119
J. Anat. (1990), 173, pp. 119-127 With 8 figures Printed in Great Britain
Early neuronal development in the spinal cord of a reptile assessed by neurofilament protein immunoreactivity E. MARTI, M. A. P. BATISTA, A. R. BELLO, A. LANCHA AND D. DAHL*
U.D.I. Biologia Celular, Facultad de Biologia, Universidad de La Laguna, Tenerife, Spain and *Department of Neuropathology, Harvard Medical School and Spinal Cord Injury Research Laboratory, West Roxbury, Veterans Administration Medical Centre, Boston, Massachussets, USA (Accepted 23 May 1990) INTRODUCTION
A few years ago a research programme was started in our department using the lizard Gallotia galloti as a model for the study of neurogenesis in reptiles. The major aim of this project was to provide data on the embryonic origin of a given neuronal population in order to reconstruct its evolutionary history and to establish homologies. Data have already been published about neurogenesis in various areas of the brain of this lizard (Diaz et al. 1990; Trujillo et al. 1987; Yanes et al. 1987, 1988 a, b, 1989). However, studies on the early stages of neurogenesis occurring in the spinal cord are lacking and, as the spinal cord is the simplest structure of the developing vertebrate central nervous system, we regard it as an ideal model for the study of these events. Intermediate filament proteins have been considered as taxonomic characters (Dahl & Bignami, 1986) and they have practical applications in the study of developmental events since they allow the cell type to be identified independently of morphological criteria (Bennet & Dillulo, 1985a; Raju, Bignami & Dahl, 1981; Tapscott, Bennet & Holtzer, 1981). The changeover from vimentin to neurofilament proteins as intermediate filaments can be taken as a sign of the onset of neuron formation (Bennet & Dillulo, 1985b; Bignami, Raju & Dahl, 1982; Tapscott et al. 1981). In this study we have combined morphological, ultrastructural and immunohistochemical data in order to provide a comprehensive study of the early steps of neurogenesis in the spinal cord of the lizard Gallotia galloti. MATERIALS AND METHODS
Gallotia galloti (Reptilia, Lacertidae) embryos were staged using the developmental table of Lacerta vivipara (Dufaure & Hubert, 1961). Early stage embryos (up to Stage 28, S.28) were removed from the oviduct and immersed in toto in either Ringer's solution, for their classification or directly in fixative solution. From S.28 onwards embryos were collected from their burrows, the eggs taken to the laboratory and there dissected. Embryos freed from their extraembryonic membranes were subjected to the same procedure as described above. From S.28 onwards the specimens were decapitated before fixation in order to allow better preservation, and from S.35 onwards, the spinal cord itself was carefully removed with the aid of a dissecting microscope. At least 6-8 embryos were killed at each embryonic stage, from S.22 to hatching.
120
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Conventional histology Tissue for conventional histology was fixed in Bouin's solution, embedded in Paraplast and sectioned at 3-10 ,um. Serial sections from the whole length of the spinal cord were stained with haematoxylin and eosin, cresyl violet or the Kluver Barrera technique. Electron microscopy Tissue for electron microscopy was fixed in 2-5 % glutaraldehyde in phosphate buffer solution (0 1 M, pH 7 2), postfixed with 2% osmium tetroxide in Millonig's buffer at 4 °C, embedded in Spurr resin and sectioned with a Reichert ultramicrotome OM-30. Semithin sections (0-5-1 jum) were stained with toluidine blue/borax (1: 1) and ultrathin sections with uranyl acetate and lead citrate (Reynolds, 1963).
