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| Identification of zebrafish shield organizer-specific genes |
| Received:March 14, 2016 |
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| DOI：10.3969/j.issn.1005-4847.2016.05.001 |
| KeyWord:Zebrafish;Shield organizer;FACS;RNA deep sequencing;in situ hybridization |
| Author | Institution |
| 万传璐 |
安徽大学生命科学学院, 合肥 |
| 闫一芳 |
中国科学院动物研究所膜生物学国家重点实验室, 北京 |
| 曹羽 |
中国科学院动物研究所膜生物学国家重点实验室, 北京 |
| 王强 |
中国科学院动物研究所膜生物学国家重点实验室, 北京 |
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| Abstract: |
| Objective During zebrafish gastrulation, dramatic movements rearrange cells into three germ layers and contribute to the formation of the shield organizer, which acts as a dorsal signal center to pattern the body axis. Global identification of shield organizer-specific genes in early gastrulas will be valuable for studying the regulatory cascades during organizer formation and body axis establishment. Methods Tg(gsc:GFP) transgenic embryos express GFP in the shield organizer, which is controlled by a 1.8 kb gsc promoter. Flow cytometry technology and RNA deep sequencing analysis were applied to isolate GFP positive cells from the Tg(gsc:GFP) transgenic embryos and systematically uncover the genes highly expressed in the dorsal organizer. Subsequently, the expression of shield organizer-specific genes was further confirmed by quantitative real-time PCR and whole mount in situ hybridization method during zebrafish embryonic development. Results GFP-positive cells exceeding 96% purity were isolated from shield-stage Tg(gsc:GFP) transgenic embryos and 657 organizer highly expressed genes were identified through RNA deep sequencing analysis. The results of in situ hybridization experiments revealed that a number of genes including KIAA1324, ripply1,twist2, isthmin1, nme4, zgc174153 and rrbp1b were expressed in shield organizer during zebrafish gastrulation. Conclusions The identification of these shield organizer-specific genes in the current study provides useful clues to explore the zebrafish developmental functions in further studies. |
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