METTL3 Modulates Ctsk+ Lineage Supporting Cranial Osteogenesis via Hedgehog

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 Sufu^fl/+ 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.