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Exogastrulation induced by heavy water in sea urchin larvae
Cell Differentiation, 8 (1979) 431--435 © Elsevier/North-Holland Scientific Publishers Ltd.
431
EXOGASTRULATION INDUCED BY HEAVY WATER IN SEA URCHIN LARVAE
MOTONORI HOSHI Institute o f Low Temperature Science, Hokkaido University. Sapporo 060 (Japan)
Accepted July 31st, 1979 Replacement of H~O with D:O in seawater causes exogastrulation in larvae of the sea urchins, Hemicentrotus pulcherrimus, Strongylocentrotus intcrmedius and S. nudus. When larvae at any stages before mesenchymal blastula stage are transferred to 40% D:O-seawater all of them develop gradually to exogastrulae and finally up to plutei with evaginated archenterons. Effects of D~O are partly reversible at limited steps of the way to exogastru[ation. Fertilisation and cleavage are not affected appreciabl~" by D~O (50% or less) except for the delay of cleavage.
Diverse effects of heavy water (D20) upon various biological systems ranging from animal behaviour to interaction between biomolecules have been reported (for reviews see Thompson, 1963; Katz and Crespi, 1970). When H~O is replaced with D20, virtually every biological process slows down and higher order structures of proteins and other molecules are generally stabilized. These changes axe owing to the solvent effects of D20 and to the effects of substitution of D for H in molecules at both the reaction site (primary isotopic substitution) and sites not directly involved in the reaction (secondary isotopic substitution} (Pittendrigh et al., 1973). Shortly after Urey (1933} isolated deuterium, Luck~ and Harvey (1935) found that D:O delayed the division of sea'urchin eggs. In the early 60's, it was shown that D20 blocked mitosis reversibly by 'freezing' or 'stabilising' mitotic apparatus of sea urchin eggs (Gross and Spindel, 1960; Marsland and Zimmerman, 1963}. Inou~ and his associates have demonstrated t h a t D20 increases reversibly the volume, length and birefringence of the mitotic spindles of sea urchin eggs, encountering different sensitivities at different mitotic stages, and t h a t it induces rapid emergence of microtubules in the spindle area (for a review see Inou~ and Sato, 1967}. They have suggested t h a t this agent favours the construction of microtubules from subunits. Recently, microtubules have called general a t t e n t i o n as among the fundamental organelles acting in karyokinesis, control of cell shape, intracellular transport, cell movement, sensory transduction and so on (for a review see Olmsted and Borisy, 1973). Tilney and Gibbs (1969} demonstrated t h a t the pseudopodia of secondary mesenchymal cells in sea urchin larvae have a
432 large number of microtubules' and microfilaments. They also found that colchicine and hydrostatic pressure, both of which cause the disintegration of the microtubules in these cells as well as others, block the second phase of invagination that is the pulling of archenteron by pseudopodia of the secondary mesenchymal cells (Dan and Okazaki, 1956; Gustafson and Kinnander, 1956 ). Since destruction of pseudopodia of secondary mesenchymal cells by various means blocks the invagination of sea urchin larvae, which results in exogastrulation (Dan and Okazaki, 1956; Okazaki, 1956), I have predicted that exogastrulation may be caused by the agents that effect (either stabilise or disintegrate) microtubules. We have obtained exogastrulae by rearing the larvae in the presence of either colchicine, vinblastine, dithiothreitol, cytochalasin B or cytochalasin C, or at low temperature (Takahashi et al., 1977}. Sulfhydryl reagents did not induce exogastrulation. Under these conditions (except for the presence of cytochalasins) microtubules are known to be affected. Cytochalasins affect the microfilaments. Here I add D20 to the list of agents which cause exogastrulation in sea urchin larvae. MATERIALS AND METHODS
Deuterated seawater Commercial heavy water (99.75 atom%) was used after double distillation. Artificial seawater made up in D20 diluted with H20-seawater (no D20 enrichment~ to yield a final concentration of 0--50%D20-seawater. The 'concentrations' of D20 henceforth reported refer to the volume percent of D20 used in making the seawater. Animals The following sea urchins were used during their breeding seasons: Hernicentrotus pulcherrimus, Strongylcentroutus intermedius and S. nudus. Gametes were collected by introducing 0.5 M KC1 into the coelom. Developing eggs or embryos, approx. 10 ~ in 50 pl, were transferred at various stages of development to 2 ml D20-seawater in a multidish (Linbro Chemicals), where t h e y were reared. In some experiments, embryos were returned to H20-seawater after a short stay in D20-seawater. At adequate time intervals, embryos were examined for cleavage and development. To see the effects of D20 on fertilisation, unfertilised eggs were inseminated in D20-seawater. RESULTS AND DISCUSSION Several lots of commercial D20 have been shown to contain an impurity causing a p h e n o m e n o n t h a t had been attributed to D20 (Huxtable and Bressler., 1974). Thus, D20 was twice distilled before use.
