Vertebrate Biology in Microgravity

Richard J Wassersug. American Scientist. Volume 89, Issue 1. Jan/Feb 2001.

Space biology is a young science, having come into existence less than a half-century ago. Yet it is almost as old as spaceflight itself, for it was a mere 30 days after Sputnik was launched in October 1957 that the Siberian dog Laika was flying laps around our planet. Since then people have visited the moon and have lived in space for more than a year. Still, our understanding of how spaceflight affects living organisms remains rudimentary.

We do know, though, that biological processes that occur above the cellular level cannot be predicted from the responses of their cells or tissues alone. For example, astronauts lose about one percent of the mineral density in their heel bones per month in space. But bone cells do not show the necessary shift in their physiology to account for this reduction when raised in tissue culture in space. Much of what we have discovered lately about whole organisms in space has led to unanticipated challenges for space biology-challenges that will have to be met in the coming decade, if we are going to make substantial progress in this field.

The truth is that we will not have conquered the space environment until we can safely complete a life cycle in that setting, and this will require us to learn much more about how creatures reproduce, grow and behave in space.

The Space Environment

Space biologists distinguish the effects of spaceflight from those of microgravity. Extended exposure to microgravity requires orbital flight, and even the gentlest of launch vehicles produces enormous amounts of noise and vibration, plus elevated G forces, until orbital velocity is achieved.

Once in orbit, machines and astronauts continue to produce vibrations that are tricky to control because there is little to damp them. The space environment also exposes animals to high-energy radiation unlike anything they experience on Earth. To control for these and other extraneous factors (for example, fluctuations in atmospheric pressure as astronauts enter and exit a spacecraft), biologists studying the effects of microgravity per se ideally need onboard centrifuges that, by rotating slowly, can expose control specimens to the level of gravity found on Earth’s surface. Such incubating centrifuges have been rarely available in the past, but are scheduled to be an integral part of the International Space Station.

Development in Space

No vertebrates have been raised from conception to sexual maturity in the absence of gravity. Thus a major focus of the initial biological research planned for the Space Station will be the growth and reproduction of such organisms.

Among amniotes, no birds or reptiles have bred in orbit, although fertilized chicken (Gallus domesticus) and quail (Coturnix coturnix) eggs have flown on several occasions. Young chick embryos have not survived. Tatsuo Suda and colleagues at Showa University recently demonstrated that, in the absence of gravity, the yolk, which is less dense than the albumin, fails to maintain proximity to the shell, which is necessary for gas exchange. Eggs launched at later developmental stages have fared better. Quail eggs that were fertilized on the ground have hatched on the Mir Space Station, but yielded hatchlings that held their heads awry and would not or could not spontaneously feed.

Experiments with mammals are equally limited and have also given mixed results. (Ever since Sally Ride’s flight in 1983, NASA has flown co-ed crews, including a husband and wife team on one mission in 1992, but no astronauts have reported getting pregnant in space.) In 1979, female rats mingled for two and a half weeks with male rats on an unmanned Russian satellite, but none returned pregnant. In 1983, rats impregnated before launch spent a brief 4.5 days on a similar satellite and were able to give birth five days later, on return to Earth.

More extensive experiments with mammalian reproduction in space were jointly sponsored by the National Institutes of Health (NIH) and NASA and were carried on the Space Shuttle in 1994 and 1995. The experiments involved pregnant rats that spent 11 and 9 days, respectively, in space and returned to Earth just before reaching term. Once back on the ground, the dams gave birth on time and to pups that were ostensibly normal, from which biologists April Ronca and Jeffrey Alberts of Indiana University concluded that microgravity does not hinder intrauterine embryogenesis. However, during delivery the flight dams had twice as many abdominal contractions as the ground controls, suggesting that more extended exposure to spaceflight could still have a detrimental effect on pregnancy, or at least the birthing process. Indeed a colleague and I found surprising changes (discussed below) in some of the muscles used by the dams to expel their offspring.

Ronca and Alberts found that the neonate pups born after one of the flights immediately showed subtle, but transient, retardation in their righting reactions when compared with Earth-bred controls. They were slow to roll over when placed upside down in water. Some did not roll over at all. This is indicative of a disturbance in the development of vestibulomotor reflexes-the system that includes structures in an animal’s inner ear that judge position and the muscles that respond to correct an animal’s stance and keep it upright. (Disturbances of this system in people are a common cause of falls and injuries.) The righting responses, however, normalized within five days of birth.

