The Transcription Factor TCFAP2C/AP-2 Cooperates with CDX2

MOLECULAR AND CELLULAR BIOLOGY, July 2010, p. 3310–3320 Vol. 30, No. 130270-7306/10/$12.00 doi:10.1128/MCB.01215-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

The Transcription Factor TCFAP2C/AP-2� Cooperates with CDX2 ToMaintain Trophectoderm Formation�

Peter Kuckenberg,1† Sandra Buhl,1† Tatiana Woynecki,1 Betina van Furden,2 Elena Tolkunova,3Friederike Seiffe,1 Markus Moser,4 Alexey Tomilin,3 Elke Winterhager,2 and Hubert Schorle1*

Department of Developmental Pathology, Institute of Pathology, University of Bonn Medical School, Sigmund-Freud-Str. 25,Bonn D-53127, Germany1; Institute of Molecular Biology, University of Duisburg-Essen, Medical School, Hufelandstr. 55,

Essen 45122, Germany2; Institute of Cytology, Russian Academy of Science, Tikhoretski Ave. 4, St. Petersburg 194064,Russia3; and Department of Molecular Medicine, Max Planck Institute of Biochemistry,

Am Klopferspitz 18, Martinsried D-82152, Germany4

Received 9 September 2009/Returned for modification 24 October 2009/Accepted 10 April 2010

In mammals, cell lineage specification is established at the blastocyst stage. At this stage, transcriptionfactor Cdx2 represses pluripotency genes, thus promoting extraembryonic trophoblast fate. Recently, tran-scription factor Gata3 was shown to act in a parallel pathway in promoting trophoblast cell fate, suggestingthat there are more factors working in the trophoblast lineage. Here, we report that the transcription factorTcfap2c is expressed at a high level in the trophectoderm and is able to induce trophoblast fate in embryonicstem cells. Trophoblast fate induced by Tcfap2c does not require Cdx2 and vice versa, suggesting that themolecules act in alternative pathways. However, both Tcfap2c and Cdx2 are required for the upregulation ofElf5, a marker of trophoblast stem cell maintenance, suggesting that both factors are required for stabletrophoblast induction. Tcfap2c-induced trophoblast-like cells are stable in long-term culture, indicating thatthey are capable of self-renewal. Tcfap2c-controlled trophoblast maintenance involves the induction of Cdx2and the repression of the pluripotency factor Nanog. Tcfap2c-induced trophoblast-like cells differentiate totrophoblast derivatives in vitro and contribute to the trophectoderm in blastocysts in vivo. Taken together, theseobservations suggest that Tcfap2c and Cdx2 cooperate to override the pluripotency program and establish theextraembryonic trophoblast maintenance program in murine embryos.

The earliest cell fate decision during mammalian develop-ment is the establishment of the first two cell lineages of theblastocyst prior to implantation. The inner cell mass (ICM)forms the embryo proper as well as extraembryonic endoder-mal components of the placenta, whereas the trophectoderm(TE) gives rise to the fetal portion of the placenta, a structureunique to mammalian development (11). Self-renewing embry-onic stem cell (ESC) and trophoblast stem cell (TSC) lineshave been derived from each of these lineages in vitro (10, 23,36). TSCs exhibit the potential to differentiate into multipletrophoblastic cell types in vitro, participate in the normal de-velopment of chimeras, and contribute exclusively to the tro-phoblastic component of the placenta in vivo (28, 36).

At the genetic level, key factors that establish and maintainthe TE lineage in the early embryo have been identified. Basedon so far unknown positional information, the Hippo signalingpathway component YAP (Yes kinase-associated protein 1, acoactivator of Tead4) is phosphorylated by LATS (large tumorsuppressor, a Ser/Thr kinase that belongs to the Ndr/LATSsubfamily of protein kinase A/PKG/PKC kinases) and becomescytoplasmic in the inner cells of the morula. In outer blas-tomeres, YAP remains in the nucleus and associates with and

activates TEAD4 (26), which in turn transactivates the expres-sion of the transcription factor CDX2 (caudal-related ho-meobox 2) (27, 40). CDX2 represses pluripotency markerssuch as Oct3/4 (Pou5f1) and Nanog (and vice versa), whichleads to the maintenance of the restricted expression of Cdx2in the TE (6, 28). CDX2 and the T-box factor EOMES (eome-sodermin) are regarded as the key transcription factors re-quired for the establishment of a functional TE. Recently, thetranscription factor Gata3 was described to regulate tropho-blast development downstream of Tead4 and in parallel withCdx2 (16, 30). Restriction of lineage potency is mediatedthrough the epigenetic regulation of Elf5, which is methylatedand silenced in the embryonic lineage but hypomethylated andexpressed in the trophoblast lineage. By forming a positive-feedback loop with Cdx2 and Eomes, Elf5 reinforces the com-mitment to the trophoblast lineage (25).

Little is known about the factors that control TSC mainte-nance and self-renewal or the molecules that control differen-tiation inhibition. Fibroblast growth factor 4 (FGF4) activationof ERK1/2 has been demonstrated to control TSC survival andself-renewal in a mitogen-activated protein kinase (MAPK)kinase kinase (MAP3K; MEKK)-dependent manner (1, 36,41). In addition, members of the transforming growth factor-�/activin subfamily have been shown to influence TSC main-tenance (9, 13).

The overexpression of Tead4, Cdx2, or Eomes in ESCs issufficient to instructively promote trophoblast fate in ESCs (26,28, 37). In addition, the activation of Ras-MAPK signalingpromotes the formation of functional TE cells from ESC cul-tures (21). There, the transcription factor Tcfap2c (AP-2�;

* Corresponding author. Mailing address: Department of Devel-opmental Pathology, Institute of Pathology, University of BonnMedical School, Sigmund-Freud-Str. 25, Bonn D-53127, Germany.Phone: 49-228-28716342. Fax: 49-228-28719757. E-mail: [email protected].

† These authors made equal contributions.� Published ahead of print on 19 April 2010.

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Mouse Genome Informatics accession number 106032) wasfound to be upregulated in TSC-like cells (21). In mice andhumans, the TCFAP2 (AP-2) family of transcription factorsconsists of five proteins, all of which have unique functionsduring mammalian development and act by controlling thebalance between proliferation and differentiation (8). Tcfap2c-deficient mice die at approximately E7.5 due to trophectoder-mal cell defects in the ectoplacental cone (2, 38). The mutantmice display reduced cell proliferation in the ectoplacentalcone, and the number of trophoblast giant cells is reduced;therefore, the trophoblast fails to establish a functional pla-centa, leading to resorption of the embryo.

