- Email: [email protected]
Satellite cells in developing spinal ganglia. An immunohistochemical study
hr. 1. Dcvl. Neuroscience. Vol.7.No.3, pp. 275-279.1989. Printed in Great Britain.
m6-574w89 $03.00+0.00 Pergamon Press plc. ISDN
SATELLITE CELLS IN DEVELOPING SPINAL GANGLIA. AN IMMUNOHISTOCHEMICAL STUDY LIBERO
LAURIOLA,*$FABRIZIOMICHEII-I,?ANTONELLACOLI,* GIULIO BIGOTI* and DOMENICOCOCCHIAS
*Department of Pathology, Universita’ Cattolica S. Cuore, Large F. Vito 10016t3 Rome, Italy, tDepartment of Anatomy, Universita’ di Bari, Italy. $Department of Public Health and Cell Biology, Universita’ “Tor Vergata”, Rome, Italy (Received
20 September
1988; in revised form 30 November
1988; accepted 6 December
1988)
Abatrae-The present immunohistochemical study investigates the presence and distribution of SIOOcontaining glial cells in the early stages of development in human spinal ganglia. From the earliest ages investigated immunoreactive cells could be detected in a continuous layer at the periphery as well as inside gangfionic rudiments in close relationship with neural elements, both at the light and ultrastructural levels. The possibility that these glial cells, exhibiting such a distinctive distribution, play a modulatory role on microenvironmental influences during maturation could be taken into account. Neither glial fibrillary acidic protein nor myelin basic protein could be detected at the ages investigated. Key words: Satellite cells, Spinal ganglia, Human development, S-100 protein, immunohistochemistry.
It is generally agreed that during the development of spinal ganglia neural crest cells differentiate along two cell lines, some becoming ganglionic neurons, others satellite cells.” Available data essentially indicate that each glial cell is related to several neural cells when satellite cells are first detectable in developing ganglia whereas in adult animals each neuronal body is usually enveloped by an individual satellite cell sheath. 5*9In order to obtain more detailed information in animal and human neurodevelopmental studies, one of the problems is to find molecular markers for the early identification of cell types which cannot be accurately identified by morphological criteria alone. In particular, a potentially useful marker in the study of the glial lineage is the S-100 antigen, originally isolated from the nervous system,* where it is located primarily in glial cells.14 The present immunohistochemical study investigates the presence and distribution of S-lOOcontaining glial cells in the early stages of development of spinal ganglia in the human embryo. EXPERIMENTAL
PROCEDURE
Ten human embryos obtained from abortions, ranging from the 7th to the 12th week of intrauterine life, were ,studied. An approximate estimate of the embryonic age by crown-rump length was performed according to Hamilton et al.’ All the embryos, Bouin-fixed, were obtained from public hospitals. For immunohistochemistry, sections (4 pm thick) were deparaffinized in xylol, treated with 0.3 hydrogen peroxide in methanol for 20 min to block endogenous peroxidase activity, and finally processed for the immunoperoxidase reaction using the unlabelled antibody PAP method.‘* Before the immunoreaction, some sections were treated with 0.1% trypsin (Difco Lab, Detroit, Michigan) in a 0.1 M CaC12 solution adjusted to pH 7.8 with NaOH. Rabbit antiserum to whole S-100, either obtained com,mercially (Dakopatts, Copenhagen, Denmark) or produced by us and characterized according to Zuckermann et al. I5 was used at dilutions varying from 1500 to 1:lOOO. In some experiments, an antiserum against the S-1OOb isotype (kindly provided by Dr K. Haglid, University of Goteborg) was used, at the same dilutions as the other anti-S-100 antisera. The specificity of the immunostaining was confirmed by replacement of the primary antiserum with non-immune rabbit serum or pre-absorption of anti-S-100 antiserum with purified antigen. Sections were also reacted with anti-glial fibrillary acidic protein (GFAP; 1:400) or anti-myelin basic protein (MBP; 1:400) antibody (both obtained from Dakopatts). The technical details of the immunostaining procedures have been reported elsewhere.‘j For electron microscopy, tissue fragments were cut on a Sorvall TC-2 sectioner and processed by the PAP method using an anti-S-100 antibody, as previously reported.‘The sections were postfixed in 1.5% osmium tetraoxide in 0.1 M phosphate buffer for 30 min, dehydrated in ethanol and 0 Author
to whom all correspondence should be addressed.
