Inhibition of MAP2K and GSK3 Signaling Promotes Bovine Blastocyst Development and Epiblast-Associated Expression of Pluripotency Factors
Inhibition of MAP2K and GSK3 Signaling Promotes Bovine Blastocyst Development and Epiblast-Associated Expression of Pluripotency Factors
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DOI：
10.1095/biolreprod.112.103390
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发表时间：
2013-03-01
影响因子：
3.6
通讯作者：
Oback, Bjoern
中科院分区：
文献类型：
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作者：
Harris, Daina;Huang, Ben;Oback, Bjoern
哺乳动物胚泡中的细胞分离成三种不同的谱系，即滋养层、下胚层和上胚层。在发育过程中，它们将分别形成胚外组织和胚胎组织。在小鼠中，只有上胚层细胞可以直接转化为培养的多能胚胎干细胞，能够形成所有的成体细胞类型。这种转化通过双重抑制（即，2i）丝裂原活化蛋白激酶激酶（Map2k），拮抗Fgf信号传导，和糖原合成酶激酶3（Gsk 3），刺激Wnt途径。我们研究了2i处理对牛囊胚谱系分离和多能性相关基因表达的影响。在存在二甲亚砜或MAP2K（0.4 μ M PD 0325901）和GSK 3（3 μ M CHIR 99021）的抑制剂的情况下培养来自受精卵（第1天）阶段的体外受精（IVF）胚胎。与溶剂对照相比，2i条件增加了牛IVF培养物中卵丘细胞的丰度，这损害了囊胚形成。去除卵丘后，2i可加速囊胚发育，并使内细胞团（ICM）和滋养层细胞数量分别增加30%和27%。这些发育和形态学变化伴随着基因表达的改变。信号抑制增加了假定的上胚层标记NANOG和SOX 2的转录，同时抑制了假定的下胚层标记GATA 4。使用显微外科囊胚解剖，我们发现NANOG和SOX 2水平的增加是ICM特有的，而不是由于滋养层中的异位表达。其他多能性相关（POU5F1，KLF4，DPPA3）或滋养层细胞富集（CDX2）基因的表达不受影响。总之，2i条件重编程牛ICM的转录谱，但不是滋养层细胞。通过将平衡从下胚层转移到上胚层相关基因表达，2i培养可以为随后的多能干细胞培养物的衍生准备牛上胚层。
Cells in the mammalian blastocyst segregate into three distinct lineages, namely, trophoblast, hypoblast, and epiblast. During development, these will form extraembryonic and embryonic tissues, respectively. In mouse, only epiblast cells can be directly converted into cultured pluripotent embryonic stem cells, capable of forming all adult cell types. This conversion is promoted by the double inhibition (i.e., 2i) of mitogen-activated protein kinase kinase (Map2k), antagonizing Fgf signaling, and of glycogen synthase kinase 3 (Gsk3), stimulating the Wnt pathway. We investigated the effect of 2i treatment on lineage segregation and pluripotency-related gene expression in bovine blastocysts. In vitro fertilized (IVF) embryos were cultured in the presence of dimethyl sulfoxide or inhibitors of MAP2K (0.4 mu M PD0325901) and GSK3 (3 mu M CHIR99021) from the zygote (Day 1) stage. Compared to vehicle controls, 2i conditions increased the abundance of cumulus cells in bovine IVF cultures, which compromised blastocyst formation. Following cumulus removal, 2i accelerated blastocyst development and increased inner cell mass (ICM) and trophoblast cell numbers by 30% and 27%, respectively. These developmental and morphological changes were accompanied by alterations in gene expression. Signal inhibition increased transcription of putative epiblast markers NANOG and SOX2 while repressing putative hypoblast marker GATA4. Using microsurgical blastocyst dissection, we found that the increase in NANOG and SOX2 levels was specific to the ICM and not due to ectopic expression in the trophoblast. Expression of other pluripotency-related (POU5F1, KLF4, DPPA3) or trophoblast-enriched (CDX2) genes was not affected. In summary, 2i conditions reprogrammed the transcriptional profile of bovine ICM but not trophoblast cells. By shifting the balance from hypoblast-to epiblast-associated gene expression, 2i culture may prime bovine epiblast for subsequent derivation of pluripotent stem cell cultures.