摘要：视网膜神经前体细胞(RPCs)通过不对称分裂产生视网膜神经节细胞(RGCs)（视网膜最早发生的神经元类型），这一过程伴随着ath5基因（又名atoh7）转录活动的出现。尽管许多已有研究报道ath5转录因子对神经前体细胞产生视网膜神经节细胞是必要的，但迄今为止，尚无研究从单个ath5等位基因位点的视角去探究RPCs进行不对称分裂时ath5等位基因的转录激活是否存在不对称性， 而此种不对称性与之后的RGCs细胞命运会否发生关联。在此项研究中，我们对受精后24-30-36-48 小时不同发育时期的斑马鱼(Danio rerio)视网膜进行了冠状冰冻切片然后分别进行了RNA荧光原位杂交(RNA-FISH)、DNA荧光原位杂交(DNA-FISH)以及两者相结合的Double荧光原位杂交实验(Double-FISH)。RNA-FISH结果显示，受精后36 h的视网膜中，ath5转录活动主要分布于视网膜中央，而随着视网膜发育进程，ath5转录活动将逐步推移至睫状边缘区。之后进行了Double-FISH实验，通过IMARIS软件进行单个细胞的三文重构，初步分析显示，RPCs进行不对称分裂时ath5等位基因的转录激活很可能存在着不对称性，可能会与神经发生中的不对称分裂及细胞命运决定（尤其是RGCs）相关联。26811
毕业论文关键词：视网膜神经前体细胞；不对称分裂；ath5转录活动；等位基因；荧光原位杂交
Allelic-Specific Transcriptional Activation of Ath5 in Neurogenic Asymmetric Cell Division in Danio Rerio
Abstract：Retinal Progenitor Cells (RPCs) can produce Retinal Ganglion Cells (RGCs), as the first type of retinal neurons, by passing through asymmetric pision. During this process, transcription activity of ath5 gene starts expression in these RPCs and their daughter cells. Although transcriptional factor ath5 has been reported to be necessary when RPCs generating RGCs, yet so far, allelic-specific transcriptional activity of ath5 gene has not been explored, which may serves as an key for answering critical question whether ath5 transcriptional activity exhibits asymmetry and whether such asymmetry associates with RGCs cell fate determination. In this study, we collect embryos of wild-type zebrafish embryos at a series of development stage of 24-30-36-48 hours post fertilization, then we obtain coronal frozen-sections of these embryos and conduct separate experiments of RNA fluorescence in situ hybridization (RNA-FISH) as well as DNA fluorescence in situ hybridization (DNA-FISH) experiments. As RNA-FISH results shows, ath5 transcriptional activity exhibits mainly in the central area of 36 hpf retinal coronal section tissues, while this transcription pattern changes and presents restricted pattern near retina ciliary marginal zone at the development stage of 48 hpf. After that, we conduct optimized dual RNA-DNA FISH experiments called Double-FISH，and analyze these data by construction 3-D structure of single cell using IMARIS software. Preliminary results indicate that allelic-specific transcriptional activity of ath5 seems asymmetric, which might associates with neurogenic asymmetric cell pision and subsequent cell fate determination，especially RGCs.
Key words: Retinal Progenitor Cells；asymmetric cell pision；ath5 transcriptional activity；gene alleles；fluorescence in situ hybridization
 目  录
摘要    3
绪论    4
视网膜基本结构    4
视网膜神经前体细胞不对称分裂    4
ath5对调控RGCs细胞命运很重要（必要条件）    5
不对称分裂的研究现状    6
1 材料与设备    7
1.1 试验材料    7
1.2 试验试剂    7
1.3 仪器    7
2 方法    7
2.1 冷冻切片    7 ath5等位基因在斑马鱼神经前体细胞不对称分裂过程中的转录激活状态:http://www.751com.cn/shengwu/lunwen_21100.html