Gene Expression Patterns 11 (2011) 334–344

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Gene Expression Patterns

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Messenger RNA and microRNA profiling during early mouse EB formation

Rashmi Tripathi a, Harpreet Kaur Saini b,⇑, Roland Rad a, Cei Abreu-Goodger b, Stijn van Dongen b,Anton J. Enright b

a Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdomb EMBL – European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, United Kingdom

a r t i c l e i n f o

Article history:Received 25 December 2009Received in revised form 21 February 2011Accepted 14 March 2011Available online 1 April 2011

Keywords:Embryonic stem cellsEBsMessenger RNAMicroRNAPluripotencyGerm layerNervous system developmentLet-7Lin28MicroRNA targetsGene ontology

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⇑ Corresponding author. Tel.: +44 1223 492676; faxE-mail address: hsaini@ebi.ac.uk (H.K. Saini).

a b s t r a c t

Embryonic stem (ES) cells can be induced to differentiate into embryoid bodies (EBs) in a synchronisedmanner when plated at a fixed density in hanging drops. This differentiation procedure mimics post-implantation development in mouse embryos and also serves as the starting point of protocols used indifferentiation of stem cells into various lineages. Currently, little is known about the potential influenceof microRNAs (miRNAs) on mRNA expression patterns during EB formation. We have measured mRNAand miRNA expression in developing EBs plated in hanging drops until day 3, when discrete structuralchanges occur involving their differentiation into three germ layers. We observe significant alterationsin mRNA and miRNA expression profiles during this early developmental time frame, in particular ofgenes involved in germ layer formation, stem cell pluripotency and nervous system development. Com-putational target prediction using Pictar, TargetScan and miRBase Targets reveals an enrichment of bind-ing sites corresponding to differentially and highly expressed miRNAs in stem cell pluripotency genes anda neuroectodermal marker, Nes. We also find that members of let-7 family are significantly down-regu-lated at day 3 and the corresponding up-regulated genes are enriched in let-7 seed sequences. Theseresults depict how miRNA expression changes may affect the expression of mRNAs involved in EB forma-tion on a genome-wide scale. Understanding the regulatory effects of miRNAs during EB formation mayenable more efficient derivation of different cell types in culture.

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1. Results and discussion

Embryonic stem (ES) cells were first established in culture fromthe inner cell mass of mouse blastocysts (Evans and Kaufman,1981; Martin, 1981). They can also differentiate into three-dimen-sional structures called EBs (EBs), which mimic many aspects ofmammalian embryos. To date most microarray based expressionstudies of differentiating EBs have focused primarily on mRNAs(Hailesellasse Sene et al., 2007; Leahy et al., 1999; Manserghet al., 2009). There has been only one study that has looked at bothmRNA and miRNA expression changes in undifferentiated and dif-ferentiated human ES cells (Lakshmipathy et al., 2007). MiRNAshave been shown to be important modulators of ES cell growthand differentiation (Marson et al., 2008; Wilson et al., 2009). Theseare a single stranded 21–23 nt family of RNAs first discovered inCaenorhabditis elegans (C. elegans) as short RNAs regulating devel-opmental timing (Lee et al., 1993; Olsen and Ambros, 1999). Theyrepress gene expression by targeting cognate messenger RNAs(mRNAs) for degradation or translational repression (Bartel,

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2004). This activity is based on miRNAs binding to complementarysites in the 30 un-translated regions (UTRs) of their target tran-scripts. The so called ‘seed-region’ of the miRNAs (2–8 nucleotides(nt)) appears to be a key specificity determinant in this interaction(Lewis et al., 2005). Analysing mRNA and miRNA expression pro-files simultaneously in an ES cell based developmental model couldhelp unravel potential regulatory effects of miRNAs on mRNAexpression during this process.

In order to avoid significant heterogeneity in EB size, differenti-ation status and gene expression (Kurosawa, 2007; Lewis et al.,2005) as often observed in suspension cultures (Mogi et al.,2009), we plated ES cells at a fixed cell density of 1000 cells perdrop using the hanging drop method (Guan et al., 1999; Kurosawa,2007; Mogi et al., 2009). The three dimensional environment pro-vided to stem cells allowed them to differentiate synchronouslyinto clusters of cells which then spontaneously differentiated intoan outer endoderm layer and an inner cell mass (Fig. 1). RNA wasisolated from undifferentiated stem cells at day 0 and days 1, 2 and3 of EB formation and hybridised on Illumina beadarrays. Illuminaexpression profiling was performed in triplicate using the R statis-tical package, with overall correlations between replicates ob-served between 0.98 and 0.99.

Fig. 1. ES cell differentiation into simple EBs in hanging drops at (a) day 1, (b) day 2 and (c) day 3. The appearance of an outer endoderm layer is visible at day 3.

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1.1. mRNA expression analysis

We obtained lists of genes with at least 2-fold change in expres-sion and adjusted p-values <0.01 at days 1–3 when tested againstday 0 (Supplementary Table S1). We found that a greater numberof genes show large expression changes by day 3 than by days 1and 2. This corresponds with increased differentiation status ofES cells at day 3 compared to earlier time points. The comparisonof day 3 against day 0 produced 1942 differentially expressedgenes, comprising 884 up-regulated and 1058 down-regulated.For visualization, we performed hierarchical clustering of onlythe most significantly differentially expressed genes with adjustedp-values <10�5 (Fig. 2). Among the up-regulated genes, a number ofgenes known to be involved in the formation of ectoderm, endo-derm and mesoderm layers were identified (Table 1a).

