p120-catenin is essential for terminal end bud function and mammary morphogenesis

Abstract

SUMMARYAlthough p120-catenin (p120) is crucial for E-cadherin function, ablation experiments in epithelial tissues from different organ systems reveal markedly different effects. Here, we examine for the first time the consequences of p120 knockout during mouse mammary gland development. An MMTV-Cre driver was used to target knockout to the epithelium at the onset of puberty. p120 ablation was detected in approximately one-quarter of the nascent epithelium at the forth week post-partum. However, p120 null cells were essentially nonadherent, excluded from the process of terminal end bud (TEB) morphogenesis and lost altogether by week six. This elimination process caused a delay in TEB outgrowth, after which the gland developed normally from cells that had retained p120. Mechanistic studies in vitro indicate that TEB dysfunction is likely to stem from striking E-cadherin loss, failure of cell-cell adhesion and near total exclusion from the collective migration process. Our findings reveal an essential role for p120 in mammary morphogenesis. RESEARCH ARTICLE p120-catenin in mammary developmentThe mammary gland provides an outstanding in vivo system for studying morphogenetic events (e.g. invasion and differentiation), as the majority of the development of this non-vital organ occurs after birth. Prior to puberty, the mammary gland exists as a rudimentary ductal tree. At the onset of puberty at ~3 weeks of age, proliferative structures at the tips of ducts, known as terminal end buds (TEBs), develop and begin to invade the surrounding stroma (Hinck and Silberstein, 2005). TEBs comprise a dynamic mass of E-cadherin-positive luminal body cells surrounded by a motile cap cell layer expressing P-cadherin (cadherin 3) (Daniel et al., 1995;Ewald et al., 2008; Hinck and Silberstein, 2005). The TEBs bifurcate repeatedly to form the ductal tree and, ultimately, the mature gland. This process, termed branching morphogenesis, concludes at ~10-12 weeks, when the TEBs have traversed the length of the fat pad and a fully developed ductal tree has formed (Cardiff and Wellings, 1999; Hennighausen and Robinson, 2005;Richert et al., 2000;. Disruption of TEBs is often associated with delayed ductal outgrowth and impaired branching morphogenesis, thus suggesting an essential function of TEBs in the overall development of the mammary gland (Jackson-Fisher et al., 2004;Kouros-Mehr et al., 2006;Lu et al., 2008;Parsa et al., 2008;Srinivasan et al., 2003;Sternlicht et al., 2006).Here, we examine the role of p120 in the developing mammary epithelium. MMTV promoter-driven Cre recombinase expression in p120 f/f mice was used to induce p120 ablation at the onset of puberty. In week 4, developing epithelial structures exhibited mosaic p120 ablation, the extent of which varied widely between mice. p120 loss in nascent ducts caused severe morphological defects (e.g. cell rounding and sloughing into the lumen), despite the presence of p120 family members, which were unable to compensate for p120 loss. p120 null cells were observed less frequently ...

Stromal matrix metalloproteinase-11 is involved in the mammary gland postnatal development

“…Thus, TEB disruption is often associated with delayed ductal outgrowth and impaired branching. 29,30 We show that the absence of MMP-11 does not intrinsically affect the cellular organization of the mammary epithelium compartment. Initial pre-pubertal rudimental ductal trees were similar regardless of the MMP-11 status.…”

“…Immunofluorescence experiments were performed, as previously described, 19,30 using primary antibodies directed against E-cadherin (Zymed Laboratories Inc., San Francisco, CA, USA), anti-aSMA (Sigma-Aldrich), anti-keratin 14 (Eurogentec, Angers, France), anti-keratin 8/18 (Spring Biosciences, Pleasanton, CA, USA), anti-CD31 (BD Bioscience, Le Pont de Claix, France), anti-vimentin (Fitzgerald Industries International, Acton, MA, USA) at recommended concentrations. After washing, slides were incubated with fluorescent Alexa-488-and Cy3-conjugated secondary antibodies (Jackson Immuno Research, West Grove, PA, USA).…”

“…Disruption of TEBs is often associated with delayed ductal outgrowth and impaired branching morphogenesis. 29,30 Accordingly, at 4 weeks of age, many distal branches of the wild-type ducts were tipped with TEBs (32.6±8.35 per gland), indicating that active ductal morphogenesis was in progress. However, in the MMP-11 À / À glands, although morphogenesis process was also initiated, there were less TEBs (22.0 ± 5.1 per gland; P ¼ 0.04; Figure 1B).…”

A strategy for tissue self-organization that is robust to cellular heterogeneity and plasticity

“…For example, ablation of key epithelial cell-cell adhesion molecules such as P-or E-cadherin during the development of the mouse mammary gland does not dramatically alter cell positioning (11,12,32). Even ablation of p120 catenin did not alter the relative positioning of MEP and LEP with respect to the tissue boundary, although it did trigger glandular thickening and lumen filling (33). On its surface, our finding that binary MEP-ECM cohesion dominates tissue self-organization seems inconsistent with several reports that integrins are dispensable for cell positioning during mouse mammary gland development (34).…”

p120-catenin controls contractility along the vertical axis of epithelial lateral membranes

“…Notably, conditional p120 KO phenotypes in mice are often striking but highly variable from one epithelial cell type to another. In many human cancers, p120 is mislocalized and/or downregulated to varying degrees, suggesting potential roles in tumor progression or metastasis Kurley et al, 2012;Smalley-Freed et al, 2010). Interestingly, in contrast to its Drosophila and C. elegans counterpart, vertebrate p120 is essential for cadherin stability.…”