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Biomedical subjects

N Zagris

Publications and source records attributed to N Zagris.

At least 19 recordsLinked to original sources

Extracellular matrix in development of the early embryo.

The extracellular matrix interacts with cells and promotes and regulates cellular functions such as adhesion, migration, proliferation, differentiation, and morphogenesis. Extracellular molecules are linked to one another by multiple binding domains and form a stable, multifunctional matrix. Cells respond to the extracellular matrix through plasma membrane receptors, which include integrin and non-integrin receptors. The regulation of these interactions requires the coordination of a multiplicity of signals both spatially and temporally.

Animals↗

Differential expression of laminin genes in early chick embryo.

The expression patterns of laminin alpha1 and beta1 genes were examined by In situ hybridization of their mRNAs in the early chick embryo from the blastoderm at stage X (morula) to the 10-somite stage. The laminin alpha1 and beta1 transcripts were found in abundance in the chick blastoderm at stage X before initiation of synthesis of the protein. Laminin polypeptides were detected shortly thereafter in embryos at stage XIII (blastula). The expression of the laminin transcripts was intense in the epiblast and in the hypoblast of embryos at stage XIII. During gastrulation (stage HH3-4), the laminin (alpha1 and beta1 cRNAs gave strong signals in the cells ingressing through the primitive streak, in the migrating mesenchymal cells and the cells of the lower layer. The expression of laminin alpha1 and beta1 genes was restricted to specific cell populations later in development. At the neurula stage (stage HH5-6), the expression of laminin transcripts was low in epithelial ectoderm and strong in chordamesoderm neural ectoderm and may implicate important developmental roles for laminin in the morphogenetic movements of neural plate bending during primary neurulation. At the 10-somite stage (stage HH10-11), the (alpha1 and beta1 cRNAs gave no signals in the neural tube, notochord, and ectoderm. The alpha1 and beta1 cRNAs gave strong signals in neural crest cells and this may indicate that the neural crest cells can produce laminin. The alpha1 cRNAs gave strong signals in the dermamyotome and no signal in the sclerotome of somites, and intense signals in the pronephric tubules. The laminin expression pattern in somites may show transient expression of the alpha1 and/or expression of a distinct alpha1 isoform in the sclerotome. The selective expression of laminin alpha1 and beta1 subunits which shows a developmentally regulated, tissue specific distribution suggests potential roles for different members of the same subfamily of genes in the developing chick embryo.

Animals↗

Activation of embryonic genome in chick.

The earliest stages of development in most animals are under the control of maternally inherited information. The initiation of embryonic gene expression has been reported at the mid-blastula in amphibians and the mid-2-cell stage to the early morula in mammals. In chick embryos, embryonic gene expression was detectable at stage X (morula) and showed marked activation at stage XIII (blastula) with a gradual increase thereafter. Synthesis of rRNA and tRNA was low at stage X and was already the major class of RNA at stage XIII in chick embryos. The observed upregulation of RNA synthesis seems to coincide with a period of extensive fine structural differentiation when the first major cellular migrations start and signal the formation of the primitive streak in the chick embryo.

Animals↗

5-Azacytidine changes gene expression and causes developmental arrest of early chick embryo.

Methylation of DNA appears to be an important maintenance mechanism for inhibiting gene expression during development in vertebrates. 5-azacytidine (5-azaC) is used as an experimental tool for demethylation and it induces differentiation in various systems. In the chick embryo, the first cellular migrations signal the onset of primitive streak and gastrula formation and result in neural induction and morphogenesis of the embryonic axis. In the present work with the early chick embryo, 5-azaC perturbs normal cellular migrations and the embryos produce an atypical short, thickened primitive streak. These embryos have the tendency to form neural tissue but the embryonic axis shows sparse identity of patterning along its length. A small percentage of embryos display formation of double embryonic axes. Blastula embryos show reduced expression of some polypeptides and express characteristic polypeptides which are not present in morula embryos normally. Under the influence of 5-azaC, blastula embryos expressed all the polypeptides which are characteristic of embryos at both the morula and the blastula stages. If 5-azaC perturbs DNA methylation as the chick embryo develops from the histologically simple blastula, then the wave of methylation which has been reported to start at the late blastula and continues during postgastrulation in vertebrate embryos does not seem to be important for the induction of mesodermal and of neural tissues, but is important for the patterning of these tissues.