275
276
L. Lauriola et al.
propylene-oxide and embedded in Epon 812. Sections of SO-90 nm thickness were prepared using an LKB Ultratome III, lightly counterstained with lead citrate and observed with a Philips EM 400. RESULTS Paraffin sections from all embryos treated with anti-S-100 antiserum exhibited, from the earliest ages investigated, the presence of immunostained cells in spinal ganglia {Fig. 1). Immunolabelled cells constituted a continuous layer at the periphery of the ganglionic rudiment, isolating the neural elements from the thin fibroblastic layer of the developing meningeal envelope, In addition, numerous immunostained cells, dendritic in shape, were also detected inside the ganglion, among neuronal elements, becoming more numerous during development (Figs 2 and 3). Immunolabelled glial cell bodies and processes were often observed in close proximity to the walls of blood vessels present in the gan~ionic anlage (Figs 1 and 4). Immun~tained cells identi~able as precursors of Schwann cells were abundantly present along both the ventral and dorsal root and spinal nerve. The ultrastructural examination clearly confirmed the presence, at the periphery of the ganglionic rudiment, of a continuous layer of immunostained cells characterized by cytoplasmic processes filled with immunoreaction product in the matrix, where few organelles were also observed (Fig. 5) In addition, immunola~lled cells, dendritic in shape, with thin cytoplasmic processes not completely encircling individual neuronal cells, were also numerous inside the ganglion. Overlapping immunohist~hemical findings were observed using an anti~~m anti-S-lob isotype, which is known to be specifically restricted to glial cells in the human brain.2 On the other hand, treatment of sections with anti-GFAP did not show the presence of labelled cells, in accordance with previous findings3 Likewise, MBP immunoreactivity was never observed in the examined samples. Counte~tai~ing of sections with toluidine blue showed that neuronal cells were unlabelled by S-100 antiserum. No immunoreactivity was observed when the sections were treated with preimmune rabbit serum or with antiserum absorbed with the related antigen (not shown). DISCUSSION The present study investigates, by S-100 immunohistochemistry, the presence and distribution of satellite cells in human spinal ganglia during the early stages of development. The peculiar distribution of the S-loo-containing cells, constituting a continuous envelope around developing spinal ganglia, as also observed in migrating sympathetic nests,? seems noteworthy. Keeping in mind that differentiating events in the peripheral nervous system are possibly influenced by su~ounding embryonic tissues and extracellular matrix molecules, the possibility that these satellite cells, exhibiting such a distinctive distribution around ganglionic rudiments, play a modulatory role on microenvironmental influences should be taken into account. In this respect, it is suggestive that S-lOOl3 has been reported to have neurite extension factor activity’ and also to be released from glial cells, i3 at least in cell cultures from tissue of the central nervous system. In the light of these reports, the possibility that the S-100 in satellite cells acts in a paracrine fashion to stimulate neurite outgrowth during development of the nervous system could also be borne in mind. With regard to labelled cells inside developing human ganglia, the present data are in agreement with reports on other animal species, also indicating that at these stages of development satellite cells do not completely encircle individual neuronal cells, unlike the adult pattern, where each ganglion cell is completely enveloped by one or more satellite cells.” Glial fibrillary acidic protein appears to be undetectable in satellite celfs of sensitive peripheral ganglia at the early stages of human development, although some peripheral glial elements have been reported to contain this antigen in adult mammals. A delayed postnatal expression of GFAP in satellite cells or differences between species or in antigenicity between central and peripheral GFAP3 could explain this discrepancy. On the other hand, the absence of MBP immunoreactivity, as we have observed, is consistent with the later occurrence of myelination in the
Satellite cells in developing spinal ganglia
Figs 1-3. Paraffin sections of human embryo spinal ganglia treated with anti-S-100 antiserum. Sections were not counterstained. Fig. 1. x 90. Intensely immunostained cells are detectable both in the ganglion and in ganglion roots. Fig. 2. x 225. Fig. 3. x 340. Immunoreactivity is confined to glial cells located in a continuous layer at the periphery as well as inside the ganglion. Cells constituting the meningeal layer as well as neurons appear to be unstained. Note immunostained glial cells in close proximity to walls of the blood vessel (Fig. 2. *).
