Neuroscience Research, 15 (1992) 221-223 © 1992 Elsevier Science Publishers Ireland, Ltd. All rights reserved 0168-0102/92/$05.00
221
NEURES 00579
Rapid communication
Gradient expression of neural cell adhesion molecule (NCAM) in the pontine migratory stream of fetal rats Katsuhiko Ono
a,
H i r o a k i A s o u b, M a s a o Y a m a d a c and A k i r a T o k u n a g a a
a Third Department of Anatomy and c Department of ~rology, Okayama University Medical School, Okayama 700, Japan, b Department of Physiology, School of Medicine, Keio University, Shinjuku, 160, Japan (Received 9 July 1992; accepted 11 August 1992)
Key words: NCAM; Monoclonal antibody; Immunoprecipitation; Pontine migratory stream; Rat
Summary Immunohistochemical localization of a neural cell adhesion molecule (NCAM) was examined in the subpial pontine migratory stream of the fetal rat with a monoclonal antibody specific for the rat NCAM, MAb-AFll. Although the pontine cell strand showed weaker expression of NCAM than the parenchymal areas of the brainstem, within the cell strand, NCAM-immunoreactivity was somewhat greater in the anterior part of the stream near the neurons' destination than the caudal part. The weak NCAM gradient in the pontine migratory stream may contribute to formation of the pontine cell strand and the basal pontine gray.
During formation of the pontine nuclei of vertebrates, the neurons are generated in the ventricular neuroepithelium of the fourth ventricle and then migrate to their destinations through the subpial part of the ponto-medullary regions (Essick, 1912; Altman and Bayer, 1978, 1987; Tan and Le Douarin, 1991). The immature pontine cells relocate along neuronal guides (Rakic, 1990; Ono and Kawamura, 1990), and thus are called neurophilic cells. In contrast to young neurons migrating along glial fibers, which have been studied extensively in the neonatal cerebellum (Fishell and Hatten, 1991), the mechanisms of neurophilic cell migration remain unclear. In the present study, as the first step in elucidating the molecular mechanisms of the neurophilic cell relocation, we examined immunohistochemical localization of a neural cell adhesion molecule (NCAM) in the pontine migratory stream.
Correspondence to: Katsuhiko Ono, PhD, c / o Dr. Urs Rutishauser, Department of Genetics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA. Fax: (216) 368 3182.
Wistar rats were mated overnight, and the day when a vaginal plug was observed was regarded as embryonic day 0 (E0). E15-E20 stage rat fetuses were taken from the uterus under deep ether anesthesia and perfused through the left ventricle with 4% paraformaldehyde in 0.1 M phosphate buffer (PB). The brains were immersed in PB containing 20% sucrose, cut sagittally or coronally with a cryostat, and thaw-mounted onto gelatin-coated glass slides. Sections were subsequently incubated with anti-NCAM monoclonal antibody (MAb-AFll, see below) for 3 h and then with FITClabeled anti-mouse IgG (CappeD, diluted 1 : 100, for 30 min at 37°C. Control sections were treated without MAb-AFll. Anti-NCAM monoclonal antibody (MAbA F l l ) was generated by immunizing mice with cell dissociate of the fetal rat brainstem. Details of cell fusion, screening and cloning of hybridomas were described previously (Kohler and Milstein, 1975; Ono et al., 1989). Western blot analysis was carried out as described previously (Yuasa et al., 1991), and MAbA F l l recognized 200-250 kDa polypeptides from fetal rat brain, and 180, 140, and 120 kDa polypeptides from
222
a
a
b
b
140120-
1
2
Fig. 1. Western blot analysis of the polypeptides from [etal rat cerebellum and brainstem (a), adult rat cerebellum (b) and adult mouse cerebellum (c). Note that MAb-AF11 does not recognize mouse brain polypeptide. Fig. 2. Western blot analysis of adult rat cerebellar polypeptides immunoprecipitated with M A b - A F l l . The polypeptides on the membrane are immunostained with either M A b - A F l l (a) or anti-NCAM polyclonal antibody (b). Figs. 3-5. M A b - A F l l immunohistochemistry with the E16 rat brainstem, arranged caudal to rostral sequence. Coronal sections. The pontine migratory stream is indicated by arrows. Note that NCAM-immunoreactivity is somewhat greater in the rostral part (Fig. 5) than in the caudal part (Fig. 3). NE, neuroepithelium. Figs. 6 and 7. Sagittal sections through the pontine migratory stream (PS) of the El9 rat. Arrows indicate direction of the stream. The basal pons (Pn in Fig. 7), the destination of the migratory pontine cells, shows stronger NCAM-immunoreactivity than the caudal part near the fourth ventricle. Bars = 100/zm.
