Fig. S2

The phenotype of cct5tf212b is caused by a mutation within cct5. Related to Figure 1.

(A, B) Administration of 100 μM standard control morpholino to 3-dpf-old larvae (morpholino control) did not reduce the birefringence in comparison to larvae that were treated with injection solution (injection control). However, a highly significant reduction in birefringence was detected with 3-dpf-old larvae treated with 100 μM of the translation-blocking morpholino cct5_ex1(+85+110). Data are mean ± SEM. ****p < 0.0001 by one-way ANOVA with Tukey’s post hoc test; n = 10. (C, D) Administration of 200 μM of the splice-altering morpholino cct5_ex5(+187-18) to 3-dpf-old larvae lead to a highly significant reduction in birefringence compared to control larvae that were injected with injection solution (injection control) or larvae that were treated with 200 μM standard control morpholino (morpholino control). Administration of 200 μM cct5_ex5(+187-18) together with 50 ng cct5 transcript significantly increased the birefringence compared to larvae that were injected only with 200 μM cct5_ex5(+187-18). Data are mean ± SEM. ****p < 0.0001 by one-way ANOVA with Tukey’s post hoc test; n = 10. (E) A schematic of the transgenic construct in Tg(cry:mCherry,-1.8cct5:GFP) shows that mCherry expression is controlled by the lens-specific αA-crystallin promoter and GFP is regulated by upstream region of cct5 ranging from -1860 to +24 bp relative to the start codon. (F) At 2 dpf, control injected Tg(cry:mCherry,-1.8cct5:GFP) showed a widespread GFP expression that was abolished in siblings treated with 100 μM cct5_ex1(+85+110); note residual control mCherry in the lens (arrowhead). Representatives of four analyzed larvae per genotype are shown (n = 4 per genotype). (G) Whereas RT-PCR performed with control 3-pdf-old larvae resulted in a 769-bp amplicon, RT-PCR with larvae injected with 200 μM cct5_ex5(+187-18) resulted in two additional amplicons: 688-bp corresponding to a deletion of 81-bp from exon 5 and 853-bp resulting from inclusion of the intron between exons 5 and 6. All amplicons were identified by sequencing. (H) cct5^{hi2972Tg/tf212b} compound heterozygotes displayed a reduction in birefringence that was similar to single cct5^hi2972Tg homozygotes. Data are mean ± SEM. ****p < 0.0001 by student’s t-test; n = 10. (I) Compared to control-injected cct5^hi2972Tg homozygotes, administration of 25 ng cct5 transcript to cct5hi2972Tg homozygotes resulted in a highly significant increase in birefringence. Data are mean ± SEM; ****p < 0.0001 by one-way ANOVA with Tukey’s post hoc test; n = 10. (J) Administration of 50 ng cct5 transcript to cct5^tf212b homozygotes resulted in a highly significant increase in birefringence compared to control-injected cct5^tf212b homozygotes. Data are mean ± SEM. ***p < 0.001 by one-way ANOVA with Tukey’s post hoc test; n = 10.