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PubMed2005

Plant lectins as carriers for oral drugs: is wheat germ agglutinin a suitable candidate?

Wheat germ agglutinin (WGA) is a plant protein that binds specifically to sugars expressed also by gastrointestinal epithelial cells. WGA is currently investigated as an anti-tumor drug and as a carrier for oral drugs. Information on whether it can cross the gastrointestinal epithelium and on its possible effects on the integrity of the epithelial layer is however scanty or lacking, and herein we address these issues. Differentiated Caco2 cells have been used as a model of polarized intestinal epithelium. WGA concentration at both the apical and the basolateral side of the epithelium has been quantified using a sensitive ELISA assay (sensitivity threshold 0.84 nM). Trans epithelial electrical resistance (TEER) has been measured to evaluate the integrity of the epithelium upon treatments with WGA. (3)H-Mannitol (182.2 Da) and FITC-dextran (3000 Da) have been used to measure the permeability of the epithelium. Cell viability has been measured by the MTT, by 7-AAD uptake, and Annexin-V binding assays. Up to a concentration of 5.6 microM, approximately 0.1% of intact WGA molecules only could cross the epithelial layer. WGA perturbed the integrity of the epithelium and increased the permeability of the tissue in a dose- and time-dependent manner. WGA did not induce cell death but increased the permeability of individual cells to 7-AAD which is normally not uptaken by viable cells. These data allowed us to define a toxicity threshold for WGA on epithelial cells. WGA suitability as a carrier for oral drugs can therefore be evaluated on a rational basis.

Caco-2 Cells↗
PubMed1978

Investigation of the effect of hydroxyurea on the cell cycle and the development of necrosis in the embryonic CNS of mice.

On day 10 of gestation pregnant mice (strain NMRI) were given an intravenous injection of 500 mg/kg Hydroxyurea (HU). Simultaneously either 5 muCi/g or 10 muCi/g 3H-thymidine (3H-Tdr; specific activity 5Ci/mmol) was administered to the animals. At various times after treatment embryos and electron microscopy. Two hours after administration of HU condensations of the chromatin structure could be detected electron microscopically in some cells. Thirty minutes later the nucleolus became smaller and denser, the cytoplasm shrank, and the cell organelles moved closer together. Three hours after application of the drug break-down of the involved cells set in. As in the autoradiograms about 98% of the counted necroses were labelled, and since labelled thymidine is almost exclusively incorporated during the S-phase, it can be stated the HU influences only metabolic processes which take place during the S-phase. From the morphological findings it can be concluded that in the case of the S-phase-specific metabolic pathway, which is influenced by HU, we are primarily dealing with DNA synthesis.

Animals↗
PubMed1978

[Effect of neuropharmacologic preparations and colchicine on morphogenetic processes in embryonal amphibian cells].

The effect of antagonists of biogenous amines (antitransmitters--AT) and colchicine on rapid morphogenetic processes in the explants of embryonic ectoderm with underlying mesoderm cut from the lateral region of Rana temporaria embryos at the late neurula-early taibud stages was studied. The normal morphogenesis of the explants consists of two successive phases: phase of contact polarization and phase of cell movement into the fragment. The high concentrations of AT inhibited completely the morphogenesis of explants but somewhat lower concentrations inhibited the second phase of morphogenesis and not only did not prevent the cell polarization but even assisted its propagation over all the fragment. The inhibiting effect of AT was relieved by 5-hydroxytryptamine which per se stimulated the morphogenesis of explants. Thus, AT exert a specific inhibition of the motility of embryonic cells but do not prevent the contact interactions responsible for cell polarization within every layer.

Amphetamines↗
PubMed1993

The murine steel panda mutation affects kit ligand expression and growth of early ovarian follicles.

