Cell type-specific alternative splicing of cytoskeletal domains governs cell fate in the developing cerebral cortex

Alternative splicing regulates over 90% of multiexon mammlian genes, but its role in specifying neural progenitor cell (NPC) fates has not been explored. Our analyses of purified mouse NPCs and neurons from developing cerebral cortices revealed hundreds of conserved and differentially spliced exons that add or remove key protein domains, especially in genes regulating the cytoskeleton. TBR2-EGFP BAC transgenic mice, generated by GENSAT and outcrossed to FVB strain for 2 generations, drives EGFP expression in the vast majority of intermediate progenitors and neurons at E14.5. We FACS sorted EGFP positive (differentiating and differentiated neurons) and negative (neural progenitors) cells from the dorsal cerebral cortices of E14.5 TBR2-EGFP. Two replicates for each population were subjected to RNA sequencing. Alternatively spliced exons between the two populations (NPCs and neurons) were identified using MISO.可变剪接（alternative splicing）调控超过90%的多外显子哺乳动物基因，但其在调控神经前体细胞（neural progenitor cell, NPC）命运中的作用尚未得到研究。我们对发育中小鼠大脑皮层中分离纯化的神经前体细胞及神经元进行分析，鉴定出数百个保守的差异剪接外显子，此类外显子可添加或移除关键蛋白质结构域，尤其在调控细胞骨架的基因中富集。由GENSAT构建的TBR2-EGFP细菌人工染色体（bacterial artificial chromosome, BAC）转基因小鼠，经与FVB品系回交两代后，可在胚胎发育第14.5天（E14.5）时，在绝大多数中间前体细胞及神经元中驱动增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）的表达。我们从该转基因小鼠E14.5天的背侧大脑皮层中，通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）分离得到EGFP阳性细胞（正在分化及已分化的神经元）与阴性细胞（神经前体细胞）。每个细胞群体设置两个生物学重复，随后进行RNA测序（RNA sequencing）。最终利用MISO软件鉴定出两个细胞群体（神经前体细胞与神经元）之间的差异可变剪接外显子。