433 No appreciable effects of D~O on fertilisation were found at the concentrations up to 50% as shown in Table I. This does not agree with a previous report t h a t fertilisation, more strictly the elevation of the fertilisation envelope of Arbacia was significantly prevented by D20 at concentrations over 20% {Gross et al., 1960). All the fertilised eggs cleaved in seawater containing 40% D20 or less, and most of them did so even in 50% D20-seawater. Table I shows, however, the delay o f cleavage in D20-seawater as reported previously (Luckd and Harvey, 1935; Gross and Spindel, 1960; Marsland and Zimmerman, 1963). In D20-seawater, embryos developed normally up to primary mesenchyme blastulae, except for a delay of development to various degrees depending upon D20 concentrations. As clearly shown in Fig. 1, replacement o f H20 with D20 caused exogastrulation. When larvae of all the species used were transferred to 40% D:O-seawater at any stages before mesenchyme blastula, all of t h e m developed to exogastrulae and later on to plutei with evaginated guts {Fig. 2). In 50% D20-seawater, t h e y developed to exogastrulae at stage 1 {Fig. 2a), but t h e y did not develop b e y o n d this stage. When primary mesenchyme blastulae were transferred to D20-seawater, the subsequent development differed between species, as shown in Fig. 1. In S. intermedius, D20 induced exogastrulation in these larvae as well as those transferred at earlier stages. In H. pulcherrimus, D20 induced exogastrulation but was far less effective than that administered at earlier stages. In the case o f S. nudus, no exogastrulae were obtained even in 50% D20-seawater. In these species, no larvae with evaginated guts were obtained by administering D20 to early gastrulae or further-developed larvae. When gastrulae were transferred to 50% D20-seawater, t h e y developed slowly to plutei in all the species studied. Effects o f D20 are similar in general to those of chilling. We have foun~ chilling o f early gastrulae causes exogastrulation (Takahashi et al., 1977).
TABLE I FERTILISATION AND CLEAVAGE OF THE EGGS OF HEMICENTROTUS PULCHERRIMUS IN D~O-SEAWATER Approximately 103 eggs were inseminated in 2 ml of D20-seawater and reared at 15°C. The final concentration of spermatozoa was 106 cells/ml. All values in percent. Concentration
Fertilisation
Development stage at 2 h post-insemination
of D20 0 3 6 12 25 50
100 100 100 100 100 99
Fertilised egg
2-cell stage
4-cell-stage
0 0 1 1 2 19
1 1 13 15 93 80
99 99 86 84 5 0
434 i
i
10
20
|
i
40
50
e-
g o
50
w .
0
0 0
30
Concentration of I~0
'/0
Fig. 1. Exogastrulation induced by D20. Eggs were fertilised and reaxed in H20-seawater. At various stages of development, embryos were transferred to 3--50% D~O-seawater and were allowed to develop. (Open symbols) blastulae just hatched were transferred to D20-seawater. (Closed symbols) primary mesenchymal blastulae were transferred to D20-seawater. (Circles) H. Pulcherrimus; (triangles) S. nudus; squares; S. intermedius. When fertilised eggs or embryos before hatching were transferred to D20-seawater, the similar curves to those of open symbols were obtained.
