Figure 4

Synergistic effect of rspo2 and rspo3 ablation on zebrafish limb development and craniofacial morphology. (A) Schematic illustrating experimental design. Targeted mutagenesis of rspo3^−/− in zebrafish was carried out using CRISPR/Cas9 gene editing. A – 20 bp deletion was bred to homozygosity. Intercross or rspo3^+/− were injected with 4 gRNAs against rspo2 and the resulting larvae were genotyped and analyzed for phenotype. (B) Whole mount ventral and lateral images of Alcian blue/Alizarin red S stained 9 dpf larvae. rspo3^−/− embryos that were rspo2 gRNA/Cas9 injected (rspo2^Δ) larvae were similar to wild-type except that rspo2^Δ larvae exhibited disrupted development of the pectoral fin. Impaired fin development was exacerbated with decreasing genetic dosage of rspo3 (black arrows, dotted yellow lines delineate fins). While craniofacial development in rspo3^−/− and rspo2^Δ larvae were largely normal, rspo3^−/−; rspo2^Δ double mutants exhibited a dysmorphic lower jaw (white arrow). Scale bar: 100 μm. (C) Quantification of pectoral fin developmental disruption. rspo2^Δ larvae tended to have disrupted development of a single pectoral fin. This effect was significantly exacerbated with decreasing genetic dosage of rspo3, as rspo3^−/−; rspo2^Δ double mutant larvae failed to develop pectoral fins altogether. (D) Quantification of angle measurements between Meckel’s cartilage (m) and palatoquadrate (pq). While rspo3^−/− and rspo2^Δ mutants had normal lower jaw morphology, rspo3^+/−; rspo2^Δ and rspo3^−/−; rspo2^Δ mutants displayed a significantly decreased angle at the Meckel’s/palatoquadrate joint. N = 10–16. p < 0.01. *Indicates significance relative to wild-type.