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Fig. 8. ß-Spectrin contributes to the maintenance of axonal connections. All embryos were stained with anti-Fas2 and anti-GFP, and contain aptGAL4 and UAS-TauMyc GFP in the background except for D-F; these embryos contain UAS-TauGFP. Anterior is up in all panels. Embryos of each genotype (left) were fixed, stained and dissected at the indicated stages (top). (A-C) yw embryos. (A) At stage 14, the Ap neurons in each segment approach the midline and make a stereotypical turn anteriorly (arrowhead). (B) At stage 15, neurons extend growth cones further anteriorly almost reaching the next segment. (C) At late stage 16, axons make one continuous bundle spanning the length of the embryo. Note that in wild-type embryos, the Ap neurons remain ipsilateral. (D-F) robo mutant embryos. In robo mutant embryos at stage 14 (D), Ap neurons do not respect the midline boundary and cross over to the other side (starred arrowhead). Later in development, axons continue to recross the midline as they extend anteriorly (E,F). (G-I) ß-spectrin^em6/Y hemizygous embryos. (G) Similar to wild-type embryos at stage 14, the Ap neurons in ß-spectrin mutants approach the midline, make an anterior turn, and do not cross the midline (arrowhead). Interestingly, in some segments, the Ap neurons also begin to extend processes slightly posteriorly (arrows). (H) At stage 15, most of the Ap neurons still respect the midline boundary, although we occasionally saw a stray axon crossing over (starred arrowhead). (I) Later in development, the Ap neurons lose sensitivity to the midline, fail to maintain their appropriate ipsilateral connections and cross the midline (starred arrowheads). Additionally, in some segments, we noticed that some axons do not extend all the way to the next segment (feathered arrowhead). (J-L) ß-spectrin^em6 hemizygous plus slit heterozygous embryos. (J) Even when one copy of slit is removed in a ß-spectrin null background, stage 14 Ap neurons still do not cross the midline but begin to extend anteriorly. (K) By stage 15, some axons already lose sensitivity to the midline (starred arrowheads). (L) By late stage 16, Ap neurons in many segments now cross the midline (starred arrowheads).

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