Therefore, I examined whether D20 was more effective at lower temperatures. I could not find any significant differences in the ability of D20 to cause exogastrulation at temperatures between 6°C and 25°C. If the larvae showing the first sign of exogastrulation, namely larvae at stage 1 in Fig. 2, were returned to H20-seawater, some of them developed to normal plutei. This suggests that effect of D20 is partly reversible at this stage. When exogastrulae at later stages were transferred to H20-seawater, none of them grew into the normal plutei. It is well established that gastrulation proceeds in two phases (Dan and Okazaki, 1956; Gustafson and Kinnander, 1956). The first phase, the autonomous invagination of the endodermal plate by deformation of cells (Gustafson and Wolpert, 1963), was not affected by D20 at concentrations used as shown in Fig. 2 (note the shallow invagination of proximal portion of evaginated guts). The second phase, the pulling of the archenteron by the pseudopodia of the secondary mesenchymal cells, might be affected to some extent by D20 less than 50%. D20 seems to affect instead the formation of secondary mesenchymes in the correct positions, because it induces exogastrulation only when administered to larvae up to the primary mesenchyme blastulae. The evaginated guts have cilia and differentiate to some extent even in
435
Fig. 2. Egogastrulation induced by D20 in the larvae o f H. pulcherrimus. Blastulae which had just hatched were transferred to 25% D20-seawater and were reared at 20 °C. Bars represent 50 um (a) Larva showing the first sign of exogastrulation (Stage 1), 6 h after the transfer; (b) Early exogastrula showing normal arrangement of primary mesenchymal cells, 20 h after the transfer; (c) Exogastrula (Prism with the evaginated gut), 30 h after the transfer; (d) Exogastrula with the differentiated gut, 35 h after the transfer. The gut contracts occasionally; (e) Early pluteus with the evaginated gut, 40 h after the transfer; (f) Pluteus with the evaginated gut, 60 h after the transfer.
D20-seawater, as shown in Fig. 2. They contract occasionally and are easily removed from b o d y b y agitating the culture medium. In conclusion the evidence presented above indicates that the partial substitution of D20 for H20 in sweater causes exogastrulation in all the urchins studied. ACKNOWLEDGEMENTS I am much obliged to Drs. Y. Nagai, T o k y o Metropolitan Institute of Gerontology, and K. Onodera, K y o t o University, for their suggestions and criticisms. I am also indebted to the directors and the staffs of the Marine Biological Stations at Misaki (University of T o k y o ) , Akkeshi (Hokkaido University) and Oshoro (Hokkaido University), where
436 part of the present study was performed. This work was supported in part by grants from the Ministry of Education, Science and Culture, Japan and from Itoh Science Foundation.
REFERENCES Dan, K. and K. Okazaki: Biol. Bull. 110, 29--42 (1956). Gross, P.R. and W. Spindel: Ann. N.Y. Acad. Sci. 9 0 , 5 0 0 - - 5 2 2 {1960}. Gustafson, T. and H. Kinnander: Exp. Cell Res. 11, 36--51 {1956). Gustafson, T. and L. Wolpert: Int. Rev. Cytol. 1 5 , 1 3 9 - - 2 1 4 (1963). Huxtable, R. and R. Bressler: J, Membrane Biol. 1 7 , 1 8 9 - - 1 9 7 (1974). Inou6, S. and H. Sato: J. Gen. Physiol. 5 0 , 2 5 9 - - 2 8 8 (1967). Katz, J.J. and H.L. Crespi: In: Isotope effects in reaction rates, eds., C.G. Collins and N.S. Bowman (Van Nostrand-Reinhold Book Co., New York) pp. 286--363 (197(}}. Luck6, B. and E.N. Harvey: J. Cell. Comp. Physiol. 5 , 4 7 3 - - 4 8 2 (1935). Marsland, D. and A.M. Zimmerman: Exp. Cell Res., 30, 23--35 (1963). Okazaki, K.: Embryologia 3, 23--36 (1956). Olmsted, J.B. and G.G. Borisy: Annu. Rev. Biochem. 42, 507--540 (1973). Pittendrigh, C.S., P.C. Caldarola and E.S. Cosbey: Proc. Natl. Acad. Sci. U.S.A. 70, 2037--2041 (1973). Takahashi, T., M. Hoshi and t~. Asahina: Dev. Growth Differ.19,131--137 (1977). Thompson, J.F.: Biological effects of deuterium (Pergamon Press, New York) {1963L Tilney, L.G. and J.R. Gibbs: J. Cell Sci. 5 , 1 9 5 - - 2 1 0 (1969). Urey, H.C.: Science 7 8 , 5 6 6 - - 5 7 1 (1933).
