Reph, a Regulator of Eph Receptor Expression Figure 1. Eph and ephrin expression patterns in the D. melanogaster larval third instar optic lobe. A1–A5) anti-HRP staining (green) reveals overall optic lobe architecture. Posterior to the morphogenetic furrow (MF), retinal axons of ommatidia (small circles, A1) in the developing eye disk (ED; A2) project through the optic stalk (OS; A2) to topographically innervate two target regions in the brain: the lamina (Lam; demarcated by the lamina furrow, LF; A3) and the medulla (A5), which lies proximal to the lamina. Axons of medulla cortical neurons (Med cortex; A5) also project topographically to form a crescent-shaped neuropil (Med n’pil; A5). Schematic representations of optic lobe architecture are shown in A1 (eye and lamina) and A4 (medulla). B1-B5) anti-Eph staining (grayscale). Across the ED, Eph expression appears uniform posterior to the MF. Within the Lam, Eph expression is highest at the posterior midline and lowest at the dorsal-ventral margins of the anterior (B2, B3; depicted schematically in B1). Within the medulla, Eph expression is again highest at the midline, diminishing along the dorsal-ventral axis (B5; depicted schematically in B4). C1– C5) anti-Ephrin staining (grayscale) reveals uniform expression of Ephrin across the developing ED (C2). Within the Lam Ephrin expression appeared relatively uniform (C3). Ephrin-specific expression patterns in the developing ED and Lam are depicted schematically in C1. Within the medulla, Ephrin expression mirrors that of Eph: highest at the midline, diminishing along the dorsal-ventral axis (C5; depicted schematically in C4). D) Summary of Eph/Ephrin expression patterns in the developing visual system (horizontal perspective, which depicts the spatial relationship of the lamina and medulla target fields). Anterior (red arrow) and dorsoventral margins (yellow arrows) are indicated. All image panels were of late third larval instar stage brains stained for HRP (anti-HRP, A2, A3, A5), Eph (anti-Eph, B2, B3, B5) or Ephrin (anti-Ephrin, C2, C3, C5). Note that A2, B2, and C2 are the same image for which individual channels have been displayed. All other images represent distinct specimens. Dorsal (D), ventral (V), anterior (A) and posterior (P) orientations (central compass) are identical for all panels. doi:10.1371/journal.pone.0037303.g001 A Dominant Modifier Screen for Eph Pathway Components We have previously shown that misexpression of a UAS-eph+ transgene disrupts optic lobe development [21] and reasoned that such transgene-mediated phenotypes might manifest as observable defects in the adult eye. After a survey of GAL4 driver and transgene combinations, we determined that the moderate rough eye phenotype of sevenless2-GAL4, UAS-ephrin+ (hereafter referred to as SE; Figure 2B) animals was best suited for a modifier screen. The SE adult eye defect exhibited little variation of penetrance and was suppressed by co-expression of a dominant-negative eph transgene (Figure 2C) or by the ephKD mutation (data not shown). In the former case, we reason that co-expression with an excess of a kinase-inactive Eph isoform would bind up ectopic Ephrin, suppressing its signaling activity. In the latter case (ephKD, see Figure 3), the eph+ locus was targeted by homologous recombina- tion to yield a kinase-defective mutant allele. In the absence of a functional Eph kinase, ectopic Ephrin expression had no effect on adult eye morphology. These observations indicate that the SE morphological phenotype is due to ectopic activation of the Eph pathway. As a preliminary test of the SE background in detecting modifier mutations, deficiency lines representing all four D. melanogaster chromosomes (Bloomington Drosophila Stock Center) were sur- veyed for genetic interaction with SE. A total of 196 deficiency lines were screened representing approximately 43% of the D. melanogaster genome. Approximately two-dozen enhancers and suppressors of the SE rough eye phenotype were identified by this approach, which gives an estimate of 50–55 interacting loci across the entire genome. To identify point mutations that acted as modifiers, ENU mutagenized animals were crossed into the SE background and the F1 progeny examined for enhancement or suppression of the SE eye phenotype (see Materials and Methods). Approximately 15,000 F1 progeny were screened in this manner; 52 lines were retained to form a collection of candidate mutants. This collection includes 24 enhancers (13 recessive lethals) and 28 suppressors (11 recessive lethals; see Figure 3D). On the basis of chromosomal location and complementation analysis the 52 mutants were found to define 40 loci, which should represent PLoS ONE | www.plosone.org 4 May 2012 | Volume 7 | Issue 5 | e37303