Lineage Choice and Differentiation in Mouse Embryos and Embryonic Stem Cells

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Developmental Biology 264 (2003) 1Ð14 www.elsevier.com/locate/ydbio Review

Lineage choice and differentiation in mouse embryos and embryonic stem cells

David A.F. Loebel, Catherine M. Watson, R. Andrea De Young, and Patrick P.L. Tam*

Embryology Unit, Children’s Medical Research Institute, Locked Bag 23, Wentworthville, NSW 2145 Australia Received for publication 21 February 2003, revised 21 May 2003, accepted 11 June 2003

Abstract The use of embryonic stem (ES) cells for generating healthy tissues has the potential to revolutionize therapies for human disease or injury, for which there are currently no effective treatments. Strategies for manipulating stem cell differentiation should be based on knowledge of the mechanisms by which lineage decisions are made during early embryogenesis. Here, we review current research into the factors influencing lineage differentiation in the mouse embryo and the application of this knowledge to in vitro differentiation of ES cells. In the mouse embryo, specification of tissue lineages requires cellÐcell interactions that are influenced by coordinated cell migration and cellular neighborhood mediated by the key WNT, FGF, and TGF␤ signaling pathways. Mimicking the cellular interactions of the embryo by providing appropriate signaling molecules in culture has enabled the differentiation of ES cells to be directed predominately toward particular lineages. Multistep strategies incorporating the provision of soluble factors known to influence lineage choices in the embryo, coculture with other cells or tissues, genetic modification, and selection for desirable cell types have allowed the production of ES cell derivatives that produce beneficial effects in animal models. Increasing the efficiency of this process can only result from a better understanding of the molecular control of cell lineage determination in the embryo. © 2003 Elsevier Inc. All rights reserved.

Keywords: FGF; WNT; BMP; Stem cell; Germ cell; Differentiation; Lineage; Pluripotent; Mouse; Embryo

Introduction ferentiation of tissue lineages in the early mouse embryo and describe the application of this knowledge to in vitro There is considerable interest in the therapeutic applica- embryonic stem (ES) cell differentiation. tions of pluripotent cells derived from embryos or adult Around the time of implantation, the mouse embryo is tissues. Consequently, much effort is being directed toward composed of three distinct cell types (Fig. 1A and B): the understanding the mechanisms for maintenance of the plu- trophectoderm, the primitive endoderm, and the inner cell ripotent state and the pathways leading to lineage speciﬁ- mass (ICM). The trophectoderm gives rise to the chorion cation. Control of these mechanisms may enable the ma- and the trophoblast, which differentiates into the giant cells, nipulation of pluripotent cells for the development of cell- the cytotrophoblast and syncytiotrophoblast of the placenta. and tissue-based replacement therapies for damaged or Primitive endoderm forms the visceral and parietal dysfunctional tissues. The ability to manipulate the differ- endoderm that lines the yolk sac cavity and the exocoelom. entiation of embryo-derived pluripotent cells requires a The ICM is organized into a pluripotent epithelial layer (the thorough understanding of the processes of lineage commit- epiblast) from which embryonic and other extraembryonic ment and differentiation of embryonic cells in vivo. In this tissues are derived (Gardner and Beddington, 1988). review, we outline the current state of knowledge of the Gastrulation is a major milestone in early postimplanta- molecular and cellular basis of the determination and dif- tion development. During gastrulation, pluripotent epiblast cells are allocated to the three primary germ layers of the * Corresponding author. Fax: ϩ61-2-96872120. embryo (ectoderm, mesoderm, and deﬁnitive endoderm) E-mail address: [email protected] (P.P.L. Tam). and the extraembryonic mesoderm of the yolk sac and

Fig. 1. (A) Mouse embryo at periimplantation (E4.75, Pou5f1 expression in the ICM), early-streak (E6.5, Otx2 expression in the epiblast), and early-bud (E7.5, Cer1 expression in the anterior definitive endoderm) stages. (B) The differentiation of trophectoderm (grey), ICM (blue), and primitive endoderm (brown) of E4.75 embryo to the extraembryonic and embryonic tissues of E7.5 embryo (germ layer tissues are color-coded, see key). (C) Proximal and distal polarity of tissue compartments. In the epiblast, progenitor populations are color-coded: blue, ectoderm; red, mesoderm; yellow, endoderm. Red arrows and bar, BMP signals and antagonists; blue arrow and bar, WNT3A signals and antagonists. (D) Colonies of embryonic and trophectodermal stem cells (white arrows) derived from the ICM and extraembryonic ectoderm, respectively (picture of TS cells provided by Dr. Janet Rossant, Samuel Lunenfeld Research Institute, ES cells provided by Ms. Melinda Hayward). amnion (Fig. 1A and B). The primary germ layers are the cific gene expression. This process is strongly influenced by progenitors of all fetal tissue lineages. The specification of cellular interactions and signaling (Fig. 1C; Tam and Be- tissue lineages is accomplished by the restriction of the hringer, 1997; Tam et al., 2000). developmental potency and the activation of lineage-spe- Primordial germ cells (PGCs) arise from a progenitor D.A.F. Loebel et al. / Developmental Biology 264 (2003) 1–14 3

