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Combined microarray analysis uncovers self-renewal related signaling in mouse embryonic stem cells.

Zhu H, Yang H, Owen MR - Syst Synth Biol (2008)

Bottom Line: Albeit the expression of individual genes in signaling pathways was not noticeably different between cells cultivated with and without LIF, at gene-set level the expression of ERK/MAPK (but not JAK/STAT) and cell cycle related genes was found significantly enriched in cells cultivated with LIF.When data at the first 7 time points were compared with data at the last 4 time points, the expression of several cell cycle related gene sets was apparently enriched in all three cell lines, indicating the active cell proliferation in the first 2 days.When that mechanism weakens, cell differentiation begins.

View Article: PubMed Central - PubMed

Affiliation: Center for Mathematical Medicine and Biology, School of Mathematical Sciences, University of Nottingham, Nottingham, NG7 2RD, UK, Hao.Zhu@nottingham.ac.uk.

ABSTRACT
Due to the limited understanding of self-renewal and pluripotency related signaling in stem cells, extracting information from genome-wide expression data is not only important but also challenging. With the combined use of two methods, we analyzed a set of microarray data at 11 time points from three mouse embryonic stem cell lines cultivated with and without leukemia inhibitory factor (LIF) for 14 days. Albeit the expression of individual genes in signaling pathways was not noticeably different between cells cultivated with and without LIF, at gene-set level the expression of ERK/MAPK (but not JAK/STAT) and cell cycle related genes was found significantly enriched in cells cultivated with LIF. This indicates that the Ras/Raf/ERK pathway, in addition to JAK/STAT, may also be a key player to carry on external LIF signal into mouse embryonic stem cells to promote self-renewal. When data at the first 7 time points were compared with data at the last 4 time points, the expression of several cell cycle related gene sets was apparently enriched in all three cell lines, indicating the active cell proliferation in the first 2 days. Compared with the slight decay of Oct4/Nanog/Sox2 during the 14 days, the expression of cell differentiation genes such as Gata4/6 underwent a drastic increase, which indicates that the upregulated expression of cell differentiation genes may better reflect the loss of self renewal than the down regulated expression of the stemness indicators Oct4, Sox2 and Nanog. Apart from differential expression and gene set enrichment analyses, a clustering algorithm was also used to classify genes into co-expression clusters. The possible regulation of two clusters, whose expression was most changed during cell culture from very low to very high, was explored. The drastic changes of these genes, including Slc39a8 which was a potential indicator of cell differentiation, in contrast the slight changes of self-renewal genes, imply that differentiation may be the default fate of stem cells and self-renewal may rely on a maintenance mechanism. When that mechanism weakens, cell differentiation begins.

No MeSH data available.


Related in: MedlinePlus

The possible regulation and regulators of the two co-expressed gene clusters with the most significantly changed expression during the 14 days in the three cell lines. Red and green colors indicate high and low expression level. The hierarchy of regulation indicates the possible relationship among regulators. The expression of each regulator is divided into two stages which exert different regulatory impacts on either other regulators or clustered genes
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Fig3: The possible regulation and regulators of the two co-expressed gene clusters with the most significantly changed expression during the 14 days in the three cell lines. Red and green colors indicate high and low expression level. The hierarchy of regulation indicates the possible relationship among regulators. The expression of each regulator is divided into two stages which exert different regulatory impacts on either other regulators or clustered genes

Mentions: An important question in microarray data analysis is how changed gene expression is regulated, under different conditions and at different times. To answer this question, genes should be clustered according to their expression, and then regulators of each cluster be identified. Judged against the control sample, 2257 genes, either differentially expressed in the three cell lines or important for developmental signaling, were chosen for clustering (see Materials and methods). Using a revised clustering method (Segal et al. 2003), which not only classifies genes into clusters of different expression patterns but also reveals their possible regulation by defined regulators, we found that the expression of a few gene clusters underwent a drastic change during the 14 days from very low (in green in Fig. 3) to very high (in red in Fig. 3). It is reasonable to postulate that the expression of these gene clusters reflects mES self-renewal and differentiation. To reveal how their drastically altered expression was regulated during the cell culture, we assumed that in the decay of self-renewal and the initiation of differentiation the expression of regulators themselves underwent a significant change, showing either a positive or a negative enrichment, and further that these regulators were transcriptional factors. From the 2257 genes we picked up 26 transcriptional factors (Table S2), which had a high enrichment score computed with the gene set enrichment analysis, and used them as regulators in the clustering program. Apart from a Max Module Number of 50, which means the genes are clustered into at most 50 clusters, all other parameters were the default values. The two clusters with the most change in gene expression during the 14 days, with the possible regulation by the chosen regulators (Fig. 3), comprised genes (including Slc39a8 which was a potential indicator of cell differentiation as aforementioned) that had very low expression at early time points but very high expression at later times. Repeated running of the program gave the similar results, revealing that the low expression at the early time points was probably because of the repression by active self-renewal genes such as Oct4, Nanog, Pcna, Myc and Smarcad1 (Chambers et al. 2003; Mitsui et al. 2003; Niwa et al. 2000), and that the high expression at the late time points was probably due to both the enhanced expression of differentiation genes (Gata4/6) and the down regulated self-renewal genes (especially Oct4, Jun and Cebpb). In contrast, the typical self-renewal genes, including Nanog, Oct4 and Sox2, only showed slight changes during the 14 days. This implies that differentiation may be the default fate of stem cells and self-renewal relies on a maintenance mechanism. When that mechanism weakens, cell differentiation begins.Fig. 3


Combined microarray analysis uncovers self-renewal related signaling in mouse embryonic stem cells.

