Maternally Expressed Research Articles

Identification of imprinted genes in the skeletal muscle of newborn piglets by high-throughput sequencing.

Imprinted genes - exhibiting parent-specific transcription - play essential roles in the process of mammalian development and growth. Skeletal muscle growth is crucial for meat production. To further understand the role of imprinted genes during the porcine skeletal muscle growth, DNA-seq and RNA-seq were used to explore the characteristics of imprinted genes from porcine reciprocal crosses. A total of 584 545 single-nucleotide variations were discovered in the DNA-seq data of F0 parents, heterozygous in two pig breeds (Yorkshire and Min pigs) but homozygous in each breed. These single-nucleotide variations were used to determine the allelic-specific expression in F1 individuals. Finally, eight paternal expression sites and three maternal expression sites were detected, whereas two paternally expressed imprinted genes (NDN and IGF2) and one maternally expressed imprinted gene (H1-3) were validated by Sanger sequencing. DNA methylation regulates the expression of imprinted genes, and all of the identified imprinted genes in this study were predicted to possess CpG islands. PBX1 and YY1 binding motifs were discovered in the promoter regions of all three imprinted genes, which were candidate elements regulating the transcription of imprinted genes. For these identified imprinted genes, IGF2 and NDN promoted muscle growth whereas H1-3 inhibited cell proliferation, corroborating the 'parental conflict' theory that paternally expressed imprinted genes assisted descendants' growth whereas maternally expressed imprinted genes inhibited it. This study discovered porcine imprinted genes in skeletal muscle and was the first to reveal that H1-3 was expressed by the maternal allele to our knowledge. Our findings provided valuable resources for the potential utilization of imprinted genes in pig breeding.

Widespread imprinting of transposable elements and variable genes in the maize endosperm

Fertilization and seed development is a critical time in the plant life cycle, and coordinated development of the embryo and endosperm are required to produce a viable seed. In the endosperm, some genes show imprinted expression where transcripts are derived primarily from one parental genome. Imprinted gene expression has been observed across many flowering plant species, though only a small proportion of genes are imprinted. Understanding how imprinted expression arises has been complicated by the reliance on single nucleotide polymorphisms between alleles to enable testing for imprinting. Here, we develop a method to use whole genome assemblies of multiple genotypes to assess for imprinting of both shared and variable portions of the genome using data from reciprocal crosses. This reveals widespread maternal expression of genes and transposable elements with presence-absence variation within maize and across species. Most maternally expressed features are expressed primarily in the endosperm, suggesting that maternal de-repression in the central cell facilitates expression. Furthermore, maternally expressed TEs are enriched for maternal expression of the nearest gene, and read alignments over maternal TE-gene pairs indicate that these are fused rather than independent transcripts.

Parent-of-origin effects on carcass traits in Japanese Black cattle.

Variances caused by the differential expression of paternally and maternally imprinted genes controlling carcass traits in Japanese Black cattle were estimated in this study. Data on marbling score (BMS), carcass weight, rib thickness, rib-eye area (REA) and subcutaneous fat thickness (SFT) were collected from a total of 13,115 feedlot steers and heifers in a commercial population. A sire-maternal grandsire model was used to analyse the data, and then, imprinting parameters were derived by replacing the genetic effect of the dam with the effect of the maternal grandsire in the imprinting model to calculate the genetic parameter estimates. The proportions of the total genetic variance attributable to imprinted genes ranged from 8.7% (SFT) to 35.2% (BMS). The remarkably large imprinting variance of BMS was mainly contributed by maternally expressed inheritance because the maternal contribution of the trait was much larger than that of the paternal trait. The parent-of-origin effect originating from maternal gene expression was also observed for REA. The results suggested the existence of genomic imprinting effects on the traits of the Japanese Black cattle. Hence, the parent-of-origin effect should be considered for the genetic evaluation of Japanese Black cattle in breeding programmes.

