Transcription Factor Gene AP-2{gamma} Essential for Early Murine Development

Transcription factor gene AP-2 ${gamma}$ belongs to a family of four closelyrelated genes. AP-2 ${gamma}$ had been implicated in multiple functionsduring proliferation and differentiation based on its expressionpattern in trophoblast, neural crest, and ectoderm cells inmurine embryos. In order to address the question of the roleof AP-2 ${gamma}$ during mammalian development, we generated mice harboringa disrupted AP-2 ${gamma}$ allele. AP-2 ${gamma}$ heterozygous mice are viable anddisplay reduced body sizes at birth but are fertile. Mice deficientfor AP-2 ${gamma}$ , however, are growth retarded and die at days 7 to9 of embryonic development. Immunohistochemical analysis revealedthat the trophectodermal cells that are found to express AP-2 ${gamma}$ fail to proliferate, leading to failure of labyrinth layer formation.As a consequence, the developing embryo suffers from malnutritionand dies. Analysis of embryo cultures suggests that AP-2 ${gamma}$ isalso implicated in the regulation of the adenosine deaminase(ADA) gene, a gene involved in purine metabolism found expressedat the maternal-fetal interface. Therefore, AP-2 ${gamma}$ seems to berequired in early embryonic development because it regulatesthe genetic programs controlling proliferation and differentiationof extraembryonic trophectodermal cells.

INTRODUCTION

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Introduction

The AP-2 transcription factor gene family consists of four differentgenes referred to as AP-2 ${alpha}$ , AP-2ß, AP-2 ${gamma}$ , and the recentlydiscovered AP-2 ${delta}$ gene (4, 15, 17, 19, 22, 37, 40). All membersof the AP-2 family share a characteristic protein structurecontaining a unique C-terminal helix-span-helix motif that mediatesprotein dimerization; together with a basic domain they areinvolved in DNA binding with varying affinity to GC-rich elements.An N-terminal proline- and glutamine-rich region mediates transcriptionalactivation (33, 36).

Despite the expression of all AP-2 genes in the extraembryonictrophoblast cells, it remained unclear if there was a redundantfunction in placental gene regulation. Gene knockout experimentswith AP-2 ${alpha}$ and AP-2ß indicate that the AP-2 proteinscarry out individual functions during mouse development. WhileAP-2 ${alpha}$ is predominantly essential for craniofacial developmentand ventral body wall closure (29, 39), lack of AP-2ßleads to polycystic kidney disease (20). Both AP-2 ${alpha}$ - and AP-2ß-deficientmice display unaltered implantation and placentation. Is AP-2 ${gamma}$ a key regulator of placental development? Prior to implantation,AP-2 ${gamma}$ is expressed in the trophoblast cells starting at day 3.5of murine development. After implantation, the expression ofAP-2 ${gamma}$ continues in the trophoblast cells and its derivatives,the primary giant cells and the diploid cells of the polar trophectoderm.With ongoing proliferation of the trophoblast cells, AP-2 ${gamma}$ isexpressed in the ectoplacental cone and the extraembryonic ectoderm.At the time of chorio-allantoic fusion, AP-2 ${gamma}$ expression is increasedin all derivatives of the trophoblast lineage (27, 30). Thus,AP-2 ${gamma}$ , together with the T-box gene Eomes (26), is the only transcriptionfactor gene found to be expressed in all trophoblast lineagesthroughout placental development. This suggests that AP-2 ${gamma}$ mightplay a role in regulating trophoblast gene expression programs.In fact, AP-2 ${gamma}$ has been shown to regulate the genes for adenosinedeaminase (ADA) (30, 31), human placental lactogen (24), andhuman chorionic gonadotropin-ß (11). Nevertheless,the function of AP-2 ${gamma}$ during murine development remained to beelucidated.

