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M Maéno

Publications and source records attributed to M Maéno.

At least 19 recordsLinked to original sources
PubMed2001

Two-step induction of primitive erythrocytes in Xenopus laevis embryos: signals from the vegetal endoderm and the overlying ectoderm.

Primitive blood cells differentiate from the ventral mesoderm blood islands in Xenopus embryos. In order to determine the tissue interactions that propagate blood formation in early embryogenesis, we used embryos that had the ventral cytoplasm removed. These embryos gastrulated normally, formed a mesodermal layer and lacked axial structures, but displayed a marked enhancement of alpha-globin expression. Early ventral markers, such as msx-1, vent-1 and vent-2 were highly expressed at the gastrula stage, while a dorsal marker, goosecoid, was diminished. Several lines of experimental evidence demonstrate the critical role of animal pole-derived ectoderm in blood cell formation: 1) Mesoderm derived from dorsal blastomeres injected with beta-galactosidase mRNA (as a lineage tracer) expressed alpha-globin when interfaced with an animal pole-derived ectodermal layer; 2) Embryos in which the animal pole tissue had been removed by dissection at the blastula stage failed to express alpha-globin; 3) Exogastrulated embryos that lacked an interaction between the mesodermal and ectodermal layers failed to form blood cells, while muscle cells were observed in these embryos. Using dominant-negative forms of the BMP-4 and ALK-4 receptors, we showed that activin and BMP-4 signaling is necessary for blood cell differentiation in ventral marginal zone explants, while FGF signaling is not essential. In ventralized embryos, inactivation of the BMP-4 signal within a localized area of the ectoderm led to suppression of globin expression in the adjacent mesoderm layer, but inactivation of the activin signal did not have this effect. These observations suggest that mesodermal cells, derived from a default pathway that is induced by the activin signal, need an additional BMP-4-dependent factor from the overlying ectoderm for further differentiation into a blood cell lineage.

Activin Receptors, Type I↗
PubMed2000

Xenopus msx-1 regulates dorso-ventral axis formation by suppressing the expression of organizer genes.

We demonstrated previously that Xmsx-1 is involved in mesoderm patterning along the dorso-ventral axis, under the regulation of BMP-4 signaling. When Xmsx-1 RNA was injected into the dorsal blastomeres, a mass of muscle tissue formed instead of notochord. This activity was similar to that of Xwnt-8 reported previously. In this study, we investigated whether the activity of Xmsx-1 is related to the ventralizing signal and myogenesis promoting factor, Xwnt-8. Whole-mount in situ hybridization showed that Xmsx-1, Xwnt-8, and XmyoD were expressed in overlapping areas, including the ventro-lateral marginal zone at mid-gastrula stage. The expression of XmyoD was induced by the ectopic expression of either Xmsx-1 or Xwnt-8 in dorsal blastomeres, and Xwnt-8 was induced by the ectopic expression of Xmsx-1. On the other hand, the expression of Xmsx-1 was not affected by the loading of pCSKA-Xwnt-8 or dominant-negative Xwnt-8 (DN-Xwnt-8) RNA. In addition, Xmsx-1 RNA did not abrogate the formation of notochord if coinjected with DN-Xwnt-8 RNA. These results suggest that Xmsx-1 functions upstream of the Xwnt-8 signal. Furthermore, the antagonistic function of Xmsx-1 to the expression of organizer genes, such as Xlim-1 and goosecoid, was shown by in situ hybridization analysis and luciferase reporter assay using the goosecoid promoter construct. Finally if Xmsx-1/VP-16 fusion RNA, which was expected to function as a dominant-negative Xmsx-1, was injected into ventral blastomeres, a partial secondary axis formed in a significant number of embryos. In such embryos, the activity of luciferase, under the control of goosecoid promoter sequence, was significantly elevated at gastrula stage. These results led us to conclude that Xmsx-1 plays a central role in establishing dorso-ventral axis in gastrulating embryo, by suppressing the expression of organizer genes.

Actins↗
PubMed2000

Involvement of BMP-4/msx-1 and FGF pathways in neural induction in the Xenopus embryo.

