Development of cell surface activity and cell surface adhesiveness in early embryos of the newt,Cynops pyrrhogaster

The development of cell surface activity and adhesiveness was examined in relation to cleavage number in early embryos of the newt, Cynops pyrrhogaster. Both large hyaline bleb formation and surface adhesiveness to substratum were manifested in presumptive ectodermal cells isolated from embryos after the eleventh cleavage (mid-blastula stage). Scanning electron microscopy of the inner surface of the blastocoelic wall (presumptive ectodermal cell layer) revealed the formation of large blebs after the eleventh cleavage. Treatment with alcian blue and lanthanum nitrate demonstrated the accumulation of an extracellular matrix (ECM) on the surface of large blebs.     

Origin of primordial germ cells in the prestreak chick embryo

The temporal and spatial pattern of segregation of the avian germline from the formation of the area pellucida to the beginning of primitive streak formation (stages VII–XIV, EG&K) was investigated using the culture of whole embryos and central and peripheral embryo fragments on vilelline membranes at stages VII–IX, immunohistological analysis of whole mount embryos and sections with monoclonal antibodies MC-480 against stage-specific embryonic antigen-1 (SSEA-1) and EMA-1, and with the culture of dispersed blastoderms at stages IX–XIV with and without an STO feeder layer. Whole embryos at intrauterine stages developed up to the formation of the primitive streak despite the absence of area pellucida expansion. Primordial germ cells (PGCs) appeared in the cultures of whole embryos and only in central fragments containing a partially formed area pellucida at stages VII–IX. When individual stage IX–XIV embryos were dispersed and cultured without a feeder layer, 25–45 PGCs/embryo were detected only with stage X–XIV, but not with stage IX blastoderms. However, the culture of dispersed cells from the area pellucida of stages IX–XIII on STO feeder layers yielded about 150 PGCs/embryo. The carbohydrate epitopes recognized by anti-SSEA-1 and EMA-1 first appeared at stage X on cells in association with polyingressing cells on the ventral surface of the epiblast and later on the dorsal surface of the hypoblast. The SSEA-1-positive hypoblast cells gave rise to chicken PGCs when cultured on a feeder layer of quail blastodermal cells. From these observations, we propose that the segregation and development of avian germline is a gradual, epigenetic process associated with the translocation of SSEA-1/EMA-1-positive cells from the ventral surface of the area pellucida at stage X to the dorsal side of the hypoblast at stages XI–XIV. © 1996 Wiley-Liss, Inc.     

Temporal-spatial expression of two Paracentrotus lividus cell surface proteins

The temporal expression of two cell surface proteins, called BEP1 and BEP4, during Paracentrosus lividus embryonic development was studied. These proteins are found in both monomeric and dimeric forms in egg and embryos and we have established that their specific form is related to their being in the cytoplasm or on the cell surface. The spatial distribution of BEP1 and BEP4 proteins in eggs and embryos was established by whole mount immunohistochemistry. These proteins are located in the animal part of unfertilized and fertilized eggs; thereafter they are much less represented in structures derived from the vegetal cells of the embryo such as the micromeres of the 16 cell stage, the primary mesenchyme of blastula and the gut of gastrula. At the prism stage BEP1 and BEP4 proteins are present to some ectodermal parts and thereafter, at the pluteus stage, to the oral region.     

Ultrastructural study of concanavalin-A binding to the surface of preimplantation mouse embryos

