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.     

Immunophenotypic properties and estrogen dependency of budding cell structures in the developing mouse mammary gland

Abstract. The initial phase of growth of the parenchymal component of the mouse mammary gland is ductal clongation, which is mainly accomplished by proliferating cells in a specialized structure termed end bud. End buds are composed of multiple layers of epithelial cells (so called body cells) which are capped by a single layer of morphologically unique cells termed cap cells.
We sought to examine the interrelationship between cap cells and other epithelial cell subclasses using a variety of antibodies to different keratin proteins and also antibodies to vimentin, actin and collagen IV. An extensive immunohistochemical characterization of the epithelial components of the developing and differentiating mammary gland demonstrated that cap cells were devoid of any immunohistochemically - detectable keratins but were positive for collagen IV. In contrast, the majority of cells in the end bud along with the luminal epithelial and myoepithelial cells were keratin positive. The body cells of the end bud were the only cells which were positive for antibody to keratin 6, a keratin which previously has been reported to be expressed in proliferating mammary epithelial cells. In addition, estrogen receptor was localized only to epithelial cells of ducts, alvcoli and body cells of end buds, but not to cap cells or myoepithelial cells. We interpret these results to suggest that cap cells are not totpotent stem cells but rather cells specialized in paving the way for ductal elongation as well as serving as precursors to myoepithelial cells.     

Epithelium-dependent extracellular matrix synthesis in transforming growth factor-beta 1-growth-inhibited mouse mammary gland

Exogenous transforming growth factor beta (TGF-beta 1) was shown in earlier studies to reversibly inhibit mouse mammary ductal growth. Using small plastic implants to treat regions of developing mammary glands in situ, we now report that TGF-beta 1 growth inhibition is associated with an ectopic accumulation of type I collagen messenger RNA and protein, as well as the glycosaminoglycan, chondroitin sulfate. Both macromolecules are normal components of the ductal extracellular matrix, which, under the influence of exogenous TGF-beta 1, became unusually concentrated immediately adjacent to the epithelial cells at the tip of the ductal growth points, the end buds. Stimulation of extracellular matrix was confined to aggregations of connective tissue cells around affected end buds and was not present around the TGF-beta 1 implants themselves, indicating that the matrix effect was epithelium dependent. Ectopic matrix synthesis was specific for TGF-beta 1 insofar as it was absent at ducts treated with other growth inhibitors, or at ducts undergoing normal involution in response to endogenous regulatory processes. These findings are consistent with the matrix-stimulating properties of TGF-beta 1 reported for other systems, but differ in their strict dependence upon epithelium. A possible role for endogenous TGF-beta 1 in modulating a mammary epithelium-stroma interaction is suggested.     

P190A RhoGAP is required for mammary gland development

P190A and p190B Rho GTPase activating proteins (GAPs) are essential genes that have distinct, but overlapping roles in the developing nervous system. Previous studies from our laboratory demonstrated that p190B is required for mammary gland morphogenesis, and we hypothesized that p190A might have a distinct role in the developing mammary gland. To test this hypothesis, we examined mammary gland development in p190A-deficient mice. P190A expression was detected by in situ hybridization in the developing E14.5 day embryonic mammary bud and within the ducts, terminal end buds (TEBs), and surrounding stroma of the developing virgin mammary gland. In contrast to previous results with p190B, examination of p190A heterozygous mammary glands demonstrated that p190A deficiency disrupted TEB morphology, but did not significantly delay ductal outgrowth indicating haploinsufficiency for TEB development. To examine the effects of homozygous deletion of p190A, embryonic mammary buds were rescued by transplantation into the cleared fat pads of SCID/Beige mice. Complete loss of p190A function inhibited ductal outgrowth in comparison to wildtype transplants (51% vs. 94% fat pad filled). In addition, the transplantation take rate of p190A deficient whole gland transplants from E18.5 embryos was significantly reduced compared to wildtype transplants (31% vs. 90%, respectively). These results suggest that p190A function in both the epithelium and stroma is required for mammary gland development. Immunostaining for p63 demonstrated that the myoepithelial cell layer is disrupted in the p190A deficient glands, which may result from the defective cell adhesion between the cap and body cell layers detected in the TEBs. The number of estrogen- and progesterone receptor-positive cells, as well as the expression levels of these receptors was increased in p190A deficient outgrowths. These data suggest that p190A is required in both the epithelial and stromal compartments for ductal outgrowth and that it may play a role in mammary epithelial cell differentiation.     

