Severe disruption of iridosome biogenesis and crystal formation in hps5 mutants. (A and B) Brightfield images of WT (A) and hps5−/− (B) zebrafish larvae at 72 hpf. Insets show lack of reflective crystals in hps5−/− eyes and reduced melanophore pigmentation compared to WT. Scale bar, 500 μm. (A′ and B′) Confocal MIPs of iridophores at 72 hpf and reflectance imaging at 7 dpf (A″ and B″) showing near-complete absence of crystal formation in hps5−/− mutants. Insets in (A″ and B″) highlight aberrant crystal aggregates in mutant iridophores. Scale bar, 10 μm. (C) Schematic of the hps5 mutation, which introduces a premature stop codon resulting in a truncated protein. Illustration created in part using https://www.biorender.com/. (D) Quantification of the percentage of eye area containing crystals in WT and hps5−/− larvae at 72 hpf. Data represent mean ± SEM. from two clutches (WT, n = 10; hps5−/−, n = 9). Significance assessed by two-way ANOVA: ***P < 0.001. n = number of total larvae measured in different biological repeats. (E) Proposed model illustrating the involvement of the endolysosomal pathway and the trans-Golgi network in iridosome biogenesis. LRO components implicated in crystal formation, including RAB32a, Ap3m2, and Hps5, are indicated in brackets.
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