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Biphasic stage sensitivity to UV suppression of gastrulation in sea urchin embryos
Cell Differemiation. 18 (1986l 45-49
45
El~vier ScientificPublishers Ireland. Ltd. CDF 00341
Biphasic stage sensitivity to UV suppression of gastrulation in sea urchin embryos S h o n a n A m e m i y a n. Shigenobu Y o n e m u r a ~, Seiichiro K i n o s h i t a ~ a n d Tsugio Shiroya 2 i Misaki Marine BiologicalStation. Unwersio"of Tokro. Miura. Kanagawa 238.02 and 2 Z~,logical In~'tuut~; Facultr o[ Scwm~: University of Tokro. Bunkyo. Tok r,, 113. Japan
(Accepted 15 July 1985)
The effects of ultraviolet light (UV) on the gastrulation of .sea urchin embffos were examined. The results suggest that gastrulation is inhibited by UV irradiation and that stage sensitivity to UV suppression of gastrulation changes biphasically: higher sensitivity at early and late blastula, and lower .sensitivity. at the mid-blastula stages. The UV-induced inhibition of gastrulation was completely reversible by subsequent exposure to visible light. .sea urchin; gastrulatiom UV light Introduction
A great number of cell-biological (Moore, 1930: Dan, 1960; Gustafson and Wolpert, 1963), ultrastructural (Amemiya et al., 1982) and molecularbiological (Galau et al., 1977: Bruskin et al., 1981) studies have been done on the gastrulation process in sea urchin embryos. Still, the mechanism of gastrulation remains to be clarified. in the present study, the effects of ultraviolet (UV) light irradiation on the gastrulation of sea urchin embryos were examined at the cellular level. The results indicate that sensitivity to UV suppression of gastrulation may change biphasically in the developmental stages from early to late blastula. Materials and Methods Sea urchin
Eggs and sperm were obtained from the sea urchin, Hemicentrotus pulcherrimus, by artificial
spawning, using a KCI solution. Inseminated eggs were cultured in filtered sea water at 20°C with gentle stirring. U V irradiation
A low pressure mercury lamp {Toshiba, GL-15, 254 nm) was used as the source of UV light. Diluted embryo suspensions in petri dishes placed 20 cm below the UV source were irradiated. The fluence rate was kept at 7.5 J/m-' per s with a UV monitor (Topcon UVR-254). Following irradiation, the embryos were incubated at 20°C in the dark. Photoreactit,ation ( P R )
For PR treatment, white fluorescent lamps (Matsushita Electric, FL20SW, ×3) were placed under a glass plate kept at 20°C as described previously ( Ejima and Shiroya, 1982). Five minutes after UV irradiation, the petri dishes containing the embryo suspensions were placed above a PR
0045-6039/86/$03.50 " 1986 ElsevierScientificPublishers Ireland. Ltd.
46 AI00
light and illuminated from underneath. The light intensity was 100 W / m 2, measured by a photocell illuminometer (Toshiba Electric, SPI-5).
Obxen,amm of UV e[fects The effects of UV irradiation on the embryos were examined morphologically, and the effects were shown as the percentage of permanent blastula or second phase of gastrulation. For quantitative analysis, aliquots were taken from suspensions of normal, UV-irradiated, and PRtreated embryos. 21 h after insemination, and fixed with 2,~ formalin in sea water. The number of embryos arrested at the permanent blastula or at the second phase of gastrulation was calculated as the percentage of the embryos fixed. Embryos in which the development was arrested at the mesenchyme blastula stage, without any indication of archenteron indentation, were identified as permanent blastula. The embryos which had developed secondary mesenchyme cells on the tip of the archenteron were identified as the second phase of gastrulation. To obtain pictures of normal. OVirradiated, and PR-treated embryos, photographs were taken of living embryos on a glass slide, under a Nikon Nomarksi differential interference microscope.
