1 Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Rm. 8009, Omaha, NE 68198-6805, USA
2 Basic Research Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bldg. 10, Room 8B07, 9000 Rockville Pike, Bethesda, MD 20892-1750, USA
3 Laboratory of Genetics and Physiology, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bldg. 8, Rm. 107, Bethesda, MD 20892-0822, USA
*Authors for correspondence (e-mail: kuwagner{at}unmc.edu and gs4d{at}nih.gov)
Accepted 18 December 2001
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SUMMARY |
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Key words: Mammary gland, Cre recombinase, Epithelium, Parity, Stem cells, Involution, Differentiation, Mouse
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INTRODUCTION |
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In each reproductive cycle, proper alveolar differentiation and milk protein synthesis require the synergistic action of lactogenic hormones and local growth factors (Hennighausen and Robinson, 1998; Hennighausen and Robinson, 2001
). Prolactin seems to play a central role in this differentiation process (Horseman et al., 1997
; Ormandy et al., 1997
). The signal transducer and activator of transcription 5 (STAT5), as a component of the prolactin-signaling pathway (Liu et al., 1997
; Miyoshi et al., 2001
), cooperates with other factors such as the glucocorticoid receptor and C/EBPß to achieve maximum expression of genes for milk proteins (Stocklin et al., 1996
; Wyszomierski and Rosen, 2001
). Very low levels of expression of genes for milk proteins can also be found in virgins, but their synthesis increases considerably during the second half of pregnancy (Robinson et al., 1995
). Interestingly, the transcriptional regulation of genes encoding milk proteins varies slightly between caseins and whey proteins. In mice, casein transcription increases rather early during pregnancy, and high levels of expression of the whey acidic protein (WAP) and
-lactalbumin is restricted to the last phase of pregnancy (i.e. mainly in lactogenesis II) (Pittius et al., 1988
; Robinson et al., 1995
). The differential upregulation of caseins and whey proteins may reflect a progression towards terminal differentiation. Beside hormones and local growth factors, proper expression of the Wap gene requires cell-to-cell contact and the formation of a closed lumen (i.e. a correct three-dimensional structure of an alveolus) (Chen and Bissell, 1989
). Therefore, the expression profile of the Wap gene is frequently applied as an indicator for advanced differentiation of mammary epithelial cells. High levels of Wap gene expression are maintained throughout lactation, but its expression declines significantly during the first phase of involution (days 1 and 2 after weaning) and reaches nearly undetectable levels during the second phase of mammary gland remodeling (i.e. 4 to 6 days after weaning the litter) (Burdon et al., 1991
; McKnight et al., 1992
).
On the basis of two paradigms [(1) high levels of WAP gene expression are restricted to differentiated mammary epithelial cells and (2) differentiated alveolar cells undergo apoptosis], it is unclear whether a WAP-promoter driven transgenic mouse model expressing Cre recombinase might be useful for studying the loss-of-function of genes in the mammary gland, in particular, tumor-susceptibility genes in multiparous and aging mouse models for human breast cancer. Unlike various other WAP-based transgenic strains (Burdon et al., 1991; McKnight et al., 1992
), we were able to identify a WAP-Cre expressing line that follows closely the temporal and spatial regulation of the endogenous WAP gene (Wagner et al., 1997
). Based on genomic alterations that the Cre recombinase engraves on another reporter transgene (i.e. recombination between loxP sites), we found that a large number of mammary epithelial cells previously expressing the WAP-Cre transgene remained in the mammary gland after complete remodeling (Wagner et al., 1997
). More importantly, these cells seem to multiply during subsequent pregnancies and, therefore, our findings contradict the generally accepted paradigms on mammary development. Clearly, our observations are not an artifact caused by a deregulated activation of the promoter of our randomly integrated WAP-Cre construct, as Ludwig and co-workers (Ludwig et al., 2001
) have recently reported similar observations in mutant mice that express Cre under the endogenous WAP promoter (WAP-Cre knock-in mutants). Based on these findings, we hypothesize that a specific number of WAP-expressing or differentiated cells bypass apoptosis and remain in the parous gland, where they can give rise to a clonal population of alveolar cells during subsequent pregnancies. Indirectly, our hypothesis implies that mammary epithelial cells from parous individuals are different from nulliparous animals in their genetic program, despite the close resemblance in their morphological appearance. To address this issue, we have used double transgenic mice carrying the WAP-Cre and the Rosa-lox-Stop-lox-lacZ (herein referred to as Rosa-lacZ) to monitor differentiation and cell survival in the developing and involuting mammary gland on the level of single cells. The main objective was to determine how many WAP-Cre expressing cells (i.e. hormone responsive and differentiated cells) are capable of resisting programmed cell death during involution. We have determined their location and growth properties in multiparous animals, in transplants and in culture. We demonstrate that these partially committed cells are not just alveolar progenitors, but they also share similarities with multipotent mammary stem cells. They can contribute to both ductal and alveolar epithelial cell types in transplants. Moreover, we show that the differentiation and survival process provides a mechanism for cell selection that is important for bypassing genetic pathways in gene deletion models, in order to revert to a mutant phenotype in subsequent pregnancies.
