RNAseq profiling of FACS-sorted zebrafish larval macrophages reveals similarities with human M1 and M2 transcriptome signatures

We used different zebrafish transgenic lines to sort macrophages, neutrophils and immature lymphoid cells from 5-6 day old zebrafish larvae and analyzed their transcriptomes. Comparison between the different transcriptomes and gene ontology analysis revealed specificities for each cell population. Comparison with previously published data showed that zebrafish larval macrophages expressed several known human M1 and M2 macrophages. Transcriptome analysis of uninfected and infected macrophages from embryos infected by of Mycobacterium marinum revealed infection induced transcriptional changes and a shift towards M1 transcriptomic signature. Overall design: Embryos were grown into egg water refresh every day and incubated for 5 or 6 days at 28°C. 0.003% 1-phenyl-2-thiourea (Sigma-Aldrich) was added after 1 day to prevent melanisation. After the incubation period, embryos were dissociated into single cell suspension by Trypsin treatment and fluorescent cells were sorted by FACS. RNA extraction and library preparation were performed as previously described. (Rougeot et al., 2014, Methods Mol Biol 1197:41-66). For infection experiments, zebrafish embryos were manually dechorionated at 24 hours post fertilization (hpf) and were infected by injection in the caudal vein of 125 colony forming unit of Mycobacterium marinum M strain expressing GFP. Infected larvae were collected for FACS sorting 5 day post infection.

我们使用不同的斑马鱼转基因品系（zebrafish transgenic lines），从5至6日龄的斑马鱼幼体中分离巨噬细胞（macrophages）、中性粒细胞（neutrophils）以及未成熟淋巴样细胞，并对其转录组（transcriptome）进行分析。对不同转录组开展比较分析并结合基因本体论（Gene Ontology, GO）分析，揭示了各细胞群体的特异性分子特征。与既往发表的研究数据比对后发现，斑马鱼幼体巨噬细胞可表达多种已被报道的人类M1型与M2型巨噬细胞相关分子特征。对感染海分枝杆菌（Mycobacterium marinum）的胚胎来源的未感染与感染巨噬细胞进行转录组分析，结果显示感染可诱导转录组发生改变，并使细胞向M1型转录组特征偏移。

实验设计总览：
将胚胎置于斑马鱼胚胎养殖用水中，每日更换养殖用水，于28℃下培养5或6日。接种1日后添加0.003%的1-苯基-2-硫脲(1-phenyl-2-thiourea, Sigma-Aldrich)以抑制色素沉着。培养周期结束后，采用胰蛋白酶(Trypsin)处理将胚胎解离为单细胞悬液，通过荧光激活细胞分选术(Fluorescence-Activated Cell Sorting, FACS)分选荧光标记细胞。RNA提取与文库构建操作参照已发表方案执行（Rougeot等人，2014年，《分子生物学方法》(Methods Mol Biol)，1197卷：41-66）。
感染实验中，于受精后24小时(hours post fertilization, hpf)手动去除斑马鱼胚胎的卵膜，通过尾静脉注射125菌落形成单位的表达绿色荧光蛋白(GFP)的海分枝杆菌M菌株感染幼体。感染后5日，收集受感染幼体用于FACS分选。