Temporal pattern of mRNA expression of zebrafish brsk2a and brsk2b at 8 stages. mRNA was extracted from whole embryos at 1 dpf, 2 dpf, 3 dpf, and brain tissues at 5 dpf, 7 dpf, 10 dpf, 1 mpf and 3 mpf. Data are shown as mean ± SEM, n = 3 for each genotype.

Generation of brsk2ab mutant zebrafish. (A) structure modelling of zebrafish brsk2a and brsk2b protein. PK represents protein kinase domain; UBA represents ubiquitin-associated domain. (B) Two CRISPR/Cas9 guide RNAs were designed to target exon 10 in brsk2a and exon 4 in brsk2b, respectively. The mutations of brsk2a and brsk2b were verified by Sanger sequencing, and computational modelling showed the proteins were truncated through SWISS-MODEL tool (https://swissmodel.expasy.org/interactive, accessed on 12 July 2023). (C) To validate the knockout efficiency, total mRNA was extracted at 3 mpf followed by reverse transcription, and qRT-PCR was conducted to validate the expression level of brsk2a, and brsk2b was decreased in brsk2ab^−/− zebrafish (n = 3 for each genotype, brsk2a, p = 0.0007; brsk2b, p = 0.0004). Data are shown as mean ± SEM, *** p < 0.001.

brsk2ab^−/− zebrafish showed a catch-up growth pattern. (A) Representative pictures of measured zebrafish. (B) The body lengths were measured at 2 dpf (WT: brsk2ab^−/−, n = 120: 131), 3 dpf (n = 105: 105), 5 dpf (n = 100: 89), 2.5 mpf (n = 19: 21), and 4 mpf (n = 14: 16). The brsk2ab^−/− larvae have shorter bodies than WT larvae at 2 dpf (WT vs. brsk2ab^−/−, 3.760 ± 0.09 vs. 3.623 ± 0.09; p < 0.0001), 3 dpf (4.329 ± 0.14 vs. 4.170 ± 0.15; p < 0.0001), and 5 dpf (5.071 ± 0.21 vs. 4.819 ± 0.22; p < 0.0001), but there was no difference in body length at 2.5 mpf (21.19 ± 1.58 vs. 20.90 ± 2.31; p = 0.646) and 4 mpf (27.57 ± 1.66 vs. 28.04 ± 1.53; p = 0.425). Data are shown as mean ± SEM, **** p < 0.0001.

The locomotor activity of brsk2ab^−/− zebrafish in different stages. (A) The trajectory of locomotion in WT and brsk2ab^−/− zebrafish in different time points. (B) The brsk2ab^−/− zebrafish presented significantly reduced swimming velocity at 5 dpf (WT vs. brsk2ab^−/−, 3.342 ± 1.63 vs. 2.229 ± 1.18; p = 0.0002). While no significant difference was observed at 14 dpf (4.046 ± 1.50 vs. 3.826 ± 1.18; p = 0.2753) and later stages (2 mpf: 5.589 ± 1.47 vs. 6.234 ± 1.75; p = 0.1087; 3 mpf: 6.309 ± 1.31 vs. 5.998 ± 1.37; p = 0.2745). At 5 dpf: n = 48: 48; at 14 dpf: n = 89: 91; at 2 mpf: n = 26: 20; and at 3 mpf: n = 25: 25. Data are shown as mean ± SEM, *** p < 0.0003.

brsk2ab^−/− zebrafish displayed gradually severe social defects as they grew up. The experimental paradigm of social preference tests at juvenile stage (A) and adult stage (B). (C) The experimental paradigm of shoaling tests at adult stage. (D) The brsk2ab^−/− zebrafish showed specious defects in social preference at 1 mpf and the SPI change was smaller than that of WT zebrafish (WT vs. brsk2ab^−/−, n = 15: 19; distance: 0.1116 ± 0.20 vs. −0.08843 ± 0.29, p = 0.0298; time: 0.1228 ± 0.16 vs. −0.0827 ± 0.32, p = 0.0327). (E) The discrepancy of SPI change was obvious at 1.5 mpf (WT vs. brsk2ab^−/−, n = 21: 23; distance: 0.5166 ± 0.46 vs. 0.03154 ± 0.40, p = 0.0005; time: 0.0160 ± 0.48 vs. −0.0307 ± 0.47, p = 0.7444). (F,H) The experimental paradigm of social preference tests in adulthood. The SPI of adult brsk2ab^−/− zebrafish was significantly decreased compared to WT fish both in distance and time ratios at 3 mpf (WT vs. brsk2ab^−/−, n = 18: 15, distance: 0.9858 ± 0.04 vs. 0.4247 ± 0.56, p = 0.0022; time: 0.9846 ± 0.05 vs. 0.3328 ± 0.66, 0.0029) and 4 mpf (WT vs. brsk2ab^−/−, n = 19: 19, distance: 0.9589 ± 0.09 vs. 0.7523 ± 0.43, p = 0.0072; time: 0.9486 ± 0.13 vs. 0.7361 ± 0.43, p = 0.0044). (G,I) The shoaling behavior tests exhibited the average inter-individual distance of brsk2ab^−/− zebrafish was smaller than that of WT fish at 3 mpf (WT vs. brsk2ab^−/−, n = 9: 10, 6.935 ± 0.99 vs. 14.07 ± 2.58, p < 0.0001) and 4 mpf (WT vs. brsk2ab^−/−, n = 12: 14, 6.785 ± 4.51 vs. 16.75 ± 3.56, p < 0.0001). Data for each genotype are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001,**** p < 0.0001.

brsk2ab^−/− zebrafish displayed escalating anxiety-like behaviors. (A) The analytical paradigm showed that the open field tank was divided into two equal zones, and the thigmotaxis was calculated using the time and distance ratio that fish spent in the peripheral zone. (B) There was no significant difference in the thigmotaxis index between WT and brska2b^−/− zebrafish at 2 mpf (WT vs. brsk2ab^−/−, n = 20: 27, distance: 0.7737 ± 0.14 vs. 0.8376 ± 0.16, p = 0.1622; time: 0.8245 ± 0.13 vs. 0.8712 ± 0.15, p = 0.2225). (C) The thigmotaxis of brsk2ab^−/− zebrafish was observed to have increased more than that of WT zebrafish at 3 mpf (WT vs. brsk2ab^−/−, n = 27: 22, distance: 0.8522 ± 0.05 vs. 0.9274 ± 0.05, p < 0.0001; time: 0.8934 ± 0.05 vs. 0.9389 ± 0.06, p = 0.0038). Data are shown as mean ± SEM s, ** p < 0.01, **** p < 0.0001.