Transcriptome profiles of zebrafish larvae from a caffeine-induced anxiety model and a high fat-diet induced obesity model [miRNA-Seq]

Obesity associates with anxiety disorders in adult humans, rodents and fish, although there is also evidence to the contrary. Moreover, it is unknown if this association is developmentally regulated, and which genes are involved. RNA-sequencing differences between caffeine-induced anxious zebrafish larvae, which exhibited greater thigmotaxis, erratic swimming and higher whole-body cortisol levels than matched controls, and high fat diet-fed obese larvae with more numerous and larger peritoneal adipocytes than standard diet controls, were revealed. There were 231 poly(A)+ genes common to both models (padj<0.05) and 75% of the transcripts upregulated (>1.5 fold) in the anxiety model were downregulated (<0.67 fold) in obese larvae. Immune system pathways were overrepresented among these inversely regulated genes. Profile analyses of RNA-seq poly(A)+ and miRNA data were carried out on two zebrafish larval models (15 days post fertilization[dpf]), one of anxiety (induced by a half hour exposure to 100 mg/liter caffeine [CAF], compared to no treatment [NO]) and the other of obesity (induced by feeding 6 dpf larvae a high fat diet [HFD, egg yolk-based] until 15 dpf, compared to a standard diet [SD]). Both models were optimized and validated, the anxiety model by behavioral and cortisol level tests and the obesity model by histology-based adiposity analysis. RNA was extracted from pools of 30 larvae per replicate (3 replicates per treatment [NO, CAF, SD, HFD]), euthanized by ice-cold water and snap-frozen in liquid nitrogen.

尽管存在相悖的研究证据，但肥胖与成年人类、啮齿类动物及鱼类的焦虑障碍存在关联。此外，目前尚不明确这种关联是否受发育调控，以及涉及哪些基因。本研究揭示了咖啡因诱导的焦虑型斑马鱼幼鱼与高脂饮食诱导的肥胖型斑马鱼幼鱼之间的RNA测序（RNA-sequencing）差异：前者相较于匹配对照组表现出更强的趋触性（thigmotaxis）、不规则游泳行为以及更高的全身皮质醇（cortisol）水平；后者则相较于标准饮食对照组拥有更多且体积更大的腹膜脂肪细胞。两个模型共鉴定出231个共有poly(A)+基因（校正后P值padj<0.05）；且焦虑模型中上调超过1.5倍的转录本中，有75%在肥胖幼鱼体内呈现下调（转录倍数小于0.67）。在这些呈反向调控的基因中，免疫系统通路显著富集。本研究针对两种斑马鱼幼鱼模型（受精后15天，15 dpf）开展了RNA-seq poly(A)+转录组与microRNA（miRNA）数据的表达谱分析：其一为焦虑模型，通过将幼鱼暴露于100 mg/L咖啡因（CAF）环境半小时构建，以未处理组（NO）作为对照；其二为肥胖模型，通过从受精后6天（6 dpf）起喂食基于蛋黄的高脂饮食（HFD）直至15 dpf构建，以标准饮食组（SD）作为对照。两种模型均经过优化与验证：焦虑模型通过行为学检测与皮质醇水平测定完成验证，肥胖模型则通过基于组织学的脂肪含量分析完成验证。实验样本为每个处理组（NO、CAF、SD、HFD）各3个生物学重复，每个重复包含30条幼鱼的混合样本；幼鱼经冰水麻醉处死之后，迅速置于液氮中冻存以提取总RNA。