Gata6 -null heterozygous mice show BAV and severe aortic insufficiency
We targeted exon 2 of the Gata6 gene, encoding the large GATA-N domain of the protein (Fig. S1A). Indels introduced by CRISPR-Cas9 editing led to a frameshift mutation giving rise to a premature termination codon at amino-acid position 291 (V291X), designated henceforth as Gata6STOP/+ (Fig. S1A, B). Histological analysis of embryonic day (E)16.5 Gata6STOP/+ mice revealed a right-non-coronary (RN) type BAV morphology, based on coronary ostia positions relative to the aortic valve leaflets (Fig. S1C), with nearly 70% (20 of 30) penetrance, and a peri-membranous ventricular septal defect (VSD) with 43% (13 of 30) penetrance (Fig. S1D).
To determine the effect of GATA6 haploinsufficiency on cardiac function, we examined 30-week-old adult mice by Spectral Doppler echocardiography of the ascending and descending aorta (Fig. 1A). Severe aortic regurgitation was observed in the ascending and especially the descending aorta in 80% (4 of 5) of Gata6STOP/+ mice (Fig. 1A, B), indicating aortic insufficiency due to valve dysfunction. Moreover, ejection fraction (EF) and fractional shortening (FS) were also reduced in Gata6STOP/+ mice (Fig. 1C), suggesting systolic dysfunction. Other parameters, ie. left ventricular diastolic volume (LV VOL; d), left ventricular systolic volume (LV VOL; s), and LV mass (LV mass C), as well as MVE/A, (flow velocity across the mitral valve), and strain (% aortic deformation), were comparable between the Gata6STOP/+ mutant and control (Fig. S2). Movat´s staining in 52-week-old mice aortic valve sections did not show any obvious differences in collagen deposition, elastin fibers or muscle mucin or fibrin composition (Fig. 1D).
BAV associates with aberrant OFT development in Gata6 haploinsufficient mice
We examined OFT morphology at E11.5 by IMARIS 3D modeling and volume rendering from IsoB4 whole-mount immunostaining (Fig. 2A; movie S1). Gata6STOP/+ mice display a shorter OFT compared to littermate controls in E11.5 Gata6STOP/+ mice (Fig. 2B; movie S2). Moreover, we measured tortuosity as a parameter of OFT curvature and found it to be unchanged (Fig. 2B). We also examined OFT caliber as it relates to the separation of the aortic and pulmonary valves by the aorticopulmonary septum (APS), which occurs between E11.5 and E12.5 (28) (Fig. 2C). Thus, OFT dimensions in E12.5 Gata6STOP/+ mice are 20% below normal and lead to narrowing of the OFT caliber (Fig. 2D). The shortened distance between primitive pulmonary and aortic valves (Fig. 2D), reduces the APS and gives rise to a more circular OFT (Fig. 2D). The major (ventral-dorsal) axis of the was also significantly reduced (Fig. 2D).
To further define the cellular mechanism causing OFT maldevelopment in Gata6STOP/+ mice, we examined cell proliferation in E9.5 hearts, specifically the cell cycle S-phase by bromodeoxyuridine (BrdU) immunostaining (Fig. 2E, E’). Proliferation was reduced in Gata6STOP/+ OFT (Fig. 2E, E’), in both myocardium and endocardium (Fig. 2F). These findings indicate that the shortened and narrowed OFT in Gata6STOP/+ mice can be ascribed to decreased myocardial and endocardial cell proliferation.
Cell-autonomous requirement for GATA6 in the SHF
We next sought to establish GATA6 requirement in cardiovascular development (29). Endothelial-specific Gata6 deletion using the Tie2 driver line results in overriding aorta with 45% (5 of 11) penetrance (Fig. S3A, B). Gata6 deletion in myocardium, endocardium (30) and epicardium (31) using the Nkx2.5Cre driver line causes fully penetrant VSD (Fig. S3C, D), and is lethal at birth (Table S1) (32). In addition, Gata6flox/flox;Nkx2.5Cre mutants show extensive hypertrabeculation and a thinner compact ventricular wall, consistent with the key roles of GATA6 in endocardium and myocardium during ventricular chamber development (32). None of these endocardial/endothelial-specific models recapitulate the BAV phenotype (Fig. S3B, D).
