Bmp2 is expressed in the AV and OFT myocardium before cushion expansion, starting at E8.5 (Sugi, Yamamura et al. 2004). In the chicken, targeting TGFβR3 in vitro inhibits endocardial cell EMT, while overexpression of TGFβR3 in non-transforming ventricular endocardial cells causes EMT (Brown, Boyer et al. 1999). Specific cleavage of the ECM components of the cardiac jelly continues at this stage, including cleavage of Versican (Kern, Twal et al. 2006).
The myocardium and proepicardium both contribute to the ECM in the subepicardial space (Tidball 1992; Kalman, Viragh et al. 1995). RNAseq libraries were generated as described without normalizations or RNA/cDNA fragmentation (Christodoulou, Gorham et al. 2011). Gene expression profiles were generated as described (Christodoulou, Gorham et al. 2011) using a Bayesian p-value (Audic and Claverie 1997).
VENOFT AVC
HH18 Chick
E11.0 Mouse
Significantly Detected Genes (reads > 10)
Spatially Enriched Gene Expression (2.00 Fold)
VEN 16
VEN77
VENOFT
Fold, p<0.001
Fold, p<0.001
Equivalent stages in the chick (HH18) and mouse (E11.0) were selected when robust EMT occurs in both the OFT and AVC, but not the VEN at this time point ( Camenisch, Molin et al. 2002 ). By identifying genes that were upregulated in both cushions in the comparison, we eliminated the influence of the epicardial cells (AVC) and neural crest cells (OFT), which are only found in a single cushion sample and therefore not in the shared gene list that we (Figure 7A) ; see Wt1 (AVC, VEN), Sox10 (OFT), BMP10 (VEN)). We see several hundred genes enriched in the cushions, consistent with the hypothesis that the AVC and OFT should share common genes involved in endothelial EMT (Figure 4D).
Therefore, genes with a significant p-value (p<.001) and >2-fold higher expression in the pads (AVC & OFT) compared to VEN are considered to be enriched in these compartments and were potential candidates for involvement in EMT. Overall, 198 genes were identified in the chicken (Appendix A) and 105 in the mouse (Appendix B) that were >2-fold higher expressed in the pads. Of note, 31 out of 105 identified in the mouse were found to be expressed in the endothelium or endothelium-derived mesenchyme.
Thus, the RNA-seq method was sensitive enough to detect changes in gene expression in the endothelium and mesenchyme when the majority of the sample originates from the myocardium. Of these, 54 genes in the chicken and 41 genes in the mouse were associated with abnormal heart development and function. Several genes known to play important roles in endocardial EMT were revealed in >1.25-fold gene lists.
In particular, small changes (<2-fold) in the expression levels of transcription factors can have a significant effect on cell behavior (Niwa, Miyazaki et al. 2000). To gain a better understanding of the genes in the >2-fold cushion enriched gene lists, we examined the associated predicted protein location, predicted protein function, and biological processes (Figure 9).
Spatially Enriched Gene Expression (1.25 Fold)
VEN 146
VEN218
Selected significantly enriched biological processes (from DAVID BP-FAT) are depicted for bird and mouse (p<0.0001 for all terms). To examine which biological processes were associated with the pillow-enriched gene lists in bird and mouse, we performed functional annotation using the Database for Annotation, Visualization, & Integrated Discovery (DAVID) software (Dennis, Sherman et al. 2003). . Consistent with common morphological processes occurring in the pads of each, we identified enriched biological processes in both bird and mouse associated with the EMT process (Figure 9C–D).
GO analysis of the >1.25-fold gene lists yielded results similar to those obtained with the >2-fold gene lists (Figure 10). Thus, GO analysis of cushion-enriched gene lists in chickens and mice identified shared processes expected for a population of endothelial cells undergoing EMT. To gain a better understanding of how the genes enriched in the cushion interact with each other, GRN analysis was performed of the chick and mouse.
Gene regulatory networks were generated with IPA software using >2-fold cushion-enriched genes in chick and mouse. Lines indicate interactions between genes observed in the literature across all systems, with solid lines reflecting a direct interaction and dashed lines indicating an indirect interaction. We observed several genes in the network that, when targeted in mice, resulted in phenotypes associated with abnormal cushion or valve development (red circles).
This high number of genes known to be important in the development of cushions or valves provides confidence that this network of genes reflects the biological processes that occur in the cushions. Thus, GRN analysis of pillow-enriched gene lists in bird and mouse provided insight into the relationships between genes known to regulate endocardial EMT and identified candidate signaling pathways for functional analysis.
HAPLN1
MEIS2 was recently reported to be required for cardiac looping in zebrafish but has no described function in valve development (Paige, Thomas et al. 2012). FOXP2 is a forkhead/winged-helix transcription factor that has been extensively studied in the context of neuronal development. It is necessary for birds to learn to sing and mutations in FOXP2 have been linked to human speech pathologies (Enard 2011).
Although some candidates have been investigated in valvulogenesis, none have a described role in endocardial EMT. A functional analysis using siRNA to target candidate genes in the well-defined chicken AVC explant collagen gel assay in vitro was used to assess any role in endocardial EMT (reviewed (DeLaughter, Saint-Jean et al.). Each of the two independent siRNA constructs targeting candidates resulted in a decrease in endocardial EMT (Figure 13 A–E).
