TGF-beta 1-induced inhibition of mouse mammary ductal growth: developmental specificity and characterization

TGF-beta 1, implanted into growing mouse mammary glands, was previously shown to inhibit ductal growth in an apparently normal and fully reversible manner. In this report we extend these findings to show that TGF-beta 1 inhibition is highly specific. In pregnant or hormone-treated mice, doses of TGF-beta 1 that were capable of fully inhibiting ductal elongation had little effect on the proliferation of lobuloalveolar structures. Additionally, the inhibitory action of TGF-beta 1 on ducts is epithelium-specific, resulting in cessation of DNA synthesis in the rapidly proliferating epithelium of mammary end buds, but does not inhibit DNA synthesis in the stroma surrounding the end buds. At the cellular level, transplant studies showed that TGF-beta 1 inhibited the regeneration of mammary ductal cells when implanted into mammary gland-free fat pads by suppressing the formation of new end buds, without inhibiting maintenance DNA synthesis in ductal lumenal epithelium; this observation indicates the potential of TGF-beta 1 to maintain patterning by suppressing adventitious lateral branching. The time-course of TGF-beta 1 inhibition of end buds was rapid, with cessation of DNA synthesis by 12 hr, followed by loss of the stem cell (cap cell) layer. The question of glandular exposure to TGF-beta 1 administered in EVAc implants was also investigated. Incorporation of TGF-beta 1 into EVAc was found not to degrade the hormone, while the release kinetics of the ligand from implants, its retention in the gland, and the demonstrable zone of exposure were consistent with observed inhibitory effects. These results support the hypothesis that TGF-beta 1 is a natural regulator of mammary ductal growth.     

Regulation of mammary morphogenesis: evidence for extracellular matrix-mediated inhibition of ductal budding by transforming growth factor-beta 1.

Branching morphogenesis in the mammary gland involves focal regions of cell proliferation, the terminal and lateral ductal buds, that exist simultaneously with extensive regions of differentiated ducts in which budding and growth are actively suppressed. Exogenous transforming growth factor-beta 1 (TGF-beta 1) has previously been shown to locally inhibit the formation and growth of mammary ductal buds. Here we report that endogenous TGF-beta 1, produced by epithelial and stromal mammary cells, forms complexes with extracellular matrix (ECM) molecules surrounding those ductal structures in which budding is inhibited. The largest amounts of immunostainable TGF-beta 1 are found in mature periductal ECM, and the least in newly synthesized ECM. In all areas of active ductal growth, where DNA-synthetic buds were forming new ductal branches, we found a highly focal loss of TGF-beta 1 from the periductal ECM at the bud-forming region of the duct. When growth of the new buds terminated, the structures again became associated with TGF-beta-rich ECM. These findings indicate that ECM must reach a certain state of maturity before it becomes associated with TGF-beta 1 and that TGF-beta 1 can be depleted selectively from the periductal ECM at focal growth points. A different type of growth point, the alveolar (secretory) buds, was also investigated. These buds are known not to be inhibited by exogenous TGF-beta 1, and we found them not to be associated with changes in ECM-bound TGF-beta 1. Our results support the concept that the periductal ECM acts as a reservoir for TGF-beta 1 that functions to maintain an open pattern of mammary branching by inhibiting ductal, but not alveolar, bud formation.     

Inhibition of mouse mammary ductal morphogenesis and down-regulation of the EGF receptor by epidermal growth factor

EGF, initially demonstrated to be a potent mitogen for a variety of cell types, has more recently been shown to inhibit proliferation of several cell lines. Few studies, however, have addressed the effects of EGF on growth and morphogenesis of tissues in vivo, particularly with regard to EGF as a possible inhibitor. We now demonstrate that EGF treatment of vigorously growing mammary ducts, administered directly to the glands by slow release plastic implants, inhibited normal ductal growth. Inhibition was restricted to the region around the implant and untreated glands in the same animal were normal, indicating direct effects of EGF. EGF-treated end buds were smaller and demonstrated reduced levels of DNA synthesis, although remnants of a stem (cap) cell layer persisted. Full inhibition of growth occurred within 3 days of implantation and required extended exposure to EGF, since treatment of 5 hr or less had no effect on ductal growth. At the lower inhibitory doses tested, growth resumed within 8 days, indicating reversibility of inhibition. No lobuloalveolar or hyperplastic response was seen. 125I-EGF autoradiography revealed that ductal growth inhibition was preceded by the disappearance of EGF receptors located in the cap cell layer of the end bud epithelium and in stromal cells adjacent to the buds. These results, in conjunction with our previous evidence demonstrating the growth-stimulatory effect by EGF on nonproliferating mammary ducts, suggest a growth regulatory role for EGF in mouse mammary ductal morphogenesis.     