d
i 60
!,
~ ~o
;' 0
~
""c
100
-
if.J0
: --: 200
. . . . . . 250
350
300
t.O0
~.50
UV DOSe ( J i m ~ )
Fig. I. UV light dose-respons~ curve. I-':mbryosin me~nchyme blastula stage (12 h after insemination at 20°C) were irradiated with various UV doses, followed hy dark cultivation for 9 h (corresponding to 21 h post-insemination; the control was in late gastrula stage), and the number of embryos which had developed to the second pha~ of gastrulation was counted. :[~ach plot is the mean value_+ standard error of three experiments carried out on different batches of embryos. ing from pre-hatching blastula to e a r l y gastrula (Fig. 2). A UV dose of 225 J / m 2 given at any time from pre-hatching (10.5 h after insemination) to the onset of primary mesenchyme cell ingression (12 h after insemination) resulted in an almost complete suppression of gastrulation, and the de-
100
Results and Discussion Embryos at the mesenchyme blastula stage (12 h after insemination at 20°C) were irradiated with UV doses between 38 and 450 J / m 2. and cultured in a dark room for 9 h (corresponding to 21 h after insemination: the control had developed to late gastrula). Then the embryos which developed to the second phase of gastrulation were counted. The second phase was inhibited in a dose-dependent manner at UV doses between 0 and 150 J / m 2 (20-s exposure at 7.5 J / m 2 per s) (Fig. I). At a higher UV dose, there was an increase in the number of embryos where development stopped before reaching the second phase. At about 200 J / m 2. the arrest was nearly 100%. The sensitivity to UV suppression of gastrulalion was examined at developmental stages rang-
A
8
!,o o
Curve I
C~ve 2
g 0
,
I ,0
n
:2
:3
:~
fs
Ig
Time ofler Insemination ( h )
Fig. 2. UV irradiation sensitivity c,f ~a urchin embryos. At each time point, the embryos were irradiated with a UV dose of 225 J / r n 2 followed by dark cultivation until 21 h after insemination, then the embryos arrested at the blastula stage were counted. The ~.lage ~nsitivity curve (represented by the open circle.,, and solid line) for the UV suppression of gastrulation can he divided into two curve.,,, I and 2. The upper part of the figure shows the normal developmental stage~.
47 I00, rz
.~_
/
• zz---
,:----I
--~-
I-. . . .
/
so T,'
"6 60
1 / /I 0
10
2O
3O
4O
50
60
II
90
Idinu|C,S FXpO~Ur~ ( |OOWlm I )
Fig. 3. Visible light dose-response curve. Embryos irradiated by UV (225 J/m 2) 12 h after inmmination were treated with visible light (I00 W/m 2) 5 min after UV irradiation for various periods of time 10-90 rain, followed by dark cultivation until 21 h post-insemination. Then the embryos which had developed 1o the second phase of gastrulation were counted. Each plot is the mean value+ standard error of three experiments carried out on different batches of embryos.
~r~
v e l o p m e n t was arrested at the time o f p e r m a n e n t b l a s t u l a f o r m a t i o n . L a t e r in d e v e l o p m e n t the effects o f UV on g a s t r u l a t i o n decreased. M o r e than 50% o f the e m b r y o s , i r r a d i a t e d 13 h after insemination (when the ingression of primary m e s e n c h y m e cells into the blastocoel had been c o m p l e t e d ) , d e v e l o p e d to gastrula. I n d e n t a t i o n was i n c o m p l e t e in s o m e cases. A f t e r this. the stage sensitivity to UV light increased sharply, reaching a m a x i m u m 13.5 h after insemination, a n d dec r e a s e d again as a result o f the onset o f invagination (15.5 h after insemination). T h e stage sensitivity for the UV s u p p r e s s i o n of g a s t r u l a t i o n m a y be d i v i d e d into two UV sensitivity curves (Fig. 2). I r r a d i a t e d e m b r y o s (225 J / m 2, 12 h after ins e m i n a t i o n ) were p u t in visible light (100 W / m 2. fluorescent light) 5 min after i r r a d i a t i o n for vari-
(:1
Fig. 4. Light microscopic photographs of normal. UV-irradiated and PR-treated embryos. Bar represents I00 p.m. (a) Normal embryo
12 h following insemination. (b) Normal embryo 21 h after insemination. (c) UV-irradiated embryo 21 h after in~,emmation. Embryo was irradiated with UV (225 J/m "~) 12 h after in,ruination followed by dark cuhivation until 21 h pod,t-insemination. Id) PR-trcated embryo 21 h after insemination. Embryos irradiated (225 J/m-" I 12 h after in,ruination were treated ~ith visible light (I(X) W/m-" } 5 rain after UV irradiation for 60 rain followed by dark cultivation for 21 h after in~,emination.
