The silk gland of the domesticated silkworm Bombyx mori, is a remarkable organ that produces vast amounts of silk with exceptional properties. Little is known about which silk gland cells execute silk protein synthesis and its precise spatiotemporal control. Here, we use single-cell RNA sequencing to build a comprehensive cell atlas of the silkworm silk gland, consisting of 14,972 high-quality cells representing 10 distinct cell types, in three early developmental stages. We annotate all 10 cell types and determine their distributions in each region of the silk gland. Additionally, we decode the developmental trajectory and gene expression status of silk gland cells. Finally, we discover marker genes involved in the regulation of silk gland development and silk protein synthesis. Altogether, this work reveals the heterogeneity of silkworm silk gland cells and their gene expression dynamics, affording a deeper understanding of silk-producing organs at the single-cell level.

The steroid hormone 20-hydroxyecdysone (20E), which is known to regulate insect molting and metamorphosis, is crucial for the normal development of silk glands (SGs) in the silkworm Bombyx mori. However, how the 20E signaling pathway and its core members function in the SG remains largely unclear. Here, we report that the orphan nuclear receptor BmHR3, a 20E-response factor, plays an essential role in regulating SG development and silk protein synthesis. First, we showed that tissue-specific BmHR3 overexpression and knockout result in severe developmental defects in posterior silk glands (PSGs). Second, we revealed that BmHR3 dysfunction in PSGs dramatically represses the transcription of silk fibroin protein-coding genes, thereby inhibiting fibroin protein synthesis. Finally, we confirmed that BmHR3 can regulate fibroin protein-coding gene expression via direct and indirect mechanisms. This study elucidates the vital function of BmHR3 in B. mori SG and provides valuable information for thoroughly understanding the regulatory roles of 20E signaling in specialized insect organs.

Silk production is a classic example of a domestication trait, yet the cell-type-specific driver of its enhancement in the silkworm Bombyx mori remains unresolved. To address this, we integrated extensive bulk RNA-seq data with a single-nucleus RNA-seq atlas of silk glands (SGs) from domestic B. mori and wild B. mandarina for deconvolution analysis. This identified phenotype-associated cell subpopulations (Scissor+ and Scissor− cells) that enrich in B. mori and B. mandarina, respectively. Transcriptomic characterization revealed that B. mori SG cells exhibit a pervasive “pro-synthesis” transcriptional state, with concerted upregulation of silk protein genes and metabolic pathways. Conversely, B. mandarina cells maintained a “protective–adaptive” state, enriched for stress response and xenobiotic metabolism genes. Pseudotime analysis further delineated the cell state transitions, pinpointing key dynamic gene expression linked to high silk yield. Our findings demonstrate that domestication reshaped the silk gland cellular landscape, promoting a systemic shift toward a synthesis-optimized cell state. This study offers a new framework at the cellular level to elucidate the evolution of complex traits under selection.