HAND2 Target Gene Regulatory Networks Control Atrioventricular Canal and Cardiac Valve Development

Abstract

The HAND2 transcriptional regulator controls cardiac development, and we uncover additional essential functions in the endothelial to mesenchymal transition (EMT) underlying cardiac cushion development in the atrioventricular canal (AVC). In Hand2-deficient mouse embryos, the EMT underlying AVC cardiac cushion formation is disrupted, and we combined ChIP-seq of embryonic hearts with transcriptome analysis of wild-type and mutants AVCs to identify the functionally relevant HAND2 target genes. The HAND2 target gene regulatory network (GRN) includes most genes with known functions in EMT processes and AVC cardiac cushion formation. One of these is Snai1, an EMT master regulator whose expression is lost from Hand2-deficient AVCs. Re-expression of Snai1 in mutant AVC explants partially restores this EMT and mesenchymal cell migration. Furthermore, the HAND2-interacting enhancers in the Snai1 genomic landscape are active in embryonic hearts and other Snai1-expressing tissues. These results show that HAND2 directly regulates the molecular cascades initiating AVC cardiac valve development.

Keywords: AVC; ChIP-seq; EMT; EndMT; RNA-seq; Snai1; cardiac cushion; endothelial to mesenchymal transition; mouse genetics; transcriptome.

Figure 1. ChIP-Seq analysis using the Hand2^3xF allele identifies the HAND2 cistrome in mouse embryonic hearts

(A, B) The majority of the genomic regions enriched in HAND2 chromatin complexes from mouse embryonic hearts at E10.5 map ≥10kb away from the closest transcription start site (TSS) and are evolutionarily conserved. (C) The top GO terms associated with HAND2 candidate targets reveal the preferential enrichment of genes functioning in cardiac development. (D) The consensus Ebox motif is most enriched by de novo motif discovery. (E) Selection of VISTA enhancers enriched in HAND2 chromatin complexes. Green intervals indicate the regions with enhancer activity (VISTA enhancer database); blue intervals highlight the regions enriched in HAND2 chromatin complexes (MACS peaks). Distances to the nearest TSS within the TAD are indicated on top. ChIP-Seq profiles of the two biological replicates (E10.5) are shown in light and dark blue, respectively. The H3K27ac ChIP-Seq profile for mouse hearts (E11.5) is shown in grey (Nord et al., 2013). The scheme at the bottom shows the placental mammal conservation (Cons) plot (PhyloP). Representative transgenic LacZ reporter embryos for VISTA enhancers associated to genes functioning in OFT and/or right ventricle development (Gata4, Myocd, Gata6 and Tbx20) are shown below. (F) ChIP-qPCR validation of the ChIP-Seq peaks (panel E) for mouse embryonic hearts at E10.5 (n=3 biological replicates) and E9.25 (n=2; mean ± SD). (G) Expression of the HAND2 targets Gata4, Myocd, Gata6 and Tbx20 in wild-type and Hand2-deficient (Hand2^Δ/Δ) embryonic hearts (E9.0–9.5). Scale bars: 100μm. oft: outflow tract, ra: right atrium, rv: right ventricle, lv: left ventricle, avc: atrioventricular canal, la: left atrium. See also Figures S1–S3 and Tables S1–S4.

Figure 2. AVC cardiac cushion agenesis in Hand2-deficient mouse embryos

(A) Distribution of the endogenous HAND2^3xF protein (using anti-FLAG antibodies, green fluorescence) during in mouse embryonic hearts at E9.5 and E10.5. Representative sagittal sections are shown. White arrows: endocardium; white arrowheads: myocardium; white asterisks: HAND2 expressing AVC cardiac cushion mesenchymal cells; black arrows: epicardium. Scale bars: 50μm. (B) Hematoxylin/Eosin staining reveals the absence of delaminating endocardial cells with mesenchymal characteristics (white asterisks) in the AVC of Hand2-deficient mouse embryos. White arrow: endocardium. Scale bars: 100μm. (C, D) Detection of the platelet endothelial cell adhesion molecule (PECAM) in the endocardium and smooth muscle actin (SMA) in the AVC myocardium of wild-type and Hand2-deficient embryos. White arrow: endocardium, white arrowhead: myocardium (panels A to D). (E) Colocalization of the HAND2^3xF (green fluorescence) with TWIST1 transcriptional regulators (red fluorescence) in the AVC of wild-type (Hand2^3xF/3xF) and Hand2-deficient (Hand2^Δ/Δ) embryos at E9.5. Colocalization is detected in delaminating mesenchymal cells (indicated by arrows, left panel), which are missing from the mutant AVC (right panel). Scale bars: 100μm. avc: atrioventricular canal, ba: branchial arches, la: left atrium, lv: left ventricle, oft: outflow tract, peo: proepicardial organ.

Figure 3. Hand2-deficient AVC endocardial cells fail to initiate EMT and mesenchymal cell migration

(A) Smooth muscle actin (red) and F-actin (green) distribution in cells that have migrated into the matrix from AVC explants of wild-type and Hand2-deficient embryos after 72hrs in culture. Scale bars: 100μm. Bottom panels show cells in proximity to the explant (i and iii) and at the far edge of migration (ii and iv). Scale bars: 20μm. (B) Quantification of the numbers of cells that migrated into the matrix from wild-type (n=10) and Hand2-deficient AVC explants (n=7). The mean ± SD (p=0.0001, Mann-Whitney test) and all individual data points are shown.