Mutations at the murine steel (Sl) locus encoding the ligand for the c-kit receptor result in defects in gametogenesis, hematopoiesis, and melanogenesis. Steel Panda (Slpan) is an allele at the Sl locus obtained by an X-ray mutagenesis protocol. Slpan/Slpan homozygotes are mildly anemic black-eyed whites with pigmented ears and scrotum; females are sterile while males are fertile. To investigate the basis of the phenotype of the Slpan mutation, the coding region of the kit ligand (KL) in Slpan/Slpan animals was characterized and shown to be identical to that from +/+ mice. RNA expression patterns in adult Slpan/Slpan mice were investigated by RNA blot analysis and RNase protection assays. KL RNA expression was shown to be reduced in several tissues including testis, lung, and kidney, to about 60% in heterozygotes and 20% in homozygous mutant mice. Intermediate effects were seen in cerebellum and spleen, while in heart and brain no change was apparent. Therefore, the Slpan mutation affects KL RNA levels in a tissue-specific manner. Histological analysis showed that the number of oocytes in neonatal homozygotes was reduced to 20% of that in heterozygotes, and that in juvenile and adult mice ovarian follicle development was arrested at the one-layered cuboidal stage, with a few exceptions. KL production by central cords of the perinatal ovary was severely reduced as shown by immunohistochemistry. In neonatal testes of homozygotes, the germ cell number was reduced to 30% of that in heterozygotes, but meiotic spermatocytes were produced on schedule in juvenile animals. Therefore, a reduced level of KL in Slpan/Slpan ovary arrests ovarian follicle development, while a similar reduction in testes has relatively little effect on spermatogonial development.

Aging↗
PubMed2002

Embryonic stem cell lines of nonhuman primates.

Human embryonic stem (ES) cell lines have opened great potential and expectation for cell therapy and regenerative medicine. Monkey and human ES cell lines, which are very similar to each other, have been established from monkey blastocysts and surplus human blastocysts from fertility clinics. Nonhuman primate ES cell lines provide important research tools for basic and applicative research. Firstly, they provide wider aspects of investigation of the regulative mechanisms of stem cells and cell differentiation among primate species. Secondly, their usage does not need clearance or permission from the regulative rules in many countries that are associated with the ethical aspects of human ES cells, although human and nonhuman embryos and fetuses are very similar to each other. Lastly and most importantly, they are indispensable for animal models of cell therapy to test effectiveness, safety, and immunological reaction of the allogenic transplantation in a setting similar to the treatment of human diseases. So far, ES cell lines have been established from rhesus monkey (Macaca mulatta), common marmoset (Callithrix jacchus), and cynomolgus monkey (Macaca fascicularis), using blastocysts produced naturally or by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). These cell lines seem to have very similar characteristics. They express alkaline phosphatase activity and stage-specific embryonic antigen (SSEA)-4 and, in most cases, SSEA-3. Their pluripotency was confirmed by the formation of embryoid bodies and differentiation into various cell types in culture and also by the formation of teratomas that contained many types of differentiated tissues including derivatives of three germ layers after transplantation into the severe combined immunodeficiency (SCID) mice. The noneffectiveness of the leukemia inhibitory factor (LIF) signal makes culture of primate and human ES cell lines prone to undergo spontaneous differentiation and thus it is difficult to maintain these stem cell colonies. Also, these ES cells are more susceptible to various stresses, causing difficulty with subculturing using enzymatic treatment and cloning from single cells. However, with various improvements in culture methods, it is now possible to maintain stable colonies of monkey ES cells using a serum-free medium and subculturing with trypsin treatment. Under such conditions, cynomolgus monkey ES cell lines can be maintained in an undifferentiated state with a normal karyotype and pluripotency even after prolonged periods of culture over 1 year. Such progress should facilitate many aspects of stem cell research using both nonhuman primate and human ES cell lines.

Animals↗
PubMed1993

Postimplantation expression patterns indicate a role for the mouse forkhead/HNF-3 alpha, beta and gamma genes in determination of the definitive endoderm, chordamesoderm and neuroectoderm.