223 adult rat cerebellum, showing embryonic-to-adult conversion (Fig. 1). Antigen for the MAb was elucidated as NCAM by the immunoprecipitation experiment (Fig. 2); anti-NCAM polyclonal antibody (Itoh et al., 1989) recognized the adult rat cerebellar polypeptides immunoprecipitated with MAb-AFll. In the ponto-medullary region of El6, the pontine migratory stream was extended from the ventricular neuroepithelium to the pontine flexure. Immunoreactivity of NCAM was weak in the pontine stream, whereas the parenchymal areas of the brainstem showed strong expression (Figs. 3-5). The neuroepithelial layer was faintly labeled with MAb-AFll. Within the pontine cell strand, NCAM immunoreactivity was somewhat greater in the rostral part near and at the basal pons than in the caudal part. The migratory stream at El9 also showed gradient localization of NCAM, as observe at El6 (Figs. 6 and 7). Control sections without MAb-AFll treatment did not show specific labeling (not shown). The present study clearly demonstrated that NCAM-immunoreactivity was weaker in the subpial migratory stream than in the parenchyma of the brainstem. In addition, the medullary migratory stream of the brainstem and the external granular layer of the cerebellum at fetal stages, both of which were composed of neurophilic migratory cells (Hynes et al., 1986; Ono and Kawamura, 1989; Tan and Le Douarin, 1991), were also weakly immunoreactive to NCAM (not shown). Namely, the specific subpial terrain with relocating neurophilic ceils showed weak NCAM immunoreactivity. It is probable that weak NCAM expression in the subpial cell makes them easy to segregate from the parenchymal areas. NCAM-immunoreactivity in the pontine migratory stream was somewhat greater in the lower stream, near the destination, than in the upper stream, near the sites of the neurons' origin, which indicates a weak gradient of NCAM along the stream. NCAM in the fetal stage is highly sialylated (Fig. 1). Recently Rutishauser demonstrated that polysialic acid (PSA) of NCAM mediates cell-cell interactions during the formation of the nervous system (Rutishauser, 1989). Although expression of NCAM seemed to be very weak in the pontine stream, the gradient of NCAM with PSA may play some functional roles in the formation of the basal pontine nuclei.
Acknowledgment We thank Ms. Kazuko Mori for her expert technical help. This study was supported by a Grant-in-Aid for Scientific Research provided by the Ministry of Education, Science and Culture, Japan, and also by a Narishige Neuroscience Grant, Tokyo.
