DEV025635 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Expression of patterning genes in wing discs with uniform Dpp pathway activity. (A,B) Triple stainings for wg, ptc-lacZ and pH-H3 in wild-type (A) and C765>dpp (B) discs. Uniform Dpp signaling leads to an expanded wing pouch, as demonstrated by the ring of Wg staining. Thus, the wing pouch in such discs overlaps with the medial and lateral areas defined in Fig. 3B. ptc-lacZ staining indicates that Hedgehog signaling is not altered in C765>dpp discs. (C,D) omb-lacZ expression in wild-type (C) and C765>dpp (D) discs. The latter genotype shows extensive misexpression of omb. (E-G) vg expression in wild-type (E), C765>dpp (F) and dpp^12/14;brk^XA double-mutant (G) discs. The vg expression domain is expanded in the overgrowing discs (E,F). (H-J) sal expression in wild-type (H), C765>dpp (I) and dpp^12/14;brk^XA double-mutant (J) discs. The sal domain widens into the expanded wing pouch similarly in C765>dpp and dpp^12/14;brk^XA double-mutant discs. The rate of sal transcription within its expression domain depends in part on a direct Dpp input (Marty et al., 2000; Barrio and de Celis, 2004). This effect explains the minor differences in the expression levels seen in I and J. Scale bars: 50 µm in A-J.

  References

  Barrio, R. and de Celis, J. F. (2004). Regulation of spalt expression in the Drosophila wing blade in response to the Decapentaplegic signaling pathway. Proc. Natl. Acad. Sci. USA 101, 6021-6026.

• Supplemental Figure S2 -

  Fig. S2. Comparison of cell proliferation and disc size between wild-type discs and discs with altered Dpp pathway activity during the mid-third instar stage. (A,B) Wild-type (A) and C765>dpp (B) discs dissected 96 hours AEL stained for pMad and pH-H3. Slight differences in the proliferation rate of medial and lateral cells can already be seen in C765>dpp discs at that stage. (C,D) Wild-type (C) and C765>dpp (D) discs 96 hours AEL assayed for proliferation with BrdU. Uniform proliferation seen in wild-type discs is lost in C765>dpp discs. (E) The size of wild-type discs compared with C765>dpp discs 96 hours AEL. Wild type, n=18; C765>dpp, n=21. Error bars indicate the 95% CIs. Scale bars: 50 µm.

• Supplemental Figure S3 -

  Fig. S3. Uniform activation of the Dpp pathway by Tkv^Q235D leads to disc overgrowth with enhanced proliferation rates in the lateral areas as compared with the medial area. (A) C765>tkv^Q235D disc. BrdU staining reveals enhanced proliferation in the lateral region compared with the medial region. (B) Disc from esg>tkv^Q235D animal. Again, BrdU staining is elevated in the lateral region. (C) The size of C765>tkv^Q235D discs and esg>tkv^Q235D discs. Discs of both genotypes overgrow to the same extent as discs in which dpp was uniformly expressed (compare with Fig. 4C). C765>tkv^Q235D, n=11; esg>tkv^Q235D, n=28. Error bars represent 95% CIs. Scale bars: 50 µm.

• Supplemental Figure S4 -

  Fig. S4. The disc size depends on brk levels. (A,B) Discs of C765>brk (A) and C765>brk,tkv^Q235D (B) animals. GFP expression shows the C765-Gal4 driver activity. (C) The size of discs of the two genotypes. Note that the difference in size, as compared with the equivalent experiment conducted with the esg-Gal4 driver (esg>brk and esg>brk,dpp), is due to the later onset of expression of the C765-Gal4 driver. C765>brk discs, n=30; C765>brk,tkv^Q235D discs, n=19. Error bars represent 95%CIs. Scale bars: 50 µm.

• Supplemental Figure S5 -

  Fig. S5. Graded Dpp pathway activity levels in the medial area of the wing disc are not required to drive cell proliferation. (A-A′′) Third instar wing disc of larvae expressing tkv^Q235D under the omb-Gal4 driver. Gal80^ts inhibited tkv^Q235D expression during embryogenesis; 24 hours AEL, larvae were shifted for 96 hours to the permissive temperature (29°C). The inset in A shows a second instar disc expressing tkv^Q235D and GFP under the omb-Gal4 driver. Gal4 function was inhibited during early embryogenesis by the Gal80^ts system; 24 hours AEL, embryos were shifted for 48 hours from 18°C to 29°C. Note that although the Dpp pathway was uniformly activated within the omb expression domain (indicated by pMad staining in A), proliferation was uniform (see BrdU staining in A′), and significant growth did occur during wing disc development (compare omb expression domain between second instar and third instar discs).