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Max Delbrueck Center for Molecular Medicine, Berlin, Germany
Received 31 March 2004/ Returned for modification 14 May 2004/ Accepted 19 August 2004
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ABSTRACT |
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and C/EBPß are related transcription factors that are important for the function of various organs in the postnatal mouse. Gene replacement and tissue culture experiments have suggested partial redundancy of both transcription factors. Here we show that mouse embryos deficient of both C/EBP and C/EBPß (C/EBPß–/–) die between embryonic day 10 (E10) and E11 and display defective placentas. In situ hybridization revealed that C/EBP and C/EBPß are coexpressed in the chorionic plate at E9.5 and later in the trophoblasts of the labyrinthine layer. In C/EBPß–/– placentas, allantoic blood vessels invaded the chorion; however, vessel expansion and development of the labyrinthine layer was impaired. Furthermore, a single copy of either C/EBP in the absence of C/EBPß or C/EBPß in the absence of C/EBP is sufficient to complete development, suggesting complementation of these C/EBPs during embryogenesis. A single copy of C/EBP in the absence of C/EBPß, however, fails to rescue survival after birth, suggesting haploinsufficiency of C/EBP in newborns. Our data thus reveal novel essential, redundant, and dosage dependent functions of C/EBPs. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The four most related members of the C/EBP family (, ß, 
, and 
) display extensive homology in their carboxyl-terminal basic and leucine zipper domains, which mediate DNA binding and dimerization, respectively. Toward the N terminus, C/EBPs are more divergent and homology is restricted to distinct peptide motifs that are involved in transactivation or regulation of the activity of C/EBPs (17, 19, 46). Although data obtained from mouse molecular genetics and expression profiling studies suggest that the functions of C/EBP and C/EBPß are rather distinct, it has also been shown that C/EBPß can partially replace C/EBP functions when expressed from the C/EBP locus (8, 16). It is not clear, however, to what extent regulation of C/EBP and C/EBPß gene expression or structural differences of the transcription factors account for redundant or divergent functions. Tissue specificity also appears to result from combinatorial interactions with unrelated transcription factors, such as c-Myb, to specify hematopoietic gene regulation (25, 27), or peroxisome proliferator activated receptor 
(PPAR) to specify adipogenic gene regulation (13, 34). Accordingly, an important issue in understanding C/EBP biology and physiology is to determine the degree of functional redundancy or specificity of individual C/EBPs.
Here we report novel sites of expression of C/EBP and C/EBPß during mouse embryogenesis. We show that at embryonic day 9.5 (E9.5) C/EBPß is expressed in the ectoplacental cone and in the chorionic plate, whereas C/EBP expression is restricted to the chorionic plate. At E10.5 C/EBP is coexpressed with C/EBPß in the labyrinthine layer of the chorioallantoic placenta. C/EBPß is found in all of the trophoblast cell lineage of the placenta. Deletion of all C/EBP and -ß alleles (C/EBPß–/–) results in embryonic developmental arrest at around E10 to E11 that is associated with a gross failure of the placenta to undergo proper morphogenesis. We also show that a single copy of either C/EBP in the absence of C/EBPß, or C/EBPß in the absence of C/EBP could rescue embryogenesis. Our data suggest essential and redundant functions of both transcription factors during early embryonic development. Furthermore, C/EBP is haploinsufficient for survival of the newborns after birth in the absence of C/EBPß.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß–/– embryos.
The C/EBP and C/EBPß knockout strains were obtained from Gretchen Darlington (Baylor College of Medicine, Houston, Tex.) and Esta Sterneck (NCI, Frederick, Md.), respectively, and have been described elsewhere (42, 43). Since the C/EBPß–/– females are sterile and the C/EBP–/– newborns die of hypoglycemia 8 h after birth (18, 42, 43), double C/EBP heterozygous mice (C/EBPß+/–) were generated and intercrossed to obtain double knockout C/EBPß–/– embryos. Genotyping was routinely performed by PCR analysis with two upper primers located either in the pGK promoter of the neomycin cassette (5'-ACGAGACTAGTGAGACGTGCTAC-3') or in the 3'-untranslated region for C/EBP (5'-GGAGGAAGATACAGGAAGTGAGAT-3') or in the coding region for C/EBPß (5'-GCTTCGAACCCGCGGACTGCAA-3') and a common lower primer located in the 3'-untranslated region of the gene of interest (C/EBP, 5'-TGGTTTAGCATAGACGTGCACACT-3'; C/EBPß, 5'-CATCTTTAAGGTGATTACTCAGGGC-3'). DNA was obtained from tail biopsies or, in the case of embryos, from yolk sacs. PCR was performed in 50-µl reaction mixtures. The cycling conditions consisted of an initial 5-min denaturating step at 95°C, followed by 40 cycles of 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C. For C/EBP, reaction products of 542 and 305 bp represent wild-type and mutant alleles, respectively. For C/EBPß, reaction products of 771 and 530 bp represent wild-type and mutant alleles, respectively. Histology, isolectin B4 staining, and in situ hybridization. Entire conceptuses and placentas were isolated from E9.5 to E14.5 from staged pregnancies. The plug day was designated day 0.5, although the embryos were staged according to standard morphological landmarks.
