Neuronal diversity and convergence in a visual system developmental atlas (scRNA-seq)

Deciphering how neuronal diversity is established and maintained requires a detailed knowledge of neuronal gene expression throughout development. In contrast to mammalian brains, the large neuronal diversity of the Drosophila optic lobes and its connectome are almost completely characterized. However, a molecular characterization of this diversity, particularly during development, has been lacking. We present novel insights into brain development through a nearly exhaustive description of the transcriptomic diversity of the optic lobes. We acquired the transcriptome of 275,000 single-cells at adult and five pupal stages, and developed a machine learning framework to assign them to almost 200 cell-types at all timepoints. We discovered two large neuronal populations that wrap neuropils during development but die just before adulthood, as well as neuronal subtypes that partition dorsal and ventral visual circuits by differential Wnt signaling throughout development. Moreover, we showed that neurons of the same type but produced days apart synchronize their transcriptomes shortly after being produced. We also resolved during synaptogenesis neuronal subtypes that converge to indistinguishable transcriptomic profiles in adults while greatly differing in morphology and connectivity. Our datasets almost completely account for the known neuronal diversity of the optic lobes and serve as a paradigm to understand brain development across species. 38 scRNA-Seq libraries (about 7000 cells each) were generated from adult and pupal (at 5 stages) Drosophila optic lobes using 10X Genomics Chromium™ Single Cell 3’ v2 protocol.

解析神经元多样性的建立与维持机制，需要详尽掌握发育全过程中神经元的基因表达特征。与哺乳动物大脑不同，果蝇（Drosophila）视神经叶（optic lobes）具备丰富的神经元多样性，其连接组（connectome）也已近乎完全表征。然而，该多样性的分子特征，尤其是发育阶段的分子特征，长期以来仍缺乏系统研究。本研究通过对果蝇视神经叶的转录组多样性（transcriptomic diversity）进行近乎全面的描述，为大脑发育研究提供了全新视角。我们采集了成虫及5个蛹期共27.5万个单细胞（single-cell）的转录组数据，并构建了机器学习（machine learning）框架，可在所有时间节点将这些细胞归类至近200种细胞类型（cell-types）。研究发现两类大型神经元群：它们在发育阶段会包裹神经纤维网，但在成年前夕发生凋亡；同时还鉴定出一类神经元亚型，其通过全程差异调控Wnt信号通路（Wnt signaling）来划分背侧与腹侧视觉环路。此外，我们证实，即便产生时间相隔数日的同类神经元，在生成后短期内即可实现转录组特征的同步化。我们还解析了突触发生（synaptogenesis）阶段的神经元亚型：这些亚型在成年个体中转录组特征趋于一致，但形态与连接模式却存在显著差异。本数据集几乎完整覆盖了已知的果蝇视神经叶神经元多样性，可作为跨物种大脑发育研究的范式模型。本研究采用10X Genomics Chromium™ 单细胞3’端v2测序方案（10X Genomics Chromium™ Single Cell 3’ v2 protocol），从成虫及5个发育阶段蛹期的果蝇视神经叶中构建了38个单细胞RNA测序（single-cell RNA sequencing, scRNA-Seq）文库，每个文库约包含7000个细胞。