Fig. 8

Functional analysis of NDD genes using F0 mutagenesis. (A–D) WISH method was performed to study mRNA expression patterns for atad3 at 1 dpf (A, A’), 2 dpf (B, B’), and 3 dpf (C, C’, C”), and for afg2b at 1 dpf (D) and 2 dpf (D’). (A, B, C, and D’), lateral view; and (A’, B’, C’, and D), dorsal view. (E –I) Representative images of morphology for uninjected (E and H), atad3 (F), afg2b (G), and cox4i1 (I) at 5 dpf. The blue line indicates head size, the red line indicates eye size, the black line indicates body length, magenta arrowheads indicate heart edema, and blue hashtags indicate missing swim bladder. Lateral view and anterior to the left. (J) Measurements of head and eye size, and body length of atad3 (n = 15), afg2b (n = 20), cox4i1 (n = 10) F0 knockouts. (K) Experimental procedures were conducted for three behavioral assays on F0 zebrafish larvae. Larvae, with or without injection at 4 dpf, were individually transferred to 96-well plates. The plates were then placed in the behavioral recording chamber on the following day (at 5 dpf) for the LDT test. Subsequently, the plates were moved to an incubator and left for an additional day for the VSR and AEBR assays. The animals' responses to each stimulus and their locomotion were tracked and analyzed using customized software. (L) A box and whisker plot was utilized to visualize the percentage of responses in F0 knockouts after VSR and AEBR assays, with a sample size of n = 24 larvae for each group. The percentage of responses to five light stimuli for VSR and the responses to 12 sound stimuli for AEBR were calculated for each larva. (M) Locomotor activities of zebrafish larvae in LDTs at 5 dpf, n = 48 larvae for each group. Larvae were habituated in the dark for 30 min, followed by three cycles of 5-min periods of light and dark. Error bars represent the mean ± SEM. D, dark period, L, light period. Blue arrows indicate the distance moved at the first minute lights on for atad3 F0 larvae, and magenta arrows indicate the increased distance moved during dark period of afg2b F0 larvae. (N) Average cumulative distance traveled by each larva during the first minute of the light period as indicated by blue arrow in (M). (O) Average cumulative distance traveled by each larva during three cycles of either light or dark periods. (P) Representative images of the uninjected control and atad3 F0 in Tg(tuba1a:nls-Kal4FF;UAS:GCaMP7a);nacre under bright-field (left panel) and fluorescent microscopy (middle panel) at 5 dpf. The schematic diagram illustrates the structure of the zebrafish larva brain at the top right panel, while the bottom right panel displays a fluorescent image of uninjected larvae treated with 15 mM pentylenetetrazole (PTZ) at 5 dpf. n = 3 larvae for each group. Dorsal view, anterior to the left. For (J, N, and O), each dot represents one larva, and error bars indicate the mean ± SD. For (J, L, and O), statistical significance was calculated by Brown–Forsthye and Welch's ANOVA with Dunnett's T3 multiple comparisons test; for (N), two-tailed unpaired Student's t test with Welch's correction: not significant (ns) P ≥ 0.05, *P < 0.05, **P < 0.01, and ****P < 0.0001. Di, diencephalon; E, eye; Tel, telencephalon; Ov, otic vesicle; Re, retina; Le, lens; Li, liver; In, intestine; Fb, forebrain; Mb, midbrain; Hb, hindbrain; TeO, optic tectum; Cb, cerebellum.