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  Dynamic transcription programs during ES cell differentiation towards 3 m% u, M4 M, s3 s$ V
   mesoderm in serum versus serum-freeBMP4 culture
   Stephen J Bruce1,2 , Brooke B Gardiner1,3,4 , Les J Burke1,4 , M Milena , _- h7 }. f+ N8 m- v) S6 k
   Gongora1,3 , Sean M Grimmond1,3,4  and Andrew C Perkins1,2,3,4
   1Institute for Molecular Bioscience, University of Queensland, Brisbane,
   Queensland, Australia& S6 k3 E( ^7 H+ k( A, T: s& `( q
   2Wesley Research Institute, Queensland, Australia
   3ARC Special Research Centre in Functional and Applied Genomics, Australia& U. t5 U3 K6 [, E' B
   4Australian Stem Cell Centre, Melbourne, Australia
   author email corresponding author email( A/ u2 b7 {/ t/ \: F! L
   BMC Genomics 2007, 8:365doi:10.1186/1471-2164-8-365
   The electronic version of this article is the complete one and can be
   found online at: http://www.biomedcentral.com/1471-2164/8/365
         Received:28 May 2007$ _7 G  ]9 y( D! r
         Accepted:10 October 2007( W* m. Q1 H+ C. u; Z, P: ~' w
         Published:10 October 2007

   © 2007 Bruce et al; licensee BioMed Central Ltd.
   This is an Open Access article distributed under the terms of the Creative
   Commons Attribution License (http://creativecommons.org/licenses/by/2.0), # N3 e  Y4 x; m1 u# K; N
   which permits unrestricted use, distribution, and reproduction in any / a8 \. H9 [0 w1 m: s& O- f
   medium, provided the original work is properly cited.
   Abstract
   Background
   Expression profiling of embryonic stem (ES) cell differentiation in the
   presence of serum has been performed previously. It remains unclear if 4 W9 r; W$ k; K1 u; c. r' m) W
   transcriptional activation is dependent on complex growth factor mixtures % L( A& c, j3 ^# L6 g* v
   in serum or whether this process is intrinsic to ES cells once the stem 5 y# d7 F9 _6 E/ Q
   cell program has been inactivated. The aims of this study were to
   determine the transcriptional programs associated with the stem cell state 1 p; x9 E% r: }7 `: A+ t0 e
   and to characterize mesoderm differentiation between serum and serum-free 7 S9 M+ M. [% q+ ~
   culture.
   Results) c! `1 ^$ z. E  U
   ES cells were differentiated as embryoid bodies in 10% FBS or serum-free % }: \* }6 R1 v$ J
   media containing BMP4 (2 ng/ml), and expression profiled using 47 K 9 j6 G/ e: m5 o
   Illumina(R) Sentrix arrays. Statistical methods were employed to define
   gene sets characteristic of stem cell, epiblast and primitive streak
   programs. Although the initial differentiation profile was similar between
   the two culture conditions, cardiac gene expression was inhibited in serum
   whereas blood gene expression was enhanced. Also, expression of many
   members of the Kruppel-like factor (KLF) family of transcription factors 7 r4 U4 v! ~6 m( S2 X: `
   changed dramatically during the first few days of differentiation. KLF2
   and KLF4 co-localized with OCT4 in a sub-nuclear compartment of ES cells, 9 `% M& X3 F6 P
   dynamic changes in KLF-DNA binding activities occurred upon
   differentiation, and strong bio-informatic evidence for direct regulation 7 Q* |4 S' Q3 D3 {3 E
   of many stem cell genes by KLFs was found.# _" G: w$ R9 b' y% d$ g- `
   Conclusion6 K- T8 C6 S) `9 w% E% ]
   Down regulation of stem cell genes and activation of epiblast/primitive
   streak genes is similar in serum and defined media, but subsequent
   mesoderm differentiation is strongly influenced by the composition of the
   media. In addition, KLF family members are likely to be important
   regulators of many stem cell genes.
   Background
   Embryonic stem (ES) cells isolated from the inner cell mass of the early $ y# F& I/ h) N1 n6 v9 e" n
   mammalian blastocyst-stage embryo retain pluripotency when cultured on 5 @5 b  v1 S8 _; K; ^
   mouse embryonic fibroblasts (MEFs) in the presence of leukemia inhibitory
   factor (LIF) [1]. In the absence of LIF and MEF attachment, ES cells $ i, [) `8 a$ A) b" Q6 ?
   spontaneously differentiate into multi-cellular aggregates termed embryoid " H( L3 Q* Q) b
   bodies (EBs). Although the spatial complexity of organogenesis is not : A  S/ W: `  R3 h+ _
   established during EB maturation, the dynamics of gene expression closely
   mimic those which characterize early stages of mouse development [2-5]. 1 Z. }( m) D: o7 A( B$ U
   Thus, ES cell differentiation is an excellent model system for the
   discovery of genes involved in developmental processes.
   Many studies have attempted to identify genes that define the stem cell 2 B& R. X2 x- M; J* X9 Y
   state by mining for genes co-expressed in ES cells and various other stem
   cell populations such as hematopoietic and neural stem cells [6,7], or ES . _6 f- t2 D& l1 g/ |1 H
   cells and trophoblast stem cells [8]. Some of these studies have been
   criticized because they were unable to define a similar cohort of stem 6 d2 {) @& |! f% F+ r' c! o
   cell genes [9]. Also, this approach is likely to find co-expression of
   'housekeeping' genes that are irrelevant to the stem cell state. More
   recently, dynamic expression profiling during ES cell differentiation has / x( S! e. E6 g6 Y! G
   been undertaken using various EB culture techniques [10,11] and the
   addition of exogenous growth factors [7]. Most of these studies followed
   differentiation for a short time period (up to 6–8 days) due to inherent 0 C; Z; m- Y  v# `5 f/ E" S
   limitations of liquid or adherent cultures cell differentiation systems.
   Various platforms have been used to determine gene expression profiles,
   including differential display, SAGE [12] and slide micro-arrays, such as
   those provided by Affymetrix, Agilent and Compugen. In this study we $ z, U' l" f- ^. F
   employed Illumina® Sentrix Mouse-6 oligo bead arrays [13], which have ~47
   K probes that are largely based on the MEEBO set of transcripts, plus ESTs , Q! j4 p$ M$ N
   discovered as part of the FANTOM2 transcriptome project [14], some 3 F& F' g1 y, r5 E6 K! m3 S
   alternatively employed exons, and a small subset of non-coding
   transcripts. This is a new, rich and sensitive platform for expression
   profiling [15].
   ES cells were differentiated in methylcellulose cultures, which allowed
   consistent EB development over 16 days in either serum [2,16] or defined / d& C4 k3 d7 M8 ~$ f1 L: c
   serum-free media containing BMP4 (2 ng/ml), termed serum-freeB4L hereafter ( E: g- O4 y8 a  h4 B- B
   [17]. The dynamics of loss of stem cell gene expression and activation of ; y- n$ t- v" }! n3 _8 h
   epiblast and primitive streak gene expression programs were similar in   I2 e5 e  N6 r
   serum and serum-freeB4L but QT clustering revealed a significant ; f, c8 ~9 W' l# l# O9 J1 {0 N( _
   difference in mesoderm outcomes from day 6 onwards. In particular, cardiac
   gene expression was greater in serum-freeB4L whereas activation of the
   blood program was enhanced in serum.
   High expression levels of Kruppel-like factor (KLF) family members: Klf2, . I2 c9 W! s; Q; P0 K
   Klf4, Klf5 and Klf9 was detected in undifferentiated ES cells, and 7 A2 o9 C/ `- ]: j% x8 l
   confirmed by quantitative real time RT-PCR, indirect immuno-fluorescence
   and DNA-binding assays. Expression was down regulated rapidly upon   o  @4 V+ s( M& o0 C. ^8 M; p
   differentiation and a different set of Klfs: Klf3, Klf16 and Klf1/Eklf,
   were activated upon ES cell differentiation. Also, KLF-binding sites were / S+ Y3 D* I/ a) }, y5 f
   markedly over represented in the proximal promoters of most stem cell
   specific genes, suggesting possible direct regulation by KLFs. We suggest
   a model in which the balance between self-renewal and differentiation is
   regulated by competitive occupancy of the proximal promoters of key stem 4 L- ^" y1 C1 ^' h  C/ g
   cell genes such as Zfp42/Rex-1, Nanog, Pou5f1(Oct4), Lefty1 and Lefty2, 4 f* L2 @' e! {9 b& z
   and others such as the Klf genes themselves.3 b7 R2 |( |2 q3 M
   Results
   Differentiation of ES cells in serum and serum-freeB4L culture$ g5 n2 x3 L8 V% b4 T
   The directed differentiation of embryonic stem cells into desirable cell
   types will depend upon the addition of specific growth factors to defined
   media and reproducible physical conditions of culture. To establish a
   baseline for such studies, we undertook a comparison of murine ES cell
   differentiation using an embryoid body (EB) methylcellulose culture system % n4 \/ h9 T4 U/ W
   containing either 10% FBS [2] or a chemically defined BSA-based media , q& ^( H5 ~! \  Z3 C. F. h/ U
   containing 2 ng/ml of BMP4, termed serum-freeB4L [17]. Triplicate
   experiments were performed in which ES cells were harvested after feeder
   depletion, and set up in parallel EB cultures in serum versus 5 Q2 A. g8 |* v1 ^7 A3 B+ M
   serum-freeB4L. RNA was collected at regular intervals up to 16 days, ! \3 P, L8 Q$ {, q  q3 K& [
   quantitative RT-PCR was performed on a panel of genes representing defined
   stages of development to establish the robustness of the culture system
   (Figure 1), and expression profiling was performed using Illumina® Sentrix - b  H& `1 |5 z6 `3 T
   Mouse-6 bead arrays (see Methods and [13]).1 Q! c$ D5 C. i$ K" i
   Figure 1. Dynamic gene expression during EB differentiation in serum and
   serum-free media containing BMP4 [2 ng/ml]. Expression of stem cell,
   primitive streak and late mesoderm genes were analysed by quantitative 8 u2 p7 [( Y) C  u4 u7 J! F: r& j
   RT-PCR in undifferentiated ES cells (0, grey bar) and in EBs collected for
   up to 16 days in serum (white bars) or defined media (black bars) from the * {9 G& j" f/ q5 n% t5 W
   same starting ES cell populations. Bars represent the means of three
   (serum) or two (serum-freeB4L) biological replicates. The Y-axis   m# f2 W# r  H( ~$ Q. n; R8 `
   represents a log scale normalized relative to the housekeeping gene HPRT.
