In the Absence of Frazzled Over-Expression of Abelson Tyrosine Kinase Disrupts Commissure Formation and Causes Axons to Leave the Embryonic CNS Joy N. Dorsten1., Bridget E. Varughese1., Stephanie Karmo 1 , Mark A. Seeger2, Mark F. A. VanBerkum1* 1 Department of Biological Sciences, Wayne State University, Detroit, Michigan, United States of America, 2 Department of Molecular Genetics and Center for Molecular Neurobiology, Ohio State University, Columbus, Ohio, United States of America Abstract Background: In the Drosophila embryonic nerve cord, the formation of commissures require both the chemoattractive Netrin receptor Frazzled (Fra) and the Abelson (Abl) cytoplasmic tyrosine kinase. Abl binds to the cytoplasmic domain of Fra and loss-of-function mutations in abl enhance fra-dependent commissural defects. To further test Abl’s role in attractive signaling, we over-expressed Abl in Fra mutants anticipating rescue of commissures. Methodology/Principal Findings: The Gal4-UAS system was used to pan-neurally over-express Abl in homozygous fra embryos. Surprisingly, this led to a significant decrease in both posterior and anterior commissure formation and induced some commissural and longitudinal axons to project beyond the CNS/PNS border. Re-expressing wild-type Fra, or Fra mutants with a P-motif deleted, revert both commissural and exiting phenotypes, indicating that Fra is required but not a specific P-motif. This is supported by S2 cell experiments demonstrating that Abl binds to Fra independent of any specific P- motif and that Fra continues to be phosphorylated when individual P-motifs are removed. Decreasing midline repulsion by reducing Robo signaling had no effect on the Abl phenotype and the phenotypes still occur in a Netrin mutant. Pan-neural over-expression of activated Rac or Cdc42 in a fra mutant also induced a significant loss in commissures, but axons did not exit the CNS. Conclusion/Significance: Taken together, these data suggest that Fra activity is required to correctly regulate Abl- dependent cytoskeletal dynamics underlying commissure formation. In the absence of Fra, increased Abl activity appears to be incorrectly utilized downstream of other guidance receptors resulting in a loss of commissures and the abnormal projections of some axons beyond the CNS/PNS border. Citation: Dorsten JN, Varughese BE, Karmo S, Seeger MA, VanBerkum MFA (2010) In the Absence of Frazzled Over-Expression of Abelson Tyrosine Kinase Disrupts Commissure Formation and Causes Axons to Leave the Embryonic CNS. PLoS ONE 5(3): e9822. doi:10.1371/journal.pone.0009822 Editor: Michael Hendricks, Harvard University, United States of America Received January 27, 2010; Accepted February 27, 2010; Published March 23, 2010 Copyright: 2010 Dorsten et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the National Science Foundation (#0416472) to M. VanBerkum. J.D. was partially supported by a Rumble Graduate Fellowship from Wayne State University and was a recipient of the William Turner Memorial Foundation Scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mvanberk@biology.biosci.wayne.edu . These authors contributed equally to this work. Introduction Attractive and repulsive guidance cues originating at or near the midline help guide the formation of the Drosophila embryonic nervous system. Receptors for these cues initiate intracellular signaling pathways that govern axon outgrowth and steering as well as formation of dendritic branching patterns [1–4]. Commissures form as axons integrate information from chemoat- tractive Netrins guiding them towards the midline and Slit- dependent repulsion preventing them from crossing [5–9]. Ectopic misexpression studies indicate that these attractive and repulsive systems mostly work independently of each other [10], but there is evidence of a fine interplay between systems at the transcriptional level [11,12] and downstream of receptors. In Drosophila, Netrins are midline attractants detected by Frazzled, a receptor expressed on most CNS neurons and in its absence many posterior commissures fail to form [5,6,13]. Fra may also have a non-cell autonomous effect guiding selected neurons at the segmental boundary [14]. A second Netrin receptor, the Down’s Syndrome Cell Adhesion Molecule, Dscam [15], is also expressed on most neurons, and mutations in fra and Dscam interact to further reduce commissure formation [16,17]. Since some commissures still form in Netrin embryos, the presence of an additional Netrin-independent attractive system has been proposed [9,16]. This is supported by evidence that Dscam may respond to a non-Netrin cue [16] and the recent identification of turtle as an important cell adhesion molecule that interacts with mutations in Netrin or fra to further reduce commissure formation [18]. At the midline, Slit-mediated repulsion prevents ipsilateral axons from crossing the midline and commissural axons from re- crossing the midline once on the contralateral side [7,8]. Slit is detected by Roundabout, a receptor expressed on most axons, and in its absence many ipsilateral axons meander across the midline; PLoS ONE | www.plosone.org 1 March 2010 | Volume 5 | Issue 3 | e9822