PTN in the Mammary Gland Figure 3. PTN protein expression in mammary glands and mammary epithelial cells (MECs). (A) Immunohistochemistry of virgin mouse mammary glands with a polyclonal anti-PTN antibody or secondary antibody only (sec. only) as a negative control. The dark brown staining shows PTN crossreactivity. Scale Bar = 50 mm. Magnified ductal areas are indicated by the red squares (Scale Bar = 25 mm) and individually magnified duct epithelial cells by the black squares (Scale Bar = 10 mm). doi:10.1371/journal.pone.0047876.g003 secreted into the extracellular matrix irrespective of PTN activity (Figure 5E) [53]. However, whereas most (,90%) of anti-PTN treated mammospheres showed detectable laminin deposition, this was observed in only a few (,20%) of the control spheres. From this, we conclude that PTN activity in MECs disrupts cell adhesion to the extracellular matrix via reduced laminin deposition and thus results in fewer spheres with correct apical organization. We speculate that PTN activity inhibits the deposition of extra-cellular matrix components such as laminin and thus its function elicits cells migration and invasion of MECs grown on a plastic surface. PTN function during mammary gland development In contrast to other organs, most mammary gland development happens after birth during puberty when the ovaries begin hormone production. In mice, the primordial mammary ductal tree starts proliferating, branching and invading the mammary fat pad after weaning at approximately three weeks of age when hormonal stimuli start. By 9 to 12 weeks, the epithelial ducts have fully invaded the fat pad and become quiescent until pregnancy occurs. Treatment of mice with the PTN blocking antibody introduced in the in vitro studies above was used to investigate PTN function during mammary gland development. The antibody KD is 1 nM and we detected serum concentrations of 1 to 10 mM in the treated mice one week after the last dose (Figure S1B). This suggests that sufficient dosing was used to block endogenous PTN. Mice treated during the crucial phase of mammary duct invasion into the fat pad from 3 to 6 weeks of age showed an increase in ductal penetration of the mammary fat pad and in the average number of branch points per duct (Figure 6A to C). In a further analysis we assessed additional well-defined functional units in the developing mammary gland, Terminal End Buds (TEBs). TEBs are highly proliferative club-shaped structures containing stem cells [54,55,56] and drive the penetration of the mammary ducts into the fat pad. Their number is high during early puberty [54] and morph into alveolar and terminal buds as the gland matures [54,57]. Mice treated with the anti-PTN antibody showed an increase in the number of Terminal Ends but not in TEBs (Figure 6D,E). The percentage of TEBs has been suggested as an indicator of the developmental stage of the mammary gland [55]. Indeed, control mice showed a 1.8-fold higher fraction of TEBs suggesting that PTN activity inhibits the differentiation of TEBs into Terminal Ends (Figure 6F). H&E staining confirmed that both treated and control groups had indistinguishable, regular mam- mary duct structure (data not shown). In mice treated with anti- PTN for an additional 3 weeks until 9 weeks of age no differences in the final ductal and branching structures of the mammary glands were observed between treatment and controls (data not shown). From these data we conclude that PTN delays ductal outgrowth, branching and terminal end formation without impacting the final gland development. PTN signaling pathways in the developing mammary gland The MAP kinase pathway is used by hormones and growth factors that control mammary gland development (for a review see [58]). Since the treatment with the PTN blocking antibody showed that PTN delays ductal outgrowth, branching and terminal end formation during pubertal development, we investigated if the enhanced maturation of the glands observed with this treatment PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e47876