Transcriptional profiling of mouse and human ES cells identifies SLAIN1,a novel stem cell gene

Claire E. Hirst a, Elizabeth S. Ng a, Lisa Azzola a, Anne K. Voss b, Tim Thomas b,Edouard G. Stanley a, Andrew G. Elefanty a,!

a Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, VIC 3800, Australiab The Walter and Eliza Hall Institute of Medical Research, Royal Parade, Parkville, VIC 3050, Australia

Received for publication 16 December 2005; accepted 20 January 2006

Abstract

We analyzed the transcriptional profiles of differentiating mouse embryonic stem cells (mESCs) and show that embryoid bodies (EBs)sequentially expressed genes associated with the epiblast, primitive streak, mesoderm and endoderm of the developing embryo, validating ESCs asa model system for identifying cohorts of genes marking specific stages of embryogenesis. By comparing the transcriptional profiles ofundifferentiated ESCs to those of their differentiated progeny, we identified 503 mESC and 983 hESC genes selectively expressed inundifferentiated ES cells. Over 75% of the mESC genes were expressed in hESC and vice versa, attesting to the underlying similarity of mESCsand hESCs. The expression of a cohort of 68 genes decreased greater than 2-fold during differentiation in both mESCs and hESCs. As well ascontaining many validated ESC genes such as Oct4 [Pou5f1], Nanog and Nodal, this cohort included an uncharacterised gene (FLJ30046), whichwe designated SLAIN1/Slain1. Slain1 was expressed at the stem cell and epiblast stages of ESC differentiation and in the epiblast, nervous system,tailbud and somites of the developing mouse embryo. SLAIN1 and its more widely expressed homologue SLAIN2 comprise a new family ofstructurally unique genes conserved throughout vertebrate evolution.© 2006 Elsevier Inc. All rights reserved.

Keywords: Embryonic stem cell; Cell differentiation; Transcriptional profiling; SLAIN1

Introduction

Embryonic stem cell lines established from the inner cellmass of preimplantation blastocysts can differentiate intoectoderm, mesoderm and endoderm (Keller, 1995; Reubinoffet al., 2000; Thomson et al., 1998), providing an opportunity tostudy early stages of mammalian development in vitro.Characterising the genes that regulate the stem cell state inboth mESCs and hESCs will facilitate the maintenance ofundifferentiated ESCs and their directed differentiation toclinically relevant lineages for cell replacement therapies ofthe future.

Several studies have addressed the hypothesis that stem cellsisolated from different sources might express a conservedcohort of ‘stemness’ genes. Although two separate reports

comparing the gene expression profiles of mESCs, neural stemcells (NSCs) and haematopoietic stem cells (HSCs) identified216 and 283 genes, respectively, only 6 genes were common toboth lists (Evsikov and Solter, 2003; Ivanova et al., 2002;Ramalho-Santos et al., 2002). When these data were comparedwith a subsequent study that identified 385 genes enriched inpopulations of ESCs, NSCs and retinal stem cells, only onegene was common to the three lists (Fortunel et al., 2003).These findings prompted the authors to question the conceptthat a universal set of ‘stemness’ genes accounted for theproperties of self-renewal and multipotency common to stemcells from different sources (Fortunel et al., 2003).

In an attempt to identify genes specific to ESCs, thetranscriptional profiles of ESCs were compared to a variety ofnon-stem cell sources including tumor samples, normal tissuesand cell lines. In this manner, anywhere from 124 to 1760cDNAs more highly expressed in ESCs were identified(Richards et al., 2004; Sperger et al., 2003; Tanaka et al.,

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! Corresponding author. Fax: +61 3 9905 0780.E-mail address: andrew.elefanty@med.monash.edu.au (A.G. Elefanty).

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2002). When gene sequences from cDNA libraries generatedfrom undifferentiated and differentiated hESCs were compared,Brandenberger et al. derived a list of 532 genes that wereincreased in undifferentiated cells (Brandenberger et al.,2004b). Highlighting the variability between published results,only 9 of this list of 532 genes were common with the‘stemness’ genes generated in the study of Ramalho-Santos etal. and only 32 with the ‘signature’ genes of Ivanova et al.(Ivanova et al., 2002; Ramalho-Santos et al., 2002).

When Bhattacharya and colleagues compared sets of genesenriched in mESCs and hESCs, they found that less than 30% oftheir 92 hESC ‘stemness’ genes were represented in publishedlists of mESCs genes (Bhattacharya et al., 2004; Ivanova et al.,2002; Ramalho-Santos et al., 2002; Tanaka et al., 2002).Similarly, a study by Sato et al. compared 918 genes enriched inundifferentiated H1 hESC to published mESC-derived data(Ramalho-Santos et al., 2002) and found that 227/541 (42%) ofthe genes with mouse orthologues were highly enriched inmESCs, also indicating a limited overlap in the gene expressionprofiles of hESC and mESC (Sato et al., 2003).

In this study, we analyzed the transcriptional profiles ofdifferentiating mESCs and demonstrate the sequential expres-sion of genes associated with embryonic epiblast, primitivestreak, mesoderm and endoderm, consistent with the hypothesisthat in vitro differentiation of ESCs recapitulates molecularchanges associated with normal embryonic development. Thesedata provided a rational framework for our prediction that theexpression of genes specific to undifferentiated mESC woulddecrease by day 4 of differentiation, the time when germ layergenes were activated. Using this criterion, a list of 503 putativemESC stem cell genes was identified and similar transcriptionalprofiling of undifferentiated and differentiated hESCs led to theidentification of an analogous population of 983 hESC putativestem cell genes. Attesting to the underlying similarity of mESCsand hESCs, greater than 75% of the mESC stem cell genes wereexpressed in undifferentiated hESCs and vice versa. A cohort of68 of these genes, whose transcription decreased greater that 2-fold during differentiation of both mESCs and hESCs, includedmany previously validated ESC stem cell genes as well as anovel gene, designated SLAIN1 (FLJ30046). In this report, weshow that SLAIN1 is expressed at the stem cell and epiblaststages of ESC differentiation and in the epiblast, nervoussystem, tailbud and somites of the developing mouse embryo.SLAIN1 and its more widely expressed homologue SLAIN2form a new family of structurally unique vertebrate genes.

Materials and methods

Culture and differentiation of mESCs and hESCs

W9.5 ES cells were cultured as previously described (Barnett and Kontgen,2001). The Slain1-!geo gene trapped mESC lines (XG385, XG752 andXH855) were obtained from the NIH sponsored NHLBI-BayGenomics (http://baygenomics.ucsf.edu/overview/projorg.html) and NCRR-Mutant Mouse Re-gional Resource Center (http://www.mmrrc.org/). The Slain1-!geo ES cell lineswere cultured under antibiotic selection (150 "g/ml Geneticin) in Knockout™DMEM supplemented with 15% Knockout™ Serum Replacement, non-essential amino acids, 2 mM Glutamine, 50 units/ml Penicillin, 50 "g/mlStreptomycin (all from Invitrogen), 50 mM 2-mercaptoethanol (Sigma), and

1000 units/ml LIF (Chemicon). Both the W9.5 and the Slain1-!geo ES cell lineswere differentiated as described (Ng et al., 2005a). Briefly, mESCs were seededat 5000 cells/ml in Differentiation Media comprising Iscove's ModifiedDulbecco's Medium (Invitrogen) supplemented with 15% FCS in non-adherentdishes for the indicated number of days. Alternatively, cells were differentiatedunder serum-free conditions in Chemically Defined Medium (Wiles andJohansson, 1999) modified as described (Ng et al., 2005a) in the presence orabsence of 10 ng/ml of BMP4 (R&D Systems). The hES2 cell line wasmaintained on monolayers of irradiated PMEFs as previously described(Reubinoff et al., 2000). hESC differentiation was induced by embryoid bodyformation in hanging drops in Differentiation Media for 5 days. Embryoidbodies were then plated on gelatin-coated dishes for a further 9 days to allowfurther differentiation and expansion.