The msx homeodomain protein is a downstream transcription factor of the bone morphogenetic protein (BMP)-4 signal and a key regulator for neural tissue differentiation. Xmsx-1 antagonizes the dorsal expression of noggin and cerberus, as revealed by in situ hybridization and reverse transcription-polymerase chain reaction assays. In animal cap explants, Xmsx-1 and BMP-4 inhibit the neural tissue differentiation induced by noggin or cerberus. A loss-of-function study using the Xmsx-1/VP-16 fusion construct indicated that neural tissue formation was directly induced by the injection of fusion ribonucleic acid, although the expression of neural cell adhesion molecule (N-CAM) in the cap was less than that in the cap injected with tBR or noggin. In contrast to the single cap assay, unexpectedly, both BMP-4 and Xmsx-1 failed to inhibit neurulation in the ectodermal explants to which the organizer mesoderm was attached. The results of cell-lineage tracing experiments indicated that the neural cells were differentiated from the animal pole tissue where the excess RNA of either BMP-4 or Xmsx-1 was injected, whereas notochord was differentiated from the organizer mesoderm. Neural tissue differentiated from BMP-4-injected ectodermal cells strongly expressed posterior neural markers, such as hoxB9 and krox20, suggesting that the posterior neural cells differentiated regardless of the existence of the BMP signal. The introduction of a dominant-negative form of the fibroblast growth factor (FGF) receptor (XFD) into the ectodermal cells drastically reduced the expression of pan and posterior neural markers (N-CAM and hoxB-9) if co-injected with BMP-4 RNA, although XFD alone at the same dose did not shut down the expression of N-CAM in the combination explants. Therefore, it is proposed that an FGF-related molecule was involved in the direct induction of posterior neural tissue in the inducing signals from the organizer mesoderm in vivo.

Animals↗
PubMed1999

Immunodetection of Xenopus bone morphogenetic protein-1 in adult and embryonic cells.

In order to analyze biochemical properties of Xenopus bone morphogenetic protein-1 (XBMP-1), rabbit antiserum (alpha-B1) was raised against a synthetic peptide (P1) corresponding to a hydrophilic N-terminal region. XBMP-1B (Xtld) synthesized in the reticulocyte lysate was successfully immunoprecipitated by this antiserum. This precipitation was completely blocked when P1 was added to the reaction, indicating that alpha-B1 recognized XBMP-1B specifically. In Western blot analysis, two distinct sizes of protein (107 and 34 kD) were detected in hind limbs in metamorphosing animals. Both proteins were detected in various adult tissues such as lung, liver, kidney, heart, muscle, intestine, brain, and testis. The mixing of the liver and muscle extracts, and the following detection of immunoreactive proteins suggested that the 34 kD band was a proteolytic product of the 107 kD protein. In the embryonic extracts from the unfertilized egg (stage 0) to swimming tadpoles (stage 40), a 63 kD protein was detected in addition to the 107 kD protein. We also showed that the 107 kD protein was much more expressed in the animal half of the unfertilized eggs than in the vegetal half, but that it was ubiquitously expressed in the gastrula embryos. We suggest that the 63 and 107 kD proteins correspond to full-length proteins encoded by XBMP-1A and XBMP-1B genes, and these proteins are expressed in embryo and in various adult tissues.

Animals↗
PubMed1998

GATA-1 inhibits the formation of notochord and neural tissue in Xenopus embryo.

The expression of GATA-1, which encodes for a hemopoietic transcription factor, initiates at gastrula stage in the Xenopus embryo (1). In order to examine a possible function of GATA-1 in dorso-ventral patterning of mesoderm and ectoderm derivatives, the synthesized RNA of GATA-1 was overexpressed in embryonic cells to assess its biological effects. In the embryos injected with GATA-1 RNA in the dorsal marginal zone at 4-cell stage, dorsal epidermis did not cover the vegetal cells so that the gastrulation was not completed. The same dose of GATA-1 RNA injected into ventral marginal zone did not influence the development, and GATA-2 RNA transcribed from the same vector had little effect, suggesting that this phenomenon is physiologically important. The morphological and immunohistochemical studies revealed that notochord and neural tissue were mostly eliminated in the embryos or the dorsal marginal zone explants after injection of GATA-1 RNA. GATA-1 also inhibited neurogenesis in animal cap explants, which was induced by the injection with noggin RNA. Northern blot analysis using dorsal marginal zone explants showed, however, that only a slight amount of alpha-globin message was induced, and cardiac alpha-actin message was retained. Therefore, GATA-1 did not convert completely the dorsal phenotype to the ventral one. Furthermore, the injection of GATA-1 RNA didnot alter the expression of early dorsal and ventral markers at the onset of gastrulation. These results suggest that GATA-1 is an potential inhibitor of the dorsalization and the neurogenesis, but it affects on the specification of dorsal tissues in relatively later steps.

