Mouse retinal cell atlas: molecular identification of sixty-three amacrine cell types

Amacrine cells (ACs) are a diverse class of interneurons that modulate input from photoreceptors to retinal ganglion cells (RGCs), rendering each RGC type selectively sensitive to particular visual features, which are then relayed to the brain. While many AC types have been identified morphologically and physiologically, they have not been comprehensively classified or molecularly characterized. We used high-throughput single-cell RNA sequencing (scRNA-seq) to profile >32,000 ACs from mice of both sexes, and applied computational methods to identify 63 AC types. We identified molecular markers for each type, and used them to characterize the morphology of multiple types. We show that they include nearly all previously known AC types as well as many that had not been described. Consistent with previous studies, most of the AC types expressed markers for the canonical inhibitory neurotransmitters GABA or glycine, but several expressed neither or both. In addition, many expressed one or more neuropeptides, and two express glutamatergic markers. We also explored transcriptomic relationships among AC types and identified transcription factors expressed by individual or multiple closely related types. Noteworthy among these were Meis2 and Tcf4, expressed by most GABAergic and most glycinergic types, respectively. Together, these results provide a foundation for developmental and functional studies of ACs, as well as means for genetically accessing them. Along with previous molecular, physiological and morphological analyses, they establish the existence of at least 130 neuronal types and nearly 140 cell types in mouse retina. Amacrine cells from retina of P19 mouse was collected and transcriptomic analysis of single cells were performed.无长突细胞（Amacrine cells, ACs）是一类高度多样的中间神经元，可调控光感受器向视网膜神经节细胞（retinal ganglion cells, RGCs）的信号输入，赋予每种RGC类型对特定视觉特征的选择性响应能力，随后这些信号被传递至大脑。尽管已有诸多AC类型通过形态学与生理学手段被鉴定，但尚未得到全面分类及分子水平的特征解析。本研究采用高通量单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）对雌雄小鼠的32000余个无长突细胞进行转录组谱分析，并通过计算方法鉴定出63种AC类型。我们为每种细胞类型鉴定了特异性分子标记，并利用这些标记对多种AC类型的形态学特征进行了表征。研究表明，这些鉴定出的AC类型几乎涵盖了所有此前已报道的AC类型，同时还包含诸多此前未被描述的新型无长突细胞。与既往研究结果一致，大多数AC类型表达经典抑制性神经递质γ-氨基丁酸（GABA）或甘氨酸的标记物，但存在部分类型既不表达这两种标记物，也可同时表达二者。此外，诸多AC类型表达一种或多种神经肽，另有2种类型表达谷氨酸能相关标记物。本研究还探究了AC类型间的转录组学关联，并鉴定出由单个或多个紧密相关的细胞类型所特异性表达的转录因子。其中值得关注的是转录因子Meis2与Tcf4，它们分别在大多数GABA能AC类型和大多数甘氨酸能AC类型中表达。综上，本研究结果为无长突细胞的发育与功能研究奠定了重要基础，同时也为其遗传靶向研究提供了可行手段。结合此前的分子、生理学及形态学分析结果，本研究证实小鼠视网膜中至少存在130种神经元类型及近140种细胞类型。本研究收集了P19日龄小鼠视网膜中的无长突细胞，并开展了单细胞转录组学分析。