| Summary: | 碩士 === 國立臺灣大學 === 分子與細胞生物學研究所 === 98 === Ras-realated nuclear protein (Ran) has been reported to be involved in cell division by modulating assembly of tubulin and regulating nucleocytoplasmic transport. However, the biological function of Ran in embryonic development is unknown. In this study, we take the advantage of zebrafish as an animal model in developmental biology to study the molecular mechanism that how Ran is involved in eyes development. Using whole mount in situ hybridization showed ran transcripts were ubiquitously expressed throughout the whole embryo before gastrula period, but restrictedly expressed in brain and eyes after the pharyngula. We injected the anitsense morpholino oligonucleotides (MO) into zebrafish embryos to specifically knockdown Ran translation, and observed defects in eyes and brain. Particularly, the occurrence of microphthalmos was extremely predominant, in which the order layers with differentiated retinal cells was not so pronounced as that in wild-type eyes. Furthermore, we observed both the BrdU signals and the expression of cyclinD1 were declined earlier in the eye filed in embryos injected with ran-MO, indicating that cell cycle exit in retina is defective in ran-morphants. No apoptotic signal in eye field was found either at early (24 hpf) stage or late (72 hpf) embryonic stage by TUNEL assay. The expressions of rx1 and pax6, which are markers of the eye field in early embryonic stage, were only slightly reduced in ran-morphants, indicating that the retinal cells of ran-morphants were mostly specified to neural progenitor cells. However, when we examined the expressions of some retinal differentiating genes such as shh, six3a and p57kip2, a gene down stream of shh, we found that these genes were not expressed in the ran-morphants either at middle (48 hpf) stage or late stage of retinal development. We also observed the retinal neuron proteins such as HuC and Neurolin were repressed. This line of evidences suggested that retinal cells in ran-morphants were undifferentiated, which leaded to failure of developing normal retinal structure. In addition, we found that there was no deltac expression in HuC- and neurolin-negative cells, even nor in ciliary marginal zone, indicating that these cells were no longer progenitor cells. In other words these cells were not only undifferentiated but also unproliferated at late stage. Furthermore, the proneural gene ath5, which is the first gene to determine the cell fate of developing ganglion cell layer, and neuroD, which is needed to determine the cell fate of amacrine cell and photoreceptor, were dramatically decreased or even unexpressed in retinae of ran-morphants. Taken together, we concluded that the loss of Ran function causes the decrease expression of proneural genes, resulting in the cell fate of retinal cells are not determined to go further differentiate in the middle and late developmental stages, which in turn, the retinal development is disrupted, although the eye field can perform specification and process to retinal progenitor cells in early developmental stage.
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