N6-methyladenosine (m6A) modification, a eukaryotic messenger RNA modification catalyzed by methyltransferase-like 3 (METTL3), plays a pivotal role in stem cell fate determination. Calvarial bone development and maintenance are orchestrated by the cranial sutures. Cathepsin K (CTSK)–positive calvarial stem cells (CSCs) contribute to mice calvarial ossification. However, the role of m6A modification in regulating Ctsk+ lineage cells during calvarial development remains elusive. Here, we showed that METTL3 was colocalized with cranial nonosteoclastic Ctsk+ lineage cells, which were also associated with GLI1 expression. During neonatal development, depletion of Mettl3 in the Ctsk+ lineage cells delayed suture formation and decreased mineralization. During adulthood maintenance, loss of Mettl3 in the Ctsk+ lineage cells impaired calvarial bone formation, which was featured by the increased bone porosity, enhanced bone marrow cavity, and decreased number of osteocytes with the less-developed cellular outline. The analysis of methylated RNA immunoprecipitation sequencing and RNA sequencing data indicated that loss of METTL3 reduced Hedgehog (Hh) signaling pathway. Restoration of Hh signaling pathway by crossing Sufufl/+ alleles or by local administration of SAG21 partially rescued the abnormity. Our data indicate that METTL3 modulates Ctsk+ lineage cells supporting calvarial bone formation by regulating the Hh signaling pathway, providing new insights for clinical treatment of skull vault osseous diseases.

中文翻译：

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METTL3 通过 Hedgehog 调节 Ctsk+ 谱系支持颅骨成骨


N6-甲基腺苷 (m6A) 修饰是一种由甲基转移酶样 3 (METTL3) 催化的真核信使 RNA 修饰，在干细胞命运决定中发挥着关键作用。颅骨的发育和维护是由颅缝协调的。组织蛋白酶 K (CTSK) 阳性颅骨干细胞 (CSC) 有助于小鼠颅骨骨化。然而，m6A 修饰在颅骨发育过程中调节 Ctsk+ 谱系细胞的作用仍然难以捉摸。在这里，我们发现 METTL3 与颅骨非破骨细胞 Ctsk+ 谱系细胞共定位，这也与 GLI1 表达相关。在新生儿发育过程中，Ctsk+谱系细胞中Mettl3的消耗延迟了缝线的形成并减少了矿化。在成年维持过程中，Ctsk+谱系细胞中Mettl3的缺失会损害颅骨骨形成，其特点是骨孔隙度增加、骨髓腔增强、骨细胞数量减少且细胞轮廓欠发达。甲基化RNA免疫沉淀测序和RNA测序数据的分析表明，METTL3的缺失减少了Hedgehog (Hh)信号通路。通过跨越 Sufufl/+ 等位基因或通过局部施用 SAG21 恢复 Hh 信号通路可部分挽救该异常。我们的数据表明，METTL3通过调节Hh信号通路来调节支持颅骨骨形成的Ctsk+谱系细胞，为颅骨穹窿骨疾病的临床治疗提供新的见解。