1 Department of Molecular, Cellular and Developmental Biology, Yale University, 266 Whitney Avenue, New Haven, CT 06511, USA
2 Departments of Craniofacial Biology, and Cellular and Structural Biology, BRB151, Campus Box C286, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262, USA
* Present address: Gene Targeting Service, Section of Comparative Medicine, Yale University School of Medicine, PO Box 208016, New Haven, CT 06520-8016, USA
Present address: Northwestern University Medical School, Childrens Memorial Hospital, 2300 Childrens Plaza, MC 204, Chicago, IL 60614, USA
Author for correspondence (e-mail: trevor.williams{at}uchsc.edu)
Accepted 14 March 2002
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SUMMARY |
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Key words: Tcfap2c mutant mouse, Postimplantation development, Extra-embryonic, Trophoblast, AP-2
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INTRODUCTION |
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The AP-2 proteins have also been linked with cell fate determination, as they are responsive to a number of signaling molecules, including cAMP and the morphogen retinoic acid (RA) (Imagawa et al., 1987; Lüscher et al., 1989
). Indeed, the mouse Tcfap2c gene (which encodes the protein AP-2
) was isolated independently, and named AP-2.2, in a screen for RA-inducible genes in murine embryonal carcinoma cells (Oulad-Abdelghani et al., 1996
).
The mouse Tcfap2c gene is expressed in the central and peripheral nervous system, ectoderm, limbs, face and mammary glands during mouse development, in a dynamic spatiotemporal pattern similar to that of the other AP-2 family members (Chazaud et al., 1996; Mitchell et al., 1991
; Moser et al., 1997b
) (J. Zhang and T. W., unpublished). Concurrent with their initial expression in the embryo, all three AP-2 genes are also expressed in the extra-embryonic trophoblast (Chazaud et al., 1996
; Moser et al., 1997b
). Importantly, Tcfap2c has been shown to be uniquely expressed in trophoblast at 6.5 d.p.c. and thus earlier in development than the other members of the family, which are expressed at 8 d.p.c. (Chazaud et al., 1996
; Moser et al., 1997b
; Shi and Kellems, 1998
). Later in embryogenesis, Tcfap2c also demonstrates strong and persistent expression in all trophoblast lineages of the chorioallantoic placenta, including the secondary giant cells, spongiotrophoblast and labyrinthine trophoblast (Sapin et al., 2000
; Shi and Kellems, 1998
).
Previous gene targeting studies have shown that either Tcfap2a or Tcfap2b (genes encoding AP-2 and AP-2ß, respectively) can be mutated without affecting placental function, although both genes have essential functions during development of the embryo proper. Tcfap2a is required for limb, eye, craniofacial, cardiovascular, skeletal and body wall development (Brewer et al., 2002
; Nottoli et al., 1998
; Schorle et al., 1996
; Zhang et al., 1996
), while Tcfap2b is required for renal epithelial cell survival during late embryogenesis (Moser et al., 1997a
). Consistent with these findings, several genes expressed in epidermal and neural crest lineages have been found to contain AP-2-binding sites as a component of their regulatory sequences (Leask et al., 1991
; Maconochie et al., 1999
). Other potential targets of AP-2 action are genes associated with extra-embryonic function, including those involved in steroid hormone biosynthesis and signaling within the placenta (Hu et al., 1996
; Johnson et al., 1997
; LiCalsi et al., 2000
; Pena et al., 1999
; Peng and Payne, 2001
; Piao et al., 1997
; Richardson et al., 2000
; Steger et al., 1993
). In particular, Shi and Kellems (Shi and Kellems, 1998
) have identified a placental-specific footprinted region, matching the AP-2 consensus site, within the placental regulatory element of the murine adenosine deaminase gene (Ada). Mutation of this AP-2 site abolished reporter gene expression in the placenta of transgenic mice. Indeed, as Tcfap2c is the most highly expressed of the AP-2 family members in the extra-embryonic tissues, it has been proposed that this gene may be required not only for placental Ada expression but also for development of the mature placenta (Shi and Kellems, 1998
).
