Frogs and space.
Using animal models to study the effects
of microgravity
Joanne Pearson
Mariama Issaka
Daniel Martínez
Edoardo Giovanni
Banyuls,
Origins of life – Life in space 2010
BACKGROUND
Since life appeared on earth, gravity has been a costant selection force. The behavior and
development of all organism as a indirect, if not direct, determinant.
The role that gravity plays in biological processes can be revealed explicity under
microgravity condition.
During the past, Xenopus laevis has been used as a model system for many different studies
about development and behavior.
Experiments were done:
Clinostat
Parabolic flights
Space flights
Orbiting Frog Otolith (1970) NASA
Frogs in Space,FRIS (1991) in the Mir station
BACKGROUND - Xenopus laevis (Anura)
The Amphibia are a special class of animals in the context of gravitation biology. They were
the frist vertebrate to become land-dwelling and to resist gravity by supporting their weight
on limbs.
Source: scienceclarified.com
BACKGROUND – The effect of microgravity
Opposit effects about microgravity and hypergravity
The structure of cells is altered with differences in the citoskeleton, apoptosis rate and
cellular response to the envrioment
Tadpoles of normal apparence emerge despite some alteration in cleavage,gastrulation and
neurolation.
The altered cell morphology and function observed in many of experiment performed in
microgravity, embrio can still develop into living organism
The tadpoles returned from space were virtually normal in behavior and morphology, with
the notable exception of position in the water column and lung size
The microgravity tadpoles tend to show a slightly stronger optomotor response
Fertilization rates were high in microgravity condition
The tadpoles returned from spaceflight metamorphosed and matured normally
REASONS & ADVANTAGES
One reason for the experiment is to see to which extent gravity is necessary for normal
development and in which range adaptive mechanisms are efficient enough at altered
gravity to guarantee a normal lifestyle/ development for the frog.
The next reason is to know if the gravity is required for normal embryonic development,
for example in the establishment of embryonic polarities.
The results will also show the interferences of gravity with vestibular functioning and
behaviour.
At last the experiment will test the sensitivity of the brain metabolism, of the neuronal
signal-response chain and of the behaviour of a frog in the micro gravity environment.
WHAT ARE THE ADVANTAGES OF AMPHIBIANS?
• External embryonic stages.
• Faster development than mammals.
• Small body size.
• High reproduction rate.
• Absence of body weight-related proprio-perception.
• Reduced influence of gravity on supporting tissues, muscles, vascular tonus
systems.
• Relatively higher sensitivity to gravity due to larger otoliths, differently
positioned sacculus-otholith membranes
(Rahman & Slenzka., 1994)
OBJECTIVE
The objective of the experiment is to look at the
development of Xenopus laevis individuals from
embryo through to a early frog making comparisons
between two different gravity conditions (1g and μg)
with a duration of almost 12 months
We will do this for 60 frogs in total, 30 under micro
gravity and 30 under 1g.
The areas of the frog we will monitor will be the
general development of embryo, inner ear,
cytoskeleton, neurons and behaviour.
HYPOTHESIS
1. Microgravity conditions alter early development of the embryo, in the embryogenesis
process, causing some malformations during the blastocoel and gastrula stages. (Black et
al., 1996) The effects seen are not reversible in 1g.
Black et al., 1996
HYPOTHESIS
2. The apoptosys rate decreases in microgravity conditions because of abnormalities in the
cytoskeleton organization inside the cell (Crawford-Young, 2006) and these effects are not
reversible in 1g.
Source: microbiologybytes.com
HYPOTHESIS
3. The development of NCC (neural crest cells) change in microgravity condition causing an
abnormality of symmetry during migration on the formation of cartilage (Olson et al.,
2010) and these changes are not reversible in 1g.
Olson et al., 2009
HYPOTHESIS
4. Changes in gravity conditions interfere with the response of the semicircular canal in
the labyrinth of Xenopus laevis related to the posture perception, and these changes are
reversible when the individuals return to normal condition.
