Histochemical organization and cellular composition of ductal buds in developing human breast: evidence of cytochemical intermediates between epithelial and myoepithelial cells

In developing human breast, terminal end buds (TEBs), lateral buds (LBs), and lobules of three to five alveolar buds (ABs) predominate in prepubertal females, whereas lobules of ABs and lobules of up to 60 ductules predominate in pubertal females. The appearance of clefts in TEBs and LBs suggests that they are precursors of ABs. In histological sections the ductal buds are composed of a heterogeneous collection of cells that include cortical and peripheral cells. The cortical cells can line small lumina in TEBs/LBs, whereas the peripheral cells which cap their distal tips are more irregular and loosely packed. Monoclonal antibodies (MAb) to epithelial milk-fat globule membranes and antiserum to epithelial membrane antigen immunocytochemically stain the cortical cells, particularly where such cells line lumina, and weakly stain the peripheral cap cells. Similar histochemical staining patterns are observed in desialylated sections with peanut lectin. Antiserum and MAb to smooth muscle actin moderately stain the peripheral cap cells, and this staining increases the closer the peripheral cells become to the myoepithelial cells of the subtending duct. Similar but weaker staining patterns are observed with antibodies to vimentin. Keratin MAb PKK2 and LP34, which stain myoepithelial cells in preference to epithelial cells in main ducts, as well as MAb to epithelium-specific keratin 18, all stain many of the cortical/luminal cells in buds and lobules of developing breast; the peripheral cap cells are relatively unstained. It is suggested that the undifferentiated peripheral cap cells show transitional forms both to the cortical epithelial cells that eventually line the lumina and to the myoepithelial cells of the subtending duct.     

Use of peanut lectin and rat mammary stem cell lines to identify a cellular differentiation pathway for the alveolar cell in the rat mammary gland.

The presence of the carbohydrate receptor for PNL has been used to identify the previously described morphological types of epithelial cell produced as the stem cell line rat mammary 25 (Rama 25) differentiates to casein secretory alveolar-like cells in vitro. Thus when cultures of the epithelial stem cell line Rama 25 are treated with neuraminidase, fluorescently-conjugated PNL fails to stain cuboidal cells, stains weakly grey cells, and stains strongly the surface of dark cells. When superconfluent cultures of Rama 25 are treated with dimethyl sulfoxide or retinoic acid and prolactin, estradiol, hydrocortisone, and insulin to induce differentiation to alveolar cells, PNL stains strongly the untreated surfaces of droplet cells and casein-secreting vacuolated cells. PNL-staining of the derivative cell lines with truncated cellular pathways, and quantitative binding of [125I]-labeled PNL to the cultured cells are consistent with this cellular staining pattern. The presence of the carbohydrate receptor for peanut lectin (PNL) has also been used to identify specific epithelial cell types in different mammary structures of the developing rat mammary gland, as they differentiate to casein secretory alveolar cells in vivo. Thus when different structures of the developing rat mammary gland are treated with neuraminidase, peroxidase-conjugated PNL fails to stain histochemically the majority of epithelial cells in ducts, stains the cytoplasm of the majority of epithelial cells in terminal end-buds (TEBs), and stains strongly the luminal surfaces of the majority of epithelial cells in alveolar buds (ABs). PNL also stains the untreated luminal surfaces of alveolar cells, whether or not the cells can be stained with a monoclonal antibody to rat beta-casein. Stimulation of mammary differentiation by an analogue of ethyl retinoate or by perphenazine causes cells in end-buds to bind PNL without the necessity for their desialylation similar to that seen in casein secretory alveoli of lactating rats. In conclusion the different interconverting cell types of Rama 25 which form a pathway to casein-secretory cells in vitro are thus equated with recognisable epithelial cell types in vivo. These results suggest that casein-secretory cells in vivo are generated by similar successive interconversions between the major epithelial cell types present in the different mammary structures in the order: ducts, TEBs, ABs, alveoli, and secretory alveoli.     

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.     

