Transcriptome analysis for tunic cells of the ascidian, Ciona intestinalis

本研究旨在为目前EvoDevo研究的重要模式生物--海鞘类动物--海鞘细胞建立分子生物学基础。尽管鳞片是该动物的主要特征器官，但镶嵌在该器官中的细胞群尚未真正被研究。本研究最初的计划由两部分组成，即(1)对鳞片细胞中表达的RNA进行解码，(2)对鳞片细胞进行分子和细胞研究。为了避免这个问题，我们对原计划进行了如下修正：(A)我们收集了被衣细胞的冰冻样本，(B)我们提供了从被衣细胞中分离RNA的持续需求-研究和(C)我们设计了一种定制的微阵列，用于未来在被衣细胞中的基因表达分析。(见MatSumae等人)。为了推进上述战略中的第二个战略，我们关注作为鳞片的主要成分的纤维素丝，它是分析鳞片细胞的障碍。在本研究的合作研究员HiRose教授的建议下，我们克服了第二个困难。我们建立了一种用玻片孵育被膜细胞的方法，将被膜细胞与被膜器官分离。这种方法使我们能够通过DAPI染色观察到被膜细胞及其核的清晰图像。我们用我们的方法从含有至少三种类型细胞的粗鳞片中分离出两种类型的鳞片细胞。我们预计，这种方法将使各种分析方法成为可能，包括肠衣菌的原位杂交和细胞培养。至于未来的前景，由于我们已经收集了用于RNA提取的衣器样本，我计划在未来两年内对衣细胞的RNA序列进行解码。

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.