We observe an up-regulation of ectodermal marker genes suchas Fgf5 and Sox4 during all 3 days, whereas Nes shows an increased

Fig. 2. Unsupervised hierarchical clustering of significantly (adj. p-value

expression at day 3. Fgf5 expression has been shown to increasedramatically in pluripotent embryonic ectoderm prior to gastrula-tion in developing mouse embryos (Hebert et al., 1991) and is oftenused as a primitive ectoderm marker in differentiating EBs. Sox4has been implicated in inducing neuronal traits in stem cells(Bergsland et al., 2006) and Nes is an intermediate filament proteinfound in neural precursor cells (Andressen et al., 2001). Coinci-dently, Nes has also been identified in a subset of hormone-nega-tive pancreatic cells with unusually extended proliferativecapacity when cultured in vitro (Zulewski et al., 2001).

Endodermal specific genes such as Nodal, Ttr, Amn and Cldn7show an increase in expression. Nodal, an antagonist of Lefty1 is ex-pressed prior to gastrulation and plays a pivotal role in endodermand mesoderm development in mouse (Zhou et al., 1993). More-over, sustained Nodal expression has also been shown to preventdifferentiation of stem cells into the definitive endoderm lineageby prolonging expression of pluripotency transcripts like Oct3/4

s <10�5) differentially expressed genes at day 3 compared to day 0.

Table 1Log2 fold change values of (a) germ layer makers and (b) pluripotency genes at days 1, 2 and 3 compared to day 0.

a)Marker Genes Germ Layer Log2 Fold-Change (adj. p-values < 0.01)

Day1 - Day0 Day2 - Day0 Day3 - Day0

Fgf5 Ectoderm 2.80 4.71 4.86Sox4 Ectoderm 1.11 1.88 1.83Nes Ectoderm �0.76 �0.38 0.77Nodal Endoderm 0.51 0.35 0.73Ttr Endoderm 1.73 3.77 4.13Amn Endoderm 1.94 2.23 2.07Cldn7 Endoderm �1.54 �0.33 0.52Foxa2 Endoderm �0.67 �0.86 �0.73Sox17 Endoderm �0.27 �0.49 �0.32Flt1 Endothelial 1.04 0.95 1.41Mixl1 Mesendoderm 0.73 0.22 0.82Eomes Mesendoderm 0.49 0.31 1.09Gsc Mesendoderm �0.20 0.23 1.49Runx1 Mesendoderm 0.68 0.65 0.98T Mesoderm 0.14 0.03 2.11Gata4 Mesoderm and Endoderm 0.54 0.29 0.15Gata6 Mesoderm and Endoderm �0.04 �0.58 �0.51Lefty1 Left-right axis formation 3.28 4.12 3.75Lefty2 Left-right axis formation 3.13 2.64 1.86

b)Pluripotency Genes Function Log2 Fold-Change (adj. p-values < 0.01)

Day1 - Day0 Day2 - Day0 Day3 - Day0

Sox2 Transcription Factor 0.72 0.09 �0.70Stat3 Transcription Factor �0.43 �0.44 �0.65Sall4 Transcription Factor 0.98 1.03 0.96Tcf7 Transcription Factor 0.13 0.33 0.69Pou5f1 Transcription Factor 1.19 1.20 0.95Nanog Transcription Factor �0.77 �2.66 �1.72Esrrb Nuclear Hormone Receptor 0.13 �1.60 �2.73Nr5a2 Nuclear Hormone Receptor 0.97 �0.61 �1.38Lin28 Translational Enhancer 0.77 1.24 1.53Lin28b Translational Enhancer 0.31 0.52 0.83Klf4 Transcriptional Activator/Repressor �0.83 �1.69 �1.64Zfp42 Transcriptional Activator 0.55 �1.26 �1.92Myc Oncogene 1.34 0.95 0.75

⁄Non-significant (adj. p-value > 0.01) log2 fold-change values are blue.

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(Takenaga et al., 2007). Interestingly, expression of Ttr at day 3 isapproximately 14-fold higher than at day 1, which coincides withthe appearance of primitive endodermal cells (Abe et al., 1996).The Amn gene that functions in gastrulation (Kalantry et al.,2001) and Cldn7, a tight junction protein are expressed in the vis-ceral endoderm (Kubota et al., 2001). However, there is a reducedexpression of Foxa2, which correlates with reduced expression ofGATA transcription factors, Gata-6 and Gata-4, suggesting the pres-ence of visceral endoderm (which derives from the inner cell massand gives rise to extra-embryonic endoderm), and absence ofdefinitive endoderm at day 3 (Matsuura et al., 2006).

Expression levels of Eomes, Mixl1, Gsc and T (Brachyury), whichplay central roles in mesendoderm (Izumi et al., 2007) are alsoup-regulated significantly at day 3, but do not show significantchanges at days 1 and 2. Eomes has been demonstrated to playan important role in early stages of mesendoderm formation andMixl1 has been shown to work during later stages (Izumi et al.,2007). T (Brachyury) is a major marker for the mesoderm and isregulated by both Eomes and Mixl1 (Izumi et al., 2007). Gsc is a spe-cific marker of mesendoderm, which is expressed in the organizerregion from which definitive endoderm arises (Tada et al., 2005).Taken together, these results imply that the formation of mesendo-derm has initiated, its further differentiation into definitive endo-derm has not yet occurred on day 3.

Local activation of the Wnt pathway in EBs has been shown toresult in differentiation of cells into mesendodermal progenitors(ten Berge et al., 2008). We used Axin-2 as a representative markerof the Wnt signalling pathway as previously described (Jho et al.,

2002) and found that it is up-regulated at all time points. Thereis more than 2-fold change in expression at day 3. These resultsfurther support our hypothesis involving commitment of embry-onic stem cells into the mesendodermal lineage at day 3.

In addition, we also find an up-regulation of lefty-related genes,Lefty1 and Lefty2, which are implicated in establishing the left–right body axis (Meno et al., 1998). These genes are maximallyup-regulated at day 2 of EB formation, and their expression subse-quently falls at day 3, although their expression remains higher atthis time point as compared to undifferentiated stem cells. Leftyproteins have been shown to be transiently expressed during hu-man EB differentiation and also appear to have significant controlover mesoderm formation (Dvash et al., 2007).

