Biomedical subjects

M Asashima

Publications and source records attributed to M Asashima.

At least 91 records · Page 5Linked to original sources

PubMed1995

Identification of a Xenopus glutamine synthetase gene abundantly expressed in the embryonic nervous system but not in adult brain.

We used a PCR-based subtraction cloning procedure with concanavalin A-treated and -untreated animal caps from stage 9 Xenopus embryos to search for genes up-regulated during early neural development. One such gene was found to encode a protein homologous to several known glutamine synthetases, and we named it xGS. Molecular hybridization studies revealed that xGS mRNA is maternally transmitted and abundantly expressed in neuroectoderm-derived tissues during the gastrula and neurula stages. The expression of xGS mRNA in the nervous system continues until the larval stages, but declines thereafter and becomes undetectable in adult brain. Considering its metabolic activity and potential neuroprotective effect against the neurotoxic substances such as glutamate and ammonia, the glutamine synthetase may play an important role in the early stages of vertebrate neural development.

Amino Acid Sequence↗

PubMed1995

Activin induces the expression of the Xenopus homologue of sonic hedgehog during mesoderm formation in Xenopus explants.

The Xenopus homologue of sonic hedgehog (Xhh) was detected in Xenopus embryos at stages 13 and 31 by RT-PCR, but it was not expressed in explants isolated from the animal hemisphere of Xenopus embryos at stage 8-9. Treatment of the animal cap with activin (1-100 ng/ml) induced the expression of Xhh. However, it was not induced by 100 ng/ml basic fibroblast growth factor (bFGF). Whole mount in situ hybridization confirmed the expression of Xhh in the animal cap treated with activin. The expression of Xhh induced by activin was not inhibited in the presence of cycloheximide, suggesting that Xhh is an early response gene induced by activin.

Activins↗

PubMed1995

Development of the otolith organs and semicircular canals in the Japanese red-bellied newt, Cynops pyrrhogaster.

The sequence in which the otoliths and semicircular canals and their associated sensory epithelia appear and develop in the newt are described. Three-dimensional reconstruction of serial sections through the otic vesicle of newt embryos from stages 31 through 58 demonstrate the first appearance, relative position and growth of the otoliths. A single otolith is first seen in stage 33 embryos (approximately 9 days old); this splits into separate utricular and saccular otoliths at stage 40 (13 days). The lateral semicircular canal is the first to appear, at stage 41 (14 days). The anterior and posterior canals appear approximately one week later and the vestibular apparatus is essentially fully formed at stage 58 (approximately 5 weeks). The data reported here will serve as ground-based controls for fertilized newt eggs flown on the International Microgravity Laboratory-2 Space Shuttle flight, to investigate the influence of microgravity on the development of the gravity-sensing organs.

Animals↗

PubMed1995

Non-invasive assessment of otolith formation during development of the Japanese red-bellied newt, Cynops pyrrhogaster.

Pre-mated adult female newts and embryos have been flown on the International Microgravity Laboratory-2 (IML-2) Space Shuttle flight in 1994 (Wiederhold et al., 1992b). With the specimens available from this flight, the calcification of otoliths, ulna, radius and backbone of the flown larvae and adult newts were analyzed. The experiments presented here studied the development of the otoliths on the ground. Otoliths of living newts, from embryo to adult, were observed in situ with the application of a new X-ray and bio-imaging analyzer system. For the establishment of this method, newts at different developmental stages were used. An imaging plate temporarily stores the X-ray energy pattern at the bio-imaging analyzer. A latent image on the imaging plate was transformed into a digital time series signal with an image reader. Acquired digital information was computed with the image processor. The processed information was recorded on film with an image recorder, in order to visualize it on an enlargement computed radiograph. To analyze development of the otoliths, photo-stimulated luminescence level was detected by an image analyzer, using transmitted X-ray photons. A single clump of otoconia could first be seen at stage 33. Stage-36 embryos first have distinguishable otoliths, with the utricle in front and saccule behind. Our results show that this X-ray method detects the otoliths equally as well as sectioning. In the newt, the mandibular/maxillary bone formed before the spine. It is suspected that for the newt embryo, living in water, feeding becomes necessary prior to support of the body.

