A Retinoic Acid:YAP1 signaling axis controls atrial lineage commitment

Abstract

Vitamin A/Retinoic Acid (Vit A/RA) signaling is essential for heart development. In cardiac progenitor cells (CPCs), RA signaling induces the expression of atrial lineage genes while repressing ventricular genes, thereby promoting the acquisition of an atrial cardiomyocyte cell fate. To achieve this, RA coordinates a complex regulatory network of downstream effectors that is not fully identified. To address this gap, we applied a functional genomics approach (i.e scRNAseq and snATACseq) to untreated and RA-treated human embryonic stem cells (hESCs)-derived CPCs. Unbiased analysis revealed that the Hippo effectors YAP1 and TEAD4 are integrated with the atrial transcription factor enhancer network, and that YAP1 is necessary for activation of RA-enhancers in CPCs. Furthermore, in vivo analysis of control and conditionally YAP1 KO mouse embryos (Sox2-cre) revealed that the expression of atrial lineage genes, such as NR2F2, is compromised by YAP1 deletion in the CPCs of the second heart field. Accordingly, we found that YAP1 is required for the formation of an atrial chamber but is dispensable for the formation of a ventricle, in hESC-derived patterned cardiac organoids. Overall, our findings revealed that YAP1 is a non-canonical effector of RA signaling essential for the acquisition of atrial lineages during cardiogenesis.

Figure 1.. scRNAseq analysis of RA^minus and RA^plus hCPCs and hCMs captures the expression of atrial and ventricular lineage genes

A) Scheme depicting the strategy for cardiac induction in hESCs. 1uM of RA treatment (from day 4 to day 7) was used to induce atrial lineages. This study focuses on analysis of D5 CPCs and D30 CMs, untreated (RA^minus) or treated with RA (RA^plus). B) scRNAseq was performed on differentiation day 5 (D5) in RA^minus and RA^plus samples (1uM RA, 24h). UMAP plot showing indicated markers. Cells are colored by cell annotation (yellow and blue) or by expression of marker genes indicated below the graphs. C) Immunostaining and quantification of the atrial and ventricular markers MYH7 and MYH6, in D30 CMs, treated as indicated (n=5, Students t-test, *p<0.05, **p<0.01). D) scRNAseq was performed on differentiation day 30 (D30) in RA^minus and RA^plus samples (1uM RA, 72h from D4-D7). UMAP plots show markers of pan-CM, smooth muscle, ventricular, and atrial CMs. Two independent differentiation experiments were pooled for D5 and D30 sequencing experiments. Abbreviations: hESCs: human embryonic stem cells, ME: mesoendodermal, Cme: cardiac mesoderm, CPC: cardiac progenitor cells, RA: Retinoic Acid, v: ventricular, a: atrial, CM:cardiomyocyte, GSK3i: GSK3 inhibitor, Wnti: Wnt inhibitor, UMAP: Uniform manifold approximation and projection.

Figure 2.. RA treatment in hCPCs triggers genome-wide opening of TEAD4 enhancers

A) snATACseq experiments were carried out in RA^minus and RA^plus day 5 cultures (hCPCs). Scheme shows chromatin accessibility changes and associated number of genes induced by RA treatment in hCPCs. B) Heatmap of RA-target genes with correlative changes in chromatin accessibility (snATACseq) and gene expression (snRNAseq) in the hCPCs. C) De novo motifs analysis showing the top motifs lost and gained in RA^plus versus RA^minus hCPCs. D) Venn diagram showing the number of total RA-induced differentially activated regions (DARs) with and without TEAD4 motifs. E) Gene Ontology analysis of genes near RA-induced TEAD4 enhancers. F) Bars graph shows top DNA motifs found in the TEAD4 enhancers. The p value (Chi-square test for independence) indicates the motifs significantly associated to TEAD4. G) TEAD4 ChIP-seq was performed in RA-treated hCPCs. Genome browser captures show TEAD4 binding on RA-induced enhancers containing TEAD4 binding motifs. Red boxes show distance of RA-TEAD4 motifs from nearest TSS. H) Summarizing scheme showing that RA signal induces the opening of enhancers containing TEAD4 (T), NR2F2 (N), GATA3 (G) and MEIS1/2 (M) binding sites. Two independent differentiation experiments were pooled for snATACseq processing. Two independent ChIP experiments were pooled for sequencing.

Figure 3.. RA induces YAP1 binding to TEAD4 enhancers in hCPCs.

