MEMO1 is required for ameloblast maturation and functional enamel formation

Coordinated mineralization of soft tissue is central to organismal form and function, while dysregulated mineralization underlies several human pathologies. Oral epithelial derived ameloblasts are polarized, secretory cells responsible for generating enamel, the most mineralized substance in the human body. Defects in ameloblast development result in enamel anomalies, including amelogenesis imperfecta. Identifying proteins critical in ameloblast development can provide insight into specific pathologies associated with enamel related disorders or more broadly, mechanisms of mineralization. Previous studies identified a role for MEMO1 in bone mineralization; however, whether MEMO1 functions in the generation of additional mineralized structures remains unknown. Here, we identify a critical role for MEMO1 in enamel mineralization. First, we identified that Memo1 is expressed in ameloblasts and that conditional deletion of Memo1 from ameloblasts results in enamel defects, characterized by a decline in mineral density and tooth integrity. Molecular profiling of ameloblasts and their progenitors in Memo1 oral epithelial mutants revealed a disruption to cytoskeletal associated genes and a reduction in late stage ameloblast markers, relative to controls. Histology revealed that the molecular defects correlated with a disruption of the apical Tome's process and the basolateral interacting, papillary layer, in Memo1 mutant ameloblasts. Finally, deletion of Memo1 from an oral epithelial ameloblast cell line, followed by cellular and molecular analyses, revealed altered cytoskeletal networks, relative to control cells. Collectively, our findings integrate MEMO1 into an emerging network of molecules important for ameloblast development and provide both in vivo and in vitro systems to further interrogate the relationship of cytoskeletal and amelogenesis-related defects. Overall design: single cell RNA-sequencing of Memo1 mutant mouse ameloblast lineages and control tissue

软组织的协同矿化是生物体形态与功能的核心基础，而矿化失调则是多种人类疾病的病理诱因。口腔上皮来源的成釉细胞（ameloblasts）是一类极化分泌细胞，负责合成人体中矿化程度最高的组织——牙釉质。成釉细胞发育异常可导致牙釉质病变，其中包括釉质发育不全（amelogenesis imperfecta）。鉴定成釉细胞发育过程中的关键蛋白，既能为釉质相关疾病的特异性病理机制提供研究思路，也可为更广范畴的矿化机制研究提供参考。既往研究已证实MEMO1在骨矿化中发挥作用，但MEMO1是否参与其他矿化结构的生成仍未明确。本研究证实MEMO1在牙釉质矿化中具有关键功能。首先，我们发现Memo1在成釉细胞中表达，且从成釉细胞中条件性敲除Memo1会引发牙釉质缺陷，表现为矿化密度与牙齿完整性下降。对Memo1口腔上皮突变体中成釉细胞及其祖细胞的分子谱分析显示，与对照组相比，其细胞骨架相关基因表达紊乱，且晚期成釉细胞标志物水平降低。组织学分析显示，在Memo1突变体成釉细胞中，分子层面的缺陷与顶端托姆斯突（Tome's process）以及基底侧相互作用的乳头层的结构紊乱相关。最后，我们对口腔上皮成釉细胞系进行Memo1敲除，随后开展细胞与分子分析，结果显示与对照组细胞相比，其细胞骨架网络发生异常改变。综上，本研究将MEMO1纳入调控成釉细胞发育的新兴分子网络，并为深入探究细胞骨架与釉质发育缺陷之间的关联提供了体内与体外研究模型。实验设计概述：对Memo1突变小鼠的成釉细胞谱系及对照组织进行单细胞RNA测序（single cell RNA-sequencing）。