We also monitored expression changes of key pluripotencytranscripts during these time-points of EB development(Table 1b). Certain transcripts like Oct4 (Pou5f1), Tcf7, Myc, Sall4,Lin28 and Lin28b are significantly up-regulated during this phase.Other transcripts like Sox2, Esrrb, Stat3, Klf4, Nanog and Rex1(Zfp42) are found to be down-regulated. Comparison of these re-sults and previously published findings involving expression anal-ysis during stem cell differentiation reveals considerable overlap inexpression trends for Sox2, Klf4, Esrrb, Nanog and Stat3 (Gloveret al., 2006; Niwa et al., 2000; Palmqvist et al., 2005). Previousstudies have reported a transient increase in Oct4 expression with-in the first 12–24 h of stem cell differentiation (Palmqvist et al.,2005). However, in our experiments, we observe a sustainedexpression of Oct4 up to 72 h of EB differentiation. For Myc, we ob-serve increased expression levels contrary to previous studies that

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reported reduced expression during stem cell differentiation(Cartwright et al., 2005). Moreover, we also observe an up-regulationof Lin28 and Lin28b transcripts during this early phase of EB devel-opment. Lin28 has been identified as one of a number of transformingfactors responsible for inducing pluripotency in transfected humanfibroblasts and its expression has also been shown to be down-reg-ulated during stem cell differentiation (Yang and Moss, 2003).Hence our finding that Lin28 expression actually increases duringinitial stages of EB formation is contrary to what one might expect.However, Mansergh et al. have recently reported increased expres-sion of pluripotency marker genes like Oct4, Nanog and Rex1 instem cells differentiating under high serum concentrations (15–20%) (Mansergh et al., 2009). We have used 15% serum concentra-tion in our differentiation media, which could promote the mainte-nance of pluripotent stem cell populations in differentiating EBs. Itis also possible that this initial burst of Oct4, Myc and Lin28 expres-sion during early EB formation gradually tapers off during laterstages. One must note that previous studies have carried out differ-entiation of ES cells after LIF withdrawal or after retinoic acid treat-ment. We have used a different methodology for differentiatingstem cells developed by Guan et al. (1999). It is possible that differ-ent procedures applied for eliciting differentiation of stem cells re-sult in alteration of the external environment in ways that wouldaffect expression signatures of key pluripotency transcripts. It isalso possible that these expression profiles are unique to this dif-ferentiating cell line. Indeed, different cell lines often exhibit variedpatterns of expression of stem cell pluripotency markers duringdifferentiation (Abeyta et al., 2004; Hailesellasse Sene et al., 2007).

Other notable genes significantly up-regulated at day 3 includegenes that are implicated in nervous system development such asSall2 (Pincheira and Donner, 2008), Zic2 (Salero and Hatten, 2007),Pou3f1 (Ryu et al., 2007), Lpar4 (Shin et al., 2007), Sox11 (Uwano-gho et al., 1995), Sort1 (Jansen et al., 2007) and Slc7a3, a brain spe-cific cationic transporter (Hosokawa et al., 1997). Among them,Sall2, Zic2, Pou3f1 and Sox11 are transcription factors that promotethe development and function of neurons. Lpar4 is expressed inneural stem/progenitor cells, and in particular, has been shownto have a range of effects including regulating morphological rear-rangements, proliferation and differentiation (Pebay et al., 2007).Interestingly, Trh shows maximum up-regulation at day 3 (Supple-mentary Table S1). Trh or thyrotropin releasing hormone is ex-pressed in pancreas and has been shown to be involved inpancreatic development (Luo et al., 2008). Trh is also expressedin nerves (Otake and Nakamura, 2000). Moreover, Nes has beenfound to be expressed in both neural progenitor cells as well aspancreatic endocrine cells (Zulewski et al., 2001). Also, the nervoussystem and pancreas have similar mechanisms of developmentthough they differ in origin (the nervous system is derived fromectoderm, while the pancreas is derived from definitive endoderm)(Kim et al., 1997; Li et al., 1999; Schwitzgebel et al., 2000). Thus,there is a possibility that EBs at day 3 consist of cells differentiatinginto neural and pancreatic lineages. However, based on expressionof germ layer marker genes, it can be noted that although day 3 EBshave ectoderm and mesendodermal layers, they lack the definitiveendoderm. Also, EBs at day 3 show high expression of many neuro-nal marker genes, while the major pancreatic marker Pdx-1 (Jons-son et al., 1994; Offield et al., 1996) and other pancreatic specificgenes (Edlund, 1998; Hebrok et al., 1999; Rouiller et al., 1991)are not significantly up-regulated. Thus, these findings togetherwith the lack of expression of pancreatic markers suggest that cer-tain cells in the EBs at day 3 are developing into the neural lineage.

Other genes such as Pim2 (Mikkers et al., 2004), Dnmt3b (en-riched in epiblast) (Bruce et al., 2007) and Emb, Clic6 (enriched inendoderm) (Sherwood et al., 2007) (Sherwood et al., 2007) are alsoup-regulated at day 3. Further, chemokine (C–X–C motif) ligand 12(Cxcl12) belonging to the CXC chemokine family is also up-regu-

lated. Cxcl12 plays an important role in hematopoiesis, as well asin vascular and cerebellar development (Guo et al., 2005). Previousstudies have also shown an increase in expression of Cxcl12 at day3–5 of EB formation, corresponding to early stages of hemangio-blast formation (day 2.5–3.5) and hematopoiesis (days 4–6) (Guoet al., 2005). We also observe an up-regulation of Flt-1, which isa known marker of hemangioblast and important for endothelialformation (Festag et al., 2007). In addition, we have also foundan up-regulation of Tal1, which is expressed in the developing vas-culature and is required for vascular modelling during embryogen-esis (Kallianpur et al., 1994). Other important markers ofendothelial such as VE-cadherin, CD41 are not found to be signifi-cantly expressed at day 3, which supports the hypothesis that theyare expressed at later stages of endothelial formation (Nikolova-Krstevski et al., 2008). Similarly, transcription factors required forhematopoiesis such as Runx1, Gata-1, HoxB4 (McKinney-Freemanet al., 2008) are not detected at day 3. These findings suggest thatday 3 EBs contain hemangioblast precursors, which are not furtherdifferentiated into hematopoietic and endothelial lineages.

