Biomedical subjects

M Asashima

Publications and source records attributed to M Asashima.

At least 19 recordsLinked to original sources

PubMed2001

Synergistic activation of the Wnt signaling pathway by Dvl and casein kinase Iepsilon.

Although casein kinase Iepsilon (CKIepsilon) has been shown to regulate the Wnt signaling pathway positively, its mode of action is not clear. In this study we show that CKIepsilon activates the Wnt signaling pathway in co-operation with Dvl. CKIepsilon and Axin associated with different sites of Dvl, and CKIepsilon and Dvl interacted with distinct regions on Axin. Therefore, these three proteins formed a ternary complex. Either low expression of Dvl or CKIepsilon alone did not accumulate beta-catenin, but their co-expression accumulated greatly. Dvl and CKIepsilon activated the transcriptional activity of T cell factor (Tcf) synergistically. Although the Dvl mutant that binds to Axin but not to CKIepsilon activated Tcf, it did not synergize with CKIepsilon. Another Dvl mutant that does not bind to Axin did not activate Tcf irrespective of the presence of CKIepsilon. Furthermore, Dvl and CKIepsilon co-operatively induced axis duplication of Xenopus embryos. These results indicate that Dvl and CKIepsilon synergistically activated the Wnt signaling pathway and that the binding of the complex of Dvl and CKIepsilon to Axin is necessary for their synergistic action.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Inhibition of the Wnt signaling pathway by the PR61 subunit of protein phosphatase 2A.

Axin, a negative regulator of the Wnt signaling pathway, forms a complex with glycogen synthase kinase-3beta (GSK-3beta), beta-catenin, adenomatous polyposis coli (APC) gene product, and Dvl, and it regulates GSK-3beta-dependent phosphorylation in the complex and the stability of beta-catenin. Using yeast two-hybrid screening, we found that regulatory subunits of protein phosphatase 2A, PR61beta and -gamma, interact with Axin. PR61beta or -gamma formed a complex with Axin in intact cells, and their interaction was direct. The binding site of PR61beta on Axin was different from those of GSK-3beta, beta-catenin, APC, and Dvl. Although PR61beta did not affect the stability of beta-catenin, it inhibited Dvl- and beta-catenin-dependent T cell factor activation in mammalian cells. Moreover, it suppressed beta-catenin-induced axis formation and expression of siamois, a Wnt target gene, in Xenopus embryos, suggesting that PR61beta acts either at the level of beta-catenin or downstream of it. Taken together with the previous observations that PR61 interacts with APC and functions upstream of beta-catenin, these results demonstrate that PR61 regulates the Wnt signaling pathway at various steps.

Animals↗

PubMed2001

Activin A induces expression of rat Sel-1l mRNA, a negative regulator of notch signaling, in rat salivary gland-derived epithelial cells.

We previously established a rat submandibular gland (SMG)-derived epithelial cell line (RSMG-1) to study the mechanism of morphogenesis in salivary gland development and regeneration. We found that activin A regulated the branching morphogenesis of RSMG-1 cells, suggesting that it is involved in SMG morphogenesis. We used a subtraction cloning procedure with activin-A-treated and untreated RSMG-1 cells to identify activin-A-induced genes. One of the genes detected encoded a rat homologue of Sel-1l (rSel-1l). rSel-1l is a mammalian homologue of C. elegans sel-1, which is a negative regulator of Notch signaling. In this study, we confirmed that activin A induces rSel-1l mRNA expression in RSMG-1 cells, and that rSel-1l is expressed in SMG acinar cells. These results suggest that activin A regulates the differentiation of RSMG-1 cells to acinar cells.

Activins↗

PubMed2001

Pluripotent cells (stem cells) and their determination and differentiation in early vertebrate embryogenesis.

