Developmental roles of Nr2f1 and Nr2f2 in the vertebrate cranial neural crest

Nr2f1 和 Nr2f2 在脊椎动物颅神经嵴中的发育作用

• 批准号: 10535559
• 负责人: David Paulding
• 金额: $ 4.04万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10535559
• 关键词: Ablation; Afferent Neurons; Alleles; Animal Model; Animals; Automobile Driving; Biological Assay; Bone structure; Branchial arch structure; Cell Culture Techniques; Cell Death; Cell Proliferation; Cell Survival; Cells; Cephalic; Characteristics; Clinic; Congenital Abnormality; Craniofacial Abnormalities; Cre driver; Data; Development; Dorsal; Embryo; Face; Family; Family member; Fellowship; Fishes; Future; Gene Family; Genes; Genetic; Histology; Human; Imaging Techniques; Immunofluorescence Immunologic; In Situ; Individual; Jaw; Knock-out; Knowledge; Laboratories; Mandible; Maxilla; Mesenchymal; Mission; Modeling; Modernization; Molecular; Morphology; Mus; Mutant Strains Mice; Mutation; National Institute of Dental and Craniofacial Research; Nature; Neural Crest; Neural Crest Cell; Nuclear Family; Nuclear Receptors; Pathway interactions; Patients; Pattern; Phenotype; Pigments; Play; Population; Publishing; Reporter; Role; Severities; Skeleton; Specific qualifier value; Structure; System; Techniques; Testing; Thinking; Training; Transgenic Organisms; Variant; Work; Zebrafish; apoAI regulatory protein-1; base; conditional mutant; craniofacial; differential expression; experience; experimental study; genetic variant; inhibitor; insight; loss of function; member; migration; mouse genetics; mutant; skeletal; skills; transcriptome sequencing

Project Summary/Abstract The Nr2f nuclear receptors are essential for the formation of the facial primordia and for patterning the upper jaw, though their specific role(s) remain incompletely defined. Zebrafish suffer a broad spectrum of phenotypes with nr2f loss of function, ranging from a striking upper-jaw-to-lower-jaw transformation in nr2f2/5 double mutants, to a severe reduction of the pharyngeal arches and an almost total loss of facial skeleton in quadruple nr2f1a/1b/2/5 and nr2f2/5/6a/6b mutants. In mice, preliminary data show that early conditional ablation of Nr2f1/2 in the cranial NC (CNC) results in a similar hypoplasticity of the pharyngeal arches and a severe reduction in the dorsal facial skeleton. The overarching hypothesis of this proposal is that the Nr2fs function in at least two discrete steps of CNC development: First, they are predicted to confer ectomesenchyme fate to a subset of CNC via activation of Twist1. Loss of Nr2f function appears to cause this population of CNC to die instead. To test this model, the fellowship candidate will compare expression of Twist1 and its downstream targets in mouse mutants and controls and attempt rescue of the fish mutant phenotypes with twist1a misexpression in the neural crest. He will perform lineage-tracing experiments in both fish and mice to determine when loss of CNC occurs and use a combination of whole mount in situ and immunofluorescence experiments to examine possible causes. The results of these experiments will be bolstered with RNA-Seq of FACS-sorted mutant mouse CNC. Completing this portion of the proposed study will add the Cre-lox conditional mutation system to his ~3 years of experience in mouse genetics, train him in zebrafish genetics, and provide broad experience in modern imaging techniques. Second, the Nr2fs are proposed to pattern post-migratory CNC ectomesenchyme to make the skeletal structures of the upper jaw distinct from those of the lower jaw. There is evidence for this later patterning role in zebrafish, but early CNC loss in the conditional mouse mutants has confounded attempts to determine conservation of function. To separate this putative patterning role from the earlier NC role, the candidate proposes to use a later-acting Cre driver to ablate Nr2f1/2 and then to examine conditional mutants for homeotic jaw phenotypes. He will also perform RNA-Seq on post-migratory CNC in these mutants to determine whether a different set of targets is dysregulated at this later stage, consistent with these Nr2f roles being discrete. Together, the proposed studies will train the candidate in a wide range of laboratory techniques and analyses, preparing him for future research as an independent PI. Consistent with the stated mission of the NIDCR, this study will add to the body of knowledge on neural crest development and craniofacial anomalies, laying the groundwork for future development of non-surgical therapies to ameliorate patient conditions.

项目总结/摘要 Nr 2f核受体对于面部原基的形成和上部结构的形成是必需的。 颚，虽然它们的具体作用仍然没有完全确定。斑马鱼具有广泛的表型 nr 2f功能丧失，范围从nr 2f 2/5双 突变，严重减少咽弓和面部骨骼几乎完全丧失四重 nr 2f 1a/1b/2/5和nr 2f 2/5/6a/6 b突变体。在小鼠中，初步数据显示，Nr 2f 1/2的早期条件性消融 在颅侧NC（CNC）导致类似的咽弓可塑性减退和严重减少， 背侧面骨该提议的首要假设是，Nr 2fs在至少两种情况下起作用。 CNC发展的离散步骤：首先，它们被预测为CNC的一个子集赋予外生间充质组织命运。 通过激活Twist 1。Nr 2f功能的丧失似乎会导致CNC群体死亡。为了验证这一 模型，奖学金候选人将比较小鼠突变体中Twist 1及其下游靶标的表达 并控制和尝试拯救神经嵴中具有twist 1a错误表达的鱼类突变表型。 他将在鱼和老鼠身上进行谱系追踪实验，以确定CNC丢失的时间， 使用整体原位和免疫荧光实验的组合来检查可能的原因。 这些实验的结果将用FACS分选的突变小鼠CNC的RNA-Seq支持。 完成拟议研究的这一部分将把Cre-lox条件突变系统添加到他大约3年的研究中。 在小鼠遗传学方面的经验，在斑马鱼遗传学方面培训他，并在现代成像方面提供广泛的经验 技术.第二，提出了Nr 2fs图案化迁移后CNC外胚间充质，以使 上颚的骨骼结构与下颚的不同。有证据表明这种后来的模式 在斑马鱼中的作用，但条件小鼠突变体中的早期CNC丢失混淆了确定 功能守恒。为了将这个假定的模式化角色与早期的NC角色分开，候选人 建议使用一个迟发性Cre驱动程序来消除Nr 2f 1/2，然后检查条件突变体的同源异型 颌骨表型他还将在这些突变体中对迁移后的CNC进行RNA-Seq，以确定是否 一组不同的靶在该后期被失调，这与这些Nr 2f作用是离散的一致。 总之，拟议的研究将培训候选人在广泛的实验室技术和分析， 为他将来作为独立私家侦探的研究做准备与NIDCR的既定使命一致， 这项研究将增加对神经嵴发育和颅面异常的认识， 为未来开发非手术疗法以改善患者状况奠定了基础。