In the study presented here, we addressed the question of therole of AP-2 ${gamma}$ using gene knockout technologies. We demonstratethat AP-2 ${gamma}$ is essential for early embryogenesis, due to the factthat the mutants display a significant retardation of growthby embryonic day 7.5 (E7.5) and are resorbed by E9.5. The lossof AP-2 ${gamma}$ leads to a reduction in cell proliferation in the extraembryonicectoderm and the ectoplacental cone; in addition, loss of AP-2 ${gamma}$ leads to a reduced number of giant cells. As a consequence thelabyrinth layer, derived from chorion and allantois, fails toform. The embryo suffers from deprivation of nutrients and,as a result, retards in growth and dies.

In addition, in vitro experiments show that the expression ofadenosine deaminase (ADA) is highly reduced in trophectodermoutgrowths of AP-2 ${gamma}$ mutant animals compared to wild-type trophectodermcells. ADA is a purine metabolic enzyme that converts cytotoxicdeoxyadenosine to deoxyinosine (1). Thus, we hypothesize thatthe reduced ADA expression leads to the accumulation of thetoxin, and to cell death. This could be a further mechanismleading to the death and resorption of the null mutant. Thework outlined here helps in understanding the molecular processescontrolled by transcription factor gene AP-2 ${gamma}$ .

MATERIALS AND METHODS

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Materials and Methods

Construction of the targeting vector. A genomic clone of murine AP-2 ${gamma}$ gene (kindly provided by PascalDollé, IGMBC, Strasbourg, France) was used to designthe targeting vector by standard recombinant techniques. A 6-kbgenomic AP-2 ${gamma}$ fragment spanning exons 2 to 7 was subcloned intopBluescript II KS (+/-). loxP sites flanked exon 5, which hadbeen shown to be critical for AP-2 function. The 5'-loxP sitecontained an additional Asp718 site at the 5' end and was insertedin intron 4 at the PstI site. A floxed selection cassette containingthe pgk-neo and the HSV-tk genes (kind gift of Peter Mombaerts[18]) mediating positive and negative selections, respectively,was inserted in intron 5 at the XbaI site.

Targeting of ES cells and generation of AP-2 ${gamma}$ -deficient mice. Linearized vector DNA was introduced by electroporation intoE14-KPA murine embryonic stem (ES) cells (kindly provided byKlaus Peter Knobeloch, FMP, Berlin, Germany) and cultured followingstandard procedures (28). After selection with G418 (activesubstance, 250 µg/ml), two correctly targeted clones of176 clones were identified by Southern blotting and subsequentlysubjected to cre-mediated loop-out reaction. Following selectionwith ganciclovir, 104 of 111 clones had lost the complete insertand thus carried loss-of-function alleles. To generate chimericmice, C57BL/6J blastocysts injected with AP-2 ${gamma}$ ^+/- ES clones weretransferred into pseudopregnant foster mice. Offspring of germline-transmitting chimeric mice were screened for the presenceof the disrupted AP-2 ${gamma}$ gene. Mice heterozygous for the disruptedAP-2 ${gamma}$ allele were mated to obtain animals homozygous for themutation.

PCR assays. Genotyping of mice and blastocysts was performed as follows.Tail biopsy or inner cell mass cells were lysed at 55°Cin 200 or 20 µl, respectively, of lysis buffer (50 mMKCl, 10 mM Tris HCl [pH 8.3], 2.5 M MgCl₂, 0.1 mg of gelatinper ml, 0.45% NP-40, 0.45% Tween 20, 1 mg of proteinase K perml) for 2 h. Boiling for 10 min inactivated the proteinase K,and 10 µl of each sample was used for PCR analysis. Threeprimers were used to detect wild-type and null-mutant alleles:P1 (5'-AACAGGTTATCATTTGGTTGGGATT-3'), P2 (5'-CAATTTTGTCCAACTTCTCCCTCAA-3'),and P3 (5'-AATAGTCAGCCACCGCTTTACT AGG-3'), which amplified 300-bp(wild-type) and 700-bp (null-allele) fragments. The followingPCR cycle profile was used: 1 cycle of 94°C for 10 min,followed by 37 cycles of 94°C for 30 s, 55°C for 30s, and 72°C for 30 s, and finally 1 cycle of 72°C for7 min. PCR products were electrophoresed on a 2% standard agarosegel and documented using Eagle-Eye gel documentation system(Stratagene, Heidelberg, Germany).

