Mechanisms responsible for tooth morphogenesis in vertebrates

摘要 牙齿畸形在人类中非常常见。在大多数情况下，它们是由于基因突变， 控制牙齿的形态发生。Runx 2基因就是这种情况，一旦突变， 锁骨颅骨发育不良（CCD）是一种常染色体显性遗传综合征，表现为桩状牙齿， 人类虽然许多因素已被确定在牙齿形态发生的过程中，目前，知之甚少 关于细胞信号如何参与牙齿形成过程中器官形状的建立。我们之前的工作 发现信号分子视黄酸（RA）是鱼类牙齿诱导的主要因素之一， 最近的数据表明RA在牙齿形态发生中也起作用。特别是鱼类和斑马鱼， 研究脊椎动物牙齿的遗传学、细胞和分子生物学以及器官发生的良好模型。的 本研究的目的是了解视黄酸在牙齿形态发生中的作用 在不同的鱼类中，建立CCD的鱼类模型，阐明Runx 2在牙齿发育过程中的作用， 形态发生，并了解其与RA信号的联系。最后，该项目提出了识别新基因的方法， 与牙齿形态发生有关，并通过基因敲除研究它们在斑马鱼中的功能。 通过在牙齿形态发生过程中将鱼胚胎和幼体暴露于外源性RA和RA抑制剂， 能够理解RA信号在鱼类牙齿形态发生中的作用机制。我们的初步 数据表明，在牙胚的不同细胞中，RA的水平受到以下因素的时间和水平的控制： RA降解酶cyp 26 b1在发育中的牙胚的细胞亚群中的表达。修改 因此，牙胚中cyp 26 b1表达的开始将改变牙齿中可用的RA水平 最终改变牙齿的形状。我们将研究顺式调节变化， cyp 26 b1在牙齿形态发生过程中表达时间的进化变化， 相关的鱼类，斑马鱼和山米诺鱼，承担显着不同形状的牙齿， 成年人和胚胎发育期间。我们手中有斑马鱼runx 2b（斑马鱼的直系同源物， 在牙胚中表达的人Runx 2基因）功能丧失突变体。这个变种人将被用来 进行牙齿形态发生的表型分析，并了解RA信号传导与 Runx 2在牙齿形态发生中的表达。为了确定在牙齿发育过程中起作用的基因， 形态发生，我们将通过细胞分选选择暴露于外源性RA信号的牙胚细胞，并比较 它们的转录组来控制处于同一发育阶段的发育中的牙细胞。由此产生的基因 差异表达的基因将通过使用CRISPR/cas9基因敲除进行表型分析。 技术.这项工作将揭示更多关于RA及其调控的基因，包括Runx 2，如何控制牙齿 发育、疾病（锁骨颅骨发育不良）和进化中的形态发生。

Abstract Misshapen teeth are highly common in humans. In most instances, they are due to genetic mutations in genes controlling the morphogenesis of teeth. This is the case for the Runx2 gene, which once mutated leads to cleidocranial dysplasia (CCD) an autosomal dominant genetic syndrome presenting with peg-like teeth in humans. While many factors have been identified in the process of tooth morphogenesis, currently, little is known about how cell signaling participates in establishing organ shape during odontogenesis. Our previous work has identified the signaling molecule retinoic acid (RA) to be one of the main actors of tooth induction in fish and recent data suggest that RA also plays a role during tooth morphogenesis. Fish and zebrafish, in particular, are good models to study genetics, cell and molecular biology, and organogenesis of the tooth in vertebrates. The objective of the proposed studies is to understand the roles played by retinoic acid during tooth morphogenesis in different fish species, to develop a fish model of CCD, to clarify the role played by Runx2 during tooth morphogenesis, and to understand its link with RA signaling. Finally, this project proposes to identify novel genes implicated in tooth morphogenesis and to study their function by gene knock-outs in zebrafish. By exposing fish embryos and larvae to exogenous RA and RA inhibitor during tooth morphogenesis we will be able to understand the mechanism of action of RA signaling during tooth morphogenesis in fish. Our preliminary data identified that the levels of RA in different cells of the tooth germ are controlled by the timing and level of expression of the RA degrading enzyme cyp26b1 in a subset of cells of the developing tooth germ. Modifying the onset of cyp26b1 expression in the tooth germ will, therefore, change the level of RA available in the tooth germ ultimately modifying the shape of the tooth. We will study the cis-regulatory changes responsible for evolutionary changes in the timing of cyp26b1 expression during tooth morphogenesis between two closely related fish species, the zebrafish and the mountain minnow, that bear dramatically different shape of teeth in adults and during embryonic development. We have in hands the zebrafish runx2b (the zebrafish ortholog of the human Runx2 gene that is expressed in the tooth germ) loss-of-function mutant. This mutant will be used to perform a phenotypic analysis of tooth morphogenesis and to understand the relationship between RA signaling and Runx2 expression during tooth morphogenesis. To identify novels genes playing a role during tooth morphogenesis, we will select by cell sorting, tooth germ cells exposed to exogenous RA signaling and compare their transcriptome to control developing tooth cells at the same developmental stage. The resulting genes differentially expressed will be subjected to phenotypic analysis by gene knock-out using the CRISPR/cas9 technology. This work will reveal more about how RA and genes it regulates, including Runx2, controls tooth morphogenesis in development, diseases (cleidocranial dysplasia) and evolution.