Mammary Gland Development Is Mediated by Both Stromal and Epithelial Progesterone Receptors

Robin C. Humphreys, John Lydon, Bert W. O’Malley and Jeffrey M. Rosen

Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A combination of a knockout mouse model, tissue transplantation, and gene expression analysis has been used to investigate the role of steroid hormones in mammary gland development. Mouse mammary gland development was examined in progesterone receptor knockout (PRKO) mice using reciprocal transplantation experiments to investigate the effects of the stromal and epithelial PRs on ductal and lobuloalveolar development. The absence of PR in transplanted donor epithelium, but not in recipient stroma, prevented normal lobuloalveolar development in response to estrogen (E) and progesterone (P) treatment. Conversely, the presence of PR in the transplanted donor epithelium, but not in the recipient stroma, revealed that PR in the stroma may be necessary for ductal development. Members of the Wnt growth factor family, Wnt-2 and Wnt-5B, were employed as molecular markers of steroid hormone action in the mammary gland stroma and epithelium, respectively, to investigate the systemic effects of E and P. Hormonal treatment of intact, ovariectomized, and PR-/- mice and mice after transplantation of PR-/- epithelium into wild type (PR+/+) stroma demonstrated that these two locally acting growth factors are regulated by independent mechanisms. Wnt-2 is acutely repressed by E alone, while Wnt-5B gene expression is induced only after chronic treatment with both E and P. Wnt 5B appears to be one of the few molecular markers of P action in the mammary epithelium. This study suggests that the regulation of mammary gland development by steroid hormones is mediated by distinct effects of the stromal and epithelial PR and differential growth factor expression.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Aberrant regulation of normal developmental pathways plays an important role in initiating and supporting mammary gland transformation. Both hormonal and developmental status are known to be important factors in the etiology of breast cancer. These hormonal and developmental cues are often mediated at the molecular level by a combination of systemic hormones and locally acting growth factors. Synergism among locally acting growth factors enhances and augments the diversity of potential signals transmitted to the epithelial and stromal components of the gland. This synergism can be stimulatory or inhibitory and can affect gene expression in the epithelium and mesenchyme.

Estrogen (E) and progesterone (P), in cooperation with pituitary hormones, are the primary systemic hormones required for the induction of proliferation and differentiation of epithelial and stromal cells leading ultimately to the formation of ductal and alveolar structures during mammary gland development. The interaction of E and P with GH, PRL, and insulin in regulating this differentiative process has been well documented (1). Steroid hormones also regulate the expression of a number of different locally acting growth factors, including members of the epidermal growth factor, insulin-like growth factor, and fibroblast growth factor (FGF) families (2, 3). Most of these growth factors exhibit localized effects due to protein stability, adhesion and residence in extracellular matrix, transport and secretion, and availability of receptor molecules. For this reason they are believed to act as local mediators of the differentiative and proliferative signals of the systemic hormones. Systemic regulation of locally acting growth factor activity allows for fine regulation of large-scale developmentally associated proliferative and differentiative functions.

Mammary gland development is dependent on physical, molecular, and often reciprocal, interactions between the stromal and epithelial compartments (4). The ability to recapitulate fully differentiated structures from a fragment of syngeneic parenchyma, and to separate and recombine epithelial and stromal compartments in vivo, makes the mammary gland an excellent model system in which to study these interactions. Evidence for this reciprocal dependence has been demonstrated in classic recombination experiments between the epithelial and stromal androgen receptor pathways (4). The specific role of the epithelial and stromal PR in the development and differentiation of the mammary gland is unclear (5, 6).

Wnt-1, the progenitor of a family of related growth factors, was discovered in mouse mammary tumors as a result of proviral activation (7). Members of the Wnt gene family are expressed in invertebrates and vertebrates where they regulate cell fate and pattern formation (8). Wnt genes, other than Wnt-1, are expressed in the mammary glands of mice in a developmentally specific pattern (9, 10, 11). The function of these endogenous Wnt genes during mammary gland development is unknown. From these studies it is apparent that Wnt gene expression is tightly regulated and is dependent on the developmental state of the mammary gland. In BALB/c mice, Wnt-2 is expressed primarily during early ductal development, 5–8 weeks postnatally, coincident with time of PR induction by E, and is markedly down-regulated at the onset of pregnancy. Conversely, Wnt-5B transcripts are detectable in the late virgin gland at 6–12 weeks of age but increase markedly during pregnancy, reaching a peak at day 18. Wnt-5B expression is localized primarily in the ductal and lobuloalveolar cells, while Wnt-2 expression is detected in the stroma (9, 11). These results suggest that E and P may play a role in regulating Wnt-2 and Wnt-5B gene expression in both the stroma and epithelium. This restricted pattern of gene expression is indicative of molecules that may be involved in the developmental processes of the gland.

In this study the progesterone receptor knockout (PRKO) mouse (12) has been used for reciprocal transplantation experiments in syngeneic mice to investigate the distinct roles of the stromal and epithelial PR in mammary ductal and alveolar development. Wnt-2 and -5B provided specific molecular markers of steroid hormone action in the mammary gland stroma and epithelium, respectively. The PRKO mouse permitted definition of the unique effects of P distinct from those mediated by E on Wnt gene expression. This experimental approach should facilitate the identification of other steroid-mediated local growth factors on mammary gland development.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Epithelial and Stromal PRs Have Separate Roles in Mammary Gland Development
The mammary gland has the unique ability to recapitulate the complete ductal and alveolar structures from a transplanted fragment of syngeneic mammary epithelium (13). This characteristic allows the analysis of interactions between epithelium and stroma in vivo. The PR is present in both epithelial and stromal compartments of the murine mammary gland (5, 14). To establish the role of the PR in each of these compartments, reciprocal transplantation experiments were performed using epithelium and stroma derived from syngeneic 129SvEv PR-/- and PR+/+ mice, respectively. PR-/- epithelium transplanted into the cleared fat pads of PR+/+ mice penetrated and filled the stroma with ductal structures (Fig. 1AGo). Interestingly, the PR-/- epithelium failed to develop alveoli and to display an increase in the number of secondary ductal branches in response to steroid hormone treatment (Fig. 1BGo). The control ipsilateral glands from the host animal responded as expected to steroid hormone treatment with alveolar proliferation (Fig. 1Go, D vs. C). This result demonstrates that the PR in the epithelium is required for normal lobuloalveolar formation and differentiation of the epithelium. In addition, the presence of PR-regulated signaling pathway in the stroma cannot compensate for the lack of PR in the epithelium. In contrast, PR+/+ epithelium transplanted into the cleared fat pad of PR-/- hosts and treated with E and P exhibited lobuloalveolar development (Fig. 2DGo). An increase in secondary branching in these E- and P-treated transplants can be clearly seen under higher magnification (Fig. 2FGo, arrow). However, an unexpected, marked reduction in the extent of ductal outgrowth was observed in these transplants after 10 weeks of growth (Fig. 2CGo), as compared with the PR+/+ (Fig. 2Go, A and B) and the PR-/- (Fig. 1AGo) epithelium transplanted into the PR+/+ stroma. The same PR+/+ epithelium transplanted in PR+/+ stroma responded as expected to steroid hormone treatment with extensive alveolar growth and an increase in secondary branching (Fig. 2BGo). The outgrowths from the PR+/+ epithelium transplanted into the PR-/- stroma also displayed unusual terminal endbuds (Fig. 2Go, C and E).



