Syk Loss in Breast Cells We conclude that the effect of heterozygotic Syk loss in mammary gland development is present in two different strains of mice. Taken together, these data indicate that Syk is haploinsuffi- cient and reduced levels can significantly impact murine mammary gland development. Increased proliferation and invasiveness of mouse mammary epithelial cells in vivo and in vitro. Following the observation of increased ductal development in Syk +/2 mice, we performed an assessment of cell proliferation in mammary gland tissues to determine whether epithelial cells were more proliferative after Syk loss. In both 10 and 12 week animals, the % positive Ki67 nuclei were significantly and dramatically increased in the heterozygote knockout Syk +/2 virgin mice compared with the wild type (Figures 6A and B). Next, we isolated primary mouse epithelial cells to determine in vitro their proliferative and invasive capacity. The percent of primary epithelial cells isolated from mammary glands of the Syk +/2 virgin females in S-phase was 2.4 fold higher than wild type animals (Figure 6C). We used a three-dimensional in vitro model consisting of primary mammary epithelial cells embedded in a reconstituted Figure 6. Heterozygous Syk knockout accelerates cell proliferation and invasiveness of mouse mammary epithelial cells. (A) Representative images illustrate sections triple stained with anti-Ki67 (red), anti-keratin14 (CK14m green), and DAPI (blue) from mammary glands of Syk +/+ wild type and Syk +/2 heterozygous mice from 12-week virgin females. Scale bars correspond to 30 mm. (B) Increased mammary epithelial cell proliferation was quantified from images illustrated in (A) in three animals for each point. (C) Primary cell cultures also exhibit increased cell proliferation. Total % of cells in S-phase was quantified in primary cells from Syk +/+ wild type and Syk +/2 heterozygous mice from 12-week virgin females. (D) Freshly isolated primary mammary epithelial cells were embedded Matrigel. Organoids were imaged at 24 hrs and show greatly enhanced size in Syk heterozygous knockdown compared with wild type glands. A typical invasive leading edge is indicated (arrow). Scale bars correspond to 50 mm. Increased cellular area (E) and fiber length (F) of organoids was assessed using Metamorph Image software. In all cases (B–F), data is represented by means +/2 SEM. P values are significant by the student’s t-test. doi:10.1371/journal.pone.0007445.g006 PLoS ONE | www.plosone.org 8 October 2009 | Volume 4 | Issue 10 | e7445