Developmental Diethylstilbestrol Exposure Alters Genetic Pathways of Uterine Cytodifferentiation
Wei-Wei Huang,
Yan Yin,
Qun Bi,
Tung-Chin Chiang,
Neysa Garner,
Jussi Vuoristo,
John A. McLachlan and
Liang Ma
Tulane/Xavier Center for Bioenvironmental Research (W.-W.H., T.-C.C., N.G., J.A.M.) and Center for Gene Therapy (J.V.), Tulane University Medical Center, New Orleans, Louisiana 70112; and Department of Cell and Molecular Biology (Y.Y., Q.B., L.M.), Tulane University, New Orleans, Louisiana 70118
Address all correspondence and requests for reprints to: Liang Ma, Division of Dermatology, Department of Medicine, Washington University, Campus Box 8123, 660 South Euclid Avenue, St. Louis, Missouri 63110. E-mail: lima{at}im.wustl edu.
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ABSTRACT
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The formation of a simple columnar epithelium in the uterus is essential for implantation. Perturbation of this developmental process by exogenous estrogen, such as diethylstilbestrol (DES), results in uterine metaplasia that contributes to infertility. The cellular and molecular mechanism underlying this transformation event is not well understood. Here we use a combination of global gene expression analysis and a knockout mouse model to delineate genetic pathways affected by DES. Global gene expression profiling experiment revealed that neonatal DES treatment alters uterine cell fate, particularly in the luminal epithelium by inducing abnormal differentiation, characterized by the induction of stratified epithelial markers including members of the small proline-rich protein family and epidermal keratins. We show that Msx2, a homeodomain transcription factor, functions downstream of DES and is required for the proper expression of several genes in the uterine epithelium including Wnt7a, PLAP, and K2.16. Finally, Msx2/ uteri were found to exhibit abnormal water trafficking upon DES exposure, demonstrating the importance of Msx2 in tissue responsiveness to estrogen exposure. Together, these results indicate that developmental exposure to DES can perturb normal uterine development by affecting genetic pathways governing uterine differentiation.
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INTRODUCTION
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THE EMBRYONIC MÜLLERIAN duct derives from the intermediate mesoderm and gives rise to the oviduct, uterus, cervix, and upper third of the vagina during mammalian embryonic development. The initial formation of the Müllerian duct requires Lim-1 because female mice lacking this homeodomain protein have no uterus (1). Subsequently, Abdominal B Hox genes are required to establish the segmental boundaries between these structures along the developing Müllerian duct as mutations in these genes result in region-specific developmental defects in the female reproductive tract (2, 3, 4). The determination of epithelial cell fate along the Müllerian duct is interesting because epithelial cells residing at different axial levels differentiate into different epithelial cell types. The uterine luminal and glandular columnar epithelia are derived from the anterior Müllerian epithelium, whereas the vaginal stratified squamous epithelium is derived from the posterior Müllerian epithelium. Classical tissue recombination experiments demonstrated that this cell fate determination event involves reciprocal interactions between the epithelium and the underlying stroma (5). The determination of uterine epithelium occurs between postnatal d 5 and 7 and is thought to be meditated by signals from the stroma. Before this time, the fate of uterine epithelium can be changed to that of vaginal epithelium when combined with vaginal mesenchyme, whereas the converse recombination results in uterine epithelial differentiation (5, 6). After postnatal d 7 (P7), the fate of uterine epithelium is determined and cannot be changed by mesenchymal cues from the vagina. Molecularly, epithelial p63 expression is induced by signals from the cervical or vaginal mesenchyme and is thought to be an "identity switch" that controls stratified epithelial differentiation in the female reproductive tract (7). Although the nature of these stromal signals is not clear at present, swapping the homeodomain of Hoxa-11 with that of Hoxa-13 resulted in the expression of a hybrid Hox protein in the uterine stroma and subsequent stratification of uterine epithelium, suggesting that the stromal signal is likely controlled by Hox proteins (8). In addition, Wnt7a/ mutants were found to develop an abnormal stratified uterine epithelium, suggesting that members of the Wnt family, and possibly other growth factors, may play a vital role in subsequent uterine epithelial differentiation (9).
One approach to uncover genes that are important for uterine development is to identify genes whose expression is altered en route to abnormal uterine differentiation. Proper uterine cytodifferentiation can be disturbed by developmental exposure to a synthetic estrogen diethylstilbestrol (DES) (10). DES was the first synthetic estrogenic compound orally administered to pregnant women (from 19471971) in an effort to preserve pregnancy, and was later found to be a teratogen for the developing fetus (11). DES exposure in humans has been associated with reproductive tract anomalies such as T-shaped uterus, vaginal adenosis, extensive ectropion, annular cervical rings in females (12, 13). In female mice, neonatal DES exposure causes uterine malformations including hypoplasia, stratification of luminal epithelium, disorganized smooth muscle and reduced endometrial glands (10, 14). DES is thought to cause these teratogenic anomalies through both an estrogen-dependent pathway and an epigenetic pathway (14). DES has a binding affinity for the estrogen receptor-
(ER-
) that is much higher than that of the naturally occurring estrogen 17ß-estradiol (15), suggesting that DES functions as a strong estrogen. Indeed, the majority of the reproductive patterning defects observed in DES-treated mice are mediated through ER-
(14). Consistently, a number of developmental control genes, including several Hox and Wnt genes that were previously shown to be potently repressed by DES during critical periods of reproductive tract patterning, failed to be repressed in ER-
knockout mice (3, 14, 16). A study comparing DES and estrogen targets revealed that these two compounds share around 45% of their target genes, while at the same time, many targets are unique to each compound (17). These data provide evidence that DES binds to ER-
to induce abnormal differentiation of the entire female reproductive tract.
