Deficient GATA6-CXCR7 signaling leads to bicuspid aortic valve

Abstract

The cardiac outflow tract (OFT) transiently links the ventricles to the aortic sac and forms the arterial valves. Abnormalities in these valves, such as bicuspid aortic valve (BAV), are common congenital anomalies. GATA6-inactivating variants cause cardiac OFT defects and BAV, but their mechanisms are unclear. We generated Gata6STOP/+ mice using CRISPR-Cas9, which show highly penetrant BAV (70%) and membranous ventricular septal defects (43%). These mice exhibited decreased proliferation and increased ISL1-positive progenitor cells in the OFT, indicating abnormal cardiovascular differentiation. Gata6 deletion with the Mef2cCre driver line recapitulated Gata6STOP/+ phenotypes, indicating a cell-autonomous role for Gata6 in the second heart field. Gata6STOP/+ mice showed reduced OFT length and caliber, associated with deficient cardiac neural crest cell contribution, which may cause valvulo-septal defects. RNA-sequencing analysis showed depletion in pathways related to cell proliferation and migration, highlighting Cxcr7 (also known as Ackr3) as a candidate gene. Reduced mesenchymal cell migration and invasion were observed in Gata6STOP/+ OFT tissue. CXCR7 agonists reduced mesenchymal cell migration and increased invasion in wild-type but not in Gata6STOP/+ explants, indicating the GATA6-dependent role of CXCR7 in OFT development and its potential link to BAV.

Keywords: ACKR3/CXCR7; Bicuspid aortic valve; Cardiac neural crest; Endocardial cushion development; GATA6; Outflow tract.

Fig. 1.

Gata6^STOP/+ mice have severe aortic insufficiency and systolic dysfunction. (A) Echocardiographic images of 30±4 week control and Gata6^STOP/+ mice hearts. Yellow boxes represent the acoustic windows. Top: normal blood flow of ascending aorta. Middle and bottom: regurgitation in ascending and descending aorta in Gata6^STOP/+ mice, respectively. Blue and red coloring represent the pulsed wave Doppler capture. Yellow arrowheads indicate retrograde flow during diastole. n=5 mice. (B) Quantification of ascending and descending aortic insufficiency. P-values were obtained by Fisher's Exact test. (C) Quantification of the percentages of left ventricular ejection fraction (EF) and fractional shortening (FS). Data are represented as mean±s.d. P-values were obtained by unpaired two-tailed Student's t-test. n=5 mice. (D) Movat's pentachromic staining in 52-week-old control and Gata6^STOP/+ mice. n=5 mice. BAV, bicuspid aortic valve; TAV, tricuspid aortic valve. Scale bar: 50 μm.

Fig. 2.

Outflow tracts of Gata6^STOP/+ mice are shorter and narrower, which is associated with reduced proliferation. (A) Whole-heart immunostaining images of E11.5 Gata6^STOP/+ and control mice. Top: the red line indicates outflow tract (OFT) linear length. The yellow line indicates length considering OFT tortuosity. Isolectin B4 (IsoB4, white) was used to label the endocardium with DAPI counterstaining (blue). Bottom: 3D OFT modeling with IMARIS software. The OFT region is rendered in yellow. Scale bar: 100 µm. (B) Quantification of OFT linear length and tortuosity measurements. Data are represented as mean±s.d. P-values were obtained by unpaired two-tailed Student's t-test. (C) Hematoxylin and Eosin staining of OFT frontal sections of E12.5 Gata6^STOP/+ and control mice. Asterisks indicate the position of the leaflets. Brackets indicate the aorticopulmonary septum (APS). Dashed circles delimit the aortic valve (AV) and pulmonary valve (PV). (D) Quantification of OFT area, inter-valve distance, circularity and major axis measured using Fiji Image software. Data are represented as means±s.d. P-values were obtained by unpaired two-tailed Student's t-test. n=6 control and 5 Gata6^STOP/+ embryos. (E) BrdU immunostaining of E9.5 Gata6^STOP/+ and control embryos. BrdU labels proliferating cells (green), α-SMA demarcates the myocardium (red), ERG demarcates endocardial cell nuclei (white), and nuclear counterstaining with DAPI is shown (blue). (E′) High magnification views of the boxed areas. Yellow arrowheads indicate BrdU⁺ myocardial cells, and white arrowheads indicate BrdU⁺ endocardial cells. (F) Quantification of the percentage of BrdU⁺ cells to DAPI⁺, SMA⁺ and ERG⁺ cells in the OFT. Data are represented as mean±s.d. P-values were obtained by unpaired two-tailed Student's t-test. n=3 control and 4 Gata6^STOP/+ embryos.

Fig. 3.

