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BOR - Papers in Press, published online ahead of print May 3, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.052407
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BIOLOGY OF REPRODUCTION 75, 324–333 (2006)
DOI: 10.1095/biolreprod.106.052407
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Analysis of Transcription Factor AP-2 Expression and Function During Mouse Preimplantation Development1

Quinton Winger 3 56 , Jian Huang 56 , Heidi J. Auman 4 7, Mark Lewandoski 8, and Trevor Williams 2 567  

Departments of Craniofacial Biology5 and Cell Developmental Biology,6 University of Colorado Health Sciences Center at Fitzsimons, Aurora, Colorado 80045 Department of Molecular, Cellular, and Developmental Biology,7 Yale University, New Haven, Connecticut 06511 Genetics of Vertebrate Development Section,8 National Cancer Institute, Frederick Cancer Research & Development Center, Frederick, Maryland 21702

ABSTRACT

Theactivating protein 2 (AP-2) transcription factor family is required for multiple aspects of mouse postimplantation development, but much less is known about the expression and possible function of these genes during the preimplantation period. In the present study, we have examined the expression of all five members of the mouse AP-2 gene family in the unfertilized oocyte and from zygote formation to the blastocyst stage of development. Four AP-2 genes are differentially expressed during the preimplantation period,Tcfap2a,Tcfap2b,Tcfap2c, andTcfap2e. Furthermore, with the exception ofTcfap2a, these genes are also expressed in unfertilized oocytes, indicating that they may be important for oogenesis, maternal-effect functions, or both. Given these findings, we have initiated studies to assess how various combinations of maternal and zygotic AP-2 gene expression might function together to regulate pre- and peri-implantation development. The present study focuses on the interplay between the expression of zygoticTcfap2aand maternal and zygoticTcfap2c. These studies indicate that zygotic, but not maternal,Tcfap2cexpression is required for normal embryogenesis. In addition, the combined loss of bothTcfap2aandTcfap2caccelerates embryonic lethality compared to the loss of either gene alone, demonstrating that genetic redundancy exists between these two AP-2 family members during the peri-implantation period of embryogenesis.

developmental biology, early development, embryo, gene regulation, trophoblast

INTRODUCTION

A number of critical cellular and physiological events have been identified during the period of mammalian reproductive development encompassing fertilization to blastocyst formation. However, the molecular analysis of mammalian pre- and peri-implantation development has, until recently, proven a particular challenge because of the limited amounts of material available for study. With the advent of new technologies, particularly microarray analysis, fresh insight is now being gained into the genes that are expressed during the transition from the unfertilized oocyte to the implanting blastocyst [1–11]. Gene targeting has also led to the identification of several critical genes that are required during this period of mouse embryogenesis [1, 6–8, 12]. Such studies have deepened our understanding of the mechanisms regulating oogenesis, fertilization, cleavage, blastocyst formation, and implantation.

It has long been realized that the mouse oocyte contains considerable quantities of protein and RNA that are activated and utilized following fertilization. In general, such molecules are subsequently degraded during the preimplantation period and replaced with zygotic RNA transcripts and proteins. This transition from a reliance on maternal to zygotic products begins with zygotic gene activation (ZGA) at the two-cell stage [13, 14]. It was initially thought that the gene expression changes happening during the ZGA were relatively widespread and promiscuous. More recently, transcript profiling of this stage of development has indicated that a more narrow set of zygotic genes is activated, especially those involved in transcription and RNA processing [2, 4, 9–11]. However, a thorough understanding of this transition with respect to many individual genes is complicated by the presence of both maternal and zygotic transcripts from the same gene from the two-cell stage onward. For such genes, the kinetics of maternal RNA decay and ZGA have been determined in only a few instances [13–18].

Similarly, the relative importance of such a gene's maternal versus zygotic transcripts has rarely been distinguished [19]. This problem is highlighted by the recent appreciation that maternal-effect genes may be more common in mammalian development than previously anticipated [1, 20–22]. Females lacking a maternal-effect gene are healthy and viable, and their oocytes undergo apparently normal development and fertilization. However, subsequent development of the zygote is defective, as a maternal gene product required for embryonic development after fertilization is absent. Some maternal-effect genes, including Zar1, Npm2, and Hsf1, exert their influence immediately after fertilization, at the oocyte-to-embryo transition, arresting development prior to the onset of the ZGA [22–24]. The absence of Nalp5 (previously known as Mater), Ube2a (previously known as HR6A), and Zfp36l2 maternal gene products usually arrests zygotes soon after, at the two-cell stage, while embryos derived from females lacking Dppa3 (previously known as stella) rarely reach the blastocyst stage [16, 21, 25, 26]. Defects due to the lack of additional maternal gene products, particularly those required for DNA repair, DNA methylation, or chromatin remodeling, can present much later in development as increased mutation loads, inappropriate gene expression, or midgestation lethality [15, 27–29]. Nevertheless, not all maternal gene products are essential for normal embryogenesis. Thus, zygotes lacking maternal E-cadherin or ß-catenin, although clearly defective, can still generate viable embryos, presumably because of the activation of the paternal allele soon after fertilization [19].

On the basis of the results obtained in mouse knockout studies, a maternal and zygotic expression component has also been proposed for the gene encoding activating protein 2 (AP-2){gamma}, Tcfap2c [30]. AP-2{gamma} is a member of the AP-2 transcription factor family that is defined by the presence of a conserved C-terminal DNA-binding and dimerization domain [31–33]. There are five AP-2 genes in the human and mouse genome, Tcfap2a, Tcfap2b, Tcfap2c, Tcfap2d, and Tcfap2e, encoding the proteins AP-2{alpha}, ß, {gamma}, {delta}, and {varepsilon}, respectively [31, 34–38]. Currently, mouse molecular genetic analyses have uncovered specific roles for Tcfap2a, Tcfap2b, and Tcfap2c in distinct developmental events. Tcfap2a is required for multiple processes, including limb, eye, craniofacial, cardiovascular, neural tube, and body wall development [39–45], while Tcfap2b is particularly important for renal epithelial cell survival [46]. Although the loss of either Tcfap2a or Tcfap2b has a dramatic impact on embryonic development and results in perinatal lethality, loss of these genes does not cause extra-embryonic tissue deficiencies, and mutant embryos are capable of normal implantation and placental development.

