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The pleurodele, an animal model for space biology studies
1996 A&. Space Rev. Vol. 17, No. 6/7, pp. (6/7)265+/7)268, Copyright 0 1995 COSPAR Printed in Great Britain. All rights resewed. 0273-1177/96 $9.50 + 0.00 0273-1177(95)00643-5
THE PLEURODELE, AN ANIMAL MODEL FOR SPACE BIOLOGY STUDIES L. Gualandris, S. Grinfeld, F. Foulquier, P. Kan and A. M. Duprat Centre de Biologie ah Dkveloppement, VMR-CNRS 9925, Vniversitt?PaulSabatier, Toulouse, France
ABSTRACT Pleurodeles waltl, an Urodele amphibian is proposed as a model for space biology studies. Our laboratory is developing three types of experiments in space using this animal: 1) in viva fertilization and development (“FERTILE” project); 2) influence of microgravity and space radiation on the organization and preservation of spacialized structures in the neurons and muscle cells (in vitro; “CELIMENE” PROJECT); 3) influence of microgravity on tissue regeneration (muscle, bone, epidermis and spinal cord).
INTRODUCTION The Urodele amphibian Pleurodeles waltz (figure 1) of the Salamandridue family, has many advantages as a subject for biological experiments in space: like other amphibians, it is relatively light (50 to 60 gr), it is sturdy and the adult lives indifferently in water or in damp air. In addition it can live without food for 3 to 4 weeks and it is very easy to manipulate (it does not jump). Other biological properties have been known for a long time and used routinely in our laboratory. Three of these properties are of interest for studies in space; 1) fertilization takes place in vivo and spermatozoa can live for several months in the cloaca of the female; 2) embryonic cells can differentiate functionally and morphogically in culture into muscle cells, neurons, melanocytes, etc...; 3) the central nervous system (spinal cord) of the adult regenerates in the same way as muscles and bones after tail section. Corresponding experiments are being developed with this aim and are described below.
1) IN VZVO FERTILIZATION
AND DEVELOPMENT
(“FERTILE”
project)
In the amphibian egg, the animal-vegetal polarity and the dorso-ventral symmetry, seem to be directly dependent on gravity. These features are determined during the very early stages of development.
Figure 1: adult pleurodele (X0.3)
Successful in vitro fertilizations have been obtained in space by Ubbels et aE 111 and later by Souza et al 121. It is now of interest to establish whether a natural in viva fertilization is possible under microgravity conditions, i.e. if the meeting and fusion of gametes also occur normally in the genital tract of the female. With the pleurodele, this experiment becomes possible in space /3/ : in this species, spermatozoa stay alive in the cloaca of the female for several months after mating and the production of a first laying. A “second laying” is then induced by the injection of luteinizing hormone-releasing hormone (LHRH). In this case, the eggs are fertilized during their passage in the cloaca by the stored spermatozoa. In the pleurodefe, embryonic development is rather slow: the one-cell stage lasts 6 hours and the neurula stage is reached after 3 days. This allows an accurate analysis of early development, and thus is of great help to determine more precisely whether microgravity provokes some abnormalities, which could be rapidly regulated. The occurrence of real fertilization (and not gynogenesis) can be proved by the utilization of cytogenetic (presence of a pericentric inversion in males /4/) and enzymatic markers enabling an early analysis of the sex-ratio in the embryos /5/. This experiment, called “FERTILE” (Fecondation et Embryogenese ReaIisCe chez un Triton XPT E$VCIdans L ‘Espace) is planned to be performed in 1996, aboard Mir station. For this experiment, we collaborate with the groups of Professors Houiilon (Paris) and Dournon (Nancy). The hardware is developed by CNES and the French firm COMAT.
2) CELIMENE
PROJECT
“CELIMENE” stands for : CELlules en Impesanteur
: Muscles Et Neurones Embryonnaires.
