ALLELE-SPECIFIC IN-SITU HYBRIDIZATION (ASISH) ANALYSIS - A NOVEL TECHNIQUE WHICH RESOLVES DIFFERENTIAL ALLELIC USAGE OF H19 WITHIN THE SAMECELL LINEAGE DURING HUMAN PLACENTAL DEVELOPMENT

Precursory studies of H19 transcription during human foetal developmen t have demonstrated maternally derived monoallelic expression, Analyse s in extra-embryonic tissues, however, have been more equivocal, with discernible levels of expression of the paternal allele of H19 documen ted in the first trimester placenta, By refining the in situ hybridiza tion technique we have developed an assay to enable the functional imp rinting status of H19 to be determined at the cellular level, This ass ay involves the use of oligonucleotide DNA probes that are able to dis criminate between allelic RNA transcripts containing sequence polymorp hisms. Biallelic expression of H19 is confined to a subpopulation of c ells of the trophoblast lineage, the extravillous cytotrophoblast, whi le the mesenchymal stroma cells maintain the imprinted pattern of mono allelic 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 funct ional imprinting, but appears to result from a developmentally regulat ed cell type-specific activation of the paternal allele. In addition, biallelic expression of H19 does not seem to affect the functional imp rinting of the insulin-like growth factor II gene, which is monoalleli cally expressed at relatively high levels in the extra-villous cytotro phoblasts, These results imply that the allelic usage of these two gen es in normal human placental development may not be directly analogous to the situation previously documented in the mouse embryo.