Cell Lineage Determination in the Mouse

* Catherine M. Watson and Patrick P.L. Tam Embryology Unit, Children’s Medical Research Institute, Locked Bag 23, Wentworthville, New South Wales, 2145, Australia

ABSTRACT. During the peri-implantation development of the mouse embryo from the blastocyst through gastrulation, Pou5f1 (OCT-4) down-regulation is closely linked to the initial step of lineage allocation to extraembryonic and embryonic somatic tissues. Subsequently, differentiation of the lineage precursors is subject to inductive tissue interactions and intercellular signalling that regulate cell proliferation and the acquisition of lineage-specific morphological and molecular characteristics. A notable variation of this process of lineage specification is the persistence of Pou5f1 activity throughout the differentiation of the primordial germ cells, which may underpin their ability to produce pluripotent progeny either as stem cells (embryonic germ cells) in vitro or as gametes in vivo. Nevertheless, intercellular signalling still plays a critical role in the specification of the primordial germ cells. The findings that primordial germ cells can be induced from any epiblast cells and that they share common progenitors with other somatic cells provide compelling evidence for the absence of a pre-determined germ line in the mouse embryo.

Key words: primordial germ cell/cardiac mesoderm/trophoblast/OCT-4

During embryonic development, progenitor cells that are Fully committed, or determined, cells can no longer be per- initially pluripotent become restricted in their potency as the suaded experimentally to change their fate. options for further differentiation are progressively limited. OCT-4 is a POU domain transcription factor encoded by the Pou5f1 gene and is expressed in the blastomeres of the cleavage stage pre-implantation embryo. Pou5f1 activity is * To whom correspondence should be addressed: Embryology Unit, maintained in the cells of the inner cell mass (ICM) of the Children’s Medical Research Institute, Locked Bag 23, Wentworthville, New South Wales, 2145, Australia. blastocyst, the epiblast of the peri-implantation embryo and Fax: +61-2-9687-2120 in the primordial germ cells (PGC) of the early organogene- E-mail: [email protected]; [email protected] sis embryo. The inner cell mass and the epiblast have been Abbreviations: Akp2, alkaline phosphatase 2, liver (TNAP tissue non- specific alkaline phosphatase); Ascl2, (Mash2) achaete-scute complex ho- shown by lineage analysis to be the sole source of progeni- molog-like (Drosophila); BMP, bone morphogenetic protein; bHLH, basic tors of all types of somatic tissues in the fetal body as well helix-loop-helix; Cdh1, E-cadherin; Cdx2, caudal type homeo box 2; Dkk1, as the PGCs (Gardner and Beddington, 1988). dickkopf homolog 1 (Xenopus laevis); eed, embryonic ectoderm develop- Female primordial germ cells give rise to oocytes, which ment; E, embryonic day; Eomes, eomesodermin homolog (Xenopus laevis); EPL, primitive ectoderm-like; Esrrb, estrogen related receptor, display the full potency to form all types of tissues of the re- beta; ES, embryonic stem; Esx1, extraembryonic, spermatogenesis, sultant conceptus upon fertilization by the sperm derived homeobox 1; FGF, fibroblast growth factor and Fgfr2 fibroblast growth from the male primordial germ cells, or when activated par- factor receptor 2; Flt, FMS-like tyrosine kinase; FOG, Friend of GATA; Foxa2, forkhead box A2; GATA, GATA-binding protein; Hand, heart and thenogenetically. Furthermore, pluripotent stem cells which neural crest derivatives expressed transcript; ICM, inner cell mass; Kit,(C- can be derived in vitro from the ICM [as embryonic stem Kit) kit oncogene; LIF, leukemia inhibiting factor; Lhx1, LIM homeobox (ES) cells] or the PGCs [as embryonic germ (EG) cells], can protein 1; MADS, MCM1, agamous, deficiens, serum response factor; contribute to every type of somatic cell and PGCs of the Mef2, myocyte enhancer factor; Mesp1, mesoderm posterior 1; Mesp2, mesoderm posterior 2; MyoD, myogenic differentiation; Nkx2-5,NK2 chimaeric embryo (see Tada and Tada this issue). transcription factor related, locus 5 (Drosophila); OCT-4, octamer-4 prod- These properties of the Pou5f1 (OCT-4) expressing cells uct of POU domain, class 5, transcription factor 1, Pou5f1 gene; Pem, are consistent with the notion that the activity of this tran- placentae and embryos oncofetal gene; PGC, primordial germ cell; Pl1, scription factor is linked to the maintenance of lineage placental lactogen 1; POU, Pit-Oct-Unc; Traf3, Tnf receptor-associated factor 3 (amn); Tbn, taube nuss; TS, trophoblast stem; Wnt, wingless- pluripotency. Of specific interest is that Pou5f1 activity is related MMTV integration site. always down-regulated in ES and EG cells upon in vitro dif-

