Effects of space flight on Xenopus laevis larval development

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 27391-32 (1995)

Effects of Space Flight on Xenopus Zaevis Larval Development

E. SNETKOVA, N. CHELNAYA, L. SEROVA, S. SAVELIEV, E. CHERDANZOVA, S. PRONYCH, AND R. WASSERSUG Institute of Biomedical Problems (E.S., N.C., L.S.), Institute of Human Morphology (S.S.), and Moscow State University (E. (2.1, Moscow, Russia; and Department of Anatomy and Neurobiology, Sir Charles lhpper Medical Building, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada (5'2, R. W)

ABSTRACT Fifty-three fertilized Xenopus laevis embryos at early tail bud stage were launched into orbit aboard a Biocosmos satellite and remained in microgravity for 11.5 days. During this period, the embryos hatched and continued to develop as free-living larvae. Forty-eight individuals survived the mission. Upon recovery these tadpoles had smaller headshodies and proportionately longer tails than ground controls. Almost all the flight animals had caudal lordosis and consequently swam in backward somersaults. Compared to ground-based controls, their notochords were significantly larger in cross-sectional area and were deformed. Caudal muscle fibers were less dense and involuted in a fashion indicative of degeneration. In contrast, cranial muscles associated with buccal pumping did not differ between the flight and control animals.

Upon landing, the flight larvae were found to be negatively buoyant and lay on the bottom when they were not swimming. They had significantly smaller lungs than controls, suggesting that they had failed to inflate their lungs in microgravity. Additionally, the branchial baskets, gill filters and thymuses all showed signs of retarded development or degeneration.

The caudal deformity that we observed in the flight X. Zaevis has been independently observed in three other space flight experiments where embryos were launched then hatched in space. In contrast, Xenopus larvae from another orbital experiment that were raised from fertilization through hatching in space did not exhibit any caudal abnormalities. These divergent results suggest that either features of the launch itself (i.e., high acceleration and vibration) or an abrupt decrease in gravity during the tail bud stage detrimentally affects musculoskeletal development in anurans. 0 1995 Wiley-Liss, Inc.

Anurans (frogs and toads) have a long history in studies on the effects of various factors on development-including the effects of space flight and microgravity (Miquel and Souza, '91; Rahmann and Slenzka, '94). Most of these investigations focused on either early embryogenesis (e.g., Miquel and Souza, '91; Ubbels et al., '90) or vestibular development (e.g., Vinnikov et al., '83; Rahmann et al., '90; Neubert et al., '94). The effect of space flight on the development of most organ systems is still unknown.

In this paper we report on the effects of space flight on organogenesis in the African clawed frog, Xenopus laevis. For this study, fertilized X. laevis eggs were flown on a Russian Biocosmos satellite. They were launched at the early tail bud stage and recovered 11.5 days later as young tadpoles.

This experiment was initially proposed to address a single question about the effect of micro-

@ 1995 WILEY-LISS. INC.

gravity on anuran lung development. On earth, newly hatched Xenopus larvae swim up to the surface to inflate their lungs with air. However, in microgravity there is no "up"; thus we reasoned that Xenopus tadpoles hatched in space might not fill their lungs in a timely fashion. Indeed, Xeno- p u s tadpoles in closed containers during parabolic flight (Wassersug, '92; Wassersug and Souza, '90) were seen bumping into air bubbles but never en- tered them (Wassersug, pers. obs.). Schmalhausen ('86, p. 240) stated that amphibians must use their lungs in order for them to develop normally. As a prelude to our space flight experiment, Xenopus tadpoles were raised on the ground in well aer-

Received December 14, 1994; revision accepted April 20, 1995. Address reprint requests to Richard Wassersug, Department of

Anatomy and Neurobiolom, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia, B3H 4H7, Canada.

22 E. SNETKOVA ET AL.

ated water, but without access to the air-water interface. From that preliminary experiment we confirmed Schmalhausen’s claim; Xenopus tadpoles that did not fill their lungs shortly after hatching had abnormally small lungs and reduced growth rates overall (Pronych and Wassersug, ’94).

Our tadpoles that hatched in space had smaller lungs and reduced body volume. However, they also revealed many other morphological differences from control larvae that developed on the ground. In this paper we report on several of the differences that we observed between the flight and control animals and discuss our results in light of other experiments on Xenopus development in space.

MATERIALS AND METHODS An adult female Xenopus Zaevis, obtained from

a colony at Moscow State University, was bred with a male X. Zaevis obtained from Nasco (Fort Atkinson, Wisconsin), a commercial supplier. Fol- lowing a standard laboratory breeding protocol (Etheridge and Richter, ,781, human chorionic go- nadotropin was used to induce ovulation, am- plexus, and fertilization in these frogs. Several hundred fertilized eggs were obtained. One hundred fifty-six of these were divided into three groups.

