Mechanisms responsible for tooth morphogenesis in vertebrates

脊椎动物牙齿形态发生的机制

• 批准号: 10298099
• 负责人: Yann Gibert
• 金额: $ 36.06万
• 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/10298099
• 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; Epithelial; Evolution; Exposure to; Fishes; Functional disorder; Gene Expression; Genes; Genetic; Genetic Diseases; Genetic study; Germ Cells; Goals; Goldfish; Growth Factor; Hand; Histologic; Human; Human Genetics; Incisor; Knock-out; Larva; Link; Mesenchymal; Mesenchyme; Minerals; Minnows; Modeling; Molar tooth; Molecular; Molecular Biology; Morphogenesis; Morphology; Mus; Mutate; Mutation; Odontoblasts; Odontogenesis; Organ; Organogenesis; Orthologous Gene; Pathway interactions; Pharmacology; Phenotype; Play; Process; Pupa; Rattus; Receptor Signaling; Regulation; Retinoids; Role; Series; Shapes; Siblings; Signal Transduction; Signaling Molecule; Structure; Syndrome; Techniques; Testing; Tooth Abnormalities; Tooth Cell; Tooth Germ; Tooth structure; Tretinoin; Ursidae Family; Vertebrates; Work; Zebrafish; base; developmental genetics; differential expression; gene function; genetic analysis; human disease; inhibitor/antagonist; loss of function; malformation; morphogens; mutant; novel; oral ectoderm; 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.

摘要 畸形牙齿在人类中非常常见。在大多数情况下，它们是由于基因的遗传突变造成的 控制牙齿的形态发生。Runx2基因就是这种情况，一旦发生突变，就会导致 锁骨颅骨发育不良(CCD)是一种常染色体显性遗传综合征，以钉状牙为表现 人类。虽然在牙齿形态发生过程中发现了许多因素，但目前对此知之甚少。 关于细胞信号如何在牙齿发育过程中参与器官形状的建立。我们之前的工作是 发现信号分子维甲酸(RA)是鱼类牙齿诱导的主要因素之一。 最近的研究表明，RA在牙齿形态发生过程中也起着重要作用。尤其是鱼和斑马鱼， 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。这个 本研究的目的是了解维甲酸在牙齿形态发生过程中的作用。 在不同鱼类中，建立Ccd的FISH模型，阐明Runx2在牙齿中所起的作用 形态发生，并了解其与类风湿关节炎信号的联系。最后，该项目建议识别新的基因。 并通过基因敲除研究其在斑马鱼牙齿形态发生中的作用。 通过在牙齿形态发生过程中将鱼类胚胎和幼虫暴露于外源RA和RA抑制剂，我们将 能够理解RA信号在鱼类牙齿形态发生过程中的作用机制。我们的预赛 数据表明，牙胚不同细胞中的RA水平受时间和水平的控制 RA降解酶cyp26b1在发育中的牙胚细胞亚群中的表达。正在修改 因此，cyp26b1在牙胚中的表达将改变牙齿中可用的RA水平 细菌最终改变了牙齿的形状。我们将研究顺式法规的变化对 牙齿形态发生过程中cyp26b1表达时间的进化变化 近亲鱼类，斑马鱼和山地小鱼，有着截然不同的牙齿形状 在成体和胚胎发育期间。我们手中有斑马鱼runx2b(斑马鱼的直系同源物 在牙胚中表达的人类Runx2基因)功能丧失突变体。这个突变体将被用来 进行牙齿形态发生的表型分析，并了解RA信号之间的关系 和Runx2在牙齿形态发生过程中的表达。识别小说中在牙齿中起作用的基因 形态发生，我们将通过细胞分选，将暴露于外源RA信号的牙胚细胞进行比较 它们的转录组控制发育中的牙齿细胞在同一发育阶段。由此产生的基因 使用CRISPR/Cas9通过基因敲除对差异表达的基因进行表型分析 技术这项工作将揭示更多关于RA及其调控基因，包括Runx2，是如何控制牙齿的 发育、疾病(锁骨颅骨发育不良)和进化中的形态发生。