123 C.M. Watson and P.P.L. Tam ferentiation and also in the somatic descendants of the inner trophoblast giant cells and the extraembryonic ectoderm cell mass and the epiblast (Pesce and Schöler, 2000). This (Rossant, 1986). The proliferation and differentiation of the raises the possibility that suppression of Pou5f1activity may polar trophectoderm have been shown to be dependent on be essential for the onset of lineage specification and cell the inductive interaction with the ICM and on the signalling differentiation (Yeom et al., 1996). Concordant with this activity of FGF4, a fibroblast growth factor protein that is prediction is the finding that absent or low level OCT-4 ex- expressedinICMderivatives. pression in ES cells biases the differentiation of the ES cells In embryos that lack FGF4 activity, the ICM degenerates, to trophectoderm (Nichols et al., 1998) which seems to re- and the trophectoderm ceases proliferation and undergoes capitulate the lack of Pou5f1 activity in the trophectoderm giant cell transformation (Feldman et al., 1994). FGF4 and of the blastocyst. OCT-4, in partnership with LIF, may act Tbn (Taube nuss) which are required for survival of the as a repressor of the activity of genes associated with ICM (Voss et al., 2000), may indirectly influence the differ- trophectoderm differentiation and an activator of other entiation of the polar trophectoderm. Actively proliferating genes that maintain pluripotency (Niwa et al., 2000). Con- cells that adopt a stable epithelial morphology have been versely, an elevation of OCT-4 expression triggers ES cell isolated from the trophectoderm in vitro inthepresenceof differentiation not to trophectoderm but instead to visceral high levels of FGFs (Tanaka et al., 1998). Removal of endoderm and mesoderm (Niwa et al., 2000). Modulation of FGF4 from the culture medium or the omission of primary OCT-4 expression in pluripotent embryonic cells therefore mouse embryonic fibroblast feeder cells from the TS culture plays a crucial role in directing their descendants along leads to differentiation of the epitheloid TS cells into specific paths of differentiation and represents the first step trophoblast giant cells (Tanaka et al., 1998). of lineage specification. FGF is also essential for trophoblast differentiation in ad- ES cells have been shown to remain pluripotent under in dition to its effect on cell proliferation (Tanaka et al., 1998; vitro culture conditions that promote the acquisition of an Rassoulazadegan et al., 2000). In the intact blastocyst, epithelial morphology (EPL cells) (Rathjen et al., 1999). If phagocytic activity is highest in the mural trophectodermal the transition from ES to EPL is equivalent to the develop- cells that are furthest from the ICM. Mural trophectoderm ment of ICM to epiblast in the peri-implantation embryo, cells continue to display characteristic phagocytic activity this may suggest that the ICM derivatives will remain pluri- when they are cultured in vitro. However, addition of FGF4 potent until the onset of gastrulation. Major decisions about and serum derived factors or co-culturing with ES cells in- lineage allocation and cell fate are made during the forma- hibits the phagocytic activity of the mural trophectoderm, tion of the germ layers during gastrulation, between E6.25– and they behave more like the polar trophectodermal cells 7.5. As cells pass through the primitive streak they are allo- that are presumably subject to a stronger influence of FGF cated to one of two germ layers, endoderm or mesoderm. in the embryo (Tanaka et al., 1998; Rassoulazadegan et al., The potency of the cells of each layer becomes restricted 2000). after passage through the streak (Davidson et al., 1999). Amongst the various cell lineages that are established in the Activity of regulatory genes is indicative of trophoblast post-gastrulation stage embryos, the PGCs are the only cells differentiation that retain OCT-4 expression and continue to express the During the differentiation of the extraembryonic transcription factor at a low level in fully grown oocytes ectoderm to trophoblastic giant cells, Fgfr2, Esrrb and Cdx2 (Pesce and Schöler, 2000). activities are down-regulated whereas Pl1 expression is In this review, we will examine the specific factors in- elevated (Table I) (Tanaka et al., 1998). Complete loss of volved in determination of cell lineages in mouse embryos Cdx2 activity results in the lack of trophoblast differentia- after their initial specification. We focus on the trophecto- tion and the mutant embryo fails to implant in the uterus derm, which is the first cell type that is allocated from (Beck et al., 1999). The expression of Eomes is critical for the OCT-4 expressing progenitor cell pool, the cardiac the differentiation of the extraembryonic ectoderm into mesoderm which is allocated from the OCT-4 expressing giant trophoblasts (Ciruna and Rossant, 1999; Russ et al., pluripotent epiblast during gastrulation and the PGC lineage 2000) and derivation of TS cells. Eomes does not require that continues to express OCT-4 during development. FGF4 in vitro to instigate differentiation into giant cells however FGF4 may be required to maintain Eomes expres- The Trophoblast Lineage sion (Russ et al., 2000). Two X-linked genes, Pem and Esx1 as well as basic Trophoblast differentiation occurs in response to FGF4 helix-loop-helix (bHLH) transcription factors such as Ascl2 signals from the ICM derivatives and Hand1 are expressed in trophectodermal tissues. Pem is expressed in extraembryonic ectoderm, ectoplacental cone, Trophectoderm forms the outer epithelial cell layer of the giant trophoblasts and chorionic ectoderm (Lin et al., 1994). blastocyst. Cells of the polar trophectoderm which overlie Esx1 is expressed in the chorionic ectoderm and the labyrin- the ICM differentiate to become the ectoplacental cone, the