The first group of 53 individuals, designated “flight animals,” was placed in a rectangular plexiglass box that had been used previously on Biocosmos satellites. The box had an internal volume of 3.0 L and was half filled with 1.5 L of boiled, aerated, standing tap water at 215°C. The oxygen content, salinity, and pH of the water in the box was measured at that time. The box was then sealed shut with a metal plate and rubber gasket. At that time the larvae were at stage 6.5-7 of Nieuwkoop and Faber (’56). The box was loaded into the satellite and launched approximately 40 hr later when the tadpoles were at Nieuwkoop and Faber stage 25, based on developmental rates among control specimens (see below). The box remained sealed until the termination of the experiment.

A second group, also of 53 individuals, was kept as a “synchronous control.” These embryos were placed in an identical box to the flight animals, but were maintained on the ground for the dura- tion of the experiment. They too were sealed in their container, with 1.5 L of the same water used for the flight animals. As ground controls, they were exposed to neither microgravity nor the vi- brations and accelerations of launch and landing.

The remaining animals were maintained as

a “free control” in a standard 30 L aquarium. The aquarium was filled with the same water used above, but was open to the air during the experiment.

The animals were not fed during the experiment although they were free to eat microorganisms that grew spontaneously in the surrounding water. The temperature in the Biocosmos satellite was monitored continuously during the flight and the synchronous control was maintained at the same temperature. The temperature at the time of launch was 19°C and ranged from 19-22°C for the first 9 days of the flight. However, during the last 2 days of the flight the temperature rose from 22°C to over 26°C.

The Biocosmos satellite was launched on De- cember 29, 1992, from Plesetsk, Russia, and was recovered near Karaganda, Kazakstan, 11.5 days later, on January 10, 1993. The flight and synchronous ground control boxes were opened at room temperature 2 hr after landing. The physicochemical features of the water were remeasured at that time (Table 1) and the behavior of the tadpoles was videotaped. Three tadpoles each from the flight and synchronous control groups were separated from the rest for further observation over the following days. Their swimming behavior was carefully observed for any indications of readaptation to the earth’s 1-G environment.

The remainder of the surviving tadpoles in the three groups (Table 2) were then fixed in 4% neu- tral buffered formaldehyde solution for detailed external morphometric analysis. The majority of the flight and synchronous ground control animals were then dehydrated in serial solutions of alco- hol, embedded in paraffin, serially sectioned at 10 p, and stained with hematoxylin and eosin. Some specimens were cut frontally and others in cross section. This was done to allow us to view and compare both cranial and caudal muscles in cross section, in reference to the long axis of the muscles themselves. A Zeiss IBAS Image Analyzer

TABLE 1. Physicochemical properties of the water i n the flight container and synchronous control at the beginning

and end of the experiment

Salinity PO2 pH (mg/L) (% saturation)

Flight container Start of experiment 8.00 343 92 End of experiment 6.45 378 80

Start of experiment 8.00 343 92 End of exDeriment 6.60 371 81

Control container

XENOPUS DEVELOPMENT IN SPACE 23

TABLE 2. External morphology of the flight animals (n = 45), synchronous ground control (n = 49) and free ground control (n = 49) animals"

Character (in mm)

Group Synchronous Free

Flight controls controls

Tbtal length Snout-vent length Head-body length Tail length Snout to front of brain Snout to front of branchial baskets Cranial length End of branchial basket to end of bodv

10.79 f 0.10 4.2 -c 0.03

3.60 f 0.03 7.14 2 0.09 0.62 2 0.01 2.04 f 0.02 1.42 2 0.01 1.60 2 0.02

10.78 2 0.06 4.6 2 0.02

4.04 2 0.02 6.70 f 0.05 0.85 f 0.01 2.56 f 0.02 1.71 r 0.01 1.48 & 0.01

11.0 2 0.07 4.9 2 0.03

4.35 2 0.02 6.65 2 0.06 0.98 +: 0.01 2.80 f 0.02 1.85 2 0.01 1.52 2 0.02

*All features differ between the larvae of the three groups a t the P < 0.025 level except where noted in the text. The sample sizes here differ from those given in the text because animals that died in the synchronous control and flight groups after landing, but before fixation, were excluded from study, and one animal was arbitrarily removed from the free control group to match sample sizes between the two control groups.

was used to calculate areas, volumes, and surface however, did not differ in terms of the relative topography for various organs and tissues ob- length of the tail compared to the body (t-test, P served in the serial sections. > 0.1). Thus the shorter head-body and propor-

tionately longer tail in the flight group is not due simply to the space flight animals being absolutely RESULTS

Survivorship and developmental stage smaller in any way, but must instead have re- Survivorship of tadpoles in the three groups

was: flight specimens 48 (91%); synchronous ground controls 52 (98%); and free ground controls 49 (98%). The differences are not statisti- cally significant (Chi-square test, P > 0.5). These are all high survivorships despite the fact that none of the specimens were actively fed during the experiment. All of the animals were at Nieu- wkoop and Faber ('56) stage 47, which corresponds to a young, free-swimming, freely feeding tadpole.

