Modeling conotruncal malformations in zebrafish embryos

描述（由申请方提供）：心脏动脉极处的心肌-平滑肌交界处发育异常导致归类为圆锥动脉干畸形的先天性缺陷，如右心室双出口和法洛四联症。我们实验室最近的工作表明，在分隔之前构建动脉极的基本元素在发育过程中高度保守。在具有分开的肺循环和体循环的心脏的发育中，心肌和平滑肌早在该区域被分隔之前就被添加到动脉极。该实验室的进一步研究明确表明，动脉极排列不良，即圆锥动脉干排列不良缺陷是由于分隔前动脉极发育异常所致。斑马鱼是一种理想的生物体，在其中研究动脉极的发展，没有分隔的混淆事件。此外，它作为遗传模型的优势使其成为研究圆锥动脉干畸形潜在基因的优秀脊椎动物。Tbx 1是与DiGeorge表型相关的基因，其中圆锥动脉干畸形是主要组成部分。斑马鱼的货车高突变体在tbxl基因上有一个无效突变，但与此突变相关的心脏缺陷从未被分析过动脉极的发育。此外，据报道FGF 8是TBX 1的下游效应物，并且我们在鸡中有证据表明流出排列对FGF 8信号传导非常敏感。因此，该提议的假设是Tbx 1通过FGF 8和其他尚未鉴定的基因调节前体对形成斑马鱼心脏动脉极的心肌和平滑肌细胞的贡献。为了了解圆锥动脉干畸形的基因-表型关系，我们需要有关于动脉极祖细胞起源和发育历史的详细信息。因此，目标1将使用细胞示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。所提出的动脉极祖细胞的细胞示踪和离散消融类型在任何其他动物模型中都是不可能的。目的2将使用斑马鱼突变体和反义吗啉技术来确定这些祖细胞在形成动脉极中对tbxl和fgf 8表达被破坏的敏感性。这些实验将提供有关在类似于DiGeorge患者的遗传背景中动脉极祖细胞的正常和异常发育的细胞和分子信息。由于DiGeorge表型的可变性，Aim 3被设计为使用候选基因方法和双杂合子的高通量筛选来鉴定tbxl功能的未知遗传修饰剂。由于动脉极是圆锥动脉干畸形的部位，因此对其发育和影响其发育的基因的详细了解将最终允许更好地预测具有此类畸形后代的风险个体。

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.