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Regular Article |
a Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208
ABSTRACT
An analysis of the pattern of expression of the mouse placental hormone prolactin-like protein A (PLP-A) has revealed that this hormone is expressed exclusively in secondary trophoblast giant cells but not in primary giant cells. Thus, PLP-A serves as a marker for a subset of giant cells. Recent results have indicated that PLP-A binds to and inhibits the activity of natural killer cells, and thus, the localized expression of PLP-A may be important for regulating the activity of this class of T lymphocytes in a restricted region of the implantation site. Previous studies indicated that the transcription factor GATA-2 is required for the trophoblast giant cell-specific expression of two other hormones in the prolactin family, placental lactogen I and proliferin. In the absence of GATA-2, PLP-A continues to be expressed, but in this mutant background, PLP-A mRNA is detected in both primary and secondary giant cells. Thus, GATA-2 contributes both to positive and negative regulation of trophoblast giant cell-specific gene expression, and this factor apparently plays an important role in generating or maintaining the distinct functions of secondary, compared with primary, trophoblast giant cells.
development, developmental biology, gene regulation, placenta, pregnancy, trophoblast
INTRODUCTION
Trophoblast cells descend from the first differentiated cells of the mammalian blastocyst, the trophectoderm. In rodents, different regions of the trophectoderm give rise to different trophoblast populations. The mural trophectoderm cells line the cavity of the blastocyst but do not contact the inner cell mass; these trophectoderm cells terminally differentiate into primary giant cells. The portion of the trophectoderm adjacent to the inner cell mass (the polar trophectoderm) gives rise to proliferating trophoblasts of the ectoplacental cone; some of these cells then undergo differentiation to form the secondary giant cells [1, 2] (see also [3] for schematic drawings of this differentiation sequence). The formation of both the primary and secondary giant cells involves the cessation of cell proliferation and the onset of a series of endoreduplicative cell cycles, leading to a highly amplified genome and a much larger cell size [1, 2]. The resulting giant cells form the interface between the maternal and embryonic compartments, establish intimate contacts with maternal decidual cells, and come into direct contact with maternal blood by displacing maternal endothelial cells from vessels that reach the implantation site [1, 2]. Thus, proper differentiation of giant cells is required for implantation, with aberrant formation of these cells leading either to a failure of the embryo to attach to the uterus or to subsequent embryonic loss due to defects in placentation.
In the mouse, the primary and secondary giant cells both express a number of placental giant cell-specific hormones, including placental lactogen I (PL-I) [4] and proliferin (PLF) [5]. On the basis of the uniform expression of these and other gene products and on the similar roles in establishing contacts with maternal tissue at the outer limits of the embryonic compartment, the primary and secondary giant cells in the mouse are clearly quite similar. Nevertheless, the pregnancy-specific glycoprotein gene rnCGM1 is differentially expressed between the primary and secondary giant cells of the rat placenta [6], indicating that these two cell types are distinguishable in at least one function.
In addition to PL-I and PLF, mouse trophoblast giant cells secrete a large number of other hormones closely related to prolactin (PRL), including the placental hormone prolactin-like protein A (PLP-A) [7, 8]. The recently described effect of this placental cytokine in inhibiting natural killer (NK) cell cytolytic activity [9] suggests that this hormone plays an important role in regulating maternal immune response to the implanted embryo. Prolactin-like protein A is expressed specifically in giant cells in early to mid gestation [7, 8], a pattern of expression similar to that of PL-I and PLF. The expression of these latter two hormones in trophoblast giant cells shares a requirement for the transcription factors GATA-2 and GATA-3 [10, 11], zinc finger proteins that were initially identified in the erythropoietic system [12] but that have subsequently been implicated in gene expression in other cell types, including T lymphocytes [13] and pituitary gonadotropes [14]. In transfected trophoblast cell cultures, these factors can directly stimulate transcription from the PL-I gene promoter, and each can activate the promoter when introduced into nontrophoblast cells [11]. In vivo, both GATA-2 and GATA-3 are required for maximal expression of PL-I and PLF, although the absence of GATA-2 has a much more pronounced effect [10].