Rossi et al., 2009
HYPOTHESIS
5. Microgravity produces changes in the optomotor behaviour of tadpols, these can be
seen to swim lower than 1g tadpols group. (Black et al., 1996; Souza et al., 1995) This
behaviour is reversible at 1g if the tadpoles are not held in microgravity for long periodes
of time. In the other case, this will be irreversible.
Pronych et al., 1996
EXPERIMENT / METHOD
EARTH
SPACE
Embryos
Fertilize
EARTH
Tadpoles
Study
Study
Adults
Study
Adults
Study
Embryos
Fertilize
Study
Tadpoles
Study
EXPERIMENT / METHOD
General
-18 h after: injection of HCG and activate sperm with suspension 1/3 modified Ringer’s
-Eggs placed on small screens, flooded with sperm suspension 10 min. into Lexam
chambers with 1/5 modified Ringer’s
-Half of chambers in 1g (centrifugue) and other half in μg (10^-3 – 10^-5 g)
Particular
-Embryos were fixed in 4% paraformalehyde and adults frogs with standard fixing mixture
-Adult frogs anesthesied with tricaine methane sulfonate, posterior dissection and
positioning in Perpex chamber
-Individuals stained with dyes like alcian Blue, masson tricrhomic, toluidin blue…
(Method adapted from Black et al., 1996; Souza et al., 1995; Rossi et al., 2009; Olson et al., 2010)
HARDWARE
Adult frogs
Tadpoles
Source: ns.umich.edu
Life support
box (LSB)
Source: vssec.vic.edu.au
Source: trekearth.com/
Eggs and sperm
Source: xlaevis.com
Frog recovery
box (FRB)
Source: vssec.vic.edu.au
HARDWARE
International
Space Station
Source: wikipedia.org
Shuttle
Source: esa.int
Kibo (希望)
Source: wikipedia.org
Researchers
CONTROLS
• All embryos have to come from the same larvae.
• The water in both systems needs to be kept at the same
temperature 18oC at the start then after 50 hours upped to
21 o C to increase rate of development. Temp in 1 gravity
and micro gravity needs to be at +- 0.25oC.
• Both the 1g and the μg embryos need to be grown under
exactly the same conditions. They need to be given the
same amount of life support using a life support box (LSB).
• All frogs are the same species, Xenopus laevis.
• Air will be pumped into the system at 100ml per min.
PRE-LAUNCH CHECKS
-The year before: frogs spawned with HCG, quantity and quality of fertilization
recorded
-3 weeks before: frogs with the best history of yielding high quality eggs were shipped
to Kennedy Space Center
-36 hours before: four female with more or less the same weight, were removed from
their aquaria and transferred to a foam-lined container, and was loaded into the LSB
-Sperm preparation: two macerated testes in a solution of modified Ringer’s .
-Sperm conservation: 19 h before was loaded into a Shuttle middeck refrigerator and
held at 4 °C until used in flight
(Method adapted from Black et al., 1996; Souza et al., 1995)
IN-FLIGHT MEASUREMENTS
Embryo:
-fixation and blue alcian stain, view general structures
-fixation and stain with immunohystochemical method, view cytoskeleton
Tadpole:
-fixation and blue alcian stain, view general structures
-optomotor behaviour test on glass with strips
-dissection and study the lungs
Adult:
-fixation of neural part, view structures
-electrophysiological measurements of labyrinth
(Method adapted from Black et al., 1996; Souza et al., 1995; Rossi et al., 2009; Olson et al., 2010)
EARLY RETRIEVAL
To bring the frogs back to earth for further measurements they need to be placed into
a Frog Recovery Box (FRB).
The FRB will be sealed but will sustain the frog’s life through special tablets that
release oxygen when mix with water.
Source: vssec.vic.edu.au
MEASUREMENTS TAKEN AFTER LANDING
After landing the micro gravity frogs and tadpoles need to be
monitored continuously to see if the affects/ deformities that have
happened due to micro gravity stay or can be reversed.
