Fig. 6. Abnormalities in the cytoskeleton of sal mutant neurones in vitro (A-C) and in situ (D-E). Tubulin staining in vitro uncovered irregularities in the distribution of tubulin along the axon. The staining did not reach the growth cone (arrow in A) and had sometimes interruptions along the axon (arrows in B). Compare with the wild-type neurones in C [double stained with anti-tubulin (in red) and anti-HRP (in green)], where tubulin staining is uniformly intense along the axons and reaches almost the distal border of the growth cone (arrow). (D) At the stage of the strong TEM phenotype (early stage 16) laser confocal microscopy showed normal spatial distribution of F-actin, tubulin, and the microtubule associated protein Futsch/22C10 in sal mutant CNS. Notice the wild-type pattern in both heterozygotes and homozygotes, with accumulation of the marker along nerve roots and major axonal tracts. Careful analysis at higher magnification revealed frequent `mild' malformations in the organisation of axonal tracts in sal null condition (not shown). (E) When the fluorescence levels were measured (see Materials and Methods) the homozygotes were found to express higher levels for Actin, and lower for Tubulin (P<0.05) and Futsch (P=0.05). Each bar represents the mean values for samples of at least six embryos each and the s.e.m. is indicated.