Maternal peripheral blood natural killer cells incorporate placenta-associated microRNAs during pregnancy.

Although recent studies have demonstrated that microRNAs (miRNAs or miRs) regulate fundamental natural killer (NK) cellular processes, including cytotoxicity and cytokine production, little is known about the miRNA-gene regulatory relationships in maternal peripheral blood NK (pNK) cells during pregnancy. In the present study, to determine the roles of miRNAs within gene regulatory networks of maternal pNK cells, we performed comprehensive miRNA and gene expression profiling of maternal pNK cells using a combination of reverse transcription quantitative PCR (RT-qPCR)-based miRNA array and DNA microarray analyses and analyzed the differential expression levels between first- and third-trimester pNK cells. Furthermore, we constructed regulatory networks for miRNA-mediated gene expression in pNK cells during pregnancy by Ingenuity Pathway Analysis (IPA). PCR-based array analysis revealed that the placenta-derived miRNAs [chromosome 19 miRNA cluster (C19MC) miRNAs] were detected in pNK cells during pregnancy. Twenty-five miRNAs, including six C19MC miRNAs, were significantly upregulated in the third- compared to first-trimester pNK cells. The rapid clearance of C19MC miRNAs also occurred in the pNK cells following delivery. Nine miRNAs, including eight C19MC miRNAs, were significantly downregulated in the post-delivery pNK cells compared to those of the third-trimester. DNA microarray analysis identified 69 NK cell function-related genes that were differentially expressed between the first- and third-trimester pNK cells. On pathway and network analysis, the observed gene expression changes of pNK cells likely contribute to the increase in the cytotoxicity, as well as the cell cycle progression of third- compared to first-trimester pNK cells. Thirteen of the 69 NK cell function-related genes were significantly downregulated between the first- and third-trimester pNK cells. Nine of the 13 downregulated NK-function-associated genes were in silico target candidates of 12 upregulated miRNAs, including C19MC miRNA miR-512-3p. The results of this study suggest that the transfer of placental C19MC miRNAs into maternal pNK cells occurs during pregnancy. The present study provides new insight into maternal NK cell functions.

Human exosomal placenta-associated miR-517a-3p modulates the expression of PRKG1 mRNA in Jurkat cells.

During pregnancy, human placenta-associated microRNAs (miRNAs) derived from the miRNA cluster in human chromosome 19 are expressed in villous trophoblasts and secreted into maternal circulation via exosomes; however, little is known about whether circulating placenta-associated miRNAs are transferred into maternal immune cells via exosomes, and modulate expression of target genes in the recipient cells. We employed an in vitro model of trophoblast-immune cell communication using BeWo cells (a human trophoblast cell line) and Jurkat cells (a human leukemic T-cell line) and investigated whether BeWo exosomal placenta-associated miRNAs can suppress expression of target genes in the recipient Jurkat cells. Using this system, we identified PRKG1 as a target gene of placenta-associated miRNA miR-517a-3p. Moreover, we demonstrated that BeWo exosomal miR-517a-3p was internalized into Jurkat cells and subsequently suppressed the expression of PRKG1 in recipient Jurkat cells. Furthermore, using peripheral blood natural killer (NK) cells in vivo, we confirmed that circulating miR-517a-3p was delivered into maternal NK cells as it was into Jurkat cells in vitro. Placenta-associated miR-517a-3p was incorporated into maternal NK cells in the third trimester, and it was rapidly cleared after delivery. Expression levels of miR-517a-3p and its target mRNA PRKG1 were inversely correlated in NK cells before and after delivery. These in vitro and in vivo results suggest that exosome-mediated transfer of placenta-associated miRNAs and subsequent modulation of their target genes occur in maternal NK cells. The present study provides novel insight into our understanding of placenta-maternal communication.

Differential expression of ADAM (a disintegrin and metalloproteinase) genes between human first trimester villous and extravillous trophoblast cells.

A disintegrin and metalloproteinases (ADAMs) are members of the metzincin family of zinc-dependent metalloproteinases that play pivotal roles in the proteolytic degradation of the extracellular matrix for cell invasion. Few studies have investigated the ADAM subtypes that are expressed in first trimester trophoblast cells. The purpose of this study was to elucidate the differential expression profiles of ADAMs between first trimester villous trophoblast cells (VTs) and extravillous trophoblast cells (EVTs). We isolated EVTs from explanted human first trimester chorionic villi and investigated the mRNA expression levels of five members of the ADAM family (ADAMTS1, ADAMTS2, ADAM10, ADAM12, and ADAM17) using real-time PCR. Chorionic villous tips were defined as first trimester VTs. Of the differentially expressed ADAM genes between first trimester VTs and EVTs, ADAMTS1 was expressed at a significantly higher level in EVTs than in VTs. In contrast, both ADAM10 and ADAM12 were expressed at significantly higher levels in VTs than in EVTs. No differences were found in the mRNA levels of ADAMTS2 and ADAM17 between the two cell types. Moreover, we demonstrated that in VTs, the expression level of ADAM12 was significantly downregulated in the late first trimester (10-13 gestational weeks) compared to the middle first trimester (7-8 weeks). These results suggest that first trimester trophoblast cells express ADAM genes in cell type- and gestational age-dependent manners. Our data provide additional insight into the functions of ADAMs in the human placenta.

