Reduced dosage of <i>β-catenin</i> provides significant rescue of cardiac outflow tract anomalies in a <i>Tbx1</i> conditional null mouse model of 22q11.2 deletion syndrome

<div><p>The 22q11.2 deletion syndrome (22q11.2DS; velo-cardio-facial syndrome; DiGeorge syndrome) is a congenital anomaly disorder in which haploinsufficiency of <i>TBX1</i>, encoding a T-box transcription factor, is the major candidate for cardiac outflow tract (OFT) malformations. Inactivation of <i>Tbx1</i> in the anterior heart field (AHF) mesoderm in the mouse results in premature expression of pro-differentiation genes and a persistent truncus arteriosus (PTA) in which septation does not form between the aorta and pulmonary trunk. Canonical <i>Wnt/β-catenin</i> has major roles in cardiac OFT development that may act upstream of <i>Tbx1</i>. Consistent with an antagonistic relationship, we found the opposite gene expression changes occurred in the AHF in <i>β-catenin</i> loss of function embryos compared to <i>Tbx1</i> loss of function embryos, providing an opportunity to test for genetic rescue. When both alleles of <i>Tbx1</i> and one allele of <i>β-catenin</i> were inactivated in the <i>Mef2c-AHF-Cre</i> domain, 61% of them (n = 34) showed partial or complete rescue of the PTA defect. Upregulated genes that were oppositely changed in expression in individual mutant embryos were normalized in significantly rescued embryos. Further, <i>β-catenin</i> was increased in expression when <i>Tbx1</i> was inactivated, suggesting that there may be a negative feedback loop between canonical Wnt and <i>Tbx1</i> in the AHF to allow the formation of the OFT. We suggest that alteration of this balance may contribute to variable expressivity in 22q11.2DS.</p></div

Histology analysis of representative embryos with the “rescue” genotype.

<p>Transverse H&E histological sections of hearts from E14.5 significantly rescued embryos (<i>Tbx1 LOF</i> with loss of one allele of <i>β-catenin</i> in the <i>Mef2c-AHF-Cre</i> domain). (A) An embryonic heart with a thin septum formed that separates the Ao and the PT (top black arrow), and a small VSD compared to the usual PTA-VSD in <i>Tbx1 LOF</i> embryos (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006687#pgen.1006687.g002" target="_blank">Fig 2C</a>). (B) Example showing a DORV (middle black arrow), with a separate Ao and PT; normal ventricular septation is present. (C) Example of a heart showing a partially rescued septation between the Ao and PT (middle black arrow) and normal septation between the two ventricles (right black arrow). (D) Rescued septation between the Ao and PT. (E) <i>Mef2c-AHF-Cre</i> lineage quantification from the same area shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006687#pgen.1006687.g002" target="_blank">Fig 2G</a> for control, <i>Tbx1 LOF</i> and rescued embryos. (F) Detection of <i>Tbx1</i> and <i>β-catenin</i> by qRT-PCR in control, <i>Tbx1 LOF</i> and rescued embryos. Statistical significance of the difference in gene expression was estimated using two-tailed t-test, FC = fold change, p values < 0.05. Error bars = standard deviation (SD). Abbreviations: RA = right atrium, RV = right ventricle, LA = left atrium, LV = left ventricle, Ao = Aorta, PT = pulmonary trunk, VS = ventricular septum, PTA = persistent truncus arteriosus, VSD = ventricular septal defect, DORV = double outlet right ventricle, OFT = outflow tract.</p

Constitutive <i>β-catenin</i> expression in the AHF promotes differentiation.