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Figure 1. Absence of Lobuloalveolar Development in Transplanted PR-/- Epithelium

PR-/- epithelium was transplanted into cleared fat pads of PR+/+ 129SvEv mice. After 10 weeks of growth, the mice were injected subcutaneously daily with E and P, and the mammary glands were collected at day 0 (A and C) and day 8 (B and D). The arrows in panels A and B denote the site of transplantation. Note that in panel A the fat pad has been penetrated with ductal epithelium after 10 weeks of growth in vivo. Also note the increase in alveolar development in the ipsilateral PR+/+ glands (C and D) after hormonal stimulation (compare panels C and D). Bar = 1.4 mm.

 


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Figure 2. The Morphological Response of PR+/+ Epithelium Transplanted into PR-/- and PR+/+ Stroma to Steroid Hormone Treatment

PR+/+ epithelium was transplanted into PR+/+ (A and B) and PR-/- (C, D, E, and F) stroma. After 10 weeks of growth, the mice were injected daily with E and P subcutaneously, and the mammary glands were collected at day 0 (A, C, and E) and day 8 (B, D, and F). Alveolar formation is evident in PR+/+ (epithelium) PR-/- (stroma) after 8 days of E and P treatment (arrow in F). Note the reduction in ductal development in PR-/-/PR+/+ (D) compared with both PR+/+ (B) and PR-/- epithelium (Fig. 1Go, A and B). PR+/+ epithelium in PR+/+ stroma responds to steroid hormone treatment with extensive alveolar growth and an increase in secondary branching (B). The arrows in A and B define the site of transplantation. Magnification in panels A, B, C, and D is defined by bar in A = 2 mm. Magnification in panels E and F is defined by bar in E = 0.75 mm. (PR-/-/PR+/+, n = 4 and PR+/+/PR+/+, n = 4).

 
E and P Treatment Represses Wnt-2 Gene Expression in the Mammary Glands of Ovariectomized (ovx) and Intact Mice
The role of E and P in regulating Wnt gene expression has been implied from the pattern of Wnt gene expression observed in normal mammary gland development (9, 10, 11). In particular, Wnt-2 and Wnt-5B display dramatic and inverse changes in gene expression levels at the onset of pregnancy. Wnt-2 appears to be expressed primarily in the mammary gland stroma, and Wnt-2 transcripts have been detected in the cleared mammary fat pad (9, 11), whereas Wnt-5B is expressed specifically in ductal and lobuloalveolar cells (11). Thus, these locally acting growth factors provide excellent molecular markers to investigate the role of steroid receptors on ductal and lobuloalveolar development. First, however, it was necessary to establish whether E and P either alone or in combination could regulate the expression of Wnt-2 and Wnt-5B in a manner analogous to that observed during mammary gland development. BALB/c mice were treated with E and P to mimic the onset of pregnancy. RNA from the mammary glands of hormonally treated and untreated, ovx, and intact mice were examined for changes in gene expression using a quantitative, RT-PCR method (9). A decrease of approximately 4-fold relative to the untreated (time zero) group in Wnt-2 gene expression was observed after E and P treatment of intact BALB/c mice (n = 3, P < 0.001, Fig. 3AGo). A 2-fold decrease (P < 0.002) in Wnt-2 gene expression is observed after only 2 days of E and P treatment. Wnt-2 gene expression decreased progressively with daily E and P treatment and remained low to day 12 (data not shown). This decrease in gene expression of Wnt-2 is not, however, as dramatic (20-fold) as that observed after the onset of pregnancy (9).



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Figure 3. The Response of Wnt-2 Gene Expression in Intact (A) and ovx (B) BALB/c Mice to E and P Treatment

A, Quantitative RT-PCR analysis of RNA from mammary glands of BALB/c mice injected daily with E and P, subcutaneously, for 8 days. EP refers to treatment with E and P for the number of days designated. Values: Molecules/nanogram RNA(MOL/NG) represent the mean ± SEM. The star denotes that EP 4 is statistically different from EP 0, P < 0.001, n = 3. B, Quantitative RT-PCR analysis of RNA from mammary glands of BALB/c mice treated with E and P, E, and vehicle beeswax implants for 1, 3, and 14 days. E refers to treatment with E alone for the number of days designated. The absolute values for the entire control group (C) were low in this experiment, possibly due to an effect of the vehicle, but did not change significantly with time. Values represent the mean ± SEM. The stars denote that EP 14 and E 14 are statistically different from EP 1 and E 1, respectively; P < 0.001, n = 3.

 
Circulating E and P can cause cyclical repression and induction of PR expression levels and possibly attenuate the molecular effects of pharmacological doses of E and P. To eliminate the effects of endogenous ovarian hormones, three groups of ovx mice were implanted subcutaneously for 14 days with beeswax pellets that contained E and P together, E alone, or carrier. The thoracic mammary glands were collected from three animals at each time point within each treatment group at 1, 3, and 14 days after implantation of the pellets. A 3-fold decrease relative to the day 1 E and P treatment group was observed in Wnt-2 expression after 3 days, increasing to 5-fold at 14 days compared with control (n = 3, P < 0.001, Fig. 2BGo). Interestingly, mice treated with E alone showed the same 5-fold decrease relative to day 1 E alone mice in Wnt-2 gene expression after 14 days (n = 3, P < 0.001).

