Gene expression in Zfp36l3 wildtype and knockout trophoblast stem cells and placenta

The ZFP36L3 protein is a rodent-specific, placenta- and yolk sac-specific member of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins. These proteins bind to AU-rich elements in target mRNAs, and promote their deadenylation and decay. We addressed the hypotheses that the absence of ZFP36L3 would result in the accumulation of target transcripts in placenta and/or yolk sac, and that some of these would be important for female reproductive physiology and overall fecundity. Mice deficient in ZFP36L3 exhibited decreased neonatal survival rates, but no apparent morphological changes in the placenta or surviving offspring. We found Zfp36l3 to be paternally imprinted, with profound parent-of-origin effects on gene expression. The protein was highly expressed in the syncytiotrophoblast cells of the labyrinth layer of the placenta, and the epithelial cells of the yolk sac. RNA-Seq of placental mRNA from Zfp36l3 KO mice revealed many significantly up-regulated transcripts, whereas there were few changes in KO yolk sacs. Many of the up-regulated placental transcripts exhibited decreased decay rates in differentiated trophoblast stem cells derived from KO blastocysts. Several dozen transcripts were deemed high probability targets of ZFP36L3; these include proteins known to be involved in trophoblast and placenta physiology. The type 1 transferrin receptor mRNA was unexpectedly decreased in KO placentas, despite an increase in its stability in KO stem cells. This receptor is critical for placental iron uptake, and its decrease was accompanied by decreased iron stores in the KO fetus, suggesting that this intrauterine deficiency might have deleterious consequences in later life. Four biological replicates were analyzed for each trophoblast stem cell condition. There are 8 different conditons: wild type and knockout undifferentiated, differentiated 0h ActD, differentiated 2h ActD, differentiated 4h ActD. Five biological replicates were analyzed for wild type and knockout placenta. Three replicates were analyzed for heterozygous placenta. There are two types of heterozygous placentas: maternal or paternal transmission of the mutant allele.ZFP36L3蛋白是CCCH型串联锌指蛋白家族中三联素蛋白（Tristetraprolin, TTP）的成员，且具有啮齿动物特异性、胎盘及卵黄囊特异性。此类蛋白可结合靶mRNA中的AU富集元件（AU-rich elements），并促进靶mRNA发生脱腺苷化与降解。本研究针对两项假说开展验证：ZFP36L3的缺失会导致靶转录本在胎盘和/或卵黄囊中积累，且其中部分转录本对雌性生殖生理及整体生育力具有重要作用。ZFP36L3敲除（Knockout, KO）小鼠的新生存活率显著降低，但胎盘及存活后代无明显形态学异常。研究发现Zfp36l3基因呈父本印记，其表达受显著的亲本起源效应调控。该蛋白在胎盘迷路层的合体滋养层细胞以及卵黄囊上皮细胞中呈高表达状态。对Zfp36l3敲除小鼠的胎盘mRNA进行RNA测序（RNA-Seq）分析后发现，存在大量显著上调的转录本，而敲除卵黄囊的转录本变化则相对较少。诸多上调的胎盘转录本在源自敲除囊胚的分化型滋养层干细胞中，降解速率显著降低。数十个转录本被确定为ZFP36L3的高可信度靶标，其中包括已知参与滋养层及胎盘生理过程的蛋白。意外的是，1型转铁蛋白受体mRNA在敲除胎盘中的表达量降低，尽管其在敲除干细胞中的稳定性有所提升。该受体对胎盘铁摄取至关重要，其表达下调伴随敲除胎儿体内铁储备的减少，这提示这种宫内铁缺乏可能会对个体后期健康产生不利影响。每种滋养层干细胞条件均设置4次生物学重复，共包含8种实验条件：野生型与敲除型未分化细胞、经放线菌素D（Actinomycin D, ActD）处理0h的分化细胞、经ActD处理2h的分化细胞、经ActD处理4h的分化细胞。野生型与敲除型胎盘样本均设置5次生物学重复，杂合子胎盘样本设置3次生物学重复；杂合子胎盘分为两类：突变等位基因经母本传递与经父本传递的杂合胎盘。