Dynamic distribution of region-specific maternal protein during oogenesis and early embryogenesis of Xenopus laevis

Summary For analysing spatial distribution of maternal proteins in an amphibian egg, monoclonal antibodies specific to certain regions were raised. One monoclonal antibody was found (MoAB Xa5B6) which reacted specifically with the animal hemisphere of the mature Xenopus laevis egg. The maternal protein that reacted with the MoAb Xa5B6 was shown to be distributed asymmetrically along the dorso-ventral axis in the upper region of the equatorial zone of the fertilized egg. At late blastula stage, the antigen protein could be observed clearly in both the marginal zone and animal cap. It was localized predominantly in mesodermal and ectodermal cells of late neurula embryos. The Xa5B6 antigen accumulated during oogenesis. The distribution pattern of maternal protein was remarkably different in the developmental stages of the oocyte. The pattern in the mature oocyte was completely different from that of the immature egg in which the antigen was located in the radial striations of the oocyte cytoplasm. After maturation, the distribution pattern changed drastically to an animal-vegetal polarization and the striation labellings were no longer observed. By Western blot examination, it was confirmed that the amounts of antigen protein were constant during early embryogenesis and the mesoectoderm contained a greater amount of antigens than the endoderm at late blastula. The antibody detected two bands of approximately 70 × 10³ and 30 × 10³ Mr by Western blot analysis. The latter molecule may possibly be a degrading moiety of the former. The results were discussed in relation to establishment of animal-vegetal (A/V) and dorso-ventral (D/V) polarization at the molecular level. Offprint requests to: A.S. Suzuki     

Effects of retinoic acid on the endoderm in Xenopus embryos

 When Xenopus embryos from mid-tailbud to early tadpole stages were exposed to retinoic acid (RA), the gut developed with an uncoiled, straight intestinal tube, morphogenesis of the liver and stomach was affected and intestinal epithelial cells developed without a brush border and alkaline phosphatase activity. However, the temporal and spatial expression pattern of XlHbox 8, the only homeobox gene expressed in the endoderm was unaffected. In lateral plate mesodermal cells the expression of α-smooth muscle (SM) actin was delayed. A similar syndrome has been reported in a study of embryos lacking functional FGF receptors in which it was proposed that the uncoiled intestinal tube and the delayed differentiation of the intestinal muscle cells are causally related. Our results support this proposition and further suggest that mesenchymal-epithelial interactions concerned with regional specification of the endoderm may be impaired resulting in other defects in the gut. Received: 3 October 1997 / Accepted: 3 February 1998     

Immunohistological localisation of monoclonal antibody R 24-recognized ganglioside Gilac2* in early chick embryos

The spatio-temporal cellular expression and biosynthesis of ganglioside Glac2 was investigated in early chick embryogenesis. For demonstration of embryonic Glac2-biosynthesis, chick embryos of stage 0 and of stages 4-5 were incubated in vitro in the presence of radioactive sugar precursors. It was found that chick embryos synthesize Glac2 as early as at the blastula stage as well as at the gastrula stage, both within the area pellucida and the area opaca. In contrast to the biosynthetical findings immunohistochemical staining of the chick embryos at various stages by aid of the mouse monoclonal antibody (mAb) R 24, specific for the immunoepitope NeuAc alpha, 8NeuAc alpha, 3Gal beta less than, as present on the ganglioside Glac2, revealed a spatio-temporal cellular pattern of expression of this ganglioside in early chick embryos. Immunohistochemical staining of the chick embryo at stage 0 shows that all cells of the embryo, the extraembryonic epiblast and the yolk endoderm included, are mAb R 24-positive. At the intermediate streak stage (stage 3), the cranial part of the deep layer, the so-called endophyll, is strongly mAb R 24-positive, whereas at the end of gastrulation (stage 5), mAb R 24-recognized epitopes appear to be restricted to a narrow band of deep-layer cells in the endophyllic crescent and to the yolk endoderm of the area opaca. At this stage, no labelling by the antibody is observed in cell layers of the future embryo. The beginning of neurulation (stage 7) is characterized by the expression of the mAb R 24-recognized epitope in the notochord, whilst the deep layer in the cranial part of the neural fold still expresses this epitope. No ecto- or mesodermal structures are stained by the antibody at this developmental stage. During further development (stage 12 and 13), mAb R 24-reactivity is restricted to the cranial part of the embryo with a preferential staining of cells of endodermal origin. At these stages, the notochord expresses mAb R 24 binding sites only in its cranial region. The spatial and temporal correlation between the presence of mAb R 24-recognized epitopes and the morphogenetic positioning of tissues may be indicative for a possible role of the ganglioside Glac2 in corresponding cellular interactions.     