Wnt-5B Expression Is Induced by E and P in ovx and Intact Mice
During normal mammary gland development, Wnt-5B expression is observed initially at 6–8 weeks in the virgin mouse and increases at the onset of pregnancy with maximal expression observed at day 16–18 of pregnancy (9, 10, 11). Wnt-5B expression increased 4-fold by day 8 of E and P treatment of intact mice as compared with the untreated (time zero) mice and was maximally induced by day 16 (P < 0.004, n = 3) as illustrated in Fig. 4AGo. Thus, the increase in Wnt-5B gene expression, which parallels that observed during midpregnancy, requires chronic E and P treatment. The pattern of Wnt-5B expression in the ovx mice (Fig. 4BGo) was similar to that observed in the intact animal but displayed a more dramatic response. Wnt-5B expression remained low at day 1 and day 3 but increased 9-fold at day 14 relative to the day 1 E- and P-treated group (P < 0.001, n = 3). In contrast to the regulation of Wnt-2, there was no significant effect of E alone on Wnt-5B expression in ovx mice. The large increase observed in Wnt-5B expression in ovx mice may reflect the sensitization of the gland to P due to the absence of endogenous hormones and a rapid induction of PR gene expression.



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Figure 4. The Response of Wnt-5B Gene Expression in Intact (A) and ovx (B) BALB/c Mice to E and P Treatment

A, Quantitative RT-PCR analysis of RNA from mammary glands of BALB/c mice injected daily with E and P, subcutaneously, for 18 days. Values represent mean ± SEM. The star denotes that EP 16 is statistically different from EP 0, P < 0.004, n = 3. B, Quantitative RT-PCR analysis of RNA from mammary glands of BALB/c mice treated with E and P beeswax implants for 1, 3, and 14 days. Values represent mean ± SEM. The star denotes that EP 14 is statistically different from EP 1; P < 0.001, n = 3.

 
Morphological Changes in Normal and Steroid Hormone-Treated Mice Correlate with the Changes in Wnt Gene Expression
Changes in Wnt-2 and Wnt-5B gene expression are coincident with morphological changes observed in the mammary gland in response to E and P treatment (Fig. 5Go). Mammary glands of 12-day E- and P-treated BALB/c mice (Fig. 5HGo) exhibit a morphology similar to that observed in an 8-day pregnant mouse. Several differences were observed, however, between the steroid hormone-treated glands and those from the normal midpregnant mouse, especially when comparing the first few days after hormone administration. For example, on the second day of treatment, transient alveolar proliferation was observed (Fig. 5FGo). However, by the fourth day these alveoli were no longer detectable, and a decrease in the amount of secondary branches was observed (Fig. 5Go). This transient alveolar budding has not been reported in mice treated with pharmacological doses of E and P but is similar to the effect observed in some strains of mice who respond to ovarian cycling by producing a transient alveolar proliferation in the mature virgin gland. This phenomena has, however, not been observed in BALB/c mice (1, 15). Surprisingly, at day 8 some major ducts displayed a ductal hypertrophy (Fig. 5GGo, arrow). This hypertrophy has been observed in mammary glands of mice implanted with hepatocyte-growth factor (HGF) and treated with E and P (16). Unlike the alveolar budding, this ductal hypertrophy was not transient and was still detectable in some glands at day 12–16 (Fig. 5HGo). Permanent alveoli appeared at day 12 and increased in number and density throughout the remainder of the treatment. This progression of morphological changes can be compared with the normal gland (1). During pregnancy, alveoli and secondary branching appear by day 4 and increase in density and number with the progression of pregnancy (Fig. 5Go, C and D).



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Figure 5. Morphology of Mammary Glands from Hormone-Treated and Pregnant Mice

The progression of morphological changes in control mammary glands in response to the onset of pregnancy (panel A, 10-week virgin; B, day 2 pregnancy; C, day 4 pregnancy; D, day 12 pregnancy) and in E- and P-treated (panel E, untreated; panel F, E + P, 2 days of treatment; panel G, E + P, 4 days of treatment; panel H, E + P, 12 days of treatment). Mammary glands isolated from control and hormonally treated mice were stained with hematoxylin as described in Materials and Methods. Note the transient alveoli in panel F. The arrow in panel G denotes the hypertrophic duct. Bar = 100 µm.

 
These morphological alterations observed in hormonally treated and normal mammary glands can be correlated with the E- and P-induced changes in Wnt-2 and Wnt-5B gene expression. The appearance of transient alveoli at day 2 coincides with the decrease in Wnt-2 expression observed in E- and P-treated and normal glands. Conversely, permanent and functionally capable alveoli appear after 4–8 days of E and P treatment, preceding the increase in Wnt-5B expression. P concentrations increase gradually during pregnancy, affecting the formation of alveoli and the induction of the differentiated alveolar phenotype (6, 17). Appropriately, the increase in Wnt-5B expression in the E- and P-treated mouse requires long-term treatment of steroid hormones mimicking the pattern of morphological and gene expression changes, observed in the pregnant gland.

Differential Regulation of Wnt-2 and Wnt-5B Gene Expression in PR-/- Mammary Glands and in PR-/- Epithelium Transplanted into PR+/+ Stroma after E and P Treatment
The response of Wnt-2 and Wnt-5B gene expression to the onset of pregnancy and exogenous E and P suggested that the P-signaling pathway might play a primary role in regulating Wnt gene expression in the mammary gland. To examine the role of the PR in Wnt gene regulation, Wnt gene expression levels were determined in PR-/- mice (12) after treatment with E and P. Wnt-5B gene expression did not change in response to E and P treatment in PR-/- mice (n = 3, Fig. 6AGo). However, the E and P repression of Wnt-2 expression was still observed but was not significant until day 8 of hormone treatment (P < 0.003, n = 3, Fig. 6BGo). This E-induced decrease in Wnt-2 gene expression was observed in the absence of any detectable changes in mammary gland morphology in the PR-/- mice.



