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

Spatial Transcriptional Profile of the Chick and Mouse Endocardial Cushions Identify Novel Regulators of Endocardial EMT in Vitro

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Spatial Transcriptional Profile of the Chick and Mouse Endocardial Cushions Identify Novel Regulators of Endocardial EMT in Vitro

Daniel M DeLaughter et al. J Mol Cell Cardiol. .
Free PMC article


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.


Figure 1
Figure 1. Spatial transcriptional profile of heart identifies cushion-enriched genes
A-B, AVC, OFT and VEN were dissected from equivalent stages of chick HH18 (A) and mouse E11.0 (B). C, RNA-seq analysis identified genes with significantly expressed (>10 reads) in each spatial region of the developing heart tube. D, Mapping genes with enriched expression (>2-fold) onto each spatial region identifies those genes with cushion-enriched expression (in red), 198 in the chick and 105 in the mouse. Scale bars - 100μM.
Figure 2
Figure 2. Spatial expression of select genes in the HH18 chick and E11.0 mouse heart tube
For each gene, expression was normalized to the region with the highest read count A, Regional expressed patterns in the mouse heart are consistent with previous observations at E10.5-E11.5. Bmp10 (VEN), Isl1 (OFT), Hand1 (VEN), Hand2 (endocardium throughout heart tube), Tbx18 and Wt1 (Epicardium attaching to AVC and migrating towards VEN), and Sox10 (neural crest in OFT and in OFT and AVC cushions). B, Expression of T-box genes in chick and mouse are consistent with previous reports (reviewed in [50]). n.d.- TBX1 has not been annotated in the chick genome and is not included in the analysis.
Figure 3
Figure 3. Gene ontology, predicted protein location, and protein function of cushion-enriched genes
A, Predicted protein function of genes identified in the mouse and chick cushion-enriched gene lists generated using IPA software. Percent of total genes with defined function is depicted. Genes with unknown function in chick (89) and mouse (44) are not shown. B, Predicted protein location of genes identified in mouse and chick cushion using IPA software. C-D, Analysis of genes found >2-fold enriched in chick and mouse AVC and OFT using DAVID software. Selected significantly enriched biological processes (from DAVID BP-FAT) are depicted for chick and mouse (p<0.0001 for all terms). Significance was based on p-values generated by DAVID software[24]. Biological processes highlighted in yellow are shared between chick and mouse.
Figure 4
Figure 4. Gene regulatory networks generated from cushion-enriched-gene lists identifies known regulators of endocardial EMT
Gene regulatory networks were generated with IPA software using >2-fold cushion-enriched genes in chick and mouse. An example mouse network is depicted. Lines denote interactions between genes observed in the literature across all systems, with solid lines reflecting a direct interaction and doted lines denoting an indirect interaction. Gene names over shapes denote nodes. Green nodes have decreased expression and red have increased expression. Additional genes were annotated based on known interactions in the literature (Bmp). Hapln1 and Meis2 nodes were added to identify potential interactions of novel candidate genes in this network. Genes were marked that are known to regulate EMT in mice in vivo (green circles), in mice in vitro (red circles), and in chick in vitro (blue circles).
Figure 5
Figure 5. Confirmation of cushion-enriched gene expression in E11.0 hearts
A-D, Whole mount in situ hybridization with probes specific for (A) Hapln1, (B) Id1, (C) Foxp2, and (D) Meis2 in E11.0 embryos in whole hearts (top) and 10 μM cryosections (bottom) revealed enriched expression in the AVC (brackets) and OFT (outlines) compared to VEN. avc- atrioventricular canal, oft- outflow tract, a- atria, ven- ventricle. Scale bars: 100μM.
Figure 6
Figure 6. Candidate genes are required for endocardial EMT in vitro
A, Representative photomicrographs of AVC explants incubated with HAPLN1-targeted siRNA. B-E, Candidate gene expression in HH16 chick embryos was knocked down with siRNA in an in vitro collagen gel assay. The mean number of cells in the collagen gel was determined and normalized to the number of cells in controls (100%). Con - GC-content matched, randomized siRNA constructs with no homology to any known chick gene, TGFβR3 - siRNA targeting a gene known to be required for EMT in vitro. a or b - independent constructs targeting indicated gene. * - p<0.01. F, Transcriptional network depicting genes known to be downstream of NF-κB which had increased expression in the AVC and VEN. See legend in Figure 4. G, NF-κB inhibition by two independent small molecule inhibitors (10 μM BMS 345541 and 2 μM SN50) resulted in a loss of function an in vitro collagen gel assay. SB431542, an ALK5 kinase inhibitor, was used as a positive control. Vehicle - 0.01% DMSO. * - p<0.01.

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