Chondrocyte stacking defects with loss of prdm3 and prdm16 can be rescued by pharmacological manipulation of Wnt/β-catenin activity or genetically in prdm3−/−;prdm16−/− double mutants. (A-B) Wild-type (wt) or prdm3−/− zebrafish embryos were treated with suboptimal doses of 0.75 µM IWR-1 or DMSO (vehicle control) from 24 to 48 hpf. Inhibitor- and vehicle-treated larvae were collected at 6 dpf and stained with Alcian Blue or Alizarin Red. (A-A‴) High magnification of chondrocytes within cartilage structures. (B) Quantification of chondrocyte organization (angle between adjacent cells) (n=at least 5 per genotype per treatment group); mean±s.d. Scale bars: 100 µm. (C-D) Wild-type (wt) or prdm16−/− embryos were treated with suboptimal doses of 0.05 µM Gsk inhibitor XV (Wnt activator) or DMSO (vehicle control) from 24 to 48 hpf. Larvae were collected at 6 dpf and stained with Alcian Blue and Alizarin Red. (C-C‴) High magnification of chondrocytes within cartilage elements. (D) Quantification of chondrocyte organization (n=at least 10 per genotype per treatment group); mean±s.d. Scale bar: 100 µm. (E-H″) prdm3+/−;prdm16+/− heterozygous fish were generated and intercrossed. Larvae were collected at 6 dpf and stained with Alcian Blue and Alizarin Red. (E-H′) Dissected and mounted viscerocranium (E-H) and neurocranium (E′-H′) from wild type (E,E′), prdm3−/− (F,F′), prdm16−/− (G,G′) and prdm3−/−;prdm16−/− double mutants (H,H′). (E″-H″) High magnification of chondrocytes in cartilage elements of wild type (E″), prdm3−/− (F″), prdm16−/− (G″) and prdm3−/−;prdm16−/− double mutants (H″). cbs, ceratobranchials; ch, ceratohyal; ep, ethmoid plate; mc, Meckel's cartilage; pq, palatoquadrate; ps, parasphenoid; tr, trabeculae. (I) Quantification of the angle between adjacent chondrocytes across wild type, prdm3−/−, prdm16−/− and prdm3−/−;prdm16−/− double mutants, as well as all other combinatorial mutants indicated; mean±s.d. Scale bars: 100 µm. **P≤0.005; ns, not significant (unpaired, two-tailed Student's t-test).
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