Flow cytometry

Embryoid bodies were dissociated using trypsin/EDTA (Invitrogen)supplemented with 1% chicken serum (Hunter). FACS-Gal analysis wasperformed as described (Elefanty et al., 1999; Fiering et al., 1991). In someexperiments, dissociated cells were stained with antibodies directed against Flk-1 (directly conjugated to PE: BD Biosciences) or E-cadherin (Zymed) detectedwith allophycocyanin-conjugated goat anti-rat IgG (BD Biosciences) prior toFACS-Gal analysis. Cells were analyzed using a FACSCalibur runningCellQuest software (Becton Dickinson).

GeneChip sample preparation and hybridisation

Biotin-labelled cRNA was prepared essentially as specified by theAffymetrix standard protocol. Briefly, total RNA was isolated from cells usingQIAShredder and RNeasy mini columns including an in-column DNase Idigestion (Qiagen). cDNAwas synthesized from 7.5 "g of total RNA using anoligo dT(24) primer containing a T7 RNA polymerase binding site. BiotinylatedcRNA was produced using the BioArray High-Yield Transcript Labeling Kit(Enzo Diagnostics). Fifteen micrograms of labelled cRNA was fragmented for35 min at 94°C in fragmentation buffer before hybridisation. Hybridisation andwashing were performed according to Affymetrix recommended protocols.

Data analysis

Raw data analysisScanned GeneChip images were analyzed using MicroArray Suite 5.0 and

additional analysis performed using Data Mining Tool 3.0 (Affymetrix).Microarray data are available at the ArrayExpress web site (http://www.ebi.ac.uk/arrayexpress/) via Accession numbers E-MEXP-303 and E-MEXP-304.

Identification of differentially expressed genesDuplicate samples of undifferentiated mESCs and d4 mEBs from two

independent differentiations were analyzed by pairwise comparisons. Samplesof undifferentiated hES2 cells (from passage 19 and 128) were compared to EBsdifferentiated for 5 days (d5 hEBs) in hanging drops or d5 hEBs that were platedonto gelatin for further 9 days (d14 hEBs). ESCGs were characterised by thefollowing criteria: (1) they were present in both baseline samples, (2) they weredecreased in at least 3 of the 4 pairwise comparisons between undifferentiatedand differentiated samples, and (3) there was at least a 2-fold decrease in 2 of the4 pairwise comparisons.

Comparison of human and mouse enriched genesOrthologues for ESCGs located on either the Human HG-U133A and B or

mouse MG-U74Av2 GeneChips were identified using a combination of theNetAffx website (http://www.netaffx.com) and BLASTn searches (http://www.ncbi.nlm.nih.gov/BLAST/).

Expression analysis

Total RNA was extracted from adult mouse organs using the RNeasy MidiKit according to the manufacturer's instructions (Qiagen). A multi-tissue RNANorthern blot containing 10 "g of total RNA for each tissue was probed with a

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random-primed radiolabelled probe (MegaPrime Labelling Kit) spanning exons4 to 9 of mouse Slain1. Semi-quantitative RT-PCR was performed as previouslydescribed (Elefanty et al., 1997). Thirty cycles of amplification were used for allthe primer sequences at the annealing temperatures shown in SupplementaryTable 4.

Whole mount in situ hybridisation

A probe coding for nucleotides 894 to 1848 of the mouse Slain1 cDNA(accession number BC079866) was cloned into pBluescriptKS. Whole mount insitu hybridisation was performed using in vitro transcribed, digoxygenin-labelled cRNA essentially as described previously (Pera and Kessel, 1997).

Results

Differentiating mouse ES cells sequentially expressdevelopmental stage-specific genes

In order to document the repertoire of genes expressed inESC and their differentiated progeny, we analyzed thetranscriptional profiles of mESCs at daily intervals over 6days of differentiation using the murine U74Av2 AffymetrixGeneChip. The microarray data were validated by semi-quantitative RT-PCR analysis of selected genes that markedsequential developmental stages in the embryo, including innercell mass (Oct4 [Pou5f1], Rex1 [Zfp42], and Gbx2), epiblast(Fgf5), primitive streak (Brachyury [T]), and haematopoieticmesoderm (Flk1 [Kdr] and !H1 globin [Hbb-b1]) (Supplemen-tary Fig. 1). These data correlated well with previously reportedpatterns of gene expression during ES cell differentiation(Keller et al., 1993; Lacaud et al., 2002; Ng et al., 2005a;Robertson et al., 2000).

GeneChip analysis revealed the concerted expression ofdifferentiation stage-specific genes in a series of distinct‘waves’ (Fig. 1). The ‘signature’ genes that have been chosento demonstrate this phenomenon have been shown by others tobe expressed at the appropriate stages in embryonic develop-ment, thus establishing a link between the data generated fromin vitro ES differentiation and the embryo. The expression ofmost validated stem cell genes such as Oct4, FoxD3, Rex1,Gbx2, and Sox2 was high in undifferentiated mESCs and hadwaned significantly by d4 of differentiation consistent with theirexpression in the early embryo (see examples in Pelton et al.,2002) (Fig. 1A). The biphasic expression of Nanog, with peaksof expression at d0 and at d3 of differentiation, mirrored itsdistribution in vivo in the inner cell mass of the preimplantationblastocyst and later in the epiblast adjacent to the elongatingprimitive streak (Hart et al., 2004). The second wave of genesthat peaked in expression at d2–3 included the epiblast marker

Fig. 1. Sequential expression of genes during mESC differentiation reflectssuccessive stages of embryological development. Relative transcript level forsets of genes indicative of the developmental stages representing (A) stem cells,(B) epiblast, (C) primitive streak, (D) ventral mesoderm, and (E) endoderm.Relative transcript levels represent the signal strength for each transcriptexpressed as a percentage of the maximal signal for that gene. (F) Expressionprofile of single genes representing each of the developmental stages shown inpanels A–E demonstrating that the temporal relationship between each stage ofESC differentiation mirrors the sequential phases of embryonic development.

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Fgf5 (Haub and Goldfarb, 1991; Hebert et al., 1991; Pelton etal., 2002) and a number of genes including Retinoid X receptorgamma (Rxrg), Occludin (Ocln), and Claudin 6 and 7 (Cldn6and 7) (Fig. 1B), which have been associated with epithelial-ization (Kubota et al., 2001; Turksen and Troy, 2001). At d3–4,we identified a third stage of differentiation that wascharacterised by a rapid, transient increase in transcription ofthe primitive streak markers such as Brachyury, Eomesodermin[Eomes], Wnt3, and Fgf8 (see examples in Ciruna and Rossant,1999; Liu et al., 1999) (Fig. 1C). Consistent with the fact thathaematopoietic and endothelial cells are the first mesodermallineages to emerge from the primitive streak (Kinder et al.,1999), we noted an upregulation in the expression of genesmarking ventral mesoderm including Flk1, Gata1-3, Runx1, Scl[Tal1], and !H1 globin in d5 and d6 EBs (see examples inElefanty et al., 1999; Levanon et al., 2001; Yamaguchi et al.,1993) (Fig. 1D and data not shown). Endodermal genes such asGata6, Sparc, Keratin 19 (Krtl-19), and Collagen type IV, alpha5 (Col4a5) were also expressed at this stage (see examples inHolland et al., 1987; Morrisey et al., 1996; Tamai et al., 2000)(Fig. 1E). However, it was not possible to unambiguouslyclassify the type of endoderm formed because many of thesegenes are expressed in both primitive and in definitiveendodermal lineages. The sequential overlapping nature of thedifferentiation stages was evident when the expression levels forindividual genes representing examples of each developmentalstage were superimposed on the same graph (Fig. 1F).

