Failure of chondrocyte convergent extension in psmb1 mutants.

(A) Alcian blue staining of 4 dpf psmb1 larvae. Mc, Meckel’s cartilage; ch, ceratohyal; pq, palatoquadrate. The asterisk indicates ceratohyal angle. Scale bars: 100 μm. (B) Imaging of sox10:kaede; psmb1^–/– larvae at 72 hpf demonstrates defects in first and second arch cartilage derivatives as well as semicircular canal formation (white arrowhead). Scale bars: 50 μm. (C) Col2a1 (magenta) and Sox9a (green) antibody staining of psmb1 larvae at 72 hpf (top, scale bars: 50 μm) and wheat germ agglutinin stain at 96 hpf (bottom, scale bars: 30 μm). (D) Quantification of chondrocyte stacking in wheat germ agglutinin stain images via analysis of the angle formed by drawing a line through 3 adjacent chondrocytes. n = 7 (+/+), 12 (+/–), 7 (–/–). (E) Quantification of chondrocyte stacking in wheat germ agglutinin stain images by analysis of length/width ratio. n = 7 (+/+), 12 (+/–), 7 (–/–). (F) Antibody stain for Sox9a at 60 hpf and 72 hpf. Scale bars: 50 μm. (G) Quantification of the number of chondrocytes present in the ceratohyal, palatoquadrate, and Meckel’s cartilages at 60 hpf in images in F. n = 7 (+/+), 13 (+/–), 7 (–/–). (H) Quantification of the number of chondrocytes present in the ceratohyal, palatoquadrate, and Meckel’s cartilages at 72 hpf in images in F. n = 7 (+/+), 11 (+/–), 5 (–/–). Data shown represent mean ± SD. Significance was calculated with 1-way ANOVA with Dunnett’s multiple comparisons test, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Chondrocyte cell death and proliferation in psmb1 mutants.

(A) Time-course study of apoptag TUNEL staining (green) and Sox9a staining (magenta) from 55–72 hpf. (B–E) Quantification of A demonstrates that cell death is not elevated in mutant chondrocytes until 72 hpf, at which point sox9a⁺TUNEL⁺ cells can be found (white arrowhead). 55 hpf: n = 10 (+/+), 16 (+/–), 6 (–/–). 60 hpf: n = 10 (+/+), 21 (+/–), 8 (–/–). 65 hpf: n = 6 (+/+), 6 (+/–), 6 (–/–). 72 hpf: n = 8 (+/+), 10 (+/–), 10 (–/–). (F) pH3 (magenta) antibody staining in sox10:kaede larvae (green) at 57 hpf, 65 hpf, and 72 hpf. (G–I) Quantification of C shows no proliferation of craniofacial chondrocytes across genotypes. 57 hpf: n = 6 (+/+), 10 (+/–), 6 (–/–). 65 hpf: n = 10 (+/+), 14 (+/–), 7 (–/–). 72 hpf: n = 7 (+/+), 11 (+/–), 9 (–/–). Scale bars: 50 μm. Data shown represent mean ± SD. Significance was calculated with 1-way ANOVA with Dunnett’s multiple comparisons test. *P < 0.05; ns, not significant.

psmb1 is dispensable for pharyngeal arch morphogenesis and patterning.

(A) Imaging of sox10:kaede embryos at 24 hpf (top, scale bars: 50 μm) did not reveal any defects with neural crest cell migration to the arches. RNAscope for hand2, dlx2a, and jag1b demonstrates normal dorsal-ventral arch patterning in psmb1 mutants at 32 hpf (bottom, scale bars: 30 μm). (B) Imaging of gutGFP embryos at 24 hpf and myod1 RNAscope at 48 hpf demonstrates that the pharyngeal arch endoderm and muscle are formed normally in psmb1 mutants. Scale bars: 50 μm. (C) pH3 antibody staining (magenta) in fli1a:EGFP embryos at 24 hpf and 48 hpf to assess neural crest cell proliferation in psmb1 mutants. Scale bars: 30 μm (D) TUNEL staining (magenta) in fli1a:EGFP embryos at 24 hpf and 48 hpf to assess neural crest cell death in psmb1 mutants. Scale bars: 20 μm. (E and F) Quantification of proliferation in pharyngeal arches 1 and 2 in images in C; normalization is to arch 1 and 2 fli1a⁺ area. (E): n = 6 (+/+), 13 (+/–), 5 (–/–). (F): n = 7 (+/+), 8 (+/–), 13 (–/–). (G and H) Quantification of cell death in pharyngeal arches 1 and 2 in images in D; normalization is to fli1a⁺ arch 1 and 2 area. (G): n = 9 (+/+), 18 (+/–), 5 (–/–). (H): n = 8 (+/+), 17 (+/–), 8 (–/–). Data shown represent mean ± SD. Significance was calculated with 1-way ANOVA with Dunnett’s multiple comparisons test, ns: not significant.

