DNA methylation in the developing marsupial embryo.

Abstract

Marsupial development differs from early development of placental mammals in that the blastocyst is unilaminar, so that both embryonic and extraembryonic cells are derived from a single layer of cells (protoderm) which faces the blastocyst cavity. Also, all cells in female marsupial conceptuses so far examined show preferential paternal X-inactivation. To test for a possible correlation between cell position, paternal X-inactivation and DNA hypomethylation, marsupial DNA preparations from three regions, embryo, vascular yolk sac and avascular yolk sac, were digested with methyl-specific restriction endonucleases, separated on agarose gels and end-labelled with 32P-dCTP. The size distribution of the fragments obtained indicated three levels of methylation: high methylation of embryonic DNA, intermediate levels of methylation of vascular yolk sac DNA and hypomethylation of avascular yolk sac DNA. The degree of methylation of repeat sequences, observed as discrete bands in end-labelled HpaII digests, was correlated with the overall methylation of tissue DNA. Thus, the difference in methylation in embryonic and extraembryonic DNA was similar to that described for the mouse conceptus, and the outside cell position of marsupial fetal precursor cells did not correlate with hypomethylation. HpaII tiny fragments, which indicate the presence of CpG-rich islands of DNA, were evident in the marsupial digests. In the mouse DNA, these islands are associated with gene transcription and provide one route to cloning of unique gene sequences.