Neural Crest Modulates FGF Signaling in the Pharynx

描述（由申请人提供）：圆锥锥体畸形是严重的先天性心脏缺陷，需要在儿童早期进行手术。出生时患有这些缺陷的婴儿发病率和死亡率都很高。胚胎流出道（一个被定义为流出血管的区域，包括与心肌的连接处，称为动脉极）的异常发育会产生这些缺陷。在最近对鸡胚胎神经嵴消融模型的研究中，我们发现在咽腹间质中有一个次级心野（SHF），它为发育中的心脏的动脉极提供心肌和平滑肌细胞。神经嵴消融后，来自SHF的心肌未添加到生长的动脉极上，这导致圆锥锥体畸形，如覆盖主动脉和双出口右心室。我们的初步研究结果表明，神经嵴消融后，心肌外流道的衰竭和随后的不对准缺陷是由于尾咽FGF信号的升高引起的：1)FGF靶基因在神经嵴消融后升高；2) FGF8最活跃的异构体Fgf8b升高；3) FGF报告细胞系在咽部腹侧表达FGF8b信号；4)阻断FGF8信号可以恢复神经嵴消融胚胎SHF中心肌的增加和流出道的正常排列。这些初步数据支持了我们的总体假设，即动脉极的正常发育取决于心脏神经嵴细胞对咽部FGF8b信号的调节。我们将检验具体假设：FGF8b升高通过影响二次心野心肌成分的增殖、迁移和/或分化导致异常动脉极发育（目的1）；神经嵴细胞通常通过吞噬FGF8蛋白和/或通过减少Fgf8b亚型的转录来抑制尾咽部的FGF8信号传导（目的2）。在目的1中，我们将把外植的SHF暴露于不同浓度的FGF8b中，并确定其对phospho-ERK、增殖、迁移、细胞死亡和分化的影响。我们将把表达FGF8b的质粒电穿孔到蛋胚的鸡咽内胚层，以将次级心脏场的发育事件与流出线排列联系起来。在目的2中，我们将确定体外FGF8b内吞作用的动力学，然后在体内阻断内吞作用，以确定其在流出线排列中的作用。最后，我们将确定神经嵴与内胚层和外胚层在控制FGF8b异构体表达中的关系。综上所述，这些研究将促进对心脏发育中神经嵴功能的机制理解，并将提供对导致圆锥锥体畸形的因素的见解。

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.