Frogs and space.Using animal models to study the effects

of microgravity

Joanne PearsonMariama IssakaDaniel MartínezEdoardo 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.

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

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.

REASONS & ADVANTAGES

• 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)

WHAT ARE THE ADVANTAGES OF AMPHIBIANS?

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.

OBJECTIVE

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

Fertilize Study Study

Embryos Tadpoles Adults Adults

Study

Fertilize

Embryos Tadpoles

Study

SPACEEARTH EARTH

StudyStudy

EXPERIMENT / METHOD

-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)

-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)

-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…

-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…

GeneralGeneral

ParticularParticular

(Method adapted from Black et al., 1996; Souza et al., 1995; Rossi et al., 2009; Olson et al., 2010)

HARDWARE

Tadpoles Adult frogs Eggs and sperm

Life support box (LSB)

Frog recovery box (FRB)

Source: vssec.vic.edu.au

Source: trekearth.com/ Source: xlaevis.comSource: ns.umich.edu

Source: vssec.vic.edu.au

HARDWARE

International Space Station

Shuttle Researchers

Kibo ( 希望 )

Source: wikipedia.orgSource: wikipedia.org

Source: esa.int

• 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.

CONTROLS

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

Embryo:-fixation and blue alcian stain, view general structures-fixation and stain with immunohystochemical method, view cytoskeleton

Adult:-fixation of neural part, view structures -electrophysiological measurements of labyrinth

Adult:-fixation of neural part, view structures -electrophysiological measurements of labyrinth

Tadpole:-fixation and blue alcian stain, view general structures-optomotor behaviour test on glass with strips -dissection and study the lungs

Tadpole:-fixation and blue alcian stain, view general structures-optomotor behaviour test on glass with strips -dissection and study the lungs

(Method adapted from Black et al., 1996; Souza et al., 1995; Rossi et al., 2009; Olson et al., 2010)

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.

EARLY RETRIEVAL

Source: vssec.vic.edu.au

MEASUREMENTS TAKEN AFTER LANDING

Stimulis cylinder, adapted from Pronych et al., 1996

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.

• 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.

POSSIBLE APPLICATIONS

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.

REFERENCESBlack, 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