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. 2022 Jan 13;11:e71256. doi: 10.7554/eLife.71256

Figure 3. Disruption of the PIPK domain of Pikfyve in zebrafish caused early-onset cataract.

(A) A schematic diagram showing the generated pikfyveΔ8 mutant allele. The underlined base pairs are the sgRNA target. The deleted base pairs are shown in dark blue while inserted ones are shown in red. The stop codon introduced in the mutant form is shown in the grey box. (B) Representative images showing the lens of sibling and pikfyveΔ8 mutants at 5 dpf. (C) Representative differential interference contrast (DIC) images showing the lens of pikfyve+/+, pikfyve+/Δ8, and pikfyveΔ8/Δ8 embryos at 3 dpf and 5 dpf. The scale bars represent 10 μm in (B) and (C). (D) Quantification of vacuole number in the lens of pikfyve+/+, pikfyve+/Δ8, and pikfyveΔ8/Δ8 embryos at 3 dpf (n=7 for pikfyve+/+; n=10 for pikfyve+/Δ8; n=7 for pikfyveΔ8/Δ8) and 5 dpf (n=7 for pikfyve+/+; n=11 for pikfyve+/Δ8; n=6 for pikfyveΔ8/Δ8). (E) Representative images of HLEB3 cells treated with DMSO or PIKFYVE inhibitor YM201636 for 4 hr. The scale bars represent 25 μm. (F) Quantification of the vacuole numbers in (E). ****, p<0.0001, Student’s t-test. All experiments were repeated three times. See Figure 3—source data 1 for details.

Figure 3—source data 1. Raw data for quantification in Figure 3D and F.

Figure 3.

Figure 3—figure supplement 1. Characterization of pikfyve-deficient zebrafish mutants.

Figure 3—figure supplement 1.

(A) Gross morphology of 5-dpf and 7-dpf siblings and pikfyveΔ8 mutants. The scale bars represent 500 μm. (B) Survival rate of pikfyveΔ8 mutants (n=48) and siblings (n=50).
Figure 3—figure supplement 1—source data 1. Raw data for quantification in Figure 3—figure supplement 1B.
Figure 3—figure supplement 2. Ectopic overexpression of PIKFYVEG1943E failed to induce cataract defect in zebrafish.

Figure 3—figure supplement 2.

(A) Schematic view of constructs in which the WT and G1943E variant form of PIKFYVE were expressed under the control of the ubiquitously expressed ubiquitin promoter. (B) Representative images showing the lens of 5-dpf WT, Tg(ubi:PIKFYVEEWT) and Tg(ubi:PIKFYVEG1943E) zebrafish embryos. The scale bars represent 20 μm. All experiments were repeated three times.
Figure 3—figure supplement 3. The G1943E variant form of PIKFYVE is less efficient to rescue the vacuole defect in pikfyve-deficient zebrafish mutants.

Figure 3—figure supplement 3.

(A) Representative images showing the lens of 5-dpf siblings, pikfyveΔ8, pikfyveΔ8;Tg(ubi:PIKFYVEWT), and pikfyveΔ8;Tg(ubi:PIKFYVEG1943E) zebrafish. The scale bars represent 20 μm. (B) Whole-mount in situ hybridization (WISH) analysis of human PIKFYVE transcripts in 2-dpf Tg(ubi:PIKFYVEWT) and Tg(ubi:PIKFYVEG1943E) zebrafish. The scale bars represent 200 μm. See Figure 3—figure supplement 3—source data 1 for details.
Figure 3—figure supplement 3—source data 1. Raw data for quantification in Figure 3—figure supplement 3B, C.