Cellular and Genetic Determinants of Pharyngeal Arch Artery Specification

描述(申请人提供)：涉及心脏出口的大动脉(例如“大血管”)的畸形是常见的先天性疾病。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两侧对称动脉产生，这些动脉经历了广泛的重塑，产生了出生时存在的复杂模式。虽然咽弓动脉(PaaS)的重塑方面已经得到了广泛的研究，但其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是不相容的，而轻微的大血管缺陷会导致先天性心血管畸形(CCM)，我们的长期目标是阐明PAA内皮的细胞来源，并识别介导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对PAA发育的遗传解剖。通过对一个新的Tg(NKX2.5：ZsHuang)斑马鱼报告系的检测，我意外地在PAA内皮细胞中发现了Zs黄色荧光。这一观察结果是没有预料到的，因为在这一人群中没有观察到NKX2.5转录本。基于这些数据，我推测PAA内皮细胞起源于较早的NKX2.5+细胞来源，在该细胞来源中一直存在Zs黄色荧光。尽管完全没有被探索过，但这一假说得到了传统的小鼠NKX2.5 cre/loxP谱系追踪的支持。因此，NKX2.5很可能在大血管的建立中起着保守的作用，但到目前为止还没有被认识到，这一作用值得进一步研究。我的初步数据还表明，NKX2.5和一个必要的TGFbeta途径组件，潜在的TGFbeta结合蛋白3(Ltbp3)，是PAA发育所必需的，但对于诱导剩余的血管形成是必不可少的。基于令人信服的初步数据，我建议检验这一假设，即来自第二心区(SHF)的ltbp3介导的TGFbeta信号促进表达NKX2.5的PAA前体细胞的内皮分化。在开发了用于照亮NKX2.5+前体及其衍生物的新试剂后，我有独特的机会直接测试这一想法。由于PAA缺陷会导致CCM或胚胎死亡，建议的研究对于开发改进的预防和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 congenital disorders. In most circumstances, the genetic basis for these abnormalities has not been identified. During embryogenesis, the great vessels arise from six pairs of bilaterally symmetric 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 congenital cardiovascular malformations (CCMs), our long-term goal is to elucidate the cellular source of PAA endothelium and to 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) zebrafish reporter line, I unexpectedly discovered ZsYellow fluorescence in PAA endothelium. This observation was not anticipated, as nkx2.5 transcripts are not observed in this population. Based on these data, I 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. My preliminary data also demonstrate that nkx2.5 and a requisite TGFbeta pathway component, Latent TGFbeta Binding Protein 3 (ltbp3), are required for PAA development, but dispensable for induction of the remaining vasculature. Based on compelling preliminary data, I propose to test the hypothesis that ltbp3-mediated TGFbeta 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, I have the unique opportunity to directly test this idea. As PAA defects cause CCMs or embryonic lethality, the proposed studies are significant for informing the development of improved preventative and therapeutic approaches.