Spatial Transcriptional Profile of the Chick and Mouse Endocardial Cushions Identify Novel Regulators of Endocardial EMT in vitro
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ABSTRACT:
PROVIDER: PRJNA184872 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR642078.fastq.gz | Fastqsanger.gz | |||
| SRR642079.fastq.gz | Fastqsanger.gz | |||
| SRR642080.fastq.gz | Fastqsanger.gz | |||
| SRR642081.fastq.gz | Fastqsanger.gz | |||
| SRR642082.fastq.gz | Fastqsanger.gz |
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Project description:Objective: We developed an unbiased strategy to identify genes important in endocardial epithelial-to-mesenchymal transformation (EMT) using a spatial transcriptional profile. Methods and Results: Endocardial cells overlaying the cushions of the atrioventricular canal (AVC) and outflow tract (OFT) undergo an EMT to yield VICs. RNA sequencing (RNA-seq) analysis of gene expression between AVC, OFT, and ventricles (VEN) isolated from chick and mouse embryos at comparable stages of development (chick HH18; mouse E11.0) was performed. EMT occurs in the AVC and OFT cushions, but not VEN at this time. 210 genes in the chick (n=1) and 105 genes in the mouse (n=2) were enriched 2-fold in the cushions. Gene regulatory networks (GRN) generated from cushion-enriched gene lists confirmed TGFβ as a nodal point and identified NF-κB as a potential node. To reveal previously unrecognized regulators of EMT four candidate genes, Hapln1, Id1, Foxp2, and Meis2, and a candidate pathway, NF-κB, were selected. In vivo spatial expression of each gene was confirmed by in situ hybridization and a functional role for each in endocardial EMT was determined by siRNA knockdown in a collagen gel assay. Conclusions: Our spatial-transcriptional profiling strategy yielded gene lists which reflected the known biology of the system. Further analysis accurately identified and validated previously unrecognized novel candidate genes and the NF-κB pathway as regulators of endocardial cell EMT in vitro. 3 separate regions of the developing heart tube were dissected and processed for RNA sequencing analysis in both HH18 chick and E11.0 ICR mouse (done in duplicate)
2013-04-16 | E-GEOD-43194 | biostudies-arrayexpress
Project description:Objective: We developed an unbiased strategy to identify genes important in endocardial epithelial-to-mesenchymal transformation (EMT) using a spatial transcriptional profile. Methods and Results: Endocardial cells overlaying the cushions of the atrioventricular canal (AVC) and outflow tract (OFT) undergo an EMT to yield VICs. RNA sequencing (RNA-seq) analysis of gene expression between AVC, OFT, and ventricles (VEN) isolated from chick and mouse embryos at comparable stages of development (chick HH18; mouse E11.0) was performed. EMT occurs in the AVC and OFT cushions, but not VEN at this time. 210 genes in the chick (n=1) and 105 genes in the mouse (n=2) were enriched 2-fold in the cushions. Gene regulatory networks (GRN) generated from cushion-enriched gene lists confirmed TGFβ as a nodal point and identified NF-κB as a potential node. To reveal previously unrecognized regulators of EMT four candidate genes, Hapln1, Id1, Foxp2, and Meis2, and a candidate pathway, NF-κB, were selected. In vivo spatial expression of each gene was confirmed by in situ hybridization and a functional role for each in endocardial EMT was determined by siRNA knockdown in a collagen gel assay. Conclusions: Our spatial-transcriptional profiling strategy yielded gene lists which reflected the known biology of the system. Further analysis accurately identified and validated previously unrecognized novel candidate genes and the NF-κB pathway as regulators of endocardial cell EMT in vitro.
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Project description:Valvular Interstitial Cells (VICs) are a common substrate for congenital and adult heart disease yet the signaling mechanisms governing their formation during early valvulogenesis are incompletely understood. We developed an unbiased strategy to identify genes important in endocardial epithelial-to-mesenchymal transformation (EMT) using a spatial transcriptional profile. Endocardial cells overlaying the cushions of the atrioventricular canal (AVC) and outflow tract (OFT) undergo an EMT to yield VICs. RNA sequencing (RNA-seq) analysis of gene expression between AVC, OFT, and ventricles (VEN) isolated from chick and mouse embryos at comparable stages of development (chick HH18; mouse E11.0) was performed. EMT occurs in the AVC and OFT cushions, but not VEN at this time. 198 genes in the chick (n=1) and 105 genes in the mouse (n=2) were enriched 2-fold in the cushions. Gene regulatory networks (GRN) generated from cushion-enriched gene lists confirmed TGF? as a nodal point and identified NF-?B as a potential node. To reveal previously unrecognized regulators of EMT four candidate genes, Hapln1, Id1, Foxp2, and Meis2, and a candidate pathway, NF-?B, were selected. In vivo spatial expression of each gene was confirmed by in situ hybridization and a functional role for each in endocardial EMT was determined by siRNA knockdown in a collagen gel assay. Our spatial-transcriptional profiling strategy yielded gene lists which reflected the known biology of the system. Further analysis accurately identified and validated previously unrecognized novel candidate genes and the NF-?B pathway as regulators of endocardial cell EMT in vitro.
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Project description:The epithelial-to-mesenchymal transition (EMT) and migration of cranial neural crest cells are critical processes that occur in the early embryo that permit proper craniofacial patterning. Disruptions in these processes not only impair development but also lead to various diseases, underscoring the need for their detailed understanding at the molecular level. The chick embryo has served historically as an excellent model for human embryonic development. While chick cranial neural crest cell EMT and migration have been characterized at the transcript level, studies at the protein level—to allow direct measurement of the active players—have not been undertaken to date. In this study, we applied mass spectrometry (MS)-based proteomics to establish a deep proteomics profile of the midbrain region during early embryonic development. We developed a proteomics method combining optimal lysis conditions and offline fractionation with nanoflow liquid chromatography coupled to high-resolution MS to analyze the tissue from this region, which identified >5,900 proteins involved in key pathways related to neural crest cell EMT and migration such as signaling, proteolysis/extracellular matrix (ECM), and transcriptional regulation. This study offers valuable insight into important developmental processes occurring in the midbrain region and demonstrates the utility of proteomics for characterization of various tissues during chick embryogenesis.
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2018-06-29 | PXD009194 | Pride
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