Showing papers on "Epiblast published in 1989"

An in situ transgenic enzyme marker for the midgestation mouse embryo and the visualization of inner cell mass clones during early organogenesis.

Abstract: In order to study the deployment of cells during gastrulation and early organogenesis, it is necessary to have an in situ cell marker which can be used to follow cell fate. To create such a marker a transgenic mouse strain, designated Tg(Act-lac Z)-1, which carries 6 copies of the Escherichia coli lac Z gene under the control of the rat beta-actin promoter, was made by pronuclear injection of DNA. Staining early postimplantation hemizygous mouse conceptuses, during gastrulation and early organogenesis, for beta-galactosidase activity shows that lac Z expression is ubiquitous and constitutive in all epiblast derivatives of the 10th day conceptus. No activity is seen in trophectoderm and primitive endoderm derivatives. Postimplantation grafts of [3H]thymidine-labelled transgenic cells establish the cell autonomy of this transgenic marker. Preliminary observations on the distribution of inner cell mass (ICM) descendant clones, identified in situ in midgestation conceptuses, confirm the pluripotency of individual ICM cells. The implications regarding patterns of cell growth in nascent fetal primordia are discussed.

Regionalisation of the mouse embryonic ectoderm: allocation of prospective ectodermal tissues during gastrulation.

Abstract: The regionalisation of cell fate in the embryonic ectoderm was studied by analyzing the distribution of graft-derived cells in the chimaeric embryo following grafting of wheat germ agglutinin—gold-labelled cells and culturing primitive-streak-stage mouse embryos. Embryonic ectoderm in the anterior region of the egg cylinder contributes to the neuroectoderm of the prosencephalon and mesencephalon. Cells in the distal lateral region give rise to the neuroectoderm of the rhombencephalon and the spinal cord. Embryonic ectoderm at the archenteron and adjacent to the middle region of the primitive streak contributes to the neuroepithelium of the spinal cord. The proximal-lateral ectoderm and the ectodermal cells adjacent to the posterior region of the primitive streak produce the surface ectoderm, the epidermal placodes and the cranial neural crest cells. Some labelled cells grafted to the anterior midline are found in the oral ectodermal lining, whereas cells from the archenteron are found in the notochord. With respect to mesodermal tissues, ectoderm at the archenteron and the distal-lateral region of the egg cylinder gives rise to rhombencephalic somitomeres, and the embryonic ectoderm adjacent to the primitive streak contributes to the somitic mesoderm and the lateral mesoderm. Based upon results of this and other grafting studies, a map of prospective ectodermal tissues in the embryonic ectoderm of the full-streak-stage mouse embryo is constructed.

Cellular contacts required for neural induction in Xenopus embryos: evidence for two signals.

Abstract: Neurogenesis begins in amphibian embryos around the time of gastrulation when a portion of the ectoderm receives an inducing signal from dorsal mesoderm. Two different proposals have been made for how ectoderm must come into contact with dorsal mesoderm in order for the inducing signal to pass between the two tissues. Induction in one proposal would require normal gastrulation movements to bring dorsal mesoderm underneath, and into apposition with, the overlying ectoderm. The inducing signal in this case would pass between dorsal mesoderm and ectoderm as apposed tissue layers. The other proposal is that induction requires only a small contact between ectoderm and dorsal mesoderm at the boundary they share before gastrulation. The inducing signal by this proposal would pass laterally across this small area of contact between mesoderm and ectoderm, perhaps before gastrulation, and spread within the ectodermal cell layer. Since it is not known to what extent neurogenesis depends on each of these proposed contacts between ectoderm and dorsal mesoderm, we have generated explants of embryonic tissue in which one or the other type of contact between mesoderm and ectoderm is favored. The amount of neural tissue formed under these various conditions was then assessed using a quantitative RNase protection assay to measure the levels of two neural-specific RNA transcripts. The results show that neural tissue forms efficiently when ectoderm and dorsal mesoderm only interact laterally within a plane of tissue. In contrast, neural tissue forms extremely poorly when ectoderm is placed experimentally in apposition with involuting, anterior-dorsal mesoderm.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of Cell Adhesion Molecule E-Cadherin in Xenopus Embryos Begins at Gastrulation and Predominates in the Ectoderm