Fig. 2. Establishment of the germ-cell lineage in prestreak to late streak mouse embryos: showing the signaling and mediating molecules that influence (A) priming, (B) specification and localization of cells derived from the proximal epiblast, and (C) commitment of the PGCs prior to and during gastrulation (Lawson et al., 1986, 1999; Tremblay et al., 1995, 2001; Hayashi et al., 1997, 2002; Tsang et al., 1999, 2001; Chang et al., 1999; Ying et al., 2000; Ying and Zhao, 2001; Mukhopadhyay et al., 2001, 2003; Chang and Matzuk, 2001; Saitou et al., 2002). population in the epiblast around E5.5 and give rise to the ment from which they were established (reviewed in mature spermatozoa and ova of adults. The germ cell lin- McLaren and Durcova-Hills, 2001). eage is unusual because it differentiates into a highly spe- Trophectodermal stem (TS) cells (Fig. 1D), with the cialized cell type, but must retain the totipotent property that molecular properties of diploid trophoblast cells, may be is required by the zygote of the next generation. The pro- isolated from the blastocyst or extraembryonic ectoderm of genitor cells of this lineage respond selectively to specific periimplantation embryos. TS cells have a restricted differ- signals promoting differentiation into PGCs. They are then entiation potential: they can form giant cells in vitro sequestered into the base of the allantois to avoid being (Tanaka et al., 1998; Yan et al., 2001) and have only been swept up into the dynamic embryonic milieu of gastrulation. observed to contribute to trophectodermal cell lineages in Eventually, they migrate to the genital ridges and display vivo. Whereas ES cells can be induced to become TS cells sex-specific differentiation before maturing into gametes. (Niwa et al., 2000), TS cells have not been shown to acquire Embryonic stem (ES) cells can be derived in vitro from the lineage potential of ES cells. As yet, there are no the ICM of the periimplantation embryo (Fig. 1D). Like the published reports of stem cells derived from the third cell ICM and the early epiblast, ES cells are capable of differ- type, the primitive endoderm of the embryo, but this re- entiating into a wide range of fetal tissues in culture (Ros- mains an exciting possibility. sant and McKerlie, 2001) or in chimeric embryos and can even form the entire fetus (Nagy et al., 1993). However, significant contribution to either the trophectoderm or vis- Maintaining pluripotency ceral endodermal lineages by ES cells has not been demonstrated. Pluripotency of embryonic cells refers to their ability to Embryonic germ cell (EG) lines can be established from differentiate into cell types of many tissue lineages. Under- premigratory (E8.0 and E8.5), migratory (E9.5), and post- standing the molecular basis of pluripotency and the con- migratory PGCs that have arrived at the genital ridge ditions required to maintain this state in the early embryo is (E11.5ÐE12.5). EG cells are pluripotent and can contribute vital for an understanding of the maintenance of pluripotent to embryonic cell lineages in chimeras (Labosky et al., cells in culture. 1994). Many of the cellular specializations of the PGC, such The transcription factor OCT4, encoded by Pou5f1, is an as pseudopodia, disappear in culture, and EG cells resemble essential regulator of preimplantation mouse development ES cells in morphology regardless of the stage of develop- and is expressed in pluripotent cells: oocytes, the ICM, early 4 D.A.F. Loebel et al. / Developmental Biology 264 (2003) 1–14 preimplantation embryos, primitive ectoderm, PGCs, and Establishment of the germ line is a multistep process ES cells (Niwa, 2001). OCT4 is essential for the formation of the ICM in vivo (Nichols et al., 1998). Epiblast cell Specification of primordial germ cells begins in the epi- differentiation is accompanied by repression of Pou5f1, blast of periimplantation embryos. PGCs continue to ex- which is mediated by the binding of germ cell nuclear factor press Pou5f1 and can restore the totipotent properties (GCNF) to the proximal promoter of Pou5f1. This results in unique to germ cells during gametogenesis. The formation the restriction of Pou5f1 expression to the germ cell lineage of the PGC population from proximal epiblast progresses (Fuhrmann et al., 2001). In vitro studies of ES cells have through multiple stages, requiring BMP signaling, cellÐcell shown that developmental regulation is effected by varying interaction, and cell movement (Fig. 2). BMP4 signaling OCT4 expression levels. Thus, repression of Pou5f1 in- from the extraembryonic ectoderm primes proximal epiblast duces trophectodermal differentiation, and elevated Pou5f1 cells to be capable of attaining either a germ cell or allantoic expression induces endodermal and mesodermal differenti- fate. ation, whereas intermediate Pou5f1 levels are required for BMP2, Ϫ4, and Ϫ8B, produced by the extraembryonic the maintenance of pluripotency of ES cells in vitro (Niwa tissues in the vicinity of the proximal epiblast of the pre- et al., 2000). streak embryo, are necessary for specification of germ cells OCT4 can act as a repressor or activator of transcription (Fig. 2A and B). Bmp4Ϫ/Ϫ mice have no PGCs, whereas depending on the presence of its cofactors (Niwa, 2001). Bmp4ϩ/Ϫ mice have reduced numbers, indicating that SOX2 is a potential coactivator for OCT4 (Ambrosetti et al., BMP4 acts in a dose-dependent manner to induce PGCs 1997). Candidate targets of OCT4 and SOX2 include Fgf4 (Lawson et al., 1999). Bmp2Ϫ/Ϫ null embryos have reduced and Hand1, which are associated with maintenance and numbers of PGCs and Bmp4ϩ/Ϫ; Bmp2ϩ/Ϫ double heterozy- differentiation of trophoblasts (Niwa, 2001; Avilion et al., gotes have fewer PGCs than either Bmp4ϩ/Ϫ or Bmp2ϩ/Ϫ 2003). The developmental defects displayed by embryos embryos. Bmp2 and Bmp4 therefore exert an additive effect lacking Sox2 function suggest that both Sox2 and Pou5f1 are on PGC specification. In contrast, Bmp2 and Bmp8b do not important for correct specification of the ICM, the primitive display any additive effects (Ying and Zhao, 2001). Ho- endoderm, and the trophectoderm (Avilion et al., 2003). modimers of BMP4 and BMP8B (Ying et al., 2000) act In culture, ES cells remain undifferentiated in the pres- synergistically to induce PGCs in the epiblast in vitro (Ying ence of the cytokine LIF. LIF acts via a receptor comprising et al., 2001). Coculture of epiblast and visceral endoderm in the LIF-specific subunit LIFR␤ and gp130 and can be trans- the presence of BMP4 leads to stronger expression of Tissue duced by the JAK-STAT signaling pathway. STAT3 seems Nonspecific Alkaline Phosphatase (TNAP), a germ cell to be the principal component of the signaling process in ES marker, in the epiblast cells adjacent to visceral endoderm cells (Raz et al., 1999). However, an uncharacterized ES relative to cultures lacking BMP4. Although it has not been cell renewal factor (ESRF) can maintain the pluripotency of tested whether the visceral endoderm in the coculture pro- ES cells without activating Stat3 (Niwa et al., 1998). vides a source of BMP2, the data are nevertheless consistent Stat3Ϫ/Ϫ embryos develop until E6.0 and then rapidly de- with a role for combined BMP2 and BMP4 action in the generate at gastrulation (Takeda et al., 1997). Until ESRF is induction of PGCs (Pesce et al., 2002). Thus, in vivo and in characterized, it will remain unclear whether both Stat3- vitro data show that BMP2, Ϫ4, and Ϫ8B proteins all play dependent and -independent pathways are critical for main- a role in PGC induction. taining pluripotency in the embryo. Close proximity of the extraembryonic ectoderm to the ES cells can differentiate into early primitive ectoderm- epiblast during a narrow window of development is essen- like (EPL) cells when cultured in conditioned medium tial for germ cell specification in vivo. Distal epiblast cells (Rathjen et al., 1999). EPL cells are a step closer to ecto- form PGCs when placed adjacent to extraembryonic dermal commitment and, unlike ES cells, EPL cells are ectoderm in vivo (Tam and Zhou, 1996) or in culture unable to contribute to tissues in chimeric embryos (Rathjen (Yoshimizu et al., 2001). The interaction with extraembry- et al., 1999; Pelton et al., 2002). EPL cells resemble the onic ectoderm is only required for PGC production by the epiblast of the embryo in that they continue to express epiblast of E5.5 embryos but not by E6.0 epiblast, and the Pou5f1, and the differences in gene expression between ES tissue requirement can be replaced by BMP4 (Fig. 2A; and EPL cells recapitulate the differences between the ICM Yoshimizu et al., 2001). and epiblast. EPL cell-derived embryoid bodies are predis- Smad1 and Smad5 are important for germ cell specifica- posed to differentiation to mesoderm (Lake et al., 2000), but tion and act downstream of BMP receptors in the germ cell can be redirected toward a neural fate by continued expo- precursors. Smad1 is expressed in epiblast and visceral sure to conditioned medium (Rathjen et al., 2002). With- endoderm during gastrulation and is expressed in about 25% drawal of conditioned medium from EPL cell culture results of proximal epiblast cells. Only a minority of these cells in reversion to an ES cell-like state, including the potential coexpress Smad5 or Ϫ8, but it is postulated that these cells for in vitro differentiation and the ability to contribute to are the PGC progenitors (Hayashi et al., 2002). Smad1Ϫ/Ϫ tissues in chimeric mice (Rathjen et al., 1999; Lake et al., embryos fail to develop an allantois and have very low 2000). numbers of PGCs, whereas heterozygotes are similar to wild D.A.F. Loebel et al. / Developmental Biology 264 (2003) 1–14 5 type embryos (Tremblay et al., 2001). Smad5Ϫ/Ϫ embryos fragilis gene. PGC progenitors need to be in the right place also have very few or no PGCs, whereas Smad5ϩ/Ϫ have at the right time to respond to local signals. Commitment to fewer PGCs than wild type embryos, showing that Smad5 is the germ-cell lineage is accomplished by spatial sequestra- also involved in germ cell specification in a dose-dependent tion of the PGCs, reinforcement by localized signals, and manner similar to BMP4 (Fig. 2B; Chang et al., 2001). induction of germ line-specific gene expression. Interactions within a community of epiblast cells may be important for germ cell specification (Fig. 2B). Induction of PGCs is more efficient in epiblast fragments containing at Specification of the primary germ layer tissues least 40 cells or in closely packed epiblast explants on feeder layers than in dissociated epiblast cells (Yoshimizu et BMP signaling is essential for the choice between al., 2001; Pesce et al., 2002). fragilis/mil1, fragilis2/mil2, ectodermal and mesodermal fates and fragilis3/mil3 are members of a gene family encoding interferon-induced transmembrane proteins, which in mice BMP signaling is required to induce mesoderm forma- comprises five members (Saitou et al., 2002; Tanaka and tion in vivo (Fig. 3, red area). Most Bmp4Ϫ/Ϫ embryos fail Matsui, 2002). This gene family is conserved in other spe- to gastrulate and form no mesoderm (Winnier et al., 1995). cies and is thought to be involved in homotypic cell adhe- Loss of Bmpr1 and Bmpr2b function also results in reduced sion and extension of cell cycle time. fragilis is highly epiblast proliferation and no formation of mesoderm (Wil- expressed in the proximal epiblast and gradually becomes son et al., 1993; Mishina et al., 1995). The similarities in the restricted to a group of cells in the posterior PS coincident phenotypes of Bmp4, Bmpr1, and Bmpr2 mutants suggest with the site where PGCs arise and TNAP and also fragilis2 that BMP4 signals predominantly through heterodimers of and Ϫ3 are expressed. fragilis expression can be induced in these receptors (Wilson et al., 1993; Mishina et al., 1995). distal epiblast cells by coculturing them with extraembry- BMP signaling activates receptor regulated SMADS 1, 5, onic ectoderm. Its expression is reduced in Bmp4ϩ/Ϫ and and 8, which interact with the common partner SMAD4 to absent in Bmp4Ϫ/Ϫ embryos, indicating a dose-dependent regulate gene expression (Miyazawa et al., 2002). Smad4 is induction of fragilis by BMP4 (Saitou et al., 2002). Thus, required for epiblast proliferation, gastrulation, and meso- fragilis, fragilis2, and fragilis3 are potentially mediators of derm formation (Yang et al., 1998; Sirard et al., 1998). cellÐcell interactions in PGC specification in the mouse For correct patterning and differentiation of the embryo, (Lange et al., 2003). the influence of BMP signaling must be modulated by PGCs must be localized to the base of the allantois antagonistic molecules. The BMP antagonists Chordin and during gastrulation to avoid a somatic cell fate. The pro- Noggin are expressed in the mouse organizer and promote gressive shift in the location of fragilis-expressing epiblast differentiation of ectodermal lineages (Fig. 3, blue area). cells is consistent with the general pattern of cell movement Chordin and Noggin have overlapping functions during during gastrulation followed by isolation of the progenitor embryogenesis and are required for anterior neural differ- PGC population to the base of the allantois, where their entiation in the embryo (Bachiller et al., 2000). proliferation is reduced relative to the somatic cells that pass BMP4 is able to influence ES cells to choose a meso- through the PS (Tam and Snow, 1981; Saitou et al., 2002). dermal rather than neurectodermal fate in culture (Fig. 4, Bmp2 is expressed just prior to and during gastrulation in red area). Treatment of ES cells with BMP4 inhibits neural the visceral endoderm overlying the ectodermÐepiblast differentiation and represses the neurectodermal marker boundary in the region where the PS forms. Its expression Pax6 and upregulates T in a concentration-dependent man- pattern also makes it a candidate for a localization signal, ner, independent of cell survival or proliferation (Johansson together with Bmp4 for the PGC progenitors (Fig. 2B; Ying and Wiles, 1995; Wiles and Johansson, 1997; Finley et al., and Zhao, 2001; Fujiwara et al., 2001). Cells expressing 1999). BMP4 induces differentiation of cells resembling fragilis congregate in the posterior PS and allantois of posteriorÐventral embryonic mesoderm, whereas dorsalÐan- E7.0ÐE7.5 late streak stage embryos. At this site, some of terior mesodermal cell types are induced by another TGF␤- the fragilis-expressing cells begin to express stella (also related molecule, Activin A (Johansson and Wiles, 1995). referred to as PGC7 and Dppa3; Saitou et al., 2002). Ex- The effectiveness of BMP4 in altering cell fate is limited to pression of stella, which encodes a putative chromosomal a period prior to the onset of neural differentiation in culture organization or RNA processing factor, may mark the emer- (Finley et al., 1999). Under different conditions, BMP4 gence of a committed PGC population (Saitou et al., 2002; promotes the differentiation of ES cells and EPL cells to Sato et al., 2002). surface ectoderm at the expense of neuroectoderm, under- Specification of the germ line is a multistep process (Fig. lining the properties of BMP4 as a suppressor of neurecto- 2). Progenitors for PGCs in the epiblast are initially primed dermal differentiation (Fig. 4, blue area; Kawasaki et al., by BMP4 signals from the extraembryonic ectoderm at 2000; Rodda et al., 2002). However, there is no direct E5.5. The specification of the PGCs is influenced by the evidence for a role for BMP4 in surface ectoderm differen- action of BMPs and SMADs and by the interactions be- tiation in mouse embryos (Fig. 3). tween cells, which may be mediated by the product of the BMP antagonists promote the acquisition of an ectoder- 6 D.A.F. Loebel et al. / Developmental Biology 264 (2003) 1–14