Zhu H, Yang H, Owen MR - Syst Synth Biol (2008)

The possible regulation and regulators of the two co-expressed gene clusters with the most significantly changed expression during the 14 days in the three cell lines. Red and green colors indicate high and low expression level. The hierarchy of regulation indicates the possible relationship among regulators. The expression of each regulator is divided into two stages which exert different regulatory impacts on either other regulators or clustered genes
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2553326&req=5

Fig3: The possible regulation and regulators of the two co-expressed gene clusters with the most significantly changed expression during the 14 days in the three cell lines. Red and green colors indicate high and low expression level. The hierarchy of regulation indicates the possible relationship among regulators. The expression of each regulator is divided into two stages which exert different regulatory impacts on either other regulators or clustered genes
Mentions: An important question in microarray data analysis is how changed gene expression is regulated, under different conditions and at different times. To answer this question, genes should be clustered according to their expression, and then regulators of each cluster be identified. Judged against the control sample, 2257 genes, either differentially expressed in the three cell lines or important for developmental signaling, were chosen for clustering (see Materials and methods). Using a revised clustering method (Segal et al. 2003), which not only classifies genes into clusters of different expression patterns but also reveals their possible regulation by defined regulators, we found that the expression of a few gene clusters underwent a drastic change during the 14 days from very low (in green in Fig. 3) to very high (in red in Fig. 3). It is reasonable to postulate that the expression of these gene clusters reflects mES self-renewal and differentiation. To reveal how their drastically altered expression was regulated during the cell culture, we assumed that in the decay of self-renewal and the initiation of differentiation the expression of regulators themselves underwent a significant change, showing either a positive or a negative enrichment, and further that these regulators were transcriptional factors. From the 2257 genes we picked up 26 transcriptional factors (Table S2), which had a high enrichment score computed with the gene set enrichment analysis, and used them as regulators in the clustering program. Apart from a Max Module Number of 50, which means the genes are clustered into at most 50 clusters, all other parameters were the default values. The two clusters with the most change in gene expression during the 14 days, with the possible regulation by the chosen regulators (Fig. 3), comprised genes (including Slc39a8 which was a potential indicator of cell differentiation as aforementioned) that had very low expression at early time points but very high expression at later times. Repeated running of the program gave the similar results, revealing that the low expression at the early time points was probably because of the repression by active self-renewal genes such as Oct4, Nanog, Pcna, Myc and Smarcad1 (Chambers et al. 2003; Mitsui et al. 2003; Niwa et al. 2000), and that the high expression at the late time points was probably due to both the enhanced expression of differentiation genes (Gata4/6) and the down regulated self-renewal genes (especially Oct4, Jun and Cebpb). In contrast, the typical self-renewal genes, including Nanog, Oct4 and Sox2, only showed slight changes during the 14 days. This implies that differentiation may be the default fate of stem cells and self-renewal relies on a maintenance mechanism. When that mechanism weakens, cell differentiation begins.Fig. 3

Bottom Line: Albeit the expression of individual genes in signaling pathways was not noticeably different between cells cultivated with and without LIF, at gene-set level the expression of ERK/MAPK (but not JAK/STAT) and cell cycle related genes was found significantly enriched in cells cultivated with LIF.When data at the first 7 time points were compared with data at the last 4 time points, the expression of several cell cycle related gene sets was apparently enriched in all three cell lines, indicating the active cell proliferation in the first 2 days.When that mechanism weakens, cell differentiation begins.

View Article: PubMed Central - PubMed

Affiliation: Center for Mathematical Medicine and Biology, School of Mathematical Sciences, University of Nottingham, Nottingham, NG7 2RD, UK, Hao.Zhu@nottingham.ac.uk.

ABSTRACT
Due to the limited understanding of self-renewal and pluripotency related signaling in stem cells, extracting information from genome-wide expression data is not only important but also challenging. With the combined use of two methods, we analyzed a set of microarray data at 11 time points from three mouse embryonic stem cell lines cultivated with and without leukemia inhibitory factor (LIF) for 14 days. Albeit the expression of individual genes in signaling pathways was not noticeably different between cells cultivated with and without LIF, at gene-set level the expression of ERK/MAPK (but not JAK/STAT) and cell cycle related genes was found significantly enriched in cells cultivated with LIF. This indicates that the Ras/Raf/ERK pathway, in addition to JAK/STAT, may also be a key player to carry on external LIF signal into mouse embryonic stem cells to promote self-renewal. When data at the first 7 time points were compared with data at the last 4 time points, the expression of several cell cycle related gene sets was apparently enriched in all three cell lines, indicating the active cell proliferation in the first 2 days. Compared with the slight decay of Oct4/Nanog/Sox2 during the 14 days, the expression of cell differentiation genes such as Gata4/6 underwent a drastic increase, which indicates that the upregulated expression of cell differentiation genes may better reflect the loss of self renewal than the down regulated expression of the stemness indicators Oct4, Sox2 and Nanog. Apart from differential expression and gene set enrichment analyses, a clustering algorithm was also used to classify genes into co-expression clusters. The possible regulation of two clusters, whose expression was most changed during cell culture from very low to very high, was explored. The drastic changes of these genes, including Slc39a8 which was a potential indicator of cell differentiation, in contrast the slight changes of self-renewal genes, imply that differentiation may be the default fate of stem cells and self-renewal may rely on a maintenance mechanism. When that mechanism weakens, cell differentiation begins.

No MeSH data available.


Related in: MedlinePlus