In contrast, Tcfap2c is required much earlier in embryogenesis-within the trophoblast cell lineage [30]. Embryos lacking Tcfap2c produce aberrant extra-embryonic tissues, fail to develop a functional placenta, and die around 7.5 days postconception (dpc) [30, 47]. The interpretation of when Tcfap2c is first required during embryogenesis is complicated, however, because Tcfap2c-null blastocysts still contain residual maternally derived AP-2{gamma} protein [30], reflecting expression of this gene in developing oocytes of the ovary [48]. These observations suggested that maternally derived AP-2{gamma} protein, obtained either directly from the unfertilized oocyte or translated from maternal Tcfap2c transcripts after fertilization, influences the development of the preimplantation embryo prior to the activation of zygotic Tcfap2c expression. Therefore, in the following analysis, we have examined when maternal Tcfap2c mRNA is lost and when zygotic mRNA expression begins during the preimplantation period of mouse development. In addition, we have used a floxed allele of Tcfap2c in combination with a Zp3-cre transgene to generate a conditional ablation of Tcfap2c in the oocyte. This approach enabled a comparison of mouse embryonic development in the absence of 1) the maternal, 2) the zygotic, or 3) both the maternal and zygotic components of Tcfap2c expression. We also demonstrate that several other members of the AP-2 gene family are coexpressed with Tcfap2c during the preimplantation period and reveal genetic redundancy between Tcfap2a and Tcfap2c for embryogenesis. Taken together, our findings support important regulatory functions for the AP-2 transcription factor family during mouse pre- and peri-implantation development.

MATERIALS AND METHODS

Animals

All animal experiments were performed in accordance with protocols approved by the University of Colorado Health Sciences Center or Yale University Animal Care and Usage Committees. FVB/NJ and C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME). Mice containing the Tcfap2c-null allele [30], the Tcfap2a floxed allele, Alflox [39], and the Zp3-cre transgene [49] have been described previously. The generation and characterization of mice harboring a conditional Tcfap2c allele, Gamflox, in which loxP sites flank exon 6, will be described elsewhere (unpublished results). For the oocyte-specific Tcfap2c knockout experiments, female mice were generated that contained the Zp3-cre transgene along with one null allele and one floxed Tcfap2c allele. The presence of a null allele ensured that just a single Cre-mediated recombination event at the Tcfap2c floxed allele would result in oocytes lacking functional AP-2{gamma}. AP-2{alpha}ki mice have been described previously and contain a null allele of Tcfap2a in which an IRES-lacZ sequence has been inserted into exon 7 [40]. Genotyping of adult mice was performed by PCR analysis of tail DNA [30, 39, 40]. Tail clips were incubated overnight at 55°C in proteinase K/lysis buffer (50 mM KCl, 10 mM Tris [pH 8.3], 2 mM MgCl2, 0.1 mg of gelatin per milliliter, 0.45% Nonidet P-40, 0.45% Tween 20, and 100 µg of proteinase K per milliliter) prior to PCR.

Detection of AP-2 Transcripts in Preimplantation Embryos

Embryos were collected from naturally mated females at time points suitable to collect two-cell, four-cell, and morula- and blastocyst-stage embryos. Unfertilized oocytes were collected from superovulated females. RNA was isolated from pools of 10 embryos/oocytes by the RNeasy isolation kit (Qiagen, Foster City, CA), and the RNA was concentrated by ethanol precipitation and subjected to DNAseI digestion (amplification-grade DNAseI, Gibco BRL, Burlington, ON, Canada) prior to reverse transcription (RT). RNA isolations were repeated three times on different pools of embryos from each stage investigated and were performed on both FVB/NJ and C57BL/6J mice. RT-PCR was performed with primers specific for each AP-2 gene, and in each case, the primer pairs were designed to anneal to sequences derived from separate exons to minimize spurious signals from genomic DNA contamination. Primers used for PCR were as follows: AP-2{alpha} 5'-AAT CTG GGC TCT TAC ACA CAC ACC-3' and 5'-ATA GGG ATG GCG GAG ACA GCA TTG-3'; AP-2ß 5'-GGC TTC TTG GGA GGA ATG TCA G-3' and 5'-CCT TCT ACC AGT GAG GTG AGT AAC G-3'; AP-2{gamma} 5'-AGG AGG TGC AGA ATG TGG ACG-3' and 5'-CTT CAC CTT CCA CGA GAC G-3'; AP-2{delta} 5'-TCC ACC ACC AGT CCT TCC ATT AC-3' and 5'-GCC GTT GAG AAC AAT CCC ACA C-3'; AP-2{varepsilon} 5'-AAG TCC CCA TTC CCT CCA AAG C-3' and 5'-TTG AGC CCA ATC TTC TCC AGC-3'. To distinguish transcripts derived from the deleted or undeleted Gamflox allele, the following primer pair was utilized for RT-PCR: forward primer, 5'-TGG AAG ACT GTC CCT GCT CAG CTC-3'; reverse primer, 5'-GGA GAA GGT CAG TGA ACT CCT TGC-3'. All PCR analyses were performed with Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA) for 35 cycles of 30 min at 94°C, 30 min at 58°C, and 30 min at 72°C. Diagnostic products of 274 bp (AP-2{alpha}), 293 bp (AP-2ß), 405 bp (AP-2{gamma}), 371 bp (AP-2{delta}), 286 bp (AP-2{varepsilon}), 271 bp (deleted Gamflox), and 416 bp (undeleted Gamflox) were visualized following agarose gel electrophoresis.

