Establishment of polarities in the oocyte of Xenopus laevis: the provisional axial symmetry of the full-grown oocyte of Xenopus laevis.

We aimed at understanding of formation and function of the "Nieuwkoop Centre" in embryonic pattern formation. Discussed are data on genesis of cytoplasmic localizations in ovarian oocytes, transient modifications of cytoskeletal structures creating cytoplasmic asymmetries in fertilized eggs, the axis determining "vegetal cortical rotation" and fate of distinct cells, as shown by injection of specific molecular markers into particular blastomeres at specific times. Egg rotation and centrifugation suggested that sperm that gravity cooperate in symmetrization of the axially symmetrical anuran egg. After fertilization in space or in a fast rotating clinostate, axis formation and embryonic development were normal although the blastocoel was transiently abnormal. Normal tadpoles came back on Earth after ovulation, fertilization and culture in space. They metamorphosed normally and got healthy Earth-born F1 offspring. We conclude that neither sperm nor gravity are required for determination of the bilateral symmetry in the embryo of Xenopus laevis. In normal development sperm and gravity, either alone or in collaboration, may overrule an initial bilaterality inherent to, the full-grown oocyte, residing in some still unidentified component(s)/or mechanisms.

Dynamic changes in the tubulin cytoskeleton during oogenesis and early development in the anuran amphibian Xenopus laevis (Daudin).

In order to find proper methods for preservation and visualization of tubulin-containing structures we applied different chemical fixation protocols, embedding and staining methods, as well as some methods of microtubule cytoskeleton preparation. As an example we studied the dynamics of such structures in growing oocytes, fertilized eggs and cleaving eggs of Xenopus laevis, using various immunofluorescence and immunoperoxidase reactions. The results were evaluated and compared with results from CLSM studies by different authors, which let us suggest some reliable protocols for preservation and visualization of various tubulin-containing structures in animal cells. The origin and progressive complexity of these structures in the developing amphibian oocyte and fertilized egg is closely related to the origin of embryonic polarities and, because of that, to embryonic pattern formation.

A method for staining of cell nuclei in Xenopus laevis embryos with cyanine dyes for whole-mount confocal laser scanning microscopy.

To study the cell cleavage pattern in experimentally treated Xenopus laevis blastulae, we devised a method to visualize all cell nuclei, whether in interphase or in a mitotic phase, in whole-mount embryos using confocal laser scanning microscopy. Optimal staining conditions were defined for the recently commercialized cyanine nucleic acid stain TO-PRO-3, which is excited by a 647-nm laser beam and fluoresces in the far red of the spectrum. This is beyond the spectral range of autofluorescence caused by most biomolecules and, in particular, by the high amount of yolk granules in these embryos. The quality of the TO-PRO-3 image was compared to that after nuclear staining with BOBO-3, another cyanine dye that fluoresces at slightly shorter wavelengths. In the proposed procedure, special attention is paid to permeabilization of the membranes to the dyes and to bleaching of the natural pigment of the embryos with maximal preservation of cellular and nuclear structures. Because of its emission maximum at 661 nm, TO-PRO-3 is a promising nuclear stain for specimens with special background problems and for multicolor fluorescence microscopy.

Transient effects of microgravity on early embryos of Xenopus laevis.

In order to study the role of gravity on the early development of the clawed toad Xenopus laevis, we performed an experiment on the Maser-6 sounding rocket launched from Kiruna (Sweden) on 4 Nov 1993. The aim was to find out whether a short period of microgravity during fertilization and the first few minutes of development does indeed result in abnormal axis formation as was suggested by a pilot experiment on the Maser 3 in 1989. On the Maser 6 we used two new technical additions in the Fokker CIS unit, viz. a 1-g control centrifuge and a video recording unit which both worked successfully. The 1-g control centrifuge was used to discriminate between the influences of flight perturbations and microgravity. After fertilization shortly before launch, one of the first indications of successful egg activation, the cortical contraction, was registered in microgravity and on earth. Analysis of the video tapes revealed that the cortical contraction in microgravity starts earlier than at 1 g on earth. After recovery of the eggs fertilized in microgravity and culture of the embryos on earth, the morphology of the blastocoel has some consistent differences from blastulae from eggs fertilized in the 1-g centrifuge of the rocket. However from the gastrula stage onward, the microgravity embryos apparently recover and resume normal development: the XBra gene is normally expressed, and histological examination shows normal axis formation.