48
ous periods of time, and cultured in the dark for 21 h after insemination. As shown in Fig. 3, PR treatment after UV irradiation caused a considerable increase in the frequency of the second phase of gastrulation. The recovery from the complete suppression was achieved by keeping the embryos in visible light for 20 to 30 min. In Fig. 4 the morphology of normal embryos is compared with UV-irradiated and PR-treated embryos. Twelve hours after insemination the primary mesenchyme cells with tadpole-like shapes ingressing into the blastocoel are evident in normal embryos (Fig. 4a). Twenty-one hours after insemination, the archenteron tip has developed secondary mesenchyme cells that extend all the way to the animal pole; this corresponds to the late gastrula stage (Fig. 4b). The development of embryos irradiated by UV (225 J/m-') 12 h after insemination followed by dark cultivation, was arrested at the permanent blastula before gastrulation, and the blastocoel was partly filled with degenerating mesenchyme cells (Fig. 4c). The embryos treated with visible light after UV irradiation developed to late gastrula with morphological features essentially the same as in normal embryos 21 h after insemination (Fig. 4d). Gastrulation is a complex cellular process, consisting of cell locomotion, cell proliferation, cell adhesion and cell reformation. According to the present experimental results, we are not able to determine which stages of the gastrulation are damaged by UV irradiation. However, the PR experimental results shown in Fig. 3. indicate that the target in the UV sensitive stage (shown by curve 1 in Fig. 2) may be nuclear DNA. It is well known that PR is principally due to photocatalytic cleavage of UV-induced pyrimidine dimers in cellular DNA (Cook, 1970: Ejima et al., 1984). The temporal nature of the UV sensitivity (curve 2. see Fig. 2) suggests that certain factors essential 1o gastrulation are activated in this period. Major differences in the pattern of protein synthesis in sea urchin embryos have been reported to appear at the beginning of gastrulation (Brandhorst. 1976: Rapraeger and Epel, 1981; Bedard and Brandhorst. 1983). Akimoto and Shiroya (in preparation) show that content of actin, apparently involved in cell locomotion in gastrulation, decreases
in UV-irradiated sea urchin embryos. The possibility that a UV-induced division skip may effect gastrulation should also be considered (Akimoto et al., 1983). During the cleavage stage of sea urchin eggs, the number of blastomeres increases exponentially, reaching a plateau at the hatching blastula stage. This plateau is maintained until late blastula, when the second burst of cell divisions starts before gastrulation (H. Fujisawa and S. Amemiya, unpublished data). The time of this second burst coincides with the UV-sensitivc stage represented by curve 2 in Fig. 2, suggesting that cell division at this stage may be effected by UV irradiation.
Acknowledgement This work was supported by Grant-in-Aid (No. 59540449) from the Ministry of Education, Science and Culture of Japan.