The HNF-3 alpha, beta and gamma genes constitute a family of transcription factors that are required for hepatocyte-specific gene expression of a number of genes, e.g. transthyretin, alpha-1 antitrypsin and tyrosine aminotransferase. These genes share a highly conserved DNA-binding domain first found in the Drosophila gene, forkhead, which is required for the normal patterning of the developing gut and central nervous system in Drosophila. In adult mouse tissues, transcripts from HNF-3 alpha and beta have been localised to the liver, intestine and lung, whereas HNF-3 gamma is found in the liver, intestine and testis. In light of the early developmental significance of forkhead in Drosophila, we have compared the patterns of expression of HNF-3 alpha, beta and gamma mRNAs during murine embryogenesis. We find that these genes are sequentially activated during development in the definitive endoderm. HNF-3 beta mRNA is expressed in the node at the anterior end of the primitive streak in all three germ layers and is the first gene of this family to be activated. Subsequently, HNF-3 alpha is transcribed in the primitive endoderm in the region of the invaginating foregut and HNF-3 gamma appears upon hindgut differentiation. These genes have different anterior boundaries of mRNA expression in the developing endoderm and transcripts are found in all endoderm-derived structures that differentiate posterior to this boundary. Therefore, we propose that these genes define regionalization within the definitive endoderm. Furthermore, differential mRNA expression of HNF-3 alpha and beta is detected in cells of the ventral neural epithelium, chordamesoderm and notochord. In the neural epithelium, expression of HNF-3 alpha and beta mRNA becomes localised to cells of the floor plate. We propose that, in addition to their characterised requirement for liver-specific gene expression, HNF-3 alpha and beta are required for mesoderm and neural axis formation. We also conclude that HNF-3 beta is the true orthologue of the Drosophila forkhead gene.

Animals↗
PubMed1997

The homeobox gene PV.1 mediates specification of the prospective neural ectoderm in Xenopus embryos.

Bone morphogenetic protein 4 (BMP4), a member of the TGF beta superfamily, has been implicated in the dorsoventral specification of both mesoderm and ectoderm. High levels of BMP4 signaling appear to specify ventral lineages, while lower levels are causally associated with the development of dorsal lineages. We have previously identified a homeobox-containing transcription factor (PV. 1) which is a likely mediator of the ventralizing effects of BMP4 in the mesoderm. Here we provide evidence that PV.1 also functions downstream of BMP4 in the patterning of ectoderm, specifying epidermal and suppressing neural gene expression. PV.1 is expressed in the prospective neuroectoderm at the time of ectodermal fate determination. BMP4 and xSmad1 (a downstream effector of BMP4) induce PV.1 in uncommitted ectoderm and the dominant negative form of the BMP4 receptor (DN-BR) blocks PV.1 expression. In animal pole explants PV.1 counteracts the neuralizing effects of chordin and the DN-BR and restores them to their original epidermal fate. To address the physiological significance of these observations we employed an animal cap transplantation system and demonstrated that overexpression of PV.1 in the prospective neuroectoderm specifically blocks neurogenesis in intact embryos. Thus, PV.1 plays an important role in the ventralization of both mesoderm and ectoderm. We have previously shown that PV.1 is also preferentially expressed in the ventral endoderm, suggesting that this transcription factor may be involved in the ventralization of all three germ layers.

Amino Acid Sequence↗
PubMed1993

The early development of major projections from caudal levels of the spinal cord to the brainstem and cerebellum in the gray short-tailed Brazilian opossum, Monodelphis domestica.

The Brazilian short-tailed opossum, Monodelphis domestica, is born 14-15 days after copulation and is available for experimentation at stages of development corresponding to those which occur in utero in placental mammals. In the present study, we took advantage of the opossum's embryology to study the development of projections from caudal levels of the spinal cord to the brainstem and cerebellum using axonal tracing methods. In all cases, a 2-3 day survival time was used for axonal transport. When injections of Fast blue (FB) were made into caudal levels of the thoracic cord at postnatal day (PD) 1 or 2, axonal labeling could not be identified at supraspinal levels. When injections were made at PD3, however, labeled axons were found in the fasciculus gracilis at caudal medullary levels, within the ventrolateral medulla and pons, within an incipient inferior cerebellar peduncle, and within the cerebellar anlage. The dorsal root origin of at least some of the axons within the fasciculus gracilis was evidenced by the transganglionic transport of cholera toxin conjugated to horseradish peroxidase from the hindlimbs. After FB injections at PD7, a few labeled axons could be traced from the fasciculus gracilis into the nucleus gracilis and from the ventrolateral pathway to the inferior olive. Generally comparable results were obtained using wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). In cases injected with FB at PD9, the pattern of brainstem labeling was adult-like. Although labeled axons were present within the cerebellum of animals injected with FB on PD3, they were limited to the marginal zone. Axonal labeling was present within an identifiable internal granular layer in cases injected with either FB or WGA-HRP at PD16, and it appeared to be limited to specific bands which foreshadowed those seen at later stages of development and in the adult animal. In some cases, labeled axons were present within the molecular layer where they were not seen in the adult animal. Our results provide a timetable for the normal development of projections from caudal levels of the spinal cord to the brainstem and cerebellum in Monodelphis and show that such development occurs postnatally rather than prenatally, as in placental mammals.