Post-uterine development in microgravity appears to be more precarious for rodents. In 1998 on what was called the “Neurolab” mission, maternal rats were flown with their nursing pups. Despite efforts in the design of the housing to keep the neonates near their mothers, many of the pups that were less than a week old at launch lost contact with their mothers in microgravity and were not rescued. Dehydration and subsequent mortality were high among the compromised young. In addition, surviving pups had chronic difficulties with their righting reflexes many weeks after their return to Earth.

At this point the greatest success in vertebrate development in space comes from work with nonamniotes: fish and amphibians. I was privileged to participate in an experiment that flew on the Space Shuttle in 1992, one that examined embryonic development in the African clawed frog, Xenopus laevis. Embryogenesis in anurans (frogs and toads) is rapid and completed externally (in contrast to amniotes, in which the embryo develops inside of the mother, often quite slowly). Anuran development is therefore easy to observe and study. Depending on the species, frogs can go from fertilization to hatching in a matter of a few days. Embryogenesis can therefore be completed during an average one-and-a-half-week Space Shuttle mission.

On Earth, gravity greatly influences early anuran development. This is because frogs’ eggs are asymmetric, with opposing heavier (more dense) and lighter (less dense) poles. The eggs may come out of the female with those poles pointing in any direction. However, immediately upon fertilization the heavier pole rotates downward, with subsequent cell divisions orthogonal to the gravitational axis. Here on earth this gravitationally dependent downward rotation of the heavy end of the egg ultimately distinguishes the head from the tail end of the embryo. Thus the core question of NASA’s Frog Embryology Experiment was whether gravity is necessary for normal frog development. In the absence of gravity would tadpoles still have a head at one end and a tail at the other?

For our experiment, adult female frogs were launched on the Shuttle and milked of their eggs by an astronaut on orbit. The eggs were fertilized with sperm that had been extracted from males prior to the launch. Subsets of the resulting embryos were fixed at various times during the flight.

When the Shuttle returned to Cape Canaveral there were many perfectly normal-looking, one-to-two day old tadpoles in the experimental chambers. That answered the key question: Gravity is not necessary for basic embryological development, at least not for Xenopus.

However we were surprised by the morphology of some of the embryos that had been preserved in orbit. We observed abnormalities in the distribution of cytoplasm in embryos at the two-cell stage and also at a later stage, when the embryo is a ball of hundreds of cells surrounding an internal cavity, called the blastocoel. Because of the initial asymmetric distribution of mass in frogs’ eggs, embryos raised on Earth under normal gravitational conditions at these particular stages continue to be asymmetric. However, without gravity this asymmetry was clearly muted. For example, the roof of the blastocoel, which is normally much thinner than the floor and only two cell layers thick in embryos raised on Earth, had four to five cell layers in the embryos raised in microgravity.

If our experiment had terminated at that stage, my colleagues and I would have been skeptical about the possibility of the resulting tadpoles looking normal. The embryos raised in microgravity, however, had the remarkable ability to regulate their development and correct for these early perturbations. How this regulation or normalization happens is not known. Minimally it must require interaction between gradients of signaling molecules and mechanical forces (that is, gravity) on the embryos.

Although the resulting tadpoles looked externally normal, they differed from control specimens in behavior. For one thing, they were initially negatively buoyant and lay on the bottom of their containers. A closer look revealed their lungs to be diminutive and uninflated. To inflate their lungs on Earth, Xenopus tadpoles must surface and take an initial bite of air shortly after hatching. The tadpoles on the Shuttle were raised in containers that had air pockets, yet they failed to fill their lungs with air. Without gravity as a clue to the location of the interface between air and water, we believe the tadpoles had nothing to help guide them to the airspace. In a ground-based experiment, my student Scott Pronych showed that Xenopus tadpoles prohibited from surfacing right after hatching exhibited the same stunted growth and high mortality as those raised in microgravity.

