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First published online 14 November 2007
doi: 10.1242/dev.012872

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Cytoplasmic domain requirements for Frazzled-mediated attractive axon turning at the Drosophila midline

Department of Neuroscience, University of Pennsylvania School of Medicine, 1113 BRB2/3, 421 Curie Blvd., Philadelphia, PA 19104, USA.

View larger version (64K): [in this window] [in a new window]   Fig. 1. fra and Netrin mutants have defects in a subset of commissural neurons. (A-C) Stage 16 embryos stained with mAb BP102 to display all axons and anti-GFP to visualize a subset of commissural neurons, the Egl neurons. Indicated genotypes also contain UAS-TauMycGFP and eagleGal4. Anterior is up. (A) In wild-type embryos, EW and EG neurons cross the midline in every segment (white arrows). (B) EW axons fail to cross the midline in many segments of Netrin mutants (starred arrow). Note the thinning of CNS commissures (arrowhead) when stained with BP102 (magenta). (C) Similar to Netrin mutants, the EW axons frequently fail to cross the midline in fra mutant embryos (starred arrow). Note the thinning of commissures (arrowhead) when stained with BP102 (magenta). (D-F) Cartoons depicting the trajectories of the EW and EG neurons in each of the indicated genotypes.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Schematic of the Fra cytoplasmic domain indicating conserved motifs. (A) Sequence of the Fra cytoplasmic domain. Red lines over the sequence indicate the conserved P1, P2 and P3 motifs as defined by Kolodziej et al. (Kolodziej et al., 1996). These sequences were deleted in the constructs shown in B. Orange lines under the sequence indicate the four PXXP motifs found in the Fra cytoplasmic domain. The blue line delimits the region deleted in the construct P3 1/2; this construct leaves the critical Fak-binding residues, defined in vertebrate DCC, intact. (B) Schematic diagram of some of the constructs used in this study. Each construct carries a six-Myc epitope at its carboxyl terminus for comparison of expression levels and localization. Ig, immunoglobulin; FN3, fibronectin type III.

View larger version (66K): [in this window] [in a new window]   Fig. 3. Fra is required cell autonomously to guide axons and the P3 motif is critical for mediating Fra attractive function. (A-D) Stage 16 embryos stained with mAb BP102 to visualize all axons, and anti-GFP to highlight the Egl neurons. Indicated genotypes also contain UAS-TauMycGFP and eagleGal4. Anterior is up. (A) EW axons fail to cross the midline in many segments of fra mutants (starred arrows). Also notice the overall thinning of CNS commissural bundles (arrowhead). (B) fra mutant defects can be rescued (arrows) by specifically expressing UAS-Fra-Myc in the Egl neurons. (C) Similar to UAS-Fra-Myc, expression of UAS-FraP1P2-Myc in the Egl neurons results in rescue of the fra guidance defects. (D) A Fra receptor lacking the P3 motif cannot rescue the guidance defects of fra mutants. (B-D, bottom panels) Anti-Myc staining (green) of embryos expressing UAS-Fra-Myc (B), UAS-FraP1P2-Myc (C) or UAS-FraP3-Myc (D) under the control of elavGal4 shows that the transgenes are expressed at comparable levels and are similarly localized to CNS axons. One segment is shown. (E) The rescuing ability of each of the constructs used in this study. Green bars indicate rescue that is comparable to that of wild type; red bars indicate a failure to rescue. Data is presented as the percentage of EW axons that fail to cross the midline. Mutant defects were scored as the complete absence or thinning of EW axons across the midline. Each bar represents an independent transgenic line (see Fig. S1 in the supplementary material; Materials and methods). A total of 60-80 embryos were scored for each genotype (or approximately 15-20 embryos for each line). Error bars indicate s.e.m. For statistical analysis, see Fig. S1 in the supplementary material.

View larger version (17K): [in this window] [in a new window]   Fig. 4. The P3 motif is not required for Frazzled multimerization. (A,B) Co-immunoprecipitation of Myc and HA-tagged Frazzled constructs expressed in Drosophila S2R+ cells. Lysates were immunoprecipitated with either anti-Myc antibody (A, and upper left in B), or anti-HA antibody (upper right B). (A) Myr-Fra-Myc co-immunoprecipitates with HA-tagged Fra constructs, including one lacking the P3 motif (lane 3). FraC-HA serves as a control (lane 5). (B) A P3 motif is not required in either construct for interaction, as Myr-FraP3-Myc binds to P3 constructs as efficiently as to those containing wild-type cytoplasmic domains (compare lanes 2, 5, 7 and 10 to lanes 1, 4, 6 and 9).