Morphology External morphology

sulted from conditions associated with space flight. Most of the flight animals exhibited some cau-

dal lordosis (hyperextension; Fig. 2). This deformity affects the ability of the tadpoles to swim normally and is discussed below in the section on behavior.

The flight animals were lighter in color than the controls. Examination under the microscope

A representative flight tadpole is shown at the top in Figure 1, above a tadpole from the synchronous control and one from the free control. The tadpoles differed in a variety of ways, sum- marized in Table 2. Many of these differences simply reflect the fact that the flight animals were smaller overall than the synchronous controls which were, in turn, smaller than the free controls. However, the flight animals had significantly longer tails (t-test, P c 0.005) and shorter and smaller bodies (P c 0.005) for their total length than did the tadpoles in either control group.

The two controls differed from each other in most features as well. These appear to be related to the fact that the free which were raised in an open tank at lower density, had access to better nutrition. The two control groups,

Fig. 1. Three representative X. Zaeuis larvae from (top) the flight group, (middle) the synchronous ground control, and (bottom) the free ground control. All tadpoles are 10.8 mm long. The flight tadpoles have proportionately smaller bodies and longer tails than control larvae. They are also more lightly colored due to aggregation of melanin in their mel- anocytes.


Fig. 2. Gross variation in flight tadpoles. In this sample of tadpoles, the caudal deformities increase from lower right to upper right in a clockwise direction. The most common morphologies are shown on the left; the least common morphologies are on the right. The majority of tadpoles show mod- erate to severe hyperextension (lordosis) of the tail. For scale, the least deformed larva (lower right) is 10.8 mm long.

showed that this was not due to lack of melano- cytes, but to aggregation of the melanin in the flight animals (T. Ostroumova and N. Luchin- skaya, personal communication). Light color in Xenopus tadpoles on earth commonly reflects mor- bidity or adaptation to the dark. The tadpoles in both the flight and synchronous control were maintained in the dark, so dark adaptation alone cannot explain these pigment differences.

Lungs The lungs in the flight group were significantly

smaller in volume than in the synchronous controls (t-test, P e 0.0006; Table 3). They had on average half (49%) the volume of those in the controls. This difference is greater than one would expect if lung size were simply proportional to body length in the flight versus control larvae.

The difference in the size and shape of the lungs is shown in Figures 3 and 4. Xenopus larvae normally have a very short, but wide stem (trachea) to their lung buds and this is the condition seen in the synchronous controls (Fig. 3). This passage was much smaller in the flight animals. In addi- tion, the lung buds, when present at all in flight animals, tended to be small and uninflated (Fig. 4).

Notochord The notochords of the flight specimens differed

from controls in two major ways. First, for the same distance along the tail, the notochord in the flight animals was, on average, 1.15 times larger in cross-sectional area than that of the synchronous controls (t-test, P c 0.05; Table 3). Secondly, the notochords were consistently deformed in the flight animals (Fig. 5). In normal Xenopus larvae the notochord has dorsal and ventral recesses for the spinal cord and the caudal artery, respectively. The lateral regions of the notochord bow outward in a symmetrical fashion. These features, shown in a control specimen in Figure 5, are grossly and variably misshapen in the flight specimens. The figures and data in Table 3 were taken from a specific region of the notochord immediately caudal to the anus. The sections, however, revealed malformation of the notochord along its length, both within the trunk region and more distally along the tail.

Given the gross deformities seen in the tails of the space flight tadpoles, it is not surprising that the notochords are malformed. After all, the notochord is the only axial element past the anal region supporting the vertebrae-less tail of Xenopus larvae (Wassersug, '89). We have not attempted to quantify and correlate the amount of curvature in the tail with the amount of notochordal malformation. It is our impression, though, that tadpoles with relatively little deformation of the tail still had grossly malformed notochords.

T B L E 3. Volumetric data on samples of the thymus, lungs and notochord from space flight us. synchronous ground control X. laevis larvae

Tissue Treatment N Mean 2 standard error Range

Thymus Flight 10 (3.95 0.40) lo4 (1.79-6.45) x lo4 Control 10 (1.52 0.12) lo5 (9.88-19.80) x lo4

Lung Flight 18 (1.08 2 0.30) x lo7 (2.78-53.60) x lo6 Control 10 (2.20 0.11) lo7 (1.64-2.60) x l o 7

Notochord Flight 13 (2.86 2 0.13) x lo6 (1.70-3.52) x lo6 Control 20 (2.49 2 0.05) x lo6 (1.92-2.83) x lo6

All volumes are in pm3 and were calculated from serial sections traced into an image analyzer. Those for the notochord do not represent the whole chord but a segment 0.5 mm long (50 sections x 10 pm) at the base of the tail, immediately caudal to the anus. All means between the space flight and controls differ significantly (see text).


