Reph, a Regulator of Eph Receptor Expression Figure 5. Reph encodes a putative nuclear protein. A) The reph1 allele, rephk8617A, was mapped by inverse PCR to the CG3920 locus, which harbors another P-element insertion l(2)k16918 (rephk16918). The reph locus spans 5.5 kb and encodes two alternatively spliced transcripts of 2.1 kb and 3.1 kb. Exons are indicated by boxes and introns by connecting lines. The insertion sites for rephk8617A (first intron of the 2.1 kb transcript) and rephk16918 (59UTR of exon 1 of the 3.1 kb transcript) are indicated on the GenBank scaffold sequence (AE003578) by red triangles. B) The 3.1 kb transcript is predicted to encode a protein 401 amino acids in length, while the 2.1 kb transcript a protein of 351 amino acids that differ only in the site of translation initiation (red arrow). A conserved nuclear localization signal sequence is indicated by the blue-shaded box. C) Reph shows weak similarities to several transcription factors although with no obvious homologs. An alignment to amino acids 361–451 of the human SPOC-D1 transcription factor (NP 653170) is shown as an example (32% identity, 67% similarity over this stretch). doi:10.1371/journal.pone.0037303.g005 sequence analysis in revealing reph’s developmental role, we turned to more functional studies. reph expression in the larval third instar visual system Developmental expression patterns of reph were analyzed via lacZ expression from the two P[lacW] enhancer trap insertions and by in situ hybridization. Reporter expression was found in the eye, lamina, and medulla of the developing visual system at the late third instar stage in patterns overlapping that of Eph (Figure 6). Like Eph, reph expression in the eye disc was most pronounced posterior to and within the morphogenetic furrow (Figure 6A9). Within the lamina, reph and Eph expression were highest at the posterior midline, diminishing in a graded fashion along the dorsoventral axis (Figures 6C and 6C9). Within the medulla, reph expression was similar to Eph, most notably expressed within the Eph-positive cell bodies of the cortex, where cytoplasmic b- galactosidase normally accumulates (Figure 6D9). Within these cortical cells, both reph and Eph expression were lowest at the dorso-ventral margins (Figures 6D9 and 6D0, yellow arrowheads). Thus, the graded pattern of reph expression in the medulla corresponds to the graded pattern of Eph expression. Additional reph expression was found in the lobula where Eph-expressing cells border the posterior face of the medulla (Figures 6D9 and 6D0). Reph lacZ expression was additionally examined in the embryonic CNS, where it over-lapped the expression of Eph (data not shown). In situ hybridizations using reph-specific probes were also performed on larval third instar brains (Figures 6E and 6F). Although the resolution was low, in situ hybridization showed similar expression patterns as for the lacZ reporter lines. Within the eye disc reph expression was found in cells posterior to and within the morphogenetic furrow (Figure 6F). Although lacZ expression appeared more uniform posterior to the morphogenetic furrow compared with RNA expression, this may be a consequence of b- galactosidase perdurability. In the optic lobe, at regions proximal to the lamina, reph expression was highest at the midline (Figure 6F) PLoS ONE | www.plosone.org 9 May 2012 | Volume 7 | Issue 5 | e37303