Fat body affects disciplined migration of tracheal progenitors.

(A) Schematic cartoon showing the migration of tracheal progenitors (red) and degenerative tracheal branches (dashed grey lines) in pupae. Fat body is shown in beige. Arrows denote anterior-posterior (A-P) axis. (B, C) Frontal section (B) and sagittal view (C) showing the relative position of fat body and tracheal progenitors. (D-J’) Migration of tracheal progenitors in control and fat body perturbation flies. (D-D’’) Migration of tracheal progenitors (red) upward from transverse connective (blue dashed lines) and along the dorsal trunk (white dotted lines) at 0hr APF (D), 1hr APF (D’), and 3hr APF (D’’). (E-E’’) Bidirectional movement of tracheal progenitors in fat body-depleted (lsp2>rpr.hid) flies. 0hr APF (E), 1hr APF (E’), 3hr APF (E’’). Arrows point to anterior movement of tracheal progenitors. (F) Bar graph showing the migration distance of tracheal progenitors. Error bars represent SEM. (G-G’) The distribution of progenitors at 2hr APF. (H-H’’) The distribution of progenitors in fat body-depleted flies at 2hr APF. (I-J’) Computer simulation depicting trajectories of progenitor migration. (I, J) Confocal images of tracheal progenitors. (I’, J’) Vectors of progenitor migration. (K) Bar graph plots the binary entropy that represents the disorderedness of migration direction of tracheal progenitors. (L) The Bernoulli random variable X showing optic flow distribution of the binarized directions in each group. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and ns indicates not significant. Scale bar: 100 μm (B, C), 200 μm (D-E’’). Genotypes: (B, C) UAS-mCD8-GFP/+; lsp2-Gal4,P[B123]-RFP-moe/+; (D-D’’, G, G’) Gal80ts/+;lsp2-Gal4,P[B123]-RFP-moe/+; (E-E’’, H, H’) UAS-rpr-hid/+;Gal80ts/+;lsp2-Gal4,P[B123]-RFP-moe/+.

Dependence of tracheal progenitors on cytokines from fat body.

(A) Top functional clusters among the differentially expressed genes of progenitors between control and fat body-depleted pupae. (B) Heatmap depicting expression levels of principal target genes of signaling pathways in L3 larvae, 0hr APF pupae and 2hr APF pupae. (C) Heatmap showing the differential expression of target genes of signaling pathways between control and fat body-depleted pupae. (D-D’’) Migration of tracheal progenitors along the dorsal trunk at 0hr APF (D), 1hr APF (D’), and 3hr APF (D’’). The white dashed line shows transverse connective. (E-E’’). Migration of tracheal progenitors in upd2RNAi flies. (F) Bar graph plots the migration distance of tracheal progenitors. Error bars represent SEM. (G) Volcano plot showing surface proteomics of tracheal epithelium (up-regulated genes with ten-fold or higher changes in red; down-regulated genes with ten-fold or higher changes in blue). (H) Top functional classes among the surface proteomics of trachea. (I) Schematic diagram depicting the working principle of the DIPF reporter. (J) The expression of DIPF reporter in tracheal progenitors. The progenitors are outlined by dashed lines. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and ns indicates not significant. Scale bar: 200 μm (D-E’’), 50 μm (J). Genotypes: (D-D’’) lsp2-Gal4,P[B123]-RFP-moe/+; (E-E’’) lsp2-Gal4,P[B123]-RFP-moe/UAS-upd2RNAi; (J) btl-Gal4/UAS-DIPF.

JAK/STAT pathway is required for the discipline of tracheal progenitor migration.

