Frazzled and Abelson Figure 4. Quantification of axons exiting to the periphery [AEP] defects. A close up of the CNS/PNS border of stage 16 embryos stained with mAb BP102 is depicted with anterior to the right. Peripheral muscles are seen by Normarski optics [A-D]. UAS transgenes are expressed using the 1407-Gal4 (1407-G4) driver line recombined onto the fra3 chromosome. [A] A fra3 heterozygote is identical to a wild-type embryo and arrows indicate lightly stained ISN (black arrow in A) and SN (white arrow in A) nerve roots. Note the absence of BP102 staining beyond the CNS/PNS boundary. AEP defects are not observed in either [A] fra3 heterozygote or [B] fra3/fra4 homozygote embryos. Homozygous fra3/fra4 embryos expressing either [C] BcrAbl or [D] Ablwt show large bundles of BP102 positive axons extending into the PNS (arrows in C, D). [E] Quantification of AEP defects per hemisegment (hs) is depicted for Abl transgenes (left) alone or in combination with Fra transgenes (right). White bars (highlighted with ‘w’) indicate fra3 1407-Gal4 heterozygote’s, black bars are fra3 1407-Gal4/fra4 homozygote’s and gray bars are fra3 1407-Gal4/fra4 homozygote’s expressing the indicated transgene. All three Abl transgenes significantly (P,0.01) enhance AEP defects, although a kinase inactive Abl is less effective. BcrAbl (middle) and Ablwt (right) defects are significantly rescued by co-expression of any of the Fra transgenes, although rescue by FraDP3 is significantly less than that observed using Frawt. Comparison to fra3 1407-Gal4/fra4 homozygote condition is indicated by *, while w compares rescue of P-motif deletion to Frawt (*,w P,0.05; **, ww P,0.01). doi:10.1371/journal.pone.0009822.g004 and 78% of PC’s [Figure 6]. We also observed no detectable difference in the AEP defects in these embryos: 29% of hemisegments in fra3/fra4 embryos expressing Ablwt had this defect compared to 26% in a robo1 heterozygote background [Figure 6]. A similar trend was seen with expression of BcrAbl in these mutant backgrounds [Figure 6]. This data suggests that an increase in Robo1-dependent repulsion is not likely to be the reason axons fail to cross at commissures or leave the CNS when Abl activity is increased in the absence of Fra. In some cases, when Netrin is detected by Unc5 it can elicit a repulsive response [60]. Thus, we sought to test whether elevated Abl activity might be enhancing the repulsive activity of midline Netrins. To do this, the C155-Gal4 insertion was recombined onto the NetDAB mutant chromosome, which is for both Netrin A and B genes [61]. Using this chromosome, BcrAbl was pan- neurally expressed in embryos that do not express either Netrin, but this still results in a loss of commissures and many axons exiting from the CNS [Figure 6B] similar to that observed in homozygous fra mutants. Thus, the Abl gain-of-function pheno- types do not appear to be the result of Netrin dependent repulsion. Fra regulates the cytoskeletal dynamics during commissure formation Besides its interactions with axon guidance receptors at the midline, Abl is a key regulator of the cytoskeletal dynamics underlying growth cone advance and steering [47,48]. Does increasing Abl activity alter the dynamics of this response and cause guidance defects? To test this idea, we sought to disrupt cytoskeletal dynamics in a fra mutant independent of Abl, but using gene mutations known to affect midline guidance. Cdc42 regulates multiple elements of the cytoskeleton, and, in Drosophila, expression of a constitutively active form of the protein (U-Cdc42V12) in a specific subset of FasII positive neurons leads to ectopic crossovers, the frequency of which are halved by a heterozygous fra mutation [37,39]. Like that observed in wild-type embryos, pan-neural expression of Cdc42V12 in a fra3 heterozygote PLoS ONE | www.plosone.org 8 March 2010 | Volume 5 | Issue 3 | e9822