Similar growth pattern of mouse mammary epithelium cultivated in collagen matrix in vivo and in vitro

Mouse mammary ductal cells cultured in type I collagen gels give rise to three-dimensional multicellular outgrowths consisting of thin spikes which are often branched, and which may have pointed or blunt ends. The significance of these spikes to normal ductal morphogenesis has been unclear, since identical structures are not known to occur in vivo; conversely, it has not been possible to maintain in gel culture the highly structured end buds which are characteristic of ductal elongation in the animal. In order to evaluate whether the pattern of radiating spikes observed in collagen gel cultures results from chemical or physical peculiarities of the culture environment, a small volume of unpolymerized type I collagen solution was injected into mammary gland-free fat pads of young adult mice. After the bubble of collagen had polymerized, an implant of mammary ductal epithelium was introduced into the center of the gel. Histological examination of the implants after 3 to 6 days of growth revealed numerous small epithelial spikes, similar to those observed in gel culture, extending into the fibrous matrix. The early stages of regeneration of mammary implants placed in gland-free fat pads were then examined without the addition of exogenous collagen. In cases where the epithelium happened to contact a fibrous region of the fatty stroma, spikes were also seen to form in these natural collagenous substrates. Whether or not exogenous collagen was used, normal end buds were formed only when epithelial spikes contacted adipocytes. It was concluded that the three-dimensional pattern of radiating tubules in collagen gels in vitro is not merely an artifact of culture, but has a counterpart in vivo whereever regenerating mammary epithelium is surrounded by fibrous stroma. A model is presented in which the pattern of epithelial outgrowth is determined by the physical characteristics of the surrounding stroma; in collagen matrix a comparatively primitive and unspecialized type of morphogenesis occurs which may not require the participation of stromal cells. In contrast, epithelial-adipocyte interactions appear to be necessary for the formation of end buds and subsequent morphogenesis of fully structured mammary ducts.     

Timp-1 is important for epithelial proliferation and branching morphogenesis during mouse mammary development.

The dynamic process of mammary ductal morphogenesis depends on regulated epithelial proliferation and extracellular matrix (ECM) turnover. Epithelial cell-matrix contact closely dictates epithelial proliferation, differentiation, and survival. Despite the fact that tissue inhibitors of metalloproteinases (Timps) regulate ECM turnover, their function in mammary morphogenesis is unknown. We have delineated the spatiotemporal expression of all Timps (Timp-1 to Timp-4) during discrete phases of murine mammary development. Timp mRNAs were abundant in mammary tissue, each displaying differential expression patterns with predominant localization in luminal epithelial cells. Timp-1 mRNA was unique in that its expression was limited to the stage at which epithelial proliferation was high. To assess whether Timp-1 promotes or inhibits epithelial cell proliferation we manipulated mammary Timp-1 levels, genetically and biochemically. Down-regulation of epithelial-derived Timp-1 in transgenic mice, by mouse mammary tumor virus promoter-directed Timp-1 antisense RNA expression, led to augmented ductal expansion and increased number of ducts (P < 0.004). In these transgenics the integrity of basement membrane surrounding epithelial ducts, as visualized by laminin-specific immunostaining, was breached. In contrast to these mice, ductal expansion was markedly attenuated in the proximity of implanted recombinant Timp-1-releasing pellets (rTIMP-1), without an increase in basement membrane deposition around migrating terminal end buds. Epithelial proliferation and apoptosis were measured to determine the basis of altered ductal expansion. Luminal epithelial proliferation was increased by 55% (P < 0.02) in Timp-1-reduced transgenic mammary tissue and, conversely, decreased by 38% (P < 0.02) in terminal end buds by implanted rTIMP-1. Epithelial apoptosis was minimal and remained unaffected by Timp-1 manipulations. We conclude that Timps have an integral function in mammary morphogenesis and that Timp-1 regulates mammary epithelial proliferation in vivo, at least in part by maintaining basement membrane integrity.     

