Spatial transcriptional profile of the chick and mouse endocardial cushions identify novel regulators of endocardial EMT in vitro

J Mol Cell Cardiol. 2013 Jun;59:196-204. doi: 10.1016/j.yjmcc.2013.03.016. Epub 2013 Apr 1.

Abstract

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 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. 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chickens
  • Endocardial Cushions / metabolism*
  • Epithelial-Mesenchymal Transition / genetics
  • Epithelial-Mesenchymal Transition / physiology*
  • Extracellular Matrix Proteins / genetics
  • Extracellular Matrix Proteins / metabolism
  • Forkhead Transcription Factors / genetics
  • Forkhead Transcription Factors / metabolism
  • Homeodomain Proteins / genetics
  • Homeodomain Proteins / metabolism
  • In Situ Hybridization
  • Inhibitor of Differentiation Protein 1 / genetics
  • Inhibitor of Differentiation Protein 1 / metabolism
  • Mice
  • Myocardium / metabolism*
  • Proteoglycans / genetics
  • Proteoglycans / metabolism
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism
  • Sequence Analysis, RNA

Substances

  • Extracellular Matrix Proteins
  • Forkhead Transcription Factors
  • Foxp2 protein, mouse
  • Homeodomain Proteins
  • Idb1 protein, mouse
  • Inhibitor of Differentiation Protein 1
  • Mrg1 protein, mouse
  • Proteoglycans
  • Repressor Proteins
  • link protein