Reph, a Regulator of Eph Receptor Expression Figure 2. A genetic screen for Eph pathway signaling molecules. A) The wild-type adult eye displays a regular ommatidial lattice. B) The rough-eye phenotype generated by expression of UAS-ephrin under control of the sevenless2-GAL4 driver (SE) used to screen for modifier mutations. C) Near-complete suppression of the SE phenotype by co-expression of a dominant-negative eph transgene (ephDN). D) Suppression of the SE phenotype by the reph1 allele. E) Suppression of the SE phenotype by rephk8617. F) Co-expression of a UAS-reph+ transgene enhances the SE phenotype. All images (206) were acquired through a digital camera attached to a Zeiss Stemi SV11 stereo dissecting microscope. Images were captured using FPG3 software. doi:10.1371/journal.pone.0037303.g002 the majority of interacting loci based on deficiency screen estimates. Eph and Ephrin mutants in the characterization of modifier mutations As a step toward clarifying the developmental roles of candidate genes that function in the Eph/Ephrin signaling pathway(s), we undertook mutagenesis of the eph and ephrin loci to generate loss-of- function alleles that could be used to establish genetic interactions with the modifier mutations. The eph and ephrin loci are located on chromosome IV, a small chromosome predicted to harbor about 81 genes. For eph, the homologous targeting method [30] was used to introduce a deletion of the kinase domain into the coding region, to create ephKD. For ephrin, we utilized local transposition to introduce a transposon into the 59 end of the gene. The ephKD mutation is predicted to be a partial loss-of-function allele, which still retains reverse-signaling and kinase-independent activities. Consistent with this notion, truncated transcripts were expressed in ephKD mutants (Figure 3 C) while ephKD phenotypes (Figure 3E) were less severe than those elicited using RNAi [21]. The ephKD mutation also resulted in increased developmental mortality and female sterility, indicative of requirements for Eph function outside of the optic lobe. The ephrinRS5 mutation also appeared to be a partial loss-of-function allele exhibiting a robust reduction, but not absence of, Ephrin expression (Figure 3H). The strategy used to generate an Eph receptor mutant with a deleted kinase domain (ephKD) is depicted in Figure 3A. The ‘donor’ construct generated a tandem partial duplication by ‘ends- in’ recombination to produce one untranslatable copy of eph and one translatable copy bearing a 39 deletion encompassing the entire kinase domain. A total of five independent strains were recovered that displayed mobilization of the donor whs gene (Figure 3B). Of these five candidates, three mapped to chromo- some IV and were demonstrated to have targeted the eph locus by Southern blot analysis. To confirm replacement of endogenous eph with the ephKD isoform, RT-PCR analysis of mRNA transcripts from the ephKD 1, 2 & 4 lines was performed (Figure 3C). Truncated transcripts predicted to encode the kinase-deleted Eph isoform were detected (Figure 3C), whereas full-length eph transcripts were not. Eph immunoreactivity was not detected in ephKD animals (compare Figures 3D9 and 3E9) by an anti-Eph antibody generated against a C-terminal peptide [21], which would be deleted in ephKD. Signaling via the Eph receptor (or ‘forward signaling’) should be abolished in the ephKD mutant, but ‘reverse signaling’ [40] via the transmembrane Ephrin, whose conserved cytoplasmic tyrosine phosphorylation site is consistent with intracellular signaling functions [36], would still be possible. Homozygous animals displayed reduced viability, with an approximately 50% develop- mental mortality. Survivors displayed normal external adult morphology. Homozygous ephKD females exhibited a 60%–90% rate of sterility. Olfactory-based learning in ephKD animals was also significantly impaired (A. Szybowski and S. Kunes, unpublished observations). Eph signaling is required for normal development of the mushroom body [22], where olfactory learning occurs. Although adult eye structure and gross retinal development were normal in ephKD animals (data not shown), at the third larval instar stage, when axonal topography is elaborated in the developing adult visual system (Figure 1), ephKD animals displayed variable defects in the centripetal projections of medulla cortical axons toward the developing neuropil (Figure 3E). These defects appeared similar in nature, albeit less severe, to those that resulted from reduction of Eph expression via RNA interference [21]. The ephKD animals displayed gaps in the crescent-shaped neuropil and ectopic axon bundles in the cell body layer surrounding the neuropil (Figures 3E and 4E). To generate a mutation at the ephrin locus, we employed local transposition of the P transposon RS5 [29] located adjacent to the ephrin locus. The ephrin locus was found to be extraordinarily refractory to P-element insertion; however, one local insertion, PLoS ONE | www.plosone.org 5 May 2012 | Volume 7 | Issue 5 | e37303