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K., and H. role of the cytoplasmic tail in rod degeneration. Finally, we showed that the cytoplasmic tail of rhodopsin might trigger rod degeneration through activating the downstream calcium signaling pathway, as drug treatment with inhibitors of intracellular calcium release prevented rod degeneration in mutants. Our results demonstrate Bay 60-7550 a previously unknown function Bay 60-7550 of the rhodopsin cytoplasmic domain during opsin-triggered photoreceptor degeneration and may open up new avenues for managing this disease. studies suggest that KIF3C, another kinesin-2 protein, may substitute for KIF3B in the kinesin-2 motor (4, 5). In addition, KIF17, another member of Bay 60-7550 the kinesin-2 family, functions as a dimer during ciliary transport (3). Kinesin-2 family members mediate protein transport in photoreceptor cells. Loss of function of KIF3A in the mouse retina interrupts the transport machinery and leads Bay 60-7550 to rapid photoreceptor cell death (6,C8). In zebrafish, mutation in gene leads to mispositioning of the basal bodies and photoreceptor cell death (9). Dysfunction of Kif3b, however, affects only a subset of cilia, and photoreceptor connecting cilium is initially absent but then partially recovered latterly (10). Although inhibition of Kif17 by dominant-negative expression or morpholino-based knockdown may affect OS development, zebrafish mutants are able to grow to adulthood with normal photoreceptor functions (10,C12). Furthermore, another study has suggested that KIF17 is dispensable for photoreceptor development (13). Photoreceptor degeneration occurs in many human genetic disorders, including retinitis pigmentosa (RP), the most common form of inherited retinal degeneration (14). Previous studies suggest that ectopic opsin accumulation leads to photoreceptor cell death and that diminishing expression of the gene can delay the degeneration of rod photoreceptors (7, 15). However, the requirement of kinesin-2 for opsin transportation remains controversial. Mutation of either the or gene results in rod degeneration together with mislocalization of rhodopsin, indicating that IFT mediates opsin transport (6, 16, 17). Further analysis using the fluorescence recovery after photobleaching method in live cells also shows that KIF3A plays a role during opsin transport (18). Conversely, recent studies suggest that opsin transport may still occur without the function of both KIF3 and KIF17 motors (13, 19, 20). The structure and function of photoreceptor cells are similar in zebrafish and humans. The zebrafish retina contains five types of photoreceptor cells, Bay 60-7550 including one rod photoreceptor cell type and four cone photoreceptor cell types. According to their absorption spectra and morphology, cone photoreceptors are classified as short single cones sensitive to ultraviolet light, long single cones sensitive to blue light, and double cones comprised of a red-sensitive and a green-sensitive cell (21, 22). Cone and rod photoreceptors form a highly ordered mosaic in the adult retina (23). Previously, we reported that rod photoreceptors degenerated in zebrafish mutants, although the underlying mechanisms are yet to be understood (10). Here we further generated zebrafish mutants and investigated rod Rabbit Polyclonal to USP13 and cone development therein. As for mutants, rod photoreceptors in mutants exhibited a faster degeneration process than cones. Further experiments demonstrated that opsins were accumulated in the apical part of cone photoreceptors, whereas most rod opsins localized to the cell membrane. We next generated a zebrafish mutant allele lacking the carboxyl-terminal cytoplasmic tail of rhodopsin and found that rods were maintained in double mutants. Furthermore, we showed that the cytoplasmic domain plays a key role during rod degeneration, as overexpression of full-length opsin or the cytoplasmic domain alone, but not the form lacking the cytoplasmic domain, accelerated rod degeneration in mutants. Finally, drug treatment with calcium channel blockers increased the rod survival ratio in mutants, suggesting that the calcium signaling pathway is involved in photoreceptor degeneration. Results Rapid rod degeneration in kif3a mutants To elucidate the mechanisms underlying the degeneration of rod photoreceptors observed following dysfunction in early zebrafish embryos (10), in this study, we further generated mutants using CRISPR/Cas9 methods (supplemental Fig. S1, mutants displayed body curvature at 3 days post fertilization (dpf). Whole-mount immunostaining with an anti-acetylated -tubulin antibody demonstrated that all cilia were absent in mutant larvae (supplemental Fig. S1, and (9,C11). The complete absence of cilia has also been described in another mutant line derived from mutants by crossing these animals to promoter (referred to here.