Integrated Analysis of Quantitative Proteome and Transcriptional Profiles Reveals the Dynamic Function of Maternally Expressed Proteins After Parthenogenetic Activation of Buffalo Oocyte

Maternal-effect genes are especially critical for early embryonic development after fertilization and until massive activation of the embryonic genome occurs. By applying a tandem mass tag (TMT)-labeled quantitative proteomics combined with RNA sequencing approach, the proteome of the buffalo was quantitatively analyzed during parthenogenesis of mature oocytes and the two-cell stage embryo. Of 1908 quantified proteins, 123 differed significantly. The transcriptome was analyzed eight stages (GV, MII, 2-cell, 4-cell, 8-cell, 16-cell, morula, blastocyst) of Buffalo using the RNA sequencing approach, and a total of 3567 unique genes were identified to be differently expressed between all consecutive stages of pre-implantation development. Validation of proteomics results (TUBB3, CTNNA1, CDH3, MAP2K1), which are involved in tight junction and gap junction, revealing that the maternal expression of the proteins possibly plays a role in the formation of cellular junctions firstly after parthenogenetic activation. Correlation and hierarchical analyses of transcriptional profiles and the expression of NPM2 and NLRP5 mRNA of buffalo in vitro developed oocytes and parthenogenetic embryos indicated that the "maternal-to-zygotic transition" (MZT) process might exist in the model of parthenogenesis, which is similar to a normally fertilized embryo, and may occur between the 8-cell to 16-cell stage. These data provide a rich resource for further studies on maternal proteins and genes and are conducive to improving nuclear transfer technology.

Mining Prognostic Significance of MEG3 in Human Breast Cancer Using Bioinformatics Analysis

Background/Aims: Maternally expressed gene 3 (MEG3) is an imprinted gene with maternal expression, which may function as a tumor suppressor by inhibiting angiogenesis. To identify the prognostic value of MEG3 in breast cancer, systematic analysis was performed in this study. Methods: To evaluate gene alteration during breast carcinogenesis, we explored MEG3 expression using the Serial Analysis of Gene Expression Genie suite and Oncomine analysis. The prognostic roles of MEG3 in breast cancer were investigated using the PrognoScan database. The heat map and methylation status of MEG3 were determined using the UCSC Genome Browser. Results: We found that MEG3 was more frequently downregulated in breast cancer than in normal tissues and this correlated with prognosis. However, estrogen receptor and progesterone receptor status were found to be positively correlated with MEG3 expression. Conversely, basal-like status, triple-negative breast cancer status, and Scarff Bloom & Richardson grade criterion were negatively correlated with MEG3 expression. Following data mining in multiple big data databases, we confirmed a positive correlation between MEG3 and heparan sulfate proteoglycan 2 (HSPG2) expression in breast cancer tissues. Conclusion: MEG3 could be adopted as a marker to predict the prognosis of breast cancer with HSPG2. However, large-scale and comprehensive research is needed to clarify our results.

Expression of Hox paralog group 13 genes in adult and developing Megalobrama amblycephala

Hox genes encode transcription factors that play a key role in specifying the body plan in most metazoans. HoxPG13 genes most probably played an important role in body length variation during the evolution of animals. This is the first report of the mRNA expression patterns of the entire Hox paralog group 13 (HoxA13a, HoxA13b, HoxB13a, HoxC13a, HoxC13b, HoxD13a) in fish. Expression was studied by qPCR in five tissues of adult Megalobrama amblycephala specimens (spleen, liver, kidney, intestine and gills) and during development (17 stages: fertilised egg to 90 days-old juveniles). Expression in tissues (for all six genes) was generally very low in gills (0.0006–0.05), spleen (0.006–0.09) and kidney (0.02–0.39); the highest in intestine (from 2.28 for HoxC13b to 244.29 for HoxC13a). During the development, a peak in expression around the hatching was observed for all six genes. Results suggest a high maternal expression of HoxA13a, and low for HoxA13ab. HoxD13a paralog exhibited the lowest expression: 0.0006–2.63 in tissues and 0.0005–1.7 during development, suggesting the possibility of a gradual loss of functionality. Expression of HoxC13 paralogs corroborates the findings in zebrafish: HoxC13b is maternally expressed and more important during the development. In adults, it was the opposite: expression was low for HoxC13b and very variable for HoxC13a (0.06–244.29). Differences in expression levels between both pairs of paralogs (Aa/Ab and Ca/Cb) indicate the possibility of the existence of some redundancy afforded by maintaining both paralogs.

