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Immunohistochemical study of glutamine synthetase expression in early glial development
Developmental Brain Research, 72 (1993) 9-14 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-3806/93/$06.00
9
BRESD 51578
Immunohistochemical study of glutamine synthetase expression in early glial development Jiro Akimoto
a, Hiroshi Itoh a, Tetsuro Miwa a and Kazuhiko Ikeda
~
Department of Neurosurgery, Tokyo Medical College, Tokyo (Japan) and b Department of Ultrastructure and lmmunohistochemistry, Tokyo Institute of Psychiatry, Tokyo (Japan)
(Accepted 22 September 1992)
Key words: Development; Gliogenesis; Radial gila; Glial fibrillary acidic protein; Glutamine synthetase; Immunohistochemistry
Early glial development in the brains of rat embryos was immunohistochemically studied using antibotides to two kinds of glial-specific proteins, glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP). The results showed that GS was first expressed in the ependymal lining of the ventral side of the neural tube in the cervical segment on Embryonic Day 14 and then in the radial glia radiating from the neural tube on Embyronic Day 16. Subsequently, GS-positive glia cells increased in number with time. On the other hand, GFAP was first expressed in the fimbria hippocampi and the pial surface on Embryonic Day 18, but no marked evidence of GFAP expression was noted in the GS-positive radial glia. These results suggested that a specific protein is expressed at each stage of early glial development. Since the radial glia was positive for GS, it was thought that GS-mediated neuronal-glial interactions may play an important role in the development of the central nervous system.
INTRODUCTION M u c h of the d e v e l o p m e n t of the central nervous system (CNS) has r e m a i n e d obscure. A l t h o u g h it is a well-known fact that n e u r o n s and glial cells develop and differentiate f r o m the ectoderm-derived neural tube f o r m e d in a very early developmental stage, great controversies have arisen as to what developmental course n e u r o n s and glial cells take to f o r m the very complicated structure of the CNS, and what mechanism is involved in the control over interactions between these cells. T h e r e have b e e n two widely a c c e p t e d theories as to developmental processes, but no conclusion has yet been drawn. T h e two theories are: the monistic theory p r o p o s e d by Fujita et al., 1°'24 and the dualistic theories p r o p o s e d first by His t3 and recently by Rakic 6'15'3° and coworkers. According to the f o r m e r theory, the neural tube is comprised of uniform epithelial matrix cells. As matrix cells repeat u p - a n d - d o w n movements, the n e u r o n s first develop from these cells, and the glial cells develop
subsequently. A c c o r d i n g to the second theory, the neurons and glial cells, respectively develop from different stem cells in the neural tube. A n increasing n u m b e r of reports supporting the latter theory have been published over the years. As stated by Rakic et al. 6'15'3°, glial cells already exist w h e n n e u r o n s begin to migrate, and they regulate n e u r o n a l differentiation by serving as a guide for migration ~8'3~. These glial cells are precisely the radial glia designated by Cajal. 4. In recent years, there have b e e n m a n y reports aimed at determining the presence or absence of these radial glia cells. As a result, the existence of radial glia has gradually b e c o m e evident t h r o u g h in vitro 8 and electron microscopic studies 7. T h e results of these studies suggest that the n e u r o n migrates by traveling on rails (processes) of the radial glia ~l, and that n e u r o n a l differentiation and cell division are controlled by signal contact between n e u r o n s and glial cells 8. With this in mind, we immunohistochemicaUy investigated radial glia by using antibodies to astrocytic markers, i.e. glial fibrillary acidic protein ( G F A P ) and glutamine synthetase (GS).
Correspondence: J. Akimoto, 6-7-1 Nishishinjyuku, Shinjyuku-ku, Tokyo 160, Japan. Fax: (81) (3) 3340-4285.