Actins↗
PubMed1997

XBMP-1B (Xtld), a Xenopus homolog of dorso-ventral polarity gene in Drosophila, modifies tissue phenotypes of ventral explants.

Previously we have isolated a Xenopus cDNA homolog of bone morphogenetic protein-1 (XBMP-1A). In the present report we describe a new cDNA clone called XBMP-1B (or Xtld) from a Xenopus embryonic library. Sequence analysis indicates that these two clones share an indentical N-terminal sequence, including a region of metalloprotease domain, three copies of a repeat first found in complement proteins C1r/s and an epidermal growth factor (EGF)-like sequence. XBMP-1B protein has an additional copy of an EGF-like sequence followed by two copies of complement 1 r/s repeat in the C-terminus. The overall protein structure predicted from the XBMP-1B sequence reveals that it encodes a protein homologous to Drosophila tolloid. Three XBMP-1 transcripts (2.9, 5.2 and 6.6 kb) were detected by northern blot analysis. However, the 2.9 kb transcript hybridized specifically with XBMP-1A and the 5.2 and 6.6 kb transcripts hybridized with XBMP-1B. In Drosophila, a major function of tolloid is to augment the activity of the decapentaplegic gene product, a close relative of tumor growth factor (TGF)-beta superfamily members, BMP-2/4. Although XBMP-1 and XBMP-4 are detected in various adult tissues of Xenopus, the expression pattern of these two genes was not tightly correlated. In the embryo, the expression of XBMP-1 increased gradually from the morula to the swimming tadpole stages. Injection of XBMP-1B RNA into the ventral blastomeres at the 4-cell stage caused an elongation of the ventral marginal zone explants and converted globin-positive blood cells to mesenchymal and muscle tissues at later stages. It was shown that XBMP-1A was less active and a 1A mutant lacking the signal sequence was inactive. Further studies revealed that injection of XBMP-1B RNA into the ventral marginal zone induced up-regulation of dorsal marginal zone markers, such as goosecoid and chordin, at the gastrulation stage. These data indicate that XBMP-1 may have a role in determining dorso-ventral patterning in Xenopus, but in a different way from the dpp/tolloid system demonstrated in Drosophila.

Amino Acid Sequence↗
PubMed1997

Xmsx-1 modifies mesodermal tissue pattern along dorsoventral axis in Xenopus laevis embryo.

This study analyzes the expression and the function of Xenopus msx-1 (Xmsx-1) in embryos, in relation to the ventralizing activity of bone morphogenetic protein-4 (BMP-4). Expression of Xmsx-1 was increased in UV-treated ventralized embryos and decreased in LiCl-treated dorsalized embryos at the neurula stage (stage 14). Whole-mount in situ hybridization analysis showed that Xmsx-1 is expressed in marginal zone and animal pole areas, laterally and ventrally, but not dorsally, at mid-gastrula (stage 11) and late-gastrula (stage 13) stages. Injection of BMP-4 RNA, but not activin RNA, induced Xmsx-1 expression in the dorsal marginal zone at the early gastrula stage (stage 10+), and introduction of a dominant negative form of BMP-4 receptor RNA suppressed Xmsx-1 expression in animal cap and ventral marginal zone explants at stage 14. Thus, Xmsx-1 is a target gene specifically regulated by BMP-4 signaling. Embryos injected with Xmsx-1 RNA in dorsal blastomeres at the 4-cell stage exhibited a ventralized phenotype, with microcephaly and swollen abdomen. Histological observation and immunostaining revealed that these embryos had a large block of muscle tissue in the dorsal mesodermal area instead of notochord. On the basis of molecular marker analysis, however, the injection of Xmsx-1 RNA did not induce the expression of alpha-globin, nor reduce cardiac alpha-actin in dorsal marginal zone explants. Furthermore, a significant amount of alpha-actin was induced and alpha-globin was turned off in the ventral marginal zone explants injected with Xmsx-1. These results indicated that Xmsx-1 is a target gene of BMP-4 signaling, but possesses a distinct activity on dorsal-ventral patterning of mesodermal tissues.