Among the down-regulated genes, we find many genes withfunctions related to cell adhesion (Laptm5 (Adra et al., 1996), Lgals3(Barondes et al., 1994), Myl4 (Fujiwara et al., 2007), intracellularsignalling (Anax3)) (Hailesellasse Sene et al., 2007) and pluripoten-cy (Fbxo15 (Tokuzawa et al., 2003), Tuba3 (D’Amour and Gage,2003), Tcfcp2l1 (Loh et al., 2006), Tex19 (Kuntz et al., 2008)). Fbxo15is highly expressed in ES cells and is regulated by Oct3/4, but is dis-pensable for ES cell self-renewal. Tex19 is a mammalian specificprotein with a restricted expression in pluripotent stem cells andgerm line. Lgals3 is required for terminal differentiation of colum-nar epithelial cells during early embryogenesis. Laptm5 is a lyso-somal-associated multispanning membrane protein preferentiallyexpressed in hematopoietic cells.

1.2. MiRNA expression analysis

After filtering out miRNAs that demonstrated low variability intheir expression, normalized expression data for 107 miRNAs wasused to generate a heatmap of all the four time points (Fig. 3). Thedendrogram shows two main groups, the first corresponding tosamples from day 0 and day 1 and the other comprising day 2and day 3. Next, we obtained lists of significantly differentially ex-pressed miRNAs at days 1–3 tested against day 0 with more than2-fold change in expression and corrected p-values <0.01 (Supple-mentary Table S2). Furthermore, we looked for up- and down-reg-ulated miRNAs, which are common between the different time-points (Fig. 4a and b).

There is only one miRNA that is continuously up-regulatedstarting from day 1, miR-106a, with log2 fold changes of 1.12,1.32 and 1.45 at days 1, 2 and 3, respectively (SupplementaryTable S2). Previous studies have analysed the expression of miR-106a during differentiating extraembryonic cells and its role inregulation of differentiation along with other members of miR-17family (Foshay and Gallicano, 2009). Interestingly in our data (Sup-plementary Table S6), other members of this family are both upand down-regulated. These results suggest differential regulationof expression of miRNAs belonging to this family. Specifically,members of this family promote cell cycle progression by function-ing as positive regulators of G1-to-S transition (Ivanovska et al.,2008). In particular, they target the gene encoding cyclin-depen-dent kinase inhibitor p21/Cdkn1a, which functions as a cycle check-point and prevents the cells from entering S phase (Ivanovska et al.,2008). Interestingly, we observe more than 2-fold decrease inexpression of Cdkn1a transcript (Supplementary Table S1) at day3 which could be caused by an increase in expression of miR-17family members.

Fig. 3. Unsupervised hierarchical clustering of normalized miRNA expression values.

Fig. 4. Venn diagrams showing the grouping of differentially expressed miRNAs that are common at different days. (a) miRNAs up-regulated at days 1–3 compared to day 0and (b) miRNAs down-regulated at days 1–3 compared to day 0.

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Only one miRNA, miR-701 is up-regulated both at days 1 and 2.As expected, there is a larger number of differentially expressedmiRNAs during the last days. We found that 10 miRNAs are up-reg-ulated at both days 2 and 3. Among them, the miR-302 familyshows the highest fold change (miR-302c shows more than 4-foldchange in expression). Members of miR-302, which have sequencesimilarity to amphibian miR-427 and teleost miR-430, have beenimplicated in establishment of mesoderm and endoderm in humanES cells by targeting Lefty genes which serve as antagonists of theNodal signalling pathway (Rosa et al., 2009). Loss of function ofmiR-302 strongly inhibits mesodermal and endodermal lineages

and expands neuroectodermal derivatives (Rosa et al., 2009). ThesemiRNAs have also been previously shown to be induced upon EScell differentiation (Houbaviy et al., 2003). Other miRNAs such asmiR-203 regulates the stemness of ES cells by repressing DeltaNp63gene (Lena et al., 2008) and miR-369-5p is specifically related tothe senescence of mesenchymal stem cells (Wagner et al., 2008).There are 3 miRNAs, miR-489, miR-539 and miR-665 that are up-regulated at day 3 only. The function of these miRNAs in ES cellshas not been documented.

Members of the miR-290 cluster of miRNAs (miR-291a-5p andmiR-292-3p) are observed to be slightly up-regulated in ES cells

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as compared to day 3 EBs (Supplementary Table S6). These miRNAsconstitute the most highly expressed cluster in ES cells (Houbaviyet al., 2003) and hence have been implicated in maintaining thepluripotent state of ES cells. Recently they have also been involvedin regulating ES cell differentiation into mesoderm and primordialgerm cell layers (Zovoilis et al., 2009). Our observation that thesemiRNAs are only slightly down-regulated on day 3 might implythat cellular differentiation is perhaps in its very early stages.