(A-D’’) Migration of tracheal progenitors along the dorsal trunk at 0hr APF, 1hr APF and 3hr APF. The white dashed line shows transverse connective. The progenitors of control (A-A’’), domeRNAi (B-B’’), hopRNAi (C-C’’), and stat92ERNAi (D-D’’) flies. (E) Bar graph showing migration distance of progenitors. Error bars represent SEM. (F-G’’) JAK inhibition causes bidirectional movement of progenitors. Migration of tracheal progenitors in the absence (DMSO-fed) (F-F’’) or in the presence of Tofacinib (JAK inhibitor) (G-G’’). (H) Bar graph showing the distance of anterior movement. Error bars represent SEM. (I-L) The expression of Stat92E-GFP in tracheal progenitors of control (I), domeRNAi (J), hopRNAi (K) and stat92ERNAi (L) flies. The progenitors are outlined by dashed lines. (M, N) The expression of Stat92E-GFP in tracheal progenitors of control (M) or upd2RNAi flies (N). (O, P) The expression of Stat92E-GFP in tracheal progenitors of DMSO-fed (control, O) or Tofacinib-treated (P) flies. Dashed lines outline tracheal progenitors. (Q) Bar graph plots relative signals of Stat92E-GFP reporter. Error bars represent SEM. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and ns indicates not significant. Scale bar: 200 μm (A-D’’, F-G’’), 50 μm (I-P). Genotypes: (A-A’’, F-G’’) btl-Gal4/+;P[B123]-RFP-moe/+; (B-B’’) btl-Gal4/+;P[B123]-RFP-moe/UAS-domeRNAi; (C-C’’) btl-Gal4/+;P[B123]-RFP-moe/UAS-hopRNAi; (D-D’’) btl-Gal4/+;P[B123]-RFP-moe/UAS-stat92ERNAi; (I) btl-Gal4,Stat92E-GFP/+; (J) btl-Gal4,Stat92E-GFP/+;UAS-domeRNAi/+; (K) btl-Gal4,Stat92E-GFP/+;UAS-hopRNAi/+; (L) btl-Gal4,Stat92E-GFP/+;UAS-Stat92ERNAi/+; (M, O, P) Stat92E-GFP/+; lsp2-Gal4/+; (N) Stat92E-GFP/+;lsp2-Gal4/UAS-upd2RNAi.

Identification of gene targets of Stat92E in Drosophila tracheal progenitors.

(A) Bubble plot represents the top functional clusters among gene targets. The establishment of planar polarity denoted in red solid box is identified with high enrichment score. (B) Top functional classes among the differentially expressed genes in larval-pupal transition. (C) ChIP-seq peaks at loci regulated by Stat92E. Scale bar: 20kb (ds, fz, stan), 5kb (fat2, vg), 1kb (fj). (D-H) Validation of gene targets of Stat92E ChIP-seq. (D-G) The expression of Ds-GFP in the tracheal progenitors of control (D), domeRNAi (E), hopRNAi (F) and stat92ERNAi (G). The progenitors are outlined by dashed lines. (H) The bar graphs plot the relative level of Ds. Error bars represent SEM. (I-M) The expression of Fj in tracheal progenitors. The expression of Fj-GFP in the tracheal progenitors of control (I), domeRNAi (J), hopRNAi (K) and stat92ERNAi (L). Dashed lines outline tracheal progenitors. (M) The bar graphs plot the relative level of Fj. Error bars represent SEM. (N-R) The level of Ft-GFP in the tracheal progenitors of control (N), domeRNAi (O), hopRNAi (P) and stat92ERNAi (Q). (R) The bar graphs plot the relative level of Ft. Error bars represent SEM. Scale bar: 50 μm (D-G, I-L, N-Q). Genotypes: (D) btl-Gal4,Ds-GFP/+; (E) btl-Gal4,Ds-GFP/+;UAS-domeRNAi/+; (F) btl-Gal4,Ds-GFP/+;UAS-hopRNAi/+; (G) btl-Gal4,Ds-GFP/+;UAS-stat92ERNAi/+; (I) btl-Gal4,Fj-GFP/+; (J) btl-Gal4,Fj-GFP/+;UAS-domeRNAi/+; (K) btl-Gal4,Fj-GFP/+;UAS-hopRNAi/+; (L) btl-Gal4,Fj-GFP/+;UAS-stat92ERNAi/+; (N) btl-Gal4/+;Ft-GFP/+; (O) btl-Gal4/+;Ft-GFP/UAS-domeRNAi; (P) btl-Gal4/+;Ft-GFP/UAS-hopRNAi; (Q) btl-Gal4/+;Ft-GFP/UAS-stat92ERNAi.

Disciplined migration requires planar cell polarity system.

