Figure 5.

α2-Chimaerin knockdown produces defects in the development of the OMS. A, Schematic representation of a lateral view of the ocular motor system in zebrafish, with anterior facing to the right. The dotted line indicates the edge of the brain. Oculomotor (III), trochlear (IV), and abducens (VI) nerves are shown in cyan, and extraocular muscles are shown in magenta. B, Schematic representation of the typical defects observed in OMS development as a result of manipulations presented here. Yellow arrows indicate ectopic branching, and white arrows indicate defasciculation. Ci, Neurite tracer extracted image following whole mount staining of the OMS of a 3 dpf wild-type zebrafish larva using anti-tubulin (cyan) and anti-myosin heavy chain (magenta). Scale bars, 100 µm. Cii, Ciii, Insets, Representative defasciculation and ectopic branching events at a higher magnification (Cii shows oculomotor main branch; Ciii shows abducens). Dii, Diii, Eii, Eiii, Whole mount staining of 3 dpf zebrafish larvae injected with 3 ng of Standard MO (Dii, Diii, Eii, Eiii) and 3 ng of chn1 MO (Eii, Eiii). In each case, Dii, Diii, Eii, and Eiii are as in C. F, Quantification of the average defasciculation and ectopic branching across the OMS, shown as a percentage. G, Quantification of the defasciculation events (percentage) analyzed in the main bundle of the OMN, and each of the four branches of the OMN, trochlear nerve (Tro), and abducens nerve (Abd). H, Quantification of the ectopic branching events (percentage) analyzed in each branch. I, Western blot detection of α2-chn in embryos injected with 3 ng of Standard MO or chn1 MO. A 65% reduction in the expression levels of α2-chn in 3 dpf zebrafish larvae (n = 3 Western blots, n = 20 injected embryos each). For phenotypic analysis: noninjected, n = 25 embryos; standard MO, n = 17 embryos; chn1 MO, n = 23 embryos. Mann–Whitney was used test for comparison of phenotypes: **p < 0.005, ***p < 0.0005. Scale bars: Ci, Di, Ei, 100 µm; Cii, Ciii, Dii, Diii, Eii, Eiii, 20 µm.