Dysregulation of TBX1 dosage in the anterior heart field results in congenital heart disease resembling the 22q11.2 duplication syndrome

Abstract

Non-allelic homologous recombination events on chromosome 22q11.2 during meiosis can result in either the deletion (22q11.2DS) or duplication (22q11.2DupS) syndrome. Although the spectrum and frequency of congenital heart disease (CHD) are known for 22q11.2DS, there is less known for 22q11.2DupS. We now evaluated cardiac phenotypes in 235 subjects with 22q11.2DupS including 102 subjects we collected and 133 subjects that were previously reported as a confirmation and found 25% have CHD, mostly affecting the cardiac outflow tract (OFT). Previous studies have shown that global loss or gain of function (LOF; GOF) of mouse Tbx1, encoding a T-box transcription factor mapping to the region of synteny to 22q11.2, results in similar OFT defects. To further evaluate Tbx1 function in the progenitor cells forming the cardiac OFT, termed the anterior heart field, Tbx1 was overexpressed using the Mef2c-AHF-Cre driver (Tbx1 GOF). Here we found that all resulting conditional GOF embryos had a persistent truncus arteriosus (PTA), similar to what was previously reported for conditional Tbx1 LOF mutant embryos. To understand the basis for the PTA in the conditional GOF embryos, we found that proliferation in the Mef2c-AHF-Cre lineage cells before migrating to the heart, was reduced and critical genes were oppositely changed in this tissue in Tbx1 GOF embryos versus conditional LOF embryos. These results suggest that a major function of TBX1 in the AHF is to maintain the normal balance of expression of key cardiac developmental genes required to form the aorta and pulmonary trunk, which is disrupted in 22q11.2DS and 22q11.2DupS.

Figure 1.

The 22q11.2 region and LCR22s. Top: a representation of the duplicated or deleted region on chromosome 22q11.2 shown with the coordinates on chromosome 22 (Mb is megabase; cen is centromere; hatched bars depicts a summary of the interval; GRCh37/hg19 assembly). A subset of the genes in the 22q11.2 region is shown. Low copy repeats on chromosome 22q11.2 (LCR22) are represented by letters A, B, C, D (sizes not at scale). Bottom: a comparison of some of the features of 22q11.2DupS and 22q11.2DS. Abbreviations: Outflow tract defect (OFT), atrial septal defect (ASD), hypoplastic left heart syndrome (HLHS), right-sided aortic arch (RAA).

Figure 2.

Percentage of cardiac anomalies in subjects with 22q11.2DupS. The pie chart indicates the relative composition of cardiac anomalies in the combined cohort. Abbreviations shown in the pie chart include: cardiac outflow tract defects [OFT: tetralogy of Fallot (TOF); persistent truncus arteriosus (PTA); ventricular septal defect (VSD); pulmonic valve (PV) stenosis; double outlet right ventricle (DORV); interrupted aortic arch (IAA); transposition of the great arteries (TGA)] and other malformations. We also indicate those with hypoplastic left heart syndrome (HLHS) and atrial septal defect (ASD). Valve malformations include mitral valve defects. Individuals with more than one defect were indicated accordingly. Complex heart defects are also indicated and this category includes defects that were not specified in the publication (52).

Figure 3.

Characterization of Tbx1 GOF mutant embryos. (A) Whole mount image of a mouse embryo at E9.5 showing the region that has been dissected for quantitative RT-PCR (qRT-PCR) experiments. (B) The micro-dissected region containing the Mef2c-AHF-Cre lineage is shown as a frontal view. (C) Quantification of Tbx1 expression levels in Tbx1 GOF and Tbx1 LOF mutant embryos as well as control embryos at E9.5 by qRT-PCR analysis. Statistical significance of the difference in gene expression was estimated using a two-tailed t-test; FC is fold change, P values <0.05. Error bars are standard deviation (SD). D) PTA phenotype observed in Tbx1 GOF embryos at E14.5. N is total number of hearts observed per group. Hematoxylin and eosin (H&E) stained histological sections of the heart of a control embryo at E14.5 with a normal cardiac OFT and ventricular septum (VS) (E–G). A Tbx1 GOF mutant embryo heart showing a PTA and VSD (H, I) as well as a hypoplastic right ventricle [RV] (J). Abbreviations: aorta (Ao), pulmonary trunk (PT), left atrium (LA), right atrium (RA), left ventricle (LV), right ventricle (RV), pharyngeal arch (PA). Control: Tbx1-GFP^f/+ and Mef2c-AHF-Cre/+; Tbx1 ^f/+, Tbx1 GOF: Mef2c-AHF-Cre/+; Tbx1-GFP^f/+, Tbx1 LOF: Mef2c-AHF-Cre/+; Tbx1 ^f/f.

