MEF2C controls segment-specific gene regulatory networks that direct heart tube morphogenesis

Abstract

The gene regulatory networks (GRNs) that control early heart formation are beginning to be understood, but lineage-specific GRNs remain largely undefined. We investigated networks controlled by the vital transcription factor MEF2C using a time course of single-nucleus RNA sequencing and ATAC sequencing in wild-type and Mef2c-null embryos. We identified a "posteriorized" cardiac gene signature and chromatin landscape in the absence of MEF2C. Integrating our multiomics data in a deep learning-based model, we constructed developmental trajectories for each of the outflow tract, ventricular, and inflow tract segments and alterations of these in Mef2c-null embryos. We computationally identified segment-specific MEF2C-dependent enhancers with activity in the developing zebrafish heart. Finally, using inferred GRNs, we discovered that the Mef2c-null heart malformations are partly driven by increased activity of the nuclear hormone receptor NR2F2. Our results delineate lineage-specific GRNs in the early heart tube and provide a generalizable framework for dissecting transcriptional networks governing developmental processes.

Keywords: cardiogenesis; developmental biology; embryogenesis; gene regulation; gene regulatory networks; heart development.

Figure 1:. MEF2C is expressed throughout the developing heart tube and its loss causes segment-specific defects.

A) Schematic of cardiac progenitors and their contributions to linear heart tube development from cardiac crescent (E7.75) to looped heart tube (E9) stage. B) Immunofluorescent staining of MEF2C (cyan) and cardiac Troponin T (cTnT, magenta) in E7.75, E8.5, and E9 WT embryos. C) Representative images of WT and Mef2c KO embryos at E7.75, E8.5 and E9. Cardiac progenitors are marked by the Smarcd3-F6-eGFP reporter transgene (green). BF, brightfield. D) Schematic of the methodology and biological insights presented in the current study. Elements of this panel were created in BioRender. B, B. (2024) https://BioRender.com/i72e213. Scale bars = 200 μm. FHF, first heart field; aSHF, anterior second heart field; pSHF, posterior second heart field; LV, left ventricle; RV, right ventricle; V, ventricle; IFT, inflow tract; OFT, outflow tract.

Figure 2:. Loss of MEF2C reduces expression of key cardiomyocyte genes across all heart tube segments and alters expression of anterior/posterior markers.

A-C) UMAPs of snRNA-seq data for cardiac progenitors, cardiomyocytes, and related mesoderm subtypes from E7.75 (A), E8.5 (B), and E9 (C) embryos labeled by cell type (left) and genotype/sample ID (right). D-F) Bar plots displaying the number of up-regulated and down-regulated genes in Mef2c KO relative to WT in cell types of interest at E7.75 (D), E8.5 (E), and E9 (F). G-I) Dot plots displaying expression of key CM genes and anterior/posterior (A/P) markers at E7.75 (G), E8.5 (H), and E9 (I). J) Fluorescence in situ hybridization of key CM genes and A/P markers in E8.5-E9 (5–10 somites) WT and Mef2c KO embryos. Note the reduced expression of CM genes Tnnt2, Ttn, Nppa, and Nkx2–5, the expanded expression of posterior markers Tbx5, Gata4, and Wnt2, and the loss of anterior OFT marker Tdgf1 in Mef2c KO embryos compared to WT. n = 5–8 WT embryos and n = 7–9 Mef2c KO embryos from 7 independent litters tested per probe. Scale bars = 200 μm. CMs, cardiomyocytes; FHF, first heart field; SHF, second heart field; JCF, juxtacardiac field; CrM, cranial mesoderm; PrxM, paraxial mesoderm; LPM, lateral plate mesoderm; SoM, somitic mesoderm; NMPs, neuromesodermal progenitors; KPs, kidney progenitors; ExM, extraembryonic mesoderm; HSCs, hematopoietic stem cells; V-CMs, ventricular cardiomyocytes; IFT-CMs, inflow tract cardiomyocytes; OFT-CMs, outflow tract cardiomyocytes; aSHF, anterior second heart field; pSHF, posterior second heart field; PostM, posterior mesoderm; PhM, pharyngeal mesoderm; MixM, mixed mesoderm; A-CMs, atrial cardiomyocytes; AVC-CMs, atrioventricular canal cardiomyocytes; Pe, proepicardium; VP, venous pole; *, Genes known to be associated with CHDs (Yang et al. 2022); #, Direct targets of MEF2C based on MEF2C ChIP-seq data (Akerberg et al. 2019).

Figure 3:. MEF2C regulates chromatin accessibility broadly throughout the heart tube and in a segment-specific manner.

A) UMAP of integrated snRNA-seq and snATAC-seq data for cardiac progenitors, cardiomyocytes, and related mesoderm subtypes at E8.5 labeled by cell types determined from snRNA-seq clustering (left) and genotype/sample ID (right). B) Bar plots displaying the number of gained and lost DARs in Mef2c KO relative to WT cell types of interest at E8.5. C) Venn diagrams displaying unique and overlapping DARs in the three heart tube segments at E8.5. D) Scatter plots displaying the relationship between the Log2 fold change (Log2FC) values of the DEG and DAR analyses for all identified Peak2Gene (P2G) links in the three heart tube segments at E8.5. Dots are colored by the P2G correlation score. E-F) Genome browser tracks displaying snATAC-seq accessibility profiles at the Myh6/Myh7 (E) and Wnt2 (F) loci for the indicated pseudobulked cell types at E8.5. DARs are highlighted and the peaks are indicated by red bars (Mef2c KO relative to WT IFT-CMs). Loops indicate P2G links, colored by the P2G correlation score. V-CMs, ventricular cardiomyocytes; IFT-CMs, inflow tract cardiomyocytes; OFT-CMs, outflow tract cardiomyocytes; aSHF, anterior second heart field; pSHF, posterior second heart field; LPM, lateral plate mesoderm; PostM, posterior mesoderm; PhM, pharyngeal mesoderm; NA, cells not available in the snRNA-seq dataset; DAR, differentially accessible region; DEG, differentially expressed gene.

