Samuel Lunenfeld Research Institute, Mount Sinai Hospital, and Department of Medical Genetics; University of Toronto
The Mash2/Ascl2 transcription factor is required for normal placental development. Only the maternal allele of Mash2 is transcribed in the developing trophoblast: maternal transmission of a null allele leads to lethality at mid-gestation in Mash2 embryos. Mash2 maps to the 1 Mb imprinted domain on distal CHR7. The imprinting of Mash2 is not, or only weakly, affected by LOI at H19 and global demethylation in Dnmtl mutant embryos. Using the Cre-loxP system, we have initiated a dissection of cis elements important for the imprinting of Mash2. We previously targeted two loxP insertions proximal of Mash2: 4.2 kb 3' of Mash2 (M2 allele) and 2.6 kb 5' of Ins2 (I2 allele), 300 kb centromeric of Mash2. Following the germline transmission of these two alleles, trans-heterozygous males (Mash2+/M2, Ins2I2/+) carrying the primary spermatocyte-specific Sycpl-Cre transgene were obtained. From these males, duplication and deletion alleles were obtained at high frequency by trans-allelic meiotic recombination. Here we report our molecular and phenotypic analysis of the 300kb deletion (delta), the structure of which was confirmed by molecular analysis and DNA FISH. No visible phenotype was observed upon paternal or maternal transmission of the deletion, which encompasses the Th gene. Surprisingly, the paternal delta and M2 alleles of Mash2 can rescue the lethality of a maternal null allele in 13 to 30% of trans heterozygotes. Evidence for LOI at Mash2 was obtained from delta/castaneus Fl hybrid placenta, where expression of the paternal delta allele was detected. We also observed an embryonic lethality in homozygous deletion embryos. This phenotype was rescued by providing exogenous L-DOPA during gestation, to bypass the requirement for the deleted tyrosine hydroxylase gene.