The TET-BMP regulatory axis in pathogenesis of CFM [RNA-Seq]

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 zebrafish model to investigate the mechanism of CFM pathogenesis. In early embryos, tet2 and tet3 are highly expressed and are essential for pharyngeal cartilage development. Single-cell RNA sequencing and genetic analyses reveal that loss of Tet2/3 impaired chondrocyte differentiation largely due to insufficient BMP signaling. Mechanistically, Tet2/3-mediated 5-hydroxymethylcytosine modification allows the 5-hydroxymethylcytosine “reader”, Sall4, to specifically bind the bmp4 promoter, thereby promoting bmp4 expression and enabling efficient BMP signaling. These findings indicate the TET-BMP regulatory axis via 5-hydroxymethylcytosine to be critical for pharyngeal cartilage development. Whole-exome sequencing of CFM patient samples show that single nucleotide polymorphisms in TET and BMP pathway genes increase the risk of CFM. Collectively, our study provides novel insights into understanding craniofacial development and CFM pathogenesis. Overall design: Total RNAs were purified using TRIzol reagent (Life Technologies, 343702). Libraries were constructed using a VAHTS Universal V8 RNA-seq Library Prep Kit for MGI (Vazyme) according to the manufacturer's instructions and then sequenced on the MGISEQ-2000 platform.

面颅微畸形（Craniofacial microsomia, CFM）是一类先天性发育缺陷，通常由胚胎鳃弓发育异常所导致。然而，CFM发病的分子基础迄今尚未完全阐明。本研究采用斑马鱼模型，探究CFM的致病机制。在早期胚胎中，tet2与tet3呈高表达状态，且对鳃弓软骨发育不可或缺。单细胞RNA测序（single-cell RNA sequencing）与遗传分析结果显示，Tet2/3的缺失会显著损害软骨细胞分化，该缺陷主要归因于BMP信号通路激活不足。机制层面，Tet2/3介导的5-羟甲基胞嘧啶（5-hydroxymethylcytosine）修饰，可使5-羟甲基胞嘧啶"读取因子"Sall4特异性结合bmp4基因启动子，进而促进bmp4的表达并维持高效的BMP信号通路活性。上述研究结果表明，经由5-羟甲基胞嘧啶介导的TET-BMP调控轴，对鳃弓软骨发育至关重要。对CFM患者样本开展的全外显子测序（whole-exome sequencing）结果显示，TET与BMP通路基因中的单核苷酸多态性（single nucleotide polymorphisms）会提升CFM的发病风险。综上，本研究为理解颅面发育及CFM的致病机制提供了全新的研究视角。整体实验设计：总RNA采用TRIzol试剂（Life Technologies, 343702）提取；依照厂商说明书，使用VAHTS Universal V8 RNA-seq Library Prep Kit for MGI（Vazyme）构建测序文库，随后在MGISEQ-2000测序平台完成测序。