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

宏观进化模式是来自自然选择还是发育限制一直争论不休。拟议的研究试图从这种外部或内部力量的角度来解释脊椎动物牙齿减少的进化趋势。鲤形鱼类（如小鱼）的口腔牙齿脱落就是这一趋势的典型。尽管有不同的喂养方式和喉齿的保留，但该群体的成员没有重新长出口腔牙齿，这表明牙齿脱落是进化的限制。描述这种损失的发展机制将检验约束假说；找到一种简单的、实验可逆的机制会反驳这种观点。先前对斑马鱼（Danio rerio）和有口腔牙齿的相关群体的基因表达的比较发现，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发育遗传途径的改变，导致牙形缺失。目的是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)确定与牙齿脱落和基因家族数进化相关的其他Dlx家族成员表达的变化。被使用的主要物种是斑马鱼和日本medaka (Oryzias latipes)，后者保留口腔牙齿。转基因方法将用于比较两个物种中Dlx2基因表达的调节，以及错误表达天然或修饰的Dlx和Fgf蛋白。将在斑马鱼、梅达卡鱼和墨西哥四环鱼（Astyanax mexicanus）中检测整个Dlx基因补体的表达（其中一些仍有待克隆）。这些物种在牙列上表现出差异，可能在它们的基因组中也存在Dlx基因的数量上。除了阐明牙齿发育的基本机制外，cypriniform口腔牙齿脱落机制的表征将允许与其他脊椎动物牙齿减少的实例进行比较。由此产生的转基因株系和构建体对于研究Dlx和Fgf基因在其他结构发育中的作用具有重要价值。该项目也将具有重要的教育价值。结果和材料将被纳入现有的进化发育生物学课程，而几名本科生（通过大学项目招募），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.