To determine the GATA6 requirement in early cardiac progenitor, we used the Mef2cCre driver line, active in anterior/secondary heart field (SHF)-derived endothelial and myocardial progenitors that give rise to the OFT, right ventricle and ventricular septum (33). Gata6flox/flox; Mef2cCre fully recapitulate of the Gata6STOP/+ BAV and VSD phenotypes (Fig. 3A), with 75% (9 of 12) and 58% (7 of 12) penetrance, respectively (Fig. 3B). These data suggest that GATA6 is required cell-autonomously in SHF-derived progenitors.
To gain insight we examined the SHF marker Islet-1 (ISL-1) (34) in Gata6STOP/+ mice. ISL-1 marks progenitors for cardiomyocytes, smooth muscle cells (SMC) and endothelial cell lineages, and is gradually switched off as progenitors incorporate into the arterial pole of the heart between E8.5 and E10.5 (35). ISL1-positive immunostaining was significantly increased in distal OFT myocardium, and marginally in endocardium of E9.5 Gata6STOP/+ hearts (Fig. 3C, D), suggesting that SHF-progenitor differentiation is deficient, whereas no differences were detected in pharyngeal mesoderm (not shown). These data indicate that defective OFT development in Gata6STOP/+ mice can be ascribed to impaired SHF- derived progenitor differentiation.
Deficient cNCCs contribution and SMC differentiation in Gata6STOP/+ OFT
Cardiac neural crest cells (cNCCs) constitute a major reservoir of mesenchyme progenitors that are required for patterning of the endocardial cushions in the OFT and aortic arch arteries (36, 37). We examined the expression of Sema3C (Fig. 4A), a marker of post-migratory cells and transcriptionally regulated by GATA6 (21, 38, 39). In situ hybridization (ISH) on OFT E12.5 Gata6STOP/+ frontal sections revealed markedly reduced Sema3C staining, suggesting reduced cNCCs presence (Fig. 4A). Moreover, GATA6 is required for neural crest cell-derived SMC differentiation (21, 40). Analysis of smooth muscle differentiation along distal, medial and proximal segments of E12.5 Gata6STOP/+ mice OFT (Fig. 4B), revealed deficient SMA + immunostaining in the APS, consistent with reduced cNCC presence and/or impaired SMC differentiation (Fig. 4C).
Gata6 regulates a pro-migratory gene signature in OFT development
For further insight, we performed RNA-seq on E11.5 Gata6STOP/+ and control OFTs (Fig. 5). EdgeR identified 113 differentially expressed genes (DEGs), of which 53 were upregulated and 60 downregulated (Fig. 5A; Table S2). Ingenuity Pathway Analysis (IPA) uncovered only a few enriched categories, ie. “Congenital anomaly of cardiovascular system” (Hand1, Foxf1) and “Differentiation of bone cells” (Postn) (Fig. 5B, D). To expand the gene categories, we performed Gene Set Enrichment Analysis (GSEA) against “HALLMARK” sets (41) (Fig. 5C). Enriched pathways were “G2M CHECKPOINT” and “KRAS SIGNALING UP” potentially highlighting a proliferative defect.
In contrast, Gata6STOP/+ OFT gene expression was depleted for functional categories like “Cell movement of neurons” (Ackr3, Erbb3), “Chemotaxis” (Sema3a, Robo2, Ntn1, Nrp2, Ntrk2), “Invasion of cells” (Diras2, Itga4), “Proliferation of endothelial cells” (Anxa8) (Fig. 5B, D). Related categories such as “Angiogenesis” (Pdgfra, Sfrp2), and “Development of epithelial tissue” (Col12a1, Lamc2) were also depleted (Fig. 5B, D). Consistent with this, GSEA revealed depleted gene sets like “EPITHELIAL MESENCHYMAL TRANSITION” (EMT) (Fig. 5C) (42), and furthermore, cellular stress pathways like “DNA REPAIR” and “P53 PATHWAY”, which is potentially linked to “OXIDATIVE PHOSPHORYLATION” (Ndor1), “GLYCOLYSIS” (Gpc1), and lipid metabolism dysfunction (ie. “CHOLESTEROL HOMEOSTASIS” (Apoe) and “ADIPOGENESIS”) (Fig. 5C, D). Moreover, depleted “MYOGENESIS” (Mstn, Mybpc1), is consistent with GATA6 requirement for myocardial differentiation (43).