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MATERIALS AND METHODS |
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Preparation and X-Gal staining of mammary gland whole mounts
Briefly, the entire inguinal mammary gland (i.e. gland 4) was spread on a glass slide and fixed for 1-2 hours in 2% paraformaldehyde, 0.25% glutaraldehyde, 0.01% NP-40 in phosphate-buffered saline (PBS). Tissues were washed repeatedly in 1xPBS and processed for X-Gal staining as described previously (Wagner et al., 1997). Mammary glands were postfixed in 10% formalin, dehydrated in 100% ethanol and placed overnight in xylene before whole-mount analysis. For the analysis of tissue sections, mammary glands were dehydrated following the X-Gal procedure, embedded in paraffin, sectioned and counterstained with Nuclear Fast Red.
Tissue transplantation
DeOme and his colleagues originally devised the technique of tissue fragment transplantation into mammary fat pads cleared from endogenous mammary epithelium (DeOme et al., 1959). The surgical procedures for clearing the fat pad of 3-week-old female mice and the method of implanting tissue fragments and cell suspensions have been described previously (Daniel et al., 1968
; Faulkin and DeOme, 1960
; Smith et al., 1980
; Smith et al., 1991
). Random fragments (
1.0 mm3) of mammary epithelium were taken from nulliparous and nonpregnant parous WAP-Cre Rosa/lacZ females. Immediately after the retrieval of the epithelium from the transgenic donors, the fragments were implanted into the cleared fat pad of 3-week-old recipients (homozygous AthymicNCr-nu females). The recipients were kept as nulliparous virgins for 7 to 12 weeks to provide sufficient time for the transplanted epithelium to penetrate the wild-type fat pad and form a ductal tree. The recipients were neither bred nor treated with hormonal supplements to prevent a secondary activation of the WAP-Cre transgene. After more than 7 weeks, the mammary glands were prepared as whole mounts and stained with X-Gal as described above.
Primary cultures, selection, X-Gal staining
Primary mammary epithelial cultures from nulliparous and parous WAP-Cre/Rosa-lacZ double transgenic females were prepared in analogy to methods described previously (Li et al., 2000; Medina and Kittrell, 2000
). WAP-Cre/Rosa-lacZ cultures were examined by X-Gal staining after 48, 72 and 96 hours. The cells were fixed for 10 to 15 minutes in chilled 0.08% glutaraldehyde in 1xPBS and washed at least three times in 1xPBS before the X-Gal staining procedure described above. The cells were stained for 24 to 48 hours at 30°C and washed twice in 1xPBS before examination.
