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A cell surface determinant expressed early on migrating avian neural crest cells
Developmental Brain Research, 9 (1983) 235-238
235
Elsevier
A cell surface determinant expressed early on migrating avian neural crest cells MICHEL VINCENT, JEAN-LOUP DUBAND and JEAN-PAUL THIERY Institut d'Embryologie du Centre National de la Recherche, Scientifique et du Colldge de France, 49, bis Avenue de la Belle Gabrielle, 94130 Nogent-Sur-Marne (France)
(Accepted March 29th, 1983) Key words: avian embryo - - neural crest - - peripheral nervous system - - monoclonal antibody - - ciliary ganglion
We have produced monoclonal antibodies against quail ciliary ganglion in an attempt to identify specificmarkers of this neural crest derivative. One of these antibodies, NC/1, recognizes supportive and neuronal cells of the peripheral nervous system and also most, if not all migrating neural crest cells. We report herein the use of NC/1 to identify crest cells during their migration to their site of final localization. In addition, this antibody may shed light on how the neural crest derived mesectoderm and the peripheral nervous system segregate from one another since the NC/1-defined antigen becomes restricted to the cells of the latter. The neural crest is a transient structure in vertebrate embryos detaching from the neural epithelium and giving rise, after an extensive intraembryonic migration, to a variety of cell types including neurons and supportive cells of the peripheral nervous system, melanocytes and many cranio-facial structures designated as mesectodermal derivatives10. It is an excellent model system to study cell migration and the progressive restriction of precursor cell capabilities during development9.13,19. Therefore, a library of stage- or cell-type-specific markers would be useful to decipher mechanisms controlling segregation of the neural crest cells into their different lineages. We thus decided to produce monoclonal antibodies against quail ciliary ganglion, a parasympathetic ganglion entirely derived from the neural crest 10. BALB/c mice were immunized with 8-dayold quail ciliary ganglia until high titer sera were obtained. Three days after an i.v. boost injection, immune mouse splenocytes were fused with Sp2/0 myeloma according to the method of K6hler and Milstein 8. Hybridoma culture supernatants were systematically tested for antibody by indirect immunofluorescence on 24-h microcultures of living cells from ciliary, sympathetic and sensory ganglia. One of the supernatants, NC/1, appeared to react strongly both with flat cells and neurons in each type of cultures (not shown). Its reactivity with living cells suggests that it recognizes a cell surface-associated anti0165-3806/83/$03.00 © 1983 Elsevier Science Publishers B.V.
gen. NC/1 did not bind either to 8-day-old quail embryo heart fibroblasts or to somitic mesenchyme (see Fig. lb) but labeled cultured trunk neural crest cells (not shown). Therefore, NC/1 defines an antigen present in vitro on trunk crest cells and on the cells of the sensory and autonomic ganglia. It was selected for further characterization: NC/1 producing hybridomas were cloned twice and thereafter grown in vitro or in ascites for large-scale production of antibody. All antibody preparations gave results identical to those obtained with the original supernatant. The specificity of this antibody was further probed by immunolabeling sections of quail embryos made at levels where crest cells had begun their migration. The antibody was found to stain migrating neural crest cells; for example, in the prosencephalon, crest cells which are known to accumulate between the ectoderm and the optic vesicle 6, were stained by NC/1 (Fig. la). Similarly, at the mesencephalic level, crest cells which migrate laterally under the ectoderm were intensely stained (Fig. lb). In slightly older embryos, at the posterior rhombencephalic level, neural crest cells that had reached the apex of the pharynx could be easily detected among the surrounding tissues, which remained unstained (Fig. 2a); at later stages, when these cells had become localized in the gut 11, they could still be labeled by NC/1 (Fig. 2b). We noticed that the periphery of the notochord, at the trunk level, was diffusely stained; NC/1 fluores-
236