Whole-mount in situ hybridization. Digoxigenin-labeled probes were generated with recombinant clonescontaining full-length cDNAs of Brachyury (T) with T7 and SP6RNA polymerase. E8 embryos were hybridized according to standardprotocols as previously described (14) (http://stratus.lifesci.ucla.edu./hhmi/derobertis/).Embryos were photographed by using a Prog.Res. digital camera(Jenoptik, Jena, Germany) fitted to a Zeiss-Axiovert microscope(Zeiss, Jena, Germany). Pictures were assembled by using AdobePhotoshop and Illustrator software.

Histological sections. Deciduae at E7.5 to E8.5 resulting from heterozygous intercrosseswere collected and fixed in 4% (wt/vol) paraformaldehyde inphosphate-buffered saline (PBS) overnight at 4°C, processed,and embedded in paraffin following standard procedures. Sectionsof 7-µm thickness were prepared, processed, and used forhematoxylin and eosin and PCNA staining.

PCNA staining. After removal of paraffin the sections were rinsed twice withdistilled H₂O, incubated for 10 min in 2 N HCl, and washed againtwice with distilled H₂O. After incubation in methanol-0.3%H₂O₂ for 20 min and washing in distilled H₂O, the sections wereblocked for 10 min in blocking buffer (0.05 M Tris HCl, 0.15M NaCl [pH 7.6], 0.5% Tween 20) containing 10% horse serum and0.5% mouse serum. After being washed twice in PBS, the sectionswere incubated with PCNA-specific antibody diluted 1:100 inblocking buffer for 1 to 2 h and rinsed twice with PBS. Anti-PCNAantibody binding was detected with horseradish peroxidase-conjugatedsecondary antibody diluted 1:50 in blocking buffer accordingto the manufacturer's instructions (Vector ABC-Elite and AECkits). The sections were counterstained with hematoxylin for30 s, embedded in Immunomount (Shandon-Lipshah, Runcorn, UnitedKingdom), and photographed.

Immunohistochemical staining of sections. After removal of paraffin the sections were rinsed twice withPBS and incubated in methanol-1% H₂O₂ for 30 min. After beingwashed three times in PBS the sections were boiled five timesin a microwave (5 min each) in 10 mM sodium citrate (pH 6) andthereafter blocked in 10% horse serum plus 2% milk powder inPBS for 30 min at room temperature. After being rinsed withPBS the sections were incubated with an antibody specific toAP-2 (1:200; Geneka, Munich, Germany) at 4°C overnight.After two washes in PBS the secondary antibody (1:400 in PBSplus 2% milk powder) was applied for 1 h. The subsequent stepsof the procedure were the same as for the PCNA assay describedabove.

Immunohistochemical staining of blastocyst cultures. The blastocyst cultures were fixed in acetone for 20 min at-20°C and washed twice in PBS. After incubation in methanol-0.3%H₂O₂ for 5 min and three washes in PBS, the cultures were blockedin PBS-3% (wt/vol) milk powder for 30 min at room temperatureand incubated with an antibody specific to ADA (1:100; SantaCruz Biotechnology) in PBS-3% milk powder at 4°C overnight.The cultures were washed in PBS and incubated with the secondaryantibody (1:400 in PBS-3% milk powder) for 1 h at room temperature.The subsequent steps of the procedure were the same as for thePCNA assay described above.

In vitro culture of blastocysts. Natural matings between heterozygous mice were used to obtainembryos of all genotypes. Embryos were staged according to thedetection of vaginal plugs resulting from the crosses (noonof day 1 of plugging was E0.5). Blastocysts at E3.5 were flushedfrom the uterine horns and cultured individually in 50 µlof ES cell medium without leukemia inhibitory factor on gelatin-coatedchamber slides (Nunc, Wiesbaden, Germany) for up to 1 week.Photographs of the cultures were taken with a Zeiss Axiocamfitted to an inverted microscope (Zeiss Axiovert), and genotypewas determined by PCR of picked inner cell mass.