Considering the links between Tcfap2c and placental function, and its potential importance in both normal development and in tumorigenesis, we investigated its biological function by generating Tcfap2c-deficient mice. Our studies demonstrate that Tcfap2c is vital to embryonic survival during the early postimplantation period, and that expression of AP-2 in the extra-embryonic membranes can rescue development of Tcfap2c mutant embryos.
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MATERIALS AND METHODS |
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Whole-mount immunocytochemistry of blastocysts
Embryos were flushed from uteri at 3.5 d.p.c. and fixed at room temperature for 30 minutes in 4% paraformaldehyde in saline buffer (pH 7.4), preincubated with 0.1% H2O2/PBS/0.2% Triton-X-100 for 20 minutes, blocked in 1% BSA for 5 minutes and incubated with 10% normal goat serum for 30 minutes at room temperature. Embryos were incubated overnight at 4°C with the anti-AP-2 rabbit polyclonal antiserum,
96, described elsewhere (Turner et al., 1998
). Subsequently, embryos were incubated for 30 minutes at room temperature with biotin-SP-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories) followed by peroxidase-conjugated Streptavidin (Jackson Immunoresearch Laboratories) and the substrate 3,5 diaminobenzidine for visualization (Aldrich). Alternatively, for fluorescent detection, embryos were incubated with AlexaFluor 488-conjugated goat anti-rabbit IgG (Molecular Probes) and counterstained for DNA with Hoechst 33342 (Molecular Probes).
Immunohistochemistry
Uteri or whole decidua were isolated in ice-cold PBS at 4.5-7.5 d.p.c., fixed in 4% paraformaldehyde at 4°C overnight, dehydrated, oriented with respect to the antimesometrial-mesometrial axis, embedded in paraffin and serially sectioned at a thickness of 5 µm. Immunostaining was performed as described (Turner et al., 1998). After thorough washing, the samples were treated with 2.5% hydrogen peroxide in methanol for 30 minutes, then blocked in 1% BSA and 10% normal goat serum. Addition of the anti-AP-2
polyclonal rabbit primary antiserum at a 1:500 dilution was followed by incubation with Biotin-SP-conjugated goat anti-rabbit IgG, peroxidase-conjugated Streptavidin, and 3,5 diaminobenzidine for visualization (Jackson ImmunoResearch; Aldrich). No staining was observed when the primary antibody was omitted, or when a blocking peptide specific for AP-2
(Turner et al., 1998
) was preincubated with the primary antibody. Note that the antisera used to study AP-2
expression recognizes an epitope at the C terminus of the protein that should be absent from any product derived from the targeted allele.
Histological analysis
Embryos from Tcfap2c+/ intercrosses were processed for histological analysis as described by Kaufman (Kaufman, 1990). Briefly, whole decidua were dissected and fixed overnight in Bouins fixative, dehydrated, oriented with respect to the antimesometrial-mesometrial axis and embedded in paraffin. Sagittal sections (5 µm) were cut and stained with Hemotoxylin and Eosin (H&E). After histological examination, embryonic tissues were scraped from slides and subjected to PCR analysis as described above.
In situ hybridization
Embryos were staged according to their morphology (Downs and Davies, 1993; Theiler, 1989
). In situ hybridization using digoxigenin-labeled RNA probes was performed on whole-mount embryos and histological sections according to the protocol described (Shen et al., 1997b
), except that whole embryos were processed in 0.74 µm mesh inserts (Costar). In situ hybridization on sections using [33P]UTP-labeled probes was performed according to the protocol described by Hogan et al. (Hogan et al., 1994
) and modified for 33P as described (Biroc et al., 1993
). The plasmid CCC36, containing a 200 bp fragment of Tcfap2c exon 7, was used to synthesize the AP-2
sense and antisense probes. This fragment, which extends from the 5' boundary of exon 7 to the ClaI site in the middle of exon 7, is absent from the targeted Tcfap2c allele. Other probes used for in situ hybridization analysis were generously provided by the following researchers: T (R. Beddington); Otx2 and Hnf3b (T. Gridley); Pl1 (Csh1 Mouse Genome Informatics) Mash2 (Ascl2 Mouse Genome Informatics), Eomes, Cdx2, Fgfr2 (J. Rossant); Ada (R. Kellems); Bmp4 (B. Hogan); and Hand1 (J. Cross).