Zygotic Activation of Tcfap2c Transcripts

A single A/G base pair polymorphism occurs between the FVB/NJ (A) and C57BL/6J (G) mouse strains within exon 4 of Tcfap2c; this sequence difference produces specific restriction enzyme sites for AlwNI and AvaII, respectively that can be used to distinguish RT-PCR fragments from the two mouse strains (Fig. 1). Following natural matings between FVB/NJ female mice and C57BL/6J males, embryos were collected at appropriate stages, and RNA was isolated from pools of 10 embryos by the RNeasy kit. RT-PCR was initially performed with the AP-2{gamma} primer pair with Platinum Taq DNA polymerase (Invitrogen) for five cycles of the following: 30 min at 94°C, 30 min at 58°C, and 30 min at 72°C. Subsequently, PCR was performed on 5 µl of product from the first round of PCR with the nested AP-2{gamma} primer pair 5'-CAC TTG CTC CTA CAC GAT CAG-3' and 5'-TAG GCA TTC CGG TGG TGA CAG-3' and the same reaction conditions. The primer pairs were designed to anneal to sequences derived from separate exons to minimize spurious signals from genomic DNA contamination. Following PCR, the products were subjected to restriction enzyme digestion with either AlwNI or AvaII and analyzed by gel electrophoresis. To block RNA polymerase transcription activity during embryonic development, the zygotes were cultured in M16 media (Sigma, St. Louis, MO) containing {alpha}-amanitin concentrations of 0, 1, 10, and 100 µg/ml until the two-cell stage, prior to RNA isolation.


Figure 1
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  		FIG. 1. Analysis of Tcfap2c zygotic gene activation. A) Top. An A:G polymorphism (bold) within exon 4 of Tcfap2c creates specific restriction enzyme recognition sites (underlined) for C57BL/6J and FVB mouse strains. This polymorphism maps to the third position of a serine codon and does not alter the predicted AP-2{gamma} protein sequence. Beneath are shown the diagnostic restriction fragment length polymorphisms in base pairs that result from this nucleotide sequence difference between strains with respect to the cDNA fragment that was amplified by PCR. B) Typical result from a restriction digest of an RT-PCR product derived from a C57BL/6J mouse (GG) or an F1 hybrid from a C57BL/6J and FVB cross (AG). C) Restriction fragment length polymorphism analysis of Tcfap2c RT-PCR products from pure FVB or F1 hybrid two-cell embryos analyzed after collection at 1.5 dpc (–) or following collection at 0.5 dpc and culture in the indicated concentrations (expressed in micrograms per milliliter) of {alpha}-amanitin ({alpha}-aman) until the two-cell stage. The 92-bp AvaII fragment (arrowhead) and the 221-bp AlwNI product (arrow) derived from the C57BL/6J allele are indicated. The sizes of DNA markers (M) in base pairs are shown to the left of the gels in B and C. Note that the 37-bp AvaII fragment is not visible in these gel images

Degradation of Maternal Tcfap2c Transcripts

Poly(A) containing RNA was isolated from single embryos by the mRNA affinity paper isolation method [50], and the mRNA was subjected to DNAseI digestion (amplification-grade DNAseI, Gibco) prior to RT. Nested RT-PCR and restriction enzyme digestion were performed as in the preceding section.

In Situ Hybridization

Mouse blastocyst-stage embryos were collected from superovulated females mated to males. Embryos were fixed overnight in 4% paraformaldehyde and washed in PBS. Embryos were then permeabilized for 30 min (150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, and 50 mM Tris [pH 8.0]) and postfixed in 4% paraformaldehyde/0.2% glutaraldehyde for 15 min. The embryos were then taken through a series of increasing sucrose percentages until they were in 30% sucrose in PBS, which was followed by an increasing percentage of OCT until embryos were in 100% OCT. The frozen samples were sectioned at 10 µM and stored at –70°C. Specific probes for the individual mouse AP-2 genes were as published [35]. Approximately 1 µg of digoxigenin-labeled sense or antisense RNA probe was incubated on the slides overnight at 70°C in hybridization buffer (50% formamide, 5x standard saline citrate, yeast tRNA [50 µg/ml], 1% SDS, and heparin [50 µg/ml]). The slides were then washed, and alkaline phosphatase (AP)-conjugated anti-digoxigenen antibody (Roche Molecular Biochemicals, Laval, PQ, Canada) was used to detect the digoxigenin-labeled RNA probe with BM purple AP substrate (Roche). Slides were counterstained with nuclear fast red by the protocol recommended by the manufacturer (Vector Laboratories, Burlingame, CA).

lacZ Staining

Embryos were stained for ß-galactosidase activity by standard procedures [51], fixed in 4% paraformaldehyde, and photographed.

Trophoblast Stem Cells

Trophoblast stem (TS) cells were established from 3.5-dpc mouse blastocysts in the presence of FGF4 and feeder cells as described [52]. Cells were maintained for 10 passages before removal of feeder cells and were then grown in the absence of feeders with conditioned media. Proliferative TS cells were induced to differentiate into giant cells by culture in the absence of FGF4 and conditioned media for 6 days. RNA was isolated from TS cells or their derivatives by the RNeasy kit, and RT-PCR was performed as described above.

RESULTS

Zygotic Activation of Tcfap2c

Previous studies had indicated that blastocysts that were genetically Tcfap2c null still contained AP-2{gamma} protein that became exhausted soon after implantation [30]. We reasoned that this protein would be derived from maternally expressed mRNA or, less likely, from paternal RNA present in the sperm. RT-PCR analysis indicated that maternal Tcfap2c mRNA did exist in wild-type oocytes and also demonstrated that no Tcfap2c RNA was present in sperm (see below and data not shown). Given these findings, we next wanted to determine when maternal mRNA was lost during development and when zygotic transcription of Tcfap2c began, as this might influence the ability of Tcfap2c-null or heterozygous embryos to complete preimplantation development. We chose wild-type mice for these analyses rather than Tcfap2c-null zygotes, since we wished to understand the general properties of Tcfap2c mRNA decay and activation rather than the special circumstances that might apply in the modified genetic background of the Tcfap2c-null zygote. We also note that transcripts derived from the wild-type versus Tcfap2c-null alleles could possess very different stabilities and that this scenario would complicate the interpretation of mRNA dynamics.

