Frazzled and Abelson Figure 5. FasII positive longitudinal axons and sema2b expressing commissural axons leave the CNS in fra mutant embryos expressing BcrAbl or Ablwt. Stage 16 embryos were stained with either mAb 1D4 against FasII [A-D] or anti-myc to visualize axons expressing the sema2b-tau-myc reporter [E-H]. Anterior is to the right. With 1D4, ISN (black arrow in A) and SN (white arrow in A) motor nerve roots are observed exiting the CNS in both [A] fra3, 1407-Gal4 heterozygote and [B] fra3, 1407-G4/fra4 homozygote embryos. Using the 1407-Gal4 (1407-G4) driver, expression of either [C] BcrAbl or [D] Ablwt in a fra3/fra4 homozygote causes additional FasII expressing axons (arrows) to leave the CNS, including axons normally within the medial most pCC/MP2 pathway (arrow in C). [E-H] A sema2b-Tau-myc reporter was used to assess a subset of commissural axons. [E] In a fra3 heterozygote, sema2b expressing neurons extend axons into the anterior commissure (white arrowhead) and then turn anteriorly to form part of the longitudinal connectives (black arrowhead). [F] In homozygous fra3/fra4 mutants most axon projections into the anterior commissure are still intact; however, extensions in the longitudinal connectives may be disrupted (arrowhead). In fra3/fra4 embryos expressing either [G] BcrAbl or [H] Ablwt the axons of sema2b expressing neurons are observed projecting towards the periphery (arrows). doi:10.1371/journal.pone.0009822.g005 also causes severe errors in axon pathway formation as commissures fuse and large gaps appear in the LC ([Figure 7A], arrowhead). Yet, quite surprisingly, when Cdc42V12 is expressed in a fra3/fra4 homozygote most commissures are absent in nearly every segment, and LC formation improves as fewer gaps are now observed [Figure 7A, D]. It is noted, however, that while over- expression of Cdc42V12 in a fra3/fra4 mutant caused a dramatic loss in commissures, even more severe than over-expression of BcrAbl or Ablwt, no axons were observed leaving the CNS. These data confirm the importance of Fra signaling during formation of commissures and point to a key role for Abl in regulating cytoskeletal elements. The severity of these Cdc42V12 phenotypes prompted us to test another protein, Rac, which also regulates actin dynamics and a constitutively active form of Rac (RacV12) also causes FasII expressing axons to ectopically cross the midline [37,39]. When RacV12 is pan-neurally expressed in wild-type embryos or a fra3 heterozygote [Figure 7B], commissures and longitudinal connec- tives appear thin and slightly disorganized. But when RacV12 is over-expressed in a homozygous fra embryo, it too yields a major reduction in commissure formation [Figure 7E]. While some axons appear to orient away from the midline ([Figure 7E}, arrow), they were never observed to cross the CNS/PNS boundary. Next, we sought to selectively elevate myosin activity in a fra mutant using a constitutively active Myosin Light Chain Kinase (U-ctMLCK) that increases myosin activity by phosphorylating the Regulatory Light Chain of myosin II [56]. Expression of ctMLCK in a subset of FasII positive axons induces ectopic midline crossing errors, which, like RacV12 and Cdc42V12, are sensitive to heterozygous removal of fra [37]. Pan-neural expression of ctMLCK in wild-type embryos leads to ectopic crossovers (i.e. fuzzy commissures) in almost every segment leading [56]. While the frequency of this defect is somewhat diminished in a fra heterozygote, many segments still display fuzzy commissures ([Figure 7C], arrow). Unlike Cdc42 or Rac, however, expression of ctMLCK in fra homozygous embryos does not reduce commissure formation beyond that normally observed in a fra mutant [Figure 7F]. Similar results were observed for all three transgenes (U-Cdc42V12, U-RacV12 and U-ctMLCK) when the pan-neural elav-Gal4 driver line was utilized (data not shown). Over-expression of RacV12 in a Netrin embryo (NetDAB) also reduces commissure formation [Figure 6B]; Cdc42 could not be examined with Netrin due to stock health. Together, the Rac and Cdc42 data suggest that, in the absence of Fra, unilateral perturbation of actin cytoskeletal dynamics is sufficient to reduce commissure formation. These data continue to highlight the critical role Fra plays in regulating cytoskeletal dynamics during guidance events at the midline, and implicate all three molecules, Abl, Rac and Cdc42, in this process. Moreover, because RacV12 and Cdc42V12 do not cause AEP defects, these data also suggest that Abl contributes unique information governing which axons may leave the CNS. Discussion Frazzled and Abelson Tyrosine kinase activity clearly cooperate during the formation of embryonic commissures. In the absence of PLoS ONE | www.plosone.org 9 March 2010 | Volume 5 | Issue 3 | e9822