Reph , a Regulator of Eph Receptor Expression in the Drosophila melanogaster Optic Lobe Richard E. Dearborn Jr.2*, Yong Dai1, Brian Reed1, Tamar Karian2, Jessica Gray1, Sam Kunes1 1 Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, United States of America, 2 Department of Pharmaceutical Sciences, Albany College of Pharmacy and Health Sciences, Albany, New York, United States of America Abstract Receptors of the Eph family of tyrosine kinases and their Ephrin ligands are involved in developmental processes as diverse as angiogenesis, axon guidance and cell migration. However, our understanding of the Eph signaling pathway is incomplete, and could benefit from an analysis by genetic methods. To this end, we performed a genetic modifier screen for mutations that affect Eph signaling in Drosophila melanogaster. Several dozen loci were identified on the basis of their suppression or enhancement of an eye defect induced by the ectopic expression of Ephrin during development; many of these mutant loci were found to disrupt visual system development. One modifier locus, reph (regulator of eph expression), was characterized in molecular detail and found to encode a putative nuclear protein that interacts genetically with Eph signaling pathway mutations. Reph is an autonomous regulator of Eph receptor expression, required for the graded expression of Eph protein and the establishment of an optic lobe axonal topographic map. These results reveal a novel component of the regulatory pathway controlling expression of eph and identify reph as a novel factor in the developing visual system. Citation: Dearborn RE Jr, Dai Y, Reed B, Karian T, Gray J, et al. (2012) Reph, a Regulator of Eph Receptor Expression in the Drosophila melanogaster Optic Lobe. PLoS ONE 7(5): e37303. doi:10.1371/journal.pone.0037303 Editor: Edward Giniger, National Institutes of Health (NIH), United States of America Received January 11, 2012; Accepted April 19, 2012; Published May 16, 2012 Copyright: 2012 Dearborn et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Institutes of Health, National Eye Institute grant EY10112 to S.K. and National Institutes of Health, National Eye Institute grant EY07030 to R.D. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Co-author Dr. Shibo Jiang is a PLoS ONE Editorial Board member, but this does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. All other authors have declared that no competing interests exist. * E-mail: richard.dearborn@acphs.edu Introduction Bi-directional signaling through receptor tyrosine kinases of the Eph family and their Ephrin ligands contributes to diverse processes during and after development including the establish- ment of topographic axon projections in the visual system [1,2], cell migration [3,4], vascular development [5] and long-term potentiation [6–8]. Crucial to deciphering the mechanisms by which this signaling pathway mediates such diverse and complex processes is a complete catalog of its pathway components. Substantial progress towards this end has been made by biochemical approaches [9]. Activation of Eph receptors and transmembrane B-class Ephrin ligands regulates cytoskeletal dynamics through the recruitment one of several SH2/SH3 adaptor proteins [10–12] and PDZ-domain proteins [13]. Ras superfamily GTPase activity [14–16] and crosstalk with other signaling pathways including MAPK [17,18], PI-3 kinase [19] and heterotrimeric G proteins [13] are involved. Nonetheless, many effector molecules may have been missed by these approaches. A complementary approach, utilizing genetic analysis, offers the possibility of additional insights into Eph signal transduction. We have undertaken this approach in D. melanogaster. The diverse activities and functional redundancy of the large vertebrate Eph and Ephrin families complicates pathway studies. The D. melanogaster genome, in contrast, encodes only a single Eph receptor and Ephrin ligand [20]. Previous work has demonstrated a conserved role for the D. melanogaster Eph pathway in the establishment of adult visual system axonal topography [21] and axon guidance within the mushroom body [22]. Within the visual system, Eph is expressed in a gradient, reminiscent of its graded retinal expression required for vertebrate retinotectal map formation, and controls the dorsoventral patterning of cortical axons that project centripetally to form the medulla neuropil [21]. Within the olfactory system, Eph is expressed by mushroom body neurons throughout development and Eph signaling regulates the guidance of individual axon branches within the mushroom body [22]. In light of these observations and the diversity of its resources for genetic analysis, D. melanogaster should serve as a valuable model system in which to identify novel components of the Eph pathway. The D. melanogaster eye has often been used as an assay in genetic screens, including those for components of receptor tyrosine kinase (RTK) pathways [23–25]. Each ommatidium of the approximately 750 that comprise the compound eye is composed of the same complement of cells: eight photoreceptor neurons (R-cells) and a set of non-neuronal accessory cells, which includes lens-secreting cone cells and pigment cells [26–28]. The photoreceptor neurons project retinotopically into distinct regions of the optic lobes: R1– R6 axons terminate in the lamina while R7 and R8 axons terminate in different layers of the medulla [28]. The finely ordered structure of the D. melanogaster visual system gives rise to a neurocrystalline lattice, which is perturbed by changes in cell numbers, altered cell differentiation or aberrant axon projections. These types of developmental defects give rise to visible phenotypes that are well-suited to enhancer/suppressor screens. We utilized this approach, conducting a genetic screen for modifiers of an eye defect associated with the ectopic expression PLoS ONE | www.plosone.org 1 May 2012 | Volume 7 | Issue 5 | e37303