(n = 17), only 12% (2/17) exhibited multiple gaps in the medulla neuropil, while 65% (11/17) had no gaps. Disruption of lobula architecture was more common in ephrinRS5 animals—24% (4/17) exhibited moderate defects and 47% (8/17) showed mild defects. As was the case with ephKD mutants, large HRP+ coritical inclusions were rare in ephrinRS5 mutants, with only 6% (1/17) of the specimens manifesting this phenotype. Thus, the ephKD phenotype was generally characterized by multiple gaps in the medulla neuropil with relatively normal lobula development, while the ephrinRS5 phenotype was somewhat reversed with a higher percentage of normal medulla development and a greater degree of lobula disruption relative to ephKD mutants. Such qualitative categorization served as a baseline for assessing genetic interac- tions between modifier mutations and the ephKD and ephrinRS5 mutants. The SE suppressor mutation, reph, interacts with ephrin and eph mutations Mutations in genes that participate in Eph/Ephrin signaling should disrupt optic lobe axonal topography as observed when eph expression patterns or levels are altered ([21] and below). Such mutations might also have additional distinct phenotypes, as a consequence of the gene’s pleiotropic or tissue-specific functions outside of the Eph pathway. The lethal SE enhancers and suppressors displayed a range of temporal lethality, spanning early embryogenesis to late pupation. We examined visual system architecture in homozygous viable and late-lethal mutants at the late third instar larval stage, the time of visual system axon outgrowth and topographic patterning. Of eleven modifier mutations representing distinct loci that were examined at this stage eight displayed defects in medulla architecture (data not shown) roughly similar to eph and ephrin mutants (Figures 3E and 3H). Since the criterion of optic lobe developmental defects was insufficient in and of itself to identify a given enhancer or suppressor as a component of the Eph/Ephrin signaling pathway, we sought to establish genetic links. To identify modifier mutations that interacted with the Eph/Ephrin pathway in the establishment of axon topography, we screened the mutant collection for suppression or enhancement of the ephrinRS5 and ephKD mutant phenotypes (Figures 3E, 3H). This strategy identified an SE suppressor, which we have named reph (regulator of eph expression, see below) as a strong interactor with Eph pathway function. reph1 and rephK8617A were strong suppressors of the SE phenotype, a phenotype induced by ectopic Eph pathway activation (Figure 2). In contrast, heterozygosity for either the reph1 or rephK8617A alleles enhanced the optic lobe axonal targeting phenotypes of the ephrinRS5 or ephKD homozygotes (Figure 4), while reph1 or rephK8617A heterozygosity in an otherwise wild type background displayed normal optic lobe development. All of the phenotypic features associated with the ephKD were exacerbated by the presence of the reph alleles. In the reph1/+; ephKD/ephKD specimens examined (Figure 4G, n = 10), 80% (8/10) had multiple gaps in the medulla neuropil and all exhibited some form of lobula disruption, 50% (5/10) of these being severe (e.g. multiple gaps, misprojections, etc.). Furthermore, large HRP+ cortical inclusions were frequently observed (8/10 of the specimens examined). For the reph1/+; ephrinRS5/ephrinRS5 genotype (Figure 4C, n = 5), 80% (4/5) exhibited multiple gaps in the medulla neuropil, 80% (4/5) had large HRP+ cortical inclusions, and 80% (4/5) mild to moderate disruption of lobula architecture. Similar phenotypes were observed for the rephK8617A allele, confirming that the genetic interaction could be attributed to the reph mutations and not the genetic background of the lines. As was the case for reph1, Reph, a Regulator of Eph Receptor Expression rephK8617A/+; ephKD/ephKD specimens (Figure 4H, n = 7) exhibited multiple gaps in the medulla neuropil (86%, 6/7), increased lobula disruption (57% or 4/7 being severe) and large HRP+ cortical inclusions (86% or 6/7 of the specimens examined). Likewise, for the rephK8617A/+; ephrinRS5/ephrinRS5 genotype (Figure 4D, n = 5), 80% (4/5) exhibited multiple gaps in the medulla neuropil, 60% (3/5) had large HRP+ cortical inclusions, and 80% (4/5) mild to moderate disruption of lobula architecture. Taken together, these data were consistent with reph functioning within the Eph/Ephrin signaling pathway. Molecular characterization of the reph locus To gain insight into the role of reph in the Eph/Ephrin pathway, we undertook a molecular characterization of the reph locus. We used deficiency mapping and complementation with P-element lethal insertion mutations to localize reph1 to Df(2L)sc19-8, which spans a small region (24C2-24D2) that contains only 13 predicted genes. Like reph1, a chromosome bearing Df(2L)sc19-8 suppressed the SE rough eye phenotype. The P-element insertion P[lacW]l(2)k16918k16918, one of four lethal insertions uncovered by Df(2L)sc19-8, displayed semi-lethality over the reph1 mutant chromosome. DNA sequencing mapped P[lacW]l(2)k16918k16918 to the reph locus, revealing that the P[lacW]l(2)k16918k16918 insertion was indeed a reph allele, which was designated rephk16918 (Figure 5A). To generate additional P-element insertions within reph, we mobilized a second insertion in the Df(2L)sc19-8 region, P[lacW]bowlk8617,and obtained one reph1 non-complementing insertion, named rephk8617A (Figure 5A). Rescued DNA sequence flanking the rephk8617A insertion matched the predicted locus, CG3920 (Flybase). The rephk8617A allele also suppressed the SE eye phenotype as a rephk8617A/+ heterozygote (Figure 2E). The reph locus is predicted to encode two mRNAs via alternative splicing (Figure 5A). rephk8617A is an insertion within the first intron of the 2.1 kb transcript, upstream of the first exon of the reph 3.1 kb transcript. The rephk16918 insertion is located 1.3 kb downstream of rephk8617A within the 59UTR of the 3.1 kb reph transcript. As a final test of the identity of reph and CG3920, we examined rescue with a UAS-reph+ transgene (see below). Pan- neural expression of UAS-reph+ with the driver elav-GAL4 [41] rescued the homozygous lethality of reph1, consistent with a requirement in the nervous system. Given that eph alleles are viable [22] these data suggest that reph has additional functions unrelated to the regulation of eph. In addition, SE animals harboring a single copy of UAS-reph+, under sevenless2-GAL4 control, displayed a more severe (enhanced) SE eye phenotype (Figure 2F). These data permit the conclusion that reph is the CG3920 locus. The predicted reph transcripts encode proteins of 351 and 401 amino acids, which differ in their translation initiation sites (Figure 5B). Reph lacks robust homology to known proteins, although database searches indicate weak similarities between Reph and a variety of transcription factors. An example of such homology to a region of the human SPOC (Spen paralog and ortholog C-terminal) domain-containing 1 protein is shown in Figure 5C. SPOC domain-containing proteins are nuclear effectors of receptor tyrosine kinase signaling in Drosophila nervous system development [42–44]. Although not itself a member of the SPOC family, Reph harbors a nuclear localization signal sequence, consistent with a putative role in transcription (Figure 5B). Although it lacks any other conserved domain features, the overall structure of Reph may be related to the IMP dehydrogenase/GMP reductase family; the significance of this structural similarity is unclear. Given the limited utility of PLoS ONE | www.plosone.org 8 May 2012 | Volume 7 | Issue 5 | e37303