GATA6 loss-of-function inhibits CM and CPC development.

(A) Schematic for in vitro CM directed differentiation cytokine-based protocol (used throughout the study except for Supplemental Fig. 2B-E). Color gradients represent relative developmental stages (white: pluripotent, grey: mesoderm, yellow: cardiac mesoderm, pink: CPCs, and red: CMs). (B) Representative flow cytometry plots for cTnT+ CMs at day 14 of cardiac differentiation for GATA6+/+, GATA6+/-, and GATA6-/- hESCs. (C) %cTnT+ CMs quantified by flow cytometry between days 13 and 16 (dots indicate independent biological replicates). Significance indicated as ****P<0.0001 by two-way ANOVA with Tukey’s multiple comparison test by genotype. There was no significant difference between clones of the same genotype when two-way ANOVA and Tukey’s multiple comparison test was performed for all six sample groups. (D) Day 6 RT-qPCR for the CPC markers indicated normalized to GATA6+/+ (n=6). (E) Day 6 RT-qPCR for heart field markers normalized to GATA6+/+ (n=4). Data represents the mean ± SEM, significance indicated as *p<0.05, **p<0.01, ***p<0.001, ns indicates not significant as determined by one-way ANOVA and Tukey’s multiple comparisons test. The labels w4, w2 (wildtype), m2, m5 (heterozygous), and m11, m14 (homozygous) refer to the isogenic wildtype and mutant hESC clones (see Supplemental Fig. 1B)

GATA6 is required for cardiac mesoderm development.

(A) Western blot time course using protein lysates from GATA6+/+ hESCs probed for GATA6 with β-actin used as a loading control. (B) Flow cytometry quantification for % KDR and PDGFRα double-positive (%K+P+) cells from days 3 to 5 of cardiac differentiation of GATA6+/+, GATA6+/-, or GATA6-/- hESCs (n≥4). Asterisks indicate statistical significance comparing GATA6-/- and WT on days 4 or 5 of cardiac differentiation. (C) Day 5 flow cytometry quantification for %K+P+ cells (n=7). D) Representative flow cytometry plots for day 5 %K+P+ cells. (E) Day 5 flow cytometry quantification (n=7) for %KDR+ (left) or PDGFRα+ (right). (F) Representative flow cytometry plots for day 2 %BRACHYURY+ cells (red) overlaid IgG stained controls (blue). (G) Quantification for day 2 or day 3 %BRACHYURY+ cells (n=4). (H) RT- qPCR for day 2 T, EOMES, MESP1, and MESP2 expression levels normalized to GATA6+/+ samples (n=6). Data represents the mean ± SEM, with significance indicated as **p<0.01, ****p<0.0001, and ns indicating not significant by two-way ANOVA (B and G) or one-way ANOVA (C, E, H, and I) with Tukey’s multiple comparison test. The labels w4, w2 (wildtype), m2, m5 (heterozygous), and m11, m14 (homozygous) refer to the isogenic wildtype and mutant hESC clones (see Supplemental Fig. 1B)

Transcriptome analysis at early mesoderm patterning stages.

(A) Gene Ontology (GO) analysis for biological process (BP) from decreased differentially expressed genes (dDEGs) identified comparing GATA6-/- to WT samples from day 2 RNA-seq data (left). Heatmap for genes related to BMP signaling from day 2 RNA- seq shown on the right. Color gradient on heatmap indicates relative gene expression levels. (B) GO analysis (BP) of increased DEGs identified comparing GATA6+/- to WT samples from day 2 RNA-seq (left). Heatmap for genes related to negative regulation of canonical WNT pathway from day 2 RNA-seq shown on the right. (C) Volcano plot for day 2 GATA6-/- sample RNA-seq gene expression data relative to WT controls. Dots represent genes, red indicates p-adj<0.05 and black indicates p-adj>0.05. (D) Gene set enrichment analysis (GSEA) (BP) of GATA6-/- cells relative to WT controls from day 3 RNA-seq data. NES indicates normalized enrichment score. (E) Heatmap for BMP signaling genes from day 3 RNA-seq data. (F) Western blots using protein lysates from GATA6+/+, GATA6+/- or GATA6-/- cells at day 2 of cardiac differentiation probed with antibodies recognizing phospho-SMAD1/5/9, phospho-SMAD2/3, total SMAD2/3, non-phospho-β-catenin, and total β-catenin with β- actin used as a loading control. (G) GSEA analysis of day 2 or day 3 RNA-seq gene expression data (GATA6-/- relative to WT) using the day 2 lateral mesoderm (relative to hESC, left) and day 3 cardiac mesoderm (relative to day 2 lateral mesoderm, right) during hESC cardiac differentiation datasets from Koh et al. (Ref. 37) NES indicates normalized enrichment score; FDR indicates false discovery rate. H) Heatmaps from RNA-seq day 2 (left) or day 3 (right) data using core enrichment genes identified in (G) for lateral mesoderm (relative to hESCs) and cardiac mesoderm (relative to lateral mesoderm). (I) Heatmap showing enriched paraxial mesoderm gene expression in the mutant cells.

