Reph, a Regulator of Eph Receptor Expression (ephKD1/ephKD1), L4 (ephKD1/+), L5 (ephKD2/+), L6 (ephKD2/ephKD2), L7 (ephKD4/+), L8 (ephKD4/ephKD4), L9 (ephKD3/ephKD3), L10 (molecular weight markers). Full-length transcript was not detected in ephKD homozygous animals (26 cycles). RT-PCR using primers for the 39-deleted isoform of Eph is shown in L11–L19, right side of gel and right diagram (primer locations indicated by arrows). Source RNA for L11–L19 was identical to L1–L9. Only the truncated Eph isoform was expressed in ephKD animals. Abbreviations: LBD (ligand-binding domain), FNIII (fibronectin type III repeats), JXM (juxtamembrane region), TK (tyrosine kinase domain), SAM (sterile alpha motif), PDZ (postsynaptic density 95/Discs-large/zona occludens-1 domain). D,D9) Eph (anti-Eph, red in D, shown alone in D9) is expressed on cortical neuron axons in wild-type third instar larvae, accumulating in a high-midline low-dorsoventral gradient in the medulla neuropil (compare anti-HRP staining, green, to anti-Eph staining, red). E,E9) Lack of Eph immunoreactivity (anti-Eph, red in E, shown alone in E9) corresponded to optic lobe defects (anti-HRP, green) in ephKD animals, manifest as gaps in the neuropil (arrowheads). F) Schematic of the 5.8 kb ephrin locus localized to the 102C2 region of chromosome IV. The ephrin gene is comprised of 5 exons (black boxes) and 4 introns (gray lines). The start codon (red arrow) and RS5 P-element insertion site (red shaded box) into 59UTR of the first exon are also indicated. G,G9) In wild-type animals, Ephrin expression (anti-Ephrin, red in G, shown alone in G9) is punctate along cortical neuron axons and concentrated in the optic lobe neuropil (anti-HRP, green in G). H,H9) In ephrinRS5 mutants, Ephrin expression is considerably reduced in the optic lobe (anti-Ephrin, red in H, shown alone in H9), resulting in neuropil defects (arrowheads; anti-HRP, green in H). White or yellow bars indicate the dorsoventral midline. Scale bar in D is 20 mm for D,D9,E,E9, G,G9, H,H9. doi:10.1371/journal.pone.0037303.g003 ephrinRS5, was obtained that disrupted the 59 UTR of the ephrin mRNA (Figure 3F). The homozygous mutant animals were viable, with normal external adult morphology. They exhibited approx- imately a 50% reduction in ephrin transcript level, as determined by quantitative RT-PCR analysis (data not shown). In wild-type animals, Ephrin is found in puncta on the axons of medulla cortical neurons, and concentrated within the neuropils of the medulla and lobula (Figures 1-C5 and 3G9). Ephrin protein was substantially reduced in the optic lobe of ephrinRS5 homozygotes (Figure 3H9). While these animals displayed normal ommatidial development (data not shown), subtle defects in optic lobe axonal topography were observed (Figure 3H, arrowheads). In the medulla, cortical axon misprojection resulted in neuropil gaps and ectopic axons in the cortical cell body layer. Photoreceptor axon projection defects were observed in the lamina, but these misprojections appeared correlated with defects in medulla architecture. The similarity of the ephrinRS5 hypomorphic pheno- type with that of ephKD is consistent with the notion that these proteins act in the same pathway. However, we must note that many unrelated activities contribute to medulla development and can yield superficially similar axon projection phenotypes. Although the anti-HRP staining used in these studies doesn’t reveal specific aspects of the optic lobe defects associated with ephKD and ephrinRS5 mutations, such as axon subsets or expression of cell adhesion molecules, it provides useful categorization based on general architectural features. To better assess and characterize these medulla phenotypes, ephKD and ephrinRS5 animals were scored based on the following criteria: the presence of gaps in the medulla neuropil (0, 1 or .1), the degree of lobula neuropil disruption (none, mild, moderate, or severe) and the presence of HRP+ cortical inclusions (yes or no). For ephKD mutants (n = 16) all exhibited at least one gap in the medulla neuropil with the majority (75%, 12/16) showing more than one such gap. Half of these animals had normal lobula architecture with 38% (6/16) exhibiting mild disruption and only 12.5% (2/16) having moderate-level defects. Large HRP+ cortical inclusions were rare, with only 12.5% (2/16) of ephKD mutants exhibiting this feature. Notably, the ephrinRS5 phenotype appeared qualitatively different based on the aforementioned criteria. In the specimens examined Figure 4. reph interacts genetically with the ephKD and ephrinRS5 mutations. In wild-type flies (A, E) the medulla neuropil (med) is distinguished by its regular crescent shape (anti-HRP staining, grayscale, all panels A–H). Mutant phenotypes could be broadly categorized as defects in the medulla neuropil (white arrowheads), HRP+ cortical inclusions (yellow arrowheads) or disruption of lobula (lob) architecture (red arrowheads). In homozygous ephrinRS5 mutants (B), subtle abnormalities were observed in the medulla neuropil and cortex. The severity of ephrinRS5 medulla defects was enhanced by a single copy of reph1 (C) or rephK8617A (D). In homozygous ephKD mutants (F) gaps in the medulla neuropil were often present. A single copy of the reph1 allele (G) or rephK8617A (H) exacerbated the ephKD mutant phenotype. doi:10.1371/journal.pone.0037303.g004 PLoS ONE | www.plosone.org 7 May 2012 | Volume 7 | Issue 5 | e37303