Analysis of extraembryonic mesodermal structure formation in the absence of morphological primitive streak

During mouse gastrulation, the primitive streak is formed on the posterior side of the embryo. Cells migrate out of the primitive streak to form the future mesoderm and endoderm. Fate mapping studies revealed a group of cell migrate through the proximal end of the primitive streak and give rise to the extraembryonic mesoderm tissues such as the yolk sac blood islands and allantois. However, it is not clear whether the formation of a morphological primitive streak is required for the development of these extraembryonic mesodermal tissues. Loss of the Cripto gene in mice dramatically reduces, but does not completely abolish, Nodal activity leading to the absence of a morphological primitive streak. However, embryonic erythrocytes are still formed and assembled into the blood islands. In addition, Cripto mutant embryos form allantoic buds. However, Drap1 mutant embryos have excessive Nodal activity in the epiblast cells before gastrulation and form an expanded primitive streak, but no yolk sac blood islands or allantoic bud formation. Lefty2 embryos also have elevated levels of Nodal activity in the primitive streak during gastrulation, and undergo normal blood island and allantois formation. We therefore speculate that low level of Nodal activity disrupts the formation of morphological primitive streak on the posterior side, but still allows the formation of primitive streak cells on the proximal side, which give rise to the extraembryonic mesodermal tissues formation. Excessive Nodal activity in the epiblast at pre‐gastrulation stage, but not in the primitive streak cells during gastrulation, disrupts extraembryonic mesoderm development.     

The endoderm gene regulatory network in sea urchin embryos up to mid-blastula stage

As the result of early specification processes, sea urchin embryos eventually form various mesodermal cell lineages and a gut consisting of fore-, mid- and hindgut. The progression of specification as well as the overall spatial organization of the organism is encoded in its gene regulatory networks (GRNs). We have analyzed the GRN driving endoderm specification up to the onset of gastrulation and present in this paper the mechanisms which determine this process up to mid-blastula stage. At this stage, the embryo consists of two separate lineages of endoderm precursor cells with distinct regulatory states. One of these lineages, the veg2 cell lineage, gives rise to endoderm and mesoderm cell types. The separation of these cell fates is initiated by the spatially confined activation of the mesoderm GRN superimposed on a generally activated endoderm GRN within veg2 descendants. Here we integrate the architecture of regulatory interactions with the spatial restriction of regulatory gene expression to model the logic control of endoderm development.     

Two antigenically distinct forms of β-1,3-glucanase in sea urchin embryonic development

The enzyme β-1,3-glucanase is contained in the unfertilized eggs of most species of sea urchin. In some species, including Lytechinus variegatus, there is also substantial activity following gastrulation, and during remaining larval development. To determine if the same form of β-1,3-glucanase is present in both unfertilized eggs and after gut differentiation, an affinity purification procedure was utilized to isolate enzyme from unfertilized Lytechinus eggs. β-1,3-Glucanase is a 70,000-Da protein in this species, similar to the molecular weight of enzyme isolated from Strongylocentrotus purpuratus. Purified enzyme was used to generate an antibody that specifically recognized a 70,000-Da protein in unfertilized eggs by Western blot analysis, and stained the cortical granules of unfertilized eggs by immunofluorescence. The antibody also specifically immunoprecipitated β-1,3-glucanase activity from egg sonicates. The antibody was used to demonstrate that the form of β-1,3-glucanase present following gastrulation is antigenically distinct from the egg form. The 70,000-Da protein recognized by the antibody was no longer present by 24 hr, but embryos of this and later stages contained substantial amounts of activity, indicating the enzyme at these stages differs from the egg-specific form. In addition, the antibody was not capable of immunoprecipitating enzyme activity from pluteus sonicates. β-1,3-Glucanase has been partially purified from pluteus stage embryos, and appears to be a complex of approximately 200,000 Da. The enzyme is specific to endoderm and appears following differentiation of the gut, suggesting that it may function in larval digestion.     

Expression of a cell surface protein during morphogenesis of the reproductive system in Manduca sexta embryos

New antibody markers have allowed more refined examinations of embryogenesis. Features are being found that were overlooked in whole and sectioned embryos stained with traditional histochemical labels. Two monoclonal antibodies that recognize two different cell surface proteins in Manduca sexta label cells of the developing reproductive system. These specific immunolabels reveal that during a brief period of Manduca embryogenesis, rudiments of both male and female genital ducts are present in a single embryo. This transient phase of genital differentiation parallels the transient indifferent stage known to occur during development of reproductive systems in many vertebrate embryos. At the end of this indifferent stage, one of the two pairs of genital ducts retracts and degenerates. The dynamic expression of the two surface proteins on cells involved in morphogenesis of both the female and male reproductive systems also suggests that these proteins are important in orchestrating the specific cellular interactions that occur between mesodermal cells of the genital ducts and the nearby ventral ectoderm.     

