sc-RNAseq of the day 4 zebrafish spinal cord. sc-RNAseq of the day 4 zebrafish spinal cord

Identification of the neuronal types that form the specialized circuits controlling distinct behaviors has benefited greatly from the simplicity offered by zebrafish. Electrophysiological studies have shown that additional to connectivity, understanding of circuitry requires identification of functional specializations among individual circuit components, such as those that regulate levels of transmitter release and neuronal excitability. In this study we use single cell RNAseq (scRNAseq) to identify molecular distinctions causal to the unique physiology of primary motoneuron (PMn) function, as well as associated specialized interneurons that are tailored specifically for mediation of the powerful escape response. Generating scRNA-seq datasets that provide both a wide overview of the cellular populations of the spinal cord and a granular view of the zebrafish motor neurons. Overall design: Cells from the spinal cords of 150 tg(SAIG213a;EGFP) zebrafish were analyzed via scRNA-seq and populations of interneurons were identified based on established markers. For examination of Motor neurons we additionally preformed FACS on cells dissociated from spinal cords of 150 tg(mnx1:GFP) zebrafish.

解析构成调控不同行为的特化神经环路的神经元类型，极大受益于斑马鱼（zebrafish）模型的简洁实验体系。电生理研究表明，除神经连接特性外，解析神经环路还需明确环路各组成成分的功能特化特征，例如调控递质释放水平与神经元兴奋性的环路组分。
本研究采用单细胞RNA测序（single cell RNAseq, scRNAseq）技术，鉴定导致初级运动神经元（primary motoneuron, PMn）功能独特生理特性的分子差异，以及特化用于介导强效逃逸反应的相关特异性中间神经元。本研究生成的scRNA-seq数据集，既可全面呈现脊髓细胞类群的整体转录组特征，又能细致展现斑马鱼运动神经元的精细表达谱。
实验设计：通过scRNA-seq分析150尾tg(SAIG213a;EGFP)转基因斑马鱼的脊髓细胞，并基于已报道的分子标记鉴定中间神经元类群。为解析运动神经元，本研究额外对150尾tg(mnx1:GFP)转基因斑马鱼解离的脊髓细胞预先开展了荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）。