Charles W. Daniel

The Mammary Gland: A Model for Development

questions with broad biological implications for the Hennighausen and colleagues, in a recent review development of many organs and tissues in a variety (1) cite evidence that more than 8,300 publications on of organisms. Much of the emergence of the mammary the development and secretions of the mammary gland gland as a model system is due to powerful experimenhad been published by 1899. The term a development,o tal techniques that are uniquely available to pracwhen used in reference to mammary biology, is broadly titioners of mouse mammary research. It is not an interpreted, since the mammary apparatus is repeatedly exaggeration to say that scientists not previously familgrowing, reshaping, destroying and renewing itself in iar with mammary research are astonished when first synchrony with reproductive statusÐ a developingo learning the power of these techniques. The interseceven in the adult. Even so 8,300 is an impressive tion of modern methods of modifying and analyzing number, particularly when considering that it is only the mouse genome with pre-existing mammary techa prelude to the avalanche of literature in the present nologies for transplantation and other manipulations, century. Historically, mammary gland research has makes the mammary gland the system of choice for been driven by a few dominant interests. One is the many studies in developmental genetics. In what other issue of infant nutrition and the need to understand system can the epithelium be rescued from moribund this marvelous milk-producing device that defines our mutants (even embryos) and transplanted, regenerating class Mammalia, a matter involving both public health a new gland in which the phenotype can be readily and agricultural interests. A second is the cancer probidentified, analyzed, and even used to create serially lem, certainly one of the most pressing health issues propagated tissue lines? These transplantation techof our time and one that particularly engages the emoniques make possible tissue recombinantsÐ genetic tions, striking, as it often does, women in the prime chimerasÐ in which the relative contributions of epiof life. And there is of course our less goal-oriented thelium and stroma can be evaluated. Mammary cells a academico interest in the basic biology of the breast, can be grown and genetically manipulated in culture, an interest which underlies and supports other research, then implanted into gland-free fat pads to regenerate and which ultimately will provide a coherent intellecnew variants. These manipulations can be carried out tual framework upon which we can base long-term on glands in adult animals, easily exposed and large progress in the understanding of both infant nutrition enough to be readily manipulated. These and other and breast disease. experimental techniques were reviewed by Medina in In the past thirty years or so another thread has the first issue of this Journal (2) and a forthcoming become increasingly prominent, one that is abundantly special issue will be devoted to a comprehensive

The Transforming Growth Factors Beta in Development and Functional Differentiation of the Mouse Mammary Gland

The transforming growth factors beta (TGF-beta) are multifunctional regulators with diverse effects on a variety of developmental processes and differentiated functions. In the mammary gland, a considerable amount of evidence has accumulated indicating that TGF-beta plays a critical role during several phases of the mammary cycle. TGF-beta regulates growth and patterning of the mammary ductal tree in the virgin mouse. During pregnancy, TGF-beta is required for alveolar development and functional differentiation, while at the same time inhibiting secretion of milk proteins. At parturition this inhibition is lifted, permitting initiation of lactation.

Characteristics of proliferative cells from young, old, and transformed WI 38 cultures.

Mitotic cells were obtained by a shake off procedure from cultures of normal WI 38 cells at various passage levels, and from SV-40 virus transformed cells. The size of all mitotic WI 38 cells was similar regardless of in vitro age, whereas cells from the monolayer displayed an age-related increase in size. Mitotic transformed cells were similar to normal in size, but no size changes were observed in transformed monolayer cells during serial passage. Ultrastructural studies of mitotic WI 38 cells revealed no consistent change in the numbers of mitochondria or lysosome-like bodies during aging in culture. Mitotic transformed cells displayed numbers of mitochondria comparable to normal cells, but lysosome-like bodies occurred less frequently. Size distribution and structural characteristic are presented in relation to the ability of cells to synthesize DNA and to divide. These results support the contention that aging in WI 38 cultures is characterized by a declining fraction of homogeneous, actively dividing cells, and an increasing fraction of heterogenous nondividers that display senescent changes.

Glycosaminoglycans in the basal lamina and extracellular matrix of serially aged mouse mammary ducts.