Here, we show that Tcfap2c is expressed in preimplantationembryos and becomes downregulated in the ICM of late blas-tocysts. Upon implantation, Tcfap2c is expressed in all TEderivatives, except syncytiotrophoblast cells of the labyrinthlayer, by up to E19.5 of murine development. In vitro, TSClines express TCFAP2C in an undifferentiated state as well asduring differentiation into TE derivatives. Induction of Tcfap2cin ESCs (28) instructs the expression of TE markers and TSCfate even in Cdx2-deficient ESCs. Upregulation of Elf5 wasdemonstrated after either CDX2- or TCFAP2C-driven promo-tion of TSC fate in ESC. However, Elf5 expression is notobserved in TSC-like cells generated from Cdx2-overexpress-ing Tcfap2c�/� ESCs or Tcfap2c-overexpressing Cdx2�/�

ESCs. Furthermore, we show that Tcfap2c-induced TSC-likecells retain their TSC character upon long-term culturing,demonstrating that Tcfap2c is sufficient to induce stable TSCfate. We demonstrate that these cells are functional based ontheir expression of markers of TE derivatives in vitro and theability to incorporate into the TE of blastocysts in vivo. Weprovide evidence that TCFAP2C induces Cdx2 and repressesNanog expression. This finding suggests that the maintenanceof TSC fate requires both TCFAP2C and CDX2 protein func-tion.

MATERIALS AND METHODS

Cell culture. MG1.19 (12) ESCs were cultured as described previously (28).All other ESCs were grown on irradiated murine embryonic fibroblasts (feeders)in standard ESC medium (Dulbecco modified Eagle medium [DMEM] supple-mented with 15% fetal calf serum [FCS], nonessential amino acids, L-glutamine,penicillin-streptomycin, ß-mercaptoethanol, 1,000 U/ml leukemia inhibitory fac-tor [LIF]). TSCs were generated and maintained as described previously (36).TSC medium consisted of RPMI 1640 supplemented with 20% FCS, 2 mML-glutamine, 1 mM sodium pyruvate, 50 �g/ml penicillin-streptomycin, and 100�M ß-mercaptoethanol CMF4H medium contained FGF4 (25 ng/ml) and hep-arin (1 �g/ml) and was prepared by adding 70% fibroblast conditioned TSCmedium (CM) to 30% TSC medium. After several passages, the TSCs weregenotyped for Tcfap2c, according to standard protocols (38). For regular differ-entiation, TSCs were cultured in TSC medium. For the induction of labyrinthdifferentiation, TSCs were cultured in TSC medium plus 25 nM trichostatin A(TSA; Sigma-Aldrich, Munich, Germany).

Western blotting. For protein analysis, a Mini-Protean electrophoresis cell andMini-Trans-Blot system (Bio-Rad, Munich, Germany) were used. Protein wasprepared using a standard protocol and was electrophoresed at 30 mA for 90min. The gel was transferred onto a polyvinylidene difluoride membrane in aBio-Rad electrotransfer chamber overnight at 30 V and 4°C. After the mem-brane was blocked with phosphate-buffered saline (PBS) supplemented with0.1% (vol/vol) Tween 20 and 5% low-fat milk powder (PBSTM), it was incubatedwith the following primary antibodies (Abs) in PBSTM: TCFAP2A (1:500; Ac-tive Motif, Rixensart, Belgium) for 2 h at room temperature; TCFAP2C (1:500)for 1 h at room temperature (clone 6E4/4; Upstate, NY); TCFAP2B, TCFAP2D,and TCFAP2E (1:5,000; M. Moser, Munich, Germany) overnight at 4°C; and�-actin (1:6,000; Dako, Hamburg, Germany) as a loading control for 1 h at room

temperature. The secondary antibodies, anti-rabbit antibody–horeseradish per-oxidase (HRP) and anti-mouse antibody–HRP (Dako), were diluted 1:500 for1 h at room temperature. Finally, the membrane was incubated in 2 ml Pierce-Super Signal West Pico chemiluminescent substrate (Perbio, Bonn, Germany),and the signal was detected using X-ray film (Kodak, Germany).

Antibodies for immunostaining. The following primary Abs and dilutions wereused for immunofluorescence: mouse anti-CDX2 (1:400; clone 88; Biocare Med-ical, Zytomed, Berlin, Germany), mouse anti-Oct3/4 monoclonal Ab (1:100;C-10; Santa Cruz, Heidelberg, Germany), rabbit anti-TCFAP2C polyclonal Ab(1:200; H77; Santa Cruz, Heidelberg, Germany), mouse anti-CDH3 monoclonalAb (1:25; clone 56C1; Neomarkers, Fremont, CA), and rabbit anti-mouseNANOG (Abcam, Cambridge, United Kingdom). Detection of primary Abs wasperformed with secondary Abs that detect mouse and rabbit IgG, conjugatedwith Alexa Fluor 488 or Alex Fluor 594 (1:400; both from Invitrogen, Karlsruhe,Germany). 4�,6-Diamidino-2-phenylindole (DAPI; Sigma-Aldrich) was used fornuclear staining. Fluorescent images of the blastocysts were captured in opticalsections using an Observer microscope (Carl Zeiss, Jena, Germany) with theapotome setting and were processed using Axiovision (version 4.7) software(Carl Zeiss). Cells were observed using an IM-DRB fluorescent microscope(Leica, Bensheim, Germany).

Histology and immunohistochemistry. Embryos were fixed in 4% neutralbuffered formalin at 4°C overnight and embedded in paraffin. For immunohis-tochemistry, 1- to 3-�m sections were incubated using the following primaryantibodies for 1 h at 37°C (4°C, overnight): TCFAP2C (1:250; 6E4; kindlyprovided by H. Hurst) and peroxidase-conjugated isolectine B4 (TSA; Sigma-Aldrich). Anti-mouse secondary antibodies (1:400; Dako) were used for 30 minat room temperature. Signals were visualized using a Vectastain ABC kit (VectorLaboratories). Sections were photographed using Diskus software (Hilden, Ger-many), and files were processed using Adobe Photoshop Elements 5 and Illus-trator software.

RT-PCR/quantitative real-time RT-PCR (qRT-PCR) analysis. Total RNA wasisolated from cells and tissues by using an RNeasy minikit (Qiagen, Hilden,Germany) or TRI reagent (Sigma-Aldrich), according to the manufacturer’sinstructions. For standard reverse transcription-PCR (RT-PCR), first-strandcDNA was synthesized from 500 ng or 1 �g of total RNA in a 20-�l reactionmixture using oligo(dT) primers and SuperScriptIII reverse transcriptase (In-vitrogen), according to the manufacturer’s recommendation. PCR was per-formed using 25 to 30 cycles of 94°C for 30 s, 55 to 62°C for 30 s, and 72°C for50 s. The primer sequences are available on request. Quantitative real-time PCRwas performed using an ABI Prism 7300 sequence detection system (AppliedBiosystems, Darmstadt, Germany) and SYBR green (Applied Biosystems), ac-cording to the manufacturer’s protocol. Equivalent amounts of cDNA generatedfrom the RT reactions were used as templates for PCR. Reactions were per-formed at least in triplicate for each sample. Expression of genes was normalizedto the expression of �-actin and glyceraldehyde-3-phosphate dehydrogenase, andthe mRNA level of each gene in untreated ESCs (control) was set equal to 1. Thereal-time PCR mixtures were processed in triplicate in a total volume of 20 �lcontaining 40 ng cDNA, 3.75 pmol gene-specific primers, and 10 �l master mix,including the SYBR green reagent (Applied Biosystems). The PCR program wascomposed of 10 s at 95°C, followed by 45 cycles of 5 s at 95°C and 35 s at 60°C.After PCR, a melting curve analysis was arranged for determination of PCRproduct specificity. For each sample, a cycle threshold (CT) value was recorded.PCRs were set up in triplicate, and the mean of the three CT values wascalculated. To determine the relative gene expression, the comparative CT

method (��CT method) was used. The level of gene expression was shown as thefold change compared to the level for the control sample. Primer sequences andreferences are available on request.

