Loss of Oct4 expression during the development of murine embryoid bodies

We describe the internal organization of murine embryoid bodies (EBs) in terms of the structures and cell types formed as Oct4 expression becomes progressively lost. This is done by making the EBs from iPS cells carrying a novel Oct4 reporter (Oct4-MerCreMer;mTmG) which is inducible, sensitive, and permanent in all cellular progeny. When these EBs are treated with tamoxifen, the Oct4 expressing cells switch from a red to a green fluorescence color, and this is maintained thereafter by all their progeny. We show that there is no specific pattern in which Oct4 is downregulated, rather it appears to be spatially random. Many of the earliest cells to lose Oct4 expression stain positive for markers of visceral endoderm (DAB2, α-fetoprotein (AFP), HNF4). These are randomly located, although if endoderm differentiation is allowed to commence before EB formation then an external layer is formed. This is true both of EBs made from the reporter iPS cells, or from an embryo-derived mouse ES line (R1 cells). Markers of the early body axis, Brachyury (BRA) and FOXA2, usually showed a concentration of positive cells in one region of the EB, but the morphology is not predictable and there are also scattered cells expressing these markers. These patterns are similar in R1 cells. Use of the Oct4 reporter showed a difference between BRA and FOXA2. BRA, which marks the early mesoderm, node and notochord, arises in Oct4 expressing cells on days 3-4. FOXA2, which marks the floor plate of the neural tube and definitive endoderm, as well as the node and notochord, arises at the same time but mostly in cells that have already lost Oct4 expression. Several clumps of cardiomyocytes are visible by days 7-8 of EB development, both in our iPS cells and in R1 cells. Using the Oct4 reporter we show that the cells forming these clumps lose Oct4 expression between days 3 and 5. Overall, our results indicate that EBs recapitulate normal development quite well in terms of the tempo of events and the appearance of specific markers, but they do not resemble embryos in terms of their morphology.     

带有穿膜结构域的转录因子蛋白Oct4和Sox2的融合表达

目的:克隆、表达及纯化带有穿膜结构域的转录因子蛋白Oct4和Sox2。方法:根据GenBank中的Oct4和Sox2基因序列,在其3’端引入穿膜结构域11R,并在其两端引入NdeⅠ和XhoⅠ酶切位点,进行全基因合成;将目的基因克隆至pET41a载体,进行酶切鉴定及测序;将所获阳性重组质粒转化感受态大肠杆菌BL21(DE3),经IPTG诱导表达后,对表达产物进行Western印迹鉴定;最后用Ni-NTA亲和层析柱对所获目的蛋白进行纯化。结果:质粒酶切鉴定结果表明带有目的基因的重组质粒构建成功;SDS-PAGE结果显示有相对分子质量约42×103和38×103的特异性蛋白表达条带,经Western印迹证实为目的蛋白;用Ni-NTA亲和层析柱纯化后,得到均一的Oct4和Sox2目的蛋白。结论:得到带有穿膜结构域的转录因子融合蛋白Oct4和Sox2,为今后安全开展诱导性多能干细胞研究奠定了基础。     

BMPs functionally replace Klf4 and support efficient reprogramming of mouse fibroblasts by Oct4 alone

Generation of induced pluripotent stem cells by defined factors has become a useful model to investigate the mechanism of reprogramming and cell fate determination. However, the precise mechanism of factor-based reprogramming remains unclear. Here, we show that Klf4 mainly acts at the initial phase of reprogramming to initiate mesenchymal-to-epithelial transition and can be functionally replaced by bone morphogenetic proteins (BMPs). BMPs boosted the efficiency of Oct4/Sox2-mediated reprogramming of mouse embryonic fibroblasts (MEFs) to ～1%. BMPs also promoted single-factor Oct4-based reprogramming of MEFs and tail tibial fibroblasts. Our studies clarify the contribution of Klf4 in reprogramming and establish Oct4 as a singular setter of pluripotency in differentiated cells.     

哺乳动物BMP4蛋白功能位点的进化踪迹分析

哺乳动物骨形态发生蛋白(BMPs)具有促进动物软骨形成,调节细胞增殖、分化及迁移的多种功能。此外,该蛋白在动物个体发育及人(Homo sapiens)肿瘤的发生、发展过程中也扮演着重要角色。本文以人源骨形态发生蛋白4(BMP4)为种子序列,利用多种生物信息学工具进行哺乳动物BMP4蛋白的序列查找及其同源蛋白的搜索,共得到具有完整结构域的同源蛋白序列72条,并以此为基础对哺乳动物BMP4的进化踪迹位点及相关的功能位点进行比较研究。结果表明,BMP4蛋白家族的TGFβ-propeptide结构域具22个全家族保守残基,而TGF-β结构域仅具84个亚家族特异性残基。人BMP4蛋白TGF-β结构域的配基结合位点主要分布于结合口袋的边缘区域。本研究可为BMP4蛋白重要功能区残基的确定及未知功能位点的预测提供信息。     

Over-expression of Oct4 in human esophageal squamous cell carcinoma

The Octamer 4 gene (Oct4) is a master pluripotency controller that has been detected in several types of tumors. Here, we examine the expression of Oct4 in human esophageal squamous cell carcinoma (ESCC). We found that punctate Oct4 protein was expressed in most (93.7%) ESCC samples but it was not observed in esophageal mucosa. Some ESCC cells had the capacity to form tumorospheres; those with an Oct4⁺-rich cell phenotype had increased proliferation and Oct4 mRNA levels compared to those of differentiated cells in culture or xenograft tumors. The over-expression of Oct4 in ESCCs suggests that it is a potential target for ESCC therapy. Oct4 could be a useful tumor marker in an immunohistochemical panel designed to differentiate between ESCC and esophageal mucosa. Expression of Oct4 in tumorospheres might indicate the presence of a population of ECSCs and its expression in xenograft tumors suggests that Oct4 is also associated with tumor metastasis.     

转录因子Oct4、Sox2和Nanog在早期胚胎发育过程中的表达调控

胚胎发育的分子机理是现今胚胎工程及发育生物学研究迫切需要了解的。由于胚胎发育的早期阶段就是胚胎干细胞阶段,因此两者在研究上有很好的重合性。通过对调节胚胎干细胞基因转录的三个具有代表性的转录因子Oct4、Sox2和Nanog的综述,展现出胚胎发育分子机理研究概况。     

Oct4-Mediated Inhibition of Lsd1 Activity Promotes the Active and Primed State of Pluripotency Enhancers

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Rex-1对Oct4转录活性的调控

Rex-1和Oct4是在多能性细胞中特异表达的转录因子,而Rex-1的生物学功能及其调控胚胎干细胞(ES细胞)多能性和分化能力的机制尚不清楚。实验探讨了Rex-1和Oct4的相互关系,利用免疫荧光实验和免疫共沉淀实验证明了Rex-1和Oct4两种蛋白共同定位于细胞核中,证明二者之间有直接的相互作用。 进一步的活性分析表明Rex-1能够抑制Oct4的转录激活活性。这些数据提供了一种新的调控Oct4活性的机制。