Biomedical subjects

Seiki Haraguchi

Publications and source records attributed to Seiki Haraguchi.

8 recordsLinked to original sources

PubMed2006

Tumor prone phenotype of mice deficient in a novel apoptosis-inducing gene, drs.

The drs gene was originally isolated as a suppressor of v-src transformation. Expression of drs mRNA is markedly downregulated in a variety of human cancer cell lines and tissues, suggesting the potential role of this gene as a tumor suppressor. Previously, we found that Drs protein associates with ASY/Nogo-B/RTN-x(S), an apoptosis-inducing protein in the endoplasmic reticulum, and sequentially activates caspases to induce apoptosis in human cancer cells without involvement of the mitochondria. In this study, we investigated the tumor suppressor function of drs and the correlation between Drs-mediated apoptosis and tumor suppression by generating a gene-knockout (KO) mouse. Between 7 and 12 months after birth, malignant tumors including lymphomas, lung adenocarcinomas and hepatomas were generated in about 30% of the drs KO mice, whereas no tumors were found in any of the wild-type mice during the same period of time. drs KO embryonic fibroblasts also showed enhanced sensitivity to transformation by v-src oncogene. Reintroduction of drs into a tumor cell line derived from the tumor of a drs KO mouse led to the suppression of tumor formation in nude mice, which was accompanied by enhanced apoptosis and the activation of caspase-9 and -3. Furthermore, introduction of drs into this cell line enhanced sensitivity to apoptosis mediated by caspase-3, -9 and -12 under low serum culture conditions. The present results thus indicate that drs contributes to the suppression of malignant tumor formation, and this suppression is closely correlated with drs-mediated apoptosis.

Adenocarcinoma↗

PubMed2006

Tbx6-mediated Notch signaling controls somite-specific Mesp2 expression.

Mesp2 is a transcription factor that plays fundamental roles in somitogenesis, and its expression is strictly restricted to the anterior presomitic mesoderm just before segment border formation. The transcriptional on-off cycle is linked to the segmentation clock. In our current study, we show that a T-box transcription factor, Tbx6, is essential for Mesp2 expression. Tbx6 directly binds to the Mesp2 gene upstream region and mediates Notch signaling, and subsequent Mesp2 transcription, in the anterior presomitic mesoderm. Our data therefore reveal that a mechanism, via Tbx6-dependent Notch signaling, acts on the transcriptional regulation of Mesp2. This finding uncovers an additional component of the interacting network of various signaling pathways that are involved in somitogenesis.

Animals↗

PubMed2005

Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi.

BACKGROUND: Studies of gene function in the mouse have relied mainly on gene targeting via homologous recombination. However, this approach is difficult to apply in specific windows of time, and to simultaneously knock-down multiple genes. Here we report an efficient method for dsRNA-mediated gene silencing in late cleavage-stage mouse embryos that permits examination of phenotypes at post-implantation stages. RESULTS: We show that introduction of Bmp4 dsRNA into intact blastocysts by electroporation recapitulates the genetic Bmp4 null phenotype at gastrulation. It also reveals a novel role for Bmp4 in the regulation the anterior visceral endoderm specific gene expression and its positioning. We also show that RNAi can be used to simultaneously target several genes. When applied to the three murine isoforms of Dishevelled, it leads to earlier defects than previously observed in double knock-outs. These include severe delays in post-implantation development and defects in the anterior midline and neural folds at headfold stages. CONCLUSION: Our results indicate that the BMP4 signalling pathway contributes to the development of the anterior visceral endoderm, and reveal an early functional redundancy between the products of the murine Dishevelled genes. The proposed approach constitutes a powerful tool to screen the functions of genes that govern the development of the mouse embryo.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Four-cell stage mouse blastomeres have different developmental properties.

Blastomeres of the early mouse embryo are thought to be equivalent in their developmental properties at least until the eight-cell stage. However, the experiments that have led to this conclusion could not have taken into account either the spatial origin of individual blastomeres or the spatial allocation and fate of their progeny. We have therefore readdressed this issue having defined cell lineages in mouse embryos undergoing different patterns of cleavage in their second division cycle. This has enabled us to identify a major group of embryos in which we can predict not only the spatial origin of each given four-cell blastomeres, but also which region of the blastocyst is most likely to be occupied by its progeny. We show that a pattern of second cleavage divisions in which a meridional division is followed by one that is equatorial or oblique allows us to identify blastomeres that differ in their fate and in their developmental properties both from each other and from their cousins. We find that one of these four-cell stage blastomeres that inherits some vegetal membrane marked in the previous cleavage cycle tends to contribute to mural trophectoderm. The progeny of its sister tend to donate cells to part of the ICM lining the blastocyst cavity and its associated trophectoderm. Chimaeras made entirely of these equatorially or obliquely derived blastomeres show developmental abnormalities in both late preimplantation and early postimplantation development. By contrast, chimaeras made from four-cell stage blastomeres from early meridional divisions develop normally. The developmental defects of chimaeras made from the most vegetal blastomeres that result from later second cleavages are the most severe and following transplantation into foster mothers they fail to develop to term. However, when such individual four-cell blastomeres are surrounded by blastomeres from random positions, they are able to contribute to all embryonic lineages. In conclusion, this study shows that while all four-cell blastomeres can have full developmental potential, they differ in their individual developmental properties according to their origin in the embryo from as early as the four-cell stage.

