Abstract：
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.
摘要：
N6-甲基腺苷（m6A）修饰，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起着举足轻重的作用。颅骨的发育和维持是由颅骨缝合线协调的。组织蛋白酶K（CTSK）阳性的颅骨干细胞（CSC）有助于小鼠颅骨骨化。然而,在颅骨发育过程中，m6A修饰在调节Ctsk谱系细胞中的作用仍然难以捉摸。这里,我们显示METTL3与颅骨非破骨细胞Ctsk谱系细胞共定位，也与GLI1表达相关。在新生儿发育过程中,Ctsk谱系细胞中Mettl3的消耗延迟了缝合线的形成并减少了矿化。在成年期间维持，Ctsk+谱系细胞中Mettl3的丢失损害了颅骨形成，其特点是骨骼孔隙率增加，增强的骨髓腔，细胞轮廓不发达的骨细胞数量减少。甲基化RNA免疫沉淀测序和RNA测序数据的分析表明METTL3的缺失降低了Hedgehog(Hh)信号通路。通过交叉Sufufl/+等位基因或通过局部施用SAG21来恢复Hh信号通路部分地挽救了该异常。我们的数据表明，METTL3通过调节Hh信号通路调节支持颅骨骨形成的Ctsk+谱系细胞，为颅骨穹窿骨病的临床治疗提供新的见解。
