Intricate MIB1-NOTCH-GATA6 Interactions in Cardiac Valvular and Septal Development

Abstract

Genome-wide association studies and experimental mouse models implicate the MIB1 and GATA6 genes in congenital heart disease (CHD). Their close physical proximity and conserved synteny suggest that these two genes might be involved in analogous cardiac developmental processes. Heterozygous Gata6 loss-of-function mutations alone or humanized Mib1 mutations in a NOTCH1-sensitized genetic background cause bicuspid aortic valve (BAV) and a membranous ventricular septal defect (VSD), consistent with MIB1 and NOTCH1 functioning in the same pathway. To determine if MIB1-NOTCH and GATA6 interact in valvular and septal development, we generated compound heterozygote mice carrying different Mib1 missense (Mib1^K735R and Mib1^V943F) or nonsense (Mib1^R530X) mutations with the Gata6^STOP/+ heterozygous null mutation. Combining Mib1^R530X/+ or Mib1^K735R/+ with Gata6^STOP/+ does not affect Gata6^STOP/+ single mutant phenotypes. In contrast, combining Mib1^V943F/+ with Gata6^STOP/+ decreases the incidence of BAV and VSD by 50%, suggesting a suppressive effect of Mib1^V943F/+ on Gata6^STOP/+. Transcriptomic and functional analyses revealed that while the EMT pathway term is depleted in the Gata6^STOP/+ mutant, introducing the Mib1^V943F variant robustly enriches this term, consistent with the Mib1^V943F/+ phenotypic suppression of Gata6^STOP/+. Interestingly, combined Notch1 and Gata6 insufficiency led to a nearly fully penetrant VSD but did not affect the BAV phenotype, underscoring the complex functional relationship between MIB1, NOTCH, and GATA6 in valvular and septal development.

Keywords: BAV; CHD; GATA6; MIB1; NOTCH1; VSD; genetic interactions.

Figure 1

Allele-specific Mib1 interaction with Gata6^STOP/+. (A) Schematic representation of the chromosome 18 region harboring Mib1 and Gata6 genes. (B) Graphical representation of the MIB1 domain organization and location of the identified MIB1 variants. MZM: Mib-Herc2 domain 1 + ZZ finger domain + Mib-Herc2 domain 2. REP: Mib Repeats 1 and 2. ANK: Ankyrin repeats 1−9. RNG: Ring domains 1−3. (C–G) H&E staining on aortic valve sections in control (C), Gata6^STOP/+ (D), Mib1^R530X/+ Gata6^+/STOP (E), Mib1^K735R/+ Gata6^+/STOP (F), and Mib1^V943F/+ Gata6^+/STOP mice (G) at E16.5. The asterisk (*) indicates the position of the leaflets. (H) Quantification of BAV percentage. Statistical significance was determined by the Chi-Square test. (I–M) H&E staining on ventricular chamber sections in control (I), Gata6^STOP/+ (J), Mib1^R530X/+ Gata6^+/STOP (K), Mib1^K735R/+ Gata6^+/STOP(L), and Mib1^V943F/+ Gata6^+/STOP mice (M) at E16.5. Arrowheads indicate VSD. rv, right ventricle. lv, left ventricle. (N) Quantification of VSD percentage. Statistical significance was determined by the Chi-Square test.

Figure 2

Mib1^V943F partially restores EMT in Gata6^STOP/+ mice. (A) Volcano plot of the transcripts detected by RNA-seq of Mib1^V943F/+ Gata6^+/STOP vs. control contrast. Significantly downregulated and upregulated genes (adjusted p-value < 0.05) are labeled in blue and red, respectively. Non-differentially expressed genes are labeled in gray. (B) Circular plot highlighting selected Ingenuity Pathway Analysis diseases and function terms enriched in the Mib1^V943F/+ Gata6^+/STOP genotype. Red dots, upregulated genes in the pathway. Blue dots, downregulated genes in the pathway. The height of the inner circle section represents the Benjamani–Hochberg (B-H) p-value < 0.05 (higher is more significant), and enrichment z-score values are color-coded from positive (red) to negative (blue). (C) Bubble plots show 24 enriched statistically significant Hallmark gene sets in Gata6^STOP/+ vs. control and Mib1^V943F/+ Gata6^+/STOP vs. control contrasts by Gene Set Enrichment Analysis (GSEA). Negative logarithm of NOM p-value < 0.1 is represented by the size of the bubble (bigger is more significant). Normalized enrichment score (NES) is color-coded from positive (red) to negative (blue). Low-color density bubbles represent the Hallmark gene set categories that are not statistically significant in this specific comparison. (D) Gene enrichment profiles for the “EMT” gene set in the comparison of Gata6^STOP/+ (NOM p-value = 0.0590; NES = −1.28) and Mib1^V943F/+ Gata6^+/STOP (NOM p-value = 0.0001; NES = 2.02). The expression intensity is plotted on a scale from red (upregulated) to blue (downregulated). (E) H&E staining on aortic valve and ventricular chamber sections in controls Notch1^KO/+, Gata6^STOP/+, and Gata6^STOP/+; Notch1^KO/+ mice at E16.5. The asterisk (*) indicates the position of the leaflets. Arrowheads indicate VSD. rv, right ventricle. lv, left ventricle. (F) Quantification of BAV and VSD percentages, respectively. Statistical significance was determined by the Chi-Square test.