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

心脏转录因子 Nkx2.7 是颅面发育的新型调节因子

基本信息

批准号：
10156452
负责人：
Caitlin Kelly Ford
金额：
$ 4.68万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10156452
关键词：
22q11.2 Adolescence Alcian Blue Alleles Anterior Arteries Articulation Binding Branchial arch structure Candidate Disease Gene Cardiac Cardiac Myocytes Cardiac development Cardiovascular system Cartilage Cell Count Cell Transplantation Cells Cessation of life ChIP-seq Clinical Congenital Abnormality Congenital Heart Defects Craniofacial Abnormalities DNA sequencing Data Data Set Defect Development DiGeorge Syndrome Dissection DsRed Embryo Endoderm Etiology Fertilization Genes Genetic Genetic Transcription Genome Head Heart Heart Abnormalities Homeobox Homologous Gene Human Image Intervention Jaw Knowledge Laboratories Link Mandible Meckel&apos s cartilage Mediating Mesoderm Microscopy Modeling Molecular Molecular Target Morphogenesis Multipotent Stem Cells Mus Muscle Muscle Development Mutate Mutation Nature Neural Crest Nonsense Mutation Overlapping Genes Patients Pediatric Cardiac Genomics Consortium Phenotype Play Population Recording of previous events Regulation Research Role Site Speech Therapy Speed Stains Starvation Structure Survival Analysis Techniques Temporomandibular Joint Testing Therapeutic Intervention Time Tissues Transgenes Transgenic Organisms Tube Ventricular Work Zebrafish absorption cell type chromatin immunoprecipitation clinical phenotype congenital heart disorder craniofacial craniofacial development critical period de novo mutation disease phenotype exome sequencing experimental study feeding genome-wide genomic data homeodomain human disease imaging platform infancy innovation insight loss of function malformation microdeletion novel null mutation operation overexpression prevent progenitor remediation single-cell RNA sequencing stem cell therapy stem cells transcription factor transcriptome sequencing

项目摘要

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.

项目概要与最常见的微缺失病症迪乔治综合征相关的临床表型 (22q11.2) 在人类中，说明了心血管和颅面形态发生之间的发育联系。最近对小鼠和斑马鱼的命运图谱研究进一步支持了这一观点，因为确定了心咽区 (CPF) 中的多能祖细胞，产生心脏、鳃肌、和咽弓动脉，通过咽弓（PA）介导。 NKX2-5 和 NKX2-6 是先天性心脏病中同源盒转录因子经常发生突变。在斑马鱼中，我们之前表明 Nkx2.5 和 Nkx2.7 在心脏发育中发挥冗余作用。使用新颖的功能丧失 nkx2.7等位基因是纯合致死的，我们首次证明nkx2.7-/-胚胎无法形成进食所需的颅面肌肉和软骨。这种发育缺陷是由于早期破坏 PA 中的基因调控。我们假设 Nkx2.7 作为必需的转录因子在通过咽弓介导的颅面发育过程中。我们将通过以下方式研究我们的模型揭示 nkx2.7+ CPF 祖细胞的组织特异性作用并评估转录靶点 nkx2.7 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、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研究 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的新型热诱导转基因，我们将过度表达 nkx2.7 以剖析其需求各种发展窗口。我们将把这些知识应用于 nkx2.7-/- 胚胎的单细胞 RNA 测序生成所有咽组织中遗传目标的可靠列表。我们还将比较那些假定的效应器来自患有先天性心脏病和颅面缺陷的患者的基因组数据。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)。总而言之，该提案将为 Nkx2.7 在心咽部的作用提供全面的了解。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缺陷患者的干细胞治疗策略和软骨。