Table_11_An Alternative Splicing Program for Mouse Craniofacial Development.xlsx

Alternative splicing acts as a fundamental mechanism to increase the number of functional transcripts that can be derived from the genome – and its appropriate regulation is required to direct normal development, differentiation, and physiology, in many species. Recent studies have highlighted that mutation of splicing factors, resulting in the disruption of alternative splicing, can have profound consequences for mammalian craniofacial development. However, there has been no systematic analysis of the dynamics of differential splicing during the critical period of face formation with respect to age, tissue layer, or prominence. Here we used deep RNA sequencing to profile transcripts expressed in the developing mouse face for both ectodermal and mesenchymal tissues from the three facial prominences at critical ages for facial development, embryonic days 10.5, 11.5, and 12.5. We also derived separate expression data from the nasal pit relating to the differentiation of the olfactory epithelium for a total of 60 independent datasets. Analysis of these datasets reveals the differential expression of multiple genes, but we find a similar number of genes are regulated only via differential splicing, indicating that alternative splicing is a major source of transcript diversity during facial development. Notably, splicing changes between tissue layers and over time are more prevalent than between prominences, with exon skipping the most common event. We next examined how the variation in splicing correlated with the expression of RNA binding proteins across the various datasets. Further, we assessed how binding sites for splicing regulatory molecules mapped with respect to intron exon boundaries. Overall these studies help define an alternative splicing regulatory program that has important consequences for facial development.

可变剪接（alternative splicing）是提升基因组可编码功能性转录本数量的核心机制，在众多物种中，其精准调控对于维持正常发育、细胞分化及生理稳态至关重要。近期研究表明，剪接因子突变会引发可变剪接紊乱，进而对哺乳动物颅面部发育造成深远影响。然而，目前尚未有研究针对面部形成关键时期内，差异剪接的动态变化按年龄、组织层或面部突起开展系统性分析。本研究借助深度RNA测序技术，对小鼠面部发育关键阶段（胚胎第10.5、11.5及12.5天）的三个面部突起的外胚层与间充质组织中的表达转录本进行了表征分析；同时针对与嗅上皮分化相关的鼻凹（nasal pit）获取了独立的表达数据集，最终累计获得60个独立样本数据集。对上述数据集的分析揭示了多个基因的差异表达现象，同时发现数量相当的基因仅通过可变剪接实现调控，这表明可变剪接是面部发育过程中转录本多样性的重要来源。值得注意的是，不同组织层间以及随时间推移发生的剪接变化，远比面部突起间的剪接变化更为普遍，其中外显子跳跃（exon skipping）是最为常见的剪接事件。随后，我们基于多组数据集分析了剪接变异与RNA结合蛋白（RNA binding protein）表达水平之间的相关性；此外还对剪接调控分子的结合位点在内含子-外显子边界处的分布特征进行了评估。综上，本研究明确了一套对颅面部发育具有重要调控作用的可变剪接调控程序。