431
EXOGASTRULATION INDUCED BY HEAVY WATER IN SEA URCHIN LARVAE
MOTONORI HOSHI Institute o f Low Temperature Science, Hokkaido University. Sapporo 060 (Japan)
Accepted July 31st, 1979 Replacement of H~O with D:O in seawater causes exogastrulation in larvae of the sea urchins, Hemicentrotus pulcherrimus, Strongylocentrotus intcrmedius and S. nudus. When larvae at any stages before mesenchymal blastula stage are transferred to 40% D:O-seawater all of them develop gradually to exogastrulae and finally up to plutei with evaginated archenterons. Effects of D~O are partly reversible at limited steps of the way to exogastru[ation. Fertilisation and cleavage are not affected appreciabl~" by D~O (50% or less) except for the delay of cleavage.
Diverse effects of heavy water (D20) upon various biological systems ranging from animal behaviour to interaction between biomolecules have been reported (for reviews see Thompson, 1963; Katz and Crespi, 1970). When H~O is replaced with D20, virtually every biological process slows down and higher order structures of proteins and other molecules are generally stabilized. These changes axe owing to the solvent effects of D20 and to the effects of substitution of D for H in molecules at both the reaction site (primary isotopic substitution) and sites not directly involved in the reaction (secondary isotopic substitution} (Pittendrigh et al., 1973). Shortly after Urey (1933} isolated deuterium, Luck~ and Harvey (1935) found that D:O delayed the division of sea'urchin eggs. In the early 60's, it was shown that D20 blocked mitosis reversibly by 'freezing' or 'stabilising' mitotic apparatus of sea urchin eggs (Gross and Spindel, 1960; Marsland and Zimmerman, 1963}. Inou~ and his associates have demonstrated t h a t D20 increases reversibly the volume, length and birefringence of the mitotic spindles of sea urchin eggs, encountering different sensitivities at different mitotic stages, and t h a t it induces rapid emergence of microtubules in the spindle area (for a review see Inou~ and Sato, 1967}. They have suggested t h a t this agent favours the construction of microtubules from subunits. Recently, microtubules have called general a t t e n t i o n as among the fundamental organelles acting in karyokinesis, control of cell shape, intracellular transport, cell movement, sensory transduction and so on (for a review see Olmsted and Borisy, 1973). Tilney and Gibbs (1969} demonstrated t h a t the pseudopodia of secondary mesenchymal cells in sea urchin larvae have a
432 large number of microtubules' and microfilaments. They also found that colchicine and hydrostatic pressure, both of which cause the disintegration of the microtubules in these cells as well as others, block the second phase of invagination that is the pulling of archenteron by pseudopodia of the secondary mesenchymal cells (Dan and Okazaki, 1956; Gustafson and Kinnander, 1956 ). Since destruction of pseudopodia of secondary mesenchymal cells by various means blocks the invagination of sea urchin larvae, which results in exogastrulation (Dan and Okazaki, 1956; Okazaki, 1956), I have predicted that exogastrulation may be caused by the agents that effect (either stabilise or disintegrate) microtubules. We have obtained exogastrulae by rearing the larvae in the presence of either colchicine, vinblastine, dithiothreitol, cytochalasin B or cytochalasin C, or at low temperature (Takahashi et al., 1977}. Sulfhydryl reagents did not induce exogastrulation. Under these conditions (except for the presence of cytochalasins) microtubules are known to be affected. Cytochalasins affect the microfilaments. Here I add D20 to the list of agents which cause exogastrulation in sea urchin larvae. MATERIALS AND METHODS
Deuterated seawater Commercial heavy water (99.75 atom%) was used after double distillation. Artificial seawater made up in D20 diluted with H20-seawater (no D20 enrichment~ to yield a final concentration of 0--50%D20-seawater. The 'concentrations' of D20 henceforth reported refer to the volume percent of D20 used in making the seawater. Animals The following sea urchins were used during their breeding seasons: Hernicentrotus pulcherrimus, Strongylcentroutus intermedius and S. nudus. Gametes were collected by introducing 0.5 M KC1 into the coelom. Developing eggs or embryos, approx. 10 ~ in 50 pl, were transferred at various stages of development to 2 ml D20-seawater in a multidish (Linbro Chemicals), where t h e y were reared. In some experiments, embryos were returned to H20-seawater after a short stay in D20-seawater. At adequate time intervals, embryos were examined for cleavage and development. To see the effects of D20 on fertilisation, unfertilised eggs were inseminated in D20-seawater. RESULTS AND DISCUSSION Several lots of commercial D20 have been shown to contain an impurity causing a p h e n o m e n o n t h a t had been attributed to D20 (Huxtable and Bressler., 1974). Thus, D20 was twice distilled before use.