Animals↗

Appearance and distribution of entactin in the early chick embryo.

Entactin is a sulfated glycoprotein of basement membranes and recent data indicate that it may play a major role in extracellular matrix (ECM) assembly and in modulating the activities of the other molecular components. We investigated the time of appearance and subsequent distribution of entactin during the earliest stages of morphogenesis and its involvement in the first major cellular migrations and interactions in the chick embryo. Entactin is first detected in the epiblast and in the hypoblast at the blastula stage. The accumulating ECM displays intense presence of entactin in the space between the epiblast and the hypoblast at late blastula. Entactin is increasingly abundant in the neural plate and in the ECM and also at least transiently in many mesodermal tissues such as the notochord, the developing heart and somites in the early chick embryo. Immunogold labeling revealed a punctate pattern of entactin distribution in the ECM during the gastrula, neurula and at later stages and at all levels within the embryo. Because of its early appearance in more than one germ layer, entactin may be important in the formation of most embryonic structures. Entactin is detected at the same developmental time and co-localizes with laminin. Antibodies to entactin do not interfere with triggering of the first major cell movements but perturb directional migration of these cells. It would seem that entactin plays a functional role in the directed migration of cells and does not seem to affect cell adhesion during the period of the first morphogenetic events in the early chick embryo.

Animals↗

N-glycosylated proteins interfere with the first cellular migrations in early chick embryo.

Cell adhesion and migration properties which are known to play a crucial role in developmental events seem to be modulated by variations in glycosylation of glycoproteins. In the chick embryo, the extracellular matrix (ECM) appears as a loose meshwork of fibrillar material in the space between the epiblast and the hypoblast shortly before the first major cell migrations start. Chick embryos treated with tunicamycin (TN), a specific inhibitor of N-linked glycosylation of proteins, show little or no ECM, diminished cell adhesion and a dramatic alteration in the architecture of the epiblast and of the hypoblast. The first major cell migrations which signal the onset of PS and gastrula formation are inhibited irreversibly in these embryos. Tunicamycin induces a substantial change in the labeling pattern with change in mobility of some polypeptides and with the induction or marked accentuation of multiple charged species (isoforms) of polypeptides different from these already present in the control blastoderm. The N-linked glycosylation of protein(s) that are synthesized during the interaction of the epiblast and of the hypoblast seem to play a critical role in cell adhesion and in the morphogenetic movements of gastrulation in the early chick embryo.

Animals↗

Distribution and functional role of laminin during induction of the embryonic axis in the chick embryo.

Laminin is a major glycoprotein of basement membranes and has been shown to promote cell adhesion, and movement of various nonepithelial cells and tumour cells. Using antibodies to laminin in paraffin sections and cultured embryos, we have studied the distribution of laminin and its involvement in the first morphogenetic events, beginning with the first extensive cellular migrations and interactions that result in the induction of the primitive streak (PS) and of the neural plate in the early chick embryo. Laminin immunogold labeling was not detected in the blastoderm at stage X. At stage XIII, laminin immunoreactivity was detected at the ventral surface of the epiblast and in the entire hypoblast. The intense labeling of the hypoblast indicated that these cells are active in laminin synthesis. Extracellular matrix (ECM) started accumulating as the first embryonic spaces were forming, before the morphogenetic movements of gastrulation were initiated. Immunogold labeling revealed a punctate pattern of laminin distribution in the ECM in the blastocoele, and in the space below the neural plate. Laminin, which is a multidomain molecule known to interact with other molecules of the ECM and with the cell surface, could serve as the scaffold for highly specific contact points of migrating cells and for the folding of epithelial sheets during this time in the developing embryo. We incubated blastoderms at stages X and XIII with laminin antibodies (1:30 dilution) for 4 h, then cultured the blastoderms further in plain egg albumin. The laminin antibodies did not interfere with triggering of PS cell movements, but perturbed the normal migration pattern of these cells. A normal PS did not form and, as a consequence, the embryonic axis was not induced.(ABSTRACT TRUNCATED AT 250 WORDS)

