SPECIAL ISSUE REVIEWS–A PEER REVIEWED FORUMCell and Molecular Regulation of the MouseBlastocystYojiro Yamanaka, Amy Ralston, Robert O. Stephenson, and Janet Rossant*Animals use diverse strategies to specify tissue lineages during development. A common strategy is topartition maternally supplied and localized lineage determinants into progenitor cells. The mouse embryoappears to use a different, more regulative strategy to specify the first three lineages: the epiblast (EPI:future embryo), the trophectoderm (TE: future placenta), and the primitive endoderm (PE: future yolk sac).These lineages are specified during two successive differentiation steps leading to formation of theblastocyst. Here, we review classic and contemporary models of early lineage specification in the mouse,and describe recent efforts to understand the molecular regulation of these events. We describe evidencethat trophectoderm differentiation bears resemblance to the process of epithelialization and describe theimportance of apical/basal protein complexes in regulating this process. Next, we present a revised modelof PE specification, and describe evidence that PE cells in the inner cell mass sort out to occupy theirultimate position on the surface of the EPI. Finally, we describe factors that reinforce these lineages andthree distinct stem cell types that can be isolated from them. Together, these mechanisms guide thedifferentiation of the first lineages of the mouse and thereby set up tissues that will be important for the firststeps of embryonic body patterning. Developmental Dynamics 235:2301–2314, 2006.© 2006 Wiley-Liss, Inc.Key words: lineage formation; morula; trophoblast; trophectoderm; primitive endoderm; epiblast; regulative;inside-outside; polarity; polarization; ICM; Nanog; Gata; Cdx2; Par; PKC; preimplantation; embryo; epithelialization;asymmetric divisionAccepted 18 April 2006INTRODUCTIONThe preimplantation mouse embryo ishighly regulative. Cells of the early em-bryo maintain the ability to change fateafter experimental manipulation or cellablation for a considerable period of de-velopment. However, by the late blasto-cyst stage, three distinct tissue lineagesare present: the epiblast (EPI), theprimitive endoderm (PE), and the tro-phectoderm (TE). Only one of these, theEPI will give rise to the fetus, yet propersegregation and development of theother two extraembryonic lineages iscrucial for the survival and even pat-terning of the embryo (reviewed in Bed-dington and Robertson, 1999; Ang andConstam, 2004; Rossant and Tam,2004). Given the importance of thesetissues, understanding the molecularmechanisms establishing these threelineages is key to understanding earlymouse development. This review willdescribe classic and recent studies ofthe mechanisms that allow establish-ment of the first three lineages of themouse: the EPI, PE, and TE.DEVELOPMENTALSTRATEGIES OF EARLYEMBRYOSOocytes of many species have a clearpolarity with distinct animal and veg-etal (A/V) poles. Maternally suppliedfactors, generically termed determi-nants, localize to particular regions ofthe egg, often forming a gradientalong the A/V axis. After fertilization,cell cleavage leads to segregation ofdeterminants such that, after a coupleof cleavages, individual blastomeresare not equivalent and have restricteddevelopmental potential. Ablation ofindividual populations is not rescuedby neighboring cell types. Often thisprocess is accompanied by a stereotyp-ical cleavage pattern, which is also im-portant for reproducible generation ofdifferent kinds of blastomeres. Thus,intrinsic differences in blastomeresProgram of Developmental Biology, Hospital for Sick Children, Toronto, Ontario, Canada*Correspondence to: Janet Rossant, Program of Developmental Biology, Hospital for Sick Children, 555 College Street, Room5079, Toronto, Ontario, M5G1X8, Canada. E-mail: [email protected] 10.1002/dvdy.20844Published online 13 June 2006 in Wiley InterScience (www.interscience.wiley.com).DEVELOPMENTAL DYNAMICS 235:2301–2314, 2006© 2006 Wiley-Liss, Inc.lead to the establishment of the firstcell lineages in the embryo.In mammals, early developmentand lineage specification has beenbest studied in the mouse, which ap-pears to use a different strategy. Themouse oocyte has no clear polarity,and no molecules are yet known thatare both important for lineage specifi-cation and exhibit a polarized distri-bution in the oocyte. Early cleavagepatterns are not stereotypic, and re-markably, all blastomeres retain thepotential to form all cell lineages untilthe eight-cell stage (reviewed in John-son and McConnell, 2004). Moreover,blastomere ablation at this stage doesnot affect the development of a normalfetus (Tarkowski, 1959; Tsunoda andMcLaren, 1983; Zernicka-Goetz,1998; Ciemerych et al., 2000). Theseobservations indicate that earlymouse development is governed by arelatively flexible developmentalprogram. Given this finding, elucida-tion of mechanisms driving early lin-eage specification was initially elu-sive.To understand the cellular originsof the first three lineages, it is im-portant to understand the morpho-logical changes that lead to blasto-cyst formation. During the first twodays of development, the embryo un-dergoes successive cleavage divi-sions to produce an eight-cell embryo(Fig. 1). At this stage, the morulastage, blastomeres increase cell– cellcontact to produce a compactedmorula. Subsequent divisions in-crease the topological complexity ofthe morula; cells can take up a posi-tion on the inside of the late morula,where they are enclosed entirely byother cells. Alternatively, cells canstay in contact with the external en-vironment, and be outside cells. Lin-eage tracing experiments suggestedthat the TE is derived mainly fromoutside cells, whereas inside cellscontribute to the inner cell mass(ICM; Fleming, 1987). Later, theICM will segregate into EPI and PE.Classical models of these processesrecently have been complemented bymolecular strategies, presenting amore complete view of the mecha-nisms by which these lineages arespecified and maintained.DEVELOPMENT OF THE TEClassical Models of TEFormationThe TE is the first tissue type to bespecified during mouse developmentand is morphologically distinct fromthe ICM by the early blastocyst stage(Fig. 1). The TE, which will form thefetal portions of the placenta, beginsas an epithelial sheet enclosing thefuture embryo, the ICM. Molecularly,TE and ICM lineages can be