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。牙齿被经典地用作研究器官发生的模型，如 牙齿，像大多数器官一样，通过上皮和间充质的相互作用而发育。此外，30 全世界65岁以上的人中有1%没有天然牙齿。因此，关于发展的知识 牙齿形成和替换的遗传学基础对于理解器官发生都具有相关性，因为 以及了解牙齿如何在体外并最终在体内再生。虽然基因研究 在小鼠和人类中，已经确定了参与牙齿发育的信号通路，但对此知之甚少 调控牙齿替换的遗传机制。鱼在成年后不断地更换牙齿，而且 为基因分析提供强大的系统。在这里，三颗松树的牙齿数量自然变化 刺鱼(Gasterbone Us Aculeatus)被用作研究基因调控的新模式系统 牙齿数量和牙齿更换。不同的刺鱼种群适应不同的饮食表现 牙齿数量上可遗传的戏剧性变化。两个不同的、独立派生的淡水种群 与祖先的海鱼相比，进化出的牙齿数量有了显著的增加。在这两个高牙齿人群中， 牙齿数量的增加是在发育后期通过加速牙齿替换速度而出现的。这个 不同的形式可以在实验室中交叉，使详细的正向遗传分析能够绘制控制因素图 牙齿数量的变化。新的基因组编辑方法允许对基因和顺式调控进行功能测试 感兴趣的元素。骨形态发生蛋白6(Bmp6)基因的顺式调节等位基因相关 在一个高牙齿的种群中进化出牙齿的增长。药理和遗传数据显示BMP 信号转导和Bmp6正向调节乳牙数目，但抑制牙齿替换。检验假说 关于牙齿形成和替换的发育和遗传学基础，有三个具体目标 建议。首先，我们将测试BMP信号和Bmp6是否调节牙齿干细胞在 BrdU与活性染料脉冲追逐标记、基因表达及药理学研究 实验。其次，我们将确定两个Bmp6增强子的上游调控因子，并确定增强子 和调节功能在牙齿发育和通过药理学、转基因和 基因组编辑实验。第三，我们将在一项独立的研究中确定牙齿进化的遗传基础 通过以下组合获得具有明显发育遗传基础的高齿淡水种群 基因作图、基因组编辑和基因表达实验。总而言之，这些结果将带来新的 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.