<p>(A) Whole mount <i>in situ</i> hybridization (WMISH) of <i>Wnt2</i> in a wild type (<i>Tbx1</i>^<i>+/+</i>) embryo at embryonic day 9.5 (E9.5) and a sagittal section from the heart region of the embryo is shown in B. (C) <i>Mef2c-AHF-Cre</i> lineage in a wild type embryo in a sagittal section. (D) Visualization of β-catenin signaling using a <i>TCF/Lef</i>:<i>H2B-GFP</i> reporter allele in a sagittal section of a wild type embryo. (E) WMISH of <i>Tbx1</i> in a wild type embryo. (F) <i>Mef2c-AHF-Cre</i> lineage shown by green fluorescence in a wild type embryo (<i>Mef2c-AHF-Cre/+;ROSA26-</i>^{<i>GFP f/+</i>}) at E9.5. Numbers denote the pharyngeal arches 1, 2 and 3. (G) Whole mount embryo showing the region that has been dissected for the experiments (rectangle in dorsal to the heart). (H) The micro-dissected region containing the <i>Mef2c-AHF-Cre</i> lineage is shown from a frontal view. (I) Comparison of global gene expression changes in micro-dissected tissues of <i>β-catenin GOF</i> and <i>β-catenin LOF</i> embryos at E9.5. Differentially expressed genes (p < 0.05 and FC > 1.5) in at least one of the two comparisons, <i>β-catenin LOF</i> vs control (x-axis) or <i>β-catenin GOF</i> vs control (y-axis), were plotted. Red dots denote cardiac differentiation genes. Abbreviations: heart (H), pharyngeal arch (PA), outflow tract (OFT), ventricle (V), atrium (A), FC = fold change.</p

Gene expression of cardiac muscle differentiation genes are normalized in rescued embryos.

<p>(A) Comparison of global expression changes. Differentially expressed genes (p < 0.05; n = 3,636) in either <i>Tbx1 LOF</i> or <i>β-catenin LOF</i> were sorted by their FC between <i>Tbx1 LOF</i> and controls (x-axis). The sorted genes were grouped (50 genes per group) and then the average expression changes for each group in the <i>Tbx1 LOF</i>, <i>β-catenin LOF</i> or rescue embryos (vs controls) were plotted in the y-axis. (B) Heatmap showing the expression of selected key genes increased in expression in <i>Tbx1 LOF</i> embryos but decreased in expression in <i>β-catenin LOF</i> embryos.</p

Congenital heart defects in <i>Tbx1 LOF</i> embryos.

<p>(A) Heart phenotype analysis of <i>Tbx1 LOF</i> embryos at E14.5 generated from two different crosses. PTA-VSD refers to a PTA associated with a ventricular septal defect (VSD) while PTA refers to hearts that did not show a VSD. Partial septation in <i>Tbx1 LOF</i> embryos means a PTA with presence of a short septum at some level of the OFT and complete septation between the ventricles. N = total number of hearts observed per group. Significance between <i>Tbx1 LOF</i> and controls calculated by Fisher’s exact test (p < 0.001). Note that <i>Mef2c-AHF-Cre;Tbx1</i>^{<i>flox/flox</i>} embryos had additional phenotypes (three with double outlet right ventricle, DORV and one with tetralogy of Fallot, TOF, as indicated); (B) H&E histological sections of the heart of a control embryo at E14.5, with a typical ventricular septum is shown in the inset on the lower right part of the image. (C) <i>Tbx1 LOF</i> embryo with a PTA-VSD. (D) <i>Mef2c-AHF-Cre</i> lineage tracing by using a <i>GFP</i> reporter allele in a control embryo at E9.5 and a representative sagittal section is shown in E. (F) <i>Mef2c-AHF-Cre</i> lineage tracing in a <i>Tbx1 LOF</i> embryo and a representative sagittal section of the embryo is shown in G. DAPI fluorescent stain to visualize nuclei and identify the tissue is shown in blue. (H) <i>Mef2c-AHF-Cre</i> lineage quantification from the area shown in the inset in G for control and <i>Tbx1 LOF</i> embryos. (I) Detection of <i>Tbx1</i> and <i>β-catenin</i> by qRT-PCR in control and <i>Tbx1 LOF</i> embryos. Statistical significance of the difference in gene expression was estimated using two-tailed t-test, FC = fold change, p values < 0.05. Error bars = standard deviation (SD). Abbreviations: aorta (Ao), pulmonary trunk (PT), left atrium (LA), right atrium (RA), left ventricle (LV), right ventricle (RV), pharyngeal arch (PA), outflow tract (OFT), 1, 2 and 3 indicate the first, second and third pharyngeal arches (PA), respectively.</p

Model for Tbx1 and <i>β</i>-catenin function in the SHF.

