基于Single Cell RNA-seq的斑马鱼神经干细胞不对称分裂调控机制研究

Asymmetric division is an important way to regulate self-renewal and differentiation of neural stem cells, which is crucial to the brain development. However, compared to the invertebrates, much less is known about the regulation of asymmetric neural stem cell division in vertebrates. The present proposal will use brain ventricle-targeted electroporation, laser-based single cell microdissection, Single Cell RNA-seq, and genetic mosaic experiments to study the asymmetric division of neural stem cells at single-cell transcriptome resolution in the developing zebrafish brain. These studies will systemically explore the differentially expressed genes in paired daughter cells which are generated from asymmetric neural stem cell division, and identify 2 or 3 key genes or signaling pathway which are significantly differentially expressed in paired daughter cells. Further studies will be carried out to investigate how these molecular players regulate the asymmetric division of neural stem cells. The present study will advance the understanding of the regulation mechanisms of asymmetric neural stem cell division.

神经干细胞的不对称分裂，是调控其自我更新和分化的一个重要途径，对于大脑的正常发育至关重要。然而相对于无脊椎动物，对于脊椎动物体内神经干细胞不对称分裂的调控机制尚缺乏系统的认识。本项目拟采用活体斑马鱼胚胎脑室电转、单细胞激光捕获、Single Cell RNA-seq 、镶嵌遗传学分析等技术，以发育中的斑马鱼神经干细胞为研究对象，在单细胞全转录组水平上，系统性地研究神经干细胞分裂所产生的成对子细胞中存在的差异表达基因，创建基因差异表达谱，鉴定2-3个显著性差异表达的关键基因或信号通路，揭示其对于神经干细胞不对称分裂的调控作用。研究结果将丰富对调控神经干细胞不对称分裂调控机制的认识。

中枢神经系统中神经干细胞基因表达的时空调控对于神经发生和相关细胞命运的决定具有重要意义。谱系示踪和突变体表型的研究已经为解决发育问题提供了很多见解，但仍缺乏系统性的神经干细胞发育状态图谱。为了解决这一问题，我们采用单细胞测序技术从受精后24小时和3天斑马鱼胚胎前脑组织中，收集了15294个神经干细胞的转录组信息。基于相关的生物信息学分析，我们全面地绘制了这一发育时间段内神经干细胞的细胞分群图谱和发育轨迹，并探究不同细胞类群之间的基因表达差异，从而揭示早期发育过程中神经干细胞的转变。通过与Bulk-seq联合分析，我们发现了多个可能参与神经发育调控的重要候选调控因子，并对ranbp1基因的调控机制进行了深入研究，发现阻断该基因的功能时中枢神经系统发育受阻。我们的工作将进一步加深对神经干细胞早期发育的了解并为探究大脑的发育提供新的参考。