Animals↗
PubMed1995

Expression of an amphibian homolog of the Eph family of receptor tyrosine kinases is developmentally regulated.

In order to study the function of tyrosine kinase receptors during Xenopus development, we have isolated Xek (Xenopus Elk-like kinase), a tyrosine kinase receptor, which shows significant homology to rat Elk and chicken cek5, members of the Eph family. Xek exists as a maternally expressed mRNA which decreases in expression at the mid blastula transition and reappears at late neurulation in Xenopus. Xek mRNA is expressed at higher levels in the anterior and dorsal regions of embryonic stages 16, 24 and 37. In adult Xenopus tissues, Xek appears to be ubiquitously expressed with higher expression observed in brain and ovary. In situ hybridization analysis demonstrates localized mRNA expression in the brain, brachial arches, trigeminal facial ganglion, and the retina of the swimming tadpole stage of development. The similarities in sequence and expression pattern suggest that Xek is an amphibian member of the Eph family and may play a role in the development or function of the central nervous system.

Amino Acid Sequence↗
PubMed1994

A truncated bone morphogenetic protein 4 receptor alters the fate of ventral mesoderm to dorsal mesoderm: roles of animal pole tissue in the development of ventral mesoderm.

The biological effects of endogenous bone morphogenetic protein 4 (BMP-4), a member of the transforming growth factor beta family, on embryonic development of Xenopus laevis were investigated by using a functionally defective mutant of the BMP-4 receptor (delta mTFR11), which blocks the BMP signaling pathway. Injection of delta mTFR11 RNA into either the animal pole area or ventral marginal cells at the two-cell stage induced a dorsal phenotype in the explant of ventral mesoderm with animal pole tissue from stage 10+ embryo, even though the normal fate of this explant is a "mesenchymal ball" containing blood cells. These explants with the dorsal phenotype contained muscle, neural tissue, eye capsule, and cement gland. Northern blot analysis showed an increase of cardiac alpha-actin mRNA and a decrease of T alpha-globin mRNA expression, providing further evidence of a conversion from ventral to dorsal phenotype. Although injection of delta mTFR11 RNA did not induce mesoderm in an animal cap culture, the same tissue injected with delta mTFR11 RNA can alter the differentiation fate of uninjected ventral mesodermal explant from ventral to dorsal type, suggesting specific interaction of animal pole tissue and prospective ventral mesoderm in vivo.

Animals↗
PubMed1994

Regulation of primary erythropoiesis in the ventral mesoderm of Xenopus gastrula embryo: evidence for the expression of a stimulatory factor(s) in animal pole tissue.

We have previously shown that the animal pole tissue from a st.10+ early gastrula Xenopus embryo stimulates the primary differentiation of erythrocytes in the ventral mesoderm in combination culture. To characterize the nature of this stimulation, various sizes and different portions of animal pole tissue were combined with the ventral mesoderm explants. The erythrocyte differentiation in terms of globin expression depended on the size of the animal pole tissue that was combined with the ventral mesoderm. No difference was observed in the potency of stimulation between the ventral and dorsal halves of animal pole tissue. We also found that animal pole tissue from as late as st.7 failed to stimulate both mRNA and protein levels of globin in the explant. Histological studies of the combination explant with st.7 animal pole tissue showed that epidermis, vesicle structure, and blood-cell-like cells developed in the explant, but very few blood cells expressed globin molecules. However, the stimulation of erythroid differentiation was restored if total (20 ng) or poly(A)+ (0.2 ng) RNA from st.10+ animal pole tissue was previously injected at the 2-cell stage and the resulting animal pole tissue at st.7 was combined with st.10+ ventral mesoderm. Erythroid differentiation was also restored by injection with 1 ng of Xenopus bone morphogenetic protein-4 (XBMP-4) RNA. The effect of an extremely small dose of poly(A)+ RNA on erythroid differentiation suggests that in addition to XBMP-4 there exist substances, expressed later than st.7 in the animal pole region, which can stimulate erythrocyte differentiation in the ventral mesoderm.

