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Cells from Rana pipiens gastrulae and arrested hybrid gastrulae show differences in adhesion to fibronectin-sepharose beads.

Experiments were performed to examine adhesion of Rana pipiens gastrula cells and arrested hybrid gastrula cells to fibronectin-Sepharose beads (FN-beads). Blastula cells from both normal and hybrid embryos show poor adhesion to FN-beads. Beginning at the early gastrula stage, however, normal cells show a progressively increasing tendency to adhere to beads. In two different arrested hybrid embryos, cells from all developmental stages lack the ability to adhere to beads. A third hybrid shows an increase and then a decrease in cell-bead adhesion. A fourth hybrid shows a late increase in cell-bead adhesion in animal-half cells and no increase at all in vegetal-half cells. Blastula-stage cells have the ability to adhere to con A-beads and two kinds of Cytodex beads but will not adhere to FN-beads. Similarly, some cells from arrested hybrid embryos lack the ability to adhere to FN-beads but will adhere to con A-beads and cytodex beads. Observations in the light and scanning electron microscope show that normal cells form lamellipodia on FN-beads and move about actively on them, much like they do in vivo on surfaces coated by fibrils containing fibronectin. For adherent hybrid cells attached to beads, one kind does so by small pseudopodia but does not move on them and another kind forms active lamellipodia at the tips of fusiform cells and moves on beads.

Animals

Cell adhesion to extracellular matrix in normal Rana pipiens gastrulae and in arrested hybrid gastrulae Rana pipiens female X Rana esculenta male.

Rana pipiens eggs fertilized by Rana esculenta sperm (ESC) hybrid embryos develop until gastrulation in control Rana pipiens embryos (PIP) and then show morphogenetic arrest. After arrest, ESC do not gastrulate but live for 5 days as blastula-like embryos. We studied the distribution of fibronectin (FN)-containing fibrils and integrin (INT) in PIP and ESC. There are many FN-fibrils in PIP organized in anastomosing networks radiating away from the center of individual cells and across intercellular boundaries. ESC have fewer fibrils compared to PIP. These fibrils are first located between cells in disorganized arrays. After arrest in ESC, when PIP are Stage 14 neurulae, many more FN-fibrils appear. INT-staining occurs in both embryos in similar patterns. In xenoplastic transplantations, we found that the extracellular matrix on the inner surface of the ESC blastocoel roof serves as a substratum for PIP cell migration. In an in vitro assay, we found more cell adhesion to FN-substrata in PIP than in ESC. Cell locomotion rates on FN-substrata were 1.70 +/- 0.85 microns/min for PIP but only 0.46 +/- 0.56 microns/min for ESC. We also found that the inner surface of the blastocoel roof from ESC can not promote cell adhesion and locomotion when Stage 11 fragments are used for conditioning but that Stage 14 fragments can deposit a FN-fibril-rich extracellular matrix which supports PIP mesodermal cell migration at a rate of 1.26 +/- 0.38 microns/min.

Animals

Nucleotides in embryos in the stages of morula, gastrula, and neurula.

Levels of nucleotides and sugar nucleotides in embryos of Bufo arenarum at the stages of morula, gastrula, and neurula have been measured. The total amounts of purine nucleoside diphosphates decreased from morula to gastrula, but increased sharply from gastrula to neurula. The levels of ADP followed this pattern, but those of GDP did not change significantly through the three stages. Purine nucleoside triphosphate levels, which had increased immediately after fertilization, remained almost constant through morula, gastrula, and neurula. As with the purine nucleoside diphosphates, the adenine nucleotide decreased from morula to gastrula, and increased from gastrula to neurula. In contrast, the level of GTP showed a sharp maximum at gastrula. The total pyrimidine nucleoside triphosphate did not change significantly from morula through neurula. As in previous stages of development, only uridine sugar nucleotides were detected. A sharp increase of the galactosyl ester of nucleotides was found at gastrula.

Adenosine Diphosphate

[Concanavalin-binding proteins and cytokeratins in different tissues of the early amphibian gastrula (Rana temporaria, Xenopus laevis)].