Little is known about the downstream targets of ER-
in this process. Nevertheless, many transcription factors including Msx and the zinc finger Krüpple-like factors (KLFs) have been shown to have important functions in epithelial differentiation. Members of the mammalian Msx homeobox gene family play critical roles in epithelial-mesenchymal interactions during the formation of many organs (18, 19). Although their role in uterine differentiation is not clear at present, both Msx1 and Msx2 have been shown to function in genetic pathways involving growth factors such as bone morphogenetic proteins and WNTs in other organs (20, 21, 22). Msx1 expression was detected in the uterine epithelium and its expression was found to be down-regulated during implantation (23). Msx2 is also expressed in the uterus (24), but its role in uterine cyto-differentiation has not been studied. KLF4 (also known as gut-enriched KLF or GKLF) is an epithelial-specific transcription factor known to be expressed in epithelium of the gastrointestinal tract, epidermis, thymus, and vascular endothelial cells and regulates epithelial differentiation (25, 26, 27, 28, 29). The phenotype of Klf4 knockout mice revealed that it is required for the establishment of skin barrier function (30). Interestingly, Small proline-rich protein 2a (Sprr2a), a precursor of the cornified cell envelope, is the only epidermal gene that is known to be a direct target of KLF4 (30, 31). However, the role of KLF4 in uterine differentiation has not been investigated.
Despite extensive research on the effect of DES on uterine patterning, the molecular mechanism underlying these developmental anomalies remains poorly understood. Here we have used a well-established neonatal DES mouse model, microarray technology and Msx2 knockout mice to delineate molecular pathways that are affected by DES exposure in the developing uterus. Our results reveal several novel DES targets in the uterine epithelium and provide a molecular basis for DES-induced uterine metaplasia.
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RESULTS
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DES Regulates Uterine Gene Expression Revealed by Microarray
To uncover the molecular pathways underlying DES-induced uterine malformations, we carried out an expression profiling experiment using a well-established neonatal DES mouse model (10). Wild-type CD-1 pups were injected daily with either corn oil (control) or DES from P15. RNAs from oil- and DES-treated P5 uteri were extracted and biotinylated cRNAs were synthesized. The cRNAs were hybridized to the Affymetrix (Santa Clara, CA) mouse genome array chip 430A2.0. A total of three microarray experiments were performed using three independent RNA preparations. Microarray results from all three experiments were subjected to significance analysis of microarrays (SAM) and threshold analysis. These analyses revealed that only 474 out of the total 22,627 probe sets present on the 430A2.0 chip were significantly changed in response to DES in all three microarray experiments. These results, together with a SAM plot, were listed in supplemental Table 1, published as supplemental data on The Endocrine Societys Journals Online web site at http://mend.endojournals.org. By subtracting repeated probe sets representing the same gene, we calculated that the 474 probe sets correspond to 183 and 244 unique genes that are significantly down- or up- regulated by DES, respectively. These genes were subjected to functional categorization analysis and the results were presented in supplemental Table 1. Our microarray study was consistent with published literatures in that it contains several previously reported DES-regulated genes (14, 17, 32, 33).
DES Alters Uterine Epithelial Cell Fate
Our analysis of the array data revealed that many genes that were strongly up-regulated by DES exposure encode enzymes and structural proteins, whereas some transcription factors and growth factors were down-regulated by DES exposure (Table 1
and supplemental Table 1). These data suggest that developmental DES exposure may have altered uterine cell fate, which is consistent with previous studies documenting morphological changes in DES exposed uterine epithelium from simple columnar to stratified epithelium (10, 14). To test this hypothesis, we examined the level of cell proliferation by bromodeoxyuridine (BrdU) incorporation into P5 uteri after oil or DES treatment. As shown in Fig. 1
, cell proliferation was markedly reduced in DES-treated uteri compared with oil-treated controls at P5 (compare A with B). The most dramatic decrease in cell proliferation was observed in the luminal epithelium, whereas a milder decrease was observed in the stroma. The reduced BrdU incorporation suggests that these uterine epithelial cells may have exited the cell cycle. Once out of the cell cycle, these cells could undergo either apoptosis or differentiation. Analysis of apoptosis using TUNEL [terminal deoxynucleotidyl transferase-mediated deoxy-UTP (uridine triphosphate) nick end labeling] assays revealed a significant number of apoptotic cells in normal uterine epithelium (Fig. 1C
, arrow). In contrast, no apoptotic cells were detected in DES-treated uterine epithelia (Fig. 1D
). Together, these results suggest that luminal epithelial cells have exited the cell cycle and undergone precocious differentiation.

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Fig. 1. Neonatal Exposure to DES Alters Uterine Epithelial Cell Fate
DES (2 µg/pup·d) was injected into neonatal pups from P1-P5, and uteri were harvested on P5 for analysis. DES exposure resulted in a significant decrease in cell proliferation especially in the luminal epithelium as assayed by BrdU incorporation (A and B). Normal uterine epithelial layer contains apoptotic cells (C, arrow), which are no longer observed in DES-treated uteri (D). On the other hand, DES-treated uteri are undergoing premature abnormal differentiation indicated by the expression of PLAP (E and F, arrows). Real-time RT-PCR showed that PLAP expression is not up-regulated by DES after 48 h but is strongly up-regulated after 5 d of DES treatment (G). Scale bars in all figures, 50 µm.
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Differentiation Markers Induced by DES
Uterine luminal epithelium undergoes abnormal differentiation when exposed to DES from P1P5 (10). Our microarray data revealed that DES induces several genes that are normally expressed at high levels in stratified epithelia. One novel candidate is the placental alkaline phosphatase (PLAP; GenBank accession no. AW319615), which is normally detected in the placental labyrinth during placentation (34, 35). To further confirm that this marker of placental epithelium was indeed up-regulated by DES, we stained oil- and DES-treated P5 uterine sections for PLAP activity. As shown in Fig. 1E
, no PLAP activity was observed in oil-treated uterus, whereas strong PLAP activity was observed in DES-treated P5 uterine epithelium (Fig. 1F
, arrows). A time course study using real-time RT-PCR revealed that PLAP expression was not up-regulated by DES after 48 h but was up-regulated in P3 (not shown) and P5 uterine epithelium and this up-regulation lasted through P11 (Fig. 1G
). Throughout these time points, PLAP expression was not detected in the wild-type uterine epithelium. These results suggest that DES-treated uterine epithelial cells have undergone abnormal differentiation.
