A mouse transgenic approach to induce ��-catenin signaling in a temporally controlled manner

Although constitutive murine transgenic models have provided important insights into β-catenin signaling in tissue morphogenesis and tumorigenesis, these models are unable to express activated β-catenin in a temporally controlled manner. Therefore, to enable the induction (and subsequent de-induction) of β-catenin signaling during a predetermined time-period or developmental stage, we have generated and characterized a TETO-ΔN89β-catenin responder transgenic mouse. Crossed with the MTB transgenic effector mouse, which targets the expression of the reverse tetracycline transactivator (rtTA) to the mammary epithelium, we demonstrate that the stabilized (and activated) form of β-catenin (ΔN89β-catenin) is expressed only in the presence doxycycline-activated rtTA in the mammary epithelial compartment. Furthermore, we show that transgene-derived ΔN89β-catenin elicits significant mammary epithelial proliferation and precocious alveologenesis in the virgin doxycycline-treated MTB/TETO-ΔN89β-catenin bitransgenic. Remarkably, deinduction of TETO-ΔN89β-catenin transgene expression (through doxycycline withdrawal) results in the reversal of these morphological changes. Importantly, continued activation of the TETO-ΔN89β-catenin transgene results in palpable mammary tumors (within 7-9?months) in the doxycycline-treated virgin MTB/TETO-ΔN89β-catenin bigenic but not in the same bitransgenic without doxycycline administration. Collectively, these mammary epithelial responses to ΔN89β-catenin expression agree with previous reports using conventional transgenesis and therefore confirm that ΔN89β-catenin functions as expected in this doxycycline-responsive bigenic system. In sum, our mammary gland studies demonstrate "proof-of-principle" for using the TETO-ΔN89β-catenin transgenic responder to activate (and then de-activate) β-catenin signaling in any tissue of interest in a spatiotemporal specific fashion.     

Developmental Stage and Time Dictate the Fate of Wnt/β-Catenin-Responsive Stem Cells in the Mammary Gland

The mammary epithelium undergoes extensive growth and remodeling during pregnancy, suggesting a role for stem cells. Yet their origin, identity, and behavior in the intact tissue remain unknown. Using an Axin2(CreERT2) allele, we labeled and traced Wnt/β-catenin-responsive cells throughout mammary gland development. This reveals a switch in Wnt/β-catenin signaling around birth and shows that, depending on the developmental stage, Axin2(+)?cells contribute differently to basal and luminal epithelial cell lineages of the mammary epithelium. Moreover, an important difference exists between the developmental potential tested in transplantation assays and that displayed by the same cell population in?situ. Finally, Axin2(+) cells in the adult build alveolar structures during multiple pregnancies, demonstrating the existence of a Wnt/β-catenin-responsive adult stem cell. Our study uncovers dynamic changes in Wnt/β-catenin signaling in the mammary epithelium and offers insights into the developmental fate of mammary gland stem and progenitor cells.     

Lactoferrin at basal side of mouse mammary epithelium derives in part from stroma cells

Lactoferrin is synthesized by glandular epithelial cells and neutrophils and is also present on both sides of the mammary epithelium. We have studied the origin of lactoferrin detected in the various compartments of mouse mammary tissue. As revealed by immunogold electron microscopy, lactoferrin is present in mammary epithelial cells and in the basal region of the epithelium, associated with connective tissue and stroma cells at all physiological stages studied. A perturbation of protein synthesis or transport after in vitro treatment with cycloheximide or brefeldin A does not abrogate lactoferrin labelling in the basal region of the epithelium. The expression of lactoferrin has also been observed in the fat pads of mammary glands from mice surgically depleted of epithelial cells. The sealing of one teat for 24 h is accompanied by an increase in both the number of stroma cells and the labelling of myoepithelial cells. Thus, the lactoferrin present in the interstitial space of the mouse mammary epithelium originates in part from stroma cells. Possible roles of lactoferrin at the basal side of the mammary epithelium are discussed.     

Loss of the peroxisome proliferation-activated receptor gamma (PPARgamma ) does not affect mammary development and propensity for tumor formation but leads to reduced fertility

The peroxisome proliferation-activated receptor gamma (PPARgamma) is expressed in many cell types including mammary epithelium, ovary, macrophages, and B- and T-cells. PPARgamma has an anti-proliferative effect in pre-adipocytes and mammary epithelial cells, and treatment with its ligands reduced the progression of carcinogen-induced mammary tumors in mice. Because PPARgamma-null mice die in utero it has not been possible to study its role in development and tumorigenesis in vivo. To investigate whether PPARgamma is required for the establishment and physiology of different cell types, a cell-specific deletion of the gene was carried out in mice using the Cre-loxP recombination system. We deleted the PPARgamma gene in mammary epithelium using WAP-Cre transgenic mice and in epithelial cells, B- and T-cells, and ovary cells using MMTV-Cre mice. The presence of PPARgamma was not required for functional development of the mammary gland during pregnancy and for the establishment of B- and T-cells. In addition, no increase in mammary tumors was observed. However, loss of the PPARgamma gene in oocytes and granulosa cells resulted in impaired fertility. These mice have normal populations of follicles, they ovulate and develop corpora lutea. Although progesterone levels are decreased and implantation rates are reduced, the exact cause of the impaired fertility remains to be determined.     

