Frazzled and Abelson Figure 6. Changes in midline repulsive activity do not account for the Abl gain-of-function phenotypes. The ability of a heterozygous loss of robo to alter the phenotypes that occur when the 1407-Gal4 (1407-G4) driver was used to pan-neurally express Ablwt or BcrAbl in fra mutants was quantified after staining embryos with mAb BP102 (as described in [Figures 1 or 4]). [A] Graphed are the percentage of embryos with missing or thin anterior (AC) and posterior (PC) commissures as well as the percentage of hemisegments (hs) exhibiting axons exiting towards the periphery (AEP) in fra3, 1407-G4/fra4 homozygote embryos (white; robo+/+) versus fra3, 1407-G4/fra4 homozygote embryos carrying one copy of a robo allele (black; robo+/2). The loss of one copy of robo has no significant affect on the frequency of these defects. [B-D] To test whether netrin-dependent repulsion is important, BcrAbl or RacV12 (as indicated at top) were over-expressed in netrin embryos (netDAB) using the pan-neural C155 Gal4 driver also on the X chromosome. [B] In a relatively severe netrin mutant, most posterior commissures (black arrowheads) are absent and several anterior commissures are thin. Most commissures are absent when either [C] BcrAbl or [D] RacV12 are over-expressed in all neurons, and, with BcrAbl, some axon bundles also project beyond the CNS/PNS boundary (arrow in C). doi:10.1371/journal.pone.0009822.g006 Fra, detection of Netrin-dependent chemoattraction is compro- mised and many posterior commissures fail to form. Both anterior and posterior commissures are absent if fra and abl activity is lost. This presumably reflects the ability of abl mutations to interact with a second Netrin receptor, Dscam, as well as Netrin independent receptors (e.g. Turtle) known to be important for commissure formation [16,18]. Finally, as most commissures are also lost when both maternal and zygotic contributions of Abl are genetically removed, it seems Abl itself is required for commissure formation [52]. Given these different observations, it seemed plausible that over-expressing Abl in fra embryos would improve commissure formation. However, instead of an improve- ment, we clearly document a major decrease in both anterior and posterior commissures and the induction of a novel phenotype whereby axons normally confined to the CNS now project into the periphery (AEP defects). It is worth emphasizing that these phenotypes occur even with the over-expression of a wild-type Abl transgene that retains its autoinhibitory domain and must be activated by endogenous mechanisms [47,49]. These phenotypes are completely dependent on the absence of Fra but not any specific P-motif, occur in the absence of Netrins as well, and are not alleviated if Robo-dependent midline repulsion is reduced. Interestingly, the loss of commissures, but not the AEP defects, is also observed when activated Rac or Cdc42 GTPases are over- expressed in a homozygous fra mutant. Taken together, we propose that during exploration of the midline, Fra is a key regulator of Abl activity and helps determine how the cytoskeletal machinery utilizes Abl. In the absence of Fra, axon outgrowth does not simply stall; but rather, axons follow a variety of aberrant trajectories away from the midline. This suggests that Fra normally competes with several other receptor systems to dictate how Abl functions to regulate the cytoskeletal machinery. While compet- PLoS ONE | www.plosone.org 10 March 2010 | Volume 5 | Issue 3 | e9822