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4分半钟，一个婴儿出生时就有出生缺陷，包括影响颅面的缺陷。 骷髅这些面部畸形导致毁容，导致长期的心理影响， 通常与身体功能障碍有关，给受影响的个体及其 家庭虽然大多数这些严重发育病理的原因仍然未知，但很明显， 神经嵴（NC）细胞的不适当的形成和分化，这些细胞通常会引起 受影响的颅面骨骼中的成骨细胞，起着很大的作用。考虑到控制不当 成骨细胞分化从NC和组织畸形所产生的，一个了解的， NC发展的分子网络对于颅面神经病变的潜在治疗或预防至关重要。 有缺陷，但还远未完成。例如，表观遗传调节因子（如microRNA）对基因表达的贡献， 颅面骨的形成仍然研究不足，尽管它们作为诊断标志物的潜力。 对于该K18应用，建议使用根据5 R 01 DE 025330生成的数据集来检查 通过特定的microRNA miR 361对NC发育进行表观遗传调控，miR 361在体外具有促成骨作用， 在体内发育的NC中表达。miR 361敲除时骨骼表型的评估 与单细胞RNA测序一起，使用创新和有效的体外分化模型， 将验证miR 361对NC-骨生成的影响。批量RNA测序 ChIP测序将鉴定在miR 361下游转录调节的NC特异性基因座。 总之，这些数据将定义这种miRNA、NC细胞中的转录激活和 它们随后的成骨细胞分化倾向。 目标/培训计划/环境：本K18应用程序的目标有两个方面：1）在设计中进行培训， 执行，特别是下一代测序数据集的分析，以及2）获得动手能力 具有骨骼表型的成像模式辅助分析的经验。这些技能的获得， 除了在整个支持年期间参加几个以生物信息学为重点的讲习班外， 申请人未来的资金和指导机会。申请人已经组建了一支强大的本地团队， 导师和区域合作者，他们在拟议的工作领域拥有丰富的经验， 培训科学家成为成功的独立调查员的记录。总而言之，拟议项目 将使申请人丰富她在干细胞生物学与生物信息学的长期跟踪记录， 表型骨骼分析，这将促进观点和增强必要的技能，是一个 成功的颅面生物学家