Early Development Of Zebrafish Eyes And Dynamic Expression Of CD34 And Vimentin

Zebrafish(Danio rerio)is a kind of freshwater teleosts which has numerous trait of small size,in vitro fertilization,transparent embryos,complete genome information,and high genetic homology with humans.Therefore,it is regarded as an important vertebrate model.In recent years,zebrafish has been widely used in the fields of embryonic development,human disease research,and drug development and screening.In the study of eye disease models,zebrafish is also one of the main choices,and the study of its histology has gradually attracted attention.But at present,more researches are on the eyes of adult fish.Although larvar fish is an important stage of the development of various tissues,organs,and system,the small size of the individual and difficulty in obtaining and fixation materials have increased the difficulty of research.There are also very few reports.In the present study,from the aspect of histomorphology,we elaborated the development of zebrafish eyes structures at 48,72,96,120 hours post fertilization,including the morphological characteristics of the eyes,the ultrastructure of the cells,and the changes of the eye structures in the four stages.At the same time,we utilized the immunofluorescence technique to explore the expression and distribution of CD34 and Vimentin in the retina of zebrafish at the first time.The results of the present research enrich the database of zebrafish early eyes development,and provide a reference for further research of eye-related disease.Experiment Ⅰ The microstructure and ultrastructure of the early development of zebrafish eyesZebrafish eyes structure and gene expression are very similar to humans,and in the field of life sciences,the use of it to construct special ophthalmic disease models is also popular.Therefore,it will help zebrafish eyes application in various fields,when we have a study of the morphological structure and ultrastructure of the zebrafish eyes in the early development.In this experiment,we used hematoxylin-eosin staining and transmission electron microscopy to observe zebrafish eyes in 4 periods,and found that the various structures of the eyes had great changes in different periods.Under the light microscope:retinal stratification and differentiation are obvious in these 4 stages.There are no obvious stratification in the 48 hpf retina,but the pigment layer has formed;In 72 hpf,part of the retina cells differentiate,and three granular layers and two plexiform layers can be observed;In 96 and 120 hpf,each layer of retina cells gradually differentiate into cells with specific structures and functions.In addition,the changes in the structure of the lens,cornea,iris,and mesenchymal cells can be observed in 4 periods.Under the transmission electron microscope,ultrastructural changes in the cornea,lens,photoreceptors and retinal pigmented epithelium can be clearly seen.In 48 hpf,the cornea has formed corneal endothelium and corneal epithelium,and the corneal stroma and other structures are gradually formed at 72-120 hpf.With the eyes development during 48-120 hpf,the lens epithelium transform from monolayer cubical epithelium to monolayer squamous epithelium,and primary lens fibers and secondary lens fibers also increase layer by layer.We can also clearly observe disc in photoreceptor outer segment,ellipsoid which is composed of numerous mitochondrias and myoid containing rough endoplasmic reticulum and other organelles in photoreceptor inner segment.Experiment Ⅱ The expression characteristics of CD34 and Vimentin in the zebrafish retinaCD34 and Vimentin play an important role in embryonic development and differentiation.We utilized the immunofluorescence technique to double-label CD34(red fluorescent)and Vimentin(green fluorescent)on the retina of larva fish,and found that the co-expressed yellow fluorescence mainly distribute in the photoreceptor layer,and the positive reaction gradually increases with the differentiation of the photoreceptor layer.The photoreceptor layer is mainly composed of the inner and outer segment of photoreceptor.But in the eyes development process,part of the pigment epithelial layer structure migrates to the photoreceptor layer.Therefore,the photoreceptor layer maybe also contains RPE structural components.During 48、72、96、120 hpf,the structure of the inner and outer segment of the photoreceptor continuously develop,and the physiological functions gradually enhanced;at the same time,RPE also continues to develop and differentiate,and continues to migrate to the photoreceptor layer,and its phagocytosis also increasingly enhance.The results may indicate that CD34 and Vimentin involve in the development and differentiation of the photoreceptor outer segment and RPE,as well as involve in the renewal of outer segment,and the expression of CD34 and Vimentin will increase as photoreceptor and RPE activities increase.Experiment Ⅲ Optimization of sample preparation and dyeing procedures of zebrafish larva eyesThe newly hatched zebrafish are small in size and difficult to obtain materials.Therefore,the entire individual larva can be used as a fixed object for subsequent paraffin embedding,sectioning,dyeing and so on.This chapter discusses the aspects of light microscopy dealing,paraffin embedding,sectioning,and HE staining of zebrafish larva,and strives to optimize the procedures to provide technical references for scholars in related research fields.The research showed that the result is the best with distinct color and clear structure,when we cut the dehydration time,controlled the transparency time to 2 min,decided the slice thickness is 5 μm,the hematoxylin staining for 40 s,and eosin for 3 min.