RESULTS

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Results

Targeted disruption of the AP-2 ${gamma}$ allele. A 6-kb genomic fragment spanning exons 2 to 7 was used to introducea floxed neo/tk cassette 3' of exon 5 as well as a solitaryloxP site 5' of exon 5 (Fig. 1A, upper panel). After electroporationinto ES cells and selection with G418, 2 correctly targetedclones of 173 analyzed were identified by Southern blot analysis(Fig. 1A, middle panel, and Fig. 1B). cre-mediated excisionremoved the region between the loxP sites, generating the AP-2 ${gamma}$ -nullallele (Fig. 1A, lower panel). A PCR assay was used to verifythe cre-mediated loop-out reaction (Fig. 1C). To generate miceharboring the AP-2 ${gamma}$ -null allele, cells were injected into C57BL/6blastocysts, resulting in several chimeric animals. Offspringof germ line-transmitting animals were tested for the presenceof the null allele. Mice heterozygous for the mutation appearedslightly growth retarded after birth but ultimately reachednormal sizes and were fertile (not shown).

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FIG. 1. (A) Targeting strategy of the AP-2 ${gamma}$ genomic locus. Schematics of the targeting vector (top), wild-type locus (upper middle), the mutated locus (lower middle), and the null allele (bottom) after cre-mediated loop-out reaction are shown. Shaded boxes indicate exons. Arrows indicate the positions of the primers (P1, P2, and P3) used for detection of the cre-mediated excision. (B) Southern blot analysis of genomic DNA isolated from ES cell clones digested with XhoI (left) and XhoI/Asp718 (right) hybridized to a 5' external probe (indicated in panel A) is shown. The 4.5-kb (wt) and 8.5-kb (mut) bands and 3.7-kb (wt) and 2.5-kb (mut) bands specific to the wild-type and the targeted alleles, respectively, confirmed homologous recombination and integration of the 5' loxP sequence. (C) The generation of the AP-2 ${gamma}$ null allele by cre-mediated loop-out reaction was checked by PCR amplification of ES cell DNA using primers detecting wild-type (wt) as well as null allele (null). Abbreviations: A, Asp 718; P, PstI; X, XhoI; wt, wild type; mut, mutant; tk, HSV-tk (herpes simplex virus thymidine kinase); neo, neomycin resistance gene.

Lack of AP-2 ${gamma}$ results in lethality during early gestation. To obtain mice homozygous for the AP-2 ${gamma}$ mutation, heterozygousanimals were mated. However, we did not recover any live- bornAP-2 ${gamma}$ -deficient animal, indicating that complete loss of AP-2 ${gamma}$ is not compatible with proper embryonic development. To determinethe nature of the phenotype as well as the time point of thelethality we dissected pregnant animals at various stages ofembryonic development. We found that the AP-2 ${gamma}$ -deficient animalswere present at expected Mendelian ratios up to 3.5 days postcoitum(of 27 blastocysts isolated, 7 [26%] were wild type, 14 [51%]were heterozygous, and 6 [23%] were deficient for the AP-2 ${gamma}$ allele),indicating that the mutation does not interfere with preimplantationdevelopment. After implantation, however, the AP-2 ${gamma}$ -deficientembryos were found to be growth retarded by day 6 of development,died, and were resorbed by day 9 of embryonic development.

AP-2 ${gamma}$ mutant embryos are reduced in size and fail to gastrulate. In order to examine the gross morphology of the mutant embryosand to compare them to their wild-type littermates, we dissectedembryos at E8.0. Figure 2A shows a mutant embryo next to a controllittermate. The mutant embryo is clearly retarded, and volumetriccalculation indicated that mutants are up to 50-fold smallerthan their littermates. At E6.5, gastrulation is initiated,resulting in the formation of the third germ layer, the mesoderm.In order to determine to what extent gastrulation is affected,we performed a whole-mount in situ analysis using Brachyury(T) as a probe (35). As seen in Fig. 2A, mesodermal cells areclearly visible in the wild-type embryo (Fig. 2A, right embryo),while almost no signal is detected on the AP-2 ${gamma}$ mutant animals(Fig. 2A, left embryo). These results indicate that the mutantembryo is retarded in overall growth and patterning processes.AP-2 ${gamma}$ , however, is not expressed in the embryo proper at thisstage of development but can be detected in the cells of theectoplacental cone and the trophectoderm-derived giant cells.Therefore, we decided to analyze histological sections of wild-typeand mutant conceptuses and found that not only the embryo properbut also the ectoplacental cone was growth retarded. (Fig. 2B and C).It is very likely that the growth retardation of theembryo results from a defect in the extraembryonic tissues.

