The TET-Sall4-BMP regulatory axis controls craniofacial cartilage development

Craniofacial microsomia (CFM) is a congenital defect that usually results from aberrant development of embryonic pharyngeal arches. However, the molecular basis of CFM pathogenesis is largely unknown. Here, we employ the zebrafish model to investigate mechanisms of CFM pathogenesis. In early embryos, tet2 and tet3 are essential for pharyngeal cartilage development. Single-cell RNA-sequencing reveals that loss of Tet2/3 impaired chondrocyte differentiation due to insufficient BMP signaling. Moreover, biochemical and genetic evidence reveals that the sequence-specific 5mC/5hmC-binding protein, Sall4, binds the promoter of bmp4 to activate bmp4 expression and control pharyngeal cartilage development. Mechanistically, Sall4 directs co-phase separation of Tet2/3 with Sall4 to form condensates that mediate 5mC oxidation on the bmp4 promoter, thereby promoting bmp4 expression and enabling sufficient BMP signaling. These findings suggest the TET-BMP-Sall4 regulatory axis is critical for pharyngeal cartilage development. Collectively, our study provides novel insights into understanding craniofacial development and CFM pathogenesis.面颅微型畸形（Craniofacial microsomia, CFM）是一类先天性发育缺陷，通常由胚胎期咽弓发育异常所导致，但其致病的分子基础迄今仍未明确。本研究利用斑马鱼模型，探究面颅微型畸形的致病机制。在早期胚胎中，tet2与tet3基因对咽弓软骨发育至关重要。单细胞RNA测序（Single-cell RNA-sequencing）结果显示，Tet2/3的缺失会因BMP信号通路激活不足而损害软骨细胞分化。此外，生化与遗传学实验证据表明，序列特异性5mC/5hmC结合蛋白Sall4可结合bmp4基因的启动子，激活bmp4的表达并调控咽弓软骨发育。从机制层面而言，Sall4介导Tet2/3与自身发生共相分离，形成凝聚体，介导bmp4基因启动子区域的5mC氧化修饰，进而促进bmp4表达、维持充足的BMP信号通路活性。上述研究结果表明，TET-BMP-Sall4调控轴对咽弓软骨发育至关重要。综上，本研究为阐明颅面发育及面颅微型畸形的致病机制提供了全新的见解。