   Error bars indicate standard deviation./ V& D2 I3 h' R, x
   Changes in gene expression were extremely reproducible across ES
   differentiation experiments with overall correlation of global gene $ _" m+ Y. F9 I
   expression for biological replicates in the order or 0.95–0.96.
   Approximately 30% of the probes were differentially expressed at one or
   more stages of EB differentiation as determined by one-way Welch ANOVA
   with multiple testing corrections using the Benjamini and Hochberg False
   Discovery Rate algorithm (7,967 probes with a p-value of < 0.01 by Welch " ^) D( ^8 c% ?+ v3 i& n
   t-test) (Figure 2A).
   Figure 2. Gene expression profiles during 16 days of EB differentiation in
   serum and serum-free media containing BMP4 [2 ng/ml]. (A) GeneSpring 0 _* O- L2 H/ A1 r" i) V, o. G
   representation of the subset of genes from the 48 K Illumina array that
   showed significant differences in gene expression at 2 or more time 6 h" z( S: ^  K- k% D+ w% O
   points. Each line represents the mean normalised expression of an - i# L8 o; D0 q" S3 e% B
   individual Illumina probe. Colours represent relative expression (red 4× 1 i$ i% }. l4 j+ t; O# t
   increased; green 0.25× reduced) compared to a gene mean of 1 (black) with # q- t  N8 t5 L9 v
   expression in undifferentiated ES cells (day 0) as the decision point for
   colour coding. The expression profiles of T (brachyury), Hba (α-globin), * f4 K( Q5 W5 _( I; V) X/ a
   and Igf2 are indicated for reference. (B) Clustering (tree) of the genes
   that showed significant (p < 0.01) changes in gene expression upon + S, x  S& E- I, o
   differentiation by One-way Welch ANOVA. Expression levels are heat map 4 i! I- ?' u1 b
   colour coded (inset) with high expression at Day 0 coloured red and low
   expression coloured green.
   To mine for genes with similar patterns of expression across the time 6 J' E# L( R5 ]  Z) }3 I- D" K
   course, hierarchical clustering was performed on the 7,967 probes and 1 b$ E$ L# [7 H3 ~, D
   represented graphically. A large percentage of these (~30%) displayed
   highest expression in undifferentiated ES cells and subsequent reduced
   expression over time (labeled stem in Figure 2B). A second major cluster . Y' M4 y) F% V" A- f
   showed marked induction after ~day 4–6 of differentiation (labeled
   mesoderm in Figure 2B).
   Putative stem cell genes sets
   Previous studies have identified genes important to the stem cell state by   A/ x0 ]! @5 F$ h! m6 W& F6 \( e
   comparing expression profiling data from different stem cell populations
   such as ES cells, haematopoietic stem cells (HSCs) and neural stem cells
   [6,18]. We used two alternative approaches to identify specific stem cell
   specific gene expression. Firstly, genes with dynamic expression patterns ! b( t( h7 @# A  ]8 ?
   which correlated closely with Oct4, Sox2 or Nanog expression profiles
   (Pearson correlation > 0.9) were determined as previously described [6,7]. 4 N/ Y6 b/ `) A* I3 X7 U
   Second, genes rapidly down regulated ≥3 fold by day 3 of differentiation,
   a time point where ES cell clonogenicity decreases dramatically, were
   determined. Not surprisingly there was very little overlap between these
   gene lists, primarily because Oct4 and Nanog expression persists during EB + v; V, H  r/ K1 n- v9 {1 d
   differentiation longer than many other stem cell genes (Figure 1). Full
   lists of genes ranked according to similarity of expression (Pearson
   correlation >0.9) with expression patterns for Oct4, Nanog or Sox2 are 7 d, {, g$ C1 p: W* p( |3 r4 p+ R
   provided in Additional files 1, 2, 3, respectively.
   Additional file 1. Oct4 gene list. The data provided lists all genes
   expressed during 16 days of embryoid body differentiation with similarity - {& g1 p7 u% K; @
   to Oct4 (Pearson correlation >0.9)." N: B2 n8 _2 M" o3 V
   Format: DOC Size: 630KB Download file3 B( D% b( l0 e) R
   This file can be viewed with: Microsoft Word ViewerAdditional file 2. * [- k0 |3 a/ k6 c
   Nanog gene list. The data provided lists all genes expressed during 16
   days of embryoid body differentiation with similarity to Nanog (Pearson + |  q, @8 ?# G) l" i
   correlation >0.9).6 d2 F4 U+ D+ q8 A' U) A
   Format: DOC Size: 111KB Download file, w( w" D$ Q- H( F6 T( i% z3 P
   This file can be viewed with: Microsoft Word ViewerAdditional file 3. Sox2 3 F9 L3 z$ v. q7 t! u, ?  t) _
   gene list. The data provided lists all genes expressed during 16 days of
   embryoid body differentiation with similarity to Sox2 (Pearson correlation 3 M3 s$ m8 H$ }9 p2 H5 R
   >0.9).4 j* [$ V0 J8 V  f; i5 b2 ]
   Format: DOC Size: 237KB Download file
   This file can be viewed with: Microsoft Word Viewer
   Only fifty-nine genes were down regulated ≥3 fold by day 3 of EB
   differentiation (Table 1). Many of these have been identified as stem cell
   markers by other groups using alternative strategies [6,7,19]. Information ' N/ S. W" X0 M8 k
   was compiled on their known expression patterns and/or gene knockout 1 u. @6 X+ E+ j& H
   phenotypes via literature searches as referenced in Table 1. Insufficient
   space exists to discuss every gene and most have been previously
   identified as ES cell enriched transcripts. Osteopontin is secreted by ( N% n6 G( V" N& |" d3 F: h7 G
   osteoblasts in the bone marrow niche where it is thought to play an
   important role in the maintenance of HSC quiescence and maintenance of 4 {# X1 [+ A7 S% }- V8 @
   'stemness' [20]. It has also been discovered by other groups as a strong
   marker of ES cells and is regulated directly by the key stem cell 8 W5 Y: ?% I4 M0 {* L
   transcription factors, Oct4 and Sox2 [21]. Thus, osteopontin is likely to
   be a useful marker of ES cell pluripotency and may play autocrine or
   paracrine roles in maintenance of stemness. The second most rapidly down
   regulated gene was estrogen related receptor β, Esrrb, which had
   previously been identified as a stem cell specific gene and shown to be
   critical for maintenance of the stem cell state [19]. F-box 15 (Fbxo15), a : ?& ^& G9 Z; [! n  z
   member of the large F-box family of genes [22] was also rapidly down
   regulated. Fbxo15 is known to be highly expressed in ES cells, is + F4 e2 L; k/ P7 D5 G, I
   regulated by Oct4 and Sox2, but is dispensable for ES cell self-renewal
   [23]. Recently, the stem cell specificity of this locus was used to screen " M: Q# y5 F4 x# ]5 T
   for cDNAs capable of transforming mouse fibroblasts into ES cell ( s6 R4 _  K( S; {8 p( {
   populations [24]. Other notable rapidly down regulated genes included the / D8 k- i0 w% A+ `5 I
   nodal inhibitors Lefty-1 and Lefty-2, and Zfp42/Rex-1, which are also well ) @; z8 x, e* O" Q- t  Z. L2 o
   established as stem cell markers [19].0 h) a3 Z) Y( g+ Q* s
   Table 1. Genes rapidly down regulated (>3 fold in 3 days) upon ES cell
   differentiation) y: A0 K$ h+ h5 o. c6 L
   Oct4, Nanog and Sox2 are not included in Table 1, even though each is 7 Z2 x. \0 m1 \* K
   required for the maintenance of ES cell pluripotency [25-27] due to their
   persistent expression during the first 3 days of EB differentiation
   (Figure 1 and 5A, and Additional file 1). Indeed, Oct4 expression is known
   to persist beyond the inner cell mass stage of development in vivo, with " ~- y7 V" D1 B+ l$ _# m0 F5 P
   transcripts detected in the epiblast and later in the primitive streak, % i9 k9 w) {5 X) \* s% G
   before it becomes further restricted to primordial germ cells [10,28]. 779
   genes with a similar pattern of expression to Oct4 (Pearson correlation of
   > 0.9) were identified (Additional file 1). As expected this list also 9 ?+ _# I1 V7 z* B
   includes many well known stem cell genes such as Sal-like 1, nodal,   d3 U9 K0 D0 Y5 t
   chromobox homolog 7 (Cbx7), Dnmt3b and Sprouty homolog 4 [19]. We also . E: D6 t: L+ P$ r3 T4 x$ ~7 y
   mined for stem cell genes by looking for similarity (Pearson correlation - f: ?7 ^7 V1 u& V3 t) @
   of > 0.9) with Nanog and Sox2 gene expression profiles. These provided . S! g; @- G1 a) A
