Abstract
BackgroundPreimplantation bovine development is emerging as an attractive experimental model, yet little is known about the mechanisms underlying trophoblast (TE)/inner cell mass (ICM) segregation in cattle. To gain an insight into these processes we have studied protein and mRNA distribution during the crucial stages of bovine development. Protein distribution of lineage specific markers OCT4, NANOG, CDX2 were analysed in 5-cell, 8–16 cell, morula and blastocyst stage embryos. ICM/TE mRNA levels were compared in hatched blastocysts and included: OCT4, NANOG, FN-1, KLF4, c-MYC, REX1, CDX2, KRT-18 and GATA6.ResultsAt the mRNA level the observed distribution patterns agree with the mouse model. CDX2 and OCT4 proteins were first detected in 5-cell stage embryos. NANOG appeared at the morula stage and was located in the cytoplasm forming characteristic rings around the nuclei. Changes in sub-cellular localisation of OCT4, NANOG and CDX2 were noted from the 8–16 cell onwards. CDX2 initially co-localised with OCT4, but at the blastocyst stage a clear lineage segregation could be observed. Interestingly, we have observed in a small proportion of embryos (2%) that CDX2 immunolabelling overlapped with mitotic chromosomes.ConclusionsCell fate specification in cattle become evident earlier than presently anticipated – around the time of bovine embryonic genome activation. There is an intriguing possibility that for proper lineage determination certain transcription factors (such as CDX2) may need to occupy specific regions of chromatin prior to its activation in the interphase nucleus. Our observation suggests a possible role of CDX2 in the process of epigenetic regulation of embryonic cell fate.
Highlights
Preimplantation bovine development is emerging as an attractive experimental model, yet little is known about the mechanisms underlying trophoblast (TE)/inner cell mass (ICM) segregation in cattle
Gene expression patterns of key lineage specific marker genes during bovine preimplantation development In order to follow lineage specification during bovine preimplantation development we have first analysed the mRNA expression profile of selected pluripotency and lineage specific markers: OCT4, NANOG, FN-1, KLF4, c-MYC, REX1, which are important for maintaining pluripotency in mouse and human and CDX2, KRT-18 which are crucial for the establishment and the maintenance of TE as well as GATA6 involved in mouse primitive endoderm (PrE) lineage segregation
Because for protein immunolocalisation we have chosen to concentrate on the 3 canonical lineage markers OCT4, NANOG and CDX2, we have checked the presence of gene specific mRNAs in 8–16 cell stage embryos, which in bovine is the time of major embryonic genome activation (Figure 1)
Summary
Preimplantation bovine development is emerging as an attractive experimental model, yet little is known about the mechanisms underlying trophoblast (TE)/inner cell mass (ICM) segregation in cattle. Sharing many similarities with human embryos, bovine embryos present an attractive model for investigating the fundamental mechanisms of During the initial stages of mammalian development two successive differentiation events lead to the segregation of the three committed cell lineages. In mice the process of TE formation initiates at the 8-cell stage, when embryos undergo compaction manifested by an increased intracellular adhesion, blastomere flattening and formation of adherens junctions [5]. Concurrent with this event, cells acquire an apical domain rich in proteins, such as the atypical protein kinase aPKC [6], the polarity protein Par3 [7] and the apical protein Erizn [8]. During the subsequent cell divisions (from 8–16 and from 16–32 cell stage) the differential inheritance of the apical, polarised surface determines either the TE or the ICM fate [9,10]
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