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.     

Laminin and beta1 integrins are crucial for normal mammary gland development in the mouse.

We have examined the role of integrin-extracellular matrix interactions in the morphogenesis of ductal structures in vivo using the developing mouse mammary gland as a model. At puberty, ductal growth from terminal end buds results in an arborescent network that eventually fills the gland, whereupon the buds shrink in size and become mitotically inactive. End buds are surrounded by a basement membrane, which we show contains laminin-1 and collagen IV. To address the role of cell-matrix interactions in gland development, pellets containing function-perturbing anti-beta1 integrin, anti-alpha6 integrin, and anti-laminin antibodies respectively were implanted into mammary glands at puberty. Blocking beta1 integrins dramatically reduced both the number of end buds per gland and the extent of the mammary ductal network, compared with controls. These effects were specific to the end buds since the rest of the gland architecture remained intact. Reduced development was still apparent after 6 days, but end buds subsequently reappeared, indicating that the inhibition of beta1 integrins was reversible. Similar results were obtained with anti-laminin antibodies. In contrast, no effect on morphogenesis in vivo was seen with anti-alpha6 integrin antibody, suggesting that alpha6 is not the important partner for beta1 in this system. The studies with beta1 integrin were confirmed in a culture model of ductal morphogenesis, where we show that hepatocyte growth factor (HGF)-induced tubulogenesis is dependent on functional beta1 integrins. Thus integrins and HGF cooperate to regulate ductal morphogenesis. We propose that both laminin and beta1 integrins are required to permit cellular traction through the stromal matrix and are therefore essential for maintaining end bud structure and function in normal pubertal mammary gland development.     

Mammary ductal elongation: differentiation of myoepithelium and basal lamina during branching morphogenesis

Elongation of mammary ducts in the immature mouse takes place as a result of rapid growth in end buds. These structures proliferate at the apex of elongating ducts and are responsible for penetration of the surrounding adipose stroma; by turning and branching, end buds give rise to the characteristic open pattern of the mammary ductal tree. We have used a variety of techniques to determine the cellular and structural basis for certain of these end bud activities, and now report the following. (1) The end bud tip is covered with a monolayer of epithelium, the "cap cells," which are characterized by a relative lack of intercellular junctions and other specialized features. (2) The cap cell layer extends along the end bud flank and neck regions where it is continuous with the myoepithelium which surrounds the subtending mature duct. A linear sequence of differentiative changes occur in the cap cells in this region as they progressively alter in shape and accumulate the cytological features of mature myoepithelium. Cap cells may therefore be defined as a stem cell population providing new myoepithelial cells for ductal morphogenesis and elongation. (3) Differentiation of cap cells into myoepithelium is associated with conspicuous changes in the basal lamina. At the tip, cap cells form a 104-nm lamina similar to that described in expanding mammary alveoli and in embryonic tissues. Along the end bud flanks the basal lamina is raised from the cell surface and extensively folded, resulting in a greatly thickened lamina, measuring as much as 1.4 microns. At the surface of the subtending ducts the lamina becomes structurally simplified and resembles that at the tip, but has a significantly greater thickness, averaging 130 nm. (4) The codifferentiation of myoepithelium and its basement membrane is associated with changes in the surrounding stroma. Undifferentiated mesenchymal-like cells attach to the surface of the basal lamina in the midportion of the end buds and become increasingly numerous in the neck region, forming a monolayer over the myoepithelial basal lamina. These stromal cells progressively differentiated into fibrocytes which participate in collagen fibrillogenesis and give rise to the fibrous components of the stroma surrounding the mature duct.     

