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.     

Fertilization of sea urchin eggs in space and subsequent development under normal conditions.

Sea urchin eggs are generally considered as most suitable animal models for studying fertilization processes and embryonic development. In the present study, they are used for determining a possible role of gravity in fertilization and the establishment of egg polarity and the embryonic axis. For this purpose, eggs of the particularly well known and suitable species Paracentrotus lividus have been automatically fertilized under microgravity conditions during the Swedish sounding rocket flights MASER IV and MASER V. It turns out, that fertilization "in Space" occurs normally and that subsequent embryonic and larval development of such eggs, continued on the ground, is normal, leading to advanced pluteus stages.     

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.     

A step in embryonic axis specification in Xenopus laevis is simulated by cytoplasmic displacements elicited by gravity and centrifugal force.

Determination of the body pattern in Xenopus embryos is known to involve at least six steps. One of these steps can be experimentally simulated by inclining the fertilized egg with respect to gravity or centrifugal force (10-30 g x 4 min, directed 90 degrees to the animal-vegetal axis). In these eggs, the dorsal structures of the body axis form from the side of the egg that was uppermost in the gravitational or centrifugal field. This topography is seen even if the sperm entry point side (the prospective ventral side in control eggs) was uppermost. In addition, conjoined twin embryos form at very high frequencies in response to certain conditions of single or double centrifugation. Cytological analysis shows that the dorsal structures invariably form from the side(s) of the egg away from which vegetal cytoplasm was displaced. This is similar to the situation in the unperturbed egg, where the subcortical cytoplasm of the vegetal hemisphere rotates some 30 degrees relative to the surface, and the dorsal structures form from the side of the egg away from which the subcortical cytoplasm moved. The displacements elicited by centrifugation probably substitute for the normal displacements brought about by the subcortical rotation. These and other data suggest that the subcortical rotation is a crucial step in the process of axis determination. The subcortical rotation is an autonomous activity of the activated egg, and can displace cytoplasm against gravity. I believe that the subcortical rotation will function normally at microgravity, and I expect that overall development and axis polarity at microgravity will be normal. This will be tested in spaceflight.     

Influence of cosmic radiation and/or microgravity on development of Carausius morosus.

Eggs of Carausius morosus were exposed to spaceflight conditions in two spaceflight missions, the German 7 day Spacelab Mission D1 and the Soviet 12.56 day Biosatellite Mission "COSMOS 1887". During spaceflight the eggs continued their development. Eggs of five different ages representing different sensitivity to radiation and different capacity to regeneration were used to investigate the influence of cosmic radiation and/or microgravity on insect development. Using the Biostack concept--eggs in monolayers sandwiched between nuclear track detectors--and the 1 g reference centrifuge of BIORACK in D1 we were able to separate effects of heavy ions of the cosmic radiation from microgravity effects and also from combined effects of these two factors in space. After retrieval, hatching rates, embryonic and larval growth kinetics and anomaly frequencies were determined. Microgravity leads to a reduced hatching rate of eggs exposed in the early stages of development. Hatching was normal in eggs which were exposed on the 1 g reference centrifuge. Hits by heavy ions caused body anomalies. The combined action of heavy ions and microgravity resulted in an unexpectedly high frequency of anomalies. These results obtained from the Spacelab Mission D1, were confirmed in an experiment onboard of COSMOS 1887. In addition to the previous analysis, embryonic development before hatching was followed which showed no major difference between flight and the ground control specimens. Since a reconfirmation of reduced hatching rates was observed in COSMOS 1887, too, the above results suggest some microgravity induced functional impairment of the hatching activity, rather than blockage in embryonic development.     

Microgravity experiments on the transition from laminar to oscillatory thermocapillary convection in floating zones

The onset of oscillatory thermocapillary convection in floating zones has been measured under microgravity in two sounding rocket experiments TEXUS and on board of the satellite SPAS-01. We report on the critical Marangoni numbers measured under μ-g and in the 1-g reference experiments     

Formation of otoconia in the Japanese red-bellied newt, Cynops pyrrhogaster.