Concanavalin A (con A), a lectin which specifically interacts with aD-mannose and aD-glucose, has a neutralizing effect on the explants of the early gastrula ectoderm of several amphibian species. Consequently, it was interesting to study con A-binding protein spectrum of the ectoderm and compare it to those of other early gastrula tissues. Animal pole ectoderm (APE), dorsal blastopore lip (DBL) and vegetal pole endoderm (VPE) were dissected from early gastrulae of Rana temporaria and Xenopus laevis. The extracts were subjected to SDS-PAGE with subsequent immunoelectroblotting on nitrocellulose membranes. The blots were sequentially treated with con A solution, horseradish peroxidase and diaminobenzidine. Spectra of the con A-binding glycoproteins were similar in APE, DBL and VPE of R. temporaria. Ten-twelve fractions with the molecular weight in the range from 30 to 150 kDa were stained in each blot. Fractions with the molecular weight of 150, 125, 104, 94 and 42 kDa showed more prominent lectin binding. Con A-binding protein spectra remained unchanged after freezing-thawing of the studied extracts, as well as after blots were treated with neuraminidase or sulphuric acid in order to remove sialic acid residues; the only exception was 42 kDa fraction. At the same time, a-methyl-D-mannoside pyranoside completely blocked con A binding by fractions of the studied extracts. In histological sections of R. temporaria early gastrula, all cells bound FITC-labelled con A. Similar data were obtained with tissues of X. laevis early gastrula. While electrophoretic pattern of X. laevis tissues drastically differed from that of R. temporaria, there were no significant differences between con A-binding protein spectra of X. laevis APE, DBL or VPE. Thus, all studied tissues of the amphibian early gastrula contain similar set of con A-binding proteins; however, only APE is capable of neutralization in response to con A action. These data favor our earlier assumption (see Mikhaĭlov et al., 1989) that con A reception and transmission of the corresponding signal do not determine the characteristics of the target cells response. APE, DBL and VPE extracts were assayed also for the presence of a protein similar to cytokeratin No. 8 characteristic of simple epithelia of mammals. Experiments were performed using immunoelectroblotting with monoclonal antibodies (mAB) against cytokeratin No. 8 from rat colon (mAB E2 and E7 kindly supplied by Dr. G. A. Bannikov). In R. temporaria embryos, cytokeratin 8 was detected in APE, but not in DBL or VPE. In X. laevis gastrulae all the tissues studied contained this cytokeratin.

Amphibians

Normal fates and states of specification of different regions in the axolotl gastrula.

A fate map was constructed for four regions of the early gastrula of Ambystoma mexicanum using orthotopic grafts from donors labelled with FLDx (fluoresceinated-lysinated-dextran). The region around the animal pole gave rise to epidermis only and did not include prospective neural plate. The dorsal marginal zone contributed to cephalic endoderm and to the whole length of the axial mesoderm (notochord and somites), the lateral marginal zone to lateroventral and somitic mesoderm, and the ventral marginal zone to lateroventral mesoderm. It was found that the dorsal marginal zone contributed relatively more to the anterior regions of the mesodermal mantle and the ventral marginal zone more to its posterior parts. The same regions of the gastrula and also vegetal yolky tissue were cultured as explants and labelled with tritiated mannose. Their glycoprotein synthesis pattern was compared to those of the neurula tissues to which they contribute in vivo. Animal pole explants synthesized large amounts of the epidermis-specific marker epimucin. Dorsal marginal zone explants did not synthesize epimucin but did make amounts of S2 and S6 indicative of mesoderm, as well as the notochord-specific markers S2.2 and S3.2. Lateral marginal zone explants showed the same pattern as the dorsal marginal zone including the two notochord-specific markers, although they do not contribute to notochord in vivo. Ventral marginal zone explants were more variable in their behaviour. Yolky tissue from the vegetal hemisphere of the gastrula or the archenteron floor of the neurula synthesized mainly polydisperse material of high molecular weight rather than discrete glycoproteins. The results indicate that at the early gastrula stage states of specification exist which correspond to the three germ layers, ecto-, meso- and endoderm. The ectodermal specification of animal pole explants is quite robust and cannot easily be changed by variation of the culture conditions. However treatment with a concentrated pellet of vegetalizing factor does induce a change to mesodermal specification, which is clearly detectable in the pattern of glycoprotein synthesis. Similar inductive interactions between different regions of the early embryo are thought to occur during normal development.