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Figure 6. The Response of Wnt Gene Expression in PR-/- Mammary Glands and in Transplanted PR-/- Epithelium into PR+/+ Stroma to Steroid Hormone Treatment

A, Quantitative RT-PCR analysis of Wnt-5B gene expression from PR-/- mammary glands after daily injections with E and P, subcutaneously, for 0, 4, and 8 days (n = 3). B, Quantitative RT-PCR analysis of Wnt-2 gene expression from PR-/- mammary glands after daily injections with E and P, subcutaneously, for 0, 4, and 8 days. The star denotes that EP 8 is statistically different from EP 0; P < 0.001, n = 3. C and D, PR-/- epithelium was transplanted into cleared fat pads of PR+/+ 129SvEv mice. After 10 weeks of growth, the mice were treated with E and P. C, Quantitative RT-PCR analysis of Wnt-5B gene expression in transplanted mammary glands after daily injections with E and P, subcutaneously, for 0, 4, and 8 days. Values represent the mean ± SEM (n = 6). D, Quantitative RT-PCR analysis of Wnt-2 gene expression in transplanted mammary glands after daily injections with E and P, subcutaneously., for 0, 4, and 8 days. Values represent the mean ± SEM. The star denotes that EP 8 is statistically different from EP 0; P < 0.013, n = 6.

 
To determine whether stromal or epithelial PRs were involved in differential Wnt gene response, the levels of Wnt transcripts were quantitated in RNA isolated from the previously described transplants. The role of the epithelium in the induction of Wnt-2 and Wnt-5B gene regulation was examined using PR-/- epithelium, transplanted into the stromal fat pad of cleared PR+/+ hosts. In these transplants no increase in the level of P-dependent Wnt-5B expression was observed after 8 days of E and P treatment (n = 6, Fig. 6CGo). To confirm that the low level of Wnt-5B expression seen in the different samples was due to the response in the epithelium and not due to absence or degradation of RNA, the expression of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was also analyzed by RT-PCR. G3PDH transcripts are readily detectable and constant throughout the treatment period (data not shown). The same 129SvEv mice containing transplanted PR-/- epithelium and treated with E and P for 8 days still possessed the ability to repress Wnt-2 expression when subjected to steroid hormone treatment (P < 0.013, n = 3, Fig. 6DGo). Interestingly, the reduction of Wnt-2 gene expression was again delayed requiring 8 days of steroid hormone treatment as observed previously in PR-/- mice.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PR Deficiency in the Stroma and Epithelium Has Distinct Effects on Mammary Gland Development
The absence of lobuloalveolar development in the PR-/- transplants into the PR+/+ fat pad after steroid hormone treatment indicates that the PR in the epithelium is required for this stage of mammary gland development. These results are consistent with previous studies showing that the absence of the PR influences lobuloalveolar development (12). Furthermore, in the reciprocal transplants, PR+/+ epithelium into PR-/- stroma (Fig. 2Go, D and E), lobuloalveolar development was observed in response to E and P, indicating that the absence of the PR in the stroma does not significantly impair the ability of the epithelium to undergo alveolar differentiation. However, ductal growth was impaired, as the PR+/+ epithelium failed to fill the PR-/- fat pad. In addition to the limited ductal development, unusual distended endbuds were present in these outgrowths. This restricted ductal growth may be due to disruption of reciprocal interactions between the stromal and epithelial compartments required for this stage of development. Less dramatic effects on ductal morphogenesis have been observed in the PR-/- mouse (12). This suggests that the PR-/- stroma, when recombined with PR+/+ epithelium in vivo, lacks the mechanism to correctly interact with and stimulate growth in the epithelium and that the stroma fails to generate signals upon which the ductal growth of the epithelium is dependent. In contrast, in the PR-/- mouse, the PR-/- epithelium, in the presence of PR-/- stroma, has adapted to the absence of reciprocal signals, and the epithelium has become independent of these inputs required for ductal growth. This hormone-dependent reciprocity of growth regulation has been observed previously in recombination experiments with wild type and TfM androgen-insensitive mammary glands. These experiments revealed that the epithelium can induce the expression of mesenchymal androgen receptors. In turn, the mesenchyme condenses around the epithelium and causes an epithelial regression (18, 19).

The PR in the stroma is expressed in a temporally distinct pattern from the epithelial receptor, has a different signaling mechanism, and affects a separate group of target genes (5). Therefore, these data support the theory of two separate functional effects of mammary gland PR based on their compartmentalization and roles in development. The unexpected results observed in transplants of PR+/+ epithelium in the PR-/- stroma suggest that there is a P-dependent stromal signal for ductal development. Recent in vivo studies of murine mammary glands treated with HGF in the presence of E and P suggest that HGF is a potential candidate second messenger for ductal growth in the mammary gland (16, 20). HGF-treated mammary glands respond to E and P by stimulating ductal growth. Interestingly, this growth factor has also been shown to regulate Wnt-5A expression (21). If this hypothesis is valid, it may be possible to rescue the PR-/- stroma defect by the direct addition of HGF. Unfortunately, because HGF knockouts are embryonic lethal and die before E16.5, no information has been obtained to date on mammary ductal development in these knockout mice (22).

Wnt-2 and Wnt-5B Gene Expression Are Regulated Independently by Steroid Hormones
This study demonstrates that two developmentally regulated Wnt genes are regulated by distinct mechanisms. The unique temporal and spatial patterns of expression of Wnt-2 and Wnt-5B suggest that these genes may play some role in the development of the mammary gland. The response of Wnt-2 and Wnt-5B to E and P treatment indicates that these genes are useful markers for the action of E in the virgin mammary gland, and for P during pregnancy, respectively. Wnt-2 gene expression is highest in the immature virgin gland of BALB/c mice and declines rapidly at the onset of pregnancy (9). In ovx and intact BALB/c mice this effect can be mimicked with the addition of pharmacological doses of E. This acute repression of Wnt-2 gene expression is correlated with the appearance of lobuloalveolar structures and the termination of ductal development. Conversely, an increase in Wnt-5B gene expression in ovx and intact mice requires chronic treatment with E and P. Ovariectomy enhances the magnitude of this response. Without the stimulation from the ovaries, the basal levels of Wnt-5B expression are probably significantly reduced, thereby allowing an enhanced response. Wnt 5B provides one of the few molecular endpoints for the action of P, and changes in Wnt 5B are coincident with lobuloalveolar development.

The results from the PR-/- studies demonstrate that Wnt-5B gene expression is induced by P and dependent on the presence of PR specifically in the epithelial compartment. E alone has no effect on Wnt-5B expression. Interestingly, the PR present in the stroma cannot compensate for the absence of PR in the epithelium for lobuloalveolar development or for induction of Wnt 5B gene expression. This result implies that P acts directly on the epithelium to induce lobuloalveolar development and, either directly or indirectly, to activate Wnt-5B gene expression. E is required for induction and maintenance of PR expression in the mammary gland (6). Therefore, it is unlikely that the regulation of Wnt-5B expression is independent of E.