Regulation of DLK1 by the maternally expressed miR-379/miR-544 cluster may underlie callipyge polar overdominance inheritance

Inheritance of the callipyge phenotype in sheep is an example of polar overdominance inheritance, an unusual mode of inheritance. To investigate the underlying molecular mechanism, we profiled the expression of the genes located in the Delta-like 1 homolog (Dlk1)-type III iodothyronine deiodinase (Dio3) imprinting region in mice. We found that the transcripts of the microRNA (miR) 379/miR-544 cluster were highly expressed in neonatal muscle and paralleled the expression of the Dlk1. We then determined the in vivo role of the miR-379/miR-544 cluster by establishing a mouse line in which the cluster was ablated. The maternal heterozygotes of young mutant mice displayed a hypertrophic tibialis anterior muscle, extensor digitorum longus muscle, gastrocnemius muscle, and gluteus maximus muscle and elevated expression of the DLK1 protein. Reduced expression of DLK1 was mediated by miR-329, a member of this cluster. Our results suggest that maternal expression of the imprinted miR-379/miR-544 cluster regulates paternal expression of the Dlk1 gene in mice. We therefore propose a miR-based molecular working model for polar overdominance inheritance.

Tissue-specific regulation of Igf2r/Airn imprinting during gastrulation.

BackgroundAppropriate epigenetic regulation of gene expression during lineage allocation and tissue differentiation is required for normal development. One example is genomic imprinting, which is defined as parent-of-origin mono-allelic gene expression. Imprinting is established largely due to epigenetic differences arriving in the zygote from sperm and egg haploid genomes. In the mouse, there are approximately 150 known imprinted genes, many of which occur in imprinted gene clusters that are regulated together. One imprinted cluster includes the maternally expressed Igf2r, Slc22a2, and Slc22a3 genes and the paternally expressed long non-coding RNA (lncRNA) Airn. Although it is known that Igf2r and Airn are reciprocally imprinted, the timing of imprinted expression and accompanying epigenetic changes have not been well characterized in vivo.ResultsHere we show lineage- and temporal-specific regulation of DNA methylation and histone modifications at the Igf2r/Airn locus correlating with differential establishment of imprinted expression during gastrulation. Our results show that Igf2r is expressed from both alleles in the E6.5 epiblast. After gastrulation commences, the locus becomes imprinted in the embryonic lineage with the lncRNA Airn expressed from the paternal allele and Igf2r restricted to maternal allele expression. We document differentially enriched allele-specific histone modifications in extraembryonic and embryonic tissues. We also document for the first time allele-specific spreading of DNA methylation during gastrulation concurrent with establishment of imprinted expression of Igf2r. Importantly, we show that imprinted expression does not change in the extraembryonic lineage even though maternal DMR2 methylation spreading does occur, suggesting distinct mechanisms at play in embryonic and extraembryonic lineages.ConclusionsThese results indicate that similar to preimplantation, gastrulation represents a window of dynamic lineage-specific epigenetic regulation in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0003-y) contains supplementary material, which is available to authorized users.

Distinct expression patterns and functions of the pygopus genes in amphioxus and zebrafish early embryogenesis

The Wnt/β-catenin transcriptional activation complex requires the adapter protein Pygopus (Pygo), which links the basal transcription machine to β-catenin to regulate the transcription of target genes. Previous studies revealed that the Pygo genes were broadly involved in the embryonic development of Drosophila, Xenopus and mouse, however, their functions in the embryonic development of amphioxus and zebrafish remain to be investigated. Here, we cloned and characterized one Pygo gene from amphioxus Branchiostoma belcheri (BbPygo) and two from zebrafish Danio rerio (DrPygo1 and DrPygo2). The spatial–temporal expression profiles showed that all these genes were maternally expressed. BbPygo expression was distributed ubiquitously in the amphioxus embryos before the gastrula stage and became restricted in the pharyngeal region in late stages. Similar to BbPygo, DrPygo2 was also expressed throughout whole embryos before the early gastrula stage, but limited in the pharyngeal region and central nervous system (CNS) in subsequent developing stages. Further knockdown of the DrPygo2 resulted in developmental defects of the body axis, CNS and cartilage segments in zebrafish. In contrast, the expression signal of the DrPygo1 gene was not detectable using the in situ hybridization method, which is consistent with its extremely low expression level detected by RT-qPCR. The expression patterns and function investigations revealed in this study suggest that the three Pygo genes probably function differentially though all of them show a conserved maternal expression pattern during the early embryonic stages of amphioxus and zebrafish.

