In the absence of frazzled over-expression of Abelson tyrosine kinase disrupts commissure formation and causes axons to leave the embryonic CNS

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.

Figure 1. Increasing Abl activity in a fra mutant reduces commissure formation.

Stage 16 embryos stained with mAb BP102 are depicted with anterior up. Indicated UAS- (U) transgenes are expressed pan-neurally using the 1407-Gal4 (1407-G4) driver line recombined onto a fra³ chromosome. For this reason, phenotypes are compared to heterozygous fra³ rather than a wild-type embryo; in most cases the heterozygote is similar to wild-type. [A] Normal anterior commissures (AC), posterior commissures (PC) and longitudinal connectives (LC) are observed in a fra³ 1407-GAL4 heterozygote stained with BP102. [B] Over-expression of BcrAbl in a fra³ heterozygote causes fuzzy commissures in most segments while [C] expression of Ablwt causes only a rare fuzzy commissure (black arrowheads). [D] Homozygous fra³/fra⁴ mutants display thinning and missing PC's (black arrowhead) in some segments but the AC remains unaffected (white arrowhead). Over-expression of either [E] BcrAbl or [F] Ablwt in a fra³/fra⁴ background enhances the degree of thinning and missing PC's (black arrowhead), and many AC's (white arrowhead) disappear. [K] Quantification of defects in the AC and PC are graphed with percent of commissures missing in a fra³ 1407-Gal4 heterozygote (white, highlighted with ‘w’), fra³ 1407-Gal4/fra⁴ homozygote (black) and fra³ 1407-Gal4/fra⁴ homozygote's expressing the indicated Abl transgene (gray). All three Abl transgenes significantly enhance the loss of PCs and both BcrAbl and Ablwt affect AC formation (*P<0.05; **P<0.01; ***P<0.001). [G] Co-expression of a Fra^wt transgene restores the fuzzy commissure defects caused by expression of BcrAbl but [H] with the Fra^ΔP3 transgene the AC and PC commissures are more distinct. Expression of [I] Fra^wt or [J] Fra^ΔP3 transgenes also rescues commissure formation when Ablwt is over-expressed in a homozygous fra mutant. [L] The distribution of segments containing fuzzy (black) or distinct (white) commissures in fra³/fra⁴ embryos co-expressing BcrAbl and each of the Fra P-motif deletion mutants is graphed.

Figure 2. BcrAbl induced crossovers depend on the P3-motif of Fra.

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 fra³ chromosome; thus, phenotypes are compared to heterozygous fra³ rather than a wild-type embryo. [A] A fra³ heterozygote exhibits a nerve cord that is essentially wild-type. Expression of [B] BcrAbl but not [C] Ablwt results in FasII axons crossing the midline incorrectly (arrowhead in B). [D] In homozygous fra³/fra⁴ mutants, the FasII fascicles may partially fuse, and small breaks in the connective may appear. Removal of both copies of fra abolishes [E] BcrAbl and [F] 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). [G] Quantification of ectopic crossing errors (per embryo) observed using BcrAbl is graphed. White bar indicates crossovers in a fra³ 1407-Gal4 heterozygote, black bar is a fra³ 1407-Gal4/fra⁴ homozygote and gray bars are fra³ 1407-Gal4/fra⁴ homozygote's expressing the indicated Fra transgene. Expression of BcrAbl in a fra³ 1407-Gal4/fra⁴ 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). [H] Re-expression of U-Fra^wt transgene restores the crossover defects (arrowhead in G) in fra³/fra⁴ embryos expressing BcrAbl, while [I] expression of Fra^ΔP3 restores about half of the ectopic crossovers.

Figure 3. Both Ablwt and BcrAbl bind to Frazzled independently of a specific P-motif.

