Frazzled and Abelson A specific P-motif of Fra is not required to interact with Abl at commissures In a previous gain-of-function assay, expression of BcrAbl in a subset of FasII expressing neurons induces ectopic crossovers, and these are enhanced by co-expression of Fra as long as the P3-motif is intact [37]. Therefore, we tested whether a specific P-motif is also required for Fra to revert the loss in commissural axon crossing. Essentially, we compared the ability of Frawt to revert the commissural defects with our previously described series of Fra mutants in which the individual P-motifs have been deleted (U- FraDP1, U-FraDP2 and U-FraDP3; [37]). All of the Fra P-motif deletion mutants were able to significantly restore commissure formation in fra3/fra4 mutants expressing Ablwt [Figure 1J, L]. In fra3/fra4 mutants expressing BcrAbl, both FraDP1 and FraDP2 were able to revert the commissure loss seen in this mutant and in fact restore the fuzzy commissure defects similar to that observed with Frawt [Figure 1L]. While expression of U-FraDP3 also restores commissure formation, the frequency of fuzzy commissures is significantly (P,0.001) reduced compared to Frawt, as many segments now exhibit a distinct AC and PC ([Figure 1H], black arrowhead, [Figure 1L]). BcrAbl specifically interacts with the P3 motif to induce ectopic crossovers The fuzzy commissure phenotype is often associated with extra axons ectopically crossing the midline between the AC and PC, a result known to occur with pan-neural expression of BcrAbl [49,57]. Ectopic crossovers of FasII expressing neurons are readily observed using the monoclonal antibody 1D4. In wild-type embryos, FasII positive neurons extend axons in three parallel bundles on either side of the midline but never cross the midline [Figure 2A]. FasII fascicles tend to fuse together or form small gaps when both copies of fra are mutated [Figure 2D]. Consistent with a previous report [49], pan-neural expression of BcrAbl causes multiple FasII positive axons to ectopically cross the midline in most segments of nearly all embryos examined ([Figure 2B] arrowhead; [quantified in Figure2G]]. Removing both copies of fra significantly (P,0.001) reduces the frequency of these crossovers Figure 2. BcrAbl induced crossovers depend on the P3-motif of Fra. Stage 16 embryos stained with mAb 1D4 against Fasciclin II (FasII) are depicted with anterior up. All UAS (U) transgenes are pan-neurally expressed using the 1407-Gal4 (1407-G4) driver line recombined onto the fra3 chromosome; thus, phenotypes are compared to heterozygous fra3 rather than a wild-type embryo. [A] A fra3 heterozygote exhibits a nerve cord that is essentially wild-type. Expression of [B] BcrAbl but not [C] Ablwt results in FasII axons crossing the midline incorrectly (arrowhead in B). [D] In homozygous fra3/fra4 mutants, the FasII fascicles may partially fuse, and small breaks in the connective may appear. Removal of both copies of fra abolishes [E] BcrAbl and [F] Ablwt induced crossover defects. In both cases, FasII expressing axons are observed projecting towards the periphery (arrows), including axons from the medial most pCC/MP2 pathway (arrow in E). [G] Quantification of ectopic crossing errors (per embryo) observed using BcrAbl is graphed. White bar indicates crossovers in a fra3 1407-Gal4 heterozygote, black bar is a fra3 1407-Gal4/fra4 homozygote and gray bars are fra3 1407-Gal4/fra4 homozygote’s expressing the indicated Fra transgene. Expression of BcrAbl in a fra3 1407-Gal4/fra4 homozygote reduces ectopic crossovers, but these are restored by co-expression of Fra transgenes (* P,0.05, or ** P,0.01). Compared to Frawt, expression of FraDP3 restores only about half of the expected crossovers (w P,0.05). [H] Re-expression of U-Frawt transgene restores the crossover defects (arrowhead in G) in fra3/fra4 embryos expressing BcrAbl, while [I] expression of FraDP3 restores about half of the ectopic crossovers. doi:10.1371/journal.pone.0009822.g002 PLoS ONE | www.plosone.org 5 March 2010 | Volume 5 | Issue 3 | e9822