References Akimoto. Y.. T. Shiroya and N. Egami: Abnormal morphogenesis of .,;caurchin embryo induced by UV partial irradiation given at cleavagestage. J. Radiat. Res. 24. 197-202 (1983). Amemiya. S.. K. Akasaka and H. Terayama: Scanning electron microscopy of gastrulation in a sea urchin (Amho('idaris cra.~xispma). J. Embryol. Exp. Morphol. 67, 27-3.5 (1982). Bedard. P.A. and B.P. Brandhorst: Patterns of protein synthesis and metal~dism during sea urchin embryogenesis. Dev. Biol. 96. 74-83 (1983). Brandhorst. B.P.: Two dimensional gel patterns of protein synthesis bel,ore and after fertilization of ~ a urchin eggs. Dev. Biol. 52. 310-317 (1976), Bruskin. A.M., A.L. Tyner. D.E, Wells, R.M. Showman and W.H. Klein: Accumulation in ¢mbryogenesis of five mRNAs enriched in the eclt~erm of the sea urchin pluteus. Dev. Biol. 87. 308-318 (19811. Cook..I.S.: Photoreactivation in animal cells. In: Photophysiolog.~, ed. A.C. Giese (Academic Press. Nov, York) pp. 191-233 ( 19701. Dan. K.: Cyto-cmbryology ol, echinoderms and amphibia. Int. Rev. ('ytol. 9. 321-367 (1960). F.jinm. Y. and T. Shiroya: Photoreactivation ass~,'iated with chromos)real abnormality in sea urchin eggs fertilized with ultraviolet-irradiated sperm. Phoh~.'hem. Photobiol. 36. 37-41 (1982). l-:.jima.Y.. M. Ikenaga and T. Shiroya: Action spectrum for pholoreactivation oi, uhraviolet-induced n)orphological
49 abnormality in sea urchin eggs. Photochem. Photobiol. 40. 461-464 (1984). Galau, G.A., E.D. Lipson. R.J. Britten and E.H. Davidson: Synthesis and turnover of polysomal mRNAs in sea urchin embryos. Cell 10, 415-432 (1977). Gustal'son. T. and L. Wolpert: The cellular basis of morphogenesis and sea urchin development. Int. Rev. Cytol. 15. 139-214 (1963).
Moore. A.R.: On the invagination of the gastrula. Protoplasma 9, 25-33 (1930). Rapraeger. A.C. and D. EI)eh The appearanceof an extracellular arylsulfatase during morphogenesis of the sea urchin Strong;,h~'entrotuspurpuratu.,~. Dev. Biol. 88, 269-278 ( 1981 ).
45
El~vier ScientificPublishers Ireland. Ltd. CDF 00341
Biphasic stage sensitivity to UV suppression of gastrulation in sea urchin embryos S h o n a n A m e m i y a n. Shigenobu Y o n e m u r a ~, Seiichiro K i n o s h i t a ~ a n d Tsugio Shiroya 2 i Misaki Marine BiologicalStation. Unwersio"of Tokro. Miura. Kanagawa 238.02 and 2 Z~,logical In~'tuut~; Facultr o[ Scwm~: University of Tokro. Bunkyo. Tok r,, 113. Japan
(Accepted 15 July 1985)
The effects of ultraviolet light (UV) on the gastrulation of .sea urchin embffos were examined. The results suggest that gastrulation is inhibited by UV irradiation and that stage sensitivity to UV suppression of gastrulation changes biphasically: higher sensitivity at early and late blastula, and lower .sensitivity. at the mid-blastula stages. The UV-induced inhibition of gastrulation was completely reversible by subsequent exposure to visible light. .sea urchin; gastrulatiom UV light Introduction
A great number of cell-biological (Moore, 1930: Dan, 1960; Gustafson and Wolpert, 1963), ultrastructural (Amemiya et al., 1982) and molecularbiological (Galau et al., 1977: Bruskin et al., 1981) studies have been done on the gastrulation process in sea urchin embryos. Still, the mechanism of gastrulation remains to be clarified. in the present study, the effects of ultraviolet (UV) light irradiation on the gastrulation of sea urchin embryos were examined at the cellular level. The results indicate that sensitivity to UV suppression of gastrulation may change biphasically in the developmental stages from early to late blastula. Materials and Methods Sea urchin
Eggs and sperm were obtained from the sea urchin, Hemicentrotus pulcherrimus, by artificial
spawning, using a KCI solution. Inseminated eggs were cultured in filtered sea water at 20°C with gentle stirring. U V irradiation
A low pressure mercury lamp {Toshiba, GL-15, 254 nm) was used as the source of UV light. Diluted embryo suspensions in petri dishes placed 20 cm below the UV source were irradiated. The fluence rate was kept at 7.5 J/m-' per s with a UV monitor (Topcon UVR-254). Following irradiation, the embryos were incubated at 20°C in the dark. Photoreactit,ation ( P R )
For PR treatment, white fluorescent lamps (Matsushita Electric, FL20SW, ×3) were placed under a glass plate kept at 20°C as described previously ( Ejima and Shiroya, 1982). Five minutes after UV irradiation, the petri dishes containing the embryo suspensions were placed above a PR
0045-6039/86/$03.50 " 1986 ElsevierScientificPublishers Ireland. Ltd.
