Verification of chicken Nanog as an epiblast marker and identification of chicken PouV as Pou5f3 by newly raised antibodies

Pluripotency is an important feature of early embryonic cells of multicellular organisms. Recent advances in stem cell research have shown that Nanog and Pou5f1 (Oct3/4) play important roles in mammalian pluripotency. However, whether these molecules exert conserved functions in other species remains unknown. Although the epiblast of the early chicken embryo would provide a useful experimental model, a lack of antibodies against chicken Nanog (cNanog) and chicken PouV/Pou5f3 (cPouV) proteins has hampered intensive investigation. Here we report newly raised polyclonal antibodies that specifically recognize cNanog and cPouV proteins. The specificity and sensitivity of the antibodies were validated by both western blotting and immunostaining with transfected 293T cells and chicken embryonic tissues. Immunohistochemistry using these antibodies revealed that cNanog protein was specifically localized in epiblastic cells and germ cells. In contrast, cPouV expression was seen almost ubiquitously. We also found that chicken epiblast‐derived colony‐forming cells that morphologically resemble mouse embryonic stem cells were cNanog‐positive, implying that these colony‐forming cells possess pluripotency. The anti‐cPouV antibody further enabled us to identify a previously unknown region at the N‐terminus of the cPouV protein containing a characteristic motif that is absent in mammalian Pou5f1. Thus, the antibodies raised in this study are useful tools for studying the functions of cNanog and cPouV at the protein level and the molecular mechanisms of chicken pluripotency.     

斑马鱼多能性因子的研究进展

哺乳动物多能性因子, 主要包括Pou5f1/Oct4、Sox2、Klf4、Nanog等转录因子, 不仅能够维持胚胎干细胞的未分化状态, 同时也参与使分化细胞重编程回多能性状态的过程。目前对脊椎动物多能性因子在体(in vivo)功能研究报道极少。斑马鱼是研究脊椎动物早期发育分化的理想模型, 它能够为多能性相关因子的功能研究提供在体环境, 因而可以更准确地了解多能性因子的作用信息。近年来, 已在斑马鱼中发现了多种哺乳动物多能性因子的同源基因, 如oct4、nanog等。文章主要介绍了斑马鱼中多能性因子的相关研究进展, 并与其它动物中的研究作一比较。     

Nanog overcomes reprogramming barriers and induces pluripotency in minimal conditions

Induced pluripotency requires the expression of defined factors and culture conditions that support the self-renewal of embryonic stem (ES) cells. Small molecule inhibition of MAP kinase (MEK) and glycogen synthase kinase 3 (GSK3) with LIF (2i/LIF) provides an optimal culture environment for mouse ES cells and promotes transition to naive pluripotency in partially reprogrammed (pre-iPS) cells. Here we show that 2i/LIF treatment in clonal lines of pre-iPS cells results in the activation of endogenous Nanog and rapid downregulation of retroviral Oct4 expression. Nanog enables somatic cell reprogramming in serum-free medium supplemented with LIF, a culture condition which does not support induced pluripotency or the self-renewal of ES cells, and is sufficient to reprogram epiblast-derived stem cells to naive pluripotency in serum-free medium alone. Nanog also enhances reprogramming in cooperation with kinase inhibition or 5-aza-cytidine, a small molecule inhibitor of DNA methylation. These results highlight the capacity of Nanog to overcome multiple barriers to reprogramming and reveal a synergy between Nanog and chemical inhibitors that promote reprogramming. We conclude that Nanog induces pluripotency in minimal conditions. This provides a strategy for imposing naive pluripotency in mammalian cells independently of species-specific culture requirements.     

The Role of Maternal pou5f3.3/oct60 Gene in the Regulation of Initial Stages of Tissue Differentiation during Xenopus laevis Embryogenesis

Russian Journal of Bioorganic Chemistry - The role of maternal Pou5f3.3/Oct60 factor of Xenopus laevis, a homolog of the pluripotency regulator of mammalian stem cells, the Oct4 protein, in early...     

Nanog基因的功能与调控研究进展

胚胎干细胞的无限增殖能力和亚全能性决定了它在再生医学、新药开发及发育生物学基础研究中具有巨大的应用前景。探索维持胚胎干细胞亚全能性的因子及其网络的调控功能成为胚胎干细胞生物学研究的热点。已研究发现多个与维持胚胎干细胞亚全能性相关的基因如Oct4, Nanog, Sox2等,其中Nanog是2003年5月末发现的一个基因,它对维持胚胎干细胞亚全能性起关键性作用,能够独立于L1F/Stat3维持ICM和胚胎干细胞的亚全能性。几年来,Nanog的生物学功能及其与 Oct4, Sox2等亚全能性维持基因之间的相互作用关系已有较为深入的研究,并发现多个调控Nanog表达的转录因子,从而进一步明晰Nanog与已知调控胚胎发育的信号通路之间的关系。本文在综述Nanog基因的表达特征和功能的基础上、重点探讨Nanog基因表达调控以及Oct4, Sox2等亚全能性维持基因之间的相互作用关系,并对未来的研究趋势予以展望。     

NPR-A regulates self-renewal and pluripotency of embryonic stem cells

Self-renewal and pluripotency of embryonic stem (ES) cells are maintained by several signaling cascades and by expression of intrinsic factors, such as Oct4, Nanog and Sox2. The mechanism regulating these signaling cascades in ES cells is of great interest. Recently, we have demonstrated that natriuretic peptide receptor A (NPR-A), a specific receptor for atrial and brain natriuretic peptides (ANP and BNP, respectively), is expressed in pre-implantation embryos and in ES cells. Here, we examined whether NPR-A is involved in the maintenance of ES cell pluripotency. RNA interference-mediated knockdown of NPR-A resulted in phenotypic changes, indicative of differentiation, downregulation of pluripotency factors (such as Oct4, Nanog and Sox2) and upregulation of differentiation genes. NPR-A knockdown also resulted in a marked downregulation of phosphorylated Akt. Furthermore, NPR-A knockdown induced accumulation of ES cells in the G1 phase of the cell cycle. Interestingly, we found that ANP was expressed in self-renewing ES cells, whereas its level was reduced after ES cell differentiation. Treatment of ES cells with ANP upregulated the expression of Oct4, Nanog and phosphorylated Akt, and this upregulation depended on NPR-A signaling, because it was completely reversed by pretreatment with either an NPR-A antagonist or a cGMP-dependent protein kinase inhibitor. These findings provide a novel role for NPR-A in the maintenance of self-renewal and pluripotency of ES cells.     

Knockdown of p53 suppresses Nanog expression in embryonic stem cells

Mouse embryonic stem cells (ESCs) express high levels of cytoplasmic p53. Exposure of mouse ESCs to DNA damage leads to activation of p53, inducing Nanog suppression. In contrast to earlier studies, we recently reported that chemical inhibition of p53 suppresses ESC proliferation. Here, we confirm that p53 signaling is involved in the maintenance of mouse ESC self-renewal. RNA interference-mediated knockdown of p53 induced downregulation of p21 and defects in ESC proliferation. Furthermore, p53 knockdown resulted in a significant downregulation in Nanog expression at 24 and 48 h post-transfection. p53 knockdown also caused a reduction in Oct4 expression at 48 h post-transfection. Conversely, exposure of ESCs to DNA damage caused a higher reduction of Nanog expression in control siRNA-treated cells than in p53 siRNA-treated cells. These data show that in the absence of DNA damage, p53 is required for the maintenance of mouse ESC self-renewal by regulating Nanog expression.