Xenopus laevis embryos can establish their spatial bilateral symmetrical body pattern without gravity.

One assumes that gravity cooperates with the sperm in the establishment of bilateral symmetry in the embryo, particularly in species with yolky eggs. However, only experiments under genuine microgravity can prove this. May 2nd 1988 on the TEXUS-17 Sounding Rocket, eggs of Xenopus laevis became the first vertebrate eggs ever successfully fertilized in Space. Fertilization was done in fully automated hardware; the experiment was successfully repeated and extended in 1989. Here we report a "Space First" from the IML-1 Space Shuttle mission (January 1992): In similar hardware and under microgravity, artificially fertilized Xenopus eggs started embryonic development. Histological fixation was pre-programmed at the time gastrulation would occur on Earth and indeed, gastrulae were fixed. Thus after fertilization in near weightlessness Xenopus embryos do develop bilaterally symmetrically, very probably cued by the sperm alone.

Developmental biology on unmanned space craft.

Even the short period (6-7 min) of real microgravity during Sounding Rocket (SR) flights has provided important basic new information about the influence of the lack of gravity on particular developmental processes. However some of the reports also clearly revealed the need of an increased number of larger samples per experiment. The SR provides a very appropriate way for testing of specific flight hardware designed for experiments on long-duration missions. It was stated that the number of flight opportunities should be extended, in order to keep scientists interested in the performance of Space experiments. To this end ESA and USSR have started to collaborate in the BIOKOSMOS-9 Mission with the aim of continuing that collaboration in the future, to provide additional unmanned flight opportunities with a duration of 10-14 days. The reports on experiments performed on Biokosmos-9 showed that such missions can be a very useful extension of flight opportunities, although several of the experimental conditions should be improved. In comparison with the 1988 COSPAR meeting, the material presented at the 1990 COSPAR Session on Developmental Biology showed considerable progress in methodology: the more descriptive phase is over, the period of the actual microgravity-experiments has started, and cell- and molecular biological approaches are increasingly applied. It also became clear that in experiments with relatively small cells (plant cells, human A431 epidermoid carcinoma cells and lymphocytes) the experimental results from flight experiments were often identical to those obtained in simulated microgravity. When applicable to the biological systems involved, users should more often consider the application of such Earth-based methods, that is not only in preparation of a real Space experiment. Finally, it was emphasized that some of the results from earlier experiments, which were assumed to demonstrate real microgravity effects, might have only seemingly been due to the microgravity conditions. Instead, they could have been caused secondarily by factors such as the lack of convection in surrounding media. It is important to test this possibility in future experiments.

Fertilization and development of eggs of the South African clawed toad, Xenopus laevis, on sounding rockets in space.

Egg rotation and centrifugation experiments strongly suggest a role for gravity in the determination of the spatial structure of amphibian embryos. Decisive experiments can only be made in Space. Eggs of Xenopus laevis, the South African clawed toad, were the first vertebrate eggs which were successfully fertilized on Sounding Rockets in Space. Unfixed, newly fertilized eggs survived reentry, and a reasonable number showed a seemingly normal gastrulation but died between gastrulation and neurulation. Only a few reached the larval stage, but these developed abnormally. In the future, we intend to test whether this abnormal morphogenesis is due to reentry perturbations, or due to a real microgravity effect, through perturbation of the reinitiation of meiosis and other processes, or started by later sperm penetration.

Developmental biology in space: why and how?

Developmental Biology is a broad rapidly developing discipline. This Workshop was restricted to a sub-field: Gravity in Embryonic Development.

Fertilization of frog eggs on a Sounding Rocket in space.

During the TEXUS-17 flight (April/May 1988) eggs of a higher organism, the anuran amphibian Xenopus laevis, have for the first time been successfully fertilized under microgravity on a Sounding Rocket. This result also implies that Life Sciences Experiments of Short Duration can be carried out on Sounding Rockets. The latter can therefore function as additional carriers for such experiments. Histological sections of the experimental material demonstrated the penetration of sperm into eggs, while SEM analysis revealed the differentiation of characteristic egg surface structures. Our TEXUS-17 experiment convincingly shows that the modified automatic experiment container, originally designed for experimental BR 52NL on the D1-mission, now functions flawlessly. Eight containers were flown in an airtight, well-isolated box (TEM 06-15), and a similar set was activated on Earth, two hours later. The analysis of the biological material is in progress.