The ability of GATA factors to regulate trophoblast-specific expression of two genes in the PRL family suggests that these factors, and in particular GATA-2, may contribute to decisions in trophoblast cell differentiation. If the GATA-2 transcription factor is a critical component for trophoblast cell differentiation and function, then it would be expected to regulate an even broader program of trophoblast-specific gene expression, including the expression of PLP-A. We therefore undertook an analysis of the placental expression pattern of PLP-A in the wild-type placenta and in the placenta in the absence of GATA-2.
MATERIALS AND METHODS
Animals
All procedures were approved by the Northwestern University Animal Care and Use Committee. Mice were maintained on a 14L:10D cycle, and food and water were freely available. Individual male mice were introduced into cages containing two female mice and remained until a vaginal plug was detected, with noon on the day of appearance of the plug designated as Gestational Day 0.5. Mice heterozygous for a mutated GATA-2 gene [15] were bred to generate homozygous mutant and littermate control conceptuses. Treatment with proteinase K and extraction with phenol and chloroform were used to isolate DNA from embryos or yolk sacs, and genotypes were determined by PCR, which produces distinctly sized products from the wild-type and mutant alleles [10].
In Situ Hybridization
Pregnant mice were sacrificed at specific gestational stages, and in each case, the uterus was immediately removed and cut between conceptuses. For wild-type matings, each conceptus and surrounding uterine tissues were then rapidly frozen on dry ice; for GATA-2 mutant matings, embryos were first removed for genotype analysis, and the remaining tissue was then rapidly frozen on dry ice. Tissue was sectioned on a cryostat to a thickness of 14 µm. The tissue sections were fixed in 5% paraformaldehyde and acetylated. Antisense and sense riboprobes for PLP-A [7], PL-I [16], PLF [17], placental lactogen II (PL-II) [18], and proliferin-related protein (PRP) [19] were generated by in vitro transcription of the corresponding cDNA clones with SP6 or T7 RNA polymerase in 20 µl with 0.5 µg linearized template DNA, 1x transcription buffer (Roche Molecular Biochemicals, Indianapolis, IN), 40 U ribonuclease inhibitor (Promega, Madison, WI), 400 µM NTPs (ATP, CTP, and GTP), 100 µM UTP, and 40 µM digoxigenin-11-UTP; reactions were carried out at 37°C for 60 min. Template DNA was removed with 1 µl RQ1 deoxyribonuclease (Promega) at 37°C for 10 min, and probes were recovered by ethanol precipitation. Hybridizations were carried out at 47°C for 12–16 h in a humidified chamber; the hybridization solution consisted of 50% formamide, 0.3 M NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, Denhardt solution (200 µg/ml each of Ficoll 400, polyvinylpyrrolidone, and BSA), 10% dextran sulfate, 10 mM dithiothreitol, 0.5 mg/ml yeast tRNA, 0.5 mg/ml poly(A) RNA, and 10 ng/ml denatured riboprobe. Following the hybridization period, slides were rinsed in 2x SSC and treated for 1 h at 37°C with 20 µg/ml RNase A. Slides were then washed at 65°C for 30 min in 0.5x SSC and for 1–2 h in 0.1x SSC. Blocking buffer (2x SSC, 0.05% Triton X-100, and 0.1% BSA) was added for 1 h, followed by 1 h at 37°C with alkaline phosphatase-conjugated antibody against digoxigenin diluted 1:500 in blocking buffer. Slides were washed at 37°C for 10 min in 100 mM Tris-HCl, pH 7.5, and 150 mM NaCl; then for 10 min in 20 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 50 mM MgCl2; and finally, in the latter solution supplemented with 250 µg/ml nitroblue tetrazolium and 225 µg/ml 5-bromo-4-chloro-3-indolyl-phosphate (Roche) until adequate staining was observed. At least five conceptuses were analyzed for each gestational stage or genotype.
