期刊
DEVELOPMENT GROWTH & DIFFERENTIATION
卷 58, 期 2, 页码 215-224出版社
WILEY
DOI: 10.1111/dgd.12263
关键词
embryonic RNA interference; multi-color fluorescence insitu hybridization; quantification; spider embryo; tyramide
资金
- Narishige Zoological Science Award
- JSPS KAKENHI [24870035, 26440130, 23370095]
- Grants-in-Aid for Scientific Research [24870035, 15K07139, 23370095, 26440130] Funding Source: KAKEN
The simultaneous and quantitative analysis of the expression of multiple genes helps to shed light on gene regulatory networks. We established a method for multi-color fluorescence insitu hybridization (mFISH) for the analysis of cell-type diversification and developmental gene regulation in the embryo of the spider Parasteatoda tepidariorum. This mFISH technique allowed quadruple staining using four types of labels for RNA probes, digoxigenin, fluorescein, biotin, and dinitrophenyl, together with different fluorescent tyramides. To validate the usability of mFISH, we conducted four experiments. First, we distinguished similar gene expression patterns with mFISH, which showed overlaps and differences in the expression domains of anterior patterning hedgehog (hh), orthodenticle (otd), and labial genes at a cellular resolution. Second, we used mFISH to identify early cell types that are internalized on the anterior side. We found that fork head-positive cells were subdivided into two cell types, 012_A08-positive endoderm cells and twist-positive mesoderm cells. Third, we quantified the ratio of expression levels of the odd-paired (opa) gene in the chelicera and pedipalp segments based on the intensity of mFISH signals. Finally, we combined mFISH with embryonic RNA interference. It was possible to identify opa knockdown cell clones and detect the specific reduction of opa and the upregulation of otd and hh expression levels in the same cell clone that formed in the head region. This study proposes that mFISH is a powerful tool for the cell-level analysis of gene regulation and quantification in the spider model.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据