Differentiation of the embryonic mammary gland

The mammary epithelium is an ectodermal derivative. As such, among the first distinctions that must be made is the differentiation of presumptive mammary epithelium from tissue that would otherwise form skin, hair follicles or other ectodermally derived structures. This differentiation occurs in at least two major stages. The first stage begins about day 10 of gestation (E10) with the establishment of the mammary streaks, two lines of epidermally derived thickened epithelium that run anterior --> posterior, symmetrically displaced off the ventral midline. These streaks represent the first morphological evidence of mammary pattern formation and differentiation.

The second stage occurs around E11 with the definition of the nipple region. Presumptive mammary epithelium forms a lens-shaped disk that becomes associated with underlying condensed mammary mesenchyme. The mammary epithelium continues to grow to form a bulb-shaped mammary bud that elongates and invades the condensed mesenchyme. Mammary epithelial cell identity is firmly established as early as E12.5 (the bud stage), as evidenced by the ability to transplant the presumptive mammary gland into a cleared fat pad and regenerate a ductal tree (CW Daniel, G Robinson, personal communication).

Invasion of the mammary fat pad precursor mesenchyme

As the mammary bud elongates into a mammary sprout, it reaches a second mesenchyme, the fat pad precursor mesenchyme, and undergoes a small amount of branching morphogenesis to form the rudimentary gland of the neonate. Several genes are known to act either at or before this critical transition to allow further growth or to control establishment of sexual dimorphism of the mouse mammary gland (eg Lef1, PThRP, PPR1) [25,26,27]. Tissue recombination experiments demonstrate that mammary mesenchyme and fat pad mesenchyme affect the growth of mammary epithelium in dramatically different ways, suggesting fundamental differences in their identities and biological properties with respect to tissue interactions (see [2]). How these differences are established is unknown.

Growth initiation, cellular differentiation and growth arrest

From birth to puberty, the gland remains rudimentary and relatively growth quiescent. At puberty, ovarian hormones stimulate rapid and invasive ductal elongation driven by growth of a structure called the terminal end bud, which consists of four to six layers of relatively undifferentiated 'body cells' and a surrounding single layer of 'cap cells'. These two populations differentiate into lumenal epithelial cells (also consisting of multiple cell types) and myoepithelial cells, respectively, as the subtending duct is formed [1,28]. Whereas a variety of individual cell types are known to exist and their developmental capacities have begun to be explored, virtually nothing is known about how these cell lineages and fates are established in the first place.

Upon reaching the limits of the fat pad at ductal maturity, ductal elongation ceases and terminal end buds regress to leave a branched system of differentiated ducts. These ducts will remain relatively quiescent as long as the animal remains virgin. How this growth control is achieved and maintained is not known, although it probably involves members of the transforming growth factor-β superfamily.

Hormonal changes during pregnancy initiate a cyclical phase of development in which there is a dramatic transition from a predominantly ductal to a predominantly lobuloalveolar gland morphology. Lobuloalveolar progenitor cells located within the ducts proliferate to form alveolar buds, which further differentiate to form the alveoli. Near midpregnancy, the alveolar epithelium acquires the capacity to produce milk proteins (the stage I transition of lactogenesis) but secretory function is inhibited. At parturition, inhibition of secretory function is released and these cells begin to secrete large quantities of milk (the stage II transition of lactogenesis). Upon weaning, milk secretion ceases and the gland involutes. During involution, most alveolar cells undergo apoptosis (programed cell death), while a residual epithelial population remodels itself back into a ductal tree to await the next pregnancy.

Of course, there are known hormonal and growth factor signals that control some of these transitions, but little is known about how epithelial and stromal cells are poised to respond to such signals or how the resultant differentiated state is maintained.

In the mouse (and human), 39 known Hox (HOX) complex genes are arranged in four paralogous gene clusters, one on each of four different chromosomes. These genes are primarily responsible for anterior-posterior patterning of the body and limbs but organ-specific functions are also known. Targeted disruption (knockout) strains exist for most Hox genes, as do a limited number of overexpressing transgenic strains. Both types of mutants have rarely been examined for mammary defects and tumors, and overall phenotypic analysis of single gene mutations has been somewhat hampered by functional compensation from paralogous genes within the complex itself. Nevertheless, it is already clear that some Hox genes are critically important for proper mammary gland development and function (Table 1).

Early immunohistochemical studies and screens based on polymerase chain reaction first detected homeobox gene expression in mammary epithelial cell lines and tumors [29,30]. Subsequently, Friedmann and coworkers identified several Hox genes that were expressed in the normal mouse mammary gland and associated neoplasias (Table 1) [31,32]. In situ hybridization demonstrated that many of these genes are expressed either in the mammary epithelium or in the periductal stroma, or both (Fig. 2).

