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

项目总结 研究：每4.5分钟就有一名婴儿出生时就有先天缺陷，包括影响颅面部的先天缺陷 骷髅。这些面部畸形会导致毁容，导致长期的心理影响，并 通常与身体功能障碍有关，给受影响的个人和他们的 家人。尽管大多数严重的发育病理的原因尚不清楚，但很明显 神经脊(NC)细胞的不适当形成和分化，这些细胞通常会引起 受影响的头面部骨骼中的成骨细胞起着很大的作用。考虑到失控之间的因果关系 从NC向成骨细胞分化及其引起的组织畸形 NC发生的分子网络对于潜在的治疗或预防颅面部疾病是至关重要的 缺陷，但还远远不完整。例如，表观遗传调节因子，如microRNAs对 颅面部骨骼的形成仍未得到充分研究，尽管它们有可能作为诊断标记物。 对于此K18应用程序，建议使用已在5R01DE025330下生成的数据集来检查 一种特殊的microRNA miR361对NC发育的表观遗传学调控作用 在体内发育中的NC中表达。MiR361基因敲除后骨骼表型的评估 结合使用创新和有效的体外分化模型的单细胞RNA测序 对人类多能干细胞的研究将验证miR361对NC成骨的影响。批量RNA测序 芯片测序将识别miR361下游转录调控的NC特定基因座。 总之，这些数据将定义这种miRNA、NC细胞中的转录激活和 他们随后的成骨细胞分化倾向。 目标/培训计划/环境：此K18应用程序的目标有两个：1)在设计中进行培训， 执行，特别是分析下一代测序数据集，以及2)获取实际操作 骨骼表型的成像模式辅助分析经验。这些技能的获得，在 除了参加几个以生物信息学为重点的研讨会外，支助年还将准备 申请者申请未来的资助和指导机会。申请人已经组建了一支强大的当地人团队 在拟议工作领域拥有丰富经验的导师和区域合作者，以及 培训科学家成为成功的独立调查人员的记录。总而言之，拟议的项目 将使申请人能够用生物信息学和生物信息学丰富她在干细胞生物学方面的长期记录 表型骨骼分析，它将培养成为一名 成功的颅面生物学家。

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.