Transcriptomic Analysis of the Zebrafish Prion Mutants Supports Conserved Cross-Species Function of the Cellular Prion Protein. Transcriptomic Analysis of the Zebrafish Prion Mutants Supports Conserved Cross-Species Function of the Cellular Prion Protein

Wild-type zebrafish larvae and our prp1ua5003/ua5003;prp2ua5001/ua5001 mutant zebrafish larvae at 3 days post fertilisation were pooled into three biological replicates each of 50 larvae. Each replicate underwent paired end 100-125 and Illumina HiSeq2500 sequencing to a depth of 40 million reads, carried out by Otogenetics (Atlanta, GA). Samples were subsequently processed using the TopHat/Cufflinks bioinformatics pipeline. Alignments were mapped to the zebrafish reference genome, GRCz10. A small amount of wild-type reads were found to contaminate two of the pooled mutant samples and were not included in subsequent analysis. In total there were 1249 genes showing at least a 50% increase (745) in transcript abundance or 50% decrease (504) decrease in transcript abundance in wild-type zebrafish larvae compared to double mutant zebrafish larvae. Subsequent gene ontology analysis shows the biological process showing genes with the biggest decrease in transcript abundance was cell adhesion, and the process with the most genes showing an increase in transcript abundance was Oxidation/Reduction. Our study finds the processes affected by prion knockdown in zebrafish is remarkably similar to other studies in early mice embryos, suggesting a conserved function of the cellular prion protein in the early development of organisms. Overall design: RNA-Sequencing analysis of 3 days post fertlisation whole wild-type and prp1-/-;prp2-/- zebrafish larvae

野生型斑马鱼幼鱼与prp1ua5003/ua5003;prp2ua5001/ua5001双突变型斑马鱼幼鱼在受精后3天时，被分为3个生物学重复组，每个重复组包含50尾幼鱼。每个重复组均采用Illumina HiSeq2500测序平台进行100~125 bp双端测序，测序深度达4000万条读段（reads），测序工作由佐治亚州亚特兰大的Otogenetics公司完成。后续使用TopHat/Cufflinks生物信息学流程对测序样本进行处理。测序比对结果均映射至斑马鱼参考基因组GRCz10。分析发现两份混合的突变样本存在少量野生型读段污染，因此未纳入后续分析流程。与双突变型斑马鱼幼鱼相比，野生型斑马鱼幼鱼中共有1249个基因的转录本丰度发生显著变化：其中745个基因转录本丰度上调至少50%，504个基因转录本丰度下调至少50%。后续基因本体（Gene Ontology, GO）富集分析显示，转录本丰度下调幅度最大的生物学过程为细胞黏附（cell adhesion），而转录本丰度上调基因数目最多的生物学过程为氧化还原（Oxidation/Reduction）。本研究发现，斑马鱼中朊蛋白敲低所影响的生物学过程与早期小鼠胚胎中的相关研究结果高度相似，提示细胞型朊蛋白在生物体早期发育过程中具有保守功能。实验设计方案：对受精后3天的全组织野生型及prp1-/-;prp2-/-双突变型斑马鱼幼鱼进行RNA测序（RNA-Sequencing）分析。