Mineralization deficit in zebrafish sox10 mutants. (A) Example images illustrating a major delay in initiation of bone mineralization in sox10 mutants between 3 and 7 dpf revealed by Alizarin Red staining. Some mineralization is present by 5 dpf but never achieves control levels before lethality at 8 dpf. (B) Schematic representation of the affected mineralized structures and their embryonic origins. (C,D) Von Kossa (C) and OsteoImage (D) staining showing absent calcium deposition and hydroxyapatite formation in sox10 mutants at 4 dpf and gradual recovery starting at 5 dpf. Arrowheads point to the opercle (op). Numbers in panels indicate the proportion of larvae of that genotype with the presented phenotype. Scale bars: 100 µm.

Normal patterns of growth and differentiation in sox10 mutant osteoblasts. (A) Reference image of a larva stained with Alcian Blue and Alizarin Red, with locations of the skeletal elements shown in B (magenta dashed box) and C (white box) highlighted. (B) Immunostaining with an anti-Sox10 antibody reveals strong expression in chondrocytes but a lack of Sox10 protein (asterisk) in mineralizing osteoblasts (sp7:GFP⁺) forming the op bone at 3 dpf. (C-C″) Example images from sequential live imaging showing normal patterns of RUNX2:mCherry, sp7:GFP and osc:GFP transgene expression in mutant osteoblasts of the op at 4 and 7 dpf. (D) Normal growth of sox10 mutant op (arrowheads) as well as other bones despite minimal calcium accumulation, revealed by live imaging of Alizarin Red-stained sp7:GFP⁺ embryos at 4 and 7 dpf. (E) Colorimetric in situ hybridizations for col10a1a and phospho1, encoding key bone matrix components, revealed no overt abnormalities in sox10 mutants at 4 dpf. op, opercle. Numbers in panels indicate the proportion of larvae of that genotype with the presented phenotype. Scale bars: 10 µm (B-C″); 100 µm (D,E).

sox10 mutants have a severe whole-body calcium deficit. (A,B) Colorimetric calcium assay reveals significantly lower levels of Ca²⁺ in sox10 mutants after mineralization begins at 3 dpf. Each data point represents a pool of 10-15 embryos. Different shapes represent biological replicates assayed on different days (unpaired t-tests; 36 hpf: P=0.580, d.f.=8; 48 hpf: P=0.083, d.f.=8; 72 hpf: P=0.091, d.f.=7; 96 hpf: P=0.0002, d.f.=6; 120 hpf: P=0.0008, d.f.=4; 144 hpf: P=0.000008, d.f.=4; 168 hpf: P=0.000005, d.f.=6). B shows an example of the colorimetric assay, showing a clear reduction in mutants. (C) Schematic of the Ca²⁺ treatment protocol. (D,D′) Increasing ambient Ca²⁺ levels to 2 or 10 mM does not rescue the mineralization deficit (D) or Ca²⁺ content (D′) (unpaired t-tests; 1 versus 2 mM: P=0.963, d.f.=2; 1 versus 10 mM: P=0.778, d.f.=2; 2 versus 10 mM: P=0.748, d.f.=2). Numbers in panels indicate the proportion of larvae of that genotype with the presented phenotype. In D′, ratios reflect the number of imaged larvae of that genotype with the presented phenotype, and dashed lines indicate the median. Significance determined by unpaired t-test. ns, not significant. Scale bars: 100 µm.

Reduction in trpv6⁺ NaR cell number in the sox10 mutants. (A) Immunostaining of a 3 dpf SOX10:Cre; ef1a: DsRed>GFP larva with an antibody against the Na⁺/K⁺ ATPase pump confirms that ionocytes do not derive from neural crest. (B) rt-PCR demonstrates that trpv6 transcription is not overtly altered at the whole-body level at 4 dpf. Each point represents a pool of 10-15 embryos (unpaired t-test; P=0.954, d.f.=4). (C-D′) Fluorescence in situ hybridizations for trpv6 (C) and igfbp5a (D) demonstrate a striking and significant reduction in NaR cell number in mutants at 4 dpf (quantified in C′,D′), with partial recovery by 7 dpf (unpaired t-tests; trpv6: 4 dpf: P<0.000001, d.f.=20; 7 dpf: P=0.0006, d.f.=17; igfbp5a: 4 dpf: P=0.0001, d.f.=23; 7 dpf: P<0.0001, d.f.=17). In B,C′,D′, dashed lines indicate the median. ns, not significant; WT, wild type. Scale bars: 100 µm.