These results establish a role for each candidate in regulating endocardial EMT in vitro and confirm that our analysis was successful in identifying genes that regulate endocardial EMT. The average number of cells in the collagen gel was determined and normalized to the number of cells in the control (100%). Randomized GC-containing compatible siRNA constructs with no homology to any known chicken TGFβR3 gene - siRNA targeting a gene known to be required for EMT in vitro.
Explants were incubated with siRNA constructs targeting TGFβR3 (positive control), HAPLN1, Foxp2, ID1, MEIS2 or a random GC-matched construct (negative control) for 48 h on a collagen gel. To obtain sufficient RNA, 15 explants were used for each siRNA construct (positive control, negative control, a, b) and repeated in triplicate for each gene targeted (HAPLN1, FOXP2, ID1, MEIS2).
Percent Expression
Compared to GAPDH
SN50 is a synthetic peptide that blocks nuclear import of the NF-κB complex itself (Lin, Yao et al. 1995). Foxp4 was also enriched in our RNA-seq data sets and can form heterodimers with FOXP2 (Li, Weidenfeld et al. 2004). Clearly, the in vitro bioassay does not always recapitulate the in vivo phenotypes (reviewed (Lencinas, Tavares et al. 2012)).
The epicardium plays an important role in the development of coronary vessels (reviewed in (Olivey, Compton et al. 2004; Olivey and Svensson 2010)). Further studies showed a significant reduction in proliferation and invasion of the epicardium and epicardially derived mesenchyme (Sanchez, Hill et al. 2011). The highly conserved 43 amino acid intracellular domain of TGFβR3 is not required for ligand presentation (Blobe, Schiemann et al. 2001), but may regulate other signaling events.
TGFβ1 and TGFβ2 promoted smooth muscle differentiation in Tgfbr3+/+ and Tgfbr3-/- cells while BMP2 did not (Hill, Sanchez et al. 2012). TGFβR3-dependent invasion stimulated by TGFβ1, TGFβ2, BMP2, HMW-HA or FGF2 has been shown to require the cytoplasmic domain of TGFβR3 in vitro (Sanchez, Hill et al. 2011). Similar results were observed in endocardial cushions where the interaction of TGFβR3 with GIPC is required to promote TGFβ2 and BMP2 dependent invasion in vitro (Townsend, Robinson et al. 2011).
Tgfbr3+/−: Immorto mice were generated as described (Austin, Compton et al. 2008) and maintained on a mixed C57BL/6 SV129 background. Tgfbr3+/+:Immorto and Tgfbr3−/−:Immorto immortalized epicardial cell lines were generated from littermates as described (Austin, Compton et al. 2008) from E11.5 embryos (Figure 16).
LARA
RV LVE13.5
We undertook a transcriptional profiling approach to examine the genes downstream of TGFβR3 in epicardial cells in vitro. -D) NF-ĸB signaling (orange circle) is a central node in representative networks generated by gene regulatory network analysis (using Ingenuity Pathway Analysis software). GRN analysis indicates that NF-ĸB signaling may be dysregulated with loss of TGFβR3 in epicardial cells.
Immortalized epicardial cells incubated with TGFβ2 or BMP2 increased NF-ĸB activity compared to VEH in Tgfbr3+/+ epicardial cells, as described (Craig, Parker et al. 2010). TGFβ2 or BMP2 ligand incubation failed to induce NF-ĸB activity in Tgfbr3 -/- cells (Figure 24B). Together, these data demonstrate that NF-ĸB signaling is dysregulated in Tgfbr3-/- epicardial cells and that NF-ĸB is required for epicardial cell invasion in vitro.
These data support the hypothesis that TGFβR3 promotes NF-ĸB activity to regulate epicardial cells. Epicardial cell invasion is also impaired in vivo and in vitro in cells lacking TGFβR3 (Sanchez, Hill et al. 2011). Tgfbr3-/- epicardial cells show dysregulated expression of genes encoding proteins found in the ECM, indicating that epicardial contributions to the ECM are altered upon loss of TGFβR3.
GRNs generated from genes differentially expressed >2-fold between Tgfbr3+/+ and Tgfbr3-/- epicardial cells in each ligand incubation group (VEH, TGFβ1, TGFβ2, BMP2) identified NF-ĸB signaling as a central hub. Previous studies have shown that TGFβR3 can inhibit NF-ĸB signaling through interaction with β-arrestin2 (You, How et al. 2009). We therefore propose that disruption of TGFβR3-regulated NF-ĸB signaling is the mechanism responsible for the loss of invasion in epicardial cells and ultimately the failure of coronary vessel development in Tgfbr3-/- embryos.
These data suggest that NF-ĸB signaling is dysregulated in Tgfbr3-/- epicardial cells and that NF-ĸB signaling is required for epicardial cell invasion in vitro. This ability of TGFβ2 and BMP2 to stimulate NF-ĸB signaling was shown to be TGFβR3 dependent in the epicardium. These data suggest that NF-ĸB may be a common downstream mediator of TGFβR3 promoted invasion into the epicardium.