Morphogenesis of the developing mammary gland: Stage-dependent impact of adipocytes

Mammary gland development is critically dependent on the interactions between the stromal and the epithelial compartments within the gland. These events are under the control of a complex interplay of circulating and locally acting hormones and growth factors. To analyze the temporal and quantitative contributions of stromal adipocytes, we took advantage of the FAT-ATTAC mice (apoptosis through triggered activation of caspase-8), a model of inducible and reversible loss of adipocytes. This loss can be achieved through the induced dimerization of a caspase-8 fusion protein. In the context of female mice, we can achieve ablation of mammary adipocytes relatively selectively without affecting other fat pads. Under these conditions, we find that adipocytes are essential for the formation of the extended network of ducts in the mammary gland during puberty. Beyond their role in development, adipocytes are also essential to maintain the normal alveolar structures that develop during adulthood. Loss of adipose tissue initiated 2 weeks after birth triggers fewer duct branching points and fewer terminal end buds (TEBs) and also triggers changes in proliferation and apoptosis in the epithelium associated with the TEBs. The reduced developmental pace that adipocyte-ablated glands undergo is reversible, as the emergence of new local adipocytes, upon cessation of treatment, enables the ductal epithelium to resume growth. Conversely, loss of local adipocytes initiated at 7 weeks of age resulted in excessive lobulation, indicating that adipocytes are critically involved in maintaining proper architecture and functionality of the mammary epithelium. Collectively, using a unique model of inducible and reversible loss of adipocytes, our observations suggest that adipocytes are required for proper development during puberty and for the maintenance of the ductal architecture in the adult mammary gland.     

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.     

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.     

Mammary gland morphogenesis is enhanced by exposure to flaxseed or its major lignan during suckling in rats

The exposure of rats to 10% flaxseed (FS) or an equivalent level of its major lignan, secoisolariciresinol diglucoside (SDG), during suckling enhances mammary gland differentiation, which protects against mammary carcinogenesis at adulthood. We determined whether this diet-induced mammary gland differentiation is mediated through the estrogenic pathway via epidermal growth factor receptor (EGFR) and estrogen receptor (ER) signaling. Rats were fed the AIN-93G basal diet (BD) from day 7 of pregnancy until delivery and then randomized to consume BD, FS, or SDG during lactation. After weaning, female offspring were fed BD throughout the experiment. At postnatal day (PND) 21 and the proestrus phase on PND 49-51, mammary glands of offspring were analyzed for morphology, cell proliferation, and expression of EGFR, epidermal growth factor (EGF), transforming growth factor-alpha, ER-alpha, and ER-beta. At PND 21, compared with the BD control, the number of terminal end buds (TEBs) and terminal ducts were increased by FS, whereas mammary epithelial cell proliferation was increased by both FS and SDG, suggesting that mammary morphogenesis was enhanced. Epithelial EGFR and stromal fibroblast EGF were increased by SDG, whereas epithelial ER-beta was decreased by FS. Conversely, at PND 49-51, a lower number of TEBs but a higher ratio of lobules to TEBs with decreased expression of EGFR or EGF was observed in both treatment groups. EGFR expression was positively associated with EGF expression and cell proliferation in TEB epithelium at PND 21. Urinary lignans of lactating dams were related to their offspring's indices of mammary gland development. In conclusion, exposure to FS or SDG during suckling enhanced mammary gland morphogenesis by modulation of EGFR and ER signaling, which led to more differentiated mammary glands at PND 49-51. The physiological outcomes of FS and SDG were similar, which suggests that SDG is partly responsible for the mammary gland differentiation effect.     

Immunocytochemical identification of cell types in human mammary gland: variations in cellular markers are dependent on glandular topography and differentiation

