Cardiac Transcription Factor Nkx2.7 is a Novel Regulator of Craniofacial Development

项目摘要 与DiGeorge综合征相关的临床表型，最常见的微缺失状况 （22q11.2）在人类，说明心血管和颅面形态发生之间的发展联系。 最近在小鼠和斑马鱼中进行的命运作图研究进一步支持了这一观点， 多能祖细胞在心咽领域（CPF），产生心脏，鳃肌， 和咽弓动脉，通过咽弓（PA）介导。NKX 2 -5和NKX 2 -6是 同源框转录因子在先天性心脏病中经常发生突变。在斑马鱼中， 显示Nkx2.5和Nkx2.7在心脏发育中起冗余作用。使用一种新的功能丧失 nkx2.7等位基因是纯合致死的，我们首次证明nkx2.7-/-胚胎不能形成 进食所必需的颅面肌肉和软骨。这种发育缺陷是由早期 破坏了肺动脉的基因调控我们推测Nkx2.7是一个重要的转录因子， 在颅面发育期间通过咽弓介导。我们将研究我们的模型， 揭示nkx2.7+ CPF祖细胞的组织特异性作用，并通过评估转录靶点 nkx2.7的表达，并评估相关的人类疾病表型。在目标1中，我们将描述 nkx2.7-/-胚胎中PA中组织特异性细胞类型的发育轨迹，受益于 创新的高速体积成像平台SCAPE显微镜。此外，我们将确定细胞- 通过使用组织特异性的细胞移植实验的nkx2.7+祖细胞的自主功能 转基因在目标2中，我们将研究Nkx2.7介导的时间和分子机制。使用 我们实验室产生了一种新的热诱导转基因，我们将过表达nkx2.7，以分析其在生长过程中的需求。 各种发展窗口。我们将把这些知识应用于nkx2.7-/-胚胎的单细胞RNA测序 以生成所有咽部组织中的基因靶点的稳健列表。我们还将比较这些假定的效应子 先天性心脏病和颅面缺陷患者的基因组数据。评估直接或 为了间接结合这些靶标，我们将使用染色质免疫沉淀和DNA测序（ChIP-seq）。 总之，该提案将提供对Nkx2.7在心咽癌中的作用的全面理解。 发育并将阐明心脏和颅面形态发生之间的关系。而且这些 研究有可能改善头部肌肉组织缺陷患者的干细胞治疗策略 和软骨。

PROJECT SUMMARY The clinical phenotypes associated with DiGeorge Syndrome, the most common microdeletion condition (22q11.2) in humans, illustrates the developmental link between cardiovascular and craniofacial morphogenesis. Recent fate mapping studies in mice and zebrafish further support this notion given the identification of a multipotent progenitor in the cardiopharyngeal field (CPF) that gives rise to the heart, branchiomeric muscles, and pharyngeal arch arteries, mediated through the pharyngeal arches (PAs). NKX2-5 and NKX2-6 are homeobox transcription factors frequently mutated in congenital heart defects. In zebrafish, we have previously shown that Nkx2.5 and Nkx2.7 play redundant roles in cardiac development. Using a novel loss-of-function nkx2.7 allele that is homozygous lethal, we demonstrate for the first time that nkx2.7-/- embryos fail to form craniofacial muscles and cartilage necessary for feeding. This developmental deficiency results from early disrupted genetic regulation in the PAs. We hypothesize that Nkx2.7 functions as an essential transcription factor during craniofacial development mediated through the pharyngeal arches. We will investigate our model by uncovering the tissue-specific roles of the nkx2.7+ CPF progenitors and by evaluating the transcriptional targets of nkx2.7 and assessing for associated human disease phenotypes. In Aim 1, we will characterize the developmental trajectories of the tissue-specific cell types in the PAs in nkx2.7-/- embryos, benefitting from an innovative, high speed, volumetric imaging platform, SCAPE microscopy. Moreover, we will determine cell- autonomous functions of nkx2.7+ progenitors through cell transplantation experiments employing tissue-specific transgenes. In Aim 2, we will investigate the temporal and molecular mechanisms mediated by Nkx2.7. Using a novel heat inducible transgene generated in our lab, we will overexpress nkx2.7 to dissect its requirement during various developmental windows. We will apply this knowledge to single cell RNA-sequencing in nkx2.7-/- embryos to generate a robust list of genetic targets in all pharyngeal tissues. We will also compare those putative effectors with genomic data from patients with congenital heart disease and craniofacial defects. To evaluate for direct or indirect binding of those targets, we will use chromatin immunoprecipitation and DNA sequencing (ChIP-seq). Altogether, this proposal will provide a comprehensive understanding of the role of Nkx2.7 in cardiopharyngeal development and will clarify the relationship between cardiac and craniofacial morphogenesis. Moreover, these studies have potential to ameliorate stem cell therapy strategies in patients with defects of the head musculature and cartilage.