Morpholino knockdown of slit2, slit3 and slit1a in wild type and yot (gli2DR) mutants. (A-J) Double labeling showing axons (anti-AT, green) and glial cells (anti-Gfap, red) in the forebrain at 28 hpf. (A) wt uninjected controls at 28 hpf and 22 hpf (inset). (B-D) Injection of MOs to slit2 (B), slit3 (C), or slit2 and slit3 together (D), into wild type resulted in defasciculation of the POC (arrowheads) and spreading of the glial bridge (brackets, lower division of bracket shows expansion beyond the bridge width seen in uninjected controls). This spreading of the glial bridge was also present prior to the appearance of POC axons (A,D, insets of POC region at 22 hpf, brackets). (E) slit1a MO-injected embryo with reduced AC and POC. Inset shows slit1a morphant with normal AC (arrowhead) and reduced POC. (F-J) yot (gli2DR) mutant embryos. (G-I) Injection of slit2 MOs (G), slit3 MOs (H), or slit2 and slit3 MOs together (I), into yot (gli2DR) mutant embryos resulted in a dramatic rescue of commissure (arrowheads) and glial bridge (arrow) formation. (J) By contrast, MO knockdown of slit1a in yot (gli2DR) mutants did not rescue POC or glial bridge formation. Dotted lines mark the optic recess. (K) Summary of results of slit MO knockdown on axons (black) and glia (red) relative to slit2/slit3 (blue) and slit1a (yellow) expression in wild type and yot (gli2DR) mutant embryos. vr, variably reduced. (L) Semi-quantitative analysis of POC formation following slit MO injections into wild type (purple bars) and yot (gli2DR) mutants (orange bars). Axon phenotypes were binned into seven categories ranging from no axons crossing (1) to a qualitatively wild-type POC (7); illustrated on left. Values represent the mean±s.e.m. All differences between injected embryos and corresponding uninjected controls are significant (Mann-Whitney U-Test, P=0.003-0.0001). No significant difference in commissure formation was found between slit2 + slit3 MO injection into wild type versus yot (gli2 DR) mutant embryos.
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