Ambystoma mexicanum

Coordinate inactivation of class III genes during the Gastrula-Neurula Transition in Xenopus.

We have identified a period during early Xenopus development when several different genes transcribed by RNA polymerase III (class III genes) are coordinately inactivated. During the late gastrula stage a major reduction in the number of active transcription complexes gives rise to a pattern of class III gene activity typical of adult somatic cells. This event is referred to as the Gastrula-Neurula Transition and involves the inactivation of genes encoding oocyte-type tRNAs and 5S RNA, along with several heterogeneous RNAs expressed during the blastula and gastrula stages of embryogenesis.

Animals

Cells from Xenopus laevis gastrulae adhere to fibronectin-sepharose beads and other lectin coated beads.

Cells from Xenopus laevis blastulae have a poorly developed ability to adhere to Sepharose beads covalently coupled to bovine plasma fibronectin (FN-beads). They do, however, have the ability to adhere to con A-beads and cytodex-1 and cytodex-3 beads. Beginning at the early gastrula stage, there is a progressively increasing ability of cells to adhere to FN-beads. Gastrula cells adhere to FN-beads by the formation of large ruffling lamellipodia. These cells can translocate on the surface of FN-beads; and when attached to both beads and the surrounding glass substratum of culture vessels, have the ability to move the beads extensively. Gastrula cells also have the ability to adhere to but not move upon con A-beads, wheat germ agglutinin-beads, and soy bean agglutinin-beads. They do not adhere significantly to Tetragonolobus purpureas agglutinin-beads. These results suggest that there are increasing numbers of fibronectin receptors present on the surface of embryonic amphibian cells during the period of gastrulation. They may explain the differential distribution of fibronectin-containing fibrils in vivo as observed by scanning electron microscopy.

Animals

[Inductive effect of the eye tissues of adult clawed toads on the gastrula ectoderm].

The inducing influence of adult eye tissues on the early gastrula ectoderm was studied in vitro. Both retina and pigment epithelium induced in the early gastrula ectoderm similar spectra of cell types, including nervous tissue, retina, pigment epithelium, lentoids, ectomesenchyme, and melanophores. It is suggested that the correspondence of these cell types with those arising at a spontaneous transdifferentiation of the isolated retina and pigment epithelium cells in vitro or at the induction of the early gastrula ectoderma by archencephalic endomesoderm during the normal development can be accounted for by that in these eye cells molecular determinants appeared as a result of induction and maintaina the stability of their differentiation and their potencies to transdifferentiation in vitro being reproduced during the lifetime of these cells.

Animals

Cinematographical study of cell migration in the opened gastrula of Ambystoma mexicanum.

The migration of inner marginal cells was studied in the Ambystoma gastrula, using scanning electron micrography and time-lapse cinemicrography. Scanning electron micrographs of gastrulae which were fixed while intact revealed that the migrating cells have flattened lamellipodia at their anterior end and a rounded cell body, which can sometimes be seen to be attached to a neighbouring cell by a slender posterior process. Films of opened gastrulae showed actively moving cells, with the same features described above. Details of their movements are reported and discussed in relation to the mechanism of gastrulation.

Ambystoma

Ambystoma maculatum gastrulae have an oriented, fibronectin-containing extracellular matrix.