Fig. 3. Frontal sections of trachea and lung buds in a representative flight X. laevis larva (right) and a synchronous ground control specimen (left). These sections were carefully selected to show the lungs at near maximal cross-sectional area. Note the expanded tracheal region in both specimens, but the virtual absence of lung buds in the flight animal. Scale bar = 0.5 mm.

Abbreviations bb branchial basket nc notochord cb gill filters on 1st and 2nd ceratobranchials t r trachea ch ceratohyal

Branchial baskets The branchial baskets of the space flight ani-

mals differed from those of the controls in both size and shape. They were smaller in the flight animals, but not disproportionately so, given the smaller headshodies of those larvae. The ceratobranchials were more transversely than longitu- dinally oriented in the flight animals than the controls (Fig. 6), leading to more spherical branchial baskets overall.

The gill filters, which arise from the ceratobranchials, were not as elaborately developed in the space flight animals as in the controls; consequently the filter clefts between the ceratobranchials were larger in the flight animals. In Xenopus, the filter rumes normally have fine tertiary and higher order folds (Wassersug, '80; see also Fig. 3 in Seale et al., '82). These higher order folds were simply not present in the space flight animals (Fig. 7).

Thymus The thymus gland in Xenopus larvae is a

small, distinct organ located midway between the eye and the ear. The thymus cells stained extremely heavily with hematoxylin in both our experimental and control tadpoles (Fig. 8). The volume of the thymus in the flight animals was on average approximately one-quarter (26%) that of the synchronous ground controls (Table 3). Several of the space flight specimens exhibited some involution of the thymus. An extreme example of either failure to develop or second- ary degeneration of the thymus is shown in Fig- ure 8, lower right.

Musculature The axial muscles of the flight larvae exhib-

ited a variety of classic abnormalities associated with muscle degeneration. These can be


Behavior

The majority of the flight tadpoles lay still at the bottom of their containers at the end of the experiment. This benthic behavior indicated that the tadpoles were negatively buoyant compared to both control groups, which did not rest on the bottom but swam continuously within the water column. Whether flight tadpoles were swimming or resting on the bottom, they seemed poorly oriented in reference to the gravity vector. For example, some would lie on their sides, others would swim upside-down.

The flight tadpoles that did swim, swam in tight, backward loops; i.e., they made backward somersaults or, what in aviation are called inside loops. The loops were not always perfect. If the tadpoles veered to the right or left, the loops became extended spirals. Even tadpoles with relatively straight tails, such as the one shown in the lower right of Figure 2, swam in inside loops.

Fig. 4. Three dimensional reconstruction of the tracheal region and lung buds in a representative flight X . laevis larva (left) and a synchronous control specimen (right). The top figures were generated by a computerized image analyzer from traced serial sections of the lungs. The bottom figures were redrawn by an artist to smooth surfaces and clarify depth. All figures are reproduced at the same magnification. The arrow through each drawing indicates the direction of the lungs in reference to the direction of the long axis in the larvae. The maximum rostrocaudal length of the trachea and lung in the control specimen (along the axis of the arrow) equals 0.75 mm.

seen in Figures 5, 9, and 10. The axial muscle fibers were infolded (Fig. 9) and widely spaced. Table 4 compares the density of the axial muscle fibers in ten space flight and synchronous ground control specimens and reveals that the fibers are less than half as numerous (48%) in the flight animals as in the controls (t-test, P c 0.0001).

Studies with mammals suggest that postural muscles are more likely to be detrimentally affected by space flight than other muscles (Roy et al., '911, and this seems to be true for our tadpoles as well. Figure 11 shows two muscles in longitudinal section from a space flight specimen. The top muscle is the orbitohyoideus, which is the primary muscle for depressing the buccal floor during respiration and feeding. It has none of the signs of degeneration that are seen in the axial muscle of the same specimen.

Regulation to normality The three flight animals that were maintained

alive initially swam as just described. However, by the third day postflight they were swimming in a more normal, midwater position (cf. Hoff and Wassersug, '861, with the head tipped downward, suggesting that they had succeeded in inflating their lungs. They were also observed swimming to the surface to take a gulp of air. These animals had moderately curved tails, like the tadpole shown in the lower left of Figure 2. They were kept alive for only 1 week because no arrange- ments were made to feed or otherwise maintain them. During this period there was no indication that their tails became any straighter.

DISCUSSION Our data reveal many behavioral and morpho-

logical differences between Xenopus larvae hatched in space and synchronous controls hatched and maintained on the ground. Since the controls were exposed to neither the microgravity of space nor the acceleration and vibration profile of launch and landing, we cannot conclude that these differences are due to microgravity per se. Never- theless the differences are profound, and they have implications to growth and development both in space and postflight.

