Modeling conotruncal malformations in zebrafish embryos

描述（申请人提供）：心脏动脉极心肌-平滑肌连接处异常发育导致先天性缺陷，如双出口右心室和法洛四联症。我们实验室最近的工作表明，在分离之前构建动脉极的基本元素在发育过程中高度保守。在肺循环和体循环分离的心脏发育过程中，心肌和平滑肌早在动脉极区分离之前就加入了动脉极。本实验室进一步的研究明确表明，动脉极畸形，即圆锥锥体畸形畸形是由于动脉极发育异常引起的。斑马鱼是研究动脉极发育的理想生物，没有分离的混杂事件。此外，它作为遗传模型的力量使它成为研究潜在的圆锥锥体畸形基因的优秀脊椎动物。Tbx1是一种与DiGeorge表型相关的基因，其中圆锥锥体畸形是其主要组成部分。斑马鱼梵高突变体在tbxl中有一个零突变，但与该突变相关的心脏缺陷从未被分析过与动脉极发育有关。此外，据报道，FGF8是TBX1的下游效应者，我们在小鸡中有证据表明，流出序列对FGF8信号传导非常敏感。因此，该提议的假设是Tbx1通过FGF8和其他尚未确定的基因调节心肌和平滑肌细胞前体的贡献，这些细胞形成斑马鱼心脏的动脉极。为了了解圆锥锥体畸形的基因-表型关系，我们需要对动脉极祖细胞的起源和发育历史有详细的了解。因此，Aim 1将使用细胞追踪和消融来识别和确定形成斑马鱼动脉极的心肌和平滑肌祖细胞的贡献。提出的动脉极祖细胞的细胞追踪和离散消融的类型在任何其他动物模型中都是不可能的。目的2将利用斑马鱼突变体和反义morpholino技术确定这些形成动脉极的祖细胞对tbxl和fgf8表达中断的敏感性。这些实验将提供关于动脉极祖细胞正常和异常发育的细胞和分子信息，其遗传背景与diggeorge患者相似。由于digegeorge表型的可变性，Aim 3被设计用于使用候选基因方法和双杂合子的高通量筛选来鉴定未知的tbxl功能遗传修饰因子。因为动脉极是圆锥锥体畸形的部位，对其发育和影响它的基因的详细了解，最终将有助于更好地预测个体的后代是否有这种畸形的风险。

DESCRIPTION (provided by applicant): Abnormal development of the myocardial-smooth muscle junction at the arterial pole of the heart leads to congenital defects classified as conotruncal malformations such as double outlet right ventricle and tetralogy of Fallot. Recent work from our lab shows that the basic elements that build the arterial pole prior to septation are highly conserved during development. In development of a heart with divided pulmonary and systemic circulations, the myocardium and smooth muscle are added to the arterial pole long before the region is septated. Further work from this lab has shown definitively that arterial pole malalignments, i.e. conotruncal malalignment defects are due to abnormal pre-septation arterial pole development. The zebrafish is an ideal organism in which to study arterial pole development without the confounding event of septation. In addition, its strength as a genetic model makes it an excellent vertebrate in which to study genes that potentially underlie conotruncal malformations. Tbx1 is a gene associated with the DiGeorge phenotype of which conotruncal malformations are a major component. The zebrafish van gogh mutant has a null mutation in tbxl, but the heart defect associated with this mutation has never been analyzed for arterial pole development. In addition, FGF8 is reported to be a downstream effecter of TBX1 and we have evidence in the chick that outflow alignment is very sensitive to FGF8 signaling. Thus, the hypothesis for this proposal is that Tbx1 through FGF8 and other as yet unidentified genes regulates the contribution of precursors to the myocardial and smooth muscle cells that form the arterial pole of the zebrafish heart. To understand the gene-phenotype relationship of conotruncal malformations we need to have detailed information about the origin and developmental history of the arterial pole progenitors. Therefore, Aim 1 will use cell tracing and ablations to identify and determine the contribution of the progenitors of the myocardium and smooth muscle that form the zebrafish arterial pole. The type of cell tracing and discrete ablations of the arterial pole progenitors that are proposed are not possible in any other animal model. Aim 2 will determine the sensitivity of these progenitors in forming the arterial pole to disrupted expression of tbxl and fgf8 using zebrafish mutants and antisense morpholino technology. These experiments will provide cellular and molecular information about normal and abnormal development of the arterial pole progenitors in a genetic background similar to that in DiGeorge patients. Because of the variability of the DiGeorge phenotype Aim 3 is designed to identify unknown genetic modifiers of tbxl function using a candidate gene approach and high throughput screening of double heterozygotes. Because the arterial pole is the site of conotruncal malformations, detailed knowledge of its development and the genes that influence it will ultimately allow better prediction of individuals at risk for having offspring with such malformations.