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Stage-specific gene expression in early chick embryo.

Inhibition of DNA replication by aphidicolin in the chick morula interferes with its progression to a normal blastula and prevents induction of the first morphogenetic cell movements of primitive streak formation. Embryos in aphidicolin synthesize some polypeptides typical of blastula but do not display all the characteristic features of morula to blastula transition. Inhibition of DNA replication interferes with the sequential synthesis of maternally coded polypeptides and with the activation of the embryonic genome in the chick embryo.

Animals↗

Extracellular matrix organized in embryonic cavities during induction of the embryonic axis in chick embryo.

Extracellular matrix (ECM) is detected as short, disorganized fibrils in the forming embryonic extracellular spaces shortly prior to the first morphogenetic cellular movements and interactions in the early chick embryo. As development progresses, the ECM is organized into an intricate network spanning the embryonic cavities. This dynamic entity undergoes relatively rapid changes in its organization pattern during the developmental period from morula to the induction of the neural plate. The ECM seems to preserve the exquisite architecture of the embryo and could guide migrating cells into defined pathways in the early embryo.

Animals↗

Stage-specific hypermutability of the regA locus of Volvox, a gene regulating the germ-soma dichotomy.

Mutation at the regA locus confers on somatic cells of Volvox (which otherwise undergo programmed death) ability to redifferentiate as reproductive cells. Stable mutations at the regA locus, but not at other loci, were induced at high frequency when embryos at one particular stage were exposed to either UV irradiation, novobiocin, nalidixic acid, bleomycin, 4-hydroxyaminoquinoline-1-oxide, 5-bromodeoxyuridine, or 5-fluorouracil. All treatments led to some mutations that were not expressed until the second generation after treatment. The sensitive period was after somatic and reproductive cells of the next generation had been set apart, but before they had undergone cytodifferentiation. Hypermutability occurs in presumptive reproductive cells (in which regA is normally not expressed) somewhat before regA normally acts in somatic cells. We postulate that hypermutability of regA in the reproductive cells at this time reflects a change of state that the locus undergoes as it is inactivated.

Bleomycin↗

Induction of gastrulation in the chick embryo.

Interaction between the epiblast and the primary hypoblast in chick blastula results in induction of the primitive streak (PS) in the epiblast. Alpha-amanitin, a specific inhibitor of poly A-containing RNA synthesis, inhibits formation of the definitive PS. This inhibition is associated with qualitative changes in the pattern of protein synthesis in the hypoblast but not in the epiblast. The protein pattern of the component areas of the epiblast shows increase in some polypeptides after treatment with alpha-amanitin. By contrast, alpha-amanitin resulted in a decrease in synthesis of several polypeptides, which are either undetectable or weakly present in the hypoblast. The alpha-amanitin-sensitive translational products of the embryonic genome that are observed in the hypoblast may have specific functions in the control of PS induction and stabilization.

Amanitins↗

Deoxycytidine reverses inhibition of morphogenesis by thymidine in young chick blastoderm.