In a follow-up experiment with Xenopus embryos on an unmanned Russian satellite, my colleagues at the Institute of Biomedical Problems and the Institute of Human Morphology in Moscow and I confirmed both the necessity for the tadpoles to fill their lungs in a timely fashion and their inability to do it in microgravity. These embryos hatched early in their flight, so they were much older on their return to Earth than were the tadpoles from NASA’s Frog Embryology Experiment. The tadpoles from the Russian satellite had passed the critical period in their development when they should have filled their lungs, and they indeed came back to Earth abnormal. Not only were they and their lungs stunted, but they exhibited a range of other malformations. Their tails, for example, curved upward. Similar results were reported from German space-flight experiments involving later-stage Xenopus embryos. Those experiments pointed toward a critical period when gravity is essential for normal vestibulomotor function and subsequent development.

NASA’s space tadpoles, in contrast, were still young enough when they came to Earth to fill their lungs. Within one week of their return, the tadpoles’ buoyancy and respiration became indistinguishable from the controls that had been raised in the normal 1G (the gravitational pull on Earth) environment. The space tadpoles subsequently grew, metamorphosed and bred normally. The later-stage Xenopus tadpoles from the Russian experiment were not so lucky None survived to metamorphosis.

Because the tadpoles from the NASA experiment were young and seemingly healthy, they were exposed to a battery of behavioral experiments. During their second day on Earth, when they had filled their lungs and were swimming in their normal off-the-bottom position, they exhibited an exaggerated tendency to follow a pattern of vertical black and white bars moved past their visual field. This strong, so-called optomotor response was interpreted as evidence that the tadpoles were relying more on visual than vestibular information to assess their position. This distinction disappeared by the following week, when we could discern no difference between the microgravity and control individuals in all aspects of their behavior.

The experiments in the developmental biology of frogs in microgravity have been followed by Japanese experiments with fire-bellied newts (Cynops pyrrhogaster) and small fish called medaka (Oryzias latipes). Both confirmed that embryogenesis for “lower” vertebrates can proceed successfully from fertilization to hatching in microgravity. However, mineral deposits in the inner ear that are a crucial component of the larval newts’ vestibular system were abnormally large. One newt maintained for nine months after the Shuttle landing held its neck in an unnaturally extended position, with its snout pointing upward.

The medaka experiment remains the only time that adult vertebrates mated freely in microgravity, thus confirming that courtship and fertilization can take place in space without human intervention. Unlike the tadpoles, the medaka larvae raised in microgravity swam normally when tested on Earth. That was surprising since medaka are similar to amphibians in that shortly after hatching they must come to the surface to fill their air bladders. In an earlier experiment, pregnant guppies (Poecilia raticulata) (guppies give birth to live young) bore larvae with empty air bladders in microgravity. As with the amphibians, ground-based studies suggest that the failure to fill the air bladder in a timely fashion severely handicaps fishes.

It is not clear how the Oryzias larvae avoided this hazard, but they were like the Xenopus tadpoles from the NASA embryology experiment; that is, they were very young at the time of Shuttle landing. Thus, they were probably still in the crucial developmental period when they could fill their air bladders and may have done so once on the ground.

Sensitive Periods in Development

Although efforts to raise vertebrates in space have been limited to very few taxa, certain patterns nevertheless emerge from the experiments reviewed above. Fertilization, where it has been studied in fish and amphibians, is not hindered. Early embryogenesis can be affected, at least in birds and frogs, but if gas-exchange problems do not emerge, as they did in birds’ eggs, embryos seem to be able to regulate their development in microgravity, as was shown in the frog development experiments. Experiments with fish, amphibians, birds and rodents demonstrated that embryos raised in microgravity produced ostensibly normal hatchlings and neonates. These immature animals may show transient vestibular problems that seem mostly to disappear when the individuals are examined in the gravitational environment of Earth.

In contrast, vestibulomotor problems may not be self-correcting if the juveniles are kept for long periods in microgravity. This has been the case for amphibians, birds and mammals. What scientists found to be true in virtually all the species studied to date is that the serious obstacles to completing a life cycle in space appear in late or postembryonic development, when the vestibulomotor system matures. For the aquatic forms, the problems identified to date have been in filling the air bladder or lungs. For the nonaquatic forms, the problems arise in feeding. In both cases the problems are severe, are of a behavioral nature related to vestibulomotor function, and hinder subsequent growth and development.

It remains to be seen whether these patterns will hold up when more species are studied. What the data do suggest, though, is that problems for vertebrates in completing their life cycle in space are not likely to be in the domain of the embryologists. Rather, they are the sort of problems that draw the attention of behaviorists and neurobiologists.

