Transcriptome analysis for tunic cells of the ascidian, Ciona intestinalis

本研究旨在建立海鞘鞘被膜细胞的分子生物学基础，海鞘是EvoDevo研究的重要模式生物。尽管被膜是该动物的主要特征器官，但迄今为止，对镶嵌在该器官上的细胞群还没有真正的研究，本研究的最初计划由两部分组成：（1）被膜细胞中表达的RNA的解码和（2）被膜细胞的分子和细胞研究。从被膜细胞中提取RNA用于NGS测序比我预期的要困难，为了避免这个问题，我们在这一时期对原计划进行了如下修改：（a）我们收集了被膜器官的冷冻样品，（B）我们提供了从被膜细胞中分离RNA的持续需求研究，（c）我们设计了一个定制的芯片用于将来的被膜细胞基因表达分析。(See Matsumae et al. 2013 in Section 13.）为了推进上述第二个策略，我们关注了被膜的主要成分纤维素丝作为被膜细胞分析的障碍。在本研究的合作研究者之一Hirose教授的建议下，我们克服了第二个困难。我们建立了一种从被膜器官中分离被膜细胞的方法。这种方法使我们能够通过DAPI染色观察被膜细胞及其核的清晰视图。我们已经证实，两种类型的被膜细胞分离的粗被膜，其中至少含有三种类型的细胞通过我们的方法。我们希望这种方法可以为包括原位杂交和细胞培养在内的多种分析方法提供可能。至于未来的前景，因为我们已经收集了用于RNA提取的被膜器官样本，我计划在未来两年内解码被膜细胞的RNA序列。

This study intend to build a molecular biological basis of tunic cells in an ascidian, Ciona intestinalis, which is now an important model organism of EvoDevo research. Although tunic is the main characteristic-organ of this animal, the group of cells which are inlaying of the organ is not really investigated to date.The original plan of this study was made up of two parts of strategy that are (1) decoding of RNAs expressed in the tunic cells and, (2) molecular and cellular study for the tunic cells. The RNA extraction from tunic cells for NGS sequencing were difficult than I have been expected.To avoid this problem, we corrected the original plan in the term as followings; (a) we collected the frozen sample of tunic-organ, (b) we provided the continued requirement-study to isolate the RNAs from tunic cells and (c) we designed a custom microarray for future analysis of gene expression in tunic cells. (See Matsumae et al. 2013 in Section 13.)In order to forward the second of abovestrategies , we were concerned about the cellulose filament which is the main component of tunic as an obstruction for analysis of tunic cells. On the advices of Prof. Hirose who is one of cooperative researcher of this study, we overcame thissecond difficulty. We established a method to isolate the tunic cells from the tunic-organ by means of incubation of tunic with a glass slide. This method allowed us to observe a clear view of the tunic-cells and its nuclei by DAPI-staining. We have verified that two types of tunic-cells are isolated from the crude tunic that contains at least three types of cells by our method. We expect that this method has made possible for variety of analytical approaches including of in situ hybridization and cell culture of C. intestinalis.As for future perspectives, because we have collected the tunic-organ sample for RNA-extraction, I'm planning for decoding the RNA sequences of tunic cells within around next two years.