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

• 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
• 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' 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

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.

项目摘要 与Digeorge综合征相关的临床表型，这是最常见的微缺失条件 （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。 鉴于A 心咽场（CPF）中的多能祖细胞产生心脏，分支肌肉， 通过咽弓（PAS）介导的咽弓动脉。 NKX2-5和NKX2-6是 同型转录因子经常在先天性心脏缺陷中突变。在斑马鱼中，我们以前有 表明NKX2.5和NKX2.7在心脏发展中起冗余作用。使用新颖的功能丧失 NKX2.7纯合致命的等位基因，我们首次证明了NKX2.7 - / - 胚胎无法形成 进食所需的颅面肌肉和软骨。这种发育不足是从早期引起的 PAS中的遗传调节破坏。我们假设NKX2.7是必不可少的转录因子 在颅面发育过程中，通过咽弓介导。我们将通过 揭示NKX2.7+ CPF祖细胞的组织特异性作用，并通过评估转录靶标 NKX2.7和评估相关的人类疾病表型。在AIM 1中，我们将表征 NKX2.7 - / - 胚胎中PAS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，探讨显微镜。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，NKX2.7+祖细胞的自主功能 转基因。在AIM 2中，我们将研究由NKX2.7介导的时间和分子机制。使用 我们实验室中产生的新型热诱导转基因，我们将过表达NKX2.7，以剖析其在期间的要求 各种开发窗口。我们将将这些知识应用于nkx2.7 - / - 胚胎中的单细胞RNA - 序列 在所有咽组织中生成强大的遗传靶标列表。我们还将比较那些推定的效应子 带有先天性心脏病和颅面缺陷患者的基因组数据。评估直接或 这些目标的间接结合，我们将使用染色质免疫沉淀和DNA测序（CHIP-SEQ）。 总之，该建议将对NKX2.7在心咽中的作用提供全面的理解 发展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。