RESULTS
As a first step in analyzing a possible requirement for the GATA-2 transcription factor in PLP-A expression, the normal pattern of expression of this gene was characterized. No PLP-A mRNA is present in the giant cells at day 6.5 of gestation (Fig. 1b), a stage at which PL-I mRNA is already evident throughout the giant cell layer (Fig. 1c). Thus, PLP-A is not synthesized during the initial formation of primary and secondary giant cells, and the PLP-A gene therefore is unlikely to be a direct target for transcription factors that are inducing trophoblast giant cell differentiation. By Day 8.5, PLP-A mRNA is readily observed (Fig. 1e), but only in the secondary giant cells; in contrast, at this developmental time, PL-I mRNA is uniformly distributed among both of these giant cell populations (Fig. 1f). Significantly, the pattern of PLP-A synthesis indicates that the secondary giant cells, the subpopulation of giant cells that forms from the ectoplacental cone trophoblasts, is functionally distinct from the morphologically identical primary giant cells which arise from the mural trophectoderm. The exclusive expression of PLP-A in the secondary giant cells continues at Day 10.5 (Fig. 1h) while PL-I mRNA is still present at high levels in both the secondary and the primary giant cells (Fig. 1i).
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Day 10.5 is approximately midgestation in the mouse, and at this stage, the placenta begins to change into a more mature structure, with the growth of the labyrinth region and the utilization of the fused chorion and allantois as a primary transporter of nutrients and wastes [2]. From Days 10.5 to 12.5, dramatic changes are also evident in giant cell gene expression, for example, the cessation of PL-I synthesis and the replacement of this hormone with newly synthesized PL-II [20]. Expression of PLP-A continues through this transition period, but again only in the secondary giant cells (Fig. 2, c and d), whereas PLF mRNA is seen at Day 12.5 in most, if not all, giant cells in both the primary and secondary giant cell regions (Fig. 2, e and f). As noted previously [7], at Day 12.5, PLP-A mRNA is observed primarily in the subset of giant cells surrounding maternal blood sinuses. Because the expressing cells come into direct contact with maternal blood, PLP-A would then be secreted preferentially at sites where the hormone would be most likely to encounter and suppress the activity of maternal NK cells.
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Placental lactogen I, PLF, and PLP-A are closely related genes that are located within a small region of chromosome 13 [7] and are all expressed specifically in giant cells at similar developmental stages. It is therefore possible that (at least in the secondary giant cells) these three genes are coordinately regulated. If so, then GATA-2 should be critically important for PLP-A expression. To test this hypothesis, the pattern of PLP-A expression was analyzed in GATA-2 null placental tissue at Gestational Day 9.5; time points beyond this stage are difficult to analyze because of the midgestational lethality of this genetic deficiency. Consistent with previous results, PL-I mRNA levels are decreased in the absence of GATA-2, based on this qualitative analysis (compare Fig. 3e and 3f). Surprisingly, in the absence of GATA-2, PLP-A expression expands into the primary giant cell zone (Fig. 3, c and d). Thus, GATA-2 appears to be required for the negative regulation of the PLP-A gene in the primary giant cells, with a consequent activation of this gene in the absence of this transcription factor.
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The expansion of PLP-A expression into all giant cells in the absence of GATA-2 raises the possibility that this transcription factor is critical for differentiating primary from secondary giant cells. Two other hormones in this family, PL-II and PRP, are also found to be asymmetrically expressed in the placenta, with these mRNAs detected exclusively in the secondary giant cell region (Fig. 3, g–j). In the absence of GATA-2 at Day 9.5, PL-II mRNA expression appears qualitatively to be reduced compared with the case of wild type, but this mRNA remains confined to the secondary giant cells (compare Fig. 3g and 3h). Thus, although GATA-2 is important for the restricted expression of PLP-A, it does not appear to be affecting all genes that are differentially expressed between the primary and secondary giant cells. Similarly, no expression of PRP in the primary giant cells is detected in the GATA-2 null tissue (Fig. 3j). Proliferin-related protein mRNA continues to be detected in the secondary giant cell region in the mutant placenta, but the mRNA distribution is more localized. Proliferin-related protein is unique among this set of genes in that it is expressed in both giant cells and diploid spongiotrophoblasts [19, 20]; the effect of the absence of GATA-2 on PRP levels is confined to the most peripheral region (the giant cells) and not to the underlying spongiotrophoblasts.
