Mechanisms responsible for tooth morphogenesis in vertebrates

脊椎动物牙齿形态发生的机制

• 批准号: 10614550
• 负责人: Yann Gibert
• 金额: $ 35.78万
• 依托单位: UNIVERSITY OF MISSISSIPPI MED CTR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-02 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10614550
• 关键词: Acids; Adult; Affect; CRISPR/Cas technology; Cell Separation; Cells; Cellular biology; Cleidocranial Dysplasia; Complex; DNA Sequence Alteration; Data; Dental; Dentition; Development; Disease; Dominant Genetic Conditions; Embryo; Embryonic Development; Enzymes; Epithelium; Evolution; Exposure to; Fishes; Functional disorder; Gene Expression; Genes; Genetic; Genetic Diseases; Genetic study; Germ Cells; Goals; Goldfish; Growth Factor; Hand; Heterozygote; Histologic; Human; Human Genetics; Incisor; Knock-out; Larva; Link; Mesenchymal; Mesenchyme; Minnows; Modeling; Molar tooth; Molecular; Molecular Biology; Morphogenesis; Morphology; Mus; Mutate; Mutation; Odontoblasts; Odontogenesis; Organ; Organogenesis; Orthologous Gene; Pathway interactions; Phenotype; Play; Process; Rattus; Receptor Signaling; Regulation; Retinoids; Role; Series; Shapes; Siblings; Signal Transduction; Signaling Molecule; Structure; Syndrome; Techniques; Testing; Thinness; Tooth Abnormalities; Tooth Cell; Tooth Germ; Tooth structure; Tretinoin; Vertebrates; Work; Zebrafish; autosome; comparison control; differential expression; gene function; genetic analysis; human disease; inhibitor; knockout gene; loss of function; malformation; mineralization; morphogens; mutant; novel; oral ectoderm; pharmacologic; teleost; transcription factor; transcriptome

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.

抽象的 畸形的牙齿在人类中非常普遍。在大多数情况下，它们是由于基因的基因突变引起的 控制牙齿的形态发生。 Runx2基因就是这种情况，曾经突变导致 cle氏司法异常增生（CCD）一种常染色体显性遗传综合征，呈现出钉状牙齿的牙齿 人类。尽管在牙齿形态发生过程中已经确定了许多因素，但目前鲜为人知 关于细胞信号如何参与牙肠发生过程中的器官形状。我们以前的工作有 确定信号传导分子视网膜酸（RA）是鱼类牙齿诱导的主要参与者之一 最近的数据表明，RA在牙齿形态发生过程中也起着作用。尤其是鱼和斑马鱼 研究脊椎动物中牙齿的遗传学，细胞和分子生物学以及器官发生的好模型。这 拟议的研究的目的是了解视黄酸在牙齿形态发生过程中扮演的作用 在不同的鱼类中，开发了CCD的鱼类模型，以阐明Runx2在牙齿中所扮演的角色 形态发生，并了解其与RA信号的联系。最后，该项目建议识别新型基因 与牙齿形态发生有关，并通过斑马鱼中的基因敲除研究其功能。 在牙齿形态发生过程中，通过将鱼类胚胎和幼虫暴露于外源性RA和RA抑制剂中，我们将是 能够理解鱼类在牙齿形态发生过程中RA信号传导的作用机理。我们的初步 数据确定牙齿胚芽不同细胞中的RA水平受到的时间和水平控制 RA降解酶CYP26B1的表达在发育中的细胞的一部分中。修改 因此，牙齿胚芽中CYP26B1表达的发作将改变牙齿中可用的RA水平 胚芽最终修饰牙齿的形状。我们将研究负责的顺式调节更改 在两个密切的牙齿形态发生过程中，CYP26B1表达时间的进化变化紧密 相关的鱼类，斑马鱼和山小鱼，在 成人和胚胎发育期间。我们掌握了斑马鱼runx2b（斑马鱼的直系同源 在牙齿胚芽中表达的人runx2基因功能丧失突变体。这个突变体将习惯 对牙齿形态发生进行表型分析，并了解RA信号传导之间的关系 牙齿形态发生过程中的Runx2表达。识别小说基因在牙齿中起作用 形态发生，我们将通过细胞分类，暴露于外源性RA信号传导的牙齿生殖细胞并进行比较 它们的转录组在同一发育阶段控制发育的牙细胞。产生的基因 差异表达将通过使用CRISPR/CAS9进行基因敲除表型分析 技术。这项工作将揭示有关其调节的RA和基因（包括Runx2）如何控制牙齿的更多信息 发育中的形态发生，疾病（克氏司法发育不良）和进化。