The nature of developmental restrictions in Xenopus laevis embryos.
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Biomedical subjects
J Heasman
Publications and source records attributed to J Heasman.
Meiotic maturation in Xenopus oocytes: a link between the cessation of protein secretion and the polarized disappearance of Golgi apparati.
We have studied the relationship between the timing of the late meiotic events that occur during progesterone-induced oocyte maturation, and intracellular protein transport. We have monitored the secretion of chick oviduct proteins from Xenopus laevis oocytes microinjected with polyadenylated mRNA and found that chick ovalbumin and lysozyme are not secreted during the second meiotic metaphase, in contrast to the earlier prophase stage. Maturation had no detectable effect on the glycosylation of ovalbumin, whereas it affected the glycosylation of chick ovomucoid. As maturation proceeded, the Golgi apparati disappeared in a polarized fashion, beginning in the vegetal half. This disappearance coincided temporally and spatially with that of the nuclear envelope. We speculate that Golgi apparatus disappearance and the block in secretion are causally related.
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Germ plasm and germ cell determination in Xenopus laevis as studied by cell transplantation analysis.
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The cytoskeleton of Xenopus oocytes and its role in development.
Much is known about determinative events in early amphibian embryos, perhaps more than any other animal group. However, as yet, little attention has been focused on the cytoarchitecture of the oocyte, and the way in which this could regulate asymmetries in the egg, which in turn could lead to developmentally important interactions. The changing cytoarchitecture of the Xenopus oocyte is described with the emphasis on the following: -firstly the polarity; the oocyte is not radially symmetrical at early stages of oogenesis, but shows marked polarity. Secondly, several cytoskeletal elements change their distribution during oogenesis, and again during maturation to form a fertilizable egg. Thirdly, monoclonal antibody methods show that the oocyte develops several asymmetries which are retained in the egg and early embryo, and may be lineage related.
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Single cell analysis of commitment in early embryogenesis.
Fate maps of amphibian embryos tell us the destination of certain areas at later stages of development. After studying Vogt's fate maps, Spemann wrote (in 1938) that "the question which at once calls for an answer is whether this pattern of presumptive primordia in the beginning gastrula is the expression of a real difference of these parts, whether they are already more or less predestined or 'determined' for their ultimate fate, or whether they are still indifferent and will not receive their determination until a later time." Until recently answers to this question have relied upon explant experiments, which indicate that by the late blastula stage ectoderm, mesoderm and endoderm regions are distinctly determined. By using a method involving single cell labelling and transplantation, it is now possible to pinpoint more accurately the time during early embryogenesis at which individual blastomeres become committed. In the vegetal pole, determination towards endoderm is a gradual process beginning during the middle blastula stage (stage 8) and completed by the beginning of gastulation (stage 10). This method offers the possibilities of comparing the committed and the uncommitted state and studying at a molecular level the mechanisms of cell determination.
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The mitochondrial cloud of Xenopus oocytes: the source of germinal granule material.
The mitochondrial cloud is a prominent mass in the cytoplasm of previtellogenic oocytes of Xenopus laevis. It is shown here that the cloud contains both mitochondria and electron-dense granulofibrillar material (GFM). Using a combination of light microscopical, fluorescence, time-lapse filming, and electron microscopical techniques, the ontogeny of these components is reported and their fate is studied. It was found that the cloud is stationary in previtellogenic stages and fragments into islands of mitochondria and GFM during stage II (using the staging system of J. N. Dumont [1972) J. Morphol. 136, 153-180). These islands become localized in the peripheral cytoplasm at one pole of the stage III oocyte. By studying successive stages, GFM was followed through oogenesis and it was found localized only at the vegetal pole of stage IV and V oocytes. Furthermore, it was found that it bears a striking resemblance in position, appearance, and associations with mitochondria to the "germinal granules" of unfertilized eggs. Germinal granules have been shown by others to become incorporated into germ-line cells. It is concluded that the GFM is the precursor of this material and that the mitochondrial cloud is the site of its accumulation and localization in the previtellogenic oocyte.
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Fates and states of determination of single vegetal pole blastomeres of X. laevis.
Vegetal pole cells of Xenopus morulae contribute progeny to all three germ layers, but from the midblastula stage onward they contribute only to the endoderm. We have investigated whether this restriction in fate reflects cell determination by implanting labeled vegetal pole cells into the blastocoels of host embryos and asking which structures later include labeled progeny. Single vegetal pole cells from the morula and also from the midblastula stage can contribute progeny to all germ layers. At the early gastrula stage the cells can contribute only to the endoderm. Thus the restriction of fate in the midblastula does not reflect cell determination. However, the cells do become determined by the beginning of gastrulation.
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Oocytes and early embryos of Xenopus laevis contain intermediate filaments which react with anti-mammalian vimentin antibodies.