Fig. 3. Specification and differentiation of germ layer derivatives in mouse embryos. Ectodermal (blue), mesodermal (red), and definitive endodermal (orange) lineages derived from the epiblast (brown) are shown, with the signaling pathways and transcription factors indicated in green. The question mark signifies the lack of direct evidence for involvement of BMP4 in surface ectoderm differentiation in mouse embryos (Harrison et al., 1995, 1999; Ahlgren et al., 1996, 1997). mal fate by ES cells (Fig. 4, blue area). Exposure of ES cells endoderm. In tissue recombination and coculture experi- to recombinant Noggin, which counteracts the mesoderm- ments, anterior ectoderm can be induced to express neural inducing effects of BMP4 (Finley et al., 1999), or transfec- markers (Otx2 and En1) by the prospective anterior (head tion with a Noggin-encoding plasmid promotes the differ- and axial) mesoderm and foregut endoderm. Inductive in- entiation of neurectodermal cells (Gratsch and O’Shea, teractions are also required to maintain expression of neural 2002). Exposure to Chordin results in reduced differentia- genes. In contrast, the posterior mesoderm and endoderm tion, with some cells retaining ES cell characteristics and a suppresses the expression of neural markers in the anterior small number differentiating to mesenchymal cell types ectoderm (Ang and Rossant, 1993; Ang et al., 1994). How- (Gratsch and O’Shea, 2002). Disruption of SMAD4-medi- ever, the nature of the inductive and suppressive activity is ated BMP/TGF␤ signal transduction enhances the ability of not known. ES cells to adopt a neural fate (Tropepe et al., 2001), which The Sry-related HMG box-containing transcription fac- is consistent with the suppression of neural differentiation tor Sox1 is expressed during early neurogenesis, becoming by BMP signaling. progressively downregulated as cells exit from mitosis (Pevny et al., 1998; Wood and Episkopou, 1999). Sox1 is Induction and differentiation of the neurectodermal upregulated in response to retinoic acid (RA)-induced neu- lineages ral differentiation in cultured ectodermal cells, and when ectopically expressed, can substitute for treatment with RA. Initial specification of neurectoderm from the ectoderm This suggests that Sox1 plays a role in initially directing of the gastrulating embryo is accomplished by inductive ectodermal cells toward a neural fate in response to external interactions with nascent mesoderm and definitive signals (Pevny et al., 1998). Fig. 4. In vitro differentiation of embryonic stem cells. Culture supplements are shown in green, culture conditions in black, and germ layer derivatives and tissue-specific markers in blue for ectodermal tissues; red for mesodermal tissues; orange for endoderm. EB, embryoid body (Yamashita et al., 1994, 2000; Wiles and Johansson, 1997, 1999; Kennedy et al., 1997; Rathjen et al., 1999; Lake et al., 2000; Kawasaki et al., 2000; Lumelsky et al., 2001; Wichterle et al., 2002; Rathjen et al., 2002; Gratsch and O’Shea, 2002; Hori et al., 2002; Yin et al., 2002). 8 D.A.F. Loebel et al. / Developmental Biology 264 (2003) 1–14

