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Most imprinted genes are concerned with embryonic development, especially placental development. Here, we identified a placenta-specific imprinted gene Qpct. Our results show that Qpct is widely expressed during early embryonic development and can be detected in the telecephalon, midbrain, and rhombencephalon at E9.5b-E11.5. Moreover, Qpct is strikingly expressed in the brain, lung and liver in E15.5. Expression signals for Qpct achieved a peak at E15.5 during placental development and were only detected in the labyrinth layer in E15.5 placenta. ChIP assay results suggest that the modification of histone H3K4me3 can result in maternal activating of Qpct.

The function of long noncoding RNAs (lncRNAs) in cell differentiation and development have begun to be revealed in recent years. However, the expression pattern and mechanisms regulating lncRNAs are largely unknown during mammalian preimplantation development. LncRNAs expressed from Dlk1-Dio3 imprinted region have been linked to pluripotency of induced pluripotent cells (iPSCs). In this study we show that these lncRNAs (Gtl2, Rian and Mirg) are first expressed at the morula stage and gradually restricted to the inner cell mass (ICM) as the embryo differentiates into the blastocyst. Analysis of DNA methylation at IG-DMR and Gtl2-DMR showed no change during preimplantation while the presence of the activating histone modification H3K4me3 increased significantly from 8-cell to blastocyst stage, which may explain the expression activation. Additionally, knockdown of transcription factors (Oct4, Sox2 and Nanog) in blastocyst reduced the expression of Gtl2, indicating pluripotency factors regulate transcription of these lncRNAs. This study provides the spatiotemporal expression and dynamic changes of lncRNAs from Dlk1-Dio3 imprinted region in mouse preimplantation stage embryos and offers insight into the potential mechanisms responsible for Gtl2 activation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Long intergenic non-coding RNAs (lincRNAs) as a key group of non-coding RNAs have gained substantial attention. Though lincRNAs have been systematically explored in various mouse tissues and cell lines, large-scale identification of lincRNAs in mouse pre-implantation embryonic development (PED) process has not be documented previously. Therefore, it is important to identify and characterize novel lincRNAs that may be involved in PED. In this paper, we performed transcriptome assembly based on published single-cell RNA-seq data during mouse PED and identified 4,438 putative lincRNAs. Combining these with Ensembl lincRNAs, we established a reference catalog of 5,808 transcribed lincRNAs in PED. We then systematically analyzed the lincRNAs in this reference catalog and revealed that the identified novel PED lincRNAs are generally comparable with known Ensembl lincRNAs in genomic aspects. In addition, the global expression patterns can be separated by zygote first cleavage division in clustering analysis and we further identified and analyzed differentially expressed lincRNAs involved in this process. The expression of lincRNAs involved in the process is negatively correlated with promoter methylation in trend. The identified lincRNAs involved in zygote first cleavage division could have important roles in mouse early embryonic development and need further functional studies. Altogether, a novel reference catalog of mouse PED lincRNAs is provided and characterized, which would be a valuable resource for further functional analyses and may help elucidate the pre-implantation regulatory mechanism.

MicroRNAs are small noncoding RNAs involved in various biological processes. We characterized the expression of miR-344-3p during mouse embryonic development. At E9.5-E10.5 and E15.5, in situ hybridization detected strong miR-344-3p signal in the central nervous system, including the cerebral cortex, hindbrain, cerebellum, thalamus, hindbrain, medulla oblongata, spinal cord, and dorsal root ganglia. Further, qRT-PCR analysis identified miR-344-3p expression at E15.5, with expression stably maintained in the brain from E12.5 to E18.5 before decreasing to relatively low levels postnatally. We also analyzed miR-344-3p expression using immunofluorescence in situ hybridization at E18.5 and within the adult brain. miR-344-3p signal was mainly detected in cortical regions surrounding the ventricular system, choroid plexus, glomerular layer of the olfactory bulb, and granular cell layer of the cerebellar cortex. Altogether, our results indicate miR-344-3p may play an important role in morphogenesis, nervous system development in the brain.

