expression of Eph in the ladder-like neuropil of the thoracic and abdominal segments. Eph expression was absent, and frequent defects in the ladder-like organization of commissural and longitudinal connectives were observed (data not shown). Howev- er, such defects were not observed in ephKD mutants or in eph mutants [22], which were usually viable. These observations suggest roles for reph both in the regulation of Eph expression and in additional activities required for embryonic CNS development and viability. In the lamina, Eph is expressed in a gradient, highest in the posterior and lowest at the anterior and dorso-ventral margins (Figure 7A0). Loss of reph activity in somatic rephk8617A clones (Figure 7B, n = 35) reduced Eph expression in the mutant cells (Figure 7B0), but not in their wild type neighbors. Photoreceptor axon projections near such clones were abnormal (red arrows, Figure 7B9) and the lamina furrow failed to extend ventrally through the clone (read arrowhead, Figure 7B9). We considered whether the loss of Eph in the lamina (Figure 7B0) might reflect failure of lamina precursor cells to differentiate [45] by staining rephk8617A somatic clones for the early differentiation marker Dachshund (Figure 7C0 and [46]). In rephk8617A somatic clones (n = 7) Dacshund was expressed normally (Figure 7D0), although lamina furrow and photoreceptor projection defects were observed. These data indicated that reph acts downstream of lamina induction. Conversely, we examined whether reph+ activity was sufficient for Eph expression by using the UAS, GAL4 system [33] to drive ectopic expression of a reph+ transgene encoding the 401 amino acid isoform of Reph (see Materials and Methods for details). Whether the smaller (351 amino acid) Reph isoform differs in activity and/or function remains to be examined. Eph is expressed in subsets of developing neurons; within the medulla, the graded pattern of Eph expression (high midline, low dorsoventral) is particularly notable (Figure 8 A0). We first examined how driving uniform reph+ expression, using the pan-neural elav-GAL4 driver [47], affected both Eph expression patterns and medulla architecture (Figure 8B). As can be seen in Figure 8B0, elav- GAL4 driven expression of the reph+ transgene effectively leveled Eph expression throughout the medulla, eliminating the charac- teristic gradient—this was most noticeable at the dorso-ventral margins. As a consequence, medulla architecture was abnormal, manifest as gaps in the medulla neuropil (arrowheads, Figure 8B9) similar to those observed when Eph/Ephrin signaling was disrupted in the ephKD and ephrinRS5 mutants. To refine the ectopic expression approach, a ‘‘FLP-out’’ activated GAL4 driver, P[tuba1.y+,CD8.GAL4 ] [48], was used to express UAS-reph+ in somatic cell clones (Figure 8C). Clones were positively marked by the co-expression of UAS-CD8::GFP [49]. In specimens harboring clones within the medulla, GFP-positive clones displayed en- hanced Eph expression and were associated with areas of large cortical inclusions (red arrowheads, Figures 8C9 and 8C0). Gaps within the medulla neuropil, an indication of axon projection defects, were also observed (yellow arrowhead, Figures 8C9and 8C0). Taken together, these data indicate that reph functions upstream of Eph as a regulator of its expression. Discussion In vertebrates, the diverse array of Eph and Ephrin family members contribute to many aspects of nervous system develop- ment, notably axon guidance [1,3,50]. Surprisingly, although expressed throughout the embryonic CNS [36,51] and larval visual system [21], the contribution of the single D. melanogaster Eph receptor/Ephrin ligand pair in terms of axon guidance and overall Reph, a Regulator of Eph Receptor Expression nervous system development remains unclear. While RNAi knock- down experiments have suggested a role for this pathway in axon guidance [21,36], analyses of an Eph allele have demonstrated little to no role in axon guidance within either the developing embryonic or larval nervous system but rather a specific requirement for mushroom body development [22]. Since the ephrinRS5 and ephKD mutants described in this paper exhibit axon guidance defects reminiscent of RNAi-induced phenotypes, how Eph/Ephrin signaling is translated into functional outcomes remains unclear but would seem to depend on the nature of the allele (e.g. null, forward-signaling inhibited, etc.). Thus, the 40 interacting loci that potentially encode Eph pathway components recovered in the genetic screen described in this paper are likely to help shed light in many areas of Eph/Ephrin signaling. One of these, reph, encodes a putative nuclear factor that evidently regulates Eph expression. Identification of Eph/Ephrin-interacting molecules via genetic screening Using the now conventional modifier screen based on an externally visible eye defect [23–25] we identified a group of candidate genes encoding components of the Eph/Ephrin signaling pathway. The SE phenotype used in this screen (Figure 2B) presumably resulted from hyper-activation of Eph signaling in the retina during development because it was suppressed by co-expression of a C-terminal truncated, domi- nant-negative Eph isoform or by the ephKD mutation. Of the 52 modifiers recovered representing 40 loci, 24 carried lethal mutations. Since eph alleles are viable [22] it is likely that the genes associated with these lethal mutations have additional developmental functions distinct from their roles within the Eph/ Ephrin signaling pathway. Although many of the mutants (8/11 loci examined, data not shown) that survived to the late third instar larval stage displayed optic lobe defects consistent with a role in the Eph pathway, such phenotypes alone could not be used to establish that the affected genes were components of the Eph/ Ephrin signaling pathway, given the diverse cellular processes and molecular pathways involved in optic lobe development. There- fore, evidence for genetic interaction between modifier mutations and Eph/Ephrin mutants was investigated. Allele-specific effects of Eph/Ephrin mutations on Drosophila nervous system development D. melanogaster encodes a single Eph receptor/Ephrin ligand pair, expressed in the embryonic CNS, the developing visual system (Figure 1) and the adult brain. Despite the simplicity of this single pairing in D. melanogaster, the role of Eph/Ephrin signaling with respect to nervous system development is surprisingly complex. Although functional as a canonical receptor/ligand pair when ectopically activated in both the embryonic CNS [22,36] and developing visual system [21], Eph and Ephrin mutations, exhibit both tissue selective and allele-specific effects, instead of revealing a generalized role. Eph-null mutations [22] and a mutation that deletes the Eph kinase domain (ephKD; Figure 3) are without discernible effect on the embryonic CNS. However, both the ephKD and ephrinRS5 alleles exhibited defects in visual system development (Figure 3). Surprisingly, prior examination of an Eph mutation did not reveal photoreceptor axon guidance defects in the developing visual system [22]. However, a requirement for Eph-signaling in the olfactory system is revealed by both Eph-null and ephKD mutants; axon guidance of mushroom body neurons is affected in the Eph-null background [22] while ephKD flies exhibit defects in olfactory-based learning (A. Szybowski and S. Kunes, PLoS ONE | www.plosone.org 11 May 2012 | Volume 7 | Issue 5 | e37303