Stem cell genes are conserved in mouse and human ESCs

Examination of global changes in gene transcription duringmESC differentiation indicated that extensive changes in geneexpression accompanied the establishment of germ layers.Specifically, 723 probe sets decreased and 704 probe setsincreased more than 2-fold when d0 and d4 samples werecompared in one experiment (Fig. 2). Because most of thevalidated stem cell genes (Fig. 1A) were significantly down-

regulated during this interval, we hypothesized that stem cellgenes could be operationally defined as genes expressed inundifferentiated ES cells whose transcription decreased in theirdifferentiated progeny.

Therefore, we nominated genes whose expression fell bygreater than 2-fold between d0 and d4 as embryonic stem cellgenes (ESCGs) (see Materials and methods). Of the 503 genesthat fulfilled this criterion, 436 (86.7%) represented namedgenes while the remaining 67 (13.3%) comprised ESTs, RIKENclones, and other uncharacterised transcripts (Fig. 3A). Wehypothesized that human orthologues of mESCGs should alsobe expressed in hESCs and should also decrease during hESCdifferentiation. Orthologues represented on the HG-U133 A andB chips were identified for 89.3% (449) of the mESCGs,comprising 94.5% (412/436) of the named genes and 55.2% (37/67) of the ESTs/RIKEN clones. Of these human orthologues,75.9% (341/449) were expressed in undifferentiated hESCs,testifying to the underlying similarity of ESCs in the two species(Fig. 3A). Expression of 34.3% (117/341) genes decreasedduring differentiation, although the signal intensity decreasedless than 2-fold in most cases. The signal of an additional 43.4%(148/341) genes remained stable, and the expression of 22.3%(76/341) genes increased (Fig. 3A and Supplementary Table 1).

A complementary experiment in which the transcriptionalprofiles of undifferentiated hESCs were compared with theirdifferentiated counterparts identified 983 hESCGs that de-creased by greater than 2-fold with differentiation (Fig. 3B).52.6% (517/983) of the hESCGs had mouse orthologuesrepresented on the MG-U74Av2 chip, and of these orthologues,79.9% (413/517) were expressed in undifferentiated mESCs(Fig. 3B). Similar to our observations with the mESCGs, 37.3%(154/413) of the hESCGs decreased to some degree duringmESC differentiation whilst the remainder either remainedunchanged (35.1%, 145/413) or increased (27.6%, 114/413)(Fig. 3B and Supplementary Table 2).

Combining the 117 mESCGs with human orthologues whoseexpression decreased during hESC differentiation (Fig. 3A)

Fig. 2. Extensive changes in gene expression accompanied the establishment of germ layers. Graph of global changes in gene expression showing the total number ofprobes sets at each day of differentiation whose signal had increased (!) or decreased (") greater than 2-fold when compared to d0 mESCs. The greatest rate ofchange in number of probe sets that conformed to the above criteria occurred between day 3 and 4 which corresponds to a time when cells are undergoing the molecularequivalent of gastrulation.

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with the 154 similarly defined hESCGs (Fig. 3B) yielded a totalof 203 genes specific to undifferentiated ESCs (udESCGs) (Fig.4A). Sixty-eight of these genes (58% of the mESCGs)decreased more than 2-fold with differentiation in both specieswhilst 86 hESCGs and 49 mESCGs downregulated by lesseramounts in both differentiated mouse and human ESCs,respectively (Fig. 4B and Supplementary Table 3). This ‘coregroup’ of 68 genes included many with validated roles inmaintaining the stem cell properties of ESCs such as Oct4,Rex1, Foxd3, Nanog, and Sox2, as well as other genes that playimportant roles in early embryonic development such as Gdf3,Nodal, Phc1, and Utf1. Classification of the remaining humanand mouse ESCGs revealed that these represented intracellularsignalling molecules, metabolic enzymes, transcription factors,and secreted factors (Fig. 4B).

The 68 core ESCGs also included three incompletelycharacterised transcripts encoding hypothetical proteinFLJ30046, HCV F-transactivated protein 1, and developmentalpluripotency associated 4 (Dppa4). Dppa4 has recently beendescribed as a gene with a similar transcriptional profile toOct4 (Bortvin et al., 2003) and, along with its humanorthologue hypothetical protein FLJ10713, has been identifiedas an ESC gene in several recent transcriptional profilingexperiments (Brandenberger et al., 2004a; Rao et al., 2004;Richards et al., 2004; Sato et al., 2003; Sperger et al., 2003).However, the other genes, FLJ30046 (with its respectivemouse orthologue 9630044O09Rik) and LOC401152 (alsoknown as HCV F-transactivated protein 1 and its mouseorthologue 1810037I17Rik), have not been previously identi-fied as potential stem-cell-specific genes. We have focussed onFLJ30046 and its mouse orthologue 9630044O09Rik, whichwe designated SLAIN1 due to the presence of a C-terminal‘SLAIN’ amino acid motif.

SLAIN1 and SLAIN2 encode related genes conserved invertebrates

Examination of public sequence databases revealed thatSLAIN1 was well conserved in vertebrates, with ESTs frommouse and human 84.8% identical at the nucleotide level.Homologues of SLAIN1 were identified in rat, chicken, cow,pig, frog, and zebrafish (Fig. 4A and Supplementary Fig. 2). Asecond highly related cluster of transcripts including5033405K12Rik and KIAA1458 in mouse and humans,respectively, were also identified and designated as SLAIN2(Supplementary Fig. 3). This gene was also highly conserved invertebrates, suggesting that SLAIN1 and SLAIN2 representedtwo members of a novel gene family (Fig. 5A).

The 8 exons of the mouse Slain1 gene spanned approxi-mately 55 kb of mouse chromosome 14 (Fig. 6). Sequenceanalysis of ESTs derived from mouse Slain1 mRNA indicatedthe presence of a splice variant in which exon 3 was omitted,resulting in a 50 amino acid internal deletion that did not alterthe reading frame. This variable splicing was confirmed by RT-PCR analysis of cDNA from mouse ESCs and adult mousebrain, from which both longer and shorter products wereobtained (data not shown).

The shorter human SLAIN1 ESTs corresponded to exons 4 to8 of the mouse Slain1 gene and mapped to human chromosome13 in the region syntenic to mouse chromosome 14. Threeupstream human SLAIN1 exons were predicted, based on thehigh degree of similarity between the human and the mouseSlain1 loci, resulting in a complete human SLAIN1 gene alsoconsisting of 8 exons spanning 65 kb of genomic sequence. Thesizes of both exons and introns were well conserved betweenthe two species, and all intron–exon boundaries conformed tothe AG/GT rule (Fig. 6). A similar genomic organization ofhuman and mouse SLAIN2 genes was observed, with homo-logues in both species consisting of 8 exons of a similar size anddistribution to those found in SLAIN1. The SLAIN2 genesspanned 75 kb of human chromosome 4 and 60 kb of thesyntenic region of mouse chromosome 5 (Fig. 6). The highdegree of similarity in the nucleotide sequence and geneticstructure suggests that SLAIN1 and 2 are members of the samegene family.

