The Maternal Environment: Influence on Early Mammalian Development

主要目的：我们的发现研究将集中于了解在着床前发育过程中能量应激代谢对河马通路直接决定细胞命运的影响。我们将确定胚泡中滋养外胚层(TE)和内细胞团(ICM)细胞系在能量代谢方面是否存在差异，以及这种差异是否影响河马信号通路的差异激活，从而导致TE分化和ICM多能性的维持。我们的假设是，在着床前胚胎中，能量代谢通过AMPK的调控通过激活河马通路来调节细胞谱系规范。*1)研究能量应激代谢对河马通路激活和TE细胞谱系规范的影响。*2)比较PI3K、AKT、mTOR和CDK4抑制对着床前胚胎河马通路激活和细胞谱系规范的影响。*3)研究缺糖和葡萄糖代谢抑制对着床前胚胎河马通路激活和细胞谱系规范的影响。*4)研究能量应激介质对着床前胚胎河马通路激活和细胞谱系规范的靶向性下调。短期和长期目标：短期目标是研究能量代谢在哺乳动物早期发育过程中的作用，并探索其调节。长期目标是了解母体和培养环境对发育中胚胎的影响，然后将这些信息应用于优化胚胎培养环境。*河马途径：河马途径由一系列蛋白激酶组成，包括Mst1/2(河马)、latsç(疣)、辅因子Sav1(萨尔瓦多)和Mob1a/b(MATS)、转录辅因子YAP1和Taz(Yki)以及转录因子tead 1-4(扇贝)。最近的研究表明，河马信号在ICM细胞中是活跃的，导致胞浆磷酸化的YAP(YAP-p)，从而阻断TEAD的活性。在外部细胞中，即TE祖细胞；河马的活性被灭活，从而允许核YAP和TEAD靶基因表达，从而导致TE细胞分化。最近的研究表明，能量应激代谢改变了河马在癌细胞中的失活。我们预测这种机制在早期胚胎中起作用，通过改变河马途径的活性，我们应该会影响早期胚胎中的细胞谱系特征。*意义：需要研究来定义支持早期开发的所有关键组件。我们的实验将揭示有关植入前发育调控的新信息。这些信息是理解环境介质对胚胎发育的影响以及揭示植入前发育过程中能量应激途径的功能作用所必需的。

Principal Objective: Our discovery research will focus on understanding the impact of energy stress metabolism on HIPPO pathway directed cell fate determination during preimplantation development. We will determine whether trophectoderm (TE) and inner cell mass (ICM) cell lineages in the blastocyst vary in their energy metabolism and whether this variation affects differential activation of HIPPO pathway signaling that results in TE differentiation and maintenance of ICM pluripotency. Our hypothesis is that energy metabolism via control of AMPK regulates cell lineage specification via hippo pathway activation during preimplantation development.***The specific objectives are:***1) To investigate the effects of energy stress metabolism on HIPPO pathway activation and TE cell lineage specification.***2) To contrast effects of PI3K, AKT, mTOR and CDK4 inhibition on HIPPO pathway activation in the preimplantation embryo.***3) To investigate glucose deprivation and inhibition of glucose metabolism on HIPPO pathway activation and cell lineage specification in the preimplantation embryo.***4) To investigate targeted downregulation of energy stress mediators on HIPPO pathway activation and cell lineage specification in the preimplantation embryo. ***Short-and Long Term Goals: The short-term goal is to investigate the role that energy metabolism exerts during early mammalian development and to explore its regulation. The long-term goal is to provide an understanding of the influences that the maternal and culture environments place on the developing embryo and then apply this information to optimize embryo culture environments. ***HIPPO pathway: The HIPPO pathway consists of a series of protein kinases including Mst1/2(Hippo); Lats ½(Warts); cofactors Sav1(Salvador) and Mob1a/b (mats); transcriptional cofactors Yap1 and Taz (Yki) and transcription factors TEAD 1-4 (Scalloped). Recent studies demonstrate that HIPPO signaling is active in ICM cells which results in cytoplasmic phosphorylated Yap (Yap-p) that blocks TEAD activity. In the outer cells ie TE progenitors; HIPPO activity is inactivated thus allowing nuclear Yap and TEAD target gene expression resulting in TE cell differentiation. Recent studies have suggested that energy stress metabolism alters HIPPO inactivation in cancer cells. We predict this mechanism is operating in early embryos and that by altering HIPPO pathway activity we should affect cell lineage specification in the early embryo. ***Significance: Research is required to define all of the vital components that support early development. Our experiments will uncover new information regarding the regulation of preimplantation development. This information is required to understand the effects of environmental mediators on embryonic development and to reveal the functional roles of energy stress pathways during preimplantation development.**