interactions amongst the collection of candidate Eph pathway genes. Reph, a novel regulator of Eph expression A suppressor mutant, designated here as reph (regulator of Eph expression; Figure 5), exhibited genetic interactions with ephKD and ephrinRS5 mutants (Figure 4), consistent with function in Eph/ Ephrin signaling. reph mapped to the region 24C2-24D2 by complementation analyses using chromosomal deficiencies and lethal P-element insertions localized reph to the CG3920 locus, which encodes two transcript isoforms (Figure 5). Ectopic expression of CG3920, in the form of a UAS-reph+ transgene, rescued reph mutant phenotypes, verifying the identity of reph as CG3920. The single most prominent feature of Reph is the presence of a nuclear localization sequence, P330RRRPSN336, suggesting a putative nuclear protein. Alignment searches indicate that Reph has similarities to a variety of transcription factors, such as the human SPOC-D1 protein (Figure 5), although no obvious homologues. Consistent with a role in transcription, Reph does appear to act as a positive regulator of Eph expression in the optic lobe. All reph alleles, and heteroallelic combinations, were found to be recessive or semi-lethal, with the majority of animals dying during embryogenesis. The viability of both Eph-null mutations [22] and ephKD mutants (Figure 3) suggests that reph has additional functions during embryonic development aside from the regulation of Eph expression. Elucidating the extent of reph’s developmental role, particularly the control of Reph expression itself, is of obvious future interest. Throughout the late third instar optic lobe, reph expression was correlated with Eph expression (Figure 6), placing Reph in the correct spatio-temporal context to regulate Eph expression. Both the necessity and sufficiency of Reph in the regulation of Eph were demonstrated via loss-of-function/gain-of-function experiments. When reph function was eliminated in somatic clones, Eph expression was reduced in a cell-autonomous fashion, whereas strong ectopic reph+ expression produced a cell-autonomous increase in Eph expression. Both perturbations resulted in developmental defects corresponding to the mutant clones (Figures 7 and 8). It is unclear to what extent these observed phenotypes generated by disruption of reph expression are attributable to changes in Eph and how much might be due to additional reph functions. The embryonic lethality of reph alleles, but not eph nulls, suggests such additional reph functions. Furthermore, reph expression within the medulla appears more References 1. Flanagan JG, Vanderhaeghen P (1998) The Ephrins and Eph receptors in neural development. Annu Rev Neurosci 21: 309–345. 2. Klein R (2001) Excitatory Eph receptors and adhesive Ephrin ligands. Curr Opin Cell Biol 13: 196–203. 3. Wang HU, Anderson DJ (1997) Eph family transmembrane ligands can mediate repulsive guidance of trunk neural crest migration and motor axon outgrowth. Neuron 18: 383–396. 4. Conover JC, Doetsch F, Garcia-Verdugo J-M, Gale NW, Yancopoulos G, et al. (2000) Disruption of Eph/Ephrin signaling affects migration and proliferation in the adult subventricular zone. Nature Neurosci 3: 1091–1097. 5. Adams RH, Diella F, Hennig S, Helmbacher F, Deutsch U, et al. (2001) The cytoplasmic domain of the ligand EphrinB2 is required for vascular morphogenesis but not cranial neural crest migration. Cell 104: 57–69. 6. Grunwald IC, Korte M, Wolfer D, Wilkinson GA, Unsicker K, et al. (2001) Kinase-independent requirement of EphB2 receptors in hippocampal synaptic plasticity. Neuron 32: 1027–1040. 7. Contractor A, Rogers C, Maron C, Henkemeyer M, Swanson GT, et al. (2002) Trans-synaptic Eph receptor-Ephrin signaling in hippocampal mossy fiber LTP. Science 296: 1864–1869. Reph, a Regulator of Eph Receptor Expression extensive than that of eph (Figure 6), which could indicate functions outside of the Eph/Ephrin signaling pathway. However, the effects of reph loss-of-function on optic lobe development were consistent with established eph mutant phenotypes. Additionally, differenti- ation of cells within reph somatic clones was normal, even though Eph expression was decreased (Figure 7). These data indicate that, at least in the optic lobe, reph is a novel regulator eph expression. Regulation of Eph/Ephrin expression Eph/Ephrin signaling has been most thoroughly characterized in terms of growth cone dynamics. Less is known, by comparison, about the regulation of Eph receptor and Ephrin ligand expression patterns. Homeobox transcription factors regulate the expression of EphA receptors during rhombomere boundary formation in the vertebrate hindbrain [54–56] and developing retina [57]. Within the optic tectum, engrailed family members regulate the expression of Ephrins [58–60]. In the retina, the T-box transcription factor Tbx5 and Vax homeodomain proteins reciprocally control the expression of B-type Eph receptors and Ephrins along the dorsal- ventral axis [61,62]. Homeobox genes also regulate EphB receptors during vascular development [63]. The identification of Reph expands the repertoire of transcriptional regulators of Eph expression. Further study of Reph function is anticipated to shed light on the regulation of Eph expression patterns, which is vital given the numerous biological processes mediated by Eph/Ephrin signaling in developing and adult tissues. We anticipate that the identification and characterization of additional genes recovered in the modifier screen will elucidate temporal-spatial specific determinants governing Eph/Ephrin signaling outcomes and facilitate a deeper understanding of the evolutionary conserved mechanisms through which this receptor/ligand pair operates. Acknowledgments We appreciate the receipt of strains and antibody reagents from Torsten Bossing and Andrea Brand (Cambridge University, UK), and the Bloomington Drosophila Stock Center. We would also like to thank the University at Albany Department of Biological Sciences Imaging Facility for use of their LSM 510Meta confocal microscope. Author Contributions Conceived and designed the experiments: RD YD SK. Performed the experiments: RD YD BR TK JG SK. Analyzed the data: RD YD SK. Contributed reagents/materials/analysis tools: RD YD JG SK. Wrote the paper: RD SK. 8. Grunwald IC, Korte M, Adelmann G, Plueck A, Kullander K, et al. 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(1999) An Eph receptor regulates integrin activity through R-ras. Proc Natl Acad Sci USA 96: 13813–13818. PLoS ONE | www.plosone.org 14 May 2012 | Volume 7 | Issue 5 | e37303