胚细胞的分离和多能外胚层的出现标志着哺乳动物胚胎中胚泡形成的最后阶段。在牛胚胎中,已经部分研究了下胚细胞的形成,证据表明,MEK信号在该谱系的分离中起着有限的作用。在这里,我们使用NANOG和SOX17的免疫荧光分析作为上胚细胞和下胚细胞的读数,探索了牛胚胎谱系分离过程中不同信号通路的作用。分别。
我们发现SOX17在16-32细胞期胚胎中开始表达,而NANOG首先从8细胞阶段检测到。SOX17首先与NANOG共表达,但是这些标记在胚泡晚期变得相互排斥。通过评估NANOG/SOX17的表达动力学,我们表明抑制MEK信号可以消除牛胚泡中的SOX17表达,不改变NANOG表达。WNT的调制,当与ERK抑制剂组合使用时,PKC和LIF不影响上皮母细胞中的NANOG表达。
这项研究表明,SOX17可以用作牛的低胚细胞的可靠早期标记,并根据其表达谱,我们表明低胚细胞在第7天的胚泡中分离。此外,使用1μM的PD0325901消除SOX17表达,而不影响上胚中的NANOG群体。WNT的调制,当与ERK抑制剂联合使用时,PKC和LIF不足以支持上母细胞中NANOG表达增强,表明应检查其他信号通路以确定其在表皮母细胞扩张中的潜在作用。
The segregation of the hypoblast and the emergence of the pluripotent epiblast mark the final stages of blastocyst formation in mammalian embryos. In bovine embryos the formation of the hypoblast has been partially studied, and evidence shows that MEK signalling plays a limited role in the segregation of this lineage. Here we explored the role of different signalling pathways during lineage segregation in the bovine embryo using immunofluorescence analysis of NANOG and SOX17 as readouts of epiblast and hypoblast, respectively.
We show that SOX17 starts to be expressed in 16-32-cell stage embryos, whereas NANOG is first detected from 8-cell stage. SOX17 is first co-expressed with NANOG, but these markers become mutually exclusive by the late blastocyst stage. By assessing the expression kinetics of NANOG/SOX17 we show that inhibition of MEK signalling can eliminate SOX17 expression in bovine blastocysts, without altering NANOG expression. Modulation of WNT, PKC and LIF did not affect NANOG expression in the epiblast when used in combination with the ERK inhibitor.
This study shows that SOX17 can be used as a reliable early marker of hypoblast in the bovine, and based on its expression profile we show that the hypoblast segregates in day 7 blastocysts. Furthermore, SOX17 expression is abolished using 1 μM of PD0325901, without affecting the NANOG population in the epiblast. Modulation of WNT, PKC and LIF are not sufficient to support enhanced NANOG expression in the epiblast when combined with ERK inhibitor, indicating that additional signalling pathways should be examined to determine their potential roles in epiblast expansion.