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

Lactation and neonatal nutrition: defining and refining the critical questions.

This paper resulted from a conference entitled "Lactation and Milk: Defining and refining the critical questions" held at the University of Colorado School of Medicine from January 18-20, 2012. The mission of the conference was to identify unresolved questions and set future goals for research into human milk composition, mammary development and lactation. We first outline the unanswered questions regarding the composition of human milk (Section I) and the mechanisms by which milk components affect neonatal development, growth and health and recommend models for future research. Emerging questions about how milk components affect cognitive development and behavioral phenotype of the offspring are presented in Section II. In Section III we outline the important unanswered questions about regulation of mammary gland development, the heritability of defects, the effects of maternal nutrition, disease, metabolic status, and therapeutic drugs upon the subsequent lactation. Questions surrounding breastfeeding practice are also highlighted. In Section IV we describe the specific nutritional challenges faced by three different populations, namely preterm infants, infants born to obese mothers who may or may not have gestational diabetes, and infants born to undernourished mothers. The recognition that multidisciplinary training is critical to advancing the field led us to formulate specific training recommendations in Section V. Our recommendations for research emphasis are summarized in Section VI. In sum, we present a roadmap for multidisciplinary research into all aspects of human lactation, milk and its role in infant nutrition for the next decade and beyond

Deletion of the nuclear localization sequences and C-terminus of PTHrP impairs embryonic mammary development but also inhibits PTHrP production.

Parathyroid hormone-related protein (PTHrP) can be secreted from cells and interact with its receptor, the Type 1 PTH/PTHrP Receptor (PTHR1) in an autocrine, paracrine or endocrine fashion. PTHrP can also remain inside cells and be transported into the nucleus, where its functions are unclear, although recent experiments suggest that it may broadly regulate cell survival and senescence. Disruption of either the PTHrP or PTHR1 gene results in many abnormalities including a failure of embryonic mammary gland development in mice and in humans. In order to examine the potential functions of nuclear PTHrP in the breast, we examined mammary gland development in PTHrP (1-84) knock-in mice, which express a mutant form of PTHrP that lacks the C-terminus and nuclear localization signals and which can be secreted but cannot enter the nucleus. Interestingly, we found that PTHrP (1-84) knock-in mice had defects in mammary mesenchyme differentiation and mammary duct outgrowth that were nearly identical to those previously described in PTHrP-/- and PTHR1-/- mice. However, the mammary buds in PTHrP (1-84) knock-in mice had severe reductions in mutant PTHrP mRNA levels, suggesting that the developmental defects were due to insufficient production of PTHrP by mammary epithelial cells and not loss of PTHrP nuclear function. Examination of the effects of nuclear PTHrP in the mammary gland in vivo will require the development of alternative animal models

<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>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

<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

<i>K14-tTA;tetO-Cre</i> deletion of <i>PTHrP</i> in the mammary gland does not impair ductal development.

<p><i>K14-tTA;tetO-Cre</i> 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 <i>K14-tTA;tetO-Cre</i> and <i>K14-tTA;tetO-Cre;PTHrP^lox/lacZ</i> mammary glands at 5 weeks of age. (E) <i>PTHrP</i> mRNA expression is decreased in <i>K14-tTA;tetO-Cre;PTHrP^lox/lacZ</i> mammary glands. (F) Ductal outgrowth was measured in Control (n = 4) and <i>K14-tTA;tetO-Cre;PTHrP^lox/lacZ</i> (n = 6) 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