psmb6 mRNA expression pattern and generation of psmb6 mutants. (A) ISH for psmb6 at 24 hpf, 36 hpf, 48 hpf, and 3 dpf. (B) Knockout of psmb6 by CRISPR-Cas9 resulted in two different types of psmb6 mutants, with deletions of 4 bp and 42 bp (five bases are located in exon 3, and thirty-seven bases were located in intron 3–4). Sanger sequencing confirmed the 4-nt and 42-nt deletion mutations. Detection of mutant knockout efficiency was performed by Western blotting. (C) psmb6 mutants displaying cerebral hemorrhage (red arrow) and cerebral necrosis (blue arrow). Scale bars: 100 μm. (D) Phenotypic rates of two types of psmb6 mutants at 4.5 dpf. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

The psmb6 mutants have midcranial nerve injury. (A) Timelapse confocal images of Tg(elavl3:EGFP) were taken to observe the neuron development in psmb6 mutants. Scale bars: 50 μm. (B) Timelapse confocal images of kdrl:eGFP; gatal:dsRed were taken to observe the vessel development and vascular integrity in psmb6 mutants. The white arrow indicates cerebral hemorrhage. Scale bars: 50 μm. (C) Statistical analysis of midbrain Tg(elavl3:EGFP) fluorescence intensity at 80 hpf by Image J. ∗∗∗∗P < 0.0001, Student's t-test. (D) Hematoxylin and eosin (H&E) staining of transversal sections for 3 dpf psmb6 mutants. Midbrain ventricle (dashed line). OPL, outer plexiform layer (triangle). Scale bars: 100 μm (left panels). Scale bars: 50 μm (middle and right panels). (E) Transmission Electron Microscopy showed dead cells (red arrow) and mitochondrial swelling (pink arrow) in 3 dpf psmb6 mutants. Lower panels show higher magnification images of the yellow boxed areas in upper panels. Outer segments (OS).Scale bars: 5 μm (upper panels), 1 μm (lower panels). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Increased apoptosis of midbrain cells in psmb6 mutants. (A) The top fifteen pathways with significant differences from RNA sequencing analysis were identified. (B) Verify sequencing results by Q-PCR at 3.5 dpf.∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, Student's t-test. (C) Detect the ubiquitinated protein by WB at 3.5 dpf, internal reference: α-β-tubulin. (D) In situ cell death detection for 3.5 dpf psmb6 mutants (red fluorescence). DAPI (blue). Scale bars: 50 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

p53 deficiency can partially restore the defects of psmb6 mutants before 3.5 dpf. (A) Morphology of psmb6 -4andp53 double mutants at indicated stages. Cerebral hemorrhage (red arrow). Scale bars: 100 μm. (B and C) Fluorescence images and fluorescence intensity quantification of Tg(elavl3:EGFP) transgenic zebrafish at 3.5 dpf. Scale bars: 50 μm ∗∗P < 0.01, Student's t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)