Dynamic transcription programs during ES cell differentiation towards mesoderm in serum versus serum-freeBMP4 culture

Abstract

Background: Expression profiling of embryonic stem (ES) cell differentiation in the presence of serum has been performed previously. It remains unclear if transcriptional activation is dependent on complex growth factor mixtures in serum or whether this process is intrinsic to ES cells once the stem cell program has been inactivated. The aims of this study were to determine the transcriptional programs associated with the stem cell state and to characterize mesoderm differentiation between serum and serum-free culture.

Results: ES cells were differentiated as embryoid bodies in 10% FBS or serum-free media containing BMP4 (2 ng/ml), and expression profiled using 47 K 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 changed dramatically during the first few days of differentiation. KLF2 and KLF4 co-localized with OCT4 in a sub-nuclear compartment of ES cells, dynamic changes in KLF-DNA binding activities occurred upon differentiation, and strong bio-informatic evidence for direct regulation of many stem cell genes by KLFs was found.

Conclusion: 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.

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 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 same starting ES cell populations. Bars represent the means of three (serum) or two (serum-free^B4L) biological replicates. The Y-axis represents a log scale normalized relative to the housekeeping gene HPRT. Error bars indicate standard deviation.

Figure 2

Gene expression profiles during 16 days of EB differentiation in serum and serum-free media containing BMP4 [2 ng/ml]. (A) GeneSpring representation of the subset of genes from the 48 K Illumina array that showed significant differences in gene expression at 2 or more time points. Each line represents the mean normalised expression of an individual Illumina probe. Colours represent relative expression (red 4× increased; green 0.25× reduced) compared to a gene mean of 1 (black) with expression in undifferentiated ES cells (day 0) as the decision point for colour coding. The expression profiles of T (brachyury), Hba (α-globin), and Igf2 are indicated for reference. (B) Clustering (tree) of the genes that showed significant (p < 0.01) changes in gene expression upon differentiation by One-way Welch ANOVA. Expression levels are heat map colour coded (inset) with high expression at Day 0 coloured red and low expression coloured green.

Figure 3

Mining for genes expressed in patterns consistent with primitive streak, and epiblast differentiation programs. (A) GeneSpring representation of epiblast and streak gene expression profiles during EB differentiation in 10% serum or serum-free^B4L 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 indicate ± SD from three biological replicates. (C) 102 genes transiently up regulated during the first 4 days of ES cell differentiation were clustered using a tree algorithm and Pearson correlation of > 0.9. The 'Brief' group represents gene with very transient expression at day 3. (D) Coding region for 8430415E04Rik. The shaded areas represent the three Heat domains.

Figure 4

QT clustering revealed sets of genes differentially expressed between serum and serum-free^B4L EB culture. QT clustering revealed nine sets of genes with similar dynamic expression profiles (Pearson Correlation coefficient of > 0.9). In sets 1–7, expression is higher in serum (black sets). In sets 8–9, expression is greater in serum-free^B4L EB culture (red sets). Some sets have been named based on the similarity of overall gene function during differentiation pathways (see Table 3).

Figure 5

Kruppel like factors (Klfs) are dynamically expressed during the first few days of ES cell differentiation. (A) GeneSpring plot of normalized gene expression for all KLFs detected during ES cell differentiation in serum or serum-free^B4L culture. Most of the genes are listed on the y-axis in order from their highest relative expression in ES cells. There is dramatic up regulation of Klf1 (Eklf) only in serum following day 6 of differentiation. Plots representing Nanog (red), Sox2 (red) and Pou5f1(Oct4) (green) are shown for comparison. 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 gene expression of six members of the KLF family by quantitative real time RT-PCR. Scheme as described for Figure 2B.

Figure 6

Kruppel like factor expression and DNA binding activity during ES cell differentiation. (A) Co-expression of Oct4 with KLF2 or KLF4 in ES cells. Indirect immuno-fluorescence shows co-localization of KLF2 and KLF4 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 shift assay showing changes in DNA binding activities at a conserved CACC 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 indicated DNA complex with a KLF2 antibody. This activity is lost upon differentiation into EBs. The identity of the CACC box binding activity in EBs denoted CAC-X, and the binding activity in ES cells denoted CAC-Y, was not definitively identified using this panel of antibodies.

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 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 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 regions of sequence conservation in the first intron and red indicates regions of sequence conservation in the 5' upstream region. rVISTA was 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).