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.     

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.     

Slit2 and netrin 1 act synergistically as adhesive cues to generate tubular bi-layers during ductal morphogenesis

Development of many organs, including the mammary gland, involves ductal morphogenesis. Mammary ducts are bi-layered tubular structures comprising an outer layer of cap/myoepithelial cells (MECs) and an inner layer of luminal epithelial cells (LECs). Slit2 is expressed by cells in both layers, with secreted SLIT2 broadly distributed throughout the epithelial compartment. By contrast, Robo1 is expressed specifically by cap/MECs. Loss-of-function mutations in Slit2 and Robo1 yield similar phenotypes, characterized by disorganized end buds (EBs) reminiscent of those present in Ntn1(-/-) glands, suggesting that SLIT2 and NTN1 function in concert during mammary development. Analysis of Slit2(-/-);Ntn1(-/-) glands demonstrates an enhanced phenotype that extends through the ducts and is characterized by separated cell layers and occluded lumens. Aggregation assays show that Slit2(-/-);Ntn1(-/-) cells, in contrast to wild-type cells, do not form bi-layered organoids, a defect rescued by addition of SLIT2. NTN1 has no effect alone, but synergistically enhances this rescue. Thus, our data establish a novel role for SLIT2 as an adhesive cue, acting in parallel with NTN1 to generate cell boundaries along ducts during bi-layered tube formation.     

Effects of epidermal growth factor,estrogen, and progestin on DNA synthesis in mammary cells in vivo are determined by the developmental state of the gland

Estrogen (E), progesterone (P), and epidermal growth factor (EGF) are involved in the growth and development of the normal mammary gland. While studies have been carried out to investigate the in vivo effects of EGF in the immature mammary gland, nothing is known about the growth effects of EGF or its potential interactions with E and/or P in the adult mammary gland. The present studies were undertaken to investigate the effects of EGF, E, and P on mammary cell proliferation in immature, peripubertal vs. adult, sexually mature mice. We have found that EGF promotes epithelial and stromal cell proliferation in both the immature and adult mammary glands. In the immature gland, the end bud epithelium is most responsive to the proliferative effects of EGF and there is no apparent interaction between EGF, E, and/or P. In contrast, in the mature gland EGF adds to the proliferative effects of E+P in the ductal epithelium resulting in more extensive ductal sidebranching. Thus these results demonstrate that the developmental state of the mammary gland determines the nature and extent of the interactions between EGF, E, and P in growth and development. © 1993 Wiley-Liss, Inc.     

Cellular composition and organization of ductal buds in developing rat mammary glands: evidence for morphological intermediates between epithelial and myoepithelial cells

In the developing rat mammary gland, terminal end buds (TEBs), lateral buds and alveolar buds represent the major sites of morphogenetic activity and cellular differentiation. The morphology and cellular composition of these buds from 20-to 22-day-old rats and cycling rats have been studied by immunocytochemical and electron microscopic techniques. The mammary buds are composed of a heterogeneous collection of cells including epithelial and myoepithelial cells, irregular loosely adherent cells, and occasional large clear cells. The irregular, loosely packed cells or cap cells are mainly situated around the periphery of the TEBs and lateral buds. "Chains" of irregularly shaped cells also extend from the peripheral cap cell layer to the center of the TEB; and, where they converge on lumina, they display microvilli and junctional complexes. At the tips of the end buds, the cap cells are of undifferentiated appearance; however, similar cells situated toward the subtending mammary ducts show a gradation in ultrastructure to that of myoepithelial cells. This change is accompanied by an increase in the amounts of immunoreactive myosin and keratin seen within the cells and a 200-fold increase in the thickness of the basement membrane. In contrast, the peripheral cells of the alveolar buds are more closely packed, contain a greater number of myofilaments, and show increased staining with antisera to myosin. We suggest that the undifferentiated cap cells do not represent a discrete cell type, since they show transitional forms to myoepithelial cells within the subtending mammary ducts, and that the tendency toward the myoepithelial phenotype is predominant in the more differentiated structures, the alveolar buds.     