For histological analyses, placentas were fixed overnight at 4°C in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Sections of 8 µm were stained with eosin and hematoxylin (H&E) according to the standard procedure. Placenta sections were stained with isolectin B4 as described elsewhere (28).
Whole-mount in situ hybridization on Vibratome slices of the placenta (70 µm) was performed as described previously (37) with antisense RNA probes for C/EBP (corresponding to the fragment from nucleotides 1451 to 2099 of mouse genomic DNA), C/EBPß (40), C/EBP (41), distal-less 3 gene (Dlx3) (24), fms-like tyrosine kinase receptor 1 (Flt-1) (23), glial cell missing 1 transcription factor (Gcm1) (3), placental lactogen-1 (PL-1) (9), PPAR (5), and the vascular endothelial growth factor (VEGF) (44).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and C/EBPß.
Genotype analysis (from E9.5 to birth) of 822 progeny from intercrosses of C/EBPß+/– yielded the expected Mendelian ratios of all genotypes at E10 (Table 1) . As expected, loss of both copies of C/EBP resulted in death shortly after birth (22, 43). Interestingly, loss of one copy of C/EBP in a C/EBPß–/– background also resulted in death of the newborns few hours after birth, whereas C/EBPß–/– animals that retain both copies of C/EBP were born alive and survived as long as the wild-type mice. This result shows that, in the absence of C/EBPß, C/EBP is haploinsufficient for survival of the newborns after birth.
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ß–/– embryos were recovered at the expected Mendelian ratio of ca. 6%. The embryos were viable and externally indistinguishable from their littermates in both size and gross morphology. At E10.25 to E11, C/EBPß–/– embryos were recovered at a lower frequency (4,55%). Few C/EBPß–/– embryos (3 of 136 [2%]) survived until E12.5. No live C/EBPß–/– embryos, however, were found at E13.5 or beyond (Table 1). These data show that a single copy of either C/EBP in the absence of C/EBPß or C/EBPß in the absence of C/EBP rescues early lethality that occurs around E10 to E11 in the absence of both factors, suggesting redundant functions of C/EBP and C/EBPß in early embryo survival.
Expression of C/EBP and C/EBPß in early placental development.
The possibility that C/EBP and C/EBPß are important in early placental development (E10 to E11) was unanticipated, since previous reports failed to detect expression of these genes in embryonic tissues earlier than E13.5 (6, 7, 20, 47). However, placental abnormalities were clearly evident in C/EBPß–/– progeny at E10. Accordingly, we reinvestigated placental C/EBP expression by in situ hybridization. At E9.5, C/EBP mRNA is expressed in the chorionic plate (Fig. 1A), whereas C/EBPß mRNA is present not only in the chorionic plate but also in the ectoplacental cone (Fig. 1B). At E10.5, C/EBP and C/EBPß mRNA are coexpressed in the trophoblasts of the labyrinthine layer (Fig. 1D and E). In addition, C/EBPß mRNA is found in the trophoblast cells of the three layers of the placenta (Fig. 1E). No C/EBP mRNA expression has been found in placental cells from E9.5 to E11.5 (Fig. 1C, F, and I), whereas C/EBP and C/EBPß mRNA are present in mouse placenta at least until E11.5 (Fig. 1G and H). Notably, neither C/EBP nor C/EBPß mRNA are expressed in the fetal endothelium permeating the presumptive labyrinth (Fig. 1A, B, D, E, G, and H) and in the yolk sac (data not shown).
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ß–/– placenta.
