METTL14 regulates chondrogenesis through the GDF5-RUNX-extracellular matrix gene axis during limb development.

m(6)A RNA methylation is essential for many aspects of mammalian development but its roles in chondrogenesis remain largely unknown. Here, we show that m(6)A is necessary for chondrogenesis and limb morphogenesis using limb progenitor-specific knockout mice of Mettl14, an essential subunit in the m(6)A methyltransferase complex. The knockout disrupts cartilage anlagen formation in limb buds with 11 downregulated proteins known to dysregulate chondrogenesis and shorten limb skeletons upon mutation in mice and humans. Further studies show a gene regulatory hierarchy among the 11 proteins. m(6)A stabilizes the transcript and increases the protein level of GDF5, a BMP family member. This activates the chondrogenic transcription factor genes Runx2 and Runx3, whose mRNAs are also stabilized by m(6)A. They promote the transcription of six collagen genes and two other chondrogenic genes, Ddrgk1 and Pbxip1. Thus, this study uncovers an m(6)A-based cascade essential for chondrogenesis during limb skeletal development.