Animals↗

PubMed2004

A novel apoptotic pathway induced by the drs tumor suppressor gene.

The drs gene was originally isolated as a suppressor against v-src transformation. Expression of drs mRNA was markedly downregulated in a variety of human cancer cell lines and tissues, suggesting that the drs gene acts as a tumor suppressor. In this study, we found that ectopic expression of the Drs protein induced apoptosis in human cancer cell lines. Analyses using deletion mutants of drs revealed that both the C-terminal region and the three consensus repeats in the N-terminal region are essential for the induction of apoptosis. Caspase-12, -9, and -3 were sequentially activated by drs, and specific inhibitors of caspase-3 and -9 suppressed drs-induced apoptosis. The release of cytochrome c from the mitochondria into the cytoplasm was not observed in apoptosis by drs, suggesting that the mitochondrial pathway does not mediate drs-induced apoptosis. Furthermore, we found that the Drs protein can interact with ASY/Nogo-B/RTN-x(S), an apoptosis-inducing protein localized in the endoplasmic reticulum, and that coexpression of these genes increased the efficiency of apoptosis. These results indicated that Drs induces apoptosis by a novel pathway mediated by ASY/Nogo-B/RTN-x(S), caspase-12, -9, and -3.

Apoptosis↗

PubMed2004

Specific gene silencing in the pre-implantation stage mouse embryo by an siRNA expression vector system.

Recently, small interfering RNAs (siRNAs) have become a powerful and widely used tool for the analysis of gene function in mammalian cells. Here we report that the microinjection of an siRNA expression vector into the nucleus is an efficient and powerful method of specific gene silencing in pre-implantation mouse embryos. We used this method to examine the expression of two genes EGFP and Oct4. Vectors encoding siRNAs targeted against EGFP or Oct4 were injected into the pronucleus or nucleus of zygotes, which were then cultured until the blastocyst stage. When the effects of RNAi were examined in blastocyst stage eggs, there was robust inhibition of the gene product in a concentration-dependent manner at both the mRNA and the protein level. The expression of other endogenous genes was not affected, showing the specificity of the vector-mediated RNAi. In addition, this method was effective for inhibiting maternally expressed mRNA. To demonstrate that RNAi of Oct4 induced a similar phenotype to that of Oct4-null embryos, the blastocysts were further cultured in ES medium. After the fourth day of culture, the embryos either had outgrown only a layer of trophoblast cells or showed developmental arrest at the blastocyst stage (>90%). Moreover, concomitant with Oct4 suppression at the blastocyst stage, we observed inhibition of Fgf4, a gene that is known to be induced downstream of Oct4 expression. Taken together, these results demonstrate that the use of siRNA expression vector is a powerful way to achieve gene silencing in the pre-implantation stage embryo.

Animals↗

PubMed2003

Conserved role of nanos proteins in germ cell development.

In Drosophila, maternally supplied Nanos functions in the migration of primordial germ cells (PGCs) into the gonad; in mice, zygotic genes are involved instead. We report the cloning and the functional analyses of nanos2 and nanos3 in mice. These genes are differentially expressed in mouse PGCs. nanos2 is predominantly expressed in male germ cells, and the elimination of this gene results in a complete loss of spermatogonia. However, nanos3 is found in migrating PGCs, and the elimination of this factor results in the complete loss of germ cells in both sexes. Hence, although mice and flies differ in their mechanisms for germ cell specification, there seems to be conserved function for nanos proteins among invertebrates and vertebrates.

Amino Acid Sequence↗

PubMed2003

nanos1: a mouse nanos gene expressed in the central nervous system is dispensable for normal development.

A mouse nanos (nanos1) gene was cloned and its function was examined by generating a gene-knockout mouse. The nanos1 gene encodes an RNA-binding protein, which contains a putative zinc-finger motif that exhibits similarity with other nanos-class genes in vertebrates and invertebrates. Although nanos1 is not detected in primordial germ cells, it is observed in seminiferous tubules of mature testis. Interestingly, maternally expressed nanos1 is observed in substantial amounts in oocytes, but the amount of maternal RNA is rapidly reduced after fertilization, and the transient zygotic nanos1 expression is observed in eight-cell embryos. At 12.5 days postcoitum, nanos1 is re-expressed in the central nervous system and the expression continues in the adult brain, in which the hippocampal formation is the predominant region. The nanos1 -deficient mice develop to term without any detectable abnormality and they are fertile. No significant neural defect is observed in terms of their behavior to date.

Amino Acid Sequence↗