Animals↗

PubMed1995

Restricted expression of Xenopus midkine gene during early development.

Midkine (MK) is a heparin-binding growth factor and forms a novel protein family together with another member, pleiotrophin (PTN)/heparin-binding growth-associated molecule (HB-GAM). A cDNA clone isolated from Xenopus laevis specifies for the Xenopus counterpart of MK (XMK), and the mode of XMK expression was studied by in situ hybridization and Northern blot analysis. XMK was first expressed at stage 11 (middle gastrula) and was located in the neural anlage at stage 12 (late gastrula). Through stage 13 to 15 (early neurula), XMK expression was restricted to the neural folds. At stage 23 (tailbud stage), XMK was predominantly localized in the brain and neural tube. At the larva stage, XMK expression was again restrictedly observed in the brain, the optic vesicles, the otic vesicle, and the spinal cord, all of which are derivatives of the neural tube, as well as in the branchial arches, derivatives of the cranial neural crest. Comparing the mode of MK expression between Xenopus and the mouse, we propose that MK plays evolutionally conserved roles in neurogenesis and development of the craniofacial architecture of ectomesenchymal origin. We also found that XMK was expressed in various adult organs; strong expression was observed in the brain, the eye and the spinal cord, all of which showed intense MK expression at the larva stage.

Amino Acid Sequence↗

PubMed1995

Comparison of mesoderm-inducing activity with monomeric and dimeric inhibin alpha and beta-A subunits on Xenopus ectoderm.

Activin possesses mesoderm-inducing activity, erythroid-differentiating activity, and follicle-stimulating hormone-releasing activity. The chemical structures of the activin molecule are formed by a combination of two beta-subunit peptides of inhibin. Inhibin is a dimer consisting of an alpha and beta subunit. To examine the mesoderm-inducing activity of these substances, we tested several configurations including: (1) two types of alpha-subunit peptide; (2) two types of inhibin A and B dimer; (3) beta A-subunit peptide monomer; (4) three types of activins A, AB and B, and (5) follistatin (activin-binding protein) by the animal cap assay using Xenopus laevis ectoderm, and by the erythroid-differentiating factor (EDF) test. Activins, which are composed of dimeric inhibin beta A- or beta B-subunit peptides, had the highest mesoderm-inducing and EDF activities. The monomeric beta A-subunit peptide exhibited mesoderm-inducing and EDF activities that were much lower than activin A. The inhibitory effect of follistatin on mesodermal induction by the beta A-subunit peptide was also lower than that of activin. Both inhibins A and B had very weak mesoderm-inducing activity and no EDF activity. The two types of inhibin alpha-subunit monomer had little mesoderm-inducing activity and no EDF activity. The mesoderm induction caused by activin A was not suppressed by the addition of the alpha-subunit monomer and inhibin. The mesoderm-inducing activity in relation to the chemical structures of the monomeric and/or dimeric inhibin alpha and beta A subunits is discussed.

Activins↗

PubMed1995

Effect of activin and lithium on isolated Xenopus animal blastomeres and response alteration at the midblastula transition.

Dorsoventral mesoderm patterning in the amphibian embryo involves a series of interactions mediated by several peptide growth factors. Animal blastomeres isolated at the 8-cell stage are useful for studying mesoderm patterning, since they contain the prospective (uninduced) mesoderm region and allow examination of the default state of animal cells. When activin is applied to these dorsal and ventral animal half explants, a competence prepattern for responding to activin is observed. In order to investigate the characteristics of prepatterning, we treated animal blastomeres with the embryo dorsalizing agent LiCl. Treatment with lithium alone did not induce normal trunk mesoderm in either blastomere. Lithium did, however, alter the competence of animal blastomeres to activin. Dorsal mesoderm was formed in the ventral blastomeres, as well as in the dorsal blastomeres. This result reveals that the early dorsoventral polarity in the animal hemisphere is not fixed. Using goosecoid(gsc) and Xwnt-8 genes as dorsal and ventral mesoderm markers, it was verified that lithium modifies the competence to activin. Unexpectedly, lithium treatment on its own resulted in gsc expression in the animal half explants. This suggests that embryo goosecoid expression may be induced by the effect of dorsal determination activity, but not by mesoderm induction. However, lithium induced also the expression of brachyury (Xbra) gene at very low levels. This would indicate the formation of dorsal-anterior mesoderm, which was not identified by the tissue observations. Expression of Xwnt-8, a ventral mesoderm marker usually induced in blastula animal caps by activin, was hardly induced in the blastomere explants. We isolated whole animal half explants at the 8-cell stage and exposed to activin at different stages. It was found that the same concentration of activin induces gsc before the midblastula stage, and induces Xwnt-8 at later stages. This suggests that the response of animal blastomeres alters depending on the stage of activin signaling.

