A mouse line carrying a lacZ transgene driven by the human EEF1A1/EF1alpha promoter was established. Although the promoter is known to show ubiquitous activity, only paternal transgene alleles were expressed, resulting in a transgene imprinting. At mid-gestation, the promoter sequence was differentially methylated, hypomethylated for paternal and hypermethylated for maternal alleles. In germline, the promoter was a typical differentially methylated region. After fertilization, however, both alleles were hypermethylated. Thus, the differential methylation of the promoter required for transgene imprinting was re-established during later embryonic development independently of the germline differential methylation. Furthermore, also a retroelement promoter closely-flanking imprinted transgene and its wild type counterpart displayed similar differential methylation during early development. The retroelement promoter was methylated differentially also in germline, but in an opposite pattern to the embryonic differential methylation. These results suggest that there might be an unknown epigenetic regulation inducing transgene imprinting independently of DNA methylation in the transgene insertion site. Then, besides CpG dinucleotides, non-CpG cytosines of the retroelement promoter were highly methylated especially in the transgene-active mid-gestational embryos, suggesting that an unusual epigenetic regulation might protect the active transgene against de novo methylation occurring generally in mid-gestational embryo.

In this study we generated RNA interference (RNAi)-mediated gene knockdown transgenic mice (transgenic RNAi mice) against the functional Inv gene. Inv mutant mice show consistently reversed internal organs (situs inversus), multiple renal cysts and neonatal lethality. The Inv::GFP-rescue mice, which introduced the Inv::GFP fusion gene, can rescue inv mutant mice phenotypes. This indicates that the Inv::GFP gene is functional in vivo. To analyze the physiological functions of the Inv gene, and to demonstrate the availability of transgenic RNAi mice, we introduced a short hairpin RNA expression vector against GFP mRNA into Inv::GFP-rescue mice and analyzed the gene silencing effects and Inv functions by examining phenotypes. Transgenic RNAi mice with the Inv::GFP-rescue gene (Inv-KD mice) down-regulated Inv::GFP fusion protein and showed hypomorphic phenotypes of inv mutant mice, such as renal cyst development, but not situs abnormalities or postnatal lethality. This indicates that shRNAi-mediated gene silencing systems that target the tag sequence of the fusion gene work properly in vivo, and suggests that a relatively high level of Inv protein is required for kidney development in contrast to left/right axis determination. Inv::GFP protein was significantly down-regulated in the germ cells of Inv-KD mice testis compared with somatic cells, suggesting the existence of a testicular germ cell-specific enhanced RNAi system that regulates germ cell development. The Inv-KD mouse is useful for studying Inv gene functions in adult tissue that are unable to be analyzed in inv mutant mice showing postnatal lethality. In addition, the shRNA-based gene silencing system against the tag sequence of the fusion gene can be utilized as a new technique to regulate gene expression in either in vitro or in vivo experiments.

Two de novo DNA methyltransferases, Dnmt3a and Dnmt3b, have been identified in humans and mice to contribute to the methylation of unmodified DNA. We recently showed a transition of de novo DNA methyltransferase expression from Dnmt3b to Dnmt3a during mouse embryogenesis and in tissue-specific stem cells, suggesting distinct functions of Dnmt3a and Dnmt3b during these processes. In this study, to characterize the functions of Dnmt3a and Dnmt3b in pluripotent stem cells, we exogenously transfected ES cells with Dnmt3a and Dnmt3b cDNAs linked to an internal ribosome entry site-green fluorescent protein gene, and then analyzed the effects of expression of these de novo DNA methyltransferases on ES cell growth and differentiation. ES cells expressing Dnmt3b showed specific downregulation of pluripotency marker genes such as Nanog and Oct 3/4. In addition, Dnmt3a-transfected ES cells showed a specific increase in mitotic index, while Dnmt3b-transfected ES cells showed a decrease in mitotic index. These results suggest that Dnmt3b has important physiological roles in the initial process of stem cell differentiation and that Dnmt3a has a function in stem cell proliferation.

Gap junctions allow the direct and bidirectional transfer of small molecules between cells. Polyamine sensitivity, which has been observed for a certain gap junction in vitro, confers rectification property to gap junction. Here we report that the polyamine sensitivity of gap junctions in vivo is crucial for skin pattern formation in zebrafish. Transgenic experiments have revealed that several connexin genes were able to rescue the spot phenotype of mutant zebrafish. Mutational analyses of the N-terminal region of connexins revealed that the ExxxE motif, a hypothetical polyamine-binding site, was important for connexin's role in pattern formation. Ectopic expression of spermidine/spermine N(1)-acetyltransferase (SSAT), a polyamine metabolic enzyme, also caused stripe pattern changes, which further indicates that the polyamine sensitivity of gap junctions is crucial. This is the first report to show that polyamine sensitivity has a physiologically relevant function and is related to skin pattern formation in animals.