The synthesis and accumulation of specific glycosaminoglycans into proteoglycans of the basal lamina and extracellular matrix is an important aspect of ductal growth and branching morphogenesis in the mouse mammary gland. The present study was undertaken to determine whether serially aged mammary gland, which has lost most of its growth potential during repeated transplantation, displays altered ability to synthesize and accumulate glycosaminoglycans into the extracellular matrix or basal lamina. Using histochemical and autoradiographic procedures coupled with enzymatic digestion, it is now shown that serially aged mammary ductal tissue synthesizes and incorporates hyaluronate into the basal lamina at the leading edge of the end bud, where growth takes place, and sulfated glycosaminoglycans are accumulated in the extracellular matrix along the end bud flanks, associated with ductal morphogenesis. These patterns of synthesis and accumulation are similar to those associated with the growth of young gland. In non-growing regions, regardless of whether growth termination resulted from serial aging or from normal growth regulatory mechanisms operating in the young gland, sulfated glycosaminoglycans were distributed in the extracellular matrix around the ductal tips. Again, the pattern was similar in young and serially aged gland. We conclude that glycosaminoglycan metabolism and distribution are related to growth status rather than tissue age, and are unlikely to be an important component of mammary senescence.

Local effects of growth factors

Regulation of mammary gland growth and morphogenesis provides a striking example of the interplay of systemic ’classical’ hormones with more recently discovered growth factors. Both are probably required for mammary morphogenesis and functional differentiation; an understanding of both is critical to understanding the problem of mammary cancer.

Regulation of cell division in aging mouse mammary epithelium.

The subject of cellular aging is a matter of considerable importance in comtemporary biology. The characteristics and parameters of these aging processes, as well as the mechanisms which underlie them, are currently investigated by a variety of techniques. At present the most widely used methodology is that of continuous cell culture, in which normal diploid tissue cells, even though provided with every known requirement and stimulus, for growth, nevertheless display an inevitable decline in proliferative capacity, followed by senescent changes and eventual loss of the culture.

Working with the Mouse Mammary End Bud

Terminal end buds (TEB) are bulbous, rapidly growing structures responsible for formation of mammary ducts during puberty. Regulation of TEB branching, turning, and extent of growth creates the characteristic pattern of the mammary tree. In this chapter we describe two methods of isolating the TEB, one by simple dissection and the other by enzymatic digestion. A procedure is proposed for the isolation of cap cells, the basal layer of the TEB that differentiates into myoepithelium and which may contribute to other mammary stem cell populations.

Slow-Release Plastic Pellets (Elvax) for Localized In Situ Treatments of Mouse and Rat Mammary Tissue

The need to define the actions of paracrine and autocrine signaling factors against a background of systemic hormones led to the development of the slow-release implant technique. Elvax40P® is an ethylene vinyl acetate copolymer. Formed into a matrix containing test materials such as hormones or growth factors, Elvax releases metered amounts and can be used to treat small zones within the mammary gland. Importantly, Elvax is generally nondenaturing to proteins and steroids and does not elicit a tissue response. To date, Elvax experiments have been used to help define the in vivo roles of a variety of growth factors and hormones. Here we describe the fabrication and surgical techniques necessary to use this material.

Expression of Hox Genes in Normal and Neoplastic Mouse Mammary Gland

The striking phenomenon of homeosis, in which one body part is substituted for another, was described and named in Drosophila more than a century ago (Bateson, 1894). Homeobox genes have been intensively studied, particularly in recent years, as advances in understanding of the molecular genetic basis for homeosis captured the interest and imagination of developmental biologists. It is now clear that homeotic genes function as master regulators, determining the developmental pathway and cell fate of individual Drosophila segments.

Mammary Growth Regulation by Transforming Growth Factor β

A considerable literature has developed around the problem of regulation of mammary development. The great majority of this work has concerned the identification of systemic mammogens, notably the ovarian steroids, pituitary peptides, adrenal corticoids, and placental factors (1). The distinguishing feature of these hormones is that they all drive mammary development towards growth or functional differentiation; that is, they act as positive regulators. The existence of these hormones has been, by and large, demonstrated in vivo by endocrine ablation surgery followed by replacement therapy, in which removal of endocrine secretion interrupted the normal development or function of the organ. Because these experiments were conducted in a physiological setting, their biological significance was never in doubt. The present situation is that the naturally occurring mammogens have been identified and described in considerable detail and, when more recent biochemical and molecular approaches are factored in, it can be said that we are developing a usable outline of the hormones driving mammary growth and function.