Animals↗
PubMed1993

Cloning and expression of cDNA encoding Xenopus laevis bone morphogenetic protein-1 during early embryonic development.

The Xenopus laevis DNA fragment encoding a protein homologous with human bone morphogenetic protein-1 (BMP-1) was amplified by polymerase chain reaction (PCR) from cDNA derived from stage 26 (st.26) embryonic RNA. Subsequently this fragment was used as a probe to isolate cDNA clones by screening of a X. laevis st.24 embryonic cDNA library. Two partial clones (22 and 63) were obtained and the missing 5'-end of the clone 22 was extended by the anchored PCR technique. The nucleotide sequence of the resulting clone (22AN) contained an open reading frame coding for a protein with 707 deduced amino acids. Three sizes of mRNA (2.9, 5.2 and 6.6 kb) were detected in blastula (st.9) and early gastrula (st.10) embryos, and in hatched tadpole (st.40), but little or no expression was observed in morula (st.7) and late gastrula (st.12) embryos, suggesting a physiological role(s) of X.laevis BMP-1 in normal embryonic development.

Amino Acid Sequence↗
PubMed1993

Murine stem cell factor stimulates erythropoietic differentiation of ventral mesoderm in Xenopus gastrula embryo.

We have reported that the animal pole cells stimulate the ventral mesoderm of early gastrula Xenopus embryo (stage 10) to differentiate into erythrocytes. To determine the molecular mechanism(s) involved in the stimulatory effect of the animal pole, ventral mesoderm explants were cultured in the presence of various defined cellular factors. In this study, we report that murine stem cell factor (SCF) stimulates globin expression at the optimum dose of 10 ng/ml. Globin expression was observed from the ventral mesoderm explants treated with SCF, but not from the dorsal mesoderm and the animal pole explants. Morphological studies of the ventral mesoderm treated with SCF showed that only a certain population of the ventral mesoderm differentiates into erythrocytes. On the other hand, coculture of ventral mesoderm and animal pole revealed the differentiation of the entire structures into mesenchyme, blood cells, and the overlying epidermis. These data suggest that SCF may play a role in the stimulation of erythrocytic differentiation, but the effect of the animal pole cells cannot be replaced with that of SCF.

Animals↗
PubMed1991

Transplantation studies on human and duck hepatocytes in athymic nude mice.

For determination of the most suitable tissue for heterotopic transplantation of exogenous hepatocytes, dissociated hepatocytes or small pieces of liver tissue were transplanted into the spleen, adipose tissue and inside the capsule of the kidney of BALB/c mice. Survival of syngeneic grafts of dissociated hepatocytes was highest in the spleen and that of pieces of liver tissue in the adipose tissue, but only the latter system was suitable for xenogeneic transplantation. Histological examination showed that a total of 50% of the human or duck liver tissue implants survived in the inguinal fat pad of athymic nude mice (BALB/c-nu). Histochemical analyses revealed that most hepatocytes transplanted into the fat pad gave positive reactions for glucose-6-phosphatase and with periodic acid-Schiff reagent at least 28 days after transplantation. Electron microscopic observation showed that these cells also maintained characteristic cellular organelles. This xenogeneic transplantation into adipose tissue should be useful in the studies on replication and infection of human hepatotropic viruses such as hepatitis B and C viruses.

Adipose Tissue↗
PubMed1990

A cDNA clone closely associated with non-A, non-B hepatitis.

A lambda gt11 cDNA library was constructed from RNA purified from hepatitis B viral surface antigen-negative human plasma with high alanine aminotransferase activity. A cDNA clone, designated as C8-2, was isolated by immunoscreening with mixed sera from non-A, non-B hepatitis (NANBH) carrier and convalescent chimpanzees. The recombinant protein produced by C8-2 reacted specifically with sera of patients in the chronic phase of NANBH. The sequence of C8-2, 269 bp, did not hybridized with any human or chimpanzee genomic DNA, and had no homology with those of primates and viruses. The existence of this sequence in RNA of possibly infectious plasma was shown by RNA blot hybridization and by Southern blot analysis of products amplified by the polymerase chain reaction. These results strongly suggest that C8-2 is derived from the agent of this viral hepatitis.