Mammalian embryonic stem cells can be obtained from the inner cell mass of blastocysts or from primordial germ cells. These stem cells are pluripotent and can develop into all three germ cell layers of the embryo. Somatic mammalian stem cells, derived from adult or fetal tissues, are more restricted in their developmental potency. Amphibian ectodermal and endodermal cells lose their pluripotency at the early gastrula stage. The dorsal mesoderm of the marginal zone is determined before the mid-blastula transition by factors located after cortical rotation in the marginal zone, without induction by the endoderm. Secreted maternal factors (BMP, FGF and activins), maternal receptors and maternal nuclear factors (beta-catenin, Smad and Fast proteins), which form multiprotein transcriptional complexes, act together to initiate pattern formation. Following mid-blastula transition in Xenopus laevis (Daudin) embryos, secreted nodal-related (Xnr) factors become important for endoderm and mesoderm differentiation to maintain and enhance mesoderm induction. Endoderm can be induced by high concentrations of activin (vegetalizing factor) or nodal-related factors, especially Xnr5 and Xnr6, which depend on Wnt/beta-catenin signaling and on VegT, a vegetal maternal transcription factor. Together, these and other factors regulate the equilibrium between endoderm and mesoderm development. Many genes are activated and/or repressed by more than one signaling pathway and by regulatory loops to refine the tuning of gene expression. The nodal related factors, BMP, activins and Vg1 belong to the TGF-beta superfamily. The homeogenetic neural induction by the neural plate probably reinforces neural induction and differentiation. Medical and ethical problems of future stem cell therapy are briefly discussed.

Animals↗

PubMed2001

Inhibition of the Wnt signaling pathway by Idax, a novel Dvl-binding protein.

In attempting to clarify the roles of Dvl in the Wnt signaling pathway, we identified a novel protein which binds to the PDZ domain of Dvl and named it Idax (for inhibition of the Dvl and Axin complex). Idax and Axin competed with each other for the binding to Dvl. Immunocytochemical analyses showed that Idax was localized to the same place as Dvl in cells and that expression of Axin inhibited the colocalization of Dvl and Idax. Further, Wnt-induced accumulation of beta-catenin and activation of T-cell factor in mammalian cells were suppressed by expression of Idax. Expression of Idax in Xenopus embryos induced ventralization with a reduction in the expression of siamois, a Wnt-inducible gene. Idax inhibited Wnt- and Dvl- but not beta-catenin-induced axis duplication. It is known that Dvl is a positive regulator in the Wnt signaling pathway and that the PDZ domain is important for this activity. Therefore, these results suggest that Idax functions as a negative regulator of the Wnt signaling pathway by directly binding to the PDZ domain of Dvl.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Dome formation and tubule morphogenesis by Xenopus kidney A6 cell cultures exposed to microgravity simulated with a 3D-clinostat and to hypergravity.

Confluent high-density cell cultures of A6 cells derived from adult male Xenopus kidney exhibit spontaneous dome-formation at 1 g. To determine whether this morphogenetic property is altered by gravity, we used a three-dimensional (3D) clinostat to subject the cells to simulated microgravity, and a centrifuge to subject them to hypergravity. We used the generation orbit control method as the new rotation control system of the 3D-clinostat, not the random method. The growth of A6 cells was significantly enhanced by hypergravity, but significantly reduced by simulated microgravity. Dome formation by A6 cells at high confluence was inhibited under simulated microgravity conditions, whereas hypergravity promoted dome formation and induced tubule morphogenesis, compared to the control at 1 g. These results indicated that changes in gravity influence the morphogenetic properties of A6 cells, such as dome formation and tubule morphogenesis. When dome formation by A6 cells at high confluence was induced spontaneously in the control 1 g culture, the gene expression of the HGF family of pleiotropic factors, such as HGF-like protein (HLP) and growth factor-Livertine (GF-l.ivertine), an epithelial serine protease of channel activating protease 1 (CAP1), and Na+, K+-adenosine triphosphatase (ATPase), increased. Simulated microgravity increased the gene expression of activin A and reduced the gene expression of HLP, GF-Livertine, CAP1, and Na+, K+-ATPase. Hypergravity, on the other hand, decreased the gene expression of activin A and increased the gene expression of HLP, GF-Livertine, CAP1, and Na+, K+-ATPase. These results suggest that the effects of gravitational changes on expression of the HGF family member gene, CAP1, and Na+, K+-ATPase gene may be important for the cell growth, tubule morphogenesis, and dome formation of A6 cells in altered

Animals↗

PubMed2001

In vitro induction systems for analyses of amphibian organogenesis and body patterning.

