NASA Grant #NAG2-1591: "The effects of
microgravity on gene expression in live zebrafish embryos."
The effects of microgravity on gene expression are poorly
understood. Initial attempts have been made to investigate the effects of
microgravity on cells in culture. It is unknown how those experiments
will relate to potential effects on gene expression in vivo. We have
begun to examine the effect of simulated-microgravity on gene expression in
specific developing organ systems in vivo using transgenic zebrafish that
express the green fluorescent protein gene (gfp) under the influence of
different promoters. The use of gfp as a ‘reporter-gene’ has two
significant advantages; you can monitor gene expression directly in a live
animal, and you can easily detect changes in morphology that might be due to
changes in expression of other genes. In preliminary experiments where
gfp gene expression was under the influence of a b-actin promoter (breeding
fish provided by Kenneth Poss), we demonstrated that simulated-microgravity
induced a 28% increase in GFP-associated fluorescence in the heart and a 16%
increase in the notochord of zebrafish embryos. We want to extend these
observations to other tissues/organs of the zebrafish embryo and we want to
examine the effects on gfp gene expression under the influence of other
promoters.
Using a bioreactor that NASA designed to simulate microgravity
for cells in culture, we will expose the transgenic zebrafish embryos to
simulated-microgravity for specific, well-controlled time periods. We
will analyze gfp gene expression by measuring the intensity and tissue specific
distribution of GFP-associated fluorescence using confocal microscopy and
de-convolution fluorescence microscopy of the live embryos and larvae. We
will determine whether simulated-microgravity has an effect on gene expression,
the tissue/organ specific nature of the effects, the minimum duration of
exposure necessary to induce an effect, and the recovery time after return to
normal gravity.
This is the first time a whole animal in vivo model will be
used and maintained alive during gene expression analysis for microgravity
related research. This research not only addresses an important issue in
space life sciences research, it clearly demonstrates the usefulness of
zebrafish with gfp constructs as a model to study the effects of microgravity
on gene expression during development.
Gillette-Ferguson, I., D.G. Ferguson, K.D. Poss and S.J. Moorman (2003) Changes
in gravitational force induce alterations in gene expression that can be
monitored in the live, developing zebrafish heart. Advances in Space
Research 32:1641-1646
Little is known about the effect of microgravity on gene expression,
particularly in vivo during embryonic development. Using transgenic
zebrafish that express the gfp gene under the influence of a b-actin promoter,
we examined the affect of simulated-microgravity on GFP expression in the
heart. Zebrafish embryos, at the 18-20 somite-stage, were exposed to
simulated-microgravity for 24 hours. The intensity of GFP fluorescence
associated with the heart was then determined using fluorescence microscopy.
Our measurements indicated that simulated-microgravity induced a 23.9% increase
in GFP-associated fluorescence in the heart. In contrast, the caudal
notochord showed a 17.5% increase and the embryo as a whole showed only an 8.5%
increase in GFP-associated fluorescence. This suggests that there are
specific effects on the heart causing the more dramatic increase. These
studies indicate that microgravity can influence gene expression and
demonstrate the usefulness of this in vivo model of ‘reporter-gene’ expression
for studying the effects of microgravity.
Shimada, N., G. Sokunbi, and S.J. Moorman (2005) Changes in gravitational force
affect gene expression in developing organ systems at different developmental
times. BMC Developmental Biology 2005, 5:10.
Background: Little is known about the affect of microgravity on gene
expression, particularly in vivo during embryonic development. Using
transgenic zebrafish that express the gfp gene under the influence of a β-actin
promoter, we examined the affect of simulated-microgravity on GFP expression in
the heart, notochord, eye, somites, and rohon beard neurons. We exposed
transgenic zebrafish to simulated-microgravity for different durations at a
variety of developmental times in an attempt to determine periods of
susceptibility for the different developing organ systems. Results: The
developing heart had a period of maximum susceptibility between 32 and 56 hours
after fertilization when there was an approximately 30% increase in gene
expression. The notochord, eye, somites, and rohon beard neurons all
showed periods of susceptibility occurring between 24 and 72 hours after
fertilization. In addition, the notochord showed a second period of
susceptibility between 8 and 32 hours after fertilization. Interestingly,
all organs appeared to be recovering by 80 hours after fertilization despite
continued exposure to simulated-microgravity. Conclusions: These results
support the idea that exposure to microgravity can cause changes in gene
expression in a variety of developing organ systems in live embryos and that
there are periods of maximum susceptibility to the effects.