Conceptual translations of the open reading frames of mouseSlain1 mRNA and the putative full-length transcript of humanSLAIN1 predicted proteins of 629 and 640 amino acids,respectively, with 86.3% identity (Fig. 5B). Mouse andhuman SLAIN2 proteins both predicted to encode proteins of581 amino acids, were 93.8% identical, and were 37.1% and36.3% identical to their respective SLAIN1 homologues (Fig.5B). Analysis of the protein sequence using MotifScan (http://myhits.isb-sib.ch/cgi-bin/motif_scan) indicated the presence ofserine-rich domains in both SLAIN1 and SLAIN2, whileSLAIN1 also contained an N-terminal proline-rich domain (Fig.5B). While proline- and serine-rich domains can mediateprotein–protein interactions, at this stage, their functionalimportance in SLAIN1 and 2 is speculative.

SLAIN1 is expressed at the ‘epiblast’ stage of ESCdifferentiation prior to gastrulation and in the embryonicepiblast and nervous system

RT-PCR analysis of Slain1 expression in differentiatingmESCs revealed maximal Slain1 mRNA levels on d2, whichdeclined by d6 of mESC differentiation. Similarly, in hESCdifferentiation, robust expression of SLAIN1 mRNA wasobserved in undifferentiated hESCs, which also waned duringhESC differentiation. These RT-PCR results were consistentwith the relative transcript levels observed in the microarraydata (Figs. 7A, B). Slain1 was also detected in adult thymus,brain, testis, and lung by Northern blot and also in bone marrowand kidney by RT-PCR (Figs. 7C and D). RT-PCR analysis ofSlain2 mRNA indicated that it was not expressed in a restrictedmanner (data not shown) and has not been investigated further.

We identified three mESC lines generated in the BayQGenomics Project (http://baygenomics.ucsf.edu/overview/projorg.html) in which gene trap vectors had integrated into the mouseSlain1 locus. Three clones (XG385, XG752, and XH855) thatcontained a !geo fusion gene inserted into intron B of Slain1 (asillustrated Fig. 8A) were obtained from the Mutant MouseRegional Resource Center (http://www.mmrrc.org/). Flowcytometry was used to determine the percentage of viable

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cells expressing !galactosidase (!gal) from the Slain1 locusduring mESC differentiation using the FACS-Gal technique(Fiering et al., 1991). Consistent with the RT-PCR analysis ofSlain1 transcription (Fig. 7A), approximately 20% of undiffer-entiated mESCs expressed Slain1-!gal, and this increased toinclude over 80% of cells at d3 of differentiation, with the slightdelay in the peak in Slain1-!gal expression as compared to thepeak in Slain1 mRNA at d2, reflecting the longer processingtime required for expression of the !gal protein (Fig. 8B). Theproportion of !gal-expressing cells diminished rapidly thereaf-ter. A similar expression profile was observed for each of thethree Slain1-!gal ESC lines (Fig. 8C).

Based on our transcriptional profiling data (Fig. 1B), thepattern of Slain1 mRNA expression suggested that it repre-sented an ‘epiblast stage’ gene. One prediction of thishypothesis was that Slain1 mRNA would be co-expressedwith E-cadherin, a marker of stem cells, epiblast, and primitivestreak (Burdsal et al., 1993; Ciruna and Rossant, 2001; Ng et al.,2005a). Indeed, all !gal-expressing cells at d3 co-expressed E-cadherin (Fig. 8D). By d4, some !gal-expressing cells were E-cadherin!, either indicating that Slain1 transcription persisted insome epiblast or primitive streak derivatives or reflecting thelong half-life of the !geo reporter gene (Fig. 8D). Analysis ofthe same cells for expression of the ventral mesoderm markerFlk1 revealed that most Flk1+ cells did not express !gal,indicating that Slain1 was not significantly expressed in ventralmesoderm.

In previous studies, it had been observed that mESCsdifferentiated in serum-free (SF) media defaulted to neurecto-dermal differentiation and supplementation of the medium withbone morphogenetic protein (BMP) 4 induced expression ofprimitive streak markers and ventrally patterned mesoderm,leading to blood cell formation (Ng et al., 2005a; Park et al.,2004; Wiles and Johansson, 1999). Therefore, we investigatedthe expression of !gal in Slain1-!gal ESCs differentiated in SFmedium in the absence and presence of BMP4 (Fig. 8E). In thepresence of BMP4, a similar profile of !gal expression wasobserved as in serum-containing cultures, with over 80% ofcells expressing !gal at d3 and fewer than 10% !gal-positive byd6. In Slain1-!gal ESCs differentiated in the absence of BMP4,however, !gal expression was maintained in over 60% of cellseven at d6. Most of these cells no longer expressed E-cadherin(data not shown), suggesting that the !gal-positive cells wereno longer at the epiblast stage and that Slain1 expressioncontinued in primitive neurectodermal cells. Consistent withthese predictions, whole mount in situ hybridisation of post-implantation mouse embryos demonstrated widespread expres-sion of Slain1 throughout the E6.5–E7.0 embryo (Figs. 9A–D)that was followed by higher levels of expression in the headfoldneurectoderm at E7.5 (Figs. 9E, F). As embryonic developmentcontinued, Slain1 mRNA was observed in the neural tube and

optic vesicles at E8.5 and then at sites of imminent neural tubeclosure in the midbrain, hindbrain, and tailbud at E9.0–E9.5 andin the dorsal aspects of the somites (Figs. 9G–L).

Discussion

We have utilised transcriptional profiling to demonstrate thesequential expression of genes representing stem cell, epiblast,primitive streak, and germ layer stages of development indifferentiating mESCs. These data extend prior observationsthat ESC differentiation represents a valid facsimile for earlypost-implantation mammalian embryogenesis by providingexpression data for a larger number of stage-specific genesthan previous studies have examined (Doetschman et al., 1985;Keller et al., 1993; Leahy et al., 1999; Palmqvist et al., 2005;Schmitt et al., 1991; Shen and Leder, 1992; Wiles andJohansson, 1999; Wiles and Keller, 1991). The concordancebetween the stage-specific expression of genes observed duringES cell differentiation and during embryonic developmentsuggests that the mechanisms that regulate and coordinate theseprocesses in vivo are, to a significant degree, operational invitro. This is an important validation of the ES differentiationsystem and gives confidence that the in vitro differentiationprocess is predictable and that findings in vitro are relevant tothe intact organism.

Our analysis indicated that, by d4 of ES differentiation,substantial changes in the repertoire of expressed genes hadoccurred, including the downregulation of known stem cellgenes (see Figs. 1 and 2). This information was used to devisean operational definition for ESC genes, using the criteria ofexpression in undifferentiated ESCs followed by downregula-tion by the time of germ layer formation. In order to morestringently define genes likely to be of broad functionalimportance in ESCs, we narrowed the inclusion criteria toonly include those mESC genes with human orthologues thatwere also restricted in expression to undifferentiated hESCs andvice versa. This defined a group of 203 genes enriched inundifferentiated ESC genes (udESCGs) that were downregu-lated 2-fold during differentiation in one species and alsodownregulated to some degree in the other (117 from mESCsand 154 from hESCs). This included a core group of 68 ESCGsthat demonstrated a greater than 2-fold reduction in expressionduring both mouse and human ESC differentiations.