The discovery that some well-known growth factors have inducing activity in embryogenesis has accelerated our understanding of embryonic induction. Relevant receptors, signal transduction pathways and patterns of gene expression have been characterized over the past decade. Amphibian embryos have provided an excellent model for analysis of embryonic induction because they are easily surgically manipulated and cultured in vitro, and with the addition of treatment with various inducing factors we have been able to control organogenesis and body patterning during early development in vitro. Activin A, a TGF-beta family protein, has a potent mesoderm-inducing activity on the isolated ectoderm called the animal cap. Activin induces animal caps to differentiate into various mesodermal and endodermal tissues, including beating hearts, in a dose-dependent fashion. Activin, in combination with retinoic acid, also induces the formation of the pronephros, a primitive embryonic kidney. The in vitro induced kidney was confirmed to function in vivo in a transplantation experiment. Furthermore, the activin-induced animal caps organize heads or trunk-and-tails in exactly the same manner as the organizer. The potential use of in vitro induction systems to further our understanding of vertebrate organogenesis and body patterning will be discussed.

Activins↗

PubMed2001

Spemann's influence on Japanese developmental biology.

The discovery of the organizer by H. Spemann and Hilde Mangold, prompted a number of studies of embryonic induction in Japan. C.O. Whitman, N. Yatsu, T. Sato, H. Oka, T. Yamada, and Y.K. Okada were the pioneers in the field of embryonic induction. T. Yamada postulated the double potential theory for embryonic induction. O. Nakamura has modified the fate map of Vogt using newt and Xenopusblastulae. T.S. Okada and G. Eguchi proposed the new concept of "transdifferentiation" based on in vitro experiments in the retina and lens. T.S. Okada is not only an excellent scientist, but he has also nurtured many active developmental biologists. M. Takeichi, from his school, discovered the cell adhesion molecle, cadherin. Nakamura and colleagues tried to determine the origin and formation of the organizer. They performed recombination experiments using the ectoderm, endoderm and mesoderm, and concluded that the phenomenon in which various mesoderm tissues are formed by the recombination of the presumptive ectoderm with endoderm was "regulation of the vegetal-animal gradient". Some groups have also tried to purify specific inducing factors. T. Yamada and colleagues isolated two different types of ribonucleoproteins. I. Kawakami and colleagues showed that the ribosome fraction has neural inducing capacity, and that the extracellular matrix contains mesodermal inducing factors. Finally Asashima and colleagues isolated and identified activin A as a MIF factor. This finding had a great influence not only in the field of developmental biology, but also in molecular biology. Using activin, Asashima's group has successfully generated various organs, tissues, trunk-tail and head structures in vitro using animal caps (undifferentiated cells). Some other important molecules such as BMP, chordin and bFGF are also being studied by young Japanese scientists.

Amphibians↗

PubMed2000

A role for Xlim-1 in pronephros development in Xenopus laevis.

Xlim-1, a LIM class homeobox gene expressed in Xenopus laevis, is one of the earliest known marker genes of pronephros development and is expressed in pronephros rudiment. In this study, we examined the role of Xlim-1 in pronephros development. Temporal expression of Xlim-1 in explants was analyzed in a series of induction assays using RT-PCR analysis. Xlim-1 was expressed 9 to 15 h after activin/retinoic acid treatment, corresponding to pronephros differentiation in explants. We further examined the role of Xlim-1 using a series of microinjection experiments. Presumptive pronephric anlagen of embryos were injected with various Xlim-1 mutants, and effects of these Xlim-1 mutants on pronephrogenesis in embryos and in explants were analyzed by RT-PCR and immunohistochemistry. Dominant-negative Xlim-1 inhibited differentiation of pronephros in activin/retinoic acid-treated animal caps. In embryos injected with a dominant-negative form of Xlim-1, development of pronephric tubules was inhibited at the late tail-bud stage. Our results suggest that Xlim-1 may not initiate differentiation of the pronephros, but that it is necessary for growth and elongation in the development of pronephric tubules.