(A-E) Migration of tracheal progenitors. The migration of progenitors in control (A-A’’), dsRNAi (B-B’’), ftRNAi (C-C’’) and fjRNAi (D-D’’) flies. (E) Bar graph plots the migration distance of anterior movement. (F-I) Level of Ft in tracheal progenitors of control (F, G) and stat92ERNAi (H-J) flies. The images show progenitors at 1hr APF (H, H’) and 2hr APF (I, I’). Ft-GFP (green) (F, H, I), phalloidin (magenta), Hoechst (blue), and merged images (F’, H’, I’). (G, J) Profile plots showing the level of Ft-GFP in control (G) and stat92ERNAi (J) flies. The levels of Ft were measured along the dotted lines in F’ or I’. Anterior (A) and posterior (P). (K) Representative traces plot the migration distance relative to the origin. (K’) Rose plot depicting the direction of cell movement. (L) Representative traces showing the movement of individual fj-KO cells relative to their origin. (L’) Rose plot depicting the movement direction of fj-KO cells. (M) Scatter plots represent the ratio (d/D) of straight-line length displacement (d) relative to the length of the migration track (D) of individual cell. Error bars represent SEM. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and ns indicates not significant. Scale bar: 200 μm (A-D’’), 50 μm (F, F’), 100 μm (H-I’). Genotypes: (A-A’’) btl-Gal4/+;P[B123]-RFP-moe/+; (B-B’’) btl-Gal4/UAS-dsRNAi;P[B123]-RFP-moe/+; (C-C’’) btl-Gal4/+;P[B123]-RFP-moe/UAS-ftRNAi; (D-D’’) btl-Gal4/UAS-fjRNAi;P[B123]-RFP-moe/+; (F, F’) btl-Gal4/+;Ft-GFP/+; (H, H’, I, I’) btl-Gal4/+;Ft-GFP/UAS-stat92ERNAi.

The production and transport of Upd2 from fat body.

(A, B) Upd2-mCherry-containing vesicles in fat body of control (DMSO-fed, A) and BFA-treated L3 larvae (B). (C) Bar graph plots the abundance of Upd2-mCherry-containing vesicles in fat body. Error bars represent SEM. (D, E) The confocal image showing the Upd2-containing vesicles in progenitors of DMSO-fed control (D) and BFA-treated flies (E). (F) Bar graph plots the abundance of Upd2-mCherry-containing vesicles in progenitors. (G) Upd2-mCherry containing vesicles (red) in fat body. (H, I) Upd2 accumulation in fat body was increased in the presence of grasp65RNAi (H) and lbmRNAi (I). (J) Bar graph plots the abundance of Upd2-mCherry-containing vesicles. Error bars represent SEM. (K-M) The Upd2 vesicles (red) in tracheal progenitors (DAPI) in control (K), grasp65RNAi (L) and lbmRNAi (M) flies. Dashed lines outline tracheal progenitors. (N) Bar graph plots the abundance of Upd2-mCherry-containing vesicles in progenitors. Error bars represent SEM. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and ns indicates not significant. Scale bar: 20 μm (A, B, G-I), 50 μm (D, E, K-M). Genotypes: (A-G, K) UAS-upd2-mCherry/+;lsp2-Gal4/+; (H, L) UAS-upd2-mCherry/UAS-grasp65RNAi;lsp2-Gal4/+; (I, M) UAS-upd2-mCherry/+;lsp2-Gal4/UAS-lbmRNAi.

The dependence of tracheal progenitors on vesicle trafficking.

