Bisphenol A alters the development of the rhesus monkey mammary gland.

The xenoestrogen bisphenol A (BPA) used in the manufacturing of various plastics and resins for food packaging and consumer products has been shown to produce numerous endocrine and developmental effects in rodents. Exposure to low doses of BPA during fetal mammary gland development resulted in significant alterations in the gland's morphology that varied from subtle ones observed during the exposure period to precancerous and cancerous lesions manifested in adulthood. This study assessed the effects of BPA on fetal mammary gland development in nonhuman primates. Pregnant rhesus monkeys were fed 400 µg of BPA per kg of body weight daily from gestational day 100 to term, which resulted in 0.68 ± 0.312 ng of unconjugated BPA per mL of maternal serum, a level comparable to that found in humans. At birth, the mammary glands of female offspring were removed for morphological analysis. Morphological parameters similar to those shown to be affected in rodents exposed prenatally to BPA were measured in whole-mounted glands; estrogen receptor (ER) α and ß expression were assessed in paraffin sections. Student's t tests for equality of means were used to assess differences between exposed and unexposed groups. The density of mammary buds was significantly increased in BPA-exposed monkeys, and the overall development of their mammary gland was more advanced compared with unexposed monkeys. No significant differences were observed in ER expression. Altogether, gestational exposure to the estrogen-mimic BPA altered the developing mammary glands of female nonhuman primates in a comparable manner to that observed in rodents.

A genome-wide association study reveals a QTL influencing caudal supernumerary teats in Holstein cattle.

Supernumerary teats represent a common abnormality of the bovine udder. A genome-wide association study was performed based on the proportion of the occurrence of supernumerary teats in the daughters of 1097 Holstein bulls. The heritability of caudal supernumerary teats without mammary gland in this study was 0.604. The largest proportion of the heritability was attributable to BTA 20. The strongest evidence for association was with five SNPs on chromosome 20, referred to as a QTL. The mode of inheritance at this QTL was dominant. These findings reveal that the occurrence of caudal supernumerary teats without mammary gland in Holstein cattle is influenced by a QTL on chromosome 20 and a polygenic part. The data support the high potential of the SNPs in the QTL region as markers for breeding against caudal supernumerary teats.

The severity of mammary gland developmental defects is linked to the overall functional status of Cx43 as revealed by genetically modified mice.

Genetically modified mice mimicking ODDD (oculodentodigital dysplasia), a disease characterized by reduced Cx43 (connexin 43)-mediated gap junctional intercellular communication, represent an in vivo model to assess the role of Cx43 in mammary gland development and function. We previously reported that severely compromised Cx43 function delayed mammary gland development and impaired milk ejection in mice that harboured a G60S Cx43 mutant, yet there are no reports of lactation defects in ODDD patients. To address this further, we obtained a second mouse model of ODDD expressing an I130T Cx43 mutant to assess whether a mutant with partial gap junction channel activity would be sufficient to retain mammary gland development and function. The results of the present study show that virgin Cx43I130T/+ mice exhibited a temporary delay in ductal elongation at 4 weeks. In addition, Cx43I130T/+ mice develop smaller mammary glands at parturition due to reduced cell proliferation despite similar overall gland architecture. Distinct from Cx43G60S/+ mice, Cx43I130T/+ mice adequately produce and deliver milk to pups, suggesting that milk ejection is unaffected. Thus the present study suggests that a loss-of-function mutant of Cx43 with partial gap junction channel coupling conductance results in a less severe mammary gland phenotype, which may partially explain the lack of reported lactation defects associated with ODDD patients.

QTL region-specific microarrays reveal differential expression of positional candidate genes of signaling pathways associated with the liability for the inverted teat defect.