References Altman, J. and Bayer, S.A. (1978) Prenatal development of the cerebellar system in the rat. II. Cytogenesis and histogenesis of the inferior olive, pontine gray and precerebellar reticular nuclei. J. Comp. Neurol., 179: 49-76. Altman, J. and Bayer, S.A. (1987) Development of the precerebellar nuclei in the rat: VI. The anterior precerebellar extramural migratory stream and the nucleus reticularis tegmenti pontis and the basal pontine gray. J. Comp. Neurol., 257: 529-552. Essick, C.R. (1912) The development of the nuclei pontis and the nucleus arcuatus in man. Am. J. Anat., 13: 25-54. Fishell, G. and Hatten, M.E. (1991) Astrotactin provides a receptor system for CNS neuronal migration. Development, 113: 755-765. Hynes, R.O., Patel, R. and Miller, R.H. (1986) Migration of neuroblast along preexisting axonal tracts during prenatal cerebellar development. J. Neurosci., 6: 867-876. Itoh, K., Asou, H., Ikarashi, Y. and Maruyama, Y. (1989) Morphological changes and neural cell adhesion molecule expression in mouse cerebrum primary cultures following long-term exposure to phorbol ester. Neurosci. Res., 6: 350-357. Kohler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 256: 495-497. Ono, K. and Kawamura, K. (1989) Migration of immature neurons along tangentially oriented fibers in the subpial part of the fetal mouse medulla oblongata. Exp. Brain Res., 78: 290-300. Ono, K. and Kawamura, K. (1990) Mode of neuronal migration of the pontine stream in fetal mice. Anat. Embryol., 182: 11-19. Ono, K., Yanagihara, M., Mizukawa, K., Yuasa, S. and Kawamura, K. (1989) Monoclonal antibody that bind to both the prenatal and postnatal astroglia in rodent cerebellum. Dev. Brain Res., 50: 154-159. Rakic, P. (1990) Principles of neural cell migration. Experientia, 46: 882-891. Rutishauser, U. (1989) Polysialic acid as a regulator of cell interactions. In R.U. Margolis and R.K. Margolis (Eds.), Neurobiology of Glycoconjugates, Plenum Press, New York and London, pp. 367-382. Tan, K. and Le Douarin, M. (1991) Development of the nuclei and cell migration in the medulla oblongata. Application of the quail-chick chimera system. Anat. Embryol., 183: 321-343. Yuasa, S., Kawamura, K., Ono, K., Yamakuni, T. and Takahashi, Y. (1991) Development and migration of Purkinje cells in the mouse cerebellar primordium. Anat. Embryol., 184: 195-212.
221
NEURES 00579
Rapid communication
Gradient expression of neural cell adhesion molecule (NCAM) in the pontine migratory stream of fetal rats Katsuhiko Ono
a,
H i r o a k i A s o u b, M a s a o Y a m a d a c and A k i r a T o k u n a g a a
a Third Department of Anatomy and c Department of ~rology, Okayama University Medical School, Okayama 700, Japan, b Department of Physiology, School of Medicine, Keio University, Shinjuku, 160, Japan (Received 9 July 1992; accepted 11 August 1992)
Key words: NCAM; Monoclonal antibody; Immunoprecipitation; Pontine migratory stream; Rat
Summary Immunohistochemical localization of a neural cell adhesion molecule (NCAM) was examined in the subpial pontine migratory stream of the fetal rat with a monoclonal antibody specific for the rat NCAM, MAb-AFll. Although the pontine cell strand showed weaker expression of NCAM than the parenchymal areas of the brainstem, within the cell strand, NCAM-immunoreactivity was somewhat greater in the anterior part of the stream near the neurons' destination than the caudal part. The weak NCAM gradient in the pontine migratory stream may contribute to formation of the pontine cell strand and the basal pontine gray.
During formation of the pontine nuclei of vertebrates, the neurons are generated in the ventricular neuroepithelium of the fourth ventricle and then migrate to their destinations through the subpial part of the ponto-medullary regions (Essick, 1912; Altman and Bayer, 1978, 1987; Tan and Le Douarin, 1991). The immature pontine cells relocate along neuronal guides (Rakic, 1990; Ono and Kawamura, 1990), and thus are called neurophilic cells. In contrast to young neurons migrating along glial fibers, which have been studied extensively in the neonatal cerebellum (Fishell and Hatten, 1991), the mechanisms of neurophilic cell migration remain unclear. In the present study, as the first step in elucidating the molecular mechanisms of the neurophilic cell relocation, we examined immunohistochemical localization of a neural cell adhesion molecule (NCAM) in the pontine migratory stream.