A number of different studies have attempted to identifygenes whose expression defines the stem cell state (Abeyta etal., 2004; Bhattacharya et al., 2004; Brandenberger et al.,2004b; Fortunel et al., 2003; Ivanova et al., 2002; Ramalho-Santos et al., 2002; Rao et al., 2004; Richards et al., 2004; Satoet al., 2003; Sperger et al., 2003; Tanaka et al., 2002). However,the lists of postulated stem cell genes generated in differentstudies contained few members in common (Palmqvist et al.,

Fig. 3. Identification of embryonic stem cell genes (ESCGs) during differentiation of (A) mouse and (B) human ES cells. 503 mouse ESCGs and 983 human ESCGswere identified based on their expression in undifferentiated cells and downregulation by at least 2-fold in differentiated progeny. Orthologues of ESCGs on human andmouse GeneChips were identified and expression in undifferentiated and differentiated cells evaluated. By these analyses, human orthologues of 117 mESCGs wereexpressed in hESCs and decreased with hESC differentiation and mouse orthologues of 154 hESCGs were expressed in mESCs and decreased with mESCdifferentiation. See text for further details.

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Fig. 4. Embryonic stem cell genes shared between mouse and human ESCs. (A) Summary of the filters used to identify the mouse and human ESCGs described in Figs.3A and B. The primary filter (1°) identified genes expressed (P) in undifferentiated ESCs (U) that decreased at least 2-fold (dec) in differentiated cells (D). Thesecondary filter (2°) identified orthologues for these genes in the reciprocal species that were present in undifferentiated cells and downregulated (lo) duringdifferentiation. (B) Venn diagram demonstrating the overlap between the 117 mouse (blue) and 154 human ESCGs (red), demonstrating that 203 genes are enriched inundifferentiated ESCs (udESC genes). This includes 68 genes that were downregulated 2-fold in both mouse and human ESCs (core ESCGs) with 49 and 86 genes thatwere downregulated to a lesser extent in mouse and human ESCs, respectively. These genes were classified by their GO Biological Process descriptions.

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Fig. 5. High degree of homology between mammalian Slain1 and Slain2 orthologues. (A) Dendrogram of mammalian Slain1 and Slain2 genes. Consensus nucleotidesequences fromHomo sapiens (Hs),Mus musculus (Mm), Rattus norvegicus (Rn), Bos taurus (Bt), and Sus scrofa (Ss) were aligned and the phylogenetic tree drawn inMegAlign 6.00 (DNASTAR). (B) Slain1 and Slain2 proteins from mouse (Mm) and human (Hs) were aligned using the Clustal W method in MegAlign 6.00(DNASTAR). Amino acids identical to mouse Slain1 are indicated by white lettering on a black background. An asterisk indicates the SLAIN motif.

Fig. 6. Slain1 and Slain2 gene loci share a conserved intron–exon structure. Nucleotide sequences of human (h) and mouse (m) Slain1 and Slain2 transcripts werealigned to corresponding genomic sequences using MegaBLAST (http://www.ncbi.nlm.nih.gov/blast/megablast.shtml) and intron–exon boundaries confirmed usingSpidey (http://www.ncbi.nlm.nih.gov/IEB/Research/Ostell/Spidey). Exon sizes (in nucleotides) are indicated within each box, and approximate intron sizes (in kb) areshown between boxes.

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2005). Therefore, it was not surprising that the 283 ‘signature’genes described by Ivanova et al. included only 5 of our 203udESCGs, that only 8 were incorporated in the 216 ‘stemness’genes of Ramalho-Santos et al., and that no genes were shared

between all three studies (Ivanova et al., 2002; Ramalho-Santoset al., 2002). Furthermore, only 3 of the ‘signature’ genesdescribed by Ivanova et al. and none of the ‘stemness’ genes ofRamalho-Santos et al. were included in our 68 core ESCGs. Thediscrepancies between these gene lists could be contributed toby the multiplicity of tissues compared in some studies,variations in the purity of the stem cell populations analyzed,and the possibility that related but not identical genes performedsimilar functions in different stem cell populations (Burdon etal., 2002; Fortunel et al., 2003; Ivanova et al., 2003). However,an equally plausible conclusion from these studies is that thereis no common set of genes whose expression defines stem cellcharacteristics for all tissue types.

We observed the greatest similarity between our data and thatreported by Palmqvist et al., who examined transcriptionalchanges occurring during the first 72 h of mESCs differentiation(Palmqvist et al., 2005). Comparison of our 503 mESCGs withtheir 187 genes ‘decreased after 72 h of LIF removal’ indicatedthat nearly 50% (93/187) of these genes were included in ourdata set. Surprisingly, only 14 genes were shared with our 68core ESCGs. This reduced overlap was in part due to the factthat the study of Palmqvist et al. omitted a number of well-characterised ESC genes such as Oct4, Nanog, and FoxD3 thatwere downregulated more slowly and thus were missed by theiruse of a 72 h endpoint (see Fig. 1).

However, comparisons of the results of other studies thatsearched for genes specifically expressed in ESCs revealed alow degree of overlap. Therefore, it was not surprising thatonly 3/25 ‘most positively significant genes’ in hESC reportedby Sperger et al., 6/66 ‘candidate hESC signature genes’ fromGolan-Mashiach et al., 4/92 genes ‘common to 6 hESC lines’identified by Bhattacharya et al., 11/227 ‘genes enriched inboth hESCs and mESCs’ from Sato et al., and 11/133‘pluripotent specific genes’ described by Rao and colleagueswere included in our list of 68 core ESCGs (Bhattacharya etal., 2004; Golan-Mashiach et al., 2005; Sperger et al., 2003). Ittranspired that Oct4 was the only gene identified by all of thesecomparisons.

It is notable that our study was the only one that directlycompared mouse and human ES cells using material cultured,harvested, and processed in the same laboratory, assayed on thesame platform type, and analyzed using the same data analysissoftware. As highlighted in recent literature, variability in themicroarray platform, labelling and hybridisation protocols, anddata analysis software contributed significantly to the lack ofconcordance observed between different groups' comparisonsof similar samples (Bammler et al., 2005; Irizarry et al., 2005;Larkin et al., 2005). It is reasonable to suggest that these

Fig. 7. Slain1 is expressed during ESC differentiation and in selected adulttissues. (A and B) RT-PCR analysis of Slain1 expression during ESCdifferentiation indicates that Slain1 expression (A) peaked at d2 and d3 ofmESC differentiation and (B) was robustly expressed in undifferentiated hESCs,correlating with the relative transcript levels calculated from GeneChip signalsfor human and mouse Slain1 probe sets. (C and D) Tissue distribution of mouseSlain1mRNA. (C) Semi-quantitative RT-PCR analysis of Slain1mRNA in adultmouse tissues compared to HPRT expression. (D) Northern blot analysis ofSlain1mRNA in adult mouse tissues with 28S and 18S RNA as loading control.

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problems contributed to the disparity between data sets reportedin other stem-cell-related gene profiling studies. In this context,it is relevant that we observed a greater than 75% concordancebetween the genes expressed in mESCs and hESCs, indicatingthat ESCs from these two species may indeed be more similarthan other studies have suggested (Bhattacharya et al., 2004;Ginis et al., 2004; Sato et al., 2003).

Furthermore, our list of genes included most of therecognized ESC genes including FGF4, FoxD3, Nanog,Nodal, Oct4, Rex1, Sox2, and Utf1 (as reviewed in (Rao,2004) and also encompassed other genes with predicted roles inmaintaining ESC pluripotency such as SFRP1, a modulator ofWnt signalling, LeftB, a member of the TGF! signallingpathway, and DNMT3a, a gene required for the establishment

and maintenance of methylation patterns in mESCs (Chen et al.,2003). This is the first study to show that this cohort of stem cellgenes is downregulated in a similar manner during both hESCand mESC differentiation, consistent with the similar patterns ofin vitro differentiation of mESC and hESC that we haveobserved (Ng et al., 2005a, 2005b).