Blastocyst outgrowths
Blastocysts from Tcfap2c+/ matings were flushed from uteri at 3.5 d.p.c. in M2 medium and individually cultured in ES cell medium without LIF containing 15% fetal bovine serum (HyClone), in 5% CO2 at 37°C, on 24-well tissue culture dishes (Falcon). On the seventh day of in vitro culture, outgrowths were photographed and subsequently removed and genotyped by PCR as described above.
Generation of Rosa26 Tcfap2c+/ and Tcfap2c/ ES cell lines and mouse chimera studies
Tcfap2c+/ mice on a 129/Sv background were bred with B6,129 hybrid mice homozygous for the ROSA ß-geo 26 gene-trap allele (ROSA 26; Jackson Laboratories) to produce Tcfap2c heterozygotes containing the ROSA26 allele. These mice were interbred; blastocysts were collected and cultured in ES medium with LIF on -irradiated mouse embryo fibroblasts for generation of ES cell lines (Robertson, 1987
). The resulting ES cell lines were genotyped by Southern blotting, stained for ß-galactosidase (ß-gal) activity and karyotyped (Hogan et al., 1994
). Tcfap2c+/ and Tcfap2c/ ES cells containing the ROSA26 allele were injected into C57BL/6J blastocysts. Resulting chimeras were either collected at various points during gestation and processed for ß-gal staining (n=123) or allowed to develop to term (n=130). For complementary blastocyst injection experiments, wild-type ES cells containing the ROSA26 insertion (provided by Elizabeth Robertson) were injected into blastocysts obtained from Tcfap2c+/ intercrosses. Resulting chimeras were collected 10.5 d.p.c., stained for ß-gal activity and examined by whole mount. Yolk sac endoderm DNA from these chimeras was isolated as described (Hogan et al., 1994
) and subjected to PCR genotyping as described above. Of all morphologically normal chimeras with high percentages of ß-gal staining, 16 were derived from wild type and 41 from Tcfap2c+/ blastocysts, respectively, while all six abnormal high percentage chimeras were derived from Tcfap2c/ blastocysts. For tetraploid-diploid aggregations, tetraploid embryos were generated from electrofusion of two-cell-stage CD-1 embryos using an Electro Cell Manipulator 2001 (BTX) (according to the manufacturers conditions) and aggregated at the four- to eight-cell stage with Tcfap2c/ ES cells (Nagy and Rossant, 1993
). Successful aggregates were transferred as blastocysts to foster mothers, and embryos were collected at 8.5, 9.5, 10.5 and 18.5 d.p.c., and processed to detect ß-galactosidase activity (n=11).
Imaging
Images were captured using 35 mm film or a SPOT II camera (Diagnostic Instruments) and manipulated in Adobe Photoshop.
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RESULTS |
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Loss of Tcfap2c causes early embryonic lethality
Intercrossing heterozygotes failed to produce live Tcfap2c/ pups out of 50 live offspring, indicating embryonic lethality (Table 1). To determine the period of lethality, gestation sites from heterozygote matings were examined at progressively earlier stages of development. Embryos from timed matings were dissected free of maternal tissues, observed by whole mount and genotyped (Fig. 1B, Fig. 2A). Tcfap2c mutants rarely survived beyond 7.5 d.p.c., but the expected ratios of Tcfap2c/ mutant embryos were recovered up until and including this timepoint (Table 1). By whole-mount examination, 7.5 d.p.c. Tcfap2c/ embryos were consistently smaller than littermates and frequently lacked organized structures such as a primitive streak (Fig. 2A, parts a-e). The extra-embryonic tissues were either underdeveloped or disorganized, and the boundary between embryonic and extra-embryonic ectoderm was often poorly defined (Fig. 2 and data not shown). On occasion, the orientation of the Tcfap2c mutants within the decidua was atypical (Fig. 2A, part c), a phenotype that is described in more detail later with reference to Fig. 4. The few Tcfap2c mutants that survived until 8.5-10.5 d.p.c. were small, often severely disorganized, and/or undergoing resorption (Table 1). One of the more organized and advanced embryos within this category is shown in Fig. 2A (panel f), a 10.5 d.p.c. Tcfap2c/ embryo in which a head fold is apparent, but the overall size and developmental stage is more typical of an 8.0 d.p.c. wild-type embryo. Taken together, these observations indicate that Tcfap2c is required for early postimplantation survival in the mouse with the major period of lethality occurring between 7.5 and 8.5 d.p.c.