Establishment of ESC carrying tamoxifen-inducible Tcfap2c. The entire openreading frame of Tcfap2c was amplified by PCR with primers mAP2gF1 (5�-GGACGCCATGTTGTGGAAAATAAC-3�) and mAP2gR (5�-GGATCCCTTCCTGTGCTTTTCCATTTT-3�), followed by subcloning into the pCR2.1 vector(Invitrogen) and digestion with EcoRI. The Tcfap2c cDNA was subcloned intopCR2.1 lacking BamHI, followed by digestion with BamHI. The ligand bindingdomain of the mutant form of mouse estrogen receptor (ER) amplified by PCRwith primers mESRF (5�-GGATCCCGAATTGAAATGGGTGCT-3�) andmESRR (5�-GGATCCTCAGATCGTGTTGGGGAAGCC-3�) was subclonedinto the pCR2.1 vector, followed by digestion with BamHI and subcloning intoBamHI of pCR2.1-Tcfap2c, resulting in the generation of pCR2.1-Tcfap2cER.After digestion with EcoRI Tcfap2cER, the cDNA was subcloned into EcoRI ofpCAG-IP (28), resulting in pCAG-Tcfap2cER-IP. Thirty micrograms of pCAG-Tcfap2cER-IP was linearized by SacI and electroporated into 1 � 107 CJ7 ESCsat 575 V/cm and 500 �F using a Bio-Rad Gene-Pulser. Twenty-four hours aftertransfection, cells were selected for 4 days using 1.4 �g/ml puromycin. Clones in

VOL. 30, 2010 Tcfap2c IN TROPHECTODERM MAINTENANCE 3311

which pCAG-Tcfap2cER-IP had integrated into the genomic DNA were selectedby their ability to undergo differentiation to TE by addition of 1 �g/ml of4-hydroxy-tamoxifen (Tx; Sigma-Aldrich).

Establishment of ESCs carrying doxycycline-inducible Tcfap2c. A gene tar-geting kit obtained from Open Biosystems (Huntsville, AL) was used to generatesingle-copy transgenic ESCs by site-specific recombination using a method de-scribed previously (3). In short, pCR2.1-Tcfap2c containing the entire openreading frame of Tcfap2c was digested with EcoRI, and the cDNA of Tcfap2c wasinserted into pBS31_tetO_promoter/simian virus 40p 5� of the tetO minimalcytomegalovirus (CMV) promoter. KH2 ESCs carrying the reverse tetracyclinetranscriptional activator (rtTA) transgene in the ROSA26 locus (3) were elec-troporated with 50 �g of pBS31_Tcfap2c and 25 �g of an expression vector forthe Flp recombinase (pCAGGS-flpE). Flp-mediated recombination of pBS31-Tcfap2c leads to the integration of Tcfap2c cDNA into the ColA1 locus of KH2ESCs. In parallel, this recombination initiates the expression of the promoter-and ATG-less hygromycin resistance cassette present in this locus. Twenty-fourhours after electroporation, 140 �g/ml hygromycin was added. Ten days later,colonies were picked and clones were screened by Southern blotting using SpeIto digest genomic DNA and a 3� internal probe also provided with the kit. Twopositive clones in which Tcfap2c expression is induced after addition of 0.5 �g/mldoxycycline for 48 h were expanded.

Production of chimeric embryos. Host embryos were obtained from C57BL/6mice and were collected at the four-cell stage. Tcfap2cER-induced ESCs andTcfap2cER-induced TSC-like cells were incubated for 10 min at 37°C in PBSsupplemented with 1 �M fluorescent dye carboxyfluorescein succinimidyl ester(CFSE; Fisher Scientific, Schwerte, Germany). The staining procedure wasstopped by adding 20% fetal bovine serum (Thermo Scientific HyClone, SouthLogan, UT). To generate chimeric embryos, 15 stained cells were injected underthe zona pelucida of four-cell-stage embryos as described previously (21). Afterinjection, the embryos were first cultured in vitro in KSOM medium lackingamino acids (15); after 24 h, they were transferred to DMEM supplemented with2 mM glutamine, 50 U/ml penicillin and streptomycin, and 20 mmol/literHEPES. They were visualized by fluorescence microscopy at the blastocyst stageusing a Leica DM-IRB (Bensheim, Germany) inverted microscope. Image pro-cessing and merging were done using ImageJ freeware (version 1.37; NationalInstitutes of Health [http://rsb.info.nih.gov/ij/]). The embryos were examined andphotographed using a Leica MZ4 stereomicroscope. Permission to performanimal experiments was granted from the Landesamt fuer Natur Umwelt undVerbraucherschutz, Recklinghausen, Germany (50.203.2-Bn12, 42/04).

Generation of ESCs deficient for Cdx2 or Tcfap2c. Heterozygous Cdx2 femaleswere superovulated and mated with heterozygous males. The mouse line was akind gift from Felix Beck (University of Leicester, Leicester, United Kingdom)(5). Blastocysts at 3.5 days postcoitum were flushed from the uteri in KSOMmedium, treated with tyrode solution (to remove the zona pellucida), and platedonto mitomycin C-inactivated mouse embryonic fibroblast cells in standard ESCmedium containing LIF. On day 4 to 5 after plating of the blastocysts, theblastocyst outgrowths that showed well-expanded ICMs with no apparent tro-phoblast compartment (a hallmark of Cdx2 deficiency) were picked and pro-cessed following a typical ESC derivation protocol (24). The newly establishedESC lines were confirmed to be Cdx2�/� by PCR genotyping (5). For thegeneration of Tcfap2c-deficient ESCs, the heterozygous Tcfap2c animals gener-ated by us (38) were used. The analogous protocol was followed to generate theTcfap2c�/� ESC lines.