Abstract 600: Maternally expressed gene 3 (MEG3) as a tumor suppressor in nasopharyngeal carcinoma

Abstract Nasopharyngeal carcinoma (NPC) is a distinctive type of head and neck cancer arising from the nasopharynx. Although rarely occurring worldwide, the incidence and mortality rates of this tumor are remarkably high among Southern China including Hong Kong. The distinct geographical distribution highlights the significance of several etiologic factors in NPC tumorigenesis, including Epstein-Barr virus (EBV) infection, genetic predisposition and intake of preserved food. However, the understanding of NPC development is still unclear. In our previous genome-wide analysis, chromosome 14q32 has been suggested as a tumor suppressor region where loss of heterozygosity (LOH) was detected in >80% of NPCs. Hence, some novel candidate genes involving in NPC development may be located within this region. By microarray study in NPC samples, consistent down-regulation of a putative tumor suppressor gene, maternally expressed gene 3 (MEG3) within the 14q32 deletion region was identified. MEG3 is an imprinted gene with preferential maternal expression and encodes for a long non-coding RNA. The silenced expression of MEG3 gene in NPC cell lines and xenografts was further confirmed by quantitative RT-PCR. Subsequent DNA methylation analysis in a panel of NPC samples demonstrated that densely methylation at a CpG island on the MEG3 promoter region was observed in all tested NPC cell lines, xenografts and 50% of primary NPCs, while normal imprinting pattern was found in non-cancerous nasopharyngeal epithelium. Interestingly, the presence of demethylation agent in culture medium could successfully rescue MEG3 expression in available NPC cell lines, indicating that promoter hypermethylation is one of the mechanisms to control MEG3 expression. On the other hand, fluorescent in situ hybridization (FISH) also showed the loss of DNA copy number at chromosome 14q32 in 60% of primary NPCs, suggesting an alternative MEG3 regulation mechanism in NPC. In our functional studies, ectopic MEG3 expression in NPC cell lines could significantly inhibit cell propagation, colony and foci formation. A series of reporter assays further suggested that MEG3 might contribute to regulate both p53 and TGF-β signaling pathways. In conclusion, we have found that MEG3 is a novel tumor suppressor gene in NPC. Further investigation the function of MEG3 in NPC pathogenesis may have potential implications to improve NPC treatment in the future. Citation Format: Wing Po Chak, Joanna Hung-Man Tong, Raymond Wai-Ming Lung, Yat Yee Chan, Kwok Wai Lo, Ka Fai To. Maternally expressed gene 3 (MEG3) as a tumor suppressor in nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 600. doi:10.1158/1538-7445.AM2014-600

IMAGe syndrome: clinical and genetic implications based on investigations in three Japanese patients

Arboleda et al. have recently shown that IMAGe (intra-uterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita and genital abnormalities) syndrome is caused by gain-of-function mutations of maternally expressed gene CDKN1C on chromosome 11p15.5. However, there is no other report describing clinical findings in patients with molecularly studied IMAGe syndrome. Here, we report clinical and molecular findings in Japanese patients. We studied a 46,XX patient aged 8·5 years (case 1) and two 46,XY patients aged 16·5 and 15·0 years (cases 2 and 3). Clinical studies revealed not only IMAGe syndrome-compatible phenotypes in cases 1-3, but also hitherto undescribed findings including relative macrocephaly and apparently normal pituitary-gonadal endocrine function in cases 1-3, familial glucocorticoid deficiency (FGD)-like adrenal phenotype and the history of oligohydramnios in case 2, and arachnodactyly in case 3. Sequence analysis of CDKN1C, pyrosequencing-based methylation analysis of KvDMR1 and high-density oligonucleotide array comparative genome hybridization analysis for chromosome 11p15.5 were performed, showing an identical de novo and maternally inherited CDKN1C gain-of-function mutation (p.Asp274Asn) in cases 1 and 2, respectively, and no demonstrable abnormality in case 3. The results of cases 1 and 2 with CDKN1C mutation would argue the following: [1] relative macrocephaly is consistent with maternal expression of CDKN1C in most tissues and biparental expression of CDKN1C in the foetal brain; [2] FGD-like phenotype can result from CDKN1C mutation; and [3] genital abnormalities may primarily be ascribed to placental dysfunction. Furthermore, lack of CDKN1C mutation in case 3 implies genetic heterogeneity in IMAGe syndrome.