Fig. 1. Transverse sections of quail embryos at the cephalic level. Formaldehyde-fixed embryos were embedded in polyethylene-glycol and sectioned as described previously 6. Sections were incubated with NC/1 hybridoma supernatant for 1 h at room temperature. After extensive washes in phosphate-buffered saline, sections were successively treated with rabbit IgG anti-mouse immunoglobulins (Nordic, Tilburg, The Netherlands) and FITC-conjugated sheep anti-rabbit immunoglobulins (Institut Pasteur, Paris). After washing, they were mounted and observed under a Leitz Orthoplan Epifluorescence microscope, a: prosencephalon, 8-somite stage. NC/1 labels most neural crest cells that remain above the neural tube and are prevented from migrating further laterally, above the optic vesicle. b: mesencephalon, 12-somite stage. Neural crest cells migrating under the ectoderm are clearly distinguishable from the surrounding mesenchyme. Some punctuate fluorescence is noticed in extraembryonic tissue. Abbreviations: ao, aortae: e, ectoderm: en, endoderm; m, mesenchyme: n. notochord; nt, neural tube', ov. optic vesicle: arrox~, front of migration. Bar = 50Hm
237
Fig. 2. Transverse sections of quail embryos at the vagal level and in the gut. a: posterior rhombencephalon, 21-somite stage embryo. Labeled neural crest cells, precursors of the enteric ganglia, are reaching the lateral ridge of the pharynx endoderm, b: gut, 4-day-old embryo. Numerous crest ceils arranged in a plexus-like structure in the splanchnopleural mesoderm are visualized by NC/1. Abbreviations: e, ectoderm; en, e ndoderm; m, mesenchyme; spm, splanchnopleural mesoderm; arrow, front of migration. Bar = 50 #m.
cence was also occasionally observed in some cells of the endoderm and of the splanchnopleural mesoderm in the developing heart (not shown). Interestingly, we found that the ectodermal placodes, which contribute to some cranial sensory ganglia, became labeled at advanced stages of neural crest cell migration. At 4 days of incubation, when most crest cells have reached their definitive locations, NC/1 also stained parts of the neural tube; in older embryos the central and the peripheral nervous systems reacted with the antibody (not shown). A number of monoclonal antibodies specific for the central and the peripheral nervous systems have been reported2,5,7A6,18. One of these antibodies, 38/D7, reacts specifically with neurons from the peripheral nervous system TM, whereas an antigen restricted to the neurons of central nervous system was defined by another monoclonal antibodyL Two too-
noclonal antibodies specific for chick ciliary ganglion neurons have been described; they were also shown to label a small proportion of mesencephalic neural crest cells in vitro, but none from the trunk level 1. The author suggested that the labeled crest cells may be the precursors of ciliary ganglion neurons. A n o t h er recently described monoclonal antibody, directed against chick sensory ganglia, reacts with neurons of the peripheral and central nervous systems and also with a small subpopulation of cultured neural crest cells 3. It was proposed that such antibodies identify early differentiating crest cells, but it has not been established yet that such cells are neuron precursors. In contrast to these reports, the NC/1 epitope is present on most, if not all, neural crest cells extremely soon after their emergence from the neural tube. We observed that cephalic crest cells, known to provide the mesectodermal derivatives in addition to
238 ganglia12,a4, are all stained during early stages of mi-
nervous system, it should be of interest to determine
gration. In contrast, after final localization, NC/1 antigen is not detected in the mesectodermal deriva-
if it has a role in neurogenesis.
tives but remains in the neural lineage. We also found
We thank N. Le D o u a r i n , S. McHanwell, J. Smith
that melanocytes are not labeled by the antibody.
and F. Dieterlen for critical reading of the manuscript and M. Denoyelle for excellent technical assis-
Thus this antibody may be a valuable reagent to define the stage at which melanocytic and mesectoder-
tance. This work was supported by grants from the
m o r e , this antibody should permit us to characterize
Centre National de la Recherche Scientifique, the Minist6re de la Recherche et de l'Industrie
some less well-defined pathways of migration despite
(81E1082) and the Institut National de la Sante et de
recent advances in the u n d e r s t a n d i n g of crest cell migratory behavior6,15,17. Experiments are in progress
la Recherche Medicale (CRL 824018). M. V. has a fellowship from Natural Sciences and Engineering
to characterize the NC/1-defined antigen. Since it appears also on ectodermal placodes and in the central
Research Council of Canada.
