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

(A) Schematic for in vitro CM directed differentiation cytokine-based protocol. 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). 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.

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 expression levels normalized to GATA6+/+ samples (n=6). (I) RT-qPCR for day 2 EOMES 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.

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).

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) 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. (B) Day 5 flow cytometry quantification for %K+P+ double-positive cells, %KDR+ single-positive cells, or %PDGFRα+ single-positive cells in GATA6-/- hESCs transduced with iLGR5 or empty vector (EV) (n=5). Significance indicated by *p<0.05 according to a two-tailed paired Student’s t-test. (C) %cTnT+ CMs from days 13-18 of cardiac differentiation quantified by flow cytometry in 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 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 one-way ANOVA (D) or two-way ANOVA (E) with Tukey’s multiple comparisons test.

CRISPR gene editing and characterization of mutant GATA6 hESC and iPSC lines.

(A) GATA6 CRISPR targeting scheme using H1 iCas9 hESCs was described in Shi et al. (Ref. 20). Two CRISPR gRNAs were used targeting the C-terminal zinc finger domain (GATA6-Cr1 and Cr2 gRNA target sequence in green, red indicates the PAM). (B) Table describing H1-GATA6-hESC clonal lines, genotype designation, and gRNA used in gene editing. Predicted protein describes mutant alleles according to the Human Genome Variation Society (HGVS) guidelines, fs indicates frameshift mutation. (C) Western blots from GATA6+/+, GATA6+/-, and GATA6-/- protein lysates at day 2 or 5 of cardiac differentiation probed for GATA6 with β-actin used as a loading control. (D) Immunofluorescence for the sarcomere marker α-Actinin and co-stained with DAPI on day 23 lactate-purified CMs. (E) Schematic for CRISPR-based correction of the GATA6 c.1071delG iPSC mutant allele to WT sequence (GATA6corr/+). A pair of gRNAs flanking the G deletion site were used for targeting with Nickase-Cas9 and a WT repair template to allow for homology directed repair (HDR) of the mutant allele. A single base substitution of G to A (indicated by brown) was used in the WT repair template to induce a silent mutation in the gRNA 2 recognition sequence to prevent additional Cas9 activity after HDR. (F) Table describing the GATA6 iPSC clonal lines. The GATA6 c.1071delG mutant allele is indicated as the protein V358Cfs (according to HGVS guidelines). (G) Immunofluorescence for the pluripotency markers NANOG or SOX2 on GATA6corr/+ or GATA61071delG/+ iPSC colonies. (H) Flow cytometry quantification for %cTnT+ CMs following cardiac directed differentiation for days 13 to 16 of GATA6corr/+ or GATA61071delG/+ iPSCs. Data represents the mean ± SEM, dots represent independent biological replicates. Significance defined as **p<0.01 using the two-tailed Student’s T-test. (I) Immunofluorescence for cTnT and co-stained with DAPI on day 23 lactate-purified iPSC-CMs. (J) Representative karyogram for GATA6corr/+ iPSCs. (K) Western blots from GATA6corr/+ or GATA61071delG/+ protein lysates at day 5 of cardiac differentiation probed for GATA6 with β-actin used as a loading control.

CPC marker gene transcriptional analysis of GATA6 WT or mutant hESCs.

(A) RT-qPCR time course for CPC markers (and GATA6) in differentiating GATA6+/+, GATA6+/-, and GATA6-/- hESCs (normalized to day 2 WT gene expression levels). (B) Schematic for in vitro CM directed differentiation CHIR protocol. (C) Principal component analysis (PCA) for pairwise comparisons of day 5 bulk RNA-seq from GATA6+/+, GATA6+/-, and GATA6-/- hESCs using the CHIR protocol (n=2). (D) Gene ontology (GO) analysis for biological process (BP) using differentially expressed genes (DEGs) from day 5 RNA-seq comparing GATA6-/- to WT. (E) Heatmap for cardiac development related genes from day 5 RNA-seq. Color gradient indicates relative gene expression level. (F) RT-qPCR time course for ALDH1A2 transcript levels in differentiating GATA6+/+, GATA6+/-, and GATA6-/- hESCs using the cytokine-based protocol.

KDR and PDGFRα cardiac mesoderm analysis at day 4.

(A) Representative flow cytometry plots at day 4 of cardiac differentiation analyzing the % KDR and PDGFRα double-positive cells (%K+P+ ) from GATA6+/+, GATA6+/-, or GATA6-/- hESCs. (B) Quantification of day 4 flow cytometry data for %K+P+ double-positive, %KDR+ single positive, or %PDGFRα+ single positive cells (n=6). Data represent the mean ± SEM, with statistical significance indicated as *p<0.05 and ns indicating not significant by one-way repeated measures ANOVA with Holm-Šídák’s multiple comparison test.