Wnt-2 gene expression was inhibited by administration of E in both PR-/- mice and in transplanted PR-/- epithelium. These results suggest that Wnt-2 gene expression is not primarily regulated by PR signaling. In the PR-/- studies there was a delay in the kinetics of the Wnt-2 response. The absence of the PR may have restricted the development of the gland in these mice and slowed the appearance of the ER, which normally appears at 4 weeks of age (23, 24). Alternatively, the absence of the PR could influence reciprocal interactions between the stroma and the epithelium, thereby preventing proper induction of ERs. ER gene expression is affected by feedback controls between E and other hormones including P (14).

Previous studies performed in Parks mice demonstrated Wnt-2 expression through midpregnancy and a repression of Wnt-2 and Wnt-5B expression after ovariectomy (11). Parks mice possess virgin lobuloalveolar development, which is absent in BALB/c mice, and it is possible that this epithelial sensitivity to estrous-associated hormones alters the regulation of Wnt-2 and Wnt-5B gene expression.

The rapid repression of Wnt-2 expression suggests the ER may be directly regulating Wnt-2. The ER is expressed in both the stroma and epithelium including the endbud (5, 14, 23, 24, 25). Interestingly, PR is induced by the ER-signaling pathway in the epithelial compartment 48 h after initial addition of E (26). This temporal delay in receptor response coincides exactly with the initial decrease observed in Wnt-2 gene expression after hormonal treatment. Therefore, the timing of this E-induced gene expression and localization of some Wnt-2 transcripts in the epithelium suggests that Wnt-2 could be regulated directly by E.

The Wnts Are Growth Factors with Pleiotropic Effects on Development
The development of the mammary gland is dependent on the interaction and cooperation of growth factors and hormones functioning through the stromal and epithelial compartments. Studies of PRL, epidermal growth factor, FGF, TGF{alpha} and ß, insulin-like growth factor, and HGF action reveal that they are regulated in specific spatial and temporal patterns and have effects on proliferation and differentiation in mammary gland development (1, 27, 28, 29, 30). The developmentally associated expression pattern, their role in the development of other organisms, biochemical characteristics, and hormonal regulation of the Wnts suggest that they are members of this complex family of locally acting growth factors.

The function of the Wnt genes in the development of the mammary gland can only be inferred from limited expression studies in vivo and in vitro and functional studies in other organisms. Wingless, the Drosophila homolog of Wnt-1, has proliferative, inductive, and cell fate determination functions (8). In addition, Wnt genes have demonstrated functional roles in Xenopus, mouse, and chicken (31, 32, 33, 34, 35, 36). These diverse studies revealed that Wnt genes can possess inductive, growth-stimulatory, and growth-restrictive functions all within a single organism.

In the mammary gland, overexpression of Wnt-1 influences the proliferation of mammary epithelium (37). The expression of Wnt-4 and Wnt-5A has been inversely correlated with proliferation in mammary epithelial cells (38). Because of its localization both within and around the highly proliferative terminal endbud, it is possible that Wnt-2 has a role in regulating proliferation in the virgin gland. The pattern of Wnt-5B expression and its dependence on the PR suggests that it interacts with cells in a more differentiated state. Interestingly, proliferation is high in the pregnant gland coincident with the increase in Wnt-5B expression. Localization of Wnt-5B transcripts to the ductal epithelium reveals it is expressed in the proper cellular location to be involved in regulating proliferation in these cells. The localization of Wnt-5B and Wnt-2 transcripts to the ductal epithelial and stromal compartments of the mammary gland (9, 11), respectively, suggest that although these two genes may have separate or even overlapping functional roles, their temporal and spatial expression patterns restrict their activity to specific stages of development. Therefore, it is probable that the expression of these Wnt genes is regulated in a specific manner to restrict their functional activities to particular developmental stages in the mammary gland.

Alteration of Wnt Gene Expression Can Transform Mammary Epithelium
In the mammary gland, ectopic expression of the Wnt genes has dramatic consequences on the transformation and development of the gland. Inappropriate expression of Wnts either temporally or spatially may result in mammary tumorigenesis. For example, Wnt-1 and Wnt-4 have been demonstrated to affect the development and transformation of the gland in vivo (37, 39, 40). Numerous other Wnts, including Wnt-2 and Wnt-5B, have in vitro transforming effects (41, 42). These in vitro transfection experiments have revealed that separate classes of Wnts exist that are distinguished by their transforming ability (43), although the properties defined in these in vitro assays do not always correspond to their effects in vivo (R. C. Humphreys and J. M. Rosen, submitted for publication).

In addition, overexpression of Wnt genes, including Wnt-2 and Wnt-5B, has been found associated with tumors in the breast and intestinal epithelium (44, 45, 46, 47). Thus, loss of regulatory control on these two Wnt genes, as with other growth factor molecules like TGF-ß and FGF (48, 49), has deleterious consequences for the development of the mammary gland. Interestingly, compartment switching of Wnt-2 expression from breast fibroblasts to tumor epithelium has been observed recently in human breast tumors (50). Therefore, there is evidence for a critical role of Wnt-2, and possibly Wnt-5B, in the transformation of the gland. Since most of the Wnt knockouts are embryonic lethals resulting in neural or kidney defects, the precise functional roles of these and other Wnt family members on normal mammary gland development will require the use of tissue-specific or regulated knockouts.

To summarize, the Wnt genes act in a cell-autonomous manner in cooperation with other growth factors and have pleuripotent effects on various developmental processes within the same organism (8). Wnt gene expression can be differentially regulated by steroid hormones in the mammary stroma and epithelium where they may act as locally acting growth factors to influence ductal and lobuloalveolar development. Hopefully, with the recent discovery of the Wnt receptor in Drosophila (51), the mechanism of Wnt action and the function of the individual Wnt family members in mammary gland development will begin to be illuminated.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals
BALB/c mice were acquired from Charles River Laboratories (Wilmington, MA) or from a breeding colony at Baylor College of Medicine, courtesy of Dr. Daniel Medina. Mice carrying the PRKO mutation in the 129SvEv inbred genetic background were used in these studies. All animals were maintained according to IACUC approved guidelines.

