Study On Zebrafish Retinal Development And Effects Of Crb2b On Photoreceptors

Retinitis pigmentosa is a hereditary blindness disease，the worldwide incidenceis19-27/100，000. The leading cause of vision loss is the progressive death ofphotoreceptor cells. The photoreceptor cells are terminally differentiated neurons，they can’t regenerate, once lost cannot be replaced. So it is very important to studyon the mechanism of retinal photoreceptor degeneration.Since the research about zebrafish artificial gynogenesis by the United StatesUniversity of Oregon geneticist George Streisinger was published in "Nature" in1981,zebrafish as an experimental animal model to study genetic development began tocause great concern of scientists. At present, zebrafish has become one of the mostimportant biological models for vertebrate development. Zebrafish retinalphotoreceptor cells consist mainly of cone cells. Zebrafish has the unique superiorityin the study of some retinal diseases, such as retinitis pigmentosa, age related maculardegeneration and so on. The zebrafish is excellent as a model animal.One key regulator of epithelial polarity is the highly conserved Crumbs proteincomplex, the central constituent of which, Crumbs (Crb), was initially discovered inDrosophila melanogaster as an essential protein for normal photoreceptormorphogenesis and zonula adherens junction formation and maintenance. There isonly one Crb gene in Drosophila. There are three vertebrate CRB–CRB1, CRB2andCRB3. CRB1gene mutation is associated with several types of autosomal recessiveinherited retinal degenerations in humans, including retinitis pigmentosa and Lebercongenital amaurosis. However, there is no clear genotype-phenotype correlation forCRB1mutations, which suggests that other components of the Crumbs proteincomplex may affect severity of retinal disease.There are five zebrafish Crb–Crb1, Crb2a, Crb2b, Crb3a and Crb3b. Inzebrafish, the two Crb2genes (Crb2a and Crb2b) have been implicated in retinadevelopmental and morphological defects. The Crb2b proteins are predominantlyexpressed at the photoreceptor inner segments in the zebrafish retina. Crb2b mediatea cell-cell adhesion function, which plays an essential role in the integrity ofphotoreceptor layer and cone mosaics. The function of zebrafish Crb2b is similar tothat of human CRB1, studying the effect of Crb2b on zebrafish photoreceptors helpsto further reveal the pathogenesis of degenerative retinal disease such as retinitispigmentosa.To study retinal development and degeneration, it is desirable to quantifyprecisely how the retina loses cells in spatial and temporal fashions. The premise is tomaster the cell numbers of different developmental stages and regions in normalretina. A direct way to quantify retinal cells is to count the number of cell nuclei underlight microscopy because each retinal cell has one cell nucleus. First, we combinedJB-4plastic embedding and Feulgen staining to visualize zebrafish retinal cell nuclei.We analyzed the effects of JB-4section thickness, extent of HCl hydrolysis, andSchiff reaction conditions on nuclear staining and visualization to optimize a protocolthat produces sensitive and specific nuclear staining of the zebrafish retina. We foundthat higher HCl concentrations and longer incubation produced stronger staining thanlower HCl concentrations and shorter incubation time. And lower HCl concentrationscan be combined with longer incubation to achieve similar staining results. We foundthat staining became stronger with longer Schiff incubation but reached the maximumafter2hour. Individual nuclei showed more clear boundaries in2μm sections than in3μm,4μm, and6μm sections. Feulgen staining of2μm thick sections was also strong and sensitive enough to distinguish the euchromatic from heterochromatic regions incone nuclei. Feulgen staining provided a more specific and stable staining thanmethylene blue-azure II and nuclear fast red staining. Feulgen staining wascompatible with whole mount in situ hybridization. In summary, we optimize aprotocol that produces sensitive and specific nuclear staining of the zebrafish retina.This simple and robust method may be utilized for cell quantification and to examinethe global chromatin organization in cone photoreceptors. Feulgen staining and wholemount in situ hybridization can be combined to visualize the nuclei and mRNAexpression patterns simultaneously.Second, we counted the numbers of ganglion cells, innernuclear layer cells,horizontal cells, rod cells and cone cells in different retinal regions of adult wildtypezebrafish. We compared the changes of cell numbers in the same retinal region ofdifferent zebrafish ages and different regions of a certain age zebrafish to find thewildtype zebrafish retinal developing characteristics. The results showed that ganglioncells, innernuclear layer cells, horizontal cells, rod cells and cone cells arrangedregularly in each retinal region. In the early stage, zebrafish retinal cells developedfrom the beginning of the periphery, then gradually extended to the center and twosides. Finally, the special regional distribution characteristics were formed. Thecentral region was the largest number of retinal cells in addition to rod cells, then thenumber of cells decreased gradually to both sides, causing the formation of normaldistribution. The posterior paracentral region was the largest number of rod cells inthe retinal anterior-posterior direction, then the number of cells decreased gradually toboth sides; the dorsal paracentral region was the largest number of rod cells in theretinal ventral-dorsal direction, then the number of cells decreased gradually to bothsides, causing the formation of abnormal distribution. The general regularity of retinalcell number temporally was that the retinal cell number decreased totally along withthe age growth of zebrafish, the number of rod cells in local regions increased in theearly stage. The experimental results provide basic data for further revealing theretinal development mechanism and studying the pathogenesis of degenerative retinopathy. In addition, the experimental results showed that the photoreceptor cellnumber presented overall down trend along with the age growth of zebrafish; and themost obvious decline happened in later period. Human age-related maculardegeneration is the photoreceptor cell degeneration disease, and it develops alongwith the age growth. The pathogenesis of age-related macular degeneration can befurther revealed through the basic data provided by the study that retinal cone cells diegradually with age, especially die largely in later period.Last, we counted the retinal cell number in different retinal regions of twodifferent age stages of pt108b adult zebrafish，and observed the spatial distribution ofpt108b transgenic zebrafish retinal cells. We compared the cell number in the sameretinal region and spatial distribution of pt108b adult zebrafish with the same age ofwildtype zebrafish to discuss the effect of Crb2b on photoreceptors. The resultsshowed that the distribution of all kinds of cells spatially in the pt108b transgeniczebrafish had no regular rules like the wildtype zebrafish. The layered structure ofpt108b transgenic zebrafish retinal cells was destroyed，cells arranged loosely，thecone cells were extremely sparse；cells arranged in disorder especially in lategrowing stage, the cone cells in the individual regions completely lost. The numberof cone cells in each retinal region from different periods in pt108b transgeniczebrafish decreased significantly compared with those in age matched wildtypezebrafish, and the number of retinal cone cells was significantly decreased with age;the number of cone cells decreased rapidly in later period, cones almost completelydisappeared in the individual regions. But in later stage, the number of other types ofpt108b transgenic zebrafish retinal cells in addition to cone cells showed differentdegrees of increase in several regions，especially the rod cells in most regions of theretina were significantly increased. The experimental results indicate that Crb2b isthe key protein in maintaining adult zebrafish normal spatial distribution of retinalcells and survival of cone cells. Suppression of Crb2b function can lead to deaths ofadult zebrafish retinal cone cells, especially mass deaths at the later stage of growthand different degrees of proliferation for retinal ganglion cells, innernuclear layer cells, horizontal cells and rod cells in later stage. In pt108b transgenic zebrafish, thefunction of Crb2b gene was inhibited, leading to progressive deaths of retinal conecells. Our animal model helps to further in-depth study the pathogenesis of retinitispigmentosa.