GATA6 CUT&RUN analysis during early mesoderm patterning.

(A) Genomic distribution for significant GATA6 binding peaks identified by GATA6 CUT&RUN at day 2 of cardiac differentiation (n=3). (B) Transcription factor motif enrichment at GATA6 bound loci. (C) GO (BP) analysis of the gene list associated with significant GATA6 binding peaks. (D) Venn diagram comparisons for day 2 GATA6 CUT&RUN identified genes (green), day 2 dDEGs (blue) and day 3 dDEGs (yellow) identified by RNA-seq (GATA6-/- relative to WT). (E) Human genome browser representations of GATA6 CUT&RUN data (blue tracks) aligned to select genes that are dDEGs identified by day 2 or 3 RNA-seq (GATA6-/- relative to WT). Orange rectangles represent approximate location for distal enhancer-like signatures (dELS) via the ENCODE Project. Asterisks indicate significant GATA6 binding peaks (p<0.003 relative to IgG controls). (F) Genes from the WNT and BMP signaling pathways that are significantly downregulated in the mutant cells compared to wildtype. Genes in Red were found by CUT&RUN to have GATA6 binding peaks in associated putative enhancers and are therefore likely to be direct targets. (G) Venn diagram comparisons for day 2 GATA6 CUT&RUN identified genes (green), day 2 dDEGs (blue, GATA6-/- relative to WT) and EOMES-CHIP-seq identified genes at day 2 of hESC-DE differentiation from Teo et al. (Ref. 46) (yellow). Inlayed heatmap indicates GATA6+/+, GATA6+/- and GATA6-/- RNA-seq gene expression data at day 2 of cardiac differentiation for the 32 triple-overlap genes.

GATA6 interactome analysis during precardiac to cardiac mesoderm patterning stages.

(A) Venn diagrams showing unique proteins identified by RIME analysis performed on GATA6+/+ hESCs at day 2 or day 4 of cardiac differentiation. Numbers in white text indicate enriched proteins identified by GATA6-RIME. G6 indicates GATA6, R indicates replicate (n=2). (B) Enriched proteins identified by GATA6 RIME (spectral count >5, unique peptides >1). (C) Venn diagram comparing day 2 (blue) and day 4 (yellow) GATA6-RIME enriched proteins. (D) GO (BP) analysis for GATA6-RIME enriched proteins on days 2 or 4. (E) Western blots for GATA6, EOMES, and SMARCC1 performed on GATA6-immunoprecipitated (G6-IP) whole-cell protein lysates from GATA6+/+ and GATA6-/- cells isolated at day 2 of cardiac differentiation. Input indicates whole-cell protein lysate controls.

Early manipulation of the WNT and BMP pathways partially rescues the CM defects in GATA6 loss-of-function hESCs.

(A) Schematic for treatment with DOX (days 1-4), CHIR (3μM, days 0-2), and/or reduced BMP4 (5ng/mL, days 0-2, indicated as “LB”) during CM directed differentiation using the cytokine-based protocol. (B) Day 5 flow cytometry quantification for %K+P+ double-positive cells, %KDR+ single-positive cells, or %PDGFRα+ single-positive cells in WT or GATA6-/- hESCs transduced with iLGR5 or empty vector (EV) (n=5). Significance indicated by *p<0.05 according to one-way anova and Tukey’s post-hoc analysis. (C) %cTnT+ CMs from days 13-18 of cardiac differentiation quantified by flow cytometry in WT or GATA6-/- cells treated with CHIR LB or vehicle (DMSO) with normal BMP4 concentration treated control (n≥6). (D) Flow cytometry at day 5 of cardiac differentiation to quantifiy %K+P+ double-positive, %KDR+ single-positive, or %PDGFRα+ single-positive cells comparing WT or GATA6-/- hESCs treated with CHIR LB with GATA6+/+ and GATA6-/- hESCs controls treated with vehicle and normal BMP4 concentration (n≥8). (E) %cTnT+ CMs from days 13-18 of cardiac differentiation quantified by flow cytometry in GATA6+/- or WT hESCs treated with CHIR (3μM) or DMSO (n≥7). Data represents the mean ± SEM, significance indicated by **p<0.01, ***p<0.001, ****p<0.0001 by two-tailed Student’s t-test (C) and two-way ANOVA (D, E) with Tukey’s multiple comparisons test.