ICE 1st

Abstract: Travelling, living and working in space is a reality. Since now 20 years, space stations are on orbit, and are quasi permanently inhabited by astronauts who can perform a large number of activities from technical operations to science experiments. On one hand, with increasing numbers of people and extended lengths of time in space, it becomes more and more important to understand more in depth the effects of space flight. On the other hand, the microgravity is a useful tool to unveil some fundamental mechanisms which are altered at the level of the cell or at the level of the organism by changes in the environment. It is known that Caenorhabditis elegans (C. elegans) can mate, reproduce and develop apparently normally during space flight (G. Nelson, p. comm.). In the first International C. elegans Experiment (ICE-first), several scientific domains will be studied. This unique opportunity provided to the science community gathered by the French space agency (CNES) and kindly offered by the European Space Agency and the Space research organisation of the Netherlands (SRON) is welcoming groups of scientists from France, Canada, Japan and United states of America. This is an international cooperative project.

Address corresponding author:
Experiment Coorinator dr. Michel Viso, CNES, Paris.
Scientific Team: dr. Nicole Buckley (Ottawa, CA), dr. Bev Girten and dr. Cassie Conley (Nasa-ARC, US), dr. Jany Vassy (CNRS. Paris, FR), dr. Ann Rose (Vancouver, CA), dr. Noriaki Ishioka (JAXA, JP) and dr. Laurent Ségalat (Lyon, FR).

Introduction

What is Caenorhabditis elegans
Caenorhabditis elegans is a nematode (Phylum Nematoda) measuring around 1mm and living naturally in soil. It is of no economic importance to man. It has reproductive, nervous, muscular, and digestive systems. It is a very simple organism mad of a fixed number of somatic cells (959). Its life span is about 2-3 weeks and in the liquid medium at 25°, the life cycle is around 5 days. The genome is totally sequenced (100 000 000 of base pairs and around 20 000 genes). Numerous mutants are available. It is used as a model system for various medical pathologies and was the subject of the 2002 Nobel Prize in Medicine or Physiology because the process of apoptosis or programmed cell death was discovered while studying C. elegans development.

Around the world many hundreds of scientists are working full time investigating its biology. C. elegans is about as primitive an organism that exists which nonetheless shares many of the essential biological characteristics that are central problems of human biology. The worm is conceived as a single cell which undergoes a complex process of development. Then lineage of the 959 cells is fixed and well known allowing a huge number of investigations in fundamental biology. Scientists study embryogenesis, morphogenesis, development, nerve function, behavior and aging. C. elegans may be handled as a micro-organism. Thus it provides the researcher with the ideal compromise between complexity and tractability.

Figure 1: The Worm!!! ©2002 Wormatlas

Studies on genome stability (Ann Rose, Canada).
It is known that incident radiation is increased several-fold during space flight and that this causes heritable changes (G. Nelson, p.comm). We will take advantage of the suitability of genetic approaches on C. elegans to address this question by analysing the distribution of an antigen (mdf-2) which is essential for the normal dicisions of the cells. An alteration of the production of this antigen will lead to the accumulation of defects including chromosome abnormalities, X-chromosome nondisjunction or loss, problems in gonad development, and embryonic lethality (Kitagawa & Rose, 1999).
Another way to evaluate the effect of radiation is to study the G-tracks wich are present in the genome. There are approximately 395 strings of nucleotides (nts) with Glutamine series (18 or greater, most within the range 20 -25 Gs), of which 134 are adjacent to coding regions. The probability of one stretch of 18 Gs occurring by chance is 1 in 64 billion. In the genome, there are nearly 400 such series. Thus, it seems unlikely that these strings have occurred by chance. Retention of G-tracks requires active enzymatic activity, which presumably is energetically costly. In the absence of DOG-1 function deletions occur spontaneously in genes containing strings of Gs. We have shown previously that the function of the dog-1 (deletion of G) is required for the maintenance of G-tracks of 18-28 nts in length (Cheung et al. 2002). Dog-1 mutants show germline as well as somatic deletions in genes containing G-tracts and display a telomere shortening phenotype.
We propose to examine strains of live worms (CC1 and Dog-1) returned from space flight for alterations in the G-tracks and compare to ground controls. The hypothesis is that the G-tracks play a functional role in maintaining genome stability and make a sensitive assay of genomic damage. We have developed primer sets to detect deletions originating in G-track and these will be used to survey G-tracks on the left end of chromosome V, the same region that Greg Nelson examined for radiation-induced mutational changes (p. comm.).