Abstract: The expression of the Ca2+-dependent epithelial cell adhesion molecule E-cadherin (also known as uvomorulin and L-CAM) in the early stages of embryonic development of Xenopus laevis was examined. E-Cadherin was identified in the Xenopus A6 epithelial cell line by antibody cross-reactivity and several biochemical characteristics. Four independent mAbs were generated against purified Xenopus E-cadherin. All four mAbs recognized the same polypeptides in A6 cells, adult epithelial tissues, and embryos. These mAbs inhibited the formation of cell contacts between A6 cells and stained the basolateral plasma membranes of A6 cells, hepatocytes, and alveolar epithelial cells. The time of E-cadherin expression in early Xenopus embryos was determined by immunoblotting. Unlike its expression in early mouse embryos, E-cadherin was not present in the eggs or early blastula of Xenopus laevis. These findings indicate that a different Ca2+-dependent cell adhesion molecule, perhaps another member of the cadherin gene family, is responsible for the Ca2+-dependent adhesion between cleavage stage Xenopus blastomeres. Detectable accumulation of E-cadherin started just before gastrulation at stage 9 1/2 and increased rapidly up to the end of gastrulation at stage 15. In stage 15 embryos, specific immunofluorescence staining of E-cadherin was discernible only in ectoderm, but not in mesoderm and endoderm. The ectoderm at this stage consists of two cell layers. The outer cell layer of ectoderm was stained intensely, and staining was localized to the basolateral plasma membrane of these cells. Lower levels of staining were observed in the inner cell layer of ectoderm. The coincidence of E-cadherin expression with the process of gastrulation and its restriction to the ectoderm indicate that it may play a role in the morphogenetic movements of gastrulation and resulting segregation of embryonic germ layers.

Changing patterns of cytokeratins and vimentin in the early chick embryo.

Abstract: The distribution of cytokeratins and vimentin intermediate filaments in the first 48 h of chick development has been determined using immunofluorescent labelling. During formation of the germ layers, cytokeratin expression is associated with the appearance of an integral epithelium (ectoderm), whereas vimentin expression is associated with cells that detach and migrate from this epithelium to form endoderm and mesoderm. Subsequently, vimentin persists in the endoderm and mesoderm and the tissues derived therefrom, such as the somites and developing heart, throughout the period of study. The appearance of cytokeratins at later stages of development occurs in some epithelia such as the ectoderm, endoderm, lateral plate and epimyocardium but not others including the neural plate, neural tube and somites. Expression of cytokeratins in endoderm and mesenchymal tissues occurs in tandem with vimentin. In conclusion, vimentin expression is related to its distribution in the epiblast before germ layer formation. Its initial appearance may be related to the motile behaviour of cells about to ingress through the primitive streak. The appearance of cytokeratin filaments, however, does not reflect germ layer derivation but rather the need for an epithelial sheet.

Transfer of dorsoventral information from mesoderm to ectoderm at the onset of limb development.

Abstract: Control of dorsoventral patterns in the chick at the prelimb stages resides in the limb mesoderm. Recombination experiments at stage 14, with dorsoventrally reversed ectoderm, result in wings with mesodermal dorsoventral polarity. Similar recombinations at stage 16 show that the ectoderm has acquired dorsoventral information and can impose this polarity on the patterns of mesodermal differentiation in the distal regions of the wing. The dorsoventral information in the ectoderm comes from the mesoderm, which transfers this information to the overlying ectoderm between stages 14 and 16. The initial dorsoventral overlying ectoderm between stages 14 and 16. The initial dorsoventral information in the ectoderm is not stable and can be reprogrammed by stage 14 mesoderm. Subsequently, there is a gradual stabilization of the ectodermal information. At the same time the mesoderm loses its capacity to reprogram dorsoventral information in the ectoderm.

Differentiation of blastocysts derived from in vitro-fertilized rhesus monkey ova.

Abstract: Six in vitro-fertilized ova were cultured for 10 to 13 days in vitro. All six had formed blastocysts with cavities, five had hatched from the zona pellucida, and one had attached to the substrate. After fixation and preparation for examination by light and electron microscopy, it was determined that all but the youngest blastocysts had developed substantial amounts of syncytial trophoblast, which morphologically resembled the syncytial trophoblast present in the first 2 days of implantation in vivo. One of the smaller blastocysts had developed syncytial trophoblast but had not hatched from the zona. All of the blastocysts showed indications of loss or inadequate development of the inner cell mass constituents, indicating that the culture conditions were suboptimal for these constituents. Apparent C-type virus particles were abundant, budding from the basal surface of the syncytial trophoblast. Because the type of trophoblast formed was that normally associated with epithelial invasion and formation of the trophoblast plate, it is suggested that such blastocysts would be useful for in vitro implantation studies as well as studies of formation of syncytial trophoblast. However, other methods should be developed for study of postimplantation embryo development. It is also noted that the inadequate differentiation of the epiblast and endoderm would not have been apparent without microscopic examination.

Distribution of extracellular matrix in the migratory pathway of avian primordial germ cells.