Pre-mated adult female newts and fertilized eggs will be flown on the International Microgravity Laboratory-2 flight, in 1994. One objective of the flight will be to observe the influence of microgravity on the development of the gravity-sensing organs in the inner ear. These organs contain sensory hair cells covered by a layer of dense stones (otoconia). Gravity and linear acceleration exert forces on these masses, leading to excitation of the nerve fibers innervating the hair cells. If the production of the otoliths is regulated to reach an optimal weight, their development might be abnormal in microgravity. Ground-based control experiments are reported describing the developmental sequence in which both the otoliths and their associated sensory epithelium and the semicircular canals appear and develop. Three-dimensional reconstruction of serial sections through the otic vesicle of newt embryos at stages 31 through 58 demonstrate the first appearance, relative position and growth of the otoliths. Reports of experiments in which fertilized frog eggs were flown on a Russian Cosmos mission conclude that the utricular otolith is increased in volume, whereas the saccular otolith maintains normal size, suggesting that at least in the utricle, the weight of the otolith might be regulated.     

Embryogenesis and organogenesis of Carausius morosus under spaceflight conditions

The influence of cosmic radiation and/or microgravity on insect development was studied during the 7 day German Spacelab Mission D1. Eggs of Carausius morosus of five stages differing in sensitivity to radiation and in capacity to regeneration were allowed to continue their development in the BIORACK 22°C incubator, either at microgravity conditions or on the 1 g reference centrifuge. Using the Biostack concept - eggs in monolayers were sandwiched between visual track detectors - and the 1 g reference centrifuge, we were able to separate radiation effects from microgravity effects and also from combined effects of these two factors in space. After retrieval, hatching rates, growth kinetics and anomaly frequencies were determined in the different test samples. The early stages of development turned out to be highly sensitive to single hits of cosmic ray particles as well as to the temporary exposure to microgravity during their development. In some cases, the combined action of radiation and microgravity even amplified the effects exerted by the single parameters of space. Hits by single HZE particles caused early effects, such as body anomalies, as well as late effects, such as retarded growth after hatching. Microgravity exposure lead to a reduced hatching rate. A synergistic action of HZE particle hits and microgravity was established in the unexpectedly high frequency of anomal larvae. However, it cannot be excluded, that cosmic background radiation or low LET HZE particles are also causally involved in damage observed in the microgravity samples.     

Early development of fern gametophytes in microgravity.

Dormant spores of the fern Ceratopteris richardii were flown on Shuttle mission STS-93 to evaluate the effects of micro-g on their development and on their pattern of gene expression. Prior to flight the spores were sterilized and sown into one of two environments: (1) Microscope slides in a video-microscopy module; and (2) Petri dishes. All spores were then stored in darkness until use. Spore germination was initiated on orbit after exposure to light. For the spores on microscope slides, cell level changes were recorded through the clear spore coat of the spores by video microscopy. After their exposure to light, spores in petri dishes were frozen in orbit at four different time points during which on earth gravity fixes the polarity of their development. Spores were then stored frozen in Biological Research in Canister units until recovery on earth. The RNAs from these cells and from 1-g control cells were extracted and analyzed on earth after flight to assay changes in gene expression. Video microscopy results revealed that the germinated spores developed normally in microgravity, although the polarity of their development, which is guided by gravity on earth, was random in space. Differential Display-PCR analyses of RNA extracted from space-flown cells showed that there was about a 5% change in the pattern of gene expression between cells developing in micro-g compared to those developing on earth.     

Leaf senescence under various gravity conditions: relevance to the dynamics of plant hormones.

