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。在最近的鸡胚神经嵴消融模型的研究中，我们发现了一个次级心脏领域（SHF）的腹咽间充质，提供心肌和平滑肌细胞的动脉极的发展心脏。在神经嵴消融后，SHF的心肌没有添加到生长的动脉极，这导致圆锥动脉干对线不良缺陷，如覆盖主动脉和右心室双出口。我们的初步结果表明，心肌附加到流出道的失败和随后的排列不良缺陷是由于神经嵴消融后尾咽中FGF信号的升高，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最活跃的亚型Fgf 8b升高; 3）FGF的报告细胞系记录腹咽中升高的FGF 8 b信号传导;和4）阻断FGF 8信号传导恢复来自SHF的心肌的添加和神经嵴消融的胚胎中流出道的正常排列。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 8b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，我们将使经处理的SHF暴露于不同浓度的FGF 8b，并确定其对磷酸化ERK、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，我们将确定体外FGF 8b内吞作用的动力学，然后在体内阻断内吞作用，以确定其在流出排列中的作用。最后，我们将确定神经嵴与内胚层和外胚层在控制FGF 8b亚型表达中的关系。总之，这些研究将促进对心脏发育中神经嵴功能的机械理解，并将提供对导致圆锥动脉干畸形的因素的深入了解。

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. 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 myocardial and smooth muscle cells to the arterial pole of the developing heart. After neural crest ablation, myocardium from the SHF is not added to the growing arterial pole and this results in conotruncal malalignment defects such as overriding aorta and double outlet right ventricle. Our preliminary results indicate that the failure of myocardial addition to the outflow tract and subsequent malalignment defects are due to elevated FGF signaling in the caudal pharynx 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) blocking FGF8 signaling restores addition of the myocardium from the SHF and normal 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 FGF8b 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 phospho-ERK, 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 determine the dynamics of FGF8b endocytosis in vitro followed by blocking endocytosis in vivo to determine its role in outflow alignment. Finally we will determine the relationship of neural crest with endoderm and ectoderm in controling FGF8b isoform expression. Together these studies will advance the mechanistic understanding of neural crest function in heart development and will provide insight into factors that cause conotruncal malformations.