STAT3 signalling drives tissue expansion during postimplantation mouse development [RNA-seq]

STAT3 has been studied extensively in the context of self-renewal of nave pluripotent mouse embryonic stem cells (mESCs). Although STAT3 is required to maintain ICM lineages when maternally eliminated, the role of STAT3 during gastrulation is unclear as the conventional knockout of Stat3 develops until E6.5. In this study, we observed that zygotic loss of Stat3 on CD1 genetic background leads to consistent developmental retardation from implantation to mid-gestation, beginning with a significant reduction in the number of epiblast cells by the time of implantation. Remarkably, mutants appear to scale normally and resemble non-affected embryos from the previous day at all postimplantation stages examined. Bulk RNA-seq analysis using isolated epiblast cells revealed that E6.5 null epiblasts were intermediate between WT E5.5 and E6.5; E7.5 null epiblasts grouped more closely with WT E6.5 than E7.5 epiblasts. Gene ontology analysis revealed lipid metabolism was compromised in Stat3 null embryos as early as E6.5. Interestingly, Socs3, downstream target of STAT3, was not induced in WT epiblast cells until E7.5. We found Y705 phospholylated STAT3 was not exist postimplantation epiblast, but S727 phoshorylated STAT3 was abundant over the time, suggesting postimplantation specific roles of S727 phosphorylated STAT3 in growth and differentiation. Our study revealed that while Stat3 null epiblast cells progress along a near normal differentiation trajectory, Stat3 deletion compromises lipid metabolism as early as E6.5. Bulk RNA-seq for post-implantation epiblast cells from WT and Stat3 null embryos; WT and Stat null embryos were collected from E5.5 to E7.5 and isolated epiblast manually. Genotype was determined using extraembryonic tissue by genomic PCR.STAT3已被广泛研究于初始态多能小鼠胚胎干细胞（naive pluripotent mouse embryonic stem cells, mESCs）的自我更新过程中。尽管母源敲除STAT3时，其对维持内细胞团（inner cell mass, ICM）谱系是必需的，但STAT3在原肠胚形成过程中的作用仍不明确，因为传统的Stat3基因敲除小鼠仅能发育至胚胎期6.5天（E6.5）。本研究中，我们观察到在CD1遗传背景下，合子型Stat3基因缺失会导致从着床到妊娠中期出现持续性发育迟缓，其起始特征为着床时上胚层（epiblast）细胞数量显著减少。值得注意的是，突变体胚胎可正常进行发育比例缩放，且在所有检测的着床后发育阶段，其形态均与前一天的未受影响胚胎相似。通过分离的上胚层细胞进行批量RNA测序（bulk RNA-seq）分析发现，E6.5天的Stat3缺失型上胚层的转录组特征介于野生型（wild type, WT）E5.5与E6.5野生型上胚层之间；E7.5天的Stat3缺失型上胚层的转录组聚类结果更接近WT E6.5上胚层，而非WT E7.5上胚层。基因本体论（gene ontology, GO）分析显示，早在E6.5天，Stat3缺失型胚胎的脂质代谢通路就已出现异常。有趣的是，STAT3的下游靶基因Socs3直到E7.5天才在野生型上胚层细胞中被诱导表达。我们发现，Y705位点磷酸化的STAT3在着床后上胚层中并未检测到，但S727位点磷酸化的STAT3在该时期大量表达，这提示S727磷酸化的STAT3在着床后阶段特异性参与胚胎生长与分化调控。本研究表明，尽管Stat3缺失型上胚层细胞的分化轨迹近乎正常，但Stat3缺失早在E6.5天就会损害脂质代谢。本数据集包含野生型与Stat3缺失型胚胎着床后上胚层细胞的批量RNA测序数据；实验收集了E5.5至E7.5天的胚胎，并手动分离其上胚层。基因型通过胚外组织的基因组聚合酶链式反应（polymerase chain reaction, PCR）进行鉴定。