Investigating the Etiology of Craniofacial and Cardiac Malformations in a Mouse Model of SF3B4-Related Syndromes

Pathogenic variants in SF3B4 are responsible for the acrofacial disorders Nager and Rodriguez Syndrome, also known as SF3B4-related Syndromes, associated with malformations in the head, face, limbs, vertebrae as well as the heart. To uncover the etiology of craniofacial malformations found in SF3B4-related syndromes, mutant mouse lines with homozygous deletion of Sf3b4 in neural crest cells (NCC) were generated. Like in human patients, these embryos had craniofacial and cardiac malformations with variable expressivity and penetrance. The severity and survival of Sf3b4 NCC mutants was modified by the level of Sf3b4 in neighbouring non NCC. RNA sequencing analysis of heads of embryos prior to morphological abnormalities showed significant changes in expression of genes forming the NCC regulatory network, as well an increase in exon skipping. We identified several key transcription factors and histone modifiers involved in craniofacial and cardiac development with increased exon skipping. Increased exon skipping was also associated with use of a more proximal branch point, as well as an enrichment in thymidine bases in the 50bp around the branch points. We propose that decrease in Sf3b4 causes changes in the expression and splicing of transcripts required for proper craniofacial and cardiac development, leading to abnormalities. To investigate the role of Sf3b4 in craniofacial development we generated mouse embryos with deletion of Sf3b4 in neural crest cells. Heads of 3 embryos prior to morphological abnormalities were pooled. 3 such pools were generated and analyzed from Sf3b4 wild type and Sf3b4 neural crest cell mutant embryos.SF3B4的致病变异是肢面发育障碍类疾病——Nager综合征（Nager Syndrome）与Rodriguez综合征（Rodriguez Syndrome，亦称SF3B4相关综合征（SF3B4-related Syndromes））——的致病根源，该类综合征伴随头面部、四肢、脊椎及心脏的畸形发育。为阐明SF3B4相关综合征患者颅面畸形的致病机制，研究人员构建了神经嵴细胞（neural crest cells, NCC）中Sf3b4纯合缺失的突变小鼠品系。与人类患者表型一致，这些胚胎同样表现出可变表现度与外显率的颅面及心脏畸形。Sf3b4神经嵴细胞突变体的表型严重程度与存活情况，可被邻近非神经嵴细胞中的Sf3b4表达水平所调控。在形态学异常出现前，对胚胎头部进行RNA测序（RNA sequencing）分析，结果显示构成神经嵴细胞调控网络的基因表达发生显著改变，同时外显子跳跃（exon skipping）事件增多。研究人员鉴定出多个参与颅面与心脏发育的关键转录因子及组蛋白修饰因子（histone modifiers），其编码转录本出现了外显子跳跃事件增加的情况。外显子跳跃事件的增加还与更靠近上游的剪接分支点（branch point）使用相关，且在分支点上下游50bp区域内存在胸腺嘧啶碱基富集现象。研究团队提出，Sf3b4表达下调会导致颅面与心脏正常发育所需转录本的表达及剪接模式发生改变，进而引发畸形发育。为探究Sf3b4在颅面发育中的作用，研究人员构建了神经嵴细胞中Sf3b4缺失的小鼠胚胎模型。研究人员收集了形态学异常出现前的3个胚胎头部样本进行混合，共构建并分析了3组野生型Sf3b4胚胎与3组Sf3b4神经嵴细胞突变胚胎的混合样本。