a,b, WT (a) and pnp4acbg20 mutant (b) larvae iridescence phenotypes. Larval gross morphology at 72 hpf; eyes with iridophores labeled (green, transgenic TDL358:GFP) and crystal reflection (gray); zoomed-in view of crystal morphology in WT and mutant conditions. Note the reduced crystal formation and altered morphology compared with the WT control (insets in a‴ and b‴). Scale bars: 500 μm (a,b), 50 μm (a′,b′,a″,b″), 10 μm (a‴,b‴) and 5 μm (insets). c, Schematic representation of the CRISPR–Cas9-mediated knockout strategy targeting the pnp4a gene. Illustration created with BioRender.com. In detail, the pnp4acbg20 mutant results in a 6-nt deletion and 1-nt insertion, leading to a 5-nt frameshift and truncated protein resulting in 150 aa instead of 291 aa. The pnp4awz19 mutant results in a 10-nt deletion and truncated protein resulting in 142 aa. d, Comparison of the average iridophore reflection per 50 μm in different WT, mutant and rescue conditions. au, arbitrary units. WT control animals were subjected to the same treatment as the pnp4acbg20 rescue condition (Methods). The mean ± s.e.m. are shown for independent biological replicates; WT, n = 7; WT control, n = 12; pnp4acbg20, n = 16; pnp4acbg20 rescue, n = 20 (two-tailed, unpaired, non-parametric Mann–Whitney test; ****P < 0.0001, **P < 0.01 and *P < 0.05). e, RT–qPCR analysis of pnp5a and pnp5b mRNA expression levels from iridophores of WT (pnp5a, n = 5; pnp5b, n = 5) or pnp4awz19 mutants (pnp5a, n = 4; pnp5b, n = 5). RNA was extracted at 6 dpf. The mean ± s.d. are shown for independent biological replicates (two-tailed, unpaired Student’s t-test; ****P < 0.0001 and ***P < 0.001).
|