[A] Stable Drosophila S2 cells were created that express an HA-tagged form of wild-type [wt] or P-motif deletion (ΔP1, ΔP2, ΔP3) mutants of Frazzled (Fra^x-HA). Frazzled was immunoprecipitated (IP) from cells using a mouse monoclonal anti-HA, and western blots were probed (IB) with guinea pig anti-Drosophila Abl (D-Abl; top) or rabbit anti-HA (bottom). Compared to the amount of Frazzled receptor pulled down, endogenous Abl binds to Frazzled independently of a P-motif. [B] Transiently transfected Ablwt or AblKN (kinase inactive) genes also bind to a myc-tagged version of wild-type Fra (Fra^wt-myc])but not to a HA-tagged receptor missing its cytoplasmic domain (Fra^ΔC-HA). The amount of Fra (middle row) and Abl (bottom row) present in the lysates is indicated. [C] Activated BcrAbl, detected by a rabbit antibody against the Bcr region, also binds to Fra-myc even if one of the P-motifs is removed. No band is observed in mock IPs from S2 cells expressing only BcrAbl (far left). The amount of Fra pulled down (middle row), and the amount of BcrAbl available in lysates (bottom row) is shown. [D] In the top panel, S2 cells were pretreated for 30 min with pervanadate and then wild-type or P-motif deletions of Frazzled were immunoprecipitated using mouse anti-myc and the degree of tyrosine phosphorylation assessed by probing a blot with mouse anti-phosphotyrosine (α-pY; top row). Compared to the amount of receptor pulled down (bottom row), the level of tyrosine phosphorylation does not appear to be diminished when a P-motif is deleted. The bottom panel assesses Fra^wt phosphorylation when Abl transgenes are co-expressed in S2 cells that were not pretreated with pervanadate. Compared to the level observed in S2 expressing only Fra^wt-myc, phosphotyrosine levels increase slightly if cells are co-transfected with Ablwt or BcrAbl, but not AblKN (bottom blot). In this blot, autophosphorylation of BcrAbl denoting its activated state is apparent (arrowhead). The position of molecular weight standards (in kDa) is indicated on the left side of each panel.

Figure 4. Quantification of axons exiting to the periphery [AEP] defects.

A close up of the CNS/PNS border of stage 16 embryos stained with mAb BP102 is depicted with anterior to the right. Peripheral muscles are seen by Normarski optics [A-D]. UAS transgenes are expressed using the 1407-Gal4 (1407-G4) driver line recombined onto the fra³ chromosome. [A] A fra³ heterozygote is identical to a wild-type embryo and arrows indicate lightly stained ISN (black arrow in A) and SN (white arrow in A) nerve roots. Note the absence of BP102 staining beyond the CNS/PNS boundary. AEP defects are not observed in either [A] fra³ heterozygote or [B] fra³/fra⁴ homozygote embryos. Homozygous fra³/fra⁴ embryos expressing either [C] BcrAbl or [D] Ablwt show large bundles of BP102 positive axons extending into the PNS (arrows in C, D). [E] Quantification of AEP defects per hemisegment (hs) is depicted for Abl transgenes (left) alone or in combination with Fra transgenes (right). White bars (highlighted with ‘w’) indicate fra³ 1407-Gal4 heterozygote's, black bars are fra³ 1407-Gal4/fra⁴ homozygote's and gray bars are fra³ 1407-Gal4/fra⁴ homozygote's expressing the indicated transgene. All three Abl transgenes significantly (P<0.01) enhance AEP defects, although a kinase inactive Abl is less effective. BcrAbl (middle) and Ablwt (right) defects are significantly rescued by co-expression of any of the Fra transgenes, although rescue by Fra^ΔP3 is significantly less than that observed using Fra^wt. Comparison to fra³ 1407-Gal4/fra⁴ homozygote condition is indicated by *, while φ compares rescue of P-motif deletion to Fra^wt (*,φ P<0.05; **, φφ P<0.01).

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).

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 null robo allele (black; robo^+/−). 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 Rac^V12 (as indicated at top) were over-expressed in netrin null embryos (net^ΔAB) 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] Rac^V12 are over-expressed in all neurons, and, with BcrAbl, some axon bundles also project beyond the CNS/PNS boundary (arrow in C).

Figure 7. Expression of Rac^V12 and Cdc42^V12 reduces commissure formation in a fra mutant.

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