Dnmt3a and Dnmt3b, which are known as functional de novo methyltransferases, are responsible for creating genomic methylation patterns during mammalian development. Recently, we have shown that specific expression of Dnmt3b in epiblast, embryonic ectoderm, hematopoietic progenitor cells and spermatogonia cells is followed by Dnmt3a expression (Watanabe D, Suetake I, Tada T, Tajima S (2002) Stage- and cell-specific expression of Dnmt3a and Dnmt3b during embryogenesis. Mech Dev 118:187–190; Watanabe D, Suetake I, Tajima S, Hanaoka K (2004) Expression of Dnmt3b in mouse hematopoietic progenitor cells and spermatogonia at specific stages. Gene Expr Patterns 5:43–49). In this study, we analyzed the expression of mouse de novo methyltransferases during development of the nervous systems. In the embryonic olfactory epithelium (OE), Dnmt3b was specifically expressed in Mash1 positive globose basal cells (i.e. transiently amplifying neural progenitor cells), while Dnmt3a was expressed in immature olfactory receptor neurons. Dnmt3b-positive cells were rarely observed in the adult OE, but were increased in regenerating OE with intranasal ZnSO4 administration. Dnmt3b was also detected in the E8.5 neural plate, E10.5 spinal cord and retina cells, while Dnmt3a was expressed in postmitotic young neurons. Furthermore, Dnmt3b was specifically expressed in ES cells, while Dnmt3a was transiently expressed during neural cell differentiation of ES cells. Dnmt3b is specifically expressed in progenitor cells during hematopoiesis, spermatogenesis and neurogenesis, suggesting an important role in the initial steps of progenitor cell differentiation. Dnmt3a is expressed in postmitotic young neurons following the Dnmt3b expression. Dnmt3a may be required for the establishment of tissue-specific methylation patterns of the genome. The coordinated expression of de novo methyltransferases from Dnmt3b to Dnmt3a suggests conserved mechanisms of de novo methylation of the genome and different functions for Dnmt3b and Dnmt3a during progenitor cell development.

In the developing mouse embryo, leftward fluid flow on the ventral side of the node determines left-right (L-R) asymmetry. However, the mechanism by which the rotational movement of node cilia can generate a unidirectional flow remains hypothetical. Here we have addressed this question by motion and morphological analyses of the node cilia and by fluid dynamic model experiments. We found that the cilia stand, not perpendicular to the node surface, but tilted posteriorly. We further confirmed that such posterior tilt can produce leftward flow in model experiments. These results strongly suggest that L-R asymmetry is not the descendant of pre-existing L-R asymmetry within each cell but is generated de novo by combining three sources of spatial information: antero-posterior and dorso-ventral axes, and the chirality of ciliary movement.

Two de novo-type DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns during development. Dnmt3b is specifically expressed in the totipotent cells of mouse early embryos and Dnmt3a, a longer form of the two isoforms, is ubiquitously expressed in mesenchyme cells after the 10 day embryo stage [Mech. Dev. 118 (2002) 187]. In the present study, we demonstrated that Dnmt3b was expressed in the nuclei of specific cells in certain tissues after the 10 day embryo stage. In fetal liver, dorsal aorta and portal vein, Dnmt3b was expressed in cells expressing CD34, indicating that the cells were hematopoietic progenitor cells. However, Dnmt3b was not expressed in the hematopoietic progenitor cells in yolk sac blood islands at 8 day embryo stage and in adult bone marrow cells. Dnmt3b was also expressed in type-A spermatogonia after birth. Dnmt3b was expressed not only in the totipotent stem cells but also in the progenitor cells the direction of differentiation of which had been already determined. On the other hand, the long form of Dnmt3a was not expressed in these hematopoietic progenitor cells in fetal liver or type-A spermatogonia, but was expressed in hepatocytes in fetal liver and type-B spermatogonia. While Dnmt3b was distributed in both the heterochromatin and euchromatin regions, Dnmt3a was specifically localized to the euchromatin region.

Inversin (Inv), a protein that contains ankyrin repeats, plays a key role in left-right determination during mammalian embryonic development, but its precise function remains unknown. Transgenic mice expressing an Inv and green fluorescent protein (GFP) fusion construct (Inv::GFP) were established to facilitate characterization of the subcellular localization of Inv. The Inv::GFP transgene rescued the laterality defects and polycystic kidney disease of Inv/Inv mice, indicating that the fusion protein is functional. In transgenic embryos, Inv::GFP protein was detected in the node monocilia. The fusion protein was also present in other 9+0 monocilia, including those of kidney epithelial cells and the pituitary gland, but it was not localized to 9+2 cilia. The N-terminal region of Inv (InvDeltaC) including the ankyrin repeats also localized to the node cilia and rescued the left-right defects of Inv/Inv mutants. Although no obvious abnormalities were detected in the node monocilia of Inv/Inv embryos, the laterality defects of such embryos were corrected by an artificial leftward flow of fluid in the node, suggesting that nodal flow is impaired by the Inv mutation. These results suggest that the Inv protein contributes to left-right determination as a component of monocilia in the node and is essential for the generation of normal nodal flow.