Among the differentially down-regulated miRNAs, there are 3miRNAs, miR-210, miR-136 and miR-376a that show a consistentdecrease in expression during days 1–3 (SupplementaryTable S2). miR-210 is an endothelial miRNA that promotes angio-genesis by inhibiting the receptor tyrosine kinase ligand Ephrin-A3 (Fasanaro et al., 2008). Four miRNAs, miR-499, miR-199b,miR-29a and miR-124a are down-regulated at days 2 and 3. Thebrain-specific miR-124a (Krichevsky et al., 2006) and miR-29a(Nelson et al., 2008) are preferentially expressed in differentiatingneurons and astrocytes, respectively, however, their expression isinduced only during the transition from neural progenitor cellsto neurons and astrocytes. The other miRNA, miR-499 is highly ex-pressed in both endothelial cells and vascular smooth muscle cells(Cui et al., 2004; Scalbert and Bril, 2008). Interestingly, there aremore miRNAs that are specifically down-regulated at day 3 only.These include miR-22, miR-146, miR-224, miR-465-3p, let-7i, let-7f, miR-23a and let-7d. We observed decreased levels of expressionfor all the members of let-7 family. Moreover, expression of let-7 inES cells is known to be post-transcriptionally regulated by Lin28,which blocks the processing of let-7 into mature sequence (Viswa-nathan et al., 2008). In our mRNA data, Lin28 is up-regulated at allday-points, with a 3-fold change at day 3, which is in agreementwith the reduced expression levels of the let-7 family. This is alsoconsistent with the previous report that Lin28 is highly expressedin the extraembryonic endoderm layer during mouse embryogen-esis and even up-regulated in stages in which the pluripotent com-partment in the embryo initiates differentiation and loses itspluripotency (Darr and Benvenisty, 2009).

Other miRNAs that show significant up-regulation but with foldchange less than 2 include miR-615, which is part of the Hox clus-ter of genes responsible for regulating spatial patterning duringdevelopment (Woltering and Durston, 2008) (SupplementaryTable S6). We also observe an up-regulation of certain miRNAsknown to be expressed in the central nervous system like miR-410, miR-495, miR-323 and miR-9. miR-323 is expressed in brainwhere it targets the semaphorin receptors which are involved inaxon guidance, angiogenesis and cell migration (John et al.,2004). miR-9 has been shown to be involved in a negative regula-tory feedback loop with nuclear receptor TLX (Zhao et al., 2009)and also demonstrates an increase in expression in our experi-ments. This miRNA is involved in neural fate determination.

A number of miRNAs previously not known to be expressed instem cells like miR-685, miR-214, miR-328, miR-221, miR-222,miR-365, miR-146b and miR-331 are found to be enriched inundifferentiated stem cells compared to day 3 (SupplementaryTable S6). miR-214 has been shown to be under the regulation ofthe transcription factor Twist-1 which has been shown to be in-volved in mesenchymal cell derivation (Bate et al., 1991). miR-338 is known to be specifically expressed in neuronal tissue andplays a role in neuronal maturation, particularly in the elaborationof structure and function of SCG axons (Aschrafi et al., 2008). Addi-tionally we also find that a number of oncomirs such as miR-196a,miR-125b, miR-27a, miR-320, miR-339, miR-328, miR-221, miR-222 and miR-331 are selectively enriched in undifferentiated EScells compared to day 3.

Our data for miRNA expression changes is largely consistentwith Lakshmipathy et al. (2007) who have published miRNAexpression changes in differentiating human ES cells. In agreement

with their data, we also observe an enrichment of miR-302 andmiR-17 family members in EBs, while members of the let-7 familyare found to be down-regulated (Fig. 4, Supplementary Table S6).miR-221 and miR-222 are also found to be enriched in ES cellscompared to differentiated EBs (Supplementary Table S6). Any dif-ferences in expression patterns observed in our analysis could beattributed to the biological differences between human and mouseES cells and different protocols used for differentiation.

1.3. Real-time PCR confirmation of mRNA and miRNA expressionresults

In order to further validate expression results derived from Illu-mina Beadarrays, real-time PCR was performed for specific mRNAand miRNA probes. Expression was quantified for days 0 and 3 ofEB formation using probes specific to Klf2, Klf4, Lefty1 and Oct4.All expression values were normalised against beta-actin and sub-sequently against day 0. As can be observed (SupplementaryFig. S1a) there is a general agreement of the expression trends ob-served for these transcripts between the Illumina platform andreal-time PCR.

For miRNAs, expression was tested using probes specific to miR-22, miR-367 and miR-211 (Supplementary Fig. S1b). These miRNAswere chosen from the upper, middle and lower range of IlluminamiRNA expression data and miR-15a was chosen as an internal ref-erence for normalising expression values. There is again a generalagreement between expression trends observed for these miRNAsusing Illumina and quantitative PCR.

1.4. GO enrichment analysis

Functional analysis of significantly differentially expressedgenes between days 0 and 3, based upon GO Biological Processterm enrichments was performed using the GOstats package in Bio-conductor. The GO analysis revealed that genes up-regulated atday 3 are enriched for terms related to ‘neurogenesis’(GO:0022008), ‘multicellular organismal development’(GO:0007275), ‘cellular developmental process’ (GO:0048869)and ‘neuron differentiation’ (GO:0030182). This finding is largelyin agreement with our observation of up-regulation of key genescorresponding to neural fate determination at day 3. Some of thenotable genes included among the list of neurogenesis relatedGO terms are Efna5 (Zimmer et al., 2007), Etv1 (Tuoc and Stoykova,2008), Fgf8 (Theil et al., 2008), Dtx1 (Cui et al., 2004), Galr2 (Hobsonet al., 2006), Rac3 (Corbetta et al., 2009), Robo1 (Nural et al., 2007),Slit3 (Knoll et al., 2003), Sall3 (Harrison et al., 2008), Nav1 (Marti-nez-Lopez et al., 2005), Socs2 (Ransome and Turnley, 2008), Nrcam(Heyden et al., 2008) and Brsk2 (Barnes et al., 2007). The completelist of enriched GO terms (adj. p-values <0.01) and the associatedgene identifiers is provided in the Supplementary material (Sup-plementary Table S3a).