10 MATERIALS AND METHODS
Embryonic Day 14
Pregnant Crj/CD rats (Japan Charles River Co., Tokyo, Japan) were used in the present study. The day of mating was counted as Day 0. Pregnant rats were anesthetized with ether and embryos were obtained at Days 10, 12, 14, 16, 18 and 20. Each embryo was immediately decapitated and head portion was fixed in a 70% ethanol solution at 4°C for 7 days. It was embedded in paraffin and frontal sections were cut at 5 txm thickness. Sections were stained with hematoxylin and eosin. For immunohistochemistry, deparaffinized sections were first treated with 0.1% H20 2 in methanol to abolish endogeneous peroxidase activity, and they were washed in phosphate-buffered saline (PBS). The sections were then incubated with rabbit polyclonal antibody raised against mouse brain G S 27 (kindly provided by M. Oda, Tokyo Metropolitan Neurological Hospital), diluted 1:400, or mouse monoclonal antibody to GFAP (DAKO, Denmark), diluted 1 :400 at room temperature for 3 h. After washing in PBS, they were incubated with biotinylated anti-rabbit lgG antibody or anti-mouse IgG antibody (Vector, USA), washed in PBS and reacted with peroxidase-conjugated streptavidin (Zymed, USA). Reaction product was visualized by diaminobenizidine and hydrogen peroxide. The sections were counterstained with hematoxylin. Immunostaining omitting incubation with primary antibody served as control.
and a structure t h o u g h t to be a m a n t l e or marginal
T h e n e u r a l tube markedly increased in thickness,
RESULTS
layer a p p e a r e d . T h e m a n t l e layer was comprised of spindle cells, which had relatively sparse c h r o m a t i n s a n d spread irregularly. GS i m m u n o r e a c t i v i t y a p p e a r e d in some of the epithelial cells of the ventral p o r t i o n of the cervical s e g m e n t in a g r a n u l a r form and in cellular processes e x t e n d i n g in a fringe-like p a t t e r n in the marginal layer. GS-positive fibers in the m a r g i n a l layer were swollen, a n d the pial surface was lined with GS-positive granules (Fig. la,b). G F A P i m m u n o r e a c t i v i t y was not observed at this stage.
Embryonic Day 16 T h e e p i t h e l i u m of the n e u r a l tube decreased in thickness, while the m a n t l e layer was thickened. Ceils c o m p o s i n g the m a n t l e layer had a r o u n d polymorphic n u c l e u s with sparse c h r o m a t i n a n d very few or n o
m a n t l e or m a r g i n a l layer was observed. No i m m u n o r e activity for G F A P a n d G S was observed at this stage
vesicles. T h e s e cells spread w i t h o u t any regular arrangement. GS-positive ceils i n c r e a s e d in n u m b e r in the epithelial cells of the v e n t r a l half of the n e u r a l tube (Fig. 2a). In the m a n t l e layer, GS-positive cells were scattered, showing radial migration. A close inspection revealed that these cells had s p i n d l e - t o - p o l y m o r p h i c nuclei, a n d
(data n o t shown).
GS-positive processes could be observed in some cells.
Embryonic Day 12 Y o u n g cells c o m p o s i n g the n e u r a l tube showed a p s e u d o s t r a t i f i e d a r r a n g e m e n t , a n d the nuclei facing the tubal cavity showed m a r k e d mitosis. No a p p a r e n t
Fig. 1. Immuno-staining for GS of rat embryo (E 14): a, cervical portion, x68; b; × 170. Counter-stained with hematoxylin. GS-positive cells (arrow) are seen in the epithelial cells at the ventral portion of neural tube. GS-positive fibers (arrowheads) are also seen both in ventral mantle layer and marginal layer.
11
Fig. 2. Immuno-staining for GS of rat embryo (E 16): a and b, cervical portion, x 170, c and d, p o n s , × 340. Counter stained with hematoxylin. GS-positive cells (arrow) are increased in number in the epithelial cell layer. In addition, scattered positive cells are seen in the ventral mantle layer, showing their migration from the epithelial layer (a and c). Radial fibers (arrowheads) are conspicuous in the mantle and marginal layers (b and d).
12
Fig. 3. Immuno-staining for GS and GFAP of rat embryo (E 18): a, cervical portion, GS, x 170; b and c, pons, GS, )<340; d, cervical portion, GFAP, x340. Counter-stained with hematoxylin. Migration of the GS-positive cells (arrow) together with radial fibers (arrowheads) are pronounced in the mantle layer (a, b and c). GFAP-positive cells and processes (*) are firstly seen at this stage in the marginal layer and pial surface (d).