Receptors for Con-A were labelled (using the peroxidase-diaminobenzidine technique) on the plasma membrane of unfertilized and fertilized mouse eggs, cleavage stage embryos, trophoblast and inner cell mass (ICM) of the blastocyst. Embryos were exposed to Con-A concentrations of 10 microgram/ml, 50 microgram/ml, or 1,000 microgram/ml and the lowest concentration was observed to be the most suitable for discerning differences between stages of embryonic development. On the surface of unfertilized and fertilized eggs and 2-cell embryos, reaction product appeared as a thin, discontinuous layer. The surface of 4- and 16-cell stage embryos had a thicker, continuous, although non-uniform, layer of the reaction product. On the surface of the cells of the late morula, and on the trophoblastic cells of the blastocyst, clustering of reaction product was observed. Cells of ICM of intact blastocyst were free of the reaction product, showing that either Con-A and/or peroxidase cannot penetrate tight junctions between trophoblastic cells. Reaction product in the form of a thin, uniform layer covered the free surface of the cells of the ICM after they had been isolated (using immunosurgery) and exposed to 50 microgram/ml of Con-A. The amount and distribution of Con-A receptors is discussed, along with their redistribution and mobility in relation to the agglutinability of preimplantation mouse embryos.     

Cell surface proteins of Drosophila. II. A comparison of embryonic and ecdysone-induced proteins

The cell-surface proteins of Drosophila embryos at gastrula and myoblast fusion stages were characterized by radioiodination and two-dimensional gel electrophoresis. Over 13% of the cell surface proteins detected in gastrula embryos were not found in myoblast fusion stage embryos or in Drosophila embryonic cell line EH34A3 cells. Nearly 18% of the cell-surface proteins detected in myoblast fusion stage embryos were evident only at that stage. Embryonic cell-surface proteins were compared with cell-surface proteins from untreated EH34A3 cells and EH34A3 cells treated with 20-hydroxyecdysone, which induces cell aggregation and the expression of "new" proteins at the cell surface (D. F. Woods and C. A. Poodry, 1983, Dev. Biol. 96, 23-31). Only one of the proteins induced by ecdysone in EH34A3 cells was detected in the NP-40 soluble fraction of radioiodinated cell lysates, even after fractionation by lectin affinity chromatography and immunoprecipitation to enrich for putative ecdysone induced proteins. However, extraction of the NP-40 insoluble pellet of embryo cells revealed one additional protein that was present both in myoblast fusion stage embryos and hormone-treated culture cells. It was concluded that except for these two proteins, the cell-surface proteins induced in cultured cell lines by treatment with 20-hydroxyecdysone are not present in significant amounts in gastrula or myoblast fusion stage embryos.     

Regular surface layer of Azotobacter vinelandii.

Washing Azotobacter vinelandii UW1 with Burk buffer or heating cells at 42 degrees C exposed a regular surface layer which was effectively visualized by freeze-etch electron microscopy. This layer was composed of tetragonally arranged subunits separated by a center-to-center spacing of approximately 10 nm. Cells washed with distilled water to remove an acidic major outer membrane protein with a molecular weight of 65,000 did not possess the regular surface layer. This protein, designated the S protein, specifically reattached to the surface of distilled-water-washed cells in the presence of the divalent calcium, magnesium, strontium, or beryllium cations. All of these cations except beryllium supported reassembly of the S protein into a regular tetragonal array. Although the surface localization of the S protein has been demonstrated, radioiodination of exposed envelope proteins in whole cells did not confirm this. The labeling behavior of the S protein could be explained on the basis of varying accessibilities of different tyrosine residues to iodination.     

Ultrastructural Study of Changes in Cell Morphology and Extracellular Matrix in Prospective Endodermal Cells of Newt Embryos (Cynops pyrrhogaster) before and during Gastrulation

The cell morphology, cell-to-cell contact behavior and extracellular matrix (ECM) of inner cells (prospective endodermal cells) of newt ( Cynops pyrrhogaster ) embryos were examined from the morula to gastrula stage by light and electron microscopy. The inner cells showed increased cell-to-cell contact from the early blastula to early gastrula stage. The cells formed blebs (5–15 μm in diameter) during the blastula stage, and started to form filopodia and lamellipodia before gastrulation. Alcian blue and lanthanum nitrate treatment revealed ECM components on the cell surface in the early blastula stage and these components increased in amount from the late blastula to early gastrula stage. It is suggested that the increase in ECM components on the cell surface may have some relation with changes in cell-to-cell contact and formation of processes on the cell surface. Besides the cell surface ECM components, glycogen-like granules were observed in intercellular spaces. From the distribution of granules in gastrulae, it is suggested that these may be important in maintaining intercellular spaces for migration of invaginating cells.     