Proteasome subunit genes are upregulated in a tissue-specific fashion in psmb1 mutants.

(A) Schematic of RNA-Seq approach for cranial sox10⁺ cells from wild-type/heterozygous versus mutant fish at 72 hpf. (B) Heatmap of normalized counts of differentially expressed genes in cranial sox10⁺ cells at 72 hpf. Wald’s test with false discovery rate correction (P adjusted < 0.05). Each sample represents cells from 25 different larvae. (C) GO enrichment analysis using ClusterProfiler identifies GO components enriched in the differentially expressed gene set. (D) Heatmap of proteasome subunit gene expression in RNA-Seq data set from sorted cranial sox10⁺ cells (E) Top: qRT-PCR for proteasome subunit genes on cDNA from dissected trunks/tails at 72 hpf. Bottom: qRT-PCR for proteasome subunit genes on cDNA from dissected heads at 72 hpf. WH, pooled wild-type/heterozygous larvae; M, mutant larvae. Data shown represent mean ± SD. Significance was calculated with unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Craniofacial muscle and tendon defects in psmb1 mutants.

(A) RNAscope to assess developmental timing and distribution of psmb1 expression. Top: psmb1 (green) expression at 24, 48, and 72 hpf. Scale bars: 50 μm, 100 μm, and 100 μm, respectively. Middle: psmb1 expression in developing pharyngeal arches and cartilage. Left and middle: psmb1 (green), fli1a (magenta), right: psmb1 (green), col2a1a (magenta). Scale bars: 50 μm. Bottom: psmb1 expression in craniofacial tissues at 72 hpf. Left: psmb1 (green), col2a1a (magenta). Middle: psmb1 (green), myod1 (magenta). Right: psmb1 (green), scxa (magenta), xirp2a (cyan). Scale bars: 30 μm. (B) Antibody stain for myosin heavy chain (MHC) at 72 hpf showing defects in craniofacial muscles in psmb1 mutants. Scale bars: 50 μm. hh, hyohyal; ih,interhyal. (C) RNAscope for myod1 at 55–72 hpf. Scale bars: 30 μm. (D) RNAscope for tnmd (green), scxa (magenta), and xirp2a (cyan) at 57–72 hpf demonstrating defects in tendon development in psmb1 mutants. Scale bars: 30 μm.

Overexpression of psmb1 in the neural crest and chondrocytes rescues mutant cartilage and tendon phenotype.

(A) Bright-field images of psmb1 wild-type, heterozygous, and mutant larvae with and without sox10:psmb1-2A-GFP transgene at 120 hpf. Scale bars: 100 μm. (B) Bright-field images of psmb1 wild-type, heterozygous, and mutant larvae with and without sox10:psmb1-2A-GFP transgene at 7 dpf. Scale bars: 100 μm. (C) Confocal imaging of sox10:mRFP, psmb1 wild-type, heterozygous, and mutant larvae with and without sox10:psmb1-2A-GFP transgene at 72 hpf. Scale bars: 50 μm. (D) Quantification of ceratohyal angle in images from C demonstrates that ceratohyal angle is almost completely restored in rescued psmb1 mutants. Data shown represent mean ± SD. n = 6, 8, 7, 22, 4, 10. Significance was calculated with 2-way ANOVA. *P < 0.05, **P < 0.01, ****P < 0.0001; ns, not significant. (E) sox10:psmb1-2A-GFP transgene substantially improves tendon phenotype, as assessed by RNAscope for tnmd at 72 hpf. Scale bars: 50 μm. (F) sox10:psmb1-2A-GFP partially rescues muscle phenotype, as assessed by antibody staining for myosin heavy chain (MHC) at 72 hpf. Scale bars: 50 μm.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.