Isolation of Mammary Glands and RNA
Number 4 (thoracic) mammary glands were removed from aged matched 6- and 8-week-old virgin BALB/c and C3H mice using standard surgical techniques. The thoracic and inguinal mammary glands from 11-week-old wild type control mice, 6-week-old 129SvEv PR-/- mice, 129SvEv PR+/+, and 129SvEv PR-/- mice with transplanted PR-deficient epithelium or wild type epithelium, respectively, were removed using standard surgical techniques. For morphological analysis, mammary glands were fixed in Tellyesniczky’s solution for 5 h and stained with hematoxylin as described previously (52). For isolation of RNA, mammary glands were homogenized in a PT2000 Polytron (Brinkmann, Westbury, NY) with RNazol (Biotecx, Houston, TX) as described by the manufacturer or homogenized in 4 M guanididium isothiocynate (Sigma, St. Louis MO) and isolated by CsCl centrifugation method. RNA was quantitated spectrophotometrically and stored at -20 C in 70% ethanol.

Construction and Transcription of cRNA Templates
Quantitative noncompetitive RT-PCR was performed as previously described (9). Complementary DNAs from Wnt-5B and Wnt-2 were prepared according to standard bacterial plasmid isolation protocols, and the DNA was purified on Qiagen (Qiagen, Chatsworth, CA) columns according to the manufacturer and isolated from the vector using unique restriction enzymes. To construct the Wnt-2 cDNA deletion template, StyI (New England Biolabs, Beverly, MA) was used to excise a fragment from bases 493–580. Digestion products were separated from the small internal fragment, religated, and subcloned into the vector pBKSII (Stratagene, La Jolla, CA). Clones were analyzed for a size difference and sequenced to confirm the location of the deletion. The Wnt-5B template was constructed in the same manner with an AvaI (New England Biolabs, Beverly MA) deletion of bases 501–576. Both templates were sequenced to confirm the orientation in the vector and the presence of an internal deletion. These constructs were used as templates for in vitro transcription reactions as described in Promega Protocols and Applications Guide, ed 2 (Promega, Madison WI). The cRNA reactions were treated with 1 U of ribonuclease-free RQ1 deoxyribonuclease in deoxyribonuclease buffer (Promega) for 60 min at 37 C and then extracted with phenol-chloroform twice and precipitated with 3 M NaAc and 100% ethanol at -20 C. The cRNA was resuspended in Tris-EDTA, quantitated spectrophotometrically, and stored at -20 C in 70% ethanol. Each template was assayed by PCR to confirm the absence of contaminating cDNA template. Optimum RT-PCR conditions for each of the templates were developed that allowed a linear response with respect to the RNA input and exhibited noncompetitive PCR.

Quantitative RT-PCR
Isolated RNA was transcribed in a reaction consisting of 1x Taq polymerase buffer (Promega), 3 mM MgCl2, 100 pmol hexanucleotide random primers (Boehringer Mannheim, Indianapolis, IN) 1.25 U of RT (GIBCO BRL, Gaithersburg MD), 1 mM of each of four deoxynucleoside triphosphates (Pharmacia, Milwaukee WI), and 20 U of RNasin (Pharmacia, Milwaukee WI) in a final reaction volume of 20 µl. Fifty nanograms, 100 ng, and 150 ng of sample RNA were added to separate RT reactions. A constant amount of cRNA template (~10,000 molecules) was added to each RT reaction as an internal standard to control for differences in RT and PCR reaction efficiency.

The primer sequences for the Wnt-2 and Wnt-5B amplifications, respectively, were:

forward: 5'-AGTCGGGAATCGGCCTTTGTTTACG-3' and reverse: 5'-AAAGTTCTTCGCGAAATGTCGGAAG-3'; forward: 5'-GACAGCGCCGCGGCCATGCGC-3' and reverse: 5'-CATTTGCAGGCGACATCAGC-3'. PCR conditions were 94 C for 1 min, 60 C for 2 min, and 72 C for 3 min, for 30 cycles and 94 C for 1 min, 65 C for 2 min, and 72 C for 3 min for 32 cycles for Wnt-2 and Wnt-5B, respectively. Primers for G3PDH were: forward: 5'-AGAGGCCTTTGCTCGAACTGGAAAG-3' and reverse: 5'-CACCAAGACGTCTGTCGCCTACTTA-3. PCR conditions were 94 C for 1 min, 60 C for 2 min, and 72 C for 3 min, for 30 cycles. All PCRs were followed by an extension at 72 C for 5 min. PCR was performed with 10 µl of each RT reaction, 2 mM magnesium chloride, 1xPCR buffer (Promega), 0.1 µCi [{alpha}-32P]dCTP (NEN DuPont, Boston, MA), 1 U of Taq polymerase (Promega, Madison WI) in a final reaction volume of 50 µl. Ten microliters of the RT-PCR products were separated on a 2% Nusieve agarose (FMC Bioproducts, Rockland, ME) gel and transferred overnight in 0.4 M NaOH to Hybond N+ nylon membrane (Amersham, Buckinghamshire, UK), and the radioactive signal was quantitated with 4–8 h exposure on a PhosphoImager (Molecular Dynamics, Sunnyvale, CA).

Steroid Hormone Treatment of Mice
All groups of mice were treated with 1 mg of P (Steris, Phoenix, AZ) and 1 µg of 17 ß-estradiol (Sigma) per day in 60 µl of sesame seed oil (Sigma) subcutaneously. Mammary glands were collected at days 0, 1, 2, 4, 8, 12, and 18. Animals were ovariectomized and allowed to regress for 4 weeks before hormone treatments were begun. Beeswax implants containing 20 µg of E and/or 20 mg of P were synthesized by adding the powdered form of the hormones to melted beeswax. The suspended hormone mixture was dropped onto dry ice to form pellets. The pellets, synthesized to deliver 1 mg P and 1 µg E/day, respectively, were implanted subcutaneously in the neck of the mice for 2 weeks. Inguinal mammary glands were collected at days 1, 3, and 14 and analyzed as described.

Transplantation Studies
Tissue fragments of 10-week-old virgin PR-/- mammary epithelium were isolated and implanted into six PR-positive 129SvEv hosts using the technique described by DeOme et al. (13). Epithelium from 129SvEv hosts was removed as described (13). In addition, tissue fragments from a 10-day pregnant 129SvEv PR+/+ mammary epithelium were isolated and implanted into four PR-/- 129SvEv hosts. Due to the limited number of PR-/- homozygote recipients and the limited extent of ductal outgrowth, these glands could not be examined for changes in Wnt gene expression. Mammary gland epithelial transplants were allowed to proliferate and penetrate the stromal fat pad for 10 weeks and then treated with steroid hormones as described above. Control experiments with wild type 129SvEv PR+/+ epithelium into cleared fat pads of three wild type 129SvEv PR+/+ mice were performed in the same manner.