Three-dimensional structure of the regular tetragonal surface layer of Azotobacter vinelandii.

Fragments of the Azotobacter vinelandii tetragonal surface (S) layer, free of outer membrane material, were obtained by treating whole cells with 100 microM EDTA. The three-dimensional structure of the S layer was reconstructed from tilted-view electron micrographs of the S-layer fragments, after computer-assisted image processing by correlation averaging. At a resolution of 1.7 nm, the S layer exhibited funnel-shaped subunits situated at one fourfold-symmetry axis and interconnected at the other fourfold-symmetry axis to form prominent cruciform linking structures. These data, in conjunction with a relief reconstruction of the surface of freeze-etched whole cells, indicated that the apex of the funnel-shaped subunit was associated with the outer membrane, while the funnel "opening" faced the environment; the cruciform linking structures were formed at the outermost surface of the S layer. Electron microscopy and image enhancement were used to compare the structure of the outer membrane-associated S layer with that of fragments of the S layer dislodged from the outer membrane. This analysis revealed an increase in the lattice constant of the S layer from 12.5 to 13.6 nm and an alteration in the position of the cruciform linking structures in the z direction. These conformational changes resulted in a reduction in the thickness of the S layer (minimum estimate, 5 nm) and an apparent increase in the size of the gaps between the subunits. In terms of the porosity of the S layer, this gave the appearance of a transition from a closed to a more open structure.     

The effects of embryo stage and cell number on the composition of mouse aggregation chimaeras

Studies with intact preimplantation mouse embryos and some types of chimaeric aggregates have shown that the most advanced cells are preferentially allocated to the inner cell mass (ICM) rather than the trophectoderm. Thus, differences between 4-cell and 8-cell stage embryos could contribute to the tendency for tetraploid cells to colonise the trophectoderm more readily than the ICM in 4-cell tetraploid<-->8 cell diploid chimaeras. The aim of the present study was to test whether 4-cell stage embryos in 4-cell diploid<-->8-cell diploid aggregates contributed equally to all lineages present in the E12.5 conceptus. These chimaeras were compared with those produced from standard aggregates of two whole 8-cell embryos and aggregates of half an 8-cell embryo with a whole 8-cell embryo. As expected, the overall contribution of 4-cell embryos was lower than that of 8-cell embryos and similar to that of half 8-cell stage embryos. In the 4-cell<-->8-cell chimaeras the 4-cell stage embryos did not contribute more to the trophectoderm than the ICM derivatives. Thus, differences between 4-cell and 8-cell embryos cannot explain the restricted tissue distribution of tetraploid cells previously reported for 4-cell tetraploid<-->8-cell diploid chimaeras. It is suggested that cells from the more advanced embryo are more likely to contribute to the ICM but, for technical reasons, are prevented from doing so in simple aggregates of equal numbers of whole 4-cell and whole 8-cell stage embryos.     

Immunological detection of cell surface galactosyltransferase in preimplantation mouse embryos

Presence of cell surface galactosyltransferase was surveyed in preimplantation mouse embryos by indirect immunofluorescence staining using an affinity-purified antibody against galactosyltransferase from human milk. Distinct fluorescence staining was observed in embryos ranging from late 8-cell stage to early blastocysts, while the embryos at other stages were stained only weakly. The cell surface enzyme was also present in F9 embryonal carcinoma cells, in a fraction of bone marrow cells of the mouse, and in a few percent of testicular sperm.     