ChIP analysis. Wild-type TSCs (2 � 106) were resuspended in 500 ml ice-coldPBS and cross-linked with 1% formaldehyde (Sigma-Aldrich) for 7 min at roomtemperature. The reaction was stopped by adding 0.1 ml 1.375 M glycine, fol-lowed by washing of the TSCs two times with PBS. The cell pellet was resus-pended in 200 �l SDS lysis buffer (0.1% SDS, 10 mM EDTA, 50 mM Tris, pH8.1), and the mixture was incubated for 10 min on ice. Sonication was performedin a Bioruptor apparatus (Diagenode, Belgium) for 10 min (high setting for 30 son and 30 s off) to achieve a fragment size between 200 and 500 bp. Afterpelleting of the cells, the supernatant was transferred to a new tube and the DNAconcentration was measured. Ten microliters of protein G Dynabeads (Invitro-gen) was preincubated with 1 �g of specific anti-TCFAP2C antibody (H77; SantaCruz) in 500 �l chromatin immunoprecipitation (ChIP) dilution buffer (0.01%SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, 167 mM NaCl, pH8.1) for 1 h. After the supernatant was removed, the beads were washed twotimes in ChIP dilution buffer. For the precipitation, 10 �g of shared DNA, 2 �gsalmon sperm DNA, and protease inhibitor were mixed in a total volume of 200�l in ChIP dilution buffer and the mixture was incubated overnight at 4°C. Onthe next day, the protein G-antibody-protein-DNA complex was washed threetimes in 0.5 ml wash buffer (0.01% Tween 20 in Tris-EDTA [TE]). The DNA waseluted by adding 110 �l elution buffer to the pelleted complex at room temper-

ature for at least 1 h. Reverse cross-linking was performed by adding 5 �lproteinase K and 10 �l 5 M NaCl and incubation at 55°C for 2 h, 65°C for 6 h,and 75°C for 2 h. Ethanol-sodium acetate-precipitated DNA was analyzed byPCR using the primer pair F (5�-CAGTCTGGGTCACCTTACAGCTTC-3�)and R (5�-GACACCAACCAAATCAGCCTATC-3�). As a control, ChIP wasperformed without antibody and an antihemagglutinin (anti-HA) antibody.

Luciferase assay. Regulatory reporter elements of the respective genes wereamplified by PCR from 129Sv genomic DNA (the primers are available onrequest), subcloned into the pCRII TOPO vector (Invitrogen), and verified bysequencing. The luciferase reporter plasmids were constructed by the introduc-tion of the regulatory elements into pGL3-Basic (Promega, Madison, WI). Forgenerating the dTCFAP2-Nanog reporter, the Nanog promoter was digested withEcoRI and XhoI and religated to delete the sequence between bp �310 and�118. A total of 75 ng of luciferase reporters and 125 ng of the expressionvectors encoding Tcfap2c (pCAG-Tcfap2c-IP) and Cdx2 (pCAG-Cdx2-IP; a giftfrom Hitoshi Niwa, RIKEN Center for Developmental Biology [CDB], Kobe,Japan) were transfected into 2.5 � 104 MG1.19 ESCs using Lipofectamine 2000(Invitrogen) with 4 ng of pRL-CMV (Promega) as an internal control. Thetransfected cells were lysed 24 h after transfection, and luciferase activity wasestimated using a dual-luciferase assay kit (Promega). The activity of theluciferase reporter cotransfected with the empty pCAGIP expression vectorwas set equal to 1.

RESULTS

Tcfap2c expression was detected in early embryos in thetrophoblast lineage. To determine the spatiotemporal expres-sion of Tcfap2c and to gain better insight into the role ofTcfap2c in early development, we analyzed the mRNA andprotein levels at various preimplantation stages. Using RT-PCR, we detected Tcfap2c mRNA in morulae and blastocysts(Fig. 1A), confirming the results presented by Winger et al.(39). Immunodetection showed that the TCFAP2C protein ispresent in all cells of the eight-cell stage and morula (data notshown). By the late blastocyst stage, the TCFAP2C signal washigh in the trophectoderm and reduced in the inner cell mass(Fig. 1B, B�, and B�). After implantation, on days E5.5 andE6.5, the TCFAP2C protein was detected in all cells of theTEC lineage, such as the extraembryonic ectoderm, which har-bors the trophoblast stem cell population. TCFAP2C was alsofound in the ectoplacental cone and in primary and secondarytrophoblast giant cells but not in the embryoblast or primitiveendoderm (Fig. 1C and D). On day E7.5, TCFAP2C persistedin all cells of the TEC lineage. The thin layer of mesothelialcells underlying the chorion and the cells of the primitiveendoderm were negative for TCFAP2C (Fig. 1E and E�). Onday E8.5, after chorioallantoic fusion, the TCFAP2C proteinwas not detected in the allantois compartments, which give riseto placental blood vessels and the umbilical cord (Fig. 1F andF�). Beginning at E11.5 and continuing to E19.5, theTCFAP2C protein was detected in the developing spongiotro-phoblast and giant cell layers (Fig. 1, G) but not in the syncy-tiotrophoblast cells of the labyrinth layer. On E19.5, giant cellsand spongiotrophoblast cells express TCFAP2C, while in thelabyrinthine layer, only a few small mononuclear cells werepositive for TCFAP2C. Immunodetection of adjacent sectionsusing isolectine B4, which detects maternal blood sinusoids,shows that TCFAP2C-positive cells remain in contact withisolectine B4-positive cells and are therefore most likely mono-nuclear giant cells. This finding is further supported by the factthat mononuclear giant cells are known to express placentallactogen II (Pl2), which has been demonstrated to be a tran-scriptional target of TCFAP2C in rat and human cells (29, 32).

Next, immunostaining of ESCs and TSCs was performed.

3312 KUCKENBERG ET AL. MOL. CELL. BIOL.

Interestingly, few cells within ESC colonies were positive forTCFAP2C (Fig. 2A and B). In contrast, TSCs displayed astrong TCFAP2C signal (Fig. 2C and D). Western blot analy-ses for the detection of all TCFAP2 isoforms demonstratedthat TCFAP2C and TCFAP2A were expressed at high levels inundifferentiated TSCs. Upon induction of TSC differentiation,the TCFAP2C and TCFAP2A signals were upregulated andpersisted up to day 13 of differentiation (the last day of anal-ysis). Also, by this time, terminally differentiated spongiotro-phoblast, syncytiotrophoblast, and giant cells were detected inculture (data not shown). In addition, the TCFAP2B proteinwas detected as soon as differentiation was initiated. In con-trast, the TCFAP2D and TCFAP2E proteins were not de-tected in TSCs (Fig. 2E). The specificities of the TCFAP2Aand TCFAP2C antibodies were confirmed using in vitro-trans-lated protein (data not shown). These data suggest a role forTCFAP2A, TCFAP2B, and TCFAP2C in TSCs.

