Neural Crest Modulates FGF Signaling in the Pharynx

描述(由申请人提供)：圆锥干畸形是一种严重的先天性心脏缺陷，需要在童年早期进行手术。出生时有这些缺陷的婴儿会有很高的发病率和死亡率。流出道(一个定义为流出血管，包括与心肌交界处的区域，称为动脉极)的异常胚胎发育会产生这些缺陷。神经脊消融术可导致多种圆锥干畸形：直接影响包括没有流出道分离，间接影响是流出道排列不齐。在最近对鸡胚胎神经脊消融模型的研究中，我们在腹侧咽间充质中发现了一个次级心电场(SHF)，它提供了成为心脏和平滑肌肌细胞的细胞，对动脉极的正常形成至关重要。在神经脊消融模型中，来自SHF的心肌没有正确地添加到生长的动脉极上，导致圆锥干不对准，并导致流出道分离失败。这些排列不齐的缺陷是两种圆锥干畸形的组成部分，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脊消融后成纤维细胞生长因子信号增强所致，这是基于本实验室进行的四项研究得出的证据：1)神经脊消融后成纤维细胞生长因子靶基因上调；2)FGF8最活跃的亚型Fgf8b升高；3)成纤维细胞生长因子报告细胞系在咽腹记录到高水平的FGF8b信号；以及4)FGF8b抗体恢复正常的环状结构，并挽救神经脊消融胚胎流出道的排列。这些初步数据支持我们的总体假设，即动脉极的正常发育依赖于心脏神经脊细胞对咽部FGF8信号的调节。我们将检验特定的假设：FGF8b升高通过影响次级心场的心肌成分的增殖、迁移和/或分化而导致动脉极发育异常(目标1)；神经脊细胞通常通过吞噬成纤维细胞生长因子蛋白和/或通过减少Fgf8b亚型的转录而抑制尾部咽部的FGF8信号(目标2)。在目标1中，我们将解释的SHF暴露于不同浓度的FGF8b，并确定其对增殖、迁移、细胞死亡和分化的影响。我们将FGF8b表达质粒电穿孔到鸡胚胎的咽部内胚层，以将次级心区的发育事件与流出序列相关联。在目标2中，我们将(截断)

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)