Region-specific gene expression in early postnatal mouse thalamus.

Previous studies in the developing mouse thalamus have demonstrated that regional identity is established during early stages of development (Suzuki-Hirano et al. J. Comp. Neurol. 2011;519:528-543). However, the developing thalamus often shows little resemblance to the anatomical organization of the postnatal thalamus, making it difficult to identify genes that might mediate the organization of thalamic nuclei. We therefore analyzed the expression pattern of genes that we have identified as showing regional expression in embryonic thalamus on postnatal days (P) 6-8 by using in situ hybridization. We also identified several genes expressed only in the postnatal thalamus with restricted expression in specific nuclei. We first demonstrated the selective expression of neurotransmitter-related genes (vGlut2, vGAT, D2R, and HTR2C), identifying the neurotransmitter subtypes of cells in this region, and we also demonstrated selective expression of additional genes in the thalamus (Steel, Slitrk6, and AI852580). In addition, we demonstrated expression of genes specific to somatosensory thalamic nuclei, the ventrobasal posterior nuclei (VP); a visual thalamic nucleus, the dorsal lateral geniculate nucleus (dLGN); and an auditory thalamic nucleus, the medial geniculate body (MGB) (p57Kip, Nr1d1, and GFRα1). We also identified genes that are selectively expressed in multiple different nuclei (Foxp2, Chst2, and EphA8). Finally, we demonstrated that several bone morphogenetic proteins (BMPs) and their inhibitors are expressed in the postnatal thalamus in a nucleus-specific fashion, suggesting that BMPs play roles in the postnatal thalamus unrelated to their known role in developmental patterning. Our findings provide important information for understanding the mechanisms of nuclear specification and connectivity during development, as well as their maintenance in adult thalamus.

Evolutionary origin of sex-related genes in the mouse brain.

With the aim of elucidating the evolutionary process of sexual dimorphism in the brain at the molecular level, we conducted genomic comparisons of a set of genes expressed in a sexually different manner in the mouse brain with all genes from other species of eukaryotes. First, seventeen protein-coding genes whose levels of mRNA expression in the brain differed between male and female mice have been known according to the currently available microarray data, and we designated these genes operationally as "sex-related genes in the mouse brain". Next, we estimated the time when these sex-related genes in the mouse brain emerged in the evolutionary process of eukaryotes by examining the presence or absence of the orthologues in the 26 eukaryotic species whose genome sequences are available. As a result, we found that the ten sex-related genes in the mouse brain emerged after the divergence of urochordates and mammals whereas the other seven sex-related genes in the mouse brain emerged before the divergence of urochordates and mammals. In particular, five sex-related genes out of the ten genes in the mouse brain emerged just before the appearance of bony fish which have phenotypic sexual dimorphism in the brain. Interestingly, three of these five sex-related genes that emerged during this period were classified into the "protein binding" function category. Moreover, all of these three genes were expected to have the functions that are related to cell-cell communications in the brain according to the gene expression patterns and/or functional information of these genes. These findings suggest that the orthologues of the sex-related genes in the mouse brain that emerged just before the divergence of bony fish might have essential roles in the evolution of the sexual dimorphism in the brain forming protein-protein interactions.

The ken and barbie gene encoding a putative transcription factor with a BTB domain and three zinc finger motifs functions in terminalia development of Drosophila.

Mutations in the ken and barbie locus are accompanied by the malformation of terminalia in adult Drosophila. Male and female genitalia often remain inside the body, and the same portions of genitalia and analia are missing in a fraction of homozygous flies. Rotated and/or duplicated terminalia are also observed. Terminalia phenotypes are enhanced by mutations in the gap gene tailless, the homeobox gene caudal, and the decapentaplegic gene that encodes a TGFbeta-like morphogen. The ken and barbie gene encodes a protein with three CCHH-type zinc finger motifs that are conserved in several transcription factors such as Krüppel and BCL-6. All defects in ken and barbie mutants are fully rescued by the expression of a wild-type genomic construct, which establishes the causality between phenotypes and the gene.