During early development of the urodele Ambystoma maculatum, the appearance and distribution of fibronectin-containing fibrillar extracellular materials were studied by immunocytochemistry. Fibronectin (FN) first appears in the early blastula (stage 7) as thin punctate fibrils on the cell surface concentrated in the marginal zone. In late blastula (stage 9), thin fibrils are found throughout the blastocoel roof. Early gastrulae (stage 10) have numerous fibrils and multifibrillar strands concentrated in the dorsal lip region and oriented preferentially along a line parallel to the dorsal lip-animal pole axis. There is a striking increase in the amount of FN fibrils during the rest of gastrulation. This FN-containing network can be transferred to plastic substrata with preservation of the preferential orientation observed in vivo. Dorsal marginal zone explants placed on such conditioned substrata show polarized outgrowth toward the animal pole region of conditioned areas when placed on the dorsal lip side or the ventral marginal zone side of conditioned substrata. This outgrowth occurs symmetrically on bovine plasma FN-coated substrata, is prevented by Fab' fragments of antibodies to FN but fails to occur on laminin coated substrata. When migrating mesodermal cells from early gastrulae are cultured on substrata conditioned by deposition of the fibrillar matrix, these cells exhibit striking contact inhibition of locomotion, a phenomenon that may explain dispersal of migrating mesodermal cells across the blastocoel roof. When leading edges of mesodermal cells collide, cells abruptly change direction. When leading edges collide with trailing edges, the trailing edges detach from the substratum and cells move apart in the direction of the leading edge.

Ambystoma

Cell lineage of zebrafish blastomeres. III. Clonal analyses of the blastula and gastrula stages.

Aspects of the early lineages of blastomeres in the embryo of the zebrafish, Brachydanio rerio have been described. Because of the optical clarity of the embryo, lineages of selected cells can be followed directly by microscopy through many cell divisions. Also, it is shown here that the fluorescent molecules fluorescein-dextran and rhodamine-horseradish peroxidase can be used as cell lineage tracers, marking the clonal progeny of founding blastomeres. The labeled cells can be easily visualized in the live embryo, and utilizing a sensitive video camera to amplify fluorescence, the same clone may be examined repeatedly while the cells divide and migrate. Cells that descend from a single blastomere remain closely associated together through the end of the blastula stage. At the time when epiboly begins (early gastrula) cells in the labeled clone scatter and become dispersed among unlabeled cells. It has been observed that there is no invariant mapping of the embryo's midline (determined by the position of the embryonic shield in the gastrula) with respect to the early planes of cleavage. This finding shows that in the zebrafish the region of the embryo that a cell will occupy is not specified by the cell's early ancestory.

Animals

[3H]serotonin binding to blastula, gastrula, prism, and pluteus sea urchin embryo cells.

1. The presence of serotonin binding sites in blastula, gastrula, prism, and pluteus embryos of the sea urchin, Arbacia punctulata, was investigated by the binding of radiolabelled serotonin to dissociated embryo cells. 2. [3H]serotonin binding sites were identified in prism, early pluteus, and advanced pluteus larvae, but not in blastula or gastrula embryos. 3. The ontogeny of [3H]serotonin binding activity closely parallels that of serotonin content as previously reported in Paracentrotus lividus embryos (Toneby, 1977a). 4. Results of this study support a regulatory role of serotonin in developmental processes in postgastrula sea urchin embryos.

Animals

Dermatan sulfate formation in gastrulae of the sea urchin Clypeaster japonicus.

Gastrullation of sea urchin embryos is arrested in sulfate-free sea water. This developmental arrest has been considered to be due to lack of sulfation of glycosaminoglycans in the extracellular matrix of the embryos. In the present study, we characterized a dermatan sulfate type component formed in gastrula-stage embryos of the sea urchin Clypeaster japonicus and examined the effects of sulfate deprivation on the formation. Glycosamino-glycans were prepared from gastrula-stage embryos incubated with [3H]acetate in normal and sulfate-free sea water. Enzymatic analyses indicated that embryos formed a glycosaminoglycan of the dermatan sulfate type which contained an N-acetylgalactosamine-6-sulfate-containing disaccharide as a major unit, plus a minor unidentified component. Under sulfate-free conditions, embryos formed an under-sulfated chondroitin/dermatan sulfate copolymer which mainly consisted of non-sulfate, glucuronic acid-containing (chondroitin) disaccharide units. These results suggest that sulfate deprivation diminishes not only the degree of sulfation but also the formation of L-iduronic acid-containing (dermatan) disaccharide units in dermatan sulfate in sea urchin embryos.

Acetates

The LIM domain-containing homeo box gene Xlim-1 is expressed specifically in the organizer region of Xenopus gastrula embryos.