One might suppose that the behavior of vertebrates in microgravity would have been well studied by now. That is not the case. Few behavioral studies have been done with young vertebrates in microgravity because the habitats for raising them in space have simply not existed. For orbital experiments one also needs special habitats that remove waste products and maintain water quality for the aquatic forms and air quality for the terrestrial forms. Furthermore, the study of vertebrates up to sexual maturity requires blocks of time longer than the two or so weeks of a Space Shuttle mission. Such long-term, high-quality habitats have not existed for either the Space Shuttle or Mir, although they are now being built for the International Space Station.

Acute Responses of Vertebrates

Relatively little neurobehavioral research has been done in microgravity with vertebrates, juvenile or otherwise. Some physiological experiments that have required in-dwelling electrodes in unconstrained animals have failed because of the twisting and turning of the animals in microgravity. In order to avoid these problems, it is now standard operating procedure to pretest animals and apparatuses slated for orbital flight with brief intervals of microgravity obtained via parabolic flight. Using an aircraft, such as NASA’s KC-135, scientists can get approximately half-minute episodes of microgravity, albeit alternated with episodes of hyper-gravity (which can rise as high as three times the gravitational pull of Earth, depending on the aircraft).

The response of animals to abrupt free-fall (that is, microgravity) is astonishingly diverse and serves as an indication that there is much we do not know about basic animal behavior in microgravity. With Tomio Naitoh at Shimane University and Masamichi Yamashita at the Institute of Space and Astronautical Science in Japan, my students and I have made an effort to survey systematically the behavioral responses of a variety of “lower” vertebrates to the acute onset of microgravity. Some examples from amphibians and reptiles demonstrate how diverse these reactions can be.

One predictable and commonly observed response of animals that find themselves in microgravity is to react as though they are upside down and to initiate repetitive righting responses. Typically, the animal rolls over and over, since in microgravity there is no vestibular confirmation that the action was successful.

Terrapins (Mauremys japonica) in microgravity hyperextend their necks and limbs and make a rowing action with their hind limbs. In air this looks very strange. However, one can elicit the same display from a turtle on Earth by placing it upside down on a surface. The rowing action allows the animal to flip itself over.

The rat snake (Elaphe quadrivirgata) responds aggressively in weightlessness. Within the first three seconds of entering microgravity, a rat snake lunged at its own body when a loop floated by its head. Presumably, with the loss of surface contact and proprioceptive stimulus, the animal could no longer distinguish self from non-self.

Most animals that can embrace something will tenaciously grasp at surfaces in microgravity. Thus on one parabolic flight a sand lizard (Lacerta agilis) grabbed its own tail between its legs and clung tightly to it as though clinging to a tree. The lizard did not let go even when the airplane began its pullout (hyper-gravity) maneuver.

Terrestrial and arboreal frogs take up a “sky diving” posture in microgravity and hold that posture as long as they do not contact a surface. When a frog falls on Earth, it assumes this posture, which keeps its center of mass below its feet, thus providing positional control. In microgravity the same pose can be taken to an extreme. One tree frog (Rhacophorus schlegelii) managed to extend and abduct its limbs so far in microgravity that they were completely above and behind its torso. If one forced a frog’s limbs into that position, one would be accused of molesting the animal. Yet in microgravity the individual frog contorted itself into this posture without the application of any external forces.

When frogs in microgravity touch a surface, they immediately initiate rolling or twisting movements, presumably to reorient themselves belly-down toward the contacted surface. In one case a marine toad (Bufo marinus) attempted this pirouette with such ferocity that it got its hind limbs twisted together and momentarily could not untangle them.

All of the responses described above are forcefully executed and suggest that vertebrates find free-fall a stressful experience warranting prompt countermeasures. However, as with most patterns in nature, if one looks hard enough one finds exceptions. The caecilian Typhlonectes sp., a limbless aquatic amphibian, stopped swimming and simply went limp with the onset of microgravity during each parabola.

Collectively these examples demonstrate that animals will do extraordinary things in microgravity. Many of these behaviors had never before been seen, and could not have been predicted from studies with the same creatures on Earth.

Furthermore, the responses described so far were from animals tested in isolation. In our earlier parabolic flight experiments, we occasionally flew animals two or more to a cage, to increase our sample size. We quickly learned that animals housed in groups interact in a wholly unnatural fashion in weightless conditions, often clinging to one another in any way possible.