RNA-seq analysis following cardiac differentiation at day 2 or day 3.

(A) Number of DEGs identified when comparing GATA6+/- and GATA6-/- samples to GATA6+/+ controls from day 2 and 3 RNA-seq data. B) PCA analysis for GATA6+/+, GATA6+/-, and GATA6-/- profiles from day 2 or day 3 RNA-seq. Data is representative of at least 2 independent biological replicates per sample. (C) Day 2 and 3 GO (BP) analysis using dDEGs identified comparing GATA6+/- to WT samples. (D) Heatmaps for OFT septum morphogenesis and cardiac development related genes from day 3 RNA-seq. Color gradient indicates relative gene expression levels. (E) Venn diagram comparisons of dDEGs (relative to WT) identified by day 2 or day 3 RNA-seq of GATA6+/- and GATA6-/- samples. (F) Volcano plots for day 2 or 3 GATA6+/- sample RNA-seq gene expression data relative to WT. Dots represent genes, red indicates p-adj<0.05 and black indicates p-adj>0.05. (G) Heatmaps for WNT and BMP related genes from day 2 RNA-seq. (H) Gene set enrichment analysis (GSEA) analysis of day 2 RNA-seq gene expression data (GATA6-/- relative to WT) using the GATA6, EOMES, and SMAD2/3 co-binding during hESC-DE differentiation dataset from Chia et al. (Ref. 22) and EOMES ME direct activation dataset from Tosic et al. (Ref. 41). NES indicates normalized enrichment score; FDR indicates false discovery rate.

GATA6 CUT&RUN peaks overlap with previously published EOMES CHIP-seq peaks.

Human genome browser representations of GATA6 CUT&RUN data at day 2 of cardiac differentiation (blue tracks) and EOMES CHIP-seq identified genes at day 2 of hESC-DE differentiation from Teo et al. (Ref. 46) (red tracks). Orange rectangles represent approximate location for distal enhancer-like signatures (dELS) via the ENCODE Project (indicated by black arrows). Asterisks indicate significant GATA6 binding peaks (p<0.003 relative to IgG controls) and significant EOMES binding peaks as defined by Teo et al. (Ref. 46).

Extended GATA6-RIME analysis.

(A) Graph depicts GATA6-RIME enriched proteins (spectral count) identified at day 2 of cardiac differentiation and their corresponding day 2 RNA-seq gene expression level significance (p-adj, GATA6-/- relative to WT). Dots indicate genes, black indicates p-adj>0.05, red indicates decreased expression level (p-adj<0.05), green indicates increased expression level (p-adj<0.5). (B) Venn diagram comparisons for GATA6-RIME enriched proteins from day 2 (D2 G6) and day 4 (D4 G6) of cardiac differentiation from the present study with day 4 GATA6-RIME (DE: D4 G6) or day 2 EOMES-RIME (DE: D2 EOMES) enriched proteins reported during hESC-DE differentiation from Heslop et al. (Ref. 21 and Ref. 39). (C) Proteins identified in the Venn diagram comparisons of RIME data described in (B).

Extended data for iLGR5 and early CHIR treatment.

(A) RT-qPCR time course for relative LGR5 expression in differentiating GATA6+/+, GATA6+/-, and GATA6-/- hESCs normalized to day 2 WT gene expression level (on left). Day 2 RT-qPCR quantification for relative LGR5 expression level normalized to WT (on right, n=6). (B) Day 4 RT-qPCR for relative LGR5 expression level (normalized to WT) in iLGR5 or EV transduced GATA6-/- hESCs treated with or without varying concentrations of DOX (30, 125, and 250 indicate ng/mL concentrations of DOX). (C) %cTnT+ CMs from days 13 to 17 of cardiac differentiation quantified by flow cytometry in GATA6-/- cells treated with CHIR (3μM) or vehicle (n≥8). (D) Flow cytometry quantification for %K+P+ double-positive cells at day 5 of cardiac differentiation from GATA6-/- hESCs treated with CHIR and vehicle treated GATA6+/+ or GATA6-/- hESCs controls (n≥3). Data represents the mean ± SEM, significance indicated by *p<0.05, **p<0.01, ***p<0.001, by two-tailed Student’s t-test (C) or one-way ANOVA with Tukey’s multiple comparisons test (A, D).

GATA6-RIME Enriched Proteins

Cell lines, primer sequences, and antibodies used.