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.     

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.     

Growth of mouse mammary gland end buds cultured in a collagen gel matrix

End buds are the growing terminal structures of the ducts in the mammary gland. They are made up of undifferentiated epithelial cells that, under the hormonal milieu of adult and pregnant females, give rise to the ducts and alveoli of the mature gland. The end buds are of interest in understanding the developmental biology of the gland. They are also likely targets for many mammary carcinogens and, therefore, of interest in understanding the biology of mammary cancer. A method was developed for isolating end buds as a pure subpopulation from collagenase and hyaluronidase digested 4-week-old C57BL/Crgl mouse mammary glands. They were cultured within a rat tail collagen gel matrix for 3 weeks and fed with media containing various combinations of sera, hormones and growth-promoting factors. Growth in response to the different media was measured by photographing individual end bud colonies over time using dark field illumination. The growth of the individual colonies was quantitated using a computer-assisted photodensitometry method of determining the colonies' mass. The end buds showed the greatest growth response (greater than 20-fold increase in 3 weeks with a minimum doubling time of 48 h) to medium supplemented with 50% equine serum and cholera toxin or with less serum (15%) and epidermal growth factor (EGF), hormones and cholera toxin. Initially, the colonies grew logarithmically, then slowed as the colony size increased. This was due, in part, to the cessation of growth by the cells in the center. This end bud culture system differs from that of dissociated mouse mammary cells in a number of ways which are discussed.     

The use of thioesterase II as a rat mammary epithelial cell-specific marker

The avidin-biotin-peroxidase complex immunoperoxidase technique was employed to determine the intercellular distribution of thioesterase II in rat mammary glands. This enzyme is responsible for shifting the product specificity of the fatty-acid synthetase enzyme complex from long to medium chain fatty acids. Thioesterase II was found exclusively in the cells lining the lumen of the ductal and alveolar structures in glands from mature virgin (150 days old) and pregnant rats. The ductal cell staining intensity was considerably less than that of the alveolar cells in the mature virgin rat glands. No immunoreactive thioesterase II was found in the stromal, adipose, vascular, or myoepithelial components of the gland in the developmental stages examined. In the glands from immature virgin rats (40-45 days old) thioesterase II was again found only in the epithelial cells lining the lumen of the ductal and end-bud structures although this layer was usually more than one cell thick. Quantitative determination of thioesterase II activity in cytosol preparations revealed similar levels in mammary fragments from enzymatically-dissociated glands obtained from mature virgins and in end buds derived from immature virgins, but somewhat higher levels in mammary structures derived from late-pregnant animals. These immunohistological and biochemical results demonstrate thioesterase II's usefulness as a mammary epithelial cell-specific marker.     

Vitamin D(3) receptor ablation alters mammary gland morphogenesis

Postnatal mammary gland morphogenesis is achieved through coordination of signaling networks in both the epithelial and stromal cells of the developing gland. While the major proliferative hormones driving pubertal mammary gland development are estrogen and progesterone, studies in transgenic and knockout mice have successfully identified other steroid and peptide hormones that impact on mammary gland development. The vitamin D(3) receptor (VDR), whose ligand 1,25-dihydroxyvitamin D(3) is the biologically active form of vitamin D(3), has been implicated in control of differentiation, cell cycle and apoptosis of mammary cells in culture, but little is known about the physiological relevance of the vitamin D(3) endocrine system in the developing gland. In these studies, we report the expression of the VDR in epithelial cells of the terminal end bud and subtending ducts, in stromal cells and in a subset of lymphocytes within the lymph node. In the terminal end bud, a distinct gradient of VDR expression is observed, with weak VDR staining in proliferative populations and strong VDR staining in differentiated populations. The role of the VDR in ductal morphogenesis was examined in Vdr knockout mice fed high dietary Ca(2+) which normalizes fertility, serum estrogen and neonatal growth. Our results indicate that mammary glands from virgin Vdr knockout mice are heavier and exhibit enhanced growth, as evidenced by higher numbers of terminal end buds, greater ductal outgrowth and enhanced secondary branch points, compared with glands from age- and weight-matched wild-type mice. In addition, glands from Vdr knockout mice exhibit enhanced growth in response to exogenous estrogen and progesterone, both in vivo and in organ culture, compared with glands from wild-type mice. Our data provide the first in vivo evidence that 1,25-dihydroxyvitamin D(3) and the VDR impact on ductal elongation and branching morphogenesis during pubertal development of the mammary gland. Collectively, these results suggest that the vitamin D(3) signaling pathway participates in negative growth regulation of the mammary gland.     