   overlapping but not identical sets of putative stem cell specific genes
   (Additional files 2, 3). Full data for the 16 days of differentiation can ) H3 g/ h1 k% U$ C1 a' m% x
   be interrogated and mined for specific differentiation outcomes via Signet
   (Login_Bruce) (see Methods).# Z& J. R! N) o5 Y6 j. K
   Tightly co-ordinated waves of gene expression suggest sequential . V' y4 ]3 u. q' M& Z- ^* W6 T. t
   activation of epiblast, primitive streak and mesoderm differentiation
   programs9 a% I! ^$ h9 m; w* `
   Many alternative differentiation programs occur simultaneously during ES 4 P3 C" r# x: W! W1 t* N* ]4 G
   cell differentiation. Therefore, it is difficult to find gene
   syn-expression patterns that identify specific programs using standard
   clustering methods. An alternative approach is to search for expression
   patterns that closely resemble genes of known function. For example, the T
   box gene, brachyury, is a well characterized specific marker of the 8 \; v5 h$ f1 K3 b: x
   primitive streak [29]. Brachyury expression is transient in vivo, a
   feature recapitulated during ES cell differentiation [2]. Using qRT-PCR, / c$ M# h, q  `- o& k; z' Q
   brachyury was induced ~1000 fold from D0 to day 4 and then rapidly ' w% Q: ~- h' R0 O4 ]2 |
   silenced to baseline levels by D6 (Figure 1). The dynamics of brachyury
   expression were similar between serum and serum-freeB4L media (Figure 1
   and 3A), but serum-free media was unable to support brachyury induction in
   the absence of BMP-4 (data not shown). Thus, BMP4 (or related factors in & w9 T% R8 C& H! a; V9 P: X
   serum such as activin) is essential for mesoderm generation. Thirty genes ; Z* |$ N% S8 D- C
   were identified with a similar expression pattern to brachyury (Pearson
   correlation >0.9) (Table 2) including many genes with established
   expression or critical functions during primitive streak formation, such + \, t3 `9 R- _6 J
   as Mix1 [30], Lim1 [31], Sp5 [32] and eomesodermin [33]. Transient ; R2 _( |! Y& w9 ?2 ~) {
   expression of many of these 'streak-specific' genes was validated by 3 G. k8 H: r6 O) d
   qRT-PCR (Figure 3B and data not shown). In all cases there was a close
   correlation between the qRT-PCR results and the profiling data (Figure . ~/ f8 \0 U2 \; c8 T  f* U2 ?  y
3A).2 j) V4 ?% m3 U* b6 N
   Figure 3. Mining for genes expressed in patterns consistent with primitive & C8 K# e( U# t" `' J& g9 C
   streak, and epiblast differentiation programs. (A) GeneSpring
   representation of epiblast and streak gene expression profiles during EB
   differentiation in 10% serum or serum-freeB4L culture. The up-regulation
   of genes between days 2–4 were similar under both conditions. (B) qRT-PCR
   analysis of Mixl, Lim1, Cdx4 and Riken clone 8430415E04Rik. The Y-axis
   represents expression relative to the housekeeping gene HPRT. Error bars 5 W& J. Z/ J' k! w" d
   indicate ± SD from three biological replicates. (C) 102 genes transiently
   up regulated during the first 4 days of ES cell differentiation were ! P4 `& K- s. K/ [3 w- K1 x7 U- u
   clustered using a tree algorithm and Pearson correlation of > 0.9. The
   'Brief' group represents gene with very transient expression at day 3. (D) 2 {; T: `( h- I# p
   Coding region for 8430415E04Rik. The shaded areas represent the three Heat
   domains.Table 2. Brachyury-like (Primitive Streak) gene list (Pearson . {; }5 y. s6 B* p* t, t
   correlation >0.9 with brachyury)
   Interestingly, four novel transcripts were identified in this list (Table 7 A* }8 E0 s. y' {0 @( {' e/ s
   2). One of these, 8430415E04Rik, was detected by two independent probes, 5 }# M' S! v- N8 b5 O$ }, {% L
   strongly supporting its 'streak-specific' expression; by qRT-PCR there was + f7 v4 q  l1 B
   ~50 fold induction from day 2 to day 3 of differentiation and a decrease
   to near baseline levels by day 6 (Figure 3B). The gene contains 24 exons,
   and the full length cDNA encodes for a protein of 868 amino acids (Figure * y: j, f+ E  d% u: c3 I  p. G8 m0 E
   3D) which is highly conserved in vertebrates (Additional file 4). It has 7 S3 H" V- O0 {4 w
   little homology to other proteins apart from three predicted HEAT domains
   or armadillo domains (grey shading in Figure 3D). Drosophila armadillo 3 s3 V. ~- m$ i5 j! M- F* J9 a
   interacts with β-catenin via its HEAT/armadillo repeat [34], which
   suggests the protein encoded by 8430415E04Rik may also be involved in
   protein-protein interactions.
   Additional file 4. Sequence alignment of RIKEN clone 8430415E04RIK. The
   data provides an alignment of RIKEN clone 8430415E04RIK using human,
   mouse, chick and zebrafish sequences./ |* f- `3 L# [" F; o2 E
   Format: DOC Size: 31KB Download file
   This file can be viewed with: Microsoft Word Viewer
   The list of primitive streak genes in table 2 is smaller than that
   previously published [10] due to the very stringent criteria used to - ?: \0 V; \7 m
   determine brachyury-like patterns of expression. Thus, to find further ; C! q2 j1 h% V; q! q/ ^9 z
   genes characteristic of epiblast, early and late primitive streak 4 x; S6 |3 ^& ]) A
   programs, probes were identified which were transiently up and down
   regulated at least two fold with peak expression at day 2, 3 or 4 of EB + b% R4 t1 v* B) e& i9 u  z
   differentiation. Gene lists were combined (102 Illumina probes) and
   clustered into epiblast, early, mid and late streak groups plus a unique 9 B; \1 I; w: I; Y* D. r/ K
   group in which expression was very transient at day 3 of differentiation : X) c& \& D, ~; o9 B7 m. ?& f
   (designated 'brief' at the bottom of Figure 3C). Also, lists were
   generated by searching for similarity of dynamic expression profiles to " R$ q1 k1 A/ q) h2 [7 p. w; ^0 X
   known genes which are accepted specific markers of specific stages of 2 Q* B: x, B/ H
   mouse development. For example, Fgf5 is specifically expressed in the
   epiblast [35], and Wnt5a is expressed in the late primitive streak [36]. ! E3 U, H, S+ U: t7 J4 ]; Y5 U- q
   The Fgf5-like (epiblast) list includes the methyltransferase, Dnmt3b, ) C' Y: C* ~  @8 V$ R
   which was massively up-regulated in the first 2 days of EB differentiation
   (Additional file 5) and is known to be essential for mesoderm 6 h% @( B; j0 j" E  ]# M
   differentiation [37]. This list also includes Pim2, Zic2, Wnt8a, the SP1   a& m9 t- j7 F' Z" A9 I
   (and possibly SP5) cofactor Crsp2, and Irf1. The Wnt5a-like (late
   primitive streak) list contains 76 genes including many homeobox genes : a) ^$ ?1 K6 P  {0 z1 g5 u
   (Hoxb2, Hoxd1, Hoxb6 and Hox8/Msx2), Cdx2, Hand1, Tbx3, Bmp4 and the VEGF $ }- y/ P' L! K3 p$ ~
   receptor Flt1 (Additional file 6). Many are expressed at E10 of mouse
   development in the remnants of the primitive streak [36], suggesting the 9 y9 L) w1 E& X1 A# T9 f
   Wnt5a-like list includes many genes known to pattern the embryo following ) M- X1 H4 P+ R5 B
   formation of the germ layers. There is a significant overlap between the 3 N* I+ @. S5 e& B; J1 }  K
   genes we identified as being epiblast-, early and late streak-specific and
   those recently identified using a similar EB differentiation system and 1 q# ]1 a3 T) Z9 H
   Affymetrix arrays [10], and by other groups [2,38]. In addition, there are
   addition novel RIKEN cDNAs in these lists, which are likely to encode for
   interesting proteins worthy of further study (Additional files 5, 6).4 r* m7 j4 B- b7 e5 G& B: @
   Additional file 5. FGF5 gene list. The data provided lists all genes
   expressed during 16 days of embryoid body differentiation with similarity * e( w4 o& x, {9 L7 j0 J" k
   to FGF5 (Pearson correlation >0.9).. H: G( ]& |& Q, t
   Format: DOC Size: 69KB Download file
   This file can be viewed with: Microsoft Word ViewerAdditional file 6.
   Wnt5a gene list. The data provided lists all genes expressed during 16
   days of embryoid body differentiation with similarity to Wnt5 (Pearson - t- A) {  l5 ]2 m
   correlation >0.9).
