Fig. 2: Cell differentiation trajectories and functional dynamics during silkworm wing disc development.

a Pseudotime trajectory analysis of Wm cells showing a bifurcated lineage pattern. b Proportional distribution of annotated cell types across the three trajectory states. c Branching plot showing Wm cell distribution along pseudotime trajectories from L5D1 to P6. d Predicted pseudotime trajectory analysis showing the differentiation trends of Wm cells. e Ridge plots showing the temporal progression of Wm, Epithelial, and Cuticle subtypes along the pseudotime axis. f Heatmap showing the expression dynamics of differentially expressed genes along the pseudotime trajectory, clustered into functional gene modules. Selected enriched pathways are highlighted. The enrichment analysis was performed via Over-Representation Analysis (ORA), with the statistical significance for each pathway evaluated using a two-sided hypergeometric test. P-values were adjusted for multiple comparisons using the Benjamini–Hochberg false discovery rate (FDR) correction. The enrichment results are visualized in a bubble plot. The color gradient of the bubbles represents the adjusted p-value (p.adjust), as indicated in the scale, with more intense color (lower p.adjust) denoting higher statistical significance. The size of the bubbles corresponds to the number of genes (Count) enriched in each pathway. Dynamic changes of Toll/Imd signaling pathway activity (g) and Hippo signaling pathway activity (h) across ten developmental stages. The x-axis represents distinct developmental stages: larval (L5D1 to L5D7), spinning (W-1, W-2), and pupal stages (P6, the sixth day of the pupa). The box plot elements are defined as follows: the center line indicates the median (50th percentile); the lower and upper bounds of the box indicate the 25th and 75th percentiles, respectively, defining the interquartile range (IQR); the whiskers extend to the minimum and maximum data points within 1.5× IQR from the respective box bounds; data points beyond the whiskers are plotted individually as outliers. A dashed horizontal line at y = 0 serves as a reference. i Regulatory network of key transcription factors (TFs) enriched in Wm cells and their predicted downstream target genes. j Quantitative RT-PCR analysis showing significant downregulation of BmRfx expression after RNAi (Data are shown from three biological replicates, with n = 30 independent wing discs used for each replicate; mean ± SD; two-tailed unpaired t test, ***p < 0.001). k Morphological effect of BmRfx gene knockdown on wing disc development. Dashed white boxes indicate the wing bud region. Trans as transverse section. Arrowheads indicate regions of morphological alteration in the wing disc following RNAi treatment. The upper panels show the overall morphology of wing discs, whereas the lower panels display transverse histological sections of the same samples. l Effects of BmRfx gene knockdown on wing morphology at the adult stage. m Quantitative RT-PCR analysis showing significant downregulation of SfRfx expression after RNAi (Data are shown from three biological replicates, with n = 20 independent wing discs used for each replicate; mean ± SD; two-tailed unpaired t test, **p < 0.01). n Morphological effect of SfRfx gene knockdown on wing disc development. Trans as transverse section. Arrowheads indicate regions of morphological alteration in the wing disc following RNAi treatment. o Effects of SfRfx gene knockdown on wing morphology at the adult stage. Cell types: Wm (wing morphogenesis). Source data are provided as a Source Data file.