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Appearance of antigenic material in gastrula ectoderm after neural induction
Cell Differentiation 1, 15-18 (1972). © North-Holland Publishing Company
APPEARANCE OF ANTIGENIC MATERIAL IN GASTRULA ECTODERM AFTER NEURAL INDUCTION M. STANISSTREET * and E. M. DEUCHAR Department of A natomy. The Medical School, University of Bristol, U.K. Accepted 10 December 1971 Using immuno-electrophoretic methods, protein extracts from dorsal (induced) and ventral (non-induced) late gastrula ectoderm of Xenopus laevis embryos have been compared. The induced ectoderm, which will differentiate into neural tissue, is found to contain higher concentrations of three antigenic components than the ventral ectoderm. This provides evidence of protein changes that occur immediately after neural induction.
The appearance of new proteins in differentiating embryonic cells has been demonstrated by immunological methods in a number of amphibian species. Flickinget and Nace (1952) were able to show that the antigenic complement of whole embryos of Rana temporaria changes during development. Spar (1953), Clayton (1953), Takayanagi (1958), Ranzi (1961) and Inoue (1961) have also followed changes in the antigenic complement of whole amphibian embryos, and all of these workers were able to demonstrate changes at the time of gastrulation. Since then Romanovsky (1964) has been able by a modification of the Ouchterlony method to show that at least one new antigen appears between blastula and early gastrula stages and two further antigens between early and late gastrula stages, in whole embryos of Rana temporaria. Relatively little work has been carried out on tissues isolated by dissection, but both Clayton (1953) and Vainio (1956) have been able to show differences between the antigens of different gastrula tissues. Particularly interesting was Clayton's additional finding that in Triturus alpestris the neural plate ectoderm contains antigens that are not present in the epidermis, at the neurula stage. She did not, however, provide evidence of any new antigens appearing in the dorsal ectoderm of the gastrula, as an immediate result of induction by the mesoderm. Since neural induction, like any other mechanism that controls differentiation processes in embryonic cells, must act by initiating the synthesis of new protein, it should be possible to show changes in antigenicity in the dorsal ectoderm at the end * New address: Department of Zoology, The University, Liverpool.
16
Appearance of antigenic material after neural induction
of gastrulation, for by that time it has received the induction stimulus and is capable of differentiating into neural tissue if isolated. So in the present study, saline-soluble proteins have been extracted from pieces of dorsal ectoderm dissected from late gastrulae of Xenopus laevis (stage 12- 12½ of Nieuwkoop and Faber, 1956) and compared with similar samples from ventral ectoderm of the same stage, using immunoelectrophoretic methods. Care has been taken to compare samples containing equal amounts of total protein, so that any apparent increase of antigen observed represents an increase in the concentration of this protein relative to other proteins in the tissue. The ectoderm extracts have been tested with antisera prepared against different embryonic stages and against some adult tissues.
MATERIAL AND METHODS Xenopus embryos were de-jellied with cysteine-papain solution (Dawid, 1965) and rinsed in Holtfreter's saline. They were either collected in batches of I00 for injection of antigens, or used for ectoderm dissections. A n tisera Antisera were produced in female rabbits by an injection regime consisting of primary intradermal injection of antigen emulsified with adjuvant, followed at 4 to 6 weeks by a secondary intravenous injection of antigen alone. Blood was taken from the marginal ear vein, and serum obtained by centrifugation. Alternate secondary injections and bleedings followed at appropriate intervals. Test antigens 100 pieces of dorsal and ventral ectoderm were dissected from late gastrulae (stage 12, Nieuwkoop and Faber, 1956), rinsed in buffered saline (pH 7.3), and transferred to small conical tubes. The volume was adjusted to 25 pt and the pieces homogenised with a rapidly spinning fine steel wire. Homogenates were stored at -35°C, then centrifuged at 15,000 g for 30 min and the supernatant removed and stored. 2/al of supernatant was used for protein determination (Lowry et al., 1951). Immunoelectrophoresis Immunoelectrophoresis was performed on microscope slides covered with 1% Ionagar in barbitone/acetate buffer (pH 8.6). 100 pieces of ectoderm were found to give sufficient supernatant (approximately 15 ~1) for one sample and the volumes of samples from the same embryos were adjusted to give equivalent total protein. Samples were separated for 3 hr at 12.5 mA and 50 V, then undiluted serum (0.1 ml) was added to the serum troughs. After 72 hr the slides were washed in 0.9% NaCI and the agar dried to a thin film and stained with Naphthalene Black in 7% acetic acid.