Correspondence to: Katsuhiko Ono, PhD, c / o Dr. Urs Rutishauser, Department of Genetics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA. Fax: (216) 368 3182.
Wistar rats were mated overnight, and the day when a vaginal plug was observed was regarded as embryonic day 0 (E0). E15-E20 stage rat fetuses were taken from the uterus under deep ether anesthesia and perfused through the left ventricle with 4% paraformaldehyde in 0.1 M phosphate buffer (PB). The brains were immersed in PB containing 20% sucrose, cut sagittally or coronally with a cryostat, and thaw-mounted onto gelatin-coated glass slides. Sections were subsequently incubated with anti-NCAM monoclonal antibody (MAb-AFll, see below) for 3 h and then with FITClabeled anti-mouse IgG (CappeD, diluted 1 : 100, for 30 min at 37°C. Control sections were treated without MAb-AFll. Anti-NCAM monoclonal antibody (MAbA F l l ) was generated by immunizing mice with cell dissociate of the fetal rat brainstem. Details of cell fusion, screening and cloning of hybridomas were described previously (Kohler and Milstein, 1975; Ono et al., 1989). Western blot analysis was carried out as described previously (Yuasa et al., 1991), and MAbA F l l recognized 200-250 kDa polypeptides from fetal rat brain, and 180, 140, and 120 kDa polypeptides from
222
a
a
b
b
140120-
1
2
Fig. 1. Western blot analysis of the polypeptides from [etal rat cerebellum and brainstem (a), adult rat cerebellum (b) and adult mouse cerebellum (c). Note that MAb-AF11 does not recognize mouse brain polypeptide. Fig. 2. Western blot analysis of adult rat cerebellar polypeptides immunoprecipitated with M A b - A F l l . The polypeptides on the membrane are immunostained with either M A b - A F l l (a) or anti-NCAM polyclonal antibody (b). Figs. 3-5. M A b - A F l l immunohistochemistry with the E16 rat brainstem, arranged caudal to rostral sequence. Coronal sections. The pontine migratory stream is indicated by arrows. Note that NCAM-immunoreactivity is somewhat greater in the rostral part (Fig. 5) than in the caudal part (Fig. 3). NE, neuroepithelium. Figs. 6 and 7. Sagittal sections through the pontine migratory stream (PS) of the El9 rat. Arrows indicate direction of the stream. The basal pons (Pn in Fig. 7), the destination of the migratory pontine cells, shows stronger NCAM-immunoreactivity than the caudal part near the fourth ventricle. Bars = 100/zm.
223 adult rat cerebellum, showing embryonic-to-adult conversion (Fig. 1). Antigen for the MAb was elucidated as NCAM by the immunoprecipitation experiment (Fig. 2); anti-NCAM polyclonal antibody (Itoh et al., 1989) recognized the adult rat cerebellar polypeptides immunoprecipitated with MAb-AFll. In the ponto-medullary region of El6, the pontine migratory stream was extended from the ventricular neuroepithelium to the pontine flexure. Immunoreactivity of NCAM was weak in the pontine stream, whereas the parenchymal areas of the brainstem showed strong expression (Figs. 3-5). The neuroepithelial layer was faintly labeled with MAb-AFll. Within the pontine cell strand, NCAM immunoreactivity was somewhat greater in the rostral part near and at the basal pons than in the caudal part. The migratory stream at El9 also showed gradient localization of NCAM, as observe at El6 (Figs. 6 and 7). Control sections without MAb-AFll treatment did not show specific labeling (not shown). The present study clearly demonstrated that NCAM-immunoreactivity was weaker in the subpial migratory stream than in the parenchyma of the brainstem. In addition, the medullary migratory stream of the brainstem and the external granular layer of the cerebellum at fetal stages, both of which were composed of neurophilic migratory cells (Hynes et al., 1986; Ono and Kawamura, 1989; Tan and Le Douarin, 1991), were also weakly immunoreactive to NCAM (not shown). Namely, the specific subpial terrain with relocating neurophilic ceils showed weak NCAM immunoreactivity. It is probable that weak NCAM expression in the subpial cell makes them easy to segregate from the parenchymal areas. NCAM-immunoreactivity in the pontine migratory stream was somewhat greater in the lower stream, near the destination, than in the upper stream, near the sites of the neurons' origin, which indicates a weak gradient of NCAM along the stream. NCAM in the fetal stage is highly sialylated (Fig. 1). Recently Rutishauser demonstrated that polysialic acid (PSA) of NCAM mediates cell-cell interactions during the formation of the nervous system (Rutishauser, 1989). Although expression of NCAM seemed to be very weak in the pontine stream, the gradient of NCAM with PSA may play some functional roles in the formation of the basal pontine nuclei.