(A-E) The expression of Stat92E-GFP in tracheal progenitors of control (A), grasp65RNAi (B), lbmRNAi (C), rab5RNAi (D) and rab7RNAi (E) flies. The progenitors are outlined by dashed lines. (F) Bar graph plots the relative expression of Stat92E-GFP. Error bars represent SEM. (G, H) The expression of Stat92E-GFP in tracheal progenitors in DMSO-fed (G) and BFA-treated (H) flies. (I) Bar graph plots the relative expression of Stat92E-GFP. Error bars represent SEM. (J) The signal of DIPF reporter in tracheal progenitors. (K) The effects of Tofacinib (JAK inhibitor) on DIPF reporter in progenitors. (L) The effects of Brefeldin A on DIPF reporter in progenitors. Dashed lines outline tracheal progenitors. (M) Bar graphs showing the signal of DIPF reporter. Error bars represent SEM. (N-O’’) Migration of tracheal progenitors in DMSO-fed flies (N-N’’) and BFA-treated flies (O-O’’). (P) Bar graph plots migration distance of anterior movement. Error bars represent SEM. (Q-U’’) Migration of tracheal progenitors at 0hr APF (Q), 1hr APF (Q’), 3hr APF (Q’’). The confocal images showing the tracheal progenitors in control (Q-Q’’), grasp65RNAi (R-R’’), lbmRNAi (S-S’’), rab5RNAi (T-T’’) and rab7RNAi (U-U’’) flies. (V) Bar graph plots the migration distance of anterior movement. Error bars represent SEM. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and ns indicates not significant. Scale bar: 50 μm (A-E, G, H, J-L), 200 μm (N-O’’, Q-U’’). Genotypes: (A, G, H) lsp2-Gal4,Stat92E-GFP/+; (B) UAS-grasp65RNAi/+;lsp2-Gal4,Stat92E-GFP/+; (C) lsp2-Gal4,Stat92E-GFP/UAS-lbmRNAi; (D) UAS-rab5RNAi/+;lsp2-Gal4,Stat92E-GFP/+; (E) UAS-rab7RNAi/+;lsp2-Gal4,Stat92E-GFP/+; (J-L) btl-Gal4/UAS-DIPF; (N-Q’’) lsp2-Gal4,P[B123]-RFP-moe/+; (R-R’’) lsp2-Gal4,P[B123]-RFP-moe/UAS-grasp65RNAi; (S-S’’) lsp2-Gal4,P[B123]-RFP-moe/+;UAS-lbmRNAi/+; (T-T’’) UAS-rab5RNAi/+;lsp2-Gal4,P[B123]-RFP-moe/+; (U-U’’) UAS-rab7RNAi/+;lsp2-Gal4,P[B123]-RFP-moe/+.

The roles of endocytic trafficking system in the transport of Upd2.

(A-D’’’) The confocal images showing the colocalization between Upd2 (red) (A, B, C, D) and Rab5 (GFP) (A’), Rab7 (GFP) (B’), Grasp65 (GFP) (C’) or Lbm (GFP). (A’’, B’’, C’’, D’’) Merged images. (D’’’) 3D high-magnification view of the boxed inset in D’’. (D’’’’) The Pearson’s correlation coefficient depicting colocalization between Lbm and Upd2 in fat body cells. (E-J) The PLA (Proximity Ligation Assay) assay showing the interaction between Upd2 and Rab5 (E, F), Rab7 (G, H) or Lbm (I, J). (K-M) Co-immunoprecipitation assay showing physical interaction between Upd2 and Rab5 (K), Rab7 (L) or Lbm (M) in larval fat body. (N-T) The expression of Lbm-pHluorin in larval fat body and progenitors of control (N, O), rab5RNAi (P, Q) and rab7RNAi (R, S) flies. Dashed lines outline tracheal progenitors. Arrowheads point to Lbm-pHluorin puncta. (T) Schematic diagram depicting Upd2-operated disciplined migration of tracheal progenitors. Scale bar: 5 μm (A-D’’, E-J), 10 μm (N, P, R), 20 μm (O, Q, S). Genotypes: (A-A’’, E, F) UAS-upd2-mCherry/+;lsp2-Gal4/UAS-GFP-rab5; (B-B’’, G, H) UAS-upd2-mCherry/+;lsp2-Gal4/UAS-GFP-rab7; (C-C’’) UAS-upd2-mCherry/UAS-grasp65-GFP;lsp2-Gal4/+; (D-D’’’’, I-L) UAS-upd2-mCherry/UAS-lbm-GFP;lsp2-Gal4/+; (N, O) UAS-lbm-pHluorin/+;lsp2-Gal4/+; (P, Q) UAS-lbm-pHluorin/UAS-rab5RNAi;lsp2-Gal4/+; (R, S) UAS-lbm-pHluorin/UAS-rab7RNAi;lsp2-Gal4/+.