Exine development in Nymphaea colorata (Nymphaeaceae)

We show a sequence of developmental events in microspores and tapetal cells in Nymphaea colorata based upon transmission and scanning electron microscopic observations. There are parallel cytoplasmic processes and surface coatings in microspores and tapetal cells. Uptake is indicated by the passage of lanthanum as a tracer from anther locule into the microspore cytoplasm and by the condition of the cytoplasmic surface of microspores. The callose envelope is not a barrier to transfer of lanthanum. During formation of the proexine glycocalyx tiny spiral elements, components of the exine substructural units, were oriented in different directions in the surface coating of microspores and tapetum. Lipoidal globules are associated with the spiral elements. After the uniform proexine stage, three regions of different exine structure and their gradations become differentiated in the sporoderm: 1) a proximal region with thick tectum and foot layer, thin columellae and a compact layer of lamellated endexine; 2) a distal pole region with separately disposed endexine lamellae; and 3) an equatorial encircling-sulcate aperture region which consists of infratectal layer, foot layer, and endexine lamellae. Based upon the presence of structurally comparable surface coats in microspores and tapetal cells, experimental uptake of lanthanum nitrate, and the co-ordinated processes in tapetum and microspores, we conclude that there is probably a reciprocal controlling influence between the microspores and the tapetum and other sporophytic tissues.     

The preimplantation mammalian embryo: characterization of intercellular junctions and their appearance during development.

Junctions in developing mammalian embryos were investigated with lanthanum tracer and freeze-fracture methods. The outermost blastomeres of mouse morulae possess focal tight junctions which become zonular and exclude lanthanum, thereby separating the “inner” cells from the maternal environment. This compartmentalization, creating a microenvironment inside the embryo, may be required for cell determination and for the accumulation of fluid during blastocoel expansion. Desmosomes appear for the first time at the blastocyst stage in the trophoblast junctional complex which also is characterized by gap junctions and a zonula occludens with underlying microfilament-like material and microtubules. Both gap and tight junctions have been visualized in freeze-fracture replicas of rabbit blastocysts. The zonula occludens forms a permeability barrier which is consistent with the high transtrophoblast electrical resistance. Mouse presumptive and mature inner cell mass (ICM) cells were associated by frequent gap junctions whereas junctional complexes were absent. Trophoblast gap and adhering junctions and cytoplasmic processes appeared to fix the ICM to one pole of the embryo and partially isolate it from the blastocoel. These findings support the idea that the ICM and trophoblast communicate upon implantation and require that the intercellular junctions between them be dissembled if the ICM is to migrate to a mesometrial position.     

Ultrastructural study of surface components of Streptococcus suis.

The presence of capsular material on cells of nine reference strains of Streptococcus suis representing serotypes 1 to 8 and 1/2 was determined by transmission electron microscopy after polycationic ferritin labeling, immunostabilization, or fixation with a combination of glutaraldehyde and lysine. All the cells of the reference strains examined were covered with a layer of capsular material whose thickness varied between 20 to 30 nm and 350 to 375 nm when examined by immunostabilization. Capsular material from cells exposed to homologous antiserum was usually thicker than that from polycationic ferritin-labeled cells or cells fixed with glutaraldehyde-lysine. Negative staining revealed detectable surface structures on S. suis strains. All strains carried peritichous, thin, and flexible fimbriae with a diameter of approximately 2 nm and a length of up to 250 nm. This study indicated that morphological differences of surface structure exist among S. suis reference strains.     

The development of chick spinal cord in tissue culture. II. Cultures of whole chick embryos

By using whole-chick-embryo cultures followed by fragment cultures of spinal-cord primordia, it was possible to reproduce in vitro the whole process of neuronal development beginning with its initiation and continuing up to and including the maturation of neurons. Normal whole embryos were developed to Hamilton-Hamburger stages 17 and 18 by growing embryos from the primitive streak stage on large (28-mm) glass rings. The advantage of whole-embryo cultures is that development can be staged accurately, which is especially important during the early stages when morphogenesis progresses very rapidly. By using such accurately staged embryos and tritiated thymidine, we have determined that some postmitotic neuronal precursor cells appear in chick embryos as early as Hamburger-Hamilton stages 4 and 5, i.e. the definitive streak stages before the neural tube has formed.     

Surface protein composition of Aeromonas hydrophila strains virulent for fish: identification of a surface array protein.

