Frazzled and Abelson Figure 7. Expression of RacV12 and Cdc42V12 reduces commissure formation in a fra mutant. Stage 16 embryos stained with the mAb BP102 are depicted with anterior up. UAS (U) transgenes are expressed pan-neurally using the 1407-GAL4 (1407-G4) driver line recombined onto the fra3 chromosome, and, as in other figures, phenotypes are compared to heterozygous fra3. Phenotypes in heterozygous fra3 embryos tend to be somewhat milder than that observed in a wild-type embryo (see text). [A] Expression of Cdc42V12 in a fra3 heterozygote background results in fused commissures [arrowhead] and large gaps in the longitudinal connectives. [B] Expression of RacV12 in a fra3 heterozygote causes a thinning of both PC (black arrowhead) and AC (white arrowhead) as well as LC (black arrow). [C] Expression of ctMLCK in a fra3 heterozygote results in fuzzy commissures (black arrowhead). Expression of both [D] Cdc42V12 and [E] RacV12 in a fra3/fra4 mutant significantly reduces commissure formation (black arrowheads) and, with expression of RacV12, some axons may orientate towards the periphery (arrow in E), but they are not observed to exit the CNS. [F] Expression of ctMLCK in a fra3/fra4 mutant does not significantly alter the PC (arrowhead) and LC defects normally observed in a fra mutant. doi:10.1371/journal.pone.0009822.g007 itors undoubtedly include other midline guidance cues, the emergence of AEP defects suggests that Fra also competes with guidance systems not normally associated with the midline. In the absence of Fra, these other receptors appear to utilize the extra Abl to alter cytoskeletal dynamics at a variety of choice points, ultimately preventing commissure formation and directing some axons out of the CNS. The Abl gain-of-function phenotype described herein occurs only if fra is absent. That is, commissures form correctly in a heterozygous fra mutant or when partially active Fra transgenes with a single P-motif deleted are re-expressed with Abl. In S2 cell immunoprecipitation experiments, both Abl and BcrAbl bind to the cytoplasmic tail of Fra independent of any specific P-motif (see also [36]). While surprising given the conservation of these P- motifs and their known importance to Fra function [32,33], the lack of P-motif specificity is consistent with our genetic rescue experiments. All three P-motif deletion mutants rescue commis- sure formation and the AEP defects elicited by over-expression of either wild-type Abl or BcrAbl in fra embryos. The ectopic midline crossovers (fuzzy commissures) observed with only BcrAbl also depend on Fra and specifically the P3-motif, confirming a previous report [37]. However, BcrAbl is not an endogenous Drosophila protein and, as discussed by others [62], the human Bcr domain may induce neomorphic phenotypes. Because BcrAbl does not preferentially bind to the P3 motif, and wild-type Abl does not elicit crossover defects, we now suspect that the ectopic crossovers are an example of a neomorphic phenotype, a hypothesis that will be extensively addressed in future work. The physical interaction between Fra and either Ablwt or BcrAbl in immunoprecipitation assays can reflect direct or indirect association between the two proteins. It is possible that our failure to observe P-motif dependence reflects the binding of Abl to multiple P-motifs or the use of scaffold proteins associated with more then one P-motif. Given that Forsthoefel et al. [36] demonstrated that the cytoplasmic tail of Fra fused to glutathio- nine-S-transferase (GST) binds to in vitro translated Abl, direct binding of Abl to Fra is clearly possible. If so, these experiments suggest that Abl may bind to Fra in the regions between P-motifs, PLoS ONE | www.plosone.org 11 March 2010 | Volume 5 | Issue 3 | e9822