Activins↗

PubMed2000

Inhibition of Wnt signaling pathway by a novel axin-binding protein.

Axin forms a complex with adenomatous polyposis coli gene product, glycogen synthase kinase-3beta (GSK-3beta), beta-catenin, Dvl, and protein phosphatase 2A and functions as a scaffold protein in the Wnt signaling pathway. In the Axin complex, GSK-3beta efficiently phosphorylates beta-catenin, which is then ubiquitinated and degraded by proteasome. We isolated a novel protein that binds to Axin and named it Axam (for Axin associating molecule). Axam formed a complex with Axin in intact cells and bound directly to Axin. Axam inhibited the complex formation of Dvl with Axin and the activity of Dvl to suppress GSK-3beta-dependent phosphorylation of Axin. Furthermore, Axam induced the degradation of beta-catenin in SW480 cells and inhibited Wnt-dependent axis duplication in Xenopus embryos. These results suggest that Axam regulates the Wnt signaling pathway negatively by inhibiting the binding of Dvl to Axin.

Adaptor Proteins, Signal Transducing↗

PubMed2000

A novel beta-catenin-binding protein inhibits beta-catenin-dependent Tcf activation and axis formation.

beta-Catenin is efficiently phosphorylated by glycogen synthase kinase-3beta in the Axin complex in the cytoplasm, resulting in the down-regulation. In response to Wnt, beta-catenin is stabilized and translocated into the nucleus where it stimulates gene expression through Tcf/Lef. Here we report a novel protein, designated Duplin (for axis duplication inhibitor), which negatively regulates the function of beta-catenin in the nucleus. Duplin was located in the nucleus. Duplin bound directly to the Armadillo repeats of beta-catenin, thereby inhibiting the binding of Tcf to beta-catenin. It did not affect the stability of beta-catenin but inhibited Wnt- or beta-catenin-dependent Tcf activation. Furthermore, expression of Duplin in Xenopus embryos inhibited the axis formation and beta-catenin-dependent axis duplication, and prevented the beta-catenin's ability to rescue ventralizing phenotypes induced by ultraviolet light irradiation. Thus, Duplin is a nuclear protein that inhibits beta-catenin signaling.

Amino Acid Sequence↗

PubMed2000

XSIP1, a member of two-handed zinc finger proteins, induced anterior neural markers in Xenopus laevis animal cap.

We characterized Xenopus SIP1 (XSIP1), Smad interacting protein, from activin-treated animal caps by differential screening. The XSIP1 is very similar to mouse SIP1 in the protein coding region including the zinc finger domain and homeodomain. The expression pattern was analyzed by RT-PCR and whole mount in situ hybridization. XSIP1 expression was initially restricted to the dorsal marginal zone in the late gastrula and was subsequently expressed at the lateral edge of neural plate and, in the tailbud stage, in the forebrain, neural tube, and eye. Overexpression of XSIP1 at the animal caps resulted in activation of anterior neural markers without mesodermal markers. Ectopic expression of XSIP1 induced enlargement of neural cells and disordered eye formation. In addition to abnormal head phenotypes, many embryos were short-tailed. Our findings suggest that XSIP1 is a transcriptional repressor, which may be involved in the activin-dependent signal pathway.

Amino Acid Sequence↗

PubMed2000

Expression of the Xenopus GTP-binding protein gene Ran during embryogenesis.

The Ran gene family encodes small GTP binding proteins that are associated with a variety of nuclear processes. We isolated a Xenopus Ran cDNA and analyzed the pattern of expression of this gene during embryogenesis. Ran is expressed maternally and later in the CNS, neural crest, mesenchyme, eyes, and otic vesicles. However, expression is not detected in the somites or the notochord.

Amino Acid Sequence↗

PubMed2000

In vitro control of organogenesis and body patterning by activin during early amphibian development.