Aging↗
PubMed1985

Protein synthetic patterns of tissues in the early chick embryo.

Tissues dissected from early chick embryos were labelled in vitro with [35S]methionine, and their patterns of polypeptide synthesis investigated using the technique of two-dimensional (2-D) polyacrylamide gel electrophoresis. Apart from providing a preliminary description of the molecular changes associated with the processes of gastrulation and segmentation in the chick embryo, this study has revealed a number of polypeptides that may be useful as markers of cell type or function. The protein synthetic patterns of hypoblast from early and late gastrulae (stages 2 and 4, respectively: Hamburger & Hamilton, 1951) and of definitive endoblast and junctional endoblast from late gastrulae all resemble one another closely, but differ markedly from that of the epiblast at either stage. The lower layer tissues are characterized by the presence of eleven polypeptides that are largely absent from the epiblast. These findings are discussed with reference to current theories on the origins of the lower layer tissues. Comparisons between the 2-D patterns for tissues dissected from gastrulae and from embryos undergoing segmentation (stage 12) have revealed ten polypeptides showing stage-specific rather than tissue-specific expression. Apart from these ten polypeptides, the 2-D patterns for epiblast and ectoderm were practically identical, and distinguishable from those of other tissues by a lack of any unique polypeptides. On the other hand, stage-4 endoblast and stage-12 endoderm differed in the expression of many polypeptides. One polypeptide was found that may be considered as a marker of mesodermal cell type, as it was present in lateral plate, segmental plate and somitic mesoderm, but not in tissues of the other germ layers. Lateral plate could be distinguished from the other mesodermal tissues in the expression of a number of polypeptides, but the similarity in the 2-D patterns for segmental plate and somites suggest that the separation of somites from the anterior end of the segmental plate is not accompanied by the synthesis of new polypeptides.

Animals↗
PubMed2001

Cell autonomous commitment to an endodermal fate and behaviour by activation of Nodal signalling.

In vertebrates the endoderm germ layer gives rise to most tissues of the digestive tract and controls head and heart morphogenesis. The induction of endoderm development relies on extracellular signals related to Nodals and propagated intracellularly by TGFbeta type I receptors ALK4/Taram-A. It is unclear, however, whether Nodal/ALK4/Taram-A signalling is involved only in the specification of endodermal precursors or plays a more comprehensive role in the activation of the endodermal program leading to the irreversible commitment of cells to the endodermal fate. Using cell transplantation experiments in zebrafish, we show that marginal cells become committed to endoderm at the onset of gastrulation and that commitment to endoderm can be reached by intracellular activation of the Nodal pathway induced by expression of an activated form of the taram-A receptor, Tar*. In a manner similar to endoderm progenitors, Tar*-activated blastomeres translocate from their initial site of implantation in the blastoderm to reach the surface of their migration substratum, the yolk syncitial layer, where they join endogenous endodermal derivatives during gastrulation and differentiate according to their anteroposterior position. We demonstrate that Nodal/Tar*-induced commitment does not rely on a secondary signal released by Tar*-expressing cells or a signal released by endogenous endoderm since Tar*-expressing wild-type cells can restore endoderm derivatives when transplanted into the endoderm-deficient mutant casanova. Likewise, the YSL does not appear essential for the maintenance of endodermal identity during gastrulation once the Nodal pathway has been activated. Thus, our results demonstrate that the activation of Nodal signalling is sufficient to commit cells both to an endodermal fate and behaviour. Wild-type endoderm implantation into casanova embryos rescues, in a non-autonomous fashion, the defective fusion of the two heart primordia in the midline, highlighting the importance of endoderm for normal heart morphogenesis.

