dhcr7−/− zebrafish show retarded growth, microcephaly, and changes in brain sterols (A) Schematic diagram for generating dhcr7−/− zebrafish using CRISPR/Cas9. (B) Relative mRNA expression of dhcr7 at 9 days post-fertilization in dhcr7+/+ and dhcr7−/− zebrafish larvae (n = 3, 6 larvae/sample). (C) Representative images and time course of body length in dhcr7+/+ (n = 12) and dhcr7−/− (n = 13) zebrafish. (D) Representative images of 1-month post-fertilization (mpf) dhcr7+/+ and dhcr7−/− zebrafish brains. (E) Measurement of the brain area in 1-mpf dhcr7+/+ (n = 5) and dhcr7−/− (n = 6) zebrafish. (F) Images of 3-mpf dhcr7+/+ and dhcr7−/− zebrafish brains. (G) Measurement of the brain area in 3-mpf dhcr7+/+ (n = 8) and dhcr7−/− (n = 5) zebrafish. The brain area adjusted for body length was calculated to assess microcephaly. (H) Heatmap images of filipin staining in 1-mpf dhcr7+/+ and dhcr7−/− zebrafish brains (n = 5 each). (I) Brain cholesterol in 1-mpf and 3-mpf dhcr7+/+ and dhcr7−/− zebrafish (n = 5 each). (J) Brain 7-DHC in 1-mpf (n = 5 each) and 3-mpf (n = 6 each) dhcr7+/+ and dhcr7−/− zebrafish. All values are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n.s: not significant. Microcephaly is the primary phenotype observed in patients with SLOS. No significant difference was found in the brain size of dhcr7−/− zebrafish at 1-mpf (Fig. 1D and E). However, differences in brain size in dhcr7−/− zebrafish became more pronounced as they grew. Their brains were significantly smaller than wild-type brains at 3-mpf (Fig. 1F and G). The levels of cholesterol and 7-DHC were assessed, and whole-mount filipin staining was performed on juvenile brains at 1-mpf. Cholesterol levels decreased significantly in dhcr7−/− zebrafish (Fig. 1H). We confirmed the decreased cholesterol in dhcr7−/− zebrafish brains using gas chromatography–mass spectrometry (GC–MS) in 1-mpf juvenile and 3-mpf adult zebrafish (Fig. 1I). In contrast, 7-DHC levels measured using liquid chromatography–mass spectrometry (LC–MS) were increased in dhcr7−/− zebrafish brains (Fig. 1J). These data are consistent with patients with SLOS, suggesting that dhcr7−/− zebrafish mimic the biochemical phenotype of human patients with SLOS. 3.2. Abnormalities in gfap+ neural stem cells in dhcr7−/− zebrafish We labeled mature neurons by mating dhcr7−/− zebrafish with huc promoter-driven Kaede fluorescent protein transgenic zebrafish [18]. The dorsal-view fluorescence intensity of mature neurons was significantly decreased in the telencephalon, optic tectum, and cerebellum of dhcr7−/− juvenile at 1-mpf, suggesting a decreased neuron density (Fig. 2A).

Decreased neurons and neural stem cells in dhcr7−/− zebrafish (A) Representative images of 1-mpf dhcr7+/+ (n = 6) and dhcr7−/− (n = 5) zebrafish brains in the Tg[huc:Kaede] background. (B) Images of 1-mpf dhcr7+/+ (n = 11) and dhcr7−/− (n = 9) zebrafish brain in the Tg[gfap:Tomato] background. (C) Images of transverse sections of 2-mpf dhcr7+/+ and dhcr7−/− zebrafish brains in the Tg[gfap:Tomato] background (red), stained with DAPI (blue). All values are presented as the mean ± SEM. *p < 0.05, **p < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Impaired myelination and autophagy in dhcr7−/− zebrafish (A) Representative Klüver–Barrera staining of transverse sections of 3-mpf dhcr7+/+ and dhcr7−/− zebrafish telencephalon and optic tectum. Arrows indicate myelin stained in the dhcr7+/+ telencephalon. (B), (C) Representative transmission electron micrographs of the telencephalon (B) and optic tectum (C) from 2-mpf dhcr7+/+ and dhcr7−/− zebrafish. Arrows indicate axonal lysosome-like structures; arrowheads indicate mitochondria with indistinct cristae, which appeared expanded. (D) Lysosome staining by Lysoprime Green in 2-mpf dhcr7+/+ and dhcr7−/− zebrafish telencephalon (n = 5 each). (E) Western blot analysis of p62 and Lc3b protein levels in 2-mpf dhcr7+/+ and dhcr7−/− zebrafish brains (n = 5 each) relative to GAPDH. All values are presented as the mean ± SEM. *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Synaptic defects and behavioral abnormalities in dhcr7−/− zebrafish (A) Transmission electron micrographs of the telencephalon from 2-mpf dhcr7+/+ and dhcr7−/−zebrafish. (B) Monoamine levels in the brains from 2-mpf dhcr7+/+ and dhcr7−/− zebrafish (n = 4 each). (C) Schematic illustration of the novel tank diving test. (D) Representative swimming traces of 2-mpf dhcr7+/+ and dhcr7−/− zebrafish for the first 2 min of the novel tank diving test. (E) Total swimming distance of 2-mpf dhcr7+/+ (n = 13) and dhcr7−/− (n = 14) zebrafish in 6 min. (F) The number of entries per min to the top half of the tank for 2-mpf dhcr7+/+ (n = 13) and dhcr7−/− (n = 14) zebrafish. (G) Swimming distance per min in the upper half of the tank for 2-mpf dhcr7+/+ (n = 13) and dhcr7−/− (n = 14) zebrafish. (H) Latency entering the tank's top half for 2-mpf dhcr7+/+ (n = 13) and dhcr7−/− (n = 14) zebrafish. (I) Schematic illustration of the mirror biting test. (J) Representative swimming traces of 2-mpf dhcr7+/+ and dhcr7−/− zebrafish for a 5 min mirror-biting test.(K) Time spent per min in the mirror area for 2-mpf dhcr7+/+ (n = 10) and dhcr7−/− (n = 9) zebrafish. All values are presented as the mean ± SEM. *p < 0.05, **p < 0.01. n.s: not significant.