A striking observation from our microarray results is the significant up-regulation in expression of two small proline-rich proteins, Sprr2a and Sprr2f by DES (Table 1
). The Sprr multigene family is clustered in a 140-kb region within the epidermal differentiation complex (36). The Sprr genes encode proteins that are important components of the cornified cell envelope, a structure synthesized during the late stages of keratinocyte differentiation. Sprr genes are subdivided into three families, Sprr1, 2, and 3, of which the Sprr2 family is the most complex and the most extensively studied. Sprr genes are differentially expressed in various squamous epithelia and in each case, their expression is strictly confined to cells committed to terminal differentiation (37). We focused our study on the regulation of Sprr2a. Sprr2a has been shown to be regulable by estrogen in adult uterus (33). However, its regulation during uterine development by DES has not been described. First, we used real-time RT-PCR to verify the microarray results and to uncover the kinetics of regulation by DES. The up-regulation of Sprr2a by DES is obvious within 48 h after DES treatment (Fig. 2A
). This up-regulation also lasted through P11, 6 d after DES withdrawal, but it is no longer expressed in either the oil or DES-treated uteri at P24 (Fig. 2A
and data not shown). Ribonuclease (RNase) protection assay using a 450 bp Sprr2a cRNA probe also confirmed the strong up-regulation in Sprr2a expression in DES-treated uteri (data not shown). We next asked in which uterine tissue layer Sprr2a was overexpressed. Sprr2a was normally found weakly expressed in the adult uterine epithelium (36). Radioactive in situ hybridization was used to examine the tissue-specific expression of Sprr2a upon DES exposure. In oil-treated samples, Sprr2a expression was barely detectable in the uterine luminal epithelium at postnatal d 1, 3, and 5 (Fig. 2
, B, D, and F). In contrast, Sprr2a expression was strongly up-regulated in the uterine luminal epithelium at P3 and P5 (arrows in Fig. 2
, E and G).

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Fig. 2. DES Up-Regulates the Expression of Sprr2a, a Member of SPRR Family
Real-time RT-PCR revealed that Sprr2a expression is up-regulated by DES within 24 h of DES exposure and this up-regulation persists at least past postnatal d 11 (A). Radioactive in situ hybridization assays showed that DES did not induce Sprr2a expression within 6 h (B and C) but did induce Sprr2a expression in the uterine luminal epithelium on P3 and P5 (DG, arrows).
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Another novel DES target revealed by our microarray analysis was epidermal keratin K2.16 (also known as mouse basic hair keratin 4), which is normally found expressed in the hair, the scale epidermis of the mouse tail, and the posterior part of the filiform papillae of the tongue (38). Expression of this hard keratin in the uterus has not been previously reported. We first used real-time RT-PCR to examine the kinetics of regulation of this gene by DES. Oil- and DES-treated uterine RNA at specific time points was reverse-transcribed and subjected to real-time PCR using oligonucleotide primers specific to K2.16. Real-time RT-PCR showed that K2.16 expression was induced by DES after 24 h and this induction lasted through P11 (Fig. 3E
). Quantification of this induction at P5 showed a change in RNA abundance equaling to a 12-fold up-regulation in DES- compared with oil-treated uteri (Fig. 3E
). This result is in agreement with our microarray data, which show an average up-regulation of 8.8-fold (Table 1
). In situ hybridization demonstrated that K2.16 was weakly expressed in the control uterine epithelium (Fig. 3
, A and C), whereas exposure to DES resulted in a dramatic up-regulation in the same tissue layer (Fig. 3
, B and D). To exclude the possibility that such an increase in expression is the result of increased uterine epithelial cell population in DES-treated uteri, we counted the number of luminal epithelial cells on 8 random cross-sections of control or DES-treated uterus at P3. The results showed that DES-treated uteri have about 25% more epithelial cells than control uteri (103.8 ± 13.4 and 83.8 ± 6.4, respectively), which is not enough to account for the fold increase in RNA level. Therefore, K2.16 expression is truly modulated by DES.

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Fig. 3. Regulation of an Epidermal Keratin, K2.16, by DES
K2.16 is weakly expressed in the uterine epithelium on P3 and P5 as assayed by in situ hybridization (A and C). Neonatal DES exposure resulted in up-regulation of this genes expression in the uterine epithelium (B and D, arrows). For in situ hybridization, signals were in red and nuclei were counterstained with Hoescht 33258 nuclear dye. Real-time RT-PCR showed the regulatory kinetics of K2.16 by DES (E).
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DES Alters the Expression of Transcription Factors
In an effort to delineate genetic pathways controlling uterine cytodifferentiation, we focused our attention on transcription factors whose function in cell-fate determination or epithelial differentiation has been demonstrated in other organs. The regulation of Sprr2a gene expression has been explored in the skin and the epithelium of other organs. The Sprr2a promoter has been isolated, and several transcription factor binding sites in this promoter have been identified (39). One candidate transcription factor that regulates Sprr2a in the skin is a KLF zinc-finger transcription factor, Klf4 (30, 31). Klf4/ mutant mice die shortly after birth because their skin cannot protect them from external assault and water loss (30). Thus, Klf4 is required for the establishment of the external barrier function of the skin during development. We asked whether Klf4 expression was also modulated by DES in the neonatal uterus. Klf4 expression was found to be up-regulated by DES in all three microarray experiments (Table 1
). Klf4 expression was slightly induced after 24 h of DES exposure and its expression was still up-regulated on P11 (Fig. 4E
). We confirmed this result with in situ hybridization and showed that Klf4 expression was specifically up-regulated in the P3 and P5 uterine epithelium (data not shown and Fig. 4
, A and B).

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Fig. 4. DES Alters the Expression of Different Transcription Factors Controlling Uterine Epithelial Differentiation
In situ hybridization showed that expression of Msx2 and Klf4 are differentially affected by DES treatment on P5. DES activates ectopic Klf4 expression (A and B, arrows), whereas repressing Msx2 expression in the uterine luminal epithelium (D and E, arrows). Regulation of Klf4 and Msx2 by DES were further confirmed real-time RT-PCR (C and F), respectively.
