Hey2 enhancer activity defines unipotent progenitors for left ventricular cardiomyocytes in juxta-cardiac field of early mouse embryo.

The cardiac crescent is the first structure of the heart and contains progenitor cells of the first heart field, which primarily differentiate into left ventricular cardiomyocytes. The interface between the forming cardiac crescent and extraembryonic tissue is known as the juxta-cardiac field (JCF), and progenitor cells in this heart field contribute to the myocardium of the left ventricle and atrioventricular canal as well as the epicardium. However, it is unclear whether there are progenitor cells that differentiate specifically into left ventricular cardiomyocytes. We have previously demonstrated that an enhancer of the gene encoding the Hey2 bHLH transcriptional repressor is activated in the ventricular myocardium during mouse embryonic development. In this study, we aimed to investigate the characteristics of cardiomyocyte progenitor cells and their cell lineages by analyzing Hey2 enhancer activity at the earliest stages of heart formation. We found that the Hey2 enhancer initiated its activity prior to cardiomyocyte differentiation within the JCF. Hey2 enhancer-active cells were present rostrally to the Tbx5-expressing region at the early phase of cardiac crescent formation and differentiated exclusively into left ventricular cardiomyocytes in a lineage distinct from the Tbx5-positive lineage. By the late phase of cardiac crescent formation, Hey2 enhancer activity became significantly overlapped with Tbx5 expression in cells that contribute to the left ventricular myocardium. Our study reveals that a population of unipotent progenitor cells for left ventricular cardiomyocytes emerge in the JCF, providing further insight into the mode of cell type diversification during early cardiac development.

ETS-dependent enhancers for endothelial-specific expression of serum/glucocorticoid-regulated kinase 1 during mouse embryo development.

Serum/glucocorticoid-regulated kinase 1 (SGK1) is predominantly expressed in endothelial cells of mouse embryos, and Sgk1 null mice show embryonic lethality due to impaired vascular formation. However, how the SGK1 expression is controlled in developing vasculature remains unknown. In this study, we first identified a proximal endothelial enhancer through lacZ reporter mouse analyses. The mouse Sgk1 proximal enhancer was narrowed down to the 5' region of the major transcription initiation site, while a human corresponding region possessed relatively weak activity. We then searched for distal enhancer candidates using in silico analyses of publicly available databases for DNase accessibility, RNA polymerase association and chromatin modification. A region approximately 500 kb distant from the human SGK1 gene was conserved in the mouse, and the mouse and human genomic fragments drove transcription restricted to embryonic endothelial cells. Minimal fragments of both proximal and distal enhancers had consensus binding elements for the ETS transcription factors, which were essential for the responsiveness to ERG, FLI1 and ETS1 proteins in luciferase assays and the endothelial lacZ reporter expression in mouse embryos. These results suggest that endothelial SGK1 expression in embryonic vasculature is maintained through at least two ETS-regulated enhancers located in the proximal and distal regions.

Synthesis and physical and biological properties of 1,3-diaza-2-oxophenoxazine-conjugated oligonucleotides.

The artificial nucleobase 1,3-diaza-2-oxophenoxazine (tCO) and its derivative G-clamp strongly bind to guanine and, when incorporated into double-stranded DNA, significantly increase the stability of the latter. As the phenoxazine skeleton is a constituent of major pharmaceuticals, we hypothesized that oligonucleotides (ONs) containing phenoxazine bases would induce property changes related to intracellular uptake and migration in tissues. In this study, we designed and synthesized a novel G-clamp-linker antisense oligonucleotide (ASO) in which a G-clamp base with a flexible linker was introduced into the 5'-end of an ASO targeting mouse long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (mMALAT1). Compared to unconjugated ASO, the G-clamp-linker ASO induced significantly more effective knockdown of mMALAT1 in mouse skeletal muscle. The ASOs conjugated with 2'-deoxyribonucleotide(s) bearing a tCO nucleobase at the 5'-end exhibited a similar knockdown effect in skeletal muscle. Thus, it may be possible to improve therapeutic effects against skeletal muscle diseases, such as muscular dystrophy, by using ONs with incorporated phenoxazine nucleobases.