Identification of imprinted genes subject to parent-of-origin specific expression in Arabidopsis thalianaseeds

BackgroundEpigenetic regulation of gene dosage by genomic imprinting of some autosomal genes facilitates normal reproductive development in both mammals and flowering plants. While many imprinted genes have been identified and intensively studied in mammals, smaller numbers have been characterized in flowering plants, mostly in Arabidopsis thaliana. Identification of additional imprinted loci in flowering plants by genome-wide screening for parent-of-origin specific uniparental expression in seed tissues will facilitate our understanding of the origins and functions of imprinted genes in flowering plants.ResultscDNA-AFLP can detect allele-specific expression that is parent-of-origin dependent for expressed genes in which restriction site polymorphisms exist in the transcripts derived from each allele. Using a genome-wide cDNA-AFLP screen surveying allele-specific expression of 4500 transcript-derived fragments, we report the identification of 52 maternally expressed genes (MEGs) displaying parent-of-origin dependent expression patterns in Arabidopsis siliques containing F1 hybrid seeds (3, 4 and 5 days after pollination). We identified these MEGs by developing a bioinformatics tool (GenFrag) which can directly determine the identities of transcript-derived fragments from (i) their size and (ii) which selective nucleotides were added to the primers used to generate them. Hence, GenFrag facilitates increased throughput for genome-wide cDNA-AFLP fragment analyses. The 52 MEGs we identified were further filtered for high expression levels in the endosperm relative to the seed coat to identify the candidate genes most likely representing novel imprinted genes expressed in the endosperm of Arabidopsis thaliana. Expression in seed tissues of the three top-ranked candidate genes, ATCDC48, PDE120 and MS5-like, was confirmed by Laser-Capture Microdissection and qRT-PCR analysis. Maternal-specific expression of these genes in Arabidopsis thaliana F1 seeds was confirmed via allele-specific transcript analysis across a range of different accessions. Differentially methylated regions were identified adjacent to ATCDC48 and PDE120, which may represent candidate imprinting control regions. Finally, we demonstrate that expression levels of these three genes in vegetative tissues are MET1-dependent, while their uniparental maternal expression in the seed is not dependent on MET1.ConclusionsUsing a cDNA-AFLP transcriptome profiling approach, we have identified three genes, ATCDC48, PDE120 and MS5-like which represent novel maternally expressed imprinted genes in the Arabidopsis thaliana seed. The extent of overlap between our cDNA-AFLP screen for maternally expressed imprinted genes, and other screens for imprinted and endosperm-expressed genes is discussed.

Drosophila Ube3a regulates monoamine synthesis by increasing GTP cyclohydrolase I activity via a non-ubiquitin ligase mechanism

The underlying defects in Angelman syndrome (AS) and autism spectrum disorder (ASD) may be in part due to basic defects in synaptic plasticity and function. In some individuals serotonin reuptake inhibitors, which decrease pre-synaptic re-uptake of serotonin, can ameliorate symptoms, as can resperidone, which blocks both dopamine and serotonin receptors. Loss of maternal UBE3A expression causes AS, while maternal duplications of chromosome 15q11.2–q13 that include the UBE3A gene cause ASD, implicating the maternally expressed UBE3A gene in the ASD phenotype. In a Drosophila screen for proteins regulated by UBE3A, we identified a key regulator of monoamine synthesis, the gene Punch, or GCH1, encoding the enzyme GTP cyclohydrolase I. Here we show that Dube3a, the fly UBE3A orthologue, regulates Punch/GCH1 in the fly brain. Over-expression of Dube3a elevates tetrahydrobiopterin (THB), the rate-limiting cofactor in monoamine synthesis while loss of Dube3a has the opposite effect. The fluctuations in dopamine levels were associated with hyper- and hypoactivity, respectively, in flies. We show that changes in Punch/GCH1 and dopamine levels do not depend on the ubiquitin ligase catalytic domain of Dube3a. In addition, both wild type Dube3a and a ubiquitination-defective Dube3a-C/A form were found at high levels in nuclear fractions and appear to be poly-ubiquitinated in vivo by endogenous Dube3a. We propose that the transcriptional co-activation function of Dube3a may regulate GCH1 activity in the brain. These results provide a connection between monoamine synthesis (dopamine/serotonin) and Dube3a expression that may explain why some individuals with ASD or AS respond better to selective serotonin reuptake inhibitors than others.