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Tcfap2c/ mutants are defective in embryonic and extra-embryonic development
To further investigate the cause of the early postimplantation lethality, Tcfap2c mutants and wild-type or heterozygous littermates were examined in greater detail through histological sections at 7.5 d.p.c. (Fig. 4). Once documented, embryonic tissue was removed from slides and genotyped. Analysis of these morphological and genotypic data indicated that a range of developmental abnormalities was present in the 7.5 d.p.c. Tcfap2c/ embryos (Table 2). All Tcfap2c/ embryos exhibited growth retardation, disruption of the maternal-embryonic interface, and defective development of the extra-embryonic tissues. One of the major trophoblast cell types, the trophoblast giant cells, was reduced in number in the mutant embryos (Fig. 4). Whereas 50 to 60 primary giant cells could be present in wild-type conceptuses, in some Tcfap2c/ embryos, as few as one to two giant cells were observed. In the majority of mutants, the extra-embryonic ectoderm was disorganized and often appeared to be either abnormally elongated or as a series of folds stacked upon one another (Fig. 4D,H, respectively; Table 2). Moreover, the exocoelomic and ectoplacental cavities usually failed to form, although the proamniotic cavity was present in the majority of Tcfap2c/ mutants (Fig. 4; Table 2). The ectoplacental cone was typically reduced in size, compact, and not well-integrated with the surrounding maternal tissues. In most cases, there were large pools of maternal blood in the decidua adjacent to, but not continuous with, the ectoplacental cone of the Tcfap2c/ conceptuses (Fig. 4). In some developmentally delayed mutants, the ectoplacental cone was absent altogether. In other examples, the primary embryonic axis (ultimately the dorsoventral axis) was defective, as the embryo was oriented atypically with respect to the extra-embryonic membranes. For example, the ectoplacental cone was frequently positioned nearly perpendicular to the epiblast (Fig. 4G and data not shown), in contrast to the slightly angled alignment observed in wild-type conceptuses (Fig. 4A,C; Table 2). This atypical orientation was also observed in whole-mount preparations of some embryos, evident as abnormal bending of the embryo accompanied by malformation of Reicherts membrane (Fig. 2A, panel c). Although the primary embryonic axis was normal in approximately 30% of mutant embryos, in many instances the extent of the defect was difficult to determine due to general disorganization.
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Occasionally, parietal endoderm/Reicherts membrane was observed to intervene abnormally between the extra-embryonic ectoderm and ectoplacental cone of mutants (Fig. 2A, part e; Table 2). Histological sections from this class of mutant indicated that Reicherts membrane appeared to encapsulate the embryo, sometimes as an abnormally thickened and continuous layer (Fig. 3e and data not shown; Table 2). By contrast, visceral endoderm was present and appeared normal in the majority of mutant embryos. The allantoic bud, while sometimes present in wild-type embryos at 7.5 d.p.c., was rarely observed in the Tcfap2c mutant embryos.
In addition to the extra-embryonic defects, we also noted abnormalities in the embryo proper. In particular, the mutant epiblast was typically smaller and retarded in development in comparison with littermates (Fig. 4). In some cases, mesoderm formed but accumulated as a large mass of loosely associated cells (Fig. 3E; Fig. 4H). Several of the mutants analyzed lacked mesoderm formation, which meant they failed to undergo gastrulation. Between 15 and 20% of mutants exhibited a more pronounced degree of disorganization, such that separate embryonic and extra-embryonic compartments were not identifiable (Fig. 4E; Table 2).