In contrast, genes down-regulated at day 3 show an enrichmentof terms corresponding to ‘positive regulation of developmentalprocess’ (GO:0051094), ‘negative regulation of biological process’(GO:0048519), ‘blood vessel development’ (GO:0001568) and‘wound healing’ (GO:004206). The complete list of enriched GOterms is provided (Supplementary Table S3b).

1.5. Analysis of miRNA binding sites in stem cell pluripotency genes

We decided to investigate whether there exists a regulatoryrelationship between differentially expressed miRNAs and pluripo-tency genes. We used computational miRNA target prediction pro-grams for this purpose. These programs mainly rely ondetermining Watson–Crick base-pairing between a miRNA andthe 30UTR of a potential target mRNA and calculating evolutionary

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conservation of the target site. Besides employing these basic rulesfor target identification, the prediction methods are diverse in theirmethodologies and performance (Bartel, 2009). We used Pictarpredictions in mouse (unpublished results, based on 17 vertebrategenomes) (Krek et al., 2005), TargetScanMouse Release 5.1 (Grim-son et al., 2007) and miRBase mouse targets v5 (Griffiths-Joneset al., 2008) to identify potential binding sites for differentially ex-pressed miRNAs in various pluripotency genes and the major neu-roectodermal marker Nes (Supplementary Tables S4). Pictar andTargetScan have been reported as the best performing target pre-diction tools as assessed with recent proteomic studies (Baeket al., 2008). miRBase Targets is also useful for detecting targetswith less strict overall base pairing (Bartel, 2009). It has beenshown that miRNA target prediction programs vary considerablyin their outcomes. This could be attributed to use of differentUTR databases, or due to intrinsic differences in the algorithmsthemselves (Bartel, 2009). Nevertheless, these tools provide biolo-gists with a set of predicted miRNA and mRNA interactions thatcan be relevant in a developmental process like formation of earlyEBs.

We searched for binding sites of the 10 most highly expressedand the 20 most differentially expressed miRNAs (10 up- and 10down-regulated) in Nes and the pluripotency genes. Among these30 miRNAs, 24 are conserved in both human and mouse.

We find that Nes is consistently predicted by TargetScan andmiRBase to be targeted by the down-regulated miR-214 as wellas by the highly expressed miR-693 and miR-24. For pluripotencygenes, we observe overlap in predictions for down-regulated miR-NAs (miR-23a, let-7d�7f�7i, miR-199 and miR-124a) among up-regulated transcripts such as Lefty1, Lin28, Lin28b and Myc. In thesecases the miRNA and its target expression are inversely correlated.In particular, let-7 is predicted to target Lin28, Lin28b and Myc tran-scripts by two or more methods. Lin28 has been previously shownto block the processing of let-7 pre-miRNAs (Viswanathan et al.,2008) and let-7 has been confirmed to target the Lin28 transcriptby a Renilla luciferase assay (Kiriakidou et al., 2004). This indicatesthe presence of a negative feedback loop operating between LIN28protein production and let-7 miRNAs. Two methods predict sitesfor the up-regulated miR-106a in Stat3 that shows reduced expres-sion upon stem cell differentiation.

Among the ten most highly expressed miRNAs, we find 13 pre-dictions supported by more than one method for Nes, Lefty1,Lin28b, Sall4, Tcf7, Stat3 and Sox2 transcripts. For Tcf7, miR-24 bind-ing sites are predicted by all 3 methods. The pluripotency tran-scripts are initially expressed at high levels in ES cells andsubsequently repressed during EB formation. The miRNAs thatare highly expressed during all phases of EB differentiation mayfine-tune the expression levels of these genes at various timepoints. Further analysis reveals that multiple transcripts are poten-tially regulated by the same miRNA entity. For example, miR-21has been shown recently to regulate stem cell pluripotency byaffecting Nanog and Sox2 expression (Singh et al., 2008). In addi-tion, binding sites of miR-21 are predicted for Lin28b, Stat3, Sox2and Tcf7 transcripts.

Fig. 5. Sylamer word enrichment analysis. The x-axis represents genes that have been rmost down-regulated at day 3 compared to day 0. The y-axis represents the �log 10 tra

While the predicted miRNA binding sites have yet to be exper-imentally validated, this gives an insight into the potential regula-tory network of miRNAs and genes during early differentiation ofEBs.

1.6. Enrichment analysis of differentially expressed mRNAs

We analyzed the enrichment of miRNA seed sequences in theranked list of genes (from up- to down-regulated) obtained bycomparing the gene expression at day 3 against day 0. Using anintegrated analysis we find that words of length 6, 7, and 8 comple-mentary to the let-7 family seed region (2–8 nt) are significantlyenriched among the up-regulated genes (adjusted p-value0.0026) (Fig. 5). We find no significant enrichment for seed regionsof other miRNA families among the up- or down-regulated part ofthe genelist. These results suggest the functional importance ofdown-regulation of let-7 during early EB formation by affectingthe expression levels of target transcripts.

1.7. Conclusions

Analysis of genes that are significantly up-regulated during EBformation reveals the expression of germ layer specific genes suchas Nes, Fgf5 and Sox4 (ectoderm markers), Nodal, Amn and Cldn7(endodermal markers) and Eomes, Brachyury, Gsc (mesendodermmarkers). Based on the expression of germ layer marker genes,we also found that the genes specific to definitive endoderm suchas Foxa2, Gata-4 and Gata-6 show reduced expression at day 3, sug-gesting that EBs at day 3 consist of ectoderm, mesendoderm andvisceral endoderm, but lack definitive endoderm. The gene expres-sion signature at day 3 also hints towards formation of neural line-age where we observe significant up-regulation of key genesimplicated in early nervous system development. This is also re-flected in the significant enrichment of GO terms correspondingto ‘neurogenesis’ and ‘nervous system development’ in the up-reg-ulated genes. The expression levels of most of the pluripotencytranscripts are consistent with previous studies except for Oct4and Myc. This difference can be attributed to this study’s focuson early EB development and the use of a different differentiationmethod. Genes down-regulated at day 3 mostly correspond to plu-ripotency markers, cell adhesion and intracellular signalling re-lated genes.