Animals↗
PubMed2004

[Stem cell research in gastroenterology].

Stem cells are undifferentiated cells capable of undergoing self-renewal and differentiation into a variety of cell types. They are derived from adult tissues (adult stem cells) as well as embryonal blastocysts (embryonic stem cells). Embryonic stem cells have pleuripotent capacity able to form tissues of all three germ layers but many ethical controversies concerning resource allocation or methods of harvesting are arising. Recently, many studies have demonstrated the multipotency of adult stem cells, but the mechanism of the plasticity remains to be determined yet. Several studies have suggested the possibilities of application of stem cells or tissue specific cells to regenerate gastroenterologic diseases such as liver cirrhosis, hepatitis, or inherited metabolic disorders. However, most of those trials are still limited to animal models, although anecdotal claims of successful therapy in humans have been reported. Even though the expectations and the promise of cell therapy are high, clinical efficacy has not been definitely demonstrated at this time. Thus, the application of cell therapy cannot be recommended to the patients outside the clinical trial setting.

Gastrointestinal Diseases↗
PubMed2006

Maternal expression of a NANOS homolog is required for early development of the leech Helobdella robusta.

The gene nanos (nos) is a maternal posterior group gene required for normal development of abdominal segments and the germ line in Drosophila. Expression of nos-related genes is associated with the germ line in a broad variety of other taxa, including the leech Helobdella robusta, where zygotically expressed Hro-nos appears to be associated with primordial germ cells. The function of maternally inherited Hro-nos transcripts remains to be determined, however. Here, the function of maternal Hro-nos is examined using an antisense morpholino (MO) knockdown strategy, as confirmed by immunostaining and western blot analysis. HRO-NOS knockdown embryos exhibit abnormalities in the distribution of micromeres during cleavage. Subsequently, their germinal bands are positioned abnormally with respect to the embryonic midline and the micromere cap, epiboly fails, and the HRO-NOS knockdown embryos die. This lethality can be rescued by injection of mRNA encoding an eGFP::HRO-NOS fusion protein. HRO-NOS knockdown embryos make their normal complements of mesodermal and ectodermal teloblasts, and the progeny of these teloblasts segregate into distinct mesodermal and ectodermal layers. These results suggest that maternal Hro-nos is required for embryonic development. However, contrary to previous suggestions, maternal inherited Hro-nos does not appear necessary for ectoderm specification.

Animals↗
PubMed1991

Changes in glycoconjugate expression during early chick embryo development: a lectin-binding study.

A selection of lectins was used to investigate developmentally regulated changes in the distribution of cell surface oligosaccharides during the gastrulation and neurulation stages of early chick embryo development. Lectins from three specificity classes were used: glucose/mannose specificity (concanavalin A [Con A], Lens culinaris agglutinin [LCA], Pisum sativum agglutinin [PSA]); N-acetylglucosamine specificity (Lycopersicon esculentum agglutinin [LEA], wheat germ agglutinin [WGA], succinylated WGA [sWGA]); N-acetylgalactosamine/galactose specificity (Dolichos biflorus agglutinin [DBA], soybean agglutinin [SBA], Sophora japonica agglutinin [SJA], Bandeiraea (Griffonia) simplicifolia lectin I [BSL I], peanut agglutinin [PNA], Artocarpus integrifolia lectin [Jacalin], Ricinus communis agglutinin-1 [RCA-1], Erythrina cristagalli lectin [ECL]). At gastrulation stages, patterns of lectin binding could be distinguished in the epiblast, mesoderm, and endoderm cell layers. The primitive streak failed to bind any of the lectins, but LEA and WGA bound to the epiblast in regions lateral to the streak, indicating the loss of some glucosamine residues medially in preparation for the ingression movements of gastrulation. Several lectins showed marked binding to the mesoderm cells after their passage through the primitive streak; these were LCA, PSA, WGA, sWGA, BSL, and most particularly PNA. Therefore, the epithelial-mesenchymal transformation from epiblast to mesoderm at the primitive streak is accompanied by cell surface oligosaccharide changes in the epiblast and mesoderm that involve all classes of lectins including the PNA-binding sequence Gal beta 1-3GalNAc. Ultrastructurally, PNA was shown to bind extracellularly to matrix fibrils. Jacalin, having the same sugar specificity as PNA, but binding to serine/threonine linked chains rather than asparagine linked chains showed no binding to the mesoderm.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Reduction of gap junctional conductance by microinjection of antibodies against the 27-kDa liver gap junction polypeptide.

