et al., 1997;Steinberg and McNutt, 1999) and links the cadherin/catenin complex to the cortical actin cytoskeleton through the binding of α-catenin (Herrenknecht et al., 1991;Nagafuchi and Tsukita, 1994). Additionally, β-catenin plays a crucial role in the canonical Wnt signalling cascade. The intracellular Wnt signal is propagated from the membrane through Dishevelled (Yanagawa et al., 1995) to downregulate glycogen synthase kinase-3β (GSK-3β) and subsequently disrupt a protein complex that includes GSK-3β, adenomatous polyposis coli (APC), axin, and members of the ubiquitination/proteasome pathway (Easwaran et al., 1999a;Ikeda et al., 1998;Kikuchi, 1999;Kishida et al., 1998;Rubinfeld et al., 1996;Salomon et al., 1997). Disruption of this complex prevents the GSK-3β-dependent phosphorylation of β-catenin on specific N-terminal serine and threonine residues, and thus protects β-catenin protein from degradation through ubiquitin-mediated proteolysis (Easwaran et al., 1999a;Rubinfeld et al., 1996). The stabilized β-catenin protein can then be transported to the nucleus where it forms complexes with members of the T-cell factor (TCF)/Lef family of HMG-box transcription factors (Behrens et al., 1996; Huber et al., 1996;Molenaar et al., 1996). Together, β-catenin and TCF proteins comprise a bipartite transcripton factor in which TCF supplies the DNA binding moiety and β-catenin provides the transactivation domain (reviewed by Barker et al., 2000). This complex activates the transcription of target genes and, in some cases, relieves the repression activity of TCF alone (reviewed by Bienz, 1998). Thus, β-catenin plays crucial roles in both epithelial cell-cell adhesion, as well as in signal transduction.Additionally, β-catenin is a point of intersection and integration of several other signalling pathways. For example, the retinoic acid receptor RAR binds to β-catenin in a retinoic acid ligand-dependent manner, not only sequestering β...