10X genomics analysis of adult and larval habenula from the gng8-GFP transgenic line. 10X genomics analysis of adult and larval 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: For larval dataset, multiple gng8-GFP labeled zebrafish brains were dissociated and habenular cells FAC sorted into PBS+1%BSA and immediately loaded into 10X chromium for cell capture. For adult dataset, 5-6 habenulae were cleanly dissected out, dissociated, filtered in PBS+BSA and immediately captured in droplets with 10x platform. Both libraries were then constructed using standard instrutions of the 10x plaform.

细胞类型与标记基因的鉴定，对于解析神经发育与神经功能至关重要，但大脑的规模与复杂性极大阻碍了细胞类型的全面发现。我们将单细胞RNA测序（single-cell RNA-seq）与大脑解剖配准技术相结合，构建了斑马鱼缰核（habenula）的综合图谱——该结构为保守的前脑中枢，参与疼痛处理与学习过程。
对约13000个缰核细胞（测序覆盖度>4倍）的单细胞转录组分析，成功鉴定出18种神经元类型与数十个标记基因。将标记基因定位至通用参考图谱，可为解剖学与功能研究提供丰富的资源，并能在厌恶刺激后将激活神经元映射至对应的神经元类型。
令人惊喜的是，尽管经历了大脑发育与功能成熟，幼体与成体斑马鱼的缰核仍保留了一致的细胞类型。本研究构建了可用于解析缰核发育与功能的基因表达图谱，并为其他脑区的全面特征分析提供了通用研究框架。
整体实验设计：对于幼体数据集，我们解离了多例经gng8-GFP标记的斑马鱼大脑，通过荧光激活细胞分选（FACS）将缰核细胞收集至含1%牛血清白蛋白（bovine serum albumin, BSA）的磷酸盐缓冲液（phosphate-buffered saline, PBS）中，并立即使用10X Chromium系统完成细胞捕获。对于成体数据集，我们精准分离出5~6个缰核组织，经解离、过滤后，在含BSA的PBS中立即通过10X平台完成液滴包埋。随后，两个测序文库均按照10X平台的标准操作流程进行构建。