Exogenous thymidine affects morphogenesis of the early chick blastoderm possibly by depleting the deoxycytidine triphosphate pool. The aim of this study is to determine whether the inhibitory action of thymidine on early chick blastoderm morphogenesis is alleviated by the removal of thymidine and/or treatment with deoxycytidine. Chick blastoderms at the full hypoblast stage develop abnormally in egg albumen containing 1.23 X 10(-3) M thymidine. Development is normal when deoxycytidine is included simultaneously in the culture medium with thymidine at equimolar concentrations. Blastoderms were cultured in egg albumen containing 15 microCi/ml thymidine [methyl-3H] or 10 microCi/ml deoxycytidine [5-3H], and 1.2 X 10(-3) M 2'-deoxycytidine or 1.23 X 10(-3) M thymidine, respectively. The culture was interrupted at timed intervals, and the amount of radioactivity associated with DNA was determined. Exogenous deoxycytidine in the culture medium caused a noticeable increase in the incorporation of 3H-thymidine, while exogenous thymidine markedly inhibited the uptake and incorporation of 3H-deoxycytidine into DNA of blastoderms. Thymidine does not inhibit the expansion of blastoderm, the migration of cells for formation of the primitive streak (PS), and the induction of axial tissues, but it interferes with the organization of these tissues to form the embryonic axis. Blastoderms show slight signs of recovery when thymidine is removed. Deoxycytidine counteracts the action of thymidine and seems to be a rate-limiting factor in normal differentiation of the early chick blastoderm.

Animals↗

Communication between primary endoderm and mesoderm for erythroblast differentiation in early chick blastoderm.

Blood islands are mesodermal cells which, in close association with the underlying endoderm, form blood cells and endothelium in the early embryo. The primary hypoblast (primary endoderm), separated from the epiblast of chick blastoderm at the full hypoblast stage, was labeled with 14C-protein hydrolysate. The labeled hypoblast was recombined with its denuded unlabeled epiblast. Radioautographs and uptake and incorporation measurements in the blood islands show that there is transport of label from the primary hypoblast to the erythroblasts or to some progenitor cell(s) of the erythroblasts in the epiblast. The observed cell communication between mesoderm and the endoderm is in concert with the description of the blood island cytoarchitecture in which mesodermal cells are making direct contact with the adjacent endodermal cells during erythroblast differentiation.

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Differentiation of chick blood island erythroblasts into erythrocytes and stability in long-term culture.

Clusters of 20-70 erythroblasts from blood islands of early chick blastoderm were cultured in serum-free chemically defined medium for a 3-month period. The erythroblast cluster produces erythroid cells and hemoglobins characteristic of the primitive and definitive erythroid cell lines. It seems there is a progenitor erythroid cell(s) in the erythroblast cluster which starts and/or continues maturing along various pathways of hemopoietic differentiation under simple culture conditions. The erythroid character of these cells is stable during the 3-month culture period.

Animals↗

Chloramphenicol inhibits hemoglobin synthesis in blood islands of young chick blastoderm.

Hemoglobin (Hb) synthesis is reduced by 1.5 to 12.4 X 10(-4) M chloramphenicol (CAP) in chick blastoderms at head process and 4-somite stages. Chloramphenicol at 18.6 X 10(-4) M results in decrease of amino acid uptake and incorporation into protein, and inhibits Hb formation completely. However, this effect can be overcome partially when blastoderms are removed from CAP-containing to plain culture medium. The morphological appearance, protein content, and behaviour in amino acid uptake and incorporation of control and CAP-treated blastoderms at the time Hb first makes its appearance can be correlated and seem to reflect changes occurring in the vascular area during the time of blood island organization and subsequent massive Hb synthesis in the simple culture system employed.

Amino Acids↗

Thymidine: inhibitor of differentiation in the young chick blastoderm in culture.

Differentiation in the young chick blastoderm is affected by thymidine at concentrations higher than 8.2 x 10(-4) M. Blastoderms at the hypoblastic island stage cultured continuously in the presence of thymidine form an atypical primitive streak which is not capable of inducing the embryonic axis. However, blastoderms with a mature streak escape the effect of thymidine and develop normally.

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