At E10, the placentas of wild-type, C/EBP–/–, and C/EBPß–/– embryos exhibit the characteristic three-layered appearance with the labyrinthine layer as the inner layer, the spongiotrophoblast in the middle, and the trophoblast giant cells forming the outer layer that interfaces with the maternal decidua (see Fig. 3A and data not shown) (35). In C/EBPß–/– placentas, the spongiotrophoblast layer and the giant cells appeared normal. The labyrinthine layer, however, was drastically reduced in thickness (compare Fig. 2D with 2A to C). The chorionic trophoblasts in C/EBPß–/– were found densely packed at the base of the placentas (Fig. 2D and 3B), whereas chorionic trophoblasts in age-matched wild-type and single-knockout placentas had already started to spread inward and participate in the formation of the labyrinthine layer, together with the angiogenic allantoic mesenchyme (Fig. 2A to C and 3A). In the C/EBPß–/–, fetal blood vessels, identified by luminal nucleated embryonic blood cells, were detected either outside of the placenta or (when in the placenta) had formed exclusively at the base where the surrounding trophoblast tissue was very compact (Fig. 2D and 3B). In contrast, in wild-type and single-knockout placentas an intricate network of fetal blood vessels had developed in juxtaposition to maternal blood sinuses (compare Fig. 2A to C and compare 3A to 2D and 3B). The elongated differentiated trophoblasts that form the hemotrichorial barrier around the fetal blood vessels were undetectable in the C/EBPß–/– placentas (compare Fig. 2A to C and D). The tight apposition of fetal blood vessels and maternal blood sinuses seen in the normal placenta is, however, a key prerequisite for the function of the labyrinth in the exchange of gas and nutrients between fetal and maternal blood.
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ß–/– concepti.
All C/EBPß–/– placentas that were analyzed at E10 were associated with live embryos, as indicated by beating hearts. Their yolk sac appeared histologically normal and well vascularized. No obvious vascularization defects were observed in these embryos. The formation of their vitellin vessels and their major arteries was similar to those of wild-type animals (data not shown). However, in the labyrinthine layer of C/EBPß–/– placentas, loss of isolectin B4 activity, a marker for the matrix surrounding the fetal vessels, was observed (Fig. 3D), whereas wild-type placentas showed strong staining in their labyrinthine layer (Fig. 3C). The chorionic plate and the yolk sac were similarly stained by isolectin B4 in both genotypes (Fig. 3C and D). Taken together, these data suggest that the vascularization of the labyrinthine layer of the placenta was impaired in C/EBPß–/– conceptuses.
Analysis of marker gene expression in C/EBPß–/– placenta.
For a more detailed characterization of the placental defect, the expression of several trophoblast markers in C/EBPß–/– and control placentas was compared. Since the expression patterns of trophoblast marker mRNAs in wild-type, C/EBP–/–/ß+/–, and C/EBP+/–/ß–/– placentas were similar (data not shown), results obtained with C/EBPß–/– placentas are compared only to wild-type placentas in Fig. 4. In situ hybridization of placental sections of C/EBPß–/– at E10 showed no difference in the expression of the spongiotrophoblast marker Flt-1 (a receptor tyrosine kinase for VEGF and placental growth factor [PlGF]) (12, 23) and of the trophoblast giant cell marker PL-1 (9) compared to the wild-type placenta. Alkaline phosphatase activity in secondary giant cells and in the chorionic plate was identical in wild-type and C/EBPß–/– placentas (data not shown). Thus, although the morphology was highly abnormal in C/EBPß–/– placentas, cytodifferentiation of giant cell and spongiotrophoblast placental cell types and expression of their specific markers still occurred. Gcm1, a marker for the branching initiation of the chorioallantoic placenta (3, 37), was slightly less expressed in C/EBPß–/– placentas, probably because of the dramatic reduction in size of the labyrinthine layer. However, the presence of Gcm1 indicates that the branching initiation of the morphogenesis could still occur.
