Regulatory Changes Mediating Emergence of Novel Neural Crest Traits

描述(申请人提供)：脊椎动物的进化与神经脊的出现密切相关，神经脊是一种迁徙和多潜能的细胞群，它产生了许多定义脊椎动物的特征，包括明确定义的头部和外周神经节。无颌鱼(包括七鳃鳗)有真正的神经脊细胞，但没有完整的神经脊衍生细胞，缺乏颌骨、交感神经节和可能的肠神经节。在这里，我们将检验一种假设，即转录因子在迁移前神经脊细胞中的新部署可能赋予了该细胞群新的发育潜力，增强了选定的神经脊衍生物的进化。与这一假设一致的是，两个转录因子Twist和Ets1在脊椎动物的迁移前神经脊细胞中表达，但七鳃鳗不表达。为了测试可能促进新脊椎动物细胞类型产生的调控联系，我们将利用RNA-SEQ和新的转基因方法来实现以下目标：目的1：七鳃鳗迁飞前脑神经、迷走神经和躯干神经脊的转录组分析：为了研究观察到的环口神经脊和颌口神经脊之间的差异，我们将对七鳃鳗胚胎中含有迁徙前神经脊细胞的颅神经管、迷走神经和躯干背部神经管进行RNA-SEQ分析。我们将通过原位杂交的方法确认基因在七鳃鳗和雏鸡中的差异表达模式，以寻找无齿鳗和颌口动物之间的差异表达。目的2：七鳃鳗和其他脊椎动物神经脊基因表达的保守调控元件的系统基因组学和功能分析：我们将确定七鳃鳗神经脊基因的调控区域，首先集中在Ets1和PHOX2B上，因为这两个基因在七鳃鳗和颌口之间存在差异表达。这些将通过在七鳃鳗和小鸡胚胎中进行转基因来进行测试。目的3：利用保守的调控元件在七鳃鳗中错误表达节口神经脊基因：我们将利用跨物种和/或内源性增强子在七鳃鳗中异位表达“缺失”的转录因子，看看它们是否促进七鳃鳗所缺乏的细胞类型的形成。

DESCRIPTION (provided by applicant): Evolution of vertebrates is intimately linked to the advent of the neural crest, a migratory and multipotent cell population that gives rise to many defining vertebrate characteristics, including a well-defined head and peripheral ganglia. Jawless fish (including the lamprey Petromyzon marinus) have bona fide neural crest cells, but do not have the full complement of neural crest derivatives, lacking jaws, sympathetic and possibly enteric ganglia. Here, we will test the hypothesis that the novel deployment of transcription factors in premigratory neural crest cells may have conferred new developmental potential onto this cell population, potentiating evolution of selected neural crest derivatives. Consistent with this hypothesis, two transcription factors, Twist and Ets1, are expressed in premigratory neural crest cells of gnathostomes but not lampreys. To test regulatory connections that may have facilitated production of novel vertebrate cell types, we will utilize RNA-seq together with novel transgenesis approaches to perform the following aims: Aim 1: Transcriptome analysis of lamprey premigratory cranial, vagal and trunk neural crest: To investigate the differences observed between cyclostome and gnathostome neural crest, we will perform RNA-seq analysis of cranial, post-otic (i.e. vagal) and trunk dorsal neural tubes containing premigratory neural crest cells from embryonic lamprey. We will confirm expression patterns of differentially expressed genes by in situ hybridization in lamprey and chick to find differentially expressed between agnathans and gnathostomes. Aim 2: Phylogenomic and functional analysis/dissection of conserved regulatory elements mediating expression of neural crest genes of lamprey and other vertebrates: We will identify regulatory regions of lamprey neural crest genes, initially focusing on Ets1 and Phox2b since they are differentially expressed between lamprey and gnathostomes. These will be tested by transgenesis in both lamprey and chick embryos. Aim 3: Utilize conserved regulatory elements to misexpress gnathostome neural crest genes in lamprey: We will utilize cross-species and/or endogenous enhancers to ectopically express "missing" transcription factors in lamprey to see if they promote formation of cell types that lamprey lack.