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Another transcription factor whose expression was shown to be modulated by DES in our microarray study is Msx2. Msx genes have been shown to play very important roles in epithelial-mesenchymal interactions during the formation of a variety of organs (18, 19). Therefore, we reasoned that Msx2 may also have important functions in uterine development, possibly mediating signal transductions across tissue layers. We observed a consistent 2-fold down-regulation of Msx2 expression upon DES exposure in two of the three microarray experiments, and the same trend was observed in the third microarray experiment. In situ hybridization assays on oil- and DES-treated P5 uteri confirmed a significant down-regulation of Msx2 in the uterine epithelium (Fig. 4
, D and E). Real-time RT-PCR showed a 3-fold reduction of Msx2 message at P5, a change that is comparable with the microarray results (Fig. 4F
). We next examined the kinetics of the regulation of Msx2 expression by DES. This analysis revealed that Msx2 expression was slightly repressed 24 h after DES exposure and repression was obvious after 48 h of DES exposure. Again, this repression was still obvious on P11. Taken together, these data provide evidence that Msx2 is another downstream target gene of DES during early uterine development.
DES Induces Abnormal Water Imbibition in Msx2/ Uterus
Although histological examination of Msx2/ uteri did not reveal any gross patterning defects (compare Fig. 5
, C with E), we asked whether removing Msx2 from the uterus would have any effect on its responsiveness to DES exposure. Msx2/ mice are viable and can be distinguished from their wild-type littermates by the curly vibrissa at birth (19, 40). Msx2/ newborn pups were injected sc with oil and DES from P1 to P5. Uteri were harvested at P5 and stained for PLAP activity. We detected a significant amount of PLAP activity in Msx2/ oil-treated uteri, suggesting that the mutant uterine epithelium is showing some molecular changes despite its normal histological appearance (compare Fig. 5A
with Fig. 1E
). Treatment with DES resulted in an up-regulation of PLAP activity in a similar fashion as in wild-type mice (Figs. 5B
and 1F
). These results on PLAP expression in wild-type and Msx2/ mouse uteri were further confirmed by real-time RT-PCR (data not shown). In wild-type mice, DES exposure does not cause an overtly abnormal phenotype in the P5 uterus, except for a slight increase in the height of uterine epithelial cells (Fig. 5
, E and F) (41). Surprisingly, a dramatic uterine hypoplasia was observed on P5 in Msx2/ uteri after DES treatment (Fig. 5D
) compared with oil controls (Fig. 5C
). In the mutants, the uterine lumen was found to be severely dilated with a significant reduction in the size of the uterine epithelial cells (Fig. 5D
, arrowheads). At the same time, we observed a reduction in the amount of stromal tissues with varying severity in these mice (Fig. 5D
, arrows). These phenotypes were observed in 14 out of 16 (87.5%) mutant uterine horns compared with only 1 out of 10 (10%) wild-type uterine horns exposed to DES. In contrast, neither wild-type (0/8) nor mutant (0/14) oil-treated control uteri exhibit these phenotypes. During dissection of DES-treated Msx2/ reproductive tracts, we observed a substantial amount of fluid trapped in the vaginal lumen. Thus, this uterine phenotype in Msx2/ mice could be the result of abnormal water trafficking in response to DES stimulation.

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Fig. 5. The Msx2/ Uterus Responds Differently to DES Exposure
Examination of PLAP activity in Msx2/ uteri at P5 revealed elevated PLAP activity compared with wild-type controls (compare Fig. 5A with 1E ). DES up-regulates PLAP activity in Msx2/ uterus (B). In wild-type uteri, DES exposure caused only subtle morphological changes on P5 in the uterus, including increased thickness and reduced contour of the luminal epithelium (E and F). The Msx2/ uterus exhibited comparable histology to that of the wild type (C). In contrast, the Msx2/ uterus exposed to DES exhibited dramatic morphological changes including enlargement of the uterine lumen, disappearance of uterine contour, and a dramatic reduction of the stromal cell population (D, arrows). In addition, luminal epithelial cell size is significantly reduced in DES-treated Msx2/ uteri compared with those in wild type (D, F, arrowheads).
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Because Wnt genes had been implicated in uterine cytodifferentiation (9), we next examined whether they constitute components of the Msx2 genetic pathway during uterine differentiation. Wnt5a and Wnt7a are normally expressed in the uterine stroma and epithelium, respectively (9). The expression of these genes was examined by real-time RT-PCR and in situ hybridization in wild-type and Msx2/ mouse uteri. Interestingly, Wnt7a expression was dramatically up-regulated in Msx2/ uterine epithelium by approximately 4-fold compared with wild-type controls, whereas Wnt5a expression was not changed (Figs. 6
, A, C, E, and G; and 7G
). We next examined the regulation of Wnt5a and Wnt7a by DES both in wild-type and in Msx2/ mice. In wild-type mice, DES significantly represses Wnt7a as assayed by in situ hybridization and real-time RT-PCR (9) (Figs. 6
, A and B, and 7G
). A similar repression in Wnt7a was observed in Msx2/ mice (Figs. 6
, C and D, and 7G
). These results suggest that Msx2 is normally required to control the proper level of uterine Wnt7a expression but is dispensable for its regulation by DES. Surprisingly, although the level of Wnt5a expression was not affected by DES in both wild-type and Msx2/ mouse uteri as assayed by real-time RT-PCR (Fig. 7G
), its expression shifted from the stroma to the epithelium in both the wild-type and Msx2/ uteri (Fig. 6
, EH). This change in expression pattern is much more robust in Msx2/ mutants than in wild-type controls. Therefore, Msx2 appears to play an important role in uterine development by controlling the expression of Wnt genes that are important for reproductive tract morphogenesis.