Antiserum to epithelial membrane antigen and three monoclonal antibodies (MAb) to milk-fat globule membranes immunocytochemically stain only epithelial cells, whereas a fourth reacts also with myoepithelial cells in inter- and intralobular ducts of human breast. Staining with peanut lectin shows a gradual increase for epithelial cells, from little or no staining in ducts through variable staining in ductules to intense staining in secretory alveoli. Antisera and MAb to vimentin, smooth-muscle actin, MAb to the common acute lymphoblastic leukemia antigen and to a glycoprotein of 135 KD stain myoepithelial cells in main ducts, but this staining is reduced in inter- and intralobular ducts and ductules. MAb to epithelial-specific keratin 18 stain a minor population of ductal epithelial cells, the major population of epithelial cells in interlobular (ILD) and extralobular terminal ducts (ETD), and epithelial cells in a minority of ductules. In lactating glands most epithelial cells in ductules are stained, but the alveolar and myoepithelial cells are unstained. Keratin MAb PKK2 and LP34 strongly stain myoepithelial cells, but only a minor population of epithelial cells in main ducts. However, these MAb stain principally the epithelial cells in ILD, ETD, and a minority of ductules. In lactating glands most epithelial cells are stained in ductules, but the myoepithelial and not the alveolar cells are stained intensely in secretory lobules. It is suggested that the unusual staining pattern of cells found principally in the ILD, ETD, and some ductules may represent regions of growth and/or subpopulation(s) of cells intermediate between epithelial and myoepithelial cells.     

p190-B RhoGAP regulates mammary ductal morphogenesis

Previous studies from our laboratory have demonstrated that p190-B RhoGAP (p190-B) is differentially expressed in the Cap cells of terminal end buds (TEBs) and poorly differentiated rodent mammary tumors. Based on these observations we hypothesized that p190-B might play an essential role in invasion of the TEBs into the surrounding fat pad during ductal morphogenesis. To test this hypothesis, mammary development was studied in p190-B-deficient mice. A haploinsufficiency phenotype was observed in p190-B heterozygous mice as indicated by decreased number and rate of ductal outgrowth(s) at 3, 4, and 5 wk of age when compared with their wild-type littermates. This appeared to result from decreased proliferation in the Cap cells of the TEBs, a phenotype remarkably similar to that observed previously in IGF-I receptor null mammary epithelium. Furthermore, decreased expression of insulin receptor substrates 1 and 2 were observed in TEBs of p190-B heterozygous mice. These findings are consistent with decreased IGF signaling observed previously in p190-B-/- mouse embryo fibroblasts. To further assess if this defect was cell autonomous or due to systemic endocrine effects, the mammary anlagen from p190-B+/+, p190-B+/-, and p190-B-/- mice was rescued by transplantation into the cleared fat pad of recipient Rag1-/- mice. Surprisingly, as opposed to 75-80% outgrowths observed using wild-type donor epithelium, only 40% of the heterozygous and none of the p190-B-/- epithelial transplants displayed any outgrowths. Together, these results suggest that p190-B regulates ductal morphogenesis, at least in part, by modulating the IGF signaling axis.     

Null mutation of peroxisome proliferator-activated receptor-interacting protein in mammary glands causes defective mammopoiesis

To investigate the role of nuclear receptor coactivator peroxisome proliferator-activated receptor-interacting protein (PRIP) in mammary gland development, we generated a conditional null mutation of PRIP in mammary glands. In PRIP-deficient mammary glands, the elongation of ducts during puberty was not affected, but the numbers of ductal branches were decreased, a condition that persisted long after puberty, indicating that the potential of ductal branching was impaired. During pregnancy, PRIP-deficient mammary glands exhibited decreased alveolar density. The lactating PRIP-deficient glands contained scant lobuloalveoli with many adipocytes, whereas the wild type glands were composed of virtually no adipocytes but mostly lobuloalveoli. As a result, PRIP mammary-deficient glands could not produce enough milk to nurse all the pups during lactation. The ductal branching of mammary glands in response to estrogen treatment was attenuated in PRIP mutant glands. Whereas the proliferation index was similar between wild type and PRIP-deficient glands, increased apoptosis was observed in PRIP-deficient glands. PRIP-deficient glands expressed increased amphiregulin, transforming growth factor-alpha, and betacellulin mRNA as compared with wild type glands. The differentiated function of PRIP-deficient mammary epithelial cells was largely intact, as evidenced by the expression of abundant beta-casein, whey acidic protein (WAP), and WDNM1 mRNA. We conclude that PRIP is important for normal mammary gland development.     

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.     

