Neural Crest Modulates FGF Signaling in the Pharynx

神经嵴调节咽部 FGF 信号传导

• 批准号: 7416729
• 负责人: Margaret Loewy Kirby
• 金额: $ 42.41万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7416729
• 关键词: Ablation; Affect; Antibodies; Aorta; Cardiac; Cell Death; Cell Line; Cells; Chick Embryo; Child; Childhood; Chimera organism; Coculture Techniques; Code; Congenital Heart Defects; Data; Defect; Depressed mood; Development; Double Outlet Right Ventricle; Ectoderm; Embryo; Embryonic Development; Endocytosis; Endoderm; Event; FGF8 gene; Failure; Fibroblast Growth Factor; Fibroblast Growth Factor 8; Floor; Gene Expression; Gene Targeting; Genetic Transcription; Heart; In Situ Hybridization; In Vitro; Infant; Investigation; Label; Laboratories; Lead; Measures; Mediating; Mesenchyme; Modeling; Morbidity - disease rate; Myocardial; Myocardium; Neural Crest; Neural Crest Cell; Operative Surgical Procedures; Pharyngeal structure; Plasmids; Polymerase Chain Reaction; Positioning Attribute; Production; Protein Isoforms; Protein Overexpression; Proteins; Quail; Regulation; Reporter; Role; Signal Transduction; Smooth Muscle Myocytes; Source; Staging; Testing; Time; Tissues; Vesicle; base; cardiogenesis; in vivo; insight; laser capture microdissection; malformation; migration; mortality; novel; research study; response

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.

描述（由申请人提供）：圆锥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。患有这些缺陷的婴儿患有显着的发病率和死亡率。流出道（定义为流出血管的区域，包括与心肌的连接处，称为动脉极）的异常胚胎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，我们在腹侧咽间充质中发现了次级心脏场（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 的心肌不会添加到生长的动脉极，这会导致圆锥动脉对线缺陷，例如主动脉和双出口右心室。我们的初步结果表明，心肌添加到流出道的失败和随后的排列缺陷是由于神经嵴消融后尾咽中 FGF 信号升高所致，基于本实验室进行的研究的四项证据：1）神经嵴消融后 FGF 靶基因升高； 2) FGF8最活跃的亚型Fgf8b升高； 3) FGF 报告细胞系记录腹侧咽部 FGF8b 信号传导升高； 4)阻断FGF8信号传导可恢复来自SHF的心肌的添加以及神经嵴去除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 FGF8b 信号传导的调节。我们将测试具体假设：FGF8b 升高通过影响次级心区心肌成分的增殖、迁移和/或分化而导致动脉极发育异常（目标 1）；神经嵴细胞通常通过 FGF 蛋白的内吞作用和/或减少 Fgf8b 同工型的转录来抑制尾咽中的 FGF8 信号传导（目标 2）。在目标 1 中，我们将外植的 SHF 暴露于不同浓度的 FGF8b，并确定其对磷酸 ERK、增殖、迁移、细胞死亡和分化的影响。我们将表达 FGF8b 的质粒电穿孔到卵内鸡胚的咽内胚层中，以将次级心脏区域的发育事件与流出对齐相关联。在目标 2 中，我们将在体外确定 FGF8b 内吞作用的动态，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 FGF8b 亚型表达中的关系。这些研究将共同​​推进对心脏发育中神经嵴功能的机制理解，并将深入了解导致圆锥干畸形的因素。