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   This file can be viewed with: Microsoft Word Viewer+ {4 K2 d/ b6 p' x7 O% }. L; c; M, q/ j
   Following the primitive streak wave of gene expression there was a " f7 f" ^& V4 [( g0 |( F
   dramatic activation of many genes (Figure 2A). Many of the most highly
   induced genes, such as globin genes, heme synthesis enzymes and
   haematopoietic transcription factors, are known to be expressed in the   X9 F" t& c, Q$ y8 J9 b# U! g
   blood. qRT-PCR for globin genes confirmed > 10,000 fold induction between + d9 G" N# K% M( y
   days 3 and 8 with a steady decline thereafter (Figure 1). Other mesoderm
   and endoderm programs are also activated at this stage, so it is difficult
   to mine for these different outcomes., k# E! n& q3 G
   Enhanced cardiac gene expression and reduced erythropoiesis in & ?, {3 j/ }: B$ y" h
   serum-freeB4L EB culture3 Q4 p4 ~. P! @: F" Q% Q
   Overall there were very strong similarities between ES cell
   differentiation in serum and serum-free cultures supplemented with 2 ng/ml
   BMP4. For example, all 29 of the primitive streak gene set, as defined
   from serum-containing cultures, were also expressed transiently and to a
   similar level in serum-freeB4L media (Figure 3A). Genes differentially
   expressed at each EB time point (Day 1–16) were pooled and subjected to QT
   clustering to find sets differentially expressed between serum and
   serum-freeB4L EB culture. Using a minimal cut-off of five genes per group, 5 P; I9 ?# w1 l0 `( b' \/ m
   nine gene sets were identified; seven showed higher expression in serum 1 T2 {, J6 w+ i& R8 T9 d7 I8 ~
   and two showed higher expression in serum-freeB4L (Table 3 and Figure 4).
   Table 3. Genes differentially expressed during ES cell differentiation in
   serum versus serum-free defined media (BMP4 2 ng/ml)Figure 4. QT 1 |5 N4 c/ [+ V5 L( q# z% z
   clustering revealed sets of genes differentially expressed between serum 9 T8 J% g' J# d1 V% I
   and serum-freeB4L EB culture. QT clustering revealed nine sets of genes
   with similar dynamic expression profiles (Pearson Correlation coefficient + L* `; F/ q# A8 o  h
   of > 0.9). In sets 1–7, expression is higher in serum (black sets). In 5 m* b8 @6 W1 \( ?
   sets 8–9, expression is greater in serum-freeB4L EB culture (red sets). 0 u7 o& |3 A( ^# c0 R) b
   Some sets have been named based on the similarity of overall gene function
   during differentiation pathways (see Table 3).- }' n7 l# f3 Z- @
   The genes in set 9 were expressed more highly in serum-freeB4L media from 5 P8 C, M9 J4 u; S* c- M; o  g; K& K
   day 6, and genes in set 8 showed up-regulation at day 12 and 16 in the - p- v) p9 m9 Z! O
   absence of serum (Figure 4). Both sets are comprised almost exclusively of $ X# h" P8 k" p9 L! }
   cardiac-specific genes such as myosin heavy chain 7 (Myh7), myosin light
   chain 7 (Myl7), the cardiac isoform of α-actin (Actc1), troponin (Tnnc2),
   tropomysin (Tpm2), the nicotinic cholinergic receptor (Chrng), and cardiac
   muscle fast twitch 1 specific ATPase (Atp2a1) (Table 3). Further + o( F. Z/ j0 n; D6 T9 J
   interrogation of the array data revealed a cohort of cardiac transcription ! c0 @% Y2 h8 J! Q: x9 ~
   factors, including ISL1, MEF2C, Bop/Smyd1 and Hand2 [39], which were 4 Q& r* P: X" D1 E9 O5 d( e/ ?
   transiently up regulated 1.4 to 3.5 fold in serum-freeB4L media compared
   to serum at day 6 (Signet Login_Bruce). Importantly, this elevated cardiac ; P" C! }6 S$ @! d
   gene expression program correlated with increased numbers of spontaneously
   beating EBs in serum-freeB4L media (data not shown). Enhanced expression
   (~100 fold) of Actc1, Myl4 and Myl7 in serum-freeB4L media compared with 2 J4 X2 G- Y/ \# |
   serum-containing media was confirmed by qRT-PCR (Figure 1). Taken , ^& `9 Z  g# F  a( Q
   together, this data strongly suggests cardiac muscle cell differentiation
   is either inhibited in serum or enhanced by the addition of BMP4 to
   serum-free culture.
   The genes in set 2 were expressed in both serum and serum-freeB4L
   conditions immediately following primitive streak formation, but expressed 9 @1 [& {/ h6 |1 Q0 d
   to higher levels and slightly earlier in serum (Table 3 and Figure 4).
   Many of these genes are globins (both embryonic and definitive) suggesting
   erythropoiesis is activated more robustly in the presence of serum (See
   Discussion). Alas2, a gene encoding the erythroid specific isoform of the : r$ A4 M7 o* u. h$ s. a
   first and rate limiting enzyme in the heme biosynthesis pathway, and the
   erythroid specific transcription factor, p45-NF-E2 are also in set 2 [40]. . c6 @( y2 C* ^4 _" M% k
   qRT-PCR for α- and βh1-globin confirmed more rapid expression in serum 3 r" Q" `! G  ^) P5 B* s' j
   versus SFB4L media (Figure 1), suggesting factors in addition to BMP4
   enhance activation of the blood program from a similar primitive streak
   platform.5 `6 G2 D+ b# A8 J# h3 y9 c
   The genes in set 3 were expressed at a similar time in serum but expressed
   at very low levels or not at all in serum-freeB4L media. Again, this set 1 ?. i* b% m2 s
   is highly enriched for erythroid specific genes such as alpha hemoglobin 4 |' n! E3 E& n; E- l
   stabilising protein (Ahsp) [41], Glycophorin A, erythroid Kruppel-like 6 |5 \, J  K- ~
   factor (EKLF/Klf1) [42] and mitochondrial solute carrier protein . J! q8 b1 b' N) Q, [
   (mitoferrin) [43] (Figure 4 and Table 3). Genes in sets 1 and 5 are
   activated more robustly at late time points in serum cultures. Most of
   these are specifically expressed in macrophages. Together, these results
   suggest delayed and less robust primitive haematopoiesis occurs in SFB4L
   culture compared with serum, an observation confirmed by less robust ' R8 H& Q7 x6 y9 L
   erythroid cell generation and 'redness' of the EBs from day 6–10 of ! o1 O+ q( \0 p) S! e( f4 ?
   differentiation (data not shown).- W7 l0 A7 I; D9 N+ Y. n
   Rapid changes in Kruppel-like factor gene expression upon ES cell
   differentiation' ?+ U  v6 }: T7 K! p
   Three Kruppel-like factor genes, Klf2, Klf4 and Klf5, were rapidly down
   regulated during the first few days of EB development (bold type in Table 6 [6 P  L6 Z8 o
   1). All are members of the KLF family of transcription factors which are 7 S6 _# \& \0 t& D& E
   characterized by a similar C-terminal domain of three C2H2 zinc fingers
   which confers binding to CACC box elements in promoters and more distant
   regulatory elements [44,45]. Klf2 and Klf4 have N-terminal transcriptional
   action domains and act primarily as transcriptional activators, whereas
   Klf5 is best known as a transcriptional repressor [46]. Until recently,
   Klf2 and Klf4 were considered to have restricted expression patterns
   [47,48] with gene knockout animals showing late developmental defects in ) r( W" r: H# X& M
   vasculogenesis and skin differentiation, respectively [49,50]. Recently ! F8 |; T8 [3 u  `
   however, Klf4 expression was noted in ES cells [19,51], and forced
   expression was shown to enhance Oct4 expression and promote self-renewal 3 T3 h! r( M  i
   [51].. E( p$ r/ n1 Z- @
   qRT-PCR confirmed the rapid down regulation of Klf2, Klf4 and Klf5 in both
   serum and serum-freeB4L differentiation conditions, although Klf5
   expression was down regulated more slowly (Figure 5A and 5B, and Table 4).
   Klf2, Klf4 and Klf5 were all re-induced from day 3–5 of differentiation as # v9 A: U5 Y$ y3 _% K8 @' g: W; S
   mesoderm and endoderm developmental pathways are activated (Figure 5A and
   5B). Sixteen of the 17 known KLFs were examined in detail throughout the ; q1 z5 X" @: f! [# s# P. @8 J
   entire differentiation time course (Table 4). Klf9 was also steadily down
   regulated (>10 fold) between ES cells and day 5 EBs in serum and 8 s( Q) v" O' Z6 v3 B) ^7 w/ v6 A
   serum-freeB4L media (Figure 5B). Klf3, a well characterized 6 E  {& y2 m% h
   transcriptional repressor [52] via its ability to recruit the 2 _- O- O; k% P
   co-repressor, CtBP [53], displayed an interesting biphasic expression 2 w+ I5 F! J# e; _
   pattern with initial rapid down regulation then re-induction from days 5–6
   (Figure 5B). In contrast, Klf16 displayed little change in expression 3 k* J. N; @, ~( \. R! i1 \. y
   levels over the 16 days of EB maturation (Figure 5B).
   Table 4. Kruppel-like factor gene expression during ES cell ) ]! f+ t# ]3 ~* X1 S( u% J% T4 Q. J0 E4 n
   differentiationFigure 5. Kruppel like factors (Klfs) are dynamically ' w! j/ P) @" M
   expressed during the first few days of ES cell differentiation. (A)
   GeneSpring plot of normalized gene expression for all KLFs detected during 0 [6 E" V0 R% |' R: p1 \* G5 I8 |/ ?