The mouse mammary gland requires the actin-binding protein gelsolin for proper ductal morphogenesis

Gelsolin is an actin-binding/severing protein expressed in intracellular and secreted forms. It is a major regulator of the form and function of the actin cytoskeleton in most all cells. Here we demonstrate that female mice with a targeted deletion of the gelsolin gene (Gsn-/-) have defects in mammary gland morphogenesis. Two distinct defects were identified in the gelsolin-null mammary gland. First, the mammary anlage from Gsn-/- mice failed to elongate at the onset of puberty and remained rudimentary until approximately 9 weeks of age, early block (Gsn-/-(EB)). Second, after the mammary epithelium had filled the mammary fat pad, a complete lack of terminal branching, or late block, was observed (Gsn-/-(LB)). The Gsn-/-(EB) was seen in 70% of Gsn-/- mice and appeared to be dependent on a modifier gene(s) in addition to the loss of gelsolin. Gsn-/-(LB) was observed in all Gsn-/- mice. Terminal end buds (TEBs) were not evident in the mammary anlage from Gsn-/-(EB) mice until approximately 9 weeks of age. Cellular proliferation in the terminal ductal regions of Gsn-/-(EB) females was detected by bromodeoxyuridine incorporation, but was less than that found in the TEBs of age-matched controls. In mice deficient for gelsolin, mammary gland architecture was unaltered at the histological level. Lobuloalveolar development was delayed in response to pregnancy in mammary glands of Gsn-/- mice but was otherwise normal. Lactation and involution in the gelsolin-null animals were similar to those of wild-type mice. Transplantation of epithelium devoid of gelsolin into a wild-type (GsnWT) mammary fat pad resulted in proper arborization of the ductal tree. Transplantation of GsnWT epithelium into the Gsn-/- fat pad recapitulated the lack of terminal branching seen in Gsn-/- females. These results indicate that gelsolin is required in the mammary stroma for proper ductal morphogenesis. Our results provide the first evidence of an actin regulatory protein affecting mammary ductal growth through stromal-epithelial communication.     

Proteomic analysis of mouse mammary terminal end buds identifies axonal growth cone proteins

Ductal morphogenesis in the mouse mammary gland occurs mainly postnatally and is driven by specialized structures at the ends of the developing ducts, the terminal end buds (TEBs), which later regress once ductal growth is complete. To identify proteins that are specifically associated with migration of TEBs we developed a novel method of isolating TEBs, which eliminated the mammary stroma. The protein expression profile of the TEBs was then compared with that of isolates taken from the 4th inguinal mammary gland of adult virgin mice using two-dimensional (2-D) gel electrophoresis and mass spectrometry (MS) analysis (matrix-assisted laser desorption/ionization and quadrupole time of flight). Following construction of an integrated protein expression database, 44 protein features which showed differential expression levels between the two sets were chosen for MS analysis. Of these, 24 gave protein annotations whereas the other 20 produced unidentified peptides. Fourteen unequivocal proteins were identified from these 24, whereas the remaining 10 matched more than one protein within a single 2-D gel feature. Several of the identified proteins were associated with the cytoskeleton and have previously been reported in axonal growth cones, suggesting that they may influence cell shape and motility within the advancing TEBs, in a similar fashion to migrating axons.     

Sialomucin complex (rat Muc4) transmembrane subunit binds the differentiation marker peanut lectin in the normal rat mammary gland

Sialomucin complex (SMC, rat Muc4) is a heterodimeric glycoprotein composed of two subunits, the mucin component ASGP-1 and the transmembrane subunit ASGP-2. SMC/Muc4 is highly expressed on the surface of 13762 rat mammary adenocarcinoma cells at approximately 100 times the level found in the lactating mammary gland. Immunocytochemical staining of SMC/Muc4 in the developing rat mammary gland is localized to the apical membrane of the ductal epithelium. This staining pattern is similar to that for peanut lectin, a differentiation marker, which binds to cells expressing the disaccharide Thomsen-Friedenreich or TF antigen. Blotting of glycoproteins expressing the TF antigen from mammary tissues with peanut lectin detects a protein matching the migration of ASGP-2. Analysis of immunoprecipitated SMC/Muc4 by peanut lectin blotting shows that the TF antigen is abundantly present on the ASGP-2 subunit, hence the similarity of staining pattern with SMC/Muc4 antisera and peroxidase-conjugated lectin in mammary tissues. The TF antigen is also present on ASGP-2 of SMC/Muc4 produced by confluent cultures of Rama 37 rat mammary epithelial stem cells after their induction to an alveolar-like phenotype with prolactin. These results indicate that the TF antigen is present on the ASGP-2 transmembrane subunit of SMC/Muc4 from phenotypically normal tissues and cells, in contrast to malignant cells whose peanut lectin-binding disaccharide is located on ASGP-1.     

