Gary B. Silberstein

Ductal morphogenesis in the mouse mammary gland: evidence supporting a role for epidermal growth factor.

Epidermal growth factor (EGF) is a potent mitogen for a variety of cells in vitro, but studies on its effects in vivo and its possible role as a natural growth regulator are few. Using slow-release plastic implants, capable of delivering EGF to small regions of the gland over a period of several days, we have shown that EGF reinitiated ductal growth and morphogenesis in growth-static glands of ovariectomized mice. The effects of implanted EGF were confined to the zone around the implant and were time and dose dependent. Unimplanted glands in the same animal were unaffected. Local effects included (1) the formation of new ductal growth points (end buds), (2) the restoration of normal end bud histomorphology and the reappearance of a stem (cap) cell layer, (3) the reinitiation of epithelial DNA synthesis, and (4) an increase in ductal diameter. No lobulo-alveolar or hyperplastic growth was seen. Competitive binding assays and autoradiography were used to characterize EGF receptor activity in growing and static glands. High and low affinity receptors were demonstrated in each tissue, while 125I-EGF autoradiography revealed differential, specific binding of the ligand to certain epithelial and stromal elements. In the epithelium, label was concentrated in the cap cells of the end buds and in myoepithelial cells of the mammary ducts. Stromal cell label was heaviest adjacent to the epithelium in the end bud flank and subtending ducts, suggesting the induction of stromal EGF receptors by mammary epithelium. Because exogenous EGF is both a mitogenic and morphogenetic factor in this tissue and can serve as a locally acting substitute for known systemic mammogens such as estrogen and prolactin, it must be considered a strong candidate for a naturally occurring mammary tissue mitogen.

TGF-β1-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.

Postnatal mammary gland morphogenesis

Mammary glands develop postnatally by branching morphogenesis creating an arborated ductal system on which secretory lobuloalveoli develop at pregnancy. This review focuses on the interrelated questions of how ductal and alveolar morphogenesis and growth are regulated in the mouse mammary gland and covers progress made over approximately the last decade. After a brief overview of glandular development, advances in understanding basic structural questions concerning mechanisms of duct assembly, elongation, and bifurcation are considered. Turning to growth regulation, remarkable progress has taken place based largely on the study of genetically engineered mice that lack or overexpress a single gene. The use of mammary glands from these and wildtype animals in sophisticated epithelial‐stromal or epithelial‐epithelial recombination experiments are reviewed and demonstrate paracrine mechanisms of action for the classical endocrine mammogens, estrogen, progesterone, growth hormone, and prolactin. In addition, IGF‐1, EGF, or related peptides, and elements of the activin/inhibin family, were shown to be necessary for ductal growth. The inhibition of ductal growth, and in particular, lateral branching, is necessary to preserve stromal space for later lobuloalveolar development. Excellent evidence that TGF‐β1 naturally inhibits this infilling, possibly by blocking hepatocyte growth factor synthesis, is reviewed along with evidence indicating that the action of TGF‐β1 is modulated by its association with the extracellular matrix. Finally, experimental approaches that may help integrate the wealth of new findings are discussed. Microsc. Res. Tech. 52:155–162, 2001.

Key stages in mammary gland development: The mammary end bud as a motile organ

In the rodent, epithelial end buds define the tips of elongating mammary ducts. These highly motile structures undergo repeated dichotomous branching as they aggressively advance through fatty stroma and, turning to avoid other ducts, they finally cease growth leaving behind the open, tree-like framework on which secretory alveoli develop during pregnancy. This review identifies the motility of end buds as a unique developmental marker that represents the successful integration of systemic and local mammotrophic influences, and covers relevant advances in ductal growth regulation, extracellular matrix (ECM) remodeling, and cell adhesion in the inner end bud. An unexpected growth-promoting synergy between insulin-like growth factor-1 and progesterone, in which ducts elongate without forming new end buds, is described as well as evidence strongly supporting self-inhibition of ductal elongation by end-bud-secreted transforming growth factor-β acting on stromal targets. The influence of the matrix metalloproteinase ECM-remodeling enzymes, notably matrix metalloproteinase-2, on end bud growth is discussed in the broader context of enzymes that regulate the polysaccharide-rich glycosaminoglycan elements of the ECM. Finally, a critical, motility-enabling role for the cellular architecture of the end bud is identified and the contribution of cadherins, the netrin/neogenin system, and ErbB2 to the structure and motility of end buds is discussed.

Glycosaminoglycans in the basal lamina and extracellular matrix of the developing mouse mammary duct

Abstract Mammary ductal elongation in the subadult virgin mouse takes place as the enlarged epithelial tip, or “end bud,” grows into the surrounding fatty stroma. By means of histochemical and autoradiographic techniques used in conjunction with enzyme susceptibility studies, we demonstrated that the basal lamina (BL) at the end bud tip is rich in hyaluronate, which is produced by a distinct outermost layer of epithelium, the “cap cells.” The posterior regions of the end bud are associated with ductal morphogenesis and tissue stabilization, and here both the epithelia and the stroma are sites of intense sulfated glycosaminoglycan (S-GAG) synthesis. Synthesis of S-GAGs was also found to extend beyond the region of collagen fibrillogenesis, and into the connective tissue between the advancing ducts, leading us to speculate that these S-GAGs play a role in the establishment of interductal spacing.

