斑马鱼胚胎胚盾特异表达基因的鉴定

目的 由于原肠期细胞的剧烈运动，在斑马鱼胚胎的背侧汇聚形成了称为胚盾（Shield Organizer）的结构，是胚胎发育的信号组织中心，在背腹轴建立和胚层诱导过程中具有关键作用。系统鉴定在胚盾特异表达的基因，可为进一步探讨胚盾形成的机制及其指导胚胎早期发育的分子机理提供参考。方法 Tg(gsc:GFP)转基因鱼在胚盾表达特异的绿色荧光。通过流式细胞分选技术分离富集GFP阳性细胞并提取总RNA进行RNA深度测序，检测可能在胚盾高水平表达的基因。然后利用荧光实时定量PCR(Quantitative real-time PCR,qRT-PCR)和原位杂交技术鉴定若干在胚盾特异表达的基因。结果 从Tg(gsc:GFP)转基因鱼胚胎中分离富集到了纯度超过96%的GFP阳性细胞，RNA深度测序的结果显示有657个基因的表达水平比整胚细胞或GFP阴性细胞高2倍以上。最后，确认了KIAA1324，ripply1，twist2，isthmin1，nme4，zgc174153，rrbp1b等7个基因在胚盾特异表达。结论 系统鉴定到了斑马鱼胚胎胚盾特异表达基因，为下一步研究这些基因的发育生物学功能奠定了基础。

Identification of zebrafish shield organizer-specific genes

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 1.8kb gsc promoter. Flow cytometry technology and RNA deep sequencing analysis were applied to isolate GFP positive cells from 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 methods 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 a number of genes including KIAA1324, ripply1, twist2, isthmin1, nme4, zgc174153 and rrbp1b are 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 their developmental functions in next studies.