Development Genetics of Tooth Number Variation in Sticklebacks

项目摘要 该项目的预期总体影响是确定潜在的发育和遗传机制， 牙齿的形成和替换牙齿已被经典地用作研究器官发生的模型， 牙齿与大多数器官一样，通过上皮-间质相互作用而发育。此外，30 全世界65岁以上的人中有10%没有天然牙齿。因此，知识的发展 牙齿形成和替换的遗传基础与理解器官发生有关， 以及了解牙齿如何在体外和最终在体内再生。虽然遗传学研究 在小鼠和人类中，已经确定了与牙齿发育有关的信号通路，但对牙齿发育的信号通路知之甚少。 牙齿替换的遗传机制鱼在成年后会不断更换牙齿， 为基因分析提供了强大的系统。在这里，自然变化的牙齿数量在threespine 棘鱼（Gasterosteus aculeatus）被用作一种新的模型系统，以了解基因如何调控 牙齿数量和牙齿更换。不同的棘鱼种群适应不同的饮食， 牙齿数量的巨大遗传变化。两个不同的，独立的淡水种群， 与祖先的海洋鱼类相比，进化出的牙齿数量大幅增加。在这两个高齿人群中， 牙齿数目的增加在发育后期通过加速的牙齿替换率而出现。的 不同的形式可以在实验室中交叉，使详细的遗传分析，以地图因素控制 齿数的变化。新的基因组编辑方法允许对基因和顺式调控基因进行功能测试 感兴趣的元素。骨形态发生蛋白6（Bmp 6）基因的顺式调节等位基因与骨形成相关。 在一个高齿人群中进化出了牙齿。药理学和遗传学数据表明BMP 信号传导和Bmp 6正调节乳牙数量，但抑制牙齿替换。来验证假设 关于牙齿形成和替换的发育和遗传基础，有三个具体目标， 提出了首先，我们将测试BMP信号传导和Bmp 6是否调节牙齿干细胞的静止， BrdU和活体染料脉冲追踪标记的牙齿替换，基因表达和药理学 实验其次，我们将鉴定两个Bmp 6增强子的上游调节子，并确定增强子的结构。 和调节剂的功能，在牙齿发育和替代药物，转基因， 基因组编辑实验第三，我们将在一个独立的研究中， 高齿淡水人口与一个独特的发展遗传基础的组合， 基因图谱、基因组编辑和基因表达实验。这些结果将共同带来新的 了解牙齿替换的发育和遗传机制。

Project Summary The expected overall impact of this project is to identify developmental and genetic mechanisms underlying tooth formation and replacement. Teeth have classically been used as a model to study organogenesis, as teeth, like most organs, develop through reciprocal epithelial-mesenchymal interactions. Furthermore, 30 percent of people worldwide over the age of 65 have no natural teeth. Thus, knowledge of the developmental and genetic basis of tooth formation and replacement has relevance both for understanding organogenesis, as well as for understanding how teeth can be regenerated in vitro and ultimately in vivo. Although genetic studies in mice and humans have identified signaling pathways involved in tooth development, less is known about genetic mechanisms regulating tooth replacement. Fish replace their teeth constantly throughout adult life, and offer powerful systems for genetic analysis. Here, natural variation in tooth number in the threespine stickleback fish (Gasterosteus aculeatus) is leveraged as a new model system to learn how genes regulate tooth number and tooth replacement. Different stickleback populations adapted to different diets exhibit dramatic heritable changes in tooth number. Two different, independently derived freshwater populations have evolved major increases in tooth number compared to ancestral marine fish. In both high-toothed populations, the tooth number increase arises late in development through an accelerated tooth replacement rate. The different forms can be crossed in the lab, enabling detailed forward genetic analyses to map factors controlling the changes in tooth number. New genome editing methods allow functional tests of genes and cis-regulatory elements of interest. A cis-regulatory allele of the Bone Morphogenetic Protein 6 (Bmp6) gene is associated with evolved tooth gain in one high-toothed population. Pharmacological and genetic data suggest BMP signaling and Bmp6 positively regulate primary tooth number, but inhibit tooth replacement. To test hypotheses about the developmental and genetic bases of tooth formation and replacement, three specific aims are proposed. First, we will test whether BMP signaling and Bmp6 regulate dental stem cell quiescence during tooth replacement by BrdU and vital dye pulse-chase labeling, gene expression, and pharmacological experiments. Second, we will identify upstream regulators of two Bmp6 enhancers and determine enhancer and regulator functions during tooth development and replacement by pharmacological, transgenic, and genome editing experiments. Third, we will identify the genetic basis of evolved tooth gain in an independently derived high-toothed freshwater population with a distinct developmental genetic basis by a combination of genetic mapping, genome editing, and gene expression experiments. Together these results will shed new light on developmental and genetic mechanisms underlying tooth replacement.