Evidence for crucial role of hindgut expansion in directing proper migration of primordial germ cells in mouse early embryogenesis

During mouse gastrulation, primordial germ cells (PGCs) become clustered at the base of the allantois and move caudally into the hindgut endoderm before entering the genital ridges. The precise roles of endoderm tissues in PGC migration, however, remain unclear. By using Sox17 mutants with a specific endoderm deficiency, we provide direct evidence for the crucial role of hindgut expansion in directing proper PGC migration. In Sox17-null embryos, PGCs normally colonize in the allantois and then a small front-row population of PGCs moves properly into the most posterior gut endoderm. Defective hindgut expansion, however, causes the failure of further lateral PGC movement, resulting in the immobilization of PGCs in the hindgut entrance at the later stages. In contrast, the majority of the remaining PGCs moves into the visceral endoderm layer, but relocate outside of the embryonic gut domain. This leads to a scattering of PGCs in the extraembryonic yolk sac endoderm. This aberrant migration of Sox17-null PGCs can be rescued by the supply of wildtype hindgut cells in chimeric embryos. Therefore, these data indicate that hindgut morphogenic movement is crucial for directing PGC movement toward the embryonic gut side, but not for their relocation from the mesoderm into the endoderm.     

Early appearance in neural crest and crest-derived cells of an antigenic determinant present in avian neurons

A monoclonal antibody (“$\text{E}\text{C8}$”) against chicken dorsal root ganglion cells has been produced. The epitope (antigenic determinant) to which this antibody binds appears in neuronal cells—of both the peripheral and central nervous systems—and in a limited number of nonneuronal cell types in avian embryos. The epitope is intracellular and is probably part of a protein as judged by its susceptibility to proteases. This epitope appears very early in neuronal development. It may be detected in brain, spinal cord, and ventral root nerve fibers of Hamburger-Hamilton stage 16 chicken embryos (51–56 hr of incubation). At this same age, $\text{E}\text{C8}\text{-}\text{immunoreactive}$ cells can be found in the neural crest migratory space between the neural tube and the somite about a day before dorsal root ganglia begin to coalesce. Since some cultured neural crest cells (but not somitic mesenchymal cells) also express this epitope, we propose that the $\text{E}\text{C8}$ monoclonal antibody identifies an early differentiating subpopulation of neural crest cells which express this putative neuronal trait soon after the time of cessation of migration in vivo.     

Properties of cells from inverted embryos ofXenopus laevis investigated by scanning electron microscopy

Summary Xenopus embryos held inverted from the one cell stage show a partial reversal of the pattern of cleavage: the blastocoel forms towards the new upper pole, and the non-pigmented cells forming the blastocoel roof are smaller than normal endoderm cells. Two properties of the cells from inverted embryos have been studied: their capacity to form cilia when cultured for 48 h, normally a property of ectoderm cells; and their scanning electron microscopical appearance when isolated and cultured for shorter periods, which differs for normal ectoderm and endoderm cells. Groups of the upper, non-pigmented cells from inverted embryos do not form cilia in a longerterm culture, whereas groups of the lower, pigmented cells do. In contrast, the scanning electron microscopical appearance of the upper, non-pigmented cells of inverted embryos is more like that of normal ectoderm cells; the appearance of lower, pigmented cells is more like that of normal endoderm. Thus the determination to form cilia is not reversed by inversion, whereas the control of cell morphology is.     

Isolation and characterization of a new class of acidic glycans implicated in sea urchin embryonal cell adhesion

Three major glycan fractions of 580 kDa (g580), 150 kDa (g150), and 2 kDa (g2) were isolated and purified from Lytechinus pictus sea urchin embryos at the mesenchyme blastula stage by gel filtration and high pressure liquid chromatography. Chemical analysis, by gas chromatography, revealed that g580 is highly sulfated and rich in N-acetylglucosamine, N-acetylgalactosamine, glucuronic acid, and fucose. The g150 fraction is less acidic than g580 and contains high amounts of amino sugars, xylose, and mannose. The g2 fraction is neutral, rich in N-acetylglucosamine, mannose, and galactose. The g580 and g150 fractions are resistant to glycosaminoglycan-degrading enzymes, indicating that they are distinct from the glycosaminoglycans. The g580 fraction resembles, with respect to chemical composition, a previously characterized 200 kDa sponge adhesion glycan (g200). The binding of the monoclonal antibody Block 2, which recognizes a repetitive epitope on g200, as well as of the anti-g580 polyclonal antibodies to both g580 and g200 indicated that these two glycans share similar antigenic determinants. The Fab fragments of the Block 2 antibody, which previously have been shown to inhibit cell adhesion in sponges, also blocked the reaggregation of dissociated sea urchin mesenchyme blastula cells. These results indicate that g580 carries a carbohydrate epitope, similar to the sponge adhesion epitope of g200, which is involved in sea urchin embryonal cell adhesion.     

