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Fig. 4. The Ankyrin-binding and PH domains are not required for axon guidance. (A-E) Late stage 16 embryos stained with the anti-Fas2 Mab. Anterior is up. (A) ß-spectrin heterozygous embryos have no Fas2-positive longitudinal bundles crossing the midline. (B) ß-spectrin (em6) hemizygous embryos display Fas2-positive axons crossing the midline (arrows). (C) ß-spectrin null hemizygous embryos ubiquitously expressing full-length ß-Spectrin are rescued for both the medial ectopic crossing defect and lateral longitudinal breaks. (D) Ubiquitously expressing a form of ß-Spectrin lacking the Ankyrin-binding domain (ßSpecank) also rescues the midline axon guidance defects seen in ß-spectrin mutant embryos. (E) Ubiquitously expressing a form of ß-Spectrin lacking the PH domain (ßSpecPH) also rescues ß-spectrin mutant midline guidance errors. (F) Quantification of ectopic Fas2-positive midline crossovers in ß-spectrin heterozygous, hemizygous mutant, and transgenic rescued backgrounds. a, em6/+ (n=15); b, em6/Y (n=13); c, em6/Y+Ub-ßSpecWT (n=9); d, em6/Y+Ub-ßSpecank (n=15); e, em6/Y + Ub-ß-SpecPH (n=20). Asterisk denotes a significant difference between genotype b and genotypes a, c, d and e (P=1.22x10^-6; two-sample Student's t-test). Error bars indicate s.e.m. (G-M) Same embryos as in B-E, stained with the polyclonal anti-Myc antibody. Anti-Myc stainings were performed at the same time and images were taken at the same confocal settings. (G) Sibling ß-spectrin mutant embryos not expressing a transgene do not stain with the Myc antibody. Ubiquitously expressed wild-type ß-Spectrin localizes to the axons (H) and to the plasma membrane at sites of cell contact (K). Ubiquitously expressed ßSpecank localizes similarly to full-length transgenic protein to the axon scaffold (I) and to the plasma membrane (L). Ubiquitously expressed ßSpecPH localizes to axons (J), although at reduced levels, but is no longer localized to the plasma membrane nor at sites of cell contact (M).

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