Altogether, the bioinformatic analysis indicates that GATA6 regulates a set of processes associated with cellular proliferation and especially migration, potentially involved in development and patterning of the OFT.
ACKR3/CXCR7 mediates GATA6 dependent migration and invasion
Endocardial cushion explant assays can recapitulate morphogenetic processes occurring in vivo, including EMT and proliferation (44, 45). Therefore, to corroborate the potential link between OFT underdevelopment and defective cell migration in Gata6STOP/+ mutants, we performed explant assays (Fig. S4A). Consistent with the assessment in the RNA-seq analysis, mesenchymal migratory and invasive behaviors in 11.5 Gata6STOP/+ explants were below normal, whereas proliferation was unchanged (Fig. S4B, C).
We then focused on Ackr3/Cxcr7 as a potential mediator of Gata6 function in the OFT. Ackr3 gene expression was found to be decreased in Gata6STOP/+ OFT (FC=-1.27, adj. p-val = 0.0196), and importantly, Cxcr7 mutant mice recapitulate valvulo-septal phenotypes found in the Gata6STOP/+ mutants (46). CXCR7, together with the chemokine receptor CXCR4 and its ligand SDF-1/CXCL12, regulates cellular migration and proliferation in many developmental and pathological settings (47).
We evaluated CXCR7 requirement for GATA6-mediated cellular motility. We tested the effect of VUF11207, a specific CXCR7 agonist that induces the cytosolic scaffolding protein β-arrestin recruitment followed by its internalization and downstream pathway activation (48). Thus, whereas supplementing WT OFT explants with 100nM had no effect (Fig. S5A, A´, B, B´, D), supplementing with 1µM of VUF11207 led to a significant dose-dependent decline in migration, defined as farthest distance migrated (Fig. S5A, A´, C, C, D). In contrast, a dose-dependent increase was found with 1µM of VUF11207 for the invasion parameter, defined as farthest distance invaded, relative to control (Fig. S5A´´, C´´, D), whereas supplementing 100nM had no effect (Fig. S5A´´, B´´, D). However, providing the explants with either 100nM or 1µM of the agonist did not result in any proliferative changes (Fig. S5D).
Next, consistent with our pilot experiment, supplementing control explants with 1µM VUF11207, resulted in decreased migration (Fig. 6A, A´, C) and increased invasion (Fig. 6A, A´´, C), confirming that migratory processes taking place in endocardial cushion development are regulated by CXCR7. However, Gata6STOP/+ explants did not respond to 1µM VUF11207 supplementation (Fig. 6B-B´´, C), suggesting that migration and invasion mediated by CXCR7 are dependent on Gata6. No effects on proliferation were observed in either Gata6STOP/+ or littermate control explants (Fig. 6C).
CXCR7 modulates CXCR4 function by sequestering CXCL12 (49, 50, 51). To determine whether CXCR7 modulation of CXCR4 is dependent on Gata6, we tested the effects of AMD3100, a dual function chemical compound known to simultaneously antagonize CXCR4 (52) and allosterically activate CXCR7 (53). Having previously defined an optimal dose-dependent AMD3100 concentration (data no shown), we found that supplementing WT OFT explants with 1µM AMD3100 did not affect migration (Fig. 6D, D´, F), but significantly increased invasion (Fig. 6D, D´´, F) and proliferation (Fig. 6D, D´, F). This suggests that CXCR4 regulates mesenchymal cell proliferation in OFT explants, while CXCR7 likely regulates migration and invasion. Importantly, supplementing Gata6STOP/+ OFT explants with 1µM AMD3100 did not alter mesenchymal cell migration, invasion, or proliferation relative to Gata6STOP/+ OFT explants in absence of AMD3100 (Fig. 6E-E´´, F). Thus, Gata6 is required for the pro-invasive effects mediated by simultaneous CXCR4 inhibition and CXCR7 activation via AMD3100, but not for pro-proliferative effects.