View larger version (57K): [in this window] [in a new window]   Fig. 5. The cytoplasmic domain of Fra is not required for proper localization. (A-E') Stage 16 embryos stained with BP102 to visualize all axons. (C'-E') Same embryos as C-E. Staining with anti-Myc or anti-HA reveals Fra receptor expression. Anterior is up. (A) Wild-type embryos stained with BP102 exhibit a ladder-like CNS scaffold with distinct thick anterior and posterior commissures. (B) Commissural bundles are thin or absent in fra mutant embryos (arrowheads). (C) Expressing a full length Fra receptor in all neurons using elavGal4 rescues fra mutant defects. (C') Wild-type Fra localizes to the axons scaffold when expressed in all neurons. (D) When a weak insert of FraC is expressed panneurally in a fra mutant background, many axons fail to cross the midline (arrowheads). (D') Despite the inability of FraC to rescue fra guidance defects, this truncated receptor is still localized to the axon scaffold as well as the wild-type full-length receptor. (E) Expression of FraCRobo67Myc does not rescue the defects seen in fra mutant embryos (arrowheads); however, in contrast to FraC, FraCRobo67Myc does not efficiently localize to axons (E') perhaps because the exongenous Robo sequence interferes with normal Fra receptor distribution.

View larger version (71K): [in this window] [in a new window]   Fig. 6. Axons expressing FraC are not attracted toward the midline. (A-E) Stage 16 embryos stained with mAb BP102 to visualize all axons (magenta) and anti-HA to reveal FraC-HA expression (green). Anterior is up. (A) Wild-type embryos stained with BP102 exhibit a ladder-like CNS scaffold. (B) Commissural bundles are thin when one copy of FraC is panneurally expressed in a wild-type background (arrowheads). (C) Axon commissures are almost completely absent when two copies of FraC are expressed. (D) Fewer axons cross the midline when one copy of FraC is expressed in a fra heterozygous background (arrowheads; compare this phenotype to the one in B). (E) When one copy of FraC is expressed in a fra homozygous mutant background, axons are no longer attracted toward the midline. (F) Diagram of the FraC-HA construct used in these experiments. (G,H) Stage 16 embryos stained with mAb BP102 and anti-GFP to highlight the Egl neurons. (G) When one copy of FraC is expressed specifically in the Egl neurons in a wild-type background, EW axons fail to cross the midline in many segments (arrow with star). (H) None of the EW axons cross the midline when FraC is expressed in a fra mutant background. Additionally, we also see defects in EG axon guidance (starred feathered arrow), a phenotype never observed in fra mutants (feathered arrows in G). Specifically, both EW and EG axons are often observed exiting the midline (carets).

View larger version (58K): [in this window] [in a new window]   Fig. 7. The FraC misexpression phenotype depends on Netrin but not Slit-Robo or Unc-5 signaling. (A,B,E-J) Stage 16 embryos stained with mAb BP102 to display all axons and anti-HA to reveal FraC-HA expression. Anterior is up. (A) Netrin mutants contain many segments with absent or thin commissures suggesting a decrease in midline attraction (arrowheads). (B) Expression of FraC cannot enhance the Netrin phenotype suggesting that the receptor depends on Netrin to generate an overexpression phenotype (compare this phenotype to the one in Fig. 5E). (C,D) Late stage 16 embryos stained with anti-FasII (1D4) to display three ipsilateral bundles of axons and anti-HA to reveal FraC-HA expression. Anterior is up. (C) Medial FasII-positive bundles frequently cross and re-cross the midline in robo mutants. (D) Fewer FasII-positive axons cross the midline when FraC is overexpressed. (E) fra, robo double mutants have many extra axons crossing the midline, though the defects are slightly less severe than robo single mutants. (F) Expression of FraC in all neurons in fra, robo double mutants causes a dramatic defect in commissure formation, although some commissures still do form relatively normally. (G) In fra, slit double mutants almost all axons collapse at the midline. (H) Expression of FraC is able to push axons laterally in a fra, slit mutant background. (I,J) The FraC misexpression phenotype is not dependent on the presence of the Unc-5 repulsive Netrin receptor.

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