Fig. 4. Egfr signalling is responsible for R8 twinning in ed^4.12. (A) Graph to show the interactions between ed^4.12 and the Egfr pathway. Genotype is plotted against the proportion of equivalence groups not resolving to single R8 cells. The line above each bar represents the standard error of the mean. Six to nine eye discs were counted of each genotype. (B-H) Confocal microscopy of third larval instar eye imaginal discs. (B,C) Suppression of R8 twinning (Sens expression) by Egfr^IK35. (B) Homozygous ed (ed^4.12/ed^4.12). (C) Homozygous ed with loss of one copy of Egfr (ed^4.12 Egfr^IK35/ed^4.12 +). (D-E'') Loss of R8 twinning in Egfr clones. (D,E) Egfr^IK35 homozygous clones in an ed^4.12/ed^4.12 background. Immunohistochemical detection of Sens (red, D,D',E,E''), ß-galactosidase (green, D,D'',E,E'') and DAPI (blue). The absence of the green ß-galactosidase staining marks the Egfr homozygous clone. Arrows indicate single R8 cells within the clone. (F,F') spi^SC2 clone in an ed^4.12/ed^4.12 background. Immunohistochemical detection of Sens (red) and nlsGFP (green). The absence of the nlsGFP marks the spi-null region, twinned R8 precursors can be seen in both the presence and absence of spi. (G,G') Egfr^IK35 clone in an sca^BP2/sca^BP2 background. Immunohistochemical detection of Sens (red) and ß-galactosidase (green). The absence of the green ß-galactosidase staining indicates the sca Egfr double homozygous clone (the rest of the disc is heterozygous for sca and Egfr and so displays no R8 phenotype). G is a more basal section than G', twins and triplets of R8s can readily be seen in the more apical sections of the clone (arrow). (H) Overexpression of pnt-P1 posterior to the morphogenetic furrow (genotype GMR-Gal4/UAS-pntP1). Immunohistochemical detection of Sens (red) and Ato (green) reveals twinned cells with R8 characteristics (arrows).

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