Several genes, including Hoxc-6, Hoxc-8, Hoxd-8, Hoxd-9 and Hoxd-10, appeared to be regulated developmentally. Most intriguing was the demonstration that expression of some homeobox genes was sensitive to manipulation of the estrogen level. Together, these data provided the first substantive clues that homeobox genes are important for normal mammary development.

Soon after, Srebrow et al [33] identified five Hox genes expressed in CID-9 mouse mammary epithelial cells. When these cells are cultured on reconstituted basement membrane in the presence of lactogenic hormones, they form alveolar-like hollow spheres and differentiate to express milk proteins. Expression of two genes, Hoxa-1 and Hoxb-7, was shown to be downregulated by exogenous basement membrane, suggesting that homeobox gene expression may be modulated in vivo by interactions between epithelial cells and components of the basement membrane. Such interactions have been shown to be critical for mammary differentiation and function, and tend to be altered on neoplastic progression [34,35,36,37].

Taking cue from these studies, Chen and Cappechi [38] demonstrated compromised mammary function in mutant mice carrying various deletions of paralogous genes Hoxa-9, Hoxb-9 and Hoxd-9. Single mutant lines disrupted for either Hoxa-9 or Hoxb-9 (heterozygous at the remaining relevant Hox loci) showed only a small decrease in newborn survival, while the Hoxd-9 disruption alone reduced survival to below 50%. Double mutant combinations showed synergistic interaction that reduced newborn survival well below the additive expected values for each single mutation, suggesting functional cooperativity. Dams with the most severely affected genotypes (Aabbdd, aaBbdd and aabbdd) demonstrated marked hypoplasia of the mammary gland after parturition with gland morphology resembling that of a midpregnant animal.

During embryogenesis, Hoxb-9 and Hoxd-9 are each expressed in condensed mammary mesenchyme surrounding the epithelial bulb, suggesting roles in mesenchyme condensation or in modulation of epithelial-mesenchymal interactions during early mammary gland development [38]. Unfortunately, in situ hybridization against Hoxa-9 and Hoxb-9 using adult tissues has not been performed. However, Friedmann has examined expression of Hoxd-9 throughout postnatal development and showed it to be expressed highly in periductal fibroblasts and ductal epithelium in the virgin, but only expressed weakly in these cell types during pregnancy. Interestingly, Hoxd-9 showed enhanced expression in developing alveolar epithelium relative to subtending ducts, consistent with its apparent role in alveolar differentiation during pregnancy.

In situ hybridization has also demonstrated expression of Hoxd-10 in both epithelium and stroma with elevated levels in the epithelium of developing and secreting alveoli. These observations suggested a role in lobuloalveolar differentiation or the transition from pregnancy to lactation. Consistent with this hypothesis, targeted disruption of Hoxd-10 led to lactation failure in a significant percentage of homozygous mutant animals ([39]; Lewis Mt, Daniel CW, unpublished results). In glands of affected animals, alveolar development progressed through late pregnancy but failed, in whole or in part, to make the transition to lactation. In severely affected animals, alveoli failed to expand and increased pup mortality was observed. Together with the data for the group 9 paralogs, these observations firmly establish a functional role for Hox genes in alveolar differentiation and function.

Msx is a small family of three related genes. In the mouse, at least two members (Msx-1 and Msx-2) appear to play roles in mediating inductive tissue interactions during organogenesis. Only Msx-1 and Msx-2 have been examined in the mouse mammary gland [40,41].

Expression of Msx-1 and Msx-2 has been demonstrated in mammary buds during embryogenesis, with both genes expressed in the epithelium [41]. In contrast, in postnatal mice, Msx-1 and Msx-2 are expressed in reciprocal tissue compartments, with Msx-1 expressed in the epithelium and Msx-2 expressed in the periductal stroma (Fig. 2). The expression of Msx-2 in the fat pad was dependent on the presence of mamary epithelium. Results are suggestive of a fundamental transition in epithelial-stromal signaling that leads to tissue compartment switching of Msx-2 expression during organogenesis.

Expression studies also suggested a potential role in mediation of hormone responses. The Msx-2 mRNA was down-regulated in glands of ovariectomized and antiestrogen treated animals, and upregulated by estrogen replacement in ovariectomized animals. So far, Msx-1, Msx-2 and Msx-1/Msx-2 targeted disruption strains have been examined for mammary defects only during embryogenesis. No defects in embryonic mammary gland development were observed in Msx-1 mutants but Msx-2 and Msx-1/Msx-2 animals reportedly showed developmental defects as early as E13.5 ([41]; Maas R, unpublished results cited in [41]).

