Cfap298ΔΔS does not rescue LR defects in zebrafish. (A) Representative images of cfap298+/− and cfap298−/− zebrafish embryos at 48 hpf, with cfap298+/− embryos displaying straight bodies and/or tails compared to cfap298−/−, which display curved bodies and/or tails. (B) Comparison of mouse wild-type Cfap298 and Cfap298ΔΔS with wild-type zebrafish cfap298 and engineered cfap298ΔΔS sequence. Area outlined in red indicates location of the cfap298ΔΔS mutation in mouse and zebrafish. (C,D) Quantification of body curvature (C) and heart position (D) observed in uninjected and injected embryos. Uninjected wild-type (n=9) and cfap298+/− (n=13) embryos were observed to have straight tails compared to cfap298−/− (n=10), which displayed statistically significant greater tail curvature (for wild type and cfap298+/−, P≥0.9999; for wild type and cfap298−/−, P≤0.0001; for cfap298+/− and cfap298−/−, P≤0.0001 by Fisher's exact test). However, for left–right defects, we did not observe a statistically significant difference between wild type, cfap298+/− or cfap298−/−, but we did observe a statistically significant difference between cfap298+/− and cfap298−/− (for wild type and cfap298+/−, P=0.4091; for wild type and cfap298−/−, P=0.0705; for cfap298+/− and cfap298−/−, P=0.0021 by Fisher's exact test). For embryos injected with 500 pg of wild-type cfap298 mRNA, we observed no significant difference between wild-type (n=16), cfap298+/−(n=36) or cfap298−/− (n=20) embryos (for wild type and cfap298+/−, P≥0.9999; for wild type and cfap298−/−, P≥0.9999; for cfap298+/− and cfap298−/−, P≥0.9999 by Fisher's exact test) as well as no significant difference in left–right defects (for wild type and cfap298+/−, P=0.6769; for wild type and cfap298−/−, P≥0.9999; for cfap298+/− and cfap298−/−, P≥0.9999 by Fisher's exact test). For embryos injected with 500 pg of cfap298ΔΔS mRNA, wild-type (n=23) and cfap298+/−(n=38) embryos were observed to have mostly straight tails compared to cfap298−/− (n=27), which displayed statistically significant greater tail curvature (for wild type and cfap298+/−, P≥0.9999; for wild type and cfap298−/−, P≤0.0001; for cfap298+/− and cfap298−/−, P≤0.0001 by Fisher's exact test) as well as significantly higher incidence in left–right defects (for wild type and cfap298+/−, P=0.2178; for wild type and cfap298−/−, P≤0.0001; for cfap298+/− and cfap298−/−, P≤0.0001 by Fisher's exact test). We also observed a significant difference in tail curvature between cfap298−/− uninjected, wild-type mRNA-injected and ΔΔS mRNA-injected embryos (for uninjected and wild-type mRNA injected, P≤0.0001; for uninjected and ΔΔS mRNA injected, P=0.0179; for wild-type mRNA injected and ΔΔS mRNA injected, P≤0.0001 by Fisher's exact test). We did not observe a significant difference in cfap298−/− uninjected and ΔΔS mRNA-injected embryos (P=0.8864 by Fisher's exact test), but we did observe a significant difference in left–right defects in cfap298−/− embryos injected with wild-type mRNA compared to uninjected embryos or embryos injected with ΔΔS mRNA (for uninjected and wild-type mRNA injected, P=0.0009; for wild-type mRNA injected and ΔΔS mRNA injected, P≤0.0001 by Fisher's exact test). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Scale bars: 500 μm.
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