DISCUSSION
The discovery that PLP-A is specifically expressed in the secondary trophoblast giant cells demonstrates that primary and secondary giant cells are functionally distinct in the mouse placenta. At least two other related hormones, PL-II and PRP, are also not produced at detectable levels in the primary giant cell population, whereas PL-I and PLF are synthesized by all the giant cells. Thus, the two populations of giant cells have overlapping but non-identical endocrine functions. Synthesis of PLP-A specifically in secondary giant cells, which are adjacent to the uterine mesometrial decidual tissue, and especially in those cells in contact with maternal blood, results in the concentration of secreted PLP-A protein at sites where it could most efficiently bind to and inhibit the function of NK cells [9], thereby protecting the genetically distinct embryonic and extra-embryonic compartments from the maternal immune system. Indeed, the restricted production of PLP-A, PL-II, and PRP, along with a pregnancy-specific glycoprotein [6], within the secondary giant cells suggests a general mechanism by which asymmetric processes such as placental implantation, invasion, and vascularization are likely to be mediated by the localized synthesis of key regulatory factors.
In addition to providing a new perspective on how the activities of PLP-A, PL-II, and PRP may be asymmetrically directed at the implantation site, these findings suggest that these three hormones can serve as useful markers to investigate how distinct primary and secondary giant cell fates arise. Because expression of the PLP-A gene is not detected by Day 6.5 of gestation, after the time that secondary giant cells first form, and because expression of PL-II and PRP is initiated even later in gestation, activation of these genes does not appear to be linked to the earliest developmental decisions in the formation of the secondary trophoblast giant cells from ectoplacental cone trophoblasts.
At least one factor that is critically important for the restricted expression of PLP-A in the secondary giant cells is the transcription factor GATA-2. In the absence of GATA-2, the clear demarcation of PLP-A expressing and nonexpressing zones in the giant cell layer disappears, with the result that the primary and secondary giant cells are more similar in the spectrum of genes they express. It therefore seemed possible that GATA-2 is an essential component of the program that functionally distinguishes primary from secondary giant cells. However, PL-II and PRP expression is still restricted to the secondary giant zone region in Day 9.5 placentas lacking GATA-2. It remains possible, because Day 9.5 represents the onset of PL-II and PRP synthesis, that at later stages, a difference in the distribution of these mRNAs would be seen between wild-type and GATA-2 mutant placentas; the lethality of the GATA-2 mutation by midgestation [15] prevents such an analysis. Thus, the data at present argue for GATA-2 having a specific function in repressing PLP-A synthesis in primary giant cells, rather than a general effect on trophoblast giant cell fate.
We had previously demonstrated that GATA-2 is a positive regulator of the early, giant cell-specific expression of the PL-I and PLF genes [10, 11]. The current results indicate that GATA-2 is also a positive regulator of the later expression of the PL-II and PRP genes in secondary giant cells. Thus, GATA-2 activates a broad program of trophoblast-specific gene expression at distinct developmental stages. Surprisingly, we also find that GATA-2 acts as a negative regulator of PLP-A expression in primary giant cells because the loss of GATA-2 results in expression of PLP-A in this cell population. The ability of a transcription factor to act both positively and negatively has many precedents, but the especially intriguing aspect in this case is that the oppositely regulated target genes are closely related and closely linked. The positive regulation of PL-I and PLF gene expression by GATA-2 results from direct binding of this factor to sites in these gene promoters [10, 11]. Negative regulation of the PLP-A gene may also be direct, but the isolation and analysis of the PLP-A gene will be required to address this possibility. A direct, cell autonomous action of GATA-2 in the primary giant cells to repress transcription is possible because GATA-2 is expressed in the placenta in both primary and secondary giant cells [11] and would be consistent with transcriptional repression by GATA factors observed during erythropoiesis [21–23].
ACKNOWLEDGMENTS
We thank Stuart Orkin and Doug Engel for making available GATA-2 mutant mice, and we thank Doug Engel for helpful discussions.
FOOTNOTES
First decision: 11 February 2000.
1 This work was supported by NIH grant HD29962, by the P30 Research Center on Fertility and Infertility at Northwestern University (HD28048), and by the Robert H. Lurie Cancer Center (CA60553). 
2 Correspondence: Daniel Linzer, Dept. of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, 2153 Sheridan Rd., Evanston, IL 60208. FAX: 847 467 1757; dlinzer{at}northwestern.edu
Accepted: March 30, 2000.
Received: January 13, 2000.
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