Protein alignment of INPP5K, Inpp5ka, and Inpp5kb highlighting conserved amino acids required for phosphatase activity. The start and end of the catalytic domain in the human protein are marked with large red arrowheads. Amino acids required for phosphatase activity have been denoted with asterisks (*). Small arrowheads indicate residues that are altered by missense variants in humans. Hs, Homo sapiens; Dr, Danio rerio

inpp5ka and inpp5kb mRNAs differ in expression levels in zebrafish larvae. A Gene expression determined by qPCR. inpp5ka is more highly expressed in whole body lysates through 5 dpf. B Larval tissues were excised from the eye, head, and tail for localized gene expression analysis excluding the area of the trunk around the yolk. C, D Expression for both inpp5ka (C) and inpp5kb (D) is low in the tail and increases in the eyes and brain. By 5 dpf, both are most highly expressed in the eyes. Results from the whole body from (A) are shown as reference. E, Finpp5ka (E) maintains higher expression levels than inpp5kb (F) in the tail/muscle and head at 30 dpf and in adult fish. Both genes are expressed at very high levels in the eyes. Values are averages ± SEM. 2-way ANOVA results for tissue and developmental timepoint (time) are also listed. *p < 0.05, **p < 0.01, ***p < 0.001

inpp5ka and inpp5kb mRNAs differ in localization at 3 dpf. A–C In situ hybridization in 3 dpf larvae shows that inpp5ka mRNA is highly expressed throughout the head and eyes. Scale bars: 500µm in (A), 100µm in (C). D–Finpp5kb expression is concentrated to the pineal gland. The pineal gland is indicated by the black arrow

Cellular distribution of inpp5ka and inpp5kb at 5 dpf by RNA scope in situ. A–Cinpp5ka shows even distribution in the brain and eye (A, B), also labeling lens cells (B). Muscle expression is sparse and at lower levels (C). D–Finpp5kb brain expression is highest in the pineal gland (arrow in D). Increased expression is also noted in the inner nuclear layer of the retina with lower expression in photoreceptors, retinal ganglion cells, and lens cells (E, also see Suppl. Figure 1B). Sparse expression is present in muscle cells (F, also see Suppl Fig. 1C). G–I Merged images including both probes (inpp5ka in green and inpp5kb in red) and DAPI to counterstain the nuclei. Scale bars: 100 µm for (A), (D), and (G), 50 µm for all other panels

Inpp5ka and Inpp5kb exhibit different phosphatase activity. A Phosphatase activity of human INPP5K, Inpp5ka, and Inpp5kb in the malachite assay. Human INPP5K and Inpp5ka demonstrate high activity for the PI(4,5)P₂ substrate. PIP₃ did not elicit activity from any isoform. B Inpp5ka is more significantly active against diC8PI(4,5)P₂ compared to Inpp5kb. Values are averages ± SEM. ***p < 0.001 following a t-test