Expression of posterior Hox genes and SHH activity are chimeric embryos, ectopic neural tubes form entirely from associated with differentiation of ventral motor neurons in Fgfr1Ϫ/Ϫ cells, which fail to fully traverse the PS and take the caudal neural tube. In the embryo, neurectoderm ini- on an ectodermal fate (Ciruna et al., 1997; Yoshikawa et al., tially has a rostral character and requires the action of RA to 1997). The phenotypes of Fgf8 and Fgfr1 mutant mice impart the caudal characteristics of spinal cord, presumably suggest a role in the migration of nascent mesoderm away by the activation of the Hox genes specific to posterior from the streak, possibly by repression of E-cadherin neural tube. Subsequently, graded SHH signaling, mediated (Yamaguchi et al., 1994; Ciruna et al., 1997; Sun et al., by the transcriptional repressor Gli3, is required to induce 1999; Ciruna and Rossant, 2001). E-cadherin prevents the motor neuron progenitors (Fig. 3, blue area; Persson et al., interaction of LEF/TCF proteins with the transcriptional 2002). coactivator ␤-catenin, disrupting WNT signaling. Seques- Several groups have reported the generation of neurec- tering E-cadherin rescues expression of a T-lacZ reporter, todermal lineages from ES cells by the provision of factors, presumably due to the restoration of the WNT signaling including FGF, SHH, and RA, that affect neural differenti- pathway (Ciruna and Rossant, 2001). However, expression ation in vivo. Using Sox1-GFP expression as a marker, Ying of Tbx6 is independent of Wnt3a (Yamaguchi et al., 1999). et al. (2003) demonstrated that differentiation of ES cells to The potential involvement of FGF signaling in directing neurectodermal lineages could be accomplished without cells down a mesodermal pathway rather than a neurecto- embryoid body formation. Addition of FGF4 stimulated ES dermal pathway has been demonstrated by the ability of cell differentiation to neurectodermal cells, and inhibition of exogenous FGF2 to induce the formation of mesoderm in FGF signaling abolished the differentiation of pluripotent explants of anterior epiblasts in culture (Burdsal et al., cells to neurectoderm (Ying et al., 2003). Treating embryoid 1998). bodies with RA and either a HH agonist or recombinant Antagonism of WNT signaling promotes neural differ- SHH allows the differentiation of ES cells to motor neurons entiation in culture. The WNT antagonist Sfrp2 is expressed in vitro, presumably by mimicking the conditions that pro- during neural differentiation of embryoid bodies. When mote the differentiation of motor neurons in vivo (Fig. 4, transfected into ES cells, Sfrp2 enhances neuronal differen- blue area; Wichterle et al., 2002). Multistep strategies in- tiation (Aubert et al., 2002). volving the application of signaling molecules (FGF8 and The earliest mesodermal derivatives to differentiate are SHH), and the upregulation of Nurr1, a neuronal differen- the endothelial and hematopoietic cells of the blood islands tiation-related transcription factor (Kim et al., 2002) or in the yolk sac. The extraembryonic mesoderm from which selection for cells that express Nestin (Nishimura et al., the vascular and hematopoietic progenitors arise is amongst 2003) have enabled the generation of dopaminergic neurons the earliest to pass through the PS (Kinder et al., 1999). It in vitro. has been demonstrated that in vitro, a single population of cells can act as precursors of both hematopoietic and endo- Molecular control of mesodermal differentiation thelial lineages (Robb and Begley, 1996; Yamashita et al., 2000). WNT3A signaling induces cells from the epiblast to Scl is required for differentiation of hematopoietic lin- adopt a paraxial mesodermal rather than a neurectodermal eages. Yolk sac hematopoiesis fails in SclϪ/Ϫ embryos fate after gastrulation (Yoshikawa et al., 1997). In (Shivdasani et al., 1995; Robb et al., 1995), and Scl is Wnt3aϪ/Ϫ embryos, epiblast cells ingressing into the PS are necessary for contribution to any primitive or definitive diverted to a neurectodermal fate instead of forming meso- hematopoietic lineage in the mouse (Porcher et al., 1996). dermal cells. Consequently, ectopic structures resembling Scl is also expressed in yolk sac endothelium, but is not neural tubes are formed in place of the somites (Yoshikawa necessary for its differentiation. et al., 1997). Scl is necessary for the early stages of in vitro differen- Regulation of genes acting downstream of WNT3A sig- tiation of mesoderm to hematopoietic lineages and acts naling requires the transcription factors Lef1 and Tcf1. during a limited time window (Elefanty et al., 1997; Rob- Mouse mutants lacking both Lef1 and Tcf1 develop ectopic ertson et al., 2000; Endoh et al., 2002). Loss of Scl affects neural tubes and are defective in paraxial mesoderm differ- genes specifically expressed during hematopoiesis, but does entiation (Galceran et al., 1999). WNT3a signaling, via not affect the expression of early mesodermal markers or Lef1/Tcf1, maintains expression of the mesodermal tran- endothelial markers (Elefanty et al., 1997). scription factor T in presumptive paraxial mesoderm Signaling from the endoderm via vascular endothelial (Yamaguchi et al., 1999). Maintenance of T expression can growth factor (VEGF) is required for endothelial and he- be rescued by a constitutively active LEF1-␤-catenin fusion matopoietic differentiation of mesoderm (Fig. 3, red area; protein (Galceran et al., 2001). Carmeliet et al., 1996; Shalaby et al., 1997; Fong et al., The WNT signaling pathway interacts with the FGF 1999). VegfϪ/Ϫ or Vegfϩ/Ϫ embryos die before birth with pathway to induce mesoderm differentiation (Fig. 3, red disrupted blood vessel formation (Carmeliet et al., 1996; area). Fgfr1 is required for expression of T and Tbx6 (Chap- Damert et al., 2002). Targeting of the VEGF receptors Flt1 man and Papaioannou, 1998; Ciruna and Rossant, 2001). In and Flk1 confirms the role of VEGF signaling in the correct D.A.F. Loebel et al. / Developmental Biology 264 (2003) 1Ð14 9 differentiation of endothelial lineages (Fong et al., 1995, that the primary function of SHH in muscle development is 1999; Shalaby et al., 1995). Flt1 regulates the differentia- as a survival or proliferation factor (Kruger et al., 2001). tion of mesoderm to hemangioblast/angioblasts (Fong et al., 1999), whereas Flk1 is required cell autonomously for cor- Definitive endoderm differentiation requires FGF, TGF␤ rect maturation of endothelial cells and migration of pre- and WNT signalling cursor cells (Shalaby et al., 1995, 1997). A role for VEGF in differentiation of hematopoietic precursors has been At present, the data relating to the roles of signaling shown by the generation of mice homozygous for a hypo- molecules in definitive endoderm differentiation are frag- morphic allele of VEGF (Damert et al., 2002). Flk1 may mentary. In zebrafish and Xenopus, a pathway of interacting mediate this function since loss of Flk1 activity also impairs factors responding to Nodal-related signaling molecules has hematopoiesis (Shalaby et al., 1995, 1997). been established, involving SMAD signal transduction and VEGF signaling can also influence the differentiation of paired-like homeodomain transcription factors acting up- hematopoietic and endothelial lineages in vitro (Fig. 4, red stream of SOX transcription factors to induce endoderm area). Blast cell colonies, which are derived from embryoid (Alexander and Stainier, 1999; Poulain and Lepage, 2002). bodies and give rise to hematopoietic cells, are induced There is some evidence that similar pathways may op- following treatment with VEGF (Kennedy et al., 1997). In erate in the mouse (Fig. 3, orange area). In chimeric mice, Ϫ/Ϫ culture, VEGF encourages ES cell-derived Flk1-expressing Smad2 ES cells are not able to contribute to the defin- cells to differentiate into endothelial cell types. Treatment itive endoderm lineages, indicating that Smad-mediated ␤ with platelet-derived growth factor (PDGF) BB induces the TGF signaling is needed for the initial specification of the same cells to differentiate into smooth muscle cells. Fur- definitive endoderm lineage (Weinstein et al., 1998; Waldrip et al., 1998; Tremblay et al., 2000). The phenotypes thermore, by treating cells with VEGF in collagen gels, Ϫ/Ϫ Ϫ/Ϫ three-dimensional vascular structures can be formed (Ya- of Sox17 and Mixl1 mice suggest that both transcrip- mashita et al., 2000). tion factors are required for the differentiation but not for formation of the definitive endoderm (Hart et al., 2002; Hedgehog signaling has the ability to promote the early Kanai-Azuma et al., 2002). Mixl1 is apparently essential for differentiation of endothelial and hematopoietic precursors the proper allocation of endoderm precursors from mesen- in tissue explants (Dyer et al., 2001). However, this does not doderm progenitors during gastrulation. Loss of Sox17 re- fully reflect the situation in vivo, as the initial stages of sults in a severe reduction in the amount of definitive endothelial and hematopoietic differentiation can occur in endoderm. Sox17-null definitive endoderm has reduced vi- the absence of IHH or its receptor, SMO (Byrd et al., 2002). ability and proliferation, resulting in occupation of the gut SMAD-mediated signal transduction is also implicated by visceral endoderm-like cells. Testing the tissue potency in differentiation of heart tissue from mesodermal precur- of the null-mutant ES cells in chimeric embryos reveals sors. Expression of the cardiac-specific transcription factor more dramatic effects of the loss-of-function of these fac- Nkx2-5 requires an enhancer element containing SMAD tors. Sox17Ϫ/Ϫ and Mixl1Ϫ/Ϫ ES cells fail to contribute to binding sites (Lien et al., 2002), suggesting that Nkx2-5, like the gut endoderm in competition with the wild type embry- its Drosophila orthologue tinman, may be regulated by onic cells (Hart et al., 2002; Kanai-Azuma et al., 2002). BMP signaling. This agrees with previous findings that Germ layer explant experiments show that differentiation Ϫ transient exposure of chick explants to BMP2 or 4in of definitive endoderm requires soluble factors originating Ϫ combination with FGF2 or 4 induces contractile cardiac from the mesoderm and ectoderm layers during gastrulation muscle differentiation and Nkx2-5 expression in nonprecar- (Fig. 3, orange area). FGF4 alone is sufficient initiate dif- diac mesoderm (Ladd et al., 1998; Barron et al., 2000). ferentiation, but additional factors provided by interacting SHH and WNT signaling are also important for further tissues are necessary for survival of the induced endoderm differentiation of paraxial mesoderm derivatives (Fig. 3, red (Wells and Melton, 2000). FGF signaling is also involved area). All skeletal muscles in the body of the mouse are later during organogenesis, where exposure of ventral fo- derived from the somites, which arise by segmentation of regut endoderm to FGF from the cardiac mesoderm inhibits the paraxial mesoderm on either side of the neural tube and ventral pancreas, the default fate, to liver formation (Deut- notochord. Wnt3a activates myogenic transcription factors sch et al., 2001). Embryos lacking ␤-catenin in the anterior MyoD and Myogenin in embryonal carcinoma cells (Ridge- PS, node, and notochord are depleted of definitive way et al., 2000). Expression of the early myogenic tran- endoderm, although the visceral endoderm is displaced nor- scription factor Myf5 is reduced in the early dermomyotome mally. In chimeric embryos, precardiac mesoderm popu- Ϫ Ϫ Ϫ Ϫ of Wnt1 / ;Wnt3 / embryos but later increases to normal lates the endodermal layer, and ectopic hearts form in levels, suggesting that WNT signaling is compensated for ␤-catenin-null embryos. This suggests the downregulation by other factors in later development (Ikeya and Takada, of ␤-catenin is required to direct cells in the PS to choose an 1998). Myf5 is regulated by SHH signaling, via GLI tran- endodermal rather than mesodermal fate (Lickert et al., scription factor binding to an SHH responsive promoter 2002) and implicates the WNT-responsive intracellular (Gustafsson et al., 2002). However, there is also evidence pathway in the formation of the definitive endoderm. How- 10 D.A.F. Loebel et al. / Developmental Biology 264 (2003) 1Ð14 ever, the signaling molecule is as yet unidentified, and there Parkinson’s disease (Kim et al., 2002; Nishimura et al., is no direct evidence for the involvement of WNT. 2003). ES cell-derived motor neurons have been grafted into Manipulation of gene expression in ES cells has revealed chick embryo spinal cords and are shown to survive and roles for several other transcription factors in endoderm differentiate correctly in vivo (Wichterle et al., 2002). Flk1- differentiation. Tcf2Ϫ/Ϫ cells are severely impaired in their expressing, ES cell-derived vascular progenitor cells were ability to contribute to the gut in chimeric mice, indicating able to differentiate into endothelial cells, after grafting into an additional requirement for Tcf2 in definitive endoderm tumor-bearing mice. The grafted cells were incorporated formation (Barbacci et al., 1999; Coffinier et al., 1999). into the developing vasculature of the tumor, demonstrating Overexpression of Foxa2 in ES cells induces genes nor- an ability to contribute to adult neovascularization (Yurugi- mally expressed in endoderm-derived tissues such as the Kobayashi et al., 2003). Pancreatic cells, produced by cul- liver, gut, and lung. However, genes involved in late ture in selective media and supplemented with FGF2, endoderm differentiation are not induced (Levinson-Dush- formed islet-like structures and, upon transplantation into nik and Benvenisty, 1997). diabetic mice, improved survival and body weight mainte- As yet there are no reports of directed in vitro differen- nance (Lumelsky et al., 2001). In vitro derived hepatocyte tiation of ES cells to early gut endoderm by manipulation of progenitors were found to undergo proper differentiation culture conditions with conditioned media or soluble fac- into parenchymal cells in the liver of recipient animals tors. However, the production of hepatic progenitors and (Ishizaka et al., 2002; Yin et al., 2002). pancreatic islet cells, which are both later endodermal de- The encouraging outcome of the above experiments in- rivatives, by directed in vitro differentiation of ES cells has dicates that directed differentiation of ES cells is possible been reported (Fig. 4, orange area; Lumelsky et al., 2001; and may be translated into therapeutic applications. How- Hori et al., 2002; Yin et al., 2002). ever, the need to rigorously validate the fidelity of cell differentiation by marker analysis has been highlighted by controversy over the ability of in vitro derived cells that Potential for therapeutic applications resemble pancreatic islets to synthesize appreciable quanti- ties of insulin (Rajagopal et al., 2003). In addition, the need For in vitro differentiated ES cells to be useful in human to eliminate undesirable cell types is illustrated by the ob- stem cell therapies, the cells must be capable of contributing servation of tumor formation in recipient animals, probably to target tissues of a recipient, without the presence of due to the presence of undifferentiated stem cells in the undifferentiated cells precipitating the formation of terato- transplanted material, despite barely detectable levels of mas. This requires homogeneous populations of differenti- Pou5f1 expression (Nishimura et al., 2003). ated cells. In the embryo, the decision of a pluripotent cell Therefore, a more thorough understanding of the pro- to progress toward a particular lineage requires the con- cesses that drive tissue differentiation in the embryo is certed efforts of inductive or inhibitory signals from tissues essential for improving the current techniques for directed in the immediate vicinity and the ability of the cell to ES cell differentiation. Better control of in vitro cell differ- interpret these signals. Replication of these conditions in entiation may therefore necessitate the replication of the in vitro can be achieved by controlling the delivery of similar vivo situation that favors specific lineage development. In combinations of growth factors to the ES cells. particular, it is necessary to reconstitute the interactions To date it has not been possible to generate truly homo- among cells within a community and between cell popula- geneous populations of differentiated cells purely by the tions in vivo. Further complications are the multiple effects manipulation of culture conditions. In the majority of cases, of single factors and the synergistic and antagonistic effects the generation of nearly homogeneous population of cells of combinations of factors. Understanding the complex tis- has been accomplished by the selection of the desired cell sue interactions and interdependence of molecular mecha- type on the basis of expression of lineage-specific markers nisms of lineage choices in vivo presents a significant chal- or transgenes, or ectopic expression of lineage-specific tran- lenge, but the outcome of embryological investigations will scription factors. However, enriching specific cell types by be crucial to the realization of stem cell-based therapies. these means is less attractive for therapeutic applications because it involves genetic modifications, which may have an undesirable and unpredictable outcome and which may Acknowledgments impair the ability of cells to undergo the full repertoire of lineage differentiation. We thank Melinda Hayward for providing the ES cells Despite difficulties in obtaining homogeneous popula- and Janet Rossant for the picture of TS cells (Fig. 1D), and tions, functional derivatives of all three primary germ layers Bruce Davidson, Leon McQuade, and Peter Rowe for com- have been successfully produced from ES cells. Differenti- ments on the manuscript. Our work is supported by the ated dopamine-producing neurons were found to incorpo- National Health and Medical Research Council (NHMRC) rate into the striatum and improve performance in behav- of Australia and Mr James Fairfax. P.P.L.T. is a NHMRC ioral tests when grafted into mice that phenocopy Senior Principal Research Fellow. D.A.F. Loebel et al. / Developmental Biology 264 (2003) 1Ð14 11

