Figure 6

Silencing AX-1 in the developing cerebellum induces aberrant trajectories of parallel fibers. (a-c,f) Parallel fibers from areas outlined in (a) were analyzed in 250-μm-thick vibratome slices taken from HH35 cerebella after downregulation of AX-1 by ex ovo RNAi at HH34 (a-c,f). (b) In the absence of AX-1, the formation of the ML was severely disturbed in the transfected area. The width of the ML was irregular and fiber density was clearly reduced (arrows). (c) Most strikingly, axons of granule cells failed to extend in a parallel manner both with respect to each other and with respect to the pial surface (open arrows). (f) The same phenotype was obtained with a different long dsRNA derived from the AX-1 cDNA (open arrows). (d) On the contralateral side of the cerebellum injected and electroporated with AX-1 dsRNA the organization of the ML and the density of the parallel fibers were indistinguishable from non-injected, age-matched control embryos. (e) As expected, no difference between non-injected (not shown) and EGFP-injected control embryos was observed. (g,h) As an independent method for AX-1 perturbation, we used antibodies to block AX-1 function at the protein level. The phenotype obtained by ex ovo RNAi was reproduced with function-blocking antibodies. (i) The effect of AX-1 silencing by ex ovo RNAi was quantified. The relative fluorescence intensity after neurofilament staining was determined as a measure for the decrease in both ML width and parallel fiber density. On average, the staining intensity of the ML on the targeted side, that is, in the absence of AX-1, was reduced by 37.8 ± 3.6% compared to the non-affected control side (AX-1 dsRNA; n = 19 slices from 9 embryos). The staining intensities did not differ between the two halves of the cerebellum in embryos electroporated with the EGFP plasmid alone (EGFP; -0.3 ± 2.8%; n = 14 slices from 6 embryos) or in non-treated control embryos (control; 1.1 ± 3.2%; n = 10 slices from 4 embryos). ***P-values were < 0.0001 for the comparison between embryos treated with AX-1 dsRNA and EGFP-expressing control embryos, and experimental embryos versus non-treated control embryos. PS, pial surface; V, ventricle. Bar: 500 μm in (a), 100 μm in (b,d,e,g), and 50 μm in (c,f,h).