Figure 4.

KO C2orf69 zebrafish phenocopy the human syndrome with spontaneous fatal seizures

(A) Exon-intron structure of the C2orf69 ortholog in zebrafish. Annotations of the two distinct germline frameshift mutations generated by CRISPR-Cas9 editing at the genome and protein levels.

(B) Developmental expression of C2orf69 during early zebrafish embryogenesis. The transcription of C2orf69 begins at 48 hpf without any detectable maternal contribution.

(C) C2orf69 KO fish are indistinguishable from WT siblings at 4 months.

(D) C2orf69 KO fish show statistically significant reduced body mass and length at 8 months.

(E) Spontaneous seizures in adult 8-month-old KO fish lead to fully penetrant lethality.

(F) Six representative swimming tracks extracted from 2 min videos of 11 dpf KO and WT larvae each show high-speed swimming bouts (red) within 5 min of exposure to 5 mM PTZ.

(G) Quantification of high-speed swim bouts in 2 min (n = 24 each). The p value of the two-sided permutation t test is <0.0001

(H) Quantification of distance swam in 2 min (n = 24 each). The p value of the two-sided permutation t test is 0.0008

(I) The percentage of KO adult fish that show tonic seizures upon exposure to 5 mM PTZ (80%) within 12 min is 2.5-fold higher compared to WT (27%). The p value of the two-sided t test is 0.03.

(J) Molecular markers from 4-month-old whole brain extracts measured by qPCR reveal constitutive CNS inflammation in KO adult fish compared to WT siblings.