433 No appreciable effects of D~O on fertilisation were found at the concentrations up to 50% as shown in Table I. This does not agree with a previous report t h a t fertilisation, more strictly the elevation of the fertilisation envelope of Arbacia was significantly prevented by D20 at concentrations over 20% {Gross et al., 1960). All the fertilised eggs cleaved in seawater containing 40% D20 or less, and most of them did so even in 50% D20-seawater. Table I shows, however, the delay o f cleavage in D20-seawater as reported previously (Luckd and Harvey, 1935; Gross and Spindel, 1960; Marsland and Zimmerman, 1963). In D20-seawater, embryos developed normally up to primary mesenchyme blastulae, except for a delay of development to various degrees depending upon D20 concentrations. As clearly shown in Fig. 1, replacement o f H20 with D20 caused exogastrulation. When larvae of all the species used were transferred to 40% D:O-seawater at any stages before mesenchyme blastula, all of t h e m developed to exogastrulae and later on to plutei with evaginated guts {Fig. 2). In 50% D20-seawater, t h e y developed to exogastrulae at stage 1 {Fig. 2a), but t h e y did not develop b e y o n d this stage. When primary mesenchyme blastulae were transferred to D20-seawater, the subsequent development differed between species, as shown in Fig. 1. In S. intermedius, D20 induced exogastrulation in these larvae as well as those transferred at earlier stages. In H. pulcherrimus, D20 induced exogastrulation but was far less effective than that administered at earlier stages. In the case o f S. nudus, no exogastrulae were obtained even in 50% D20-seawater. In these species, no larvae with evaginated guts were obtained by administering D20 to early gastrulae or further-developed larvae. When gastrulae were transferred to 50% D20-seawater, t h e y developed slowly to plutei in all the species studied. Effects o f D20 are similar in general to those of chilling. We have foun~ chilling o f early gastrulae causes exogastrulation (Takahashi et al., 1977).
TABLE I FERTILISATION AND CLEAVAGE OF THE EGGS OF HEMICENTROTUS PULCHERRIMUS IN D~O-SEAWATER Approximately 103 eggs were inseminated in 2 ml of D20-seawater and reared at 15°C. The final concentration of spermatozoa was 106 cells/ml. All values in percent. Concentration
Fertilisation
Development stage at 2 h post-insemination
of D20 0 3 6 12 25 50
100 100 100 100 100 99
Fertilised egg
2-cell stage
4-cell-stage
0 0 1 1 2 19
1 1 13 15 93 80
99 99 86 84 5 0
434 i
i
10
20
|
i
40
50
e-
g o
50
w .
0
0 0
30
Concentration of I~0
'/0
Fig. 1. Exogastrulation induced by D20. Eggs were fertilised and reaxed in H20-seawater. At various stages of development, embryos were transferred to 3--50% D~O-seawater and were allowed to develop. (Open symbols) blastulae just hatched were transferred to D20-seawater. (Closed symbols) primary mesenchymal blastulae were transferred to D20-seawater. (Circles) H. Pulcherrimus; (triangles) S. nudus; squares; S. intermedius. When fertilised eggs or embryos before hatching were transferred to D20-seawater, the similar curves to those of open symbols were obtained.