Tmem100-BAC-EGFP mice to selectively mark and purify embryonic endothelial cells of large caliber arteries in mid-gestational vascular formation.

Embryonic vascular development is achieved through the complex arrays of differentiation, proliferation, migration and mutual interaction of different cell types, and visualization as well as purification of unique cell populations are fundamental in studying its detailed mechanisms using in vivo experimental models. We previously demonstrated that Tmem100 was a novel endothelial gene encoding a small transmembrane protein, and that Tmem100 null mice showed embryonic lethality due to severe impairment of vascular formation. In the present study, we generated an EGFP reporter mouse line using a 216 kb genomic region containing mouse Tmem100 gene. A novel line designated as Tmem100-BAC-EGFP mice precisely recapitulated the Tmem100 expression profile at the mid-gestational stage, which was highly enriched in endothelial cells of large caliber arteries in mouse embryos. FACS experiments demonstrated that Tmem100-BAC-EGFP mice served to selectively purify a specific population of arterial endothelial cells, indicating their usefulness not only for the research concerning Tmem100 expression and function but also for comparative analysis of multiple endothelial cell subgroups in embryonic vascular development.

Expression of Hey2 transcription factor in the early embryonic ventricles is controlled through a distal enhancer by Tbx20 and Gata transcription factors.

Development of multi-chambered heart is associated with spatio-temporal regulation of gene expression. A basic helix-loop-helix transcription factor Hey2 is specifically expressed in the embryonic mouse ventricles and is indispensable for ventricular myocyte differentiation, compartment identity and morphogenesis of the heart. However, how Hey2 transcription is precisely regulated in the heart remains unclear. In this study, we identified a distal Hey2 enhancer conserved in the mouse and human to possess specific transcriptional activity in ventricular free wall myocytes at the looping stage of cardiac development. Deletion of the enhancer significantly decreased endogenous Hey2 expression in the ventricular myocardium but not in other tissues of mouse embryos. Mutation/deletion of the conserved binding sites for T-box and Gata proteins, but not NK-2 proteins, abolished the enhancer activity, and Tbx20 null mice completely lost the enhancer activity in the embryonic ventricles. Luciferase reporter analysis suggested that the ventricular enhancer activity was controlled by Tbx20 through its DNA binding and cooperative function with cardiac Gata proteins. These results delineate a regulatory mechanism of ventricular Hey2 expression and help fully understand molecular cascades in myocardial cell differentiation and cardiac morphogenesis during embryonic development.

Importance of endothelial Hey1 expression for thoracic great vessel development and its distal enhancer for Notch-dependent endothelial transcription.

Thoracic great vessels such as the aorta and subclavian arteries are formed through dynamic remodeling of embryonic pharyngeal arch arteries (PAAs). Previous work has shown that loss of a basic helix-loop-helix transcription factor Hey1 in mice causes abnormal fourth PAA development and lethal great vessel anomalies resembling congenital malformations in humans. However, how Hey1 mediates vascular formation remains unclear. In this study, we revealed that Hey1 in vascular endothelial cells, but not in smooth muscle cells, played essential roles for PAA development and great vessel morphogenesis in mouse embryos. Tek-Cre-mediated Hey1 deletion in endothelial cells affected endothelial tube formation and smooth muscle differentiation in embryonic fourth PAAs and resulted in interruption of the aortic arch and other great vessel malformations. Cell specificity and signal responsiveness of Hey1 expression were controlled through multiple cis-regulatory regions. We found two distal genomic regions that had enhancer activity in endothelial cells and in the pharyngeal epithelium and somites, respectively. The novel endothelial enhancer was conserved across species and was specific to large-caliber arteries. Its transcriptional activity was regulated by Notch signaling in vitro and in vivo, but not by ALK1 signaling and other transcription factors implicated in endothelial cell specificity. The distal endothelial enhancer was not essential for basal Hey1 expression in mouse embryos but may likely serve for Notch-dependent transcriptional control in endothelial cells together with the proximal regulatory region. These findings help in understanding the significance and regulation of endothelial Hey1 as a mediator of multiple signaling pathways in embryonic vascular formation.

A role of Hey2 transcription factor for right ventricle development through regulation of Tbx2-Mycn pathway during cardiac morphogenesis.