Studies on muscle growth and survival (Catharine Conley/Beverly Girten, USA; and Laurent Ségalat, France).
Microgravity has an important impact on muscle physiology and growth. C. elegans has muscles which are analogous to vertebrates in terms of composition and basic organisation. Investigations on C. elegans would benefit from a large number of mutants available in this organism. In this first study, emphasis would be put on two sets of genes : First, we will investigate the localization of Tropomodulin proteins and other contractile proteins of muscle. In mammals, Tropomodulins display altered expression in response to muscle under- or over-loading. Worms have two Tropomodulin genes, thus the data obtained would provide preliminary results concerning the appropriateness of worms as a model for Tropomodulin involvement in muscle atrophy (Strain CC1). Second, we will analyse the phenotype of animals mutant for genes encoding protein involved in muscle survival. One such protein is dystrophin, the product of the gene mutated in Duchenne Muscular Dystrophy, an inherited disease in which patients suffer from a progressive muscle necrosis. We will also study the effect of microgravity on other mutations affecting C. elegans muscle survival (including MyoD, perlecan, titin). Two mutants will be flown: LS541 and LS761. The effects of microgravity will be analysed by immucytochemistry and microscopy. For one set of experiment, the worms will be issued from an "egg prep" allowing a slow development still the launch and to fix by the end of the flight "old" adult worms (Scenario C).

Whole-genome microarray analysis of responses to spaceflight in C.elegans (Catharine Conley, Stuart Kim USA)
Spaceflight is suspected or has been recognized to produce specific physiological responses, including radiation damage repair in response to cosmic radiation, and muscle atrophy in response to microgravity-induced unweighting. A number of additional physiological phenomena have been reported, such as immune dysfunction and altered aging, that are not well understood at the cellular or molecular level. Microarray analysis is an excellent method of screening for both known and novel genes that show altered expression in response to a particular treatment.
The proposed experiment involves performing RNA expression analysis using a microarray designed to probe for nearly every gene in the genome. Although microarray analysis does not provide detailed information concerning the function of any particular gene, its acts as an efficient indicator of which genes might be interesting to study further, because they show altered expression in response to the treatment.
Two hypotheses that can be tested directly using microarray data obtained from spaceflown worms are a) that radiation-repair genes will be up-regulated, and b) that genes involved in muscle specification and contractility will be down-regulated. The analysis does not provide information concerning the specific role of any of these genes, but does confirm that genes we predict to show altered expression based on data from mammals are indeed responsive in worms. Additional hypotheses concerning worm 'immune' function and aging can also be tested, by determining the expression of genes known to be involved in those physiological processes. These results would provide excellent preliminary data for proposing additional spaceflight experiments to examine specific genes in detail. Whole-genome analyses provide a useful resource to the entire scientific community, as any researcher considering a particular line of study can check microarray data to determine whether their genes of interest respond to treatment. For this ICE flight, we propose to obtain RNA from worms that have been exposed to spaceflight and returned to earth alive. This experiment will require samples for RNA analyses to be frozen within 1-2 hrs of the return to Earth. RNA will be analysed using the whole-genome microarray developed by the Kim lab in the Stanford Genome Center. Data from the microarray analysis will be made publicly available in the Stanford Microarray Database. The worms will be prepared from a CC1 mixed stage population, kept at 18°C till the launch and after and which will be recover alive for immediate treatment (Scenario B).

Morphometry of larval C. elegans development during spaceflight. (Catherine Conley, Beverly Girten, USA)
Spaceflight is thought to affect mammalian development at specific 'critical periods' during infancy. Although worm cultures have grown successfully during several previous spaceflights, it is not yet known whether spaceflight exerts non-lethal effects on worm development.
In liquid CeMM, the media proposed for use in the ICE flight, larvae shed cuticles as they molt and progress to the next developmental stage. The length of the shed cuticles is indicative of the length of the larvae at the time when they molt, and thus can be used as a metric for larval development. For this experiment , we propose to measure the range of lengths exhibited by shed cuticles in media from cultures that have been returned alive. The distribution of length data will indicate the number and progression of larval moults during development in space. The Conley lab has already determined the normal progression of development in CeMM on Earth (manuscript in press), and this will be the first analysis of C. elegans develoment in space. The worms will be prepared from a mixed stage population, kept at 18°C till the launch and after and will be recover alive for immediate treatment (Scenario B).

