Table2_Cross-species single-cell landscapes identify the pathogenic gene characteristics of inherited retinal diseases.xlsx

IntroductionInherited retinal diseases (IRDs) affect ∼4.5 million people worldwide. Elusive pathogenic variants in over 280 genes are associated with one or more clinical forms of IRDs. It is necessary to understand the complex interaction among retinal cell types and pathogenic genes by constructing a regulatory network. In this study, we attempt to establish a panoramic expression view of the cooperative work in retinal cells to understand the clinical manifestations and pathogenic bases underlying IRDs.MethodsSingle-cell RNA sequencing (scRNA-seq) data on the retinas from 35 retina samples of 3 species (human, mouse, and zebrafish) including 259,087 cells were adopted to perform a comparative analysis across species. Bioinformatic tools were used to conduct weighted gene co-expression network analysis (WGCNA), single-cell regulatory network analysis, cell–cell communication analysis, and trajectory inference analysis.ResultsThe cross-species comparison revealed shared or species-specific gene expression patterns at single-cell resolution, such as the stathmin family genes, which were highly expressed specifically in zebrafish Müller glias (MGs). Thirteen gene modules were identified, of which nine were associated with retinal cell types, and Gene Ontology (GO) enrichment of module genes was consistent with cell-specific highly expressed genes. Many IRD genes were identified as hub genes and cell-specific regulons. Most IRDs, especially the retinitis pigmentosa (RP) genes, were enriched in rod-specific regulons. Integrated expression and transcription regulatory network genes, such as congenital stationary night blindness (CSNB) genes GRK1, PDE6B, and TRPM1, showed cell-specific expression and transcription characteristics in either rods or bipolar cells (BCs). IRD genes showed evolutionary conservation (GNAT2, PDE6G, and SAG) and divergence (GNAT2, MT-ND4, and PDE6A) along the trajectory of photoreceptors (PRs) among species. In particular, the Leber congenital amaurosis (LCA) gene OTX2 showed high expression at the beginning of the trajectory of both PRs and BCs.ConclusionWe identified molecular pathways and cell types closely connected with IRDs, bridging the gap between gene expression, genetics, and pathogenesis. The IRD genes enriched in cell-specific modules and regulons suggest that these diseases share common etiological bases. Overall, mining of interspecies transcriptome data reveals conserved transcriptomic features of retinas across species and promising applications in both normal retina anatomy and retina pathology.

引言
遗传性视网膜疾病（IRDs）影响全球约450万人。超过280个基因中的致病变体难以捉摸，与一种或多种IRDs的临床形式相关联。构建一个调控网络，以理解视网膜细胞类型和致病基因之间的复杂相互作用，是必要的。在本研究中，我们试图建立视网膜细胞合作工作的全景表达视图，以理解IRDs的临床表现和致病基础。
方法
采用单细胞RNA测序（scRNA-seq）数据，对来自3种物种（人类、小鼠和斑马鱼）的35个视网膜样本（包括259,087个细胞）进行了跨物种的比较分析。使用生物信息学工具进行加权基因共表达网络分析（WGCNA）、单细胞调控网络分析、细胞间通讯分析和轨迹推断分析。
结果
跨物种比较揭示了单细胞分辨率的共享或物种特异性基因表达模式，例如，在斑马鱼Müller胶质细胞（MGs）中特异性高表达的stathmin家族基因。确定了13个基因模块，其中9个与视网膜细胞类型相关，且模块基因的基因本体（GO）富集与细胞特异性高表达基因一致。许多IRD基因被鉴定为枢纽基因和细胞特异性调控子。大多数IRDs，尤其是视网膜色素变性（RP）基因，富集在视杆细胞特异性调控子中。整合表达和转录调控网络基因，如先天性静止性夜盲症（CSNB）基因GRK1、PDE6B和TRPM1，在视杆细胞或双极细胞（BCs）中表现出细胞特异性表达和转录特征。IRD基因在物种间光感受器（PRs）的轨迹上表现出进化保守性（GNAT2、PDE6G和SAG）和分化（GNAT2、MT-ND4和PDE6A）。特别是，Leber先天性黑蒙（LCA）基因OTX2在PRs和BCs轨迹的起始阶段表现出高表达。
结论
我们确定了与IRDs密切相关的分子途径和细胞类型，架起了基因表达、遗传学和发病机制之间的桥梁。富集在细胞特异性模块和调控子中的IRD基因表明，这些疾病具有共同的病因基础。总体而言，跨物种转录组数据的挖掘揭示了视网膜在物种间保守的转录组特征，并在正常视网膜解剖学和视网膜病理学中具有潜在的应用价值。