A basic helix-loop-helix transcription factor Hey2 is expressed in the ventricular myocardium and endocardium of mouse embryos, and Hey2 null mice die perinatally showing ventricular septal defect, dysplastic tricuspid valve and hypoplastic right ventricle. In order to understand region-specific roles of Hey2 during cardiac morphogenesis, we generated Hey2 conditional knockout (cKO) mice using Mef2c-AHF-Cre, which was active in the anterior part of the second heart field and the right ventricle and outflow tract of the heart. Hey2 cKO neonates reproduced three anomalies commonly observed in Hey2 null mice. An earliest morphological defect was the lack of right ventricular extension along the apico-basal axis at midgestational stages. Underdevelopment of the right ventricle was present in all cKO neonates including those without apparent atresia of right-sided atrioventricular connection. RNA sequencing analysis of cKO embryos identified that the gene expression of a non-chamber T-box factor Tbx2 was ectopically induced in the chamber myocardium of the right ventricle. Consistently, mRNA expression of the Mycn transcription factor, which was a cell cycle regulator transcriptionally repressed by Tbx2, was down regulated, and the number of S-phase cells was significantly decreased in the right ventricle of cKO heart. These results suggest that Hey2 plays an important role in right ventricle development during cardiac morphogenesis, at least in part, through mitigating Tbx2-dependent inhibition of Mycn expression.

Crystallographic Structure of Novel Types of AgI -Mediated Base Pairs in Non-canonical DNA Duplex Containing 2'-O,4'-C-Methylene Bridged Nucleic Acids.

Metal-mediated base pairs have widespread applications, such as in DNA-metal nanodevices and sensors. Here, we focused on their sugar conformation in duplexes and observed the crystallographic structure of the non-canonical DNA/DNA duplex containing 2'-O,4'-C-methylene bridged nucleic acid in the presence of AgI ions. The X-ray crystallographic structure was successfully obtained at a resolution of 1.5 Å. A novel type of AgI -mediated base pair between the N1 positions of anti-conformation of adenines in the duplex was observed. In the central non-canonical region, a hexad nucleobase structure containing AgI -mediated base pairs between the N7 positions of guanines was formed. A highly bent non-canonical structure was formed at the origin of AgI -mediated base pairs in the central region. The bent duplex structure induced by the addition of AgI ions might become a powerful tool for dynamic structural changes in DNA nanotechnology applications.

2',4'-BNA/LNA with 9-(2-Aminoethoxy)-1,3-diaza-2-oxophenoxazine Efficiently Forms Duplexes and Has Enhanced Enzymatic Resistance*.

Artificial nucleic acids are widely used in various technologies, such as nucleic acid therapeutics and DNA nanotechnologies requiring excellent duplex-forming abilities and enhanced nuclease resistance. 2'-O,4'-C-Methylene-bridged nucleic acid/locked nucleic acid (2',4'-BNA/LNA) with 1,3-diaza-2-oxophenoxazine (BNAP (BH )) was previously reported. Herein, a novel BH analogue, 2',4'-BNA/LNA with 9-(2-aminoethoxy)-1,3-diaza-2-oxophenoxazine (G-clamp), named BNAP-AEO (BAEO ), was designed. The BAEO nucleoside was successfully synthesized and incorporated into oligodeoxynucleotides (ODNs). ODNs containing BAEO possessed up to 104 -, 152-, and 11-fold higher binding affinities for complementary (c) RNA than those of ODNs containing 2'-deoxycytidine (C), 2',4'-BNA/LNA with 5-methylcytosine (L), or 2'-deoxyribonucleoside with G-clamp (PAEO ), respectively. Moreover, duplexes formed by ODN bearing BAEO with cDNA and cRNA were thermally stable, even under molecular crowding conditions induced by the addition of polyethylene glycol. Furthermore, ODN bearing BAEO was more resistant to 3'-exonuclease than ODNs with phosphorothioate linkages.

Enhanced duplex- and triplex-forming ability and enzymatic resistance of oligodeoxynucleotides modified by a tricyclic thymine derivative.