13 In the marginal layer, many GS-positive fibers extending in a fringe-like pattern were observed, and the pial surface was covered with a layer of GS-positive lining (Fig. 2b). In the more rostral region between the mesencephalon and the pons, many GS-positive cells radiated from the epithelium of the neural tube and spread over the mantle layer. GS-positive fibers running radially between intercellular spaces were more conspicuous at this stage (Fig. 2c,d). G F A P immunoreactivity was still not seen at this stage.
Embryonic Day 18 GS immunoreactivity was seen in some epithelial cells of the neural tube, which practically formed a single layer. GS immunoreactivity was also detected in the cells radially spreading in the mantle layer as well as in bipolar processes of these cells. Distal processes extended to the marginal layer and spread in the dendroid form at the pial surface, forming a GS-positive lining (Fig. 3a). In the pons, GS immunoreactivity was observed in the subependymal cells, and also in cell processes, which resembled a fringe-like pattern and seemed to be weaving in between intercellular spaces (Fig. 3b,c). G F A P immunoreactivity was first observed at this stage, showing a fringe-like pattern of cell processes in the marginal layer and pial surface in the cervical segment. G F A P immunoreactivity was also evident in the fimbria hippocampi and the pial surface in the same region (Fig. 3d).
nature of radial glia. G F A P immunoreactivity was first observed in cells and their fibers on rat Embryonic Day 18, which is consistent with previous reports 3'~4. GS was confirmed by Norenberg et al. 2'25'26'27 to be a protein existing only in astrocytes, at least in the brain and has been used as a marker protein for astrocytes. However, few studies have been performed with respect to the, GS expression in the course of gliogenesis tg. In the present study, GS immunoreactivity was first observed in cells composing the epithelium of the neural tube in the cervical segment on Embryonic Day 14, and then in fibers which radiated from the neural tube. The positive cells and fibers increased in number by time. Based on the time of the appearance of these cells and their morphology, the GS-positive cells were considered to be radial glia. The data of our immunohistochemical study of rat embryo accord well with those of previous immunohistochemical studies of chick optic tectum ~6 and lizard embryo ~4, in that the appearance of GS immunoreactivity preceeds that of G F A P immunoreactivity in radial glia. In the mouse embryonic period 1'5, GS enzymic activity became detectable on Embryonic Day 16 and increased by time 2s'3:. Studies by in situ hybridization 2°'21, showing the appearance of GS m R N A on Embryonic Day 14, support the immunohistochemical data. The main functions of GS include inactivation of the excitatory neurotransmitter glutamic acid and detoxication of ammonium 17"25'32. It may be possible that such functions are required in the microenvironment of the developing nervous tissue, where the radial glia interact with migrating neurons along their fibers.
DISCUSSION REFERENCES
It is known that long fibers radiating from the neural tube exist in higher vertebrates at the time of development of the CNS. Golgi's stain can reveal the fiber containing cells known as radial glia, epithelial cells or spongioblasts, which are thought to appear temporarily during the development of the C N S 7. Rakic et al. 3°'3~ stated that this cell type, the radial glia designated by Cajal 4, play an essential role in the development of the CNS as a guide for the migration of nerve cells. Recent s t u d i e s 22'23"29'31, revealed that the radial fibers appear at an earlier stage of development than previously considered. Electron microscopic studies 7'~ have shown that radial glia share features with astrocytes, and that nerve cells migrate along its processes ~'12'~s, thus providing evidence of n e u r o n a l glial interaction in the nervous tissue development. The present immunohistochemical study was carried out using the astrocyte-specific markers G F A P and GS as indicators in order to gain more information on the
