Hypothesis: Development of the equilibrium receptor system depends on gravity. Rationale: Molecular positioning cues, trophic/inhibitory factors, and stimulus linked patterned electrical activity all play roles in establishing precise central connections in developing sensory systems. In the equilibrium receptor system, the relative contributions different cues, including gravity, play in the development of the system remain unknown. However, an understanding of the development of the equilibrium receptor system is critical to understanding abnormal development of the system seen in birth defects or its abnormal function after trauma. To date, it has only been possible to test hypotheses about the role of gravity vs. other developmental cues in the development of equilibrium receptor systems in the microgravity environment of Space Shuttle missions. It would be advantageous to be able to perform such experiments on earth. NASA designed the Rotating Wall Perfused Vessel (RWPV) for culturing cells in a simulated microgravity environment on earth. Replacing the culture medium with fresh water adapts the RWPV to accommodate eggs from aquatic vertebrates, such as zebrafish. Therefore, it should be possible to use the RWPV to test, on earth, the effects of a microgravity environment on developing equilibrium receptor systems. Our preliminary data on zebrafish indicate that this is indeed the case. Objectives: The zebrafish nervous system has become a well accepted model for developmental neurobiology studies. Therefore, zebrafish are used to determine whether, and to what extent, the development of their equilibrium receptor systems depend on gravity. Zebrafish eggs are placed in the RWPV before the vestibular end-organs begin to develop. The eggs are maintained in the RWPV until they hatch. Deficits in the animals' ability to maintain equilibrium orientation and morphological differences between the equilibrium receptor systems of experimental and control hatchlings, will suggest that the proper development of the equilibrium receptor system is dependent on gravity. To demonstrate a deficit in the ability to maintain equilibrium orientation, the ability to maintain appropriate equilibrium orientation during swimming under normal gravity conditions and the integrity of compensatory eye reflexes are evaluated. To demonstrate morphological changes in the equilibrium receptor systems, the morphology of vestibular end organs, at light and transmission electron microscopic levels and the afferent projection patterns from the sensory epithelia into the CNS are evaluated. These observations are compared for animals that developed in a simulated microgravity environment and animals that developed in a normal gravity environment.
Stimulus dependence of the development of the zebrafish
(Danio rerio) vestibular system.
Stephen J. Moorman*, Charlotte Burress, Rodolfo Cordova, and Joshua
Slater
It has been suggested that stimulus dependence is a general feature
of all developing sensory systems. We tested this idea for the developing
zebrafish vestibular system using a bioreactor NASA designed to simulate
microgravity for cells in culture on earth. We replaced the culture
medium with aquarium water and maintained zebrafish eggs/hatchlings in
the bioreactor for either 72 or 96 hours post-fertilization. These
experimental animals displayed a swimming behavior that was indistinguishable
from the control animals when illuminated from above. However, when
illuminated from below, experimental animals swam not only dorsal surface
up but also laying on their side, they corkscrewed, swam vertical loops,
and occasionally even swam upside down. When incubated in the bioreactor
for 96 hours, the saccular otolith was significantly smaller than normal
suggesting that otolith development was either delayed or slower than normal.
When incubated in the bioreactor for 72 hours, some animals were missing
one or more otoliths. In contrast, control animals all had 2 otoliths
on each side. This supports the idea that otolith development was
delayed. Immediately upon removal from the bioreactor at 96 hours,
experimental animals showed some signs of compensatory eye rotation, but
with a much less clear relationship between the orientation of the eye
and the direction of gravity than the age-matched control animals.
This difference was still obvious one day later. These results support
the idea that development of the vestibular system in zebrafish is dependent
on the presence of the normal stimulus the system is designed to detect.
Abstracts:
Moorman, S.J., J. Slater, R. Cordova, and C. Burress (1997) Development of the zebrafish vestibular system depends on normal gravity. Abstract in Proceedings of Society for Neurosciences, vol 23.
Articles:
Moorman, S.J., J. Slater, R. Cordova, and C. Burress (1998) Stimulus dependence of the development of the zebrafish (Danio rerio) equilibrium receptor system. J. Neurobiology (in press)
Moorman, S.J. and J.E. Anwah (1998) Counter-rotation of the zebrafish (Danio rerio) eye in response to relative changes in light position. Submitted to: IOVS
S.J., R. Cordova, C. Burress, and M. Cao (1997) The 'Critical Period'
for development of the zebrafish (Danio rerio) equilibrium receptor
system. Manuscript in preparation.
(last revised 22 July 1998)