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RESULTS |
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Using our double transgenic mice to label differentiated and apoptosis-resistant cells permanently, we have demonstrated for the first time that cells previously expressing an alveolar differentiation marker (i.e. WAP) can contribute to the formation of primary and secondary ducts. It remains to be determined in a more detailed study whether these cells give rise to all ductal epithelial subtypes, including myoepithelial cells. Preliminary studies on histological sections of the transplants show that the blue cells are mainly localized in the luminal epithelium of large ducts (Fig. 3G), small ducts (Fig. 3H) and terminal end buds (Fig. 3I).
The functional memory of the mammary epithelium
Transplantation models are powerful tools with which to study mammary development in mutant mice that exhibit complex phenotypes or embryo lethality after day E12.5 (Robinson et al., 2000; Robinson et al., 2001
). In the majority of experiments that use transplantation models, studying the intrinsic effects of a targeted mutation on mammogenesis is required. Our findings on the selective outgrowth of X-Gal-positive cells and their descendants in transplants suggest that there might be differences in transplantation models when mutant mammary epithelial cells originate from nulliparous or parous donors. We postulate that in successive pregnancies a subset of mammary epithelial cells undergo a rigorous selection process. It has been frequently reported that mouse models with a targeted mutation are able to compensate for the loss of an important gene in consecutive lactation periods (Liu et al., 1998
; Ormandy et al., 1997
). The genetic pathways involved in the compensation might be different for each mouse model. For example, the lack of Stat5a can be compensated through upregulation of Stat5b (Liu et al., 1998
), or the loss of one functional allele of the prolactin receptor (Prlr) can be compensated through upregulation of the wild-type allele (P. Kelly, personal communication) or through the downregulation of SOCS1 (Lindeman et al., 2001
). However, what is the general mechanism for cell selection that results in a functional mammary gland? According to the current theory, alveolar self-renewal that originates only from a naïve stem cell population during each pregnancy cycle does not provide a genetic instruction for the reversal of a mutant phenotype, as differentiated cells that adapt to a mutant situation are lost during the involution phase. We have crossed WAP-Cre/Rosa-lacZ double transgenic mice with heterozygous Prlr mutants (Ormandy et al., 1997
) to address whether our findings on the newly identified mammary epithelial cell population in parous animals provide a general mechanism for alveolar self-renewal and reversal of a mutant phenotype in successive lactations. We have analyzed mammary differentiation and the distribution of X-Gal-positive cells in three consecutive post-partum periods (Fig. 4). The loss of one functional Prlr gene inhibited alveolar development (Fig. 4A), and lactation could not be established after the first pregnancy cycle. Nevertheless, a limited number of X-Gal-positive cells still remained in the mammary gland of involuted WAP-Cre/Rosa-lacZ/Prlr+/ triple mutant mice (Fig. 4B). A significant increase in the number of differentiated alveolar cells was observed at the end of the second gestation period, and many more blue cells did not undergo apoptosis after remodeling was completed (Fig. 4D). More than 50% of the triple mutant mice were unable to nurse their litter after the second gestation period. Lactation and normal development was restored during the third pregnancy cycle (Fig. 4E,4F). Our observations suggest that the newly identified population of epithelial cells in parous mammary glands might be the basis for a general mechanism that facilitates self-renewal of the alveolar compartment in consecutive lactation cycles. The blue cells that did not undergo apoptosis during the involution phase might serve as the functional memory of the mammary epithelium. Again, this could be a universal mechanism for the positive selection of cells that learn how to bypass an altered signaling pathway, but the adaptation of specific compensatory factors to bypass a particular targeted mutation might be different in each mutant mouse strain.
