Developmental Genetics of the Pharyngeal Apparatus

描述(申请人提供)：咽部器官发育到头面部、胸腺、甲状旁腺和心脏流出道(OFT)涉及复杂的基因相互作用，使它们对遗传和环境侮辱高度敏感。肌-心-面综合征/DiGeorge综合征与22q11基因缺失有关，其特征是咽部器官的衍生性缺陷。Tbx1是一种T-box转录因子，也是人类VCFS/DGS的基因，在小鼠中失活时，与相同结构中的缺陷有关。该综合征患者最严重的缺陷是OFT缺陷。TBX1在咽器官的多个组织中表达，包括OFT发育所需的第二心区(SHF)间充质。小鼠是识别OFT发育基因的很好的模式生物，因为它的形态发生的许多方面与人类相同。为了确定TBX1在SHF中受调控的遗传途径，我们对TBX1-/-和野生型小鼠胚胎的尾咽区域进行了基因表达谱分析。SHF的关键标志Isl1在Tbx1-/-突变体中下调，而第一心野(FHF)的后部规范所需的基因，如Raldh2、Gata4和Tbx5，以及心房特异性肌肉收缩基因的子集，在异位表达。在功能获得突变体中，伴随着SHF所致心脏缺陷的相反的表达变化发生在TBX1中。基于这些数据，我们假设TBX1正向调节SHF细胞的增殖并限制其在咽尾间充质中的过早分化。在微阵列中发现了几个对维甲酸途径重要的基因、转录因子和新基因。我们建议用缺失的、有条件的功能丧失和功能获得的突变小鼠胚胎来鉴定最有生物学意义的基因，以便在SHF中建立TBX1下游的遗传途径。为了确定TBX1的直接下游靶基因，将在哺乳动物细胞中使用报告系统。在VCFS/DGS和小鼠突变体中，组织间的相互作用是神经脊细胞(NCC)迁移和OFT分离所必需的。最近，22q11.2缺失的另一个基因crk1被发现在Tbx1下游介导Fgf8信号转导。为了在SHF和NCC中验证这一假说，将分析分别具有泛中胚层和神经脊CRK1失活的胚胎，以确定成纤维细胞生长因子反应基因的变化表达和SHF基因的特征。利用微阵列研究和条件突变体鉴定的基因，我们将能够在SHF和NCC中剖析TBX1的遗传途径，以便从咽部器官发育OFT。叙述：这项研究与公共卫生的相关性在于，这项计划将使我们能够找到导致出生缺陷的基因。通过在模型生物中采用遗传学方法，我们可以获得在人类身上无法获得的见解。

DESCRIPTION (provided by applicant): The development of the pharyngeal apparatus into the craniofacial region, thymus, parathyroid and cardiac outflow tract (OFT) involves complex gene interaction, making them highly sensitive to genetic and environmental insults. Velo-cardio-facial syndrome/DiGeorge syndrome is associated with hemizygous 22q11 deletions and characterized by defects in the derivatives of the pharyngeal apparatus. Tbx1, a T-box transcription factor, and the gene for VCFS/DGS in humans, is associated with defects in the same structures when inactivated in the mouse. The most serious defect in patients with the syndrome is OFT defects. Tbx1 is expressed in the multiple tissues in the pharyngeal apparatus including the second heart field (SHF) mesenchyme required for OFT development. The mouse serves as an excellent model organism to identify genes for OFT development because many aspects of its morphogenesis are shared with humans. To identify Tbx1 regulated genetic pathways in the SHF, we performed gene expression profiling of the caudal pharyngeal region in Tbx1-/- and wild type mouse embryos. Isl1, a key marker for the SHF, among selected others, were downregulated in Tbx1-/- mutants, while genes required for posterior specification of the first heart field (FHF), such as Raldh2, Gata4, and Tbx5, as well as a subset of atrial-specific muscle contractile genes were ectopically expressed. Opposite expression changes concomitant with SHF-derived cardiac defects occurred in TBX1 gain-of- function mutants. Based upon these data, we hypothesize that Tbx1 positively regulates SHF cell proliferation and restricts premature differentiation in the caudal pharyngeal mesenchyme. Several genes important for the retinoic acid pathway, transcription factors and novel genes were found in the microarrays. We propose to characterize the most biologically interesting genes in order to build the genetic pathway downstream of Tbx1 in the SHF using null, conditional loss- and gain-of-function mutant mouse embryos. To identify direct downstream target genes of Tbx1, reporter systems will be used in mammalian cells. Tissue interactions are required for neural crest cell (NCC) migration and OFT septation, defective in VCFS/DGS and mouse mutants. Recently, Crkl, another gene deleted on 22q11.2, was shown to mediate Fgf8 signaling downstream of Tbx1. To test this hypothesis in the SHF and in NCCs, embryos with pan-mesodermal and neural crest inactivation of Crkl, respectively, will be analyzed for altered expression of Fgf responsive genes and SHF genes characterized. Using genes identified by microarray studies and conditional mutants we will be able to dissect the genetic pathway of Tbx1 in the SHF and NCCs for OFT development from the pharyngeal apparatus. Narrative: The relevance of this research to public health is that this program will enable us to find genes that cause birth defects. By taking genetics approaches in model organisms, we can obtain insights that would otherwise not be possible in humans.