of Ephrin during eye development. More than two-dozen essential loci that either enhanced or suppressed this defect were identified. We also describe the isolation and molecular characterization of one such gene, reph, which is found to be a novel regulator of Eph expression in the developing D. melanogaster nervous system. Materials and Methods Modifier Screen for Eph Signaling Mutants The eye phenotype that served as the basis for the modifier screen was produced by sevenless2-GAL4, UAS-ephrin. Stable stocks of sevenless2-GAL4, UAS-ephrin were generated by recom- bination and balanced over CyO, hereafter referred to as SE (sevenless2-GAL4, UAS-ephrin), in preparation for the modifier screen. To match as closely as possible the genetic background of SE for the screen, sevenless2-GAL4 animals were isogenized and tested for endogenous modifiers of the SE phenotype; no endogenous modifiers were present in the isogenized sevenless2- GAL4 stock. Isogenized sevenless2-GAL4 males were mutagenized following a 12-hour starvation by transfer to vials containing sucrose-soaked cotton impregnated with 3 mg/ml of ethyl-nitrosourea (ENU). After 12 hours of feeding on the sucrose/ENU food source, mutagenized males were transferred, in two sequential rounds, to normal food for 4-hour periods to allow for grooming and removal of external ENU contamination and subsequently mated with SE females in bottles at a ratio of 5 mutagenized males per 50 SE females. A total of 50 mutagenized males were crossed to 500 SE females. After incubation at 25uC, F1 progeny were examined for the presence of dominant enhancers or suppressors of the SE phenotype and scored. Candidate lines were back- crossed to SE to track the mutation and expand the line. Mutations were localized to individual chromosomes by subse- quent crosses to balancer stocks (FM-7 for the X-chromosome, y+CyO for chromosome II, Tb/Sb for chromosome III and CiD for chromosome IV) and tracing of segregation of the modifier phenotype with the balancers. After localization to a particular chromosome, candidate mutants were back-crossed to SE to confirm the presence of the modifier mutation in the balanced stock. Mapping of Point Mutations to Gene Loci To identify loci harboring point mutations in the Eph collection, lethal lines were crossed to chromosomal deficiency lines (Bloomington Drosophila Stock Center) and assessed for non- complementation. Successively smaller deficiencies were screened until the smallest obtainable deficiency-defined non- complementing region was identified. Next, P-element lethals within the target region were crossed to the SE modifier lethal and assessed for non-complementation. For SE modifier lethals in which available P-element lines failed to resolve the locus, local P-elements were hopped (see below) to generate a collection of new regional-specific P-element insertions. These de novo P- element hops were then individually crossed to the SE modifier lethal under scrutiny and assessed for non-complementation. Once a non-complementing line was identified, the locus of insertion was determined by inverse PCR from the P-element ends (see below). P-element Insertional Mutagenesis and Inverse PCR Analysis of Insertion Loci Quiescent P-elements were mobilized by crossing target lines (see below) to flies of the genotype [w1118; sp/CyO; D2-3,Sb/TM6B] and incubating at 25uC. In most cases, mobilization of resident P- Reph, a Regulator of Eph Receptor Expression elements by the D2-3 transposase involves duplication of the existing P-element followed by hopping of the duplicated element. Successfully induced hops therefore manifest themselves in F1 offspring as darker eye pigmentation owing to the presence of two copies of the w+ marker carried by the P-element. For identification of the reph locus two lethal P-element lines, p[K08617]/CyO and p[K16918]/CyO, localized to a small deficiency non-complementing to the reph1 mutant, were used as starting lines to produce a new collection of local hops. Individual F1 hops from these lines were crossed directly to balanced reph1 mutants and assessed for non-complementation. Non-comple- menting lines were then subjected to inverse PCR analysis to determine the locus of insertion. For generation of the ephrin hypomorph, ephrinRS5, an RS5 P-element insertion on chromosome IV [29] was mobilized. Candidate local hops mapping to chromosome IV were further analyzed by Southern blot of the ephrin locus. Insertions within the ephrin locus revealed by Southern blot were then subjected to inverse PCR to determine the precise location of the P-element insertion. Inverse PCR was performed essentially as described (E.J. Rehm, BDGP, http://www.fruitfly.org/about/methods/inverse.pcr. html), but is summarized here. Flies were homogenized in 16 PBS/0.5 mg/ml Proteinase K (25 ml per fly) in eppendorf tubes, incubated at room temperature for 30 minutes and subsequently heat-inactivated at 100uC for 5 minutes. The homogenate was then spun down and the supernatant collected. 10 ml of genomic extract was then cut with one of three restriction enzymes: Sau3A I, HinP1 I or Msp I. Qiagen column-cleaned digests were then circularized and used as template for PCR under the following conditions: 406 cycles, 3 min extension, 50uC annealing temper- ature. The following primer pairs were used in the inverse PCR reactions: A) pLac1 59 CACCCAAGGCTCTGCTCCCACATT 39 & pLac4 59 ACTGTGCGTTAGGTCCTGTTCATTGTT 39 and B) Sp1 59 ACACAACCTTTCCTCTCAACAA 39 & Splac2 59 GAATTCACTGGCCGTCGTTTTACAA 39. Successfully amplified bands were gel purified using a Qiagen column, sub- cloned into Topo according to manufacturers’ protocol (Invitro- gen) and used to transform competent cells. Mini-preps (Qiagen) obtained from cells transformed with Topo vector containing inverse PCR inserts were used as template for sequencing reactions (ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit). Sequences obtained from inverse PCR fragments were then BLASTed against the D. melanogaster genome to identify the site of P-element insertion. Targeted Homologous Recombination to Generate Kinase-Dead Eph Animals: DNA constructs and fly crosses Targeted homologous recombination was performed essential- ly as described by Rong and Golic [30]. A 6.0 kb EcoRI fragment of the eph genomic region was first cloned into pBS(KS). Subsequently, an I-SceI recognition site was synthesized as two oligos and cloned into a unique site in exon 6 of the eph gene. The I-Sce-I modified eph genomic fragment was then cloned into the Not I site of the P-element vector pP[.whs.N.} to produce the donor construct pP[.whs.ephKD.] for eph targeting. The donor construct was then transformed into flies [31]. Recombination was induced by crossing y1w1; [donor]/y+CyO; [70FLP], [70 I- Sce1]/TM3,Sb,e flies to y1w67c23 flies and heat shocking. Progeny were then screened for w+CyO or changed w+ eye color and test- crossed to map the w+ marker. Animals with w+ mapping to the chromosome IV were examined via Southern blot to verify the targeting event. PLoS ONE | www.plosone.org 2 May 2012 | Volume 7 | Issue 5 | e37303