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

• 批准号: 10693325
• 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
• 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/10693325
• 关键词: Affinity Chromatography; Alleles; Ameloblasts; Amelogenesis; Amelogenesis Imperfecta; Award; Basement membrane; Biomimetics; Breeding; CRISPR/Cas technology; Clustered Regularly Interspaced Short Palindromic Repeats; 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; Mass Spectrum Analysis; Membrane; Mentors; Michigan; Minerals; Modeling; Molecular; Molecular Analysis; Mus; Mutation; Neonatal; Odontoblasts; 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; autosome; biomineralization; career; career development; conditional knockout; enamelin; experience; improved; insight; light microscopy; malformation; member; mineralization; mouse model; prevent; research and development; skills; tool

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.

项目摘要/摘要 我的职业目标是研究生物矿化的法规，特别是在搪瓷和牙本质形成中 发展和病理观点。我一直在研究非合成齿状发生 由DSPP基因突变引起的不完美fecta，以及由A中的突变引起的障碍症 基因谱。在这里，我提议研究成成成成婚的矿物色带 牙釉质形成中的附着/伸长络合物。 BM相关基因COL17A1，LAMA3和LAMB3中的单等位基因缺陷引起常染色体显性 人类中的症状不完美。它们在分泌的阿不成细胞中强烈表达，并随着本地化而定位 未观察到BM结构的搪瓷矿化前部。这些发现导致了以下假设 地下膜附着复合物的蛋白质是关键成分 延伸和东方牙釉质的成成木母细胞特异性矿物色带附着/伸长络合物 矿物质矿物质的丝带在分泌作用的阶段。 提出了三个具体目标（SA）。 SA1：确定成成布特异性矿物的关键成分 野生型（WT）小鼠中的色带附着/伸长络合物。 BM相关组件的定位 将在光和电子显微镜水平上定义，它们之间的蛋白质相互作用将是 探索。 SA2：通过有条件敲出Lama3，确定牙釉质形成过程中的LAMA3的功能 在成成木中的表达。已建立的amelx-icre鼠标模型将用于有条件地删除 LAMA3在可用LAMA3FL鼠标模型的成成布中的表达。分子和超微结构 将进行分析。 SA3：生成COL17A1条件敲除鼠标模型，并确定 搪瓷形成中XVII胶原型的功能。使用Easi-easi-house将生成Col17a1fl小鼠 CRISPR技术。小鼠将用amelx-icre小鼠分子和超微结构饲养 表征。该提案的完成将提高我们对搪瓷形成的理解，棚屋 淡淡的膜生成的治疗选择，并为牙釉质仿生学提供见解。这 COL17A1FL小鼠还将成为其他器官和组织中XVII胶原蛋白型研究的关键工具。 从培训的角度来看，该奖项将为我的独立性和开放场所奠定坚实的基础 未来的研究与合作。从科学上讲，我将开发电子显微镜，蛋白质组学的技能 分析和转基因小鼠产生，并增强我在研究设计和开发中的能力。 在专业上，我将提高我在科学沟通，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金记录的教职员工组成。这 密歇根大学的研究基础和支持性培训环境具有全面而强大的研究基础。 他们将共同将我转变为生物矿化领域的独立研究者。