The miRNA expression signature reveals that miR-302 and miR-17 clustered family members are highly expressed at day 3 com-pared to day 0. Previous studies have also shown that the miR-302 family is exclusively expressed during early embryogenesisand controls mesendodermal fate specification through the Nodaland Lefty networks. miR-106a, which belongs to the miR-17 family,is up-regulated at days 1–3 and its target p21 is found to be down-regulated. Numerous oncomirs like miR-382, miR-323, miR-365,miR-196a, miR-125b, miR-27a, miR-382 and miR-323 also showdifferences in their expression patterns. All the members of thelet-7 family are found to be down-regulated at day 3, consistentwith their regulation by Lin28. Interestingly, our Sylamer analysis

anked according to their fold-change expression values from most up-regulated tonsformed significance p-value (positive for enrichment and negative for depletion).

R. Tripathi et al. / Gene Expression Patterns 11 (2011) 334–344 341

shows the enrichment of let-7 seed sequence in the genes up-reg-ulated at day 3 of EB formation. These results suggest that the let-7family of miRNAs may cause large-scale changes in the expressionprofile during early EB formation.

The miRNAs binding sites predictions provide an insight intomiRNA and mRNA interactions that may be essential in stem celldifferentiation and lineage formation. Experimental validation oftargets and subsequent perturbation of key miRNAs regulatingdevelopmental and pluripotency markers by knock-down orknock-out experiments may increase the efficiency of derivationof different lineages as has been demonstrated for miR-302 familyof miRNAs (Rosa et al., 2009).

2. Experimental procedures

2.1. ES cell culture

AB2.2 wild-type ES cells were maintained on SNL76/7 feedercell layers mitotically inactivated by c-irradiation (Ramirez-Soliset al., 1993). ES cells were grown in M15 medium without LIF(Knockout Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco/Invitrogen)), supplemented with 15% foetal bovine serum (FBS,Gibco/Invitrogen), 2 mM L-glutamine, 50 U/ml penicillin, 40 lg/ml streptomycin and 100 lM b-mercaptoethanol (BME). Cells werecultured at 37 �C with 5% CO2. ES cell medium was changed daily.Upon reaching 80–85% confluence, ES cells were ready for passag-ing. The media was changed about 2 h before passaging. After 2 h,media was aspirated and the plate was washed once with PBS. Twomilliliter of trypsin was added to each 90-mm plate. The plate wasincubated in a tissue culture incubator at 37 �C for 15 min. Eightmilliliter of fresh M15 media was added to each plate. After dis-persing the cells by pipetting them up and down vigorously, thecell suspension was distributed equally to three 90-mm feederplates. The plates were incubated in a tissue culture incubator at37 �C and 5% CO2.

2.2. EB formation

ES cells grown on 90-mm plates up to 80% confluence were fed2–3 h before trypsinization. The plates were washed in PBS andtrypsinized for 15 min. The cells were re-suspended in 10 mlM15 medium and counted using a hemocytometer. The cells werediluted in Differentiation Medium (For 100 ml Knockout Dul-becco’s Modified Eagle’s Medium DMEM, Gibco/Invitrogen),25 ml FBS (Gibco/Invitrogen), 1.25 ml 200 mM 100� L-glutaminestock (Gibco/Invitrogen), 1.25 ml 10 mM BME stock (10 mM) and1.25 ml 100� nonessential amino acids (NEAA) stock (Gibco/Invit-rogen) were added to a final concentration of 1000 cells per 20 ll(Guan et al., 1999; Kurosawa, 2007). Twenty microliter drops wereplated on the lids of 140-mm plates, which were carefully invertedover the petri-dishes filled with approximately 15 ml PBS. Thedishes were carefully placed in the incubator at 37 �C and 5%CO2. EB formation was closely monitored daily.

2.3. Total RNA isolation from ES cells and EBs and Illumina bead arrays

ES cells and differentiating EBs at days 1, 2 and 3 were pelletedand total RNA was isolated using mirVana miRNA isolation kit(Ambion) according to the manufacturer’s protocol. RNA isolatedfrom samples including mRNA and miRNA was processed for miR-NA and mRNA expression using Illumina Bead Arrays (Sentrix arrayMatrix Universal Probe Set 5A, corresponding to miRBase version9.1 for miRNA expression profiling and MouseWG-6 version 1.1for mRNA expression profiling) according to the manufacturer’sprotocol. Raw data was obtained and analysed as described below.

2.4. mRNA expression analysis

Raw intensities were obtained using Illumina BeadStudio (ver-sion 3.1.8). The sample gene profile file was imported into statisti-cal package R and processed using the Bioconductor package lumi(Du et al., 2008). The raw intensities were first transformed to scalethe variance across the intensity range using log2 and then quantilenormalized. Microarray quality control was performed using thearrayQualityMetrics package (Kauffmann et al., 2009). To detect dif-ferentially expressed genes, the normalized expression values wereanalyzed using linear models with limma (Smyth, 2004). Signifi-cance of differential expression was tested by an empirical Bayesmoderated t-test and adjusted for multiple testing using Benjaminiand Hochberg’s (BH) method (Benjamini and Hochberg, 1995). Thelists of differentially expressed probes with adjusted p-values<0.01 and greater than 2-fold-change in either direction were ob-tained for each day compared against day 0. The list of up- anddown-regulated genes between days 3 and 0 were used for GeneOntology (GO) analysis. GO analysis was performed using the con-ditional hyperGTest function from the Bioconductor GOstats pack-age (Falcon and Gentleman, 2007). To obtain the gene universe,we removed probes without Entrez Gene identifiers and also fil-tered out genes with low variability (interquartile range,IQR < 0.5). Lists of unique Entrez Gene identifiers were evaluatedfor enrichment of ‘‘Biological Process’’ terms, using GO mappingsdefined in the Bioconductor org.Mm.eg.db annotation library. OnlyGO categories of at least the fourth level and with adjusted p-val-ues <0.01 were considered. The raw mRNA (.txt) file is available atArrayExpress (accession number E-TABM-620) (Parkinson et al.,2007).