A novel cysteine-rich motif, named LIM, has been identified in the homeo box genes lin-11, Isl-1, and mec-3; the mec-3 and lin-11 genes determine cell lineages in Caenorhabditis elegans. We isolated LIM class homeo box genes from Xenopus laevis that are closely related to lin-11 and mec-3 in the LIM and homeo domains. This paper deals with one of these genes, Xlim-1. Xlim-1 mRNA is found at low abundance in the unfertilized egg, has a major expression phase at the gastrula stage, decreases, and rises again during the tadpole stage. In adult tissues the brain shows the highest abundance, by far, of Xlim-1 mRNA. The maternal and late expression phases of the Xlim-1 gene suggest that it has multiple functions at different stages of the Xenopus life cycle. In the gastrula embryo, Xlim-1 mRNA is localized in the dorsal lip and the dorsal mesoderm, that is, in the region of Spemann's organizer. Explant experiments showed that Xlim-1 mRNA is induced by the mesoderm-inducer activin A and by retinoic acid, which is not a mesoderm inducer but affects patterning during Xenopus embryogenesis; application of activin A and retinoic acid together results in synergistic induction. The structure, inducibility, and localized expression in the organizer of the Xlim-1 gene suggest that it has a role in establishing body pattern during gastrulation.

Activins

Experimental analysis of the extension of the dorsal marginal zone in Pleurodeles waltl gastrulae.

The capacity for extension of the dorsal marginal zone (DMZ) in Pleurodeles waltl gastrulae was studied by scanning electron microscopy and grafting experiments. At the onset of gastrulation, the cells of the animal pole (AP) undergo important changes in shape and form a single layer. As gastrulation proceeds, the arrangement of cells also changes in the noninvoluted DMZ: radial intercalation leads to a single layer of cells. Grafting experiments involving either AP or DMZ explants were performed using a cell lineage tracer. When rotated 90 degrees or 180 degrees, grafted DMZ explants were able to involute normally and there was extension according to the animal-vegetal axis of the host. In contrast, neither single nor bilayered explants from AP involutes completely, and neither extends when grafted in place of the DMZ. Furthermore, when inside of the host, these AP grafts curl up and inhibit the closure of the blastopore. Once transplanted to the AP region, the DMZ showed no obvious autonomous extension. DMZs cultured in vitro showed little extension and this only from the late gastrula stage onward. Removal of blastocoel roof blocked involution to a varied extent, depending on the developmental stage of the embryos. From these results, it is argued that differences could well exist in the mechanism of gastrulation between anuran and urodele embryos. That migrating mesodermal cells play a major role in urodele gastrulation is discussed.

Animals

Transient expression of XMyoD in non-somitic mesoderm of Xenopus gastrulae.

XMyoD is the earliest marker of muscle development in Xenopus embryos and is expressed in presumptive somites in the late gastrula. In the early gastrula, in situ hybridization showed XMyoD transcripts in precursors of both muscle and non-muscle mesoderm. Embryos ventralized by UV irradiation made no muscle, but expressed XMyoD transiently. Embryo explants that differentiated ventral mesoderm also expressed XMyoD transiently. These results show that the initiation of XMyoD expression is not sufficient to convert cells to muscle and suggest that XMyoD is expressed in response to a general mesodermalizing signal; expression is stabilized and enhanced only in muscle precursors that have received a dorsalizing signal.

Animals

A fate map of superficial and deep circumblastoporal cells in the early gastrula of Pleurodeles waltl.

We have determined the fate of presumptive mesodermal cells in the early Pleurodeles waltl gastrula. We labeled all cells in a gastrula with RLDx cell lineage tracer and superficial cells with 125I and then grafted small pieces of the marginal zone orthotopically into unlabeled host embryos. Labeled progeny were identified in sectioned embryos at the tail bud stage. The use of double-labeled grafts allowed us to study the relative contributions by superficial and deep cells to different derivatives. We found that the presumptive regions are generally distributed according to classical fate maps for urodeles but that the boundaries between presumptive regions are indistinct, due to extensive intermingling between cells at the edges of grafted regions. We have shown that there is a high dorsal to low ventral gradient of mixing between superficial and deep cells.

Animals