Figure 4.

Changing the Tbx1 dosage in both directions has opposite affects on the Mef2c-AHF-Cre lineage. Representative sagittal sections showing the Mef2c-AHF-Cre lineage using a GFP reporter allele as well as DAPI fluorescent stain to visualize nuclei and identify the tissue is shown in blue. (A) E9.5 control, (B) Tbx1 GOF and (C) Tbx1 LOF embryo sections. (D–F) Higher magnification images of the rectangular areas shown in A, B and C, respectively. (G) E10.5 control, (H) Tbx1 GOF and (I) Tbx1 LOF embryo sections. Quantification of the Mef2c-AHF-Cre lineage from the area shown in the inset for each type of embryo at E9.5 (J) and E10.5 (K). Only one type of control is shown. Control for Tbx1 GOF: Mef2c-AHF-Cre/+; Rosa26^GFP/+, Tbx1 GOF: Mef2c-AHF-Cre/+; Tbx1-GFP^f/+, control for Tbx1 LOF: Mef2c-AHF-Cre/+; Tbx1 ^f/+; Rosa26^GFP/+, Tbx1 LOF: Mef2c-AHF-Cre/+; Tbx1 ^f/f; Rosa26^GFP/+. Asterisks indicate P value <0.05.

Figure 5.

Proliferation of AHF. (A–J) Proliferation of AHF in controls, Tbx1 GOF and Tbx1 LOF embryos. Immunofluorescence images of sagittal sections to visualize the AHF lineage (GFP, green) and cell proliferation [anti-phospho Histone H3 (Ser10); red]; in control and Tbx1 GOF embryos are shown. DAPI fluorescent stain to visualize nuclei and identify the tissue is shown in blue. (D–F) Higher resolution images provide a snapshot of how the cells were counted for statistical analysis. Statistical analysis was performed to determine whether cell proliferation was the same or different between groups of embryos by two-tailed t-test, P value <0.05. Error bars = standard deviation (SD). Abbreviations: AHF (anterior heart field), right ventricle (RV). Control for Tbx1 GOF: Mef2c-AHF-Cre/+; Rosa26^GFP/+, Tbx1 GOF: Mef2c-AHF-Cre/+; Tbx1-GFP^f/+, control for Tbx1 LOF: Mef2c-AHF-Cre/+; Tbx1^f/+; Rosa26^GFP/+, Tbx1 LOF: Mef2c-AHF-Cre/+; Tbx1 ^f/f; Rosa26^GFP/+. Asterisks indicate P value <0.05.

Figure 6.

Gene expression changes in the AHF from Tbx1 GOF versus LOF embryos at E9.5. (A and B) Comparison of representative gene expression changes in the microdissected AHF of Tbx1 GOF and Tbx1 LOF embryos at E9.5. Plotted are differentially expressed genes (P < 0.05 and FC > 1.5) comparing Tbx1 LOF and Tbx1 GOF versus controls by qRT-PCR analysis (FC is fold change).

Figure 7.

Model for Tbx1 dosage dependent regulation of AHF genes important for OFT septation. In the model, we compare the Tbx1 mutant (left) to control embryos at E9.5. The upper left triangle represents the Tbx1 expression pattern in the AHF that will migrate into the cardiac OFT. Left panel: depicts how altered dosage of Tbx1 results in imbalanced AHF gene expression, which causes a PTA. Right panel: Tbx1 dosage in the AHF of a control embryo. We show that Tbx1 is a key regulator of gene expression for normal cardiac OFT formation resulting in a normal aorta and pulmonary trunk.