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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, ßSpec
ank 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.
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