Shimada, N. and S.J. Moorman (2006) Changes in gravitational force cause
changes in gene expression in the lens of developing zebrafish.
Developmental Dynamics 235:2686-2694
Gravity has been a constant physical factor during the evolution and
development of life on Earth. We have been studying effects of
simulated-microgravity on gene expression in transgenic zebrafish embryos
expressing gfp under the influence of gene-specific promoters. In this
study, we assessed the effect of microgravity on the expression of the heat
shock protein 70 (hsp70) gene in lens during development using transgenic
zebrafish embryos expressing gfp under the control of hsp70
promoter/enhancer. Hsp70:gfp expression was up-regulated (45%) compared
with controls during the developmental period that included the
lens-differentiation stage. This increase was lens specific since the
whole embryo showed only a 4% increase in gfp expression. Northern blot and in
situ hybridization analysis indicated that the hsp70:gfp expression
recapitulated endogenous hsp70 mRNA expression. Hypergravity exposure
also increased hsp70 expression during the same period. In situ hybridization
analysis for two lens-specific crystallin genes revealed that neither micro-
nor hypergravity affected the expression level of βB1-crystallin, a non-hsp
gene used as a marker for lens differentiation. However, hypergravity
changed the expression level of αA-crystallin, a member of the small hsp gene
family. TUNEL assay analysis showed that altered-gravity decreased
apoptosis in lens during the same period and the decrease correlated with the
up-regulation of hsp70 expression, suggesting that elimination of nuclei from
differentiating lens fiber cells was suppressed probably through hsp70
up-regulation. These results support the idea that Δg influences hsp70
expression and differentiation in lens-specific and developmental period
specific manners and that hsp family genes play a specific role in the response
to Δg.
Moorman, S.J., N. Shimada, G. Sokunbi, and C. Pfirrmann (2007) Simulated-Microgravity Induced Changes in Gene Expression in Zebrafish Embryos Suggest that the Primary Cilium is Involved in Gravity Transduction. Gravitational and Space Biology 20:79-86
Gravity has been a constant physical factor during the evolution and development of life on Earth. We have been studying effects of simulated-microgravity on gene expression in transgenic zebrafish embryos expressing gfp under the influence of gene-specific promoters. We have looked at a number of different genes expressed in a variety of different organ systems. For instance, we have looked at beta-actin expression in the heart, eye, notochord and rohon beard neurons, hsp70 expression in the lens, alpha-A1 and beta-B1 crystallin expression in the lens, and fli1 expression in the heart and blood vessels. Different organs and cell types show periods of maximum susceptibility during developmental periods that coincide with specific developmental events. The organ-specific developmental events correlate with periods when primary cilia are playing organ-specific developmental roles. In the notochord, each primary cilium is positioned to function as a ‘strain gauge’ to monitor the stresses associated with bending of the notochord in response to forces such as gravity. Unloading the notochord by placing the embryos in a simulated-microgravity environment causes more dramatic changes in gene expression than those seen in any other tissue. The developing cardiovascular system looses its susceptibility to simulated-microgravity induced changes in gene expression as the primary cilium of the endothelial cell becomes a flow sensor in the lumen of the blood vessels. Rohon beard neurons show simulated-microgravity induced changes in the variability of gene expression levels that can be explained by a change in the balance between the canonical and non-canonical Wnt pathways, pathways that are influenced by the primary cilium. A search of the cilia-related proteome reveals a link between the primary cilium and hsp70 expression, which might explain the simulated microgravity induced change in hsp70 expression in the developing lens. The ubiquitous nature of the primary cilium as a cell organelle suggests that gravity sensing might be a general feature of all vertebrate cells where the primary cilium has not been co-opted for another sensory function.
Moorman, S.J. and A.Z. Shorr (2008) The primary cilium as a gravity sensor and regulator of transcriptional noise. Developmental Dynamics
Circumstantial evidence has suggested that the primary cilium might function as a gravity sensor. Direct evidence of its gravity sensing function has recently been provided by studies of rohon beard neurons. These neurons showed changes in the variability of gene expression levels that are linked to the cyclic changes in the Earth’s gravitational field due to the Sun and Moon. These cyclic changes also cause the tides. Rohon beard neurons, after the primary cilia have been selectively destroyed, no longer show changes in gene expression variability linked to the cyclic changes in Earth’s gravitational field. After the neurons regrow their primary cilia, the link between variability in gene expression levels and the Earth’s changing gravitational field returns. This suggests two new functions for the primary cilia, detecting the cyclical changes in the Earth’s gravitational field and transducing those changes into changes in the variability (stochastic nature) of gene expression.
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