First, consider the lungs. The small and typi- cally uninflated lungs in our flight specimens support the hypothesis that tadpoles that hatch in space may be unable to orient toward an air


Fig. 5. Cross sections of the tail from three representative flight X . laeuis larvae compared to a synchronous ground control (upper left). These sections are all from the base of the tail, just caudal to the anus. The spinal cord can be seen in each section dorsal to the notochord. Note the gross asym- metries in the notochord of the flight animals compared to that of the control. Scale bar = 0.1 mm.

bubble and properly fill their lungs (see Souza et al., '95). An alternative hypothesis is that the difference we observed was due to a more direct effect of microgravity on pulmonary tissue. We believe this is unlikely, since embryonic mouse lung tissue grown in tissue culture on the US Space Shuttle was essentially normal (Spooner et al., '94). The presence of any air within the lungs of the flight tadpoles may reflect pulmonary ven- tilation that occurred between the time that the tadpoles returned to earth and when they were

fixed postflight. Ground-based studies, where X. Zaevis larvae have been prohibited from filling their lungs for a few days or weeks, indicate that Xenopus larvae can survive and grow without pulmonary respiration, but that they have decreased growth rates, increased mortality, and delayed metamorphosis (Pronych and Wassersug, '94; Wassersug and Murphy, '87). Our space flight results with X. Zaevis suggest that otherwise normal aquatic amphibians may be similarly handicapped in space flight because of the inability to venti- late their lungs.

The decrease in the size of the thymus in our flight tadpoles also may have deleterious implications to long term growth and well-being of amphibians in space. The thymus in anurans, as in mammals, is a critical component of the developing immune system. The changes that we observed in the thymus of flight tadpoles are severe enough that it is difficult to imagine that they could have occurred solely in the few hours between reentry and fixation on the ground. There is growing, although not consistent, evidence that space flight alters immune cell function tcf. Nash et al., '92; Sonnenfeld et al., '92; and Chapes et al., '92 for recent reviews). Our space flight tadpoles had slightly lower survivorship than control specimens. Yet the difference was not great enough to conclude that the flight animals were directly immunocompromised at the time of fixation. The results do suggest, however, that it might be worth assessing the immunological status of amphibian larvae raised on future space flights.

The decreased body size and proportionately longer tails in our space flight tadpoles are difficult to explain. The small size of the abdomen in the flight larvae may reflect starvation, except that the controls were not so severely affected and, like the flight animals, were unfed during the experiment. It may be that the microorganisms, which could multiply in the tank as a source of food for the tadpoles, were themselves growing at a lower rate in the flight container than in the ground control tanks. Unfortunately, no effort was made to sample the water during or after the flight for microorganisms.

As indicated in Table 1, the physicochemical properties of the water in both the flight container and the controls differed very little at the end of the experiment. In both containers, the pH and pOz went down slightly whereas the salinity rose slightly. These shifts are neither unexpected nor extreme. Thus, it is unlikely that differences in the morphology of the flight tadpoles can be ac-


Fig. 6. Frontal sections of the left branchial basket in a representative flight X . Zaeuis larva (right) and a synchronous ground control specimen (left). Rostra1 is toward the top. These sections were selected to show the baskets near

maximal cross-sectional area, although the section on the left is slightly more dorsal than the one on the right. The branchial baskets are round and the ceratobranchials more transversely oriented in the flight specimen. Scale bar = 0.5 mm.

counted for as a direct result of these changes in water chemistry.

The shape changes in the branchial baskets and the reduced density of the gill filters in the flight specimens may reflect the overall deterioration in these animals. At the same time, since the density of the gill filters correlates with the ability of the larvae to filter-feed (Wassersug, 'SO), the reduced gill filters may be part of the cause for the larvae's poor condition.

The head in tot0 is proportionately smaller in our flight animals than in the controls (Fig. 1). The literature on the effects of altered gravity on cranial/ neural development in aquatic vertebrates has re- cently been reviewed by Rahmann and Slenzka ('94). At one extreme, hypergravity (of 2 to 4 G) in- duces a 15% reduction in total brain volume in the cichlid fish Oreochromis mossambicus, with the most pronounced shrinkage in the optic tectum and

cerebellum (Slenzka et al., '90; Rahmann et al., '92). But the majority of the studies to date with post- metamorphic fish and amphibians in hypo-G-4- ther simulated on a clinostat or from actual space flight-have found few gross effects. The same is not true for premetamorphic Xenopus embryos. Neff et al. ('93) report enlarged heads and eyes in X. Zaeuis embryos raised on a clinostat. Similarlx Souza et al. ('94) found significantly larger brain ventricles, head and eyes in hatchling X. Zaeuis larvae raised in microgravity on the US Space Shuttle. These results do not necessarily contradict our results, since our tadpoles were approximately 1 week older than those in either the Neff et al. or Souza et al. studies. The differences do underline, though, the fact that the effects of altered gravity on amphibian organogenesis may differ greatly depending on the developmental stage examined (see also Malacinski, '90; Ubbels, '92).