Antibody raised against isolated rat liver gap junctions was microinjected into coupled cells in culture to assess its influence on gap junctional conductance. A rapid inhibition of fluorescent dye transfer and electrical coupling was produced in pairs of freshly dissociated adult rat hepatocytes and myocardial cells as well as in pairs of superior cervical ganglion neurons from neonatal rats cultured under conditions in which electrotonic synapses form. The antibodies have been shown by indirect immunofluorescence to bind to punctate regions of the plasma membrane in liver. By immunoreplica analysis of rat liver homogenates, plasma membranes, and isolated gap junctions resolved on NaDodSO4/polyacrylamide gels, binding was shown to be specific for the 27-kDa major polypeptide of gap junctions. This and similar antibodies should provide a tool for further investigation of the role of cell-cell communication mediated by gap junctions and indicate that immunologically similar polypeptides comprise gap junctions in adult mammalian cells derived from all three germ layers.

Animals↗
PubMed1980

Isolation and genetic characterization of cell-lineage mutants of the nematode Caenorhabditis elegans.

Twenty-four mutants that alter the normally invariant post-embryonic cell lineages of the nematode Caenorhabditis elegans have been isolated and genetically characterized. In some of these mutants, cell divisions fail that occur in wild-type animals; in other mutants, cells divide that do not normally do so. The mutants differ in the specificities of their defects, so that it is possible to identify mutations that affect some cell lineages but not others. These mutants define 14 complementation groups, which have been mapped. The abnormal phenotype of most of the cell-lineage mutants results from a single recessive mutation; however, the excessive cell divisions characteristic of one strain, CB1322, require the presence of two unlinked recessive mutations. All 24 cell-lineage mutants display incomplete penetrance and/or variable expressivity. Three of the mutants are suppressed by pleiotropic suppressors believed to be specific for null alleles, suggesting that their phenotypes result from the complete absence of gene activity.

Animals↗
PubMed1975

[Action of microbial flora of the digestive tract on the metabolism of bile acids in the rat (author's transl)].

An isotopic balance is established in rats receiving a regular feed intake of [4-14-C]cholesterol so that various chemical species of bile acids have the same specific activity. This property is used to study bile acids distribution in the rat liver, digestive tract and fecal excretion. Bile acids are separated by thin-layer chromatography, radioactivity is determined by liquid scintillation, and the mass by 3-hydroxysteroid dehydrogenase action. The resulting comparative study made between the germ-free rat (axenic rat) and the rat exposed to microbes ("holoxenic" or conventional rat) receiving a semi-synthetetic feed, shows the influence excercised on the metabolism by the microbial flora of the digestive tract. This study confirms that the axenic rat compared to its holoxenic homologue has a higher bile acids pool and a lower fecal excretion. At all levels of the digestive tract (small intestine and the whole caecum and large intestine), probably as well as in the liver, the total amount of bile acids which is observed in the axenic rat is about twice the amount observed in the holoxenic rat, but fecal excretion is decreased by 20%. Values obtained by this method are higher than those previously observed by other authors using gas-liquid chromatography or [14-C]cholic acid isotopic dilution. This study also confirms that cholic and beta-muricholic acids are the main bile acids in the axenic rat and in addition establishes that in this animal bile acids composition is complex and varies from the small intestine to the feces. Besides cholic, alpha- and beta-muricholic, chenodeoxycholic and ursodeoxycholic acids, unidentified chemical species constitute 21% of the whole in the feces. Comparing the compositions observed in axenic and holoxenic rats in this experiment, it could not be determined if the relative activity of the two pathways of bile acid biosynthesis is deeply or only slightly changed by the presence of microbial flora. This is because of a large fraction of unknown composants in the feces of the axenic rat and the extreme complexity in the feces of the holoxenic rat.

