Neural Crest Modulates FGF Signaling in the Pharynx

描述（由申请方提供）：圆锥动脉干畸形是严重的先天性心脏缺陷，需要在儿童早期进行手术。出生时有这些缺陷的婴儿具有显著的发病率和死亡率。流出道（一个被定义为流出血管的区域，包括与心肌的交界处，称为动脉极）的异常胚胎发育产生这些缺陷。神经嵴消融导致各种圆锥动脉干畸形：直接影响包括流出道分隔缺失，间接影响是流出道排列不良。在最近的鸡胚神经嵴消融模型的研究中，我们已经发现了一个次级心脏领域（SHF）的腹咽间充质，提供细胞，成为心脏和平滑肌细胞的动脉极的正常形成必不可少的。在神经嵴消融模型中，来自SHF的心肌没有适当地添加到生长的动脉极，导致除了流出道分隔失败之外的圆锥干排列不良。对线不良缺陷是两种圆锥动脉干畸形的组成部分，称为右心室双出口和法洛四联症。我们的初步结果表明，这些畸形是由于神经嵴消融后FGF信号升高，这是基于本实验室进行的四项研究的证据：1）神经嵴消融后FGF靶基因升高; 2）FGF 8最活跃的同种型Fgf 8b升高; 3）FGF的报告细胞系记录腹咽中升高的FGF 8b信号;和4）FGF 8b抗体在神经嵴消融的胚胎中恢复正常的成环并挽救流出道的排列。这些初步数据支持我们的总体假设，即动脉极的正常发育取决于心脏神经嵴细胞对咽部FGF 8信号传导的调节。我们将测试特定的假设：升高的FGF 8b通过影响次级心脏区域的心肌成分的增殖、迁移和/或分化而导致异常的动脉极发育（目的1）;神经嵴细胞通常通过FGF蛋白的内吞作用和/或通过减少FGF 8b亚型的转录而抑制尾咽中的FGF 8信号传导（目的2）。在目标1中，我们将暴露于不同浓度的FGF 8b，并确定其对增殖，迁移，细胞死亡和分化的影响。我们将FGF 8b表达质粒电穿孔到卵内鸡胚的咽内胚层中，以将次级心脏领域的发育事件与流出对齐相关联。在目标2中，我们将（截短）

DESCRIPTION (provided by applicant): Conotruncal malformations are severe congenital cardiac defects that require surgery early in childhood. Infants born with these defects suffer from significant morbidity and mortality. Abnormal embryonic development of the outflow tract (a region defined as the outflow vessels including the junction with the myocardium, called the arterial pole) produces these defects. Neural crest ablation causes a wide variety of conotruncal malformations: direct effects include absence of outflow septation, and indirect effects are malalignment of the outflow tract. In recent studies of the neural crest ablation model in chick embryos, we have discovered a secondary heart field (SHF) in the ventral pharyngeal mesenchyme that provides cells that become cardiac and smooth muscle myocytes essential to the normal formation of the arterial pole. In the neural crest-ablation model, myocardium from SHF is not added properly to the growing arterial pole resulting in malalignment of the conotruncus in addition to failure of outflow septation. The malalignment defects are a component of two conotruncal malformations called double outlet right ventricle and tetralogy of Fallot. Our preliminary results indicate that these malformations are due to elevated FGF signaling after neural crest ablation based on four pieces of evidence from investigations performed in this laboratory: 1) FGF target genes are elevated after neural crest ablation; 2) Fgf8b, the most active isoform of FGF8 is elevated; 3) a reporter cell line for FGF registers elevated FGF8b signaling in the ventral pharynx; and 4) FGF8b antibody restores normal looping and rescues alignment of the outflow tract in neural crest-ablated embryos. These preliminary data support our overall hypothesis that normal development of the arterial pole depends on the regulation of FGF8 signaling in the pharynx by cardiac neural crest cells. We will test the specific hypotheses: that elevated FGF8b leads to abnormal arterial pole development by affecting proliferation, migration and/or differentiation of the myocardial component of the secondary heart field (aim 1); neural crest cells normally depress FGF8 signaling in the caudal pharynx by endocytosis of the FGF protein and/or by decreasing the transcription of the Fgf8b isoform (aim 2). In aim 1, we will expose explanted SHF to various concentrations of FGF8b and determine its effect on proliferation, migration, cell death and differentiation. We will electroporate an FGF8b expressing plasmid into the pharyngeal endoderm of chick embryos in ovo to correlate developmental events in the secondary heart field with outflow alignment. In aim 2, we will (truncated)