A) ChIP-qPCR analysis of indicated proteins in RA^minus and RA^plus hCPCs. The analyzed gene regions are depicted in the X axis. Note that RA induces binding of YAP1 to TEAD4- enhancers on APOA1, APOA2 and LBH genes but does not affect binding to the Hippo target gene CTGF (n=4, p<**0.01, ***0.001). B) Volcano plot shows YAP1 bound peaks (from ChIP-seq; number of overlapping peaks are indicated) on RA regulated enhancers (snATAC-seq). C) snATACseq was performed in RA-treated YAP1 KO hCPCs and compared to WT. Scheme depicts the number of chromatin regions that change accessibility in the absence of YAP1. D) Heatmap shows the chromatin accessibility (peak intensity) of RA-induced TEAD4 enhancers in WT and YAP1 KO RA^plus hCPCs. E) Top motifs found in the DNA motif analysis of the 7066 regions that lost accessibility in the RA-treated YAP1 KO hCPCs compared to WT. F) Summarizing scheme shows that YAP1 is necessary for the opening of RA-enhancers in hESC-CPCs. Two independent differentiation replicates were pooled for sequencing.

Figure 4.. YAP1 regulates lineage genes in RA^plus hCPCs

A) UMAP distribution of RA^plus WT and YAP1 KO Day 5 cultures, colored by sample identity. The two main populations identified corresponding to endoderm (Foxa2+) and CPCs (Pdgfra+) are highlighted. B) Heatmap shows the differentially expressed genes in the YAP1 KO CPCs versus WT from scRNAseq analysis (Log2 FC>[0.5], adj p<0.01). C) Western Blot shows expression of HAND1 in WT and YAP1 KO D5 CPCs untreated (−) or treated with RA for 24h (+). Actin was used as loading control. Three biological independent replicates are shown. D) GO analysis of DEGs in the YAP1 KO vs WT RA^plus hCPCs. Heart development-related categories are highlighted. E) YAP1 ChIP-seq analysis were performed in WT RA^plus hCPCs. Genome browser captures show YAP1 binding distribution on the indicated genes. Two independent ChIP experiments were pooled for sequencing.

Figure 5.. YAP1 is required for atrial chamber development in hESC-derived patterned cardiac organoids

A) Scheme outlines the cardiac induction protocol, adapted from Volmert at al., Nat Comm. 2023. The D30 organoids have (A) atrial, (V) ventricular, and (E) proepicardium structures, as indicated in the scheme. B) WT and YAP1 KO H1 hESCs were differentiated to cardiac organoids as shown in (A). Representative bright field images are shown, taken at the indicated days. C) Graphs show quantification of the area (left), shape (middle) and length (right) of the WT and YAP1 KO organoids on the indicated days of differentiation. Two differentiation experiments were done with similar results and 7–8 organoids were analyzed. D) Immunofluorescence images of three representative WT and YAP1 KO day 30 organoids stained with DAPI (blue), the ventricular marker MYL3 (red), and the atrial marker NR2F2 (green). Scale bars, 200μm E) Quantification of NR2F2+ and MYL3+ areas and organoid cross-section area. Students t-test (n=7–8, Students t-test).

Figure 6.. cYap1 deletion in vivo alters the transcriptome of the SHF and Vitamin A/RA pathway.

A) Breeding scheme used to generate cYap1 KO and heterozygous control embryos, and a simplified scheme of a E7.75 embryo. B) scRNAseq was performed on 10 cYap1 KO and 7 control E7.75 embryos. Unbiased clustering identified 19 clusters in these embryos, that were annotated as indicated in 6E using known markers (see also Figure S5E). C) UMAP showing Yap1 expression levels in control and cKO embryos. Red arrowhead marks the extraembryonic (ExE) (Sox2⁻, Rhox5⁺). D) UMAP shows space distribution of normalized number of cells in control and cYap1 KO embryos. E) Dotplot graph showing the number of DEGs in cYap1 KO vs control embryos in each cluster. Note that the bigger the dot the more DEGs in a given population (abs(Log2FC)>0.25, adj. p value<0.05). F) Heatmap shows DEGs in the SHF of cYap1 KO vs control embryos. Genes relevant for heart development are highlighted. Genes in red are directly involved in SHF patterning and RA activity. UMAPs showing the expression levels of critical SHF genes. G) Scheme summarizing the effect of Yap1 depletion in the SHF. H) UMAPs showing results from unbiased SCPA analysis on Vitamin A/RA signaling terms indicated in the vertical orange boxes. Significant changes in pathway activity are displayed in orange color. The grey color indicates no enrichment. The name of the populations with significant changes are indicated.

Figure 7.. Scheme summarizing results. YAP1 is a downstream effector of Vit A/RA signaling essential for the acquisition of atrial lineages.

RA signaling induces YAP1 binding to TEAD4 and NR2F2-containing enhancers in CPCs, and YAP1 opens the chromatin and activates atrial genes (on the left, hCPCs). The activity of YAP1 is necessary for the acquisition of posterior trajectories in CPCs (“orange dots”, embryo model in the middle) and the formation of their derived structures, such an atrial chamber (on the right, organoid models).