Immunohistochemistry Tissue for immunohistochemistry was fixed in Bouin's Hollande solution, embedded in Paraplast and sectioned at 7-10 ,um. Sections were mounted on poly-L-lysine-coated slides (Huang et al. 1983) and all sections were stained following a slight modification of the peroxidase anti-peroxidase method of Sternberger (1979). Details of the procedure are described elsewhere (Marti et al. 1987). Neurofilament triplet protein antisera characteristics have been previously determined (Dahl et al. 1984). All the reagents were diluted with phosphate buffered saline (PBS) (0-1 M, pH 7-4). Neurofilament protein antisera dilutions were 1: 500 to 1: 1000, goat anti-rabbit IgG dilution was 1: 200 and peroxidase anti-peroxidase (PAP) dilution was 1: 300. Immunoreaction was revealed with 01 % (w/v) 3,3'-diaminobenzidine tetrahydrochloride (DAB) solution in PBS containing 0-025 % hydrogen peroxide. Controls of immunoreaction To ensure method specificity, several slides were incubated with nonimmune rabbit serum in each batch of immunostained sections. The specificity of the neurofilament triplet protein antisera was confirmed by lack of immunostained product when control sections were incubated with antisera preabsorbed with 150 K neurofilament protein (natural avian) (0- 1 nmol antigen/ml diluted antisera). RESULTS
The events occurring during neurogenesis, from neural tube closure to hatching, as expected, were found to be similar along the length of tube, showing only time differences. We shall therefore describe in detail only those events occurring at the cervical level. Fig. 1. S.23 neural tube. The lateral walls are formed by a pseudostratified epithelium, whereas the roof and floor plates (rp, fp) are formed by a single cell layer. cc, central canal; nc, neural crest. Wax section. Toluidine blue. Bar, 10 ,um. Fig. 2. S.26 neural tube in which numerous mitoses (arrowheads) are present at the central canal border. The lateral walls are formed by a thick ventricular or mantle zone (vz) surrounded by a thin cell free layer, the marginal zone (mz). cc, central canal; n, notochord; rp, roof plate. Wax section. Haematoxylin eosin. Bar, 10 /zm. Fig. 3. Floor plate of the S.26 neural tube showing wedge-shaped cells contacting both the external and the internal (central canal) limits of the neural tube. Apical cytoplasmic processes are strongly attached by cell junctions. (arrowheads). Thick arrowheads point to residual masses with a laminar content. cc, central canal. Bar, 1 #tm.
Neurogenesis in the reptilian spinal cord
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J. Anat. (1990), 173, pp. 119-127 With 8 figures Printed in Great Britain
Early neuronal development in the spinal cord of a reptile assessed by neurofilament protein immunoreactivity E. MARTI, M. A. P. BATISTA, A. R. BELLO, A. LANCHA AND D. DAHL*
U.D.I. Biologia Celular, Facultad de Biologia, Universidad de La Laguna, Tenerife, Spain and *Department of Neuropathology, Harvard Medical School and Spinal Cord Injury Research Laboratory, West Roxbury, Veterans Administration Medical Centre, Boston, Massachussets, USA (Accepted 23 May 1990) INTRODUCTION
A few years ago a research programme was started in our department using the lizard Gallotia galloti as a model for the study of neurogenesis in reptiles. The major aim of this project was to provide data on the embryonic origin of a given neuronal population in order to reconstruct its evolutionary history and to establish homologies. Data have already been published about neurogenesis in various areas of the brain of this lizard (Diaz et al. 1990; Trujillo et al. 1987; Yanes et al. 1987, 1988 a, b, 1989). However, studies on the early stages of neurogenesis occurring in the spinal cord are lacking and, as the spinal cord is the simplest structure of the developing vertebrate central nervous system, we regard it as an ideal model for the study of these events. Intermediate filament proteins have been considered as taxonomic characters (Dahl & Bignami, 1986) and they have practical applications in the study of developmental events since they allow the cell type to be identified independently of morphological criteria (Bennet & Dillulo, 1985a; Raju, Bignami & Dahl, 1981; Tapscott, Bennet & Holtzer, 1981). The changeover from vimentin to neurofilament proteins as intermediate filaments can be taken as a sign of the onset of neuron formation (Bennet & Dillulo, 1985b; Bignami, Raju & Dahl, 1982; Tapscott et al. 1981). In this study we have combined morphological, ultrastructural and immunohistochemical data in order to provide a comprehensive study of the early steps of neurogenesis in the spinal cord of the lizard Gallotia galloti. MATERIALS AND METHODS
Gallotia galloti (Reptilia, Lacertidae) embryos were staged using the developmental table of Lacerta vivipara (Dufaure & Hubert, 1961). Early stage embryos (up to Stage 28, S.28) were removed from the oviduct and immersed in toto in either Ringer's solution, for their classification or directly in fixative solution. From S.28 onwards embryos were collected from their burrows, the eggs taken to the laboratory and there dissected. Embryos freed from their extraembryonic membranes were subjected to the same procedure as described above. From S.28 onwards the specimens were decapitated before fixation in order to allow better preservation, and from S.35 onwards, the spinal cord itself was carefully removed with the aid of a dissecting microscope. At least 6-8 embryos were killed at each embryonic stage, from S.22 to hatching.
