<i>reph</i> loss-of-function suggests cell autonomous regulation of Eph expression.

<p>A requirement for <i>reph</i> in optic lobe development was assessed by generating homozygous somatic <i>reph^k8617A</i> clones by the FLP, FRT method. Mutant clones were marked by loss of expression of an <i>arm-lacZ</i> reporter (anti-βgal, red in B, blue in C and D). Overall optic lobe architecture was revealed by anti-HRP staining (green color in all panels, shown alone in A′ and B′). A,A′,A″) A wild type specimen illustrating the normal distribution of Eph expression in the lamina. B,B′,B″) A specimen harboring a homozygous <i>reph^k8617A</i> clone (white or yellow outlines) along the ventral margin of the lamina displayed reduced Eph expression (anti-Eph, blue in B, shown alone in B″) and incomplete LF formation (red arrowhead in B′). To assess potential pleiotrophic effects of reph^k8617A on lamina development, reph^k8617A clones were stained for dachshund, a marker of lamina neurogenesis (anti-Dac, red color in C and D, shown alone in C″ and D″). C,C′,C″) A wild type specimen highlighting Dac expression in the lamina. D,D′,D″) In the specimen shown, a large reph^k8617A clone encompassed most of the ventral lamina (yellow outline) indicated by loss of lac-Z staining (blue). Dac expression was normal within the clone. Dorsal is up and ventral is down in all panels. Abbreviations: lamina (lam), lamina furrow (LF), lobula (lob). Yellow bars in A″–D″ indicate the position of the midline.</p

Ectopic misexpression of <i>reph</i> up-regulates Eph expression in the developing visual system.

<p>To determine whether <i>reph⁺</i> was sufficient for Eph expression, the UAS, GAL4 system was used to drive expression of a <i>UAS-reph⁺</i> transgene in cell-specific patterns or within somatic clones. GAL4-expressing cells and clones were positively marked by membrane-bound GFP expressed from a <i>UAS-CD8::GFP</i> transgene (green color in B and C; shown alone in B′. The axonal architecture was visualized by staining with anti-HRP (green in A, red in C). A,A′,A″) A wild type specimen showing the medulla and its characteristic high midline-low dorsoventral gradient of Eph (anti-Eph, blue in A, shown alone in A″). B,B′,B″) Expression of <i>UAS-reph⁺</i> in cortical neurons using an <i>elav-GAL4</i> driver flattens the Eph gradient, notably at the dorsoventral margins (anti-Eph, blue in B, shown alone in B″). Defects in medulla development seen as gaps in the neuropil (yellow arrowheads in B′,B″) result from this up-regulation of Eph expression. C,C′,C″) Several <i>UAS-reph⁺</i> cortical clones (red arrowheads in C′,C″) generated using a flip-out <i>tubGAL4</i> driver can be seen in this specimen. Within these clones, Eph expression (anti-Eph, shown alone in C″) was up-regulated. The enhanced Eph expression was associated with defects manifest as large HRP⁺ cortical inclusions. Disruption of the normal Eph expression pattern also affected medulla neuropil development (yellow arrowhead in C′, C″). Abbreviations: lobula (lob), medulla neuropil (med n).</p

Isolation of <i>eph</i> and <i>ephrin</i> mutants.

