Co-participation of paternal and maternal genomes before the blastocyst stage is not required for full-term development of mouse embryos.
Co-participation of paternal and maternal genomes before the blastocyst stage is not required for full-term development of mouse embryos.
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DOI：
10.1093/jmcb/mjv055
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发表时间：
2015-08
影响因子：
5.5
通讯作者：
Xin Li;Jia-qiang Wang;Leyun Wang;Haifeng Wan;Yufei Li;Tianda Li;Yukai Wang;L. Shuai;Yihuan Mao-Yihu
中科院分区：
文献类型：
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作者：
Xin Li;Jia-qiang Wang;Leyun Wang;Haifeng Wan;Yufei Li;Tianda Li;Yukai Wang;L. Shuai;Yihuan Mao-Yihu
所有哺乳动物的发育都是从精子和卵子受精成二倍体开始的。母本和父本基因组在它们对印记基因表达的不同贡献方面在功能上是不等同的，并且它们是互补的，并且对于胚胎和出生后发育都是必需的（麦格拉思和Solter，1984; Surani等人，1986年）。众所周知，卵裂球的细胞核采用“Rabl构型”，通过该构型，父本和母本基因组在相同的细胞核内在空间上分离，直到8细胞阶段（Mayer et al.，2000; Santenard等人，2010年）。先前的研究表明，通过在4细胞阶段融合雄核发育和孤雌发育的单倍体卵裂球重建的二倍体胚胎可以产生活的后代，这表明在8细胞阶段之前，在同一个细胞核内，父本基因组不一定与母本基因组相关（Renard et al.，1991年）。然而，还没有完全阐明这两个基因组的参与是否需要在以后的胚胎阶段的全面发展。最近，我们和其他人已经分别从仅含有精子或卵母细胞基因组的单亲单倍体胚泡中衍生出小鼠雄激素发生和孤雌生殖单倍体胚胎干细胞（ahESC和phESC）（Leeb和Wutz，2011; Li等人，2012年; Yang等人，2012年）。我们假设，如果从ahESCs和phESCs的融合胚胎干细胞（fESCs）的建立，母亲和父亲的基因组之间的相互作用将在fESCs生成过程中完全消除。因此，胚胎干细胞的发育潜力可以反映出在胚泡阶段之前亲本基因组的共同参与是否对小鼠胚胎发育至关重要。因此，我们试图建立一种新的方法来利用父母的基因组共同参与小鼠的足月发育的早期发育的要求。首先，我们分别如所述衍生小鼠ahESC和phESC系（Leeb和Wutz，2011; Li等人，2012年）。ahESCs和phESCs分别稳定表达绿色荧光蛋白（GFP）和红色荧光蛋白（RFP），并通过几轮荧光激活细胞分选（FACS）保持高比例的单倍体成分。为了产生二倍体fESC，我们通过FACS分选单倍体ahESC和phESC（均在第10代下），将它们与聚乙二醇（PEG）融合，然后分选具有GFP和RFP双阳性表达的二倍体细胞以从单细胞集落衍生亚细胞系（指定为fESC系的第1代）（图1A）。我们在三个独立的实验中产生了30个细胞系，并随机选择了四个细胞系，命名为AP 1 -1，AP 2 -1，AP 3 -2和AP 4 -5，用于进一步分析。这些fESC系保持了经典的mESC形态，并且对GFP和RFP均呈阳性（图1 B）。简单序列长度多态性（SSLP）分析证实了ahESC的129遗传起源和phESC的C57遗传起源的存在（图1C）。此外，这些细胞系保持具有38+ XX小鼠染色体的完整核型（图1D）。与正常二倍体mESC类似，这些细胞表现出碱性磷酸酶染色的阳性信号（补充图S1 A），并表达典型的多能标志物，包括Oct 4、Nanog、Sox 2和SSEA-1（图1 E和补充图S1 B）。这些结果表明，我们可以通过小鼠ahESC和phESC的融合获得稳定的小鼠fESC。接下来，我们通过分析体外分化和体外培养来研究fESCs的发育潜力。
Dear Editor, The development of all the mammalian species begins from fertilization of a sperm and an egg into a diploid state. The maternal and paternal genomes are not functionally equivalent in term of their differential contributions to the imprinting gene expressions, and they are complementary and both essential for embryonic and postnatal development (McGrath and Solter, 1984; Surani et al., 1986). It is well known that the nucleus of blastomeres adopt a ‘Rabl configuration’by which the paternal and maternal genomes separate spatially within the same nucleus until the 8-cell stage (Mayer et al., 2000; Santenard et al., 2010). Previous studies showed that diploid embryos reconstituted by fusion of androgenetic and parthenogenetic haploid blastomeres at the 4-cell stage can produce living offspring, indicating that the paternal genome is not necessarily associated with the maternal genome within the same nucleus before the 8-cell stage (Renard et al., 1991). However, it has not been fully elucidated whether the participation of both genomes is required in later embryonic stages for full-term development. Recently, we and others have derived the mouse androgenetic and parthenogenetic haploid embryonic stem cells (ahESCs and phESCs) from the uniparental haploid blastocysts containing only the sperm or oocyte genome, respectively (Leeb and Wutz, 2011; Li et al., 2012; Yang et al., 2012). We hypothesize that if the fused ESCs (fESCs) from ahESCs and phESCs were established, the interaction between the maternal and paternal genomes would be completely eliminated during the fESC-generation process. Then the developmental potential of the fESCs could reflect whether co-participation of parental genomes before the blastocyst stage is essential for mouse embryonic development. Hence, we sought to establish a new way to exploit the requirement of co-participation of parental genomes in early development for mouse full-term development. Firstly, we derived mouse ahESC and phESC lines as described, respectively (Leeb and Wutz, 2011; Li et al., 2012). The ahESCs and phESCs stably expressed green florescence protein (GFP) and red florescence protein (RFP), respectively, and maintained high percentage of haploid component through several rounds of fluorescence-activated cell sorting (FACS). To generate diploid fESCs, we sorted the haploid ahESCs and phESCs (both under passage 10) by FACS, fused them with polyethylene glycol (PEG), and then sorted diploid cells with double-positive expression of GFP and RFP to derive sub-cell-lines from single cell colony (assigned as passage 1 for fESC lines)(Figure 1 A). We generated 30 cell lines in three independent experiments, and randomly chose four cell lines, named AP1-1, AP2-1, AP3-2, and AP4-5, for further analyses. These fESC lines maintained a classical mESC morphology and were positive for both GFP and RFP (Figure 1 B). Simple sequence length polymorphism (SSLP) analysis confirmed the presence of the 129 genetic origin of ahESCs and C57 genetic origin of phESCs (Figure 1 C). Further, these cell lines kept an intact karyotype with 38+ XX mouse chromosomes (Figure 1 D). Similar to normal diploid mESCs, these cells exhibited positive signals for alkaline phosphatase staining (Supplementary Figure S1A), and expressed typical pluripotent markers including Oct4, Nanog, Sox2, and SSEA-1 (Figure 1 E and Supplementary Figure S1B). These results demonstrate that we can derive stable mouse fESCs through fusion of mouse ahESCs and phESCs. Next, we examined the developmental potential of the fESCs by analyzing the differentiation in vitro and …