Figure 4. Transcriptome analysis identifies the transcriptional targets of HAND2 in the AVC

(A) Heat map of the differentially expressed genes (DEGs) identified by comparing the transcriptomes of wild-type and Hand2-deficient AVCs from mouse embryonic hearts at E9.25–E9.5 (n=4 and n=3 biological replicates were analyzed for mutant and wild-type AVCs, respectively). DEGs are genes whose expression is significantly changed (≥1.5-fold) between wild-type and mutant samples (p<0.05) (B) The box plot shows the number of HAND2 ChIP-Seq peaks in the TADs harboring genes with unchanged, down- or up-regulated expression in mutant AVCs, respectively. The TADs of genes with altered expression encode more HAND2-interacting genomic regions. To account for the different numbers of genes and HAND2 ChIP-Seq peaks per TAD, peak counts were normalized as numbers of peaks per gene for each TAD. (C) GO enrichment analysis for biological processes for the 167 down-regulated and 372 up-regulated genes (in mutant AVCs) that contain HAND2 ChIP-Seq peaks in their TADs. (D) GO analysis to identify the HAND2 target genes with annotated functions in EMT processes and AV cushion morphogenesis. (E) Mouse embryonic hearts isolated at E9.25–E9.5 were used for ChIP-qPCR validation of the most prominent HAND2 ChIP-Seq peaks in the TADs of the genes shown in panel D (n=4 using two biological replicates, p≤0.05). See also Figure S4 and Table S5 and S6.

Figure 5. Whole mount in situ hybridization reveals spatial changes in some of the differentially expressed HAND2 target genes

(A, B) Unaltered expression of Bmp2 (panel A) and Hey2 (panel B) indicates that the AVC domain is established correctly in Hand2-deficient hearts. (C, D) Ectopic expression of the Hey1 (panel C) and Hhex transcriptional regulators (panel D) in the mutant AVC corroborated their up-regulation detected by RNA-Seq analysis. (E-F) Loss of Twist1 (panel E), Msx1 (panel F), Sox9 (panel G) and Has2 (panel H) from the mutant AVC is in agreement with their transcriptional down-regulation detected by RNA-Seq analysis. (I) Alcian blue staining of glycosaminoglycans shows the reduced deposition of extra-cellular matrix in the cardiac jelly of Hand2-deficient mouse embryos. Right panels show the enlargements indicated by frames in the left panels. Gene names in black: unaltered, red: increased, blue: reduced transcript levels as determined by RNA-Seq analysis (Figure 4). Scale bars: 100μm. See also Figure S5.

Figure 6. Re-expression of Snai1 in Hand2-deficient AVC explants partially restores mesenchymal cell migration and regulation of Snai1 expression by enhancers enriched in HAND2 chromatin complexes

(A) Snai1 expression is lost from the endocardium of Hand2-deficient hearts. (B) Upper panels: Hand2-deficient AVC explants were infected either with GFP (control) or SNAI1-GFP adenovirus to re-express SNAI1Smaples were analyzed 72 hours after infection. All infected cells that migrated into the matrix are marked by GFP expression (green). Smooth muscle actin (SMA, red) was detected to reveal cellular morphology. Lower panel: Quantitation of cell migration in Hand2-deficient AVC explants. The mean ±SD and all individual data points are shown. The observed increase in mesenchymal cells in SNAI1-GFP infected explants is significant (p= 0.0093, Mann-Whitney test). Scale bars: 100μm. (C) Scheme of the mouse Snai1 TAD (red, HiC-data). The HAND2 ChIP-Seq profiles in the heart (this study), Hand2-expressing tissues (eT) and limb buds (Lb(Osterwalder et al., 2014) are shown below together with the H3K27ac profile in developing hearts (E11.5, Nord et al., 2013). The Snai1 TAD boundaries are marked by CTCF-binding regions in opposite orientation (Gomez-Marin et al., 2015). Green bars indicate the HAND2 target CRMs located +14kb, +45kb and +57kb that were analyzed by LacZ reporter assays in transgenic founder embryos. (D) Snai1 transcript distribution in wild-type mouse embryos (E10.5). (E, F) representative transgenic founder embryos (E10.5) show the activity of LacZ reporter constructs encoding the +45kb and +57kb CRMs, respectively. The upper panels depict whole embryos, dissected hearts and forelimb buds. The lower panels show sagittal sections at the level of the heart. The boxed areas indicate the enlargements shown in the right panels. Asterisks: AVC cardiac cushion mesenchyme; as: aortic sac; avc: atrioventricular canal; ba: branchial arch; la: left atria; lv: left ventricle; oft: outflow tract; ra: right atria; rv: right ventricle. Scale bars: 200μm. See also Figure S5 and S6.

Figure 7. Scheme depicting the interactions among positively regulated HAND2 targets genes

The HAND2 GRN was constructed using Ingenuity pathway analysis in combination with manual annotation (Chen et al., 2008). Arrows indicate transcriptional up-regulation and direct up-regulation by HAND2 transcriptional complexes is indicated in green. Broken grey lines direct protein-protein interactions. This graph represents the simplest possible scheme to illustrate the relevant direct interactions.