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and Dlx3 (5, 24). In addition, C/EBP proteins are implicated in the regulation of angiogenic factors such as VEGF (29). We therefore examined the expression of Dlx3, PPAR, and VEGF as potential downstream C/EBP target genes in placental development. As shown in Fig. 4, at E10 PPAR mRNA expression is similar in all layers of C/EBPß–/– and wild-type placentas, except in the labyrinthine layer of C/EBPß–/– placentas due to its reduction or absence (compare the labyrinthine layer of C/EBPß–/– placentas to the wild-type placentas). Dlx3 and VEGF expression is restricted to the chorionic plate in C/EBPß–/– placentas, whereas both are expressed in the labyrinthine layer of wild-type placentas (Fig. 4). VEGF production by trophoblast giant cells is not affected in C/EBPß–/– placentas compared to wild-type placentas (data not shown). These data indicate that the structural abnormalities observed in C/EBPß–/– placentas are associated with altered expression of specific markers of the labyrinthine layer. These results also show that C/EBP and C/EBPß are involved in placental development either independently of or downstream of PPAR. |
DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß–/– conceptuses reveal a novel function of C/EBP and C/EBPß transcription factors in mouse placental development. C/EBPß–/– embryos die at ca. E10 to E11 when the placenta starts functioning as the site of gas and nutrient exchange between fetal and maternal blood. In situ hybridization data showed that the spatial and temporal distribution of C/EBP and C/EBPß mRNA supports a functional role of both transcription factors in placenta. They are coexpressed at E9.5 in the chorionic plate and at E10.5 in the labyrinthine trophoblast cells. The placenta-restricted expression of C/EBP and C/EBPß and the early lethal effect resulting from the absence of both factors in C/EBPß–/– embryos strongly suggests an essential role of C/EBPs in placentogenesis.
Lethality during the postimplantation period can be the result of either defects in yolk sac or chorioallantoic placentas (10). Because C/EBP and C/EBPß are not expressed in the yolk sac (data not shown) and C/EBPß–/– yolk sacs are histologically normal and because mouse mutants displaying similar phenotypes die at ca. E10.5 from placental deficit (15), our data suggest that the lethality of C/EBPß–/– embryos is probably due to placental defects.
Evidence for redundant functions of C/EBP and C/EBPß in the liver and in hematopoiesis was previously reported in studies using a gene replacement strategy (8, 16). Our data confirm and extend these observations by showing that, in the absence of C/EBPß, C/EBP is haplosufficient for placentogenesis but haploinsufficient for survival of the newborns after birth. Furthermore, in the absence of C/EBP, C/EBPß is haplosufficient for placentogenesis.
The epithelial compartment of the placenta is derived from the trophoblast cell lineage, whereas the stromal and vascular compartments are derived from extraembryonic mesoderm, specifically the allantoic mesoderm in the mouse. We show that C/EBPß mRNA is found in the three trophoblast cell types—giant cells, spongiotrophoblasts, and trophoblasts of the labyrinthine layer—and that C/EBP is found in trophoblasts of the labyrinthine layer. In addition, their mRNAs were also expressed in the trophoblast stem cells of the chorionic plate and in the trophoblast stem cell line (TS) kept under undifferentiated conditions (data not shown). In humans, C/EBP and C/EBPß are also localized in the trophoblast cells during pregnancy (4). Thus, these data strongly suggest a role of C/EBP and C/EBPß in trophoblast cell lineage. The absence of both C/EBP and C/EBPß in early embryogenesis results in a defective placental development. Histological analyses of C/EBPß–/– placentas show the absence of extensive villous branching that creates the labyrinth. This abnormal phenotype was completely penetrant, since all of the E10 C/EBPß–/– placentas examined, but none of the wild-type and other genotype controls, exhibited the same cellular anomalies. In C/EBPß–/– placentas, a defect in trophoblast cell growth and maturation could account for the defective labyrinthine layer formation. C/EBP and C/EBPß have already been implicated in branching morphogenesis of mammary epithelial cells (32, 39). It is tempting to propose a similar role for C/EBP and C/EBPß in the branching morphogenesis of the placenta. Gcm1, the marker for branching morphogenesis initiation (3, 37), is still detectable in C/EBPß–/– placentas, indicating that C/EBP and C/EBPß are not required for the initiation of branching morphogenesis but probably for its progression. Branching morphogenesis and progression (also called chorioallantoic morphogenesis) are prerequisites for proper vascularization of the placenta (1, 11). Thus, the absence of vascularization of the labyrinth of the C/EBPß–/– placentas, visualized by the lack of isolectin B4 activity, is most likely a consequence of the block in the progression of chorioallantoic morphogenesis. This hypothesis is supported by the observation that all other vascular processes in the C/EBPß–/– embryos appear to be normal, including the establishment of yolk sac circulation, the formation of vitellin vessels and major arteries, and chorioallantoic fusion. Therefore, placental vascularization appears to be dependent on both C/EBP- and C/EBPß-mediated trophoblast functions.