We designed and synthesized an artificial nucleic acid, [3-(1,2-dihydro-2-oxobenzo[b][1,8]naphthyridine)]-2'-deoxy-D-ribofuranose (OBN), with a tricyclic structure in a nucleobase as a thymidine analog. Oligodeoxynucleotides (ODNs) containing consecutive OBN displayed improved duplex-forming ability with complementary single-stranded (ss) RNA and triplex-forming ability with double-stranded DNA in comparison with ODNs composed of natural thymidine. OBN-modified ODNs also displayed enhanced enzymatic resistance compared with ODNs with natural thymidine and phosphorothioate modification, respectively, due to the structural steric hindrance of the nucleobase. The fluorescence spectra of OBN-modified ODNs showed sufficient fluorescence intensity with ssDNA and ssRNA, which is an advantageous feature for fluorescence imaging techniques of nucleic acids with longer emission wavelengths than bicyclic thymine (bT).

Placental labyrinth formation in mice requires endothelial FLRT2/UNC5B signaling.

The placental labyrinth is the interface for gas and nutrient exchange between the embryo and the mother; hence its proper development is essential for embryogenesis. However, the molecular mechanism underlying development of the placental labyrinth, particularly in terms of its endothelial organization, is not well understood. Here, we determined that fibronectin leucine-rich transmembrane protein 2 (FLRT2), a repulsive ligand of the UNC5 receptor family for neurons, is unexpectedly expressed in endothelial cells specifically in the placental labyrinth. Mice lacking FLRT2 in endothelial cells exhibited embryonic lethality at mid-gestation, with systemic congestion and hypoxia. Although they lacked apparent deformities in the embryonic vasculature and heart, the placental labyrinths of these embryos exhibited aberrant alignment of endothelial cells, which disturbed the feto-maternal circulation. Interestingly, this vascular deformity was related to endothelial repulsion through binding to the UNC5B receptor. Our results suggest that the proper organization of the placental labyrinth depends on coordinated inter-endothelial repulsion, which prevents uncontrolled layering of the endothelium.

Oligonucleotides Containing Phenoxazine Artificial Nucleobases: Triplex-Forming Abilities and Fluorescence Properties.

1,3-Diaza-2-oxophenoxazine ("phenoxazine"), a tricyclic cytosine analogue, can strongly bind to guanine moieties and improve π-π stacking effects with adjacent bases in a duplex. Phenoxazine has been widely used for improving duplex-forming abilities. In this study, we have investigated whether phenoxazine and its analogue, 1,3,9-triaza-2-oxophenoxazine (9-TAP), could improve triplex-forming abilities. A triplex-forming oligonucleotide (TFO) incorporating a phenoxazine component was found to show considerably decreased binding affinity with homopurine/homopyrimidine double-stranded DNA, so the phenoxazine system was considered not to function as either a protonated cytosine or thymine analogue. Alternatively, a 9-TAP-containing artificial nucleobase developed by us earlier as a new phenoxazine analogue functioned as a thymine analogue with respect to AT base pairs in a parallel triplex DNA motif. The fluorescence of the 9-TAP moiety was maintained even in triplex (9-TAP:AT) formation, so 9-TAP might be useful as an imaging tool for various oligonucleotide nanotechnologies requiring triplex formation.

Silver(I)-Ion-Mediated Cytosine-Containing Base Pairs: Metal Ion Specificity for Duplex Stabilization and Susceptibility toward DNA Polymerases.

Spectroscopic characterization of AgI -ion-mediated C-AgI -A and C-AgI -T base pairs found in primer extension reactions catalyzed by DNA polymerases was conducted. UV melting experiments revealed that C-A and C-T mismatched base pairs in oligodeoxynucleotide duplexes are specifically stabilized by AgI ions in 1:1 stoichiometry in the same manner as a C-C mismatched base pair. Although the stability of the mismatched base pairs in the absence of AgI ions is in the order C-A≈C-T>C-C, the stabilizing effect of AgI ions follows the order C-C>C-A≈C-T. However, the comparative susceptibility of dNTPs to AgI -mediated enzymatic incorporation into the site opposite templating C is dATP>dTTP≫dCTP, as reported. The net charge, as well as the size and/or shape complementarity of the metal-mediated base pairs, or the stabilities of mismatched base pairs in the absence of metal ions, would be more important than the stability of the metallo-base pairs in the replicating reaction catalyzed by DNA polymerases.