1 Berl, S., Glutamine synthetase. Determination of its distribution in brain during development, Biochemistry 5 (1966) 916-921. 2 Bell, K.P. and Norenberg, M.D., Glutamine synthetase. Glial localization in brain, Science 195 (1976) 1356-1358. 3 Bignami, A. and Dahl, D., Astrocyte-specific protein and neuroglial differentiation. An immunofluorescence study with antibodies to the glial fibrillary acidic protein, J. Comp. Neurol. 153 (1979) 27-38. 4 Cajal, R.Y.S., Histologie du Syst~me Nerl'eux de l'Homme et des Vertdbr~s Vol. H, reprinted by: Consejo Superior de lnvestigaciones Cientificas, Paris, 1955. 5 Caldani, M., Rolland, B., Fages, C, and Tardy, M., Glutamine synthetase activity during mouse brain development, Experientia, 38 (1982), 1199-1202. 6 Cameron, R.S. and Rakic, P., Glial cell lineage in the cerebral cortex: a review and synthesis, Glia, 4 (1991) 124-137. 7 Choi, B.H. and Lapham, L.W., Radial glia in the human fetal cerebrum. A combined Golgi, immunofluorescent and electron microscopic study. Brain Res. 148 (1978) 295-311. 8 Culican, S.M., Baumrind, N.K., Yamamoto, M. and Pearlman, A., Cortical radial glia. Identification in tissue culture and evidence for their transformation to astrocytes, J. Neurosci., 10 (1990) 684-692. 9 Edmondson, J.C. and Hatten, M.E., Glial-guided granule neuron
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migration in vitro. A high-resolution time lapse video microscopic study, Z Neurosci., 7 (1987) 1928-1934. Fujita, S., An autoradiographic study on the origin and fate of the subpial glioblast in the embryonic chick spinal cord, Z Comp. Neurol., 124 (1965) 51-60. Gadisseux, J.F., Kadhim, H.J., Bosch, P.A., Caviness, V.S. and Evrard, P,, Neuron migration within the radial fiber system of the developing murine cerebrum, An electron micrscopic autoradiographic analysis, Det~. Brain Res., 52 (1990) 39-56. Hatten, M.E., Riding the glial monorail. A common mechanism for glial-guided neuronal migration in different regions of the developing mammalian brain, Trends Neurosci., 13 (1990) 179-184. His, W., Die Entwicklung des menschlichen Gehirns wiihrend der ersten Monate, S. Hirzel, Leipzig, 1904. Kamada, H., ltoh, T., Hyogo, T. et al., Immunohistochemical study of developing rat embryo-localization of vimentin, GFAP, neurofilament protein within rat embryo central nervous system, Brain Nerce, 40 (1988) 2ll-218. Levitt, P., Cooper, M.L. and Rakic, P., Coexistence of neuronal and glial precursor cells in the cerebral ventricular zone of the fetal monkey: an ultra structural immunoperoxidase analysis, J. Neurosci., 1 (1981) 27-39. Linser, P.J. and Perkins, M., Gliogenesis in the embryonic avian optic tectum. Neuronal-glial interactions influence astroglial phenotype maturation, Dez,. Brain Res., 31 (1987) 277-290. Lomneth, R., Bkaily, G., Sperelakis, N., Liwnicz, B.H. and Gruenstein, E., Electrophysiological and biochemical characterization of a continuous human astrocytoma cell line with many properties of well-differentiated astrocytes, Brain Res., 486 (1989) 95107. Mackay, R.D.G., The origin of cellular diversity in the mammalian central nervous system, Cell 58 (1989) 815-821. Monzon-Mayor, M., Yanes, C., Tholey, G., Barry, J.D. and Gombos, G., Immunohistochemical localization of glutamine synthetase in mesencephalon and telencephalon of the lizard gallotia galloti during ontogeny, Glia 3 (1990) 81-97. Mearow, K.M., Mill, J.F. and Vitkovic, L., The ontogeny and
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localization of glutamine synthetase gene expression m ~at brain. Mol. Brain Res., 6 (1989) 223-232. Mearow, K.M., Mill, J.F. and Freese, E., Neuron-gliat interactions involved in the regulation of glutamine synthetase. Glia 3 (1990) 385-392. Mission, J.P., Edwards, M.A., Yamamoto, M. and Caviness Jr., V.S., Mitotic cycling of radial glial cells of the fetal murine cerebral wall. A combined autoradiographic and immunohistochemical study, Det,. Brain Res., 38 (1988) 183-190. Mission, J.P., Edwards, M.A., Yamamoto, M. and Caviness Jr., V.S., Identification of radial gila cells within the developing murine central nervous system. Studies based upon a new immunohistochemical marker, Det'. Brain Res., 44 (1988) 95-108. Miyake, T., Fukuyama, R. and Fujita, S., Differentiation of martrix cells and its histochemical aspects in early neurogenesis, Acta Histochem. Cytochem., 18 (1985) 77-83. Norenberg, M.D., The distribution of glutamine synthetase in the rat central nervous system, Z Histochern. Cytochem., 27 (1979) 756-762. Norenberg, M.D., Fine structural localization of glutamine synthetase in astrocytes of rat brain. Brain Res., 161 (1979) 303-310. Oda, M., Ikeda, K. and Yamamoto, T., Immunohistochemistry of glutamine synthetase of the mouse and human brain, Neuropathol (Tokyo), 4 (1983) 19-30. Patel, A.J.,Hunt, A. and Tahournin, C.S.M., Regional development of glutamine synthetase activity in the rat brain and its association with the differentiation of astrocytes, Det,. Brain Res., 8 (1983) 31-37. Raft, M.C., Glial cell diversification in the rat optic nerve, Science, 243 (1989) 1450-1455. Rakic, P., Neuronal-glial interaction during brain development, Trends Neurosci., 4 (1988) 184-187. Rakic, P., Specification of cerebral cortical areas, Science, 241 (1988) 170-176. Tardy, M., Rolland, B., Fages, C. and Caldani, M., Astroglial cells. Glycocorticoid target cells in the brain, Clin. Neuropharrnacol., 7 (1984) 296-302.