The inverted teat defect is the most common disorder of the mammary complex in pigs. It is characterized by the failure of teats to protrude from the udder surface, preventing normal milk flow and thus limiting the rearing capacity and increasing the risk of mastitis. The inverted teat defect is a liability trait with a complex mode of inheritance. We previously identified QTL for inverted teats. As a complementary approach that integrates map-based efforts to identify candidate genes for the inverted teat defect with function-driven expression analysis, application-specific microarrays were constructed that cover 1525 transcripts mapping in QTL regions on pig chromosomes 2, 3, 4, 6 and 11. About 950 transcripts were expressed in epithelial and mesenchymal teat tissue. The expression of three categories of teats was compared: normal teats of both non-affected and affected animals and inverted teats of affected animals. In epithelium and mesenchyme, 62 and 24 genes respectively were significantly differentially expressed (DE). The majority of biofunctions to which a significant number of DE genes were assigned are related to the following: (1) cell maintenance, proliferation, differentiation and replacement; (2) organismal, organ and tissue development; or (3) genetic information and nucleic acid processing. Moreover, the DE genes belong almost exclusively to canonical pathways related to signaling rather than metabolic pathways. This is in line with findings obtained by genome-wide catalogue microarrays. This study adds another piece to the puzzle of the etiology of inverted teats by indicating that causal genetic variation leading to the disorder is likely among the genes encoding for members of the signaling cascades of growth factors.

Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation.

The composite structure of the mammalian skull, which forms predominantly via intramembranous ossification, requires precise pre- and post-natal growth regulation of individual calvarial elements. Disturbances of this process frequently cause severe clinical manifestations in humans. Enhanced DNA binding by a mutant MSX2 homeodomain results in a gain of function and produces craniosynostosis in humans. Here we show that Msx2-deficient mice have defects of skull ossification and persistent calvarial foramen. This phenotype results from defective proliferation of osteoprogenitors at the osteogenic front during calvarial morphogenesis, and closely resembles that associated with human MSX2 haploinsufficiency in parietal foramina (PFM). Msx2-/- mice also have defects in endochondral bone formation. In the axial and appendicular skeleton, post-natal deficits in Pth/Pthrp receptor (Pthr) signalling and in expression of marker genes for bone differentiation indicate that Msx2 is required for both chondrogenesis and osteogenesis. Consistent with phenotypes associated with PFM, Msx2-mutant mice also display defective tooth, hair follicle and mammary gland development, and seizures, the latter accompanied by abnormal development of the cerebellum. Most Msx2-mutant phenotypes, including calvarial defects, are enhanced by genetic combination with Msx1 loss of function, indicating that Msx gene dosage can modify expression of the PFM phenotype. Our results provide a developmental basis for PFM and demonstrate that Msx2 is essential at multiple sites during organogenesis.

Generation of ERα-floxed and knockout mice using the Cre/LoxP system.

Estrogen receptor alpha (ERα) is a nuclear receptor that regulates a range of physiological processes in response to estrogens. In order to study its biological role, we generated a floxed ERα mouse line that can be used to knock out ERα in selected tissues by using the Cre/LoxP system. In this study, we established a new ERα knockout mouse line by crossing the floxed ERα mice with Cre deleter mice. Here we show that genetic disruption of the ERα gene in all tissues results in sterility in both male and female mice. Histological examination of uterus and ovaries revealed a dramatically atrophic uterus and hemorrhagic cysts in the ovary. These results suggest that infertility in female mice is the result of functional defects of the reproductive tract. Moreover, female knockout mice are hyperglycemic, develop obesity and at the age of 4 months the body weight of these mice was more than 20% higher compared to wild type littermates and this difference increased over time. Our results demonstrate that ERα is necessary for reproductive tract development and has important functions as a regulator of metabolism in females.

Genome-wide association study uncovers four QTL predisposing to supernumerary teats in cattle.

Supernumerary teats (hyperthelia, SNTs) are a common abnormality of the bovine udder with a medium to high heritability and a postulated oligogenic or polygenic inheritance pattern. SNTs not only negatively affect machine milking ability but also act as a reservoir for bacteria. A genome-wide association study was carried out to identify genes involved in the development of SNTs in the dual-purpose Fleckvieh breed. A total of 2467 progeny-tested bulls were genotyped at 43 698 single nucleotide polymorphisms, and daughter yield deviations (DYDs) for 'udder clearness' (UC) were used as high-heritability phenotypes. Massive structuring of the study population was accounted for by principal components analysis-based and mixed model-based approaches. Four loci on BTA5, BTA6, BTA11 and BTA17 were significantly associated with the UC DYD. Three associated regions contain genes of the highly conserved Wnt signalling pathway. The four QTL together account for 10.7% of the variance of the UC DYD, whereas the major fraction of the DYD variance is attributable to chromosomes with no identified QTL. Our results support both an oligogenic and a polygenic inheritance pattern of SNTs in cattle. The identified candidate genes permit insights into the genetic architecture of teat malformations in cattle and provide clues to unravel the molecular mechanisms of mammary gland alterations in cattle and other species.

