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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要 Nr2f 核受体对于面部原基的形成和上部图案的形成至关重要下巴，尽管它们的具体作用仍未完全确定。斑马鱼具有广泛的表型 nr2f 功能丧失，从 nr2f2/5 double 中显着的上颌到下颌转变突变体，四倍体中咽弓严重减少，面部骨骼几乎完全丧失 nr2f1a/1b/2/5 和 nr2f2/5/6a/6b 突变体。在小鼠中，初步数据表明 Nr2f1/2 的早期条件消融颅内 NC (CNC) 会导致类似的咽弓发育不全以及咽弓严重减少面部背侧骨骼。该提案的总体假设是 Nr2fs 至少在两个 CNC 开发的离散步骤：首先，预计它们将赋予 CNC 的一个子集外胚充质命运通过激活 Twist1。 Nr2f 功能的丧失似乎会导致 CNC 群体死亡。为了测试这个模型中，奖学金候选人将比较 Twist1 及其下游靶标在小鼠突变体中的表达并控制并尝试拯救神经嵴中扭曲1a错误表达的鱼突变表型。他将在鱼和小鼠身上进行谱系追踪实验，以确定 CNC 何时丢失以及结合使用原位整体安装和免疫荧光实验来检查可能的原因。这些实验的结果将得到 FACS 分选突变小鼠 CNC 的 RNA 测序的支持。完成拟议研究的这一部分将在他约 3 年的研究中添加 Cre-lox 条件突变系统小鼠遗传学方面的经验，对他进行斑马鱼遗传学方面的培训，并提供现代成像方面的丰富经验技术。其次，Nr2fs被提议对迁移后的CNC外胚充质进行图案化，以使得上颌的骨骼结构与下颌的骨骼结构不同。有证据表明这种后来的模式斑马鱼中的作用，但条件小鼠突变体中的早期 CNC 丢失已经混淆了确定的尝试功能守恒。为了将这个假定的模式角色与早期的 NC 角色区分开来，候选人建议使用后期作用的 Cre 驱动程序来消除 Nr2f1/2，然后检查条件突变体的同源异型颌表型。他还将对这些突变体的迁移后 CNC 进行 RNA 测序，以确定是否在此后期阶段，一组不同的目标失调，这与这些 Nr2f 角色的离散性一致。总之，拟议的研究将培训候选人广泛的实验室技术和分析，为他作为独立 PI 的未来研究做好准备。与 NIDCR 的既定使命一致，研究将丰富有关神经嵴发育和颅面异常的知识体系，奠定为未来开发非手术疗法以改善患者状况奠定基础。