Generation of znf536 KO zebrafish and growth phenotype analysis.

A Generation of the znf536 KO line. Red asterisk (upper panel) and yellow box (lower panel) indicate the sgRNA target region. B Schematic representation of predicted proteins in the WT and KO. Blue boxes indicate the zinc finger C2H2 domains. C Genotype ratio for larval stage zebrafish from heterozygous incrosses (6–20 dpf) (n = 1433). D Representative images of znf536 WT and KO zebrafish at 3 and 6 mpf adult stages. n = 14 for WT, n = 44 for Het, and n = 9 for KO (3 mpf); n = 9 for KO (6 mpf). Scale bar: 10 mm.

Food competition test.

A Example image of rotifers in the intestine of zebrafish at 14 dpf. The arrowhead or dashed line indicates rotifers in the gastrointestinal tract. Scale bar: 200 µm. B Categorization of rotifer consumption by genotype. n = 16 for WT, n = 36 for Het, and n = 9 for KO.

Decreased anxiety-like behavior in znf536 KO zebrafish.

A Representative heatmaps from the novel tank assay. The red color indicates increased dwell time and blue indicates decreased. B Quantified data for total time spent in the bottom zone. C Average speed of locomotion. n = 15 for WT, n = 16 for Het, and n = 13 for KO. Data was presented as mean ± standard error of the mean (S.E.M.). Statistical significance was determined by the Kruskal–Wallis tests with post-hoc Dunn’s multiple comparisons tests. ns, no significance; p*** < 0.001.

Impairments of social interaction in znf536 KO zebrafish.

A Representative heatmap of the social interaction assay. The red color indicates increased dwell time and blue indicates decreased. Solid lines indicate a transparent divider and dashed lines indicate the very close zone (2 cm from the social cue region). B Quantified data for duration within the very close zone. n = 11 for WT, n = 19 for Het, and n = 23 for KO. Data was presented as mean ± standard error of the mean (S.E.M.). Statistical significance was determined by Kruskal–Wallis test with post-hoc Dunn’s multiple comparisons test. ns, no significance; p**** < 0.0001. C Representative image from the shoaling assay. Individual adult fish were pointed by arrows. D Quantified data for the average of the inter-fish distance of the shoaling assay. The test was performed 3 times. For each trial, 3 fish of each genotype were used.

Anatomical and histological analysis of adult brains.

A, B Representative images of znf536 KO adult zebrafish at 11 mpf and their isolated brains. Arrows indicate the reduced size of the cerebellum in KO zebrafish. n = 7 for WT and n = 6 for KO. Scale bar: 10 mm (left panels) and 500 µm (right panels). C, D Brain section images at positions indicated by dashed lines in A, B. Serial sections were carefully examined to compare relative planes. C”’, D”’ Magnification of marked areas in C’, D’ (dashed box). ImageJ analysis C’, D’ showed that the relative size of valvular cerebelli of znf536 KO zebrafish (average 7.8% of the whole brain, n = 4) was reduced, compared to that of WT zebrafish (average 9.6%, n = 3). CCe corpus cerebelli, NLV nucleus lateralis valvulae, PGZ periventricular gray zone of optic tectum, TeV tectal ventricle, TL torus longitudinalis, Val lateral division of valvular cerebelli, Vam medial division of valvular cerebelli. Scale bar: 500 µm.

Defects in cerebellar structure and function in znf536 KO zebrafish.

A, B”’ Reduced neuronal activity in KO zebrafish monitored by c-fos expression after novel tank assay, especially in the cerebellum (arrowheads). n = 3 for WT and n = 3 for KO. C Expression of Purkinje cell markers (aldoca, pvalb7, ca8) were specifically affected in the Va region. n = 3 for WT and n = 3 for KO for each marker. D Confocal images of myelinated bundles in Tg[mbp:mgfp]::znf536 KO brain, focused in the Va region. A unique circular structure (asterisk) was identified within the Va. n = 2 for WT and n = 2 for KO. CCe corpus cerebelli, Dm medial zone of the dorsal telencephalic area, TeO optic tectum, Va valvular cerebelli. Scale bars: 500 µm.