The processing variables that control the occurrence of breakthrough phenomenon in sandwich injection molding were investigated. Particular attention was paid to the conditions that would yield an optimum sandwich molded part. The effects of variation of core volume ratio that would yield optimum core of shot size, injection speed and the skin/core viscosity ratio were examined. The flow morphology at the various stages of flow was also explored. The study was conducted with a bar-flow mold mounted on a twin-barrel injection machine fitted with a common nozzle. It was noted that the core material flow distance depends on the core cylinder screw metering position (core shot size). At the stage at which the core material completely penetrated through the skin material (breakthrough), four distinct regions of flow could be identified. The flow distance of the core material is controlled by the injection speed of the core material while the injection speed of the skin material is of little significance. In order to increase the flow distance of the core material, it is effective to increase the core material injection speed albeit limited by an optimum value of screw metering position. It was found that the flow distance of the core material was longer when the core material viscosity was lower than that of the skin material but breakthrough occurred much earlier so core volume is limited. Breakthrough failed to occur when the viscosity of the core material was higher than that of the skin material. It was found that optimum core volume and core distribution occurred when skin/core viscosities were intermediate. The morphology of the skin material leading to breakthrough was also analyzed.

The flow behavior of skin and core materials in co-injection molding was investigated to understand the flow patterns that could lead to core breakthrough in sequential and simultaneous injection molding. A spiral-flow mold was used and the core material was loaded with a black master batch to facilitate the monitoring of the core material. PC/ABS blend was used as the skin material and ABS was the core material. It was revealed irrespective of simultaneous injection time, sandwich injection molding display the four flow regions identified earlier i. e. primary injection region, core advance region, core expansion region and core breakthrough region. The flow length of core expansion region lengthened with increasing simultaneous injection time of skin and core material while the overall injection time decreased. It was also observed that the core became thinner and wider with increasing simultaneous injection time.

The processing conditions that exert influence on the occurrence of breakthrough during sandwich injection molding were investigated. A spiral flow mold mounted on a twin-barrel injection machine fitted with a single nozzle was used. The effect of mold cavity thickness and the effects of skin material cylinder temperature on skin material viscosity, melt strength, and drawing ratio were considered. Scanning electron microscopy was used to study the morphology of the skin material at the flow front. It was found that the flow length at core expansion region increased and breakthrough phenomenon was delayed as the thickness of the mold cavity increased. When cylinder temperature is increased, the viscosity is lowered and the flow front of the skin material extends with greater ease when pushed by the core material, thus, a greater core volume can be accommodated. As the flow front progresses, the tendency is that the skin material becomes thinner Polishing and etching followed by SEM examination of the skin material at the flow front revealed that the skin material is oriented perpendicular to the flow direction and the extent of orientation increases with increasing cylinder temperature. This further supported the observation that the deformation ratio and drawing ratio increased with increasing cylinder temperature.

Key Points Left–right (L/R) asymmetry provides a unique opportunity to study cellular and molecular mechanisms of asymmetry generation. Symmetry breaking in the mouse might involve unidirectional fluid flow around the node, which is generated by rotational movement of the monocilia. Transforming growth factor-β-related factors, Nodal and Lefty2, act as asymmetric signals. Nodal is a left-side determinant, whereas Lefty2 is an antagonist that restricts the duration and the site of Nodal action. Nodal and Lefty2 might comprise a reaction–diffusion system that amplifies L/R differences. The theoretical model describes two diffusible molecules, one of which is an activator that stimulates both its own synthesis and the synthesis of its partner, which is an inhibitor. The model requires that the inhibitor diffuses more rapidly than the activator. Midline structures are required to separate the left and the right halves of embryos. Lefty1 functions as a specific midline barrier. A transcription factor Pitx2 mediates Nodal signals and is responsible for generating the left-side morphology of many visceral organs. Although Nodal, the Lefty proteins and Pitx2 have conserved roles among vertebrates, diverse strategies might have been adopted for setting up asymmetric Nodal expression.