The vertebrate makorin ubiquitin ligase gene family has been shaped by large-scale duplication and retroposition from an ancestral gonad-specific, maternal-effect gene

BackgroundMembers of the makorin (mkrn) gene family encode RING/C3H zinc finger proteins with U3 ubiquitin ligase activity. Although these proteins have been described in a variety of eukaryotes such as plants, fungi, invertebrates and vertebrates including human, almost nothing is known about their structural and functional evolution.ResultsVia partial sequencing of a testis cDNA library from the poeciliid fish Xiphophorus maculatus, we have identified a new member of the makorin gene family, that we called mkrn4. In addition to the already described mkrn1 and mkrn2, mkrn4 is the third example of a makorin gene present in both tetrapods and ray-finned fish. However, this gene was not detected in mouse and rat, suggesting its loss in the lineage leading to rodent murids. Mkrn2 and mkrn4 are located in large ancient duplicated regions in tetrapod and fish genomes, suggesting the possible involvement of ancestral vertebrate-specific genome duplication in the formation of these genes. Intriguingly, many mkrn1 and mkrn2 intronless retrocopies have been detected in mammals but not in other vertebrates, most of them corresponding to pseudogenes. The nature and number of zinc fingers were found to be conserved in Mkrn1 and Mkrn2 but much more variable in Mkrn4, with lineage-specific differences. RT-qPCR analysis demonstrated a highly gonad-biased expression pattern for makorin genes in medaka and zebrafish (ray-finned fishes) and amphibians, but a strong relaxation of this specificity in birds and mammals. All three mkrn genes were maternally expressed before zygotic genome activation in both medaka and zebrafish early embryos.ConclusionOur analysis demonstrates that the makorin gene family has evolved through large-scale duplication and subsequent lineage-specific retroposition-mediated duplications in vertebrates. From the three major vertebrate mkrn genes, mkrn4 shows the highest evolutionary dynamics, with lineage-specific loss of zinc fingers and even complete gene elimination from certain groups of vertebrates. Comparative expression analysis strongly suggests that the ancestral E3 ubiquitin ligase function of the single copy mkrn gene before duplication in vertebrates was gonad-specific, with maternal expression in early embryos.

At Least Ten Genes Define the Imprinted Dlk1-Dio3 Cluster on Mouse Chromosome 12qF1

BackgroundGenomic imprinting is an exception to Mendelian genetics in that imprinted genes are expressed monoallelically, dependent on parental origin. In mammals, imprinted genes are critical in numerous developmental and physiological processes. Aberrant imprinted gene expression is implicated in several diseases including Prader-Willi/Angelman syndromes and cancer.Methodology/Principal FindingsTo identify novel imprinted genes, transcription profiling was performed on two uniparentally derived cell lines, androgenetic and parthenogenetic primary mouse embryonic fibroblasts. A maternally expressed transcript termed Imprinted RNA near Meg3/Gtl2 (Irm) was identified and its expression studied by Northern blotting and whole mounts in situ hybridization. The imprinted region that contains Irm has a parent of origin effect in three mammalian species, including the sheep callipyge locus. In mice and humans, both maternal and paternal uniparental disomies (UPD) cause embryonic growth and musculoskeletal abnormalities, indicating that both alleles likely express essential genes. To catalog all imprinted genes in this chromosomal region, twenty-five mouse mRNAs in a 1.96Mb span were investigated for allele specific expression.Conclusions/SignificanceTen imprinted genes were elucidated. The imprinting of three paternally expressed protein coding genes (Dlk1, Peg11, and Dio3) was confirmed. Seven noncoding RNAs (Meg3/Gtl2, Anti-Peg11, Meg8, Irm/“Rian”, AK050713, AK053394, and Meg9/Mirg) are characterized by exclusive maternal expression. Intriguingly, the majority of these noncoding RNA genes contain microRNAs and/or snoRNAs within their introns, as do their human orthologs. Of the 52 identified microRNAs that map to this region, six are predicted to regulate negatively Dlk1, suggesting an additional mechanism for interactions between allelic gene products. Since several previous studies relied heavily on in silico analysis and RT-PCR, our findings from Northerns and cDNA cloning clarify the genomic organization of this region. Our results expand the number of maternally expressed noncoding RNAs whose loss may be responsible for the phenotypes associated with mouse pUPD12 and human pUPD14 syndromes.