Retinoic acid-mediated patterning of the pre-pancreatic endoderm in Xenopus operates via direct and indirect mechanisms

Early patterning of the endoderm as a prerequisite for pancreas specification involves retinoic acid (RA) as a critical signalling molecule in gastrula stage Xenopus embryos. In extension of our previous studies, we made systematic use of early embryonic endodermal and mesodermal explants. We find RA to be sufficient to induce pancreas-specific gene expression in dorsal but not ventral endoderm. The differential expression of retinoic acid receptors (RARs) in gastrula stage endoderm is important for the distinct responsiveness of dorsal versus ventral explants. Furthermore, BMP signalling, that is repressed dorsally, prevents the formation of pancreatic precursor cells in the ventral endoderm of gastrula stage Xenopus embryos. An additional requirement for mesoderm suggests the production of one or more further pancreas inducing signals by this tissue. Finally, recombination of manipulated early embryonic explants, and also inhibition of RA activity in whole embryos, reveal that RA signalling, as it is relevant for pancreas development, operates simultaneously on both mesodermal and endodermal germ layers.     

EMA,an epithelial membrane-associated antigen during early development and morphogenesis ofXenopus laevis

Summary We raised monoclonal antibodies against a membrane fraction ofXenopus neurulae in order to detect tissue-specific cell-surface markers. Here we describe a monoclonal antibody that recognizes an epithelial membrane-associated antigen (EMA) in immunohistological stainings. The tissue-specific and membrane-associated antigen detected in immunohistological stainings could serve as useful marker in epithelium differentiation and membrane organization of the early embryo. In tadpoles and adults EMA was found in specific epithelial tissues derived from different germ layers such as kidney, skin, gut, pancreas, epiphysis and choroid plexus. In the cleaving embryo this antibody stained newly formed membranes between blastomeres from the two-cell stage onwards. Cytoplasmic staining in large oocytes and early embryos was also observed. The possibility that the cytoplasmic signal represents a maternal store of membrane material is discussed.     

Pathways of avian neural crest cell migration in the developing gut

The NC-1 and E/C8 monoclonal antibodies recognize a similar population of neural crest cells as they migrate from vagal levels of the neural tube and colonize the branchial arch region of 2- to 3-day-old chicken embryos. Some of these immunoreactive cells then appear to enter the gut mesenchyme on the third day of incubation just caudal to the third branchial cleft. After entering the gut, these cells migrate in a rostral-caudal direction, using primarily the superficial splanchnic mesodermal epithelium of the gut as a substratum. The antigen-positive cells remain preferentially associated with the splanchnopleure. Few antigenic cells enter the mesenchyme surrounding the endoderm at anterior levels whereas they are found throughout the mesenchyme when nearing the umbilicus. At postumbilical levels, immunoreactive cells are distributed on both sides of the differentiating muscle layer but not within it. Although fibronectin immunoreactivity can be found throughout the wall of the gut, there is no apparent relationship between the distribution of fibronectin and the location of the immunoreactive cells. These results suggest that a mechanism more complex than a mere interaction with fibronectin may account for migration of crest-derived cells in the gut.     

Embryonic development and metamorphosis of the scyphozoan <Emphasis Type="Italic">Aurelia</Emphasis>

We investigated the development of Aurelia (Cnidaria, Scyphozoa) during embryogenesis and metamorphosis into a polyp, using antibody markers combined with confocal and transmission electron microscopy. Early embryos form actively proliferating coeloblastulae. Invagination is observed during gastrulation. In the planula, (1) the ectoderm is pseudostratified with densely packed nuclei arranged in a superficial and a deep stratum, (2) the aboral pole consists of elongated ectodermal cells with basally located nuclei forming an apical organ, which is previously only known from anthozoan planulae, (3) endodermal cells are large and highly vacuolated, and (4) FMRFamide-immunoreactive nerve cells are found exclusively in the ectoderm of the aboral region. During metamorphosis into a polyp, cells in the planula endoderm, but not in the ectoderm, become strongly caspase 3 immunoreactive, suggesting that the planula endoderm, in part or in its entirety, undergoes apoptosis during metamorphosis. The polyp endoderm seems to be derived from the planula ectoderm in Aurelia, implicating the occurrence of “secondary” gastrulation during early metamorphosis.