Our core list of 68 ESCGs included three incompletelycharacterised transcripts encoding hypothetical proteinFLJ30046, LOC401152 (gene designation HCV F-transacti-vated protein 1), and developmental pluripotency associated 4(Dppa4). Dppa4 was recently identified along with Dppa2,Dppa3 (also known as Stella or PCG7), and Dppa5 (Esg1) in ascreen for genes with similar transcriptional profiles to Oct4(Bortvin et al., 2003).

Fig. 8. Expression of Slain1 during mESC differentiation. (A) Cartoon of mouse Slain1 gene structure indicating the insertion site of the En2 !geo cassette in intron B.(B) Time course of !galactosidase expression in differentiating XG385 Slain1-!geo gene trapped ESCs as determined by FACS-Gal. The percentage of !gal-positivecells is shown in the lower right of each plot. (C) Graph showing the percentage (mean ± SD) of !gal-positive cells for all three Slain1-!geo ESC lines throughoutdifferentiation. (D) Correlation of E-cadherin and Flk-1 with !gal expression in differentiating Slain1-!geo ESCs. (E) Differentiation of Slain1-!geo ESCs in serum-free (SF) media supplemented with (+) or without (!) BMP4.

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In this study, we focussed our analysis on FLJ30046(9630044O09Rik), a gene that we subsequently designatedSLAIN1 (Slain1). The presence of serine- and proline-richdomains within the protein sequence suggests that Slain1 mayinteract with other proteins, but the nature of these interactions isnot yet clear. In vitro differentiation of Slain1-!geo gene trappedmESC lines demonstrated a peak of Slain1 expression at d2–3(epiblast stage), in keeping with the microarray and PCR data.Further analysis of !galactosidase activity in these linesindicated that Slain1 expression was downregulated in Flk-1expressing ventral mesoderm but persisted in ESCs differenti-ating towards neurectodermal in SF medium in the absence ofgrowth factors. The applicability of these results to the earlymouse embryo was demonstrated by in situ hybridisation, whichdemonstrated widespread Slain 1 expression in the pregastrula-tion embryo and, subsequently, the developing nervous systemand somites. In the adult, Slain1 mRNA was found in brain,thymus, testis and lung by Northern blot and also in bonemarrow and kidney by RT-PCR, indicating that Slain1expression was not restricted to ectodermal derivatives.

The localisation of Slain1 to mouse chromosome 14 placedthe gene within the piebald coat colour-spotting locus that

spans an 18 cM interval surrounding the endothelin B receptor(Ednrb) gene. Deletion complementation analysis has definedseveral critical regions in this locus that delineated distinctphenotypes of respiratory distress syndrome, skeletal patterningdefects and spinal cord malformations (Peterson et al., 2002).The Slain1 gene appears to lie within a 1.3 cM deletion regiondesignated as Ednrbs-1Acrg (Welsh and O'Brien, 2000). TheEdnrbs-1Acrg mutant mice exhibited predominantly skeletalpatterning defects with a range of abnormalities including failedtailbud development, reduced somite numbers, and neural tubedefects including underdeveloped and open neural folds (Welshand O'Brien, 2000). The expression of Slain1 in these regionsof the post-gastrulation mouse embryo makes it a possiblecandidate for the mutant gene. Targeted mutation of the Slain1locus in mice is now required to clarify its role as candidate forthis syndrome.

In summary, the inclusion of most of the known ESC genesin our ‘core’ ESCG set vindicates the strategy taken to identifystem-cell-enriched genes and supports the comparison oftranscriptional profiles of closely related cell types as a meansof identifying genes that mark different stages of developmentor cellular differentiation.

Fig. 9. Slain1 is expressed throughout the E6.5–E7 embryo and subsequently in the neural tube, tailbud, and somites of the post-implantation mouse embryo atE8.5–E9.5. Whole mount in situ hybridisation of mouse embryos hybridised with sense (A, E, G) or anti-sense (B–D, F, H–L) Slain1 probes corresponding tobases 894 to 1848 of the mouse Slain1 cDNA (accession number BC079866). At E6.5, Slain1 is expressed throughout the embryo (A–D), at E7.5, there isenhanced expression in the headfolds (E, F), and from E8.5 to E9.5, in the optic vesicle (ov), neural tube (nt), midbrain (mb), hindbrain (hb), tailbud (tb) and thedorsal aspects of the somites (so).

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Acknowledgments

This work was supported by the Australian Stem Cell Centre,the National Health and Medical Research Council of Australia,ES Cell International and the Juvenile Diabetes ResearchFoundation. A.G.E. is a NHMRC Senior Research Fellow.

Appendix A. Supplementary data

Supplementary data associated with this article can be foundin the online version at doi:10.1016/j.ydbio.2006.01.023.

References

Abeyta, M.J., Clark, A.T., Rodriguez, R.T., Bodnar, M.S., Pera, R.A., Firpo,M.T., 2004. Unique gene expression signatures of independently-derivedhuman embryonic stem cell lines. Hum. Mol. Genet. 13, 601–608.

Bammler, T., Beyer, R.P., Bhattacharya, S., Boorman, G.A., Boyles, A.,Bradford, B.U., Bumgarner, R.E., Bushel, P.R., Chaturvedi, K., Choi, D.,Cunningham, M.L., Deng, S., Dressman, H.K., Fannin, R.D., Farin, F.M.,Freedman, J.H., Fry, R.C., Harper, A., Humble, M.C., Hurban, P.,Kavanagh, T.J., Kaufmann, W.K., Kerr, K.F., Jing, L., Lapidus, J.A.,Lasarev, M.R., Li, J., Li, Y.J., Lobenhofer, E.K., Lu, X., Malek, R.L.,Milton, S., Nagalla, S.R., O'Malley, J.P., Palmer, V.S., Pattee, P., Paules,R.S., Perou, C.M., Phillips, K., Qin, L.X., Qiu, Y., Quigley, S.D., Rodland,M., Rusyn, I., Samson, L.D., Schwartz, D.A., Shi, Y., Shin, J.L., Sieber,S.O., Slifer, S., Speer, M.C., Spencer, P.S., Sproles, D.I., Swenberg, J.A.,Suk, W.A., Sullivan, R.C., Tian, R., Tennant, R.W., Todd, S.A., Tucker, C.J.,Van Houten, B., Weis, B.K., Xuan, S., Zarbl, H., 2005. Standardizing globalgene expression analysis between laboratories and across platforms. Nat.Methods 2 (5), 329–330.

Barnett, L.D., Kontgen, F., 2001. Gene targeting in a centralized facility.Methods Mol. Biol. 158, 65–82.

Bhattacharya, B., Miura, T., Brandenberger, R., Mejido, J., Luo, Y., Yang, A.X.,Joshi, B.H., Ginis, I., Thies, R.S., Amit, M., Lyons, I., Condie, B.G.,Itskovitz-Eldor, J., Rao, M.S., Puri, R.K., 2004. Gene expression in humanembryonic stem cell lines: unique molecular signature. Blood 103,2956–2964.

Bortvin, A., Eggan, K., Skaletsky, H., Akutsu, H., Berry, D.L., Yanagimachi, R.,Page, D.C., Jaenisch, R., 2003. Incomplete reactivation of Oct4-relatedgenes in mouse embryos cloned from somatic nuclei. Development 130,1673–1680.

