RNA-seq analysis of Slc39a8 (neo/neo) versus wild-type mice: yolk sac, placenta and fetal liver, kidney, lung, heart and cerebellum

Slc39a8 encodes ZIP8, a ubiquitous divalent cation/bicarbonate symporter expressed in pluripotent mouse embryonic stem cell; ZIP8 influxes Zn2+, Mn2+ and Fe2+. Slc39a8(neo/neo) knockdown mice globally exhibit 10-15% of wild-type ZIP8 mRNA and protein levels, and show a pleiotropic phenotype of stunted growth, neonatal lethality, multi-organ dysmorphogenesis, and dysregulated hematopoiesis manifested as severe anemia. Herein we performed transcriptomics of GD13.5 yolk sac and placenta, and GD16.5 liver, kidney, lung, heart and cerebellum, comparing Slc39a8 (neo/neo) with wild-type. Meta-data analysis of differentially-expressed genes revealed 29 unique genes from all tissues -- having enriched GO categories associated with hematopoiesis and hypoxia and KEGG categories of complement, response to infection, and the coagulation cascade -- consistent with dysregulated hematopoietic stem cell fate. Based on transcription factor (TF) profiles in the JASPAR database, and searching for TF-binding sites enriched by Pscan, numerous genes encoding zinc-finger TFs and associated with hematopoietic stem cell functions were identified. We conclude that, in this mouse model, deficient ZIP8-mediated Zn2+ transport affects zinc-finger (e.g. GATA proteins) and other transcription-factors (e.g. TAL1) predominantly in yolk sac, strongly supporting the observed dysmorphogenesis and anemia phenotype. mRNA samples of Slc39a8(+/+) and Slc39a8 (neo/neo) yolk sacs and placentas at GD13.5, fetal liver, kidney, lung, heart and cerebellum at GD16.5 were profiled in triplicate using Illumina HiSeq.Slc39a8编码ZIP8，这是一种广泛表达于多能小鼠胚胎干细胞的普遍性二价阳离子/碳酸氢盐协同转运蛋白；ZIP8可介导Zn²+、Mn²+与Fe²+的跨膜内流。Slc39a8(neo/neo)敲低小鼠的整体ZIP8 mRNA及蛋白表达水平仅为野生型小鼠的10%~15%，并呈现多效性表型：生长迟缓、新生致死、多器官形态发生异常，以及以重度贫血为临床表现的造血功能失调。本研究对妊娠第13.5天（GD13.5）的卵黄囊、胎盘，以及妊娠第16.5天（GD16.5）的肝脏、肾脏、肺脏、心脏与小脑开展转录组测序分析，比较Slc39a8(neo/neo)小鼠与野生型小鼠的基因表达差异。对差异表达基因进行元数据分析后，从所有检测组织中共筛选得到29个独特的差异基因，这些基因的富集基因本体（Gene Ontology, GO）功能类别与造血过程、缺氧应答相关，而富集的京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路则涉及补体系统、感染应答与凝血级联反应，这与造血干细胞命运调控异常的结论相符。基于JASPAR数据库中的转录因子（Transcription Factor, TF）谱信息，并通过Pscan软件富集转录因子结合位点，本研究鉴定出大量编码锌指转录因子且与造血干细胞功能密切相关的基因。本研究认为，在该小鼠模型中，ZIP8介导的Zn²+转运缺陷主要在卵黄囊中影响锌指类转录因子（如GATA家族蛋白）及其他转录因子（如TAL1）的功能，有力佐证了观测到的多器官形态发生异常与贫血表型。本研究以三重复的方式，使用Illumina HiSeq测序平台对GD13.5时Slc39a8(+/+)与Slc39a8(neo/neo)小鼠的卵黄囊、胎盘，以及GD16.5时的胎肝、肾脏、肺脏、心脏与小脑的mRNA样本进行了转录组测序分析。