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

项目摘要/摘要 我的职业目标是研究生物矿化的规律，特别是牙釉质和牙本质的形成。 无论是发育角度还是病理角度。我一直在研究非综合征性牙本质形成 由DSPP基因突变引起的釉质发育不全，以及由 基因图谱。在这里，我提议研究成釉细胞特有的矿物带 牙釉质形成中的附着/伸长复合体。 BM相关基因COL17A1、LAMA3和LAMB3的单等位基因缺陷导致常染色体显性遗传 人类的成釉发育不完全。它们在分泌型成釉细胞中强烈表达，并定位于 釉质矿化前锋，未观察到BM构造。这些发现导致了这样的假设 基底膜附着复合体的蛋白质是 成釉细胞特有的矿物质带附着/延长复合体，延伸和定向釉质 成釉作用分泌期矿化前沿的条带。 提出了三个具体目标(SA)。SA1：确定成釉细胞特有矿物质的关键成分 野生型(WT)小鼠的带状附着/延长复合体。BM相关组件的国产化 在光学和电子显微镜水平上，将定义它们之间的蛋白质相互作用 探索过了。SA2：通过有条件地敲除LAMA3来确定LAMA3在釉质形成过程中的作用 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移除 Lama3f1小鼠模型成釉细胞中lama3的表达。分子和超微结构 将执行分析。SA3：建立COL17A1条件性基因敲除小鼠模型并确定 XVI型胶原在牙釉质形成中的作用一只Col17a1fl小鼠将使用EASI- CRISPR技术。用Amelx-iCre小鼠培育小鼠进行分子和超微结构研究 人物刻画。这一建议的完成将促进我们对釉质形成的理解 阐明釉质发育不全的治疗方案，并为釉质仿生学提供见解。这个 Col17a1fl小鼠也将成为研究其他器官和组织中XVI型胶原的关键工具。 从训练的角度来看，这个奖项将为我的独立和开放场地奠定坚实的基础 未来的研究和合作。在科学上，我将发展电子显微镜、蛋白质组学方面的技能 分析，转基因小鼠的产生，并增强我的研究设计和开发能力。 在专业方面，我将提高我在科学交流、实验室管理和指导方面的能力。 我的导师团队由拥有长期导师经验和资助记录的教职员工组成。这个 密歇根大学拥有全面和强大的研究基础和支持性的培训环境。 他们一起，将促进我转变为生物矿化领域的独立调查员。

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.