Once frogs return to the Earth, we will observe their swimming
performance and position, then they will be tested for their
tendency to track a moving stimulus:
The tadpoles were first housed in water and allowed 3.5 min to
acclimate to the vial before being tested for their tendancy to track
a moving stimulus by using a stimulus cylinder (60°s¯¹,1 min in the
clockwise and counterclockwise direction)
The both groups will be examined on an optical microscopy to view
important structures and to compare with the normal group.
We will examine the labyrinth with an electrophysiological method.
Stimulis cylinder, adapted
from Pronych et al., 1996
POSSIBLE APPLICATIONS
•
One of the main reasons we have decided to run this experiment again is because
all experiments need to be ran more than once by different labs to prove there
validity.
•
If we study the affects of microgravity on the inner ear of a frog we are able to use
this information to understand why humans are affected by micro gravity. In the
future we may be able to use this information to create something that is able to
help the human ear adapt to the conditions in space and therefore orientation
may not be affected and movement will be easier.
•
The information that we collect with regards to the cytoskeleton will be used to
help use understand bone development in micro gravity. It is important of us to
understanding bone development to help astronauts in the future and know how
humans will be affected by the microgravity.
•
By monitoring the effects of neural crest cells (NCC) we will be able to see what
affects they have on the skull and visceral skeleton. If we know why they are
effected we can try to adapt these cells so the skeleton and skull develops
correctly.
EXPECTED RESULTS
Cytoskeleton: We expect changes in the group of tadpoles with μg that decreases the
apoptosys rate and causes some malformations , like in the extremities or macrocephale.
Obviously, these changes are not reversible when the frogs return to normal gravity.
Early development: We expect blastocoel roof comprised with more cell layers in the μg
group and a different position of blastopore lip. These changes will not be reversible in
normal gravity condition.
Neural: there are not statistical support to say that exist an asymmetry in the formation of
cartilage, but the cartilage will be smaller in the μg group than the normal gravity group.
Labyrinth: We expect a decrease in frecuency of the peaks at response of perception of
position in microgravity, and these changes will be reversible in normal conditions with
some time of recuperation.
Behaviour: We expect some change in the altitude where the tadpoles swim, and this
change will be reversible if the μg-tadpoles are not held there for long periods of time.
REFERENCES
Black, S.; Larkin, K.; Jacqmotte, N.; Wassersug, R.; Pronych, S.; Souza, K.; Regulative Development of
Xenopus laevis in Microgravity. 1996. Advanced Space Research.17 :209-217
Black, S.; Larkin, K.; Jacqmotte, N.; Wassersug, R.; Proynch, S.; Souza, K. Regulative development of
Xenopus laevis in microgravity. 1996. Advanced Space Research. 17:209-217
Crawford-young, S.J. Effects of Microgravity on Cell Cytoskeleton and Embryogenesis. 2006.
International journal of development biology. 50:183-191
Rossi, M.L.; Rubbini,G.; Gioglio, L.; Martini, M.; Fesce, R. Exposure to Reduced Gravity Impairs
Junctional Transmission at the Semicircular Canal in the Frog Labyrinth. 2009. American Physiological
Society, 298:439-452
Olson, M.W.; Wiens, J.D.; Gaul, L.T.; Rodriguez, M.; Hauptmeier, L.C. Xenopus Development from late
Gastrulation to Feeding Tadpole in Simulated Microgravity. 2010. International journal of development
biology. 54:167-174
Pronych, S.; Souza, K.; Neff, A.; Wassersug, R. Optomotor behaviour in Xenopus laevis tadpoles as a
measure of the effect of gravity on visual and vestibular neural integration. 1996. The Journal of
Experimental Biology. 199:2689-2701
Yamashita, M.; Izumi-Kurotani, A.; Mogami, Y.; Okuno, M.; Naitoh, T.; Wassersug, R.J. The Frog in Space
(FRIS) Experiment Onboard Space Station Mir: Final Report and Follow on Studies. 1997. Biological
Science in space. 11:313-320