2.5. miRNA expression analysis

For the miRNA arrays, raw intensities were obtained from Illu-mina BeadStudio (version 3.2.6). The miRNA sample gene profilefile was read into R using the Bioconductor package lumi. Thelog2 transformation was applied to the data and quantile normal-ized. The quality of arrays was assessed using arrayQualityMetrics.A contrast matrix defining all the pair-wise comparisons betweendifferent day points was subsequently fit, using limma. Further,the data was filtered to exclude probes that were unexpressedand those with low variability (IQR < 0.5). The filtered data wasused for unsupervised hierarchical clustering, using a distancemeasure of 1 minus the Pearson correlation coefficient betweensamples. For visualizing the expression values using a heatmap,the values for each probe were first centered by subtracting themean expression value across samples. Differential expressionwas assessed using an empirical Bayes moderated t-statistic, andp-values were adjusted for multiple testing using the BH method.MiRNAs with adjusted p-values less than 0.01 and greater than2-fold change were considered to be differentially expressed. Theraw miRNA (.txt) file is available at ArrayExpress (accession num-ber E-TABM-621) (Parkinson et al., 2007).

2.6. QPCR for selected mRNAs

Real-time PCR analysis was carried out to verify expression re-sults obtained with the Illumina expression platform. Four pluripo-tency transcripts, Klf4, Klf2, Oct4 and Lefty1 and Beta-actin as apositive control were selected. Ten microliter RNA (containing1 lg of sample RNA) was incubated for 10 min with 1 ll OligodTprimer (Promega). First strand reverse transcription was carriedout by adding 4 ll FS buffer, 2 ll DTT, 1 ll dNTPs (10 mM each),1 ll RNase inhibitor and 1 ll Superscript Reverse Transcriptase tothe mixture. The reaction was incubated at 42 �C for 60 min. Thereaction was stopped by heating the mixture to 95 �C for 5 min.

342 R. Tripathi et al. / Gene Expression Patterns 11 (2011) 334–344

Eighty-five microliter of distilled water was added to dilute thereaction mixture. The probes and primers are described (Supple-mentary Table S5). The concentration of the primers and probeswas adjusted to 25 and 4 lM, respectively. Reactions were set upin duplicate per reverse-transcription reaction sample using12.5 ll of Absolute Blue QPCR ROX mix, 1.0 ll forward primer,1.0 ll reverse primer, 0.5 ll probe, 5.0 ll water and 5.0 ll RT reac-tion in ABI Prism 7900HT Applied Biosystems real-time PCR ma-chine. Negative controls containing water instead of RT reactionwere set up as well.

2.7. QPCR for selected miRNAs

The first step cDNA synthesis reaction was carried out by mak-ing a master mix consisting of 0.15 ll 100 mM dNTPs, 1.0 ll re-verse transcriptase, 1.5 ll 10� buffer, 0.19 ll RNase inhibitor perreaction and 4.16 ll nuclease free water. Master mix (7.7 ll) wasmixed with 5.5 ll of sample RNA (diluted to 10 ng/ll) in a polypro-pylene tube. Three microliter of miRNA specific primer was trans-ferred to each well of PCR plate and mixed with 12 ll of RT mastermix. The PCR plate was loaded onto the thermal cycler and thereaction was incubated at 16 �C for 30 min, 42 �C for 30 min,85 �C for 5 min and stored at 4 �C.

The second step cDNA synthesis involved taking 1.33 ll of eachcDNA reaction, 1.0 ll of Taqman assay, 10 ll of Taqman 2� Univer-sal Master Mix of 7.67 ll of water. Each reaction was set up induplicates. The program was set up as follows: Step1: 50 �C 20;Step2: 95 �C 100; followed by 40 cycles of Step 3: 95 �C 0.150; Step4: 60 �C 10.

2.8. Identification of miRNAs targeting key pluripotency transcripts

The EnsEMBL transcripts for Nes and pluripotency genes werescanned for the presence of miRNA binding sites using Pictar pre-dictions in mouse (unpublished results, based on 17 vertebrategenomes) (Krek et al., 2005), TargetScanMouse Release 5.1 (Grim-son et al., 2007) and miRBase mouse targets v5 (Griffiths-Joneset al., 2008). For Pictar predictions, the best rodent score was used.For TargetScan and miRBase, the best Total Context Score and p-va-lue-og were used respectively.

2.9. Sylamer word enrichment analysis

Full details of Sylamer are provided elsewhere (van Dongenet al., 2008). In brief, the algorithm assesses enrichment and/ordepletion of nucleotide words of specific length that are compli-mentary to segments of the seed region (positions 2–8 nt) of miR-NAs in the 30UTRs of ranked genes. Significance is calculated usinghypergeometric statistics. In this study we combine Sylamer signif-icance scores for different segments of a seed region to obtain asingle significance score for each group of miRNAs sharing thesame seed region. This approach integrates signals from differentword lengths and increases method sensitivity compared to a stan-dard Sylamer analysis (Supplementary Fig. S2).

Acknowledgements

This study was funded by Wellcome Trust. R.T. was supportedby Cambridge Commonwealth Trust and the Wellcome Trust. Wewould like to thank Prof. Allan Bradley for providing us with guid-ance and laboratory support. We would like to thank Dr. Peter Ellisfor performing the Illumina Beadarray experiments.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.gep.2011.03.004.

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