Figure 4:. Each heart tube segment exhibits a distinct MEF2C-depedent developmental trajectory.

A) Schematic of multimodal Models for Integrated Regulatory Analysis (MIRA) pipeline. B) UMAP of outflow tract lineage cells plotted by MIRA topic models labeled by cell type (top) and genotype (bottom). C) Pseudotime plot of outflow tract lineage cells. D) Lineage trajectory stream plots for outflow tract lineage cells labeled by cell type (top) and genotype (bottom). E) Stream plots displaying the flow of gene expression (top) and chromatin accessibility (bottom) topics in the outflow tract lineage cells. Examples of the top genes and TF binding motifs for dynamic topics are labeled. F-I) Same as (B-E), but for ventricular lineage cells. J-M) Same as (B-E) and (F-I), but for inflow tract lineage cells.

Figure 5:. Candidate regulatory elements with MEF2C-dependent chromatin accessibility display enhancer activity in zebrafish.

A) Schematic of selection process to identify candidate MEFC-dependent enhancers from integrated snRNA-seq and snATAC-seq data. B) Genome browser tracks displaying snATAC-seq accessibility profiles, MEF2C ChIP-seq occupancy profiles (Akerberg et al. 2019), and H3K27ac ChIP-seq occupancy profiles (Nord et al. 2013) at example loci containing IFT-specific (left), V-specific (middle), and OFT-specific (right) candidate enhancers (yellow highlights). C) Genome browser tracks displaying snATAC-seq accessibility profiles, MEF2C ChIP-seq occupancy profiles (Akerberg et al. 2019), and H3K27ac ChIP-seq occupancy profiles (Nord et al. 2013) at the Myh6/Myh7 locus, which contains two candidate enhancers with IFT-specific altered accessibility (yellow highlights, MVEB1 and MVEB2) that overlap with regions found in the VISTA Enhancer Browser database (Visel et al. 2007). D) Images of E11.5 mouse embryos from the Vista Enhancer Browser database (Visel et al. 2007) demonstrating positive enhancer activity of regions that overlap with candidates MVEB1 and MVEB2. E) Schematic of the Tol2 transgenesis assay used to screen candidate enhancers in zebrafish. Elements of this panel were created in BioRender. B, B. (2024) https://BioRender.com/h83r503. F) Representative ventral view images of Tg(cmlc2:mCherry) zebrafish embryos at 72 hours post-fertilization (hpf) injected with candidate enhancers that demonstrated positive activity in the heart. Boxed area in the representative brightfield image (left) indicates the anatomical region of interest captured in the fluorescent images. G) Schematic representation of the observed onset of enhancer activity for candidate enhancers that demonstrated positive activity in the heart. H) Representative ventral view images of Tg(cmlc2:mCherry) zebrafish embryos at 24 and 72 hpf injected with MVEB2:eGFP or MVEB6:eGFP reporter constructs. Scale bars = 100 μm.

Figure 6:. Loss of MEF2C induces an overactive posteriorized gene regulatory network that is partially rescued by reduced NR2F2 dosage.

A) TF binding motif enrichment analysis for gained DARs in Mef2c KO relative to WT IFT-CMs at E8.5. B) Pie charts showing the proportion of lost or gained DARs (Mef2c KO relative to WT) containing NR and MEF2C motifs or only NR motifs. C) Odds ratio analysis for NR2F2 target genes (Rouillard et al. 2016) amongst DEGs up-regulated in Mef2c KO IFT-CMs. p-value calculated using Fisher’s exact test. D) Ridge plot displaying module scores for up-regulated NR2F2 targets in Mef2c KO and WT heart tube segments. E) Inferred GRNs constructed for Mef2c KO and WT IFT-CMs at E8.5 and E9. Boxed region of E8.5 WT GRN is shown at higher magnification in (F). F) Schematic of in silico simulated Mef2c KO in the E8.5 WT GRN. G) Results of GRN validation displaying the high accuracy (74%) of predicted relative to measured gene expression changes at E9. H) Visualizations of subnetworks consisting of 12 cardiac TFs and the top 100 DEGs within the WT and Mef2c KO E8.5 IFT-CM GRNs. I) Visualization of direct NR2F2 interactions in the WT and Mef2c KO E8.5 IFT-CM GRNs. Direct interactions that occur upon Mef2c KO are highlighted. #, mis-regulated DEG in Mef2c KO IFT-CMs at E8.5; *, Direct target of NR2F2 (Rouillard et al. 2016). J-K) Immunofluorescent staining of cardiac Troponin T (cTnT, green) in representative E9.5 embryos (18–24 somites) collected from Mef2c^+/−;Nr2f2^+/− to Mef2c^+/− crosses. Boxed regions in (J) are shown at higher magnification in (K). Arrows point to the ventricle, which is expanded in Mef2c^−/−;Nr2f2^+/− embryos compared to Mef2c^−/− embryos. Asterisks mark the atria, which are better developed and have undergone more looping in Mef2c^−/−;Nr2f2^+/− embryos compared to Mef2c^−/− embryos. n=5 Mef2c^−/− and n=7 Mef2c^−/−;Nr2f2^+/− embryos from 7 independent litters. Scale bars = 200 μm.