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

项目总结/摘要 我的职业目标是研究生物矿化的规律，特别是在牙釉质和牙本质的形成， 从发展和病理的角度来看。我一直在研究非综合征性牙本质形成 由DSPP基因突变引起的牙釉质发育障碍，以及由A基因突变引起的牙釉质发育障碍。 基因谱。在这里，我提议研究成釉细胞特有的矿物带 附着/延伸复合体在釉质形成中的作用。 BM相关基因COL 17 A1、LAMA 3和LAMB 3的单一等位基因缺陷导致常染色体显性遗传 人类的牙釉质发生它们在分泌型成釉细胞中强烈表达，并定位于沿着 釉质矿化前沿，其中没有观察到BM结构。这些发现引出了一个假设， 基底膜附着复合物的蛋白质是细胞膜的关键组分。 成釉细胞特异性矿物带附着/延伸复合体，其延伸和定向釉质 在釉质形成的分泌阶段，在矿化前沿的带状物。 提出了三个具体目标（SA）。SA 1：确定成釉细胞特定矿物质的关键成分 在野生型（WT）小鼠中的带状附着/延伸复合物。BM相关组件的本地化 在光学和电子显微镜水平将被定义，蛋白质之间的相互作用将被定义。 探讨了SA 2：通过条件性敲除Lama 3来确定Lama 3在釉质形成期间的功能 在成釉细胞中表达。建立的Amelx-iCre小鼠模型将用于条件性去除 可用的Lama 3fl小鼠模型的成釉细胞中的Lama 3表达。分子和超微结构 将进行分析。SA 3：生成Coll 7a 1条件性敲除小鼠模型并确定Col 17 a1基因敲除小鼠模型的表达。 XVII型胶原在釉质形成中的作用。将使用Easi-Mouse生成Col 17 a1 fl小鼠。 CRISPR技术。小鼠将与Amelx-iCre小鼠一起繁殖，以进行分子和超微结构检查。 特征化这项提案的完成将推进我们对釉质形成的理解， 阐明了釉质发育不全的治疗方案，并为釉质仿生学提供了见解。的 Col 17 a1 fl小鼠也将成为研究其他器官和组织中XVII型胶原的重要工具。 从培训的角度来看，这个奖项将为我的独立性和开放的场地奠定坚实的基础， 未来的研究与合作。作为科学家，我将在电子显微镜，蛋白质组学 分析和转基因小鼠的产生，并加强我在研究设计和开发方面的能力。 实习期间，我将提高我在科学交流，实验室管理和指导方面的能力。 我的导师团队由具有持续指导经验和资助记录的教师组成。的 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。 总之，他们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.