46 AI00
light and illuminated from underneath. The light intensity was 100 W / m 2, measured by a photocell illuminometer (Toshiba Electric, SPI-5).
Obxen,amm of UV e[fects The effects of UV irradiation on the embryos were examined morphologically, and the effects were shown as the percentage of permanent blastula or second phase of gastrulation. For quantitative analysis, aliquots were taken from suspensions of normal, UV-irradiated, and PRtreated embryos. 21 h after insemination, and fixed with 2,~ formalin in sea water. The number of embryos arrested at the permanent blastula or at the second phase of gastrulation was calculated as the percentage of the embryos fixed. Embryos in which the development was arrested at the mesenchyme blastula stage, without any indication of archenteron indentation, were identified as permanent blastula. The embryos which had developed secondary mesenchyme cells on the tip of the archenteron were identified as the second phase of gastrulation. To obtain pictures of normal. OVirradiated, and PR-treated embryos, photographs were taken of living embryos on a glass slide, under a Nikon Nomarksi differential interference microscope.
d
i 60
!,
~ ~o
;' 0
~
""c
100
-
if.J0
: --: 200
. . . . . . 250
350
300
t.O0
~.50
UV DOSe ( J i m ~ )
Fig. I. UV light dose-respons~ curve. I-':mbryosin me~nchyme blastula stage (12 h after insemination at 20°C) were irradiated with various UV doses, followed hy dark cultivation for 9 h (corresponding to 21 h post-insemination; the control was in late gastrula stage), and the number of embryos which had developed to the second pha~ of gastrulation was counted. :[~ach plot is the mean value_+ standard error of three experiments carried out on different batches of embryos. ing from pre-hatching blastula to e a r l y gastrula (Fig. 2). A UV dose of 225 J / m 2 given at any time from pre-hatching (10.5 h after insemination) to the onset of primary mesenchyme cell ingression (12 h after insemination) resulted in an almost complete suppression of gastrulation, and the de-
100
Results and Discussion Embryos at the mesenchyme blastula stage (12 h after insemination at 20°C) were irradiated with UV doses between 38 and 450 J / m 2. and cultured in a dark room for 9 h (corresponding to 21 h after insemination: the control had developed to late gastrula). Then the embryos which developed to the second phase of gastrulation were counted. The second phase was inhibited in a dose-dependent manner at UV doses between 0 and 150 J / m 2 (20-s exposure at 7.5 J / m 2 per s) (Fig. I). At a higher UV dose, there was an increase in the number of embryos where development stopped before reaching the second phase. At about 200 J / m 2. the arrest was nearly 100%. The sensitivity to UV suppression of gastrulalion was examined at developmental stages rang-
A
8
!,o o
Curve I
C~ve 2
g 0
,
I ,0
n
:2
:3
:~
fs
Ig
Time ofler Insemination ( h )
Fig. 2. UV irradiation sensitivity c,f ~a urchin embryos. At each time point, the embryos were irradiated with a UV dose of 225 J / r n 2 followed by dark cultivation until 21 h after insemination, then the embryos arrested at the blastula stage were counted. The ~.lage ~nsitivity curve (represented by the open circle.,, and solid line) for the UV suppression of gastrulation can he divided into two curve.,,, I and 2. The upper part of the figure shows the normal developmental stage~.
