Peg5/Neuronatin is an imprinted gene located on sub-distal chromosome 2 in the mouse.

We have established a systematic screen for imprinted genes using a subtraction-hybridization method with day 8.5 fertilized and parthenogenetic embryos. Two novel imprinted genes, Peg1/Mest and Peg3, were identified previously by this method, along with the two known imprinted genes, Igf2 and Snrpn. Recently three additional candidate imprinted genes, Peg5-7 , were detected and Peg5 is analyzed further in this study. The cDNA sequence of Peg5 is identical to Neuronatin, a gene recently reported to be expressed mainly in the brain. Two novel spliced forms were detected with some additional sequence in the middle of the known Neuronatin sequences. All alternatively spliced forms of Peg5 were expressed only from the paternal allele, confirmed using DNA polymorphism in a subinterspecific cross. Peg5/Neuronatin maps to sub-distal Chr 2, proximal to the previously established imprinted region where imprinted genes cause abnormal shape and behavior in neonates.     

Paternal expression of a novel imprinted gene, Peg12/Frat3, in the mouse 7C region homologous to the Prader-Willi syndrome region.

Paternally expressed imprinted genes (Pegs) were systematically screened by comparing gene expression profiles of parthenogenetic and normal fertilized embryos using an oligonucleotide array. A novel imprinted gene, Peg12/Frat3, was identified along with 10 previously known Pegs. Peg12/Frat3 is expressed primarily in embryonic stages and might be a positive regulator of the Wnt signaling pathway. It locates next to the Zfp127 imprinted gene in the mouse 7C region, which has syntenic homology to the human Prader-Willi syndrome region on chromosome 15q11-q13, indicating that this imprinted region extends to the telomeric side in the mouse.     

Analysis of imprinted murine <Emphasis Type="Italic">Peg3</Emphasis> locus in transgenic mice

Peg3 is an imprinted gene exclusively expressed from the paternal allele. It encodes a C₂H₂ type zinc-finger protein and is involved in maternal behavior. It is important for TNF-NFkB signaling and p53-mediated apoptosis. To investigate the imprinting mechanism and gene expression of Peg3 and its neighboring gene(s), we used a 120 kb Peg3-containing BAC clone to generate transgenic mice. The BAC clone contains 20 kb of 5 and 80 kb of 3 flanking DNA, and we obtained three transgenic lines. In one of the lines harboring one copy of the transgene, Peg3 was imprinted properly. In the other two lines, Peg3 was expressed upon both maternal and paternal transmission. Imprinted expression was linked to the differential methylation of a region (DMR) upstream of the Peg3 gene. A second, maternally expressed gene, Zim1, present on the transgene was expressed irrespective of parental inheritance in all lines. These data suggest that, similar to other imprinted genes within domains, Peg3 and Zim1 are regulated by one or more elements lying at a distance from the genes. The imprinting of Peg3 seen in one line may reflect the presence of a responder sequence. Concerning the expression of the Peg3 transgene, we detected appropriate expression in the adult brain. However, this was not sufficient to rescue the maternal behavior phenotype seen in Peg3 deficient animals.     

Expression of imprinted Igf2 and Peg1/Mest genes in postimplantation parthenogenetic mouse embryos treated with transforming growth factor alpha in vitro

The effect of transforming growth factor alpha (TGFt) on the expression of imprinted Igf2 and Peg1/Mest genes was studied in diploid parthenogenetic embryos (PEs) of (CBA x C57BL/6)F1 mice during the postimplantation period of embryogenesis. The PEs were treated with TGFalpha in vitro at the morula stage and, after they developed to the blastocyst stage, were implanted into the uterus of false-pregnant females. On the tenth day of pregnancy, the PEs were explanted for subsequent in vitro culturing for 24 or 48 h. The expression of the imprinted Igf2 and Peg1/Mest genes was studied by means of whole mount in situ hybridization using digoxigenin-labeled antisense RNAs. The expression of the imprinted Igf2 and Peg1/Mest genes was studied in embryos on the tenth day of in utero development before culturing and after 24 and 48 h of culturing in vitro. The expression of Igf2 before culturing was detected only in the brain of 60% of PEs on the tents day of pregnancy (the 21-to 25-somite stages); while the Peg1/Mest expression was not detected at all. In control (not treated with TGFalpha) PEs, neither gene was expressed at the same 21- to 25-somite stages. After 24 h of culturing, the Igf2 expression was detected in the brain of 71% of PEs at the 30- to 35-somite stages, while the Peg1/Mest expression was not detected. In control (untreated) PEs, neither imprinted gene was expressed at the 30- to 35-somite stage. After 48 h of culturing, Igf2 was expressed in the regions of the brain, developing jaws, heart, liver, and somites of all TGFalpha-treated PEs at the 40- to 45-somite stages; and Peg1/Mest was expressed in the brain, heart, and liver of these embryos. In control (untreated) PEs, neither Igf2 nor Peg1/Mest was expressed at these stages The expression patterns of the imprinted Igf2 and Peg1/Mest genes in PEs at the most advanced developmental stages (40-45 somites) and in normal (fertilized) embryos at the same stages were similar; however, their expression rate in PEs was substantially lower than in normal embryos. These data indicate that exogenous TGFalpha can reactivate the expression of the two imprinted genes, modulating the effects of genomic imprinting in such a way that the PE development is improved and substantially prolonged.     

Expression patterns of the novel imprinted genes Nap1l5 and Peg13 and their non-imprinted host genes in the adult mouse brain

Recent work has implicated imprinted gene functioning in neurodevelopment and behaviour and defining the expression patterns of these genes in brain tissue has become a key prerequisite to establishing function. In this work we report on the expression patterns of two novel imprinted loci, Nap1l5 and Peg13, in adult mouse brain using in situ hybridisation methods. Nap1l5 and Peg13 are located, respectively, within the introns of the non-imprinted genes Herc3 and the Tularik1 (T1)/KIAA1882 homologue in two separate microimprinted domains on mouse chromosomes 6 and 15. These 'host' genes are highly expressed in brain and consequently we were interested in assessing their expression patterns in parallel to the imprinted genes. The brain expression of all four genes appeared to be mainly neuronal. The detailed expression profiles of Nap1l5 and Peg13 were generally similar with widespread expression that was relatively high in the septal and hypothalamic regions, the hippocampus and the cerebral cortex. In contrast, there was some degree of dissociation between the imprinted genes and their non-imprinted hosts, in that, whilst there was again widespread expression of Herc3 and the T1/KIAA1882 homologue, these genes were also particularly highly expressed in Purkinje neurons and piriform cortex. We also examined expression of the novel imprinted genes in the adrenal glands. Nap1l5 expression was localised mainly to the adrenal medulla, whilst Peg13 expression was observed more generally throughout the adrenal medulla and the outer cortical layers.