Whole Mount Staining and Sectioning
The whole gland staining was carried out essentially as described (25) except that glands were stained for only 2 h in hematoxylin.


    ACKNOWLEDGMENTS
 
The authors thank Dr. Susanne Krnacik for providing the RNA for the ovariectomy experiments and for critical reading of the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Jeffrey M. Rosen, Baylor College of Medicine, Houston, Texas 77030.

This work was supported by NIH Grant CA-64255 and Grant DAMD17-94-J-4253 from the Department of Defense (to J.M.R.).

Received for publication September 9, 1996. Revision received November 14, 1996. Accepted for publication November 21, 1996.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Imagawa W, Yang J, Guzman R, Nandi S 1994 Control of Mammary Gland Development. In: Knobrl E, Neill JD (eds) The Physiology of Reproduction. Raven Press, New York, pp 1033–1063
  2. Daniel CW, Silberstein GB 1987 Postnatal development of the rodent mammary gland. In: Neville MC, Daniel CW (eds) The Mammary Gland. Plenum Press, New York, pp 3–36
  3. Vonderhaar BK 1988 Regulation of development of the normal mammary gland by hormones and growth factors. Cancer Treat Res 40:251–266[Medline]
  4. Cunha GR, Hom YK 1996 Role of mesenchymal-epithelial interactions in mammary gland development. J Mammary Gland Biol Neoplasia 1:21–35[Medline]
  5. Haslam SZ, Shyamala G 1981 Relative distribution of estrogen and progesterone receptors among the epithelial, adipose, and connective tissue components of the normal mammary gland. Endocrinology 108:825–830[Abstract]
  6. Haslam SZ 1988 Acquisition of estrogen-dependent progesterone receptors by normal mouse mammary gland. Ontogeny of mammary progesterone receptors. J Steroid Biochem 31:9–13[CrossRef][Medline]
  7. Nusse R, van OA, Cox D, Fung YK, Varmus H 1984 Mode of proviral activation of a putative mammary oncogene (int-1) on mouse chromosome 15. Nature 307:131–136[Medline]
  8. Klingensmith J, Nusse R 1994 Signaling by wingless in Drosophila. Dev Biol 166:396–414[CrossRef][Medline]
  9. Bühler TA, Dale TC, Kieback C, Humphreys RC, Rosen JM 1993 Localization and quantification of Wnt-2 gene expression in mouse mammary development. Dev Biol 155:87–96[CrossRef][Medline]
  10. Gavin BJ, McMahon AP 1992 Differential regulation of the wnt gene family during pregnancy and lactation suggests a role in postnatal development of the mammary gland. Mol Cell Biol 12:2418–2423[Abstract]
  11. Weber-Hall SJ, Phippard DJ, Niemeyer CC, Dale TC 1994 Developmental and hormonal regulation of Wnt gene expression in the mouse mammary gland. Differentiation 57:205–214[CrossRef][Medline]
  12. Lydon JP, DeMayo FJ, Funk CR, Mani SK, Hughes AR, Montgomery CJ, Shyamala G, Conneely OM, O’Malley BW 1995 Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities. Genes Dev 9:2266–2278[Abstract]
  13. DeOme KB, Faulkin LJ, Bern HA, Blair PB 1958 Development of mammary tumors from hyperplastic alveolar nodules transplanted into gland-free mammary pads of female C3H mice. Cancer Res 19:515–519
  14. Haslam SZ 1989 The ontogeny of mouse mammary gland responsiveness to ovarian steroid hormones. Endocrinology 125:2766–2772[Abstract]
  15. Vonderhaar BK 1984 Hormone and growth factors in mammary gland development. In: Veneziale CM (ed) Control of Cell Growth and Proliferation. Van Nostrand-Reinhold, Princeton, NJ, pp 11–33
  16. Jones FE, Jerry JJ, Guarino BC, Andrews GC, Stern DF 1996 Heregulin induces in vivo proliferation and differentiation of mammary epithelium into secretory lobuloalveoli. Cell Growth Differ 7:1031–1038[Abstract]
  17. Murr SM, Stabenfeldt GH, Bradford GE, Geschwind II 1974 Plasma progesterone during pregnancy in the mouse. Endocrinology 94:1209–1211[Medline]
  18. Heuberger B, Fitzka I, Wasner G, Kratochwil K 1982 Induction of androgen receptor formation by epithelium-mesenchyme interaction in embryonic mouse mammary gland. Proc Natl Acad Sci USA 79:2957–2961[Abstract]
  19. Kratochwil K 1987 Tissue combination and organ culture studies in the development of the embryonic mammary gland. In: Gwatkin RBL (ed) Developmental Biology: A Comprehensive Synthesis. Plenum Press, New York, pp 315–334
  20. Yang Y, Spitzer E, Meyer D, Sachs M, Niemann C, Hartmann G, Weidner KM, Birchmeier C, Birchmeier W 1995 Sequential requirement of hepatocyte growth factor and neuregulin in the morphogenesis and differentiation of the mammary gland. J Cell Biol 131:215–226[Abstract]
  21. Huguet EL, Smith K, Bicknell R, Harris AL 1995 Regulation of Wnt5a mRNA expression in human mammary epithelial cells by cell shape, confluence, and hepatocyte growth factor. J Biol Chem 270:12851–12856[Abstract/Free Full Text]
  22. Schmidt C, Bladt F, Goedecke S, Brinkmann V, Zschlesche W, Sharpe M, Gherardl E, Birchmeier C 1995 Scatter factor/hepatocyte growth factor is essential for liver development. Nature 373:699–702[CrossRef][Medline]
  23. Muldoon TG 1978 Characterization of mouse mammary tissue estrogen receptors under conditions of differing hormonal backgrounds. J Steroid Biochem 9:485–494[CrossRef][Medline]
  24. Hunt ME, Muldoon TG 1977 Factors controlling estrogen receptor levels in normal mouse mammary tissue. J Steroid Biochem 8:181–186[CrossRef][Medline]