As with other organs, data from the mammary gland are consistent with a role in mediation of tissue interactions. Because homozygous Msx-1 and Msx-2 mutations are embryonic lethal, analyses of Msx gene function in the adult mammary gland are largely absent. Transplantation rescue experiments and gland reconstitution assays using embryonic tissue should be extremely useful in further examining Msx gene function in the adult mammary gland [42,43,44].

Aristaless-like

The Aristaless-like gene Alx-4 is expressed in mesenchymal condensations of tissues whose development is dependent on epithelial-mesenchymal interactions [45]. In the mammary gland, Alx-4 expression was associated primarily with actively condensing stroma at the neck of terminal end buds (Fig. 2). Alx4 mutants failed to complement Strong's luxoid (lst) mutants and the two strains show similar defects [46,47]. Interestingly, mammary gland of lst mice appear normal, as do other ventral (but not dorsal) structures in which Alx-4 is expressed [45,46,47], although it is not clear to what extent the mammary glands from these animals were analyzed.

At least five members of the Iroquois-related homeobox (IRX) gene family are expressed in the human mammary gland [48]. One member, IRX-2, was shown to be expressed in discrete epithelial cell lineages being found in lumenal epithelium of both ducts and alveoli, but not myoepithelium. IRX-2 also showed developmental regulation: expression was enhanced in terminal buds and terminal lobules of the immature gland but uniformly distributed in mature glands. During lactation, some alveolar epithelial cells (~18%) showed reduced levels of mRNA relative to adjacent strongly expressing cells. Uniform epithelial expression was reestablished upon involution. Mutational analyses of these genes are underway in several laboratories.

Both Engrailed-related (En) En-1 and En-2 have been examined in the mouse mammary gland by Northern hybridization [32]. En-2 was not detected in any tissue examined; in contrast, En-1 was expressed at virtually constant levels through early pregnancy but was not detectable during late pregnancy or lactation. Homozygous En-1 mutations are embryonic lethal. Because neither embryonic glands nor epithelial transplants have been examined, it remains possible that En-1 serves a function in the mammary gland near the early→late pregnancy transition [49,50,51].

Several other homeobox genes have been identified either in intact mammary glands of from cDNA library screens. These include members of the POU domain containing, MOX, and MEIS gene families ([52,53,54]; Lewis MT, Daniel CW, unpublished results). No functional or detailed expression data are yet available. Given the known importance of the POU and MEIS gene families in development and cancer, these should be among the first to be evaluated for mammary function.

Expression of all eight normally expressed homeobox genes examined (Hoxb-6, Hoxb-7, Hoxc-6, Hoxc-8, Hoxd-4, Hoxd-8, Hoxd-9 and Hoxd-10) was lost on neoplastic progression in a selected population of mouse hyperplasias and tumors [32]. Conversely, three genes not normally expressed were activated in subsets of tumors (Hoxa-1, Hoxd-3 and Hoxd-12).

Thus far, it appears that loss of function mutations simply affect developmental progression but that gain of function mutations impact neoplastic progression unpredicatable ways. For example, loss of function mutations of Hoxa-9/Hoxb-9/Hoxd-9 or of Hoxd-10 resulted in developmental failure, rather than tumor formation. As for gain of function mutations, whereas overexpression of human HOXA-10 in MCF7 cells promoted cell cycle arrest in G1, overexpression of murine Hoxb-7 in SkBr3 mammary cancer cells caused increased proliferation and decreased growth factor dependency [57]. These observations suggest that the consequences of a given Hox gene mutation cannot be predicted a priori, but will depend entirely on the nature of the mutation and on the battery of downstream genes misregulated as a result.

HSIX1 is expressed toward the end of S phase of the cell cycle in MCF7 cells [22,58]. Overexpression of HSIX1 in these cells abolished the G2 cell cycle checkpoint in response to irradiation, leading to inappropriate entry into mitosis. Interestingly, very low expression of HSIX1 was observed in the normal mammary gland, but elevated expression was observed in 44% of primary breast cancers and in an astonishing 90% of metastatic lesions. These data represent the only known involvement of homeobox genes in mammary cell cycle control at the G2 checkpoint. Given that loss of this and other checkpoints is associated with mammary cancer [59,60,61,62], these data represent the strongest indication to date that homeobox genes may contribute to neoplastic progression in the mammary gland.

Msx-1 expression was maintained in all tumors examined [40]. In contrast, Msx-2 became undetectable in neoplasias. Given its reportedly critical role in embryonic mammary gland development, loss or alteration of stromal Msx-2 function in the adult mammary gland might be expected to have profound effects on interactions between the neoplastic epithelium and its associated stroma.

Similar to the expression pattern observed for Msx-1, IRX-2 expression was also maintained in all tumor types examined [48]. However, in some tumors, there was evidence of either increased expression or altered ratios of the two known transcripts. Since the IRX-2b transcript lacks the homeobox found in the IRX-2a transcript, target gene regulation may be expected to be altered.