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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

Network connections of lineage-enriched transcription factors in different cardiovascular cell types. LEG groups are color-coded and lineage-specificity was shown using the same bar charts as in Fig. 3. The connection of transcription factors was built based on IPA (Ingenuity Pathway Analysis, Ingenuity Systems). (a) Multipotential cardiovascular progenitor. (b) Cardiomyocyte. (c) Smooth muscle cell. (d) Endothelial cell.
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f4: Network connections of lineage-enriched transcription factors in different cardiovascular cell types. LEG groups are color-coded and lineage-specificity was shown using the same bar charts as in Fig. 3. The connection of transcription factors was built based on IPA (Ingenuity Pathway Analysis, Ingenuity Systems). (a) Multipotential cardiovascular progenitor. (b) Cardiomyocyte. (c) Smooth muscle cell. (d) Endothelial cell.

Mentions: Transcription factors are interconnected thus forming networks to specify lineage commitment and cellular function in a temporal manner18. Thus, we sought to dissect the early process of human cardiovascular development by analyzing the transcription factor networks (TFNs) using the Lineage-Enriched Transcription Factors (LETFs). It has been previously demonstrated that NANOG, SOX2, OCT4 are among the central TFNs in hESCs1920 and we observed the same result by using Ingenuity IPA to analyze the ES specific TFs (data not shown). Additionally, we built the lineage-specific TFNs in MCP, CM, SM and EC (Fig. 4). TFNs of each single lineage indicated the dominant gene programs controlling lineage-specific biofunctions, whereas the TFN of “MCP&CM” reflected the dynamic TFN transition during cardiovascular development and TFN of “SM&EC” revealed commonly shared mechanisms during vascular cell formation. For example, we found the MCP-specific TFN was centered by jun proto-oncogene (JUN), FOS and related genes (JUNB, FOSB, JUND, ATF3 and EGR genes) (Fig. 4a), indicating the high proliferative potential of MCPs as previously described21. Sub networks of TFNs in cardiac (such as HAND1/2, GATA4 and ISL1) and hematopoietic (such as the HOXB genes) developments were also present in MCP, indicating the plasticity of the MCP in its differentiation potential. The expression level of some cardiac specific TFs, such as HAND2 and GATA4, remained high in both “MCP” and “CM” (Fig. 4a, b, yellow color labeled genes), indicating that these transcription factors may function at stages of both heart progenitor formation and CM specification. We term these early onset TFs as “early” cardiac TFs. Transcriptions of CM-specific cardiac TFs, such as MEF2C and NKX2.5, were initiated during the differentiation from MCPs to CMs and were maintained high in CM, indicating their vital role during CM fate specification (Fig. 4b). We term these late onset TFs as “late” cardiac TFs. Therefore, the dynamic transitions of TFNs from the proliferation-controlling TFs in MCP (Blue genes in Fig. 4a) to “early’ cardiac TFs (Yellow genes in Fig. 4a,b) and subsequently to “late’ cardiac TFs (Orange genes in Fig. 4b) revealed the underlying programs controlling human CM development. Interestingly, we found MEF2C, GATA4 and HAND2 centralized the TFNs of MCP and CM, which is consistent with their essential roles in directly reprogramming fibroblasts into cardiomyocytes (Fig. 4b)6722. Almost no TFs were co-enriched in MCP&SM or in MCP&EC, indicating a more profound transcriptional change during the early segregation of MCP towards vascular cell fate than to cardiac muscle fate. However, many TFs are enriched in both SM and EC, such as cyclin-dependent kinase inhibitor 2A (CDKN2A), CCND1 and RUNX1 (Fig. 4c,d), indicating the common regulatory mechanism during vasculogenesis. Therefore, TFNs, which were built by the LETFs, could globally reveal the temporally transcriptional and biofunctional changes during human cardiovascular development.


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)

Network connections of lineage-enriched transcription factors in different cardiovascular cell types. LEG groups are color-coded and lineage-specificity was shown using the same bar charts as in Fig. 3. The connection of transcription factors was built based on IPA (Ingenuity Pathway Analysis, Ingenuity Systems). (a) Multipotential cardiovascular progenitor. (b) Cardiomyocyte. (c) Smooth muscle cell. (d) Endothelial cell.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4440522&req=5

f4: Network connections of lineage-enriched transcription factors in different cardiovascular cell types. LEG groups are color-coded and lineage-specificity was shown using the same bar charts as in Fig. 3. The connection of transcription factors was built based on IPA (Ingenuity Pathway Analysis, Ingenuity Systems). (a) Multipotential cardiovascular progenitor. (b) Cardiomyocyte. (c) Smooth muscle cell. (d) Endothelial cell.
Mentions: Transcription factors are interconnected thus forming networks to specify lineage commitment and cellular function in a temporal manner18. Thus, we sought to dissect the early process of human cardiovascular development by analyzing the transcription factor networks (TFNs) using the Lineage-Enriched Transcription Factors (LETFs). It has been previously demonstrated that NANOG, SOX2, OCT4 are among the central TFNs in hESCs1920 and we observed the same result by using Ingenuity IPA to analyze the ES specific TFs (data not shown). Additionally, we built the lineage-specific TFNs in MCP, CM, SM and EC (Fig. 4). TFNs of each single lineage indicated the dominant gene programs controlling lineage-specific biofunctions, whereas the TFN of “MCP&CM” reflected the dynamic TFN transition during cardiovascular development and TFN of “SM&EC” revealed commonly shared mechanisms during vascular cell formation. For example, we found the MCP-specific TFN was centered by jun proto-oncogene (JUN), FOS and related genes (JUNB, FOSB, JUND, ATF3 and EGR genes) (Fig. 4a), indicating the high proliferative potential of MCPs as previously described21. Sub networks of TFNs in cardiac (such as HAND1/2, GATA4 and ISL1) and hematopoietic (such as the HOXB genes) developments were also present in MCP, indicating the plasticity of the MCP in its differentiation potential. The expression level of some cardiac specific TFs, such as HAND2 and GATA4, remained high in both “MCP” and “CM” (Fig. 4a, b, yellow color labeled genes), indicating that these transcription factors may function at stages of both heart progenitor formation and CM specification. We term these early onset TFs as “early” cardiac TFs. Transcriptions of CM-specific cardiac TFs, such as MEF2C and NKX2.5, were initiated during the differentiation from MCPs to CMs and were maintained high in CM, indicating their vital role during CM fate specification (Fig. 4b). We term these late onset TFs as “late” cardiac TFs. Therefore, the dynamic transitions of TFNs from the proliferation-controlling TFs in MCP (Blue genes in Fig. 4a) to “early’ cardiac TFs (Yellow genes in Fig. 4a,b) and subsequently to “late’ cardiac TFs (Orange genes in Fig. 4b) revealed the underlying programs controlling human CM development. Interestingly, we found MEF2C, GATA4 and HAND2 centralized the TFNs of MCP and CM, which is consistent with their essential roles in directly reprogramming fibroblasts into cardiomyocytes (Fig. 4b)6722. Almost no TFs were co-enriched in MCP&SM or in MCP&EC, indicating a more profound transcriptional change during the early segregation of MCP towards vascular cell fate than to cardiac muscle fate. However, many TFs are enriched in both SM and EC, such as cyclin-dependent kinase inhibitor 2A (CDKN2A), CCND1 and RUNX1 (Fig. 4c,d), indicating the common regulatory mechanism during vasculogenesis. Therefore, TFNs, which were built by the LETFs, could globally reveal the temporally transcriptional and biofunctional changes during human cardiovascular development.

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