Syk Loss in Breast Cells Figure 5. Enhanced branching morphogenesis in mammary glands of Syk +/2 mice. (A) Syk protein is decreased in mammary gland extracts (minus the mammary gland lymph node) and spleen from 12-week virgin Syk +/2 heterozygous compared with Syk +/+ wild type females. Spleen extracts were used as positive control. (B) The average number of buds were counted in mammary glands from Syk +/+ wild type (black bars) and Syk +/2 heterozygous mice (gray bars) from 3-, 6-, 8-, 10-, 12-week virgin and early and late pregnancy females (EP and LP, respectively). P value was significant by the student’s t-test for 6-, 8-, 10-, 12-week virgin females and early- and late-pregnancy females (n = 3 animals per time point). In a separate experiment, mammary glands from 10 additional wild type and 10 heterozygous knockout Syk mice were examined at 8 weeks confirming the increase in bud formation after partial Syk loss (B, inset). Data are represented as means from three mice +/2 SEM except for B, inset, where n = 10 mice. (C) Representative images of mammary glands from Syk +/+ wild type and Syk +/2 heterozygote knockout mice. Scale bars correspond to 150 mm in all panels. (D) The mean distance was determined from lymph node to the distal end of ducts in mammary glands from Syk +/+ wild type (black bars) and Syk +/2 heterozygous mice (gray bars) at 10-day and 3-,6-,8-,10, 12-week virgin and early and late pregnancy females (n = 3 animals per time point). P value is significant by the student’s t-test for 3-, 6-, 8-week virgin females and late-pregnancy females. Data are represented as means +/2 SEM for three glands each. doi:10.1371/journal.pone.0007445.g005 female mammary glands at different ages. In strong agreement with the evident change in morphology of Syk +/2 mice on a 129 background, mammary glands from virgin Syk +/2 female mice displayed more abundant ductal branches and end buds compared with the wild type glands as shown in the whole mount analysis at 3, 6, 8, 10, 12 weeks and pregnancy (EP, early pregnancy, E6.5 and LP, late pregnancy, E16.5) (Figures 5B and 5C). Next, we found that the distance from lymph node to TEB in the mammary gland was significantly increased in the Syk +/2 mice compared to that of WT mice during pre-puberty (3 weeks), puberty (6 and 8 weeks) as well as in late pregnancy glands (Figure 5D). The differences were not significant at other time points. We next analyzed mammary gland morphology of pubertal and multiparous mice to determine if Syk loss affects end bud morphology or results in epithelial hyperplasia and abnormal ducts or end buds. In 10 week animals, the branches and end buds of Syk +/2 mice formed lumens similar to the wild type animals, although in the heterozygote mammary glands, many more terminal ends formed along the length and tips of the ducts (Figure S7B, X-Y, 10 wk). Similarly, glands from late pregnancy (16.5) Syk +/2 mice had many more lobules (Figure S7B, X-Y, LP). The lobules, like those from the wild type pregnant females, were hollow balls, however, the size and density of formation was significantly enhanced compared with wild type (Figure S7B, X- Y view, LP). PLoS ONE | www.plosone.org 7 October 2009 | Volume 4 | Issue 10 | e7445