9
BRESD 51578
Immunohistochemical study of glutamine synthetase expression in early glial development Jiro Akimoto
a, Hiroshi Itoh a, Tetsuro Miwa a and Kazuhiko Ikeda
~
Department of Neurosurgery, Tokyo Medical College, Tokyo (Japan) and b Department of Ultrastructure and lmmunohistochemistry, Tokyo Institute of Psychiatry, Tokyo (Japan)
(Accepted 22 September 1992)
Key words: Development; Gliogenesis; Radial gila; Glial fibrillary acidic protein; Glutamine synthetase; Immunohistochemistry
Early glial development in the brains of rat embryos was immunohistochemically studied using antibotides to two kinds of glial-specific proteins, glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP). The results showed that GS was first expressed in the ependymal lining of the ventral side of the neural tube in the cervical segment on Embryonic Day 14 and then in the radial glia radiating from the neural tube on Embyronic Day 16. Subsequently, GS-positive glia cells increased in number with time. On the other hand, GFAP was first expressed in the fimbria hippocampi and the pial surface on Embryonic Day 18, but no marked evidence of GFAP expression was noted in the GS-positive radial glia. These results suggested that a specific protein is expressed at each stage of early glial development. Since the radial glia was positive for GS, it was thought that GS-mediated neuronal-glial interactions may play an important role in the development of the central nervous system.
INTRODUCTION M u c h of the d e v e l o p m e n t of the central nervous system (CNS) has r e m a i n e d obscure. A l t h o u g h it is a well-known fact that n e u r o n s and glial cells develop and differentiate f r o m the ectoderm-derived neural tube f o r m e d in a very early developmental stage, great controversies have arisen as to what developmental course n e u r o n s and glial cells take to f o r m the very complicated structure of the CNS, and what mechanism is involved in the control over interactions between these cells. T h e r e have b e e n two widely a c c e p t e d theories as to developmental processes, but no conclusion has yet been drawn. T h e two theories are: the monistic theory p r o p o s e d by Fujita et al., 1°'24 and the dualistic theories p r o p o s e d first by His t3 and recently by Rakic 6'15'3° and coworkers. According to the f o r m e r theory, the neural tube is comprised of uniform epithelial matrix cells. As matrix cells repeat u p - a n d - d o w n movements, the n e u r o n s first develop from these cells, and the glial cells develop
subsequently. A c c o r d i n g to the second theory, the neurons and glial cells, respectively develop from different stem cells in the neural tube. A n increasing n u m b e r of reports supporting the latter theory have been published over the years. As stated by Rakic et al. 6'15'3°, glial cells already exist w h e n n e u r o n s begin to migrate, and they regulate n e u r o n a l differentiation by serving as a guide for migration ~8'3~. These glial cells are precisely the radial glia designated by Cajal. 4. In recent years, there have b e e n m a n y reports aimed at determining the presence or absence of these radial glia cells. As a result, the existence of radial glia has gradually b e c o m e evident t h r o u g h in vitro 8 and electron microscopic studies 7. T h e results of these studies suggest that the n e u r o n migrates by traveling on rails (processes) of the radial glia ~l, and that n e u r o n a l differentiation and cell division are controlled by signal contact between n e u r o n s and glial cells 8. With this in mind, we immunohistochemicaUy investigated radial glia by using antibodies to astrocytic markers, i.e. glial fibrillary acidic protein ( G F A P ) and glutamine synthetase (GS).