Fig. 7. Frontal sections of a portion of a ceratobranchial in a representative flight X. laevis larva (bottom) and a synchronous control specimen (top). These sections show the folding pattern of the gill filters. Note the less extensive folding of the filter ruffles in the space flight vs. the control specimen. Scale bar = 0.1 mm.

Perhaps the most profound histological difference between our space flight X. laevis specimens and the controls is in the axial musculoskeletal system. The literature on the effects of space flight on muscle in rodents is voluminous (for a lead into the recent literature see articles in Journal of Ap- plied Physiology, Suppl. 1992, 73(2):333-1068). Whereas there is disagreement as to the cause of damage, there is no question that space flight is associated with muscular deconditioning in mammals, and that not all striated muscle is equally affected (Roy et al., '91). The axial muscle in our specimen shows many signs of degeneration, consistent with what has been reported for postural muscle in mammals. The cranial muscles of our tadpole, however, do not show the same deterioration. It is doubtful that this difference between the muscles in the head and in the tail of our flight specimens is due simply to differences in loading. The tadpoles in space would be neutrally buoy-

Fig. 8. Frontal sections of the thymus from three representative flight X . laeuis larvae compared to a synchronous ground control (upper left). These sections were selected to show the thymus in maximal cross-sectional area. The thymus in the flight groups was constantly smaller and, in the extreme (lower right), it is involuted. Scale bar = 0.1 mm.

ant and would not have had to swim to maintain their position in the water. However, videotapes of Xenopus tadpoles made in space on a 1992 NASA Space Shuttle mission (Wassersug, pers. obs.; see below), indicated that the animals were active in microgravity Thus, even in microgravity both head and tail muscles were likely working against the mechanical resistance of the water, either to propel water into the mouth or the tadpole through the water.

On the other hand, it is likely that the malformation of the notochord and the degeneration of the surrounding musculature are related, although not necessarily in a direct causal fashion. Our work is the first to show detrimental effects of space flight on the notochord. In retrospect, given the severe hyperextension of the tails in our


Fig. 9. Cross hemi-sections of a portion of the tail from a synchronous ground control X . laeuis larva (top) compared to a flight specimen (bottom). These sections are both from near the base of the tail, caudal to the anus. Dorsal is to the left. The notochords in the two sections are aligned. Note the decreased density of muscle fibers in the tail of the flight animal, as well as the involuted, degenerative nature of that musculature. Scale bar = 50 pm. See also Figure 5.

flight specimens, it would have been remarkable if they had not had some gross defects in their skeletal system. The specific deformation leading to the hyperextended tail in our space flight tadpoles is not known, but the deformity and its effect on the swimming of the tadpoles-forcing them to swim in backward somersaults-is in- triguing in light of results from other recent space flight experiments.

In two investigations separate from this one, X. laevis eggs were fertilized in space. In one (NASA's 1991 Space Shuttle mission IML-11, no effort was made to raise the embryos through hatching, but in the other (NASA's 1992 Space Shuttle mission SL-J), the embryos hatched in space and came to earth as freely swimming anuran tadpoles (Souza et al., '95). Surprisingly, those tadpoles showed neither axial deformity nor backward loop swimming. In both space and postflight on earth, they showed no tendency to swim in backward loops.

Fig. 10. Frontal (longitudinal) hemi-section of a portion of the notochord and surrounding axial muscle from a synchronous ground control (top) X. Zaeuis larva compared to a flight specimen (bottom). These sections are both from near the end of the body and base of the tail. Rostra1 is to the left. The notochords in the two sections are aligned. Scale bar = 0.1 mm. Compare to the cross section of the tail in approximately the same region (Fig. 9).

Their tails were straight and not grossly differ- ent from controls. On the same SL-J mission, one of us (WW) sent up small culture flasks with prefertilized X. laevis eggs. Those eggs were launched at approximately the same stage as our Biocosmos embryos, and like our embryos, they hatched in space. In contrast to the other SL-J tadpoles, which began their embryonic life in space, these tadpoles exhibited the same hyperextended tails and backward somersaults seen in the Biocosmos specimens. The same deformity in

TABLE 4. Number of muscle fibers from the base of the tail in a standardized, rectangulal; sampling quadrat of

1634.53 um2 area

Treatment N Mean f standard error Range

Flight 10 10.80 * 0.67 7.67-13.67 Control 10 22.43 f 0.78 19.33-27.0

The differences in muscle fiber density are significant (t-test, P < 0.OOOl).


dition, however, does not afflict Xenopus tadpoles that complete all of their embryogenesis in microgravity. Together these results suggest that some aspect of the launch is detrimental to the embryos. Whether this is the vibration of the launch, the acceleration of the launch, or the abrupt decrease in gravity when orbit is reached is uncertain. A variety of ground-based studies have examined the effect of hyper- gravity and mechanical disturbance on Xeno- pus embryogenesis (e.g., see Ubbels e t al., '90; Neff et al., '93, for recent references) but none has reported the abnormalities that we have observed in our Biocosmos tadpoles or that were seen in the other space flight experiments where Xenopus eggs were fertilized before launch. These studies do not rule out hyper-G or vibration as causes of the deformities. More research in this area may be warranted-par- ticularly to assess the developmental stages most sensitive to these stresses.