  25. Daniel CW, Silberstein GB, Strickland P 1987 Direct action of 17 beta-estradiol on mouse mammary ducts analyzed by sustained release implants and steroid autoradiography. Cancer Res 47:6052–6057[Abstract]
  26. Shyamala G, Ferenczy A 1984 Mammary fat pad may be a potential site for initiation of estrogen action in normal mouse mammary glands. Endocrinology 115:1078–1081[Abstract]
  27. Knight CH, Peaker M 1982 Development of the mammary gland. J Reprod Fertil 65:521–536[CrossRef][Medline]
  28. Nandi S 1959 Hormonal control of mammogenesis and lactogenesis in the C3H/He Crgl mouse. In: Stern C, Benson S, Quay W (eds) University of California Berkeley Publications in Zoology. University of California Press, Berkeley, pp 1–128
  29. Topper YJ, Freeman CS 1980 Multiple hormone interactions in the developmental biology of the mammary gland. Physiol Rev 60:1049–1106[Free Full Text]
  30. Plaut K, Ikeda M, Vonderhaar BK 1993 Role of growth hormone and insulin-like growth factor-I in mammary development. Endocrinology 133:1843–1848[Abstract]
  31. Cui Y, Brown JD, Moon RT, Christian JL 1995 Xwnt-8b: a maternally expressed Xenopus Wnt gene with a potential role in establishing the dorsoventral axis. Development 121:2177–2186[Abstract/Free Full Text]
  32. Du SJ, Purcell SM, Christian JL, McGrew LL, Moon RT 1995 Identification of distinct classes and functional domains of Wnts through expression of wild-type and chimeric proteins in Xenopus embryos. Mol Cell Biol 15:2625–2634[Abstract]
  33. Augustine KA, Liu ET, Sadler TW 1995 Interactions of Wnt-1 and Wnt-3a are essential for neural tube patterning. Teratology 51:107–119[Medline]
  34. Hollyday M, McMahon JA, McMahon AP 1995 Wnt expression patterns in chick embryo nervous system. Mech Dev 52:9–25[CrossRef][Medline]
  35. Yoshioka H, Ohuchi H, Nohno T, Fujiwara A, Tanda N, Kawakami Y, Noji S 1994 Regional expression of the Cwnt-4 gene in developing chick central nervous system in relationship to the diencephalic neuromere D2 and a dorsal domain of the spinal cord. Biochem Biophys Res Commun 203:1581–1588[CrossRef][Medline]
  36. McMahon AP, Bradley A 1990 The wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain. Cell 62:1073–1085[Medline]
  37. Edwards PA, Hiby SE, Papkoff J, Bradbury JM 1992 Hyperplasia of mouse mammary epithelium induced by expression of the Wnt-1 (int-1) oncogene in reconstituted mammary gland. Oncogene 7:2041–2051[Medline]
  38. Olson DJ, Papkoff J 1994 Regulated expression of Wnt family members during proliferation of C57mg mammary cells. Cell Growth Differ 5:197–206[Abstract]
  39. Lin TP, Guzman RC, Osborn RC, Thordarson G, Nandi S 1992 Role of endocrine, autocrine, and paracrine interactions in the development of mammary hyperplasia in Wnt-1 transgenic mice. Cancer Res 52:4413–4419[Abstract]
  40. Bradbury JM, Edwards PA, Niemeyer CC, Dale TC 1995 Wnt-4 expression induces a pregnancy-like growth pattern in reconstituted mammary glands in virgin mice. Dev Biol 170:553–563[CrossRef][Medline]
  41. Bradley RS, Brown AM 1995 A soluble form of Wnt-1 protein with mitogenic activity on mammary epithelial cells. Mol Cell Biol 15:4616–4622[Abstract]
  42. Blasband A, Schryver B, Papkoff J 1992 The biochemical properties and transforming potential of human wnt-2 are similar to wnt-1. Oncogene 7:153–161[Medline]
  43. Wong GT, Gavin BJ, McMahon AP 1994 Differential transformation of mammary epithelial cells by Wnt genes. Mol Cell Biol 14:6278–6286[Abstract]
  44. Huguet EL, McMahon JA, McMahon AP, Bicknell R, Harris AL 1994 Differential expression of human Wnt genes 2, 3, 4, and 7B in human breast cell lines and normal and disease states of human breast tissue. Cancer Res 54:2615–2621[Abstract]
  45. Iozzo RV, Eichstetter I, Danielson KG 1995 Aberrant expression of the growth factor Wnt-5A in human malignancy. Cancer Res 55:3495–3499[Abstract]
  46. Lejeune S, Huguet EL, Hamby A, Poulsom R, Harris AL 1995 Wnt5a cloning, expression, and upregulation in human primary breast cancers. Clin Cancer Res 1:215–222[Abstract]
  47. Vider BZ, Zimber A, Chastre E, Prevot S, Gespach C, Estlein D, Wolloch Y, Tronick SR, Gazit A, Yaniv A 1996 Evidence for the involvement of the Wnt 2 gene in human colorectal cancer. Oncogene 12:153–158[Medline]
  48. MacArthur CA, Shankar DB, Shackleford GM 1995 Fgf-8, activated by proviral insertion, cooperates with the Wnt-1 transgene in murine mammary tumorigenesis. J Virol 69:2501–2507[Abstract]
  49. Shackleford GM, MacArthur CA, Kwan HC, Varmus HE 1993 Mouse mammary tumor virus infection accelerates mammary carcinogenesis in Wnt-1 transgenic mice by insertional activation of int-2/Fgf-3 and hst/Fgf-4. Proc Natl Acad Sci USA 90:740–744[Abstract]
  50. Dale TC, Weber-Hall SJ, KS, Huguet EL, Jayatilake H, Gusterson BA, Shuttleworth G, O’Hare M, Harris AL 1996 Compartment switching of WNT-2 expression in human breast tumors. Cancer Res 56:4320–4323[Abstract]
  51. Bhanot P, Brink M, Samos CH, Hsieh J-C, Wang Y, Macke JP, Andrew D, Nathans J, Nusse R 1996 The new member of the frizzled family from Drosophila functions as a wingless receptor. Nature 382:225–230[CrossRef][Medline]
  52. Humphreys RC, Krajewska M, Krnacik S, Jaeger R, Weiher H, Krajewski S, Reed JC, Rosen JM 1996 Apoptosis in the terminal endbud of the murine mammary gland: a mechanism of ductal morphogenesis. Development 122:4013–4022[Abstract/Free Full Text]