Tsr and Yki act in interconnected pathways to regulate tracheal tube elongation. (A–E) Loss of function of tsr (B) causes convoluted DT, similar to loss of yki (C). (D) This phenotype is enhanced in tsr^k05633; yki^B5 double mutants. Scale bar: 50 µm. (E) Quantification of DT length of WT (n = 10), yki^B5 (n = 11), tsr^k05633 (n = 9), and tsr^k05633; yki^B5 (n = 8) mutants. The DT of tsr^k05633; yki^B5 double mutants is significantly longer than that of yki^B5 and tsr^k05633 single mutants. (F–M) Tracheal expression of either Tsr (H and L) or Yki (I and M) using btl-Gal4 rescues DT elongation defects of tsr^k05633 (H and I) and yki^B5 (L and M) mutants. Scale bar: 20 µm. (N) Relative expression of tsr mRNA in WT, yki^B5 and tsr^k05633 mutant embryos at stage 17. tsr mRNA levels are not significantly altered in the absence of yki. 200 embryos were used per genotype. Three biological replicates were performed per genotype. (O) Relative expression of yki mRNA in WT, yki^B5, and tsr^k05633 mutant embryos of stage 17. yki mRNA levels are not significantly altered in the absence of Tsr. 200 embryos were used per genotype. Three biological replicates were performed per genotype. (P) Western blot of protein lysates from WT, yki^B5, and tsr^k05633 mutant embryos at stage 17. Note that the protein levels of Tsr and Yki are reduced in the respective other mutant. (Q and R) Quantification of the immunoblot in (P) using Fiji, based on the intensity of Yki (Q) and Tsr (R) protein, normalized to the loading control (α-Tubulin; n = 3). (S) Relative expression of diap1 mRNA in WT, yki^B5, and tsr^k05633 mutant embryos at stage 17. Results were normalized to an endogenous control (actin-5C). Note that Diap1 is significantly down-regulated in yki mutants but significantly up-regulated in tsr mutants. 200 embryos were used per genotype. Three biological replicates were performed per genotype. Error bars represent SEM.