277
278
L. Lauriola et al.
Fig. 4. Paraffin section of human embryo spinai ganglia treated with anti-S-100 antiserum. The section was not counterstained. Note immunostained cells detectable in the gangiionic root (arrow). x 225. Fig. 5. Electron micrograph of human embryo spinal ganglion treated with anti-S-100 antiserum. Immunoreactivity is detectable in the cytoplasm of a satebite cell located at the periphery of the ganglionic rudiment. Original magnification x 4600.
Satellite cells in developing spinal ganglia
279
human peripheral nervous system (14th-18th week of intrauterine life).” In this respect, any possible relationship between S-100 expression and the appearance of MBP remains open to investigation. Acknowledgements-The assistance.
authors thank Mr P. Baldassarti for editorial assistance and Mr A. Rinelli for technical
REFERENCES I. Hamilton W. J., Boyd J. D. and Mossman H. W. (1962) Human Embryology, pp. 119-134. Williams and Wilkins, Baltimore. 2. Isobe T.. Takahashi K. and Okuyama T. (1984) S-100 protein is present in neurons of the central and peripheral nervous system. J. Neurochem. 43, 1494-1496. 3. Jessen K. K., Thorpe R. and Mirsky R. (1984) Molecular identity, distribution and heterogeneity of glial fibrillary acidic protein: an immunoblotting and immunohistochemical study of Schwann cells, enteric glia and astrocytes. 1. Neurocytol. 13, 187-200. 4. Kligman D. and Marshak D. R. (1985) Purification and characterization of a neurite extension factor from bovine brain. Proc. nam Acad. Sci., U.S.A. 82, 7136-7139. 5. Krajci D. (1975) Ontogenic development of the relation between neurons and satellite cells in spinal ganglia. Folia Morphol. (Praha) 21, 139-141. 6. Lauriola L., Maggiano N., Sentinelli S., Michetti F. and Cocchia D. (1985) Satellite cells in the normal human adrenal gland and in pheochromocytomas. An immunohistochemical study. Virchows Arch. (Cefl ParhoL) 49, 13-21. 7. Lauriola L., Sentinelli S., Maggiano N., Michetti F. and Cocchia D. (19%) Glial-like cells in sympathetic neural crest derivatives during human embryogenesis. Detection by S-100 immunohistochemistry. Devf &ah Res. 28,69-74. 8. Moore B. W. (l%5) A soluble protein characteristics of the nervous svstem. BioDhvs. Res. Commun. 19.739-744. 9. Pannese E. (1969) Electron microscopical study on the development of-satellite ceiliheath in spinal ganglia. J. camp. Neurol. 135, 381422. 10. Pannese E. (1981) The satellite cells of the sensory ganglia. In Advances in Anatomy and Embryo Cell Biology, Vol. 65, Springer-Verlag, Berlin. 11. Shaw-Dunn J. (1970) Developing myelin in human peripheral nerve. Scot. Med. 1. 15, 108-117. 12. Stemberger L. A., Hard Ph Jr, Cuculis J. J. and Meyer H. G. (1970) The unlabelled antibody enzyme method of immunochemistry. Preparation and properties of soluble antigen-antibody complex (horseradish neroxidaseantihorseradish peroxidase) and its use in-identification of spirochetes. J. Hi.s&hem.~ Cytochem. l&31$333. 13. Van Eldik L. J. and Zimmer D. B. (1987) Secretion of S-100 from rat C6 glioma cells. Brain Res. 436,367-370. 14. Zonuely-Neurath C. E. and Walker W. A. (1980) Nervous system-specific proteins: 14-3-2 protein, neuron-specific enolase, and S-100 protein. In Proreins ofrhe Nervous Sysrem (eds Bradshow R. A. and Schneider D. M.), pp. l-57. Raven Press, New York. IS. Zuckerman J. E., Herschman H. R. and Levine L. (1970) Appearance of a brain specific antigen (the S-100 protein) during human foetal development. /. Neurochem. 17.247-251.