Mouse Pde4d is located on chromosome 13 and serves many functions in important physiological processes involving cyclic adenosine monophosphate. In this study, imprinting analysis indicated that Pde4d exhibits a dynamic and specific allelic expression pattern during embryo development. This showed paternal-origin sex bias in embryonic day 9.5 (E9.5) whole embryos and placenta, and biallelic expression in the major embryonic organs and placenta at E15.5. In situ hybridization determined the spatiotemporal expression pattern of Pde4d in mouse embryos from the mid- to late-embryonic stages. This demonstrated that Pde4d was widely expressed in the neural tissues, including the forebrain, midbrain, hindbrain, and neural tube, at the mid-embryonic stage. By the late-embryonic stage, Pde4d was extensively detected throughout the developing organism, including in the liver, brain, lung, kidney, and tongue. In addition, methylation analyses indicated that tissue-specific CpG methylation of the Pde4d promoter was correlated with Pde4d mRNA expression in major E15.5 tissues. Furthermore, stage-specific CpG methylation of the Pde4d promoter was associated with gene expression in the liver at three developmental stages. Our results suggest that Pde4d might serve specific biological functions in regulating the development process of the mouse embryo, and that CpG methylation of the Pde4d promoter may play an important role in regulating Pde4d at a transcriptional level., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Long non-coding RNAs (lncRNAs) as a key group of non-coding RNAs have gained widely attention. Though lncRNAs have been functionally annotated and systematic explored in higher mammals, few are under systematical identification and annotation. Owing to the expression specificity, known lncRNAs expressed in embryonic brain tissues remain still limited. Considering a large number of lncRNAs are only transcribed in brain tissues, studies of lncRNAs in developmental brain are therefore of special interest. Here, publicly available RNA-sequencing (RNA-seq) data in embryonic brain are integrated to identify thousands of embryonic brain lncRNAs by a customized pipeline. A significant proportion of novel transcripts have not been annotated by available genomic resources. The putative embryonic brain lncRNAs are shorter in length, less spliced and show less conservation than known genes. The expression of putative lncRNAs is in one tenth on average of known coding genes, while comparable with known lncRNAs. From chromatin data, putative embryonic brain lncRNAs are associated with active chromatin marks, comparable with known lncRNAs. Embryonic brain expressed lncRNAs are also indicated to have expression though not evident in adult brain. Gene Ontology analysis of putative embryonic brain lncRNAs suggests that they are associated with brain development. The putative lncRNAs are shown to be related to possible cis-regulatory roles in imprinting even themselves are deemed to be imprinted lncRNAs. Re-analysis of one knockdown data suggests that four regulators are associated with lncRNAs. Taken together, the identification and systematic analysis of putative lncRNAs would provide novel insights into uncharacterized mouse non-coding regions and the relationships with mammalian embryonic brain development.

Non-coding RNAs (ncRNAs) Meg8 and Irm were previously identified as alternatively splicing isoforms of Rian gene. Ascertaining ncRNAs spatiotemporal expression patterns is crucial for understanding the physiological roles of ncRNAs during tissue and organ development. In this study in mouse embryos, we focused on the developmental regulation expression of imprinted macro ncRNAs, Meg8 and Irm by using in situ hybridization and quantitative real-time RT-PCR (QRT-PCR). The in situ hybridization results showed that Meg8 and Irm were expressed in the developing brain at embryonic day 10.5 (E10.5) and E11.5, while Irm expression signals were strikingly detected in the somite, where Meg8 expression signals were undetectable. By E15.5, they were expressed in brain, tongue, liver, lung and neuroendocrine tissues, while Irm displayed more restricted expression in tongue and skeletal muscle than Meg8. Furthermore, quantitative analysis confirmed that they were highly expressed in tongue and brain at E12.5, E15.5 and E18.5. These results indicated that Meg8 and Irm might be coordinately expressed and functionally correlated in diverse of organs. Notably, Irm was more closely associated with morphogenesis of skeletal muscle in contrast to Meg8 during embryonic development., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Mouse Trmt112, the homologous gene of yeast Trm112 (tRNA methyltransferase 11-2), was initially cloned from RIKEN with uncertain function. The yeast TRM112 is now known to play important roles in RNA methylation. Here, we studied the expression of Trmt112 by in situ hybridization and quantitative real-time RT-PCR (QRT-PCR). A higher expression level of Trmt112 was observed in the brain and nervous system by whole mount in situ hybridization from embryonic day 10.5 (E10.5) to E11.5. At later developmental stages E13.5 and E16.5, abundant expression was prominently found in various organs and tissues including developing brain, nervous system, thymus, lung, liver, intestine, kidney, and cartilage. Furthermore, Trmt112 was persistently expressed from E9.5 to E18.5 on whole embryos and highly expressed in multiple organs at E12.5, E15.5 and E18.5 by QRT-PCR. These results showed that Trmt112 gene was highly and ubiquitously expressed during mouse embryonic development, implying that it might be involved in the morphogenesis of diverse organs and tissues and numerous physiological functions.