Interaction between Oct3/4 and Cdx2 Determines Trophectoderm DifferentiationIntroductionResultsOverexpression of Cdx2 in ES Cells Induces Trophectoderm DifferentiationInducible Activation of Cdx2 in ES Cells Triggers Proper Differentiation to the TE LineagePlacental Contribution of ES-Derived TS Cells Generated by Activation of Cdx2ERReciprocal Inhibition of Transcriptional Activities between Cdx2 and Oct3/4Cdx2 and Oct3/4 Form a Repressor ComplexCdx2 Function Is Dispensable for TE DifferentiationCdx2 Is Required for Self-Renewal of TS CellsEomeso Induces TE Differentiation in the Absence of Cdx2Maintenance of Cdx2 May Work as a Trigger in the Early EmbryoDiscussionExperimental ProceduresCell Culture and TransfectionProduction of Chimeric EmbryosLuciferase AssayRNA Preparation and Real-Time PCR AnalysesFACS AnalysisImmunoprecipitation and ChIPImmunostaining and FRET AnalysisSupplemental DataAcknowledgmentsReferencesInteraction between Oct3/4 and Cdx2Determines TrophectodermDifferentiationHitoshi Niwa,1,2,3,*Yayoi Toyooka,1Daisuke Shimosato,1,2Dan Strumpf,4Kadue Takahashi,1Rika Yagi,1and Janet Rossant41Laboratory for Pluripotent Cell Studies, RIKEN Center for Developmental Biology (CDB), 2-2-3 Minatojima-minamimachi, Chu-o-ku,Kobe, Hyogo 650-0047, Japan2Laboratory for Development and Regenerative Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunokicho, Chu-o-ku,Kobe, Hyogo 650-0017, Japan3CREST (Core Research for Evolutional Science and Technology), Japan Science and Technology Agency, Honcho 4-1-8, Kawaguchi,Saitama 332-0012, Japan4Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada*Contact: [email protected] 10.1016/j.cell.2005.08.040SUMMARYTrophectoderm (TE), the first differentiatedcell lineage of mammalian embryogenesis,forms the placenta, a structure unique tomammalian development. The differentiationof TE is a hallmark event in early mammaliandevelopment, but molecular mechanismsunderlying this first differentiation eventremain obscure. Embryonic stem (ES) cellscan be induced to differentiate into the TElineage by forced repression of the POU-family transcription factor, Oct3/4. We showhere that this event can be mimicked byoverexpression of Caudal-related homeo-box 2 (Cdx2), which is sufficient to generateproper trophoblast stem (TS) cells. Cdx2is dispensable for trophectoderm differenti-ation induced by Oct3/4 repression butessential for TS cell self-renewal. In preim-plantation embryos, Cdx2 is initially co-expressed with Oct3/4 and they form a com-plex for the reciprocal repression of theirtarget genes in ES cells. This suggeststhat reciprocal inhibition between lineage-specific transcription factors might be in-volved in the first differentiation event ofmammalian development.INTRODUCTIONTrophectoderm (TE) is the first differentiated cell lineage toarise in mammalian embryogenesis. Mouse zygotes cleavethree times to generate 8-cell stage embryos. After the thirdcleavage, the blastomeres undergo a morphological changeknown as compaction. The next round of cell divisions tendsto occur along the apical-basal axis of the blastomeres, re-sulting in the formation of a 16-cell morula consisting of smallinner cells enclosed within larger outer cells. Most of theouter cells are then epithelialized and become TE, whereasthe inner cells go on to generate the inner cell mass (ICM)in blastocysts (Fleming, 1987). Cell-fate analyses revealedthat the ICM gives rise to all of the embryonic cells and theextraembryonic endoderm, whereas TE forms the embry-onic portion of the placenta, a structure unique to mamma-lian development (Pedersen et al., 1986; Fleming, 1987).Therefore, the differentiation of TE can be regarded as a hall-mark event in mammalian early development.We previously reported that the pluripotent embryonicstem (ES) cells derived from the ICM can be induced to un-dergo differentiation toward the TE lineage by forced repres-sion of a POU-family transcription factor, Oct3/4, whereas itsoverexpression induces differentiation mainly to extraembry-onic endoderm (Niwa et al., 2000). It had previously beensuggested that mouse ES cells possess limited ability toform TE and extraembryonic endoderm because they con-tribute to these lineages at low frequencies when they are in-jected into blastocysts to generate chimeric embryos (Bed-dington and Robertson, 1989); but our study conclusivelydemonstrated that ES cells can be caused to differentiateinto these extraembryonic lineages by controlling the func-tion of genes involved in these differentiation events.It was recently shown that Caudal-related homeobox 2(Cdx2) is involved in TE formation at the blastocyst stage inmice. The expression of Cdx2 in the pre- and early post-implantation embryos is tightly restricted in the TE lineage,especially in its proliferating population (Beck et al., 1995).Detailed analysis of mutant embryos lacking zygotic Cdx2showed that they do form blastocyst-like structures withTE-like cells (Strumpf et al., 2005). However, these mutantblastocysts never implant due to the abnormality of theseCell 123, 917–929, December 2, 2005 ª2005 Elsevier Inc. 917TE-like cells, which ectopically express genes characteristicof pluripotent stem cells, such as Oct3/4 and Nanog, butlack the expression of TE marker genes. These data clearlyindicated the importance of Cdx2 in TE, but it has yet to berevealed whether the function of this gene is essential for ini-tiating the differentiation of TE or for its functional maturation.In this report, we used an in vitro system of the mouse EScells for modeling the differentiation to TE to determine thefunctions of Cdx2 in TE differentiation and maintenance;we found that the activation of Cdx2 is sufficient to inducedifferentiation toward the TE lineage. We also show a pivotalrole for the interaction between Oct3/4 and Cdx2 in both theestablishment and maintenance of the TE lineage.RESULTSOverexpression of Cdx2 in ES Cells InducesTrophectoderm DifferentiationUsing an episomal expression vector system, we found thatoverexpression of Cdx2 directed morphological differentia-tion to TE similar to that induced by Oct3/4 repression (Fig-ure 1A) but distinct from that induced by Oct3/4 (Figure 1E)or Gata6 overexpression (Figure 1F; Fujikura et al., 2002).The efficiencies of induction of differentiation were compara-ble in these three genes (Figure 1J), indicating that theseevents were not an anomalous