Therefore, we utilized a wild-type background for this analysis, and we took advantage of a single A/G base pair polymorphism that occurs in exon 4 of Tcfap2c between the FVB/NJ and C57BL/6J strains of mice. In FVB/NJ mice, both alleles have the adenosine residue (AA), whereas both alleles in the C57Bl/6J strain have a guanosine (GG). This polymorphism fortuitously creates alternate restriction enzyme sites that can be used to distinguish RT-PCR products derived from the two strains (Fig. 1, A and B). With respect to the timing of ZGA, FVB/NJ female mice (AA) were bred to C57Bl/6J male mice (GG) to derive zygotes that were 100% polymorphic at this position (AG). RT-PCR followed by restriction enzyme analysis was then used to assay for the presence of transcripts derived from the paternal G allele at various time points following fertilization. The appearance of a 92-bp AvaII fragment diagnostic of paternal C57Bl/6J chromosome-derived Tcfap2c transcripts could be readily visualized as early as the two-cell-stage embryo, collected at 1.5 dpc (Fig. 1C, compare lanes FVB and FVB x C57, arrowhead). The presence of paternal transcripts can also be inferred from the increased intensity of the full-length 221-bp RT-PCR product after AlwNI digestion (Fig. 1C, arrow), since the corresponding region of the C57Bl/6J allele is not a target for this enzyme. In contrast, no paternal Tcfap2c mRNA was detected by RT-PCR in fertilized oocytes collected at 0.5 dpc (data not shown).

To investigate this paternal allele activation phenomenon further, fertilized oocytes were collected at 0.5 dpc and allowed to develop to the two-cell stage in vitro in the presence of varying concentrations of {alpha}-amanitin to block new transcription. This analysis demonstrated that embryos cultured in the presence of only a low concentration (1 µg/ml) of this inhibitor generated RT-PCR products from the paternal G allele (Fig. 1C). In contrast, two-cell embryos that had been cultured in an {alpha}-amanitin concentration of 10 or 100 µg/ml showed a significant reduction or complete loss of the paternal transcript, respectively, as revealed by the decreased intensity of both the 92-bp AvaII fragment and the 221-bp AlwNI fragment (Fig. 1C). Maternally derived transcripts were readily detected in all instances, indicating that the {alpha}-amanitin had not produced a generalized effect on RNA stability. Thus, when ZGA was blocked by {alpha}-amanitin, we were unable to detect any mRNA derived from the paternal allele. These results confirm the absence of paternal Tcfap2c mRNA derived from sperm itself and also reveal that zygotic Tcfap2c transcription is activated coincident with the ZGA by the two-cell stage of mouse development.

Maternal Message Degradation

To complement the analysis of paternal allele activation, we next examined the time course of maternal Tcfap2c mRNA degradation. These studies also took advantage of the A/G polymorphism present between FVB/NJ and C57BL/6J mouse strains. F1 hybrid females (AG) were generated, and these were then backcrossed to C57BL/6J (GG) males. Subsequently, embryos were collected at various stages prior to implantation and individually genotyped to identify only those that possessed both alleles derived from C57BL/6J (GG). Such GG zygotes provided a means to follow the fate of maternal transcripts derived from the A allele. Even as early as the four-cell stage and at all time points thereafter, RT-PCR and restriction enzyme analysis indicated that all GG embryos contained little or no mRNA derived from the A allele (Fig. 2 and data not shown). Specifically, the 221-bp PCR fragment was refractory to AlwNI digestion, whereas AvaII generated mainly the 92-bp G-specific fragment and not the 137-bp A derivative (arrow). In contrast, embryos collected at the two-cell stage did have a maternal message contribution from the A allele (data not shown). Therefore, maternally derived Tcfap2c mRNA is still present at the two-cell stage, but its degradation is largely complete by the four-cell stage.


Figure 2
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  		FIG. 2. Analysis of Tcfap2c maternal transcript elimination. Restriction fragment length polymorphism analysis of Tcfap2c RT-PCR products from four-cell embryos derived from a C57BL/6J FVB F1 hybrid female crossed with a C57BL/6J male. The GG embryo has inherited two C57BL/6J Tcfap2c alleles—one paternal and one maternal. The AG embryo has inherited a maternal FVB Tcfap2c (A) allele and a paternal C57BL/6J Tcfap2c (G) allele. The 137-bp AvaII fragment (double-headed arrow) that is diagnostic of the FVB A allele is not observed in the GG embryo, indicating loss of the maternal transcript. The sizes of DNA markers (M) in base pairs are shown to the left

Together, the results of the maternal and zygotic Tcfap2c transcript analysis indicate that Tcfap2c mRNA is normally present continuously throughout preimplantation development, from the unfertilized oocyte until the blastocyst stage and beyond. As maternal Tcfap2c mRNA becomes degraded between the two- and four-cell stages, the transcription of the zygotic copies of Tcfap2c begins at the two-cell stage, coincident with the ZGA. The finding that maternal Tcfap2c mRNA is present until after the two-cell stage at 1.5 dpc is also consistent with our previous observation that Tcfap2c-null blastocysts still contain AP-2{gamma} protein—although this is depleted soon after implantation [30].