Abstract: The appearance and distribution of extracellular matrix (ECM) was documented along the migratory route of chicken primordial germ cells (PGCs). The antimouse embryonal carcinoma cell antibody, EMA-1, was used to label PGCs (Urven et al.: Development 103:299-304, 1988). Antibodies against laminin, fibronectin, chondroitin sulfate proteoglycan and collagen type IV were used to label extracellular matrix components. When the PGCs emerged from the epiblast, all four ECM molecules were restricted principally to the basement membrane of the epiblast. Chondroitin sulfate was also located between hypoblast cells during this period. In late germinal crescent stages, when the PGCs entered the lumina of blood vessels, the same ECM molecules were more widespread in the mesoderm and in extracellular spaces. In addition, laminin and collagen type IV were identified on lateral surfaces of ectodermal cells at this stage. When the germ cells moved through the mesenchyme into the germinal ridge, the ECM molecules were found around mesenchymal cells, and, in the cases of laminin, fibronectin and collagen type IV, in the basement membranes of the germinal ridge epithelia. Because the appearance of these ECM components is temporally and spatially correlated with the movement of PGCs, we suggest that early PGC migration may depend on their timely appearance.

Shaping and bending of the avian neural plate as analysed with a fluorescent-histochemical marker.

Abstract: Shaping and bending of the neural plate are cardinal events of neurulation. These processes are initiated in avian embryos shortly after the onset of gastrulation and concluded concomitantly with the completion of gastrulation. The epiblast undergoes extensive morphogenetic movements during gastrulation and neurulation, but the directions, distances, rates, mechanisms and roles of such rearrangements are largely unknown. To begin to understand these morphogenetic movements, we have mapped regional displacements of the epiblast by injecting a fluorescent-histochemical marker into selected prenodal, nodal and postnodal levels of the blastoderm. Lateral epiblast regions (600 microns lateral to the midline and consisting primarily of surface epithelium) are displaced craniomedially, medial regions (300 microns lateral to the midline and consisting of neural plate and preingressed mesoderm) predominantly medially, and midline regions (consisting of neural plate and primitive streak) predominantly caudally. Displacements within the avian neural plate parallel those previously described for the amphibian neural plate. Furthermore, similar tissue displacements occur within the prenodal and postnodal levels of the avian epiblast despite the fact that neurulation is occurring in the former and gastrulation in the latter. Finally, our results show that ectodermal rudiments contained within a single cross-sectional level of the embryo are a composite of cells derived from multiple craniocaudal and mediolateral levels. Thus, regional tissue displacements are important events to consider in the analysis of the early morphogenesis of axial and paraxial organ rudiments derived from the epiblast.

Invasion of a basement membrane matrix by chick embryo primitive streak cells in vitro.

Abstract: At the time of gastrulation in the chick embryo the upper epiblast layer penetrates its own basement membrane at the primitive streak so that its cells may invade the underlying tissue space. In so forming the primary mesoderm, the cells undergo a concomitant epithelial-to-mesenchymal transformation. In this study, epiblast tissue has been explanted onto a basement membrane gel in order to examine its invasive potential. Fully ingressed primary mesoderm cells were able to penetrate the gel as individual cells, during the course of which the texture of the gel was disrupted. By contrast, epiblast tissue taken from the immediate vicinity of the primitive streak penetrated the gel, but only as a coherent tongue of cells and without gel disruption. These tongues of cells did not undergo the epithelial-to-mesenchymal transformation, and consequently spread as a epithelial sheet when replated on glass. Thus, the absence of gel disruption correlated with the failure of transformation, suggesting that these two events may be linked and that they may require in situ cell interactions for their manifestation. Tissue from the lateral epiblast failed to penetrate the gel. Instead, this tissue either spread on the gel surface or rounded up into a hollow sphere with the basal surface of the cells innermost. In the former case, despite the cell spreading, no lamina densa was organized beneath the sheet, but in the latter case polarity reversal occurred with the formation of a new lamina densa at the cell-gel interface.(ABSTRACT TRUNCATED AT 250 WORDS)

Patterns of expression of a 15K beta-D-galactoside-specific lectin during early development of the avian embryo.

