E-GEOD-43194 - Spatial Transcriptional Profile of the Chick and Mouse Endocardial Cushions Identify Novel Regulators of Endocardial EMT in vitro

Status
Released on 16 April 2013, last updated on 25 April 2013
Organism
Gallus gallus, Mus musculus
Samples (9)
Protocols (3)
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)
Experiment type
RNA-seq of coding RNA 
Contacts
Daniel DeLaughter <geo@ncbi.nlm.nih.gov>, Christine E Seidman, Daniel M DeLaughter, Danos C Christodoulou, HS Baldwin, Jamille Y Robinson, Joey V Barnett, Jon G Seidman
MINSEQE
Exp. designProtocolsVariablesProcessedSeq. reads
Files
Investigation descriptionE-GEOD-43194.idf.txt
Sample and data relationshipE-GEOD-43194.sdrf.txt
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