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Dephrin, a transmembrane ephrin with a unique structure, prevents interneuronal axons from exiting the Drosophila embryonic CNS

Wellcome Trust Cancer Research UK Institute and Department of Genetics, Cambridge University, Tennis Court Road, Cambridge, CB2 1QR, UK

View larger version (54K): [in a new window]   Fig. 1. Dephrin is a membrane protein with a cytoplasmic tail similar to B class ephrins. (A) Structural comparison between Dephrin, a vertebrate B class and an A class ephrin. The C terminus of Dephrin includes two predicted transmembrane domains (grey box). In contrast to all other ephrins, Dephrin also has a predicted transmembrane domain in front of its ephrin domain (black box) and no obvious signal sequence (hatched box in ephrin B1 and A1). Numbers are amino acid positions. Lines at the top denote different parts of Dephrin included in the indicated constructs. (B) ClustalW alignment of the most C-terminal amino acids of Dephrin and a B class ephrin. The tyrosine residue at the –3 position from the C terminus (star) is conserved in Dephrin. (C,D) The ephrin domain of Dephrin is extracellular. (C) Antisera against the ephrin domain of Dephrin bind to the surface of non-permeabilised Drosophila S2 cells in vivo. (D) Treatment of S2 cells with doublestranded (ds)Dephrin RNA greatly diminishes the antibody signal. (E) Incubation of S2 cells with dsDephrin RNA significantly reduces the expression of Dephrin. Anti-Dephrin recognizes two strong bands in lysates from untreated S2 cells (control). In dsDephrin RNA treated cells (RNAi), the bands are nearly absent. Protein mass in kDa (x103) on the left, exposure time on the right. Actin served as loading control. (F-I) The C terminus of Dephrin is protected from proteinase digestion. Proteinase K is not able to digest the C-terminal GFP tag in Dephrin-GFP-expressing embryonic clones (F, red) but the proteinase destroys the extracellular ephrin domain (G, red). Without proteinase incubation, anti-GFP (H, red) and anti-Dephrin (I, red) bind to their antigen at the membrane. Clones in F-I were stained without detergent. Dephrin-GFP fluoresces strongly only in the cytoplasm. (J,K) The N terminus of Dephrin (aa 1-202) is necessary for membrane localisation of the protein. In S2 cells transfected with full length Dephrin (Dephrin), the protein is localized at the membrane and in cytoplasmic vesicles (J). Expression of an N-terminal truncated form of Dephrin (DephrinN-Term) in S2 cells results in a diffuse cytoplasmic distribution of the protein (K). The high level expression of DephrinN-Term obscures endogenous DEphrin at the membrane. (L) The C terminus of Dephrin (aa418-aa652) serves as membrane anchor. S2 cells were transfected with a C-terminal truncation of Dephrin (DephrinC-Term) or full length Dephrin (Dephrin). Only cells expressing DephrinC-Term showed a strong accumulation of the protein in the medium.

View larger version (50K): [in a new window]   Fig. 2. Dephrin is cleaved at the N terminus and shows no alternative translation. (A) Western blots of embryonic lysate reveal a band around 75 kDa, the predicted size of Dephrin and a band at 52 kDa. (B,C) Mosaic clones were generated by injection of a UAS plasmid carrying a fusion of GFP to the N terminus (B, GFP-Dephrin) or the C terminus (C, Dephrin-GFP) of Dephrin. In embryonic cells expressing GFP-Dephrin, the GFP (green) is mainly localised in the cytoplasm, whereas Dephrin (red) accumulates at the membrane (B). The different subcellular localisation of the two parts of the fusion protein indicates a cleavage at the N terminus. In cells expressing Dephrin-GFP (C), the GFP always colocalizes with Dephrin (yellow). Clones were stained in the presence of detergent. (D) Schneider cells transfected with a Dephrin-GFP fusion express a protein of 100 kDa (75 kDa of Dephrin + 27 kDa of GFP) and a second band of 80 kDa (52 kDa of Dephrin + 27 kDa of GFP). Left lane, unfused GFP; right lane, Dephrin-GFP (E) Dephrin shows no alternative initiation of translation. Anti-GFP reveals only one band in extracts from Schneider cells transfected with a plasmid encoding the N terminus of Dephrin (aa1-210) fused to GFP.

View larger version (92K): [in a new window]   Fig. 3. Dephrin is expressed during gastrulation and in the embryonic CNS. (A) During gastrulation, Dephrin expression concentrates at the invaginating cephalic furrows (thin arrows) and at the invaginating mesoderm (thick arrow). (B) After germband retraction, expression of Dephrin is restricted to neuronal cell bodies. No protein is detectable on axonal tracts (arrowheads). (C) Expression of DEph, the Eph-like kinase in Drosophila, is restricted to axons and absent from the cell bodies. (D) Expression in muscles (Gal4 line 24B) of the secreted DephrinC-Term, which has an intact receptor binding domain, shows an accumulation of the truncated form (green, -Dephrin) along axons (red, BP102). Horizontal views; anterior to the left.