Generation of lobuloalveolar development from isolated rat mammary ducts and end buds

Implantation of excised bud-free ductal fragments (DUCTS), terminal end buds (TEBs), or alveolar buds (ABs) from virgin mammary glands of Wistar-Furth rats into interscapular fat pads of syngeneic female rats produces, after 16 weeks, complete ductal outgrowths including TEBs and ABs. Treatment of the recipient rats with perphenazine for 1 day or mating them after 12 weeks and then isolating the resultant outgrowths after 16 weeks produces significantly larger outgrowths than those from untreated hosts. The outgrowths consist of distended ducts and lobules or distended ducts and alveoli, respectively. Histochemical and immunocytochemical staining of the outgrowths with reagents that depict epithelial, myoepithelial, and lactating alveolar cells (peanut lectin alone, monoclonal and polyclonal antibodies to rat caseins) indicate similar cell compositions and arrangements for all outgrowths irrespective of their source; these are also similar to the mammary glands of the perphenazine-stimulated or lactating hosts. There is one major difference: the degree of staining of peanut lectin alone and the anti-caseins is greater for outgrowths produced by the ABs and TEBs than for those produced by the DUCTs. DUCT implants left for 1 year after cessation of lactation of the hosts are still stained appreciably by peanut lectin alone and by the anti-caseins, particularly the luminal secretions. Therefore, the complete morphogenetic and cell differentiating ability for generating mammary glands is present in bud-free ducts, but this ability can be enhanced in TEBs/ABs or abnormally expressed at ectopic sites.     

BMP4 and PTHrP interact to stimulate ductal outgrowth during embryonic mammary development and to inhibit hair follicle induction

The mammary glands develop initially as buds arising from the ventral embryonic epidermis. Recent work has shed light on signaling pathways leading to the patterning and formation of the mammary placodes and buds in mouse embryos. Relatively little is known of the signaling pathways that initiate branching morphogenesis and the formation of the ducts from the embryonic buds. Previous studies have shown that parathyroid hormone-related protein (PTHrP; also known as parathyroid hormone-like peptide, Pthlh) is produced by mammary epithelial cells and acts on surrounding mesenchymal cells to promote their differentiation into a mammary-specific dense mesenchyme. As a result of PTHrP signaling, the mammary mesenchyme supports mammary epithelial cell fate, initiates ductal development and patterns the overlying nipple sheath. In this report, we demonstrate that PTHrP acts, in part, by sensitizing mesenchymal cells to BMP signaling. PTHrP upregulates BMP receptor 1A expression in the mammary mesenchyme, enabling it to respond to BMP4, which is expressed within mesenchymal cells underlying the ventral epidermis during mammary bud formation. We demonstrate that BMP signaling is important for outgrowth of normal mammary buds and that BMP4 can rescue outgrowth of PTHrP(-/-) mammary buds. In addition, the combination of PTHrP and BMP signaling is responsible for upregulating Msx2 gene expression within the mammary mesenchyme, and disruption of the Msx2 gene rescues the induction of hair follicles on the ventral surface of mice overexpressing PTHrP in keratinocytes (K14-PTHrP). Our data suggest that PTHrP signaling sensitizes the mammary mesenchyme to the actions of BMP4, triggering outgrowth of the mammary buds and inducing MSX2 expression, which, in turn, leads to lateral inhibition of hair follicle formation within the developing nipple sheath.     

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.