Effects of mesenchyme on epithelial tissue architecture revealed by tissue recombination experiments between the submandibular gland and lung of embryonic mice

Lung epithelium during morphogenesis maintains a sheet structure of polarized cells lining a lumen, in which E-cadherin, β-catenin and tight junctional proteins are localized at the cell–cell contact sites. On the other hand, the submandibular gland epithelium at early stages of development forms a non-cavitated mass of cells where E-cadherin/β-catenin are present on the entire cell surfaces and tight junctional proteins are almost absent or weakly scattered. In the present study, tissue recombination experiments were performed between the two organs to explore roles of mesenchyme in the architectural development of the epithelium. Homotypic recombinants of both submandibular gland and lung showed the tissue architecture as observed in the intact organs. In contrast, 11-day lung epithelium cultured with 13-day submandibular mesenchyme formed multilayers of cells with the lumen being less visible. It was accompanied by redistribution of E-cadherin/β-catenin along the entire cell surfaces and by an irregular distribution of tight junctional proteins. A similar redistribution of these molecules was observed in 15-day lung epithelium cultured with the submandibular mesenchyme, although the epithelial sheet structure lining the lumen was formed. On the other hand, the tissue architecture of submandibular gland epithelium was little affected by lung mesenchyme, although the epithelium was flattened and showed branching morphogenesis.     

Loss of connexin 26 in mammary epithelium during early but not during late pregnancy results in unscheduled apoptosis and impaired development

Gap junctions are intercellular channels that are formed by the protein family of connexins (Cxs). In mammary tissue, Cx26 and Cx32 are present in the secretory epithelium and Cx43 is localized in the myoepithelium. The expression of Cx26 and Cx32 is induced during pregnancy and lactation, respectively, thus suggesting unique roles for them in the functional development of the gland. The requirement for these connexins was explored using several strains of genetically altered mice: mice with an inactivated Cx32 gene, mice in which the Cx43 gene had been replaced with the Cx32 gene (Cx43KI32 mice) and mice in which the Cx26 gene was specifically ablated in mammary epithelium at different stages of development using Cre-loxP-based recombination. Normal mammary development was obtained in Cx32-null mice and in Cx43KI32 mammary tissue. In contrast, loss of Cx26 in mammary epithelium before puberty resulted in abrogated lobulo-alveolar development and increased cell death during pregnancy, which was accompanied by impaired lactation. Loss of Cx26 in mammary epithelium during the later part of pregnancy did not adversely interfere with functional mammary development. These results demonstrate that the presence of Cx26 is critical during early stages but not during the end of pregnancy when the tissue has completed functional differentiation. Cx26 is considered a tumor suppressor gene and Cx26-null mammary tissue was evaluated after five pregnancies. No hyperproliferation or hyperplasia was observed, suggesting that Cx26 does not function as a tumor suppressor.     

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.     

Mammary gland development in transforming growth factor beta1 null mutant mice: systemic and epithelial effects

The cytokine-transforming growth factor beta1 (TGFB1) is implicated in development of the mammary gland through regulation of epithelial cell proliferation and differentiation during puberty and pregnancy. We compared mammary gland morphogenesis in virgin Tgfb1(+/+), Tgfb1(+/-), and Tgfb1(-/-) mice and transplanted Tgfb1(+/+) and Tgfb1(-/-) epithelium to determine the impact of TGFB1 deficiency on development. When mammary gland tissue was evaluated relative to the timing of puberty, invasion through the mammary fat pad of the ductal epithelium progressed similarly, irrespective of genotype, albeit fewer terminal end buds were observed in mammary glands from Tgfb1(-/-) mice. The terminal end buds appeared to be normal morphologically, and a comparable amount of epithelial proliferation was evident. When transplanted into wild-type recipients, however, Tgfb1(-/-) epithelium showed accelerated invasion compared with Tgfb1(+/+) epithelium. This suggests that the normal rate of ductal extension in Tgfb1(-/-) null mutant mice is the net result of impaired endocrine or paracrine support acting to limit the consequences of unrestrained epithelial growth. By adulthood, mammary glands in cycling virgin Tgfb1(-/-) mice were morphologically similar to those in Tgfb1(+/+) and Tgfb1(+/-) animals, with a normal branching pattern, and the tissue differentiated into early alveolar structures in the diestrous phase of the ovarian cycle. Transplanted mammary gland epithelium showed a similar extent of ductal branching and evidence of secretory differentiation of luminal cells in pregnancy. These results reveal two opposing actions of TGFB1 during pubertal mammary gland morphogenesis: autocrine inhibition of epithelial ductal growth, and endocrine or paracrine stimulation of epithelial ductal growth.     