Effects of simulated microgravity and hypergravity on the senescence of oat leaf segments excised from the primary leaves of 8-d-old green seedlings were studied using a 3-dimensional (D) clinostat as a simulator of weightlessness and a centrifuge, respectively. During the incubation with water under 1-g conditions at 25 degrees C in the dark, the loss of chlorophyll of the segments was found dramatically immediately after leaf excision, and leaf color completely turned to yellow after 3-d to 4-d incubation. In this case kinetin (10 micromolar) was effective in retarding senescence. The application of simulated microgravity conditions on a 3-D clinostat enhanced chlorophyll loss in the presence or absence of kinetin. The loss of chlorophyll was also enhanced by hypergravity conditions (ca. 8 to 16 g), but the effect was smaller than that of simulated microgravity conditions on the clinostat. Jasmonates (JAs) and abscisic acid (ABA) promoted senescence under simulated microgravity conditions on the clinostat as well as under 1-g conditions. After 2-d incubation with water or 5-d incubation with kinetin, the endogenous levels of JAs and ABA of the segments kept under simulated microgravity conditions on the clinostat remained higher than those kept under 1-g conditions. These findings suggest that physiological processes of leaf senescence and the dynamics of endogenous plant hormone levels are substantially affected by gravity.     

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.     

Amphibian egg cytoplasm response to altered g-forces and gravity orientation

Elucidation of dorsal/ventral polarity and primary embryonic axis development in amphibian embryos requires an understanding of cytoplasmic rearrangements in fertile eggs at the biophysical, physiological, and biochemical levels. Evidence is presented that amphibian egg cytoplasmic components are compartmentalized. The effects of altered orientation to the gravitational vector (i.e., egg inversion) and alterations in gravity force ranging from hypergravity (centrifugation) to simulated microgravity (i.e., horizontal clinostat rotation) on cytoplasmic compartment rearrangements are reviewed. The behavior of yolk compartments as well as a newly defined (with monoclonal antibody) non-yolk cytoplasmic compartment, in inverted eggs and in eggs rotated on horizontal clinostats at their buoyant density, is discussed.     

Possible mechanism of microgravity impact on Carausius morosus ontogenesis.

Experiments aboard "Spacelab-D1" and "Cosmos-1887" revealed an adverse effect of space flight on Carausius morosus embryos. The main influencing factor for stick insect eggs turned out to be microgravity, while the contribution of HZE particles of cosmic radiation was relatively low. Flight experiments indicated an increased vulnerability of stick insect eggs to microgravity at intermediate stages of development, that could support the "convection" hypothesis.     

Effect of microgravity on primordial germ cells (PGCs) in silk chicken offspring (Gallus gallus domesticus)

Primordial germ cells (PGCs), precursors of germline cells, display a variety of antigens during their migration to target gonads. Here, we used silk chicken offspring (Gallus gallus domesticus) embryos subjected to space microgravity to investigate the influence of microgravity on PGCs. The ShenZhou-3 unmanned spaceship carried nine fertilized silk chicken eggs, named the flight group, returned to Earth after 7 days space flight. And the control group has the same clan with the flight group. PGCs from flight and control group silk chicken offspring embryos were examined during migration by using two antibodies (2C9 and anti-SSEA-1), in combination with the horseradish peroxidase detection system, and using periodic acid-Schiff’s solution (PAS) reaction. After incubation for about 30 h, SSEA-1 and 2C9 positive cells were detected in the germinal crescent of flight and control group silk chicken offspring embryos. After incubation of eggs for 2–2.5 days, SSEA-1 and 2C9 positive cells were detected in embryonic blood vessels of flight and control group silk chicken offspring embryos. After incubation of eggs for 5.5 days, PGCs in the dorsal mesentery and gonad could also be identified in flight and control group silk chicken offspring embryos by using SSEA-1 and 2C9 antibodies. Based on location and PAS staining, these cells were identified as PGCs. Meanwhile, at the stage of PGCs migration and then becoming established in the germinal ridges, no difference in SSEA-1 or 2C9 staining was detected between female and male PGCs in flight and control group silk chicken offspring embryos. Although there were differences in the profiles of PGC concentration between male and female embryos during the special circulating stage, changing profile of PGCs concentration was similar in same sex between flight and control group offspring embryos. We concluded that there is little effect on PGCs in offspring embryos of microgravity-treated chicken and that PGC development appears to be normal.     

