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

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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.