Conditional Gene Targeting of Tcfap2c in Oocytes

The presence of maternal Tcfap2c mRNA and AP-2{gamma} protein in the unfertilized oocyte as well as in the zygote raised the possibility that Tcfap2c has a maternal-effect function in addition to its role during the postimplantation period of development. To address this hypothesis, we utilized Cre-LoxP technology to generate mouse oocytes lacking a functional Tcfap2c gene. These experiments utilized an available floxed allele of Tcfap2c, Gamflox (unpublished results), in combination with a Zp3-cre transgene that directs Cre-mediated recombination only during oocyte growth [49]. Female mice were generated that contained one Tcfap2c-null allele and one floxed allele in combination with the Zp3-cre transgene. These Tcfap2c-null/Gamflox+Zp3-cre mice were healthy and viable, since the floxed Tcfap2c allele remains unrecombined in the majority of embryonic and extra-embryonic tissues, allowing essentially normal development. The combination of one Tcfap2c-null allele and one floxed allele ensures that only a single Cre-mediated recombination event is required to generate cells lacking a functional Tcfap2c gene.

Oocytes were subsequently collected from the females following superovulation and tested for the presence of transcripts derived from either the unrecombined or recombined floxed Tcfap2c allele by RT-PCR. In all pools of oocytes tested, Zp3-cre expression had been sufficient to completely remove the floxed portion of the gene during oogenesis so that no wild-type Tcfap2c mRNA remained in the oocytes (Fig. 3). Having established that oocytes were generated in the absence of Tcfap2c, we next tested whether a maternal component was required for zygote formation and development. For these experiments, Tcfap2c-null/Gamflox+Zp3-cre females were mated to wild-type males. Resultant litter sizes were similar to those obtained with standard wild-type matings, and all offspring were viable and had a normal morphology. Subsequently, 19 of these offspring were genotyped at weaning to ascertain the type of Tcfap2c allele present as well as the occurrence of the Zp3-cre transgene. The following genotypes were obtained: three mice had inherited the Tcfap2c-null allele and the Cre transgene; five mice had inherited the Tcfap2c-null allele without the Cre transgene; six mice had inherited the deleted floxed allele along with the Cre transgene; and the remaining five mice had inherited the deleted floxed allele but not the Cre transgene. The data indicate that all four expected genotypes were obtained and that the numbers of each genotype were consistent with mendelian genetic ratios. Also, there was no trace of the undeleted floxed allele in the offspring, demonstrating the efficacy of the Zp3-cre transgene. Moreover, since the Zp3-cre gene was absent in 5 of the 11 pups containing the deleted floxed allele, the Cre-mediated recombination event must have occurred prior to fertilization. Thus, these particular five offspring were clearly derived from oocytes lacking Tcfap2c, i.e., from oocytes that had contained one Tcfap2c-null allele and one deleted floxed allele. These results indicate that the oocytes that grow in the absence of Tcfap2c are functional and can give rise to viable offspring.


Figure 3
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  		FIG. 3. Deletion of Tcfap2c in the oocyte by the Zp3-cre transgene. Left: RT-PCR analysis of RNA from pools of oocytes derived from mice with one Gamflox and one Tcfap2c-null allele (flox/null) that either lack (–) or possess (+) the Zp3-cre transgene. Product sizes are shown to the right in base pairs. Right: Diagram of the various Tcfap2c alleles and the expected product sizes. Arrows show the position of the PCR primers. The Cre-deleted Gamflox allele is missing 145 bp corresponding to LoxP flanked exon 6 sequences. The Tcfap2c-null allele lacks the portion of exon 7 that contains the downstream PCR primer and so does not yield an RT-PCR product (NA) with this protocol

Next, we considered that the expression of Tcfap2c from the paternal allele in the zygote might rescue preimplantation development, since such paternal expression occurs by the two-cell stage. To test this possibility, the females were bred with heterozygous Tcfap2c-null males to generate a series of embryos lacking both maternal and zygotic Tcfap2c. We have previously shown that embryos lacking only zygotic Tcfap2c die shortly after implantation [30]. Therefore, we focused our analysis of the combined loss of both maternal and zygotic Tcfap2c to the preimplantation period. Following timed matings, 10 blastocysts with a normal gross morphology were collected from two females (data not shown). Genotype analysis indicated that 7 of the 10 blastocysts were derived from zygotes lacking both a maternal and zygotic contribution of Tcfap2c. These studies demonstrate that Tcfap2c is not required for preimplantation mouse development. Postimplantation viability of embryos lacking both maternal and zygotic Tcfap2c expression is discussed below in reference to Tcfap2a and Tcfap2c double-null embryos.

Preimplantation Expression of the AP-2 Gene Family

The absence of a major Tcfap2c maternal effect led us to examine the expression of the other members of the AP-2 gene family during preimplantation development. Since the AP-2 proteins have a similar molecular organization and DNA-binding specificity, we believed that the loss of Tcfap2c could be compensated for by the expression of one or more family members within the oocyte and/or after fertilization. Therefore, mature oocytes and preimplantation-stage embryos were collected for RT-PCR analysis to determine which AP-2 transcription factor genes were expressed during this period of development (Fig. 4). Intriguingly, four of the five AP-2 genes, Tcfap2a, Tcfap2b, Tcfap2c, and Tcfap2e, which encode AP-2{alpha}, ß, {gamma}, and {varepsilon}, respectively, were expressed during preimplantation mouse development. Only Tcfap2d failed to produce a detectable mRNA during this period, although we detected Tcfap2d transcripts later in embryogenesis (Fig. 4).


