DEV015206 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Phosphorylation of Gro correlates with FGFR pathway activation. (A-C) Lateral view of stage 12 embryonic tracheal branches, marked by GFP (A, green). This embryo is double stained with αpGro (B,C; blue) and αdpERK (A,C; red). Three tracheal branches are outlined by broken lines. dpERK and pGro are co-expressed in the posterior lateral migrating tip cells of the tracheal branches (arrowhead).

• Supplemental Figure S2 -

  Fig. S2. Real-time PCR quantification of maternally expressed Gro, Gro^AA and Gro^DD transgenes. The protein levels of the transgenic Gro variants used in Fig. 4, 5 and S3 could not be directly monitored, because the αGro antibody does not detect Gro^AA and Gro^DD on western blots, and because tagging compromises the ability of Gro to repress in vivo (data not shown). Instead, we used QPCR to determine transgenic transcription levels. Fold changes presented are relative to the expression of native Gro. No signal is detected in negative control embryos expressing lacZ. Similar RNA expression levels were measured in this and other independent experiments.

• Supplemental Figure S3 -

  Fig. S3. Gro^AA and Gro^DD exert differential effects on expression of knirps and on terminal morphology. (A-D) Spatial distribution of kni RNA transcripts in stage 4 embryos maternally expressing Gro (B) or mutant derivatives (C,D). (A) Control. The posterior boundary of kni expression shifts towards the pole in Gro- (B) and Gro^AA (C)-expressing embryos, but not in Gro^DD embryos (D). (A-D) Black arrows indicate the same relative position in all embryos. (E-H) Cuticular phenotypes of larvae brought about by the expression of Gro and its variants. Expression of all Gro variants leads to low hatching rates (Gro 16.8%, n=148; Gro^AA 13.6%, n=176; Gro^DD 28%, n=175). Filzkörper, a posterior terminal structure, is malformed and reduced in size in Gro (F) and Gro^AA (G) larvae, but appears normal in Gro^DD larvae (H), compared with control (E). (E-H) Arrows indicate filzkörper material in the plane of focus.