The double-stranded RNA-binding protein, PACT, is required for postnatal anterior pituitary proliferation.

PACT is a double-stranded RNA-binding protein that also binds and activates the latent protein kinase, PKR, which plays a major role in cellular antiviral defense in mammals. For evaluating PACT's contribution to the innate immune system, Pact(-/-) mice have been generated; these mice exhibit notable developmental abnormalities including microtia, with craniofacial, ear, and hearing defects. Here we report that, in addition, Pact(-/-) mice had smaller body size and fertility defects, both of which were caused by defective pituitary functions. Pact(-/-) mice exhibited anterior pituitary lobe (AL) hypoplasia, which developed postnatally, when the second phase of pituitary expansion occurs. Among the 5 cell types in AL, the numbers of corticotrophs, gonadotrophs, and somatotrophs were equally decreased in Pact(-/-) mice with a greater impact on lactotrophs and a lesser impact on thyrotrophs. PACT mRNA and protein were highly expressed in the pituitary of wild-type (Wt) mice during the postnatal wave of AL proliferation, the same period in which the hypoplasia developed in Pact(-/-) mice. During this time, the pituitaries of Pact(-/-) mice did not exhibit significantly increased apoptosis compared with Wt mice but showed a decrease in cell proliferation. The inhibition of cell proliferation observed in vivo could be recapitulated in vitro in GH3 somato/lactotroph and LbetaT2 gonadotroph cell lines; knockdown of PACT expression with siRNA diminished the rate of proliferation of these cells. Our study revealed a physiologically significant role for PACT in cell proliferation and an essential role of a dsRNA-binding protein in mammalian pituitary expansion.

MMTV-Cre transgenes can adversely affect lactation: considerations for conditional gene deletion in mammary tissue.

CRE-loxP-mediated inactivation and activation of genes in mouse mammary epithelium have been widely used to study genetic pathways in normal development and neoplastic transformation in vivo. In 1997, we generated three distinct mouse lines carrying an identical MMTV-Cre transgene (lines A, D, and F). Because the presence of CRE recombinase can adversely affect the physiology of nonmammary cells, we explored whether transgenic females display lactational defects. Whereas dams from line D nurse their pups and display overtly normal mammary development, line A shows some impairment during lactation and females from line F completely fail to nurse their litters. The ability to nurse a litter correlates with the extent of alveolar development and differentiation. This study demonstrates the importance of including appropriate "Cre-only" controls and provides guidelines to avoid problems in data interpretation.

Morphological and molecular effects of overexpressed GH on mice mammary gland.

Growth Hormone (GH) plays crucial roles in mammary gland development and growth, and its upregulation has been associated with breast cancer promotion and/or progression. To ascertain how high GH levels could promote mammary tissue oncogenic transformation, morphological characteristics and the expression of receptors involved in mammary growth, development and cancer, and of mitogenic mediators were analyzed in the mammary gland of virgin adult transgenic mice that overexpress GH. Whole mounting and histologic analysis evidenced that transgenic mice exhibit increased epithelial ductal elongation and enlarged ducts along with deficient branching and reduced number of alveolar structures compared to wild type mice. The number of differentiated alveolar structures was diminished in transgenic mice while the amount of terminal end buds (TEBs) did not differ between both groups of mice. GH, insulin-like growth factor 1 (IGF1) and GH receptor mRNA levels were augmented in GH-overexpressing mice breast tissue, as well as IGF1 receptor protein content. However, GH receptor protein levels were decreased in transgenic mice. Fundamental receptors for breast growth and development like progesterone receptor and epidermal growth factor receptor were also increased in mammary tissue from transgenic animals. In turn, the levels of the proliferation marker Ki67, cFOS and Cyclin D1 were increased in GH-overexpressing mice, while cJUN expression was decreased and cMYC did not vary. In conclusion, prolonged exposure to high GH levels induces morphological and molecular alterations in the mammary gland that affects its normal development. While these effects would not be tumorigenic per se, they might predispose to oncogenic transformation.