Figure 4
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  		FIG. 4. AP-2 gene family expression. RT-PCR analysis of AP-2 transcripts during preimplantation development (A) and in embryo-derived stem cells (B). The size of the PCR products in base pairs is shown to the right of each panel. M, DNA ladder; O, unfertilized oocyte; 2C, two-cell embryo; 4C, four-cell embryo; Mor, morula; Bl, blastocyst; –, negative control, +, positive control (an E9.5 mouse embryo); F, mouse embryo fibroblasts; TS, trophoblast stem cell; ES, embryonic stem cell

The observation that four AP-2 family members are expressed during the preimplantation period suggests that these genes have a heretofore unsuspected role in oogenesis, ZGA, or blastocyst development. However, the temporal pattern of expression of these AP-2 genes was distinct. Tcfap2a transcripts were not detected in the oocyte, two-cell, four-cell, or morula-stage embryo and appeared only at the blastocyst stage. Expression of the Tcfap2a locus was further analyzed at the blastocyst stage by in situ hybridization, as was the Tcfap2a lacZ KI allele that we had previously generated ([40]; Fig. 5). These studies indicated that Tcfap2a expression was concentrated in the trophectoderm, although lower levels of expression may also occur in the inner cell mass. In contrast to the induction of Tcfap2a observed as preimplantation development proceeded, Tcfap2b transcripts were detected in the oocyte, two-cell, and four-cell embryos but were not detected at later stages (Figs. 4 and 5). This result presumably indicates that the Tcfap2b mRNA is maternally derived and is quickly degraded after fertilization. Unlike Tcfap2c, Tcfap2b mRNA is not replenished by zygotic transcription during preimplantation development.


Figure 5
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  		FIG. 5. Analysis of AP-2 family gene expression in blastocysts. A, C–F) RNA in situ hybridization of sectioned mouse blastocysts with the indicated probes. A purple-blue color corresponds to a positive signal for the specific RNA assayed, while the pink color derives from the nuclear fast red counterstain. B) Whole-mount X-gal staining of a heterozygous Tcfap2a lacZ KI blastocyst (+/KI, left) and a wild-type control (+/+, right). The blue color corresponds to a positive signal from the Tcfap2a lacZ KI allele. icm, inner cell mass; t, trophectoderm. Original magnification x100

Although Tcfap2c and Tcfap2e transcripts were detected throughout the preimplantation period, they also showed expression differences. Tcfap2c expression remained reasonably abundant at all stages, and transcripts were readily detected in both the trophectoderm and inner cell mass at the blastocyst stage (Fig. 5D). In this context, we have previously examined AP-2{gamma} protein expression in mouse blastocysts by immunocytochemistry [30]. These latter studies indicated that all cell nuclei within the trophectoderm and inner cell mass contained similar amounts of AP-2{gamma} protein, which correlates with the presence of comparable Tcfap2c transcript levels in both of these compartments (Fig. 5D). With respect to Tcfap2e, transcripts were most abundant at the earlier stages and were so greatly depleted by the blastocyst stage that they were difficult to visualize by in situ hybridization (Fig. 5F). Together, these findings indicate that transcripts derived from Tcfap2b, Tcfap2c, and Tcfap2e are present within the unfertilized oocyte. After fertilization, as Tcfap2b mRNA levels decline, only two genes are expressed by the morula stage of development, Tcfap2c and Tcfap2e. However, Tcfap2a is induced by the blastocyst stage of development, so that transcripts from three AP-2 genes are again present.

We extended the analysis of AP-2 gene expression in cell types present at the blastocyst stage by studying expression in both wild-type embryonal stem (ES) cell- and TS-cell derivatives (Fig. 4B). TS cells were cultured in vitro, and RT-PCR was used to determine the expression of AP-2 factors both in proliferative and differentiated TS cells. Transcripts from the same three AP-2 genes that were previously detected in the blastocyst-stage embryo were detected in proliferating TS cells, namely Tcfap2a, Tcfap2c, and Tcfap2e. This pattern of expression was unchanged when TS cells were stimulated to differentiate into giant cells by growth in the absence of FGF4 without feeders or conditioned media for 6 days (data not shown). The same three AP-2 genes were also expressed in wild-type ES cells along with Tcfap2b, presaging the expression of this latter gene within the embryo proper soon after implantation. Thus, four of five AP-2 genes were expressed within ES cells, with only Tcfap2d transcripts absent. Taken together, these data show that there is considerable opportunity for genetic redundancy between the AP-2 family of transcription factors during oogenesis and preimplantation development.

Tcfap2a and Tcfap2c Double-Null Embryos

Given the overlapping patterns of AP-2 expression during the transition from unfertilized oocyte to blastocyst, we wished to dissect potential genetic redundancy between the four family members that are transcribed during this period, Tcfap2a, Tcfap2b, Tcfap2c, and Tcfap2e. In previous studies, we have uncovered genetic redundancy between Tcfap2a and Tcfap2b for cranial neural tube closure and midgestation viability (unpublished results). Therefore, we considered it likely that such redundancy would also occur for the AP-2 gene family during oogenesis and preimplantation development. Unfortunately, at the present time, mouse strains with the appropriate conditional alleles are not available to study redundancy between Tcfap2b, Tcfap2c, and Tcfap2e in the unfertilized oocyte. Similarly, in the absence of a floxed Tcfap2e allele, it is not possible to examine the combined role of AP-2{alpha}, AP-2{gamma}, and AP-2{varepsilon} in the blastocyst. The one combination of AP-2 family interactions that we could examine, though, was between Tcfap2a and Tcfap2c from the blastocyst stage of development onward.

To determine if redundancy existed between the two major AP-2 factors expressed at the blastocyst stage of development, embryos were produced that lacked both Tcfap2a and Tcfap2c. Female mice containing the Zp3-cre transgene that were also homozygous for both Tcfap2a and Tcfap2c floxed alleles were mated to males that were heterozygous for both the Tcfap2a- and Tcfap2c-null alleles. All embryos from this mating scheme should inherit a deleted copy of both Tcfap2a and Tcfap2c from the Zp3-cre transgenic mother. The ultimate zygotic genotype then depends upon the inheritance of a wild-type or null copy of these two AP-2 genes from the sire. Thus, the inheritance of paternal Tcfap2a- and Tcfap2c-null alleles would yield zygotes that were effectively homozygous null for both AP-2 genes. Similarly, the inheritance of one Tcfap2a-null allele and the Tcfap2c wild-type allele from the sire would generate embryos homozygous null for Tcfap2a and heterozygous for Tcfap2c (Table 1). The expected percentages of the various genotypes after fertilization are as follows: 25% both Tcfap2a and Tcfap2c null (DKO); 25% Tcfap2a null, Tcfap2c heterozygous; 25% Tcfap2a heterozygous, Tcfap2c null; and 25% both Tcfap2a and Tcfap2c heterozygous.


