over-expression of Robo can also reduce commissure formation [10,19,20]. Two additional Robo-family members also help guide axons at the midline and/or position them within the longitudinal connective [21–24]. Genetic elimination of commissureless (comm.) prevents commissures from forming, and Comm appears to play a major role in attenuating Robo-dependent repulsion [20,25,26]. Comm is a membrane protein expressed in commissural neurons and its expression may be partially regulated by Fra as commissural axons first extend towards midline [12]. Comm is known to bind to Robo receptors to help clear it from the membrane via endocytosis and sorting pathways [27–30], although more recent data suggest that Comm may also initiate intracellular signaling pathways that attenuate midline repulsion independent of its ability to clear Robo from the membrane [31]. Like most cell surface receptors, both Robo and Fra use conserved motifs within their cytoplasmic domains to signal information to the cytoskeleton. Fra has three evolutionarily conserved P-motifs (P1, P2 and P3) in its cytoplasmic domain and it appears that P3 is particularly important for Fra signaling in vivo [32,33]. Signaling pathways originating from these motifs are thought to govern key aspects of the cytoskeletal dynamics underlying axon extension and steering [33–37]. Similarly, Robo initiates a repulsive response via activation of several signaling pathways initiated from one of the four (CC0, CC1, CC2 and CC3) conserved domains [38–46]. Of particular interest here is the Abelson tyrosine kinase signaling pathway, as both Abl itself and some of its key substrates (Ena) have been implicated in both Robo and Fra signaling. Abl is a multifunctional tyrosine kinase that serves to link membrane receptors to actin dynamics underlying cell movement [47,48]. In Drosophila, zygotic loss-of-function mutants of abl display mild defects in the axon scaffold including ectopic midline crossing errors [40,41,49]. These crossing errors are enhanced if robo itself is decreased or if mutations in genes known to interact with Robo are introduced [38,41,44]. Crossover defects and/or fused commissures are also common phenotypes observed when abl mutants are combined with mutations in a variety of genes implicated in the regulation of cytoskeletal dynamics (e.g. [38,39,44,50,51]). The observation that Abl binds to and phosphorylates the cytoplasmic tail of Robo led to the suggestion that Abl is a key regulator of actin dynamics during the transduction of midline repulsive cues [41,49]. However, other data suggest Abl’s role is not confined to the transduction of midline repulsion. When the zygotic and maternal contribution of abl is eliminated, commissures are missing and gaps appear in the longitudinal connectives [52]. Increasing levels of Abl activity also interact with heterozygous robo mutants to induce ectopic crossovers, and overexpression of Abl in a comm mutant, experiencing high levels of midline repulsion, actually improves commissure formation [49]. This suggests that increasing Abl activity could enhance midline attraction. Indeed, Abl has been linked to the transduction of midline attractive cues. In GST-pull down and immunoprecipitation assays, Abl binds to the cytoplasmic tail of Fra and, when Abl is expressed in S2 cells with Fra, the tyrosine phosphorylation levels of Fra increase [36]. In vivo, very few commissures form in fra, abl double mutants [36]. Interestingly, abl mutations also enhance the degree of commissure loss observed in combination with mutations in Dscam [16], turtle [18], amalgam and its receptor neurotactin [53], fasciclin I [54], and midline fasciclin [55]. Together, this suggests that Abl plays multiple roles at the midline as it aids in the transduction of both Netrin dependent and independent attractive cues. To further elucidate Abl’s role, we sought to elevate Abl activity in embryos that are for fra. It was reasoned that if Abl is working with Fra to Frazzled and Abelson potentiate attractive signaling and a loss of abl in a fra mutant enhances commissural defects, then over-expression of Abl in a fra mutant should partially rescue commissure formation. However, as described herein, we observed the opposite: fra commissural defects are dramatically enhanced as both anterior and posterior commissures fail to form and this is associated with a large number of CNS axons projecting beyond the CNS/PNS border. To investigate the mechanism by which Abl may be causing this paradoxical interaction with fra, we manipulate midline repulsive elements and other genes known to regulate cytoskeletal dynamics. Our results suggest that in order to respond correctly to midline attraction, Fra is required to regulate Abl activity, in part, to direct the cytoskeletal dynamics underlying movement across the midline. In the absence of Fra, Abl appears to perform these functions downstream of other guidance receptors to induce a variety of projection errors, including trajectories away from the CNS. Methods Stocks Stocks were raised on conventional cornmeal and molasses based media at room temperature or 25uC. All of the stocks used in this study have been fully characterized in previous studies. The Fra deletion mutant transgenes (UAS-FraDP1, UAS-FraDP2, UAS- FraDP3 and UAS-Frawt) and the 1407-Gal4 driver on chromosome II were described [32,37]. Other constructs were described as follows: Ablwt, AblKN and BcrAbl [49], constitutively active MLCK [56], RacV12 and Cdc42V12 [39] and sema2b-tau-myc P- element (Bloomington Stock Center; [21,22]). The phenotypes generated by these transgenes when expressed in wild-type embryos have been described in these reports. Here, all phenotypes are compared to a fra heterozygote with a 1407- Gal4 driver to control for the presence of this driver chromosome and emphasize the change that occurs in the homozygote. Any differences between wild type and a fra heterozygote are briefly described in the text. Conventional breeding strategies and/or recombination experiments are used to combine gene mutations (e.g., fra4), the Gal4 driver and/or UAS transgenes in the same fly. Stocks were confirmed by complementation tests and/or PCR screening for P-elements as described previously [32]. To identify the genotypes of stained embryos, b-galactosidase (b-gal) marked balancer chromosomes are present in all final stocks. Histology: Standard methods are used to collect eggs at 25uC and stain them with mAb BP102 or mAb 1D4 against Fasciclin II (FasII) and the HRP-linked goat anti-mouse secondary antibody. The genotype of the embryos was established by X-Gal staining that identified balancer chromosomes. Mutant phenotypes in the axon scaffold were scored and at least three replicates were analyzed using a Student’s t-test. Loss of commissures and ectopic crossovers were assessed as previously described [37], while AEP defects were scored based on the presence of a BP102 stained axon beyond the CNS/PNS boundary. Normally BP102 stains only CNS axons, and while motor nerve routes within CNS neuropile may be lightly stained, they are usually devoid of staining once in the PNS. Thus, BP102 staining of axons beyond the CNS/PNS boundary is a readily quantifiable phenotype. To observe Sema2b- expressing commissural axons we introduced a sema2b-Tau-myc P-element (Bloomington stock center; [21,22]) into the back- ground of our fra3 1407-Gal4 driver line and used an antibody against the myc epitope to detect tau-myc expression. Expression constructs HA epitope tagged Frawt and FraDC transgenes cloned into the metallothionine vector (pMT) were described previously [36]. PLoS ONE | www.plosone.org 2 March 2010 | Volume 5 | Issue 3 | e9822