REFERENCES
l l Le Douarin, N. M. and Teillet, M. A., The migration of neural crest cells to the wall of the digestive tract in avian embryo, J. Embryol. exp. Morphol., 30 (1973)31-48. 12 Le Li~vre, C. S., R61e des cellules m6sectodermiques issues des cr6tes neurales c6phaliques dans la formation des arcs branchiaux et du squelette visc6ral, J. Embryol. exp. Morphol., 31 (1974) 453--477. 13 Le Li~vre, C. S., Schweizer, G. G., Ziller, C. M. and Le Douarin, N. M., Restriction of developmental capabilities in neural crest cell derivatives as tested by in vivo transplantation experiments, Develop. Biol., 77 (1980) 362-378. 14 Noden, D. M., An analysis of the migratory behavior of avian cephalic neural crest cells, Develop. Biol., 42 (1975) 106--130. 15 Rovasio, R. A., Delouv6e, A., Yamada, K. M., Timpl, R. and Thiery, J. P., Neural crest cell migration: requirements for exogenous fibronectin and high cell density, J. Cell Biol., 96 (1983) 462-474. 16 Schachner, M., Cell type-specific surface antigens in the mammalian nervous system, J. Neurochem., 39(1982) 1-8. 17 Thiery, J. P., Duband, J. L. and Delouv6e, A., Pathways and mechanisms of avian trunk neural crest cell migration and localization, Develop. Biol., 93 (1982) 324--343. 18 Vulliamy, T., Rattray, S. and Mirsky, R., Cell-surface antigen distinguishes sensory and autonomic peripheral neurones from central neurones, Nature (Lond.), 291 (1981) 418-420. 19 Weston, J. A., Motile and social behavior of neural crest cells. In R. Bellairs, A. Curtis and G. Dunn (Eds.), Cell Behaviour, Cambridge University Press, Cambridge, 1982, pp. 429-470.
mal cell lines emerge from the neural crest. Further-
1 Barald, K. F., Monoclonal antibodies to embryonic neurons: cell-specific markers for ciliary ganglia. In N. C. Spitzer (Ed.), Neuronal Development, Plenum Press, New York, 1982, pp. 101-119. 2 Barnstable, C. J., Monoclonal antibodies which recognize different cell types in the rat retina, Nature (Lond.), 286 (1980) 231-235. 3 Ciment, G, and Weston, J. A., Early appearance in neural crest and crest-derived cells of an antigenic determinant present in avian neurons, Develop. Biol., 93 (1982) 355-367, 4 Cochard, P. and Coltey, P., Cholinergic traits in the neural crest: acetylcholinesterase in crest cells of the chick embryo, Develop. Biol., in press. 5 Cohen, V. and Selvendram, S. Y., A neuronal cell-surface antigen is found in the CNS but not in peripheral neurones, Nature (Lond.), 291 (1981) 421-423. 6 Duband, J. L. and Thiery, J. P., Distribution of fibronectin in the early phase of avian cephalic neural crest cell migration, Develop. Biol., 93 (1982) 308-323. 7 Eisenbarth, G. S., Walsh, F. S. and Nirenberg, M., Monoclonal antibody to a plasma membrane antigen of neurons, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 4913-4917. 8 KOhler, G. and Milstein, C., Continuous cultures of fused cells secreting antibody of predefined specificity, Nature (Lond.), 256 (1975) 495-497. 9 Le Douarin, N. M., The ontogeny of the neural crest in avian embryo chimaeras, Nature (Lond.), 286 (1980) 663-669. 10 Le Douarin, N. M., The Neural Crest, Cambridge University Press, Cambridge, 1982.
