Unlike wild-type Abl, BcrAbl specifically interacts with Fra to induce ectopic crossovers as long as the P3 motif is present. To ask if this genetic interaction is reflected in a specific binding of BcrAbl to the P3 motif of Fra, we transiently transfected S2 cell lines expressing myc-tagged versions of wild-type or P-motif deletion mutants of Fra with a BcrAbl transgene. Frawt or its P-motif deletions were immunoprecipitated using the C-terminal myc-tag and BcrAbl binding assessed using a commercial antibody against the Bcr component. Like wild-type Abl, BcrAbl clearly binds to Fra independently of a P-motif [Figure 3C]; thus, the selective genetic interaction between BcrAbl and the P3 motif of Fra is not reflected in differential binding properties. Forsthoefel et al. [36] demonstrated that increased expression of Abl in S2 cells also led to increased phosphorylation of Fra, so we next asked if Fra phosphorylation is altered by removal of a P- motif. Fra or its P-motifs were immunoprecipitated from stable S2 cell lines using the C-terminal myc-tag and assessed for phosphotyrosine levels by western blot. Considering the amount of receptor precipitated, phosphorylation of Fra is largely unaffected by deletion of P1 or P2, while deletion of P3 may increase the signal ([Figure 3D], top). In the blot shown, cells were pretreated with pervanadate (,30 min) to increase signal intensity and to demonstrate the lack of P-motif dependence. Phosphory- lation is also observed in the absence of pervanadate treatment, although at a much lower level (see bottom panel [Figure 3D]). Phosphorylation of Frawt increases slightly if cells are transiently transfected with wild-type or BcrAbl transgenes, and it remains similar to S2 cells alone with AblKN, presumably reflecting the endogenous Abl activity. BcrAbl binding to Fra, as well as its hyperactivation, is confirmed in this blot by the presence of an autophosphorylated BcrAbl band at the top of the gel (arrowhead). Together, these immunoprecipitation assays indicate that both Abl and BcrAbl binding to Fra and the state of Fra phosphorylation are independent of any specific P-motif. This is consistent with the ability of the deletion mutants to rescue commissure formation when Abl or BcrAbl is over-expressed in a fra mutant. Identifying neurons that exit the CNS Expression of Abl in a fra mutant also results in dozens of axons exiting toward the periphery (AEP defects), and, surprisingly, this defect is readily observed using BP102. Generally considered a marker of all CNS axons, BP102 clearly labels AC, PC and LC axons, and lightly stains ISN and SN nerve routes within the CNS, but it does not normally stain axons beyond the CNS/PNS boundary. Therefore, in mutant embryos, dense BP102 staining of axons beyond the CNS/PNS boundary clearly delineates a novel phenotype, herein termed an AEP defect (Axon Exiting to Periphery). Admittedly, no information on the identity of the exiting axons is provided with BP102, but this is addressed below. Consistent with the known BP102 staining pattern, in wild-type and fra3/fra4 mutant embryos we do not observe any AEP defects [Figure 4A, B & E]. When BcrAbl is expressed in a fra heterozygote, we observe minimal AEP defects and none in the same background with Ablwt [Figure 4E]. However, nearly a third of hemi-segments in embryos expressing BcrAbl or Ablwt in the absence of Fra exhibit AEP defects as many axons expressing the BP102 epitope actually extend past the CNS/PNS boundary ([Figure 4C & D], arrows). AEP defects are present in less than 10% of embryos over-expressing the kinase dead version of Abl (AblKN); this is significantly (P,0.01) less than either Ablwt or BcrAbl [Figure 4E]. Like the commissural axon defects, the AEP defects are rescued by re-expression of Frawt (P,0.01), FraDP1 (P,0.01) or FraDP2 (P,0.05). A small (6%) but significant (P,0.01) frequency of AEPs is still observed if FraDP3 is over- Frazzled and Abelson expressed [Figure 4E]. Therefore, Fra and Abl also interact to regulate which axons may leave the CNS. In our analysis of the fuzzy commissure defects, we noted that many of the longitudinal axons expressing FasII also project past the CNS/PNS boundary ([Figure 2E, F]; arrows). 1D4 stains both longitudinal axons that remain in the CNS and motor neurons, notably the segmental (SN) and inter-segmental (ISN) nerve bundles that exit the CNS to innervate muscles in the periphery ([Figure 5A], arrowheads). While the projection of some motor neurons is known to require Fra [13,58], the motor nerve roots within the CNS are mostly intact ([Figure 5B], arrowheads). However, expressing either BcrAbl or Ablwt in a fra3/fra4 mutant causes FasII positive longitudinal axons that normally remain in the CNS to exit. This also includes axons from the medial pCC/ MP2 fascicle, which pioneers the longitudinal connective and normally does not exit the CNS ([Figure 5C], arrow; [59]). The fact that this fascicle is observed exiting the CNS is particularly interesting since the medial pCC/MP2 is normally the least affected in a fra mutant [32]. It is also clear that while many of the AEP defects track with the motor neurons forming the SN and ISN nerve roots, others exit with seemingly no association ([Figure 5C, D], arrows). Because we cannot readily distinguish motor neurons from other FasII positive axons we did not attempt to quantify the AEP phenotype using 1D4. Given the reduction in commissures in fra3/fra4 mutant embryos expressing either Ablwt or BcrAbl, we made use of the reporter construct, sema2b-tau-myc, to specifically examine the axon projections of a small subset of commissural axons. This reporter labels the axons of a subset of neurons in abdominal segments five through eight that project axons into the anterior commissure and then turn anteriorly to form a part of the media-lateral longitudinal connective [Figure 5E] [21]. In fra3/fra4 mutants, most sema2b axons still cross in the anterior commissure, consistent with this being the least affected commissure in fra mutants, and, while small breaks in the longitudinal projections can be observed, no axons leave the CNS ([Figure 5F], arrowhead). However, when Fra is absent, expressing BcrAbl or Ablwt causes these axons to project out of the CNS and into the periphery [Figure 5G, H]. In both of these mutants, sema2b axons exit at multiple points in their trajectory; some cross the midline and then exit, others exit while extending longitudinally, and still others do not cross the midline but, rather, extend directly away from the midline [Figure 5G, H]. Thus, increased Abl activity appears to affect guidance decisions at several choice points, and, surprisingly, some of these are severe enough to drive these axons out of the CNS. Decreasing midline repulsion does not rescue guidance defects Both too much and too little Abl causes ectopic midline crossing errors when Robo-dependent repulsion is reduced [42,49], and Fra activity may regulate the level of Commissureless, which in turn governs Robo surface accumulation (Yang et al., 2009). Thus, elevating Abl activity in a fra mutant might have shifted the balance of attractive and repulsive signals at the midline to reduce commissure formation and/or allow axons to leave the CNS. To test this idea, we introduced one copy of a robo1 allele (effectively reducing Robo1 dependent repulsion by half) into our fra mutants expressing either BcrAbl or Ablwt. Surprisingly, decreasing Robo- dependent repulsion had no observable effect on either of the phenotypes induced by Abl activity [Figure 6]. In a fra homozygote expressing Ablwt, thinning or missing AC’s were seen in 60% of segments and 74% of segments had defects in the PC. Introduction of a robo1 allele did not significantly change these numbers as commissures were thinning or missing in 50% of AC’s PLoS ONE | www.plosone.org 7 March 2010 | Volume 5 | Issue 3 | e9822