Correspondence: J. Akimoto, 6-7-1 Nishishinjyuku, Shinjyuku-ku, Tokyo 160, Japan. Fax: (81) (3) 3340-4285.
10 MATERIALS AND METHODS
Embryonic Day 14
Pregnant Crj/CD rats (Japan Charles River Co., Tokyo, Japan) were used in the present study. The day of mating was counted as Day 0. Pregnant rats were anesthetized with ether and embryos were obtained at Days 10, 12, 14, 16, 18 and 20. Each embryo was immediately decapitated and head portion was fixed in a 70% ethanol solution at 4°C for 7 days. It was embedded in paraffin and frontal sections were cut at 5 txm thickness. Sections were stained with hematoxylin and eosin. For immunohistochemistry, deparaffinized sections were first treated with 0.1% H20 2 in methanol to abolish endogeneous peroxidase activity, and they were washed in phosphate-buffered saline (PBS). The sections were then incubated with rabbit polyclonal antibody raised against mouse brain G S 27 (kindly provided by M. Oda, Tokyo Metropolitan Neurological Hospital), diluted 1:400, or mouse monoclonal antibody to GFAP (DAKO, Denmark), diluted 1 :400 at room temperature for 3 h. After washing in PBS, they were incubated with biotinylated anti-rabbit lgG antibody or anti-mouse IgG antibody (Vector, USA), washed in PBS and reacted with peroxidase-conjugated streptavidin (Zymed, USA). Reaction product was visualized by diaminobenizidine and hydrogen peroxide. The sections were counterstained with hematoxylin. Immunostaining omitting incubation with primary antibody served as control.
and a structure t h o u g h t to be a m a n t l e or marginal
T h e n e u r a l tube markedly increased in thickness,
RESULTS
layer a p p e a r e d . T h e m a n t l e layer was comprised of spindle cells, which had relatively sparse c h r o m a t i n s a n d spread irregularly. GS i m m u n o r e a c t i v i t y a p p e a r e d in some of the epithelial cells of the ventral p o r t i o n of the cervical s e g m e n t in a g r a n u l a r form and in cellular processes e x t e n d i n g in a fringe-like p a t t e r n in the marginal layer. GS-positive fibers in the m a r g i n a l layer were swollen, a n d the pial surface was lined with GS-positive granules (Fig. la,b). G F A P i m m u n o r e a c t i v i t y was not observed at this stage.
Embryonic Day 16 T h e e p i t h e l i u m of the n e u r a l tube decreased in thickness, while the m a n t l e layer was thickened. Ceils c o m p o s i n g the m a n t l e layer had a r o u n d polymorphic n u c l e u s with sparse c h r o m a t i n a n d very few or n o
m a n t l e or m a r g i n a l layer was observed. No i m m u n o r e activity for G F A P a n d G S was observed at this stage
vesicles. T h e s e cells spread w i t h o u t any regular arrangement. GS-positive ceils i n c r e a s e d in n u m b e r in the epithelial cells of the v e n t r a l half of the n e u r a l tube (Fig. 2a). In the m a n t l e layer, GS-positive cells were scattered, showing radial migration. A close inspection revealed that these cells had s p i n d l e - t o - p o l y m o r p h i c nuclei, a n d
(data n o t shown).
GS-positive processes could be observed in some cells.
Embryonic Day 12 Y o u n g cells c o m p o s i n g the n e u r a l tube showed a p s e u d o s t r a t i f i e d a r r a n g e m e n t , a n d the nuclei facing the tubal cavity showed m a r k e d mitosis. No a p p a r e n t
Fig. 1. Immuno-staining for GS of rat embryo (E 14): a, cervical portion, x68; b; × 170. Counter-stained with hematoxylin. GS-positive cells (arrow) are seen in the epithelial cells at the ventral portion of neural tube. GS-positive fibers (arrowheads) are also seen both in ventral mantle layer and marginal layer.
11
Fig. 2. Immuno-staining for GS of rat embryo (E 16): a and b, cervical portion, x 170, c and d, p o n s , × 340. Counter stained with hematoxylin. GS-positive cells (arrow) are increased in number in the epithelial cell layer. In addition, scattered positive cells are seen in the ventral mantle layer, showing their migration from the epithelial layer (a and c). Radial fibers (arrowheads) are conspicuous in the mantle and marginal layers (b and d).