<p>In the model, the triangle represents the <i>Mef2c-AHF-Cre</i> lineage and the <i>Tbx1</i> expression pattern, before migrating into the heart tube at E9.5. Left panel: <i>Tbx1</i> expression is strongest in the AHF and weakest in the posterior SHF (pSHF) while <i>Wnt/β-catenin</i> expression is opposite. Left panel depicts a possible double negative feedback loop between the two genes in the SHF, required for normal cardiac outflow tract (OFT) formation. Middle panel shows increased differentiation in the AHF when <i>Tbx1</i> is inactivated or <i>β-catenin</i> is constitutively active in the AHF. This results in premature differentiation within this tissue. Right panel depicts the rescue genotype in which both alleles of <i>Tbx1</i> and one allele of <i>β-catenin</i> was inactivated in the AHF. Significant rescue of heart defects was obtained. Abbreviations: A = anterior, P = posterior, OFT = outflow tract.</p

Phenotype analysis of significantly rescued <i>Tbx1 LOF</i> embryos.

<p>Phenotypes in embryos in which one <i>β-catenin</i> loss of function allele to either <i>Mef2c-AHF-Cre/+;Tbx1</i>^<i>f/-</i> embryos (A) or to <i>Mef2c-AHF-Cre/+;Tbx1</i>^<i>f/f</i> embryos (B) was done to lower the dosage of <i>β-catenin</i> within the AHF. Significant rescue (p < 0.001, Fisher’s exact test) was obtained in both sets of double mutant embryos. Middle panels show the percent within the groups with PTA (C and C’), DORVor TOF associated with a VSD or PTA without VSD (dark grey) and those ones with partial septation or a normal heart (light grey). Partial septation in <i>Tbx1 LOF</i> embryos means a PTA with presence of a short septum at some level of the OFT (D and D’) and complete septation between the ventricles (8/50) while partial septation in the rescue genotype embryos (34/56) means a range of noticeably less severe phenotypes including: a longer partial OFT septation (E and E’) and either complete septation between ventricles (15/56) or VSD (10/56) or normal OFT (F and F’) with a VSD (4/56) or normal heart [5/56] (F and F’). Abbreviations: aorta (Ao), pulmonary trunk (PT), right ventricle (RV), outflow tract (OFT), PTA (persistent truncus arteriosus).</p

Opposing <i>β-catenin</i> and <i>Tbx1</i> conditional mutants have same effect on expression of pro-differentiation genes in the AHF.

<p>(A) Comparison of global gene expression changes in the micro-dissected AHF of <i>β-catenin GOF</i> and <i>Tbx1 LOF</i> embryos at E9.5. Plotted are differentially expressed genes (p < 0.05 and FC > 1.5) in at least one of the two comparisons, <i>Tbx1 LOF</i> vs controls (x-axis) or <i>β-catenin GOF</i> vs controls (y-axis). (B) Comparison of global gene expression changes in the micro-dissected AHF from <i>Tbx1 GOF</i> and <i>β-catenin LOF</i> embryos at E9.5. Plotted are differentially expressed genes (p < 0.05 and FC > 1.5) in at least one of the two comparisons, <i>Tbx1 GOF</i> vs controls (x-axis) or <i>β-catenin LOF</i> vs controls (y-axis). Red dots denote cardiac differentiation genes. (C) qRT-PCR analysis of <i>Tbx1 LOF</i> versus <i>β-catenin</i> LOF (FC = Fold Change) of selected genes demonstrating the opposite gene expression changes in these mutant embryos.</p