Mammary epithelial‐specific knockout of the ephrin‐B2 gene leads to precocious epithelial cell death at lactation

The family of Eph receptor tyrosine kinases and their membrane bound ligands, the ephrins, are involved in a wide variety of morphogenic processes during embryonic development and adult tissue homeostasis. Receptor‐ligand interaction requires direct cell–cell contact and results in forward and reverse signaling originating from the receptor and ligand, respectively. We have previously shown that EphB4 and ephrinB2 are differentially expressed during the development of the adult mammary parenchyma. Overexpression of EphB4 in the mammary epithelium of transgenic mice leads to perturbations in mammary epithelial morphology, motility and growth. To investigate the role of ephrinB2 signaling in mammary gland biology, we have established transgenic mice exhibiting conditional ephrinB2 knockout in the mammary epithelium. In homozygote double transgenic CreLox mice, specific knockout of ephrinB2 occurred in the mammary epithelium during the first pregnancy‐lactating period. Abolishing ephrinB2 function led to severe interference with the architecture and functioning of the mammary gland at lactation. The morphology of the transgenic lactating glands resembled that of involuting controls, with decreased epithelial cell number and collapsed lobulo‐alveolar structures. Accordingly, massive epithelial cell death and expression of involution‐specific genes were observed. Interestingly, in parallel to cell death, significant cell proliferation was apparent, suggestive of tissue regeneration.     

Cre-mediated gene deletion in the mammary gland.

To delete genes specifically from mammary tissue using the Cre-lox system, we have established transgenic mice expressing Cre recombinase under control of the WAP gene promoter and the MMTV LTR. Cre activity in these mice was evaluated by three criteria. First, the tissue distribution of Cre mRNA was analyzed. Second, an adenovirus carrying a reporter gene was used to determine expression at the level of single cells. Third, tissue specificity of Cre activity was determined in a mouse strain carrying a reporter gene. In adult MMTV-Cre mice expression of the transgene was confined to striated ductal cells of the salivary gland and mammary epithelial cells in virgin and lactating mice. Expression of WAP-Cre was only detected in alveolar epithelial cells of mammary tissue during lactation. Analysis of transgenic mice carrying both the MMTV-Cre and the reporter transgenes revealed recombination in every tissue. In contrast, recombination mediated by Cre under control of the WAP gene promoter was largely restricted to the mammary gland but occasionally observed in the brain. These results show that transgenic mice with WAP-Cre but not MMTV-Cre can be used as a powerful tool to study gene function in development and tumorigenesis in the mammary gland.     

An InCytes from MBC Selection: Regulation of Mammary Gland Branching Morphogenesis by EphA2 Receptor Tyrosine Kinase

Eph receptor tyrosine kinases, including EphA2, are expressed in the mammary gland. However, their role in mammary gland development remains poorly understood. Using EphA2-deficient animals, we demonstrate for the first time that EphA2 receptor function is required for mammary epithelial growth and branching morphogenesis. Loss of EphA2 decreased penetration of mammary epithelium into fat pad, reduced epithelial proliferation, and inhibited epithelial branching. These defects appear to be intrinsic to loss of EphA2 in epithelium, as transplantation of EphA2-deficient mammary tissue into wild-type recipient stroma recapitulated these defects. In addition, HGF-induced mammary epithelial branching morphogenesis was significantly reduced in EphA2-deficient cells relative to wild-type cells, which correlated with elevated basal RhoA activity. Moreover, inhibition of ROCK kinase activity in EphA2-deficient mammary epithelium rescued branching defects in primary three-dimensional cultures. These results suggest that EphA2 receptor acts as a positive regulator in mammary gland development, functioning downstream of HGF to regulate branching through inhibition of RhoA. Together, these data demonstrate a positive role for EphA2 during normal mammary epithelial proliferation and branching morphogenesis.     

Direct and indirect roles for β-catenin in facultative basal progenitor cell differentiation

The conducting airway epithelium is maintained and repaired by endogenous progenitor cells. Dysregulated progenitor cell proliferation and differentiation is thought to contribute to epithelial dysplasia in chronic lung disease. Thus modification of progenitor cell function is an attractive therapeutic goal and one that would be facilitated by knowledge of the molecular pathways that regulate their behavior. We modeled the human tracheobronchial epithelium using primary mouse tracheal epithelial cell cultures that were differentiated by exposure to the air-liquid-interface (ALI). A basal cell subset, termed facultative basal cell progenitors (FBP), initiate these cultures and are the progenitor for tracheal-specific secretory cells, the Clara-like cell, and ciliated cells. To test the hypothesis that β-catenin is necessary for FBP function, ALI cultures were generated from mice homozygous for the Ctnb(flox(E2-6)) allele. In this model, exons 2-6 of the β-catenin gene are flanked by LoxP sites, allowing conditional knockout of β-catenin. The β-catenin locus was modified through transduction with Adenovirus-5-encoding Cre recombinase. This approach generated a mosaic epithelium, comprised of β-catenin wild-type and β-catenin knockout cells. Dual immunostaining and quantitative histomorphometric analyses demonstrated that β-catenin played a direct role in FBP-to-ciliated cell differentiation and that it regulated cell-cell interactions that were necessary for FBP-to-Clara-like cell differentiation. β-catenin was also necessary for FBP proliferation and long-term FBP viability. We conclude that β-catenin is a critical determinant of FBP function and suggest that dysregulation of the β-catenin signaling pathway may contribute to disease pathology.