As a final note, the deformities we observed have not been reported in Rana eggs that were fertilized on the ground and completed development, through hatching, in microgravity (Young and Tremor, '68). Those embryos, however, were launched at a much younger stage than either those raised in flasks during NASA's SL-J mission o r our tadpoles from the Bio- cosmos mission.

ACKNOWLEDGMENTS Fig. 11. Longitudinal sections of cranial (above) vs. axial

(below) muscles from a space flight X. laeuis larva. The muscle shown above is the orbitohyoideus muscle, which depresses the buccal floor. The muscle shown below is from the caudal region of the trunk near the developing kidneys (visible in the upper left). Whereas the axial muscle has degenerated, the cranial muscle has not. Scale bar = 0.1 mm. Compare to Figures 9 and 10.

the tail and the abnormal swimming behavior have now been seen in X. Zaeuis larvae from a third experiment, which flew on NASA's 1993 Space Shuttle D-2 mission (Neubert et al., '94; see their Fig. 2). Once again, the eggs were fertilized on the ground and launched as embryos that hatched in space.

In conclusion, Xenopus embryos tha t are launched during certain critical, but yet t o be identified, embryonic stages develop severe abnormalities in their axial musculoskeletal system and are consequently handicapped in their ability to swim along a straight path. This con-

This research was supported by the Institute of Biomedical Problems in Russia, the Canadian Space Agency, and the Natural Science and Engineering Research Council of Canada. We thank N. Besova, S. Black, Luc Bourque, Monika Fejtek, Susan Hall, Ian Mobbs, Alan Mortimer, Leena 'Ibmi and Steve Whitefield for their support and assistance.

LITERATURE CITED Chapes, S.K., D.R. Morrison, J.A. Guikema, M.L. Lewis, and

B.S. Spooner (1992) Cytokine secretion by immune cells in space. J. Leukoc. Biol., 52:104-110.

Etheridge, A.L., and S. Richter (1978) Xenopus laeuis: Rear- ing and breeding the African clawed frog. Nasco, Fort Atkinson, Wisconsin, 14 pp.

Hoff, K.vS., and R.J. Wassersug (1986) The kinematics of swimming in larvae of the clawed frog, Xenopus laevis. J. Exp. Biol., 222:l-12.

Malacinski, G.M. (1990) Reproduction, development, and growth of animals in space. In: Fundamentals of Space Bi- ology. M. Asashima and G.M. Malacinski, eds. Japan Scien- tific Societies Press, Tokyo, pp. 123-138.

Miquel, J., and K.A. Souza (1991) Gravity effects on reproduction, development, and aging. Adv. Space Biol. Med., 1:71-97.


Nash, PV., I.V. Konstantinova, B.B. Fuchs, A.L. Rakhmilevich, A.T. Lesnyak, and A.M. Mastro (1992) Effect of spaceflight on lymphocyte proliferation and interleukin-2 production. J. Appl. Physiol., 73(Suppl.):186S-l90S.

Neff, A.W., H. Yokota, H.M. Chung, M. Wakahara, and G.M. Malacinski (1993) Early amphibian (anuran) morphogen- esis is sensitive to novel gravitational fields. Dev. Biol.,

Neubert, J., W. Briegleb, A. Schatz, B. Bromeis, A. Linke- Hommes, H. Rahmann, K. Slenzka, E. Horn, K. Esseling, and C. Sebastian (1994) Spacelab mission D-2 experiment statex “Gravity perception and neuronal plasticity.” Com- parative investigations of near weightlessness effects on the development and function of the gravity perceiving system of two waterliving vertebrates (Xenopus laeuis Daudin, Oreochromis mossambicus). In: Proc. 5th Eur. Symp. on “Life Sciences Research in Space,” ESA Publications Divi- sion, Noordwijk, The Netherlands, pp. 77-81.

Nieuwkoop, PD., and J. Faber (1956) Normal table of Xeno- pus laeuis (Daudin). North Holland Publishing Co., Amster- dam, The Netherlands, 243 pp.

Pronych, S., and R.W. Wassersug (1994) Lung use and development inXenopus laevis tadpoles. Can. J. Zool., 72:738-743.

