Tbx1 and canonical Wnt signaling in the second heart field

 描述(申请人提供)：编码T-box转录因子的TBX1单倍体缺陷是导致DiGeorge综合征/心面部畸形/22q11.2缺失综合征(22q11DS)心脏缺陷的主要原因。大约70%的22q11DS患者有心血管异常，主要是圆锥干型，由心脏流出道(OFT)的形成异常引起。在小鼠发育早期，TBX1在咽器官的整个中胚层都有表达。在小鼠中，TBX1的一个等位基因失活会导致轻度缺陷和低外显，但两个等位基因的失活会导致永久性动脉干(PTA)完全外显。来自E8.5-10.5的含有第二心前区(ASHF)中胚层的远端咽器的基因图谱表明，TBX1促进细胞增殖并限制过早分化。我们使用MEF2C-AHF-CRE驱动器在aSHF中灭活了TBX1，发现所有胚胎在出生时都死于PTA。基因图谱显示了与TBX1缺失突变小鼠胚胎相同的一组基因。与Tbx1不同，aSHF中规范的Wnt信号促进细胞分化。为了测试TBX1和典型的Wnt信号之间是否有相反的作用，我们在aSHF中灭活了TBX1的两个等位基因和一个？-catenin等位基因，发现心脏缺陷得到了显著的挽救。在此基础上，我们假设在tbx1和aSHF中典型的Wnt/？-catenin信号之间存在负反馈机制。为了测试这一点，我们将定义TBX1之间的上位关系 和？-catenin，评估细胞增殖与分化的关系，以及进行CHIP-SEQ以确定直接转录的靶基因。有趣的是，一些表达增加的基因，如WNT2和GATA6，是后部SHF(PSHF)的特异性基因，是形成心脏流入道所必需的。由于微阵列不能区分基因表达和细胞群体的变化，我们假设在aSHF中TBX1的缺失，除了具有组织自主功能外，还可能导致周围pSHF细胞的扩张，也影响心脏发育。这一假说将通过pSHF谱系追踪和带有Wnt2和GATA6的TBX1的遗传研究来验证。22q11DS患者表现出不同的心血管异常表型。该程序中描述的一些基因可能会作为遗传修饰物。此外，由于通过改变TBX1突变小鼠的Wnt信号来挽救心脏缺陷，对这一过程的更好的机制理解可能在未来转化为22q11DS患者的新药物疗法。

 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.