Regulation of mammary morphogenesis: Evidence for extracellular matrix-mediated inhibition of ductal budding by transforming growth factor-β1☆

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.

Elvax 40p implants: sustained, local release of bioactive molecules influencing mammary ductal development.

Elvax 40P (EVX), an ethylene vinyl-acetate copolymer, has been well characterized as an implant material that causes no inflammatory response and is capable of the sustained, local release of a wide variety of undenatured macromolecules in vivo. To investigate the usefulness of this material in developmental studies we examined the effect of EVX implants containing either deoxycorticosterone acetate (DCA) or testicular hyaluronidase on alveolar differentiation and ductal growth in the mouse mammary gland. DCA implants produced localized alveolar differentiation on ducts, while implants containing Thase caused basal lamina disruption at the ducts growing tip, resulting in epithelial dysplasias. We conclude that EVX implants allow assessment of the primary (nonsystemic) effects of biologically active molecules on developing tissue and should therefore have a variety of interesting experimental uses.

The role of TGF-β in patterning and growth of the mammary ductal tree

Evidence that transforming growth factor beta (TGF-β)4 influences pattern formation in the developing mammary gland and negatively regulates ductal growth is reviewed. In the mouse, overexpression of TGF-β transgenes during puberty reduces the rate of growth of the ductal tree and simplifies the pattern of arborization, while expression during pregnancy also interferes with lactation. Expression studies in the normal mouse gland indicate that TGF-β is synthesized in the mammary epithelium, with the three isoforms showing somewhat different spatial and temporal distributions. Exogenous TGF-β applied directly to the glandin situ inhibits epithelial cell division within hours, and strongly stimulates extracellular matrix synthesis over a longer time course. Normal human breast cells as well as certain breast cancer cell lines also secrete TGF-β and are themselves inhibited by it, suggesting an autoregulatory feedback circuit, that in some cases appears to be modulated by estradiol. Taken together, the evidence suggests a model in which growth and patterning of the mammary ductal tree are regulated, at least in part, by TGF-β operating through an autocrine feedback mechanism and by paracrine circuits associated with epithelial-stromal interactions.

Tumour-stromal interactions. Role of the stroma in mammary development.

Mammary development depends on branching morphogenesis, namely the bifurcation and extension of ductal growth points (end buds) and secretory lobules into a more or less fatty stroma. Because breast carcinomas are overwhelmingly ductal in origin, this review focuses on stromal influences guiding postnatal ductal development and there is only the briefest account of the role of embryonic stroma (mesenchyme). The stroma as the necessary target for endocrine mammogens and the source of stimulatory growth factors is described and the importance of mammary epithelium-induced modifications of the periductal stroma is emphasized. Evidence is presented that if they are to grow, end buds must condition proximal fatty stroma by recruiting white blood cells as well as inducing stromal cell division and, possibly, estrogen receptors. The induction of a fibrous stromal tunic around the end bud is described and its likely role as a complex ductal morphogen is discussed; a possible role in growth inhibition is also considered. Although the signals governing fibrotic induction, ductal morphogenesis, and growth inhibition are unknown, a role for transforming growth factor-β is highly likely and is discussed. Finally, a need for new conceptual and experimental approaches to understanding stromal-epithelial signaling is discussed.

The Pursuit of Truth in the Company of Friends

This past August mammary gland biology lost a great scientist and friend, Charles W. Daniel. Charles was born in Annapolis, Maryland on June 16th, 1933, where his father attended the Naval Academy. Postwar the family lived in Panama and Shanghai, settling in New Mexico when Charles was a teenager. Charles then attended the University of New Mexico, graduating with a double major in math and literature. He graduated with a Masters in Zoology in 1960, from the University of Hawai’i, and returned to the Mainland to begin a Ph.D. program at the University of California, Berkeley in the Zoology Department. At that time the Zoology Department was closely affiliated with the Cancer Research Genetics Laboratory (CRGL) founded by Dr. Kenneth B. DeOme, guided also by endocrinologist Howard Bern [1]. CRGL was the home of the BWest Coast Mammary Biology Group^ and it was there that Charles began what would become his life’s work and calling as a mammary gland biologist. By 1959, DeOme and his group, working with the mouse, had developed the epithelium-free, ‘cleared‘ fat pad as an in vivomammary ‘culture’ system that could be used to investigate the growth, developmental and tumorigenic potential of mammary epithelial cells. As proof-of-principle, fragments of hyperplastic alveolar nodules were transplanted to cleared fat pads and shown to develop into mammary tumors [2]. In 1965, Charles’ completed his Ph.D. studies using the DeOme technique to show that outgrowths of mouse mammary cells from primary cell culture, while producing some normal ducts, displayed a variety of culture-induced abnormalities [3]. Coincident with Charles’ completed Ph.D. the newest campus of the University of California opened in Santa Cruz. Its mandate, to foster novel approaches to teaching and research. The mammary transplant system was clearly novel and * Gary B. Silberstein gsilbers@ucsc.edu