235
Elsevier
A cell surface determinant expressed early on migrating avian neural crest cells MICHEL VINCENT, JEAN-LOUP DUBAND and JEAN-PAUL THIERY Institut d'Embryologie du Centre National de la Recherche, Scientifique et du Colldge de France, 49, bis Avenue de la Belle Gabrielle, 94130 Nogent-Sur-Marne (France)
(Accepted March 29th, 1983) Key words: avian embryo - - neural crest - - peripheral nervous system - - monoclonal antibody - - ciliary ganglion
We have produced monoclonal antibodies against quail ciliary ganglion in an attempt to identify specificmarkers of this neural crest derivative. One of these antibodies, NC/1, recognizes supportive and neuronal cells of the peripheral nervous system and also most, if not all migrating neural crest cells. We report herein the use of NC/1 to identify crest cells during their migration to their site of final localization. In addition, this antibody may shed light on how the neural crest derived mesectoderm and the peripheral nervous system segregate from one another since the NC/1-defined antigen becomes restricted to the cells of the latter. The neural crest is a transient structure in vertebrate embryos detaching from the neural epithelium and giving rise, after an extensive intraembryonic migration, to a variety of cell types including neurons and supportive cells of the peripheral nervous system, melanocytes and many cranio-facial structures designated as mesectodermal derivatives10. It is an excellent model system to study cell migration and the progressive restriction of precursor cell capabilities during development9.13,19. Therefore, a library of stage- or cell-type-specific markers would be useful to decipher mechanisms controlling segregation of the neural crest cells into their different lineages. We thus decided to produce monoclonal antibodies against quail ciliary ganglion, a parasympathetic ganglion entirely derived from the neural crest 10. BALB/c mice were immunized with 8-dayold quail ciliary ganglia until high titer sera were obtained. Three days after an i.v. boost injection, immune mouse splenocytes were fused with Sp2/0 myeloma according to the method of K6hler and Milstein 8. Hybridoma culture supernatants were systematically tested for antibody by indirect immunofluorescence on 24-h microcultures of living cells from ciliary, sympathetic and sensory ganglia. One of the supernatants, NC/1, appeared to react strongly both with flat cells and neurons in each type of cultures (not shown). Its reactivity with living cells suggests that it recognizes a cell surface-associated anti0165-3806/83/$03.00 © 1983 Elsevier Science Publishers B.V.
gen. NC/1 did not bind either to 8-day-old quail embryo heart fibroblasts or to somitic mesenchyme (see Fig. lb) but labeled cultured trunk neural crest cells (not shown). Therefore, NC/1 defines an antigen present in vitro on trunk crest cells and on the cells of the sensory and autonomic ganglia. It was selected for further characterization: NC/1 producing hybridomas were cloned twice and thereafter grown in vitro or in ascites for large-scale production of antibody. All antibody preparations gave results identical to those obtained with the original supernatant. The specificity of this antibody was further probed by immunolabeling sections of quail embryos made at levels where crest cells had begun their migration. The antibody was found to stain migrating neural crest cells; for example, in the prosencephalon, crest cells which are known to accumulate between the ectoderm and the optic vesicle 6, were stained by NC/1 (Fig. la). Similarly, at the mesencephalic level, crest cells which migrate laterally under the ectoderm were intensely stained (Fig. lb). In slightly older embryos, at the posterior rhombencephalic level, neural crest cells that had reached the apex of the pharynx could be easily detected among the surrounding tissues, which remained unstained (Fig. 2a); at later stages, when these cells had become localized in the gut 11, they could still be labeled by NC/1 (Fig. 2b). We noticed that the periphery of the notochord, at the trunk level, was diffusely stained; NC/1 fluores-
236
