Structural and molecular characterization of ube3a mutant zebrafish. (a) Schematic representation of the zebrafish ube3a gene structure showing the 11 exons. The point mutation (T > A) is located in exon 3. (b) Genotyping results showing the WT and mutant alleles. The T > A mutation introduces a unique site difference, allowing the differentiation between WT and mutant alleles. (c) Quantitative real-time PCR (RT—qPCR) analysis of ube3a mRNA relative expression (normalized) at 2, 10 days post-fertilization (dpf) and in adult brain tissue (N = 3 for WT and mutant, each). A reduction in ube3a mRNA levels is observed in mutant fish at larval stages. (d) Western blot analysis of the adult brain. The data are presented as the mean ± SEM. ns: non-significant, *P < 0.05.

Embryonic development and growth abnormalities in ube3a mutants. (a) Representative images of zebrafish embryos from 4 h post-fertilization (hpf) to 8 hpf, showing the progression of epiboly in WT and mutant embryos. (b) Quantitative analysis of blastoderm thickness at 5 hpf (N = 11 for WT, N = 9 for mutant), and percentage of epiboly progression at 6 hpf (N = 11 for WT, N = 11 for mutant), 7 hpf (N = 11 for WT, N = 10 for mutant), and 8 hpf (N = 11 for WT, N = 10 for mutant). (c) Hatching rate between WT and mutants each 2 h from 48 hpf to 60 hpf. (d) Representative cardiac/yolk-sac edema in 4 dpf ube3a mutant embryos. Note that the severe cardiac/yolk-sac defects were observed in ube3a mutants, and at low frequency in WT clutches (see Fig. 2e), including AB strain, and were typically non-viable Left: cardiac/yolk-sac edema; right: cardiac/yolk-sac edema and body curvature. (e) Percentage of developmental abnormalities observed between 2–5 dpf. (f) Survival rate of WT and mutant zebrafish. (g) Interorbital distance at 4 dpf in WT (N = 31) and mutants (N = 23), representative images (left) and quantification (right). (h) Distance between the optic tecta, in WT (N = 7) and mutants (N = 11), representative images (left) and quantification (right). (i) Body length at 4 dpf, in WT (N = 24) and mutants (N = 22), representative images (left) and quantification (right). The data are presented as the mean ± SEM. ns: non-significant, *P < 0.05, **P < 0.01 and ***P < 0.001.

Locomotor and stereotypic behavioral analysis in WT and ube3a mutants. (a) Representative locomotor traces for WT and mutant fish in the open-field test. (b) Average distance traveled by WT (N = 18) and mutants (N = 18), representative traces (left) and quantification (right). (c) Stereotypic movements in adult WT (N = 13) and mutants (N = 12) with schematic representation and quantification: back-and-forth swimming (top left), figure eight-like patterns (top right), small circling (bottom left), and walling behavior (bottom right). The data are presented as the mean ± SEM. ns: non-significant, **P < 0.01.

Social interaction and aggressiveness in WT and ube3a mutant zebrafish. (a) Schematic representation of the social interaction test. (b) Representative trajectory diagrams of WT (left) and mutant (right) zebrafish during the social interaction test. (c) Percentage of time spent near conspecifics for WT (N = 12) and mutants (N = 12) ube3a. (d) Schematic representation of the kin preference test. (e) Representative trajectory diagrams of WT (left) and mutant (right) zebrafish during the kin preference test. (f) Percentage of time spent near non-kin individuals (left), and near kin individuals (right) for WT (N = 12) and mutants (N = 11) ube3a. (g) Schematic diagram of the shoaling test. (h) Mean distance to neighbors (left) and proportion of time spent with at least one neighbor (right) between WT (N = 12) and mutants (N = 12). (i) Representative trajectory diagrams of WT (top) and mutant (bottom) zebrafish in the mirror attack test (aggression test). (j) Number of attacks directed at the mirror (left), average speed (middle), and distance traveled during the mirror test (right) for WT (N = 12) and mutants (N = 11) ube3a. The data are presented as the mean ± SEM. ns: non-significant, **P < 0.01, ***P < 0.001 and ***P < 0.0001. Schematic representations were created with BioRender.com.

Differential downregulation of gene expression in ube3a mutant zebrafish larvae revealed by RNA-seq. (a) Cnet plot of significantly downregulated biological processes in ube3a larvae mutants (top 10 GO terms). (b) Cnet plot of significantly downregulated cellular components in ube3a larvae mutants (top 10 GO terms). (c) Cnet plot of significantly downregulated molecular functions in ube3a larvae mutants (top 10 GO terms). (d) Top 10 GO terms for the three ontologies in ube3a larvae mutants with their enrichment score.

Differential upregulation of gene expression in ube3a mutant zebrafish adults revealed by RNA-seq. (a) Cnet plot of significantly upregulated biological processes in ube3a adult mutant brains (top 10 GO terms). (b) Cnet plot of significantly upregulated cellular components in ube3a adult mutant brains (top 10 GO terms). (c) Cnet plot of significantly upregulated molecular functions in ube3a adult mutant brains (top 10 GO terms). (d) Top 10 GO terms for the three ontologies in ube3a adult mutant brains with their enrichment score.

Transcriptional regulation in WT and ube3a mutant zebrafish RNA-seq. Differential expression of zebrafish E3 ubiquitin ligases as presented in Supplemental Table 1. The data represent transcripts per million (TPM) from our RNA-seq data of ube3a WT and mutant adult brains (N = 3). The data are presented as truncated violin plots with individual values. ns: non-significant, *P < 0.05.