Parathyroid Hormone-Related Protein Is Not Required for Normal Ductal or Alveolar Development in the Post-Natal Mammary Gland

PTHrP is necessary for the formation of the embryonic mammary gland and, in its absence, the embryonic mammary bud fails to form the neonatal duct system. In addition, PTHrP is produced by the breast during lactation and contributes to the regulation of maternal calcium homeostasis during milk production. In this study, we examined the role of PTHrP during post-natal mammary development. Using a PTHrP-lacZ transgenic mouse, we surveyed the expression of PTHrP in the developing post-natal mouse mammary gland. We found that PTHrP expression is restricted to the basal cells of the gland during pubertal development and becomes expressed in milk secreting alveolar cells during pregnancy and lactation. Based on the previous findings that overexpression of PTHrP in cap and myoepithelial cells inhibited ductal elongation during puberty, we predicted that ablation of native PTHrP expression in the post-natal gland would result in accelerated ductal development. To address this hypothesis, we generated two conditional models of PTHrP-deficiency specifically targeted to the postnatal mammary gland. We used the MMTV-Cre transgene to ablate the floxed PTHrP gene in both luminal and myoepithelial cells and a tetracycline-regulated K14-tTA;tetO-Cre transgene to target PTHrP expression in just myoepithelial and cap cells. In both models of PTHrP ablation, we found that mammary development proceeds normally despite the absence of PTHrP. We conclude that PTHrP signaling is not required for normal ductal or alveolar development

MMTV-Wnt1 and -ΔN89β-Catenin Induce Canonical Signaling in Distinct Progenitors and Differentially Activate Hedgehog Signaling within Mammary Tumors

Canonical Wnt/β-catenin signaling regulates stem/progenitor cells and, when perturbed, induces many human cancers. A significant proportion of human breast cancer is associated with loss of secreted Wnt antagonists and mice expressing MMTV-Wnt1 and MMTV-ΔN89β-catenin develop mammary adenocarcinomas. Many studies have assumed these mouse models of breast cancer to be equivalent. Here we show that MMTV-Wnt1 and MMTV-ΔN89β-catenin transgenes induce tumors with different phenotypes. Using axin2/conductin reporter genes we show that MMTV-Wnt1 and MMTV-ΔN89β-catenin activate canonical Wnt signaling within distinct cell-types. ΔN89β-catenin activated signaling within a luminal subpopulation scattered along ducts that exhibited a K18+ER−PR−CD24highCD49flow profile and progenitor properties. In contrast, MMTV-Wnt1 induced canonical signaling in K14+ basal cells with CD24/CD49f profiles characteristic of two distinct stem/progenitor cell-types. MMTV-Wnt1 produced additional profound effects on multiple cell-types that correlated with focal activation of the Hedgehog pathway. We document that large melanocytic nevi are a hitherto unreported hallmark of early hyperplastic Wnt1 glands. These nevi formed along the primary mammary ducts and were associated with Hedgehog pathway activity within a subset of melanocytes and surrounding stroma. Hh pathway activity also occurred within tumor-associated stromal and K14+/p63+ subpopulations in a manner correlated with Wnt1 tumor onset. These data show MMTV-Wnt1 and MMTV-ΔN89β-catenin induce canonical signaling in distinct progenitors and that Hedgehog pathway activation is linked to melanocytic nevi and mammary tumor onset arising from excess Wnt1 ligand. They further suggest that Hedgehog pathway activation maybe a critical component and useful indicator of breast tumors arising from unopposed Wnt1 ligand

Parathyroid Hormone-Related Protein Specifies the Mammary Mesenchyme and Regulates Embryonic Mammary Development

Parathyroid Hormone related Protein (PTHrP) is a critical regulator of mammary gland morphogenesis in the mouse embryo. Loss of PTHrP, or its receptor, PTHR1, results in arrested mammary buds at day 15 of embryonic development (E15). In contrast, overexpression of PTHrP converts the ventral epidermis into hairless nipple skin. PTHrP signaling appears to be critical for mammary mesenchyme specification, which in turn maintains mammary epithelial identity, directs bud outgrowth, disrupts the male mammary rudiment and specifies the formation of the nipple. In the embryonic mammary bud, PTHrP exerts its effects on morphogenesis, in part, through epithelial-stromal crosstalk mediated by Wnt and BMP signaling. Recently, PTHLH has been identified as a strong candidate for a novel breast cancer susceptibility locus, although PTHrP’s role in breast cancer has not been clearly defined. The effects of PTHrP on the growth of the embryonic mammary rudiment and its invasion into the dermis may, in turn, have connections to the role of PTHrP in breast cancer

Parathyroid Hormone Related Protein (PTHrP) Regulates Estrogen Receptor Expression

Parathyroid hormone related protein (PTHrP) is required for specification of the mammary mesenchyme during embryonic development. In WT mice, the mammary mesenchyme consists of 3-4 layers of stromal cells that express ER. Overexpression of PTHrP in the basal layer of the epidermis, results in ectopic ER expression in the ventral dermis. Conversely, loss of PTHrP results in ablation of ER expression. Deletion of Wnt pathway components such as beta-catenin also abolishes ER expression. These data suggest that PTHrP-induced Wnt signaling regulates ER expression. To determine if PTHrP similarly mediates ER expression in breast cancer and bone, we used RNA harvested from osteoblasts treated with PTHrP. Semi-quantitative PCR was used to analyze ER gene expression and to demonstrate that PTHrP treatment results in a 2-fold increase in the levels of ER. We also used immunofluoresence to demonstrate that treatment of T47D breast cancer cells with PTHrP increases ER levels. Taken together, out studies demonstrate that PTHrP regulates ER expression in bone and breast and that PTHrP could be used as a therapeutic agent to in the treatment of postmenopausal osteoporosis and ER-negative breast cancer