   ES cell differentiation in serum or serum-freeB4L culture. Most of the ' e4 {5 W4 e5 r
   genes are listed on the y-axis in order from their highest relative 1 }0 Y, n/ n8 i) C& F
   expression in ES cells. There is dramatic up regulation of Klf1 (Eklf) * b) F4 b1 U0 x% Q
   only in serum following day 6 of differentiation. Plots representing Nanog # i/ @- q! a4 \% i6 b9 d
   (red), Sox2 (red) and Pou5f1(Oct4) (green) are shown for comparison. 4 e; G3 i; e% D' K% ~* L
   Pou5f1 and Nanog gene expression persists at high levels for 2–3 days
   after Klf2, Klf4 and Klf5 are down regulated. (B) Validation of changes in ! f+ ?' g2 n4 A: ~  r" N
   gene expression of six members of the KLF family by quantitative real time 1 L7 j$ F: s: f0 o- e2 R8 f) D
   RT-PCR. Scheme as described for Figure 2B.
   Klf2 and Klf4 co-localize with OCT4 in the nuclei of ES cells (Figure 6A). * ]/ S% N: U- a' J% F( T+ x
   Interestingly, all three proteins were found to preferentially reside in a
   nuclear sub-compartment (possibly nucleoli for transcription factories), : S3 m1 c. K6 |7 f: ?( j
   suggesting possible co-involvement in a protein complex or network. Also,
   endogenous KLF2 was detected by specific CACC box DNA-binding activity in 3 m" t! I% j1 y7 O# X1 c7 s
   undifferentiated ES cells, which was lost upon EB differentiation (Figure
   6B). In contrast, endogenous ES cell KLF4 DNA binding activity was not
   detected using the p18INK4c promoter CACC box sequence, although
   recombinant KLF4 was shown to bind to this probe (data not shown). SP1 and ' x" `) ]2 \, J7 Y5 v* v0 X; T
   SP3 are ubiquitously expressed CACC-box binding proteins, which are
   dominant in ES cell nuclear extracts (Figure 6B). We hypothesize KLF2,
   KLF3, KLF4, KLF5 and KLF6 compete with SP1 family members for DNA binding
   to key cis elements in various stem cell genes to regulate expression in
   positive and negative fashions (see Discussion).8 n. s: s; B1 c1 y6 ~
   Figure 6. Kruppel like factor expression and DNA binding activity during : s2 n9 J: {/ p5 ]% z
   ES cell differentiation. (A) Co-expression of Oct4 with KLF2 or KLF4 in ES 6 y$ l; I+ G$ D2 p3 ~
   cells. Indirect immuno-fluorescence shows co-localization of KLF2 and KLF4 , @/ B) ~3 ?7 ?; z! I
   with Oct4 in sub-nuclear compartments (possibly nucleoli). Individual
   confocal images for OCT4, KLF2, KLF4, and DAPI are shown with the
   corresponding composite image. Scale bar 40 μm. (B) Electro-mobility gel 8 Z3 o4 z1 ?6 g! l/ |5 D) ^" |3 s
   shift assay showing changes in DNA binding activities at a conserved CACC : \; v3 p7 E3 j# @' P3 N
   box site in the p18-INK4c gene promoter. Nuclear extracts were generated
   from ES cells or EBs differentiated for four days in serum. Super-shifts
   were performed with specific antisera for SP1, SP3, KLF2, KLF3, and KLF4
   (See Methods). There is strong binding of endogenous Sp1 to the CACC
   element in ES cells and EB cells. KLF2 DNA-binding activity is present in
   ES cells as determined by a specific inhibition of binding of the   A( L! C' |* ~; }0 ?8 _# F: K
   indicated DNA complex with a KLF2 antibody. This activity is lost upon + Z5 n3 S4 B0 M% F( @
   differentiation into EBs. The identity of the CACC box binding activity in - }- m+ C3 X+ W1 a( s  r+ ]4 l
   EBs denoted CAC-X, and the binding activity in ES cells denoted CAC-Y, was + X# I1 _! a9 M4 N, F1 I( }, |
   not definitively identified using this panel of antibodies.% K9 Z3 k7 d; |
   Enrichment of KLF-binding cis elements in the proximal promoters of stem
   cell specific genes
   To investigate whether Kruppel-like factors might directly regulate stem ) l' e9 G9 [( [( E5 p1 W. d+ k
   cell gene expression, and therefore play a role in the maintenance or loss
   of 'stemness', the presence of KLF transcription factor binding sites 4 P) R+ a9 |* m; D& ?' N
   (TFBS) within the proximal promoters of all genes in Table 1 was
   determined. Although a position weighted matrix (PWM) for KLF4 binding
   site specificity has been published [54], this site does not resemble the
   CACC box sequences known to bind other KLFs. Since KLF4 has identical 8 ]! ^) _, m! a* w2 R/ x1 d
   DNA-binding residues in each of its three C-terminal C2H2 zinc fingers to
   other members of the Kruppel-like factor family [45], it should bind to
   similar sequences. Based on the crystal structure of the zinc fingers of
   Zif268 and SP1 bound to DNA [55,56], and testing of specific binding of
   CACC box variants to recombinant KLF1, KLF3 and KLF17 [45,52,57,58], a
   generic KLF TFBS has been proposed. Although a SELEX experiment for KLF1 # o. g  @6 q' T: w1 A# S* t1 h
   binding has not been published, experiments from our group suggest a C at   ?2 c& e$ {/ j. h! |) z% s% t
   position 1, an A or C at position 3, an A at position 5, and an A, T or C
   at position 9 of the 9 bp consensus provides enhanced affinity for KLF1 6 n9 f) w9 \5 f+ j8 J7 A/ c
   binding. Taken together, these studies have enabled the development of a
   PWM (called here KLF-A) that should predict KLF4, KLF2 and other KLF TFBSs
   (see Methods and Additional file 7).
   Additional file 7. Position Weighted Matrices. Provides published data on
   position weighted matrices for Oct, Sox, KLF-A, KLF4, Nanog, E-box, Gata-1
   and their variants.
   Format: DOC Size: 116KB Download file+ w  L7 g3 o, A: ?3 u
   This file can be viewed with: Microsoft Word Viewer& N- o- N/ {8 I, W
   Clover [59] was used to determine if defined TFBSs were statistically 5 }( `. Z+ L# |+ j( A5 s1 B
   over-represented in the stem cell lists. We searched for PWMs for KLF-A,
   KLF4 [54], octamer, extended octamer, nanog, and Oct-Sox binary sites as " n' k9 \" X7 B; g& E
   well as negative control sites (gata-1 and E-box) in sequences 2 kb
   upstream of the TSSs of stem cell genes (Table 1), versus 2 kb of
   sequences upstream of the entire murine transcriptome (See Additional file 7 I' \# Y5 {6 O* F8 _7 r
   7 for details of PWMs). There was significant over-representation of KLF-A - C' @& _% H, J
   TFBS within the first 2 kb of promoter sequences from the stem list gene 8 u2 Z& G% z# N! g) @- Z
   list (p value of < 0.01) (Figure 7A), as well as over-representation of $ D5 W6 J  a. m* B7 o# ^
   octamer sites and extended Oct4-binding sites as defined from ChIP-PET
   data [60]. No statistically significant over-representation of KLF4 [54], 4 w' ~8 Q4 |$ ]( I
   NANOG, GATA1 or E-box TFBSs were identified. All genes in the rapidly down
   regulated gene list have at least one KLF-A type CACC site, or octamer or
   Oct4 [60] TFBS. A full list of the promoters with highlighted sites is
   available on request. Together, this data strongly suggests direct 4 w" h1 H/ x. X% h9 h; T- H
   transcriptional regulation of many stem cell genes by KLFs.