m6-574w89 $03.00+0.00 Pergamon Press plc. ISDN
SATELLITE CELLS IN DEVELOPING SPINAL GANGLIA. AN IMMUNOHISTOCHEMICAL STUDY LIBERO
LAURIOLA,*$FABRIZIOMICHEII-I,?ANTONELLACOLI,* GIULIO BIGOTI* and DOMENICOCOCCHIAS
*Department of Pathology, Universita’ Cattolica S. Cuore, Large F. Vito 10016t3 Rome, Italy, tDepartment of Anatomy, Universita’ di Bari, Italy. $Department of Public Health and Cell Biology, Universita’ “Tor Vergata”, Rome, Italy (Received
20 September
1988; in revised form 30 November
1988; accepted 6 December
1988)
Abatrae-The present immunohistochemical study investigates the presence and distribution of SIOOcontaining glial cells in the early stages of development in human spinal ganglia. From the earliest ages investigated immunoreactive cells could be detected in a continuous layer at the periphery as well as inside gangfionic rudiments in close relationship with neural elements, both at the light and ultrastructural levels. The possibility that these glial cells, exhibiting such a distinctive distribution, play a modulatory role on microenvironmental influences during maturation could be taken into account. Neither glial fibrillary acidic protein nor myelin basic protein could be detected at the ages investigated. Key words: Satellite cells, Spinal ganglia, Human development, S-100 protein, immunohistochemistry.
It is generally agreed that during the development of spinal ganglia neural crest cells differentiate along two cell lines, some becoming ganglionic neurons, others satellite cells.” Available data essentially indicate that each glial cell is related to several neural cells when satellite cells are first detectable in developing ganglia whereas in adult animals each neuronal body is usually enveloped by an individual satellite cell sheath. 5*9In order to obtain more detailed information in animal and human neurodevelopmental studies, one of the problems is to find molecular markers for the early identification of cell types which cannot be accurately identified by morphological criteria alone. In particular, a potentially useful marker in the study of the glial lineage is the S-100 antigen, originally isolated from the nervous system,* where it is located primarily in glial cells.14 The present immunohistochemical study investigates the presence and distribution of S-lOOcontaining glial cells in the early stages of development of spinal ganglia in the human embryo. EXPERIMENTAL
PROCEDURE
Ten human embryos obtained from abortions, ranging from the 7th to the 12th week of intrauterine life, were ,studied. An approximate estimate of the embryonic age by crown-rump length was performed according to Hamilton et al.’ All the embryos, Bouin-fixed, were obtained from public hospitals. For immunohistochemistry, sections (4 pm thick) were deparaffinized in xylol, treated with 0.3 hydrogen peroxide in methanol for 20 min to block endogenous peroxidase activity, and finally processed for the immunoperoxidase reaction using the unlabelled antibody PAP method.‘* Before the immunoreaction, some sections were treated with 0.1% trypsin (Difco Lab, Detroit, Michigan) in a 0.1 M CaC12 solution adjusted to pH 7.8 with NaOH. Rabbit antiserum to whole S-100, either obtained com,mercially (Dakopatts, Copenhagen, Denmark) or produced by us and characterized according to Zuckermann et al. I5 was used at dilutions varying from 1500 to 1:lOOO. In some experiments, an antiserum against the S-1OOb isotype (kindly provided by Dr K. Haglid, University of Goteborg) was used, at the same dilutions as the other anti-S-100 antisera. The specificity of the immunostaining was confirmed by replacement of the primary antiserum with non-immune rabbit serum or pre-absorption of anti-S-100 antiserum with purified antigen. Sections were also reacted with anti-glial fibrillary acidic protein (GFAP; 1:400) or anti-myelin basic protein (MBP; 1:400) antibody (both obtained from Dakopatts). The technical details of the immunostaining procedures have been reported elsewhere.‘j For electron microscopy, tissue fragments were cut on a Sorvall TC-2 sectioner and processed by the PAP method using an anti-S-100 antibody, as previously reported.‘The sections were postfixed in 1.5% osmium tetraoxide in 0.1 M phosphate buffer for 30 min, dehydrated in ethanol and 0 Author
to whom all correspondence should be addressed.