The surface protein composition of members of a serogroup of Aeromonas hydrophila which exhibit high virulence for fish was examined. Treatment of whole cells of representative strain A. hydrophila TF7 with 0.2 M glycine buffer (pH 4.0) resulted in the release of sheets of a tetragonal surface protein array. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis showed that this sheet material was composed primarily of a protein of apparent molecular weight 52,000 (52K protein). A 52K protein was also the predominant protein in glycine extracts of other members of the high-virulence serogroup. Immunoblotting with antiserum raised against formalinized whole cells of A. hydrophila TF7 showed the 52K S-layer protein to be the major surface protein antigen, and impermeant Sulfo-NHS-Biotin cell surface labeling showed that the 52K S-layer protein was the only protein accessible to the Sulfo-NHS-Biotin label and effectively masked underlying outer membrane (OM) proteins. In its native surface conformation the 52K S-layer protein was only weakly reactive with a lactoperoxidase 125I surface iodination procedure. A UV-induced rough lipopolysaccharide (LPS) mutant of TF7 was found to produce an intact S layer, but a deep rough LPS mutant was unable to maintain an array on the cell surface and excreted the S-layer protein into the growth medium, indicating that a minimum LPS oligosaccharide size was required for A. hydrophila S-layer anchoring. The 52K S-layer protein exhibited hear-dependent SDS-solubilization behavior when associated with OM, but was fully solubilized at all temperatures after removal from the OM, indicating a strong interaction of the S layer with the underlying OM. The native S layer was permeable to 125I in the lactoperoxidase radiolabeling procedure, and two major OM proteins of molecular weights 30,000 and 48,000 were iodinated. The 48K species was a peptidoglycan-associated, transmembrane protein which exhibited heat-modifiable SDS solubilization behaviour characteristic of a porin protein. A 50K major peptidoglycan-associated OM protein which was not radiolabeled exhibited similar SDS heat modification characteristics and possibly represents a second porin protein.     

Disabled-2 is an epithelial surface positioning gene

The formation of the primitive endoderm layer on the surface of the inner cell mass is one of the earliest epithelial morphogenesis in mammalian embryos. In mouse embryos deficient of Disabled-2 (Dab2), the primitive endoderm cells lose the ability to position on the surface, resulting in defective morphogenesis. Embryonic stem cells lacking Dab2 are also unable to position on the surface of cell aggregates and fail to form a primitive endoderm outer layer in the embryoid bodies. The cellular function of Dab2, a cargo-selective adaptor, in mediating endocytic trafficking of clathrin-coated vesicles is well established. We show here that Dab2 mediates directional trafficking and polarized distribution of cell surface proteins such as megalin and E-cadherin and propose that loss of polarity is the underlying mechanism for the loss of epithelial cell surface positioning in Dab2-deficient embryos and embryoid bodies. Thus, the findings indicate that Dab2 is a surface positioning gene and suggest a novel mechanism of epithelial cell surface targeting.     

Ultrastructure of the Bacteroides nodosus cell envelope layers and surface.

The surface structure and cell envelope layers of various virulent Bacteroides nodosus strains were examined by light microscopy and by electron microscopy by using negative staining, thin-section, and freeze-fracture-etch techniques. Three surface structures were described: pili and a diffuse material, both of which emerged from one or both poles of the bacteria (depending on the stage of growth and division), and large rodlike structures (usually 30 to 40 nm in diameter) associated with a small proportion of the bacterial population. No capsule was detected. The cell envelope consisted of four layers: a plasma membrane, a peptidoglycan layer, an outer membrane, and an outermost additional layer. The additional layer was composed of subunits, generally hexagonally packed with center-to-center spacing of 6 to 7 nm. The outer membrane and plasma membrane freeze-fractured through their hydrophobic regions revealing four fracture faces with features similar to those of other gram-negative bacteria. However, some unusual features were seen on the fracture faces of the outer membrane: large raised ring structure (11 to 12 nm in diameter) on cw 3 at the poles of the bacteria; complementary pits or ring-shaped depressions on cw 2; and small raised ring structures (7 to 8 nm in diameter) all over cw 2.     

