Resumen de Study of the role of Gata6 in valve development and disease and its potential interaction with the NOTCH pathway
Bicuspid aortic valve (BAV) is the most common congenital heart defect, affecting up to 0.5 to 2% of the population. It consists in the presence of two asymmetrical leaflets instead of three symmetrical ones. This malformation is more prevalent in males than in females, shows dominant inheritance, and incomplete penetrance. Individuals with BAV are more at risk of having valve stenosis, and calcific aortic valve disease (CAVD) and usually require valve replacement. Previous genome wide association studies have shown an association with chromosome region 18q with BAV. This region harbors the Gata6 and Mib1 genes. More recent studies have shown that Gata6 loss of function mutants display RL-type BAV, the most common morphology found in humans.
NOTCH1 mutations have been associated with familial BAV and studies from our laboratory have shown that conditional deletion of Mib1 or Jag1 in endocardium and myocardium results in dysmorphic aortic valves and BAV. So, we wanted to elucidate the role of Gata6 in the aortic valve development and disease. Moreover, we studied the relationship between Gata6 and Mib1/NOTCH in this pathology.
First, we generated Gata6STOP/+ mouse line by CRISPR-Cas9 technology, with 70% BAV and 43% ventricular septal defect (VSD). Defective outflow tract (OFT) development is one of the most important causes for BAV, therefore we characterized Gata6STOP/+ OFT and they show a defect on OFT development, displaying a smaller caliber and shorter length at E12.5. Since OFT is originated from second heart field (SHF), we observed a SHF differentiation defect and a proliferation defect at E9.5, suggesting those as a potential causes for the smaller size of the OFT. We also studied cardiac neural crest cells (cNCCs), necessary for the correct septation of the OFT, and its contribution to the OFT is reduced, leading to OFT septation defects. To understand which cell type is responsible for the BAV phenotype, we obtained a previously generated conditional Gata6flox allele and inactivated this gene in the endocardium and endothelium using theTie2-Cre driver, in the myocardium and endocardium with the Nkx2.5-Cre driver, and in the second heart field (SHF) with the Mef2c-AHF-Cre driver.
The Gata6flox; Mef2cAHF-Cre line recapitulates the BAV and VSD phenotypes of the Gata6 standard knockout, suggesting the important role of Gata6 in SHF at early stages of OFT development. Moreover, to elucidate the molecular mechanisms underlying the BAV phenotype, we performed bulk RNA-Seq of the OFT at E11.5. We found that an altered transcriptional landscape in Gata6STOP/+ mutant involving cellular migratory processes. We focused on Ackr3, which encodes the CXCR7 receptor.
Cxcr7-/- mice recapitulate Gata6STOP/+ phenotype of BAV and VSD. Ex vivo assays with explanted E11.5 Gata6STOP/+ OFT tissue demonstrated that cellular migration and invasion were decreased relative to controls. Supplementing WT OFT explants with CXCR7 agonists or with the CXCL12 ligand decreases cell migration. Taken together the data suggest that CXCR7 could mediate Gata6 function in OFT morphogenesis.
We also generated double heterozygous mice with Mib1 missense and non-sense mutations in a Gata6 haploinssuficient background, and examined their BAV phenotype. To study if NOTCH pathway interacts with Gata6, we generated double heterozygous Gata6STOP/+; Notch1KO/+ mice and we observed BAV phenotype in Gata6 mutants is NOTCH-independent, besides, VSD penetrance was increased, suggesting a common regulation between Gata6 and NOTCH in ventricular septal formation.
This doctoral thesis contributes to increase our mechanistic understanding of BAV formation and may contribute to the design of therapies to prevent this pathology
