Figure 5.

Figure 5.

G0 mosaic and F2 germline zebrafish targeting of olig2. (A–H) Initial olig2 targeting experiments were performed in G0 embryos. Representative images obtained by stereomicroscope (A–D) and confocal microscopy (E–H) for Uninjected (A, E), Scrambled guide injected (B, F), or olig2-targeting Guide-injected (C–D, G–H) G0 HGj4A zebrafish at 72 hpf. Two images are provided for guide-injected fish, to demonstrate variability in G0 targeting outcomes. Stereomicroscope images for each treatment group correspond to the confocal images obtained from the same fish in the panel below. Motor neurons in the abducens nucleus (dashed white circled region) appear absent by stereomicroscopy (C, D) or variably reduced (G, H) by confocal microscopy, and abducens nerves appear thin (G;blue arrow) or absent (G, H; red arrows) compared to normal (E, F; white arrows) by confocal microscopy. (I) Barplots showing the percentages of zebrafish exhibiting wild-type-like (WT-like), malformed, or absent CN6 motor neuron nuclei, as scored under the dissecting stereomicroscope in G0 zebrafish at 72 hpf. Total numbers of fish in each group are given above the corresponding bar. Data are shown from three experimental replicates. Pearson's 3 × 3 χ² test with 4 degrees of freedom; χ² = 114.34, 95.77, 102.24 for each of three experimental replicates; P < 2.2e⁻¹⁶. (J) Kaplan-Meier survival curves demonstrating the relative survival probabilities of scramble-injected (blue line) and guide-injected (pink line) zebrafish over the first 72 hours of life. Number at risk below the plot provides counts of surviving embryos in each group taken every 24 hpf over a 72 hpf period. Relative survival probabilities of olig2-targeting and scrambled gRNA-injected embryos were compared by the log-rank test. Displayed data were derived from a single experimental replicate, but measurements were taken for three experimental replicates, all of which showed the same trend. (K, L) Representative images from wild-type or F2 germline olig2^−/− mutants (c.496_499delAGTT, p.(Ser166ValfsTer93)); key as noted for A–H).