The Influence of Housing

Bizarre behaviors such as these have implications for interpreting results from more elaborate and serious orbital experiments. Because so many animals resist free-fall by grasping what they can, animals that are housed in groups can be expected to hang onto each other. One of the best-documented effects on mammals of prolonged microgravity exposure is bone demineralization and skeletal-muscle atrophy. These effects are believed to be the result of unloading of the musculoskeletal system when the animals are free of gravity. However, unloading may not take place in microgravity if animals are housed together.

My colleagues and I first became aware of this problem from a paradoxical result we obtained in muscles from the adult rats in one of the NIH-NASA studies mentioned earlier. The animals were housed five to a chamber. Surprisingly the animals’ external oblique muscles, which help rotate the torso, appeared to hypertrophy rather than atrophy. Scientists studying the behavior of the same rats independently showed that, rather than floating freely in space, the rats continually crawled over the walls and over each other. An animal would put its feet on other rats, but would pull away when another animal touched it. The result was something akin to a dynamic, biotic jungle gym. The in-flight video showed that the rats in space rotated their torsos seven times more often than did Earth-based controls. Our subsequent study of the external oblique muscles of rats flown in isolation yielded the more traditional and expected result of muscle atrophy.

In a similar vein, the effect of housing animals singly versus in groups has since been shown to alter bone density. Spaceflight reduced bone mass by seven percent in rats housed singly on a nine-day mission, but had little effect on rats housed in groups on the same flight.

Integrative Space Biology

In retrospect, the above discoveries seem so obvious that one wonders why it required some four decades and dozens of experiments to uncover them. There are several explanations for why integrative space biology in general has not advanced faster. Some of these are self-evident and strictly logistical. Others are more subtle, but perhaps more substantive.

From a logistical perspective, building appropriate habitats and hardware for studying large and complex organisms in space is not easy Only in the last decade or so has it become pro forma to bring a video camera along when there are animals on board. Viewing the animals, though, or subjecting them to physiological tests has always been tricky. Before the International Space Station, no spacecraft, Mir included, has had the room or equipment onboard for anything but relatively simple studies with highly confined vertebrates.

The restricted room aboard spacecraft and the very high uplift costs have left space agencies in the past little choice but to fly organisms in minimal-volume containers and at abnormally high densities, which, as noted above, can distort experimental results. The alternative available to space biologists-to fly fewer organisms, thus reducing their sample size-is not conducive to good science either.

The difficulty in maintaining organisms alive in space has often, I believe, made in vitro experiments seem more appealing to mission managers. Indeed, many cell-culture experiments can be flown in the volume of one experiment with live rodents. And, with droves of biologists waiting to get their experiments on orbit and very few flight opportunities, it has been politically expedient for NASA and other space agencies to select for flight many small- rather than a few large-volume experiments. This is in spite of the fact that evidence to date suggests that cells and tissues in space alone may not reflect how whole organisms respond in the same environment.

Perhaps NASA and other space agencies fear that research with animals in space may invite whimsical comment, but such fears are probably unjustified. The mere fact that this research receives such attention testifies to the fact that it is interesting and comprehensible to the lay public. The response of whole organisms to space, be they anuran larvae or astronauts, can be more easily understood by the public at large than most experiments in cell biology.

A subtler problem is that the basic research in animal husbandry, which is a necessary step toward more elegant studies in integrative space biology, seems so antiquated. For most of modem biology, raising animals is a matter-of-fact task and not the primary mission. What is easily forgotten is that all of the exciting work being done in modern biology laboratories would not be possible without the tremendous amount of research in animal care and reproductive biology undertaken in the last century.

Of course the long-term goal of integrative space biology is not animal husbandry. Managing to keep animals alive and growing in space is simply a prelude to studying how microgravity and other factors in space affect life. The work to date with vertebrates in space repeatedly indicates that there are crucial, but not always obvious, periods when gravity is necessary for normal development. Research with vertebrates also indicates both direct and indirect pathways whereby vestibular function interacts with the function of other systems, such as respiration in aquatic vertebrates and feeding in birds and mammals. These observations suggest a multitude of studies in developmental and neural physiology that can be done in microgravity, given enough time, space and equipment. When the International Space Station becomes fully functional, biologists should finally have the sort of orbiting laboratory that will allow for real progress in integrative space biology.