Salamsnder(Pleurodeleafin Space
cm=7
The objective of this experiment is to define the effect of microgravity and space radiation on the organization and preservation of highly specialized cell structures, such as myofibrils in the striated muscle cells or axons in neurons. These observations will allow us to understand the influence of such external factors as intracytoplasmic organization, cell radiations and gravity on cell morphogenesis, morphogenesis, cell multiplication and cell adhesion. In addition, these expe~ments will provide essential data about general effects of microgravity and will contribute to better know the dangers to be met in space and thus, to aid in the prevention of these dangers. In particular, radiation is known to impair neural morphogenesis: our model enables this problem to be studied in culture and to relate it to organogenesis in the whole embryo. The amphibian embryonic cell is well adapted for space experiments: it only needs very simple culture conditions to differentiate functionally and morphologically. These cells contain their own nutritive substances; they are cultured in sealed recipients, in saline medium that does not need changing for 3 weeks at 20°C and for longer at lower temperatures, Under these conditions, it is possible to obtain a phenotypic and functional differentiation, from determined but undifferentiated cells 161 (Figure 2). During the laboratory preparation phase of the space experiment, it was shown that the embryonic cells in culture could stay for several weeks at 5°C. During this time their development stopped, but it continued normally when the cells were placed at 20°C. This property is of importance in space, to stop cell evolution during launching periods for example. This experiment will be embarked in the automatic facility IBIS developed by CNES, which permits the incubation of cell cultures and whole embryos, and automatic fixations at preprogrammed times.
&me 2: Pleurodele embryonic neurons (N), fibroblastic, cells @I) after 10 days in culture at 2O’C. Bar = 50 pm.
chord (C), pigment (P), and muscle
L. Gualandris et al.
WWfJ
3) INFLUENCE
OF GRAVITY ON TAIL REGENERATION
The central nervous system (spinal cord) of adult Urodeles regenerates in the same way as muscles and bones after tail section /7/. These potentialities of regeneration in a Vertebrate are of great. interest to study from the following points of view: - structural organization of these different tissues; - cell proliferation, which is very active in these regenerating tissues; - the analysis of expression and function of specific molecules involved in neural, muscle or bone differentiation. The different specificities of the pleurodele described above, enable this study to be performed in space relatively easily. This was done in January 1993 aboard the Russian satellite Bion 10. Results are discribed in the reference /8/.
AKNOWLEDGEMENTS
These studies are supposed by the Centre National d’Etudes Spatiales.
REFERENCES
1. G.A. Ubbels, M. Reijnen, J. Meijerink and J. Narraway, Xenopus Laevis embryos can establish their spatial bilateral symmetrical body pattern without gravity, Adv. Space Res., 14,8, 257-269 (1994). 2. K. Souza, conference in the Sth Eur. Symp. m “‘Li$?Sciences Reseu~ch in Spice’; Arc~cho~, France, 26 Sept.-ISt Oct. 1993. 3. S. Grinfeld, C. Doumon, C. Houillon, A. Bautz and A.M. Duprat, In vivo fertilization and development in microgravity using pleurodele (ZEUS project), Adv. Space Res., 14, 8, 305307 (1994). 4. A. Jaylet, Modification du caryotype par une inversion pkricentrique i%Y&athomozygote chez l’amphibien Urodkle Pleurodeles waltlii Michahelles, Chromosoma, 35,288-299 (197 1). 5. V. Ferrier, F. Gasser, A. Jaylet and C. Cayrol, A genetic study of various enzyme polymorphisms in Pleur~deLes ~~ltlii (Urodele amphibian). II. Peptidases : demons~ation of sex linkage. Biochem. Genet., 2 1, 535-549 (1983).
6. A.M.Duprat, J.P.Zalta and J.C. Beetschen, Action de I’actinomycine D sur la differentiation de divers types cellulaires embryonnaires de l’amphibien Pleurodeles waltlii en culture in vitro, Exp. Cell Res., 43,358-366 (1966). 7. S. Grinfeld, F.Foulquier, V. Mitashov, N. Bruchlinskaya and A.M. Duprat, Tissue regeneration in space (spinal cord, muscle and bone) in the amphibian Pleurodeles w&l, in : Proc. 5th Eur. Symp. on “Life Sciences in space, Arcachan, France 1993 (ESA SP-366, August 1994).