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FIG. 2. AP-2 ${gamma}$ -deficient embryos are severely retarded in development. (A) Whole-mount in situ staining for the primitive streak marker Brachyury (T) of a wild-type conceptus (right) and an AP-2 ${gamma}$ -deficient conceptus (left) at E8.0 of murine development. The blue staining indicates cells expressing Brachyury (arrows). (B to E) Histological sections of wild-type (wt) and AP-2 ${gamma}$ -deficient (-/-) embryos in utero at E7.5 of murine development. Sagittal section through the decidua (dc) harboring a wild-type (B) and an AP-2 ${gamma}$ -deficient embryo (C). Note the reduced size of the ectoplacental cone in panel C compared to that in panel B (epc; arrow). PCNA staining of wt conceptus (D) and AP-2 ${gamma}$ -deficient (-/-) conceptus (E) at E7.5 of development. Note the difference in proliferation (red signal) in the ectoplacental cone and in the extraembryonic ectoderm for the wild-type conceptus and for the mutant. Arrowheads mark erythrocytes corresponding to forming blood lacunas indicative of the beginning of resorption of the mutant conceptus. Abbreviations: hf, head fold; wt, wild type; dc, decidua; epc, ectoplacental cone. Scale bars, 500 µm (A to C) and 100 µm (D and E).

Reduced proliferation of trophectodermal cells in AP-2 ${gamma}$ mutant conceptuses. Most of the extraembryonic tissues are derived from trophectodermalcells. Trophectodermal cells are among the first specializedcells which arise from the fertilized egg. In mice, there arethree principal subtypes of trophoblast cells known: (i) thecells of the extraembryonic ectoderm that are the self-renewingstem cells, (ii) the intermediate population of the ectoplacentalcone, and (iii) the giant cells surrounding the conceptus thatare in direct contact with the maternal decidua (9). Since AP-2 ${gamma}$ is found expressed in all cells of the trophectodermal lineageat the stage spanning E6.5 to E9.5 (27, 30), we decided to analyzethese cells in more detail. Recently, it was shown that onepossible function of AP-2 genes might be the regulation of cellularproliferation by suppression of genes implicated in differentiationand apoptotic processes (23). We used paraffin sections andPCNA immunohistochemistry to detect cell proliferation. Figure2D shows that the majority of cells of the ectoplacental conein the control animal are positive for PCNA staining. The AP-2 ${gamma}$ -deficientconceptus, on the other hand, displays highly reduced PNCA staining(Fig. 2E) in cells of the ectoplacental cone, indicating thatlack of AP-2 ${gamma}$ results in reduced proliferation of extraembryonictissue.

AP-2 ${gamma}$ -deficient embryos display a reduced number of giant cells and fail to develop the labyrinth layer. Beginning from day 7.5 of development, the labyrinth layer formsas a consequence of a concerted interaction of chorion and allantois(9). Since extraembryonic ectodermal cells expressing AP-2 ${gamma}$ contributeto the chorion, we investigated the sizes and structures ofthe extraembryonic tissues by using a pan-AP-2 antibody at day8.0 of development (30). The pan-AP-2 antibody detects all AP-2isoforms and can therefore be used on wild-type and AP-2 ${gamma}$ -deficientmice to stain all derivatives of the trophectodermal lineage.As seen in Fig. 3, there were fewer trophectodermal giant cellsseen in AP-2 ${gamma}$ mutant animals (Fig. 3B) than in the wild-typelittermate (Fig. 3A). Furthermore, the mutant embryo failedto form a labyrinth layer (Fig. 3D), which was clearly detectableat this stage in the wild-type animal (Fig. 3C).

