Alternative splicing is frequent during early embryonic development in mouse

Background Alternative splicing is known to increase the complexity of mammalian transcriptomes since nearly all mammalian genes express multiple pre-mRNA isoforms. However, our knowledge of the extent and function of alternative splicing in early embryonic development is based mainly on a few isolated examples. High throughput technologies now allow us to study genome-wide alternative splicing during mouse development. Results A genome-wide analysis of alternative isoform expression in embryonic day 8.5, 9.5 and 11.5 mouse embryos and placenta was carried out using a splicing-sensitive exon microarray. We show that alternative splicing and isoform expression is frequent across developmental stages and tissues, and is comparable in frequency to the variation in whole-transcript expression. The genes that are alternatively spliced across our samples are disproportionately involved in important developmental processes. Finally, we find that a number of RNA binding proteins, including putative splicing factors, are differentially expressed and spliced across our samples suggesting that such proteins may be involved in regulating tissue and temporal variation in isoform expression. Using an example of a well characterized splicing factor, Fox2, we demonstrate that changes in Fox2 expression levels can be used to predict changes in inclusion levels of alternative exons that are flanked by Fox2 binding sites. Conclusions We propose that alternative splicing is an important developmental regulatory mechanism. We further propose that gene expression should routinely be monitored at both the whole transcript and the isoform level in developmental studies. 25 samples were analyzed. Developmental stages e8.5, e9.5 and e11.5 (embryos from all 3, placenta for only e9.5 and e11.5). 5 biological replicates for each.背景 可变剪接（alternative splicing）已知可提升哺乳动物转录组（transcriptome）的复杂度，因为几乎所有哺乳动物基因均可表达多种前mRNA（pre-mRNA）剪接异构体。然而，目前学界对于早期胚胎发育中可变剪接的发生范围与功能的认知，主要仅基于少数孤立的研究案例。如今，高通量（high-throughput）技术已使我们能够在全基因组层面研究小鼠发育过程中的可变剪接。 结果 本研究采用剪接敏感型外显子微阵列（splicing-sensitive exon microarray），对胚胎发育第8.5、9.5及11.5天的小鼠胚胎与胎盘组织中的可变异构体表达进行了全基因组分析。研究结果显示，可变剪接与异构体表达在不同发育阶段及组织中广泛存在，其发生频率与全转录本表达的变异程度相当。在本研究样本中发生可变剪接的基因，显著富集于关键发育过程相关的功能通路中。最后，本研究发现多个RNA结合蛋白（RNA binding protein）——包括推定的剪接因子（putative splicing factor）——在样本中呈现出差异表达与差异剪接特征，提示这类蛋白可能参与调控异构体表达的组织特异性与时间特异性变异。本研究以已被充分表征的剪接因子Fox2为例，证实Fox2表达水平的变化可用于预测侧翼带有Fox2结合位点的可变外显子的包含水平变化。 结论 本研究提出，可变剪接是一类重要的发育调控机制。本研究进一步建议，在发育生物学研究中，应常规同时从全转录本与异构体层面监测基因表达情况。本研究共分析25份样本：发育阶段涵盖e8.5、e9.5及e11.5天（其中3个阶段均采集胚胎样本，仅e9.5与e11.5天采集胎盘样本），每个条件设置5次生物学重复。