Identification of zebrafish shield organizer-specific genes
Received:March 14, 2016  
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KeyWord:Zebrafish;Shield organizer;FACS;RNA deep sequencing;in situ hybridization
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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      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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