   Figure 7. KLF and octamer binding sites are highly enriched in stem cell
   gene promoters. (A) A Clover analysis was used to identify
   over-represented transcription factor binding sites within the promoter
   sequences of all stem cell genes identified in Table 1. Representative
   gene promoters are shown, indicating KLF-A binding sites (pink), octamer # C, ~% n5 m8 `+ Z
   sites (ATGCWAAT) (green) and extended Oct4 binding sites (Oct4-Loh)(cyan)
   [60]. (B) Clover output for 2 kb of promoter sequence of murine
   Zfp42/Rex1. The positions and sequences corresponding to PWMs for KLF-A,
   octamer and Oct4 occupancy sites are indicated in the table and colour 5 N1 C8 L: n& S0 N
   coded in the sequence. The positions are relative to the transcriptional
   start site. (C) ECR Browser output of conserved sequence identity between
   mouse and rat in the promoter and part of the first intron of the Zfp42
   gene. The blue arrows indicate the direction of transcription. Yellow
   indicates the extent of the first (non-coding) exon, pink indicates : G+ M- X6 s& M- M% l, g
   regions of sequence conservation in the first intron and red indicates
   regions of sequence conservation in the 5' upstream region. rVISTA was , w2 n$ {* s5 }" ]* x
   used to find all of the KLF-A and octamer sites in the murine gene (Murine
   only) and conserved sites between mouse and rat (Conserved mouse, rat).0 c, v9 q2 X" X' n
   The Zfp42/Rex1 gene promoter is presented as an example of output from the
   Clover program (Figure 7B). It contains five KLF-A, one octamer and three ( }3 M& k: o4 b4 l* y
   OCT4 TFBS. The two CACC box elements within the first 250 base pairs of 5 Z9 _9 k! |; o/ I0 h
   the TSS represent classical extended KLF-binding sites [42,61]. These
   regions are the same as previously reported [62] although their functional 1 y: I) P: ]& e/ M0 `6 }9 z
   importance has not been determined. Interestingly, two of the three KLF-A
   TFBS were evolutionarily conserved between the mouse and rat gene 3 ?. j: Y7 }4 f+ X+ f$ v
   promoters (Figure 7C). Also, two further conserved CACC sites at -1.4 and
   -1.5 kb were identified within 500 bp of extended evolutionary / S6 U- c6 P- {6 R4 f3 w
   conservation (>70% identity) (Figure 7C). This might act as a KLF 5 f" u& f! M, Z( u4 @4 w
   dependent enhancer. Most of the stem cell genes in list 1 have conserved 2 Y( V9 Z4 F: [" h9 F6 N0 r2 U/ {
   CACC box elements in their promoters [63-66]. In some cases these have 8 W) S/ C4 s% \" C4 M1 f
   been reported to bind the ubiquitous SP1, but our Clover analysis suggests . W. |+ O* l0 ?- u* ~4 W" B' n
   they are also likely to bind KLFs (see Discussion).  ~8 K6 h2 l( ]" R
   Discussion
   Expression profiling of murine ES cell differentiation was undertaken over & S$ Q: h" W$ `- u' m9 k$ m7 j1 i) G- ^
   a 16-day time course. We compared gene expression in methylcellulose
   cultures containing serum versus chemically defined media containing LIF 3 }7 m* D) f6 X$ Y
   (1 U/ml) and low concentrations of BMP4 (2 ng/ml) [67]. The Illumina®
   Sentrix Mouse 6 bead array provided a sensitive and detailed platform for : w& r6 b; B3 N, U
   analysis of dynamic gene expression. Using various data mining approaches,
   lists of stem cell-enriched genes and genes that are induced during the in
   vitro equivalent of epiblast and primitive streak stages of $ [& l8 z3 p2 e; S
   differentiation were generated. In combination with other ES cell . u0 \' M4 Q  f( l0 I$ M
   profiling studies [7], our detailed expression data provides a useful 5 O, u+ y! w+ |. e
   resource for future reverse genetic approaches (i.e. siRNA knockdown) to
   study the function of these genes during ES cell differentiation and in
   vivo development. We also found a number of previously uncharacterized
   cDNAs (RIKEN clones) which could play important roles during development.
   Importantly, the loss of pluripotency, measured by Oct4 gene expression,
   was comparable between serum and serum-freeB4L EB culture, following a
   predicted decrease over the first 6 days. Surprisingly, Oct4 gene 8 |" f$ K% Z0 X0 i; o. g6 `4 j
   expression gradually increased following day 6, an observation independent ' q4 S2 C" x1 q  U0 s1 K7 i0 o
   of the cell lines used (data not shown). It remains undetermined if . ]# C4 c. P/ x" ]
   expansion of Oct4 positive ES cells persists as undifferentiated
   populations within our EB culture system. Previous studies have identified
   the development of Oct4 positive primordial germ cells (PGC) following 12
   days of ES cell differentiation [68-70], suggesting the Oct4 profile may
   alternatively represent the expansion of non-ES cell populations. Although
   the expansion of some undifferentiated ES cells is possible, the array
   profile does not suggest global persistence or up-regulation of ES gene
   expression late in the EB program. Also, markers of mesoderm induction
   such as brachyury and Mixl, do not show persistent up-regulation following
   peak expression at day 4, suggesting EBs are unlikely to harbour cells . r7 q9 {- o2 {7 R
   which are delayed or arrested from entering the differentiation program.
   We found significant differences in cardiac gene expression during EB
   differentiation in serum and serum-freeB4L culture. Our current 7 D. s' Z6 n/ I
   understanding of cardiac development provides possible insights into this
   observation. Briefly, in vivo studies have revealed BMPs secreted from the + W* Q! B+ d3 k
   anterior lateral plate, and ill-defined signals from anterior primitive
   endoderm, are key inducers of cardiac development [71-73]. The
   administration of recombinant BMP2 or BMP4 to chick explant cultures
   induces cardiac differentiation in non-cardiogenic mesoderm [71] and Bmp2
   knockout animals develop cardiac abnormalities [74]. Conversely, Wnts 8 }! D: L3 n% F- x5 Y$ t
   secreted by the neural tube are strong suppressors of cardiogenesis.
   Together, these opposing signals act to establish the borders of the heart
   field [71,75]. By mimicking the environment that establishes cardiogenesis 7 G1 B4 j7 |6 I- \- C) i& i
   in vivo, assays capable of directing cardiomyocyte production from ES & w( M: Z) }$ h
   cells have been established. BMPs can efficiently enhance the cardiac $ h! N5 P3 o6 p4 r3 A4 U
   program when added to EB culture [76,77], whereas BMP inhibition . a# D7 A$ u! P6 ]& L* G
   drastically suppresses this outcome. In our hands cultures supplemented - \( V2 E6 A# S; b
   with BMP4 (2 ng/ml) supported cardiomyocyte maturation (as determined by $ U8 W3 w6 z: u6 \
   expression of cardiac specific genes) with greatest expression detected at
   Day 8–10. This correlated with increased numbers of spontaneously beating 5 ~$ m' ^+ v& L! d
   EBs, and the timing of initial spontaneous contractions during murine
   embryogenesis [78]. The cardiac program was significantly reduced in
   culture containing 10% serum, an observation supported by a number of ' y# W1 O8 H+ m4 S. d# H
   other studies [79,80]. This suggests the constituents of serum are
   inhibitory to cardiomyocyte development. Although a detailed assessment of $ b! a. L5 C4 Y2 |  H4 p9 Z+ N
   cardiomyocyte differentiation was not the focus of this analysis, the . y- {/ b3 q6 e* l' [* j
   array output, qRT-PCR profiles and morphological observations described
   supports the usefulness of this assay in future investigations. In
   addition, the defined constituents of serum-freeB4L media provides an : j0 X7 ]! p  v$ o, _% U
   excellent opportunity to identify additional recombinant factors required
   to further expand cardiac progenitor cell production from EBs.6 v' [" L) W/ x/ X; q! w1 ], A4 O
   In contrast, the hematopoietic program was more pronounced within EBs
   grown in 10% serum than BMP4 alone (Figure 1 and 4). In particular, EKLF * `0 S0 g/ ]  c, e  C0 B( G7 @
   (Klf1) expression was significantly reduced in serum-free media 9 a" e* {% l2 O
   supplemented with BMP4 (2 ng/ml) compared with serum (Figure 5A and Table / l) [( U# l* f3 k) d. M# u, i
   4). EKLF is essential for regulation of a large cohort of erythroid 9 _& w0 |1 O$ h5 h
   specific genes [81,82], many of which were identified in sets 2 and 3
   (Figure 4). Thus, it is likely that reduced EKLF expression in SFB4L media
   directly results in inhibition of a cascade of erythroid gene expression.
   Previous work showed the addition of BMP (at 5 ng/ml) to serum-free ES + Y9 x6 t, }0 _5 A
   cell culture induces EKLF expression and restores hematopoietic cell
   differentiation [83]. Although a weak hematopoietic program was observed 9 q) Z. C# _% A. u
   in SFB4L culture, the concentration of BMP4 used was less than that used
   by Adelman et al. Thus, we suggest a robust cardiac (anterior-ventral $ x( ]( ~! }3 X3 D* ?
   mesoderm) gene expression program is induced by low conentrations of BMP4 . g! `7 ^( O7 g1 n+ Q- B. j8 d( p
   (or other BMPs), whereas a robust blood gene expression program 7 e5 C+ `8 U. {( F8 [. P
   (posterior-ventral mesoderm) requires >2 ng/ml of BMP4 or additional 8 Z$ b# e! x  ]6 G* \
   growth factors. Recently, mesoderm derived progenitor cell populations for
   hematopoietic and cardiac lineages were studied during EB development
   using ES cells in which GFP is targeted to the brachyury locus [80]. 6 I2 f" X' k7 c" i" U
   Within day 3.25 EBs, GFP+Flk1+ cells were shown to represent hematopoietic / X4 Y0 U9 Y; i. \
   precursors, whereas GFP+Flk1- cells were significantly enriched for
   cardiac progenitor populations. It would be interesting to determine if 9 n1 |6 |; \) Z# |5 }
   the ratio of Flk1+/Flk1- within the brachyury positive population is 4 x8 l1 f+ W" t/ t. z" t
   altered between our two culture conditions.
   A number of the Kruppel-like factor family of transcription factors were
   dynamically expressed in the first few days of ES cell differentiation. It $ \3 D2 b- S$ Y& r) M& k
   was initially surprising to find highly enriched Klf2 and Klf4 expression
   in ES cells, since both are considered markers of terminally , l7 E" V7 ?- U; H
   differentiated cell types such as skin [50], gut [84], vascular smooth
   muscle [49,85] and lymphocytes [86]. However, recent evidence suggests
   KLFs may regulate stem cell function, since Klf4 is enriched in ES cells
   [19] and forced over-expression within these cells can maintain 5 R' C8 I" j( j
   pluripotency in the absence of LIF [51]. Furthermore, Klf4 can bind the 4 S& f/ o) L% ]# s5 m) ~; i5 d
   Lefty1 gene core promoter co-operatively with Oct4 and Sox2 [65]. Although
   Lefty 1 and 2 are best characterized as repressors of nodal, acting to , k8 Y) f  O$ D2 X2 w8 H
   regulate left-right patterning [87], our results and those of others,
   suggest a possible redundant role for lefty proteins during the
   maintenance of ES cell pluripotency [65]. Similarly to the Oct4 expression ; u) [. i. h% \
   profile, many of the KLF family members also increased in expression late 0 _5 X* P7 v: ?
   in the differentiation program. As mentioned, the KLFs are expressed in ; C+ ?+ X. A; g6 ]' Z) j
   diverse tissues during development. It is therefore expected that the * {& q# O4 @& [7 f2 K) Z; A  b0 I7 K
   profiles obtained reflect the generation and early specification of + ~" t0 z) _& `9 s1 s9 g# Q! W
   mesoderm and endoderm cells following primitive streak gene activation at
   day 4.
