Ameloblast-specific mineral ribbon attachment/elongation complex in enamel formation

牙釉质形成中成釉细胞特异性矿物带附着/伸长复合物

基本信息

批准号：
10523565
负责人：
TIAN LIANG
金额：
$ 10.16万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10523565
关键词：
Affinity Chromatography Alleles Ameloblasts Amelogenesis Amelogenesis Imperfecta Award Basement membrane Biomimetics CRISPR/Cas technology Co-Immunoprecipitations Collaborations Collagen Type XVII Communication Complex DSPP gene Defect Dental Enamel Dental Enamel Hypoplasia Dentin Dentin Formation Dentinogenesis Imperfecta Development Distal Electron Microscopy Enamel Formation Environment Excision Exhibits Faculty Fostering Foundations Funding Generations Genes Goals Human Human Genetics ITGB4 gene Immunohistochemistry In Situ Hybridization Inner Enamel Epithelium Integrin beta4 Ions Junctional Epidermolysis Bullosa Knockout Mice Knowledge LAMC2 gene Laboratories Laminin Lead Light Mass Spectrum Analysis Membrane Mentors Michigan Minerals Modeling Molecular Molecular Analysis Mus Mutation Neonatal Odontogenesis Organ Organogenesis Pathologic Phenotype Process Proteins Proteomics Records Regulation Research Research Design Research Personnel Research Project Grants Role Scanning Electron Microscopy Skin Solid Techniques Therapeutic Intervention Tissues Training Transgenic Mice Universities Wild Type Mouse ameloblastin biomineralization career career development conditional knockout enamelin experience improved insight light microscopy malformation member mineralization mouse model prevent research and development tool

项目摘要

PROJECT SUMMARY/ABSTRACT My career goal is to study the regulations of biomineralization, particularly in enamel and dentin formation, from both developmental and pathological perspectives. I have been working on non-syndromic dentinogenesis imperfecta caused by mutations in the DSPP gene, and amelogenesis imperfecta caused by the mutations in a spectrum of genes. Here, I proposed to study the ameloblast-specific mineral ribbon attachment/elongation complex in enamel formation. Single allelic defects in BM-associated genes COL17A1, LAMA3, and LAMB3 cause autosomal dominant amelogenesis imperfecta in humans. They are strongly expressed in secretory ameloblasts, and localize along the enamel mineralization front, where no BM structure is observed. These findings lead to the hypothesis that the proteins of the basement membrane attachment complex are critical components of the ameloblast-specific mineral ribbon attachment/elongation complex that extends and orients enamel ribbons at the mineralization front during the secretory stage of amelogenesis. Three specific aims (SA) are proposed. SA1: Identify the critical components of the ameloblast-specific mineral ribbon attachment/elongation complex in wild-type (WT) mice. The localization of BM-associated components at the light and electron microscopy levels will be defined, and protein interactions among them will be explored. SA2: Determine the function of LAMA3 during enamel formation by conditionally knocking out Lama3 expression in ameloblasts. An established Amelx-iCre mouse model will be used to conditionally remove Lama3 expression in the ameloblasts of an available Lama3fl mouse model. Molecular and ultrastructural analyses will be performed. SA3: Generate a Col17a1 conditional knockout mouse model and determine the function of type XVII collagen in enamel formation. A Col17a1fl mouse will be generated using the Easi- CRISPR technology. Mice will be bred with Amelx-iCre mouse for molecular and ultrastructural characterization. The completion of this proposal will advance our understanding of enamel formation, shed light on the treatment options of amelogenesis imperfecta, and provide insights for enamel biomimetics. The Col17a1fl mouse will also become a critical tool for studies of type XVII collagen in other organs and tissues. From a training perspective, this award will set a solid foundation for my independence and open venues for future research and collaborations. Scientifically, I will develop skillsets in electron microscopy, proteomic analysis, and transgenic mouse generation, and strengthen my abilities in research design and development. Professionally, I will improve my abilities in scientific communication, laboratory management and mentoring. My mentor team consists of faculty members with sustained mentoring experience and funding records. The University of Michigan has a comprehensive and robust research basis and a supportive training environment. Together, they will foster my transition into an independent investigator in the field of biomineralization.

项目概要/摘要我的职业目标是研究生物矿化的调控，特别是牙釉质和牙本质的形成，从发展和病理的观点。我一直致力于非综合征性牙本质发生 DSPP 基因突变引起的不全症，以及 a 基因突变引起的釉质形成不全症基因谱。在这里，我提议研究成釉细胞特异性矿物带牙釉质形成中的附着/伸长复合体。 BM 相关基因 COL17A1、LAMA3 和 LAMB3 中的单一等位基因缺陷导致常染色体显性遗传人类的釉质形成不完善。它们在分泌性成釉细胞中强烈表达，并沿着牙釉质矿化前沿，未观察到 BM 结构。这些发现导致了这样的假设：基底膜附着复合物的蛋白质是细胞的重要组成部分成釉细胞特异性矿物带附着/伸长复合物，可延伸和定向牙釉质在釉质形成的分泌阶段，矿化前沿的带状结构。提出了三个具体目标（SA）。 SA1：识别成釉细胞特异性矿物质的关键成分野生型（WT）小鼠中的丝带附着/伸长复合物。 BM相关组件的本地化在光学和电子显微镜水平上将被定义，并且它们之间的蛋白质相互作用将被确定探索过。 SA2：通过有条件敲除Lama3来确定LAMA3在牙釉质形成过程中的功能在成釉细胞中表达。已建立的 Amelx-iCre 小鼠模型将用于有条件地去除 Lama3 在可用 Lama3fl 小鼠模型的成釉细胞中的表达。分子和超微结构将进行分析。 SA3：生成Col17a1条件敲除小鼠模型并确定 XVII 型胶原蛋白在牙釉质形成中的功能。将使用 Easi- 生成 Col17a1fl 小鼠 CRISPR技术。小鼠将与 Amelx-iCre 小鼠一起培育用于分子和超微结构表征。该提案的完成将增进我们对牙釉质形成、脱落的理解阐明釉质生成不全的治疗方案，并为牙釉质仿生学提供见解。这 Col17a1fl小鼠也将成为研究其他器官和组织中XVII型胶原蛋白的重要工具。从培训的角度来看，这个奖项将为我的独立性奠定坚实的基础，并为我提供开放的场地。未来的研究和合作。在科学方面，我将发展电子显微镜、蛋白质组学方面的技能分析，转基因小鼠生成，并增强我的研究设计和开发能力。在专业上，我将提高自己在科学交流、实验室管理和指导方面的能力。我的导师团队由具有持续指导经验和资助记录的教师组成。这密歇根大学拥有全面而强大的研究基础和支持性的培训环境。他们将共同促进我转变为生物矿化领域的独立研究者。