Molecular marker analysis of Tcfap2c mutant embryos
To investigate the defects in the embryonic organization of the Tcfap2c mutants, we examined the expression of pertinent marker genes at 7.5 d.p.c. by RNA in situ hybridization. To determine whether mesoderm formation is initiated in the Tcfap2c mutants, we examined the expression of the early mesoderm lineage marker T (Brachyury) (Wilkinson et al., 1990). In mutants that exhibited T expression, transcripts were either confined to a small posterior region of the proximal epiblast or localized in a pattern comparable with that of wild-type littermates, in which the nascent mesoderm was marked along the primitive streak (Fig. 5A,B). Wild-type embryos expressed the later mesoderm marker Hnf3b in the node at 7.5 d.p.c., while some Tcfap2c mutants expressed Hnf3b in a location corresponding to the anterior primitive streak, even in the absence of a distinct primitive streak (Fig. 5C,D) (Sasaki and Hogan, 1993
). Otx2, a homeodomain-containing transcription factor that is initially expressed in the anterior visceral endoderm and epiblast and becomes restricted to the anterior epiblast by the end of gastrulation, was also expressed in an anterior pattern similar to that of wild-type embryos (Fig. 5E,F) (Simeone et al., 1993
). Thus, mesoderm induction can occur and anteroposterior markers are expressed in appropriate domains, despite the morphological abnormalities in the mutant epiblast. These findings are consistent with the ability of some mutant embryos to develop to the headfold stage (Fig. 2A, part f).
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The second set of experiments addressed whether or not the embryonic retardation and disorganization observed in the Tcfap2c/ mutants was a secondary consequence of the defects in the extra-embryonic compartment. We thus carried out a converse chimera analysis, in which wild-type, ROSA26-marked ES cells were injected into blastocysts from Tcfap2c+/ matings, in order to assess the developmental potential of the wild-type ES cells in the context of an Tcfap2c mutant host environment. We collected chimeras up to 10.5 d.p.c., as this was the latest timepoint to which the Tcfap2c/ embryos had persisted and would allow us to address whether or not early placentation had been rescued by the presence of wild-type cells. For each chimera, we retrospectively genotyped the host blastocyst by PCR analysis of the DNA from yolk-sac endoderm, which contains little if any contribution from ES cells (Beddington and Robertson, 1989). Chimeras derived from either wild-type or heterozygous host blastocysts were morphologically wild type (Fig. 8D). By contrast, chimeras with Tcfap2c mutant extra-embryonic tissues showed variable phenotypes that were morphologically similar to Tcfap2c mutants recovered from natural matings, even with extensive contribution from wild-type ES cells, as judged by staining for ß-galactosidase activity (Fig. 8E and data not shown). Some of these chimeras were very small and lacked any obvious organization, although one had developed a head structure with an open neural tube (Fig. 8E). Nevertheless, this embryo was considerably smaller than wild-type littermates, and the head and trunk were underdeveloped and malformed for this stage of development, resembling the low percentage of Tcfap2c/ mice that survive until 10.5 d.p.c. (Fig. 2A, part f). In all cases, no evidence of a placenta was associated with chimeras derived from Tcfap2c/ blastocysts. Together, these findings demonstrate that there is an essential requirement for Tcfap2c within the extra-embryonic membranes for normal development of the mouse embryo beyond the early postimplantation period.