124 Cell Lineages in the Mouse thine trophoblast (Li et al., 1997). In heterozygous Esx1+/– tained on a feeder layer and in clumps of a critical density. mouse embryo, placental development is unaffected when This strongly suggests that community effects, which may the mutant allele is paternally inherited due to preferential be mediated by cell-cell interactions, may influence PGC inactivation of the paternally inherited X chromosome in specification at this stage. In contrast, epiblast cells from extraembryonic tissues. However, when heterozygous gastrulating embryos (at E6.5–E6.75) can give rise to PGCs Esx1+/– embryos carry the mutant allele on the maternal even when they are cultured as dissociated cells without X-chromosome, development of the labyrinthine and further feeder support, indicating that cell-cell interaction is spongiotrophoblasts becomes abnormal (Li and Behringer, no longer required and the PGCs can differentiate autono- 1998). Pem–/– mouse embryos show no discernible pheno- mously (Yoshimizu et al., 2001). typic abnormality in placental tissues (Pitman et al., 1998). The functional redundancy may be due to Esx1 and Pem BMP signalling plays a key role in PGC specification having overlapping functions. Hand1 is involved in differ- BMP signalling emanating from the extraembryonic entiation of trophoblast cells into giant cells (Cserjesi et al., ectoderm is one of the first molecular cues known to be 1995; Fan et al., 1999), and Ascl2 is implicated in specifica- involved in PGC specification. Bmp4lacZneo and Bmp8b null tion of diploid spongiotrophoblast cells and is critical for homozygous embryos lacked PGCs and heterozygous normal placental development (Guillemot et al., 1994). embryos had a reduced number of PGCs (Lawson et al., In summary, descendants of blastomeres that are allocat- 1999; Ying et al., 2000), suggesting that BMPs act in a dose ed to the trophectodermal lineage down-regulate Pou5f1 dependant manner to specify epiblast cells for the PGC and then respond positively to the FGF-mediated mitogenic lineage. The decrease in PGC number was due to a reduc- activity emanating from the ICM derivatives. The differen- tion in the founding population and not the lack of cell pro- tiation of the trophectoderm, however, is clearly subject to liferation. Bmp2, expressed in the visceral endoderm, and non-autonomous interaction with other embryonic cell Bmp4, expressed in the extraembryonic ectoderm, have an types. Differentiation of the trophectoderm to giant tropho- additive effect because embryos heterozygous for null blast cells takes place when the level of FGF signalling di- mutations in both Bmp2 and Bmp4 had fewer PGCs than minishes due to the increase in the distance from the source embryos that were heterozygous for either Bmp2 or Bmp4 of signalling in the ICM derivatives as the extraembryonic (Ying and Zhao, 2001). BMP activity alone, however, is not ectoderm grows in size. The molecular mechanism of the sufficient to induce or maintain the PGC population. In specification of trophectodermal progenitors is not known embryos that lack Lhx1 and Foxa2 activity, PGCs are either but is coupled with the tissue-specific regulation of OCT-4 absent or severely reduced in number despite the presence expression. of Bmp4 activity (Tsang et al., 2001).

