| Inherited photoreceptors diseases are often seen clinically, such as retinitis pigmentosa, Leber's congenital amaurosis and cone-rod dystrophy. It is easy to diagnose, but there is no effective treatment nowadays. Though a lot of virulence genes have been discovered, the exact pathogenic mechanism is still unknown. In order to find the exact pathogenic mechanism, a good animal model is very important.The zebrafish, Danio rerio, offers several distinct advantages as a genetic and embryological model system, including the external fertilization, rapid development and optical clarity of its embryos. Being a vertebrate, the zebrafish has a notochord, blood, heart and vasculature, kidney and optical systems that share many features with corresponding human systems. The zebrafish system bridges the gap between fruit fly/worm and mouse/human genetics, making it feasible to address issues of early development, organ formation, integrative physiology, pharmacology and complex disease. The retina of the zebrafish is very similar to mammal, including human. The retina of the zebrafish is laminated, including neural retina and pigment epithelium. There are three distinct layers and seven major cells in neural retina, six kinds of neuron and Muller's glial cell. The photoreceptors of the zebrafish have the similar shape of human, including cell body and dish-shaped outer segments. So the zebrafish is a main animal model used to study the photoreceptors.CRX gene was first found in 1997, it is a kind of photoreceptors specific transcription factor, localized on chromosome 19q 13.3. In mammal, CRX is mainly expressed in developing and mature rods and cones, weakly expressed in inner nuclear layer, it is also expressed in apophysis ossium. CRX protein recognizes a lot of DNA sequences located in the photoreceptor promoter regions, such as rhodopsin gene, cone cGMP phosphodiesterase β subunit, inter photoreceptors pigment connectin, et al. CRX protein can combine and activate the promoters of these genes. CRX protein can control the expression of the photoreceptor genes. CRX gene is also found in the zebrafish, in the retina and apophysis ossium. Mutations of the CRX genecan induce cone-rod dystrophy, late onset retinitis pigmentosa and congenital amaurosis.In 2000, Nasevicius first used morpholinos to knock down gene expression in zebrafish. Morpholinos can inhibit the translation from the start. Morpholinos microinjection can produce the phenotype of gene mutation. It is a kind of fast and effective gene knock down method, and generally used in the zebrafish study.Retinoic acid is a liposolubal micromolecule. It comes from Vitamin A and is needed in the development of central nerve system and retina. In normal developed zebrafish, retinoic acid is abundant in abdominal part of the retina, where the development of cones and rods is obvious. This means retinoic acid is effective in cone and rod development and eye development. Retinoic acid can also promote the development of the photoreceptors.Part I The retina and photoreceptors development of the zebrafish larvaObjectiveTo study the development of the retina and photoreceptors in zebrafish. To identify that it is feasible to use zebrafish to study retina and photoreceptors. Materials and methodsPrepare zebrafish photoreceptors RNA probe of opsin Rho, SWS1 and SWS2. Collect 72, 96 and 120hpf zebrafish larva, prepare eye histological slice and whole mount in-situ hybridization.Results1. 72hpf, the retina of the zebrafish is laminated, including three cells layers and two plexiform layers. From outer to inner, there are photoreceptors layer, outer plexiform layer, inner nuclear layer, inner plexiform layer and neural ganglion cells layer. 96hpf and 120hpf, the arrangement of the cell layer are further regular, the outer segments of the photoreceptors are further matured, plexiform layers widened. Vitreous cavity starts to emerge between lens and retina.2. 72hpf, the photoreceptors express opsin, the expression range of opsin Rho is more than SWS1 and SWS2. The expressed opsins are mainly in the nasal retina. 96hpf and 120hpf, the expressions of the three opsins are increased, and the expression regions are also increased.ConclusionIt is feasible to use zebrafish as a model to study retina and photoreceptors.Part II The development of photoreceptors after knock-down of CRX gene in zebrafishObjectivesTo study the zebrafish photoreceptors outer segments development and opsin expression after CRX gene knock-down.Materials and methodsCRX-MOs was microinjected to the zygote of the zebrafish. 72 and 96hpf, prepare eye histological slice, electron microscope sample and whole mount in-situ hybridization to study the photoreceptors development and opsin expression.Results1. There was no obvious phenotype change after CRX-MOs microinjection.2. There was no obvious histological change in optical microscope observation after CRX-MOs microinjection.3. 72hpf, there were outer segments development in control group and blank group, but no obvious outer segment was found in CRX group by electron microscope observation. 96hpf, the outer segment of the control and blank group were further developed, the outer segments were found in CRX group, but were smaller than the other two groups.4. 72hpf, opsin Rho, SWS1 and SWS2 were expressed in control and blank group, but the expressions were obviously decreased in CRX group. 96hpf, the expressions of three ospins were further increased in control and blank groups. In CRX group, the expressions were increased compared to 72hpf, but still less than control and blank group.ConclusionCRX gene can influence the development of outer segment of the photoreceptors and opsins expression in zebrafish.Part III The protectant effect of all-trans retinoic acid for CRX gene knockdown model in zebrafishObjectivesTo study if the exogenous all-trans retinoic acid can promote the photoreceptor outer segments development and opsin expression after CRX gene knock-down in zebrafish.Materials and methodsAfter CRX-MOs microinjection, 48hpf, 1×10-7 M all-trans retinoic acid was added into the culture solution. The control group added the same level of the RA dissolvent. Eye histological slice, electron microscope sample and whole mount in-situ hybridization were studied to observe the photoreceptor outer segments development and opsin expressions.Results1. 72hpf, there was no obvious phenotype change among groups. 96hpf, edema was found in CRX+RA group.2. There was no obvious change in retina histology.3. Under electron microscope, 72hpf, outer segments were found in control and CRX+RA groups, but not in CRX group.4. 72hpf, the expressions of opsin Rho, SWS1 and SWS2 can be found in control and CRX+RA groups, but little were found in CRX group.ConclusionAll-trans retinoic acid can promote photoreceptor outer segments development and opsin expression in zebrafish after CRX gene knock down.
|
PDF Full Text Request