Duplicated zebrafish (Danio rerio) inositol phosphatases inpp5ka and inpp5kb diverged in expression pattern and function

Abstract

One hurdle in the development of zebrafish models of human disease is the presence of multiple zebrafish orthologs resulting from whole genome duplication in teleosts. Mutations in inositol polyphosphate 5-phosphatase K (INPP5K) lead to a syndrome characterized by variable presentation of intellectual disability, brain abnormalities, cataracts, muscle disease, and short stature. INPP5K is a phosphatase acting at position 5 of phosphoinositides to control their homeostasis and is involved in insulin signaling, cytoskeletal regulation, and protein trafficking. Previously, our group and others have replicated the human phenotypes in zebrafish knockdown models by targeting both INPP5K orthologs inpp5ka and inpp5kb. Here, we show that inpp5ka is the more closely related orthologue to human INPP5K. While both inpp5ka and inpp5kb mRNA expression levels follow a similar trend in the developing head, eyes, and tail, inpp5ka is much more abundantly expressed in these tissues than inpp5kb. In situ hybridization revealed a similar trend, also showing unique localization of inpp5kb in the pineal gland and retina indicating different transcriptional regulation. We also found that inpp5kb has lost its catalytic activity against its preferred substrate, PtdIns(4,5)P₂. Since most human mutations are missense changes disrupting phosphatase activity, we propose that loss of inpp5ka alone can be targeted to recapitulate the human presentation. In addition, we show that the function of inpp5kb has diverged from inpp5ka and may play a novel role in the zebrafish.

Keywords: Gene duplication; INPP5K; Inositol phosphatase; Zebrafish.

Fig. 1

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

Fig. 2

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, F inpp5ka (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

Fig. 3

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–F inpp5kb expression is concentrated to the pineal gland. The pineal gland is indicated by the black arrow

Fig. 4

Cellular distribution of inpp5ka and inpp5kb at 5 dpf by RNA scope in situ. A–C inpp5ka 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–F inpp5kb 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

Fig. 5

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