Previous studies have shown that Xenopus oocytes possess a cortical shell, which includes actin-containing microfilaments and cytokeratin-containing intermediate filaments. In this paper we show that oocytes of Xenopus laevis also contain filaments throughout their cytoplasm which are stained by several anti-vimentin antibody preparations. We also show that dramatic changes in pattern of these filaments occur during oocyte differentiation, first during vitellogenesis, and then during maturation of the oocyte to form an egg.
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Further analysis of the effect of ultra-violet irradiation on the formation of the germ line in Xenopus laevis.
Ultra-violet (u.v.) irradiation of the vegetal pole of newly fertilized eggs has three documented effects: reduction of primordial germ cells (PGCs), cytological damage to the vegetal hemisphere and disruption of the normal mechanism by which the vegetal yolk mass induces the formation of the dorsal axis of the embryo. In this study, we find that 90 degrees rotation of the egg for various periods after irradiation rescues the dorsal axial structures but does not restore the number of PGCs found in the dorsal mesentery of the gut; neither is there any correlation between reduced numbers of PGCs and disruption of cleavage at the vegetal pole. We therefore conclude that the effect on the germ line is separate from the other two phenomena. Secondly, 90 degrees rotation of non-irradiated eggs was found to significantly reduce germ cell numbers migrating in the dorsal mesentery of the gut.
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Effects of the substratum on the migration of primordial germ cells.
It is now clear from work on defined cell types on artificial substrates that various chemical and physical inhomogeneities in the substrates can guide cell locomotion. It is also becoming clear that less well defined inhomogeneities in living cell substrates can guide the normal locomotion of embryonic migratory cells in vivo. The primordial germ cells (p.g.cs) of early anuran amphibian embryos are proving a useful model for the study of cell migration. When isolated from the embryo and cultured on living cellular substrate, p.g.cs become oriented by the shapes of the underlying cells or by their stress fibre cytoskeleton, or both. A combination of scanning and transmission electron microscopy in vivo shows a clearly aligned cellular substrate for p.g.c. migration along part of their route. Furthermore, we find that the glycoprotein fibronectin is involved in p.g.c. adhesion, which suggests a link between orientation of the substrate cells and p.g.c. guidance.
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Contact relations and guidance of primordial germ cells on their migratory route in embryos of Xenopus laevis.
This paper describes the relationship between primordial germ cells (p.g.cs) and the substrate over which they migrate in early embryos of the anuran amphibian Xenopus laevis. P.g.cs migrate from the embryonic gut to the dorsal body wall along the dorsal mesentery at the earliest swimming stage. Our earlier papers have described the way in which p.g.cs move in vitro. In this work we have studied the shape and cytoarchitecture of both p.g.cs and the coelomic epithelial cells (c.e.cs) over which they migrate. We have concentrated on three aspects of the morphology of these cells: first the shapes of the c.e.cs and the way that they affect the shapes of the p.g.cs; secondly the presence of adhesion plaques between the two types of cell; and thirdly the arrangement of cytoskeleton elements. The results show that c.e.cs in the dorsal mesentery are orientated cranio-caudally while those on the dorsal body wall and at the junction with the mesentery are arranged transversely, at 90 degrees to the cranio-caudal plane. P.g.cs are found in both elongated and rounded state. Where elongated, they are always in the same plane as the c.e.cs with which they are associated. The implications of this are discussed. Adhesion plaques between p.g.cs and c.e.cs are shown both by disaggregation studies and transmission electron microscope studies. Plaques are associated with the well defined microfilamentous cytoskeleton of c.e.cs, but only with a sparse array of filaments in p.g.cs. The only parts of p.g.cs where filaments are regularly found are their filopodia, which are generally seen on elongated p.g.cs in longitudinal section. We suggest on the basis of this work that p.g.cs have a dispersed cytoskeleton except during filopod extension, that they move by forming direct adhesion plaques with c.e.cs, and that c.e.cs provide a firm orientated support and possible guide to p.g.c. movement.
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Primordial germ cells of Xenopus embryos: the role of fibronectin in their adhesion during migration.
Primordial germ cells (PGCs) of Xenopus laevis are highly migratory. The last section of their migratory pathway is through the dorsal mesentery of the tadpole gut. This in vivo pathway is rich in fibronectin, a glycoprotein that promotes cell adhesion and migration in vitro. Isolated PGCs are associated with cells from the mesentery and with fibronectin. Treatment with trypsin removes both the mesentery cells and the fibronectin. The PGCs do not appear to resynthesize detectable fibronectin in vitro. In contrast, cultured adult mesentery epithelial cells synthesize large amounts of fibronectin and lay it down in subcellular fibrils that align with intracellular microfilament bundles. PGCs plated on cultured mesentery cell layers adhere to them, elongate and align with the microfilament bundles of the mesentery cells. PGCs adherent to mesentery cell layers are closely associated with fibronectin; moreover, F(ab)2 fragments of anti-Xenopus fibronectin IgG inhibit the adhesion and spreading of PGCs on the mesentery. These results indicate that PGCs can adhere to mesentery cells via fibronectin produced by the latter cells and suggest that fibronectin may be involved in the migration of PGCs in vivo.
