which is, in fact, where most of the tyrosine residues within the cytoplasmic domain of Fra reside [13,36]. Moreover, in S2 cells, tyrosine phosphorylation of Fra is not affected by removal of the P1 or P2 motif and may actually increase when P3 is removed. This is intriguing as removal of the P3-motif is known to significantly affect Fra signaling in vivo [32,33] and the FraDP3 transgene is the least capable of rescuing the AEP defects caused by Ablwt or BcrAbl expression. Immunoblots of Fra phosphory- lation in the absence of pervanadate pretreatment also suggest the steady-state level of tyrosine phosphorylation may be relatively low, or highly dynamic. S2 cells are known to express tyrosine phosphatases that antagonize Abl activity for some substrates [63,64], and antagonistic action between Abl and several phosphatases during nerve cord development has been document- ed [50,51,65–67]. Since tyrosine phosphorylation of vertebrate DCC is required for attractive responses, axon outgrowth and orientation of the axon (e.g. [68–74]), it will be important to systematically assess how Fra and Abl physically interact to regulate each other’s activity during midline guidance. Both of the phenotypes observed when Abl activity is elevated in a fra mutant, the loss of commissures and the exiting of CNS axons to the periphery, suggest these embryos are experiencing an excess of midline repulsion. During commissure formation, Slit depen- dent repulsion prevents commissural axons from crossing unless Commissureless prevents the Slit receptor Robo from accumulat- ing on the cell surface [9,75]. Before commissural axons extend towards the midline, Fra activity may help increase Comm expression [12] so Comm levels are expected to be reduced in fra mutants leading to an increase in Robo-dependent repulsion. Since increasing Robo activity in a fra embryo is sufficient to reduce commissure formation [10], it was important to test whether an excess of Robo-dependent repulsion underlies the Abl over-expression phenotypes. However, our introduction of one allele of robo (Robo1) had no discernable affect on the Abl gain-of-function phenotypes, even though, in previous work, elevating Abl activity in a heterozygous robo mutant induces ectopic midline crossing errors [49]. Given the absence of even a minimal suppression, it seems unlikely that the loss of commissures and/or AEP defects noted in our mutants reflects an increase in Robo-dependent midline repulsion. While two other Robo receptors, Robo2 and Robo3, operate during midline guidance [21–24,75] and could conceivably contribute to these Abl phenotypes, neither of these receptors have the conserved CC3 cytoplasmic domain known to be important for Abl binding to Robo1 [45,75]. Moreover, while certain Netrin receptors (e.g. Unc5; [60]) can also elicit a repulsive response, both commissure loss and AEP defects still occur when Abl is over-expressed in a Netrin mutant. This provides strong evidence that abnormal signaling by other Netrin receptors is not responsible for these phenotypes. In terms of the AEP defects, which also point to excess repulsion, it is worth noting that axons do not leave the CNS in a commissureless mutant experiencing very high levels of Slit- dependent repulsion [20,25,49], nor do they appear evident in published figures of fra Dscam double mutants, or even fra Dscam abl triple mutants [16]. While the identity of all the axons leaving the CNS has not been established, we have confirmed that at least two subtypes of CNS axons are exiting: both FasII expressing interneurons and sema2b commissural axons. FasII axons do not leave the CNS when midline repulsion is elevated in a commissureless mutant [20,25,49], and while the level of Abl activity affects the trajectory of FasII interneurons, in most cases altering Abl activity leads to midline crossing errors rather than an exit from the CNS [42,49,51]. Over-expressing Abl in a fra mutant also Frazzled and Abelson affects several different guidance decisions by Sema2b-expressing commissural axons. While the cues guiding these neurons are not well understood, the spectrum of defects observed both before and after they cross the midline suggest that these neurons are responding to more then just midline repulsion. Thus, if a repulsive mechanism is functioning, as initially suggested by the phenotype, the origin of the signal remains to be determined. Indeed, our data using activated forms of Rac and Cdc42 suggest that the primary defect lies in the ability of growth cones to properly regulate actin dynamics underlying axon outgrowth and steering. This could involve both attractive and repulsive systems. Proper axon guidance also requires concerted regulation of the cytoskeletal dynamics underlying axon outgrowth and steering [76]. Like most guidance receptors, Fra, or its vertebrate and C. elegans homologues, is known to initiate signaling pathways affecting cytoskeletal dynamics [34,37,77,78]. Abl is also a key regulator of actin dynamics in vertebrate cells [47,48,79–81] and of the development of the Drosophila nervous system. Mutations in abl interact with several cytoskeletal regulators to affect axon pathway formation: kette [82], capulet [40], chicadee (Profilin; [51]), enabled [42,83] and trio [36,84,85]. Thus, in the absence of Fra- dependent regulation, does elevated Abl activity affect the cytoskeletal machinery to indirectly cause a reduction in commissures and AEP defects? Here, we tested this basic concept by expressing in fra mutants other key regulators of cytoskeletal dynamics known to affect midline guidance [37,39,56]. Surpris- ingly, over-expression of activated Rac and Cdc42 in a fra mutant replicates the loss of commissure phenotype (but not the AEP defects) observed with Abl. The Cdc42 result is most intriguing as expression in a wild-type or heterozygous fra embryo results in fused commissures and gaps in the longitudinal connectives. Yet, upon complete removal of Fra, commissures do not form and the longitudinal connectives reform. Thus, in the absence of Fra, commissure formation, but not AEP defects, appears to be particularly sensitive to manipulation of actin-based processes. It is possible that our manipulation of Cdc42 and Rac activity in a fra mutant is affecting shared processes related to actin polymeriza- tion. For example, in vertebrate studies, Cdc42 and Abl work in parallel to regulate actin polymerization (e.g. [86–89]) and Abl may activate Rac in response to cell adhesion [87,90,91]. If so, our data suggest that in the absence of Fra activity these key regulators are being used by other surface receptors to regulate actin dynamics in a manner that ultimately prevents commissure formation. This is certainly consistent with the number of guidance systems that have been linked to these regulators [35,92–94] and the scope of guidance defects detected in our embryos. Minimally, the Cdc42 and Rac data continue to highlight the degree and importance of Fra-dependent regulation of cytoskeletal dynamics, especially actin-based processes, during commissure formation. Moreover, they point to a highly competitive process between multiple surface receptors and the cytoskeletal machinery where Fra is a major player. While competition between midline attractive and repulsive cues has been recognized [2,10], in our experiments, midline repulsive activity had no affect on the Abl phenotypes. Therefore, it seems likely that Fra is competing with several other receptor systems whose presence (but not identity) has been uncovered by over- expression of Abl, Rac or Cdc42 in homozygous fra embryos. Which guidance events are being affected has not yet been determined, but a few candidates exist. In addition to fra and Dscam [16,36], loss-of-function mutations in abl interact with the cell-cell adhesion molecules neurotactin and amalgam [53], fasI [54], midline-fasciclin [55] and turtle [18] to reduce commissure formation and some of these are fairly ubiquitously expressed in the nerve PLoS ONE | www.plosone.org 12 March 2010 | Volume 5 | Issue 3 | e9822