<p>A) The 10.2 kb <i>eph</i> locus (topmost diagram) consists of 14 exons (boxes; white = UTRs, black = coding sequences) and 13 introns (lines), localized to the 102D2-D5 region of chromosome IV. The core donor construct (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037303#s2" target="_blank">Methods</a> for details) consisted of <i>eph</i> genomic sequences (blue) lacking exons 1–4 (deleting the 5′UTR, start codon, signal sequence and a portion of the exoplasmic domain) and a 3′ region lacking the kinase domain and terminal 3′ sequences. An <i>I-SceI</i> site was engineered into the middle of exon 6 (red shading). The core construct was placed upstream of a white gene marker (<i>w^hs</i>, green shading), the whole being bracketed by FLP recognition target sequences (FRT, purple shading) and inserted into the transformation vector. ‘Ends-in’ recombination induced by FLP and <i>I-SceI</i> resulted in partial tandem duplication of the <i>eph</i> locus (bottom-most diagram). B) Southern blot analysis of five candidate <i>eph^KD</i> targeting events. <u>Lane 1</u>: molecular weight markers. <u>Lanes 2–8</u>: <i>NotI</i> digests of genomic DNA derived from: L2 (Canton S, control), L3 (Donor line, control), L4 (<i>eph^KD</i>1), L5 (<i>eph^KD</i>2), L6 (<i>eph^KD</i>3), L7 (<i>eph^KD</i>4) and L8 (<i>eph^KD</i>5). The lower molecular weight band (non-mobilized donor construct) was absent from <i>eph^KD</i> lines 1, 2 & 4 indicating successful homologous recombination. <u>Lanes 9–15</u>: <i>BglII</i> digests of genomic DNA derived from: L9 (Canton S, control), L10 (Donor line, control), L11 (<i>eph^KD</i>1), L12 (<i>eph^KD</i>2), L13 (<i>eph^KD</i>3), L14 (<i>eph^KD</i>4) and L15 (<i>eph^KD</i>5). The convergence of distinct donor and endogenous <i>eph</i> bands into a single band due to homologous recombination is clearly evident in the <i>eph^KD</i>1, 2 & 4 lines (L11, L12 and L14). C) RT-PCR using primers for the full-length <i>eph</i> transcript (L1–9, left side of gel and left diagram; primer locations indicated by arrows). L1 (Canton S, control), L2 (Donor line, control), L3 (<i>eph^KD</i>1/<i>eph^KD</i>1), L4 (<i>eph^KD</i>1/+), L5 (<i>eph^KD</i>2/+), L6 (<i>eph^KD</i>2/<i>eph^KD</i>2), L7 (<i>eph^KD</i>4/+), L8 (<i>eph^KD</i>4/<i>eph^KD</i>4), L9 (<i>eph^KD</i>3/<i>eph^KD</i>3), L10 (molecular weight markers). Full-length transcript was not detected in <i>eph^KD</i> homozygous animals (26 cycles). RT-PCR using primers for the 3′-deleted isoform of Eph is shown in L11–L19, right side of gel and right diagram (primer locations indicated by arrows). Source RNA for L11–L19 was identical to L1–L9. Only the truncated Eph isoform was expressed in <i>eph^KD</i> animals. <u>Abbreviations</u>: LBD (ligand-binding domain), FNIII (fibronectin type III repeats), JXM (juxtamembrane region), TK (tyrosine kinase domain), SAM (sterile alpha motif), PDZ (postsynaptic density 95/Discs-large/zona occludens-1 domain). D,D′) Eph (anti-Eph, red in D, shown alone in D′) is expressed on cortical neuron axons in wild-type third instar larvae, accumulating in a high-midline low-dorsoventral gradient in the medulla neuropil (compare anti-HRP staining, green, to anti-Eph staining, red). E,E′) Lack of Eph immunoreactivity (anti-Eph, red in E, shown alone in E′) corresponded to optic lobe defects (anti-HRP, green) in <i>eph^KD</i> animals, manifest as gaps in the neuropil (arrowheads). F) Schematic of the 5.8 kb <i>ephrin</i> locus localized to the 102C2 region of chromosome IV. The <i>ephrin</i> gene is comprised of 5 exons (black boxes) and 4 introns (gray lines). The start codon (red arrow) and RS5 P-element insertion site (red shaded box) into 5′UTR of the first exon are also indicated. G,G′) In wild-type animals, Ephrin expression (anti-Ephrin, red in G, shown alone in G′) is punctate along cortical neuron axons and concentrated in the optic lobe neuropil (anti-HRP, green in G). H,H′) In <i>ephrin^RS5</i> mutants, Ephrin expression is considerably reduced in the optic lobe (anti-Ephrin, red in H, shown alone in H′), resulting in neuropil defects (arrowheads; anti-HRP, green in H). White or yellow bars indicate the dorsoventral midline. Scale bar in D is 20 µm for D,D′,E,E′, G,G′, H,H′.</p

A genetic screen for Eph pathway signaling molecules.

<p>A) The wild-type adult eye displays a regular ommatidial lattice. B) The rough-eye phenotype generated by expression of <i>UAS-ephrin</i> under control of the <i>sevenless2-GAL4</i> driver (SE) used to screen for modifier mutations. C) Near-complete suppression of the SE phenotype by co-expression of a dominant-negative <i>eph</i> transgene (<i>eph^DN</i>). D) Suppression of the SE phenotype by the <i>reph¹</i> allele. E) Suppression of the SE phenotype by <i>reph^k8617</i>. F) Co-expression of a <i>UAS-reph⁺</i> transgene enhances the SE phenotype. All images (20×) were acquired through a digital camera attached to a Zeiss Stemi SV11 stereo dissecting microscope. Images were captured using FPG3 software.</p

<i>reph</i> expression correlates with Eph expression in the developing optic lobe.

<p>Expression patterns for <i>reph</i> in the developing retina, lamina and medulla were determined using a lac-Z reporter associated with <i>reph^k8617A</i> and by in situ hybridization. Staining for HRP (anti-HRP, green, A,C,D) was used to visualize general cellular architecture. In the developing retina, <i>reph</i> (anti-β-gal, red in A, shown alone in A′) was expressed primarily in cells within and posterior to the MF, which correlated well with Eph expression (anti-Eph, B). In the lamina, <i>reph</i> was expressed in a high-midline, low-dorsoventral gradient (C′, demarcated by yellow arrowheads), as was the case for Eph (C″). In the medulla, <i>reph</i> expression within cortical neurons diminished at the dorsoventral margins (D′, yellow arrowheads) as was the case for Eph expression (D″). In situ hybridization (controls are shown in E) supported the <i>lacZ</i> reporter data. In the developing retina <i>reph</i> expression was found in cells within and posterior to the MF, while within the lamina region <i>reph</i> expression was highest at the midline (F, yellow arrowheads). Abbreviations: cortex (ctx), eye disc (ED), lamina (lam), lobula (lob), medulla cortex (med c), medulla neuropil (med n), morphogenetic furrow (MF).</p

Eph and ephrin expression patterns in the D. melanogaster larval third instar optic lobe.