Amino Acid Sequence↗
PubMed1988

Effective production of mouse monoclonal antibodies against influenza virus by repeated intrasplenic immunization.

Two immunization techniques that enable production of mouse monoclonal antibodies were evaluated in terms of small quantities of antigen. Various amounts of purified influenza A virus particles were applied either for in vitro sensitization in cultured splenocytes or for intrasplenic immunization, followed by hybridization of the immunized cells with mouse myeloma cells. Hybridomas producing specific antibodies for influenza viral proteins were detected by enzyme-linked immunosorbent assay when more than 50 micrograms of antigens was used for the in vitro immunization method, and at least 5 micrograms was necessary for a single intrasplenic immunization. On the other hand, as little as 60 ng of antigen administered in two intrasplenic injections was sufficient to produce specific hybridomas. Two out of six randomly selected monoclonal antibodies obtained using the repeated intrasplenic immunization method were IgG and the other four were IgM. Immunoprecipitation analyses revealed that the recognized antigens involved a viral inner protein (nucleocapsid protein), as well as an envelope glycoprotein (hemagglutinin). We conclude that immunization by two direct injections of antigen into the spleen is the most effective method for sensitization with nanogram quantities of insoluble antigen such as influenza viruses.

Animals↗
PubMed1987

Analysis of allotolerance in thymectomized Xenopus restored with semiallogeneic thymus grafts.

The triploid J strain Xenopus laevis (MHC haplotype, j/j/j) were thymectomized as early larvae, and in the adult stage each animal was given a pair of thymuses or lymphocytes from semiallogeneic diploid donor frogs (j/k). The alloreactivity of host frogs was restored to third-party donors in terms of skin graft rejection and mixed leukocyte reaction, but there was specific unresponsiveness against the k haplotype. Grafting of heavily irradiated (10,000 rads) thymuses also restored host reactivity with induction of tolerance to the k haplotype. In the latter frogs, all thymic and splenic lymphocytes were of host origin. Injection of splenocytes from the restored frogs into secondary thymectomized J frogs not only restored immunocompetence but also transferred specific tolerance to the k haplotype. Injection of lymphocytes from the restored frogs to normal frogs failed to transfer specific tolerance by both in vivo and in vitro immune tests. The results suggest that a selective deletion of the T cell population reactive to the k haplotype was maintained in the T-cell-restored frogs.

Animals↗
PubMed1987

Tolerance induced by grafting semi-allogeneic adult skin to larval Xenopus laevis: possible involvement of specific suppressor cell activity.

Major histocompatibility complex (MHC)-homozygous Xenopus laevis were rendered tolerant to semi-allogeneic antigens by grafting skins of adult frogs during larval stages (larvally induced tolerance), and this tolerant state was compared with the tolerance induced in early thymectomized frogs by the grafting of semi-allogeneic nonlymphoid thymuses (thymus-reconstituted tolerance). In contrast to a total inability of thymus-reconstituted frogs both to reject skins and to exhibit a mixed leukocyte reaction (MLR) against the semi-allogeneic donor, larvally induced tolerant frogs showed a strong MLR against leukocytes of the tolerizing skin donor (split tolerance). Breakdown of the tolerant state in thymus-reconstituted frogs were easily accomplished by inoculation with syngeneic splenocytes, but this breakdown was extremely difficult to achieve in frogs with larvally induced tolerance. The injection of splenocytes from larvally induced tolerant frogs into normal frogs significantly suppressed semi-allogeneic graft rejection in the latter group; no suppression was obtained when splenocytes from thymus-reconstituted frogs were used. In addition, in the thymectomized frogs, recovery of allograft rejection capacity against the pertinent semi-allogeneic antigens were suppressed by the injection of splenocytes from larvally induced tolerant frogs, with the degree of suppression depending on the splenocyte dose. These results indicate that the larvally induced tolerant state is maintained by specifically induced suppressor cells affecting the in vivo allograft response but not the MLR.

Animals↗