Brandenberger, R., Khrebtukova, I., Thies, R.S., Miura, T., Jingli, C., Puri, R.,Vasicek, T., Lebkowski, J., Rao, M., 2004a. MPSS profiling of humanembryonic stem cells. BMC Dev. Biol. 4, 10.

Brandenberger, R., Wei, H., Zhang, S., Lei, S., Murage, J., Fisk, G.J., Li, Y., Xu,C., Fang, R., Guegler, K., Rao, M.S., Mandalam, R., Lebkowski, J., Stanton,L.W., 2004b. Transcriptome characterization elucidates signaling networksthat control human ES cell growth and differentiation. Nat. Biotechnol. 22,707–716.

Burdon, T., Smith, A., Savatier, P., 2002. Signalling, cell cycle and pluripotencyin embryonic stem cells. Trends Cell Biol. 12, 432–438.

Burdsal, C.A., Damsky, C.H., Pedersen, R.A., 1993. The role of E-cadherin andintegrins in mesoderm differentiation and migration at the mammalianprimitive streak. Development 118, 829–844.

Chen, T., Ueda, Y., Dodge, J.E., Wang, Z., Li, E., 2003. Establishment andmaintenance of genomic methylation patterns in mouse embryonic stemcells by Dnmt3a and Dnmt3b. Mol. Cell. Biol. 23, 5594–5605.

Ciruna, B.G., Rossant, J., 1999. Expression of the T-box gene eomesoderminduring early mouse development. Mech. Dev. 81, 199–203.

Ciruna, B., Rossant, J., 2001. FGF signaling regulates mesoderm cell fatespecification and morphogenetic movement at the primitive streak. Dev. Cell1, 37–49.

Doetschman, T.C., Eistetter, H., Katz, M., Schmidt, W., Kemler, R., 1985. The invitro development of blastocyst-derived embryonic stem cell lines:

formation of visceral yolk sac, blood islands and myocardium. J. Embryol.Exp. Morphol. 87, 27–45.

Elefanty, A.G., Robb, L., Birner, R., Begley, C.G., 1997. Hematopoietic-specificgenes are not induced during in vitro differentiation of scl-null embryonicstem cells. Blood 90, 1435–1447.

Elefanty, A.G., Begley, C.G., Hartley, L., Papaevangeliou, B., Robb, L., 1999.SCL expression in the mouse embryo detected with a targeted lacZ reportergene demonstrates its localization to hematopoietic, vascular, and neuraltissues. Blood 94, 3754–3763.

Evsikov, A.V., Solter, D., 2003. Comment on “‘Stemness’: transcriptionalprofiling of embryonic and adult stem cells” and “a stem cell molecularsignature”. Science 302, 393 (author reply 393).

Fiering, S.N., Roederer, M., Nolan, G.P., Micklem, D.R., Parks, D.R.,Herzenberg, L.A., 1991. Improved FACS-Gal: flow cytometric analysisand sorting of viable eukaryotic cells expressing reporter gene constructs.Cytometry 12, 291–301.

Fortunel, N.O., Otu, H.H., Ng, H.H., Chen, J., Mu, X., Chevassut, T., Li, X.,Joseph, M., Bailey, C., Hatzfeld, J.A., Hatzfeld, A., Usta, F., Vega, V.B.,Long, P.M., Libermann, T.A., Lim, B., 2003. Comment on “‘Stemness’:transcriptional profiling of embryonic and adult stem cells” and “a stem cellmolecular signature”. Science 302, 393 (author reply 393).

Ginis, I., Luo, Y., Miura, T., Thies, S., Brandenberger, R., Gerecht-Nir, S., Amit,M., Hoke, A., Carpenter, M.K., Itskovitz-Eldor, J., Rao, M.S., 2004.Differences between human and mouse embryonic stem cells. Dev. Biol.269, 360–380.

Golan-Mashiach, M., Dazard, J.E., Gerecht-Nir, S., Amariglio, N., Fisher, T.,Jacob-Hirsch, J., Bielorai, B., Osenberg, S., Barad, O., Getz, G., Toren, A.,Rechavi, G., Itskovitz-Eldor, J., Domany, E., Givol, D., 2005. Designprinciple of gene expression used by human stem cells: implication forpluripotency. FASEB J. 19, 147–149.

Hart, A.H., Hartley, L., Ibrahim, M., Robb, L., 2004. Identification, cloning andexpression analysis of the pluripotency promoting Nanog genes in mouseand human. Dev. Dyn. 230, 187–198.

Haub, O., Goldfarb, M., 1991. Expression of the fibroblast growth factor-5 genein the mouse embryo. Development 112, 397–406.

Hebert, J.M., Boyle, M., Martin, G.R., 1991. mRNA localization studies suggestthat murine FGF-5 plays a role in gastrulation. Development 112, 407–415.

Holland, P.W.H., Harper, S.J., McVey, J.H., Hogan, B.L.M., 1987. In vivoexpression of mRNA for the Ca++-binding protein SPARC (osteonectin)revealed by in situ hybridization. J. Cell Biol. 105, 473–482.

Irizarry, R.A., Warren, D., Spencer, F., Kim, I.F., Biswal, S., Frank, B.C.,Gabrielson, E., Garcia, J.G., Geoghegan, J., Germino, G., Griffin, C.,Hilmer, S.C., Hoffman, E., Jedlicka, A.E., Kawasaki, E., Martinez-Murillo,F., Morsberger, L., Lee, H., Petersen, D., Quackenbush, J., Scott, A., Wilson,M., Yang, Y., Ye, S.Q., Yu, W., 2005. Multiple-laboratory comparison ofmicroarray platforms. Nat. Methods 2, 345–350.

Ivanova, N.B., Dimos, J.T., Schaniel, C., Hackney, J.A., Moore, K.A.,Lemischka, I.R., 2002. A stem cell molecular signature. Science 298,601–604.

Ivanova, N.B., Dimos, J.T., Schaniel, C., Hackney, J.A., Moore, K.A., Ramalho-Santos, M., Yoon, S., Matsuzaki, Y., Mulligan, R.C., Melton, D.A.,Lemischka, I.R., 2003. Response to comments on “‘Stemness’: transcrip-tional profiling of embryonic and adult stem cells” and “a stem cellmolecular signature”. Science 302, 393d.

Keller, G.M., 1995. In vitro differentiation of embryonic stem cells. Curr. Opin.Cell Biol. 7, 862–869.

Keller, G., Kennedy, M., Papayannopoulou, T., Wiles, M.V., 1993. Hemato-poietic commitment during embryonic stem cell differentiation in culture.Mol. Cell. Biol. 13, 473–486.

Kinder, S.J., Tsang, T.E., Quinlan, G.A., Hadjantonakis, A.K., Nagy, A., Tam, P.P., 1999. The orderly allocation of mesodermal cells to the extraembryonicstructures and the anteroposterior axis during gastrulation of the mouseembryo. Development 126, 4691–4701.

Kubota, H., Chiba, H., Takakuwa, Y., Osanai, M., Tobioka, H., Kohama, G.,Mori, M., Sawada, N., 2001. Retinoid X receptor alpha and retinoic acidreceptor gamma mediate expression of genes encoding tight-junctionproteins and barrier function in F9 cells during visceral endodermaldifferentiation. Exp. Cell Res. 263, 163–172.

13C.E. Hirst et al. / Developmental Biology xx (2006) xxx–xxx

ARTICLE IN PRESS

Lacaud, G., Gore, L., Kennedy, M., Kouskoff, V., Kingsley, P., Hogan, C.,Carlsson, L., Speck, N., Palis, J., Keller, G., 2002. Runx1 is essential forhematopoietic commitment at the hemangioblast stage of development invitro. Blood 100, 458–466.

