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Comparative transcriptomic analysis of multiple cardiovascular fates from embryonic stem cells predicts novel regulators in human cardiogenesis.

Li Y, Lin B, Yang L - Sci Rep (2015)

Bottom Line: Furthermore, GEPA analysis revealed the MCP-specific expressions of genes in ephrin signaling pathway, positive role of which in cardiomyocyte differentiation was further validated experimentally.By using RNA-seq plus GEPA workflow, we also identified stage-specific RNA splicing switch and lineage-enriched long non-coding RNAs during human cardiovascular differentiation.Overall, our study utilized multi-cell-fate transcriptomic comparison analysis to establish a lineage-specific gene expression map for predicting and validating novel regulatory mechanisms underlying early human cardiovascular development.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Biology, University of Pittsburgh School of Medicine, 530 45th Street, Rangos Research Center, Pittsburgh, PA 15201.

ABSTRACT
Dissecting the gene expression programs which control the early stage cardiovascular development is essential for understanding the molecular mechanisms of human heart development and heart disease. Here, we performed transcriptome sequencing (RNA-seq) of highly purified human Embryonic Stem Cells (hESCs), hESC-derived Multipotential Cardiovascular Progenitors (MCPs) and MCP-specified three cardiovascular lineages. A novel algorithm, named as Gene Expression Pattern Analyzer (GEPA), was developed to obtain a refined lineage-specificity map of all sequenced genes, which reveals dynamic changes of transcriptional factor networks underlying early human cardiovascular development. Moreover, our GEPA predictions captured ~90% of top-ranked regulatory cardiac genes that were previously predicted based on chromatin signature changes in hESCs, and further defined their cardiovascular lineage-specificities, indicating that our multi-fate comparison analysis could predict novel regulatory genes. Furthermore, GEPA analysis revealed the MCP-specific expressions of genes in ephrin signaling pathway, positive role of which in cardiomyocyte differentiation was further validated experimentally. By using RNA-seq plus GEPA workflow, we also identified stage-specific RNA splicing switch and lineage-enriched long non-coding RNAs during human cardiovascular differentiation. Overall, our study utilized multi-cell-fate transcriptomic comparison analysis to establish a lineage-specific gene expression map for predicting and validating novel regulatory mechanisms underlying early human cardiovascular development.

No MeSH data available.


Related in: MedlinePlus

GEPA predicted differentiation-associated alternative RNA splicing and lineage-enriched long non-coding RNAs during human cardiovascular differentiation. (a) Genes with exon changes detected by RNA-seq during human cardiovascular differentiation. Locations of the genes in Hg18 human genome assembly were shown along with total exon number (No. exons) and number of exons with changes (Exon diff.). (b) Differential use of the exon 3 of FGFR1 during human cardiovascular differentiation. Stacks of sequencing reads in the five lineages were shown. Distances of the exons are NOT the actual intron lengths. Change in the expression of exon 3 was highlighted by a red frame. Primers to verify the expression of the exon 3 were indicated by red or blue arrows. (c) Semi-quantitative RT-PCR validation of the exon 3 usage during human cardiovascular differentiation. (d) Lineage-enriched long non-coding RNAs during human cardiovascular differentiation identified by GEPA. To indicate the lineage-specificity, the relative expression was shown as percent in the sum of all the cell types in the heatmap. (e) qRT-PCR validation of the lineage-enriched expressions of lncRNAs.
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f7: GEPA predicted differentiation-associated alternative RNA splicing and lineage-enriched long non-coding RNAs during human cardiovascular differentiation. (a) Genes with exon changes detected by RNA-seq during human cardiovascular differentiation. Locations of the genes in Hg18 human genome assembly were shown along with total exon number (No. exons) and number of exons with changes (Exon diff.). (b) Differential use of the exon 3 of FGFR1 during human cardiovascular differentiation. Stacks of sequencing reads in the five lineages were shown. Distances of the exons are NOT the actual intron lengths. Change in the expression of exon 3 was highlighted by a red frame. Primers to verify the expression of the exon 3 were indicated by red or blue arrows. (c) Semi-quantitative RT-PCR validation of the exon 3 usage during human cardiovascular differentiation. (d) Lineage-enriched long non-coding RNAs during human cardiovascular differentiation identified by GEPA. To indicate the lineage-specificity, the relative expression was shown as percent in the sum of all the cell types in the heatmap. (e) qRT-PCR validation of the lineage-enriched expressions of lncRNAs.