165. PLASTICITY IN IMPRINTING OF BOVINE IGF2R CORRELATES WITH TOTAL EXPRESSION LEVELS IN FETAL TISSUES

The multifunctional insulin-like growth factor 2 receptor (IGF2R) facilitates endocytosis and subsequent clearance or activation of a variety of ligands involved in cell growth and motility. Thus, the IGF2R gene has a major role in embryonic development and fetal growth. Murine Igf2r is subject to genomic imprinting and maternally expressed in peripheral fetal tissues. However, data on imprinting of IGF2R in human is still controversial with biallelic expression, partial imprinting, and monoallelic expression reported for fetal tissues [1, 2, 3]. Data from additional species may help to understand fetal IGF2R expression. The bovine is similar to human in that it is outbred and monotocous. We have analysed bovine Day 153 fetuses (55% to term, n=40) with Bos primigenius indicus and B. p. taurus genetics to determine the imprinting status of IGF2R in fetal brain, liver and skeletal muscle. Sequencing of PCR amplicons from IGF2R exon 48 revealed a polymorphic microsatellite and 14 SNPs. These were used to identify 15 heterozygous fetuses informative for imprinting analysis. We found biallelic expression of IGF2R in fetal brain and predominantly maternal expression in fetal liver and skeletal muscle. However, we observed considerable plasticity in imprinting in liver and skeletal muscle with paternal expression levels of 7%-21% and 4%-21%, respectively. Fetal liver samples with B. p. indicus maternal genetics showed significantly higher mean paternal expression levels than those with B. p. taurus maternal genetics (P<0.05). Real-time qPCR showed a significant relationship between imprinting and total IGF2R expression level within both tissues (P<0.05). Our data indicate plasticity in imprinting of IGF2R that could fine tune expression levels in fetal tissues.

Association study of the 15q11‐q13 maternal expression domain in Japanese autistic patients

Chromosome 15q11-q13 has been a focus of genetic studies of autism susceptibility, because cytogenetic abnormalities are frequently observed in this region in autistic patients. An imprinted, maternally expressed gene within the region may have a role in autistic symptomatology. In the present study, we investigated the association between autism and the maternal expression domain (MED) in the region, containing the UBE3A and ATP10C genes, and the upstream imprinting center (IC), which mediates coordinate control of imprinted expression throughout the region. We analyzed 41 single nucleotide polymorphisms (SNPs) in 166 patients with autism and 416 controls from a Japanese population. As a result, a statistically significant difference after correction for multiple testing was observed between the patients and controls in the genotypic distribution of SNP rs7164989 (SNP8 in this study) located in SNRPN, whose promoter corresponds to the IC (P = 0.018, corrected for multiple testing). In the analysis of a four-marker haplotype located in ATP10C, a statistically significant difference after correction for multiple testing was observed in the frequency of one haplotype between male patients and controls (permutation P = 0.033, corrected for multiple testing). Thus, the present study may suggest the association between autism and the MED or the upstream IC in chromosome 15q11-q13 in the Japanese population.