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  		TABLE 1. Genotypes of embryos from crosses between Alflox/Alflox; Gamflox/Gamflox; Zp3-cre females and males that were heterozygous for the Tcfap2a LacZ KI allele and the Tcfap2c-null allele

When 3.5-dpc embryos were collected from these matings, all four possible genotypes were detected at approximately the expected mendelian frequencies (Table 1). The majority of 3.5-dpc-collected embryos were still at the morula stage, but ~25% had formed blastocysts. The DKO genotype was found in both morula- and blastocyst-stage embryos. These results are consistent with our previous findings that the combined loss of maternal and zygotic Tcfap2c does not affect preimplantation development. Furthermore, these new data demonstrate that even in the absence of Tcfap2c, the further reduction or loss of Tcfap2a does not appear to affect the zygote prior to 3.5 dpc.

We have previously shown that embryos lacking zygotic Tcfap2c can survive until 7.5 dpc, but they die soon after due to defects in the extra-embryonic tissues [30]. We therefore collected embryos at 7.5 dpc to determine how the various Tcfap2a and Tcfap2c allelic combinations affected viability compared with the Tcfap2c-null phenotype (Table 1). In contrast to the data obtained with the 3.5-dpc embryos, at this later stage, we observed a significant reduction in the number of DKO embryos compared to the other three possible genotypes. Thus, the combined loss of both Tcfap2a and Tcfap2c led to an earlier lethality than the loss of either Tcfap2a or Tcfap2c alone, with the majority of concepti failing to survive to 7.5 dpc. We note that, since three DKO concepti were observed at 7.5 dpc, the absence of Tcfap2a and Tcfap2c does not preclude blastocyst implantation. We therefore hypothesize that these two AP-2 genes perform redundant functions at the blastocyst stage or soon after implantation to maintain normal development. On the basis of our analysis of Tcfap2a and Tcfap2c expression during the peri-implantation period, these functions most likely reside within the trophoblast cell lineage.

There was no significant difference between the frequencies of the three other genotypes obtained at 7.5 dpc (Table 1). Thus, when compared to mice heterozygous for both Tcfap2a and Tcfap2c, the loss of an additional allele of either Tcfap2a or Tcfap2c did not significantly affect the frequency of mutant embryos isolated. These findings strongly suggest that embryos lacking any three of the four wild-type Tcfap2a and Tcfap2c alleles are capable of surviving to at least the same stage of development as Tcfap2c-null mice. Note that we have not performed an extensive morphologic analysis of these embryos in comparison to the Tcfap2c-null mice—in large part because the phenotype of the Tcfap2c-null mouse alone is variable [30]. Therefore, it remains possible that certain aspects of peri-implantation development and function can be altered by the loss of additional AP-2 alleles. Nevertheless, it is apparent that both alleles of both Tcfap2a and Tcfap2c need to be lost before significant differences in the timing of lethality are observed in comparison to the Tcfap2c-null mice.

The data in Table 1 are also pertinent to the influence of maternal and zygotic Tcfap2c expression on developmental outcome. In the present study, we were able to isolate embryos at 7.5 dpc that lacked both maternal and zygotic Tcfap2c (Table 1)—and it should also be noted that these embryos also lacked one allele of Tcfap2a. The morphology of such embryos was similar to the previously described Tcfap2c-null phenotype in that they were smaller and more disorganized than their wild-type counterparts. These data indicate that the combined loss of both maternal and zygotic Tcfap2c does not greatly alter the viability of embryos when compared to the loss of zygotic Tcfap2c alone. Thus, we conclude that even though maternally derived Tcfap2c mRNA and protein are expressed in the unfertilized oocyte and preimplantation embryo, it is the zygotic component that is critical for early development.

DISCUSSION

Previous studies of the mouse have demonstrated that several members of the AP-2 family of transcription factors are required during the postimplantation period for important aspects of organogenesis and morphogenesis. However, the expression and function of these genes prior to implantation were less clear and prompted our present analysis. In the present study, we showed that the majority of genes encoding the AP-2 family of transcription factors are expressed during mouse preimplantation development (see Fig. 6 for summary).


Figure 6
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  		FIG. 6. Summary of AP-2 gene family expression during mouse preimplantation development. The transcript profiles are shown for the genes encoding AP-2{alpha}, AP-2ß, AP-2{gamma}, AP-2{delta}, and AP-2{varepsilon}. The results for Tcfap2c, encoding AP-2{gamma}, are divided into a maternal component (m {gamma}) and a zygotic component (z {gamma}). The data are derived from the present study and from the data mining of microarray studies [2, 5, 9–11]

Transcripts derived from Tcfap2b, Tcfap2c, and Tcfap2e are present in unfertilized oocytes. In support of these findings, Tcfap2c and Tcfap2e transcripts can be readily detected by in situ hybridization within developing oocytes of the ovary ([48]; unpublished results). Expression of the three other AP-2 genes is not apparent in the ovary by nonradioactive in situ hybridization, suggesting that the levels of Tcfap2b transcripts in oocytes are much lower than those of Tcfap2c and Tcfap2e (unpublished results). After fertilization, any Tcfap2b transcripts are quickly lost until the gene is reactivated once more in the embryo following gastrulation. In contrast, transcripts derived from Tcfap2c and Tcfap2e are found throughout preimplantation development. With respect to Tcfap2c, we further demonstrate that these transcripts are initially maternally derived but that they are then replaced by zygotic transcripts beginning at the two-cell stage. The Tcfap2a gene is not expressed maternally but becomes active during blastocyst development, most prominently in the trophoblast compartment. Only the expression of Tcfap2d was undetectable throughout this developmental window.

