Fig. 7: The diversification of tracheal cells.

a Heatmap showing gene expression over pseudotime for PC, DB, and DT cells. Dashed boxes denote the Notch targets, components of enhancer of split complex, and cut (ct). Asterisks point to genes participating in protein glycosylation. b–d Representative traces showing the expression patterns of Notch-responsive genes in PC, DB, and DT cells. The expression levels of bs (b), kni (c), peb (d) decreased from multipotent PC and DB cells to terminal differentiated DT cells. e Cell maturation map of tracheoblasts estimated by RNA velocity. f, i Cytotrace plots showing the state of differentiation of tracheal cells from ND-fed (f) or HSD-fed (i) flies. g, h UMAP visualization of tracheal cell types in ND (g) or HSD (h). j–l Lineage-tracing analyses of kni-expressing DB cells (j), Cut- (k), or Esg-positive (l) progenitors. kni-Gal4, ct-Gal4, or esg-Gal4-driven G-TRACE flies were fed on ND. k, l Cells that are neither red nor green are not cut- or esg-expressing progenitor origin. DAPI (blue). m–o Lineage-tracing analysis of kni-expressing DB cells (m), Cut- (n) or Esg-positive (o) progenitors. kni-Gal4, ct-Gal4, or esg-Gal4-driven G-TRACE flies were exposed to HSD. DAPI (blue). j–o Each experiment was repeated independently with similar results for three times. p Scatter plot representing the differentially expressed genes in tracheal cells exposed to ND or HSD. q Bar graph depicting functional clusters among the differentially expressed genes between ND and HSD groups. Hypergeometric test and False Discovery Rate (FDR) adjustment were used. Genotypes: j, m kni-Gal4/+;UAS-G-trace/+. k, n ct-Gal4/UAS-G-trace. l, o esg-Gal4/UAS-G-trace. DT dorsal trunk, DB dorsal branch, TC transverse connective, ASP air sac primordium, VB visceral branch, SB spiracular branch, PC progenitor cells, LT lateral trunk, GB ganglionic branches, ND normal diet, HSD high-sugar diet. Scar bars: 50 μm (j–o).