In the Absence of Frazzled Over-Expression of Abelson Tyrosine Kinase Disrupts Commissure Formation and Causes Axons to Leave the Embryonic CNS

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

Changes in midline repulsive activity do not account for the Abl gain-of-function phenotypes.

<p>The ability of a heterozygous loss of <i>robo</i> to alter the phenotypes that occur when the 1407-Gal4 (1407-G4) driver was used to pan-neurally express Ablwt or BcrAbl in <i>fra</i> mutants was quantified after staining embryos with mAb BP102 (as described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009822#pone-0009822-g001" target="_blank">Figures 1</a> or 4]). [<b>A</b>] 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 <i>fra³, 1407-G4/fra⁴</i> homozygote embryos (white; <i>robo^+/+</i>) versus <i>fra³, 1407-G4/fra⁴</i> homozygote embryos carrying one copy of a null <i>robo</i> allele (black; <i>robo^+/−</i>). The loss of one copy of <i>robo</i> has no significant affect on the frequency of these defects. [<b>B-D</b>] To test whether <i>netrin</i>-dependent repulsion is important, BcrAbl or Rac^V12 (as indicated at top) were over-expressed in <i>netrin</i> null embryos (<i>net^ΔAB</i>) using the pan-neural C155 Gal4 driver also on the X chromosome. [<b>B</b>] In a relatively severe <i>netrin</i> mutant, most posterior commissures (black arrowheads) are absent and several anterior commissures are thin. Most commissures are absent when either [<b>C</b>] BcrAbl or [<b>D</b>] Rac^V12 are over-expressed in all neurons, and, with BcrAbl, some axon bundles also project beyond the CNS/PNS boundary (arrow in C).</p

BcrAbl induced crossovers depend on the P3-motif of Fra.

<p>Stage 16 embryos stained with mAb 1D4 against Fasciclin II (FasII) are depicted with anterior up. All UAS (U) transgenes are pan-neurally expressed using the 1407-Gal4 (1407-G4) driver line recombined onto the <i>fra³</i> chromosome; thus, phenotypes are compared to heterozygous <i>fra³</i> rather than a wild-type embryo. [<b>A</b>] A <i>fra³</i> heterozygote exhibits a nerve cord that is essentially wild-type. Expression of [<b>B</b>] BcrAbl but not [<b>C</b>] Ablwt results in FasII axons crossing the midline incorrectly (arrowhead in B). [<b>D</b>] In homozygous <i>fra³/fra⁴</i> mutants, the FasII fascicles may partially fuse, and small breaks in the connective may appear. Removal of both copies of <i>fra</i> abolishes [<b>E</b>] BcrAbl and [<b>F</b>] Ablwt induced crossover defects. In both cases, FasII expressing axons are observed projecting towards the periphery (arrows), including axons from the medial most pCC/MP2 pathway (arrow in E). [<b>G</b>] Quantification of ectopic crossing errors (per embryo) observed using BcrAbl is graphed. White bar indicates crossovers in a <i>fra³</i> 1407-Gal4 heterozygote, black bar is a <i>fra³</i> 1407-Gal4/<i>fra⁴</i> homozygote and gray bars are <i>fra³</i> 1407-Gal4/<i>fra⁴</i> homozygote's expressing the indicated Fra transgene. Expression of BcrAbl in a <i>fra³</i> 1407-Gal4/<i>fra⁴</i> homozygote reduces ectopic crossovers, but these are restored by co-expression of Fra transgenes (* P<0.05, or ** P<0.01). Compared to Fra^wt, expression of Fra^ΔP3 restores only about half of the expected crossovers (φ P<0.05). [<b>H</b>] Re-expression of U-Fra^wt transgene restores the crossover defects (arrowhead in G) in <i>fra³/fra⁴</i> embryos expressing BcrAbl, while [<b>I</b>] expression of Fra^ΔP3 restores about half of the ectopic crossovers.</p

FasII positive longitudinal axons and <i>sema</i>2b expressing commissural axons leave the CNS in <i>fra</i> mutant embryos expressing BcrAbl or Ablwt.

<p>Stage 16 embryos were stained with either mAb 1D4 against <i>FasII</i> [<b>A-D</b>] or anti-myc to visualize axons expressing the <i>sema</i>2b-<i>tau</i>-myc reporter [<b>E-H</b>]. 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 [<b>A</b>] <i>fra³,</i> 1407-Gal4 heterozygote and [<b>B</b>] <i>fra³, 1407-G4/fra⁴</i> homozygote embryos. Using the 1407-Gal4 (1407-G4) driver, expression of either [<b>C</b>] BcrAbl or [<b>D</b>] Ablwt in a <i>fra³/fra⁴</i> homozygote causes additional FasII expressing axons (arrows) to leave the CNS, including axons normally within the medial most pCC/MP2 pathway (arrow in C). [<b>E-H</b>] A <i>sema2b</i>-Tau-myc reporter was used to assess a subset of commissural axons. [<b>E</b>] In a <i>fra³</i> heterozygote, <i>sema</i>2b expressing neurons extend axons into the anterior commissure (white arrowhead) and then turn anteriorly to form part of the longitudinal connectives (black arrowhead). [<b>F</b>] In homozygous <i>fra³/fra⁴</i> mutants most axon projections into the anterior commissure are still intact; however, extensions in the longitudinal connectives may be disrupted (arrowhead). In <i>fra³/fra⁴</i> embryos expressing either [<b>G</b>] BcrAbl or [<b>H</b>] Ablwt the axons of <i>sema</i>2b expressing neurons are observed projecting towards the periphery (arrows).</p

Expression of Rac^V12 and Cdc42^V12 reduces commissure formation in a <i>fra</i> mutant.

<p>Stage 16 embryos stained with the mAb BP102 are depicted with anterior up. UAS (U) transgenes are expressed pan-neurally using the <i>1407-GAL4</i> (1407-G4) driver line recombined onto the <i>fra³</i> chromosome, and, as in other figures, phenotypes are compared to heterozygous <i>fra³</i>. Phenotypes in heterozygous <i>fra³</i> embryos tend to be somewhat milder than that observed in a wild-type embryo (see text). [<b>A</b>] Expression of Cdc42^V12 in a <i>fra³</i> heterozygote background results in fused commissures [arrowhead] and large gaps in the longitudinal connectives. [<b>B</b>] Expression of Rac^V12 in a <i>fra³</i> heterozygote causes a thinning of both PC (black arrowhead) and AC (white arrowhead) as well as LC (black arrow). [<b>C</b>] Expression of ctMLCK in a <i>fra³</i> heterozygote results in fuzzy commissures (black arrowhead<b>)</b>. Expression of both [<b>D</b>] Cdc42^V12 and [<b>E</b>] Rac^V12 in a <i>fra³/fra⁴</i> mutant significantly reduces commissure formation (black arrowheads) and, with expression of Rac^V12, some axons may orientate towards the periphery (arrow in E), but they are not observed to exit the CNS. [<b>F</b>] Expression of ctMLCK in a <i>fra³/fra⁴</i> mutant does not significantly alter the PC (arrowhead) and LC defects normally observed in a <i>fra</i> mutant.</p