Lrp5 in Mammary Stem Cells Figure 3. Lrp6 is co-expressed by Basal Mammary Epithelial Cells. A) BALB/c virgin MECs were stained with CD24, CD49f, and Lrp6 prior to FACS analysis. MECs were divided into Lrp6+/high and Lrp6-/low, and these populations were overlaid onto the CD24/CD49f profile (Lrp6+ in blue, Lrp6- in red). B) Basal MECs were analyzed as described in Fig. 2b, to test the magnitude of Lrp6 expression relative to CD24/CD49f. C) Lrp6+, Lrp6-, and whole population (WP) MEC fractions were isolated by FACS and subsequently stained for K5 and K8 to determine their cellular phenotype. doi:10.1371/journal.pone.0006594.g003 MECs from Lrp52/2 mice using flow cytometry. Though it was difficult to quantify basal cell depletion in vivo (Fig. 1), flow cytometric analysis revealed a two-fold depletion of basal cells with respect to total epithelial cells (Fig. 6A). The frequency of basal cells in the total mammary epithelial cell population decreased from approximately 4.2 to 2.2 basal cells per 10 epithelial cells. In contrast, basal cell frequency increased in C57Bl6 MMTV-Wnt1 (MMTV-Wnt1) hyperplastic MECs, suggesting that Lrp5-depen- dent Wnt signaling is important for normal differentiation of basal cells. To confirm this result, we assessed the level of expression of lineage-specific markers using biochemical analysis. Lysates were prepared from primary MECs isolated from Lrp5 +/+, Lrp5 2/2, and MMTV-Wnt1 mice (to examine the loss- and gain of function conditions). There was less (0.36) K5 expressed in Lrp52/2 glands, and slightly more (1.2) in glands from MMTV-Wnt1 mice (Fig. 6B). A similar pattern was observed for K5 mRNA expression in isolated Lrp5 2/2 and MMTV-Wnt1 MECs as well as from E10-12 embryos (Figure S5). Microarray analysis of Lrp5 +/+ and 2/2 MECs further corroborated the relative depletion of cells with the basal phenotype in Lrp5 2/2 MECs, where the basal markers K5 and p63 were found to be significantly down- regulated 0.57- and 0.68-fold, respectively, compared to Lrp5+/+ MECs (Fig. 6C). In addition, analysis of markers previously characterized to define the MRU sub-population were found to be relatively down-regulated in Lrp5 2/2 MECs (Figure S6). Analysis of Lrp5 +/+ and 2/2 MECs in culture showed that a significantly higher number of morphologically abnormal basal cells appeared in Lrp5 2/2 MECs. The flattened, stressed morphology is illustrated best by staining for smooth muscle actin (SMA) (Fig. 6D). To determine whether these abnormal basal cells in Lrp5 2/2 MECs were senescent, we examined the expression of p16Ink4a in cultured primary MECS. mRNA levels of p16Ink4a were significantly up-regulated (3.5x) in Lrp5 2/2 MECs (Fig. 6E). To find out whether this reflected specific changes in the basal lineage, cells were purified from these populations, and the luminal and basal cells cultured independently. p16Ink4a expression was significantly increased in both the basal and luminal cells of Lrp5 2/2 mammary glands (Fig. 6F). Another basal marker that showed significantly attenuated expression in these glands is p63 (a p53 family member). Expression of this gene is under the control of two distinct promoters, and the properties of each product are functionally associated with different stages of differentiation. Thus, a full length TA-p63 isoform may regulate p53-dependent transcription and has been associated with senescent cells [36,37]. In contrast, expression of the truncated DN- p63 isoform, which suppresses p53 function, is associated with proliferative cell compartments [37,38]. mRNA isolated from MECs from Lrp5 +/+, 2/2, and MMTV-Wnt1 mice showed higher levels of levels of DN-p63 in MMTV-Wnt1 MECs, and very low TA-p63. Lrp52/2 MECS showed a reciprocal pattern, with relatively higher TA-p63 (Fig. 6G). Loss of Lrp5 does not Affect the Wnt Signaling Transactivation Response of Cultured Mammary Epithelial Cells Using qPCR, we evaluated the relative expression of Lrp5 and Lrp6 in Lrp52/2 mice, to determine whether there was a compensatory expression pattern for Lrp6 in the absence of Lrp5. The amount of Lrp6 mRNA was unchanged in Lrp52/2 MECs PLoS ONE | www.plosone.org 5 August 2009 | Volume 4 | Issue 8 | e6594