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Fig. 6. Altered Wnt7a and Wnt5a Expression in Msx2/ Uteri
Wnt7a and Wnt5a expression was assayed by in situ hybridization both in wild-type and Msx2/ uteri with neonatal oil or DES exposure. Wnt7a expression was up-regulated in Msx2/ uteri treated with oil, whereas DES repressed its expression both in wild-type and knockout mice. Wnt5a was found expressed in uterine stroma in oil-treated samples. DES dramatically shifted its expression to the uterine luminal epithelium in Msx2/ uteri, but to a lesser extent in the wild-type uteri. Quantitative real-time RT-PCR confirmed these observations (Fig. 7G ).
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Fig. 7. Msx2 Is Required for the Regulation of K2.16 Expression, But Not Required for the Regulation of Klf4 and Sprr2a by DES
The basal level of K2.16 expression in Msx2/ uterine epithelia is nearly undetectable and treatment with DES failed to up-regulate K2.16 expression in the uterine epithelium (A and B). Regulation of Klf4 and Sprr2a by DES was preserved in Msx2/ mutants (CF). These results were confirmed by quantitative real-time RT-PCR in wild-type and Msx2/ mouse uteri on P5 (G). The differences in expression between oil- and DES-treated samples were calculated based on the differences of -Cts as specified in Materials and Methods.
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Because Msx2 has been reported to function as a transcriptional repressor (42), we asked whether Msx2 is required for the regulation of some of the abnormally expressed DES-induced differentiation markers. The expression of K2.16 in Msx2/ uteri was examined by in situ hybridization and real-time RT-PCR. Figure 7
shows that K2.16 expression was no longer detected in the Msx2/ uterine epithelium, in contrast to the weak expression detected in the wild-type uterine epithelium (Fig. 7A
compared with Fig. 3C
). Moreover, K2.16 expression failed to be up-regulated by DES in Msx2 / mutant mice (Fig. 7B
). This result was confirmed by quantitative real-time RT-PCR (Fig. 7G
). In contrast, the regulation of Sprr2a and Klf4 expression by DES was not significantly affected in the Msx2/ uterine epithelium (Fig. 7
, CF). These results were confirmed by RNase protection assays (not shown). Examination of cellular proliferation using BrdU incorporation in Msx2/ uteri in the presence and absence of DES revealed that DES continues to repress luminal epithelial proliferation in the absence of Msx2 (not shown).
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DISCUSSION
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Neonatal DES exposure leads to patterning defects along the axial length of the Müllerian duct. These defects include posterior transformation of the oviduct and uterus, vaginal cornification, and adenosis. The majority of these defects were no longer observed in ER-
knockout mouse, suggesting that DES is acting as an estrogen and regulates downstream target genes through ER-
(14). Another nuclear hormone receptor, ER-related receptor ß (ERRß), has recently been shown to be an important target for DES during trophoblast stem cell proliferation and differentiation (43). However, our microarray results showed that ERRß is not expressed in either the P5 oil- or DES-treated uterine samples, excluding a functional role for ERRß in DES-induced uterine metaplasia. The molecular events downstream of ER-
, however, are not clear at present.
Cell Fate Changes Induced by DES
Developmental DES exposure results in squamous metaplasia in the adult uterine epithelium and leads to reduced fertility presumably by interfering with embryo-maternal interactions during implantation (10, 44). Yet, the molecular and genetic changes that lead to these developmental defects are not clear. To address this question, we used oligonucleotide-based microarray technology to identify genetic pathways in uterine development that are affected by DES. Because we used RNA from the whole uterus for this experiment, the results should include gene expression changes in all three uterine tissue layers, which include the epithelium, the stroma, and the muscle. Using in situ hybridization, we have identified several DES-regulated genes whose expression is confined to the uterine epithelium. These include transcription factors, growth factors, as well as structural proteins. The uterine epithelial cell fate has not yet been determined on P5 and can be induced to adopt that of the vaginal epithelium (5). Our data suggest that DES exposure alters uterine epithelial cell fate on P5 as evidenced by the premature exit of DES-treated cells from the cell cycle and the abnormal expression of multiple stratified epithelial markers. DES-exposed uterine luminal epithelial cells exhibited drastically reduced levels of proliferation and an absence of apoptosis compared with control uteri at this stage. Similar observations have been made by others in the uteri of young mice neonatally treated with DES (41). The inhibitory effect of DES on uterine cell cycle could significantly reduce the uterine somatic stem cell population that may account for the later severe uterine hypoplasia phenotype in these mice. The changes in cell cycle are accompanied by an elevated expression of several differentiation markers including PLAP, Sprrs, and keratins suggesting that uterine epithelial cells have undertaken a different developmental pathway.
Sprr2a is a differentiation marker of squamous epithelia. It was originally isolated as a UV-regulated gene, which is normally expressed in squamous epithelial cells of the skin, esophagous, and vaginal epithelium (45). Sprr2a expression is associated with epidermal barrier function, which protects the body against water loss and environmental insults such as microbial infections (46, 47). Upon colonization of germ-free mice with Bacteroides thetaiotaomicron, a normal mouse intestinal microflora, Sprr2a expression was induced 205-fold suggesting that this protein participates in fortifying the intestinal epithelial barrier in response to bacterial colonization (47). However, the role of Sprr2a in the developing uterus is not clear at this point. K2.16 is a keratin normally expressed at high levels in stratified epithelium such as the scale epidermis and the tongue epithelium (38). Expression of other epithelial keratins that are specific to the stratified epithelia, such as K1.13, was also increased in DES exposed uteri in all three microarray experiments (Table 1
). The expression of K1.13 has been detected in the tongue and in the forestomach epithelium (48). The fact that DES exposure strongly up-regulates the expression of stratified epithelial markers such as Sprr2a, K2.16, K1.13, and K2.8 supports our conclusion that developmental DES exposure changes the cell-fate of the uterine epithelial cells and induced subsequent metaplastic changes in the uterine epithelium. Such a change in cell fate may be accompanied by changes in cell-cell contact and cell-matrix contact. Indeed, we observed an array of cell junction proteins whose expression is modulated by DES from our microarray data (supplemental Table 1). The up-regulation of Sprr2a and K2.16 persisted through P11, well beyond the normal window for cell fate determination of the uterine epithelium indicating that these uterine epithelial cells may have permanently changed their cell fate and may not be reprogrammed by the stroma. The induction of Lactoferrin, a milk component and complement component factors C3 (GenBank accession no. K02782) and 1 (GenBank accession no. NM_007574) in the uterus by DES further support the above hypothesis (Table 1
and supplemental Table 1). Together, these results present a molecular basis for DES-induced premature abnormal differentiation of the uterine epithelium and suggest that DES is a differentiation factor capable of altering uterine epithelial cell fate.