Cytoskeleton and gravity at work in the establishment of dorso-ventral polarity in the egg of Xenopus laevis.

The establishment of polarities during early embryogenesis is essential for normal development. Amphibian eggs are appropriate models for studies on embryonic pattern formation. The animal-vegetal axis of the axially symmetrical amphibian egg originates during oogenesis and foreshadows the main body axis of the embryo. The dorso-ventral polarity is epigenetically established before first cleavage. Recent experiments strongly suggest that in the monospermic eggs of the anuran Xenopus laevis both the cytoskeleton and gravity act in the determination of the dorso-ventral polarity. In order to test the role of gravity in this process, eggs will be fertilized under microgravity conditions during the SL-D1 flight in 1985. In a fully automatic experiment container eggs will be kept under well-defined conditions and artificially fertilized as soon as microgravity is reached; eggs and embryos at different stages will then be fixed for later examination. Back on earth the material will be analysed and we will know whether fertilization under microgravity conditions is possible. If so, the relation of the dorso-ventral axis to the former sperm entry point will be determined on the whole embryos; in addition eggs and embryos will be analysed cytologically.

Evidence for a functional role of the cytoskeleton in determination of the dorsoventral axis in Xenopus laevis eggs.

A normal table of events of the first cleavage period in the fertilized egg (cf. Gerhart, 1980) has been completed (cf. Table I) by studying external and internal features. Through a cytological study of eggs fixed after video time-lapse observation such features can directly be correlated and it has been shown that the first postfertilization wave (PFW) reflects spermaster growth, which causes rearrangements of animal yolk material. This may, in conjunction with the interaction of the spermaster rays with the cortex, define, in time as well as in space, the asymmetric cortical contraction which we suppose to evoke asymmetry in the animal hemisphere by formation of the vitelline wall (Pasteels, 1964) and in the vegetal hemisphere by formation of the Vegetal Dorsalising Centre (Kirschner et al. 1981). Neither prick-activated eggs nor fertilized eggs incubated in vinblastine develop a spermaster. Under these conditions abnormal cytoplasmic segregation may be directed by gravity alone. For normal development the activated egg must in some way, for instance through the sperm centriole, organize microtubule assembly into a monaster. The centriole acts as a microtubule-organizing centre in structuring the egg's cytoskeleton, and through this directs localization of the various yolk components, in time as well as in space. In egg rotation experiments performed under appropriate conditions, the cytoskeleton is disturbed and yolk rearranges under gravity till a new equilibrium is established which determines a new dorsoventral polarity. Such experiments also show that neither the dorsal cytoplasm nor the grey crescent cortex act as the ultimate dorsal determinants, since their localization is unaltered upon rotation, whereas the overall yolk distribution is significantly changed.

An immunocytochemical method for the visualization of tubulin-containing structures in the egg of Xenopus laevis.

Tubulin can be isolated and purified from Xenopus laevis eggs through modification of Olmstedt's (1970) tubulin isolation method, viz. by repeating the vinblastin precipitation step after resuspension of the sediment in a detergent-containing stabilizing medium. By this we overcome the deleterious influence of the yolk granules in the isolation procedure. From 11 of Xenopus laevis eggs 25 mg VB-paracrystals can be obtained. The apparent molecular weight of the purified tubulin is 52,800. Antiserum against the purified Xenopus VB-paracrystals, raised in 2 Chinchilla rabbits, cross-reacts in immunodiffusion tests in agar gels with rat brain tubulin and with tubulin isolated from Xenopus laevis eggs by the described procedure. Specific indirect fluorescence staining and appropriate control reactions reveal that cilia of Tetrahymena pyriformis, cytoplasmic networks in cultured mouse Leydig cells, as well as mitotic spindles and nuclear regions in paraffin sections of Xenopus laevis blastulae, react with the antibodies against Xenopus laevis egg tubulin as well as with monoclonal antibodies against pig brain tubulin. These results provide additional evidence for the view that tubulin antibodies are neither species nor tissue specific and show that under appropriate conditions tubulin containing structures can be visualized in paraffin sections.

