Fig. 1. ß-Spectrin is required in neurons for midline axon guidance. (A, top) Expanded 5' region of the ß-spectrin genomic locus showing the location of each p-element insert. (Bottom) 1, Wild-type ß-Spectrin (ßSpecWT) protein including functional domains (ABD, actin-binding domain; ank, Ankyrin-binding domain; PH, pleckstrin homology domain); 2, ßSpecank replaces segment 16 (the 15th repeat) of ß-Spectrin with segment 13 of -Spectrin; 3, ßSpecPH places a stop codon at the beginning of the PH domain in ß-Spectrin. (B-D) Stage 16 embryos stained with monoclonal antibody (Mab) BP102 to reveal the CNS axon scaffold. Anterior is up. (B) ß-spectrin (em6) heterozygous embryos exhibit a wild-type ladder-like CNS architecture. (C) ß-spectrin (em6) hemizygous embryos display fused anterior and posterior commissures reflecting a reduction in midline repulsion. (D) Rescue of ß-spectrin (em6) phenotype by expressing wild-type ß-Spectrin in all post-mitotic neurons with elavGAL4. (E-H) Late stage 16 embryos stained with anti-Fas2 Mab to reveal longitudinal axon pathways. Anterior is up. (E) ß-spectrin heterozygous embryos have no Fas2-positive axon bundles crossing the midline. (F) ß-spectrin (em6) hemizygous embryo. Medial ectopic Fas2-positive bundles cross the midline (arrows) and are closer together [distance between medial fasicles: wild-type, 13.7±1.37 µm; ßspec(G0108), 8.9±1.27 µm; compare E and F, dashed line]. Lateral Fas2-positive bundles contain longitudinal breaks (arrowheads). (G) ß-spectrin mutant embryo expressing full-length wild-type ß-Spectrin in all neurons using elavGAL4. Note complete rescue of medial and lateral longitudinal axon guidance defects. (H) ß-spectrin em6 embryo expressing full-length wild-type ß-Spectrin in midline glia using simGAL4. ß-spectrin mutant phenotypes in the longitudinal pathways (arrows and arrowheads) are still observed. (I) Quantification of ectopic crosses per embryo in genotypes: a, em6/+ (n=15); b, em6/Y (n=18); c, em6/Y,UAS-ßSpec/+,elavGAL4/+ (n=15); d, em6/Y,UAS-ßSpec/+,simGAL4/+ (n=11); e, em21/Y (truncation) (n=18); f, strong p-element allele G0108/Y (n=14); g, presumptive hypomorphic p-element allele G0074/Y (n=13); h, presumptive hypomorphic p-element allele G0198/Y (n=14). Asterisk denotes significant difference in genotypes b and a, and b and c (P=8.027x10^-7; two-sample Student's t-test). There is no significant difference between genotypes b and d. Error bars indicate s.e.m. (J,K) Stage 15 embryos stained with polyclonal anti-ß-Spectrin antibody. Anterior is up. (J) Wild-type ß-Spectrin protein localizes to the plasma membrane around every cell of the CNS neuropil and to the axon scaffold (brackets). (K) ß-Spectrin protein levels are severely reduced in ß-spectrin (em6) mutant embryos. (L) ß-spectrin (G0108) mutant embryo stained with an anti-wrapper antibody showing the appropriate number and location of the midline glia.