Spatio-temporal expression pattern of tnfaip1 in zebrafish during early development. (A) qPCR detection of the relative expression level of tnfaip1 mRNA during zebrafish embryo development, with 18SrRNA as an internal control. (B) Whole mount in situ hybridization to detect tnfaip1 expression during zebrafish embryo development. n = 15 per group; scale bar, 500 μm. (C) High magnification of tnfaip1 expression in zebrafish 24, 48, and 72 hpf anterior organs. n = 15 per group; scale bar, 250 μm.

Generation of the tnfaip1 mutant in zebrafish using the CRISPR/Cas9 system. (A) Gene structure of tnfaip1 and location of CRISPR/Cas9 targets (red arrow), Target 1 and Target 2 are two knockout target sgRNAs, with the specific target sequence shown in black and the PAM (NGG) sequence in red. (B) The DNA sequencing peak and sequence of the tnfaip1 mutant. DNA sequencing of the corresponding genomic region of zebrafish tnfaip1 revealed a 160 bp deletion mutation. (C) The tnfaip1⁻¹⁶⁰ mutation resulted in a gene frame shift and early termination of the encoded protein. This mutation resulted in a protein truncated to 30 aa. The asterisks mark the termination codon. aa: amino acids. (D) Identification of genotype by DNA electrophoresis. +/+, wild type; +/−, heterozygote; −/−, homozygote. (E) Relative expression of tnfaip1 mRNA at 72 hpf detected by qPCR and 18SrRNA as an internal control. “*” represents p < 0.05, analyzed with unpaired two-tailed t-tests. (F) In situ hybridization to detect expression of tnfaip1 in the mutants. (G) Decreased protein levels in tnfaip1^−/− embryos at 72 hpf as measured by Western blot. Tubulin as an internal control.

Mutation of tnfaip1 in zebrafish affects early embryonic development. (A) Phenotypic analysis of 48–96 hpf wild-type sibling and tnfaip1^−/− embryonic development. Scale bar: 750 μm. (B) Statistical analysis of 48–96 hpf wild-type sibling and tnfaip1^−/− embryo body lengths. n = 30. “**” represents p < 0.01, analyzed with unpaired two-tailed t-tests. (C) Statistical analysis of 48–96 hpf wild-type sibling and tnfaip1^−/− embryo eye sizes, n = 30. “**” represents p < 0.01, analyzed with unpaired two-tailed t-tests.

Tnfaip1 mutation in zebrafish results in reduced expression of neuronal marker genes. (A) In situ hybridization assay for the 72 hpf wild-type sibling and tnfaip1^−/− embryos neuronal marker gene ccnd1. Yellow arrows indicate the retinal; white arrows indicate the tectum proliferative zone; black arrows indicate the hindbrain. Scale bar: 250 μm. (B) In situ hybridization assay for the 72 hpf wild-type sibling and tnfaip1^−/− embryos neuronal marker gene tuba1b. Red arrow indicates the retinal. Scale bar: 250 μm. (C) In situ hybridization assay for the 72 hpf wild-type sibling and tnfaip1^−/− embryos neuronal marker gene neurod1. Red asterisks indicate the brain; yellow arrows indicate the retinal. Scale bar: 250 μm.

Identification of differentially expressed genes between siblings and tnfaip1^−/−. (A) Volcano plot showing the overall differences in upregulated and downregulated genes in the mutants. Some differentially expressed genes are highlighted in the figure. DEGs, differentially expressed genes. (B,C) qPCR validation of differentially expressed genes associated with embryonic development in siblings and tnfaip1^−/−. “*” represents p < 0.05, “**” represents p < 0.01, analyzed with unpaired two-tailed t-tests. (D) Gene Ontology (GO) enrichment analysis of differentially expressed genes from siblings and tnfaip1 mutants. (E) KEGG pathway enrichment analysis of differentially expressed genes in siblings and tnfaip1 mutants.