Transcription factor Mef2c is required for B cell proliferation and survival after antigen receptor stimulation.

Calcineurin is required for B cell receptor (BCR)-induced proliferation of mature B cells. Paradoxically, loss of NFAT transcription factors, themselves calcineurin targets, induces hyperactivity, which suggests that calcineurin targets other than NFAT are required for BCR-induced proliferation. Here we demonstrate a function for the calcineurin-regulated transcription factor Mef2c in B cells. BCR-induced calcium mobilization was intact after Mef2c deletion, but loss of Mef2c caused defects in B cell proliferation and survival after BCR stimulation in vitro and lower T cell-dependent antibody responses and germinal center formation in vivo. Mef2c activity was specific to BCR stimulation, as Toll-like receptor and CD40 signaling induced normal responses in Mef2c-deficient B cells. Mef2c-dependent targets included the genes encoding cyclin D2 and the prosurvival factor Bcl-x(L). Our results emphasize an unrecognized but critical function for Mef2c in BCR signaling.

1,3,9-Triaza-2-oxophenoxazine: An Artificial Nucleobase Forming Highly Stable Self-Base Pairs with Three AgI Ions in a Duplex.

Metal-mediated base pairs (MMBPs) formed by natural or artificial nucleobases have recently been developed. The metal ions can be aligned linearly in a duplex by MMBP formation. The development of a three- or more-metal-coordinated MMBPs has the potential to improve the conductivity and enable the design of metal ion architectures in a duplex. This study aimed to develop artificial self-bases coordinated by three linearly aligned AgI ions within an MMBP. Thus, artificial nucleic acids with a 1,3,9-triaza-2-oxophenoxazine (9-TAP) nucleobase were designed and synthesized. In a DNA/DNA duplex, self-base pairs of 9-TAP could form highly stable MMBPs with three AgI ions. Nine equivalents of AgI led to the formation of three consecutive 9-TAP self-base pairs with extremely high stability. The complex structures of 9-TAP MMBPs were determined by using electrospray ionization mass spectrometry and UV titration experiments. Highly stable self-9-TAP MMBPs with three AgI ions are expected to be applicable to new DNA nanotechnologies.

Serum/glucocorticoid-regulated kinase 1 as a novel transcriptional target of bone morphogenetic protein-ALK1 receptor signaling in vascular endothelial cells.

Bone morphogenetic protein 9 (BMP9)/BMP10-ALK1 receptor signaling is essential for endothelial differentiation and vascular morphogenesis. Mutations in ALK1/ACVRL1 and other signal-related genes are implicated in human vascular diseases, and the Alk1/Acvrl1 deletion in mice causes severe impairment of vascular formation and embryonic lethality. In the microarray screen to search for novel downstream genes of ALK1 signaling, we found that the mRNA and protein expression of serum/glucocorticoid-regulated kinase 1 (SGK1) was rapidly up-regulated by the BMP9 stimulation of cultured human endothelial cells. The increase in SGK1 mRNA was completely blocked by the transcriptional inhibitor actinomycin D and significantly suppressed by the siRNA treatment against the co-SMAD transcription factor SMAD4. Upon the BMP9 treatment of endothelial cells, phosphorylated SMAD1/5/9 bound to a consensus site upstream of the SGK1 gene, which was necessary for BMP9-dependent increment of the luciferase reporter activity driven by the SGK1 proximal enhancer. The Sgk1 mRNA expression in mouse embryos was enriched in vascular endothelial cells at embryonic day 9.0-9.5, at which Sgk1 null mice showed embryonic lethality due to abnormal vascular formation, and its mRNA as well as protein expression was clearly reduced in Alk1/Acvrl1 null embryos. These results indicate that SGK1 is a novel target gene of BMP9/BMP10-ALK1 signaling in endothelial cells and further suggest a possibility that down-regulation of the Sgk1 expression may be involved in the mechanisms of vascular defects by the ALK1 signaling deficiency.

2'-O,4'-C-Methylene-Bridged Nucleic Acids Stabilize Metal-Mediated Base Pairing in a DNA Duplex.

