Enamel atlas: systems-level amelogenesis tools at multiple scales

摘要 牙釉质缺陷，无论是先天的、后天的还是环境的，都会带来巨大的成本。 对社会也有深刻的心理影响。尽管在过去十年中取得了重大进展，但 形成釉质的发育过程，也就是所谓的釉质发生，目前仍知之甚少。我们有 确定了至少两个延误进展的因素，我们建议在本申请中解决这两个因素。 一种是现有的小鼠试剂，它们为理解遗传调控提供了基本模型 成釉作用存在缺陷，阻碍了体内机制的剖析。另一个挑战是新的 关于牙釉质的纳米结构和相组成的信息已经开始浮出水面 模型没有考虑到这一点。获得强大的新基因方法的能力，“组学”技术 因此，材料表征方法创造了前所未有的机会，以产生复杂的 新的工具将有助于将釉质发生研究推向新的水平。 我们建议充分利用这些最新的技术进步和补充的专业知识 我们的团队创建了一套集成的、多模式的工具和参考材料。具体来说，我们将生成 创新的小鼠试剂，包括釉质发生阶段特定的Cre驱动程序、记者和有条件的 组成牙釉质基质的关键结构和蛋白水解物的敲除和敲入模型，它 将启用一个工作流来分析转录(使用RNA测序)和翻译(使用蛋白质组学)，地址为 在特定的发育阶段，甚至在单细胞的基础上(使用单细胞RNA测序)。组织和 细胞级分子图谱的补充将是对结构、组成、 以及在重叠长度尺度上形成和成熟牙釉质的力学性能。通过定位基因 表达，指定局部蛋白质组，并定量评估这些干扰的影响 在釉质的材料属性上，我们将打造一个赋能釉质发生的平台 研究人员，帮助描绘疾病的机制，并为开发新的 干预方法。

ABSTRACT Enamel defects, whether congenital, acquired, or environmental in origin, are associated with a significant cost to society and also have profound psychological impacts. Despite significant progress over the last decade, the developmental process that gives rise to enamel, known as amelogenesis, remains poorly understood. We have identified at least two factors that have delayed progress, and which we propose to address in this application. One is that existing mouse reagents, which provide the primary model for understanding genetic regulation of amelogenesis, have deficiencies that hinder dissecting the mechanisms in vivo. Another challenge is that new information regarding the nanostructure and phase composition of enamel have begun to emerge that prior models did not take into account. The ability to access powerful new genetic approaches, “omics” techniques and materials characterization methods therefore creates unprecedented opportunities to generate sophisticated new tools that will help push amelogenesis research to the next level. We propose to take full advantage of these recent technical advances and of the complementary expertise of our team to create an integrated, multi-modal set of tools and reference materials. Specifically, we will generate innovative mouse reagents, including amelogenesis-stage specific Cre drivers, reporters and conditional knockout and knock-in models of key structural and proteolytic players that constitute the enamel matrix, which will enable a workflow to profile transcription (using RNA sequencing) and translation (using proteomics) at specific developmental stages, and even on a single cell basis (using single-cell RNA sequencing). Tissue and cell-level molecular profiling will be complemented by an in-depth characterization of the structure, composition, and mechanical properties of forming and mature enamel at overlapping length scales. By mapping gene expression, specifying local proteomes, and quantitatively assessing impact of the perturbations at each of these levels on the materials properties of enamel, we will create a platform that will empower amelogenesis researchers, help delineate mechanisms of disease, and lay the groundwork to enable the development of new approaches of intervention.