View larger version (20K): [in a new window]   Fig. 4. DEph binds to Dephrin. (A) Drosophila S2 cells were incubated with medium derived from cell cultures expressing the extracellular part of DEph fused to GFP at the C terminus (DEphex-GFP). Owing to the endogenous expression of Dephrin, DEphex-GFP in the medium binds to the surface of untreated S2 cells (control) and the cells can be stained with -GFP. (B) Lowering the expression of Dephrin by incubating S2 cells with dsDephrin RNA (Dephrin RNAi) abolishes the binding of DEphex-GFP, confirming the specificity of the binding. (C) Control incubation of S2 Cells with dsDEph RNA (DEph RNAi) does not interfere with the binding of DEphex-GFP.

View larger version (142K): [in a new window]   Fig. 5. Loss of Dephrin results in the abnormal exit of interneuronal axons from the CNS. We used a transformant line expressing tau-GFP to primarily label the projections of two interneurones (MP2 neurones, Gal4 line CY27). The axons of these neurones form a fascicle that extends in parallel on both sides of the ventral midline (dotted line; E,F). (A,B) Reducing the expression of Dephrin causes the interneuronal axons to exit the CNS (A, star) and severely disrupts the axonal scaffold (B, BP102). (C,D) DEph RNAi causes similar phenotypes as Dephrin RNAi. The interneuronal axons exit the CNS (C, star) or fasciculate loosely with each other. The axonal scaffold (D) is also severely disrupted. (E,F) Injection of dsCFP RNA rarely interferes with the projections of the MP2 neurones (E) or with the general layout of the axonal scaffold (F). Horizontal views of stage 17 embryos, anterior to the left.

View larger version (84K): [in a new window]   Fig. 6. Ectopic expression of Dephrin causes axonal repulsion. (A,B) Ectopic expression of Dephrin in midline cells (A) causes a severe thinning of the commissures (arrowhead) but does not interfere with the determination of midline glia (black, anti-Wrapper). As in wild type (B), midline glia still tightly enwrap the commissural fibres. (C,D) Ectopic expression of Dephrin in midline cells prevents axons from crossing the ventral midline. The lineage of one neural precursor includes glial cells and three contralateral projecting neurones (star). In embryos with ectopic midline expression of Dephrin (C), the projections of these neurones (thin arrow) are stalled at the midline. In a 1-hour younger wild-type embryo (D), the axons (thin arrow) have already crossed the midline. (E,F) Axonal repulsion by ectopic Dephrin does not depend on Slit or Robo1. Ectopic expression of Dephrin in midline cells of slit, robo1 double mutants (E, purple, arrow) is able to push axons (FasII, brown) out of the midline. In slit, robo1 double mutants (F), the longitudinal tracks collapse at the midline. (G,H) Ectopic expression of Dephrin in longitudinal glia cells (G, thick arrow) causes breaks in the axonal scaffold (green, BP102). Longitudinal glia cells expressing GFP (H, thick arrow) do not perturb the formation of the axonal scaffold (brown, BP102). Horizontal views of stage 17 embryos (except D, stage 16); anterior to the left. Dotted line marks the midline.

View larger version (117K): [in a new window]   Fig. 7. Repulsion by Dephrin depends on DEph. (A,B) Injection of dsGFP RNA (A) increases the severity of the axonal phenotype caused by ectopic Dephrin in midline cells (compare with 6A). Commissures are often lost completely. In contrast, lowering the expression of DEph by DEph RNAi (B) allows axons (red) to cross over ectopic midline sources of Dephrin (green). Injection of dsDEph RNA into embryos with ectopic midline expression of Dephrin frequently restores the axonal scaffold to normal. Horizontal views of stage 17 embryos; anterior to the left.

View larger version (80K): [in a new window]   Fig. 8. The role of Dephrin/DEph signalling during CNS development in Drosophila embryos. (A,B) In stage 12 of embryogenesis, the first interneuronal axons project away from the midline (dotted line) and extend up to the lateral border (line) of the CNS (arrow labels growth cone in A). After reaching the border, the axons turn and continue to extend in parallel to the midline (B). At this stage anti-Futsch (brown) labels the axons of both MP2 neurones and anti-Odd (black) labels the nucleus of dMP2 and the MP1 neurones. (C,D) Anti-Dephrin staining at stage 13 shows that the MP2 axons (arrow labels growth cone) extend along a thin Dephrin-free channel (C, MP2 neurones are labelled by the expression of tau-GFP driven by the GAL4 driver CY27). D shows Dephrin expression only. (E) We propose that primary neurones project away from the ventral midline (pink) owing to the secretion of the long range repellent Slit (orange). When the growth cones reach the lateral border they are repelled by Dephrin (green). This repulsion induces growth cones, which carry the receptor DEph (blue), to turn and confines axon extension to within the CNS. Horizontal views, anterior to the left