Phosphatidylinositol synthase is required for lens structural integrity and photoreceptor cell survival in the zebrafish eye

The zebrafish lens opaque (lop) mutant was previously isolated in a genetic screen and shown to lack rod and cone photoreceptors and exhibit lens opacity, or cataract, at 7 days post-fertilization (dpf). In this manuscript, we provide four different lines of evidence demonstrating that the lop phenotype results from a defect in the cdipt (phosphatidylinositol (PI) synthase; CDP-diacylglycerol--inositol 3-phosphatidyltransferase) gene. First, DNA sequence analysis revealed that the lop mutant contained a missense mutation in the lop open reading frame, which yields a nonconservative amino acid substitution (Ser-111-Cys) within the PI synthase catalytic domain. Second, morpholino-mediated knockdown of the cdipt-encoded PI synthase protein phenocopied the cdiptlop/lop mutant, with abnormal lens epithelial and secondary fiber cell morphologies and reduced numbers of photoreceptors. Third, microinjection of in vitro transcribed, wild-type cdipt mRNA into 1-4 cell stage cdiptlop/lop embryos significantly reduced the percentage of larvae displaying lens opacity at 7 dpf. Fourth, a cdipt retroviral-insertion allele, cdipthi559, exhibited similar lens and retinal abnormalities and failed to complement the cdiptlop mutant phenotype

Using the Tg(nrd:egfp)/albino Zebrafish Line to Characterize In Vivo Expression of neurod

In this study, we used a newly-created transgenic zebrafish, Tg(nrd:egfp)/albino, to further characterize the expression of neurod in the developing and adult retina and to determine neurod expression during adult photoreceptor regeneration. We also provide observations regarding the expression of neurod in a variety of other tissues. In this line, EGFP is found in cells of the developing and adult retina, pineal gland, cerebellum, olfactory bulbs, midbrain, hindbrain, neural tube, lateral line, inner ear, pancreas, gut, and fin. Using immunohistochemistry and in situ hybridization, we compare the expression of the nrd:egfp transgene to that of endogenous neurod and to known retinal cell types. Consistent with previous data based on in situ hybridizations, we show that during retinal development, the nrd:egfp transgene is not expressed in proliferating retinal neuroepithelium, and is expressed in a subset of retinal neurons. In contrast to previous studies, nrd:egfp is gradually re-expressed in all rod photoreceptors. During photoreceptor regeneration in adult zebrafish, in situ hybridization reveals that neurod is not expressed in Müller glial-derived neuronal progenitors, but is expressed in photoreceptor progenitors as they migrate to the outer nuclear layer and differentiate into new rod photoreceptors. During photoreceptor regeneration, expression of the nrd:egfp matches that of neurod. We conclude that Tg(nrd:egfp)/albino is a good representation of endogenous neurod expression, is a useful tool to visualize neurod expression in a variety of tissues and will aid investigating the fundamental processes that govern photoreceptor regeneration in adults

Immunohistochemical evidence for multiple photosystems in box jellyfish

Cubomedusae (box jellyfish) possess a remarkable visual system with 24 eyes distributed in four sensory structures termed rhopalia. Each rhopalium is equipped with six eyes: two pairs of pigment cup eyes and two unpaired lens eyes. Each eye type probably captures specific features of the visual environment. To investigate whether multiple types of photoreceptor cells are present in the rhopalium, and whether the different eye types possess different types of photoreceptors, we have used immunohistochemistry with a range of vertebrate opsin antibodies to label the photoreceptors, and electroretinograms (ERG) to determine their spectral sensitivity. All photoreceptor cells of the two lens eyes of the box jellyfish Tripedalia cystophora and Carybdea marsupialis displayed immunoreactivity for an antibody directed against the zebrafish ultraviolet (UV) opsin, but not against any of eight other rhodopsin or cone opsin antibodies tested. In neither of the two species were the pigment cup eyes immunoreactive for any of the opsin antibodies. ERG analysis of the Carybdea lower lens eyes demonstrated a single spectral sensitivity maximum at 485 nm suggesting the presence of a single opsin type. Our data demonstrate that the lens eyes of box jellyfish utilize a single opsin and are thus color-blind, and that there is probably a different photopigment in the pigment cup eyes. The results support our hypothesis that the lens eyes and the pigment cup eyes of box jellyfish are involved in different and specific visual tasks

Age-Related Cone Abnormalities in Zebrafish with Genetic Lesions in Sonic Hedgehog

PURPOSE. Sonic hedgehog (Shh) signaling is essential for photoreceptor differentiation and retinal cell survival in embryonic zebrafish. The study was conducted to determine whether adult heterozygous carriers of mutant alleles for the shh gene display retinal abnormalities. METHODS. Retinal cryosections from young, middle-aged, and senescent wild-type and sonic-you ϩ/Ϫ (syu ϩ/Ϫ ) zebrafish were probed with retinal cell type-specific markers. Contralateral retinal flatmounts from these fish, and from adult albino zebrafish subjected to light-induced photoreceptor damage followed by regeneration, were hybridized with blue cone opsin cRNA for quantitative analysis of the blue cone pattern. Retinal expression of shh mRNA was measured by quantitative RT-PCR. RESULTS. Regions of cone loss and abnormal cone morphology were observed in the oldest syu ϩ/Ϫ zebrafish, although no other retinal cell type was affected. This phenotype was agerelated and genotype-specific. Cone distribution in the oldest syu ϩ/Ϫ zebrafish was predominantly random, as assessed by measuring the short-range pattern, whereas that of wild-type fish and the younger syu ϩ/Ϫ zebrafish was statistically regular. A measure of long-range pattern revealed atypical cone aggregation in the oldest syu ϩ/Ϫ zebrafish. The light-treated albino zebrafish displayed random cone patterns immediately after light toxicity, but showed cone aggregation on regeneration. Retinas from the syu ϩ/Ϫ fish showed reduced expression of shh mRNA compared with those of wild-type siblings. 9 In addition, Shh stimulates proliferation of rat Müller glia and the generation of rhodopsin-expressing cells in vitro, and in vivo subsequent to retinal damage. CONCLUSIONS. The The use of anamniote vertebrate models has uncovered important roles for Hedgehog signaling in the development of rod and cone photoreceptors and the RPE. In zebrafish embryos, reduction in Shh signaling through molecular, pharmacologic, or genetic techniques results in multiple retinal abnormalities including reduced rod and cone photoreceptor differentiation. Many inherited retinal degenerative diseases, including those related to age, are characterized by rod and/or cone photoreceptor death. 18 As a complement to the mouse models, zebrafish offer several advantages for studying retinal disorders. For example, zebrafish have a duplex retina with a substantial number of cone photoreceptors arranged in a precise mosaic that can be quantitatively assessed. 23 Zebrafish are also a model for aging and age-related disease