47 I00, rz
.~_
/
• zz---
,:----I
--~-
I-. . . .
/
so T,'
"6 60
1 / /I 0
10
2O
3O
4O
50
60
II
90
Idinu|C,S FXpO~Ur~ ( |OOWlm I )
Fig. 3. Visible light dose-response curve. Embryos irradiated by UV (225 J/m 2) 12 h after inmmination were treated with visible light (I00 W/m 2) 5 min after UV irradiation for various periods of time 10-90 rain, followed by dark cultivation until 21 h post-insemination. Then the embryos which had developed 1o the second phase of gastrulation were counted. Each plot is the mean value+ standard error of three experiments carried out on different batches of embryos.
~r~
v e l o p m e n t was arrested at the time o f p e r m a n e n t b l a s t u l a f o r m a t i o n . L a t e r in d e v e l o p m e n t the effects o f UV on g a s t r u l a t i o n decreased. M o r e than 50% o f the e m b r y o s , i r r a d i a t e d 13 h after insemination (when the ingression of primary m e s e n c h y m e cells into the blastocoel had been c o m p l e t e d ) , d e v e l o p e d to gastrula. I n d e n t a t i o n was i n c o m p l e t e in s o m e cases. A f t e r this. the stage sensitivity to UV light increased sharply, reaching a m a x i m u m 13.5 h after insemination, a n d dec r e a s e d again as a result o f the onset o f invagination (15.5 h after insemination). T h e stage sensitivity for the UV s u p p r e s s i o n of g a s t r u l a t i o n m a y be d i v i d e d into two UV sensitivity curves (Fig. 2). I r r a d i a t e d e m b r y o s (225 J / m 2, 12 h after ins e m i n a t i o n ) were p u t in visible light (100 W / m 2. fluorescent light) 5 min after i r r a d i a t i o n for vari-
(:1
Fig. 4. Light microscopic photographs of normal. UV-irradiated and PR-treated embryos. Bar represents I00 p.m. (a) Normal embryo
12 h following insemination. (b) Normal embryo 21 h after insemination. (c) UV-irradiated embryo 21 h after in~,emmation. Embryo was irradiated with UV (225 J/m "~) 12 h after in,ruination followed by dark cuhivation until 21 h pod,t-insemination. Id) PR-trcated embryo 21 h after insemination. Embryos irradiated (225 J/m-" I 12 h after in,ruination were treated ~ith visible light (I(X) W/m-" } 5 rain after UV irradiation for 60 rain followed by dark cultivation for 21 h after in~,emination.
48
ous periods of time, and cultured in the dark for 21 h after insemination. As shown in Fig. 3, PR treatment after UV irradiation caused a considerable increase in the frequency of the second phase of gastrulation. The recovery from the complete suppression was achieved by keeping the embryos in visible light for 20 to 30 min. In Fig. 4 the morphology of normal embryos is compared with UV-irradiated and PR-treated embryos. Twelve hours after insemination the primary mesenchyme cells with tadpole-like shapes ingressing into the blastocoel are evident in normal embryos (Fig. 4a). Twenty-one hours after insemination, the archenteron tip has developed secondary mesenchyme cells that extend all the way to the animal pole; this corresponds to the late gastrula stage (Fig. 4b). The development of embryos irradiated by UV (225 J/m-') 12 h after insemination followed by dark cultivation, was arrested at the permanent blastula before gastrulation, and the blastocoel was partly filled with degenerating mesenchyme cells (Fig. 4c). The embryos treated with visible light after UV irradiation developed to late gastrula with morphological features essentially the same as in normal embryos 21 h after insemination (Fig. 4d). Gastrulation is a complex cellular process, consisting of cell locomotion, cell proliferation, cell adhesion and cell reformation. According to the present experimental results, we