Reduced crystal production in rab32a and ap3m2 mutants, with distinct morphological alterations in rab32a mutants. (A–C) Brightfield images comparing wild type (WT) (A) rab32a−/− (B), and ap3m2−/− (C) larvae at 72 hpf. Insets highlight decreased iris reflectivity in rab32a−/− and ap3m2−/− mutants relative to WT. ap3m2−/− mutants also display reduced melanophore pigmentation. Scale bar, 500 μm. Reflectance imaging (A′–C′) and confocal MIPs of iridophores (A″–C‴) from WT and mutants at 72 and 96 hpf. Both mutants exhibit reduced crystal accumulation compared to WT. Insets in A′ and B′ highlight shorter and distorted crystals in rab32a−/− mutants, while ap3m2−/− mutants maintain WT-like crystal morphology. Scale bar, 10 μm. (D and F) Schematic of CRISPR-Cas9 targeting strategy for rab32a and ap3m2. rab32a−/− mutants harbor frameshift-inducing mutations leading to premature stop codons; ap3m2−/− mutants carry a 4-nt deletion resulting in truncation. Diagrams partially generated using BioRender.com. (E and G) Quantification of the percentage of eye area occupied by crystals in WT siblings versus mutants at 72 and 96 hpf. Data represent mean ± SEM. from ≥3 independent clutches. (E) 72 hpf: WT, n = 17; rab32a−/−, n = 18. 96 hpf: WT, n = 18; rab32a−/−, n = 21. (G) 72 hpf: WT, n = 21; ap3m2−/−, n = 31. 96 hpf: WT, n = 18; ap3m2−/−, n = 17. Significance determined by two-way ANOVA: ***P < 0.001; **P < 0.01; *P < 0.05. n = number of total larvae measured in different biological repeats.
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