CoVE: A Colony Visualization System for Animal Pedigrees

CoVE is a novel, scalable, interactive tool that can be used to visualize and manage large colonies of laboratory animals. Effective management of large colonies of with multiple individual attributes and complicated breeding schemes represents a significant data management challenge in the biological sciences. Currently available software either provides databases for record keeping or generates basic pedigrees but not both. Thus, there is a pressing need for an integrated colony management system that provides a repository for the data and addresses the visualization challenge presented by complex genealogical data. We present CoVE, a colony visualization tool that provides an overview of the entire colony, clusters individuals based on Gender, Litter or Genotype, and provides an individual view of any animal for detailed examination. We demonstrate that CoVE provides an efficient way to manage, generate and view complex pedigree of real world genealogical data from animal colonies, annotated with details of individual attributes. It enables interactive tracing of lineages and identification of censored subjects in tumor studies

<i>PTHrP</i> expression during embryogenesis.

<p>LacZ staining of <i>PTHrP^+/</i>^lacZ embryos at (A) E11, (B) E12, (C) E15.5 and (D) birth. (A) At E11, β-galactosidase staining was observed in the mammary placodes but not the surrounding mesenchyme. (B) By E12.5, intense staining was observed in all five buds. Interestingly, lacZ positive “tails” were observed from each bud (double arrowhead) (B, C). Single arrowheads indicate mammary placodes and buds. (D) <i>PTHrP^lacZ</i> expression remains restricted to the mammary epithelial cells throughout embryonic and neonatal development.</p

<i>MMTV-Cre</i> deletion of <i>PTHrP</i> in the mammary gland does not impair ductal development.

<p>(A) MMTV-Cre activity is heterogeneous in the pubertal gland. Whole-mount Xgal staining of a 5 week old gland. Sections of stained glands demonstrating that luminal and myoepithelial cells in the ducts (C), as well as body and cap cells of TEBs (B) are targeted for recombination. (D) Whole-mounts of mammary glands from <i>MMTV-cre</i> and <i>MMTV-cre;PTHrP^lox/lacZ</i> mammary glands at 5 weeks of age. (E) <i>PTHrP</i> mRNA expression is decreased in <i>MMTV-cre;PTHrP^lox/lacZ</i> mammary glands. (F) Ductal outgrowth was measured in Control (n = 6) and <i>MMTV-cre;PTHrP^lox/lacZ</i> (n = 12) as % of fat pad filled.</p

Loss of <i>PTHrP</i> has no Effect on Alveolar Development.

<p>Whole-mount analysis and histological H&E sections of control (A, D, G, J), MMTV-CKO (B,E,H,K) and K14-CKO (C,F,I,L) mice during late pregnancy (A–F) and lactation (G–L).</p

Effects of <i>PTHrP</i> deletion on cell turnover in terminal end buds during puberty.

<p>(A) End bud cell proliferation in 5-week-old mice as defined by the percentage of epithelial cells incorporating EdU within TEBs of control, <i>K14-tTA;tetO-Cre;PTHrP^lox/lacZ</i> and <i>MMTV Cre;PTHrP^lox/lacZ</i> mice. (B) Apoptosis was measured by TUNEL staining in the TEBs of control, <i>K14-tTA;tetO-Cre;PTHrP^lox/lacZ</i> and <i>MMTV Cre;PTHrP^lox/lacZ</i> mice. Red bars represent the baseline rates of proliferation and apoptosis in 5-week-old placebo-treated mice and blue bars represent rates of proliferation and apoptosis in 5-week-old mice treated with exogenous estradiol and progesterone for 48 h. EdU incorporation was significantly greater in each group of mice treated with hormones. There were no differences in the response to hormones in control mice as compared to the two types of CKO mice. There were no differences in apoptosis among control or CKO mice at baseline versus treated with hormones.</p

<i>PTHrP</i> expression during postnatal mammary gland development.

<p>(A) At the onset of puberty (3 weeks), <i>PTHrP^lacZ</i> expression is seen throughout the ductal tree. (B) As development ensues, –mount Xgal staining is evident in the ducts and TEBs at 5 weeks, specifically in the myoepitheial cells and the cap cells (C). By 8 weeks, when TEBs have regressed, LacZ expression is restricted to myoepithelial cells in the ducts (D). During late pregnancy (E–G), LacZ expression is seen in the ducts and is also evident in the developing alveoli. During lactation (H–J), LacZ is expression is seen in the milk secreting cells. High levels of <i>PTHrP^lacZ</i> staining remain in the ducts and the alveoli during lactation. (K) Developmental survey of PTHrP mRNA expression in whole mammary glands as measured by qRT-PCR. PTHrP mRNA is expressed at low levels in whole mammary glands throughout virgin postnatal development and throughout pregnancy. At the onset of lactation, PTHrP levels increase, and at involution return to virgin levels. wks = weeks; P = pregnancy day; L = lactation day; I = involution day. Relative expression: 5 weeks = 1. H&E staining (G, J).</p