Tcfap2c function is indispensable for TSC establishmentand maintenance. Tcfap2c-deficient mice display a defect introphectoderm development, as determined by gene-knockoutstudies (2, 38); therefore, we tried to generate TSCs fromTcfap2c-deficient embryos for detailed analyses. Here, 22 blas-tocysts from Tcfap2c/� intercrosses were cultured under TSC

proliferation-permissive conditions (36). All isolated blasto-cysts attached to the dish and initiated characteristic trophec-todermal outgrowths. The outgrowths from Tcfap2c/ andTcfap2c/� blastocysts were indistinguishable, whereas theoutgrowths from Tcfap2c�/� embryos showed an altered mor-phology, consistent with the findings of previous studies (2, 38)(data not shown). After passage 5, genotyping indicated thatwe obtained 13 Tcfap2c/, 5 Tcfap2c/�, and 4 Tcfap2c�/�

TSC lines. However, the cell lines heterozygous or homozy-gous for the Tcfap2c deletion could not be maintained. Thesemutant cells ceased to proliferate, and large, flattened cellsappeared, which is indicative of differentiation (data notshown). Consequently, after 10 passages, genotyping revealedthat only TSC lines wild type for Tcfap2c alleles were estab-lished (n 13). Because Tcfap2c�/� ESCs were derived fromblastocysts (described later), these results suggested thatTcfap2c is not essential for initial outgrowth. Instead, Tcfap2cmay be required for the maintenance of TSCs but is dispens-able for the derivation of ESC cultures. Moreover, even areduction in the gene dosage to one intact allele compromisedTS maintenance.

Expression of Tcfap2c in ESCs induces expression of TECmarkers. It has been shown that the expression of TEC marker

FIG. 1. Tcfap2c in early embryogenesis and TCFAP2 proteins in the trophectoderm. (A) Semiquantitative RT-PCR analysis for Tcfap2c inmorulae, blastocysts, and undifferentiated TSCs. RNA was isolated from pools of morulae or blastocysts and analyzed by RT-PCR. (B, B�, and B�)Immunofluorescent localization of TCFAP2C in E3.5 blastocysts. The nuclei of cells stained with DAPI (B) and TCFAP2C antibody (B�) and themerged picture (B�) are shown. (C to H) Immunohistochemical localization of TCFAP2C in embryo sections and embryonic stages are indicated.(I) Immunohistochemical localization of isolectine B4. Abbreviations: ExE, extraembryonic ectoderm; Emb, embryoblast; GC and Gc, giant cell;Ch, chorion; PrE, primitive endoderm; Me, mesothelium; Al, allantois; La, labyrinth; Sp, spongiothrophoblast.


genes Tead4, Cdx2, and Eomes is sufficient to promote a TSC-like fate in ESCs. Next, we examined the ability of Tcfap2c topromote trophoblast differentiation in ESCs. We generatedESCs stably expressing a Tx-inducible fusion between Tcfap2cand a modified ligand binding domain of the estrogen receptor(Tcfap2cER). These cells remained ESCs when they weregrown under ESC conditions without Tx (Fig. 3A) and under-went random differentiation under TSC conditions without Tx(Fig. 3B). Using trophoblast stem cell culture conditions(CMF4H) (36) and Tx, TSC-like cultures were derived fromTcfap2cER-induced ESCs (Fig. 3D). Tcfap2cER-induced ESCsadopted the morphology of TSC colonies, displayed enlargedand multiple nuclei, and expressed the TE surface markercadherin 3 (CDH3; pCadherin) (Fig. 3E and F). Consistentwith the findings of previous studies (28), changing CMF4H-Txmedium to differentiation medium consisting of Tx alone inTSC medium without FGF4 and heparin leads to large, flat-tened cuboidal cells resembling trophoblast giant cells (Fig.3C). Quantitative RT-PCR analysis revealed that the expres-sion of the ESC markers Oct3/4, Nanog, and Stella was reducedand that the TEC markers Cdx2, Eomes, Bmp4, Gata3,Tcfap2a, Hand1, Pl1, and Pl2 were induced. Also, the marker

for mesenchyme differentiation, Brachyury, was reduced andthe primitive endoderm markers, Gata6 and Hnf4b, were notdetected, indicating that Tcfap2c expression is sufficient topromote trophoblast fate in ESCs (Fig. 3J, green bars). Ofnote, during differentiation, few colonies displaying the ESCmorphology remained; however, they were still positive forNANOG, while the surrounding cells expressed the TSCmarker CDX2 (data not shown). Cells expressing both CDX2and NANOG or CDX2 and OCT3/4 were not observed (datanot shown). In addition, two ESC lines harboring doxycycline-inducible Tcfap2c were established. Most experiments per-formed with Tx-inducible Tcfap2c lines were repeated with thedoxycycline-inducible lines (data not shown) to exclude theside effects from tamoxifen or doxycycline and showed identi-cal results.

The caudal related homeoprotein CDX2 has been reportedto play a central role in TEC specification in vivo and in TSCinstruction in ESC-based systems (28, 37). To examine theepistatic relationship between Cdx2 and Tcfap2c, we estab-lished Cdx2�/� ESCs that conditionally express Tcfap2ER(Cdx2�/� Tcfap2cER). After treatment with Tx under TSCconditions, multiple independent clones exhibited trophoblast-like morphologies (Fig. 3G) and expressed CDH3 (Fig. 3H andI). Quantitative real-time PCR experiments showed that thepluripotency markers Oct3/4 and Nanog were reduced and thatthe markers Eomes, Bmp4, Gata3, Tcfap2a, Hand1, Pl1, andPl2 were induced to levels lower than those in Cdx2 wild-typecells (Fig. 3J, compare the red and purple bars). These resultsindicated that Tcfap2c acts independently of Cdx2 to induceTE fate.

Next, a Tx-inducible Cdx2 transgene (Cdx2ER, obtainedfrom H. Niwa) was introduced into wild-type and Tcfap2c�/�

ESCs (28). Again, after treatment with Tx, the cultures exhib-ited trophoblast-like morphologies and expressed TSC mark-ers (data not shown). qRT-PCR was performed to analyze thelevels of Cdx2 and Tcfap2c in the Tx-inducible cell lines used inthe experiments. Of note, neither the Tcfap2c nor the Cdx2levels achieved by the Tcfap2cER or Cdx2ER transgenereached levels comparable to the endogenous Tcfap2c andCdx2 levels in TSCs (data not shown). Taken together, theseexperiments indicated that Tcfap2c is able to promote thegeneration of TSC-like cells from ESCs in a Cdx2-independentmanner, and vice versa.

After the embryonic and trophoblast cell lineages were spec-ified, a stable maintenance of lineage identity is ensured byepigenetically imposed cellular memory (33). This lineage re-striction is primarily mediated by the transcription factor Elf5.Elf5 is hypomethylated and expressed in the TE in late blas-tocysts, is methylated, and is repressed in the ICM (25). ELF5maintains expression of the trophoblast stem cell gene Cdx2(and Eomes), reinforcing trophoblast cell fate. During TSCdifferentiation, the ELF5 signal is very rapidly lost (25).