Complete focal adhesion kinase deficiency in the mammary gland causes ductal dilation and aberrant branching morphogenesis through defects in Rho kinase-dependent cell contractility.

The adult, virgin mammary gland is a highly organized branched ductal network comprising two major cell types: myoepithelial and luminal epithelial cells. To study the role and mechanism of focal adhesion kinase (FAK)-mediated signaling in mammary gland development and differentiation, we used a conditional Fak-knockout mammary epithelial cell (MEC) transplantation model. Conditional Cre recombinase (Cre)-mediated Fak deletion in primary cultured MECs isolated from FAK(lox/lox)/Rosa26Cre-ERT2 donor mice caused loss of FAK in all mammary cells. Transplantation of Fak-knockout MECs in a cleared mammary fat pad of immune-deficient recipient mice resulted in development of new but dilated virgin ducts with a disrupted myoepithelial and luminal epithelial cell multilayer and aberrant ductal morphogenesis during pregnancy. In the absence of FAK, MECs spread poorly, showed enhanced Rho kinase (ROCK)-mediated cytoskeletal contractility, and failed to respond to receptor-mediated cytoskeletal remodeling. Likewise, FAK deficiency fully inhibited branching morphogenesis of mammary gland organoids in a ROCK-dependent manner. Altogether these data suggest a model in which FAK coordinates contractile forces in MECs to maintain the bilayered cellular organization of myoepithelial and luminal epithelial cells in ducts, thus allowing proper mammary gland development and function.

A brain-specific homeobox gene, Bsx, is essential for proper postnatal growth and nursing.

To investigate in vivo roles of a murine hypothalamic homeobox gene, Bsx, we generated and analyzed two mutant alleles, Bsx(DeltaHD) and Bsx(lacZ). Bsx(DeltaHD) lacks the homeodomain, and Bsx(lacZ) is an insertion of a lacZ reporter gene. Bsx-lacZ expression was detected in the hypothalamus and pineal gland and reiterates Bsx expression. Bsx homozygous mutant mice were born at the expected Mendelian ratio, but their growth was impaired. Offspring from Bsx homozygous mutant females exhibited a low survival rate due to a nursing defect. Mammary glands of the mutant females developed normally during pregnancy; however, they involuted quickly after parturition. These results demonstrate that Bsx is required for postnatal growth and maintenance of lactating mammary glands. Thus, mouse Bsx is likely involved in systemic control of suppression of apoptosis of postpartum mammary epithelial cells.

MMTV promoter-regulated caveolin-1 overexpression yields defective parenchymal epithelia in multiple exocrine organs of transgenic mice.

Caveolin-1 (Cav-1) is a major structural protein of caveolae, specialized plasma membrane invaginations that are involved in a cell-specific fashion in diverse cell activities such as molecular transport, cell adhesion, and signal transduction. In normal adult mammals, Cav-1 expression is abundant in mesenchyme-derived cells but relatively low in epithelial parenchyma. However, epithelial Cav-1 overexpression is associated with development and/or progression of many carcinomas. In this study, we generated and characterized a transgenic mouse model of Cav-1 overexpression under the control of a mouse mammary tumor virus (MMTV) long terminal-repeat promoter, which is predominantly expressed in specific epithelial cells. The MMTVcav-1(+) transgenic mice were fertile, and females bore litters of normal size with no obvious developmental abnormalities. However, by age 11months, the MMTVcav-1(+) mice demonstrated overtly different phenotypes in multiple exocrine organs when compared with their nontransgenic MMTVcav-1(-) littermates. Cav-1 overexpression in MMTVcav-1(+) mice produced organ-specific abnormalities, including hypotrophy of mammary glandular epithelia, bronchiolar epithelial hyperplasia and atypia, mucous-cell hyperplasia in salivary glands, elongated hair follicles and dermal thickening in the skin, and reduced accumulation of enzymogen granules in pancreatic acinar cells. In addition, the MMTVcav-1(+) transgenic mice tended to have a greater incidence of malignant tumors, including lung and liver carcinomas and lymphoma, than their MMTVcav-1(-) littermates. Our results indicate that Cav-1 overexpression causes organ-specific, age-related epithelial disorders and suggest the potential for increased susceptibility to carcinogenesis.