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Discussion |
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Contiguous regions of the mammary epithelium in the human breast are clonally derived as determined by X chromosome inactivation patterns (Tsai et al., 1996), indicating the presence of a pluripotent precursor. This observation has been confirmed and extended to include normal tissues surrounding mammary atypia and tumors in situ (Deng et al., 1996
; Lakhani et al., 1999
; Rosenberg et al., 1997
). In murine mammary glands, considerable evidence supports the existence of locally dispersed multipotent epithelial stem cells (reviewed by Smith and Chepko, 2001
). The multipotent epithelial population comprises three subtypes: mammary stem cells (which produce all epithelial cell types, including ducts and alveoli), ductal-limited precursors (which produce only branching ducts) and lobule-limited precursors (which produce only secretory lobules). All of these multipotent epithelial cells are found in clonally derived mammary outgrowths, indicating they arise from a mammary stem cell (Kordon and Smith, 1998
). The multipotent epithelial cells in human and murine glands are associated with cellular populations, which express luminal epithelial markers rather than the phenotypic markers associated with myoepithelial cells (Smalley et al., 1999
; Stingl et al., 1998
; Stingl et al., 2001
). These findings are consistent with our observations presented in this report. Some Wap-expressing mammary epithelial cells survive post-lactational involution and persist in a luminal niche. Subsequently, upon the succeeding pregnancy, they proliferate to help form new secretory acini. However, in transplants this parity-induced epithelial population shows the property of self-renewal, and these cells maintain themselves at regular intervals among the luminal epithelium of the extending ductal branches. They also orientate themselves within the body of the growing terminal end bud. Thus, these cells express certain features of multipotent stem cells, i.e. the property of self-renewal and the ability to divide symmetrically. The dissociation of involuted mammary glands from WAP-Cre/Rosa-lacZ double transgenic animals and the transplantation of the dispersed epithelial cells into the cleared fat pad of recipients demonstrate that this parity-induced epithelial population can re-associate with other cells and produce complete branching mammary ducts. However none of the structures produced in these studies were entirely composed of blue cells, therefore these cells may not be capable of producing all of the epithelial cell types in mammary ducts by themselves. Hence, these parity-induced mammary epithelial cells seem to lack the most important feature of mammary stem cells: the innate capacity to produce diverse progeny such as ductal myoepithelial cells.
The appearance of epithelial cells committed to secretory differentiation and capable of proliferation among the parous mammary epithelium provides a buffer population, which may protect the depletion of primary mammary stem cells from the population as a result of mitotic activity. Serial transplantation shows evidence that mammary epithelium from an aged, multiparous female is equivalent to that from young pubertal females with respect to longevity and growth potential (Young et al., 1971). The rescue of normal secretory development and lactation in the prolactin receptor heterozygous knockout mice is an example of the buffering capacity provided by the survival and proliferative capacity of the parity-induced epithelial population.
A question of interest is the significance of the appearance of partially committed, proliferation-competent cells in the parous mammary gland and the increased resistance to mammary tumorigenesis compared to the nulliparous gland. Women, regardless of ethnicity, who have undergone a full-term pregnancy before 20 years of age have one-half the risk of developing breast cancer compared with nulliparous women (MacMahon et al., 1970). Parous rats and mice also have a greatly reduced susceptibility to chemically induced mammary tumorigenesis when compared with their nulliparous siblings (Medina and Smith, 1999
; Russo and Russo, 1996
; Welsch, 1985
). The mechanism(s) for this protective effect have not been defined. The most widely accepted explanation, offered by Russo and Russo (Russo and Russo, 1996
), is that the protection is afforded by the pregnancy-induced differentiation of the target structures for carcinogenesis: terminal end buds and duct termini. More recently, it has been suggested that the hormonal milieu of pregnancy affects the developmental state of a subset of mammary epithelial cells and their progeny, which result in persistent differences in their response to carcinogenic challenge. These changes are reflected in the muted proliferative response to carcinogen exposure by the affected cells and the appearance of a sustained expression of nuclear p53 in the hormone-treated epithelium. The proliferation block and the induction of p53 occur both in rats and in mice and support the generality of this hypothesis (Sivaraman et al., 1998
; Sivaraman et al., 2001
). The ability to identify and subsequently isolate cells from the parity-induced subpopulation of epithelial cells from multiparous mice offers an opportunity to evaluate various aspects of the differences between parous and nulliparous mouse mammary glands.
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ACKNOWLEDGMENTS |
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K. U. W. was funded in part by the Department of Defense grant DAMD17-00-1-0641.
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