Figure 3. Isolation of eph and ephrin mutants.

A) The 10.2 kb eph 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 Methods for details) consisted of eph 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-SceI site was engineered into the middle of exon 6 (red shading). The core construct was placed upstream of a white gene marker (w^hs, 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-SceI resulted in partial tandem duplication of the eph locus (bottom-most diagram). B) Southern blot analysis of five candidate eph^KD targeting events. Lane 1: molecular weight markers. Lanes 2–8: NotI digests of genomic DNA derived from: L2 (Canton S, control), L3 (Donor line, control), L4 (eph^KD1), L5 (eph^KD2), L6 (eph^KD3), L7 (eph^KD4) and L8 (eph^KD5). The lower molecular weight band (non-mobilized donor construct) was absent from eph^KD lines 1, 2 & 4 indicating successful homologous recombination. Lanes 9–15: BglII digests of genomic DNA derived from: L9 (Canton S, control), L10 (Donor line, control), L11 (eph^KD1), L12 (eph^KD2), L13 (eph^KD3), L14 (eph^KD4) and L15 (eph^KD5). The convergence of distinct donor and endogenous eph bands into a single band due to homologous recombination is clearly evident in the eph^KD1, 2 & 4 lines (L11, L12 and L14). C) RT-PCR using primers for the full-length eph transcript (L1–9, left side of gel and left diagram; primer locations indicated by arrows). L1 (Canton S, control), L2 (Donor line, control), L3 (eph^KD1/eph^KD1), L4 (eph^KD1/+), L5 (eph^KD2/+), L6 (eph^KD2/eph^KD2), L7 (eph^KD4/+), L8 (eph^KD4/eph^KD4), L9 (eph^KD3/eph^KD3), L10 (molecular weight markers). Full-length transcript was not detected in eph^KD 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 eph^KD animals. Abbreviations: 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 eph^KD animals, manifest as gaps in the neuropil (arrowheads). F) Schematic of the 5.8 kb ephrin locus localized to the 102C2 region of chromosome IV. The ephrin 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 ephrin^RS5 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′.