Smart-seq2 analysis of larval zebrafish habenula from the gng8-GFP transgenic line. Smart-seq2 analysis of larval zebrafish habenula from the gng8-GFP transgenic line

The identification of cell types and marker genes is critical for dissecting neural development and function, but the size and complexity of the brain has hindered the comprehensive discovery of cell types. We combined single-cell RNA-seq with anatomical brain registration to create a comprehensive map of the zebrafish habenula, a conserved forebrain hub involved in pain processing and learning. Single-cell transcriptomes of ~13000 habenular cells (>4x coverage) identified 18 neuronal types and dozens of marker genes. Registration of marker genes onto a common reference atlas created a rich resource for anatomical and functional studies and enabled the mapping of active neurons onto neuronal types following aversive stimuli. Strikingly, despite brain growth and functional maturation, cell types were retained between the larval and adult habenula. This study provides a gene expression atlas to dissect habenular development and function and offers a general framework for the comprehensive characterization of other brain regions. Overall design: gng8-GFP zebrafish heads were dissected, dissociated and FAC sorted into 96 well plates. Single cell libraries were generated in batches of 384 cells using Smart-seq2. A total of 22 gng8-GFP fish were dissected in 3 batches and 384 cells were processed from each using Smart-seq2.细胞类型与标记基因的鉴定，是解析神经发育与功能的关键环节，但大脑的规模与复杂性，阻碍了细胞类型的系统性发现。我们将单细胞RNA测序（single-cell RNA-seq）与脑解剖配准技术相结合，构建了斑马鱼缰核（habenula）的综合图谱——缰核是一类保守的前脑枢纽，参与疼痛处理与学习过程。对约13000个缰核细胞开展单细胞转录组分析（测序覆盖度>4倍），共鉴定出18种神经元类型与数十种特异性标记基因。将标记基因配准至通用参考图谱后，可为解剖学与功能研究提供丰富的资源，并可实现厌恶性刺激后激活神经元与对应神经元类型的映射分析。值得注意的是，尽管伴随大脑生长与功能成熟，幼虫期与成年期斑马鱼缰核的细胞类型仍保持稳定。本研究不仅提供了可用于解析缰核发育与功能的基因表达图谱，还为其他脑区的全面表征提供了通用研究框架。实验总体设计：取gng8-GFP转基因斑马鱼头部进行解剖、解离，经FAC分选（Fluorescence-Activated Cell Sorting）后接种至96孔板；采用Smart-seq2技术，以每批384个细胞的规模构建单细胞文库。本研究共分3批解剖22尾gng8-GFP斑马鱼，每批均分选384个细胞并通过Smart-seq2完成文库构建。