The molecular basis of scale development highlighted by a single-cell atlas of Bicyclus anynana butterfly pupal forewings. The molecular basis of scale development highlighted by a single-cell atlas of Bicyclus anynana butterfly pupal forewings

Butterfly wings display a diversity of cell types, including large polyploid scale cells, yet the molecular basis of such diversity is poorly understood. To explore scale cell diversity at a transcriptomic level, we employ single-cell RNA sequencing of ∼5,200 large cells (>6 μm) from 22.5- to 25-h male pupal forewings of the butterfly Bicyclus anynana. Using unsupervised clustering, followed by in situ hybridization, immunofluorescence, and CRISPR-Cas9 editing of candidate genes, we annotate various cell types on the wing. We identify genes marking non-innervated scale cells, pheromone-producing glandular cells, and innervated sensory cell types. We show that senseless, a zinc-finger transcription factor, and HR38, a hormone receptor, determine the identity, size, and color of different scale cell types and are important regulators of scale cell differentiation. This dataset and the identification of various wing cell-type markers provide a foundation to compare and explore scale cell-type diversification across arthropod species. Overall design: Large cells (>6 µm) from 22.5-25-hour male pupal forewings of the butterfly Bicyclus anynana were isolated using Fluorescence Activated Cell Sorting (FACS) and analyzed using scRNA-seq

蝴蝶翅膀展现出丰富的细胞类型多样性，其中涵盖大型多倍体鳞片细胞，但此类细胞多样性的分子机制仍未得到充分阐释。为从转录组层面解析鳞片细胞的多样性，我们针对22.5至25小时龄的雄性蛹期碧翠丝眼蝶（Bicyclus anynana）前翅中约5200个直径大于6μm的大型细胞，采用单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）开展研究。通过无监督聚类分析，结合原位杂交（in situ hybridization）、免疫荧光（immunofluorescence）以及候选基因的CRISPR-Cas9基因编辑技术，我们对翅膀上的各类细胞类型进行了注释鉴定。本研究鉴定出了可标记非神经支配鳞片细胞、信息素分泌腺细胞以及受神经支配感觉细胞类型的特征基因。研究表明，锌指转录因子senseless与激素受体HR38，可调控不同鳞片细胞类型的身份、大小与色泽，是鳞片细胞分化的关键调控因子。本数据集及各类翅膀细胞类型标记基因的鉴定结果，为比较并探索节肢动物类群间鳞片细胞类型的多样化演化提供了重要研究基础。总体实验设计：通过荧光激活细胞分选（Fluorescence Activated Cell Sorting, FACS）分离得到碧翠丝眼蝶（Bicyclus anynana）22.5至25小时龄雄性蛹期前翅中的直径大于6μm的大型细胞，随后采用单细胞RNA测序（scRNA-seq）进行分析。