Targeted Homologous Recombination to Generate Kinase-Dead Eph Animals: Molecular Analyses of Targeting Efficacy Southern blot analyses were performed according to standard protocols [32] using probes targeting the I-Sce-I cut site of the donor construct. In addition, RT-PCR experiments were performed on mRNA isolated from putative ephKD animals to verify the presence of predicted truncated transcripts. Generation and misexpression of P[UAS-reph] and P[UAS- ephrin] transgenes A 2 kb EcoRI/XhoI fragment of reph containing the entire coding region for the 401 amino acid isoform was excised from a pOT2 cloning vector (GH05923, Drosophila Genomics Resource Cen- ter, Indiana University) and inserted into the transformation vector pUAST [33] to generate P[UAS-reph]. Likewise, for P[UAS- ephrin], the full-length coding region was amplified by PCR and inserted into pUAST. Transformation of D. melanogaster was performed as described by Rubin and Spradling [31]. Patterned expression of the P[UAS-reph] and P[UAS-ephrin] transgenes was accomplished using the UAS-GAL4 system [33]. All crosses were grown at 25uC and immunohistochemical analyses performed on late third instar larvae. The following crosses were used: (A) y,hsFLP122;;UAS-reph X y,w; UAS-CD8::GFP, tuba1.y+, CD2.GAL4/ y+CyO, (B) sevenless2-GAL4 X UAS-ephrin, (C) sevenless2-GAL4, UAS- ephrin/y+CyO X UAS-reph (‘.’ indicates the position of an FRT site in the respective construct). Mosaic Analysis of reph function in the Developing Optic Lobe Mosaic analysis was carried out as described by Xu and Rubin [34]. Larvae were subjected to heat shock at 37uC for 60– 75 minutes 24–36 hours after hatching to induce expression of an hsFLP transgene. After growth at 20uC, larvae were dissected at late larval third instar stage and processed for immunohistochem- ical analysis. The following crosses and strains were used in the experiments described: y,hsFLP122; P{arm-lacZ}42D, P{FRT}42D/P{y+},CyO X y,w; P{lacW}reph, P{FRT}43D/ P{y+}, CyO Immunocytochemistry Immunocytochemically staining was performed essentially as described in [35]. Primary antibodies were used at the following dilutions: rabbit anti-Eph 1:200 [21], rabbit anti-Ephrin 1:200 [36], goat FITC or Cy3 anti-HRP (Cappel) 1:200, mouse anti-bgal (Promega) 1:100. Secondary antibodies were used at the following dilutions: Cy3 or Cy5 goat anti-mouse (Jackson Immunochemical, Inc.) 1:100, Cy3 or Cy5-goat anti-rabbit (Jackson) 1:500, HRP- conjugated goat anti-mouse IgG (Jackson) 1:100. Specimens were viewed on a Zeiss LSM510 META confocal microscope. RNA localization by tissue in situ hybridization In situ hybridization was performed on larval third instar specimens as referenced in [21]. A pOT2 vector containing a full- length reph cDNA (GH05923, Drosophila Genomics Resource Center, Indiana University) was used as template to prepare both sense (BglII linearized, Sp6 RNA polymerase transcription) and antisense (XhoI linearized, T7 RNA polymerase transcription) digoxigenin-labeled probes (Riboprobe Combination System- Sp6/T7, Promega). Hybridized specimens were developed following incubation with anti-digoxigenin-alkaline phosphatase conjugated antibodies (mouse anti-digoxin, Sigma). Bright-field images were captured with a CCD camera. Reph, a Regulator of Eph Receptor Expression Results Eph and Ephrin Expression Patterns in the Developing Visual System We have previously shown that the Eph protein is expressed on the axons of photoreceptor neurons, medulla and lobula cortical neurons and the membranes of lamina neuron precursors. The medulla cortex and axonal projections establish a three-dimen- sional neuropil that provides topographically arrayed synaptic targets for R7 and R8 photoreceptor axons and lamina axons. Eph antigen on medulla axons displayed a dorsoventral gradient, high at the midline toward the dorsal and ventral poles (Figure 1; [21]). Moreover, we found that disturbing this gradient resulted in topographic medulla axon targeting defects [21]. In contrast, Eph antigen on photoreceptor axons appeared uniform across the dorsoventral axis of their retinotopic projections into the brain. The D. melanogaster genome also encodes a single Ephrin ortholog, which is most similar to vertebrate Ephrin B class ligands [36]. While RNAi interference approaches have suggested a role for Eph/Ephrin signaling in neuronal development, notably within the embryonic CNS [21,36], analyses of Eph mutants did not detect embryonic CNS defects but rather a specific role for Eph/Ephrin signaling in the developing mushroom body [22]. The disparity between these results suggests a complex role for Eph/Ephrin signaling during Drosophila development that would benefit from additional characterization, specifically analyses of Ephrin expression patterns within the larval brain. Like Eph, the expression of its ligand Ephrin was uniform across the dorsoventral columns of ommatidia posterior to the morpho- genetic furrow (Figures 1-B2 and 1-C2). Within the lamina, retinal axons distribute themselves retinotopically along the dorsoventral and anteroposterior axes, forming a crescent-shaped target field (Figure 1-A1 and 1-A3). Retinal axons arrive in this field in a posterior to anterior temporal order, in concert with the temporal dynamics of photoreceptor cell differentiation in the retina. The axons from each dorsoventral column of photoreceptor neurons arrives in the target field contemporaneously, and distribute themselves retinotopically on the dorsoventral axis of the lamina. Eph expressed on the membranes of developing lamina neurons displayed gradients on both axes; high dorsoventral midline and high posterior to low anterior (Figure 1-B3; [21]). Ephrin expression, in contrast, appeared relatively uniform (Figure 1- C3). In the medulla ganglion, the target for R7/R8 photoreceptor axons, cortical axons project topographically into a crescent- shaped neuropil, which requires cues conveyed by the pattern of Eph expression [21]. As was the case for the developing eye disc, Eph and Ephrin were expressed in similar gradients on axons of medulla cortical neurons—high midline, low dorso-ventral (Figures 1-B5 and 1-C5, respectively). Co-expression of Eph receptors and Ephrin ligands has been observed in a number of tissues [22,37,38] and suggests that the mechanism by which Eph/ Ephrin signaling regulates axon guidance is more complex than simple gradient-mediated interactions. Such co-expression would permit both forward and reverse signaling within the same cell, as well as for both trans and cis regulation of Eph/Ephrin complexes [39]. Thus, the effects of Eph/Ephrin signaling within a given cell may depend on the coordination of these various regulatory possibilities. Given the extensive expression of Eph and Ephrin in the larval third instar visual system (summarized in Figure 1D) and observations that disruption of Eph expression affects axon topography [21] we reasoned that the developing D. melanogaster visual system might provide an amenable model to screen for components of the Eph/Ephrin signaling pathway. PLoS ONE | www.plosone.org 3 May 2012 | Volume 7 | Issue 5 | e37303