Fig. 2. The presumptive R8 cell requires Spitz-mediated activation of the Egfr pathway to differentiate in sens mutants. Third instar eye imaginal discs are presented with posterior to the left in this and subsequent figures. (A-C,E-G) Mutant tissue is negatively marked by the absence of GFP (green). (A-C) Neuronal differentiation is indicated by an antibody to Elav (red). (A) Single R8 neurons (overlap with Sens, blue) are detected in rho-1 rho-3 mutant tissue, which lack all activation of the Egfr pathway. This suggests that R8 differentiation does not require Egfr activation. (B) Clusters of variable numbers of neurons are detected in sens mutant tissue. (C) Neurons are not detected in rho-1 rho-3 sens mutant tissue except at the clonal border, where non autonomous effects cause photoreceptor differentiation. This suggests that neuronal differentiation of the pre-R8 as an R2/R5 cell in sens mutants is dependent on Egfr activation. (D) spi sens double mutant tissue is identified by the absence of ß-gal (blue) and outlined with the dotted line. (D') sens mutant tissue is also marked by the absence of Sens (green). Elav (red) marks neurons. (D'') Overlay of D and D'. Tissue that lacks both spi and sens function does not contain Elav-positive cells except near the borders of the clone, where non autonomous function of spi is sufficient to induce some neuronal differentiation. Differentiation of the pre-R8 as an R2/R5 cell in sens mutant tissue is therefore also dependent on spi function. (E-F) R8 selection (pre-R8) is marked by sca-lacZ (red). (E) Pre-R8s are selected in rho-1 rho-3 mutant tissue. (F) Pre-R8s are still selected in rho-1 rho-3 sens mutant tissue, indicating that the loss of neuronal differentiation in these mutants is not secondary to a failure of R8 selection. (G) Boss (red), a marker for R8 differentiation, is absent in rho-1 rho-3 sens ro mutant tissue, suggesting that the R8 rescue seen in sens ro double mutants cannot occur when Egfr signaling is absent.