Transcriptome Landscapes of Mammalian Embryonic Cells

Abstract

This thesis describes research on gene expression profiles from different embryonic stages and cell types to identify genes involved in pluripotency or differentiation in bovine and porcine cells. The results are compared with data from other mammals. RNA expression profiles of morula and blastocyst stage embryos were compared which resulted in a list of more than 3000 genes differentially expressed between these two embryonic stages. Since the blastocyst is composed of the pluripotent ICM and the extraembryonic trophectoderm (TE), transcriptomes of these cell types were compared. Indeed, the expression levels of core pluripotency genes (NANOG, SOX2 and POU5F1) were higher in the ICM than in the TE and a number of other transcription factors were found to be expressed at higher levels in the ICM. To increase expression levels of NANOG in the ICM, MAPK expression was inhibited during embryo culture and the expression profile compared with control ICM not exposed to MAPK inhibition. The results of these experiments identified the bovine ICM as pluripotent and this pluripotency cannot be maintained by MAPK-inhibition alone. Culture media reported to keep human pluripotent cells in a naïve state or rewind primed human ES cells towards the naïve state, could also be used to maintain naïve pluripotency in mouse ES cells. This suggests that the state of the embryo is less relevant and the medium is suitable to maintain pluripotency in embryonic cells from multiple species. Therefore, bovine embryos were cultured in three of these media from the morula stage to the blastocyst stage and expression profiles compared to farther identify genes involved in bovine pluripotency. X-chromosome inactivation, initiated by the long non coding RNA XIST, is an indication for the primed state opposed to the active state of both X chromosomes in female embryonic stem cell lines. In the “naïve” media, XIST expression levels were reduced. Expression levels of genes involved in early differentiation were also found to be reduced but also expression levels of genes indicative of a naïve state were reduced. The culture of dissected ICM or whole blastocysts from bovine or porcine embryos in these media did however not result in the generation of true ES cell lines, indicating that self-renewal was not initiated. Other strategies such as Wnt inhibition remained unsuccessful as well. The protein Dickkopf 3 (DKK3) has been reported to increase MYF5 expression in zebra fish myogenic stem cells. MYF5 is involved in myogenesis leading to the formation of myofibres and muscle. We cultured porcine muscle satellite cells in the presence of several concentrations of the DKK3 protein. A small expression level increase of skeletal muscle differentiation markers was observed and also the expression levels of genes involved in the myoblast fusion process were elevated. During 5 days of culture in the presence of DKK3 fibre formation improved and myotube diameter increased as compared with cultures without DKK3.