next-generation sequencing approaches to identify genotype-phenotype relationships during miRNA tuning of neural crest osteogenesis

新一代测序方法可识别神经嵴成骨过程中 miRNA 调节过程中的基因型与表型关系

• 批准号: 10579800
• 负责人: Nicole Isolde zur Nieden
• 金额: $ 12.7万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10579800
• 关键词: Affect; Area; Aryl Hydrocarbon Receptor; Award; Bioinformatics; Cartilage; Cells; Cephalic; ChIP-seq; Congenital Abnormality; Craniofacial Abnormalities; DNA Binding; Data; Data Set; Defect; Development; Diagnosis; Early Diagnosis; Educational workshop; Embryo; Embryonic Development; Environment; Environmental Protection; Epigenetic Process; Etiology; Face; Family; Fostering; Functional disorder; Funding; Future; Genetic; Genetic Transcription; Genotype; Goals; Human; In Vitro; Individual; K-18 conjugate; Knock-out; Knockout Mice; Knowledge; Lateral; Mediating; Mentors; Mesoderm; MicroRNAs; Modeling; Molecular; Molecular Mechanisms of Action; Morphology; Mus; Neural Crest; Neural Crest Cell; Nuclear; Osteoblasts; Osteogenesis; Outcome; Paraxial Mesoderm; Pathology; Phenotype; Play; Pluripotent Stem Cells; Populations at Risk; Prevention; Process; Proteins; Psychological Impact; Publishing; Regulation; Reporting; Repression; Research; Research Personnel; Research Project Summaries; Rest; Role; Scientist; Signal Transduction; Skeleton; Time; Tissues; Training; Transcriptional Activation; Work; bone; calcification; career; craniofacial; craniofacial bone; craniofacial development; design; diagnostic biomarker; diagnostic strategy; epigenetic regulation; experience; gene regulatory network; genetic counselor; human pluripotent stem cell; imaging modality; in vivo; innovation; knock-down; malformation; neuroregulation; next generation sequencing; novel; osteoblast differentiation; osteogenic; osteoprogenitor cell; overexpression; prevent; promoter; psychologic; response; single-cell RNA sequencing; skeletal; skeletogenesis; skills; stem cell biology; trait; transcription factor; transcriptome sequencing; transcriptomics; vertebrate embryos

PROJECT SUMMARY Research: Every 4 ½ minutes, a baby is born with a birth defect, including such that affect the craniofacial skeleton. These facial malformations cause disfigurement leading to long-term psychological impact and are often associated with physical dysfunction imposing tremendous burden on the affected individuals and their families. Although the causes for most of these severe developmental pathologies are still unknown, it is clear that the inappropriate formation and differentiation of neural crest (NC) cells, the cells that normally give rise to the osteoblasts in the affected craniofacial skeleton, plays a large part. Given the causality between miscontrolled osteoblast differentiation from the NC and the tissue malformations arising from it, an understanding of the molecular networks underlying NC development is crucial for the potential treatment or prevention of craniofacial defects, yet far from complete. For instance, the contribution of epigenetic regulators, such as microRNAs to the formation of craniofacial bones remains understudied, despite their potential as diagnostic markers. For this K18 application it is proposed to use data sets generated already under 5R01DE025330 to examine the epigenetic regulation of NC development by a specific microRNA, miR361, which is pro-osteogenic in vitro and expressed in the developing NC in vivo. Assessment of skeletal phenotypes when miR361 is knocked out together with single cell RNA-sequencing using an innovative and effective in vitro differentiation model based on human pluripotent stem cells will validate the impact of miR361 on NC-osteogenesis. Bulk RNA sequencing and ChIP sequencing will identify NC-specific loci that are transcriptionally regulated downstream of miR361. Together, these data will define the relationship between this miRNA, transcriptional activation in NC cells and their subsequent propensity for osteoblast differentiation. Goals/Training plan/environment: The goals for this K18 application are two-fold: 1) to train in the design, execution and specifically the analysis of next-generation sequencing data sets, and 2) to acquire hands-on experience with imaging-modality-assisted analysis of skeletal phenotypes. The attainment of such skills, in addition to attending several workshops focused on bioinformatics throughout the year of support will prepare the applicant for future funding and mentoring opportunities. The applicant has assembled a strong team of local mentors and regional collaborators who have extensive experience in the area of the proposed work and a strong record of training scientists to become successful independent investigators. In summary, the proposed project will allow the applicant to enrich her long-standing track record in stem cell biology with bioinformatics and phenotypic skeletal analysis, which will foster the perspectives and augment the skills necessary to be a successful craniofacial biologist.

项目摘要 研究：每4½分钟，一个婴儿天生就有出生缺陷，其中包括影响颅面 骨骼。这些面部畸形会导致长期心理影响的毁容，并且是 通常与身体功能障碍有关，对受影响的个体及其 家庭。尽管大多数这些严重发育性病变的原因仍然未知，但很明显 神经rest（NC）细胞的不当形成和分化，通常会产生的细胞 受影响的颅面骨骼中的成骨细胞起着很大的作用。考虑到错误控制之间的因果关系 成骨细胞与NC的分化以及由此产生的组织畸形，对 NC发展的基础分子网络对于潜在的治疗或预防颅面至关重要 缺陷，但距离远远不够。例如，表观遗传调节剂（例如microRNAS）对 颅面骨骼的形成仍然被了解，散发其作为诊断标记的潜力。 对于此K18应用程序，建议使用根据5R01DE025330生成的数据集来检查 特定microRNA MiR361对NC发育的表观遗传调节，该Mir361在体外是促骨的基础 在发育中的NC体内表达。当mir361淘汰时骨骼表型的评估 以及使用创新且有效的体外分化模型的单细胞RNA测序 关于人多能干细胞将验证miR361对NC稳态发生的影响。散装RNA测序 芯片测序将识别由miR361下游转录调节的NC特异性局部。 这些数据一起将定义这种miRNA，NC细胞中的转录激活与 他们随后对成骨细胞分化的承诺。 目标/培训计划/环境：此K18应用程序的目标是两个方面：1）在设计中训练， 执行，特别是对下一代测序数据集的分析，以及2）获取动手 具有成像模式辅助分析的骨骼表型的经验。获得这样的技能， 在整个支持一年中，还要参加几个专注于生物信息学的研讨会，还将准备 未来资金和心理机会的申请人。申请人组成了一个强大的本地团队 在拟议工作领域拥有丰富经验的导师和区域合作者 培训科学家的记录成为成功的独立研究人员。总而言之，拟议的项目 将允许申请人通过生物信息学和 表型骨骼分析，这将促进观点并增强成为一个必要的技能 成功的颅面生物学家。