Zebrafish (Danio rerio) catecholaminergic neuron RNA-seq based transcript profiles

This project aimed at identifying developmental stage specific transcript profiles for catecholaminergic neurons in embryos and early larvae of zebrafish (Danio rerio). Catecholaminergic neurons were labeled using transgenic zebrafish strains to drive expression of GFP. At stages 24, 36, 72 and 96 hrs post fertilization, embryos were dissociated and GFP expressing cells sorted by FACS. Isolated RNAs were processed using either polyA selection and libray generation or NanoCAGE. This is the first effort to determine stage specific mRNA profiles of catecholaminergic neurons in zebrafish. Catecholaminergic neurons were labeled by four different strategies: (1) 24 hrs old embryos: we used the ETvmat2:GFP transgenic line (Wen et al. 2007). Visualization of monoaminergic neurons and neurotoxicity of MPTP in live transgenic zebrafish. Dev Biol. 2008 Vol 314 p84-92) which at this early stage labels catecholaminergic neurons in posterior tuberculum and locus coeruleus; (2) 24 hrs old embryos: we used Tg(otpb.A:egfp)zc48 transgenic line (Fujimoto et al. Identification of a dopaminergic enhancer indicates complexity in vertebrate dopamine neuron phenotype specification. Dev Biol 2011, Vol 352, p393–404) which at this stage label ventral diencephalic dopaminergic neurons and some preoptic neurons. (3) For 72 and 96 hrs old zebrafish larvae we used a th:GFP BAC transgenic lines that labels catecholaminergic neurons (Tay et al., Comprehensive catecholaminergic projectome analysis reveals single-neuron integration of zebrafish ascending and descending dopaminergic systems. Nat Comms 2011 Vol 2, 171; also: T. Leng and W. Driever, unpublished). (4) for the 36 and 48 hrs old zebrafish larvae we used a th:Gal4VP16 driver and UAS:EGFP responder transgenic line system to label catecholaminergic cells (Fernandes et al., Deep brain photoreceptors control light-seeking behavior in zebrafish larvae. Curr Biol. 2012 Vol 22 DOI 10.1016/j.cub.2012.08.016). We used the different transgenic lines, because lines (3) and (4) do not efficiently label catecholaminergic neurons at early stages, while lines (1) and (2) also have GFP expression in several other non-catecholaminergic populations at later stages of development. Embryos were dissociated and catecholaminergic neurons were FACS sorted from GFP-tagged zebrafish (Manoli and Driever, 2012, Cold Spring Harbor Protoc. DOI 10.1101/pdb.prot069633). RNA was either processed for NanoCAGE, or mRNA was isolated and amplified. cDNA was sequenced by Illumina technique. This data submission is a series of data files consisting of three independent experiments with diffrent RNA-Seq depth: Samples 1-4 (NanoCage): Samples 5-8 (RNA-Seq high read numbers), and SAmples 9-12 (RNA-Seq low read numbers).

本研究旨在鉴定斑马鱼（Danio rerio）胚胎及早期幼体中儿茶酚胺能神经元的发育阶段特异性转录本谱。研究通过转基因斑马鱼品系驱动绿色荧光蛋白（GFP）的表达，以标记儿茶酚胺能神经元。在受精后24、36、72和96小时四个阶段，研究人员对胚胎进行解离，并通过荧光激活细胞分选（Fluorescence Activated Cell Sorting，FACS）分离表达GFP的细胞。分离得到的RNA采用polyA富集建库或NanoCAGE技术进行处理。本研究是首次针对斑马鱼儿茶酚胺能神经元的阶段特异性mRNA谱开展的系统性分析。

研究采用四种不同策略标记儿茶酚胺能神经元：
1. 24小时龄胚胎：使用ETvmat2:GFP转基因品系（Wen等，2007：活体转基因斑马鱼中单胺能神经元的可视化及MPTP的神经毒性，《发育生物学》2008年第314卷，第84-92页），该品系在该早期阶段可标记后脑结节与蓝斑中的儿茶酚胺能神经元；
2. 24小时龄胚胎：使用Tg(otpb.A:egfp)zc48转基因品系（Fujimoto等，《脊椎动物多巴胺神经元表型特化的复杂性：多巴胺能增强子的鉴定》，《发育生物学》2011年第352卷，第393–404页），该品系在该阶段可标记腹侧间脑多巴胺能神经元及部分视前神经元；
3. 72和96小时龄的斑马鱼幼体：使用th:GFP细菌人工染色体（Bacterial Artificial Chromosome，BAC）转基因品系，可特异性标记儿茶酚胺能神经元（Tay等，《全面儿茶酚胺能投射组分析揭示斑马鱼上行和下行多巴胺能系统的单神经元整合机制》，《自然-通讯》2011年第2卷，第171页；另有：T. Leng与W. Driever，未发表数据）；
4. 36和48小时龄的斑马鱼幼体：使用th:Gal4VP16驱动子与UAS:EGFP应答子组成的转基因系统标记儿茶酚胺能细胞（Fernandes等，《深部脑光感受器调控斑马鱼幼体的趋光行为》，《当代生物学》2012年第22卷，DOI: 10.1016/j.cub.2012.08.016）。

之所以选用多种转基因品系，是因为品系3和4无法在发育早期高效标记儿茶酚胺能神经元，而品系1和2在发育后期会在多种非儿茶酚胺能细胞群中出现GFP异位表达。

研究人员通过解离胚胎，从GFP标记的斑马鱼细胞中经FACS分离得到儿茶酚胺能神经元（Manoli与Driever，2012，《冷泉港实验方案》，DOI: 10.1101/pdb.prot069633）。提取的RNA分别通过NanoCAGE技术处理，或分离mRNA并进行扩增。最终通过Illumina测序技术对cDNA进行测序。

本次提交的数据包含一系列数据文件，涵盖三组独立实验，其RNA测序深度各不相同：样本1-4（NanoCAGE组）、样本5-8（高测序读长RNA-Seq组）及样本9-12（低测序读长RNA-Seq组）。