Upregulation of anti-hypercalcemic hormone stc1a in sox10 mutants. (A) Volcano plot of whole-body bulk RNAseq shows stc1a as significantly upregulated in the sox10 mutants at 4 dpf (P-value cutoff: 0.05; fold change cutoff: 1.25). Red arrow points to stc1a. (B,C) Both semi-quantitative rt-PCR (B) and in situ hybridization (C) detect a robust upregulation of stc1a mRNA in sox10 mutants at 4 dpf (unpaired t-test; P<0.0001, d.f.=4). Red boxes in C indicate the regions shown at higher magnification on the right. Numbers in panels indicate the proportion of larvae of that genotype with the presented phenotype. Scale bars: 100 µm. (D,D′) The increase in stc1a transcript levels is due at least in part to an increase in the number of stc1a⁺ cells in sox10 mutant corpuscles, first detected at 45 hpf and resolving at 7 dpf (unpaired t-tests; 36 hpf: P=0.640, d.f.=11; 45 hpf: P=0.00009, d.f.=8; 72 hpf: P=0.002, d.f.=6; 96 hpf: P=0.00003, d.f.=10; 168 hpf: P=0.0007, d.f.=10. ns, not significant; WT, wild type. Scale bars in D: 10 µm.

sox10⁺ crest-derived cells surround the CS and are missing in sox10 mutants. (A) Schematic of the corpuscles' position and surrounding environs. The glands are positioned ventral to the dorsal aorta, dorsal to the pronephros, and flanking the posterior cardinal vein (pcv). sox10⁺ lineage cells branch off neighboring axon tracts (lined with sox10⁺ Schwann cells and Schwann cell precursors) to contact the corpuscles. (B) Control 72 hpf larva doubly transgenic for fli1:EGFP (magenta; labeling vasculature) and her6:mCherry (green; labeling the corpuscles). Top image is a single optical section from the lateral perspective showing endothelial cells wrapping around the CS. Bottom image is a maximum intensity projection rotated orthogonally to show the close interaction of the CS with the pcv. (C) Single optical section showing sox10:DsRed⁺ cells in close contact with the Tp1:VenusPEST⁺ corpuscles (outlined) by 50 hpf. (D,E) Whole-mount live imaging of SOX10:Cre B>R; Tp1:VenusPEST fish showing sox10⁺ lineage cells surrounding the CS at earlier stages (58 and 72 hpf), nearby at 96 and 168 hpf, and absent in the sox10 mutants. The entire fish is shown in D to highlight the deficiency of trunk NCCs (magenta) with the box indicating the region shown in E. (E′) Volume measurements of control and mutant Tp1:VenusPEST⁺ CS between 58 and 168 hpf. Only the right CS, closest to the microscope lens, was measured. (unpaired t-tests; 58 hpf: P=0.0001, d.f.=16; 72 hpf: P=0.00001, d.f.=24; 96 hpf: P=0.003, d.f.=19; 168 hpf: P=0.068, d.f.=10). In E′, dashed lines indicate the median. ns, not significant. Scale bars: 10 µm (B,D,E); 100 µm (C).

stc1a is epistatic to sox10 in control of systemic calcium content. (A) Brightfield images of sox10 and stc1a controls and mutants at 4 dpf. Double mutants phenocopy the loss of pigment (arrows) and the inner ear malformations (asterisks) of single sox10 mutants and the cardiac edema of the stc1a mutant (arrowheads). (B-C′) Loss of stc1a on the sox10 mutant background improves mineralization (B) and the number of trpv6⁺ ionocytes (C) at 4 dpf, quantified in C′ (unpaired t-test; P=0.0207, d.f.=18). In A,B, numbers in panels indicate the proportion of larvae of that genotype with the presented phenotype. (D) Calcium quantification shows an increase (unpaired t-test; P=0.143, d.f.=6) in calcium levels in sox10^−/−; stc1a^−/− compared to sox10^−/−. (E) Quantitation of mineralization levels in sox10; stc1a clutches grouped based on the intensity of Alizarin Red staining. There was a significant increase in the proportion of double mutants with detectable mineralization compared with sox10 single mutants (χ²; P=0.0206, d.f.=3). In C′-E, ‘control’ includes wild-type and heterozygous larvae. Dashed lines indicate the median. Scale bars: 100 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.