The Primordial Germ Cells Lineage-specific factors involved in germ cell differentiation PGC progenitors are specified in response to localized PGCs are the only cells to continue expressing OCT-4 signals after gastrulation. Pou5f1 activity in PGCs may be essential Clonal lineage analysis has shown that only proximal epi- for the appropriate regulation of genes necessary to preserve blast cells contribute to the PGC pool. Lineage restriction pluripotency and to prohibit differentiation of PGCs into so- has not occurred at this time because any given clone can matic cells (Table I) (Pesce and Schöler, 2000). Germ cell contribute descendants simultaneously to the PGC pool and DNA is hypomethylated relative to somatic cells and specif- the allantois and extraembryonic mesoderm (Lawson and ic methylation of imprinted alleles is erased (Razin and Hage, 1994; McLaren, 2000). Cells from the distal epiblast Shemer, 1995). In E12.5 PGCs, tissue-specific and imprint- (that are fated to form neuroectoderm) when transferred to ed genes are unmethylated and de novo methylation of these the proximal epiblast amongst the PGC precursors could genes is first observed at E15.5 (Razin and Shemer, 1995). contribute to germ cell lineage. Therefore, any cells within Enucleated mouse oocytes into which male E14.5–16.5 the epiblast can become PGCs if they are at the appropriate PGC nuclei were transplanted formed small embryos with site for specification (Tam and Zhou, 1996). This supports abnormal placentae and died at E9.5. These embryos could the concept that no pre-determined and exclusive PGC not be rescued by transfer of the inner cell mass of the nu- progenitors exist in the mouse epiblast. clear-transfer cloned embryo to a tetraploid blastocyst host, In an in vitro study, PGCs developed only from E5.5 indicating that the abnormal placenta was not the only cause (pre-gastrulation stage) epiblast that was co-cultured with of embryonic lethality. Molecular and expression analyses extraembryonic ectoderm, demonstrating that interaction of imprinted genes suggest that the absence of gametic im- between tissues is required for PGC specification (Yoshimi- prints may underpin the reduced developmental potency zu et al., 2001). At E6.0 dissociated proximal epiblast cells (Kato et al., 1999). will differentiate to form PGCs only when they are main- Akp2 (TNAP), which is expressed in migrating and early

125 C.M. Watson and P.P.L. Tam

Table I. A SUMMARY OF THE LOCATION AND MOLECULAR PROPERTIES OF THE PROGENITORS AND DESCENDANTS OF THE TROPHOBLAST, PRIMORDIAL GERM CELLS AND HEART MESODERM LINEAGES IN THE MOUSE EMBRYO

Differentiation Progenitor Specification or Commitment Trophoblast1 Timing E2.5 E3.5 E6.5–8.5 Location Trophectoderm- Polar trophectoderm associ- Extraembryonic ectoderm, outside cells of ated with the inner cell ectoplacental cone, chorion, blastocytst mass trophoblast giant cells Gene expression Down-regulation of Pem, Eomes, Fgf4, Fgfr2, Hand1, Eomes, Esx1, Ascl2, Pl1 Pou5f1 Esrrb, Cdx2, Pl1

Primordial Germ Cells2 Timing E5.5 E6.5 E7.0-8.5 Location Proximal epiblast Junction between visceral Ventral posterior part of the and definitive endoderm, primitive streak, the endoderm induced by extraembryonic of the hindgut and the adjacent ectoderm splanchnic mesoderm Gene expression Pou5f1, RespondtoBMPsignals, Akp2, Kit, Pem, Lhx1, Foxa2, hypomethylated retain hypomethylation and Pou5f1 genome Pou5f1 activity

