DEV038406 Supplementary Material

Files in this Data Supplement:

• Supplemental Appendix -

• Supplemental Figure S1 -

  Fig. S1. Comparison of the expression of Dpp/Brk, Hpo/Warts and dMyc in M/+ and M⁺ territories. The Dpp/Brk gradient is an important growth regulator in the wing disc (Affolter and Basler, 2007; Burke and Basler, 1996; Martin et al., 2004; Schwank et al., 2008) and has also been implicated in cell competition (Moreno et al., 2002). To monitor Dpp activity levels in the overgrowing M⁺ clones, we used an antibody that recognizes the phosphorylated (active) form of the Dpp transducer Mad (pMad), an antibody against the Dpp target gene product Spalt (Barrio and de Celis, 2004) and also an anti-Brk antibody (Tanimoto et al., 2000). After examining a large number of clones we did not find any significant differences comparing M⁺ clones with surrounding M/+ cells. The Hpo/Warts pathway also plays an important role in growth, and there are some suggestions that it might have a role in cell competition (Tyler et al., 2007). Upregulation of Expanded and nuclear localisation of Yki are widely used as read-outs of the hippo pathway (Dong et al., 2007; Hamaratoglu et al., 2006). We have examined the expression of those three markers in M⁺ clones but failed to detect changes in their activity levels with respect to M/+ cells. Finally, we compared the dMyc activity levels in the two types of cells. This is an important comparison because it has been suggested that the confrontation of cells with dissimilar levels of dMyc is a key issue in triggering cell competition (de la Cova et al., 2004; Moreno and Basler, 2004). Using an anti-dMyc antibody we failed to find a difference between M⁺ and M/+ cells. (A,A′) pMad expression in a disc containing a large M⁺ clone, labelled by loss of GFP. There is no indication of a difference in pMad levels. (B,B′) The expression levels of Spalt (Sal) are not modified in M⁺ clones. (C-F′) Similarly, Brk (C,C′), dMyc (D,D′), Yorkie (E,E′) and Expanded (F,F′) are unaffected.

• Supplemental Figure S2 -

  Fig. S2. Size of 24 and 48 hour BFP M⁺ clones in M⁺ and M/+ discs screened in adult wings. (A-D) 24 hour; (E-H) 48 hour. The left column shows the time of induction at the corresponding developmental stages. The middle column shows the average size of the clones measured as the number of mwh trichomes per clone and the third column includes some characteristic clones. Error bars are standard errors; *P<0.05.

• Supplemental Figure S3 -

  Fig. S3. Schematic of the method to estimate the proliferation rate in different stages and also to count the number of cells in the mature wing disc. (A) The arrows (red for the wild type and blue for M/+) mark the length of time between heat shock and fixation. The position of the arrow indicates the developmental stage. (B) Numerical data obtained in the different experiments. (C) One image of the multiple confocal sections of UAS-GFP/His2-RFP;hh-Gal4 that were taken. (D) A grid of orthogonal sections was disposed in order to delimitate between 40 and 50 'cubes' as discrete units of counting. (E) In each of the four sides of the cubes, dimensions (in terms of number of cells) were measured at three or four different points. (F) With all these measures, the average length, width and depth were multiplied to achieve a 'volume' of cells for a particular cube. (G) This was repeated for each cube in each of the measured discs. Cell counting of anterior and posterior were performed separately, but are not represented in the figure for simplicity.

• Supplemental Figure S4 -

  Fig. S4. Additional results from computer simulations modifying different parameters. (A) Predictions for the average M⁺ territory of a M/+ discs containing a variable number of early M⁺ clones in the absence of cell competition. (B) Comparison of the computer prediction and the real data for late (72 hours BFP) clones. Note the very good agreement between the two sets of data. (C) Table of results of a number of simulations for various apoptosis parameters, the time needed for the disappearance of the apoptotic cell and the frequency of apoptotic cells at the borders of M⁺ clones. The number in the left column indicates the file number in the supplementary simulation folder in the http://bacterio.cbm.uam.es:8080/scherrera/simulations.zip file.

  References

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  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.

  Burke, R. and Basler, K. (1996). Dpp receptors are autonomously required for cell proliferation in the entire developing Drosophila wing. Development 122, 2261-2269.

  Hamaratoglu, F., Willecke, M., Kango-Singh, M., Nolo, R., Hyun, E., Tao, C., Jafar-Nejad, H. and Halder, G. (2006). The tumour-suppressor genes NF2/Merlin and Expanded act through Hippo signalling to regulate cell proliferation and apoptosis. Nat. Cell Biol. 8, 27-36.

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  Schwank, G., Restrepo, S. and Basler, K. (2008). Growth regulation by Dpp: an essential role for Brinker and a non-essential role for graded signaling levels. Development 135, 4003-4013.

  Tanimoto, H., Itoh, S., ten Dijke, P. and Tabata, T. (2000). Hedgehog creates a gradient of DPP activity in Drosophila wing imaginal discs. Mol. Cell 5, 59-71.