Fig. 1. Transverse sections of quail embryos at the cephalic level. Formaldehyde-fixed embryos were embedded in polyethylene-glycol and sectioned as described previously 6. Sections were incubated with NC/1 hybridoma supernatant for 1 h at room temperature. After extensive washes in phosphate-buffered saline, sections were successively treated with rabbit IgG anti-mouse immunoglobulins (Nordic, Tilburg, The Netherlands) and FITC-conjugated sheep anti-rabbit immunoglobulins (Institut Pasteur, Paris). After washing, they were mounted and observed under a Leitz Orthoplan Epifluorescence microscope, a: prosencephalon, 8-somite stage. NC/1 labels most neural crest cells that remain above the neural tube and are prevented from migrating further laterally, above the optic vesicle. b: mesencephalon, 12-somite stage. Neural crest cells migrating under the ectoderm are clearly distinguishable from the surrounding mesenchyme. Some punctuate fluorescence is noticed in extraembryonic tissue. Abbreviations: ao, aortae: e, ectoderm: en, endoderm; m, mesenchyme: n. notochord; nt, neural tube', ov. optic vesicle: arrox~, front of migration. Bar = 50Hm
237
Fig. 2. Transverse sections of quail embryos at the vagal level and in the gut. a: posterior rhombencephalon, 21-somite stage embryo. Labeled neural crest cells, precursors of the enteric ganglia, are reaching the lateral ridge of the pharynx endoderm, b: gut, 4-day-old embryo. Numerous crest ceils arranged in a plexus-like structure in the splanchnopleural mesoderm are visualized by NC/1. Abbreviations: e, ectoderm; en, e ndoderm; m, mesenchyme; spm, splanchnopleural mesoderm; arrow, front of migration. Bar = 50 #m.
cence was also occasionally observed in some cells of the endoderm and of the splanchnopleural mesoderm in the developing heart (not shown). Interestingly, we found that the ectodermal placodes, which contribute to some cranial sensory ganglia, became labeled at advanced stages of neural crest cell migration. At 4 days of incubation, when most crest cells have reached their definitive locations, NC/1 also stained parts of the neural tube; in older embryos the central and the peripheral nervous systems reacted with the antibody (not shown). A number of monoclonal antibodies specific for the central and the peripheral nervous systems have been reported2,5,7A6,18. One of these antibodies, 38/D7, reacts specifically with neurons from the peripheral nervous system TM, whereas an antigen restricted to the neurons of central nervous system was defined by another monoclonal antibodyL Two too-
noclonal antibodies specific for chick ciliary ganglion neurons have been described; they were also shown to label a small proportion of mesencephalic neural crest cells in vitro, but none from the trunk level 1. The author suggested that the labeled crest cells may be the precursors of ciliary ganglion neurons. A n o t h er recently described monoclonal antibody, directed against chick sensory ganglia, reacts with neurons of the peripheral and central nervous systems and also with a small subpopulation of cultured neural crest cells 3. It was proposed that such antibodies identify early differentiating crest cells, but it has not been established yet that such cells are neuron precursors. In contrast to these reports, the NC/1 epitope is present on most, if not all, neural crest cells extremely soon after their emergence from the neural tube. We observed that cephalic crest cells, known to provide the mesectodermal derivatives in addition to
238 ganglia12,a4, are all stained during early stages of mi-
nervous system, it should be of interest to determine
gration. In contrast, after final localization, NC/1 antigen is not detected in the mesectodermal deriva-
if it has a role in neurogenesis.
tives but remains in the neural lineage. We also found
We thank N. Le D o u a r i n , S. McHanwell, J. Smith
that melanocytes are not labeled by the antibody.
and F. Dieterlen for critical reading of the manuscript and M. Denoyelle for excellent technical assis-
Thus this antibody may be a valuable reagent to define the stage at which melanocytic and mesectoder-
tance. This work was supported by grants from the
m o r e , this antibody should permit us to characterize
Centre National de la Recherche Scientifique, the Minist6re de la Recherche et de l'Industrie
some less well-defined pathways of migration despite
(81E1082) and the Institut National de la Sante et de
recent advances in the u n d e r s t a n d i n g of crest cell migratory behavior6,15,17. Experiments are in progress
la Recherche Medicale (CRL 824018). M. V. has a fellowship from Natural Sciences and Engineering
to characterize the NC/1-defined antigen. Since it appears also on ectodermal placodes and in the central
Research Council of Canada.
