daw1^b1403 mutants exhibit motile cilia-associated defects. (A) The 418 amino acid Daw1 protein with short N-terminal domain (NTD) and large β-propeller composed of WD40 repeats. daw1^b1403 deletes two conserved residues, I140 and A141. (B) Human DAW1 X-ray structure (Protein Data Bank: 5NNZ) showing I140 and A141 location. (C-E) 3D reconstitutions of µCT data. Most daw1^b1403 mutants showed normal spines (D), but 20% exhibited curves (E), compared with control (C). (C′-E′) Quantitation of curvature showed curved daw1^b1403 mutants had a non-stereotyped pattern (E′), compared with control (C′) and non-curved daw1^b1403 mutants. (F) Top: representative image of 25 hpf larvae showing the heart (dotted oval) on top of the yolk (dotted line). Bottom: daw1^b1403 mutants exhibited randomized heart laterality including left (L; green), middle (M; orange) and right (R; magenta)-facing hearts (n=91 mutants and 98 sib controls; ***P=4.84×10⁻²¹, chi square). (G) Top: otic vesicles at 25 hpf showed abnormal phenotypes in daw1^b1403 mutants [2N (green) – two normal; 3F (orange) – three otoliths, posterior two fused; 3S (magenta) – three separate otoliths. Bottom: quantification of n=116 mutants and 132 sibs; ***P=1.31×10⁻¹², chi square. (H) CTD in daw1^b1403 mutants was fully penetrant at 25 hpf. Scale bar: 0.5 mm (H).

Body axis curvature self-corrects after delayed onset cilia motility. (A-E) The first 5 days of development show daw1^b1403 mutants self-correct curves. (A′-E′) SuperPlots of body angle, θ (inset in A′). Pairwise comparisons used two-way ANOVA adjusted for multiple comparisons. (F-I) TICS of CC cilia motility at different time points. (F′-I′) SuperPlots of the proportion of the CC covered by cilia motility (motile area). ***P<0.001, **P<0.01, *P<0.05 (two-tailed unpaired t-tests). ns, not significant. Scale bars: 0.5 mm (A-E); 5 µm (F-I).

daw1^b1403 encodes a loss-of-function protein. (A-C) Lateral views of controls and daw1 and cfap298 crispants. (A′-C′) SuperPlots of body angle quantitation. Pairwise comparisons used two-way ANOVA adjusted for multiple comparisons. ***P<0.001, **P<0.01, *P<0.05. ns, not significant. (D) Expression of Daw1^WT but not Daw1^b1403 rescues the body curves of daw1 crispants. Scale bars: 0.5 mm (A-D).

Self-correction of curves can occur throughout developmental stages. (A-C) Top: TICS of CC cilia motility of 25 hpf cfap298^tm304 mutants raised at 30°C (A), then 52 hpf after maintaining 30°C (B) or downshifting to 22°C at 24 hpf (C). Bottom: SuperPlots of motility data. Insets: axis straightening occurred in downshifted embryos but not those maintained at 30°C. (D) Schematic of temperature (T) shifts. Embryos were raised at 30°C until either 1, 2, 3, 4 or 5 dpf, then downshifted to 22°C, inducing cilia motility. Body angles were assessed until 7 dpf. (E) Body angles across 1-7 dpf after temperature downshifts on days 1-5 (D1-D5). (F) Summary showing zebrafish larvae self-correct curves after robust CC cilia motility onsets. Distance around the sundial represents later times of motility onset in distinct conditions. ****P<0.0001, *P<0.05 (two-tailed unpaired t-tests). Scale bars: 5 µm (A-C).

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