Using ESCs harboring the Tcfap2cER- and Cdx2ER-induc-ible alleles, we asked if Elf5 expression is activated during thepromotion of the TSC-like morphology. Both transgenes wereable to induce Elf5 in ESCs that were wild type for the Cdx2and the Tcfap2c loci. However, the Elf5 signal was very weak inCdx2�/� Tcfap2cER or Tcfap2c�/� Cdx2ER cells (Fig. 4A),and this result was confirmed by qRT-PCR analyses (Fig. 4B).Induction of Elf5 by Tcfap2c or Cdx2 overexpression was ac-

FIG. 2. Expression of TCFAP2C in cell culture. (A to D) Photomi-crographs of ESC colonies (A and B) and TSC colonies (C and D). (Aand C) Immunohistochemical detection of TCFAP2C (green). (B andD) Bright-field photograph. The red circles in panels A and B indicatethe locations of the ESC colonies. (E) Western blot detecting allTCFAP2 proteins in TSCs grown in CMF4H and following differen-tiation after the removal of heparin and FGF4 (2 to 13 days). The blotfor the positive control (Co) is shown in he last column. Bars, 50 �m.


companied by trophoblast differentiation, as shown by the ap-pearance of giant cells and the upregulation of Hand1 (Fig.4B). The lack of the early TSC marker Eomes (Fig. 3J) and thelow level of Elf5, along with morphological evidence, indicatethat the TSC-like cells lacking Cdx2 or Tcfap2c are not able tomaintain the undifferentiated TSC-like state (Fig. 3H). Inagreement with this notion, all previous attempts to derive

permanent TSCs from Cdx2�/� or Tcfap2c�/� blastocysts re-peatedly failed (35; data not shown). These results suggestedthat the maintenance of undifferentiated TSC-like cells, asmeasured by Elf5 expression, requires both Tcfap2c and Cdx2.Next, we analyzed if Tcfap2cER-induced TSC-like cells arestable in culture. To this end, we treated Tcfap2cER-inducedESCs for the first 10 days with Tx to initiate TSC-like cultures.

FIG. 3. Tcfap2c induces TSC fate in a Cdx2-independent manner. (A) Colony morphology of Tcfap2c-induced ESCs cultured in ESCmedium. (B) Tcfap2cER-induced ESCs cultured for 7 days in CM4H without Tx. (C) Formation of giant cells from Tcfap2cER-ESC afterthe addition of Tx in TSC medium without FGF4 and heparin. (D to F) In vitro culture of Tcfap2cER-induced ESCs under CMF4H and Txconditions for 7 days. (F) Expression of the TS marker CDH3 (placental cadherin) was detected with anti-CDH3 antibody (green), and thenuclei were stained with DAPI (blue). (G to I) Culture of Cdx2�/� Tcfap2cER-induced ESCs under CMF4H and Tx conditions for 7 days.(A to I) Scale bars, 50 �m. (J) Quantitative RT-PCR analysis of indicated marker gene expression in ESCs (blue), TSCs (red), Tcfap2cER-induced ESCs (green), and Cdx2�/� Tcfap2cER-induced ESCs (purple). Total RNA was prepared from each pool at day 7 after activationwith Tx. The markers analyzed are indicated.


Thereafter, the cells were kept in CMF4H medium without Tx.Here, multiple colonies with a TSC-like morphology were de-rived and maintained for more than 10 passages. Interestingly,the compact TSC-like cultures generated by Tcfap2c inductionwere always surrounded by large, flat, multinucleated cells(Fig. 4C and D), which indicates ongoing giant cell differenti-ation.

Tcfap2c-induced TSCs have the ability to differentiate alongthe trophectodermal lineage in vitro and in vivo. Quantitativereal-time PCR analyses showed that the TSC-like cells pro-duced by Tcfap2cER induction expressed markers of undiffer-entiated extraembryonic ectoderm (ExE) (Mash2), trophoblastgiant cells (Pl1), and spongiotrophoblast cells (Tbpba). How-ever, the level of Mash2 in Tcfap2cER-induced TSC-like cellswas much lower than that in Cdx2ER-induced TSC-like cellsand the levels of markers of differentiated TE (Pl1 and Tbpba)were much higher (Fig. 5A). This finding is consistent with themorphological data (Fig. 4C and D), since the Tcfap2cER-induced TSC-like cells tend to differentiate more easily thanCdx2ER-induced cultures. We demonstrated earlier thatTcfap2c is expressed in all cells of the extraembryonic lineageexcept syncytiotrophoblast cells. If the Tcfap2-induced TSC-like cells resemble true TSCs, one would expect the cells toalso differentiate into syncytiotrophoblast cells. Alternatively,Tcfap2c induction might generate precommitted TSC-likecells, which would be unable to differentiate into syncytiotro-phoblast cells. TSC-like cells were first generated by Tx-medi-ated Tcfap2c induction. Thereafter, Tx was omitted, and theTSC-like cultures were treated with TSA to promote syncytial-ization in vitro (22). The Tcfap2c-induced TSC-like cells ex-

pressed the syncytiotrophoblast marker Syncytin A (Fig. 5B),indicating that their differentiation potential is not restrictedand that they can be considered true TSCs.

We next addressed if TSC-like cells generated by the expres-sion of Tcfap2c in ESCs are functional in vivo. We labeledTSC-like cells induced by Tcfap2cER with the CFSE fluores-cent dye and injected them into four-cell-stage embryos tofollow their fate in blastocysts. Of a total number of 18 injectedembryos, 8 developed to the blastocyst stage. All cells contrib-uted to the TE but not the ICM (Fig. 5C). Injection of controlTcfap2cER-induced ESCs that had been cultured in ESC me-dium without Tx contributed to the ICM (of 10 ESCs injected,6 survived, 5 showed ICM staining, and 1 showed no staining)(Fig. 5D). This result suggests that Tcfap2c-induced TSC-likecells resemble TE cells in their ability to integrate into thetrophectoderm of blastocysts.

TCFAP2C and CDX2 regulate each other, and TCFAP2Crepresses Nanog. Transfac and GenBank analyses were usedto determine the locations of TCFAP2C binding sites in thepromoter elements of Cdx2, Tcfap2c, Eomes, FgfrIIc, Nanog,and Oct3/4. Next, we evaluated the TCFAP2C transactiva-tion of promoter elements of Cdx2, Tcfap2c, Eomes, andplacenta-specific FgfrIIc and compared the results to those

FIG. 4. Maintenance of TSC fate requires Tcfap2c and Cdx2.(A) RT-PCR analysis detecting Elf5 in TSC-like cells with the indi-cated genotype produced by adding Tx for 7 days compared to the Elf5signal in ESCs and TSCs. (B) Quantitative real-time PCR of thesame cells detecting Elf5 and Hand1. Photomicrograph of TSC-likecolonies which had been induced by Tx in Tcfap2cER-induced ESCsfor 7 days in CMF4H. Afterwards, cells were grown in CMF4Hwithout Tx. The colony at passage 5 is shown as a bright field (C).Expression of the TSC marker cadherin3 (pCadherin) was detectedwith the anti-CDH3 antibody (green), and the nuclei were stainedwith DAPI (blue) (D).

