Fig. 3. Bnl FGF pathway gene expression and function in rib mutants. (A) In situ hybridization for bnl FGF mRNA (blue) in stage 12 heterozygous rib⁺ embryo (rib^P7/CyO,ftz-lacZ). (Out-of-focus brown staining is immunostaining for ß-galactosidase from ftz-lacZ.) (B) Same view of rib^P7 homozygote. bnl mRNA expression pattern is not grossly affected. Arrowheads, bnl expression domains 3', 3, 4, 5/7 (Sutherland et al., 1996). (C) In situ hybridization for btl FGFR mRNA in stage 12 heterozygous rib⁺ embryo (rib^P7/CyO,ftz-lacZ). (D) Same view of rib^P7 homozygote. Tracheal branch outgrowth is stalled but btl mRNA is expressed normally. (E) Four tracheal metameres of wild-type embryo double stained for the 2A12 tracheal lumenal antigen (brown) and DSRF/blistered, a Bnl-induced gene (blue). DSRF expression is induced in cells at the ends of primary branches (bracket, arrowheads). (F) Same view of rib^ex12 embryo. Tracheal branches do not grow out normally and DSRF expression is not induced (asterisks). (G) UAS-bnl/+;69B-GAL4/+ embryo stained for DSRF. Ectopic bnl expression induces DSRF expression throughout the tracheal system. (H) UAS-bnl/+; rib^ex12; 69B-GAL4/+ embryo stained for DSRF. Although the rib mutation blocks branch outgrowth, it does not prevent bnl induction of DSRF expression throughout the trachea. (To aid in embryo genotyping, this preparation was also stained for ß-galactosidase expressed from a TM3 Ubx-lacZ balancer chromosome in the cross, and hence has higher background staining than G.) (I) UAS-bnl/+; 69B-GAL4/+ embryo stained for diphospho-ERK. Ectopic Bnl expression activates ERK throughout the tracheal system. (J) Same view of UAS-bnl/+; rib^ex12; 69B-GAL4/+ embryo. Although the rib mutation blocks branch outgrowth, it does not prevent bnl-induced phosphorylation of ERK throughout the trachea. Scale bars: in D, 20 µm for A-D; in F, 10 µm for E,F; in J, 20 µm for G-J.