Single-cell and spatial transcriptomics define 20E-driven developmental reprogramming in silkworm wing disc

Abstract The dynamic cellular transitions, spatial organization, and hormonal regulatory mechanisms of insect wing development are incompletely understood. Here, we reported an integrated spatially resolved single-cell atlas of the silkworm (Bombyx mori) wing disc across 10 developmental time points, investigating the dynamics of 12 cell types. Wing morphogenesis (Wm) cells were characterized as central progenitors with bifurcated differentiation toward epithelial and cuticle lineages, regulated by lineage-specific transcription factors. Time-resolved snRNA-seq revealed a hierarchical, time-dependent transcriptional reprogramming cascade. Functional modules and signaling pathways were activated with temporal and spatial precision, with Wm cells acting as early signaling hubs. 20-Hydroxyecdysone (20E) accelerated fate transitions and transcriptional maturation, mimicking in vivo transitions within hours. Integrating 20E concentration dynamics, morphology, and gene expression, we proposed a five-stage gene transition model to outline wing disc development from plasticity to maturation, highlighting 20E signal dynamics and providing a framework for understanding 20E-driven organogenesis in holometabolous insects.