The 2'-O,4'-C-methylene-bridged or locked nucleic acid (2',4'-BNA/LNA), with an N-type sugar conformation, effectively improves duplex-forming ability. 2',4'-BNA/LNA is widely used to improve gene knockdown in nucleic acid based therapies and is used in gene diagnosis. Metal-mediated base pairs (MMBPs), such as thymine (T)-HgII -T and cytosine (C)-AgI -C have been developed and used as attractive tools in DNA nanotechnology studies. This study aimed to investigate the application of 2',4'-BNA/LNA in the field of MMBPs. 2',4'-BNA/LNA with 5-methylcytosine stabilized the MMBP of C with AgI ions. Moreover, the 2',4'-BNA/LNA sugar significantly improved the duplex-forming ability of the DNA/DNA complex, relative to that by the unmodified sugar. These results suggest that the sugar conformation is important for improving the stability of duplex-containing MMBPs. The results indicate that 2',4'-BNA/LNA can be applied not only to nucleic acid based therapies, but also to MMBP technologies.

Effective gene silencing activity of prodrug-type 2'-O-methyldithiomethyl siRNA compared with non-prodrug-type 2'-O-methyl siRNA.

Small interfering RNAs (siRNAs) are an active agent to induce gene silencing and they have been studied for becoming a biological and therapeutic tool. Various 2'-O-modified RNAs have been extensively studied to improve the nuclease resistance. However, the 2'-O-modified siRNA activities were often decreased by modification, since the bulky 2'-O-modifications inhibit to form a RNA-induced silencing complex (RISC). We developed novel prodrug-type 2'-O-methyldithiomethyl (MDTM) siRNA, which is converted into natural siRNA in an intracellular reducing environment. Prodrug-type 2'-O-MDTM siRNAs modified at the 5'-end side including 5'-end nucleotide and the seed region of the antisense strand exhibited much stronger gene silencing effect than non-prodrug-type 2'-O-methyl (2'-O-Me) siRNAs. Furthermore, the resistances for nuclease digestion of siRNAs were actually enhanced by 2'-O-MDTM modifications. Our results indicate that 2'-O-MDTM modifications improve the stability of siRNA in serum and they are able to be introduced at any positions of siRNA.

Syntheses of prodrug-type 2'-O-methyldithiomethyl oligonucleotides modified at natural four nucleoside residues and their conversions into natural 2'-hydroxy oligonucleotides under reducing condition.

We previously reported that reducing-environment-responsive prodrug-type small interfering RNA (siRNA) bearing 2'-O-methyldithiomethyl (2'-O-MDTM) uridine exhibits efficient knockdown activity and nuclease resistance. In this report, we describe the preparation of 2'-O-MDTM oligonucleotides modified not only at uridine but also at adenosine, guanosine and cytidine residues by post-synthetic modification. Precursor oligonucleotides bearing 2'-O-(2,4,6-trimethoxybenzylthiomethyl) (2'-O-TMBTM) adenosine, guanosine, and cytidine were reacted with dimethyl(methylthio)sulfonium tetrafluoroborate to form 2'-O-MDTM oligonucleotides in the same manner as the oligonucleotide bearing 2'-O-TMBTM uridine. Furthermore, the oligonucleotides bearing 2'-O-MDTM adenosine, guanosine, and cytidine were efficiently converted into corresponding natural 2'-hydroxy oligonucleotides under the cytosol-mimetic reducing condition.

Synthesis and thermal stabilities of oligonucleotides containing 2'-O,4'-C-methylene bridged nucleic acid with a phenoxazine base.

We designed and synthesized a novel artificial 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA/LNA) with a phenoxazine nucleobase and named this compound BNAP. Oligodeoxynucleotide (ODN) containing BNAP showed higher binding affinities toward complementary DNA and RNA as compared to ODNs bearing 2',4'-BNA/LNA with 5-methylcytosine or 2'-deoxyribonucleoside with phenoxazine. Thermodynamic analysis revealed that BNAP exhibits properties associated with the phenoxazine moiety in DNA/DNA duplexes and characteristics associated with the 2',4'-BNA/LNA moiety in DNA/RNA duplexes.