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FIG. 3. AP-2 ${gamma}$ -deficient embryos show decreased numbers of giant cells and fail to develop a labyrinth layer. Immunohistochemical staining of sagittal sections through the decidua of a wild-type (wt) (A) and an AP-2 ${gamma}$ -deficient embryo (-/-) (B) at E8.0 of murine development using a pan-AP-2 antibody is shown. Note the lack of secondary giant cells in the null mutant compared to the wild type (arrowheads in panel A). The AP-2 ${gamma}$ -deficient embryo (D) fails to form the functional labyrinth anlage (la), which is clearly visible in the control embryo (C). Scale bar, 100 µm.

Taken together, these results indicate that loss of AP-2 ${gamma}$ leadsto a deficiency of proliferation in extraembryonic tissues.Lack of AP-2 ${gamma}$ cannot be compensated by the other known membersof the AP-2 gene family, indicating a unique role for AP-2 ${gamma}$ incontrolling the development of extraembryonic tissues.

Cultured trophoblast cells lose epithelial morphology. AP-2 ${gamma}$ is detected as early as the blastocyst stage in the cellsof the trophectoderm. We sought to establish an in vitro systemin order to further analyze the cellular and molecular consequencesof the AP-2 ${gamma}$ deficiency. For this purpose, heterozygous micewere mated, the resulting embryos were flushed from the uterinehorns at day 3.5 of gestation, and blastocyst microdrop cultureswere initiated. The cultures were checked daily, and photographswere taken. A total of 27 blastocyst cultures were initiated,monitored, and genotyped. We found that the trophoblast cellsof the AP-2 ${gamma}$ -deficient animals were not distinguishable fromthe control cultures. However, after 48 h, when the trophoblastcells had attached onto the tissue culture dish, their morphologychanged and the epithelial morphology was lost after 5 daysin culture (Fig. 4A and B). Since it is known that AP-2 genesare involved in the regulation of e-cadherin (10), a moleculeinvolved in cell adhesion, we decided to check the AP-2 ${gamma}$ -deficientcultures with an antibody specific to e-cadherin. However, wecould not see any difference in staining intensity (data notshown). Thus, cultured trophoblast cells derived from AP-2 ${gamma}$ -deficientblastocysts display a change in morphology; this differenceis not based on altered levels of e-cadherin in these cells.

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FIG. 4. AP-2 ${gamma}$ -deficient blastocyst outgrowths show changed morphology. Photographs of outgrowths of wild-type (wt) (A) and AP-2 ${gamma}$ -deficient (-/-) (B) blastocysts after 5 days in culture are shown. Trophectodermal cells (te) and inner cell mass (icm) of AP-2 ${gamma}$ -deficient blastocysts display altered morphology when cultured in vitro. (C and D) Reduced expression of ADA in AP-2 ${gamma}$ -deficient blastocyst outgrowths. Trophectodermal cells of a wt culture (C) and an AP-2 ${gamma}$ -deficient culture (-/-) (D) were incubated with an ADA-specific antibody. Cells expressing ADA display brown cytoplasmic staining. Nuclei were counterstained with hematoxylin (blue). The trophectodermal cells of the control were positive for ADA expression (C), whereas in the AP-2 ${gamma}$ -deficient cells the staining was much reduced (D). Abbreviations: icm, inner cell mass; te, trophectodermal cells.

