The Role of Dlx Genes in the Development and Evolution of the Teleost Fish Dentition

项目摘要长期以来，人们一直在争论宏观进化模式是自然选择的结果还是发展限制的结果。这项拟议的研究试图用这种外力或内力来解释脊椎动物牙齿减少的进化趋势。鲤形鱼(如小鲤鱼)口腔牙齿的缺失就是这种趋势的典范。尽管有不同的喂养方式和咽喉牙齿的保留，但该组成员中没有人恢复口腔牙齿，这表明牙齿缺失是进化的制约因素。表征这种缺失的发展机制将检验约束假说；找出一种简单的、实验可逆的机制将反对该假说。先前对鲤形斑马鱼(Danio Rerio)及其近缘组与口腔牙齿的基因表达进行的比较发现，牙齿缺失与Dlx2转录因子表达之间存在关联。这一结果将被扩展到进一步表征导致鲤形牙齿缺失的发育遗传途径的修改。目的：1)确定DLX表达进化的机制，2)确定DLX功能丧失和获得的后果，3)研究成纤维细胞生长因子(FGF)信号通路作为DLX表达和口腔牙齿丧失的潜在原因，以及4)确定与牙齿丧失和基因家族数进化相关的DLX家族其他成员的表达变化。将被使用的主要物种是斑马鱼和日本青竹(Oryzias Latipe)，后者保留口腔牙齿。转基因方法将被用来比较Dlx2基因在两个物种中的表达调控，并错误表达天然或修饰的DLX和FGF蛋白。DLX基因的全部补体(其中一些仍有待克隆)的表达将在斑马鱼、青竹和墨西哥斑马鱼(Astianax Micianus)中进行检测。这些物种在牙列上表现出差异，可能在它们基因组中存在的DLX基因的数量上也存在差异。除了阐明牙齿发育的基本机制外，对鲤鱼形口腔牙齿缺失机制的表征将使我们能够与其他脊椎动物牙齿减少的例子进行比较。产生的转基因株系和构建体将对研究DLX和成纤维细胞生长因子基因在其他结构发育中的作用有价值。该项目也将具有重大的教育价值。结果和材料将被纳入一门现有的进化发育生物学课程，而几名本科生(通过大学项目招收)、两名研究生和一名博士后研究员将在执行该项目时接受研究培训。

PROJECT SUMMARY Whether macroevolutionary patterns result from natural selection or developmental constraints has long been debated. The proposed research seeks to explain in terms of such external or internal forces an evolutionary trend toward tooth reduction in vertebrates. Loss of oral teeth in cypriniform fishes (e.g. minnows) serves as a model of this trend. No member of the group has regained oral teeth, despite diverse feeding modes and retention of throat teeth, suggesting tooth loss as a constraint on evolution. Characterizing the developmental mechanism of this loss will test the constraint hypothesis; identifying a simple, experimentally-reversible mechanism would argue against it. Previous comparisons of gene expression in the cypriniform zebrafish (Danio rerio) and related groups with oral teeth identified an association between absence of teeth and of Dlx2 transcription factor expression. This result will be extended to further characterize modifications of developmental genetic pathways resulting in cypriniform tooth loss. Objectives are to 1) identify the mechanism of Dlx expression evolution, 2) identify consequences of loss and gain of Dlx function, 3) investigate Fibroblast growth factor (Fgf) signaling pathways as potential causes of loss of Dlx expression and oral teeth, and 4) identify any changes in expression of additional Dlx family members associated with tooth loss and evolution of gene family number. The primary species to be employed are the zebrafish and the Japanese medaka (Oryzias latipes), the latter of which retains oral teeth. Transgenic approaches will be used to compare regulation of Dlx2 gene expression in the two species and to misexpress native or modified Dlx and Fgf proteins. Expression of the entire complement of Dlx genes (some of which remain to be cloned) will be examined in the zebrafish, medaka and Mexican tetra (Astyanax mexicanus). These species exhibit differences in dentition and probably in the number of Dlx genes present in their genomes as well. Characterization of the mechanism of cypriniform oral tooth loss will allow comparison with other instances of vertebrate dental reduction, in addition to elucidating fundamental mechanisms of tooth development.Transgenic lines and constructs generated will be valuable for studying the role of Dlx and Fgf genes in the development of other structures. The project will have significant educational value as well. Results and materials will be incorporated into an existing course in Evolutionary Developmental Biology, while several undergraduate students (recruited through universty programs), two graduate students, and a post-doctoral researcher will receive research training while executing the project.