   Based on the likely identical binding specificity of the KLF family,
   established transcriptional activation and repression roles of certain
   family members [44], and bioinformatic evidence of a high prevalence of ' i9 `) l) _, r# S% s, {
   KLF TFBSs in many stem cell genes, we propose KLF competition for * d2 g  w3 U  W  N7 L9 D
   occupancy of these CACC box elements might determine self-renewal versus 5 O  u& y/ p+ r9 K
   differentiation of ES cells. According to this model, high levels of KLF2 1 V$ k3 Z8 G( W$ B. o$ V' c# O9 W6 X
   and KLF4 expression in undifferentiated ES cells would lead to occupancy
   of CACC box elements in promoters of stem cell genes such as Pou5f1/Oct4, 9 R6 N  Q6 ^8 r1 f( B% o/ d
   Nanog, Esrrb, Zfp42/Rex1, Lefty1 and Lefty2. Furthermore, the Klf2 and " c( {. e8 ?5 U4 e1 [, R. v2 G
   Klf4 genes themselves have CACC box elements in their proximal promoters
   (Figure 7A), suggesting a positive feedback loop within ES cells is
   likely. Interestingly, gene knockout and lentiviral shRNA gene knockdown
   of Klf2 or Klf4, does not lead to an obvious stem cell defect
   [19,49,50,85], suggesting these two KLFs may have functionally redundant
   roles during the maintenance of pluripotency. Future analysis of KLF2/KLF4 ) r7 c: `( f8 V9 Q. c
   double knockout ES lines or the knockdown of both proteins using RNAi
   technologies will be necessary to validate this hypothesis. Upon ES cell 0 T1 G# n2 T0 y: P+ Q
   differentiation, down regulation of Klf2 and Klf4 was very rapid, whereas + v% M" j* G4 L
   down regulation of Oct4 and Nanog was delayed for one to two days. We % ]1 ?4 [6 H! ]2 K/ g
   suggest loss of KLF2 and KLF4 binding to Pouf1/Oct4, Nanog, Esrrb and 6 O& e4 B2 D; s" x5 R
   perhaps other stem cell promoters, could be directly responsible for their
   down regulation. Moreover, Klf5 was down regulated more slowly, and Klf3 2 v1 i2 b, g1 p; W- k
   was up-regulated in the first two days of differentiation, suggesting
   these KLFs may function primarily as transcriptional repressors at the
   Pouf1/Oct4, nanog, Zfp42 and Esrrb gene promoters. Once this
   differentiation driving transcription network is activated we suggest 2 K# [6 X; ~7 @5 e( Q# @5 W
   KLF3, KLF5 and KLF9 can accelerate loss of the stem cell state by directly ) A9 c( `) _. t9 L* }) Q
   repressing expression of the Klf2 and Klf4 genes themselves. Therefore, a ( ^7 r% D! ^8 E6 D$ c8 e' W
   combination of cross regulation of transcriptional outputs, and 1 ^8 U: P8 K' \, S
   competition for occupancy of key stem cell promoters by KLF proteins with
   differing biochemical properties but identical DNA-binding capacities, may 1 j4 \8 }1 X. L
   determine self-renewal versus differentiation outcomes. Again validation
   of this model requires functional analysis. It is important to note we ! |% f5 e$ p; j+ |( W
   have not proven occupancy of stem cell gene promoters by the KLFs in vivo.
   Chromatin immuno-precipitation (ChIP) experiments in ES cells and
   differentiated populations are essential to validate this model, however
   ChiP grade antibodies are not currently available.
   This scenario is not necessarily limited to ES cell differentiation. A
   similar program may take place in many adult stem and progenitor cells 3 o5 h% u5 N) q6 ]# F7 L
   populations. Certainly competition between Klf4 and Klf5 has been 7 t3 o. n8 x8 d. V
   suggested to regulate cell growth [88], and Klf1 and Klf3 compete for
   binding and have opposing effects in blood cells [89]. Furthermore, an ) a1 V" H$ o1 N
   imbalance between occupancy of key stem cell or proliferation gene 9 X& v& q; }# U+ x) V
   promoters by KLFs, with differing activating and repressing functions, 2 _' c1 d3 @* v$ J0 l0 J# ^' q
   could be responsible for the development or progression of many common
   forms of cancer. For example, loss of hetero-zygosity for Klf4 and Klf5 5 K4 z  C  M/ g: Y1 L. g! z: R
   has been found in colon, stomach, breast, prostate and liver cancers ! t5 j0 V6 P6 f, B2 M* k
   [90,91]." w9 p" z, x! c' L
   In addition to the proposed complex transcriptional interplay between KLFs ; O4 X) ~- I6 }. p: X2 U
   and stem cell gene promoters, direct protein-protein interactions and ! _  F4 O* B' o
   protein networks involving KLFs are possible. We showed sub-nuclear
   localization of KLF2 and KLF4 with OCT4 in ES cells (Figure 6A), and KLF4 , l" f1 m! @. u- m
   can co-operate with OCT4 and SOX2 to drive expression of the Lefty1 core
   promoter [65]. Also, very recent biochemical purification of , O; n9 a; V. k! K
   NANOG-interacting proteins discovered KLF4 as part of a NANOG-OCT4 network 7 a7 W7 E: I! |" T  `" `
   in ES cells (Stuart Orkin, personal communication). This is also
   consistent with the ability of KLF4 to co-operate with OCT4, SOX2 and
   c-MYC to drive de-differentiation of fibroblasts into ES-like cells [24].
   Conclusion
   Defining the genetic regulation of ES cell self renewal and
   differentiation will be instrumental for the development of future cell
   based therapies. Using 47 K Illumina® Sentrix bead arrays, the
   differential expression of genes during 16 days of ES cell differentiation + ?  T  f& s3 p" _
   was determined. Hierarchical gene clustering and correlation statistical 8 c# k7 {% J# h3 y$ U( H
   analyses lead to the identification of a small cohort of genes which
   define the stem cell state. Historically, ES cell differentiation is 5 A9 g3 L) ^6 A# R
   achieved in culture media containing 10–15% FBS. A direct comparison
   between ES cell differentiation in serum and a serum-free media was ( @1 s+ e4 Z- n, Y- f. @0 ?
   undertaken. Surprisingly, global gene expression profiles were comparable * ~! L) ]/ u' W! n7 e7 m  S% e3 l0 x
   between culture conditions, with the exception of mesoderm derived cardiac
   and haematopoietic transcripts. These results support the use of defined ; Q/ @' G. @1 v7 i
   serum-free approaches for the directed differentiation of ES cell / f6 ^$ _5 Z# k( n% C. H7 g. Y
   commitment. Expression of many KLF family members were enriched in ES ) v1 g( ]$ a  F9 G* z: y: x5 |% Z
   cells and rapidly down regulated upon differentiation. KLF2 and KLF4 were + z1 f4 j8 B4 l: E  b" B+ ?3 \
   co-localisation with OCT4 in ES cell nuclei and KLF transcription factor   a  O4 G" Z0 Z0 N
   binding sites were over-represented within the promoters of the putative
   ES cell gene list (p < 0.01). Taken together, this data strongly suggests 6 o4 g8 I9 ^8 S) t3 ^( g; o
   KLF family members regulate the maintenance of ES cell pluripotency.6 ]1 N. }  A3 D: q: O
   Methods1 e# k% I8 @5 e
   ES cell culture, embryoid body formation and immunohistochemistry
   W9.5 ES cell differentiation was performed as described (Bruce et al., . d/ N. p4 o. l' Y: T+ p
   Differentiation, in press). For immuno-fluorescence, feeder depleted W9.5 & k8 H3 e1 ^. y- O' H2 s8 _$ J, i
   ES cells were seeded onto sterile gelatin-coated glass cover slips in ES . [3 y  K* W/ u8 D' y4 d
   cell media, fixed in 4% PFA for 10 min then washed in PBS. Cover slips
   were boiled in 10 mM citric acid (pH 6.4) for 10 min, washed in PBS and 1 u- c$ \, a2 [- _0 U+ `, o
   permeabilized in 0.18% triton X-100 in PBS for 10 mins. For OCT4/KLF4 3 W5 J8 M0 E3 k. p
   detection, the cover slips were blocked in 1% BSA then incubated with * U+ k4 X5 w6 S/ {6 L6 m
   rabbit polyclonal antibody raised against OCT4 (Abcam #ab19857) and goat : w" H, W; |1 p7 S$ [9 \
   Anti-mouse KLF4 (R&D Systems #AF3158) overnight at 4°C, then incubated in + m* H0 O" g# r  A2 \( @
   goat anti-rabbit Alexa-488 (1:400) (Molecular Probes #A11008) and donkey
   anti-goat Alexa-647 (1:400) for 1 hour. For co-detection of OCT4/KLF2, the
   Zenon Rabbit IgG labelling kit was used (Molecular Probes #Z-25308). 1 μg
   of KLF2 rabbit polyclonal antibody (Santa Cruz SC-28675) was labeled with % q# s: U1 X' b, h
   Alexa647 Fab fragment following the manufacturer's instructions. Cover
   slips already stained for OCT4 (Alexa488) were incubated with labeled KLF2 - c9 O9 ~5 X- z4 J& q4 m
   for 1 hr then washed and post-fixed in 4% PFA to ensure strong signal   m4 O: y4 v9 G% m1 h% y# D2 o
   detection. DAPI 1:5000 (Molecular Probes #D3571) was used to detect
   nuclear localization. Cover slips were mounted on SuperFrost® Plus slides
   (Menzel-Glaser) with VectaShield mounting media (Vector Laboratories
   H-1000). Confocal microscopy was performed on LSM 510 META Carl Zeiss
   microscope system.