Activins↗

PubMed1995

Control of the embryonic body plan by activin during amphibian development.

Embryonic induction plays an important role in establishing the fundamental body plan during early amphibian development. The factors mediating this embryonic induction have, however, only recently been discovered. In the mid-1980's, certain peptide growth factors belonging to the FGF and TGF-beta families were found to have a mesoderm-inducing effect on isolated Xenopus blastula ectoderm. The study of embryonic induction subsequently expanded rapidly and knowledge at the molecular level has gradually accumulated. One of these peptide growth factors, activin, a member of the TGF-beta superfamily, is present maternally in the Xenopus early embryo and induces various mesodermal and endodermal tissues in isolated presumptive ectoderm. After exposure of presumptive ectoderm to activin, many genes are expressed in the same manner as in normal embryogenesis. Ectoderm treated with activin can induce a complete secondary embryo, the same as the organizer does in transplantation experiments. These findings suggest that activin is one of the first induction signals responsible for establishing the embryonic body plan in early amphibian development. In this article we shall review to what extent we can control the embryonic body plan in vitro, referring to some significant findings in this field.

Activins↗

PubMed1995

Cytopathologic observations of the lung of adult newts (Cynops pyrrhogaster) on-board the space shuttle, Columbia, during the Second International Microgravity Laboratory experiments.

Four adult female Japanese newts, Cynops pyrrhogaster, were carried for 15 days aboard the orbiting space shuttle, Columbia, in July of 1994, as part of the Second International Microgravity Laboratory, IML-2 aquatic animal experiments. These previously fertilized newts, after stimulation with chorionic gonadotropin by a spaceflight adapted injection procedure, deposited numerous eggs for study of early development during weightlessness. The primitive saccular lungs of the two newts which survived the spaceflight revealed by TEM marked pulmonary cytopathologic changes including basal laminar separation, microvillar degeneration, and cytoplasmic granular changes in the primary granulated pneumocytes. Also, intracellular edema in the pulmonary collagenous matrix and vacuolar changes in the ciliated pulmonary lining cell type and in vascular endothelial cells were observed. These changes, triggered by the spaceflight, and not seen in controls also relying on respiration via the skin, may reflect a chronic mild hypoxia as it is known that newts undergoing oviposition are subject to increased oxygen demand.

Animals↗

PubMed1994

HMG-X, a Xenopus gene encoding an HMG1 homolog, is abundantly expressed in the developing nervous system.

We used a PCR-based subtraction cloning procedure with Concanavalin A-treated and untreated animal caps from stage 9 Xenopus embryos to search for genes the expression of which is induced during neurogenesis. One of these genes was found to encode a homolog of mammalian HMG 1 and 2, hence named HMG-X. HMG-X mRNA was maternally transmitted, up-regulated in neuroectoderm-derived tissues throughout early development, and eventually down-regulated in all adult tissues examined except ovary. Our data suggest that we have identified a gene for a member of the HMG1/2 family that could have an important role in neurogenesis.

Amino Acid Sequence↗

PubMed1994

Localization of activin and follistatin proteins in the Xenopus oocyte.

We found a binding protein for activin and follistatin in serum from female Xenopus laevis and identified it as vitellogenin, which is synthesized in the liver and transported into yolk platelets. Then, we investigated the localization of activin and follistatin proteins in early Xenopus oocytes (stage 6) by electron microscopic immunolabeling with gold colloidal particles. The protein molecules were found to be localized uniformly in oocyte yolk platelets, but not in other cytoplasmic organelles. These findings suggest a novel role of yolk platelets as a reservoir for inductive signals transported by vitellogenin in the differentiation and patterning of cells in Xenopus embryos.