In the process of amphibian development, an embryonic body plan is established through cell division, sequential gene expression, morphogenesis and cell differentiation. The mechanism of body patterning is complex and includes multiple induction events. Activin, a TGF-beta family protein, can induce several kinds of mesodermal and endodermal tissues in animal cap explants in a dose-dependent manner. In a recent study of the role of activin in organogenesis, we succeeded in raising a beating heart by treating animal caps with a high concentration of activin. Activin also participates in kidney organogenesis in combination with retinoic acid. An embryonic kidney induced by activin and retinoic acid in vitro can function in vivo when it is transplanted into a larva in which pronephros rudiments have already been removed. Further, the activin-treated animal caps clearly show organizer actions that are closely related to body patterning along the anteroposterior axis. These experiments will help to serve as a model system for understanding organogenesis and body patterning at the cellular and molecular levels.

Activins↗

PubMed2000

Work in progress: the renaissance in amphibian embryology.

Various historical eras in the distant as well as the recent past of amphibian embryology are briefly reviewed. The concepts which emerged from the early years matured, then were laid to rest for several decades. A resurgence, driven by key discoveries with peptide growth factors, and fueled by modern molecular biology methods, is underway. The future for several amphibian research projects should be promising since interest in basic concepts remains strong, and application of frontier methodologies is yielding novel findings.

Amphibians↗

PubMed2000

Expression cloning of Xantivin, a Xenopus lefty/antivin-related gene, involved in the regulation of activin signaling during mesoderm induction.

In a screening for activin-responsive genes, we isolated a Xenopus lefty/antivin-related gene, called Xantivin (Xatv). In the animal cap assay, the expression of Xatv was induced by activin signaling, and in the embryo, by nodal-related genes. Overexpression of Xatv in the marginal zone caused suppression of mesoderm formation and gastrulation defects, and inhibited the secondary axis formation induced by Xnr1 and Xactivin, suggesting that Xatv acted as a feedback inhibitor of activin signaling. However, in the animal cap, Xatv failed to antagonize Xnr1 and Xactivin. This result suggested that Xatv has different responses in the marginal zone and in the animal region, and antagonizes to a higher degree activin signaling in the marginal zone.

Activins↗

PubMed2000

Cloning and expression pattern of a Xenopus pronephros-specific gene, XSMP-30.

The first step in kidney development is the formation of the pronephros which is derived from mesoderm. Xenopus is an appropriate model to study this process since the pronephros can be efficiently induced in animal cap explants by treatment with activin and retinoic acid (RA). Using this in vitro system, we isolated a Xenopus homologue of SMP-30 (Senescence marker protein-30), which is a Ca(2+)-binding protein that is highly conserved in vertebrates. This gene, termed XSMP-30, was found to be selectively expressed in pronephric tubules from the late tadpole stage, by whole mount in situ hybridization. Furthermore XSMP-30 was expressed in animal caps treated with both activin and RA, a condition in which the pronephros is formed in vitro. These data indicate that XSMP-30 is a specific marker for the pronephros.

Activins↗

PubMed2000

The evolutionarily conserved porcupine gene family is involved in the processing of the Wnt family.

The Drosophila segment polarity gene product Porcupine (Porc) was first identified as being necessary for processing Wingless (Wg), a Drosophila Wnt (Wnt) family member. Mouse and Xenopus homologs of porc (Mporc and Xporc) were identified and found to encode endoplasmic reticulum (ER) proteins with multiple transmembrane domains. In contrast with porc, four different types of Mporc and Xporc mRNA (A-D) are generated from a single gene by alternative splicing. Mporc mRNA is differentially expressed during embryogenesis and in various adult tissues, demonstrating that the alternative splicing is regulated to synthesize the specific types of Mporc. In transfected mammalian cells, all Mporc types affect the processing of mouse Wnt 1, 3A, 4, 6, and 7B but not 5A. Furthermore, all Mporc types are co-immunoprecipitated with various Wnt proteins. These results suggest that Mporc may function as a chaperone-like molecule for Wnt. Interestingly, all Mporc types can substitute for Porc, as they are able to rescue the phenotypes of Drosophila porc embryos. Consistent with this observation, Mporc, like Porc, modifies the processing of Wg expressed in mammalian cells. These results demonstrate that the porc gene family encodes the multitransmembrane ER proteins, which are evolutionarily well conserved and involved in processing the Wnt family.

Acyltransferases↗