Genetic Pathways Affected by DES
We have focused our analyses on the DES regulation of two transcription factors, Klf4 and Msx2, whose functions in epithelial differentiation were revealed by knockout and overexpression studies (19, 30, 40). Our data show that these two factors constitute key regulators in the emerging genetic pathways affected by neonatal DES exposure. Klf4 is normally expressed in the suprabasal layer of the stratified epidermis and is thought to be involved in the control of cell cycle progression (27, 30). Overexpression of Klf4 in cell culture inhibited cell proliferation by creating a G1/S block (49). This is accomplished by activation of the P21 promoter (50) while suppressing the cyclin D1 promoter (51). In our system, Klf4 expression was up-regulated by DES in the uterine epithelium, and, consistently, cyclin D1 expression was down-regulated by DES treatment (Table 1
). These results suggest that the DES-induced cell cycle exit in the uterine epithelium could be explained by the up-regulation of Klf4, which may create a G1/S block in the cell cycle. In the developing skin, Klf4 is a direct regulator of Sprr2a expression and in both Klf4/ mutant and claudin 6 transgenic mice skin, in which Klf4 expression is decreased, altered Sprr2a expression was also observed (30, 52). Similarly in the uterus, both Klf4 and Sprr2a are up-regulated by DES exposure and have the same regulatory profile. Thus, our data are consistent with existing evidence and suggest that Sprr2a up-regulation by DES could be mediated by Klf4 in the uterine epithelium. In the genetic pathway regulated by developmental DES exposure in uterus, Sprr2a, therefore, may reside downstream of Klf4 (Fig. 8
).

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Fig. 8. A Model for DES-Affected Genetic Pathways in Uterine Epithelial Differentiation
DES functions mainly through ER to induce uterine metaplasia. DES represses Msx2 expression in the uterine epithelium. In turn, Msx2 is required for both the basal and DES-induction of a differentiation marker K2.16. PLAP is not expressed in the uterine epithelium at P5 but is expressed in the Msx2/ uterus, indicating that Msx2 functions to repress PLAP expression. Msx2 is required to repress Wnt7a in the uterine epithelium. On the other hand, Msx2 is not required for DES to repress Wnt7a expression and for DES to activate PLAP. Wnt5a expression pattern changes from the stroma to the epithelium upon DES treatment. This switch is much more pronounced in Msx2/ suggesting that Msx2 normally plays a role in repressing Wnt5a expression in the uterine epithelium. Another genetic pathway activated by DES includes that of Klf4 and Sprr2a. This pathway is separate from that of Msx2 because neither the basal expression nor the DES regulation of these genes was altered in Msx2/ mutants. The arrows in this figure do not indicate a direct transcriptional link between these genes but rather reflect an epistatic relationship.
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Msx genes are required for signal transductions across tissue layers during organogenesis in many organs (18, 19). In the hair follicle, Msx2 is required for the proper expression of Foxn1, a wing-helix transcription factor that controls hair cortex differentiation (40). Msx2/ mice exhibit differentiation defects in all three layers of the hair shaft, suggesting that Msx2 function is required for cell-fate determination during hair differentiation. Msx2 expression is subjected to hormonal regulation in the mammary gland, where estradiol is required to maintain high levels of Msx2 expression in the mammary epithelium (53). In the developing uterus, however, DES represses Msx2 expression in the epithelium. Therefore, estrogen appears to differentially regulate Msx2 in different organs. Msx2 appears to be a critical regulator of uterine cytodifferentiation because the expression of several uterine markers was found altered in Msx2/ mice. These include two differentiation markers, K2.16 and PLAP, and a signaling molecule, Wnt7a. No K2.16 expression was observed in the Msx2/ uterine epithelium indicating that Msx2 is required for the basal uterine expression of K2.16. On the other hand, a significant amount of PLAP activity was observed in the Msx2/ uterine epithelium compared with wild-type controls, suggesting that Msx2 is normally required to repress uterine PLAP expression. Altered expression of these two markers suggest that these genes normally function downstream of Msx2 during uterine morphogenesis (Fig. 8
). We stress, however, that the genetic relationship depicted here does not suggest a direct link between these genes, but rather reflects an epistatic pathway. In fact, DES does not necessarily have to regulate other genes through Msx2 as discussed later in this section.
Studies in a variety of organisms have suggested that Wnt genes are important regulators of many cellular processes including cell behavior, adhesion and polarity (for review, see Ref. 54). The uterus in the Wnt7a/ mutants exhibits epithelial stratification and smooth muscle disorganization (9). Neonatal DES exposure has been shown to repress Wnt7a during uterine cytodifferentiation suggesting that down-regulation of Wnt7a by DES may be a prerequisite for abnormal cell differentiation (16). Recently, Wnt signaling has been implicated in regulating the proliferation and lineage specification of somatic stem cells in adult hair follicles (55, 56). In light of these findings, altered Wnt signaling in the uterus may affect the behavior of resident uterine stem cells in response to differentiation signals. Wnt genes can function both upstream and downstream of Msx1 and Msx2 (21, 22). Here we show that Wnt7a functions downstream of Msx2 in the developing uterine epithelium (Fig. 8
), and its expression is elevated in Msx2/ uterus. Thus, Msx2 may be required to control Wnt7a expression at a fixed level in the uterus to ensure proper uterine morphogenesis. These results suggest that Msx2 is a key regulator for maintaining a proper uterine-like gene expression profile.
