Studies On Apoptosis And Molecular Differentiation During Early Eye Development Of Toad, Bufo Raddei Stranch

The retinal development of vertebrate is a complicated morphology and molecular biology process. There is cell differentiation and trandifferentiation when optic cup forms into adult retina. But the biological mechanism of the morphological changes when presumptive retinal pigmented epithelium (PRPE) and presumptive neural retina (PNR) froms is not clear, and the interrelated studies is rarely. In order to study the apoptosis and molecular differentiation in development of optic vesicle, morphological and immunochemistry technology were used.The present study mainly focused on the toad, Bufo raddei Strauch. Firstly, morphologic structure of optic vesicle was observed by haematoxylin-eosin (HE) staining. Then, the TUNEL staining was used to detect apoptosis in PRPE and PNR. At last, in order to study the molecular differentiation in optic vesicle, glial fibrillary acidic protein (GFAP) and RT97and important enzyme in pigment synthesize-tyrosinase (Tyr) were used to peculiarly mark the different proteins in the development of optic vesicle by immunochemistry technology.The morphological staining results indicated that the morphologic structure of PRPE and PNR changed distinctly in development of optic vesicle. The evaginations of the diencephalon formed optic vesicle. Optic vesicle push out and connect with the surface ectoderm. PRPE thinned, corresponding PNR thicked. Apoptosis was found in the developmental stages of optic vesicle, but density of apoptotic cells gradually decreased in the process of optic vesicle development. At same developmental stage, the density of apoptotic cells was more in ventral and anterior PNR respectively (P<0.01). The density of apoptotic cells in dorsal-ventral axis of retina was more than that in anterior-posterior axis, in addition, the density of apoptotic cells in PRPE was more than in PNR (P<0.01).The results showed the same trend of GFAP and RT97. The expression level of GFAP and RT97was more at formation of optic vesicle (FOV,0h) in PRPE, but less in PNR. At11h after FOV, the expression level of GFAP and RT97increased in PNR, and decreased to the lowest in PRPE. The expression level of GFAP and RT97were more in ventral and anterior PNR respectively, and more in anterior-posterior axis than in dorsal-ventral axis at the same developmental stage (P<0.01). At the stage of FOV, the expression level of Tyr was lower in PRPE and more in PNR. The expression level of Tyr increased in PRPE and decreased in PNR at late developmental stages. At the dorsal-ventral axis of retina, the expression level of Tyr was more in ventral PRPE (P<0.01). The expression level of Tyr was more in anterior PRPE at the early developmental stages of optic vesicle (P<0.01), but was more in posterior PRPE at late developmental stages (P<0.01). Compared with the stage of FOV, the expression levels of GFAP, RT97and Tyr were significantly different (P<0.01).Apoptosis is a normal biological event which formed a spatio-temporal pattern in retinal development of amphibian. At dorsal-ventral axis of optic vesicle, the density of apoptotic cells was higher than that in anterior-posterior axis of optic vesicle. It suggested that apoptosis might play a role in dorsal-ventral axis of optic vesicle. The density of apoptotic cells was higher in PRPE that in PNR at the same developmental stage and PRPE becomes thinner than PNR. Therefore the apoptosis might be one of the reasons to explain this morphological differentiation, especially during early eye development.The differences between expression levels of GFAP and RT97and Tyr showed that molecular differentiation occurred in development of optic vesicle. Both PNR and PRPE come from the same tissue, so GFAP, RT97and Tyr expressed in them at first. But the expression level of GFAP, RT97and Tyr was different. All the data suggest that different expression level of three proteins may be due to the different positions of PNR and PRPE in the optic vesicle. The intersection of expression curves of all three proteins was at5-6h after FOV, it is the time that optic vesicle directly contact with the covered ectoderm. From the point of molecular differentiation, the special molecular differentiation starts at the time after the contact of optic vesicle with ectoderm. We suggest that three differential proteins begin to show the differentiation of different cell type in retinal development. After the moment of intersection, neurons, gilal cells and pigment cells become to express special proteins according to their future function, rather than their orientation.