Early Lineage Segregation between Epiblast and Primitive Endoderm in Mouse Blastocysts through the Grb2-MAPK PathwaySummaryIntroductionResults"Salt and Pepper" Expression of Nanog and Gata6 in the ICM of E3.5 BlastocystsLabeled Single ICM Cells in Early Blastocysts Give Rise to Either EPI or PE at E5.5Lineage Segregation Is Not Based on Either Blastomere History or Position in the E3.5 ICMReceptor Tyrosine Kinase Signaling Is Required for Segregation of Lineages in the ICMDiscussionExperimental ProceduresMicroinjection of mRNA into Single Cells of Preimplantation EmbryosImmunosurgery and Dissociation of ICMLipophilic Dye LabelingWhole-Mount ImmunostainingFluorescent Whole-Mount In Situ HybridizationConfocal MicroscopyGenotyping of BlastocystsSupplemental DataAcknowledgmentsReferencesDevelopmental Cell 10, 615–624, May, 2006 ª2006 Elsevier Inc. DOI 10.1016/j.devcel.2006.02.020Early Lineage Segregation between Epiblastand Primitive Endoderm in Mouse Blastocyststhrough the Grb2-MAPK PathwayClaire Chazaud,1,2,5Yojiro Yamanaka,1,3,5Tony Pawson,1and Janet Rossant1,3,4,*1Samuel Lunenfeld Research InstituteMount Sinai HospitalToronto, Ontario M5G1X5Canada2Inserm, U384University AuvergneClermont-Fd, F63000France3Program of Developmental BiologyHospital for Sick ChildrenToronto, Ontario M5G1L7CanadaSummaryIt has been thought that early inner cell mass (ICM) isa homogeneous population and that cell position inthe ICM leads to the formation of two lineages, epiblast(EPI) and primitive endoderm (PE), by E4.5. Here, how-ever, we show that the ICM at E3.5 is already heteroge-neous. The EPI- and PE-specific transcription factors,Nanog and Gata6, were expressed in the ICM in a ran-dom ‘‘salt and pepper’’ pattern, as early as E3.5, ina mutually exclusive manner. Lineage tracing showedpredominant lineage restriction of single ICM cells atE3.5 to either lineage. In embryos lacking Grb2 whereno PE forms, Gata6 expression was lost and all ICMcells were Nanog positive. We propose a model inwhich the ICM develops as a mosaic of EPI and PE pro-genitors at E3.5, dependent on Grb2-Ras-MAP kinasesignaling, followed by later segregation of the progen-itors into the appropriate cell layers.IntroductionBy the time of implantation, three distinct cell types areapparent in the mouse blastocyst which give rise to sep-arate cell lineages in later development. Epiblast (EPI)gives rise to the entire fetus as well as extraembryonicmesoderm. Trophectoderm (TE) gives rise to all the tro-phoblast cell types that make up the majority of the fetalpart of the placenta, and the primitive endoderm (PE)forms the extraembryonic endoderm layers of the vis-ceral and parietal yolk sacs. The TE and PE extraembry-onic lineages are required to support the growth of themammalian fetus in the uterine environment and aresources of signals to the EPI to initiate axial patterning(Ang and Constam, 2004; Beddington and Robertson,1998; Lu et al., 2001; Rossant and Tam, 2004).By the late blastocyst, experimental chimera studieshave indicated that the three lineages are restricted totheir future fate (Gardner, 1985; Rossant et al., 2003)but the mechanisms leading to the initial segregationof the three lineages are still not well understood. Atthe eight-cell stage, all blastomeres retain the potentialto form all cell lineages (Johnson and McConnell,2004). Following compaction, the blastomeres becomepolarized and subsequent cell divisions generate out-side polar cells and inside apolar cells, dependentupon the symmetric or asymmetric cell divisions of thepolarized cells (Johnson et al., 1986). Polar cells remainoutside and will form the TE, while apolar cells becomeenclosed by the polarized outer epithelium and form theinner cell mass (ICM). This segregation is accompaniedby segregated expression of key transcription factors(TFs) required to establish cell fate, such as Oct4 (Nich-ols et al., 1998) and Cdx2 (Chawengsaksophak et al.,1997; Strumpf et al., 2005) for ICM and TE, respectively.Correct segregation of the two lineages is also depen-dent upon cell adhesion, involving E-cadherin (De Vrieset al., 2004; Riethmacher et al., 1995), and the epithelialpolarity pathway, involving genes such as Par3 andaPKC (Plusa et al., 2005).Much less is known about the segregation of EPI andPE within the ICM. By E4.5, the PE layer is clearly mor-phologically distinct. When E4.5 EPI or PE cells weredissociated and injected into other blastocysts, theirchimeric contributions were restricted to their own line-ages (Gardner, 1982, 1984; Gardner and Rossant, 1979),indicating lineage restriction by this stage. Isolated E3.5ICMs form PE over their exterior surface (Hogan andTilly, 1978; Rossant, 1975), suggesting that EPI/PE seg-regation might also depend on some positional effects,with cells located in the inside of ICM becoming EPI andthe cells lining the blastocoel becoming PE.Recent genetic evidence has implicated Nanog andGata family transcriptional factors in specifying EPI ver-sus PE fate. Nanog, a homeodomain protein, was iden-tified as a gene highly expressed in ES cells (Mitsui et al.,2003), which could substitute for LIF to promote self-re-newal of ES cells (Chambers et al., 2003). Disruption ofthis gene leads to failure to maintain pluripotency in EScells in vitro and peri-implantation lethality with failureof EPI formation in vivo (Mitsui et al., 2003). In both EScells and ICMs from null mutants, loss of Nanog resultsin all cells taking up a parietal endoderm-like fate (Mitsuiet al., 2003). This suggests that levels of Nanog expres-sion are important for the EPI/PE fate decision. In a com-plementary manner, overexpression of Gata6 or a closefamily member Gata4 is sufficient to transform ES cellsto PE lineages (Fujikura et al., 2002) and ES cells lackingGata6 or Gata4 cannot form a functional visceral endo-derm layer in in vitro embryoid body cultures (Koutsour-akis et al., 1999; Morrisey et al., 1998; Soudais et al.,1995). In vivo, Gata4 and Gata6 mutants initiate PE line-age formation, but fail to form functional visceral endo-derm in postimplantation embryos (Koutsourakis et al.,1999; Morrisey et al., 1998; Soudais et al., 1995). Gata4and Gata6 may be redundantly required for PE formationin vivo.In this study, we have carefully examined the expres-sion of Nanog and Gata6 in early development and have*Correspondence: [email protected] address: http://www.sickkids.ca/rossant/5These authors contributed equally to this work.shown