Heart mesoderm3 Timing E6.5 E7.0–7.5 E7.75–8.0 Location Posterior lateral Progenitors in expanding Cardiogenic mesoderm in the epiblast mesodermal layer anterior region of the embryo Gene expression Down-regulation of Mesp1, Mesp2, Gata4 & 6, Gata4 and 6, Hand1, Hand2, Pou5f1 Nkx2-5, Hand1 & 2, BMP Mef2a, c and d activity 1 Feldman et al., 1994; Yeom et al., 1996; Tanaka et al., 1998 2 Pesce et al., 1998, Pitman et al., 1998; Anderson et al., 2000, Tsang et al., 2001 3 Srivastava et al., 1997; Tam et al., 1997; Charron, and Nemer 1999; Kitajima et al., 2000; Kinder et al., 2001 post-migratory PGCs, is not essential for germ cell specifi- heart mesoderm can first be delineated in the posterior later- cation despite its PGC-specific activity (MacGregor et al., al region of the OCT-4 expressing epiblast of the mouse 1995). Although forced expression of Pem in ES cells embryo at early gastrulation. These epiblast cells ingress can inhibit cell differentiation and points to a role in main- through the primitive streak and join the mesoderm layer. taining pluripotency in the PGCs (Fan et al., 1999), Pem As the heart mesoderm precursors migrate to a cranial desti- activity alone is not required for PGC differentiation in vivo nation in the embryo, they begin to express genes such (Pitman et al., 1998). as Gata4, Flt1 (endothelium) and Nkx2-5 (myocardium) As the epiblast cells destined to become PGCs become (Table I) (Tam and Schoenwolf, 1999; Kinder et al., 2001). committed to the germ cell lineage they acquire the ability to differentiate oblivious to the tissue interactions and tissue Genes involved in determination of heart mesoderm specific signals presented to them. They respond to some as Mesp1 and Mesp2 are transcription factors containing yet unidentified homing signals from the precursor of the identical bHLH motifs (Kitajima et al., 2000). Deletion of genital ridge. PGCs have been shown to preferentially Mesp1 but not Mesp2 results in abnormal heart develop- migrate to genital ridges in culture in preference to other ment due to delayed mesoderm migration. Embryos that tissues (Godin et al., 1990). lack both Mesp1 and Mesp2 activity fail to form any mesoderm because of the abnormal movement of the mesoderm The Heart Mesoderm progenitors in the primitive streak. This is reminiscent of the mesodermal defects of the eed and Traf3 mutant embry- Heart progenitors follow the morphogenetic movement os (Faust et al., 1995; Tomihara-Newberger, et al., 1998). of gastrulation Compound Mesp1–/–;Mesp2–/– mutant cells are excluded from the hearts of chimaeric embryos, suggesting the Mesp Fate mapping studies have shown that progenitors of the

126 Cell Lineages in the Mouse has a cell-autonomous function in cardiac cell differentiation (Kitajima et al., 2000). Conclusion: Events of Lineage Restriction Induction of myocardial differentiation has been shown Modulation of the differentiation pathway by OCT-4 to require positive BMP signalling and the suppression of WNT activity in the cardiogenic mesoderm of the embryo. Pou5f1 is an important gene in the regulation of initial Bmp2 is expressed in mesoderm in the region which gives cell potency restriction and lineage allocation and continua- rise to the myocardium. Deletion of Bmp2 results in embry- tion of the cycle of totipotency (Yeom et al., 1996). Pou5f1 os with disorganized cardiac tissues, delayed myocardial expression is down-regulated at the onset of trophoblast differentiation, or formation of an ectopic heart in the exo- lineage allocation, mesoderm allocation and the control of coelom (Zhang and Bradley, 1996). Expression of Wnt3a its regulation is switched to a PGC specific enhancer as the and Wnt8 in Xenopus and Wnt3a and Wnt8c in the chick PGC lineage is specified to ensure pluripotency in the block the expression of heart-specific genes, while con- mature oocyte. Loss of Pou5f1 expression results in ICM versely, the activity of Wnt antagonists (Dkk1 and Crescent) cells that have been subverted to a trophoblast differentia- initiate myocardial differentiation. Similarly, inhibition of tion pathway. Failure to develop an ICM means that polar Wnt gene activity permits myocardial induction by BMP trophectoderm stem cells do not proliferate due to lack of signals in frog and chicken explants (Marvin et al., 2001; FGF4 signal from the ICM and the embryos die (Nichols et Schneider and Mercola, 2001; Tzahor and Lassar, 2001). It al., 1998). In cattle and pigs OCT-4 protein is detected in is not known if the modulation of BMP and WNT activity the ICM and trophectoderm indicating that its regulation in mayplayasimilarroleinmyocardialdeterminationinthe these species differs somewhat from the mouse, as do some mouse embryo, but cells that are not fated for heart meso- aspects of their pre-implantation development (Kirchhof derm can be induced to differentiate into myocardium only et al., 2000). Therefore OCT-4 plays a critical role in when they have reached the cardiogenic sites in the anterior maintaining the ICM lineage in mice, which in turn is region of the embryo (Tam and Schoenwolf, 1999). necessary for the differentiation of the trophoblast.