In cultured cells, the maintenance-type DNA methyltransferase (Dnmt1) is highly expressed during the proliferation stage. In the present study, we detected significant expression of Dnmt1 protein in the nuclear fraction of mouse small intestine. From its mobility in SDS polyacrylamide gel electrophoresis and the specific antibodies against the somatic cell-type Dnmt1, Dnmt1 was determined as a somatic cell type. Immunofluorescence study revealed that the Dnmt1 was highly expressed in the proliferating stem cells in crypts, and was localized in the nuclei. The present results indicate that the expression of Dnmt1 in vivo is also under the control of cell proliferation as in cultured cells.

The gene encoding the insulin-like growth-factor type-2 receptor (Igf2r) is maternally expressed and imprinted1. A CpG island in Igf2r intron 2 that carries a maternal-specific methylation imprint2 was shown in a transgenic model to be essential for Igf2r imprinting and for the production of an antisense RNA from the paternal allele3. We report here that the endogenous region2 is the promoter for this antisense RNA (named Air, for antisense Igf2r RNA) and that the 3′ end lies 107,796 bp distant in an intron of the flanking, but non-imprinted4,5, gene Mas1.

The proper folding of newly synthesized membrane proteins in the endoplasmic reticulum (ER) is required for the formation of functional mature proteins. Calnexin is a ubiquitous ER chaperone that plays a major role in quality control by retaining incompletely folded or misfolded proteins1,2,3,4,5. In contrast to other known chaperones such as heat-shock proteins, BiP and calreticulin, calnexin is an integral membrane protein1,6. Calmegin is a testis-specific ER protein that is homologous to calnexin7,8,9. Here we show that calmegin binds to nascent polypeptides during spermatogenesis, and have analysed its physiological function by targeted disruption of its gene. Homozygous-null male mice are nearly sterile even though spermatogenesis is morphologically normal and mating is normal. In vitro, sperm from homozygous-null males do not adhere to the egg extracellular matrix (zona pellucida), and this defect may explain the observed infertility. These results suggest that calmegin functions as a chaperone for one or more sperm surface proteins that mediate the interactions between sperm and egg. The defective zona pellucida-adhesion phenotype of sperm from calmegin-deficient mice is reminiscent of certain cases of unexplained infertility in human males.

This review has surveyed the current literature on random and imprinted monoallelic expression from the unusual perspective of considering that these two forms of monoallelic expression (MAE) may be regulated by similar mechanisms. The true extent of genes which show MAE is not yet known. Imprinted MAE is thought to involve 0.1–0.2% of mammalian genes, while the number of genes in the random MAE category cannot at present be predicted since very few genes have been examined with assays that distinguish parental transcripts in single cells. The perspective adopted in this review is speculative since only one gene (XIST) is known which displays both random and imprinted MAE. However, a recent report that H19 may also show random allelic expression, suggests that MAE should be further investigated as a gene regulation mechanism in mammals.

Fas is a 45-kDa membrane protein that transduces an apoptotic signal. The mouse lymphoproliferation (lpr) mutation is a leaky mutation of Fas. In this study, we examined lymphocyte development in Fas-null mice generated by gene targeting. The Fas-/- mice progressively accumulated abnormal T cells (Thy1+, B220+, CD4-, and CD8-) and developed lymphadenopathy and splenomegaly, which were much more accelerated and pronounced than those in lpr mice. In addition, the Fas-null mice showed lymphocytosis, accompanied by lymphocytic infiltration in the lungs and liver. The number of apparently normal B cells also increased, and large amounts of immunoglobulins, including anti-DNA antibodies, were produced. Thymic clonal deletion, assessed by deletion of T cells reactive to mouse endogenous superantigens, was apparently normal in the Fas-/- mice, whereas the peripheral clonal deletion of mature T cells against a bacterial superantigen was impaired. These results suggested that Fas plays a decisive role in peripheral clonal deletion but not in negative selection in the thymus.

Fas is a cell-surface protein of 45 kDa. Binding of the Fas ligand (FasL) to Fas induces apoptosis in Fas-bearing cells. Analysis of mouse mutants in either Fas or FasL have indicated that the Fas system is involved in apoptosis of lymphocytes. To identify the cells expressing Fas, mouse spleen sections were analyzed by in situ hybridization and immunohistochemistry. Fas mRNA was detected in the T sells of the inner region of the periarterial lymphatic sheath and the inner lumen of the marginal zone. The cells in the outer region of the periarterial lymphatic sheath weakly expressed Fas mRNA, whereas it was abundant in the B cells of germinal centers. Immunizing mice with keyhole limpet hemocyanin induced formation of many germinal centers in the spleen. The B cells in the activated germinal center expressed abundant Fas and underwent apoptosis. The in vitro activation of splenocytes with lipopolysaccharide induced Fas expression in B cells and the B cells became sensitive to the Fas-mediated apoptosis. These results suggest that Fas is involved in the activation-induced death of B cells.