Influence of microgravity on mitogen binding and cytoskeleton in Jurkat cells.

The effects of microgravity on Jurkat cells--a T-lymphoid cell line--was studied on a sounding rocket flight. An automated pre-programmed instrument permitted the injection of fluorescent labelled concanavalin A (Con A), culture medium and/or fixative at given times. An in-flight 1 g centrifuge allowed the comparison of the data obtained in microgravity with a 1 g control having the same history related to launch and re-entry. After flight, the cells fixed either at the onset of microgravity or after a or 12 minute incubation time with fluorescent concanavalin A were labelled for vimentin and actin and analysed by fluorescence microscopy. Binding of Con A to Jurkat cells is not influenced by microgravity, whereas patching of the Con A receptors is significantly lower. A significant higher number of cells show changes in the structure of vimentin in microgravity. Most evident is the appearance of large bundles, significantly increased in the microgravity samples. No changes are found in the structure of actin and in the colocalisation of actin on the inner side of the cell membrane with the Con A receptors after binding of the mitogen.     

Kinetics of amyloplast movement in cress root statocytes under different gravitational loads.

In order to investigate the movement of a statolith complex along the longitudinal axis of root cap statocytes under different mass accelerations, a series of experiments with Lepidium sativum L. in an automatically operating centrifuge during the Bion-11 satellite flight and on a centrifuge-clinostat have been performed. During spaceflight, roots were grown for 24 h under root-tip-directed centrifugal 1-g acceleration, then exposed to microgravity for 6, 12 and 24 min and chemically fixed. During the first 6 min of microgravity, the statoliths moved towards the cell center with a mean velocity of 0.31 +/- 0.04 micrometers/min, which decreased to 0.12 +/- 0.01 micrometers/min within subsequent 12-24 min period. The mean relative position of the statolith complex in respect to the distal cell wall (% of total cell length) increased from 24.0 +/- 0.5% in 1 g-grown roots to 38.8 +/- 0.8% in roots exposed for 24 min to microgravity, but remained smaller than in roots grown continuously in microgravity (48.0 +/- 0.7%). The properties of the statolith movement away from the distal pole of the statocyte were studied in roots grown for 24 h vertically under 1 g and then placed for 6 min on a fast rotating clinostat (50 rpm) or 180 degrees inverted. After 2 min of both treatments, the mean relative position of the statoliths increased by about 10% versus its initial position. Later on, the proximal displacement of amyloplasts slowed down under simulated weightlessness, while it proceeded at a constant velocity under 1 g inversion. In roots grown on the clinostat and then exposed to 1 g in the longitudinal direction, amyloplast sedimentation away from the central region of statocyte was similar at the beginning of distal and proximal 6-min 1-g stimulation. However, at the end of this period statolith displacement was more pronounced in proximal direction as compared to distal. It is proposed that statolith position in the statocyte of a vertical root is controlled by the force of gravity, however, the intracellular forces, first of all those generated by the network of the cytoskeleton, are manifested when an usual orientation of the organ is changed or the statocytes are exposed to microgravity and clinorotation.     

Performance of a simple Closed Aquatic Ecosystem (CAES) in space.