275
276
L. Lauriola et al.
propylene-oxide and embedded in Epon 812. Sections of SO-90 nm thickness were prepared using an LKB Ultratome III, lightly counterstained with lead citrate and observed with a Philips EM 400. RESULTS Paraffin sections from all embryos treated with anti-S-100 antiserum exhibited, from the earliest ages investigated, the presence of immunostained cells in spinal ganglia {Fig. 1). Immunolabelled cells constituted a continuous layer at the periphery of the ganglionic rudiment, isolating the neural elements from the thin fibroblastic layer of the developing meningeal envelope, In addition, numerous immunostained cells, dendritic in shape, were also detected inside the ganglion, among neuronal elements, becoming more numerous during development (Figs 2 and 3). Immunolabelled glial cell bodies and processes were often observed in close proximity to the walls of blood vessels present in the gan~ionic anlage (Figs 1 and 4). Immun~tained cells identi~able as precursors of Schwann cells were abundantly present along both the ventral and dorsal root and spinal nerve. The ultrastructural examination clearly confirmed the presence, at the periphery of the ganglionic rudiment, of a continuous layer of immunostained cells characterized by cytoplasmic processes filled with immunoreaction product in the matrix, where few organelles were also observed (Fig. 5) In addition, immunola~lled cells, dendritic in shape, with thin cytoplasmic processes not completely encircling individual neuronal cells, were also numerous inside the ganglion. Overlapping immunohist~hemical findings were observed using an anti~~m anti-S-lob isotype, which is known to be specifically restricted to glial cells in the human brain.2 On the other hand, treatment of sections with anti-GFAP did not show the presence of labelled cells, in accordance with previous findings3 Likewise, MBP immunoreactivity was never observed in the examined samples. Counte~tai~ing of sections with toluidine blue showed that neuronal cells were unlabelled by S-100 antiserum. No immunoreactivity was observed when the sections were treated with preimmune rabbit serum or with antiserum absorbed with the related antigen (not shown). DISCUSSION The present study investigates, by S-100 immunohistochemistry, the presence and distribution of satellite cells in human spinal ganglia during the early stages of development. The peculiar distribution of the S-loo-containing cells, constituting a continuous envelope around developing spinal ganglia, as also observed in migrating sympathetic nests,? seems noteworthy. Keeping in mind that differentiating events in the peripheral nervous system are possibly influenced by su~ounding embryonic tissues and extracellular matrix molecules, the possibility that these satellite cells, exhibiting such a distinctive distribution around ganglionic rudiments, play a modulatory role on microenvironmental influences should be taken into account. In this respect, it is suggestive that S-lOOl3 has been reported to have neurite extension factor activity’ and also to be released from glial cells, i3 at least in cell cultures from tissue of the central nervous system. In the light of these reports, the possibility that the S-100 in satellite cells acts in a paracrine fashion to stimulate neurite outgrowth during development of the nervous system could also be borne in mind. With regard to labelled cells inside developing human ganglia, the present data are in agreement with reports on other animal species, also indicating that at these stages of development satellite cells do not completely encircle individual neuronal cells, unlike the adult pattern, where each ganglion cell is completely enveloped by one or more satellite cells.” Glial fibrillary acidic protein appears to be undetectable in satellite celfs of sensitive peripheral ganglia at the early stages of human development, although some peripheral glial elements have been reported to contain this antigen in adult mammals. A delayed postnatal expression of GFAP in satellite cells or differences between species or in antigenicity between central and peripheral GFAP3 could explain this discrepancy. On the other hand, the absence of MBP immunoreactivity, as we have observed, is consistent with the later occurrence of myelination in the
Satellite cells in developing spinal ganglia
Figs 1-3. Paraffin sections of human embryo spinal ganglia treated with anti-S-100 antiserum. Sections were not counterstained. Fig. 1. x 90. Intensely immunostained cells are detectable both in the ganglion and in ganglion roots. Fig. 2. x 225. Fig. 3. x 340. Immunoreactivity is confined to glial cells located in a continuous layer at the periphery as well as inside the ganglion. Cells constituting the meningeal layer as well as neurons appear to be unstained. Note immunostained glial cells in close proximity to walls of the blood vessel (Fig. 2. *).
