GSE43194
Spatial Transcriptional Profile of the Chick and Mouse Endocardial Cushions Identify Novel Regulators of Endocardial EMT in vitro
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 TGFß 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. 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-?B pathway as regulators of endocardial cell EMT in vitro. Overall design: 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)