Reduced dosage of beta-catenin genetically rescues intracardiac anomalies in a Tbx1 conditional null mouse model of 22q11.2 deletion syndrome

Approximately 60-70% of patients with 22q11.2 deletion syndrome (22q11.2DS; velo-cardio-facial syndrome/DiGeorge syndrome) have cardiac outflow tract anomalies including persistent truncus arteriosus (PTA) as the most severe defect. Among the genes in the 22q11.2 region, TBX1, encoding a T-box transcription factor is a major candidate for cardiovascular malformations and its inactivation in mice results in a PTA. To identify novel signaling mechanisms that function downstream, we found that Tbx1 restricts canonical Wnt signaling in the pharyngeal apparatus. To test for tissue specificity within the pharyngeal apparatus, we inactivated Tbx1 in the anterior portion of the secondary heart field (AHF) mesoderm using the Mef2c-AHF-Cre allele and observed a full penetrant PTA (n = 30). Tbx1 promotes progenitor cells but restricts differentiation whereas Wnt signaling, in the AHF, promotes cardiomyocyte differentiation. To determine whether Tbx1 and canonical Wnt signaling act in opposing pathways, both alleles of Tbx1 and one beta-catenin allele were inactivated in the AHF and 85% of them (n = 35) showed partial or complete rescue. The antagonistic function of the two pathways was further confirmed by gene expression profiling, indicating that these two pathways provide a key balance in the AHF to prevent premature differentiation of progenitor cells prior to reaching the cardiac outflow tract. We inactivatedTbx1 and beta-catenin allele to identify function of Tbx1 and beta-catenin in the anterior portion of the secondary heart field (AHF) mesoderm. We also inactivated both alleles of Tbx1 and one beta-catenin alleles (rescue design) to determine whether Tbx1 and canonical Wnt signaling act in opposing pathways约60%~70%的22q11.2缺失综合征（22q11.2 deletion syndrome, 22q11.2DS；又称腭心面综合征/迪乔治综合征）患者存在心脏流出道畸形，其中以永存动脉干（persistent truncus arteriosus, PTA）为最严重的缺陷类型。在22q11.2区域的基因中，编码T盒转录因子（T-box transcription factor）的TBX1是心血管畸形的主要候选致病基因，且小鼠体内该基因的失活可诱导出永存动脉干（PTA）表型。为鉴定TBX1下游发挥功能的新型信号调控机制，我们发现Tbx1可在咽区（pharyngeal apparatus）中抑制经典Wnt信号通路。为验证该调控作用在咽区的组织特异性，我们利用Mef2c-AHF-Cre等位基因，在次级心区（secondary heart field, AHF）前体中胚层的前部区域内失活Tbx1，结果显示所有受试个体（n=30）均出现完全外显的永存动脉干表型。Tbx1可促进祖细胞增殖并抑制其分化，而次级心区（AHF）内的Wnt信号通路则可促进心肌细胞（cardiomyocyte）分化。为探究Tbx1与经典Wnt信号通路是否通过拮抗通路发挥调控作用，我们在次级心区（AHF）内同时失活Tbx1的两个等位基因与一个β-连环蛋白（beta-catenin）等位基因，结果显示85%的受试个体（n=35）表现出部分或完全的表型挽救。两条通路的拮抗调控功能进一步通过基因表达谱分析（gene expression profiling）得到验证，结果表明二者在次级心区（AHF）中形成关键的平衡机制，可阻止祖细胞在抵达心脏流出道前发生过早分化。本研究中，我们分别在次级心区（AHF）前体中胚层的前部区域内失活Tbx1与β-连环蛋白等位基因，以明确二者的功能；同时构建了同时失活Tbx1双等位基因与单个β-连环蛋白等位基因的挽救实验模型，以验证Tbx1与经典Wnt信号通路的拮抗调控关系。