M. STANISSTREET and E.M. D E U C H A R
17
N +
V +
Cathode
Anode
Fig. 1. Photograph of immunoelectrophoresis run of supernatant proteins of neural and ventral ectoderm from late gastrulae tested against antiserum to neurula supernatant. Arrows indicate arcs which appear stronger in neural ectoderm. N - neural ectoderm V - ventral ectoderm.
N +
V
Cathode
Anode Fig. 2. Drawing o f slide shown in fig. 1. Symbols as in fig. 1.
18
Appearance of antigenic materialafter neural induction
RESULTS Seven separate pairs of protein samples from dorsal and ventral gastrula ectoderm were compared, testing them against: (a) antiserum to homogenates of neurulae (stage 16, Nieuwkoop and Faber, 1956); (b) antiserum to supernatants of neurulae; (c) antiserum to adult Xenopus brain homogenates. Tests against antiserum to adult brain were unsatisfactory owing to the low titre of the antiserum. Results of tests (a) and (b) were comparable between experiments. The runs against antiserum to neurula homogenate showed no differences between dorsal and ventral ectoderm, but in those with antiserum to saline-soluble proteins of neurulae, three arcs consistently showed up more strongly in the dorsal ectoderm extract. Fig. I is a photograph of a pair of electrophoretograms from one experiment, and the comparison o f arcs shown in the two types of ectoderm is set out diagrammatically in fig. 2. Only those differences that were consistent in all experiments have been pointed out in the figures. We conclude from these results that one of the earliest features of neural differentiation in reponse to induction by the dorsal gastrula mesoderm in Xenopus laevis is an increase in concentration of three components of the antigenic proteins in the dorsal ectoderm. This biochemical change is recognizable before there is any sign of histological differentiation in the neural ectoderm. Further antigenic differences occurring at later stages in embryonic differentiation are being studied (Stanisstreet, unpublished). It is hoped also to correlate the protein changes in gastrula ectoderm with observations on RNA synthesis in this tissue (Thomas and Deuchar, 1971). ACKNOWLEDGEMENTS M. Stanisstreet was in receipt of an M.R.C. scholarship during the period of this work.
REFERENCES Clayton, R.M.: J. Embryol. Exptl. Morphol. 1, 25-42 (1953). Dawid, I.B.: J. Mol. Biol. 12,581-589 (1965). Flickinger, R.A. and G.W. Nace: Exptl. Cell Res. 3, 393-405 (1952). Inoue, K.: J. Embryol. Exptl. Morphol. 9,563-585 (1961), Lowry, O.H., N.J. Rosebrough, A.J. Farr and R.J. Randall: J. Biol. Chem. 193, 265275 (1951). Nieuwkoop, P.D. and J. Faber: A Normal Table of Xenopus laevis Daudin (North-Holland, Amsterdam) 251 pp. (1956). Ranzi, S.: in: Symposium on the Germ Cells and Earliest Stages of Development, ed. S. Ranzi (Fond. Baselli, Milan) pp. 604-613 (1961). Romanovsky, A.: Folia Biol., Cracow 10, 1-11 (1964). Spar, I.L.: J. Exptl. Zool. 123,467-497 (1953). Takayanagi, A.: J. Jnzen. Med. Soc., Japan 60,701-729 (1958). Thomas, N. and E.M. Deuchar: Acta Embryol. Exptl., in press (1971). Vainio, T.: Ann. Med. Exptl. Biol. Fenn. 34, 71-79 (1956).