In contrast with the required role of Msx2 in maintaining the proper uterine pattern of gene expression during development, its function is largely dispensable for the DES regulation of several molecular markers examined in this study except for K2.16. DES continues to repress uterine Wnt7a expression in Msx2/, suggesting that DES is repressing Wnt7a either directly or through an Msx2-independent pathway. Similarly, PLAP up-regulation by DES does not require Msx2 either. On the other hand, K2.16 failed to be up-regulated by DES in Msx2/ uteri, which probably reflects a requirement for Msx2 to confer its basal expression in the uterine epithelium. Meanwhile, the basal expression of Klf4 and Sprr2a is not changed in Msx2/ uteri compared with wild-type controls, and DES can activate the expression of these two genes in Msx2/, suggesting that these genes constitute a separate genetic pathway from that of the Msx2s (Fig. 8
). Interestingly, DES induces a shift in Wnt5a expression pattern from the stroma to the epithelium both in wild-type and in Msx2/ uteri. Recently, Wnt5a has been shown to be required for gland formation in the developing uterus (57). This may explain why developmental DES exposure delays and reduces uterine gland formation. In the Msx2/ uterus, stromal Wnt5a is more significantly reduced by DES compared with wild-type, which might also account for the loss of stromal tissues observed in DES-treated Msx2/ uterus. Meanwhile, ectopic Wnt5a expression in the luminal epithelium may contribute to the abnormal morphology of the uterine epithelium as overexpressing Wnt5a in melanoma cells has been shown to result in actin reorganization, increased cell adhesion and invasiveness (58). Therefore, the observed shift of Wnt5a expression could result in changes in uterine epithelial morphology, eventually leading to its transformation into stratified epithelium.
It is intriguing that, upon DES treatment, Msx2/ uteri exhibit an O-shape luminal phenotype accompanied by a reduction of the stromal tissue layer. We attributed this phenotype to the abnormal water trafficking in the Msx2/ reproductive tract. Estrogen can stimulate uterine fluid uptake from the capillaries, termed water imbibition, and also the retention of water in the uterine stroma (59). However, in Msx2/ DES-treated uteri, water appears to be transported into the uterine lumen rather than being retained in the stroma. Three things could explain this phenotype. First, there might be a change in tight junctions in the Msx2/ uterine epithelium, such that water can freely travel across the uterine epithelium. Secondly, the reduction of the stromal layer in Msx2/ uteri may reduce the ability of the stroma to retain water. Finally, there might be changes in the expression of water channel proteins, termed aquaporins, in Msx2/ uteri upon DES-treatment. Aquoporin 3 is induced in wild-type uterus upon DES exposure (Table 1
). Several aquoporin proteins are expressed in the uterus and are involved in water imbibition (60). Loss of expression of these molecules results in phenotypes that are associated with abnormal water trafficking (61). Interestingly, Wnt7a mutant uteri have recently been shown to also exhibit a similar O-shape morphology after DES exposure, further suggesting a connection between Msx and Wnt genes in uterine morphogenesis (62). These scenarios, however, are not mutually exclusive and may all contribute to the observed abnormal water imbibition in Msx2/ uteri. Future work will focus on differentiating these possibilities. In sum, our data revealed several novel DES targets that are organized in a novel genetic pathway governing uterine cytodifferentiation.
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MATERIALS AND METHODS
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Animal Treatments
All animals were handled according to National Institutes of Health guidelines and in compliance with an institutional approved animal protocol. CD-1 mice were purchased from Charles River Breeding Laboratory (Wilmington, MA). Four pairs of CD-1 mice were mated to generate newborn pups. Msx2 knockout mice were generated as described previously (19). Msx2/ mutants were on a CD-1 genetic background for more than five generations. DES (Sigma-Aldrich, St. Louis, MO) was dissolved in ethanol at 10 mg/ml, diluted 100-fold in corn oil. At birth (P0), female pups were separated from male pups and were injected with corn oil (20 µl) or 1 mg/kg·d (2 µg/pup·d) DES (20 µl/pup) from P1-P5. Injected mice were killed 6 h after the last injection, and uteri were collected by making one incision slightly posterior to the utero-tubal junction and another just anterior to the common cervical canal. This dissection technique only isolates uterine tissues, sparing oviduct, cervix and vagina. Dissected uterine tissues were either fixed in 4% paraformaldehyde for in situ hybridization or homogenized for RNA extraction unless specified otherwise.
Microarrays and Data Analysis
Ten pups in each group treated with either oil or DES daily were killed at P5. Uteri were dissected, pooled, and homogenized in a solution provided in the STAT-60 RNA isolation kit and total RNA was isolated after the manufacturers protocol (Tel-Test, Inc., Friendswood, TX). Experimental procedures for GeneChip were performed according to the Affymetrix GeneChip Expression Analysis Technical Manual. In brief, using 5 µg of total RNA as template and the T7-(deoxythymidine) 24 primer, double-stranded cDNA was synthesized using the Superscript Choice System (Invitrogen Life Technologies, Carlsbad, CA). cDNA was purified using phenol/chloroform extraction with the Phase Lock Gel (Eppendorf Scientific, Westbury, NY) and concentrated by ethanol precipitation. In vitro transcription was performed to produce biotin-labeled cRNA using a BioArray HighYield RNA Transcription Labeling Kit (Enzo Diagnostics, Farmingdale, NY) according to manufacturers instructions. Biotinylated cRNA was cleaned using the RNeasy Mini Kit (QIAGEN, Valencia, CA), fragmented to 50200 nucleotides, and hybridized to Affymetrix 430A2.0 array chips. After being washed, the array was stained with streptavidin-phycoerythrin (Molecular Probes, Eugene, OR). The staining signal amplified by binding of biotinylated antiastreptavidin (Vector Laboratories, Burlingame, CA) was secondarily stained with streptavidin-phycoerythrin, and was then scanned on a HP GeneArray Scanner. The expression data were analyzed using Affymetrix MicroArray Suite version 5.0 and SAM. To reduce the false discovery rate, probe sets that were present in at least four of the six (66%) total chips were filtered out and subjected to SAM analysis to reveal significantly changed probe sets (SAM Users guide and technical document). The false discovery rate for this analysis is 5.36%. However, this analysis left out genes that went from absence (A) to presence (P) and vice versa in response to DES. We therefore supplemented the SAM gene list with genes at the threshold of detection (from A -> P or P -> A) upon DES exposure. Comparison of the signal intensities between the DES-treated samples and oil-treated samples was denoted as fold change. Functional categorization of the array data was carried out using Function Express.