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The invasion of cultured cell layers and intact epithelia.
This paper concerns the invasive behaviour of a migratory embryonic cell type, the primordial germ cell (PGC) of the anuran amphibian Xenopus laevis. Scanning electron microscopy of isolated PGCs shows that they invade layers of cells derived from adult Xenopus mesentery, and the epithelium of the intact mesentery itself. This invasion is at least partly due to the stimulation by the PGCs of process formation in both cultured and intact epithelial cells. These broad lamellipodia move over the surface of the PGCs and eventually enclose them. We have documented previously that active invasion by the PGC also takes place, though the relative roles of these two components of the invasive process are unknown. We also demonstrate that these properties are not mediated by any contaminating cellular or extracellular material on the isolated PGCs, since their removal by trypsin does not alter the invasive behaviour.
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Evidence for substrate guidance of primordial germ cells.
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Animals↗
Electron microscopic studies on the structure of motile primordial germ cells of Xenopus laevis in vitro.
Primordial germ cells (PGCs) of Xenopus laevis have been isolated from early embryos and kept alive in vitro, in order to study the structural basis of their motility, using the transmission and scanning electron microscope. The culture conditions used mimicked as closely as possible the in vivo environment of migrating PGCs, in that isolated PGCs were seeded onto monolayers of amphibian mesentery cells. In these conditions we have demonstrated that: (a) No significant differences were found between the morphology of PGCs in vitro and in vivo. (b) Structural features involved in PGC movement in vitro include (i) the presence of a filamentous substructure, (ii) filipodial and blunt cell processes, (iii) cell surface specializations. These features are also characteristic of migratory PGCs studied in vivo. (c) PGCs in vitro have powers of invasion similar to those of migrating PGCs in vivo. They occasionally become completely surrounded by cells of the monolayer and, in this situation, bear striking resemblance to PGCs moving between mesentery cells to the site of the developing gonad in stage-44 tadpoles. We conclude that as far as it is possible to assess, the behaviour of isolated PGCs in these in vitro conditions mimics their activities in vivo. This allows us to study the ultrastructural basis of their migration.
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The formation of the gonadal ridge in Xenopus laevis. I. A light and transmission electron microscope study.
In Xenopus laevis tadpoles, between stages 44 and 49 (Nieuwkoop & Faber, 1956), the primordial germ cells (PGCs) migrate from the dorsal mesentery of the gut to the site of the presumptive GD gonadal ridge. This paper describes the process at the light- and electronmicroscope levels. The PGCs in the mesentery, which at first are very large and yolk-laden, seem to lie entirely within the cellular matrix of the mesentery, although this is not obvious in light micrographs. Where the PGCs bulge out into the coelomic cavity, they stretch the somatic cell covering to a thin, cytoplasmic layer. The somatic cells of the mesentery are held together around them at this stage by well-differentiated desmosomes. At this, and subsequent stages, the PGCs have cytoplasmic processes, roughly the size of microvilli, which are irregularly distributed over their surfaces, and which are inserted between surrounding somatic cells. Whether these processes play any role in locomotion or exploration of the substrate is uncertain. As the PGCs move laterally from the root of the mesentery to the presumptive gonadal ridge, the coelomic lining cells which cover them, initially with a very thin squamous layer, differentiate to form the cuboidal cells of the germinal epithelium. Several interesting ultrastructural features of these cells, and the PGCs, are described, particularly in the light of their surface interaction. In the light of the morphological data presented here, particularly of the cell surfaces involved, we conclude that both active locomotion by the PGCs and passive movement by the morphogenetic movements of the cells around them contribute to the establishment of the early gonadal ridge.
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The formation of the gonadal ridge in Xenopus laevis. II. A scanning electron microscope study.
This paper studies the surface morphology of the developing gonadal ridge in X. laevis between stages 44 and 49 (Nieuwkoop & Faber, 1956). During this period the primordial germ cells (PGCs) move laterally from the dorsal mesentery of the gut to the position of the presumptive gonadal ridge. As they do so the coelomic lining cells lateral to the mesentery differentiate into a specialized, longitudinally orientated band, stretching nearly the full length of the dorsal mesentery on each side. The PGCs migrate beneath this band of cells, which thus becomes the germinal epithelium of the gonadal ridge. We have demonstrated by irradiation experiments that this specialized band of cells can differentiate independently, in the absence of the PGCs.
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