Mentions: Alternative splicing produces diversities in protein output of the genome. It could play a role as regulatory mechanism in cell fate decision. A well established example is that the protein isoforms of FOXP1 gene produced by alternative splicing can have differential regulatory activities in maintaining pluripotency or promoting differentiation25. To identify the alternative splicing during human cardiovascular differentiation, we used GEPA to analyze the lineage-enrichment pattern of the splicing isoforms of the genes. We found a number of genes have differential expression patterns of the isoforms (Fig. 7a). Among them, a typical example is the differential usage of the exon 3 of Fibroblast Growth Factor Receptor 1 (FGFR1) gene during human cardiovascular differentiation (Fig. 7b). In early stage of differentiation (ES and MCP) sequencing reads of exon 3 are at similar levels as adjacent exons. However, exon 3 level is much lower compared to adjacent exons when the ES cells differentiated into SMs and ECs, in spite of an overall decrease of FGFR1 level in those cells (Fig. 7b). To validate the differential usage of exon 3 of FGFR1, we designed two primer pairs to detect the exon 3 present and exon 3 absent isoforms (Listed in Supplementary Table. 3). PCR using primers 1 produce a 496 bp product indicating the isoform with exon 3, or a 229 bp product without exon 3. PCR primers 2 locate in exon 3 and produce a 134 bp product only if exon 3 exists (Fig. 7b). Consistent with the RNA-seq, RT-PCR detected progressive decrease of FGFR1 isoform with exon 3 during the differentiation of hESC towards cardiovascular cell fates. FGFR1 isoform absent of exon 3 were not detectable in ES and MCP, while clearly detected in CMs, SMs and ECs (Fig. 7c). These results indicate the expression change of FGFR1 isoforms by skipping exon 3, which encode 89 amino acids, during the stage of MCP to cardiovascular lineage differentiation. This result suggests that our RNA-seq plus GEPA workflow could predict stage-associated splicing switch of genes during human cardiovascular differentiation.


Comparative transcriptomic analysis of multiple cardiovascular fates from embryonic stem cells predicts novel regulators in human cardiogenesis.

Li Y, Lin B, Yang L - Sci Rep (2015)

