Regulatory Changes Mediating Emergence of Novel Neural Crest Traits

描述（由申请人提供）：脊椎动物的进化与神经嵴的出现密切相关，神经嵴是一种迁移性和多能性细胞群，可产生许多定义脊椎动物的特征，包括明确的头部和外周神经节。无颌鱼（包括七鳃鳗Petromyzon marinus）有真正的神经嵴细胞，但没有完整的神经嵴衍生物，缺乏颌骨，交感神经节和可能的肠神经节。在这里，我们将测试的假设，即新部署的转录因子在前迁移神经嵴细胞可能赋予了新的发展潜力，这个细胞群，增强选定的神经嵴衍生物的演变。与这一假设相一致，两个转录因子，扭曲和Ets 1，表达在premigrating神经嵴细胞的有颌类，但不是七鳃鳗。为了测试可能促进新型脊椎动物细胞类型产生的调控联系，我们将利用RNA-seq和新型转基因方法来实现以下目标：目标1：七鳃鳗迁移前颅神经嵴、迷走神经嵴和躯干神经嵴的转录组分析：为了研究在环口动物和颌口动物神经嵴之间观察到的差异，我们将进行颅，耳后（即迷走神经）和躯干背神经管含有胚胎七鳃鳗的前迁移神经嵴细胞。我们将通过原位杂交的方法在七鳃鳗和鸡中确定差异表达基因的表达模式，以发现无颌类和有颌类之间的差异表达。 目标二：介导七鳃鳗和其他脊椎动物神经嵴基因表达的保守调控元件的系统基因组和功能分析/解剖：我们将确定七鳃鳗神经嵴基因的调控区域，最初集中在Ets 1和Phox 2b，因为它们在七鳃鳗和颌口动物之间差异表达。这些将通过在七鳃鳗和鸡胚胎中的转基因进行测试。 目标3：利用保守的调控元件错误表达gnathostome神经嵴基因七鳃鳗：我们将利用跨物种和/或内源性增强子异位表达“失踪”的转录因子七鳃鳗，看看他们是否促进七鳃鳗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.