Note added in proof. Two recent papers have described Ciruna, B., Rossant, J., 2001. FGF signaling regulates mesoderm cell fate transcription factor, Nanog, that can maintain ES cells in an specification and morphogenetic movement at the primitive streak. undifferentiated state independent of the LIF/STAT3 path- Dev. Cell 1, 37Ð49. Ciruna, B.G., Schwartz, L., Harpal, K., Yamaguchi, T.P., Rossant, J., 1997. way (Chambers et al. (2003) Cell 113, 643Ð55; Mitsui et al. Chimeric analysis of fibroblast growth factor receptor-1 (Fgfr1) func- (2003) Cell 113, 631Ð42). tion: a role for FGFR1 in morphogenetic movement through the primitive streak. Development 124, 2829Ð2841. Coffinier, C., Thepot, D., Babinet, C., Yaniv, M., Barra, J., 1999. Essential role for the homeoprotein vHNF1/HNF1 beta in visceral endoderm References differentiation. Development 126, 4785Ð4794. Damert, A., Miquerol, L., Gertsenstein, M., Risau, W., Nagy, A., 2002. Ahlgren, U., Jonsson, J., Edlund, H., 1996. The morphogenesis of the Insufficient VEGFA activity in yolk sac endoderm compromises pancreatic mesenchyme is uncoupled from that of the pancreatic epi- haematopoietic and endothelial differentiation. Development 129, thelium in IPF1/PDX1-deficient mice. Development 122, 1409Ð1416. 1881Ð1892. Ahlgren, U., Pfaff, S.L., Jessell, T.M., Edlund, T., Edlund, H., 1997. Deutsch, G., Jung, J., Zheng, M., Lora, J., Zaret, K.S., 2001. A bipotential Independent requirement for ISL1 in formation of pancreatic mesen- precursor population for pancreas and liver within the embryonic chyme and islet cells. Nature 385, 257Ð260. endoderm. Development 128, 871Ð881. Alexander, J., Stainier, D.Y., 1999. A molecular pathway leading to Dyer, M.A., Farrington, S.M., Mohn, D., Munday, J.R., Baron, M.H., endoderm formation in zebrafish. Curr. Biol. 9, 1147Ð1157. 2001. Indian hedgehog activates hematopoiesis and vasculogenesis and Ambrosetti, D.C., Basilico, C., Dailey, L., 1997. Synergistic activation of can respecify prospective neurectodermal cell fate in the mouse em- the fibroblast growth factor 4 enhancer by Sox2 and Oct-3 depends on bryo. Development 128, 1717Ð1730. protein-protein interactions facilitated by a specific spatial arrangement Elefanty, A.G., Robb, L., Birner, R., Begley, C.G., 1997. Hematopoietic- of factor binding sites. Mol. Cell. Biol. 17, 6321Ð6329. specific genes are not induced during in vitro differentiation of scl-null Ang, S.L., Conlon, R.A., Jin, O., Rossant, J., 1994. Positive and negative embryonic stem cells. Blood 90, 1435Ð1447. signals from mesoderm regulate the expression of mouse Otx2 in Endoh, M., Ogawa, M., Orkin, S., Nishikawa, S., 2002. SCL/tal-1-depen- ectoderm explants. Development 120, 2979Ð2989. dent process determines a competence to select the definitive hemato- Ang, S.L., Rossant, J., 1993. Anterior mesendoderm induces mouse En- poietic lineage prior to endothelial differentiation. EMBO J. 21, 6700Ð grailed genes in explant cultures. Development 118, 139Ð149. 6708. Aubert, J., Dunstan, H., Chambers, I., Smith, A., 2002. Functional gene Finley, M.F., Devata, S., Huettner, J.E., 1999. BMP-4 inhibits neural screening in embryonic stem cells implicates Wnt antagonism in neural differentiation of murine embryonic stem cells. J. Neurobiol. 40, 271Ð differentiation. Nat. Biotechnol. 20, 1240Ð1245. 287. Avilion, A.A., Nicolis, S.K., Pevny, L.H., Perez, L., Vivian, N., Lovell- Fong, G.H., Rossant, J., Gertsenstein, M., Breitman, M.L., 1995. Role of Badge, R., 2003. Multipotent cell lineages in early mouse development the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular depend on SOX2 function. Genes Dev. 17, 126Ð140. endothelium. Nature 376, 66Ð70. Bachiller, D., Klingensmith, J., Kemp, C., Belo, J.A., Anderson, R.M., Fong, G.H., Zhang, L., Bryce, D.M., Peng, J., 1999. Increased hemangio- May, S.R., McMahon, J.A., McMahon, A.P., Harland, R.M., Rossant, blast commitment, not vascular disorganization, is the primary defect in J., De Robertis, E.M., 2000. The organizer factors Chordin and Noggin flt-1 knock-out mice. Development 126, 3015Ð3025. are required for mouse forebrain development. Nature 403, 658Ð661. Fuhrmann, G., Chung, A.C., Jackson, K.J., Hummelke, G., Baniahmad, A., Barbacci, E., Reber, M., Ott, M.O., Breillat, C., Huetz, F., Cereghini, S., Sutter, J., Sylvester, I., Scholer, H.R., Cooney, A.J., 2001. Mouse 1999. Variant hepatocyte nuclear factor 1 is required for visceral germline restriction of Oct4 expression by germ cell nuclear factor. endoderm specification. Development 126, 4795Ð4805. Dev. Cell 1, 377Ð387. Barron, M., Gao, M., Lough, J., 2000. Requirement for BMP and FGF Fujiwara, T., Dunn, N.R., Hogan, B.L., 2001. Bone morphogenetic protein signaling during cardiogenic induction in non-precardiac mesoderm is specific, transient, and cooperative. Dev. Dyn. 218, 383Ð393. 4 in the extraembryonic mesoderm is required for allantois develop- Burdsal, C.A., Flannery, M.L., Pedersen, R.A., 1998. FGF-2 alters the fate ment and the localization and survival of primordial germ cells in the of mouse epiblast from ectoderm to mesoderm in vitro. Dev. Biol. 198, mouse. Proc. Natl. Acad. Sci. USA 98, 13739Ð13744. 231Ð244. Galceran, J., Farinas, I., Depew, M.J., Clevers, H., Grosschedl, R., 1999. Ϫ Ϫ Ϫ Byrd, N., Becker, S., Maye, P., Narasimhaiah, R., St Jacques, B., Zhang, Wnt3a / -like phenotype and limb deficiency in Lef1( / Ϫ Ϫ Ϫ X., McMahon, J., McMahon, A., Grabel, L., 2002. Hedgehog is re- )Tcf1( / ) mice. Genes Dev. 13, 709Ð717. quired for murine yolk sac angiogenesis. Development 129, 361Ð372. Galceran, J., Hsu, S.C., Grosschedl, R., 2001. Rescue of a Wnt mutation by Carmeliet, P., Ferreira, V., Breier, G., Pollefeyt, S., Kieckens, L., Gertsen- an activated form of LEF-1: regulation of maintenance but not initia- stein, M., Fahrig, M., Vandenhoeck, A., Harpal, K., Eberhardt, C., tion of Brachyury expression. Proc. Natl. Acad. Sci. USA 98, 8668Ð Declereq, C., Pawling, J., Moons, L., Collen, D., Risau, W., Nagy, A., 8673. 1996. Abnormal blood vessel development and lethality in embryos Gardner, R.L., Beddington, R.S., 1988. Multi-lineage “stem” cells in the lacking a single VEGF allele. Nature 380, 435Ð439. mammalian embryo. J. Cell Sci. Suppl. 10, 11Ð27. Chang, C., Holtzman, D.A., Chau, S., Chickering, T., Woolf, E.A., Gratsch, T.E., O’Shea, K.S., 2002. Noggin and chordin have distinct Holmgren, L.M., Bodorova, J., Gearing, D.P., Holmes, W.E., Brivan- activities in promoting lineage commitment of mouse embryonic stem lou, A.H., 2001. Twisted gastrulation can function as a BMP antago- (ES) cells. Dev. Biol. 245, 83Ð94. nist. Nature 410, 483Ð487. Gustafsson, M.K., Pan, H., Pinney, D.F., Liu, Y., Lewandowski, A., Ep- Chang, H., Huylebroeck, D., Verschueren, K., Guo, Q., Matzuk, M.M., stein, D.J., Emerson Jr., C.P., 2002. Myf5 is a direct target of long- Zwijsen, A., 1999. Smad5 knockout mice die at mid-gestation due to range Shh signaling and Gli regulation for muscle specification. Genes multiple embryonic and extraembryonic defects. Development 126, Dev. 16, 114Ð126. 1631Ð1642. Harrison, K.A., Thaler, J., Pfaff, S.L., Gu, H., Kehrl, J.H., 1999. Pancreas Chang, H., Matzuk, M.M., 2001. Smad5 is required for mouse primordial dorsal lobe agenesis and abnormal islets of Langerhans in Hlxb9- germ cell development. Mech. Dev. 104, 61Ð67. deficient mice. Nat. Genet. 23, 71Ð75. Chapman, D.L., Papaioannou, V.E., 1998. Three neural tubes in mouse Harrison, S.M., Dunwoodie, S.L., Arkell, R.M., Lehrach, H., Beddington, embryos with mutations in the T-box gene Tbx6. Nature 391, 695Ð697. R.S., 1995. Isolation of novel tissue-specific genes from cDNA librar- 12 D.A.F. Loebel et al. / Developmental Biology 264 (2003) 1Ð14

