Cellular and Genetic Determinants of Great Vessel Morphogenesis

描述（由申请人提供）：涉及心脏外大动脉的畸形(例如：“大血管”)是先天性心血管缺陷（CCDs）的常见原因。在大多数情况下，这些异常的遗传基础尚未确定。在胚胎发育过程中，大血管由嵌入咽弓内的六对双侧对称动脉形成，这些动脉经过广泛的重塑，形成了出生时的复杂形态。尽管重塑方面已被广泛研究，但咽弓动脉（PAAs）的发育起源和调节其规范的遗传程序仍然难以捉摸。由于严重的大血管缺陷与生命不相容，而轻微的缺陷会导致冠心病，我们的长期目标是阐明PAA内皮的细胞来源，并确定介导PAA建立的遗传途径，以潜在地识别新的人类疾病基因。斑马鱼模型生物允许无与伦比的实时可视化和PAA发育的遗传解剖。通过检测一种新的Tg（nkx2.5:: zyellow）报告系，我们在PAA内皮中发现了zyellow荧光。这一观察结果令人惊讶，因为在该人群中未检测到nkx2.5转录本。基于这些数据，我们假设PAA内皮来源于早期的nkx2.5+细胞来源，其中z黄荧光持续存在。尽管这一假设完全未被探索，但传统的小鼠nkx2.5 cre/loxP谱系追踪支持了这一假设。因此，nkx2.5很可能在大血管形成中起着保守的作用，但迄今为止尚未被认识到，这需要进一步的研究。我们的初步数据还表明，nkx2.5和一种必需的TGF2途径成分，潜伏TGF2结合蛋白3 (LTBP3)，是PAA发展所必需的，但对于诱导剩余的血管系统是必不可少的。基于令人关注的初步数据，包括遗传谱系追踪、功能丧失和基因表达研究，我建议验证ltbp3介导的第二心场（SHF） TGF2信号促进表达nkx2.5的PAA祖细胞内皮分化的假设。我们已经开发出新的试剂来照亮nkx2.5+祖细胞及其衍生物，我们的实验室有独特的机会直接测试这一假设。由于PAA缺陷会导致CCDs或胚胎致死，因此所提出的研究对改进治疗方法具有重要意义。

DESCRIPTION (provided by applicant): Malformations involving the large arteries that exit the heart (e.g. "the great vessels") are common causes of congenital cardiovascular defects (CCDs). In most circumstances, the genetic basis for these abnormalities has not been identified. During embryogenesis, the great vessels arise from six pairs of bilaterally symmetrical arteries embedded within the pharyngeal arches that undergo extensive remodeling to produce the complex pattern present at birth. Although the remodeling aspects have been extensively studied, the developmental origin of pharyngeal arch arteries (PAAs) and the genetic programs regulating their specification remain elusive. As severe great vessel defects are incompatible with life and milder deficiencies cause CCDs, our long-term goal is to elucidate the cellular source of PAA endothelium and identify genetic pathways mediating PAA establishment to potentially identify novel human disease genes. The zebrafish model organism allows for unparalleled real-time visualization and genetic dissection of PAA development. Through examination of a novel Tg(nkx2.5::ZsYellow) reporter line, we discovered ZsYellow fluorescence in PAA endothelium. This observation was surprising as nkx2.5 transcripts are not detected in this population. Based on these data, we postulate that PAA endothelium derives from an earlier nkx2.5+ cellular source in which ZsYellow fluorescence has persisted. Although completely unexplored, this hypothesis is supported by traditional nkx2.5 cre/loxP lineage tracing in mice. Thus, it is likely that nkx2.5 plays a conserved, yet heretofore unrecognized, role in great vessel establishment that warrants further investigation. Our preliminary data also demonstrate that nkx2.5 and a requisite TGF2 pathway component, Latent TGF2 Binding Protein 3 (LTBP3), are required for PAA development, but dispensable for induction of the remaining vasculature. Based on compelling preliminary data that include genetic lineage tracing, loss-of-function, and gene expression studies, I propose to test the hypothesis that LTBP3-mediated TGF2 signaling from the second heart field (SHF) promotes endothelial differentiation of nkx2.5-expressing PAA progenitors. Having developed new reagents for illuminating nkx2.5+ progenitors and their derivatives, our lab has the unique opportunity to directly test this hypothesis. As PAA defects cause CCDs or embryonic lethality, the proposed studies are significant for improved therapeutic approaches. PUBLIC HEALTH RELEVANCE: The great arteries are large blood vessels that carry blood away from the heart. Severe great artery defects are incompatible with life and milder deficiencies cause congenital cardiovascular malformations (CCMs). The long-term goal of our research is to elucidate the cellular source of the great arteries and identify new genes required for their formation to potentially identify novel human disease loci.