Reph, a Regulator of Eph Receptor Expression Figure 8. Ectopic misexpression of reph up-regulates Eph expression in the developing visual system. To determine whether reph+ was sufficient for Eph expression, the UAS, GAL4 system was used to drive expression of a UAS-reph+ transgene in cell-specific patterns or within somatic clones. GAL4-expressing cells and clones were positively marked by membrane-bound GFP expressed from a UAS-CD8::GFP transgene (green color in B and C; shown alone in B9. The axonal architecture was visualized by staining with anti-HRP (green in A, red in C). A,A9,A0) A wild type specimen showing the medulla and its characteristic high midline-low dorsoventral gradient of Eph (anti-Eph, blue in A, shown alone in A0). B,B9,B0) Expression of UAS-reph+ in cortical neurons using an elav-GAL4 driver flattens the Eph gradient, notably at the dorsoventral margins (anti-Eph, blue in B, shown alone in B0). Defects in medulla development seen as gaps in the neuropil (yellow arrowheads in B9,B0) result from this up-regulation of Eph expression. C,C9,C0) Several UAS-reph+ cortical clones (red arrowheads in C9,C0) generated using a flip-out tubGAL4 driver can be seen in this specimen. Within these clones, Eph expression (anti-Eph, shown alone in C0) was up-regulated. The enhanced Eph expression was associated with defects manifest as large HRP+ cortical inclusions. Disruption of the normal Eph expression pattern also affected medulla neuropil development (yellow arrowhead in C9, C0). Abbreviations: lobula (lob), medulla neuropil (med n). doi:10.1371/journal.pone.0037303.g008 transmembrane B-type Ephrins [52,53], a class that may include the D. melanogaster Ephrin [36]. Thus, activation of Ephrin signaling while simultaneously inhibiting Eph signaling, as is the case for the ephKD allele, may produce developmental outcomes distinct from failure to activate either arm of the receptor/ligand pair as would be the case for Eph-null mutations. Indeed, the fact that Eph and Ephrin can signal independently even within the same cell [38] supports the notion that integration of these distinct signaling arms may be critical determinants of Eph/Ephrin signaling outcomes. Defects in visual system development for ephKD mutants were qualitatively similar to, albeit less severe than, the loss-of-function phenotype induced by RNAi knockdown of Eph [21], the most consistent of which were defects in cortical axon guidance (Figure 3). There were additional defects noted with ephKD, such as reduced viability and sterility; these phenotypes likely were not detected in the prior analysis because they are also outcomes of microinjection, which was used to introduce double-stranded RNA. A synthesis of these observations indicates that cortical axon guidance in the developing optic lobe requires the kinase- dependent ‘forward’ signaling activity of Eph in the context of simultaneous Ephrin activation. The abundance of cortical axon guidance defects associated with putative Eph signaling mutants (data not shown) supports this conclusion. Roles for Eph signaling beyond axon guidance during nervous system development await further elucidation. The slightly weaker, similar phenotypes observed in the mutant ephrinRS5 (Figure 3) are consistent with the notion that Ephrin plays the role of ligand in the optic lobe, inducing Eph kinase activity. While detailed characterization of the ephKD and ephrinRS5 mutants remains, their moderate pheno- types were well-suited to the purpose of screening for genetic PLoS ONE | www.plosone.org 13 May 2012 | Volume 7 | Issue 5 | e37303