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DISCUSSION |
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The process of implantation begins with the attachment and integration of the embryonic trophoblast cells into the maternal endometrium (reviewed by Cross et al., 1994; Rinkenberger et al., 1997
). During the early postimplantation period, access to the maternal circulation is vital to the growth and survival of the embryo prior to the formation of the placenta. This connection is facilitated by the trophoblast cells, Reicherts membrane and the parietal endoderm, which form a primitive diffusion barrier to allow nutrients and gases to nourish the embryo without direct exposure to maternal blood. The absence of AP-2
impacts upon the development of all of these vital extra-embryonic structures during this critical period. Tcfap2c joins a small group of molecules that are essential for development during the early postimplantation period and have been proven to act within the extra-embryonic membranes (for reviews, see Copp, 1995
; Kupriyanov and Baribault, 1998
; Rinkenberger et al., 1997
). Several of these are likely to act within the visceral endoderm, including Smad4, HNF4, GATA6 and huntingtin (Chen et al., 1994
; Dragatsis et al., 1998
; Duncan et al., 1997
; Koutsourakis et al., 1999
; Sirard et al., 1998
). Others, such as HAND1, Ets2 and merlin, appear to affect specific aspects of trophoblast development, such as the differentiation of giant cells or the formation of extra-embryonic ectoderm (McClatchey et al., 1997
; Riley et al., 1998
; Yamamoto et al., 1998
). Tcfap2c would fall into this latter category; however, the particular combination of defects in all trophoblast lineages, the changes in trophoblastic gene expression, and the defective parietal endoderm makes the Tcfap2c/ mutants distinctive.
A striking aspect of the Tcfap2c knockout mice is the paucity of giant cells present either in utero or in blastocyst outgrowths. Despite the smaller population of giant cells in the Tcfap2c mutants compared with wild-type conceptuses, the mutant embryos are nonetheless able to attach to the uterine lining, induce a decidual reaction and implant. The Tcfap2c mutant trophoblast cells still produce transcripts for placental lactogen 1, a hormone that is characteristically secreted by giant cells. Moreover, expression of the putative AP-2 target gene Ada can also be detected in the Tcfap2c-null primary giant cells. Previous studies have shown that an Ada transgene was dependent on the presence of an AP-2 binding site for placental expression and implicated AP-2 in this regulation (Shi and Kellems, 1998
). We speculate that other AP-2 family members could compensate for the loss of zygotic AP-2
and thus maintain Ada expression. Alternatively, it is possible that the regulation of the endogenous Ada gene in its normal chromosomal context is more complex than that of the AP-2-dependent transgene. Although the Tcfap2c/ primary giant cells express expected markers, there may be subtle differences in the properties of the mural trophectoderm that affect implantation among some mutants, as 10-15% of mutant embryos are misoriented in the decidua. In this regard, the atypical positioning of the Tcfap2c mutants with respect to the AM-M axis is reminiscent of the phenotype observed in Fgfr2/ embryos, which are positioned randomly as implanting blastocysts within the uterine crypt (Arman et al., 1998
).
Following implantation, the trophectoderm overlying the ICM continues to proliferate and gives rise to the extra-embryonic ectoderm. Precursors in the extra-embryonic ectoderm will populate the ectoplacental cone, which expands into the deciduum to establish intimate connections with the maternal vasculature. In the majority of Tcfap2c mutants, the extra-embryonic ectoderm is either underdeveloped or disorganized. In addition, the ectoplacental cone is usually small and compact, and it does not appear to extend normally into the maternal tissues. Often, the ectoplacental cone is positioned inappropriately such that it alters the linearity of the primary axis, or the future dorsoventral axis. The abnormal phenotype in the extra-embryonic ectoderm may be mechanically related to the defects in the ectoplacental cone. For example, if the ectoplacental cone fails to migrate, the extra-embryonic ectoderm may continue to grow but be confined from elongating and collapse upon itself, resulting in the folded sheet appearance. In some cases, Reicherts membrane may serve to further obstruct the expansion of the ectoderm, as when the membrane is removed during dissection, the ectoderm rapidly expands as though under pressure (O. L., unpublished).
Studies in cell culture and in mouse chimeras provide evidence that a stem cell population exists in the extra-embryonic ectoderm, giving rise to precursor cells in the ectoplacental cone that form secondary giant cells (Rossant et al., 1978; Tanaka et al., 1998
). These stem cells express the FGFR2 receptor, and proliferate partly in response to an FGF4 signal originating in the epiblast. We find that although Fgfr2 expression is present in the Tcfap2c-null extra-embryonic compartment, the putative FGF-responsive effector molecules Cdx2 and Eomes are downregulated. The uncoupling of signaling between FGFR2 and these effectors by the absence of AP-2
could reflect either the loss or reduction of specific cells, or that AP-2
has a direct effect on the expression of these transcription factors. Notwithstanding, lowered expression of Cdx2 and Eomes would be expected to alter the functioning of the stem cell population and limit the number of cells contributing to the extra-embryonic ectoderm and the ectoplacental cone, a situation that typifies the Tcfap2c-mutant phenotype. A smaller pool of stem cells in the mutant would be consistent with the paucity of secondary giant cells within or around the ectoplacental cone that also occurs in the Tcfap2c mutants.