277
278
L. Lauriola et al.
Fig. 4. Paraffin section of human embryo spinai ganglia treated with anti-S-100 antiserum. The section was not counterstained. Note immunostained cells detectable in the gangiionic root (arrow). x 225. Fig. 5. Electron micrograph of human embryo spinal ganglion treated with anti-S-100 antiserum. Immunoreactivity is detectable in the cytoplasm of a satebite cell located at the periphery of the ganglionic rudiment. Original magnification x 4600.
Satellite cells in developing spinal ganglia
279
human peripheral nervous system (14th-18th week of intrauterine life).” In this respect, any possible relationship between S-100 expression and the appearance of MBP remains open to investigation. Acknowledgements-The assistance.
authors thank Mr P. Baldassarti for editorial assistance and Mr A. Rinelli for technical
REFERENCES I. Hamilton W. J., Boyd J. D. and Mossman H. W. (1962) Human Embryology, pp. 119-134. Williams and Wilkins, Baltimore. 2. Isobe T.. Takahashi K. and Okuyama T. (1984) S-100 protein is present in neurons of the central and peripheral nervous system. J. Neurochem. 43, 1494-1496. 3. Jessen K. K., Thorpe R. and Mirsky R. (1984) Molecular identity, distribution and heterogeneity of glial fibrillary acidic protein: an immunoblotting and immunohistochemical study of Schwann cells, enteric glia and astrocytes. 1. Neurocytol. 13, 187-200. 4. Kligman D. and Marshak D. R. (1985) Purification and characterization of a neurite extension factor from bovine brain. Proc. nam Acad. Sci., U.S.A. 82, 7136-7139. 5. Krajci D. (1975) Ontogenic development of the relation between neurons and satellite cells in spinal ganglia. Folia Morphol. (Praha) 21, 139-141. 6. Lauriola L., Maggiano N., Sentinelli S., Michetti F. and Cocchia D. (1985) Satellite cells in the normal human adrenal gland and in pheochromocytomas. An immunohistochemical study. Virchows Arch. (Cefl ParhoL) 49, 13-21. 7. Lauriola L., Sentinelli S., Maggiano N., Michetti F. and Cocchia D. (19%) Glial-like cells in sympathetic neural crest derivatives during human embryogenesis. Detection by S-100 immunohistochemistry. Devf &ah Res. 28,69-74. 8. Moore B. W. (l%5) A soluble protein characteristics of the nervous svstem. BioDhvs. Res. Commun. 19.739-744. 9. Pannese E. (1969) Electron microscopical study on the development of-satellite ceiliheath in spinal ganglia. J. camp. Neurol. 135, 381422. 10. Pannese E. (1981) The satellite cells of the sensory ganglia. In Advances in Anatomy and Embryo Cell Biology, Vol. 65, Springer-Verlag, Berlin. 11. Shaw-Dunn J. (1970) Developing myelin in human peripheral nerve. Scot. Med. 1. 15, 108-117. 12. Stemberger L. A., Hard Ph Jr, Cuculis J. J. and Meyer H. G. (1970) The unlabelled antibody enzyme method of immunochemistry. Preparation and properties of soluble antigen-antibody complex (horseradish neroxidaseantihorseradish peroxidase) and its use in-identification of spirochetes. J. Hi.s&hem.~ Cytochem. l&31$333. 13. Van Eldik L. J. and Zimmer D. B. (1987) Secretion of S-100 from rat C6 glioma cells. Brain Res. 436,367-370. 14. Zonuely-Neurath C. E. and Walker W. A. (1980) Nervous system-specific proteins: 14-3-2 protein, neuron-specific enolase, and S-100 protein. In Proreins ofrhe Nervous Sysrem (eds Bradshow R. A. and Schneider D. M.), pp. l-57. Raven Press, New York. IS. Zuckerman J. E., Herschman H. R. and Levine L. (1970) Appearance of a brain specific antigen (the S-100 protein) during human foetal development. /. Neurochem. 17.247-251.