Abstract: We have determined, by immunohistochemical and biochemical techniques, the distribution of an endogenous beta-D-galactoside-binding lectin between the early primitive streak stage and the 5th day of embryonic development of the chick. The lectin, which was purified from the pectoral muscle of 16-day-old chick embryos, migrates on SDS-PAGE as a single polypeptide of relative molecular mass 15 x 10(3). Antibodies to this pure lectin interact with the 15K (K = 10(3) M(r)) polypeptide as well as with a 6.5K polypeptide; this second component appears to be antigenically related to the 15K lectin, as antibodies affinity purified on the 15K band recognize both polypeptides. In early stages of development, lectin immunoreactivity was present in most cells of the epiblast and hypoblast in the region of the primitive streak, while towards the edge of the area pellucida the epiblast was stained less intensely. During gastrulation, strong immunoreactivity was present also in migrating cells and in the mesoblast, while at the margin of the area pellucida the epiblast was negative. Up to the 10-somite stage, lectin immunoreactivity was present in the somites, neural tube and presumptive cardiac region; the non-neural ectoderm and the extracellular matrix were not labeled; the predominant immunoreactive component at this stage of development was the 6.5K polypeptide. Later in development, the lectin immunoreactivity gradually disappeared from the dermamyotome and nervous system to reappear conspicuously as soon as a differentiated myotome could be detected. Immunoreactivity was very high in the myotome, skeletal and cardiac muscles and transient in smooth muscles. The only region of the nervous system that continued to express the lectin throughout development was the trigeminal (semilunar) ganglion; in all regions of the nervous system, the lectin immunoreactivity disappeared early in development to be re-expressed only much later. The lining epithelium of the digestive tract and other endodermal derivatives expressed the lectin transiently. In the extraembryonic membranes, immunoreactivity to the lectin was observed in the yolk sac and in both layers of the amnion. The striking regulation of the expression of this endogenous lectin suggests that its functions are linked to cell proliferation and/or to the selective expression of a developmentally-timed cell phenotype.

Immunohistochemical analysis of the segregation process of the quail germ cell lineage.

Abstract: An antiserum against quail 7 day gonadal germ cells was found to react specifically with gonadal germ cells of both sexes. Transverse sections from a range of early quail developmental stages were submitted to the antibody PAP reaction. Blastodiscs from the earliest uterine stages (II to X E.G. & K) reacted very strongly, while the overall reaction gradually decreased in older blastoderms. At stage XIII both epiblast and hypoblast were weakly stained, but some large, PGC-like cells stained intensively. During gastrulation (PS formation) the reaction of the epiblast disappears quicker than that of the hypoblast. The newly formed mesoderm and entoderm do not react at all and the reaction gradually becomes limited mainly to the PGCs and somewhat to the primary hypoblast which is moving into the germinal crescent. The widely spread reaction at the early stages is thus gradually being restricted to the PGCs.

Diferenciación de Cristalinos en Explantes de Embrión de Polio en Ausencia de Hipoblasto y Vesícula Optica1

Abstract: Summary In vitro studies of lens formation in chicken embryo in the absence of hypoblast and optic vesicle In vitro studies of lens formation in chick embryo have suggested the action of two factors leading the lens induction in the cephalic ectoderm in the absence of optic vesicle: preliminary instructive specific stimulus (homotypic endo-mesoderm) and permissive unspecific stimulus (heterotypic mesenchymes). In order to detect the true capacities of tissues that exert this influence in the cultural condition, series of in vitro experiments were planned. Exclusion experiments: explants including presumptive lens ectoderm were cultured previous to a progressive exclusion of adjacent tissues to trigger lens formation (endoderm, mesoderm and neural tissue), from stage 1 to 7 of Hamburger/Hamilton. Recombinant experiments: Recombinations of caudal epiblast with cephalic hypoblast from blastoderms stages 3, 4 and 5; and recombinations of cardiac mesoderm stage 7 with trunk ectoderm stage 11, were cultured in close association. Lens and lentoids were formed in the presumptive lens ectoderm, even when endoderm, neural tissue and optic vesicle were excluded, but always in presence of subjacent mesoderm. Observation of cephalic epiblast after to be separated mechanically from the underlying tissues showed that the presumptive cardiac mesoderm remains in contact with the epiblast. Beside the cardiac area was capable of forming lens bodies in contact with the trunk ectoderm. It was concluded that the cardiac mesoderm is able to exert a instructive specific stimulus and a permissive unspecific stimulus during in vitro lens formation.

Adhesion and fusion of the extraembryonic epiblast

Abstract: A tissue culture model system has been devised to examine the attachment, expansion, and fusion of epithelial cell sheets A normal embryonic epithelial tissue, the extraembryonic epiblast of the chick, is isolated mechanically and cultured on its natural substratum, the vitelline membrane This persistently migratory tissue has distinct adhesive and non-adhesive regions A serum-free chemically-defined culture medium has been formulated that permits determination of the effects of individual growth and trophic factors Attachment of transferred epiblasts is dependent upon the presence of mineralocorticoids in the medium This suggests that fluid transport is required for the cell sheet to make its initial attachment to the culture substratum Expansion of the cell sheet following attachment, and the fusion of epiblasts advancing toward each other, does not require the presence of mineralocorticoid No exogenous adhesive glycoproteins are required for attachment, expansion, or fusion Antibody localization shows that endogenous laminin is present on the attachment surface of the specialized adhesive edge region of the extraembryonic epiblast Following fusion of confronted epiblasts into one coherent cell sheet, the laminin disappears Throughout these studies the adhesive and non-adhesive regions of the epiblast are identified by their characteristic distributions of actin microfilaments, localized using rhodamine-phalloidin staining