APPEARANCE OF ANTIGENIC MATERIAL IN GASTRULA ECTODERM AFTER NEURAL INDUCTION M. STANISSTREET * and E. M. DEUCHAR Department of A natomy. The Medical School, University of Bristol, U.K. Accepted 10 December 1971 Using immuno-electrophoretic methods, protein extracts from dorsal (induced) and ventral (non-induced) late gastrula ectoderm of Xenopus laevis embryos have been compared. The induced ectoderm, which will differentiate into neural tissue, is found to contain higher concentrations of three antigenic components than the ventral ectoderm. This provides evidence of protein changes that occur immediately after neural induction.
The appearance of new proteins in differentiating embryonic cells has been demonstrated by immunological methods in a number of amphibian species. Flickinget and Nace (1952) were able to show that the antigenic complement of whole embryos of Rana temporaria changes during development. Spar (1953), Clayton (1953), Takayanagi (1958), Ranzi (1961) and Inoue (1961) have also followed changes in the antigenic complement of whole amphibian embryos, and all of these workers were able to demonstrate changes at the time of gastrulation. Since then Romanovsky (1964) has been able by a modification of the Ouchterlony method to show that at least one new antigen appears between blastula and early gastrula stages and two further antigens between early and late gastrula stages, in whole embryos of Rana temporaria. Relatively little work has been carried out on tissues isolated by dissection, but both Clayton (1953) and Vainio (1956) have been able to show differences between the antigens of different gastrula tissues. Particularly interesting was Clayton's additional finding that in Triturus alpestris the neural plate ectoderm contains antigens that are not present in the epidermis, at the neurula stage. She did not, however, provide evidence of any new antigens appearing in the dorsal ectoderm of the gastrula, as an immediate result of induction by the mesoderm. Since neural induction, like any other mechanism that controls differentiation processes in embryonic cells, must act by initiating the synthesis of new protein, it should be possible to show changes in antigenicity in the dorsal ectoderm at the end * New address: Department of Zoology, The University, Liverpool.
16
Appearance of antigenic material after neural induction
of gastrulation, for by that time it has received the induction stimulus and is capable of differentiating into neural tissue if isolated. So in the present study, saline-soluble proteins have been extracted from pieces of dorsal ectoderm dissected from late gastrulae of Xenopus laevis (stage 12- 12½ of Nieuwkoop and Faber, 1956) and compared with similar samples from ventral ectoderm of the same stage, using immunoelectrophoretic methods. Care has been taken to compare samples containing equal amounts of total protein, so that any apparent increase of antigen observed represents an increase in the concentration of this protein relative to other proteins in the tissue. The ectoderm extracts have been tested with antisera prepared against different embryonic stages and against some adult tissues.
MATERIAL AND METHODS Xenopus embryos were de-jellied with cysteine-papain solution (Dawid, 1965) and rinsed in Holtfreter's saline. They were either collected in batches of I00 for injection of antigens, or used for ectoderm dissections. A n tisera Antisera were produced in female rabbits by an injection regime consisting of primary intradermal injection of antigen emulsified with adjuvant, followed at 4 to 6 weeks by a secondary intravenous injection of antigen alone. Blood was taken from the marginal ear vein, and serum obtained by centrifugation. Alternate secondary injections and bleedings followed at appropriate intervals. Test antigens 100 pieces of dorsal and ventral ectoderm were dissected from late gastrulae (stage 12, Nieuwkoop and Faber, 1956), rinsed in buffered saline (pH 7.3), and transferred to small conical tubes. The volume was adjusted to 25 pt and the pieces homogenised with a rapidly spinning fine steel wire. Homogenates were stored at -35°C, then centrifuged at 15,000 g for 30 min and the supernatant removed and stored. 2/al of supernatant was used for protein determination (Lowry et al., 1951). Immunoelectrophoresis Immunoelectrophoresis was performed on microscope slides covered with 1% Ionagar in barbitone/acetate buffer (pH 8.6). 100 pieces of ectoderm were found to give sufficient supernatant (approximately 15 ~1) for one sample and the volumes of samples from the same embryos were adjusted to give equivalent total protein. Samples were separated for 3 hr at 12.5 mA and 50 V, then undiluted serum (0.1 ml) was added to the serum troughs. After 72 hr the slides were washed in 0.9% NaCI and the agar dried to a thin film and stained with Naphthalene Black in 7% acetic acid.
