To evaluate the developmental distribution of adrenergic cells in vivo, we inserted the Cre-recombinase gene into the locus encoding for the epinephrine biosynthetic enzyme phenylethanolamine n-methyltransferase (Pnmt) and crossed these Pnmt-Cre mice with ROSA26 reporter (R26R) mice to activate LacZ (encoding ␤-galactosidase) expression in cells that were selectively derived from the adrenergic lineage. Our data show the following: (1) Insertion of Cre-recombinase into the Pnmt locus created a functional knockout of Pnmt expression with concomitant loss of epinephrine in homozygous PnmtCre/Cre mice; (2) Despite the reduction in Pnmt expression and epinephrine production in PnmtCre/Cre mice, these mice were viable and fertile, with no apparent developmental defects; (3) When crossed with R26R mice, Pnmt-Cre activation of LacZ expression faithfully recapitulated Pnmt expression in vivo; and (4) LacZ expression was activated in substantial numbers of pacemaking, conduction, and working cardiomyocytes.

Understanding the molecular basis of monoallelic expression as observed at imprinted loci is helpful in understanding the mechanisms underlying epigenetic regulation. Genomic imprinting begins during gametogenesis with the establishment of epigenetic marks on the chromosomes such that paternal and maternal chromosomes are rendered distinct. During embryonic development, the primary imprint can lead to generation of secondary epigenetic modifications (secondary imprints) of the chromosomes. Eventually, either the primary imprints or the secondary imprints interfere with transcription, leading to parent-of-origin-dependent silencing of one of the two alleles. Here we investigated several aspects pertaining to the generation and functional necessity of secondary methylation imprints at the Igf2/H19 locus. At the H19 locus, these secondary imprints are, in fact, the signals mediating paternal chromosome-specific silencing of that gene. We first demonstrated that the H19 secondary methylation imprints are entirely stable through multiple cell divisions, even in the absence of the primary imprint. Second, we generated mouse mutations to determine which DNA sequences are important in mediating establishment and maintenance of the silent state of the paternal H19 allele. Finally, we analyzed the dependence of the methylation of Igf2DMR1 region on the primary methylation imprint about 90 kilobases away.