Figure 1

Expression of IC2-regulated genes in DelTel7/+ at E9.5. A) Diagram of distal mouse chromosome 7 (Chr 7) region showing the locations of the imprinting centres 1 (IC1) next to H19 and 2 (IC2) in intron 10 of the Kcnq1 gene, as well as the protein-coding genes known to be regulated by IC2 (Osbpl5, Phlda2, Cdkn1c, Kcnq1, Tssc4, Cd81, and Ascl2). This entire IC2-regulated cluster is deleted in the DelTel7 allele in which an array of telomere repeats (Tel array) was introduced distal of Ins2 [36]. B) PHLDA2 immunohistochemistry (IHC) on sections of wild type (+/+) and maternal hemizygous (DelTel7/+) placentae at E9.5. PHLDA2 IHC was performed on two placentae of each genotype. The PHLDA2 protein, localized to the labyrinth of the wild type placenta, is absent in DelTel7/+ mutants. Note the lack of spongiotrophoblast and expanded giant cell layer in the DelTel7/+ placenta. Scale bar: 1 mm. sp: spongiotrophoblast, lab: labyrinth, Gi: giant cells. C) qRT-PCR analysis of the imprinted genes Phlda2, Ascl2, and Cdkn1c, in E9.5 DelTel7/+ placentae reveals virtually no expression from the intact paternal allele. Placentae were assessed at E9.5 before the embryonic lethality of the DelTel7/+ observed at around E10.5. qRT-PCR was performed on five DelTel7/+ placentae and five wild type placentae with three technical replicates per individual placentae. Expression is relative to the reference housekeeping gene, Peptidylprolyl isomerase A (Ppia). The error bar is the standard deviation from five biological replicates, assayed in triplicates. p < 0.02 for each qRT-PCR (t-test, unpaired, two-tailed).