Bai Qing, Wei Xiangyun, Burton Edward A
Department of Neurology, University of Pittsburgh, Pittsburgh, PA, USA.
Neurosci Lett. 2009 Jan 16;449(3):252-7. doi: 10.1016/j.neulet.2008.10.101. Epub 2008 Nov 5.
We recently cloned the zebrafish neuronal enolase-2 gene and showed that a 12-kb eno2 promoter element was sufficient to drive transgene expression widely in CNS neurons in vivo from 48h post-fertilization through adulthood. The aim of the present study was to establish the expression pattern of the 12-kb eno2 promoter element in the zebrafish visual system. Endogenous eno2 mRNA was detected in the developing retina from 2 days post-fertilization (dpf), and by 12dpf was localized to the retinal ganglion cell, inner and outer nuclear layers. Similar to endogenous eno2, GFP expression in the retina of Tg(eno2:GFP) larvae was first evident at 2dpf, and by 12dpf intense GFP expression was seen in the retinal ganglion cell and photoreceptor layers, with weaker expression in the inner nuclear layer. We identified cell types expressing the eno2 promoter element by using two complementary strategies: (i) double label immunofluorescence analysis of Tg(eno2:GFP) zebrafish, and (ii) generation of double transgenic zebrafish expressing red fluorescent protein under transcriptional control of the 12-kb eno2 promoter and GFP under a rod- or cone-specific promoter. The 12-kb eno2 promoter was expressed in retinal ganglion cells, amacrine cells, including a subset that co-expressed tyrosine hydroxylase, and rod photoreceptors. These data suggest that abnormalities of vision should be sought in transgenic models of diseases generated using this promoter. Owing to the specific expression of fluorescent reporters in neuronal subpopulations, Tg(eno2:GFP) and Tg(eno2:mRFP) zebrafish may be useful for studies of retinal lamination, neuronal differentiation and synapse formation in the visual system.
我们最近克隆了斑马鱼神经元烯醇化酶-2基因，并表明一个12kb的eno2启动子元件足以在体内驱动转基因从受精后48小时到成年期在中枢神经系统神经元中广泛表达。本研究的目的是确定12kb eno2启动子元件在斑马鱼视觉系统中的表达模式。在受精后2天（dpf）的发育视网膜中检测到内源性eno2 mRNA，到12dpf时，其定位于视网膜神经节细胞、内核层和外核层。与内源性eno2相似，Tg(eno2:GFP)幼虫视网膜中的GFP表达在2dpf时首次明显可见，到12dpf时，在视网膜神经节细胞和光感受器层中可见强烈的GFP表达，在内核层中表达较弱。我们通过两种互补策略鉴定了表达eno2启动子元件的细胞类型：（i）对Tg(eno2:GFP)斑马鱼进行双标记免疫荧光分析，以及（ii）生成双转基因斑马鱼，其在12kb eno2启动子的转录控制下表达红色荧光蛋白，在视杆或视锥特异性启动子下表达GFP。12kb eno2启动子在视网膜神经节细胞、无长突细胞（包括共表达酪氨酸羟化酶的一个亚群）和视杆光感受器中表达。这些数据表明，在使用该启动子产生的疾病转基因模型中应寻找视力异常情况。由于荧光报告基因在神经元亚群中的特异性表达，Tg(eno2:GFP)和Tg(eno2:mRFP)斑马鱼可能有助于视觉系统中视网膜分层、神经元分化和突触形成的研究。
