Modified whole-mount in situ hybridisation and immunohistochemistry protocols without removal of the vitelline membrane in the appendicularian Oikopleura dioica
Modified whole-mount in situ hybridisation and immunohistochemistry protocols without removal of the vitelline membrane in the appendicularian Oikopleura dioica
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改良的整体原位杂交和免疫组织化学方案，无需去除阑尾 Oikopleura dioica 中的卵黄膜
DOI：
10.1007/s00427-017-0588-1
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发表时间：
2017
影响因子：
2.4
通讯作者：
Nishida Hiroki
中科院分区：
文献类型：
--
作者：
Onuma Takeshi A.;Matsuo Masaki;Nishida Hiroki
非蝌蚪类的Oikopleura dioicae是一种在其一生中保持蝌蚪形状的无脊椎脊索动物。其简单透明的身体，不变的细胞谱系，快速的发展和可用的基因组和转录组资源使其成为发育生物学研究的一个有前途的模式生物。然而，对O.由于人工去除卵黄膜的费力和耗时的过程，雌雄异体受到限制，因为孵化前胚胎的去卵黄化导致正常发育的失败。因此，在这项研究中，改良的程序开发的整装原位杂交（WISH）和免疫组织化学（WIHC）。该方案能够快速检测mRNA或蛋白质，而无需对每个标本进行手动检测。关键步骤是在固定前用0.05%的放线菌素E对活胚的卵黄膜进行简单处理。处理2分钟对于反义RNA探针和抗体穿透卵黄膜是最佳的。该WISH方案适用于显色和荧光酪胺信号放大反应。使用新的协议，我们发现了8个基因的组织特异性表达的尾肌，躯干表皮，心脏，咽，食道，胃或鳃开口的发展幼虫。该方法还允许使用抗FLAG抗体通过WIHC检测外源FLAG标记的组蛋白增强的绿色荧光蛋白。这项研究提供了一个有用的和方便的工具，在这个简单的脊索动物模型研究空间和时间的基因表达模式，并应有利于处理大量的遗传数据，从转录组为基础的方法和其他技术，如治疗与化学抑制剂。
The appendicularianOikopleura dioicais a planktonic chordate that retains a tadpole shape throughout its life. Its simple and transparent body, invariant cell lineages, fast development and available genome and transcriptome resources make it a promising model organism for research in developmental biology. However, large-scale analysis of gene expression inO. dioicais limited owing to the laborious and time-consuming process of manual removal of the vitelline membrane, because devitellinisation of pre-hatching embryos causes failure of normal development. Therefore, in this study, modified procedures were developed for whole-mount in situ hybridisation (WISH) and immunohistochemistry (WIHC). This protocol enables rapid mRNA or protein detection without a manual devitellination step for each specimen. The critical procedure is brief treatment of the vitelline membrane of living embryos with 0.05% actinase E before fixation. Two minutes of treatment was optimal for the penetration of antisense RNA probes and antibodies through the vitelline membrane. This WISH protocol was applicable for chromogenic and fluorescent tyramide signal amplification reactions. Using the new protocol, we found eight genes with tissue-specific expression in the tail muscle, trunk epidermis, heart, pharynx, oesophagus, stomach or gill openings of developing larvae. This procedure also allowed for the detection of exogenous FLAG-tagged histone-enhanced green fluorescent protein by WIHC using anti-FLAG antibody. This study provides a useful and convenient tool for studying spatial and temporal gene expression patterns in this simple chordate model and should facilitate handling large amounts of genetic data from transcriptome-based approaches and other techniques such as treatments with chemical inhibitors.
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影响因子：
2.7
作者：
Bassham, S;Postlethwait, J
通讯作者：
Postlethwait, J
Oikopleura dioica 成虫的内部和外部形态：扫描电镜研究
DOI：
--
发表时间：
2015
期刊：
影响因子：
--
作者：
Takeshi A. Onuma;Miho Isobe;Hiroki Nishida
通讯作者：
Hiroki Nishida
阑尾 Oikopleura dioica 幼虫发育过程中的长距离细胞迁移
DOI：
--
发表时间：
2013
期刊：
影响因子：
--
作者：
Kishi K;Onuma TA;Nishida H
通讯作者：
Nishida H
阑尾 Oikopleura longicauda 中的肌肉肌动蛋白基因和肌肉细胞：尾索动物肌肉组织之间的系统发育关系。
DOI：
10.1002/1097-010x(20000815)288:2
发表时间：
2000
期刊：
The Journal of experimental zoology
影响因子：
--
作者：
A. Nishino;Y. Satou;M. Morisawa;N. Satoh
通讯作者：
N. Satoh