Smad2 and smad3 Are Required for Mesendoderm Induction by Transforming Growth Factor-β/Nodal Signals in Zebrafish

The transforming growth factor-beta ligands Nodal, activin, and Vg1 play important roles in mesendoderm induction and patterning during vertebrate embryogenesis. These ligands are believed to transduce the signal through the receptor-activated transcription factors Smad2 and Smad3. However, the roles of smad2/3 genes in development of zebrafish embryos are largely unknown because the presence of multiple smad2/3 genes and their maternal expression have hampered the investigation of their developmental roles. We generated potent and specific dominant-negative forms of zebrafish Smad2, Smad3a, and Smad3b by mutating multiple amino acids. Overexpression of these mutants abolished mesendoderm induction by ectopic Nodal signaling in zebrafish embryos. Expression of dominant-negative smad2/3 abrogated Smad2/3 activities in wild-type embryos and caused various mesendodermal defects similar to those in Nodal-deficient embryos. Smad2/3-deficient cells transplanted into the blastodermal margin of wild-type hosts preferentially differentiated into ectodermal tissues rather than mesendodermal tissues, supporting the idea that response of cells to mesendoderm inducers requires Smad2/3 activities. Interference with Smad2/3 activities in Zoep, Moep, and MZoep mutant embryos resulted in more severe mesendodermal defects. Thus, our data reveal that Nodal signaling and mesendoderm induction depend on Smad2/3 and suggest that transforming growth factor-beta signals other than Nodal also contribute to Smad2/3 signaling and embryonic patterning.

Monoallelic expression of nine imprinted genes in the sheep embryo occurs after the blastocyst stage

The preimplantation embryos of a range of mammals can be susceptible to disruptions in genomic imprinting mechanisms, resulting in loss of imprinting. Such disruptions can have developmental consequences involving foetal and placental growth such as Beckwith-Wiedemann syndrome in humans and large offspring syndrome in sheep. Our objective was to investigate the dynamics of establishing monoallelic expression of individual sheep imprinted genes post-fertilisation. Semi-quantitative RT-PCR was used to amplify cDNA from the sheep blastocyst, day 21 foetus and day 21 chorioallantois, to compare expression levels between biparental and parthenogenetic embryos in order to indicate allelic expression status. In common with other mammals, IGF2, PEG1 and PEG3 were paternally expressed in the day 21 conceptus, while H19, IGF2R, GRB10 and p57KIP were maternally expressed. Interestingly, GNAS was maternally expressed in the foetus, but paternally expressed in the chorioallantois at day 21. Overall, the imprinting of ovine GRB10 and IGF2R was comparable with mouse but not with human. Contrary to the trophoblast-restricted maternal expression in both mouse and human, SASH2 (sheep homologue of Mash2/HASH2) was expressed in the ovine foetus and was biallelically expressed in the chorioallantois. Differential methylation of the H19 CTCF III upstream region and IGF2R DMR2 in the chorioallantois revealed predominantly paternal and maternal methylation respectively, indicating conservation of these imprinting regulatory regions. In blastocysts, IGF2R, GRB10 and SASH2 were expressed biallelically, while the other genes were not detected. Thus, for the majority of ovine imprinted genes examined, monoallelic expression does not occur until after the blastocyst stage.

Molecular characterization of common carp (Cyprinus carpio) Sonic Hedgehog and discovery of its maternal expression

Sonic hedgehog (Shh), one of important homologous members of the hedgehog (Hh) family in vertebrates, encodes a signaling molecule that is involved in short- or long-range patterning processes during embryogenesis. In zebrafish, maternal activity of Hh was found to be contributing to the formation of primary motoneurons. However, we found that all of the known Hh members were not maternally expressed in zebrafish. In the present study, full-length cDNA of common carp (Cyprinus carpio) Shh (cShh) was gained by degenerate reverse-transcription PCR (RT-PCR) and rapid amplification of cDNA ends. Sequence comparison shows that cShh coding sequence shares 93.4% identity with zebrafish Shh coding sequence, and their corresponding protein sequences have 91.9% similarity. Comparative analysis of Shh genomic sequences and Hh protein sequences from different species revealed that the genomic structures of Hh are conserved from invertebrate to vertebrate. In contrast to zebrafish Shh, cShh transcripts were detectable from one-cell stage by RT-PCR analysis. Whole mount in situ hybridization verified the maternal expression of Shh in common carp, which is, to our knowledge, the first report of that in vertebrates, suggesting that Shh might be responsible for the maternal Hh activity in common carp.