WNT signals are required for the initiation of hair follicle development.

Hair follicle morphogenesis is initiated by a dermal signal that induces the development of placodes in the overlying epithelium. To determine whether WNT signals are required for initiation of follicular development, we ectopically expressed Dickkopf 1, a potent diffusible inhibitor of WNT action, in the skin of transgenic mice. This produced a complete failure of placode formation prior to morphological or molecular signs of differentiation, and blocked tooth and mammary gland development before the bud stage. This phenotype indicates that activation of WNT signaling in the skin precedes, and is required for, localized expression of regulatory genes and initiation of hair follicle placode formation.

Netrin-1/neogenin interaction stabilizes multipotent progenitor cap cells during mammary gland morphogenesis.

Netrin-1 and its receptors play an essential role patterning the nervous system by guiding neurons and axons to their targets. To explore whether netrin-1 organizes nonneural tissues, we examined its role in mammary gland morphogenesis. Netrin-1 is expressed in prelumenal cells, and its receptor neogenin is expressed in a complementary pattern in adjacent cap cells of terminal end buds (TEBs). We discovered that loss of either gene results in disorganized TEBs characterized by exaggerated subcapsular spaces, breaks in basal lamina, dissociated cap cells, and an increased influx of cap cells into the prelumenal compartment. Cell aggregation assays demonstrate that neogenin mediates netrin-1-dependent cell clustering. Thus, netrin-1 appears to act locally through neogenin to stabilize the multipotent progenitor (cap) cell layer during mammary gland development. Our results suggest that netrin-1 and its receptor neogenin provide an adhesive, rather than a guidance, function during nonneural organogenesis.

Requirement of a myocardin-related transcription factor for development of mammary myoepithelial cells.

The mammary gland consists of a branched ductal system comprised of milk-producing epithelial cells that form ductile tubules surrounded by a myoepithelial cell layer that provides contractility required for milk ejection. Myoepithelial cells bear a striking resemblance to smooth muscle cells, but they are derived from a different embryonic cell lineage, and little is known of the mechanisms that control their differentiation. Members of the myocardin family of transcriptional coactivators cooperate with serum response factor to activate smooth muscle gene expression. We show that female mice homozygous for a loss-of-function mutation of the myocardin-related transcription factor A (MRTF-A) gene are unable to effectively nurse their offspring due to a failure in maintenance of the differentiated state of mammary myoepithelial cells during lactation, resulting in apoptosis of this cell population, a consequent inability to release milk, and premature involution. The phenotype of MRTF-A mutant mice reveals a specific and essential role for MRTF-A in mammary myoepithelial cell differentiation and points to commonalities in the transcriptional mechanisms that control differentiation of smooth muscle and myoepithelial cells.

Acute myeloid leukemia-associated Mkl1 (Mrtf-a) is a key regulator of mammary gland function.

Transcription of immediate-early genes--as well as multiple genes affecting muscle function, cytoskeletal integrity, apoptosis control, and wound healing/angiogenesis--is regulated by serum response factor (Srf). Extracellular signals regulate Srf in part via a pathway involving megakaryoblastic leukemia 1 (Mkl1, also known as myocardin-related transcription factor A [Mrtf-a]), which coactivates Srf-responsive genes downstream of Rho GTPases. Here we investigate Mkl1 function using gene targeting and show the protein to be essential for the physiologic preparation of the mammary gland during pregnancy and the maintenance of lactation. Lack of Mkl1 causes premature involution and impairs expression of Srf-dependent genes in the mammary myoepithelial cells, which control milk ejection following oxytocin-induced contraction. Despite the importance of Srf in multiple transcriptional pathways and widespread Mkl1 expression, the spectrum of abnormalities associated with Mkl1 absence appears surprisingly restricted.

Steroid receptor coactivator 2 is critical for progesterone-dependent uterine function and mammary morphogenesis in the mouse.