M. STANISSTREET and E.M. D E U C H A R
17
N +
V +
Cathode
Anode
Fig. 1. Photograph of immunoelectrophoresis run of supernatant proteins of neural and ventral ectoderm from late gastrulae tested against antiserum to neurula supernatant. Arrows indicate arcs which appear stronger in neural ectoderm. N - neural ectoderm V - ventral ectoderm.
N +
V
Cathode
Anode Fig. 2. Drawing o f slide shown in fig. 1. Symbols as in fig. 1.
18
Appearance of antigenic materialafter neural induction
RESULTS Seven separate pairs of protein samples from dorsal and ventral gastrula ectoderm were compared, testing them against: (a) antiserum to homogenates of neurulae (stage 16, Nieuwkoop and Faber, 1956); (b) antiserum to supernatants of neurulae; (c) antiserum to adult Xenopus brain homogenates. Tests against antiserum to adult brain were unsatisfactory owing to the low titre of the antiserum. Results of tests (a) and (b) were comparable between experiments. The runs against antiserum to neurula homogenate showed no differences between dorsal and ventral ectoderm, but in those with antiserum to saline-soluble proteins of neurulae, three arcs consistently showed up more strongly in the dorsal ectoderm extract. Fig. I is a photograph of a pair of electrophoretograms from one experiment, and the comparison o f arcs shown in the two types of ectoderm is set out diagrammatically in fig. 2. Only those differences that were consistent in all experiments have been pointed out in the figures. We conclude from these results that one of the earliest features of neural differentiation in reponse to induction by the dorsal gastrula mesoderm in Xenopus laevis is an increase in concentration of three components of the antigenic proteins in the dorsal ectoderm. This biochemical change is recognizable before there is any sign of histological differentiation in the neural ectoderm. Further antigenic differences occurring at later stages in embryonic differentiation are being studied (Stanisstreet, unpublished). It is hoped also to correlate the protein changes in gastrula ectoderm with observations on RNA synthesis in this tissue (Thomas and Deuchar, 1971). ACKNOWLEDGEMENTS M. Stanisstreet was in receipt of an M.R.C. scholarship during the period of this work.
REFERENCES Clayton, R.M.: J. Embryol. Exptl. Morphol. 1, 25-42 (1953). Dawid, I.B.: J. Mol. Biol. 12,581-589 (1965). Flickinger, R.A. and G.W. Nace: Exptl. Cell Res. 3, 393-405 (1952). Inoue, K.: J. Embryol. Exptl. Morphol. 9,563-585 (1961), Lowry, O.H., N.J. Rosebrough, A.J. Farr and R.J. Randall: J. Biol. Chem. 193, 265275 (1951). Nieuwkoop, P.D. and J. Faber: A Normal Table of Xenopus laevis Daudin (North-Holland, Amsterdam) 251 pp. (1956). Ranzi, S.: in: Symposium on the Germ Cells and Earliest Stages of Development, ed. S. Ranzi (Fond. Baselli, Milan) pp. 604-613 (1961). Romanovsky, A.: Folia Biol., Cracow 10, 1-11 (1964). Spar, I.L.: J. Exptl. Zool. 123,467-497 (1953). Takayanagi, A.: J. Jnzen. Med. Soc., Japan 60,701-729 (1958). Thomas, N. and E.M. Deuchar: Acta Embryol. Exptl., in press (1971). Vainio, T.: Ann. Med. Exptl. Biol. Fenn. 34, 71-79 (1956).