Allele-specific in situ hybridization (ASISH) analysis: a novel technique which resolves differential allelic usage of H19 within the same cell lineage during human placental development

Abstract

Precursory studies of H19 transcription during human foetal development have demonstrated maternally derived monoallelic expression. Analyses in extra-embryonic tissues, however, have been more equivocal, with discernible levels of expression of the paternal allele of H19 documented in the first trimester placenta. By refining the in situ hybridization technique we have developed an assay to enable the functional imprinting status of H19 to be determined at the cellular level. This assay involves the use of oligonucleotide DNA probes that are able to discriminate between allelic RNA transcripts containing sequence polymorphisms. Biallelic expression of H19 is confined to a subpopulation of cells of the trophoblast lineage, the extravillous cytotrophoblast, while the mesenchymal stroma cells maintain the imprinted pattern of monoallelic expression of H19 throughout placental development. This data demonstrates that the low level of paternal H19 expression previously detected in normal human placenta is not due to a random loss of functional imprinting, but appears to result from a developmentally regulated cell type-specific activation of the paternal allele. In addition, biallelic expression of H19 does not seem to affect the functional imprinting of the insulin-like growth factor II gene, which is monoallelically expressed at relatively high levels in the extra-villous cytotrophoblasts. These results imply that the allelic usage of these two genes in normal human placental development may not be directly analogous to the situation previously documented in the mouse embryo.