12
Fig. 3. Immuno-staining for GS and GFAP of rat embryo (E 18): a, cervical portion, GS, x 170; b and c, pons, GS, )<340; d, cervical portion, GFAP, x340. Counter-stained with hematoxylin. Migration of the GS-positive cells (arrow) together with radial fibers (arrowheads) are pronounced in the mantle layer (a, b and c). GFAP-positive cells and processes (*) are firstly seen at this stage in the marginal layer and pial surface (d).
13 In the marginal layer, many GS-positive fibers extending in a fringe-like pattern were observed, and the pial surface was covered with a layer of GS-positive lining (Fig. 2b). In the more rostral region between the mesencephalon and the pons, many GS-positive cells radiated from the epithelium of the neural tube and spread over the mantle layer. GS-positive fibers running radially between intercellular spaces were more conspicuous at this stage (Fig. 2c,d). G F A P immunoreactivity was still not seen at this stage.
Embryonic Day 18 GS immunoreactivity was seen in some epithelial cells of the neural tube, which practically formed a single layer. GS immunoreactivity was also detected in the cells radially spreading in the mantle layer as well as in bipolar processes of these cells. Distal processes extended to the marginal layer and spread in the dendroid form at the pial surface, forming a GS-positive lining (Fig. 3a). In the pons, GS immunoreactivity was observed in the subependymal cells, and also in cell processes, which resembled a fringe-like pattern and seemed to be weaving in between intercellular spaces (Fig. 3b,c). G F A P immunoreactivity was first observed at this stage, showing a fringe-like pattern of cell processes in the marginal layer and pial surface in the cervical segment. G F A P immunoreactivity was also evident in the fimbria hippocampi and the pial surface in the same region (Fig. 3d).
nature of radial glia. G F A P immunoreactivity was first observed in cells and their fibers on rat Embryonic Day 18, which is consistent with previous reports 3'~4. GS was confirmed by Norenberg et al. 2'25'26'27 to be a protein existing only in astrocytes, at least in the brain and has been used as a marker protein for astrocytes. However, few studies have been performed with respect to the, GS expression in the course of gliogenesis tg. In the present study, GS immunoreactivity was first observed in cells composing the epithelium of the neural tube in the cervical segment on Embryonic Day 14, and then in fibers which radiated from the neural tube. The positive cells and fibers increased in number by time. Based on the time of the appearance of these cells and their morphology, the GS-positive cells were considered to be radial glia. The data of our immunohistochemical study of rat embryo accord well with those of previous immunohistochemical studies of chick optic tectum ~6 and lizard embryo ~4, in that the appearance of GS immunoreactivity preceeds that of G F A P immunoreactivity in radial glia. In the mouse embryonic period 1'5, GS enzymic activity became detectable on Embryonic Day 16 and increased by time 2s'3:. Studies by in situ hybridization 2°'21, showing the appearance of GS m R N A on Embryonic Day 14, support the immunohistochemical data. The main functions of GS include inactivation of the excitatory neurotransmitter glutamic acid and detoxication of ammonium 17"25'32. It may be possible that such functions are required in the microenvironment of the developing nervous tissue, where the radial glia interact with migrating neurons along their fibers.
DISCUSSION REFERENCES
It is known that long fibers radiating from the neural tube exist in higher vertebrates at the time of development of the CNS. Golgi's stain can reveal the fiber containing cells known as radial glia, epithelial cells or spongioblasts, which are thought to appear temporarily during the development of the C N S 7. Rakic et al. 3°'3~ stated that this cell type, the radial glia designated by Cajal 4, play an essential role in the development of the CNS as a guide for the migration of nerve cells. Recent s t u d i e s 22'23"29'31, revealed that the radial fibers appear at an earlier stage of development than previously considered. Electron microscopic studies 7'~ have shown that radial glia share features with astrocytes, and that nerve cells migrate along its processes ~'12'~s, thus providing evidence of n e u r o n a l glial interaction in the nervous tissue development. The present immunohistochemical study was carried out using the astrocyte-specific markers G F A P and GS as indicators in order to gain more information on the
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