appears to be widespread in embryonic tissues and is essential for embryonic development. Mammary development fails in the absence of Lrp6; both epithelial outgrowth of the placode and the formation of the host adipose tissue is affected [22]. The role of Lrp6 in adult tissues is unclear, but loss of function mutations have been linked with human cases of coronary artery disease [23]. In contrast, Lrp5 mice are viable, although they exhibit defects in bone ossification and vascularization of the eye [24,25]. In adult tissues, Lrp5 mRNA and protein levels are high and widely expressed in tissues such as bone, pancreas, central nervous system, and in phagocytic cells [21,26]. Loss of function mutations have been associated with heritable cases of osteoporosis as well as Type I diabetes [27,28]. In the mammary gland, Wnt signaling is required for specification and outgrowth of the mammary rudiment from the embryonic skin [16], and a Wnt reporter strain shows high Wnt signaling activity at this stage [15,29]. Since inhibition of Wnt signaling prevents gland formation [15], it has been difficult to determine the functional role of Wnt signaling in later and adult stages of mammary gland development. Wnt signaling has been shown to be important not only to the maintenance of stem/progenitor compartments in gut, but in a number of other cell lineages. These include hematopoetic and embryonic stem cells [30,31,32]. Specifically, several compo- nents of the canonical Wnt signaling pathway have been found to be expressed in both embryonic and hematopoetic stem cell populations. Moreover, treatment with Wnt ligands or down- stream activation of the Wnt signaling pathway inhibits differentiation and promotes self-renewal of these cells [30,31]. Studies published in 2006 [33,34] showed that subpopulations of basal mammary cells could be isolated from the total population, that show enhanced regenerative capacity when assayed in vivo (described by their ability to regenerate a mammary tree when transferred to a host cleared fat pad). A single cell from this population was sufficient to recreate a whole gland, and they were coined somatic mammary stem cells. These subpopulations are separated by their high expression of both CD24 and CD49f (a6 integrin, or CD29, b1 integrin), but their purity is unlikely to be higher than 5%. Neither of these markers alone is useful for the identification of stem cells, or indeed resolution of whole mammary epithelial cell populations. Therefore, the behavior of the cells that are key to the growth or regeneration of glands has not yet been described. It has become a high priority to find a molecule (preferably one functionally involved in determining stemness) that is a specific marker of stem cell function, for their evaluation during normal and pathogenic development. Previously, we showed that Lrp5 mammary glands, though grossly normal (albeit developmentally delayed), were remarkably resistant to Wnt1-induced tumor development [29]. This resis- tance occurred despite the presence of Lrp6, and served to focus our attention on the specific functions of Lrp5. Lrp5 glands were almost devoid of regenerative potential when tested by in vivo stem cell assay. Here, we show that both Lrp5 and -6 proteins are expressed in the basal epithelial cell population. We also show that the loss of Lrp5 does not significantly affect the response of cultured mammary epithelial cells (MECs), tested with an in vitro Wnt reporter assay. The absence of Lrp5 generates a selective loss of the basal cell population, though the function of mammary glands is entirely preserved. Furthermore, the cells tend to become senescent in culture. In addition, we find that cells expressing high levels of Lrp5 co-localize with the CD24/CD49f double- positive stem cell-enriched fraction and have enhanced stem cell function in vivo. Lrp5 in Mammary Stem Cells Results Lrp5 and 6 Expression is Localized to Basal Mammary Epithelial Cells Lrp5 and Lrp6 proteins contain .70% amino acid sequence homology [21]. Prior studies have shown no clear patterns of expression of Lrp5 and -6, depending upon the antibody reagents used, the assay, and the fixation conditions. To ensure that our analyses can resolve Lrp5 and Lrp6, we transfected HEK293 cells with constructs coding for either Lrp5 or Lrp6. Western blotting, immunofluorescence, and flow cytometry staining of transfected 293 cells found both Lrp5 and Lrp6 antibodies were specific to each receptor (Figure S1). By immunofluorescent assay, quiescent virgin glands showed specific staining of Lrp5 in basal epithelial cells (determined by their co-expression of either keratin 5 (K5) or smooth muscle actin (SMA); Fig. 1), but not luminal cells. Interestingly, we did observe specific staining in the stroma of Lrp5 +/+ mammary glands, which was absent in Lrp5 2/2 glands. There was high background staining of the adipocytes in these paraffin-embedded samples, demonstrated by a persistent staining pattern in these cells in Lrp52/2 glands, where the specific basal cell-associated stain is absent. Overall, ductal development and morphogenesis in Lrp52/2 mammary glands is almost normal (a little delayed and hypo-branched; Lindvall et al 2006). These immunohistochemical data show that the relative cellular architecture and arrangement is also normal. In frozen sections of mammary glands from pregnant mice, we confirmed that the morphogenesis and growth typical of this stage is normal, and that the basal cell pattern of expression was continued (co-localization with basal markers, and exclusion from luminal, keratin 8 (K8)-positive cells). Northern analysis of mRNA and Western analysis of proteins from embryos and isolated MECs confirmed the loss of Lrp5 expression in these mice (Fig. 1B). Lrp5 mRNA was expressed at similar levels in glands from both virgin and pregnant mice. In the absence of Lrp5, there was no significant compensatory upregula- tion of Lrp6 (at the mRNA level) To verify that Lrp5 was expressed in basal cells, we used the flow cytometric analysis of BALB/c MECs described by Stingl et al, separating basal and luminal cells by their dual staining with CD24 and CD49f [34]. Analysis of Lrp5 expression in addition to CD24 and CD49f revealed that Lrp5 expression was predomi- nantly localized to the basal lineage (Fig. 2A). Though the intensity of Lrp5 staining was much lower in C57Bl6 MECs, analysis of this strain also showed that only basal cells expressed Lrp5, and that this staining was absent in the corresponding C57Bl6 Lrp5 2/2 MECs (Figure S2). The expression of Lrp5 was not equal in all basal cells; indeed the non-regenerative myoepithelial population (relatively lower CD24/CD49f; Stingl et al 2006) had low Lrp5 expression, whereas the regenerative mammary repopulating units (MRU) fraction had higher Lrp5 expression levels (Fig. 2A and B). Isolation of cell fractions by flow cytometry, followed by staining of sorted populations with antibodies to keratin 5 (K5) and -8 (K8) confirmed that the accuracy of gating for the basal and luminal cell populations was high. Likewise, isolation and staining of the Lrp5- positive and -negative populations for K5 and K8 showed that the Lrp5-positive population consists predominantly of basal cells and the Lrp5 negative population contains mostly luminal cells (Fig. 2C). Staining of BALB/c MECs with anti-Lrp6, in addition to CD24 and CD49f, demonstrated that Lrp6 is also predominantly localized to the basal cell lineage (Fig. 3A) and the magnitude of PLoS ONE | www.plosone.org 2 August 2009 | Volume 4 | Issue 8 | e6594