DEV01724 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Effects of wingless and Notch signalling on wing development. Third instar larval discs carrying a lacZ reporter for the expression of Wingless, expressing different transgenes. (A) Wild-type disc showing the wing pouch (inner circle, wp), the hinge region (outer circle, hng) and the notum (nt). The stripe bisecting the wing pouch is the wing margin. (B) Disc expressing dppGAL4 UASTNotch. Notice that the wing pouch region is smaller (see text for details). (C) Disc expressing dppGAL4 UASWingless. Notice the enlargement of the hinge region, as has been described before. (D) Disc expressing dppGAL4 UASWingless UASTNotch. Notice that TNotch completely suppresses the effects of UASWingless. (E) Disc expressing dppGAL4 UASArmadillo^S10. In addition to the expansion of the hinge, there are several winglets developing in the region of the notum (arrow). (F) Disc expressing dppGAL4 UASArmadillo^S10 UASTNotch. Notice the very effective, albeit not complete in this example, suppression of the effects of UASArmadillo^S10.

• Supplemental Figure 2 -

  Fig. S2. Modulation of Wnt signalling by Notch during the patterning of the larval cuticle. (A) Wild-type pattern of the larval cuticle at the end of embryogenesis. The cuticle exhibits reiterated sets of denticles that demarcate the anterior of each segment; the posterior part of each segment is devoid of denticles and this phenotype requires Wingless signalling. (B-E) Ventral cuticles of larvae expressing UASArmadillo^s10 (B,D), or UASArmadillo^s10 together with UASTNotch (C,E), under the control of armGAL4 at different temperatures. The activity of GAL4 is temperature dependent and elicits more expression of the genes under GAL4 UAS control at the higher temperature; this effect can be seen in the penetrance of the phenotypes (F-H). (B-D) Cuticles representative of the phenotypes caused by expression of Arm^s10 (B) or Arm^s10 together with TNotch (C) at 25°C. At this temperature Arm^s10 consistently elicits the naked cuticle fate (see also F). TNotch suppresses the effects of Arm^s10 and allows the development of denticles (arrow). The effect of TNotch is more pronounced at 18°C (D,E), when the effects of Arm^s10 are weaker. At 18°C most embryos expressing Arm^s10 develop some denticles, however they tend not to cross the midline (see arrowhead in D). At this temperature the effects of TNotch on Arm^s10 are manifested in the appearance of denticles across the midline (see arrowhead in E). (F-H) Quantitative analysis of the effects of TNotch on Arm^s10 at different temperatures (25°C, 22°C and 18°C). (F,G) At 25°C and 22°C the effects of Arm^s10 were assessed by the number of embryos with three or less segments with denticles, regardless of whether they crossed the midline or not. Notice that at both temperatures (25°C, 22°C) embryos that express TNotch have more segments with denticles. (F,H) At 18°C most embryos expressing Arm^s10 have segments with denticles because of the low expression of Arm^s10. In this case we evaluated the number of embryos with denticle belts, i.e. embryos in which the denticles cross the midline. As in G, we chose three as an arbitrary threshold for our quantification. In this instance we also counted the number of embryos with naked cuticle. TNotch can suppress the effects of Arm^s10.

• Supplemental Figure 3 -

  Fig. S3. Third instar larval wing discs expressing full length Armadillo under the control of enGAL4. At the beginning of the third larval instar (ea L3), although engrailed is expressed throughout the posterior compartment (indicated by region of intense green) Armadillo protein is only visible in a very small domain near the dorsoventral compartment boundary which coincides with the expression of Wingless at this time (Klein et al., 2000). Later on (mid/late L3) Armadillo is visible throughout the wing pouch but still its highest levels are at the dorsoventral boundary where the levels of Wingless are highest.

  Reference

  Klein, T., Seugnet, L., Haenlin, M. and Martinez Arias, A. (2000). Two different activities of Suppressor of Hairless during wing development in Drosophila. Development 127, 3553-3566.

• Supplemental Figure 4 -

  Fig. S4. In the absence of Notch the stabilisation of Armadillo does not require Wingless signalling. Confocal section through the wing pouch of a third instar larval wing disc expressing UASArmadillo under the control of ptcGAL4 (see text for details). (A) Armadillo expression. (B) Wingless expression. (C) GFP indicating the position of Notch mutant cells through the absence of GFP. (D) Merged image of A, B and C. GFP is green; Armadillo protein is red. The stripe of Wingless expression (blue) indicates the position of the dorsoventral boundary. Notice that there is a gap, probably produced by the absence of Notch function early in wing development and that a clone of Notch mutant cells (arrow) which lies in the path of the disc maintains high levels of expression of Armadillo (red).

• Supplemental Figure 5 -

  Fig. S5. Notch protein was immunoprecipitated from wild-type embryos and the presence of associated proteins was assessed by western blot. In Notch immunoprecipitate that contained Armadillo (data not shown) no detectable association of E-cadherin (rat monoclonal DCAD2) or nuclear lamin (mouse monoclonal T47) proteins with Notch was observed.