AP-2 ${gamma}$ -deficient trophoblast cells display reduced expression of ADA. ADA is an essential enzyme of the purine metabolism, which isfound enriched at the maternal-fetal interface throughout postimplantationdevelopment. Its cellular function is to convert adenosine,a toxic product, into inosine. ADA is expressed in maternaldecidual cells, in embryo-derived trophoblast cells, and ingiant cells lining the implantation chamber of the embryo (1).Since blocking experiments of ADA resulted in severe growthretardation and death of the embryos by E10.5 (6, 38) and ADAhas been reported to be regulated by AP-2 transcription factors,we tested whether the level of embryonic ADA is affected byloss of AP-2 ${gamma}$ . Because of the close proximity of giant trophoblastcells and the secondary deciduum, it would have been difficultto assess the relative pattern and level of zygotically derivedADA expression in the gestational site. Therefore, we decidedto determine embryonic ADA levels in embryo culture using anantibody to ADA. As seen in Fig. 4C and D, AP-2 ${gamma}$ -deficient trophoblastcells had significantly reduced levels of ADA. This findingsuggests that one molecular consequence of the AP-2 ${gamma}$ deficiencyis the lack of zygotically derived ADA expression. It was previouslyreported that the trophoblast cells are more resistant to lackof ADA than are the cells of the embryo proper (1-3). In fact,upon closer morphological examination, all inner cell mass outgrowthsderived from AP-2 ${gamma}$ -deficient blastocyst cultures displayed analtered morphology indicative of differentiation and apoptoticprocesses (Fig. 4A and B).

DISCUSSION

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Discussion

In the study presented here, we have addressed the functionalrole of transcription factor AP-2 ${gamma}$ by using homologous recombinationin murine ES cells to generate and analyze mice lacking thistranscription factor. We provide the first genetic evidencethat AP-2 ${gamma}$ plays an essential role in early postimplantationdevelopment. The phenotypic alterations seen are in agreementwith the expression pattern of AP-2 ${gamma}$ , namely in the trophoblastgiant cells, the ectoplacental cone, and the extraembryonicectoderm.

Animals lacking AP-2 ${gamma}$ display severe growth retardation, die,and are resorbed up to day 9.5 of murine embryonic development.Whole-mount in situ analysis reveals that the embryos do notgastrulate and therefore fail to form mesoderm. This failureis most likely a secondary consequence of the growth retardation,since there is no expression of AP-2 ${gamma}$ in the embryo proper atthis point of murine development.

Several studies have shown that AP-2 ${gamma}$ is expressed in trophectodermalcells as early as the blastocyst stage and is maintained inall derivatives of trophoblast cells, i.e., the primary andsecondary giant cells as well as the cells of the ectoplacentalcone (27, 30). Our studies show that the preimplantation andearly postimplantation development of AP-2 ${gamma}$ -deficient animalsis not affected, indicating that other molecules might compensatefor the function. While all derivatives of the trophoblast cellsin AP-2 ${gamma}$ -deficient animals are formed, both the embryo and theextraembryonic tissues are severely growth retarded. This growthretardation is based on a reduced proliferation of the cellsof the ectoplacental cone and a reduced number of giant cells.A role for AP-2 genes in regulating proliferation has been publishedby us and others (7, 23). AP-2 has been shown to activate theproliferative markers c-erbB2 and c-erbB3 (7) and to repressgenes which induce differentiation and apoptosis such as Stra13, Mtd (or Bok-1), and KLF-4 (23).

While these experiments demonstrate that AP-2 ${gamma}$ is essential forthe development of extraembryonic tissues, the function of thisgene in the embryo proper remains to be elucidated. Studiesshow that AP-2 ${gamma}$ is expressed in migrating neural crest cellsand in cells of the basal layer of the skin (5). Since we havebeen constructing ES cells harboring a conditional allele, experimentsthat address the role of AP-2 ${gamma}$ in the embryo proper or even specifictissues will be possible (34).

The in vitro experiments performed here indicate that AP-2 ${gamma}$ -deficienttrophoblasts display markedly reduced levels of ADA. ADA-deficientmice suffer from severely disturbed purine metabolism and dieperinatally (16, 32). Treatment of mice with the potent ADAinhibitor 2'-deoxycoformycin results in a phenocopy of the AP-2 ${gamma}$ mutant (2, 3, 12, 13), which supports the idea of AP-2 ${gamma}$ regulatingADA (6).

