The molecular basis of macrochaete diversification highlighted by a single-cell atlas ofBicyclus anynanabutterfly pupal forewings

Abstract Butterfly wings display a large diversity of cell types, including scale cells of different colors, shapes, and sizes, yet the molecular basis of such diversity is poorly understood. Scales are single-cellular projections homologous to sensory bristles in other arthropods, and different scale types are often found intermingled on the wing, alongside other sensory and epidermal cell types, making the analysis of the complete transcriptomes of each scale type difficult to achieve. To explore scale cell diversity at a transcriptomic level we employed single-cell RNA sequencing of ∼5200 large cells (>6 µm) from 22.5-25-hour male pupal forewings of the butterfly Bicyclus anynana . Using single-cell unsupervised, transcriptome clustering, based on shared and differentially expressed genes, followed by in situ hybridization, immunofluorescence, and CRISPR/Cas9 editing of candidate cell type specific genes, we annotate various cell types on the wing. We identify the molecular identities of non-innervated scale cells at various stages of differentiation, innervated sensory cell types, and pheromone producing glandular cells. We further 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. Graphical abstract