Microtubules and microfilaments (Jany Vassy, France)
From previous space flown and ground based experiments it was demonstrated that the microtubules are sensitive to the gravity level in cultured cells. It is of major interest to study this phenomenon in a model organism like Ceonorhabditis elegans. The classical strain N2 will be used but further investigations could help determining other strain of interest to challenge several hypotheses.
The microtubules and the microfilaments in dividing cells and in epithelia will be particularly studied using the appropriate antibodies for immunolocalisation and then using a confocal microscope. To achieve this goal the samples will be frozen upon the recovery and then treated with methanol and then air dried in the laboratory. This will be the first analysis of Tubuline and F-Actine in space flown C. elegans. The worms will be prepared from a mixed stage population, kept at 18°C till the launch and after and will be recover alive for immediate treatment.

Effect of space flight on cell migration and muscle cell in C. elegans development. (Hiroaki Kagawa, Noriaki Ishioka, Japan)
C. elegans has two muscle tissues; pharynx for feeding and body wall muscle for locomotion. The both correspond to heart and skeletal muscle of vertebrates. Recently we found that muscle filament gene defect affect not only muscle function but also muscle development. Additionally these mutant animals have abnormal distal tip cell migration during the worm development. Abnormal cell migration can easily be seen under microscope.
For this experiment, we use wild-type and thick filament abnormal mutant (unc-15), which produce muscle filament but decreased function and have abnormal morphology of distal tip cells.

Studies on germ line development including meiotic chromosomal dynamics and germ cell apoptosis under microgravity condition (Atsushi Higashitani, Noriaki Ishioka, Japan)
In C. elegans, the sequence of changes in chromosomal morphology during meiotic prophase 1, the oocyte maturation and the germ cell apoptosis can be observed, and the molecular mechanisms underlying these phenomena can be investigated with genetic approaches. We will analyze the effects of microgravity on these phenomena using the N2 wild-type and ced (cell death) mutants (at least two mutants, ced-1 and ced-3) strains. This experiment will require 100 to 1000 animals in each strain at mixed developmental stages, in addition to 1G control of each sample in space) fixed in flight for microscopic observations staining with DAPI and several antibodies (histone H3 phosphorylation and methylation, activated MAPK etc.).

Analysis of the aging related protein aggregation and sarcomere integrity (Shuji & Yuko Honda, Noriaki Ishioka Japan)
To examine the effects of space on protein-folding homeostasis in muscle cells, we will analyze the aggregation of polyglutamine (polyQ) in body wall muscle cells, using transgenic C. elegans (N2; Punc-54)expressing polyQ-YFP (yellow fluorescent protein) and also daf-2(e1370) lifespan-extension mutant. We will also analyze sarcomere orientation in the muscle of transgenic C. elegans(N2; Punc-54) expressing GFP (green fluorescent protein) in body wall muscle cells.

Description of the Experiment
Generally, C. elegans is growing on agar plates on a special medium (NGM). To fly the animals have to be adapted to the liquid medium specially provided by NASA (CeMM). The various strains will be prepared in the laboratory of the investigators.
Four days before the launch the strains will be prepared for the flight by the investigators in the facility of the GSBMS (Groupement scientifique pour la Biologie et la Médecine spaciale) in Toulouse and then put in their containers, ready for the grouns and space travel.
Then the samples will be transported by hand carriers to the launch pad in Baikonour, transferred in Kubik with the other experiment and kept at 18°C. Three days after the launch, the samples will be transferred to the Kubik with the centrifuge and three small containers will be placed on it. By the last day of the flight, 4 containers will be fixed by a simple operation of the astronaut. Then he will pack the 8 containers with the others to have then returned to the ground in Soyouz.
Right upon the arrival the containers will be opened and some will be filmed to evaluate the behaviour of the animals. The small bags containing the culture of the worms will be then either frozen or refrigerated till their return in Toulouse two days after the landing. The scientist will then begin their scientific work

Acknowledgement
The science team acknowledges the space agencies which are allowing this flight opportunity as "l'Agence Spatiale Canadienne" (ASC), the "Centre National d'Etudes Spatiales" (CNES), the European Space Agency (ESA), the Japan Aerospace Exploration Agency (JAXA), the National Aeronautic and Space Administration (NASA), and the Space Research Organisation of the Netherlands (SRON).