Analysis In Vitro of Capacity of Fetal Fat Pad to Support Mammary Gland Embryogenesis

Fourteen-day fetal mammary fat pad precursor tissue (FP) has the capacity to support various fetal epithelia allowing them to accomplish their characteristic development in vivo , without their own mesenchyme (1). This capacity decreases with age of fetal fat pad and is lost postnatally. To analyse the molecular mechanism of such interaction, a method for in vitro duplication of organogenesis is necessary. In the present paper, a co-culture system of fetal epithelium with prospective mammary fat pad is described. The explanted mammary epithelium started budding, then grew out forming branched mammary ducts with end buds. Ultrastructurally, the developing ductal structures exhibited the typical mammary gland morphogenesis.
³H-Thymidine incorportion assessed by autoradiography showed that the mammary gland morphogenesis in vitro was due to the proliferation of epithelial cells, not merely to a change of the shape of the epithelium. This supportive capacity of 14-day FP also decreased with aging; explanted mammary epithelium did not grow into 17-day FP. When insoluble, non-living biomatrix was used in place of living FP the epithelium grew into the matrix but the resulting structures lacked characteristic morphology of epithelium on living fetal FP. The difference of capacity between 14-day and 17-day tissues was also lost.     

TGF-beta, c-Cbl, and PDGFR-alpha the in mammary stroma

Transforming growth factor-beta (TGF-beta) is thought to regulate ductal and lobuloalveolar development as well as involution in the mammary gland. In an attempt to understand the role TGF-beta plays during normal mammary gland development, and ultimately cancer, we previously generated transgenic mice that express a dominant-negative TGF-beta type II receptor under control of the metallothionine promoter (MT-DNIIR). Upon stimulation with zinc sulfate, the transgene was expressed in the mammary stroma and resulted in an increase in ductal side branching. In this study, mammary gland transplantation experiments confirm that the increase in side branching observed was due to DNIIR activity in the stroma. Development during puberty through the end buds was also accelerated. Cbl is a multifunctional intracellular adaptor protein that regulates receptor tyrosine kinase ubiquitination and downregulation. Mice with a targeted disruption of the c-Cbl gene displayed increased side branching similar to that observed in MT-DNIIR mice; however, end bud development during puberty was normal. Transplantation experiments showed that the mammary stroma was responsible for the increased side branching observed in Cbl-null mice. Cbl expression was reduced in mammary glands from DNIIR mice compared to controls and TGF-beta stimulated expression of Cbl in cultures of primary mammary fibroblasts. In addition, both TGF-beta and Cbl regulated platelet-derived growth factor receptor-alpha (PDGFR alpha) expression in vivo and in isolated mammary fibroblasts. The hypothesis that TGF-beta mediates the levels of PDGFR alpha protein via regulation of c-Cbl was tested. We conclude that TGF-beta regulates PDGFR alpha in the mammary stroma via a c-Cbl-independent mechanism. Finally, the effects of PDGF-AA on branching were determined. Treatment in vivo with PDGF-AA did not affect branching making a functional interaction between TGF-beta and PDGF unlikely.     

Colony stimulating factor-1 is required to recruit macrophages into the mammary gland to facilitate mammary ductal outgrowth

Mammary gland development initiates postnatally with the development of terminal end buds (TEBs) at the end of the rudimentary ducts. These grow out through the fat pad and bifurcate to lay down the rudimentary ductal tree. At the initiation of their development, TEBs recruit to their surrounding stroma a substantial population of macrophages. Using mice homozygous for a null mutation in the gene for the macrophage growth factor, colony stimulating factor-1 (CSF-1), that are severely depleted in macrophages, we demonstrated that CSF-1-regulated macrophages are required for normal branching morphogenesis in the mammary gland. However, these mice have a pleiotropic phenotype as a result of the generalized macrophage deficiency. To test that the effect of the mutation observed in the mammary gland was organ-autonomous, we developed a tetracycline-binary system whereby CSF-1 was specifically expressed in the mammary epithelium under the regulation of the MMTV-promoter. This restored mammary macrophage populations but not those in other tissues and corrected the branching morphogenesis defect. Inhibition of CSF-1 expression by tetracycline treatment for varying periods suggested that CSF-1-regulated macrophages are required throughout early mammary gland development. These data show that macrophages acting locally are required for branching morphogenesis of the mammary gland.     