Larkin, J.E., Frank, B.C., Gavras, H., Sultana, R., Quackenbush, J., 2005.Independence and reproducibility across microarray platforms. Nat.Methods 2, 337–344.

Leahy, A., Xiong, J.W., Kuhnert, F., Stuhlmann, H., 1999. Use of developmentalmarker genes to define temporal and spatial patterns of differentiation duringembryoid body formation. J. Exp. Zool. 284, 67–81.

Levanon, D., Brenner, O., Negreanu, V., Bettoun, D., Woolf, E., Eilam, R., Lotem,J., Gat, U., Otto, F., Speck, N., Groner, Y., 2001. Spatial and temporalexpression patterns of Runx3 (Aml2) and Runx1 (Aml1) indicates non-redundant functions during mouse embryogenesis. Mech. Dev. 109, 413–417.

Liu, P., Wakamiya, M., Shea, M.J., Albrecht, U., Behringer, R.R., Bradley, A.,1999. Requirement for Wnt3 in vertebrate axis formation. Nat. Genet. 22,361–365.

Morrisey, E.E., Ip, H.S., Lu, M.M., Parmacek, M.S., 1996. GATA-6: a zincfinger transcription factor that is expressed in multiple cell lineages derivedfrom lateral mesoderm. Dev. Biol. 177, 309–322.

Ng, E.S., Azzola, L., Sourris, K., Robb, L., Stanley, E.G., Elefanty, A.G., 2005a.The primitive streak gene Mixl1 is required for efficient haematopoiesis andBMP4-induced ventral mesoderm patterning in differentiating ES cells.Development 132, 873–884.

Ng, E.S., Davis, R.P., Azzola, L., Stanley, E.G., Elefanty, A.G., 2005b. Forcedaggregation of defined numbers of human embryonic stem cells intoembryoid bodies fosters robust, reproducible hematopoietic differentiation.Blood 106 (5), 1601–1603.

Palmqvist, L., Glover, C.H., Hsu, L., Lu, M., Bossen, B., Piret, J.M.,Humphries, R.K., Helgason, C.D., 2005. Correlation of murine embryonicstem cell gene expression profiles with functional measures of pluripotency.Stem Cells 23, 663–680.

Park, C., Afrikanova, I., Chung, Y.S., Zhang, W.J., Arentson, E., Fong Gh, G.,Rosendahl, A., Choi, K., 2004. A hierarchical order of factors in thegeneration of FLK1- and SCL-expressing hematopoietic and endothelialprogenitors from embryonic stem cells. Development 131, 2749–2762.

Pelton, T.A., Sharma, S., Schulz, T.C., Rathjen, J., Rathjen, P.D., 2002.Transient pluripotent cell populations during primitive ectoderm formation:correlation of in vivo and in vitro pluripotent cell development. J. Cell Sci.115, 329–339.

Pera, E.M., Kessel, M., 1997. Patterning of the chick forebrain anlage by theprechordal plate. Development 124, 4153–4162.

Peterson, K.A., King, B.L., Hagge-Greenberg, A., Roix, J.J., Bult, C.J.,O'Brien, T.P., 2002. Functional and comparative genomic analysis of thepiebald deletion region of mouse chromosome 14. Genomics 80, 172–184.

Ramalho-Santos, M., Yoon, S., Matsuzaki, Y., Mulligan, R.C., Melton, D.A.,2002. “Stemness”: transcriptional profiling of embryonic and adult stemcells. Science 298, 597–600.

Rao, M., 2004. Conserved and divergent paths that regulate self-renewal inmouse and human embryonic stem cells. Dev. Biol. 275, 269–286.

Rao, R.R., Calhoun, J.D., Qin, X., Rekaya, R., Clark, J.K., Stice, S.L., 2004.Comparative transcriptional profiling of two human embryonic stem celllines. Biotechnol. Bioeng. 88, 273–286.

Reubinoff, B.E., Pera, M.F., Fong, C.Y., Trounson, A., Bongso, A., 2000.Embryonic stem cell lines from human blastocysts: somatic differentiation invitro. Nat. Biotechnol. 18, 399–404.

Richards, M., Tan, S.P., Tan, J.H., Chan, W.K., Bongso, A., 2004. Thetranscriptome profile of human embryonic stem cells as defined by SAGE.Stem Cells 22, 51–64.

Robertson, S.M., Kennedy, M., Shannon, J.M., Keller, G., 2000. A transitionalstage in the commitment of mesoderm to hematopoiesis requiring thetranscription factor SCL/tal-1. Development 127, 2447–2459.

Sato, N., Sanjuan, I.M., Heke, M., Uchida, M., Naef, F., Brivanlou, A.H., 2003.Molecular signature of human embryonic stem cells and its comparison withthe mouse. Dev. Biol. 260, 404–413.

Schmitt, R.M., Bruyns, E., Snodgrass, H.R., 1991. Hematopoietic developmentof embryonic stem cells in vitro: cytokine and receptor gene expression.Genes Dev. 5, 728–740.

Shen, M.M., Leder, P., 1992. Leukemia inhibitory factor is expressed by thepreimplantation uterus and selectively blocks primitive ectoderm formationin vitro. Proc. Natl. Acad. Sci. U. S. A. 89, 8240–8244.

Sperger, J.M., Chen, X., Draper, J.S., Antosiewicz, J.E., Chon, C.H., Jones,S.B., Brooks, J.D., Andrews, P.W., Brown, P.O., Thomson, J.A., 2003.Gene expression patterns in human embryonic stem cells and humanpluripotent germ cell tumors. Proc. Natl. Acad. Sci. U. S. A. 100,13350–13355.

Tamai, Y., Ishikawa, T., Bosl, M.R., Mori, M., Nozaki, M., Baribault, H.,Oshima, R.G., Taketo, M.M., 2000. Cytokeratins 8 and 19 in the mouseplacental development. J. Cell Biol. 151, 563–572.

Tanaka, T.S., Kunath, T., Kimber, W.L., Jaradat, S.A., Stagg, C.A., Usuda,M., Yokota, T., Niwa, H., Rossant, J., Ko, M.S., 2002. Gene expressionprofiling of embryo-derived stem cells reveals candidate genesassociated with pluripotency and lineage specificity. Genome Res 12,1921–1928.

Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J.,Marshall, V.S., Jones, J.M., 1998. Embryonic stem cell lines derived fromhuman blastocysts. Science 282, 1145–1147.

Turksen, K., Troy, T.C., 2001. Claudin-6: a novel tight junction molecule isdevelopmentally regulated in mouse embryonic epithelium. Dev. Dyn. 222,292–300.

Welsh, I.C., O'Brien, T.P., 2000. Loss of late primitive streak mesoderm andinterruption of left–right morphogenesis in the Ednrb(s-1Acrg) mutantmouse. Dev. Biol. 225, 151–168.

Wiles, M.V., Johansson, B.M., 1999. Embryonic stem cell development in achemically defined medium. Exp. Cell Res. 247, 241–248.

Wiles, M.V., Keller, G., 1991. Multiple hematopoietic lineages develop fromembryonic stem (ES) cells in culture. Development 111, 259–267.

Yamaguchi, T.P., Dumont, D.J., Conlon, R.A., Breitman, M.L., Rossant, J.,1993. flk-1, an flt-related receptor tyrosine kinase is an early marker forendothelial cell precursors. Development 118, 489–498.

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