Cell-autonomous Gata6 requirement in the secondary heart field. (A) Hematoxylin and Eosin staining of sections of the aortic valve (right) and ventricles (left) from E16.5 Gata6^flox/flox;Mef2c^Cre and control mice. Asterisks indicate the position of the leaflets. The black arrowhead indicates ventricular septal defect (VSD). lv, left ventricle; rv, right ventricle. (B) Quantification of the percentage of embryos with bicuspid aortic valve (BAV) and VSD. P-values were obtained by Fisher exact test. n=9 control and 12 Gata6^flox/flox;Mef2c^Cre embryos. TAV, tricuspid aortic valve. (C) Fluorescence immunostaining on E9.5 Gata6^STOP/+ and control OFT sections. ISL1 labeling marks secondary heart field progenitors (green), αSMA demarcates the myocardium (red), IsoB4 demarcates the endocardium (white), and nuclear counterstaining with DAPI is shown (blue). (C′) Higher magnification views of the boxed areas. (D) Quantification of the percentage of ISL1⁺ cells to total, SMA⁺ and IsoB4⁺ cells in the OFT. Data are represented as mean±s.d. P-values were obtained by unpaired two-tailed Student's t-test. n=5 control and 6 Gata6^STOP/+ embryos.

Fig. 4.

Reduced contribution of cardiac neural crest cells to OFT septation in Gata6^STOP/+ mice. (A) In situ hybridization of Sema3c in E12.5 control and Gata6^STOP/+ OFT sections. Yellow arrowheads indicate the absence of post-migratory cardiac neural crest cells in the mutant OFT. n=3 mice. (B) Schematic of distal to proximal OFT sectioning. AV, aortic valve; PV, pulmonary valve; SMC, smooth muscle cell, EC, endocardial cushion. (C) Fluorescence immunostaining for α-SMA (red) for myocardium and smooth muscle, and ERG (white) for endocardial cell nuclei, and nuclear counterstaining with DAPI (blue) in E12.5 Gata6^STOP/+ and control OFT sections. Asterisks indicate the position of the leaflets. White brackets indicate the aorticopulmonary septum (APS). White arrowheads indicate narrowed APS. n=3 mice.

Fig. 5.

Depleted cellular motility processes in E11.5 Gata6^STOP/+ OFT. (A) Volcano plot of the genes detected in the RNA-seq analysis. Relevant differentially regulated genes are indicated. Significantly downregulated and upregulated genes (adjusted P-value <0.05) are labelled in blue and red, respectively. Non-differentially expressed genes are labelled in grey. FC, fold change. (B) The circle plot highlights ten Ingenuity Pathway Analysis disease and function terms enriched in Gata6^STOP/+ mice. Red and blue dots indicate upregulated and downregulated genes in the pathway, respectively. The heights of the inner circle sections are associated with Benjamini–Hochberg (B-H) P-values <0.05 (taller, more significant), and enrichment z-scores values are color coded from positive (orange) to negative (blue). (C) The bubble plot shows 12 enriched Hallmark gene sets by gene set enrichment analysis (y-axis). The negative logarithm of the nominal (NOM) P-value <0.1 is represented by the size of the bubble (bigger, more significant). Normalized enrichment scores (NES) are color coded from positive (red) to negative (blue). The percentage of leading edge genes is represented in the x-axis. OXPHOS, oxidative phosphorylation; XENOB. MB., xenobiotic metabolism. (D) Heatmap representation of the differentially expressed genes for each of the samples analyzed (adjusted P-value <0.05) of E11.5 Gata6^STOP/+ OFT versus controls. The expression intensity is plotted on a scale from red (upregulated) to blue (downregulated). CV, cardiovascular; EMT, epithelial-mesenchymal transition.

Fig. 6.

CXCR7 mediates GATA6 regulation of mesenchymal cell migration. (A,B,D,E) E11.5 control and Gata6^STOP/+ explants supplemented with 1 μM VUF11207 (A,B) or 1 μM AMD3100 (D,E). (A′,B′,Dʹ,Eʹ) Magnification of the boxed areas in A,B,D,E of the body of the explant and outwardly migrating mesenchymal cells. (A″,B″,D″,E″) Two-dimensional orthogonal views of the explants showing mesenchymal cell invasion into the collagen gel. White arrowheads indicate invading cells. Proliferating cells (green) are indicated by yellow arrowheads. α-SMA demarcates the mesenchyme (red). Nuclear counterstaining with DAPI is shown (blue). (C,F) Quantification of mesenchymal migration, invasion and proliferation following treatment with 1 μM VUF11207 (C) or 1 μM AMD3100 (F). Data are represented as mean±s.d. P-values were obtained by unpaired two-tailed Student's t-test. For C, n=12 control DMSO, 8 control VUF11207, 5 Gata6^STOP/+ DMSO, 3 Gata6^STOP/+ VUF11207. For F, n=8 control carrier, 7 control AMD3100, 9 Gata6^STOP/+ carrier, 7 Gata6^STOP/+ AMD3100.