8. S. Grinfeld, F. Foulquier, V. Mitashov, N. Bruchlinskdia and A.M. Duprat, Amphibian tail regeneration in space : effect on the pigmentation of the blastema, this issue.
THE PLEURODELE, AN ANIMAL MODEL FOR SPACE BIOLOGY STUDIES L. Gualandris, S. Grinfeld, F. Foulquier, P. Kan and A. M. Duprat Centre de Biologie ah Dkveloppement, VMR-CNRS 9925, Vniversitt?PaulSabatier, Toulouse, France
ABSTRACT Pleurodeles waltl, an Urodele amphibian is proposed as a model for space biology studies. Our laboratory is developing three types of experiments in space using this animal: 1) in viva fertilization and development (“FERTILE” project); 2) influence of microgravity and space radiation on the organization and preservation of spacialized structures in the neurons and muscle cells (in vitro; “CELIMENE” PROJECT); 3) influence of microgravity on tissue regeneration (muscle, bone, epidermis and spinal cord).
INTRODUCTION The Urodele amphibian Pleurodeles waltz (figure 1) of the Salamandridue family, has many advantages as a subject for biological experiments in space: like other amphibians, it is relatively light (50 to 60 gr), it is sturdy and the adult lives indifferently in water or in damp air. In addition it can live without food for 3 to 4 weeks and it is very easy to manipulate (it does not jump). Other biological properties have been known for a long time and used routinely in our laboratory. Three of these properties are of interest for studies in space; 1) fertilization takes place in vivo and spermatozoa can live for several months in the cloaca of the female; 2) embryonic cells can differentiate functionally and morphogically in culture into muscle cells, neurons, melanocytes, etc...; 3) the central nervous system (spinal cord) of the adult regenerates in the same way as muscles and bones after tail section. Corresponding experiments are being developed with this aim and are described below.
1) IN VZVO FERTILIZATION
AND DEVELOPMENT
(“FERTILE”
project)
In the amphibian egg, the animal-vegetal polarity and the dorso-ventral symmetry, seem to be directly dependent on gravity. These features are determined during the very early stages of development.
Figure 1: adult pleurodele (X0.3)
Successful in vitro fertilizations have been obtained in space by Ubbels et aE 111 and later by Souza et al 121. It is now of interest to establish whether a natural in viva fertilization is possible under microgravity conditions, i.e. if the meeting and fusion of gametes also occur normally in the genital tract of the female. With the pleurodele, this experiment becomes possible in space /3/ : in this species, spermatozoa stay alive in the cloaca of the female for several months after mating and the production of a first laying. A “second laying” is then induced by the injection of luteinizing hormone-releasing hormone (LHRH). In this case, the eggs are fertilized during their passage in the cloaca by the stored spermatozoa. In the pleurodefe, embryonic development is rather slow: the one-cell stage lasts 6 hours and the neurula stage is reached after 3 days. This allows an accurate analysis of early development, and thus is of great help to determine more precisely whether microgravity provokes some abnormalities, which could be rapidly regulated. The occurrence of real fertilization (and not gynogenesis) can be proved by the utilization of cytogenetic (presence of a pericentric inversion in males /4/) and enzymatic markers enabling an early analysis of the sex-ratio in the embryos /5/. This experiment, called “FERTILE” (Fecondation et Embryogenese ReaIisCe chez un Triton XPT E$VCIdans L ‘Espace) is planned to be performed in 1996, aboard Mir station. For this experiment, we collaborate with the groups of Professors Houiilon (Paris) and Dournon (Nancy). The hardware is developed by CNES and the French firm COMAT.
2) CELIMENE
PROJECT
“CELIMENE” stands for : CELlules en Impesanteur
: Muscles Et Neurones Embryonnaires.
