Tbx1 and canonical Wnt signaling in the second heart field

 描述（由申请人提供）：编码T-box转录因子的TBX 1单倍不足主要导致DiGeorge综合征/腭心面综合征/22q11.2缺失综合征（22 q11 DS）的心脏缺陷。大约70%的22 q11 DS患者有心血管异常，主要是圆锥干型，由心脏流出道（OFT）形成异常引起。TBX 1在小鼠早期发育过程中在咽器的中胚层中表达。在小鼠中，Tbx 1的一个等位基因的失活导致轻度缺陷，具有低迁移率，但是Tbx 1的两个等位基因的失活导致具有完全迁移率的持续性动脉干（PTA）。从E8.5-10.5，包含前第二心脏领域（aSHF）中胚层的远端咽器的基因分析表明，Tbx 1促进细胞增殖和限制过早分化。我们使用Mef 2c-AHF-Cre驱动程序灭活了aSHF中的Tbx 1，并发现所有胚胎在出生时都死于PTA。基因分析显示，Tbx 1无效突变小鼠胚胎的基因相同。与Tbx 1相反，aSHF中的经典Wnt信号传导促进细胞分化。为了测试Tbxl和经典Wnt信号传导之间是否存在相反的作用，我们使aSHF中的Tbxl的两个等位基因和β-连环蛋白的一个等位基因失活，并发现心脏缺陷的显著挽救。基于此，我们假设在aSHF中Tbx 1和经典Wnt/β-连环蛋白信号传导之间存在负反馈机制。为了测试这一点，我们将定义Tbx 1 和β-连环蛋白，评估细胞增殖与分化，以及进行ChIP-seq以鉴定直接转录靶基因。有趣的是，一些表达增加的基因，如Wnt 2和Gata 6，对后SHF（pSHF）是特异性的，并且是形成心脏流入道所必需的。由于微阵列无法区分基因表达与细胞群体变化，我们假设，在aSHF中Tbx 1的损失，除了具有组织自主功能，可能会导致周围的pSHF细胞的扩张，也影响心脏发育。这一假设将通过pSHF谱系追踪和Tbx 1与Wnt 2和Gata 6的遗传研究来检验。22 q11 DS患者表现出心血管异常的可变表型表达。在这个程序中描述的一些基因可以作为遗传修饰剂。此外，由于心脏缺陷是通过改变Tbx 1突变小鼠中的Wnt信号来挽救的，因此对这一过程的更好机制理解可能在未来转化为22 q11 DS患者的新型药物治疗。

 DESCRIPTION (provided by applicant): Haploinsufficiency of TBX1, encoding a T-box transcription factor is largely responsible for heart defects in DiGeorge syndrome/velo-cardio-facial syndrome/22q11.2 deletion syndrome (22q11DS). Approximately 70% of 22q11DS patients have cardiovascular anomalies, mostly of the conotruncal type, caused by abnormalities in the formation of the cardiac outflow tract (OFT). Tbx1 is expressed throughout the mesoderm of the pharyngeal apparatus during early mouse development. Inactivation of one allele of Tbx1 in mice results in mild defects with low penetrance, but inactivation of both alleles of Tbx1 results in a persistent truncus arteriosus (PTA) with complete penetrance. Gene profiling of the distal pharyngeal apparatus containing the anterior second heart field (aSHF) mesoderm, from E8.5-10.5, suggested that Tbx1 promotes cell proliferation and restricts premature differentiation. We inactivated Tbx1 in the aSHF using the Mef2c-AHF-Cre driver and found that all embryos died at birth with a PTA. Gene profiling revealed the same set of genes as for Tbx1 null mutant mouse embryos. In contrast to Tbx1, canonical Wnt signaling in the aSHF promotes cell differentiation. To test whether there are opposing effects between Tbx1 and canonical Wnt signaling, we inactivated both alleles of Tbx1 and one allele of ß-catenin in the aSHF and found significant rescue of the heart defects. Based upon this, we hypothesize that there is a negative feedback mechanism that exists between Tbx1 and canonical Wnt/ß-catenin signaling in the aSHF. To test this, we will define the epistatic relationship between Tbx1 and ß-catenin, evaluate cell proliferation versus differentiation, as well as perform ChIP-seq to identify direct transcriptional target genes. Interestingly, some of the genes increased in expression, such as Wnt2 and Gata6, are specific to the posterior SHF (pSHF) and are required to form the cardiac inflow tract. Since the microarrays could not distinguish between gene expressions versus cell population changes, we hypothesize that loss of Tbx1 in the aSHF, in addition to having tissue autonomous functions, may cause an expansion of the surrounding pSHF cells, also affecting heart development. This hypothesis will be tested by pSHF lineage tracing and genetic studies of Tbx1 with Wnt2 and Gata6. The 22q11DS patients show variable phenotypic expressivity of cardiovascular anomalies. Some of the genes characterized in this program may serve as genetic modifiers. Moreover, because heart defects are rescued by altering Wnt signaling in Tbx1 mutant mice, a better mechanistic understanding of this process may be translated in the future to novel drug therapies on 22q11DS patients.