ies representing the individual tissue constituents of the gastrulating the generation of primordial germ cells in the mouse embryo. Genes mouse embryo. Development 121, 2479Ð2489. Dev. 13, 424Ð436. Hart, A.H., Hartley, L., Sourris, K., Stadler, E.S., Li, R., Stanley, E.G., Lawson, K.A., Meneses, J.J., Pedersen, R.A., 1986. Cell fate and cell Tam, P.P., Elefanty, A.G., Robb, L., 2002. Mixl1 is required for axial lineage in the endoderm of the presomite mouse embryo, studied with mesendoderm morphogenesis and patterning in the murine embryo. an intracellular tracer. Dev. Biol. 115, 325Ð339. Development 129, 3597Ð3608. Levinson-Dushnik, M., Benvenisty, N., 1997. Involvement of hepatocyte Hayashi, H., Abdollah, S., Qiu, Y., Cai, J., Xu, Y.Y., Grinnell, B.W., nuclear factor 3 in endoderm differentiation of embryonic stem cells. Richardson, M.A., Topper, J.N., Gimbrone Jr., M.A., Wrana, J.L., Falb, Mol. Cell. Biol. 17, 3817Ð3822. D., 1997. The MAD-related protein Smad7 associates with the TGF- Lickert, H., Kutsch, S., Kanzler, B., Tamai, Y., Taketo, M.M., Kemler, R., beta receptor and functions as an antagonist of TGFbeta signaling. Cell 2002. Formation of multiple hearts in mice following deletion of 89, 1165Ð1173. ␤-catenin in the embryonic endoderm. Dev. Cell 3, 171Ð181. Hayashi, K., Kobayashi, T., Umino, T., Goitsuka, R., Matsui, Y., Kita- Lien, C.L., McAnally, J., Richardson, J.A., Olson, E.N., 2002. Cardiac- mura, D., 2002. SMAD1 signaling is critical for initial commitment of specific activity of an Nkx2-5 enhancer requires an evolutionarily germ cell lineage from mouse epiblast. Mech. Dev. 118, 99. conserved Smad binding site. Dev. Biol. 244, 257Ð266. Hori, Y., Rulifson, I.C., Tsai, B.C., Heit, J.J., Cahoy, J.D., Kim, S.K., 2002. Lumelsky, N., Blondel, O., Laeng, P., Velasco, I., Ravin, R., McKay, R., Growth inhibitors promote differentiation of insulin-producing tissue 2001. Differentiation of embryonic stem cells to insulin-secreting struc- from embryonic stem cells. Proc. Natl. Acad. Sci. USA 99, 16105Ð tures similar to pancreatic islets. Science 292, 1389Ð1394. 16110. McLaren, A., Durcova-Hills, G., 2001. Germ cells and pluripotent stem Ikeya, M., Takada, S., 1998. Wnt signaling from the dorsal neural tube is cells in the mouse. Reprod Fertil. Dev. 13, 661Ð664. required for the formation of the medial dermomyotome. Development Mishina, Y., Suzuki, A., Gilbert, D.J., Copeland, N.G., Jenkins, N.A., 125, 4969Ð4976. Ueno, N., Behringer, R.R., 1995. Genomic organization and chromo- Ishizaka, S., Shiroi, A., Kanda, S., Yoshikawa, M., Tsujinoue, H., somal location of the mouse type I BMP-2/4 receptor. Biochem. Bio- Kuriyama, S., Hasuma, T., Nakatani, K., Takahashi, K., 2002. Devel- phys. Res. Commun. 206, 310Ð317. opment of hepatocytes from ES cells after transfection with the HNF- Miyazawa, K., Shinozaki, M., Hara, T., Furuya, T., Miyazono, K., 2002. 3beta gene. FASEB J. 16, 1444Ð1446. Two major Smad pathways in TGF-beta superfamily signalling. Genes Johansson, B.M., Wiles, M.V., 1995. Evidence for involvement of activin Cells 7, 1191Ð1204. A and bone morphogenetic protein 4 in mammalian mesoderm and Mukhopadhyay, M., Shtrom, S., Rodriguez-Esteban, C., Chen, L., Tsukui, hematopoietic development. Mol. Cell. Biol. 15, 141Ð151. T., Gomer, L., Dorward, D.W., Glinka, A., Grinberg, A., Huang, S.P., Kanai-Azuma, M., Kanai, Y., Gad, J.M., Tajima, Y., Taya, C., Kurohmaru, Niehrs, C., Izpisua Belmonte, J.C., Westphal, H., 2001. Dickkopf1 is M., Sanai, Y., Yonekawa, H., Yazaki, K., Tam, P.P., Hayashi, Y., 2002. required for embryonic head induction and limb morphogenesis in the Depletion of definitive gut endoderm in Sox17-null mutant mice. De- mouse. Dev. Cell 1, 423Ð434. velopment 129, 2367Ð2379. Mukhopadhyay, M., Teufel, A., Yamashita, T., Agulnick, A.D., Chen, L., Kawasaki, H., Mizuseki, K., Nishikawa, S., Kaneko, S., Kuwana, Y., Downs, K.M., Schindler, A., Grinberg, A., Huang, S.P., Dorward, D., Nakanishi, S., Nishikawa, S.I., Sasai, Y., 2000. Induction of midbrain Westphal, H., 2003. Functional ablation of the mouse Ldb1 gene results dopaminergic neurons from ES cells by stromal cell-derived inducing in severe patterning defects during gastrulation. Development 130, activity. Neuron 28, 31Ð40. 495Ð505. Kennedy, M., Firpo, M., Choi, K., Wall, C., Robertson, S., Kabrun, N., Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W., Roder, J.C., 1993. Keller, G., 1997. A common precursor for primitive erythropoiesis and Derivation of completely cell culture-derived mice from early-passage definitive haematopoiesis. Nature 386, 488Ð493. embryonic stem cells. Proc. Natl. Acad. Sci. USA 90, 8424Ð8428. Kim, J.H., Auerbach, J.M., Rodriguez-Gomez, J.A., Velasco, I., Gavin, D., Nichols, J., Zevnik, B., Anastassiadis, K., Niwa, H., Klewe-Nebenius, D., Lumelsky, N., Lee, S.H., Nguyen, J., Sanchez-Pernaute, R., Bank- Chambers, I., Scholer, H., Smith, A., 1998. Formation of pluripotent iewicz, K., McKay, R., 2002. Dopamine neurons derived from embry- stem cells in the mammalian embryo depends on the POU transcription onic stem cells function in an animal model of Parkinson’s disease. factor Oct4. Cell 95, 379Ð391. Nature 418, 50Ð56. Nishimura, F., Yoshikawa, M., Kanda, S., Nonaka, M., Yokota, H., Shiroi, Kinder, S.J., Tsang, T.E., Quinlan, G.A., Hadjantonakis, A.K., Nagy, A., A., Nakase, H., Hirabayashi, H., Ouji, Y., Birumachi, J., Ishizaka, S., Tam, P.P., 1999. The orderly allocation of mesodermal cells to the Sakaki, T., 2003. Potential use of embryonic stem cells for the treat- extraembryonic structures and the anteroposterior axis during gastru- ment of mouse parkinsonian models: improved behavior by transplan- lation of the mouse embryo. Development 126, 4691Ð4701. tation of in vitro differentiated dopaminergic neurons from embryonic Kruger, M., Mennerich, D., Fees, S., Schafer, R., Mundlos, S., Braun, T., stem cells. Stem Cells 21, 171Ð180. 2001. Sonic hedgehog is a survival factor for hypaxial muscles during Niwa, H., 2001. Molecular mechanism to maintain stem cell renewal of ES mouse development. Development 128, 743Ð752. cells. Cell Struct. Funct. 26, 137Ð148. Labosky, P.A., Barlow, D.P., Hogan, B.L., 1994. Mouse embryonic germ Niwa, H., Burdon, T., Chambers, I., Smith, A., 1998. Self-renewal of (EG) cell lines: transmission through the germline and differences in pluripotent embryonic stem cells is mediated via activation of STAT3. the methylation imprint of insulin-like growth factor 2 receptor (Igf2r) Genes Dev. 12, 2048Ð2060. gene compared with embryonic stem (ES) cell lines. Development 120, Niwa, H., Miyazaki, J., Smith, A.G., 2000. Quantitative expression of 3197Ð3204. Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES Ladd, A.N., Yatskievych, T.A., Antin, P.B., 1998. Regulation of avian cells. Nat. Genet. 24, 372Ð376. cardiac myogenesis by activin/TGFbeta and bone morphogenetic pro- Pelton, T.A., Sharma, S., Schulz, T.C., Rathjen, J., Rathjen, P.D., 2002. teins. Dev. Biol. 204, 407Ð419. Transient pluripotent cell populations during primitive ectoderm for- Lake, J., Rathjen, J., Remiszewski, J., Rathjen, P.D., 2000. Reversible mation: correlation of in vivo and in vitro pluripotent cell development. programming of pluripotent cell differentiation. J. Cell Sci. 113, 555Ð J. Cell Sci. 115, 329Ð339. 566. Persson, M., Stamataki, D., te, W.P., Andersson, E., Bose, J., Ruther, U., Lange, U., Saitou, M., Western, P., Barton, S., Surani, M., 2003. The Ericson, J., Briscoe, J., 2002. DorsalÐventral patterning of the spinal Fragilis interferon-inducible gene family of transmembrane proteins is cord requires Gli3 transcriptional repressor activity. Genes Dev. 16, associated with germ cell specification in mice. BMC Dev. Biol. 3, 1. 2865Ð2878. Lawson, K.A., Dunn, N.R., Roelen, B.A., Zeinstra, L.M., Davis, A.M., Pesce, M., Gioia, K.F., De Felici, M., 2002. Derivation in culture of Wright, C.V., Korving, J.P., Hogan, B.L., 1999. Bmp4 is required for primordial germ cells from cells of the mouse epiblast: phenotypic D.A.F. Loebel et al. / Developmental Biology 264 (2003) 1Ð14 13