Rahmann, H., and K. Slenzka (1994) Influence of gravity on early development of lower aquatic vertebrates. In: Proc. 5th Eur. Symp. on “Life Sciences Research in Space,” ESA Publications Division, Noordwijk, The Netherlands, pp. 147-152.

Rahmann, H., K. Slenzka, and R. Hilbig (1990) Effect of hyper-gravity on the swimming behavior of aquatic vertebrates. In: Proc. 4th Eur. Symp. on “Life Sciences Research in Space,” ESA Publications Division, Noordwijk, The Neth- erlands, pp. 253-263.

Rahmann, H., K. Slenzka, K.H. Kortje, and R. Hilbig (1992) Synaptic plasticity and gravity: Ultrastructural, biochemical and physico-chemical fundamentals. Adv. Space Res., 12:(1)6341)72.

Roy, R.R., K.M. Baldwin, and V.R. Edgerton (1991) The plasticity of skeletal muscle: Effects of neuromuscular activity. In: Exercise and Sports Science Reviews. J. Holloszy, ed. Williams and Wilkins, Baltimore, Vol. 19, pp. 269-312.

Schmalhausen, 1.1. (1986) Factors of Evolution: The Theory of Stabilizing Selection. University of Chicago Press, Chi- cago, Illinois, 297 pp.

Seale, D.B., K. Hoff, and R. Wassersug (1982) Xenopus laeuis larvae (Amphibia, Anura) as model suspension feeders. Hydrobiologia, 87:161-169.

Slenzka, K., R. Anken, A. Bauerle, K.H. Kortje, U. Paulus, and H. Rahmann (1990) Morphological, electronmicro-

155:2 70-2 74.

scopical and biochemical aspects of hyper-gravity conditions during early ontogenetic development of cichlid fish. In: Proc. 4th Eur. Symp. on “Life Sciences Research in Space,” ESA Publications Division, Noordwijk, The Netherlands, pp.

Sonnenfeld, G., A.D. Mandel, I.V. Konstantinova, W.D. Berry, G.R. Taylor, A.T. Lesnyak, B.B. Fuchs, andA.L. Bakhmilevich (1992) Spaceflight alters immune cell function and distribu- tion. J. Appl. Physiol., 73(Suppl.):191S-l95S.

Souza, K., S . Black, R.J. Wassersug, and M.D. Ross (1994) The effects of spaceflight on amphibian fertilization, development and behavior. In: Proceedings of “In Space ’94,” Japan Space Utilization Promotion Center, Tokyo,

Souza, K., S. Black, and R. Wassersug (1995) Amphibian development in the virtual absence of gravity. Proc. Natl. Acad. Sci. USA, 92:1975-1978.

Spooner, B.S., l? Hardman, and A. Paulsen (1994) Gravity in mammalian organ development: Differentiation of cultured lung and pancreas rudiments during spaceflight. J. Exp.

Ubbels, G.A. (1992) Developmental biology on unmanned space craft. Adv. Space Res., 12:(1)117-(1)122.

Ubbels, G.A., W. Berendsen, S . Kerkvliet, and J. Narraway (1990) Fertilization of Xenopus eggs in space. In: Proc. 4th Eur. Symp. on “Life Sciences Research in Space,” ESA Publications Division, Noordwijk, The Netherlands,

Vinnikov, Ya.A., O.G. Gazenko, D.V. Lychakov, and L.R. Pal’mbakh (1983) Razvitiye Vestibulyarnogo Apparata v Usloviyakh Nevesomosti. Zhurnal Obshchey Biologii,

Wassersug, R. (1980) Internal oral features of tadpoles from eight anuran families: functional, systematic, evolutionary and ecological considerations. Misc. Publ. Mus. Nat. Hist., U. Kansas, No. 68:l-146.

Wassersug, R.J. (1989) Locomotion in amphibian larvae (or “Why aren’t tadpoles built like fishes?”). Am. Zool., 29:65-84.

Wassersug, R.J. (1992) The basic mechanics of ascent and descent by anuran larvae (Xenopus laevis) . Copeia,

Wassersug, R.J., and A.M. Murphy (1987) Aerial respiration facilitates growth in suspension-feeding anuran larvae (Xe- nopus laeuis). Exp. Biol., 46:141-147.

Wassersug, R.J., and K.A. Souza (1990) The bronchial diver- ticula of Xenopus larvae. Natunvissenschaften, 77:443-445.

Young, R.S., and J.W. Tremor (1968) The effect of weightlessness on the dividing egg of Rana pipiens. Bioscience,

321-328.

pp. 113-136.

ZOO^., 269:212-222.

pp. 249-254.

44:147-163.

1992:890-894.

18:609-615.

Date post:	06-Nov-2023
Category:	Documents
Upload:	independent
View:	0 times
Download:	0 times

Effects of space flight on Xenopus laevis larval development

Documents