<p>A1–A5) anti-HRP staining (green) reveals overall optic lobe architecture. Posterior to the morphogenetic furrow (MF), retinal axons of ommatidia (small circles, A1) in the developing eye disk (ED; A2) project through the optic stalk (OS; A2) to topographically innervate two target regions in the brain: the lamina (Lam; demarcated by the lamina furrow, LF; A3) and the medulla (A5), which lies proximal to the lamina. Axons of medulla cortical neurons (Med cortex; A5) also project topographically to form a crescent-shaped neuropil (Med n'pil; A5). Schematic representations of optic lobe architecture are shown in A1 (eye and lamina) and A4 (medulla). B1-B5) anti-Eph staining (grayscale). Across the ED, Eph expression appears uniform posterior to the MF. Within the Lam, Eph expression is highest at the posterior midline and lowest at the dorsal-ventral margins of the anterior (B2, B3; depicted schematically in B1). Within the medulla, Eph expression is again highest at the midline, diminishing along the dorsal-ventral axis (B5; depicted schematically in B4). C1–C5) anti-Ephrin staining (grayscale) reveals uniform expression of Ephrin across the developing ED (C2). Within the Lam Ephrin expression appeared relatively uniform (C3). Ephrin-specific expression patterns in the developing ED and Lam are depicted schematically in C1. Within the medulla, Ephrin expression mirrors that of Eph: highest at the midline, diminishing along the dorsal-ventral axis (C5; depicted schematically in C4). D) Summary of Eph/Ephrin expression patterns in the developing visual system (horizontal perspective, which depicts the spatial relationship of the lamina and medulla target fields). Anterior (red arrow) and dorsoventral margins (yellow arrows) are indicated. All image panels were of late third larval instar stage brains stained for HRP (anti-HRP, A2, A3, A5), Eph (anti-Eph, B2, B3, B5) or Ephrin (anti-Ephrin, C2, C3, C5). Note that A2, B2, and C2 are the same image for which individual channels have been displayed. All other images represent distinct specimens. Dorsal (D), ventral (V), anterior (A) and posterior (P) orientations (central compass) are identical for all panels.</p

<i>reph</i> interacts genetically with the <i>eph^KD</i> and <i>ephrin^RS5</i> mutations.

<p>In wild-type flies (A, E) the medulla neuropil (med) is distinguished by its regular crescent shape (anti-HRP staining, grayscale, all panels A–H). Mutant phenotypes could be broadly categorized as defects in the medulla neuropil (white arrowheads), HRP+ cortical inclusions (yellow arrowheads) or disruption of lobula (lob) architecture (red arrowheads). In homozygous <i>ephrin^RS5</i> mutants (B), subtle abnormalities were observed in the medulla neuropil and cortex. The severity of <i>ephrin^RS5</i> medulla defects was enhanced by a single copy of <i>reph¹</i> (C) or <i>reph^K8617A</i> (D). In homozygous <i>eph^KD</i> mutants (F) gaps in the medulla neuropil were often present. A single copy of the <i>reph¹</i> allele (G) or <i>reph^K8617A</i> (H) exacerbated the <i>eph^KD</i> mutant phenotype.</p

Reph encodes a putative nuclear protein.

<p>A) The <i>reph¹</i> allele, <i>reph^k8617A</i>, was mapped by inverse PCR to the CG3920 locus, which harbors another P-element insertion l(2)k16918 (<i>reph^k16918</i>). The <i>reph</i> 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 <i>reph^k8617A</i> (first intron of the 2.1 kb transcript) and <i>reph^k16918</i> (5′UTR 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).</p

Conserved essential genes restricted to actinomycetes.

(A) and (B) Transposon insertion data for diphtheriae_00818 and diphtheriae_01854 genes with the respective domains of unknown function (DUF) DUF501 (PF04417) and DUF3073 (PF11273) displayed beneath. Transposon insertion sites are represented by vertical black bars, capped at a frequency of 1. (C) Distribution of homologs of diphtheriae_00818 and diphtheriae_01854 within representative genomes of the Actinobacteria phylum. Bacillus subtilis was used as an outgroup for construction of the tree; only bootstrap values under 100 are shown on the tree. Species are shaded by Class. The presence of a homolog, identified by BLASTP, is indicated by a coloured circle. (TIF)</p

Calculation of essential genes.

(A) Comparison of the insertion index scores of two technical replicates of the transposon mutant library. (B) Bi-modal distribution of the total insertion index scores for the transposon library. The exponential distribution fit to the left mode includes the essential genes (red), and the gamma distribution fit to the right mode captures the nonessential genes (blue). (TIF)</p