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

项目摘要/摘要 Nr2f核受体对于面部原基的形成和上颌骨的构型是必不可少的。 下巴，尽管他们的具体角色(S)仍然没有完全定义。斑马鱼的表型范围很广 Nr2f功能丧失，范围从nr2f2/5 Double的显著上颌骨到下颌骨转变 突变型，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，初步数据显示早期条件消融Nr2f1/2 在颅骨NC(NC)导致类似的咽弓发育不良，并严重减少 面部背部骨骼。这一提议的首要假设是Nr2f至少在两个 数控系统发展的离散步骤：首先，它们被预测将赋予数控系统的一个子集以异于传统的命运 通过激活Twist1。Nr2f功能的丧失似乎反而导致了这一群体的NC死亡。为了测试这一点 模型中，联谊会候选人将比较Twist1及其下游靶标在小鼠突变体中的表达 并控制和尝试拯救神经脊中有twist1a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丢失的时间和 使用整装原位实验和免疫荧光实验相结合的方法来检查可能的原因。 这些实验的结果将得到FACS分选突变小鼠CnC的RNA-Seq的支持。 完成拟议研究的这一部分将使Cre-lox条件突变系统增加到他~3年的 在老鼠遗传学方面的经验，在斑马鱼遗传学方面的培训，并在现代成像方面提供广泛的经验 技巧。其次，提出了用Nr2f来塑造后迁移的数控外切组织，使 上颌的骨骼结构不同于下颌骨的。有证据证明这种后来的模式 在斑马鱼中的作用，但早期条件小鼠突变体中的cnc丢失已经混淆了确定 功能守恒。为了将这个假定的模式化角色与较早的NC角色分开，候选人 建议使用后期作用的Cre驱动程序来去除Nr2f1/2，然后检查条件突变的同源异型 颌骨表型。他还将在这些突变体中对迁移后的NC进行RNA-Seq，以确定 在这个较晚的阶段，另一组不同的目标受到失调，这与这些Nr2f角色是离散的一致。 总之，拟议的研究将对候选人进行广泛的实验室技术和分析方面的培训， 为未来的研究做好准备，成为一名独立的私家侦探。与NIDCR宣布的使命相一致，这 研究将增加关于神经脊发育和颅面异常的知识，奠定 为未来非手术疗法的发展奠定基础，以改善患者的病情。

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.