FIG. 5. Tcfap2c-induced TSC-like cells differentiated in vitro andcontributed to the trophectoderm in vivo. (A) TSC-like cells wereproduced in CMF4H and Tx for 10 days. Expression of the indi-cated markers was examined using quantitative real-time PCR afteractivation of TCFAP2C (Tcfap2cER) and compared to that afteractivation of CDX2 (Cdx2ER). (B) Expression of Gcm1, Syncytin A,Mash2, and Tpbpa in TSC-like cells promoted by expression ofTcfap2c, depending on the presence of the histone deacetylaseinhibitor TSA (TSA), compared to that under conditions whereTSA was omitted (�TSA). �dox, no doxycycline was added.(C) Tcfap2cER TSC-like cells produced by culture in CMF4H andTx, labeled with the fluorescent, lipophilic dye CSFE, injected un-der the zona pellucida of four to eight cell embryos, and examinedat the blastocyst stage. The green signal indicates the location of thecells in the murine TE. No signal is detected in the ICM. The imageis representative of images for 18 injected embryos. (D) Controlexperiment showing the location of Tcfap2cER-induced ESCs (keptin ESC medium without Tx) to the ICM. The red dotted linesindicate the ICM-blastocele border.


of CDX2-mediated transactivation. Luciferase assays infeeder-free MG1.19 ESCs (12) showed that TCFAP2C wasable to activate Cdx2 and its own promoter, whereas it hadlittle effect on Eomes and the FgfrIIc promoter (Fig. 6A,black bars); however, the levels of activation were lowerthan those from CDX2-mediated transactivation (Fig. 6A,white bars). When we analyzed the effect of TCFAP2C onthe promoters of pluripotency markers, we found that theNanog reporter construct was repressed (the value for thecontrol was set equal to 1) by TCFAP2C and CDX2. Incontrast, Oct3/4 displayed only mild repression byTCFAP2C (Fig. 6B, black bars). Deletion of the three pu-tative binding sites for TCFAP2 at positions �313, �294,and �202 in the Nanog promoter (relative to the position ofATG) resulted in a moderate loss of repression, indicatingthat TCFAP2C may act in a direct manner and an indirectmanner on the Nanog promoter. Next, we assessed the re-cruitment of Tcfap2c to these sites via ChIP analyses. Son-icated chromatin containing �300-bp fragments of DNA,which had been subjected to IP using antibodies specific forTcfap2c, were subjected to PCR using the depicted primers(Fig. 6C, arrowheads P1 and P2). This method allowed us toamplify a fragment of the Nanog promoter between nucle-otides �397 and �167 that contains the three putativeTCFAP2C binding sites. ChIP with an anti-TCFAP2C anti-body showed protein-DNA complex formation with theNanog promoter element, indicating that TCFAP2C boundto the sites, suggesting direct transcriptional regulation (Fig.

6D, lane Tcfap2c). Thus, TCFAP2C regulates the trophec-toderm markers Cdx2 and Tcfap2c and represses the pluri-potency marker Nanog.

DISCUSSION

Our results demonstrate a novel role for Tcfap2c in theestablishment and maintenance of the TE. Tcfap2c is ex-pressed in the TE of late blastocysts and in all cells of the TElineage except syncytiotrophoblast cells. Furthermore, Tcfap2cand Tcfap2a are expressed in TSC cultures. Tcfap2c-deficientblastocysts did not give rise to self-renewing TSCs but werecapable of ESC formation, indicating an essential role forTcfap2c in the maintenance of TSC fate in vitro. Tcfap2c issufficient for the induction of TSC-like fate when it is ex-pressed in ESCs in FGF4, heparin, and fibroblast-conditionedmedium, even in the absence of Cdx2. We showed thatTcfap2c-induced TSC-like cells are functional TE cells basedon their expression of markers of TE derivatives in vitro andthe ability to integrate into the trophectoderm layer of blasto-cysts in vivo. The induction of Tcfap2c in ESCs for 10 days wassufficient to generate stable and self-renewing TSC-like celllines that were maintained for more than 10 passages underregular TSC conditions. However, loss of either Tcfap2c orCdx2 leads to the failure of Elf5 to be upregulated in TSC-likecells and their subsequent differentiation. We provide evidencethat TCFAP2C represses Nanog and activates Cdx2. These

FIG. 6. Transactivation of CDX2 and TCFAP2C and the regulation of Nanog. (A) Luciferase assays of reporter constructs containing thepromoter elements of Cdx2, Tcfap2c, Eomes, and Fgfr2, as indicated, in MG1.19 ESCs. The reporters were cotransfected with the expressionvectors for Tcfap2c and Cdx2. The activity of the luciferase reporter with the empty pCAGIP was set equal to 1. (B) Luciferase assays of reporterconstructs containing promoter elements of Nanog, Oct3/4, and a mutational construct of the Nanog promoter where the three TCFAP2C bindingsites are deleted using MG1.19 ESCs. Reporters were cotransfected with the expression vectors for Tcfap2c and Cdx2. The activity of the luciferasereporter with the empty pCAGIP was set equal to 1. (C) Schematic representation of the Nanog promoter, showing the location of the TCFAP2binding motifs (open diamonds) and their location relative to ATG (numbers). Arrowheads indicate the positions of the P1 and P2 primers usedfor PCR amplification. The 5� untranslated region starts at position �191). (D) ChIP assay for the TCFAP2 binding site in the Nanog promoter.Sonicated chromatin samples prepared from TSCs were immunoprecipitated with a TCFAP2C-specific antibody. The genomic DNA fragmentcontaining the indicated region of the Nanog promoter was amplified by PCR. Lanes: M, 100-bp marker; Tcfap2c, IP with TCFAP2C; no, IPwithout TCFAP2C; HA, IP done using anti-HA antibody as a control; in, input control before IP.


results indicate that TE maintenance depends on the presenceof both Cdx2 and Tcfap2c.

Several transcription factors such as Cdx2 and Tead4 weredetected in murine preimplantation stages and were shown tobe required for the specification of the trophectoderm celllineage (35, 40). Here, we demonstrate that Tcfap2c is ex-pressed in mouse embryos prior to implantation at the morulaand blastocyst stage by RT-PCR analysis. We showed that theoverexpression of Tcfap2c in ESCs led to the induction of aTSC-like fate, as demonstrated by the upregulation of Cdx2 aswell as other markers indicative of undifferentiated TSCs.These cells are stable in culture because they can be passagedfor a prolonged time without the loss of undifferentiated TSCmorphology. Also, they differentiate and become all of thederivatives of the TE, as demonstrated in vitro and in vivo.Taken together, the results of these experiments indicate a rolein TSC specification and maintenance. The following in vivodata argue against the specification scenario: (i) the TCFAP2Cprotein is downregulated in the ICM of late blastocysts only,when the TE is already specified, and (ii) mutants for Cdx2 andEomes arrest and die at about the blastocyst stage (5, 34).Tcfap2c�/� embryos are formed and implant into the uterus,indicating that Tcfap2c is not essential for TE specification.Tcfap2a/Tcfap2c double mutant blastocysts can be observed;therefore, a functional redundancy in TE specification betweenthose two molecules can be ruled out as well (39). We favor theinterpretation, that Tcfap2c has a subordinate role to Cdx2 andEomes in specification of the TE.