We have shown that AP-2 is present as a maternally derived protein in preimplantation embryos and have also determined that maternal AP-2
transcripts are present in unfertilized oocytes (Q. W., unpublished). The extent to which maternal mRNAs and proteins are involved in the development of the early mouse embryo is unknown. However, it has been known for some time that maternal transcripts can be translated into proteins that persist beyond the time of initiation of zygotic transcription (West and Flockhart, 1989
). More recently, two maternal effect genes have been identified, Hsf1 and Mater, whose products are required for mouse development beyond the zygote and the two-cell stage, respectively (Christians et al., 2000
; Tong et al., 2000
). Rescue by maternal proteins or transcripts has been proposed in the study of several mutants that show later defects than expected based on expression patterns (Haegel et al., 1995
; Larue et al., 1994
; Meagher and Braun, 2001
; Reithmacher et al., 1995
; Riley et al., 1998
; Shen-Li et al., 2000
). Similarly, it is possible that maternal contribution in the Tcfap2c mutant mouse masks an earlier requirement for AP-2
during pre- or peri-implantation development. Indeed, the lethality in the Tcfap2c/ embryos occurs soon after maternally derived protein is exhausted, when zygotic expression would normally be expected to replenish AP-2
protein levels. Based on these findings, we predict that maternal AP-2
mRNA and/or protein will have an important role in early embryogenesis. We also hypothesize that maternal stores of AP-2
could account for the variability we observe in the Tcfap2c-mutant phenotype during the postimplantation period. Specifically, the phenotype might vary depending upon the quantity of AP-2
within a particular fertilized oocyte, and its rate and extent of depletion prior to implantation. An alternative possibility is that phenotypic variability is due to the outbred genetic background of the Tcfap2c mutant mice.
The early lethality of Tcfap2c mutants precluded a direct assessment of whether Tcfap2c played a role in later embryonic or extra-embryonic development. Through the generation of chimeras by blastocyst injection, we found that Tcfap2c/ ES cells could widely contribute to many tissues of morphologically normal adults, suggesting that it is not required for later embryonic or adult viability. Although Tcfap2c does not appear to have a major unique function in the embryo proper, we predict that there will be a continuing requirement for this gene in the development of the extra-embryonic membranes, particularly within the chorioallantoic placenta. The unique and abundant expression of AP-2 in the trophoblast derivatives throughout placental development and its ability to regulate genes important in later placental function lend support to this idea. The extra-embryonic tissues are affected early in the mutant, before the formation of the chorioallantoic placenta, which begins at 9 d.p.c. with the fusion of the allantois to the chorion. Future targeted mutagenesis studies will be required to test the later requirement of Tcfap2c in the mature placenta. The fact that Tcfap2c plays no role individually in the embryo proper highlights the divergent functions among the members of the AP-2 transcription factor family. The disruption of either the Tcfap2a or Tcfap2b gene results in perinatal lethality in the mouse embryo; while Tcfap2b has a primary role in kidney morphogenesis, Tcfap2a affects multiple developmental programs, including formation of the neural tube, eye, face, forelimbs, body-wall and cardiovascular system (Brewer et al., 2002
; Moser et al., 1997a
; Nottoli et al., 1998
; Schorle et al., 1996
; Zhang et al., 1996
). The divergent developmental events influenced by the individual AP-2 family members contrasts with the observation that they bind to the same consensus sequence to activate transcription. Moreover, these transcription factors share overlapping patterns of gene expression during embryogenesis, including within the trophoblast lineages from 8 d.p.c. onwards. Therefore, it is possible that there are also redundant roles for the AP-2 gene family in the regulation of embryonic and extra-embryonic development that remain to be uncovered.
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ACKNOWLEDGMENTS |
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