are not able to determine which stages of the gastrulation are damaged by UV irradiation. However, the PR experimental results shown in Fig. 3. indicate that the target in the UV sensitive stage (shown by curve 1 in Fig. 2) may be nuclear DNA. It is well known that PR is principally due to photocatalytic cleavage of UV-induced pyrimidine dimers in cellular DNA (Cook, 1970: Ejima et al., 1984). The temporal nature of the UV sensitivity (curve 2. see Fig. 2) suggests that certain factors essential 1o gastrulation are activated in this period. Major differences in the pattern of protein synthesis in sea urchin embryos have been reported to appear at the beginning of gastrulation (Brandhorst. 1976: Rapraeger and Epel, 1981; Bedard and Brandhorst. 1983). Akimoto and Shiroya (in preparation) show that content of actin, apparently involved in cell locomotion in gastrulation, decreases
in UV-irradiated sea urchin embryos. The possibility that a UV-induced division skip may effect gastrulation should also be considered (Akimoto et al., 1983). During the cleavage stage of sea urchin eggs, the number of blastomeres increases exponentially, reaching a plateau at the hatching blastula stage. This plateau is maintained until late blastula, when the second burst of cell divisions starts before gastrulation (H. Fujisawa and S. Amemiya, unpublished data). The time of this second burst coincides with the UV-sensitivc stage represented by curve 2 in Fig. 2, suggesting that cell division at this stage may be effected by UV irradiation.
Acknowledgement This work was supported by Grant-in-Aid (No. 59540449) from the Ministry of Education, Science and Culture of Japan.
References Akimoto. Y.. T. Shiroya and N. Egami: Abnormal morphogenesis of .,;caurchin embryo induced by UV partial irradiation given at cleavagestage. J. Radiat. Res. 24. 197-202 (1983). Amemiya. S.. K. Akasaka and H. Terayama: Scanning electron microscopy of gastrulation in a sea urchin (Amho('idaris cra.~xispma). J. Embryol. Exp. Morphol. 67, 27-3.5 (1982). Bedard. P.A. and B.P. Brandhorst: Patterns of protein synthesis and metal~dism during sea urchin embryogenesis. Dev. Biol. 96. 74-83 (1983). Brandhorst. B.P.: Two dimensional gel patterns of protein synthesis bel,ore and after fertilization of ~ a urchin eggs. Dev. Biol. 52. 310-317 (1976), Bruskin. A.M., A.L. Tyner. D.E, Wells, R.M. Showman and W.H. Klein: Accumulation in ¢mbryogenesis of five mRNAs enriched in the eclt~erm of the sea urchin pluteus. Dev. Biol. 87. 308-318 (19811. Cook..I.S.: Photoreactivation in animal cells. In: Photophysiolog.~, ed. A.C. Giese (Academic Press. Nov, York) pp. 191-233 ( 19701. Dan. K.: Cyto-cmbryology ol, echinoderms and amphibia. Int. Rev. ('ytol. 9. 321-367 (1960). F.jinm. Y. and T. Shiroya: Photoreactivation ass~,'iated with chromos)real abnormality in sea urchin eggs fertilized with ultraviolet-irradiated sperm. Phoh~.'hem. Photobiol. 36. 37-41 (1982). l-:.jima.Y.. M. Ikenaga and T. Shiroya: Action spectrum for pholoreactivation oi, uhraviolet-induced n)orphological
49 abnormality in sea urchin eggs. Photochem. Photobiol. 40. 461-464 (1984). Galau, G.A., E.D. Lipson. R.J. Britten and E.H. Davidson: Synthesis and turnover of polysomal mRNAs in sea urchin embryos. Cell 10, 415-432 (1977). Gustal'son. T. and L. Wolpert: The cellular basis of morphogenesis and sea urchin development. Int. Rev. Cytol. 15. 139-214 (1963).
Moore. A.R.: On the invagination of the gastrula. Protoplasma 9, 25-33 (1930). Rapraeger. A.C. and D. EI)eh The appearanceof an extracellular arylsulfatase during morphogenesis of the sea urchin Strong;,h~'entrotuspurpuratu.,~. Dev. Biol. 88, 269-278 ( 1981 ).