Since mice deficient in C/EBP, C/EBPß, C/EBP, C/EBP, and the compound C/EBPß survive at least until birth (see reference 31 and references therein) and since C/EBP was absent in the placental tissue at the studied stages, our data also suggest that C/EBP and C/EBPß are the major C/EBPs in placental morphogenesis, in particular in the formation of the labyrinthine layer. Moreover, placental morphogenesis occurred normally in mice that retained one allele of either C/EBP in the absence of C/EBPß, or C/EBPß in the absence of C/EBP, but not after removal of both transcription factors. These results clearly show that C/EBPs are indispensable during embryogenesis and that C/EBP and C/EBPß are redundant during placental development. How C/EBP and C/EBPß repress or activate gene expression in placentogenesis still remains to be determined. Our data imply that C/EBP or C/EBPß homo- or heterodimers are required or, alternatively, that C/EBP or C/EBPß are equivalent components of other gene regulatory protein complexes during placentogenesis. AP-1 family members JunB or FRA1 are potential candidates since they may heterodimerize with C/EBP proteins (26) and they are required for proper placental morphogenesis (36, 38).
Several other transcription factors have been found to be critical for the development of the placental labyrinthine layer after initiation of morphogenesis (11, 35). Among these transcription factors, the nuclear hormone receptor PPAR was an interesting potential C/EBP target because it is activated by C/EBP and/or C/EBPß at the onset of adipogenic differentiation (33, 34). In addition, PPAR, C/EBP, and C/EBPß are coexpressed in the placenta. The expression level of PPAR, however, was found to be similar in both C/EBPß–/– and control placentas, indicating that activation of PPAR does not rely on C/EBP functions during early placentogenesis. This possibility is supported by the fact that PPAR1 is the only isoform expressed in the placenta, whereas PPAR2, initiated from an alternative promoter, is the effector of C/EBPs in adipogenesis (5).
Recent studies suggested that C/EBP and C/EBPß regulate basal Dlx3 promoter activity in placental cells (14). Moreover, Dlx3-deficient embryos die around E10 due to a defect in labyrinthine layer development (24). The data presented here, however, show that Dlx3 is still expressed in C/EBPß–/– placentas but only in the chorionic plate. Consistently, all of the markers used to label the labyrinthine layer (Gcm1, Dlx3, and VEGF) showed reduced expression that is most likely a secondary effect of defective formation of the labyrinthine layer.
The molecular mechanism underlying the prevention of placental labyrinth formation in C/EBPß–/– mice await further studies. In this context, it is important to note that C/EBPß has been identified as an important target of cyclin D1 in many human tumors (21), suggesting a proliferation-inducing function in addition to the differentiation-inducing activity of C/EBPs. Moreover, it has been shown that C/EBP and C/EBPß regulate the production of growth factors and cytokines that sustain proliferation (2, 30, 45). The placenta is a major source of growth factors, cytokines, and hormones that affect both the mother and the fetus. We therefore speculate that the absence of both C/EBP and C/EBPß might have several additional effects on embryogenesis once the placental dysfunction could be rescued. However, the low frequency of double-knockout generation (ca. 6%) currently precludes the efficient analysis of chimeric embryos that would be obtained by aggregating C/EBPß–/– embryonic stem cells derived from C/EBPß+/– intercrosses with wild-type tetraploid embryos (35). Thus, the question of molecular mechanisms and additional embryonic C/EBP functions will therefore have to await the generation of conditional knockout mice.
In conclusion, we report here the earliest developmental role of C/EBPs in the mouse. A concerted function of C/EBP and C/EBPß occurs at postimplantation stages during the generation of a functional chorioallantoic placenta. The death of C/EBPß–/– embryos is most likely due to the failure to establish a proper placental circulation. Recently, it has been reported that C/EBP, C/EBPß, and C/EBP are expressed in human placenta during pregnancy (4). Therefore, it would be important to determine whether dysregulation of C/EBP gene expression correlates with a pathological phenotype in human placentogenesis.
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ACKNOWLEDGMENTS |
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+/– mice. We thank T. Müller and C. Birchmeier (Max Delbrueck Center for Molecular Medicine, Berlin, Germany) for valuable discussions and comments on the manuscript. We thank the members of Leutz lab for discussions. We are also grateful to E. Sterneck, U. Borgmeyer, D. Riethmacher, M. Soares, R. Evans, and E. E. Voest for providing cDNA probes and to J. Rossant for providing the TS cell line that served as a valuable control during the preparation of the manuscript. |
FOOTNOTES |
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