Generation of serpini1^−/− zebrafish via CRISPR-CAS9 technique. (A): Schematic representation of CRISPR/Cas9 target sites in zebrafish gene serpini1. The 19 bp target site is located on the exon4 of the serpini1 gene. (B): DNA sequencing results of the wild-type (WT) and homozygote serpini1^−/−. Serpini1^−/− mutation leads to five-nucleotide deletion in the fourth exon. (C): Protein comparison for WT and serpini1^−/− mutation. The serpini1^−/− caused a frameshift mutation, truncated from 415 aa into a 271 aa protein. (D). mRNA level of serpini1 in serpini1^−/− compared to WT. The data are presented as the mean ± SEM, N = 3 trails. Statistical analyses were performed with Student’s t-test, ****p < 0.0001 compared to WT.

The developmental morphology defects of WT and serpini1^−/− zebrafish under CoCl₂ induced hypoxia damage. (A) Survival rates of WT under the different concentration of CoCl₂: 0, 1, 10, 20, 50 mM from 12 hpf to 96 hpf. The data are presented as the mean ± SEM, n = 200 per group. Statistical analyses were performed with one-way ANOVA, followed by Dunnett’s multiple comparisons, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to WT at the same timepoint. Dash line indicates of 50% survival rate. (B) Typical morphological defects under the treatment of 10 mM CoCl₂ in WT and serpini1^−/− zebrafish at 4dpf; short arrow indicates pericardial edema; long arrow shows spine deformation and asterisk shows small eye deformation. (C) Hatch rate at 48 hpf under the treatment of 10 mM CoCl₂ between WT and serpini1^−/− group. The data are presented as the mean ± SEM, n = 200 per group; three trails for the experiment. Statistical analyses were performed with Student’s t-test, **p < 0.01 compared to WT. (D) Survival rate at 96 hpf under the treatment of 10 mM CoCl₂ between WT and serpini1^−/− group. The data are presented as the mean ± SEM, n = 200 per group; three trails for the experiment. Statistical analyses were performed with Student’s t-test, **p < 0.01 compared to WT. (E–G) Percentage of pericardial edema, spine deformation, and small eye and brain deformation in WT and serpini1^−/− group under the treatment of 10 mM CoCl₂. The data are presented as the mean ± SEM, n = 200 per group; three trails for the experiment. Statistical analyses were performed with Student’s t-test, **p < 0.01, ***p < 0.001.

Neuroserpin deficient zebrafish showed reduced locomotor activities and more neurons loss in diencephalon area under CoCl₂ induced hypoxic injury. (A) Representative graph for distance traveled in 40 min for WT and serpini1^−/− zebrafish. (B) Quantification of average distance moved per minute in WT and serpini1^−/− under 10 mM CoCl₂ induced injury. The data are presented as the mean ± SEM, n = 96 per group. Statistical analyses were performed with Student’s t-test, ***p < 0.001, ns: no significant, compared with WT in the same CoCl₂ concentration. (C) Images of neurons in diencephalon in Tg (Huc: RFP) and Tg (serpini1^−/−-HuC: RFP) under 10 mM CoCl₂ induced injury. Scale bar:100 um. (D) Quantification of average fluorescence intensity of neurons in diencephalon area in Tg (Huc: RFP) and Tg (serpini1^−/−-HuC: RFP) under 10 mM CoCl₂ induced injury. The data are presented as the mean ± SEM, n = 6 per group. Statistical analyses were performed with Student’s t-test, **p < 0.01, ns: no significant, compared with WT in the same CoCl₂ concentration.

More severe vascular malformation in neuroserpin deficient zebrafish under CoCl₂ induced hypoxic injury. (A) Image of vessels of Tg (Fli1: EGFP) and Tg (serpini1^−/−-Fli1: EGFP) with or without 10 mM CoCl₂ induced injury. Tg (Fli1: EGFP) and Tg (serpini1^−/−-Fli1: EGFP) show normal vascular morphology in brain region including central artery (CtA), primordial hindbrain channel (PHBC), and primary head sinus (PHS). CoCl₂ caused vascular malformation both in Tg (Fli1: EGFP) and Tg (serpini1^−/−-Fli1: EGFP) under 10 mM CoCl₂-induced injury. More reduced vascular branches in brain area were observed in Tg (serpini1^−/−-Fli1: EGFP) group compared to Tg (Fli1: EGFP). Scale bar:100 um. White arrow indicates brain vessels. Dash white square box shows reduction of normal vessels in CoCl₂ treated groups. (B) Quantification of average fluorescence intensity of vessels in brain area in Tg (Fli1: EGFP) and Tg (serpini1^−/−-Fli1: EGFP) under 10 mM CoCl₂ induced injury. The data are presented as the mean ± SEM, n = 6 per group. Statistical analyses were performed with Student’s t-test, ***p < 0.001, ns: no significant, compared with WT in the same CoCl₂ concentration.

Apoptosis and oxidative stress were more severely enhanced in serpini1^−/− zebrafish group under CoCl₂ induced hypoxic injury. (A) AO staining of the apoptotic cells in the live image of WT and serpini1^−/− zebrafish under the treatment of 10 mM CoCl₂ at 4dpf. Scale bar:500 um. (B) MDA content in WT and serpini1^−/− under 10 mM CoCl₂ induced injury. The data are presented as the mean ± SEM, n = 100 per group. Three trails for the experiment. Statistical analyses were performed with Student’s t-test, ***p < 0.001 compared with WT, ns: no significant. (C) The level of GSH-Px in WT and serpini1^−/− under 10 mM CoCl₂ induced injury. The data are presented as the mean ± SEM, n = 100 per group. Three trails for the experiment. Statistical analyses were performed with Student’s t-test, **p < 0.01 compared with WT, ns: no significant. (D) The level of CAT in WT and serpini1^−/− under 10 mM CoCl₂ induced injury. The data are presented as the mean ± SEM, n = 100 per group. Three trails for the experiment. Statistical analyses were performed with Student’s t-test, ***p < 0.001 compared with WT, ns: no significant. (E) The level of SOD in WT and serpini1^−/− under 10 mM CoCl₂ induced injury. The data are presented as the mean ± SEM, n = 100 per group. Three trails for the experiment. Statistical analyses were performed with Student’s t-test, ***p < 0.001 compared with WT, ns: no significant.