120
E. MARTI AND OTHERS
Conventional histology Tissue for conventional histology was fixed in Bouin's solution, embedded in Paraplast and sectioned at 3-10 ,um. Serial sections from the whole length of the spinal cord were stained with haematoxylin and eosin, cresyl violet or the Kluver Barrera technique. Electron microscopy Tissue for electron microscopy was fixed in 2-5 % glutaraldehyde in phosphate buffer solution (0 1 M, pH 7 2), postfixed with 2% osmium tetroxide in Millonig's buffer at 4 °C, embedded in Spurr resin and sectioned with a Reichert ultramicrotome OM-30. Semithin sections (0-5-1 jum) were stained with toluidine blue/borax (1: 1) and ultrathin sections with uranyl acetate and lead citrate (Reynolds, 1963).
Immunohistochemistry Tissue for immunohistochemistry was fixed in Bouin's Hollande solution, embedded in Paraplast and sectioned at 7-10 ,um. Sections were mounted on poly-L-lysine-coated slides (Huang et al. 1983) and all sections were stained following a slight modification of the peroxidase anti-peroxidase method of Sternberger (1979). Details of the procedure are described elsewhere (Marti et al. 1987). Neurofilament triplet protein antisera characteristics have been previously determined (Dahl et al. 1984). All the reagents were diluted with phosphate buffered saline (PBS) (0-1 M, pH 7-4). Neurofilament protein antisera dilutions were 1: 500 to 1: 1000, goat anti-rabbit IgG dilution was 1: 200 and peroxidase anti-peroxidase (PAP) dilution was 1: 300. Immunoreaction was revealed with 01 % (w/v) 3,3'-diaminobenzidine tetrahydrochloride (DAB) solution in PBS containing 0-025 % hydrogen peroxide. Controls of immunoreaction To ensure method specificity, several slides were incubated with nonimmune rabbit serum in each batch of immunostained sections. The specificity of the neurofilament triplet protein antisera was confirmed by lack of immunostained product when control sections were incubated with antisera preabsorbed with 150 K neurofilament protein (natural avian) (0- 1 nmol antigen/ml diluted antisera). RESULTS
The events occurring during neurogenesis, from neural tube closure to hatching, as expected, were found to be similar along the length of tube, showing only time differences. We shall therefore describe in detail only those events occurring at the cervical level. Fig. 1. S.23 neural tube. The lateral walls are formed by a pseudostratified epithelium, whereas the roof and floor plates (rp, fp) are formed by a single cell layer. cc, central canal; nc, neural crest. Wax section. Toluidine blue. Bar, 10 ,um. Fig. 2. S.26 neural tube in which numerous mitoses (arrowheads) are present at the central canal border. The lateral walls are formed by a thick ventricular or mantle zone (vz) surrounded by a thin cell free layer, the marginal zone (mz). cc, central canal; n, notochord; rp, roof plate. Wax section. Haematoxylin eosin. Bar, 10 /zm. Fig. 3. Floor plate of the S.26 neural tube showing wedge-shaped cells contacting both the external and the internal (central canal) limits of the neural tube. Apical cytoplasmic processes are strongly attached by cell junctions. (arrowheads). Thick arrowheads point to residual masses with a laminar content. cc, central canal. Bar, 1 #tm.
Neurogenesis in the reptilian spinal cord
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