Acknowledgment We thank Ms. Kazuko Mori for her expert technical help. This study was supported by a Grant-in-Aid for Scientific Research provided by the Ministry of Education, Science and Culture, Japan, and also by a Narishige Neuroscience Grant, Tokyo.
References Altman, J. and Bayer, S.A. (1978) Prenatal development of the cerebellar system in the rat. II. Cytogenesis and histogenesis of the inferior olive, pontine gray and precerebellar reticular nuclei. J. Comp. Neurol., 179: 49-76. Altman, J. and Bayer, S.A. (1987) Development of the precerebellar nuclei in the rat: VI. The anterior precerebellar extramural migratory stream and the nucleus reticularis tegmenti pontis and the basal pontine gray. J. Comp. Neurol., 257: 529-552. Essick, C.R. (1912) The development of the nuclei pontis and the nucleus arcuatus in man. Am. J. Anat., 13: 25-54. Fishell, G. and Hatten, M.E. (1991) Astrotactin provides a receptor system for CNS neuronal migration. Development, 113: 755-765. Hynes, R.O., Patel, R. and Miller, R.H. (1986) Migration of neuroblast along preexisting axonal tracts during prenatal cerebellar development. J. Neurosci., 6: 867-876. Itoh, K., Asou, H., Ikarashi, Y. and Maruyama, Y. (1989) Morphological changes and neural cell adhesion molecule expression in mouse cerebrum primary cultures following long-term exposure to phorbol ester. Neurosci. Res., 6: 350-357. Kohler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 256: 495-497. Ono, K. and Kawamura, K. (1989) Migration of immature neurons along tangentially oriented fibers in the subpial part of the fetal mouse medulla oblongata. Exp. Brain Res., 78: 290-300. Ono, K. and Kawamura, K. (1990) Mode of neuronal migration of the pontine stream in fetal mice. Anat. Embryol., 182: 11-19. Ono, K., Yanagihara, M., Mizukawa, K., Yuasa, S. and Kawamura, K. (1989) Monoclonal antibody that bind to both the prenatal and postnatal astroglia in rodent cerebellum. Dev. Brain Res., 50: 154-159. Rakic, P. (1990) Principles of neural cell migration. Experientia, 46: 882-891. Rutishauser, U. (1989) Polysialic acid as a regulator of cell interactions. In R.U. Margolis and R.K. Margolis (Eds.), Neurobiology of Glycoconjugates, Plenum Press, New York and London, pp. 367-382. Tan, K. and Le Douarin, M. (1991) Development of the nuclei and cell migration in the medulla oblongata. Application of the quail-chick chimera system. Anat. Embryol., 183: 321-343. Yuasa, S., Kawamura, K., Ono, K., Yamakuni, T. and Takahashi, Y. (1991) Development and migration of Purkinje cells in the mouse cerebellar primordium. Anat. Embryol., 184: 195-212.