Although the essential involvement of the progesterone receptor (PR) in female reproductive tissues is firmly established, the coregulators preferentially enlisted by PR to mediate its physiological effects have yet to be fully delineated. To further dissect the roles of members of the steroid receptor coactivator (SRC)/p160 family in PR-mediated reproductive processes in vivo, state-of-the-art cre-loxP engineering strategies were employed to generate a mouse model (PR(Cre/+) SRC-2(flox/flox)) in which SRC-2 function was abrogated only in cell lineages that express the PR. Fertility tests revealed that while ovarian activity was normal, PR(Cre/+) SRC-2(flox/flox) mouse uterine function was severely compromised. Absence of SRC-2 in PR-positive uterine cells was shown to contribute to an early block in embryo implantation, a phenotype not shared by SRC-1 or -3 knockout mice. In addition, histological and molecular analyses revealed an inability of the PR(Cre/+) SRC-2(flox/flox) mouse uterus to undergo the necessary cellular and molecular changes that precede complete P-induced decidual progression. Moreover, removal of SRC-1 in the PR(Cre/+) SRC-2(flox/flox) mouse uterus resulted in the absence of a decidual response, confirming that uterine SRC-2 and -1 cooperate in P-initiated transcriptional programs which lead to full decidualization. In the case of the mammary gland, whole-mount and histological analysis disclosed the absence of significant ductal side branching and alveologenesis in the hormone-treated PR(Cre/+) SRC-2(flox/flox) mammary gland, reinforcing an important role for SRC-2 in cellular proliferative changes that require PR. We conclude that SRC-2 is appropriated by PR in a subset of transcriptional cascades obligate for normal uterine and mammary morphogenesis and function.

Association of parathyroid hormone-like hormone (PTHLH) and its receptor (PTHR1) with the number of functional and inverted teats in pigs.

Parathyroid hormone-like hormone gene (PTHLH) and its receptor, parathyroid hormone/ parathyroid hormone-like hormone receptor 1 (PTHR1), play a role in epithelial mesenchymal interactions during growth and differentiation of different tissues and anatomic structures, including teats. Therefore, PTHLH and PTHR1 were evaluated as functional candidate genes for their effects on number and shape of teats in pigs. In particular, focus was on the occurrence and number of inverted teats, the most frequent and economically relevant teat developmental defect in pigs. For this purpose, association and linkage of the PTHLH gene and the PTHR1 gene with inverted teat defect and the total number of teats and inverted teats were studied in an experimental Duroc and Berlin Miniature pig (DUMI) population. Polymorphism C1819T of PTHR1 was significantly associated with inverted teat phenotype (p = 0.014), total number of teats (p = 0.047) and was close to significance with the number of inverted teats (p = 0.078). Polymorphism C375T of PTHLH was close to significance with the inverted teat phenotype (p = 0.122) and showed no significant association with the total number of teats (p = 0.621) and the number of inverted teats (p = 0.256) in the DUMI population. Association analyses were also performed for combined effects of PTHLH and PTHR1 in order to address potential interaction, however, revealed no indication of effects of interaction. The function, position and the association shown here promote PTHR1 as a candidate gene for number of teats and in particular for affection by and number of inverted teats.

Dual role for miR-34a in the control of early progenitor proliferation and commitment in the mammary gland and in breast cancer.

The role of the tumour-suppressor miR-34 family in breast physiology and in mammary stem cells (MaSCs) is largely unknown. Here, we revealed that miR-34 family, and miR-34a in particular, is implicated in mammary epithelium homoeostasis. Expression of miR-34a occurs upon luminal commitment and differentiation and serves to inhibit the expansion of the pool of MaSCs and early progenitor cells, likely in a p53-independent fashion. Mutant mice (miR34-KO) and loss-of-function approaches revealed two separate functions of miR-34a, controlling both proliferation and fate commitment in mammary progenitors by modulating several pathways involved in epithelial cell plasticity and luminal-to-basal conversion. In particular, miR-34a acts as endogenous inhibitor of the Wnt/beta-catenin signalling pathway, targeting up to nine upstream regulators at the same time, thus modulating the expansion of the MaSCs/early progenitor pool. These multiple roles of miR-34a are maintained in a model of human breast cancer, in which chronic expression of miR-34a in triple-negative mesenchymal-like cells (enriched in cancer stem cells-CSCs) could promote a luminal-like differentiation programme, restrict the CSC pool, and inhibit tumour propagation. Hence, activation of miR-34a-dependent programmes could provide a therapeutic opportunity for the subset of breast cancers, which are rich in CSCs and respond poorly to conventional therapies.