We have shown that loss of AP-2 ${gamma}$ leads to a reduction of theproliferative capacity of extraembryonic tissues. Furthermore,in vitro cultures show that AP-2 ${gamma}$ -deficient blastocyst culturesdisplay lack of ADA, an enzyme essential for detoxificationof adenosine and deoxyadenosine. The data we presented herelead us to a model which pinpoints AP-2 ${gamma}$ as a key player in trophoblastdevelopment. Lack of AP-2 ${gamma}$ leads to reduction of proliferationand lack of expression of ADA in the extraembryonic cells. Asa consequence the embryo proper suffers from malnutrition andintoxication leading to growth retardation and subsequentlydeath and resorption by E9.5. However, the set of genes beingregulated by AP-2 ${gamma}$ has yet to be determined.

With this study three of four AP-2 genes in mice were deletedby gene knockout strategies. While animals were mostly unaffectedin the heterozygous state, the respective null mutants displayedeither cranio-abdominoschisis (29, 39), polycystic kidneys (20),or failure of the trophoblast cells, i.e., lethal phenotypes.In light of the fact that all AP-2 genes are expressed in overlappingpatterns (21, 22) and display a high degree of homology on theDNA and protein levels, it is surprising that the single genedeletions result in such diverse and extensive phenotypes. Thisshows that there are specific functions for individual AP-2genes which cannot be carried out by the other AP-2 genes.

Defects in placentation have also been reported for mice withknockouts of other genes such as Eomes (26), Hand 1 (25), andMash2 (8). While some defects are quite similar to the AP-2 ${gamma}$ knockout phenotype, there are also clear differences. The T-boxgene Eomes is found expressed throughout the differentiationof the trophoblast lineages. Loss-of-function studies revealedthat Eomes is required for the differentiation of trophectodermand the formation of trophoblast stem cells (26). In contrastto AP-2 ${gamma}$ -deficient trophectodermal cells, Eomes-deficient blastocystcultures fail to form trophectodermal outgrowths in vitro, suggestingthat this gene acts earlier in extraembryonic development. Itremains to be seen if Eomes acts upstream of AP-2 ${gamma}$ in the cascadeof transcription factors.

Mice deficient in the basic helix-loop-helix transcription factorHand1 are found to be growth retarded by day 7.5 of developmentdue to a block in trophoblast giant cell differentiation anda smaller ectoplacental cone (25). Thus, Hand1 seems to be requiredfor a specific subset of trophectodermal derivatives. Mash2knockout mice die from placental failure at day 10 of development;the spongiotrophoblast cells and their precursors are absent,and chorionic ectoderm is reduced (8). Mash2 is expressed inthe ectoplacental cone, the chorion, and their derivatives butis absent in primary and secondary giant cells and the allantois.Thus, the expression pattern of Mash2 is only partially overlappingwith AP-2 ${gamma}$ , making it unlikely that this gene is a potentialtarget molecule.

The experiments described above help us to understand the molecularprocesses underlying placental development in mice. In an evolutionarycontext, the rodent and human placentations are quite similar;therefore, results from murine models that elucidate the molecularbasis of placental development should facilitate the designof strategies to reduce fetal loss caused by placental dysfunctionsuch as preeclampsia and intrauterine growth restriction inhumans.

ACKNOWLEDGMENTS

We thank Peter Mombaerts for the pLTNL selection cassette, PascalDolle for the AP-2 ${gamma}$ genomic clone, Rolf Kemler for the e-cadherinantibody, Martin Blum for the Brachyury probe, and Ivan Horakand Klaus Peter Knobeloch for the KPA-ES cells. We thank AndreaJacob, Norma Howells, Judith Dahmen, Inge Heim, and Yvonne Petersenfor excellent technical assistance and maintenance of the animalcolony. Very special thanks to Richard "Ritchie" Jägerfor critically reading the manuscript.

This work was supported by a grant of the Deutsche Forschungsgemeinschaftto H.S. (DFG no. 503-3).

FOOTNOTES

* Corresponding author. Mailing address: Institute for Pathology, Department of Developmental Pathology, University of Bonn, Sigmund-Freud Strasse 25, 53127 Bonn, Germany. Phone: 49 228 287 6342. Fax: 49 228 287 5030. E-mail: Hubert.Schorle{at}ukb.uni-bonn.de.

REFERENCES

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