Parathyroid hormone-related protein is not required for normal ductal or alveolar development in the post-natal mammary gland - PubMed (original) (raw)

Parathyroid hormone-related protein is not required for normal ductal or alveolar development in the post-natal mammary gland

Kata Boras-Granic et al. PLoS One. 2011.

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. PTHrP expression during embryogenesis.

LacZ staining of PTHrP+/ 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) PTHrPlacZ expression remains restricted to the mammary epithelial cells throughout embryonic and neonatal development.

Figure 2

Figure 2. PTHrP expression during postnatal mammary gland development.

(A) At the onset of puberty (3 weeks), PTHrPlacZ 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 PTHrPlacZ 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).

Figure 3

Figure 3. MMTV-Cre deletion of PTHrP in the mammary gland does not impair ductal development.

(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 MMTV-cre and MMTV-cre;PTHrPlox/lacZ mammary glands at 5 weeks of age. (E) PTHrP mRNA expression is decreased in MMTV-cre;PTHrPlox/lacZ mammary glands. (F) Ductal outgrowth was measured in Control (n = 6) and MMTV-cre;PTHrPlox/lacZ (n = 12) as % of fat pad filled.

Figure 4

Figure 4. K14-tTA;tetO-Cre deletion of PTHrP in the mammary gland does not impair ductal development.

K14-tTA;tetO-Cre activity is specific to the myoepithelial cells of the ducts and the cap cells of TEBs (A, B). (A) Whole-mount Xgal staining of a 4 week old gland. (B) Section of stained gland demonstrating that myoepithelial cells are targeted for recombination in the ducts. (C) Whole-mounts of mammary glands from K14-tTA;tetO-Cre and K14-tTA;tetO-Cre;PTHrPlox/lacZ mammary glands at 5 weeks of age. (E) PTHrP mRNA expression is decreased in K14-tTA;tetO-Cre;PTHrPlox/lacZ mammary glands. (F) Ductal outgrowth was measured in Control (n = 4) and K14-tTA;tetO-Cre;PTHrPlox/lacZ (n = 6) as % of fat pad filled.

Figure 5

Figure 5. Effects of PTHrP deletion on cell turnover in terminal end buds during puberty.

(A) End bud cell proliferation in 5-week-old mice as defined by the percentage of epithelial cells incorporating EdU within TEBs of control, K14-tTA;tetO-Cre;PTHrPlox/lacZ and MMTV Cre;PTHrPlox/lacZ mice. (B) Apoptosis was measured by TUNEL staining in the TEBs of control, K14-tTA;tetO-Cre;PTHrPlox/lacZ and MMTV Cre;PTHrPlox/lacZ 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.

Figure 6

Figure 6. Cell lineage is not disrupted by PTHrP deletion in the mammary gland.

Expression of the luminal marker, Gata3 and myoepithelial marker, K14, showed normal mammary gland architecture in both PTHrP MMTV-CKO (C) and K14-CKO (D) as compared to controls (B). (A) IgG control staining with secondary antibodies.

Figure 7

Figure 7. Loss of PTHrP has no Effect on Alveolar Development.

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).

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