Cell Proliferation, Apoptosis, and Alkaline Phosphatase Assays
Cell proliferation was assayed using a BrdU Labeling and Detection Kit (Roche Molecular Biochemicals, Indianapolis, IN); apoptosis was analyzed by a TUNEL assay. Pups were injected sc with 40 µl of undiluted BrdU labeling reagent 2 h before they were killed. Uteri were then harvested and fixed in Carnoy fixative solution (acetic acid:ethonal:chloroform, 1:6:3) overnight at 4 C. After dehydration through a graded ethanol series and clearing in xylene, uteri were embedded in paraffin and sectioned at a thickness of 10 µm. Immunodetection of BrdU was performed following the manufacturers protocol. For apoptosis assays, uterine samples were fixed in 4% paraformaldehyde, and 10-µm paraffin sections were generated. TUNEL assays on these paraffin sections were performed using the In Situ Cell Death Kit (Roche Diagnostics Corp.). Alkaline phosphatase assays were carried out by incubating paraformaldehyde-fixed slides with nitro-blue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate p-toluidene salt or Boehringer Mannheim purple after hydration.
RNase Protection Assay and in Situ Hybridization
For RNase protection assays, probes were synthesized using RT-PCR. First-strand cDNA was synthesized using the GeneAMP RNA PCR Core Kit (Roche Diagnostics Corp.) using total RNA isolated from DES-injected neonatal mice as a template. PCR was then performed with the specific primers shown in Table 2
. PCR products were cloned into the PCR4-TOPO vector (Invitrogen Life Technologies, Carlsbad, CA) and sequenced. The ribosomal protein L19 (RPL19) probe was cloned as described previously (3) and the ß-actin probe was provided in the RPA II kit (Ambion, Austin, TX). Antisense probes were synthesized with either T7 or T3 RNA polymerase after digestion with restriction enzymes as specified (Table 2
). These probes were labeled with either 32P-UTP or 35S-UTP for RNase protection assays or in situ hybridization, respectively. The same amount of RPL19 probe was added to each reaction to serve as a loading control in RNase protection assays as previously described (3). Uterine total RNA (5 µg) was hybridized overnight at 48 C with 32P-UTP-labeled antisense cRNA probes for each gene assayed. After digestion with 20 µg/ml RNase A and 1.5 µg/ml RNase T1, protected fragments were precipitated and separated on a 6% denaturing polyacrylamide gel and band intensities were quantified by a phosphorimager (Bio-Rad, Hercules, CA). Radioactive in situ hybridization was performed by hybridizing 35S-labeled cRNA probes to uterine sections as previously described (63).
Quantitative Real-Time RT-PCR
PCR was performed and analyzed using an iCycler iQ real-time PCR detection system (Bio-Rad). Total RNA was reverse-transcribed into cDNA using iScript cDNA synthesis kit (Bio-Rad). Primers were designed by the Beacon Designer software (Bio-Rad), and their sequences were listed in Table 2
. SyBr green was used in amplification reactions, and the fluorescence intensity was detected with the charge-coupled device detector of iCycler. The relative amount of PCR products was quantified using the comparative threshold cycle (Ct) method and was expressed as n-fold difference between DES-treated and oil control samples. The PCR conditions were as follows: 95 C for 2 min followed by 40 cycles of 20 sec at 95 C and 60 sec at 60 C. A melting curve reaction was carried out by increasing the temperature from 7095 C at a speed of 0.5 C /10 sec. Under these conditions, only one peak was detected in each reaction indicating a single PCR product. All PCR products were subsequently cloned and their sequences were verified. Each PCR experiment was carried out in triplicates on two separate pooled RNA samples from uteri of two or more pups and average Cts were obtained. To measure the relative amount of PCR products, the Ct of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was subtracted from the Cts of genes of interest to derive
Ct. The
CT of the DES-treated samples was compared with the
CT of the oil control samples and the difference was assigned as 
CT. The fold change between the two samples was then calculated as 2
CT.
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ACKNOWLEDGMENTS
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We would like to thank Drs. Ken Muneoka, Carol Burdsal, and YiPing Chen for helpful comments on the manuscript, and Siteman Cancer Center Bioinformatics Core for help with microarray data analysis.
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FOOTNOTES
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This work was supported by National Institutes of Health Grants 1F30ESO1211801 (to W.-W.H.) and R21ES11708 and R01HD41492 (to L.M.), a National Science Foundation Grant IBN-0131316 (to L.M.), and Department of Energy Grant DE-FC26-00NT40843 (to J.A.M.).
Present address for J.V.: Biocenter Oulu, University of Oulu, Aapistie 7, 90220 Oulu, Finland.
Present address for W.W.-H., Y.Y., and L.M.: Division of Dermatology, Department of Medicine, Washington University, St. Louis, Missouri 63110.
First Published Online December 9, 2004
Abbreviations: BrdU, Bromodeoxyuridine; Ct, comparative threshold cycle; DES, diethylstilbestrol; ER-
, estrogen receptor-
; ERRß, ER-related receptor ß; KLF, Krüpple-like factor; P7, postnatal d 7; PLAP, placental alkaline phosphatase; RNase, ribonuclease; SPRR, small proline-rich protein; SAM, significance analysis of microarrays; UTP, uridine triphosphate; TUNEL, terminal deoxynucleotodyl transferase-mediated deoxy-UTP nick end labeling.
Received for publication April 15, 2004.
Accepted for publication November 30, 2004.
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