Effect of sodium dodecyl sulfate on polar lobe formation and function in Ilyanassa obsoleta embryos

Polar lobes, anucleate vegetal pole protrusions formed by Ilyanassa obsoleta embryos, serve as a mechanism for shunting morphogenetic determinants to one cell during the first two cleavages. Polar lobe material becomes segregated in the CD cell during first cleavage and in the D cell during second cleavage, resulting in a very unequal four-cell stage. Larval structures including external shell, foot, operculum, statocysts, and eyes develop only when polar lobe material is present. Treatment with the anionic detergent sodium dodecyl sulfate (SDS) before and during the first cleavage inhibited polar lobe formation and equalized cleavage, as the lobe material was distributed to two cells. No polar lobes formed during second clevage in SDS-equalized embryos, and the four-cell stage consisted of four equal cells with reduced cell contacts. SDS inrreversibly inhibited polar lobe formation without affecting cytokinesis. Although 27% of the larvae from SDS-equalized embryos had one or more lobe-dependent structures duplicated, morphogenesis was impaired: more than 40% of such larvae failed to form shell and/or statocysts. When cells were separated after equalized first cleavage and raised as pairs, the pairs of resulting larvae duplicated lobe-dependent structures with the same frequency as whole equalized embryos. Possible explanations for impaired morphogenesis in SDS-treated embryos are discussed.     

Stage-specific inhibition of Xenopus embryogenesis by aprotinin, a serine protease inhibitor.

We examined the effects of various protease inhibitors on Xenopus laevis embryogenesis. Aprotinin, a serine protease inhibitor, was found to inhibit embryogenesis markedly, but other protease inhibitors had virtually no effect. The inhibitory effect of aprotinin was specific for embryos at the blastula or gastrula stage. These results suggest that an aprotinin-sensitive protease involved in embryonic development is secreted from the embryos or appears on the surface of embryonic cells at these stages. We found that various serine proteases are in fact secreted from the embryos with their development and that some of them are sensitive to aprotinin.     

Patterns of expression of the JIM4 arabinogalactan-protein epitope in cell cultures and during somatic embryogenesis in Daucus carota L.

Spatiotemporal patterns of expression of the cell-surface arabinogalactan-protein epitope defined by monoclonal antibody JIM4 (J.P. Knox et al., 1989, Development 106, 47–56) have been characterized by indirect immunofluorescence during the process of somatic embryogenesis in Daucus carota L. The JIM 4 epitope (J4e) occurred on cells established in culture from hypocotyl explants which appeared to derive, at least in part, from the epidermal cells of the hypocotyl. Cultures maintained in the presence of 2,4-dichlorophenoxyacetic acid developed proembryogenic masses of which only infrequent cells at the surface expressed J4e. Sub-culture at a low cell density and withdrawl of the synthetic auxin resulted in an increase in J4e expression in most surface cells and most abundantly in surface layers of cells at the future shoot end of developing embryos. The transition to heart-shaped embryos occurred concurrently with the expression of J4e by groups of cells beneath the developing cotyledons, at the junction of the future root and shoot. At this stage, J4e was also expressed by a single well-defined layer of cells at the surface of the embryos. Advancement to the mature torpedo stage was accompanied by the expression of the epitope on cells forming two regions of the future stele and of cells associated with the cotyledonary provascular tissue characteristic of the carrot seedling. At this stage there was substantially less expression of the marker antigen by epidermal cells, although infrequent expression by isolated cells of the epidermis was maintained. The correlation of J4e expression with the development and distinction of plant tissue patterns during somatic embryogenesis indicates a role for plasma-membrane arabinogalactan proteins in these processes.Abbreviations AGP arabinogalactan protein - 2,4-D 2,4-di-chlorophenoxyacetic acid - J4e JIM 4 epitope - PEM proembryogenic mass We thank Andrew Davis for photographic assistance and Roger Pennell for useful discussions.