REFERENCES
l l Le Douarin, N. M. and Teillet, M. A., The migration of neural crest cells to the wall of the digestive tract in avian embryo, J. Embryol. exp. Morphol., 30 (1973)31-48. 12 Le Li~vre, C. S., R61e des cellules m6sectodermiques issues des cr6tes neurales c6phaliques dans la formation des arcs branchiaux et du squelette visc6ral, J. Embryol. exp. Morphol., 31 (1974) 453--477. 13 Le Li~vre, C. S., Schweizer, G. G., Ziller, C. M. and Le Douarin, N. M., Restriction of developmental capabilities in neural crest cell derivatives as tested by in vivo transplantation experiments, Develop. Biol., 77 (1980) 362-378. 14 Noden, D. M., An analysis of the migratory behavior of avian cephalic neural crest cells, Develop. Biol., 42 (1975) 106--130. 15 Rovasio, R. A., Delouv6e, A., Yamada, K. M., Timpl, R. and Thiery, J. P., Neural crest cell migration: requirements for exogenous fibronectin and high cell density, J. Cell Biol., 96 (1983) 462-474. 16 Schachner, M., Cell type-specific surface antigens in the mammalian nervous system, J. Neurochem., 39(1982) 1-8. 17 Thiery, J. P., Duband, J. L. and Delouv6e, A., Pathways and mechanisms of avian trunk neural crest cell migration and localization, Develop. Biol., 93 (1982) 324--343. 18 Vulliamy, T., Rattray, S. and Mirsky, R., Cell-surface antigen distinguishes sensory and autonomic peripheral neurones from central neurones, Nature (Lond.), 291 (1981) 418-420. 19 Weston, J. A., Motile and social behavior of neural crest cells. In R. Bellairs, A. Curtis and G. Dunn (Eds.), Cell Behaviour, Cambridge University Press, Cambridge, 1982, pp. 429-470.
mal cell lines emerge from the neural crest. Further-
1 Barald, K. F., Monoclonal antibodies to embryonic neurons: cell-specific markers for ciliary ganglia. In N. C. Spitzer (Ed.), Neuronal Development, Plenum Press, New York, 1982, pp. 101-119. 2 Barnstable, C. J., Monoclonal antibodies which recognize different cell types in the rat retina, Nature (Lond.), 286 (1980) 231-235. 3 Ciment, G, and Weston, J. A., Early appearance in neural crest and crest-derived cells of an antigenic determinant present in avian neurons, Develop. Biol., 93 (1982) 355-367, 4 Cochard, P. and Coltey, P., Cholinergic traits in the neural crest: acetylcholinesterase in crest cells of the chick embryo, Develop. Biol., in press. 5 Cohen, V. and Selvendram, S. Y., A neuronal cell-surface antigen is found in the CNS but not in peripheral neurones, Nature (Lond.), 291 (1981) 421-423. 6 Duband, J. L. and Thiery, J. P., Distribution of fibronectin in the early phase of avian cephalic neural crest cell migration, Develop. Biol., 93 (1982) 308-323. 7 Eisenbarth, G. S., Walsh, F. S. and Nirenberg, M., Monoclonal antibody to a plasma membrane antigen of neurons, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 4913-4917. 8 KOhler, G. and Milstein, C., Continuous cultures of fused cells secreting antibody of predefined specificity, Nature (Lond.), 256 (1975) 495-497. 9 Le Douarin, N. M., The ontogeny of the neural crest in avian embryo chimaeras, Nature (Lond.), 286 (1980) 663-669. 10 Le Douarin, N. M., The Neural Crest, Cambridge University Press, Cambridge, 1982.