   RNA extraction, cDNA synthesis and real-time RT-PCR data analysis" I& O/ N7 ^& B1 n' y+ u2 M
   Total RNA was made using TRIzol (Invitrogen), and cDNA was synthesized ) h8 _/ r1 U3 u& U
   from 2 μg of DNase1-treated total RNA using Superscript III (Invitrogen)
   and oligo-DT12–15 (Promega) according to the manufacturer's instructions.
   Quantitative RT-PCR (qRT-PCR) was carried out using the Applied Biosystems $ I$ p' ]: ^5 j4 W  a
   SYBR-green dye system and 7500 Real Time Cycler in 96-well plates. Cycling
   variables were as follows: 50°C for 2 minutes, 95°C for 10 minutes, then 0 n1 c  k8 {4 E+ t- ]
   40 cycles of 15-second denaturation at 95°C and 1-minute at appropriate % B" Q4 L* p6 c, q0 B( h6 L- `
   annealing temperatures, optimized for each set of primers based on ( d4 C8 M9 G3 ]0 v: Z0 K
   dissociation curves (Additional file 8). Expression levels were normalized
   to HPRT as determined from the ratio of delta CT values. Mean of relative . n7 h8 O2 c: x0 U1 n8 B
   expression ± SD was determined from three biological replicates.
   Sequencing of gel-purified amplicons was performed to ensure correct * B3 s3 g' `- C$ A% {& K# t
   product amplification.) D. Q$ ?  y& y' k; v* R+ N
   Additional file 8. Oligonucleotide sequences used for qRT-PCR analysis.
   Lists the oligonucleotide sequences and cycling conditions used for the # Z" G+ s* q1 J+ T  |
   qRT-PCR analysis.7 @9 B' e& ~3 }2 F5 {
   Format: DOC Size: 48KB Download file
   This file can be viewed with: Microsoft Word Viewer2 S3 |" W8 e+ w9 B
   Micro-array hybridization and analysis% M4 i# x/ x! I2 J3 o# R: O& F9 `7 p
   Expression profiling was performed using Sentrix Mouse-6 Expression , k. Q5 K' T# A1 U7 \
   BeadChip arrays from Illumina®. RNA was assessed for integrity using the
   Agilent Bioanalyzer 2100 and RNA integrity (RIN) scores above 9.5 were
   present in all samples. Amplification was performed with 500 ng of total & ^  M4 N$ k7 F% F
   RNA using the Illumina TotalPrep RNA Amplification kit (Ambion) with a 12 ) ^. X* M: P. n, q
   hour in vitro transcription reaction period. The quantity and quality of
   biotin-UTP incorporated cRNA was also assessed on the Agilent Bioanalyzer
   2100. Amplified cRNA (1500 ng per array) was hybridized to Mouse-6.v1 8 m+ K" v' Y) {
   BeadChip arrays according to the manufacturer guidelines and detected with
   Fluorolink Streptavidin-Cy3 (Amersham Biosciences). Arrays were scanned
   using the Illumina BeadStation Scanner. The raw intensity values obtained
   for the scanned array images were compiled using the proprietary
   BeadStudio v1.5.1.3 software and imported into GeneSpring GX v7.3.1
   (Agilent). A mouse Illumina probe set was defined in the GeneSpring
   Workgroup using the Illumina targetIDs as the unique identifiers and
   annotated according to array content files supplied by illumina®. Data 1 ~3 T3 `: `- L$ G
   normalization was performed by first setting all measurements less than . Q7 O; R' O- ?
   0.01 to 0.01, then applying per chip normalization to the 50th percentile,
   and per gene normalization to the median.( |% d) b* c6 U6 s- r+ \
   From an interpretation which included the three FBS experiments, a
   non-parametric Welch ANOVA (where variances were not assumed equal) was
   performed on all 46,120 probes to find a subset of genes whose expression
   varied significantly throughout the differentiation time course. A 4 R; X! q; X8 R, R
   Benjamini and Hochberg False Discovery Rate multiple testing correction : b$ W# q  N* u0 U5 D5 B
   was applied to reduce the number of false positives. This yielded a set of
   7,967 probes that showed statistically significant (p < 0.05) differences
   in expression by Welch t-test. This approach simplified running the data
   mining algorithms to find syn-expression patterns by removing genes such
   as housekeeping genes which did not display differential expression, or & W  O0 ?: d  i! B. y4 R  F
   genes which did not reach a threshold of expression at any time points.9 ^8 P8 N9 L9 H* N3 Y& O6 G
   Genome interpretations were generated in GeneSpring GXv7.3.1 (Agilent) in 0 S3 A1 Q  x  Z6 M0 C6 k
   which the mean relative expression level for each gene was represented at " ~6 }' w9 U% I
   each time point relative to the average of all time points. A number of 8 B& q, k' i5 }. s" M" Z
   statistical approaches were used to find syn-expression patterns. A - a8 T* i  A8 y, L
   Pearson correlation (between 0.9 and 1.0) was performed to find genes with & B$ Q& C& l2 G5 f
   similar expression profiles to Pouf1/Oct4, nanog, sox2, fgf5, wnt5 and
   brachyury. Hierarchical clustering was performed using the gene by gene   Z" S0 A/ X2 {& u+ Y) S
   and group by group 'tree' algorithm within the GeneSpring (Agilent)
   program. This compares each probe with every other in the lists to " J& I0 k( B- E5 s/ n4 }
   generate similarity statistics and a tree representation of similarity % R; h4 K4 H- a6 c- D; ~, ]
   based on a Pearson correlation. Also, quality threshold (QT) clustering
   [92] was used to define distinct subsets of co-expressed genes. The entire $ o1 Z" C6 G& D" S* F, g& |( X
   data set for the experiments is available via GEO as well as via our
   instance of Genespring (contact corresponding author).
   Electro-mobility gel shift assays (EMSA)
   Nuclear extracts were made from undifferentiated ES cells and embryoid
   bodies grown for four days in 10% serum. Electromobility gel shift assays
   (EMSA) were performed as previously described [61] using double stranded   ]& G  r* t6 @/ {
   oligonucleotide probes corresponding to an extended CACC site in the
   p18INK4c promoter (sense strand 5'-gttgggcggggcgtgggcggggcc-3') (Tallack,
   et al., submitted). Supershifts were performed with specific antibodies 7 d9 W! M9 v* f; w  j- t8 m
   raised against SP1 (Santa Cruz SC-059x), SP3 (Santa Cruz SC-644x), KLF2 7 P" a) `- Z4 P* h9 q0 ~
   (Santa Cruz SC-28675), KLF3 [52] and KLF4 (Santa Cruz SC-12538x).1 \  n  P/ t& e' M0 k! |" c
   Bioinformatic searches for CACC sites and other over represented elements ; D  }2 Y2 ~2 r3 F
   in promoters of stem cell genes
   The data-mining tool Biomart (accessed via Ensembl release 36 of the mouse
   genome), was used to collate 2 kb of sequences upstream of the
   transcriptional start sites (TSSs) of all genes listed in Table 1. The
   Clover program, run via the MotifViz web interface [59], was used to
   search for over-representation of position weighted matrices (PWMs) for 2 x$ ~1 [8 b; C4 v, `; Y
   octamer [93], Oct4 [60], Oct-Sox [60], KLF4 [54], E-box, GATA-1 [94] and * U7 `8 u* |  b
   CACC (KLF-A) elements in these promoter sets. The PWMs are listed in
   Additional file 7. The ECR Browser [95] was used to determine phylogenetic 2 V+ e) z0 @0 t" V( `1 {, M
   conservation of cis elements between mouse, rat and human genomes." k7 F) M, I4 V, S* \
   Abbreviations- I$ z; s$ \  _2 \+ f, O
   BSA, bovine serum albumin; EB, embryoid body; EMSA, electromobility gel
   shift assay; ES, embryonic stem; FBS, fetal bovine serum; KL,
   Fkruppel-like factor; LIF, leukemia inhibitory factor; MEF, mouse % }# Z( Y6 l& h3 }) c) Y) z% \. z
   embryonic fibroblasts; PFA, paraformaldehyde; PWM, position weighted 4 l! u+ Y  E. d8 I0 o3 c' U! @1 k
   matrix; TFBS, transcription factor binding sites.0 D& ~) \* Q: Y; i' |3 |! d
   Competing interests
   The author(s) declares that there are no competing interests.  L; s* L5 ~" @! F% k
   Authors' contributions
   SB established the EB differentiation assay, collected and purified RNA
   samples, designed and carried out the quantitative RT-PCR studies and
   drafted the manuscript. BG carried out the micro array protocol. LB $ P; R- p% v/ u
   Carried out experimentation of mesoderm specific gene expression. MG + Z5 f1 l0 _1 ^
   performed the array statistical analysis and annotation. SG participated
   in the design of the study and performed the statistical analysis. AP
   conceived the study, and participated in its design and coordination and
   helped to draft the manuscript. All authors read and approved the final
   manuscript.

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