Our interpretation suggests that Tcfap2c primarily plays arole in the maintenance of the TSC. In support of this hypoth-esis, no permanent Tcfap2c-deficient TSC line (i.e., passage 10and higher) was established. It is known that the expression ofElf5 in the TE creates a positive-feedback loop with Cdx2 andEomes and reinforces the commitment to the trophoblast lin-eage (25). A lack of Elf5 expression renders the TSC labile andprone to differentiation. TSC-like cells generated by the over-expression of Tcfap2c express Elf5, suggesting the induction ofthe TSC maintenance program. Also, as mentioned above, theTcfap2c-induced TSC-like cultures grow for an extended timeas compact colonies that express the TSC marker CDH3, fur-ther strengthening the role of Tcfap2c in TSC maintenance.Interestingly, the overexpression of Cdx2 in Tcfap2c�/� ESCsor the overexpression of Tcfap2c in Cdx2�/� ESCs led to theinduction of TSC fate, but Elf5 was not expressed. The cells failto establish morphologically distinct TSC colonies and initiatedifferentiation into TE derivatives. Therefore, we concludethat both CDX2 and TCFAP2C are required to transactivateElf5, which in turn protects TSCs from differentiation.

This scenario is supported by in vivo data. For example,extraembryonic tissues that are in contact with the Fgf4-pro-ducing ICM of the blastocyst and the epiblast at the egg cyl-inder stage, namely, the polar TE (pTE) and the ExE, maintainTSCs (20, 36). Elf5-deficient embryos implant, but Cdx2 andEomes expression cannot be maintained in the TSC compart-ment of the ExE; therefore, the embryos die (7). Similarly, theExE of Tcfap2c-deficient mice at E7.5 lacks the expression ofCdx2 and Eomes (2). We speculate that the Tcfap2c mutantsmay have depleted their TSC population, resulting in reducedcell numbers in the ectoplacental cone, as well as reducednumbers of giant cells.

On the molecular level, we showed that TCFAP2C transac-tivated the TSC marker Cdx2 and repressed the pluripotencygene Nanog. Hence, Tcfap2c can be integrated in the frame-work of genes regulating TE fate, such as Gata3, Cdx2, Eomes,and Elf5. The concerted action of the repression of Oct3/4 andNanog (6, 28) and the Tcfap2c-mediated repression of Nanogresult in the promotion of TE fate. Both Cdx2 and Tcfap2cactivate each other and are required to establish Elf5 expression.In addition, Tcfap2c and Cdx2 may transactivate an overlap-ping set of target genes to orchestrate TE lineage differentia-tion. In addition, Kidder and Palmer recently applied ChIP-on-chip analysis to TSCs and showed that TCFAP2C binds tothe promoter of Gata3, Tead4, and Elf5, indicating a possiblecross-regulation between the factors responsible for the spec-ification and maintenance of TE fate (19).

Activation of the Ras-MAPK pathway in ESCs inducedTSC-like cells, which resulted in the transcriptional upregula-tion of Tcfap2c and Cdx2 and a reduction in the Nanog proteinlevel (21). We hypothesize that Ras-MAPK-induced repres-sion of Nanog and the upregulation of Cdx2 may be mediatedin part by TCFAP2C. Induction of the Ras-MAPK pathway inTcfap2c-deficient ESCs should shed light on this molecularcascade. In addition to Tead4 (27), Wnt signaling is able totrigger Cdx2 expression and TE lineage differentiation in ESCs(14). Data from Xenopus show that Tcfap2c is also induced byWnt signaling (42); therefore, expression of both Cdx2 andTcfap2c may be initiated in response to Wnt signaling.

Why is Tcfap2c expression not restricted to pTE and ExEbut can be observed in all TE derivatives except the syncy-tiotrophoblast cells during development? This occurrence maybe due to an additional role for Tcfap2c in promoting prolif-eration/endoreduplication during differentiation. Data fromtransgenic mice show that the overexpression of Tcfap2c re-sults in enhanced proliferation in mammary gland epithelialcells (18). We speculate that this role in supporting prolifera-tion may also be required during differentiation of the TElineage. In addition, the formation of the syncytiotrophoblast,which is negative for TCFAP2C, requires exit from the cellcycle, supporting this notion (17). Also, TCFAP2C levels arehigh in human cytotrophoblast cells, but TCFAP2C is down-regulated upon terminal differentiation in vitro (31).

The schematic in Fig. 7 shows our current model. Tead4activates Gata3 (30) and Cdx2 (25), both initiate Eomesexpression, and Cdx2 represses Oct3/4 and Nanog. Oct3/4,Sox2, and Nanog stimulate each other (4), and Nanog andOct3/4 repress Cdx2 (6, 28). Cdx2 and Elf5 induce eachother, leading to the fixation of the TE lineage (25). Tcfap2cand Cdx2 activate each other, and TCFAP2C repressesNanog. Both TCFAP2C and CDX2 are required to establishElf5 expression, and the expression of ELF5 leads to thestabilization of CDX2 levels (25). Together, the expressionof these factors maintains the pool of self-renewing TSCs inthe trophectoderm, the ExE, and the ectoplacental cone(Fig. 7B, green boxes). During induction of differentiationin the chorion, the labyrinth, trophoblast giant cells, and thespongiotrophoblast, the expression of Cdx2 and, thereafter,the expression of Elf5 are lost. In this situation, TCFAP2Csupports proliferation/endoreduplication in the TE deriva-tives (Fig. 7B, yellow boxes). Tcfap2c must be downregu-lated before cell cycle exit can occur, which is required for


syncytiotrophoblast differentiation (Fig. 7B, red box). Elu-cidation of the programs regulated by Tcfap2c in the TElineage will help in deciphering the genes governing TEmaintenance and differentiation.

ACKNOWLEDGMENTS

We are indebted to our colleagues of the Schorle group for helpfuldiscussions and suggestions and to Angela Egert and Andrea Jaegerfor skillful technical assistance. We thank Hitoshi Niwa for plasmidspCAGIP-Cdx2 and pCAGIP-Cdx2ER, Max Gassmann (Institute ofVeterinary Physiology, University of Zurich Zurich, Switzerland) forMG1.19 ESCs, Felix Beck for Cdx2-deficient mice, and Shinya Ya-manaka (Kyoto University, Kyoto, Japan) for the plasmid containingthe Nanog promoter sequence.

This study was supported by DFG grants 503-6 and 503-7 and StemCell Network NRW grants to H.S.

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The Transcription Factor TCFAP2C/AP-2 Cooperates with CDX2