GEPA predicted differentiation-associated alternative RNA splicing and lineage-enriched long non-coding RNAs during human cardiovascular differentiation. (a) Genes with exon changes detected by RNA-seq during human cardiovascular differentiation. Locations of the genes in Hg18 human genome assembly were shown along with total exon number (No. exons) and number of exons with changes (Exon diff.). (b) Differential use of the exon 3 of FGFR1 during human cardiovascular differentiation. Stacks of sequencing reads in the five lineages were shown. Distances of the exons are NOT the actual intron lengths. Change in the expression of exon 3 was highlighted by a red frame. Primers to verify the expression of the exon 3 were indicated by red or blue arrows. (c) Semi-quantitative RT-PCR validation of the exon 3 usage during human cardiovascular differentiation. (d) Lineage-enriched long non-coding RNAs during human cardiovascular differentiation identified by GEPA. To indicate the lineage-specificity, the relative expression was shown as percent in the sum of all the cell types in the heatmap. (e) qRT-PCR validation of the lineage-enriched expressions of lncRNAs.
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f7: GEPA predicted differentiation-associated alternative RNA splicing and lineage-enriched long non-coding RNAs during human cardiovascular differentiation. (a) Genes with exon changes detected by RNA-seq during human cardiovascular differentiation. Locations of the genes in Hg18 human genome assembly were shown along with total exon number (No. exons) and number of exons with changes (Exon diff.). (b) Differential use of the exon 3 of FGFR1 during human cardiovascular differentiation. Stacks of sequencing reads in the five lineages were shown. Distances of the exons are NOT the actual intron lengths. Change in the expression of exon 3 was highlighted by a red frame. Primers to verify the expression of the exon 3 were indicated by red or blue arrows. (c) Semi-quantitative RT-PCR validation of the exon 3 usage during human cardiovascular differentiation. (d) Lineage-enriched long non-coding RNAs during human cardiovascular differentiation identified by GEPA. To indicate the lineage-specificity, the relative expression was shown as percent in the sum of all the cell types in the heatmap. (e) qRT-PCR validation of the lineage-enriched expressions of lncRNAs.
Mentions: Alternative splicing produces diversities in protein output of the genome. It could play a role as regulatory mechanism in cell fate decision. A well established example is that the protein isoforms of FOXP1 gene produced by alternative splicing can have differential regulatory activities in maintaining pluripotency or promoting differentiation25. To identify the alternative splicing during human cardiovascular differentiation, we used GEPA to analyze the lineage-enrichment pattern of the splicing isoforms of the genes. We found a number of genes have differential expression patterns of the isoforms (Fig. 7a). Among them, a typical example is the differential usage of the exon 3 of Fibroblast Growth Factor Receptor 1 (FGFR1) gene during human cardiovascular differentiation (Fig. 7b). In early stage of differentiation (ES and MCP) sequencing reads of exon 3 are at similar levels as adjacent exons. However, exon 3 level is much lower compared to adjacent exons when the ES cells differentiated into SMs and ECs, in spite of an overall decrease of FGFR1 level in those cells (Fig. 7b). To validate the differential usage of exon 3 of FGFR1, we designed two primer pairs to detect the exon 3 present and exon 3 absent isoforms (Listed in Supplementary Table. 3). PCR using primers 1 produce a 496 bp product indicating the isoform with exon 3, or a 229 bp product without exon 3. PCR primers 2 locate in exon 3 and produce a 134 bp product only if exon 3 exists (Fig. 7b). Consistent with the RNA-seq, RT-PCR detected progressive decrease of FGFR1 isoform with exon 3 during the differentiation of hESC towards cardiovascular cell fates. FGFR1 isoform absent of exon 3 were not detectable in ES and MCP, while clearly detected in CMs, SMs and ECs (Fig. 7c). These results indicate the expression change of FGFR1 isoforms by skipping exon 3, which encode 89 amino acids, during the stage of MCP to cardiovascular lineage differentiation. This result suggests that our RNA-seq plus GEPA workflow could predict stage-associated splicing switch of genes during human cardiovascular differentiation.

Bottom Line: Furthermore, GEPA analysis revealed the MCP-specific expressions of genes in ephrin signaling pathway, positive role of which in cardiomyocyte differentiation was further validated experimentally.By using RNA-seq plus GEPA workflow, we also identified stage-specific RNA splicing switch and lineage-enriched long non-coding RNAs during human cardiovascular differentiation.Overall, our study utilized multi-cell-fate transcriptomic comparison analysis to establish a lineage-specific gene expression map for predicting and validating novel regulatory mechanisms underlying early human cardiovascular development.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Biology, University of Pittsburgh School of Medicine, 530 45th Street, Rangos Research Center, Pittsburgh, PA 15201.

ABSTRACT
Dissecting the gene expression programs which control the early stage cardiovascular development is essential for understanding the molecular mechanisms of human heart development and heart disease. Here, we performed transcriptome sequencing (RNA-seq) of highly purified human Embryonic Stem Cells (hESCs), hESC-derived Multipotential Cardiovascular Progenitors (MCPs) and MCP-specified three cardiovascular lineages. A novel algorithm, named as Gene Expression Pattern Analyzer (GEPA), was developed to obtain a refined lineage-specificity map of all sequenced genes, which reveals dynamic changes of transcriptional factor networks underlying early human cardiovascular development. Moreover, our GEPA predictions captured ~90% of top-ranked regulatory cardiac genes that were previously predicted based on chromatin signature changes in hESCs, and further defined their cardiovascular lineage-specificities, indicating that our multi-fate comparison analysis could predict novel regulatory genes. Furthermore, GEPA analysis revealed the MCP-specific expressions of genes in ephrin signaling pathway, positive role of which in cardiomyocyte differentiation was further validated experimentally. By using RNA-seq plus GEPA workflow, we also identified stage-specific RNA splicing switch and lineage-enriched long non-coding RNAs during human cardiovascular differentiation. Overall, our study utilized multi-cell-fate transcriptomic comparison analysis to establish a lineage-specific gene expression map for predicting and validating novel regulatory mechanisms underlying early human cardiovascular development.

No MeSH data available.


Related in: MedlinePlus