Salamsnder(Pleurodeleafin Space
cm=7
The objective of this experiment is to define the effect of microgravity and space radiation on the organization and preservation of highly specialized cell structures, such as myofibrils in the striated muscle cells or axons in neurons. These observations will allow us to understand the influence of such external factors as intracytoplasmic organization, cell radiations and gravity on cell morphogenesis, morphogenesis, cell multiplication and cell adhesion. In addition, these expe~ments will provide essential data about general effects of microgravity and will contribute to better know the dangers to be met in space and thus, to aid in the prevention of these dangers. In particular, radiation is known to impair neural morphogenesis: our model enables this problem to be studied in culture and to relate it to organogenesis in the whole embryo. The amphibian embryonic cell is well adapted for space experiments: it only needs very simple culture conditions to differentiate functionally and morphologically. These cells contain their own nutritive substances; they are cultured in sealed recipients, in saline medium that does not need changing for 3 weeks at 20°C and for longer at lower temperatures, Under these conditions, it is possible to obtain a phenotypic and functional differentiation, from determined but undifferentiated cells 161 (Figure 2). During the laboratory preparation phase of the space experiment, it was shown that the embryonic cells in culture could stay for several weeks at 5°C. During this time their development stopped, but it continued normally when the cells were placed at 20°C. This property is of importance in space, to stop cell evolution during launching periods for example. This experiment will be embarked in the automatic facility IBIS developed by CNES, which permits the incubation of cell cultures and whole embryos, and automatic fixations at preprogrammed times.
&me 2: Pleurodele embryonic neurons (N), fibroblastic, cells @I) after 10 days in culture at 2O’C. Bar = 50 pm.
chord (C), pigment (P), and muscle
L. Gualandris et al.
WWfJ
3) INFLUENCE
OF GRAVITY ON TAIL REGENERATION
The central nervous system (spinal cord) of adult Urodeles regenerates in the same way as muscles and bones after tail section /7/. These potentialities of regeneration in a Vertebrate are of great. interest to study from the following points of view: - structural organization of these different tissues; - cell proliferation, which is very active in these regenerating tissues; - the analysis of expression and function of specific molecules involved in neural, muscle or bone differentiation. The different specificities of the pleurodele described above, enable this study to be performed in space relatively easily. This was done in January 1993 aboard the Russian satellite Bion 10. Results are discribed in the reference /8/.
AKNOWLEDGEMENTS
These studies are supposed by the Centre National d’Etudes Spatiales.
REFERENCES
1. G.A. Ubbels, M. Reijnen, J. Meijerink and J. Narraway, Xenopus Laevis embryos can establish their spatial bilateral symmetrical body pattern without gravity, Adv. Space Res., 14,8, 257-269 (1994). 2. K. Souza, conference in the Sth Eur. Symp. m “‘Li$?Sciences Reseu~ch in Spice’; Arc~cho~, France, 26 Sept.-ISt Oct. 1993. 3. S. Grinfeld, C. Doumon, C. Houillon, A. Bautz and A.M. Duprat, In vivo fertilization and development in microgravity using pleurodele (ZEUS project), Adv. Space Res., 14, 8, 305307 (1994). 4. A. Jaylet, Modification du caryotype par une inversion pkricentrique i%Y&athomozygote chez l’amphibien Urodkle Pleurodeles waltlii Michahelles, Chromosoma, 35,288-299 (197 1). 5. V. Ferrier, F. Gasser, A. Jaylet and C. Cayrol, A genetic study of various enzyme polymorphisms in Pleur~deLes ~~ltlii (Urodele amphibian). II. Peptidases : demons~ation of sex linkage. Biochem. Genet., 2 1, 535-549 (1983).
6. A.M.Duprat, J.P.Zalta and J.C. Beetschen, Action de I’actinomycine D sur la differentiation de divers types cellulaires embryonnaires de l’amphibien Pleurodeles waltlii en culture in vitro, Exp. Cell Res., 43,358-366 (1966). 7. S. Grinfeld, F.Foulquier, V. Mitashov, N. Bruchlinskaya and A.M. Duprat, Tissue regeneration in space (spinal cord, muscle and bone) in the amphibian Pleurodeles w&l, in : Proc. 5th Eur. Symp. on “Life Sciences in space, Arcachan, France 1993 (ESA SP-366, August 1994).
8. S. Grinfeld, F. Foulquier, V. Mitashov, N. Bruchlinskdia and A.M. Duprat, Amphibian tail regeneration in space : effect on the pigmentation of the blastema, this issue.