Murine progesterone receptor expression in proliferating mammary epithelial cells during normal pubertal development and adult estrous cycle. Association with eralpha and erbeta status.

The ovarian steroids estrogen and progesterone are important in directing the normal growth and development of the mouse mammary gland. Previously, we have demonstrated that the majority of proliferating mammary epithelial cells do not express estrogen receptor-alpha (ERalpha). In this study we examined the relationship between progesterone receptor (PR) expression and proliferation in mammary epithelial cells using simultaneous immunohistochemistry for progesterone receptor (PR) and tritiated thymidine [(3)H]-Tdr) autoradiography. Results showed that the majority (>80%) of mammary epithelial cells labeled with [(3)H]-Tdr were PR-positive in the terminal end buds (TEBs) of pubertal mice and the ducts of pubertal and adult mice. Whereas the majority of mammary epithelial cells were also PR-positive, the basal cell population, which comprises the minority of mammary epithelial cells in the mammary ducts, was predominantly PR-negative. Nevertheless, the PR-positive phenotype remained the major proliferating cell type in the basal population. These findings suggest that the progesterone signaling pathway is involved in the proliferation of basal cell populations, potentially directing formation of tertiary side branching during pubertal development and alveolar bud formation in adult glands. A proportion of the basal cells exhibited weak expression of ERbeta, suggesting that the role of ERbeta in mediating normal estrogen-induced responses should be further studied. (J Histochem Cytochem: 47:1323-1330, 1999)     

Identification of Cell Types in the Developing Goat Mammary Gland

The goat was chosen as the model system for investigating mammary gland development in the ruminant. Histological and immunocytochemical staining of goat mammary tissue at key stages of development was performed to characterize the histogenesis of the ruminant mammary gland. The mammary gland of the virgin adult goat consisted of a ductal system terminating in lobules of ductules. Lobuloalveolar development of ductules occurred during pregnancy and lactation which was followed by the regression of secretory alveoli at involution. The ductal system was separated from the surrounding stroma by a basement membrane which was defined by antisera raised against laminin and Type IV collagen. Vimentin, smooth-muscle actin and myosin monoclonal antisera as well as antisera to cytokeratin 18 and multiple cytokeratins stained a layer of myoepithelial cells which surround the ductal epithelium. Staining of luminal epithelial cells by monoclonal antibodies to cytokeratins was dependent on their location along the ductal system, from intense staining in ducts to variable staining in ductules. The staining of epithelial cells by monoclonals to cytokeratins also varied according to the developmental status of the goat, being maximal in virgin and involuting glands, lowest at lactation and intermediate during gestation. In addition, cuboidal cells, situated perpendicular to myoepithelial cells and adjacent to alveolar cells in secretory alveoli, were also stained by cytokeratin monoclonal antibodies and antisera to the receptor protein, erbB-2, in similar fashion to luminal epithelial cells. These results demonstrate that caprine mammary epithelial cell differentiation along the alveolar pathway is associated with the loss of certain types of cytokeratins and that undifferentiated and secretory alveolar epithelial cells are present within lactating goat mammary alveoli.     

Localization of vimentin in myoepithelial cells of the rat mammary gland

Summary Myoepithelial cells in the virgin rat mammary gland have been shown to contain vimentin, using a polyclonal antiserum to vimentin purified from hamster fibroblasts. This antiserum has been shown to be specific for vimentin by immunoblotting and ELISA techniques. Similar results were obtained with a monoclonal antibody to vimentin. In the mammary glands of pregnant rats, the staining with vimentin antibodies is much weaker in the myoepithelial cells of the developing alveolar buds than in the main ducts. Similarly, in lactating glands, the staining of myoepithelial cells is much weaker in the secretory alveoli than in lactiferous sinuses. In each case, staining with antivimentin co-localizes with staining with polyclonal antisera to callous keratin (which specifically stain myoepithelial cells in the rat mammary gland).     

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.