induction and growth control by Bmp4 signalling. Mech. Dev. 112, Stat3 gene leads to early embryonic lethality. Proc. Natl. Acad. Sci. 15Ð24. USA 94, 3801Ð3804. Pevny, L.H., Sockanathan, S., Placzek, M., Lovell-Badge, R., 1998. A role Tam, P.P., Behringer, R.R., 1997. Mouse gastrulation: the formation of a for SOX1 in neural determination. Development 125, 1967Ð1978. mammalian body plan. Mech. Dev. 68, 3Ð25. Porcher, C., Swat, W., Rockwell, K., Fujiwara, Y., Alt, F.W., Orkin, S.H., Tam, P.P., Goldman, D., Camus, A., Schoenwolf, G.C., 2000. Early events 1996. The T cell leukemia oncoprotein SCL/tal-1 is essential for de- of somitogenesis in higher vertebrates: allocation of precursor cells velopment of all hematopoietic lineages. Cell 86, 47Ð57. during gastrulation and the organization of a meristic pattern in the Poulain, M., Lepage, T., 2002. Mezzo, a paired-like homeobox protein is paraxial mesoderm. Curr. Top. Dev. Biol. 47, 1Ð32. an immediate target of Nodal signalling and regulates endoderm spec- Tam, P.P., Snow, M.H., 1981. Proliferation and migration of primordial ification in zebrafish. Development 129, 4901Ð4914. germ cells during compensatory growth in mouse embryos. J. Embryol. Rajagopal, J., Anderson, W.J., Kume, S., Martinez, O.I., Melton, D.A., Exp. Morphol. 64, 133Ð147. 2003. Insulin staining of ES cell progeny from insulin uptake. Science Tam, P.P., Zhou, S.X., 1996. The allocation of epiblast cells to ectodermal 299, 363. and germ-line lineages is influenced by the position of the cells in the Rathjen, J., Haines, B.P., Hudson, K.M., Nesci, A., Dunn, S., Rathjen, gastrulating mouse embryo. Dev. Biol. 178, 124Ð132. P.D., 2002. Directed differentiation of pluripotent cells to neural lin- Tanaka, M., Kasahara, H., Bartunkova, S., Schinke, M., Komuro, I., Ina- eages: homogeneous formation and differentiation of a neurectoderm gaki, H., Lee, Y., Lyons, G.E., Izumo, S., 1998. Vertebrate homologs population. Development 129, 2649Ð2661. of tinman and bagpipe: roles of the homeobox genes in cardiovascular Rathjen, J., Lake, J.A., Bettess, M.D., Washington, J.M., Chapman, G., development. Dev. Genet. 22, 239Ð249. Rathjen, P.D., 1999. Formation of a primitive ectoderm like cell pop- Tanaka, S.S., Matsui, Y., 2002. Developmentally regulated expression of ulation, EPL cells, from ES cells in response to biologically derived mil-1 and mil-2, mouse interferon-induced transmembrane protein like factors. J. Cell Sci. 112, 601Ð612. genes, during formation and differentiation of primordial germ cells. Raz, R., Lee, C.K., Cannizzaro, L.A., d’Eustachio, P., Levy, D.E., 1999. Gene Expr. Patterns 2, 297Ð303. Essential role of STAT3 for embryonic stem cell pluripotency. Proc. Tremblay, K.D., Dunn, N.R., Robertson, E.J., 2001. Mouse embryos lack- Natl. Acad. Sci. USA 96, 2846Ð2851. ing Smad1 signals display defects in extra-embryonic tissues and germ Ridgeway, A.G., Petropoulos, H., Wilton, S., Skerjanc, I.S., 2000. Wnt cell formation. Development 128, 3609Ð3621. signaling regulates the function of MyoD and myogenin. J. Biol. Chem. Tremblay, K.D., Hoodless, P.A., Bikoff, E.K., Robertson, E.J., 2000. 275, 32398Ð32405. Formation of the definitive endoderm in mouse is a Smad2-dependent Robb, L., Begley, C.G., 1996. The helixÐloopÐhelix gene SCL: implicated process. Development 127, 3079Ð3090. in T-cell acute lymphoblastic leukaemia and in normal haematopoietic Tremblay, K.D., Saam, J.R., Ingram, R.S., Tilghman, S.M., Bartolomei, development. Int. J. Biochem. Cell Biol. 28, 609Ð618. M.S., 1995. A paternal-specific methylation imprint marks the alleles Robb, L., Lyons, I., Li, R., Hartley, L., Kontgen, F., Harvey, R.P., Metcalf, of the mouse H19 gene. Nat. Genet. 9, 407Ð413. D., Begley, C.G., 1995. Absence of yolk sac hematopoiesis from mice Tropepe, V., Hitoshi, S., Sirard, C., Mak, T.W., Rossant, J., van Der, K.D., with a targeted disruption of the scl gene. Proc. Natl. Acad. Sci. USA 2001. Direct neural fate specification from embryonic stem cells: a 92, 7075Ð7079. primitive mammalian neural stem cell stage acquired through a default Robertson, S.M., Kennedy, M., Shannon, J.M., Keller, G., 2000. A tran- mechanism. Neuron 30, 65Ð78. sitional stage in the commitment of mesoderm to hematopoiesis requir- Tsang, T.E., Khoo, P.L., Jamieson, R.V., Zhou, S.X., Ang, S.L., Behringer, ing the transcription factor SCL/tal-1. Development 127, 2447Ð2459. R., Tam, P.P., 2001. The allocation and differentiation of mouse pri- Rodda, S.J., Kavanagh, S.J., Rathjen, J., Rathjen, P.D., 2002. Embryonic mordial germ cells. Int. J. Dev. Biol. 45, 549Ð555. stem cell differentiation and the analysis of mammalian development. Tsang, T.E., Kinder, S.J., Tam, P.P., 1999. Experimental analysis of the Int. J. Dev. Biol. 46, 449Ð458. emergence of leftÐright asymmetry of the body axis in early postim- Rossant, J., McKerlie, C., 2001. Mouse-based phenogenomics for model- plantation mouse embryos. Cell Mol. Biol. 45, 493Ð503. ling human disease. Trends Mol. Med. 7, 502Ð507. Waldrip, W.R., Bikoff, E.K., Hoodless, P.A., Wrana, J.L., Robertson, E.J., Saitou, M., Barton, S.C., Surani, M.A., 2002. A molecular programme for 1998. Smad2 signaling in extraembryonic tissues determines anteriorÐ the specification of germ cell fate in mice. Nature 418, 293Ð300. posterior polarity of the early mouse embryo. Cell 92, 797Ð808. Sato, M., Kimura, T., Kurokawa, K., Fujita, Y., Abe, K., Masuhara, M., Weinstein, M., Yang, X., Li, C., Xu, X., Gotay, J., Deng, C.X., 1998. Yasunaga, T., Ryo, A., Yamamoto, M., Nakano, T., 2002. Identifica- Failure of egg cylinder elongation and mesoderm induction in mouse tion of PGC7, a new gene expressed specifically in preimplantation embryos lacking the tumor suppressor smad2. Proc. Natl. Acad. Sci. embryos and germ cells. Mech. Dev. 113, 91Ð94. USA 95, 9378Ð9383. Shalaby, F., Ho, J., Stanford, W.L., Fischer, K.D., Schuh, A.C., Schwartz, Wells, J.M., Melton, D.A., 2000. Early mouse endoderm is patterned by L., Bernstein, A., Rossant, J., 1997. A requirement for Flk1 in primitive soluble factors from adjacent germ layers. Development 127, 1563Ð and definitive hematopoiesis and vasculogenesis. Cell 89, 981Ð990. 1572. Shalaby, F., Rossant, J., Yamaguchi, T.P., Gertsenstein, M., Wu, X.F., Wichterle, H., Lieberam, I., Porter, J.A., Jessell, T.M., 2002. Directed Breitman, M.L., Schuh, A.C., 1995. Failure of blood-island formation differentiation of embryonic stem cells into motor neurons. Cell 110, and vasculogenesis in Flk-1-deficient mice. Nature 376, 62Ð66. 385Ð397. Shivdasani, R.A., Mayer, E.L., Orkin, S.H., 1995. Absence of blood for- Wiles, M.V., Johansson, B.M., 1997. Analysis of factors controlling pri- mation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL. mary germ layer formation and early hematopoiesis using embryonic Nature 373, 432Ð434. stem cell in vitro differentiation. Leukemia 11 (Suppl. 3), 454Ð456. Sirard, C., de la Pompa, J.L., Elia, A., Itie, A., Mirtsos, C., Cheung, A., Wiles, M.V., Johansson, B.M., 1999. Embryonic stem cell development in Hahn, S., Wakeham, A., Schwartz, L., Kern, S.E., Rossant, J., Mak, a chemically defined medium. Exp. Cell Res. 247, 241Ð248. T.W., 1998. The tumor suppressor gene Smad4/Dpc4 is required for Wilson, V., Rashbass, P., Beddington, R.S., 1993. Chimeric analysis of T gastrulation and later for anterior development of the mouse embryo. (Brachyury) gene function. Development 117, 1321Ð1331. Genes Dev. 12, 107Ð119. Winnier, G., Blessing, M., Labosky, P.A., Hogan, B.L., 1995. Bone mor- Sun, X., Meyers, E.N., Lewandoski, M., Martin, G.R., 1999. Targeted phogenetic protein-4 is required for mesoderm formation and pattern- disruption of Fgf8 causes failure of cell migration in the gastrulating ing in the mouse. Genes Dev. 9, 2105Ð2116. mouse embryo. Genes Dev. 13, 1834Ð1846. Wood, H.B., Episkopou, V., 1999. Comparative expression of the mouse Takeda, K., Noguchi, K., Shi, W., Tanaka, T., Matsumoto, M., Yoshida, Sox1, Sox2 and Sox3 genes from pre-gastrulation to early somite N., Kishimoto, T., Akira, S., 1997. Targeted disruption of the mouse stages. Mech. Dev. 86, 197Ð201. 14 D.A.F. Loebel et al. / Developmental Biology 264 (2003) 1Ð14