Activins↗

PubMed1994

Identification of activins A, AB, and B and follistatin proteins in Xenopus embryos.

There are several lines of evidence that activin is a crucial molecule for mesoderm induction in early Xenopus embryos. However, it is not known what kind and what amounts of activin proteins exist in cleavage stage embryos. Three isoforms of activins, A, AB, and B, were demonstrated to be present at least in part as a complex with abundant follistatin, an activin-binding protein, in early Xenopus embryos (stage 1-5). These results suggest that activin stored in eggs has an important role for mesoderm induction during early Xenopus embryogenesis and that follistatin can modulate the function of activin in signaling developmental changes.

Activins↗

PubMed1994

Formation of otoconia in the Japanese red-bellied newt, Cynops pyrrhogaster.

Pre-mated adult female newts and fertilized eggs will be flown on the International Microgravity Laboratory-2 flight, in 1994. One objective of the flight will be to observe the influence of microgravity on the development of the gravity-sensing organs in the inner ear. These organs contain sensory hair cells covered by a layer of dense stones (otoconia). Gravity and linear acceleration exert forces on these masses, leading to excitation of the nerve fibers innervating the hair cells. If the production of the otoliths is regulated to reach an optimal weight, their development might be abnormal in microgravity. Ground-based control experiments are reported describing the developmental sequence in which both the otoliths and their associated sensory epithelium and the semicircular canals appear and develop. Three-dimensional reconstruction of serial sections through the otic vesicle of newt embryos at stages 31 through 58 demonstrate the first appearance, relative position and growth of the otoliths. Reports of experiments in which fertilized frog eggs were flown on a Russian Cosmos mission conclude that the utricular otolith is increased in volume, whereas the saccular otolith maintains normal size, suggesting that at least in the utricle, the weight of the otolith might be regulated.

Acceleration↗

PubMed1994

Ultrastructure of the olfactory organ of the newt, Cynops pyrrhogaster.

The ultrastructure of the nasal sacs of the Japanese newt, Cynops pyrrhogaster, was studied by scanning and transmission electron microscopy. The paired nasal sacs of the newt are dorsoventrally flattened with a lateral nasal sinus off the main cavity of each sac. Throughout each sac is a series of ridges and grooves. In the main cavity, sensory epithelium with ciliated and microvillous receptor cells lines the grooves, and a thin, ciliated non-sensory epithelium lines the ridges. Secretory glands are present in the lamina propria. In the lateral nasal sinus, the ridges are lined with a thick, non-ciliated sensory epithelium that lacks glands. This region resembles and may function as a primitive vomeronasal organ.

Animals↗

PubMed1994

Control of cell differentiation and morphogenesis in amphibian development.

We reviewed cell differentiation and morphogenesis by mesoderm-inducing factors during amphibian embryogenesis. Recently, two kinds of growth factors, activin and FGF, have been identified as influential candidates for natural mesoderm-inducing factor in amphibian development. These factors are present in early Xenopus embryos. In particular, activin has been shown to induce many kinds of mesodermal tissues in a dose-dependent manner. Activin-treated ectodermal sheet (animal cap) acts as an organizer causing gene expression, mesoderm formation and functional events such as secondary axis formation. Follistatin, an activin-specific binding protein, also present in the early Xenopus embryo, makes a complex with activin. Follistatin protein exerts no inducing activity of Xenopus animal cap. Endogenous follistatin may, however, play the role of an activin regulation factor. Endogenous actions of activin and FGF were studied using injection of their receptor mRNAs. Disruption of the FGF signaling pathway by its non-functional dominant negative receptors produced trunk and tail defects. In the case of activin, an embryo cannot form axial structures. Animal-half blastomeres from the late 8-cell stage Xenopus embryo respond to activin, and there are prepatterns in ventral and dorsal cells from very early stages. The timing of mesoderm induction during development and the relationship between the inducing factors and competent cells are discussed in this report. Differentiation of tissues and organized formation of organs can be understood as a system of serial inductive reactions originating from the organizer. We have attempted to construct a model of organizer formation based on the results of recent studies.

Activins↗

PubMed1993

Isolation and characterization of Xenopus follistatin and activins.