The ultrastructure of the dorsal yolk-free cytoplasm and the immediately surrounding cytoplasm in the symmetrized egg of Xenopus laevis.

Cytoplasmic segregation and subsequent dorsad displacement of the segregated cytoplasm lead to symmetrization of the egg of Xenopus laevis. At 60 min post-fertilization (p.f.) the 'dorsal yolk-free cytoplasm' (DYFC) is located in the dorso-animal part of the egg. Its ultrastructure and that of the immediately surrounding cytoplasm have been studied with transmission electron microscopy (TEM). The endoplasmic reticulum (ER) within the DYFC consists of single or paired cisternae and many small vesicles, both with moderately dense contents. Numerous particles, presumably ribosomes and glycogen, are present together with many mitochondria and some Golgi structures. The fraction of total yolk-free area occupied by mitochondria in the DYFC is about three times that in the adjacent cytoplasm. The number of cytoplasmic vesicles per unit area of cytoplasm is far larger in the DYFC than in the surrounding area. The morphological characteristics of the DYFC at 60 min p.f. suggest that it represents a region of high metabolic activity. Since it is located in the dorso-animal quadrant of the uncleaved egg, it may be partly responsible for a difference in metabolism between the dorsal and the ventral side of the egg, and hence may play an essential role in the determination of dorso-ventrality.

Changes of the external and internal pigment pattern upon fertilization in the egg of Xenopus laevis.

External and internal pigment shifts in Xenopus laevis eggs were studied between fertilization and first cleavage. Externally visible, constant features are: (1) the 'activation contraction', a pigment shift towards the animal side taking place between 5 and 15 min post fertilization (p.f.) and (2) the concentration of the pigment around the sperm entrance point leading to the formation of the grey crescent at the opposite side of the egg. Hence, in Xenopus the grey crescent is not formed by rotation of the pigmented cortical layer with respect to the internal egg mass. Histological examination reveals that during the activation contraction the pigment is mainly concentrated in the cortical cytoplasm. Except in the region around the sperm entrance point, from 15 min p.f. onwards, the pigment progressively disperses through the subcortical layer and part of it even moves more deeply into the egg. After fusion of the pronuclei (45--60 min p.f.) the pigment in the subcortical layer forms aggregates. During the pigment shift the yolk-free cytoplasm is displaced dorsally and is ultimately found opposite the sperm entrance point. Thin fibrillar structures in the yolk-free cytoplasm progressively orient themselves parallel to the dorso-ventral plane, and from 40 min p.f. onwards towards the pronuclei. These observations are discussed in connexion with cinematographic observations by Hara, Tydeman & Hengst (1977).

The formation of the mesoderm in urodelean amphibians : IV. Qualitative evidence for the purely "ectodermal" origin of the entire mesoderm and of the pharyngeal endoderm.

Xenoplastic recombinations of animal and vegetative parts ofAmbystoma mexicanum and Triturus alpestris blastulae, and similar recombinations of parts of3H-thymidinelabelled and unlabelledAmbystoma mexicanum blastulae demonstrate convincingly that the vegetative part (zone IV, see Nieuwkoop, 1969a) of such a recombinate does not contribute to mesoderm formation, but exclusively forms endodermal derivatives. In contrast, the animal cap of the blastula (zones I.II)-which only gives rise to atypical ectoderm if isolated-not only furnishesall the ecto-neurodermal derivatives, butall the mesodermal structures of the developing recombinate as well, and finally to a varying extent forms additional endodermal structures in the recombinate.In the recombinates endodermization of the ectodermal cap occurred at the anterior end of the invaginated archenteron-corresponding to the presumptive pharyngeal endoderm -, and along the dorsal side of the endodermal tube, while an endoderm-like epithelium is formed at the boundary between the caudal endoderm and the ectoderm (proctodaeum formation). These results suggest that in normal development also endodermization occurs in the "ectodermal half" of the egg. This occurs particularly on the dorsal side, leading to the formation of the presumptive pharyngeal endoderm situated above the dorsal blastoporal groove.These experiments show that the vegetative "half" of the amphibian blastula is firmly determined as the future endoderm, whereas the animal "half" is still virtually undetermined and pluripotent.