Therefore, I examined whether D20 was more effective at lower temperatures. I could not find any significant differences in the ability of D20 to cause exogastrulation at temperatures between 6°C and 25°C. If the larvae showing the first sign of exogastrulation, namely larvae at stage 1 in Fig. 2, were returned to H20-seawater, some of them developed to normal plutei. This suggests that effect of D20 is partly reversible at this stage. When exogastrulae at later stages were transferred to H20-seawater, none of them grew into the normal plutei. It is well established that gastrulation proceeds in two phases (Dan and Okazaki, 1956; Gustafson and Kinnander, 1956). The first phase, the autonomous invagination of the endodermal plate by deformation of cells (Gustafson and Wolpert, 1963), was not affected by D20 at concentrations used as shown in Fig. 2 (note the shallow invagination of proximal portion of evaginated guts). The second phase, the pulling of the archenteron by the pseudopodia of the secondary mesenchymal cells, might be affected to some extent by D20 less than 50%. D20 seems to affect instead the formation of secondary mesenchymes in the correct positions, because it induces exogastrulation only when administered to larvae up to the primary mesenchyme blastulae. The evaginated guts have cilia and differentiate to some extent even in
435
Fig. 2. Egogastrulation induced by D20 in the larvae o f H. pulcherrimus. Blastulae which had just hatched were transferred to 25% D20-seawater and were reared at 20 °C. Bars represent 50 um (a) Larva showing the first sign of exogastrulation (Stage 1), 6 h after the transfer; (b) Early exogastrula showing normal arrangement of primary mesenchymal cells, 20 h after the transfer; (c) Exogastrula (Prism with the evaginated gut), 30 h after the transfer; (d) Exogastrula with the differentiated gut, 35 h after the transfer. The gut contracts occasionally; (e) Early pluteus with the evaginated gut, 40 h after the transfer; (f) Pluteus with the evaginated gut, 60 h after the transfer.
D20-seawater, as shown in Fig. 2. They contract occasionally and are easily removed from b o d y b y agitating the culture medium. In conclusion the evidence presented above indicates that the partial substitution of D20 for H20 in sweater causes exogastrulation in all the urchins studied. ACKNOWLEDGEMENTS I am much obliged to Drs. Y. Nagai, T o k y o Metropolitan Institute of Gerontology, and K. Onodera, K y o t o University, for their suggestions and criticisms. I am also indebted to the directors and the staffs of the Marine Biological Stations at Misaki (University of T o k y o ) , Akkeshi (Hokkaido University) and Oshoro (Hokkaido University), where
436 part of the present study was performed. This work was supported in part by grants from the Ministry of Education, Science and Culture, Japan and from Itoh Science Foundation.
REFERENCES Dan, K. and K. Okazaki: Biol. Bull. 110, 29--42 (1956). Gross, P.R. and W. Spindel: Ann. N.Y. Acad. Sci. 9 0 , 5 0 0 - - 5 2 2 {1960}. Gustafson, T. and H. Kinnander: Exp. Cell Res. 11, 36--51 {1956). Gustafson, T. and L. Wolpert: Int. Rev. Cytol. 1 5 , 1 3 9 - - 2 1 4 (1963). Huxtable, R. and R. Bressler: J, Membrane Biol. 1 7 , 1 8 9 - - 1 9 7 (1974). Inou6, S. and H. Sato: J. Gen. Physiol. 5 0 , 2 5 9 - - 2 8 8 (1967). Katz, J.J. and H.L. Crespi: In: Isotope effects in reaction rates, eds., C.G. Collins and N.S. Bowman (Van Nostrand-Reinhold Book Co., New York) pp. 286--363 (197(}}. Luck6, B. and E.N. Harvey: J. Cell. Comp. Physiol. 5 , 4 7 3 - - 4 8 2 (1935). Marsland, D. and A.M. Zimmerman: Exp. Cell Res., 30, 23--35 (1963). Okazaki, K.: Embryologia 3, 23--36 (1956). Olmsted, J.B. and G.G. Borisy: Annu. Rev. Biochem. 42, 507--540 (1973). Pittendrigh, C.S., P.C. Caldarola and E.S. Cosbey: Proc. Natl. Acad. Sci. U.S.A. 70, 2037--2041 (1973). Takahashi, T., M. Hoshi and t~. Asahina: Dev. Growth Differ.19,131--137 (1977). Thompson, J.F.: Biological effects of deuterium (Pergamon Press, New York) {1963L Tilney, L.G. and J.R. Gibbs: J. Cell Sci. 5 , 1 9 5 - - 2 1 0 (1969). Urey, H.C.: Science 7 8 , 5 6 6 - - 5 7 1 (1933).