Yamaguchi, T.P., Bradley, A., McMahon, A.P., Jones, S., 1999. A Wnt5a Ying, Q.L., Stavridis, M., Griffiths, D., Li, M., Smith, A., 2003. Conver- pathway underlies outgrowth of multiple structures in the vertebrate sion of embryonic stem cells into neuroectodermal precursors in ad- embryo. Development 126, 1211Ð1223. herent monoculture. Nat. Biotechnol. 21, 183Ð186. Yamaguchi, T.P., Harpal, K., Henkemeyer, M., Rossant, J., 1994. fgfr-1 is Ying, Y., Liu, X.M., Marble, A., Lawson, K.A., Zhao, G.Q., 2000. Re- required for embryonic growth and mesodermal patterning during quirement of Bmp8b for the generation of primordial germ cells in the mouse gastrulation. Genes Dev. 8, 3032Ð3044. mouse. Mol. Endocrinol. 14, 1053Ð1063. Yamashita, H., Ten Dijke, P., Franzen, P., Miyazono, K., Heldin, C.H., Ying, Y., Qi, X., Zhao, G.Q., 2001. Induction of primordial germ cells 1994. Formation of hetero-oligomeric complexes of type I and type II from murine epiblasts by synergistic action of BMP4 and BMP8B receptors for transforming growth factor-beta. J. Biol. Chem. 269, signaling pathways. Proc. Natl. Acad. Sci. USA 98, 7858Ð7862. 20172Ð20178. Ying, Y., Zhao, G.Q., 2001. Cooperation of endoderm-derived BMP2 and Yamashita, J., Itoh, H., Hirashima, M., Ogawa, M., Nishikawa, S., Yurugi, extraembryonic ectoderm-derived BMP4 in primordial germ cell gen- T., Naito, M., Nakao, K., Nishikawa, S., 2000. Flk1-positive cells eration in the mouse. Dev. Biol. 232, 484Ð492. derived from embryonic stem cells serve as vascular progenitors. Na- Yoshikawa, Y., Fujimori, T., McMahon, A.P., Takada, S., 1997. Evidence ture 408, 92Ð96. that absence of Wnt-3a signaling promotes neuralization instead of Yan, J., Tanaka, S., Oda, M., Makino, T., Ohgane, J., Shiota, K., 2001. paraxial mesoderm development in the mouse. Dev. Biol. 183, 234Ð Retinoic acid promotes differentiation of trophoblast stem cells to a 242. giant cell fate. Dev. Biol. 235, 422Ð432. Yoshimizu, T., Obinata, M., Matsui, Y., 2001. Stage-specific tissue and Yang, Y., Guillot, P., Boyd, Y., Lyon, M.F., McMahon, A.P., 1998. cell interactions play key roles in mouse germ cell specification. De- Evidence that preaxial polydactyly in the Doublefoot mutant is due velopment 128, 481Ð490. to ectopic Indian Hedgehog signaling. Development 125, 3123Ð Yurugi-Kobayashi, T., Itoh, H., Yamashita, J., Yamahara, K., Hirai, H., 3132. Kobayashi, T., Ogawa, M., Nishikawa, S., Nishikawa, S.I., Nakao, K., Yin, Y., Lim, Y.K., Salto-Tellez, M., Ng, S.C., Lin, C.S., Lim, S.K., 2002. 2003. Effective contribution of transplanted vascular progenitor cells AFP(ϩ), ESC-derived cells engraft and differentiate into hepatocytes derived from embryonic stem cells to adult neovascularization in in vivo. Stem Cells 20, 338Ð346. proper differentiation stage. Blood 101, 2675.