Animals↗
PubMed2005

An experimental study on astrocytes promoting production of neural stem cells derived from mouse embryonic stem cells.

BACKGROUND: The production of neural stem cells (NSCs) derived from embryonic stem (ES) cells was usually very low according to previous studies, which was a major obstacle for meeting the needs of clinical application. This study aimed at investigating whether astrocytes could promote production of NSCs derived from ES cells in vitro. METHODS: Mouse ES cells line-D3 was used to differentiate into NSCs with astrocytes as inducing stromal cells by means of three-stage differentiation procedure. Another group without astrocytes served as control. The totipotency of ES cells was identified by observation of cells' morphology and formation of teratoma in severe combined immunodeficiency disease (SCID) mice. The quantity and purity of NSCs derived from ES cells were analyzed using clonogenic assay, immunohistochemical staining and flow cytometry assay. The plasticity of NSCs was detected by differentiating test. Octamer-binding transcription factor 4 (Oct-4) and nestin, the specific marker genes of ES cells and NSCs respectively, were detected continuously using reverse transcription-polymerase chain reaction (RT-PCR) method to monitor the process of cell differentiation. RESULTS: The ES cells of D3 line could maintain the ability of differentiating into cellular derivations of all three primary germ layers after continuous passage culture. At the end of two-stage of inducing process, 23.2 +/- 3.5 neurospheres per plate formed in astrocyte-induced group and only 0.8 +/- 0.3 per plate in the control group (clonogenic assay, P < 0.01), and the ratio of nestin positive cells was (50.2 +/- 2.8)% in astrocyte-induced group and only (1.4 +/- 0.5)% in the control group (flow cytometry, P < 0.01). With the induction undergoing, the expression of Oct-4 gradually decreased and then disappeared, while the expression of nestin was increased step by step, and the ratio of nestin positive cells was up to 91.4% by the three-stage differentiation. The nestin positive cells could be further induced into neurons, astrocytes, and oligodendrocytes in differentiating medium supplemented with fetal calf serum. The results of differentiating test showed that the ratio of NF-200 and NSE positive cells was (42.7 +/- 2.6)% in astrocyte-induced group and only (11.2 +/- 1.8)% in the control group (P < 0.01). CONCLUSIONS: Astrocytes can not only increase the production of NSCs derived from ES cells but also promote the differentiation of NSCs toward neuronal lineage.

Animals↗
PubMed2000

The role of the yolk syncytial layer in germ layer patterning in zebrafish.

Formation of the three germ layers requires a series of inductive events during early embryogenesis. Studies in zebrafish indicate that the source of these inductive signals may be the extra-embryonic yolk syncytial layer (YSL). The characterization of genes encoding the nodal-related factor, Squint, and homeodomain protein, Bozozok, both of which are expressed in the YSL, suggested that the YSL has a role in mesendoderm induction. However, these genes, and a second nodal-related factor, cyclops, are also expressed in the overlying marginal blastomeres, raising the possibility that the marginal blastomeres can induce mesendodermal genes independently of the YSL. We have developed a novel technique to study signaling from the YSL in which we specifically eliminate RNAs in the YSL, thus addressing the in vivo requirement of RNA-derived signals from this region in mesendoderm induction. We show that injection of RNase into the yolk cell after the 1K cell stage (3 hours) effectively eliminates YSL transcripts without affecting ubiquitously expressed genes in the blastoderm. We also present data that indicate the stability of existing proteins in the YSL is unaffected by RNase injection. Using this technique, we show that RNA in the YSL is required for the formation of ventrolateral mesendoderm and induction of the nodal-related genes in the ventrolateral marginal blastomeres, revealing the presence of an unidentified inducing signal released from the YSL. We also demonstrate that the dorsal mesoderm can be induced independently of signals from the YSL and present evidence that this is due to the stabilization of (&bgr;)-catenin in the dorsal marginal blastomeres. Our results demonstrate that germ layer formation and patterning in zebrafish uses a combination of YSL-dependent and -independent inductive events.

Animals↗