Developmental Genetics of the Pharyngeal Apparatus

描述（由申请人提供）：咽器在颅面区、胸腺、甲状旁腺和心流出道（OFT）的发育涉及复杂的基因相互作用，使其对遗传和环境损伤高度敏感。Velo-cardio-facial综合征/DiGeorge综合征与半合子22q11缺失有关，其特征是咽器衍生物的缺陷。Tbx1是一种T-box转录因子，也是人类VCFS/DGS的基因，在小鼠体内失活时，与相同结构的缺陷有关。该综合征患者最严重的缺陷是OFT缺陷。Tbx1在咽部的多个组织中表达，包括OFT发育所需的第二心野（SHF）间质。小鼠作为鉴定OFT发育基因的优秀模式生物，因为其形态发生的许多方面与人类共享。为了确定Tbx1在SHF中调控的遗传途径，我们对Tbx1-/-型和野生型小鼠胚胎的尾咽区进行了基因表达谱分析。在Tbx1-/-突变体中，SHF的关键标志物Isl1被下调，而第一心野（FHF）后规范所需的基因，如Raldh2、Gata4和Tbx5，以及心房特异性肌肉收缩基因的一个子集被异位表达。相反的表达变化伴随shf衍生的心脏缺陷发生在TBX1功能获得突变体中。基于这些数据，我们假设Tbx1正调控SHF细胞增殖并限制尾咽间质的过早分化。在微阵列中发现了几个对维甲酸通路重要的基因、转录因子和新基因。我们建议描述生物学上最有趣的基因，以便在SHF中建立Tbx1下游的遗传途径，使用无效的、条件丧失和获得功能的突变小鼠胚胎。为了鉴定Tbx1的直接下游靶基因，报告系统将在哺乳动物细胞中使用。神经嵴细胞（NCC）迁移和OFT分离需要组织相互作用，这在VCFS/DGS和小鼠突变体中是缺陷的。最近，另一个在22q11.2上缺失的基因Crkl被证明可以介导Tbx1下游的Fgf8信号。为了在SHF和ncc中验证这一假设，我们将分别分析Crkl泛中胚层失活和神经嵴失活的胚胎中Fgf应答基因和SHF特征基因的表达变化。利用微阵列研究和条件突变体鉴定的基因，我们将能够解剖Tbx1在SHF和ncc中导致咽部器官OFT发育的遗传途径。旁白：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。通过在模式生物中采用遗传学方法，我们可以获得在人类中不可能获得的见解。

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.