Targeting expression of a transforming growth factor beta 1 transgene to the pregnant mammary gland inhibits alveolar development and lactation.

Transforming growth factor-beta 1 (TGF-beta 1) possesses highly potent, diverse and often opposing cell-specific activities, and has been implicated in the regulation of a variety of physiologic and developmental processes. To determine the effects of in vivo overexpression of TGF-beta 1 on mammary gland function, transgenic mice were generated harboring a fusion gene consisting of the porcine TGF-beta 1 cDNA placed under the control of regulatory elements of the pregnancy-responsive mouse whey-acidic protein (WAP) gene. Females from two of four transgenic lines were unable to lactate due to inhibition of the formation of lobuloalveolar structures and suppression of production of endogenous milk protein. In contrast, ductal development of the mammary glands was not overtly impaired. There was a complete concordance in transgenic mice between manifestation of the lactation-deficient phenotype and expression of RNA from the WAP/TGF-beta 1 transgene, which was present at low levels in the virgin gland, but was greatly induced at mid-pregnancy. TGF-beta 1 was localized to numerous alveoli and to the periductal extracellular matrix in the mammary gland of transgenic females late in pregnancy by immunohistochemical analysis. Glands reconstituted from cultured transgenic mammary epithelial cells duplicated the inhibition of lobuloalveolar development observed in situ in the mammary glands of pregnant transgenic mice. Results from this transgenic model strongly support the hypothesis that TGF-beta 1 plays an important in vivo role in regulating the development and function of the mammary gland.     

Postnatal mammary gland development requires macrophages and eosinophils

Interactions between mammary epithelial and mesenchymal cells including fibroblasts and adipocytes are crucial for the proper postnatal development of the mammary ductal tree. Often overlooked, however, are the migrant cells that enter tissues at different stages of development. In this paper we identify two such cell types, macrophages and eosinophils, that are recruited around the growing terminal end buds (TEBs) during postnatal development. An important role for leukocytes in mammary gland ductal outgrowth is first demonstrated by depleting mice of leukocytes using sub-lethal (gamma)-irradiation. This treatment results in a curtailment of mammary gland epithelial development that is completely rescued by bone-marrow transplantation, concurrent with a restoration of macrophage and eosinophil recruitment around the growing ducts. Using mice homozygous for a null mutation in the gene for CSF1 (Csfm(op)/Csfm(op)), the major growth factor for macrophages, we show that in the absence of CSF1, the population of macrophages in mammary glands is depleted. In this mutant, the formation of TEBs, their outgrowth into the fat pad and the branching of the resultant ducts are all impaired. Similarly, by using mice homozygous for a null mutation in the gene for eotaxin, a major chemokine for local recruitment of eosinophils in tissue, we identify eotaxin as the necessary and sufficient chemokine responsible for eosinophil recruitment around TEBs. In the absence of eosinophils, mammary gland branch formation and to a lesser extent TEB formation are reduced. Our data show that CSF1-regulated macrophages, in collaboration with eotaxin-regulated eosinophils, have essential and complementary functions in regulating the branching morphogenesis of the mammary gland.     

In utero exposure to bisphenol A alters the development and tissue organization of the mouse mammary gland

Exposure to estrogens throughout a woman's life, including the period of intrauterine development, is a risk factor for the development of breast cancer. The increased incidence of breast cancer noted during the last 50 years may have been caused, in part, by exposure of women to estrogen-mimicking chemicals that are released into the environment. Here, we investigated the effects of fetal exposure to one such chemical, bisphenol A (BPA), on development of the mammary gland. CD-1 mice were exposed in utero to low, presumably environmentally relevant doses of BPA (25 and 250 microg/kg body weight), and their mammary glands were assessed at 10 days, 1 mo, and 6 mo of age. Mammary glands of BPA-exposed mice showed differences in the rate of ductal migration into the stroma at 1 mo of age and a significant increase in the percentage of ducts, terminal ducts, terminal end buds, and alveolar buds at 6 mo of age. The percentage of cells that incorporated BrdU was significantly decreased within the epithelium at 10 days of age and increased within the stroma at 6 mo of age. These changes in histoarchitecture, coupled with an increased presence of secretory product within alveoli, resemble those of early pregnancy, and they suggest a disruption of the hypothalamic-pituitary-ovarian axis and/or misexpression of developmental genes. The altered relationship in DNA synthesis between the epithelium and stroma and the increase in terminal ducts and terminal end buds are striking, because these changes are associated with carcinogenesis in both rodents and humans.     

