Enamel atlas: systems-level amelogenesis tools at multiple scales

摘要 牙釉质缺陷，无论是先天性的，后天性的，还是环境性的，都与巨大的成本有关 对社会也有深刻的心理影响。尽管过去十年取得了重大进展， 形成釉质的发育过程，称为釉质形成，仍然知之甚少。我们有 我们发现至少有两个因素拖延了进展，我们建议在本申请中解决这两个因素。 一个是现有的小鼠试剂，它提供了理解基因调控的主要模型， 釉质发生，有缺陷，阻碍解剖机制在体内。另一个挑战是， 关于釉质的纳米结构和相组成的信息已经开始出现， 模型没有考虑到。获得强大的新基因方法，“组学”技术的能力 因此，材料表征方法创造了前所未有的机会， 新的工具，将有助于推动釉质发生研究到一个新的水平。 我们建议充分利用这些最新的技术进步和 我们的团队致力于创建一套集成的、多模式的工具和参考资料。具体来说，我们将生成 创新的小鼠试剂，包括牙釉质形成阶段特异性Cre驱动程序、报告程序和条件 构成釉质基质的关键结构和蛋白水解参与者的敲除和敲入模型， 将使工作流程能够在2015年对转录（使用RNA测序）和翻译（使用蛋白质组学）进行分析， 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 细胞水平的分子分析将通过对结构，组成， 和机械性能的形成和成熟的釉质在重叠的长度尺度。通过基因定位 表达，指定局部蛋白质组，并定量评估扰动对每个这些蛋白质组的影响。 水平上的材料性能的釉质，我们将创建一个平台， 研究人员，帮助阐明疾病的机制，并奠定基础，使新的发展， 干预的方法。

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.