DEV026054 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. fas2 loss-of-function phenotype in the wing. (A) Control adult wing in Minute(O)^SP/+ background. (B,C) Wings containing unmarked clones of fas2^eb112. Extra wing vein material can be seen. (B′,C′) An enlargement of the wings in B and C with the extra wing vein material marked by red arrows.

• Supplemental Figure S2 -

  Fig. S2. fas2 hypomorphic wings are proportionally longer. Graph plots the mean values with one standard deviation (s.d.) as error bars. (*) Comparing wing ratios of fas2^eb112/fas2^e76 flies (only females viable) grown at 18°C with yw wild-type control females, also grown at 18°C. Wing ratio of fas2^eb112/fas2^e76 flies is 2.24±0.05 (mean±s.d.), whereas that of the control is 2.13±0.03. A Student’s t-test confirms that these two populations have significantly different wing ratios (P<0.0001). (**) Comparing wing ratios of flies over-expressing the wild-type EGFR in the wing with wild-type yw flies. Again, female wings grown at 18°C were used for consistency with fas2^eb112/fas2^e76 flies. Ratios of C765-Gal4, UAS-EGFR flies grown at 18°C (2.18±0.02) is significantly different from yw controls (P<0.0001), also grown at 18°C (2.13±0.03).

• Supplemental Figure S3 -

  Fig. S3. Fas2 does not detectably inhibit Notch, Wingless, Hedgehog, Dpp and other RTK signalling. (A) When a dominant-negative form of Suppressor of Hairless, Su(H)DN, is overexpressed in the eye, Notch signalling is reduced to give a rough eye. This was not modified by heterozygosity for fas2 (A′). (B) Heterozygous Delta (Dl^rev10) flies have reduced Notch signalling and ectopic vein material, but this was not affected by halving fas2 dose (B′). (C) Heterozygous Hairless (Hairless^P141) flies have increased Notch signalling and shorter L5 veins in the wing. The actual (a) and theoretical (b) L5 lengths were measured, and the relative L5 length was calculated (a/b), for each wing, to take into account changes in wing sizes. (C′) When fas2 levels were halved no significant change in relative L5 length was detected (see D). (D) Graph comparing the relative L5 lengths of control Hairless^P141 wings with that of flies also heterozygous for fas2. Plotted are mean values with one standard deviation error bars. Student’s t-test confirmed that there was not a significant difference between the relative L5 lengths. (E) F76e flies have increased Wingless signalling as a result of Armadillo being stabilised. The small eye phenotype was not significantly modified by heterozygosity for fas2 (E′). (F) Overexpressing Wingless in the eye hyperactivates the pathway to give a rough eye, but this was not modified when fas2 was reduced by half (F′). (G) Overexpressing the intracellular domain of E-cadherin (cadi) in the posterior part of the wing by engrailed-Gal4 sequesters Armadillo and reduces Wingless signalling. This gives the ‘ripped’ posterior compartment phenotype shown. This phenotype was not significantly modified when fas2 levels were halved (G′). (H) When a dominant negative form of Smoothened (Smo5A) is over-expressed in the wing by C765-Gal4, Hedgehog signalling is reduced, and L3 and L4 veins fuse as a result. This phenotype was not modified upon heterozygosity for fas2 (H′). (I) Activating the FGFR pathway by overexpressing an activated form of the receptor breathless, λ-btl, together with dof, gives a rough eye at 18°C. (I′) GMR>λ-btl, dof /+ rough eye was suppressed when heterozygous for fas2 (at 18°C). (J) Table to summarise interaction of fas2 with Torso signalling. trk⁵ is a hypomorphic allele of trk, a Torso ligand, which causes loss of posterior segments. fas2^e76 reduces levels of Fas2 by 90%. Data in each column refer to actual numbers (percentage in brackets) of embryos terminating in A7 or A8 abdominal segment, or wild type (containing all segments A1-A8 plus terminal structures). Using χ² test, for trk⁵ versus fas2^e76;trk⁵: df=2, χ²=1.71, P=0.425; hence, there is no significant modification of the trk⁵ phenotype by fas2. (K,K′) fas2^eb112 clones in the wing, marked by lack of GFP, do not affect phosphorylation of the Dpp transducer Mad, as assayed using anti-phospho-Mad.