Mast cells contribute to the stromal microenvironment in mammary gland branching morphogenesis

The stromal microenvironment regulates mammary gland branching morphogenesis. We have observed that mast cells are present in the mammary gland throughout its postnatal development and, in particular, are found around the terminal end buds and ductal epithelium of the pubertal gland. Mast cells contribute to allergy, inflammatory diseases, and cancer development but have not been implicated in normal development. Genetic and pharmacological disruption of mast cell function in the mammary gland revealed that mast cells are involved in rapid proliferation and normal duct branching during puberty, and this effect is independent of macrophage recruitment, which also regulates mammary gland development. For mast cells to exert their effects on normal morphogenesis required activation of their serine proteases and degranulation. Our observations reveal a novel role for mast cells during normal pubertal development in the mammary gland.     

Members of the fatty acid binding protein family are differentiation factors for the mammary gland

Mammary gland development is controlled by systemic hormones and by growth factors that might complement or mediate hormonal action. Peptides that locally signal growth cessation and stimulate differentiation of the developing epithelium have not been described. Here, we report that recombinant and wild-type forms of mammary-derived growth inhibitor (MDGI) and heart-fatty acid binding protein (FABP), which belong to the FABP family, specifically inhibit growth of normal mouse mammary epithelial cells (MEC), while growth of stromal cells is not suppressed. In mammary gland organ culture, inhibition of ductal growth is associated with the appearance of bulbous alveolar end buds and formation of fully developed lobuloalveolar structures. In parallel, MDGI stimulates its own expression and promotes milk protein synthesis. Selective inhibition of endogenous MDGI expression in MEC by antisense phosphorothioate oligonucleotides suppresses appearance of alveolar end buds and lowers the beta-casein level in organ cultures. Furthermore, MDGI suppresses the mitogenic effects of epidermal growth factor, and epidermal growth factor antagonizes the activities of MDGI. Finally, the regulatory properties of MDGI can be fully mimicked by an 11-amino acid sequence, represented in the COOH terminus of MDGI and a subfamily of structurally related FABPs. This peptide does not bind fatty acids. To our knowledge, this is the first report about a growth inhibitor promoting mammary gland differentiation.     

Wnt5a is required for proper mammary gland development and TGF-beta-mediated inhibition of ductal growth

Transforming growth factor-beta (TGF-beta) plays an essential role in growth and patterning of the mammary gland, and alterations in its signaling have been shown to illicit biphasic effects on tumor progression and metastasis. We demonstrate in mice that TGF-beta (Tgfbeta) regulates the expression of a non-canonical signaling member of the wingless-related protein family, Wnt5a. Loss of Wnt5a expression has been associated with poor prognosis in breast cancer patients; however, data are lacking with regard to a functional role for Wnt5a in mammary gland development. We show that Wnt5a is capable of inhibiting ductal extension and lateral branching in the mammary gland. Furthermore, Wnt5a(-/-) mammary tissue exhibits an accelerated developmental capacity compared with wild-type tissue, marked by larger terminal end buds, rapid ductal elongation, increased lateral branching and increased proliferation. Additionally, dominant-negative interference of TGF-beta signaling impacts not only the expression of Wnt5a, but also the phosphorylation of discoidin domain receptor 1 (Ddr1), a receptor for collagen and downstream target of Wnt5a implicated in cell adhesion/migration. Lastly, we show that Wnt5a is required for TGF-beta-mediated inhibition of ductal extension in vivo and branching in culture. This study is the first to show a requirement for Wnt5a in normal mammary development and its functional connection to TGF-beta.