Commitment to myocardial differentiation Inductive versus autonomous lineage specification GATA transcription factors, which are zinc finger DNA Inductive interaction plays a key role in the specification binding proteins, are important in cardiac development. of the somatic and germ cell lineages. Trophoblast progeni- Gata-6 is expressed in cardiac precursors and subsequently tor cells are maintained in response to FGF4 signals re- in myocardial cells (Charron and Nemer, 1999). Gata4 is ceived from the ICM in much the same way as epiblast cells expressed in the precursors of the cardiac mesoderm ahead are primed to form PGCs in response to BMP signals from of other cardiac specific transcription factors and interacts the extraembryonic ectoderm and the myocardium differen- with FOG (Friend of GATA) proteins and other nuclear tiate in response to BMP and suppressed WNT signals. proteins such as NKX2-5 (Crispino et al., 2001). No lineage-specific molecular determinants have yet The expression of Gata4, and Gata6 in pre-cardiogenic been identified for the three cell lineages considered in this mesoderm overlaps with that of Nkx2-5 and Mef2a,c,andd review. Common to the somatic lineages is the down- (Myocyte enhancer factor) (Edmondson et al., 1994; Srivas- regulation of OCT-4 expression at the initial stage of tava et al., 1997). Nkx2-5 expression coincides with mor- lineage specification, which releases the cells from the phological changes at the visible onset of commitment constraint of pluripotency. Removal of OCT-4, except in when cells change from mesenchyme to cuboidal epithelial PGCs, followed by signalling from nearby tissues leads to cells (Harvey, 1996). Ablation of Nkx2-5 causes failure of initiation of tissue specific gene expression programs. In the heart tube looping but has no impact on the formation of trophoblast lineage, Eomes has been demonstrated to serve myocardium (Tanaka et al., 1999). Myocyte enhancer factor a critical role in trophoblast differentiation and maintenance 2 (Mef) transcription factors are expressed in cardiac pre- of trophoblast stem cells whereas Hand1 is involved in cursors and differentiated cardiomyocytes and interact with giant cell differentiation and Ascl2 in spongiotrophoblast bHLH factors and contain a MADS box (MCM1, agamous, development. In the heart lineage, the morphogenetic deficiens, serum response factor). Mef2c is expressed first at experience of the precursors during gastrulation has little E7.75followedbyMef2a and Mef2d 12 hours later (Lin et impact on the commitment to myocardial differentiation. al., 1997). Mef2c deficient embryos did not survive past The most crucial factor is the interplay between WNT E10.5 and exhibited reduced heart muscle contractility. antagonists and BMP signalling. Once specified, a suite of Mef2c deficient mice down-regulated cardiac muscle genes are expressed to ensure the correct differentiation and specific genes by 4-5 fold and up-regulated Mef2b at E9.5. patterning of the developing heart. BMP signalling is Ablation of Mef2c has revealed its critical role in regulating crucial but not sufficient for the specification of the gene expression during cardiac myogenesis (Lin et al., PGCs. The maintenance of OCT-4 expression does not 1997). seem to be incompatible with the acquisition of lineage-

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