Generation of dnmt3aa and dnmt3ab gene knockout (KO) zebrafish by using TALEN (transcription activator-like effector nuclease) genome-editing tool. (A) The location of TALEN right arm, spacer, and left arm on targeting zebrafish dnmt3aa and dnmt3ab genes are showed. (B) The Sanger sequencing dendrogram showed the sequence of wildtype, heterozygotes, and homozygotes of dnmt3aa (upper panel) and dnmt3ab (lower panel) mutants. (C) Three-dimension model predictions on the structure of wild type and Dnmt3aa and Dnmt3ab mutated proteins.

Morphometric and DNA methylation level comparison for wild types, dnmt3aa, and dnmt3ab gene knockout zebrafish. (A) Morphologies of six-month-old wild types, dnmt3aa, and dnmt3ab knockout zebrafish. Female fish is shown in the upper panel and male fish in the bottom panel. (B) Morphometric analysis of wild types, dnmt3aa, and dnmt3ab gene knockout zebrafish by principal component analysis (PCA) method. (C) Schematic diagram showing the biochemical pathway on key proteins involving cytosine DNA methylation. Comparison of 5-hmC (D), 5-mC (E), and 5-hmC/5-mC ratio (F) between wild types, dnmt3aa, and dnmt3ab knockout zebrafish. The data are expressed as the mean with S.E.M. and analyzed by one-way ANOVA followed by Fisher’s LSD post hoc test (n = 6 for wild type; n = 4 for dnmt3aa KO fish; n = 5 for dnmt3ab KO fish).

Comparison of behavior endpoints among the wild-type, dnmt3aa, and dnmt3ab mutant fish during a 30-min novel tank exploration test. (A) Average speed, (B) freezing time movement ratio, (C) time in bottom duration, (D) number of entries to the top, (E) latency to enter the top, and (F) total distance traveled in the top fish tank were analyzed. The data are expressed as the median with interquartile range and analyzed by two-way ANOVA (n = 30 for wild type; n = 7 for dnmt3aa KO fish; n = 30 for dnmt3ab KO fish; ** p < 0.01, *** p < 0.005, ****, p < 0.0001). (G–I) The locomotion trajectories of a single fish of wild type, dnmt3aa KO, and dnmt3ab KO fish, right after introduced to a novel environment. (J–L) The locomotion trajectories of a single fish of the wild type, dnmt3aa KO, and dnmt3ab KO fish after 15 min of novel tank exposure.

Comparison of mirror biting behavior endpoints among wild-type, dnmt3aa, and dnmt3ab mutant fish. (A) Mirror biting time percentage, (B) longest duration in the mirror side, and (C) average speed were analyzed. The data are expressed as the median with interquartile range and analyzed by Mann–Whitney test (n = 30 for wild type; n = 10 for dnmt3aa KO fish; n = 30 for dnmt3ab KO fish; * p < 0.05, **** p < 0.0001). (D–F) The locomotion trajectories of a single fish of wild type, dnmt3aa KO, and dnmt3ab KO fish, respectively, during the mirror biting test. The mirrors were put on the right side of the fish tank and the mirror biting zones were highlighted with yellow color.

Comparison of predator avoidance behavioral endpoints among the wild-type, dnmt3aa, and dnmt3ab KO mutant fish. (A) Approaching predator time percentage, (B) average distance to the separator, and (C) average speed were analyzed. The data are expressed as the median with interquartile range and analyzed by Mann–Whitney test (n = 30 for wild type; n = 10 for dnmt3aa KO fish; n = 12 for dnmt3ab KO fish; * p < 0.05, ** p < 0.01, ****, p < 0.0001). (D–F) The locomotion trajectories of a single fish of wild type, dnmt3aa KO, and dnmt3ab KO fish, respectively, during the predator avoidance test. The predator fish were put on the left side of the fish tank and the approaching predator zones were highlighted with yellow color.

Comparison of conspecific behavior endpoints among the wild-type, dnmt3aa, and dnmt3ab mutant fish. (A) Average speed, (B) interaction time percentage, (C) longest duration in separator side, and (D) average distance to separator were analyzed. The data are expressed as the median with interquartile range and analyzed by Mann–Whitney test (n = 30 for wild type; n = 7 for dnmt3aa KO fish; n = 12 for dnmt3ab KO fish; ** p < 0.01). (E–G) The locomotion curve trajectories of a single fish of the wild type, dnmt3aa KO, and the dnmt3ab KO fish, respectively, during the social interaction test. The conspecific interaction zones were highlighted with yellow color.

Comparison of shoaling behavior endpoints among the wild-type, the dnmt3aa, and the dnmt3ab mutant fish. (A) Average inter-fish distance, (B) average shoal area, and (C) average nearest neighbor distance, and (D) average nearest neighbor distance were analyzed. Three fish were used in one group (shoal) for the shoaling test. The data are expressed as the median with interquartile range and analyzed by Mann–Whitney test (n = 30 for wild type; n = 9 for dnmt3aa KO fish; n = 12 for dnmt3ab KO fish; ** p < 0.01, *** p < 0.001, ****, p < 0.0001). (E–G) The locomotion trajectories of three fish of the wild type, the dnmt3aa KO, and the dnmt3ab KO fish, respectively, during the shoaling test.

The circadian rhythm locomotor activity for wild-type, dnmt3aa, and dnmt3ab mutant fish. (A,B) Comparison of the average speed and meandering, respectively, between wild-type, dnmt3aa, and dnmt3ab KO fish during the light and dark cycles. Comparisons of the average speed (C,F), average angular velocity (D,G), and meandering (E,H) in light and dark cycles, respectively, were demonstrated. Data are presented as median with interquartile range and analyzed by Mann–Whitney test (n = 28 for wild type; n = 18 for dnmt3aa KO fish; n = 18 for dnmt3ab KO fish; * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.0001).

Comparison of color preference ranking and index between wild type, dnmt3aa KO, and dnmt3ab KO mutant zebrafish: (A) green vs. blue combination; (B) green vs. yellow combination; (C) red vs. blue combination; (D) green vs. red combination; (E) red vs. yellow combination; and (F) blue vs. yellow combination. Data were analyzed with one-way ANOVA followed by Tukey post-hoc test. The data were presented as mean ± S.E.M. (n = 24, ** p < 0.01, **** p < 0.0001).

Comparison of short-term memory between wild type, dnmt3aa KO, and dnmt3ab KO mutant zebrafish. (A) The latency of fish swimming into the dark chamber after electrical shock was given in three training sessions. (B) The latency of fish swimming into the dark chamber one day after training sessions. Data were analyzed with two-way ANOVA followed by Tukey post hoc test. The data are presented with mean ± S.E.M. (n = 15 for WT and dnmt3ab KO fish; n = 16 for dnmt3aa KO fish, * p < 0.05, ** p < 0.01).

STRING protein interaction networking analysis between the (A) WT and dnmt3aa, and (B) WT and dnmt3ab KO zebrafish gene sets. (A) Red node represented protein involving in neuronal development; blue node represented DNA binding proteins; green node represented nucleic proteins. (B) Red node represented ABC transporter-related proteins; blue node represented proteins that associated metabolic pathways.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.