Xenopus follistatin and activins were purified from a Xenopus laevis cell line (XTC-F1) by four purification steps consisting of consecutive affinity chromatography on dextran sulfate-Sepharose and Sulfate Cellulofine, fast protein liquid chromatography gel permeation, and reverse-phase high performance liquid chromatography (HPLC). Our results thus obtained indicated that almost equimolar amounts of activins A, AB, and B were found to be present as a complex with follistatin (activin-binding protein) in the conditioned medium of XTC-F1 cells. Reverse-phase HPLC of the complex gave Xenopus follistatin and activins A, AB, and B. The purified Xenopus follistatin showed four major bands in a molecular mass range from 34 to 39 kDa by SDS-polyacrylamide gel electrophoresis under nonreducing conditions. The ability of each form of the protein to specifically bind activin was determined by activin-binding assay and ligand blotting analysis. Each protein was found to have the same NH2-terminus and its sequence was very homologous to that of mammalian follistatin. Several criteria including immunoblotting analysis and various functional assays revealed the existence of three isoforms of activins A, AB, and B in Xenopus, as in mammals. Xenopus activins significantly induced both ventral and dorsal mesoderm in explants of Xenopus blastula cells that would otherwise form epidermis. In a dose-dependent manner of each isoform of activin, the induced explants were able to differentiate into blood-like cells, coelomic epithelium, mesenchyme, muscle, and notochord. The induction patterns of three Xenopus activins were essentially the same. The mesoderm induction by the purified Xenopus activins was shown to be inhibited stoichiometrically by the purified Xenopus follistatin. These results indicate that Xenopus XTC-F1 cells secrete several molecular forms of follistatin/activin-binding protein and three isoforms of activins AB and B in addition to activin A.

Activins↗

PubMed1993

Competence prepattern in the animal hemisphere of the 8-cell-stage Xenopus embryo.

Activins are known to be potentially important regulators in Xenopus developmental processes. It has been shown that activins exist maternally in the egg and can induce mesodermal tissues in blastula animal cap explants. However, the blastula ectoderm is known to possess a predisposed local response pattern to activin, and the process of the prepatterning is not understood. We isolated animal hemispheres from late 8-cell-stage embryos and treated them briefly with activin A. Expression of the muscle-specific actin gene was induced after a 30-min activin treatment, even when it was followed by treatment with follistatin, an activin-specific binding protein. This suggests that the animal-half blastomeres become competent to activin A before the 16-cell stage. In the normal embryo, the 8-cell-stage animal dorsal blastomeres populate neural ectoderm and most of the dorsal lip of the gastrula blastopore, the region of Spemann's organizer, and are the major progenitor for dorsal mesodermal tissues. When the dorsal and ventral animal-half blastomeres of the 8-cell stage were isolated and treated with activin independently, significant differences in tissue differentiation were observed. Dorsal blastomeres gave rise to trunk and tail structures containing dorsal mesoderm, whereas the ventral blastomere explants formed spheres containing solely ventral mesoderm. Further, both muscle actin transcription and goosecoid transcription were induced primarily in dorsal blastomeres. Our results suggest that a competence prepattern of response to activin exists as early as the 8-cell stage.

Activins↗

PubMed1993

Expression of GTP-binding protein gene drg during Xenopus laevis development.

To study the genes which may play a role in the development of the vertebrate central nervous system (CNS) using a subtraction cloning approach, we previously identified a set of novel genes which are predominantly expressed in the mouse embryonic CNS and down-regulated during development. One of these genes, drg, encodes a novel 41 kilodalton GTP-binding protein (DRG), which is highly expressed in the embryonic CNS and shows remarkable evolutionary conservation. To study the biological role of this protein during Xenopus embryonic development, we cloned the Xenopus drg cDNA (Xdrg). The predicted Xenopus DRG protein (XDRG) is more than 95% identical to the mouse DRG. Analysis of Xdrg expression by Northern blots, whole-mount in situ hybridization and RNA-PCR revealed the presence of varying levels of transcript for this gene in embryos and adult tissues. Among the three mRNA species detected by Northern hybridization, two smaller ones show temporally regulated expression patterns during embryonic development.

Amino Acid Sequence↗