Development of the zebrafish vestibular system.
Moorman, S.J., C. Burress, R. Cordova
, and J. Slater (1999) Stimulus dependence of the development of the zebrafish
(Danio rerio) vestibular
system. J Neurobiology 38:247-258
ABSTRACT:
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.
Riley, B. and S.J. Moorman (2000) Development
of utricular otoliths, but not saccular otoliths, is necessary for vestibular
function and survival in zebrafish. J. Neurobiology 43:329-337
ABSTRACT:
We have been studying the consequences of embryonic vestibular dysfunction
caused by the monolith (
mnl) mutation in zebrafish.
mnl is a dominant mutation that
specifically inhibits formation of utricular otoliths. However, briefly immobilizing
mnl/ mnl
embryos in agarose with the otic vesicle orientated at certain angles selectively
induces or prevents formation of utricular and/or saccular otoliths. With
this noninvasive technique, we generated six phenotypic classes of mnl
/mnl mutants, designated S-S,
U-U, U-S, S-US, U-US, and US-US, depending on which otoliths are present
on each side (U, utricular otolith; S, saccular otolith). All mnl
/mnl larvae survived through
day 10 of development. Thereafter, S-S larvae showed a rapid decline, probably
because of starvation, and none survived to adulthood. Survival rates in
all other classes of mnl/
mnl larvae (those having at least
one utricular otolith) were close to normal. The presence or absence of utricular
otoliths also correlated with vestibular function during early larval development,
as measured by three criteria: First, unlike wild-type larvae, S-S mutant
larvae showed
almost no detectable counter-rotation of the eyes when tilted tail up or tail
down. Second, 95% of S-S mutant larvae never acquired the ability to maintain
a balanced dorsal-up posture. Third, although most wild-type larvae responded
to gentle prodding by swimming in a straight line, S-S larvae responded by
swimming in rapid circles, showing sudden and frequent changes in direction
(“zigzagging”), and/or rolling and spiraling. All other phenotypic classes
of mnl/mnl
larvae behaved normally in these assays. These data demonstrate that bilateral
loss of utricular otoliths disrupts the ability to sense gravity, severely
impairs balance and motor coordination, and is invariably lethal. The presence
of a utricular otolith in at least one inner ear is necessary and sufficient
for vestibular function and survival. In contrast, saccular otoliths are
dispensable for these functions.
Moorman, S.J., R. Cordova, S.A. Davies
(2002) A Critical Period for functional
development of the zebrafish (Danio rerio
) vestibular system. Developmental
Dynamics 223:285-291.
ABSTRACT:
We have determined a critical
period for vestibular development in zebrafish by using a bioreactor designed
by NASA to simulate microgravity for cells in culture. A critical period
is defined as the briefest period of time during development when stimulus
deprivation results in long lasting or permanent sensory deficits. Zebrafish
eggs were collected within 3 hours of being laid and fertilized. In experiment
1, eggs were placed in the bioreactor at 3, 24, 30, 36, 48, or 72 hours post-fertilization
(hPF) and maintained in the bioreactor until 96 hPF. In experiment 2, eggs
were placed in the bioreactor immediately after they were collected and maintained
in the bioreactor until 24, 36, 48, 60, 66, 72, or 96 hPF. Beginning at 96
hPF, all larvae had their vestibulo-ocular reflexes (VOR) evaluated once
each day for 5 days. Only larvae that hatched from eggs that were placed
in the bioreactor before 30 hPF in experiment 1 or removed from the bioreactor
later than 66 hPF in experiment 2 had VOR deficits that persisted for at
least 5 days. These data suggest a critical period for vestibular development
in the zebrafish that begins before 30 hPF and ends after 66 hPF. To confirm
this, zebrafish eggs were placed in the bioreactor at 24 hPF and removed
at 72 hPF. VORs were evaluated in these larvae once each day for 5 days beginning
at 96 hPF. These larvae had VOR deficits that persisted for at least 5 days.
In addition, larvae that had been maintained in the bioreactor from 24 to
66 hPF or from 30 to 72 hPF, had only temporary VOR deficits. In a final experiment,
zebrafish eggs were placed in the bioreactor at 3 hPF and removed at 96 hPF
but the bioreactor was turned off from 24 hPF to 72 hPF. These larvae had
normal VORs when they were removed from the bioreactor at 96 hPF. Taken as
a whole, these data support the idea that there is a critical period for functional
maturation of the zebrafish vestibular system. The developmental period identified
includes the timeframe during which the vestibular primary afferent neurons
are born, innervate their central and peripheral targets, and remodel their
central projections.
Abstract for Grant #NAG2-1356 from
NASA.
The response of the developing vestibular
system to exposure to a microgravity environment during the time frame prior
to otic placode induction through functional maturation remains largely unknown.
Recently, it has become possible to use a NASA designed bioreactor
to test, on earth, the effects of a simulated microgravity environment on
the developing zebrafish vestibular system.
AIM #1:
Determine the ‘critical period’ for zebrafish vestibular development.
The narrowest window of time during which exposure to a microgravity
environment leads to irreversible vestibular deficits and the shortest period
of time during which exposure to a normal gravity environment leads to normal
vestibular development will be identified.
Vestibular deficits will be demonstrated by an inability to maintain
appropriate equilibrium orientation during swimming under normal gravity
conditions and by inappropriate compensatory eye reflexes.
AIM #2:
Determine the role of gravity in establishing the afferent projection
patterns from the macular sensory epithelia into the CNS of zebrafish.
The central projections in the zebrafish hatchlings will be directly
assessed by labeling the afferent axons by injecting the lipophilic dye,
DiI, into the maculae of anesthetized zebrafish hatchlings.
The pattern of the projections to the hindbrain will be examined
using confocal microscopy of labeled hatchlings.
The arborization patterns of the primary afferents of normal zebrafish
will be compared with those of bioreactor animals from Specific Aim #1.
Aim #3:
Determine whether microgravity induces altered expression of genes
involved in the development of the zebrafish vestibular system.
Total RNA will be extracted from experimental and control embryos
and probed for expression of genes such as dlx-3
, msx-C and msx-D
by reverse-transcription polymerase chain reaction (RT-PCR) analysis, Northern
blot analysis, and in situ hybridization.
Our long-term goal is to understand how the vestibular system will
develop in the absence of gravity and in gravitational fields of different
strengths.
Abstract for Grant # DC 03531-01 from
the National Institute on Deafness and Other Communications Disorders.
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.
(last revised 6 May 2002)
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