Recently, a number of studies have analyzed global gene expression in the developing oocyte and preimplantation embryo. Several such studies have documented the expression of AP-2 gene family members at specific developmental time points. Tcfap2e transcripts were found in the oocyte, increasing markedly during the primordial-to-primary follicle transition but declining after fertilization at the onset of the ZGA [5, 10]. Tcfap2b RNAs showed a transient increase at the single-cell stage, presumably reflecting polyadenylation of maternal transcripts [10]. Tcfap2c transcripts were found at high levels in oocytes and then declined immediately after fertilization [2, 9–11]. Subsequently, Tcfap2c expression began to rise again by the four-cell stage and peaked by the eight-cell stage, remaining high thereafter. These findings are consistent with our data concerning the expression profiles of the various AP-2 gene family members. In addition, the biphasic nature of Tcfap2c expression observed in the microarray studies reflects the switch between maternal and zygotic Tcfap2c transcripts that we have documented in this study.

Several studies have indicated that there is a preference for TATA-less promoters to be differentially utilized during the preimplantation period [14]. In light of this observation, it is notable that the two AP-2 genes that are clearly activated during preimplantation development, Tcfap2a and Tcfap2c, both rely on TATA-less basal promoters for their expression. Tcfap2c is the only AP-2 gene strongly activated during the ZGA, and its promoter relies on an initiator element, along with binding sites for Sp1 and Sp3, for its transcriptional activity [53–55]. Tcfap2a is expressed by the blastocyst stage and requires an initiator element as well as an octameter element for promoter activity [56]. Tcfap2b is the only other AP-2 gene whose promoter has been mapped to date, and this has been shown to contain a canonical TATA box upstream of the major initiation site [53]. In the future, it will be of interest to ascertain if the promoters of Tcfap2d and Tcfap2e are also TATA-containing since—like Tcfap2b—these two genes are not highly expressed between the two-cell and blastocyst stage of development.

To date, three of the five mouse AP-2 genes have been mutated by gene targeting: Tcfap2a, Tcfap2b, and Tcfap2c [30, 43, 45–47]. Although all three genes are required for appropriate embryonic development, only Tcfap2c is essential for the early postimplantation viability of the embryo, particularly within the extra-embryonic lineages [30, 47]. However, the role of Tcfap2c in pre- and peri-implantation development was obfuscated by our observations that maternal mRNA and protein are present up to the four-cell and blastocyst stage, respectively (the present study; [30]). Therefore, in the present study, we investigated whether this maternal Tcfap2c contribution could influence embryonic development by deleting Tcfap2c in the oocytes with a Zp3-cre transgene. Our findings demonstrate that oogenesis, fertilization, and embryonic development occur normally after loss of maternal Tcfap2c. Moreover, there is no obvious defect in preimplantation development, even when the absence of the maternal Tcfap2c component is combined with loss of this gene in the zygote. Our results further suggest that embryos lacking both maternal and zygotic Tcfap2c die at approximately the same stage as embryos lacking only the zygotic component, although we cannot exclude the possibility that the additional loss of the maternal gene product would cause an alteration in the phenotype. Thus, our data clearly demonstrate that it is the zygotic Tcfap2c component that is critical for early development, despite the expression of Tcfap2c in the oocyte.

Despite our findings that maternal Tcfap2c is not essential in the fertilized embryo, we cannot exclude a requirement for the expression of Tcfap2c in oogenesis prior to the activity of the Zp3-cre transgene, and further studies will be needed to assess this possibility. The presence of transcripts derived from Tcfap2b and Tcfap2e in the unfertilized oocytes also provides ample opportunity for functional redundancy between Tcfap2c and these other AP-2 genes with respect to a maternal effect. In fact, we have previously found that genetic redundancy does exist between Tcfap2a and either Tcfap2b or Tcfap2c with respect to cranial neural tube closure defects in transheterozygote embryos. The loss of either one allele of Tcfap2b or Tcfap2c exacerbates the low incidence of neural tube closure defects seen with Tcfap2a heterozygotes alone (unpublished results; [57]). In the present study, we also found that redundancy occurs between Tcfap2a and Tcfap2c for development of the peri-implantation embryo, because embryos lacking both genes generally die earlier than embryos lacking either gene alone [30, 43, 45, 47]. Given the high expression levels of Tcfap2a in the blastocyst trophectoderm and the requirement of Tcfap2c in extra-embryonic tissues [30], we strongly suspect that they are acting together in these lineages. We also noted that Tcfap2e is still presumably expressed at a low level in the Tcfap2a/Tcfap2c-null blastocysts and may be sufficient to compensate partly for the loss of the other two family members in blastocyst development and implantation. Thus, new mutant alleles of Tcfap2b and Tcfap2e will be required to assess the overall requirement of the AP-2 gene family for oogenesis and preimplantation development. Nevertheless, our present studies clearly reveal that the AP-2 transcription factors are expressed in dynamic patterns before and after fertilization of the oocyte and that functional redundancy occurs between these proteins during at least the peri-implantation period.

ACKNOWLEDGMENTS

We are grateful to Weiguo Feng for assistance with the RNA in situ hybridization experiments. The authors thank members of the Williams laboratory for helpful discussions and assistance and Kristin Artinger for a critical reading of the manuscript.

FOOTNOTES

1 Supported by a Lalor fellowship award to Q.W., a grant from the Cancer League of Colorado to J.H., a University of Colorado Cancer Center Seed grant award to T.W., and Utah Agricultural Experiment Station project UTA00493 to Q.W. Back

2 Correspondence: Trevor Williams, Mailstop 8120, P.O. Box 6511, Aurora, CO 80045. FAX: 303 724 4580; trevor.williams{at}uchsc.edu Back

3 Current address: Department of Animal, Dairy, and Veterinary Sciences, Utah State University, Logan, UT 84322. Back

4 Current address: Skirball Institute, NYU School of Medicine, New York, NY 10016. Back

Received: 13 March 2006.

First decision: 4 April 2006.

Accepted: 20 April 2006.

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biolreprod.106.052407v1
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