Transcriptome Sequencing During Mouse Brain Development Identifies Long Non-Coding RNAs Functionally Involved in Neurogenic Commitment

Transcriptome analysis of somatic stem cells and their progeny is fundamental to identify new factors controlling proliferation versus differentiation during tissue formation. Here we generated a combinatorial, fluorescent reporter mouse line to isolate proliferating neural stem cells, differentiating progenitors and newborn neurons that coexist as intermingled cell populations during brain development. Transcriptome sequencing revealed numerous novel long non-coding (lnc)RNAs and uncharacterized protein-coding transcripts identifying the signature of neurogenic commitment. Importantly, most lncRNAs overlapped neurogenic genes and shared with them a nearly identical expression pattern suggesting that lncRNAs control corticogenesis by tuning the expression of nearby cell fate determinants. We assessed the power of our approach by manipulating lncRNAs and protein-coding transcripts with no function in corticogenesis reported to date. This led to several evident phenotypes in neurogenic commitment and neuronal survival indicating that our study provides a remarkably high number of uncharacterized transcripts with hitherto unsuspected roles in brain development. Finally, we focussed on one lncRNA, Miat, whose manipulation was found to trigger pleiotropic effects on brain development and aberrant splicing of Wnt7b. Hence, our study suggests that lncRNA-mediated alternative splicing of cell fate determinants controls stem cell commitment during neurogenesis. “LncRNAs control neurogenesis” Aprea, Prenninger, Dori, Monasor, Wessendof, Zocher, Massalini, Ghosh, Alexopoulou, Lesche, Dahl, Groszer, Hiller, Calegari, The EMBO Journal (In Press) Overall design: mRNA profiles of Proliferating Progenitors, Differentiating Progenitors and Neurons from lateral cortex of E14.5 mouse embryos. Each cell type in three biological replicates.

体细胞干细胞及其子代的转录组分析，是鉴定组织形成过程中调控细胞增殖与分化的新型关键因子的核心研究基础。本研究构建了一款组合式荧光报告小鼠品系，可用于分离脑发育过程中共存于混杂细胞群中的增殖型神经干细胞、分化型祖细胞与新生神经元。转录组测序分析揭示了大量新型长链非编码RNA（long non-coding RNA，lncRNA）与未表征的蛋白编码转录本，明确了神经发生定向分化的特征谱。值得注意的是，多数lncRNA与神经发生基因区域重叠，且二者表达模式近乎一致，这提示lncRNA通过调控邻近细胞命运决定因子的表达来调控大脑皮层发育。我们通过靶向操控迄今为止尚未见皮层发育功能报道的lncRNA与蛋白编码转录本，验证了本研究方法的效能。该操作在神经发生定向分化与神经元存活过程中引发了多种显著表型，表明本研究鉴定出大量未表征的转录本，其在脑发育中发挥着此前未被认知的功能。最后，我们聚焦于其中一款lncRNA Miat，研究发现对其进行操控可引发脑发育的多效性效应以及Wnt7b的异常剪接。综上，本研究提示，lncRNA介导的细胞命运决定因子可变剪接，可调控神经发生过程中的干细胞定型。"长链非编码RNA调控神经发生" 作者：Aprea、Prenninger、Dori、Monasor、Wessendof、Zocher、Massalini、Ghosh、Alexopoulou、Lesche、Dahl、Groszer、Hiller、Calegari，发表于《The EMBO Journal》（已接收待刊）实验整体设计：对E14.5小鼠胚胎外侧皮层来源的增殖型祖细胞、分化型祖细胞与神经元进行mRNA表达谱分析，每种细胞类型设置3次生物学重复。