A simple Closed Aquatic Ecosystem (CAES) consisting of single-celled green algae (Chlorella pyrenoidosa, producer), a spiral snail (Bulinus australianus, consumer) and a data acquisition and control unit was flown on the Chinese Spacecraft SHENZHOU-II in January 2001 for 7 days. In order to study the effect of microgravity on the operation of CAES, a 1 g centrifuge reference group in space, a ground 1 g reference group and a ground 1 g centrifuge reference group (1.4 g group) were run concurrently. Real-time data about algae biomass (calculated from transmission light intensity), temperature, light and centrifugation of the CAES were logged at minute intervals. It was found that algae biomass of both the microgravity group and the ground 1 g-centrifuge reference group (1.4 g) fluctuated during the experiment, but the algae biomass of the 1 g centrifuge reference group in space and the ground 1 g reference group increased during the experiment. The results may be attributable to influences of microgravity and 1.4 g gravity on the algae and snails metabolisms. Microgravity is the main factor to affect the operation of CAES in space and the contribution of microgravity to the effect was also estimated. These data may be valuable for the establishment of a complex CELSS in the future.     

A direct approach to the study of the effect of gravity on axis formation in birds.

A system has been developed to enable the normal development of aborted very early uterine avian embryos, outside the female's uterus. The shell-less aborted egg was put into a foster shell of a sister egg, previously laid by the same female. The empty space between the shell and aborted egg was filled with artificial uterine fluid. The reconstructed eggs were incubated at 42 degrees C for 30 hours in a vertical position. The atmosphere contained a high concentration of CO2 (8-10%). At the termination of the 30 h the eggs were transferred to incubation at 37 degrees C in normal atmospheric conditions. Normal development has been recorded for a certain percentage of eggs incubated up to 12 days. In other cases abnormalities, arrested development or development of extraembryonic membranes only, without a sign of an embryonic axis, have been observed. The three important conclusions from the above experiments were: 1. It is possible to develop a closed, self-contained system, disconnected from the female's body, that would support the development of early uterine embryos. 2. The incidence of embryo-less extraembryonic membranes in such a system, is correlated with the degree of detachment of the "yolk" from the outer envelopes. 3. Such a system can be further developed into an experiment suited for microgravity conditions which will be an alternative to an experiment with live birds. The experiment will be aimed at testing the importance of gravity in changing the radially symmetrical avian blastoderm into a bilaterally symmetrical blastoderm.     

Cell fusion in space: plasma membrane fusion in human fibroblasts during short term microgravity.

During short-term microgravity in sounding rocket experiments (6 min.) the cytoskeleton undergoes changes and therefore it is possible that cell processes which are dependent on the structure and function of the cytoskeleton are influenced. A cell fusion experiment, initiated by a short electric pulse, was chosen as a model experiment for this sounding rocket experiment. Confluent monolayers of primary human skin fibroblasts, grown on coverslips, were mounted between two electrodes (distance 0.5 cm) and fused by discharging a capacitor (68 micro F; 250 V; 10 msec) in a low conductive medium. During a microgravity experiment in which nearly all the requirements for an optimal result were met (only the recovery of the payload was delayed) results were found that indicated that microgravity during 6 minutes did not influence cell fusion since the percentage of fused products did not change during microgravity. Within the limits of discrimination using morphological assays microgravity has no influence on the actin/cortical cytoskeleton just after electrofusion.     

Plant reproduction systems in microgravity: experimental data and hypotheses.

Elucidation of the possibilities for higher plants to realize complete ontogenesis, from seed to seed, and to propagate by seeds in microgravity, is a fundamental task of space biology connected with the working of the CELSS program. At present, there are results of only 6 spaceflight experiments with Arabidopsis thaliana, an ephemeral plant which will flower and fruit in orbit. Morphogenesis of generative organs occurs normally in microgravity, but unlike the ground control, buds and flowers mainly contain sterile elements of the androecium and gynoecium which degenerate at different stages of development in microgravity. Cytological peculiarities of male and female sterility in microgravity are similar to those occurring naturally during sexual differentiation. Many of the seed formed in microgravity do not contain embryos. Hypotheses to explain abnormal reproductive development in microgravity are: 1) nutritional deficiency, 2) insufficient light, 3) intensification of the influence of the above-mentioned factors by microgravity, 4) disturbances of a hormonal nature, and 5) the absence of pollination and fertilization. Possible ways for testing these hypotheses and obtaining viable seeds in microgravity are discussed.