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Do radial glia give rise to both astroglial and oligodendroglial cells?
119
Developmental Brain Research, 8 (1983) 119 130 Elsevier Biomedical Press
D o R a d i a l G l i a G i v e Rise to B o t h A s t r o g l i a l a n d O l i g o d e n d r o g l i a l Cells?* BEN H. CHOI, RONALD C. KIM and LOWELL W. LAPHAM
Division of Neuropathology, Department of Pathology, University of California lrvine, California College of Medicine, Irvine, CA 92 717 and (1.. W. I.. ) Universi(v of Rochester Medical Center. Rochester. N Y 14642 ( U. S. A. ) (Accepted November 9th. 19821
Key words: gliogenesis - human fetus - spinal cord - radial glia - astroglia - oligodendroglia - myelinogenesis
The development and differentiation of gliai cells in the human fetal spinal cord was studied by correlative electron microscopic and immunohistochemical analysis in 20 embryos and fetuses between 6 and 17 weeks of ovulation age. Gliogenesis is characterized initially by the formation of radial glia and astroglia and subsequently, when myelination is about to begin, by the formation of oligodendroglia. The radial glial origin of astroglial cells has previously been demonstrated. The finding of'transitional' ceils with cytological, ultrastructurai and immunohistochemical features intermediate between those of astroglial and oligodendroglial cells andthe close relationship that develops between astroglial cells and axons just prior to the onset of mvelination suggest that oligodendroglia may also be derived from radial glial cells, either directly or through intermediate astroglial forms. INTRODUCTION
With the advent of immunohistochemical methods using specific markers for glial cells and with the correlative use of Golgi methods, radioautography and electron microscopy (EM), our understanding of the genesis and differentiation ofneuroglial ceils in the developing central nervous system (CNS) has improved greatly in recent years. For example, it has been demonstrated that radial glial cells are identifiable in the developing human fetal spinal cord (HFSC) as early as 6 weeks of ovulation age, at which time they exhibit the Golgi, immunohistochemical and EM features of astroglial cells6. It has also been shown, by EM and by immunohistochemistry (using antiserum to the astrocyte-specific glial fibrillary acidic protein (GFAP)), that neuronal and glial precursor cells coexist in the cerebral ventricular zone of the fetal monkey Iz. Thus it has become apparent that neurons and glial cells are generated concomitantly in the ventricular zone of the early devel-
oping CNS. This view is contrary to the previously held belief that gliogenesis takes place much later in ontogenetic development, i.e. after neurons have been generated from germinal matrix cells. Considerable debate exists still regarding the cell of origin and the mode of differentiation of oligodendroglial cells in the developing CNS. Evidence has been presented to indicate early divergence of astroglial and oligodendroglial cell lines25-27.32in the developing rat optic nerve; however, the timing of this event and the cells from which the oligodendroglia originated were not clearly defined. The difficulty is due primarily to limitations in our ability to classify embryonic and fetal CNS cells accurately. The purpose of this study was to investigate the development and differentiation of neuroglial cells in the early HFSC, particularly during the period of myelinogenesis, with the combined use of EM and immunohistochemistry, and to clarify the relationships between radial glia, astroglia and oligodendroglia.
* Presented in part at the 57th Annual Meeting of the American Association of Neuropathologists in Vancouver, Canada, June, 1981. 0165-3806/83/0000 0000/$03.00 '-~1983 Elsevier Science Publishers
120 MATERIALS AND METHODS
Twenty HFSC obtained from aborted embryos and fetuses received as surgical specimens were used for this study. The ovulation ages of the specimens ranged from 6 to 17 weeks (Table !). The spinal cords were removed by the dorsal approach as soon as the specimens were received. Spinal cord sections obtained from various levels with the aid of a dissecting microscope were placed into appropriate fixatives for EM 5, rapid Golgi 2~ and Goigi-Cox 33 staining and paraffin embedding. Younger embryos were serially sectioned in toto in the transverse plane and processed in a similar manner. For GFAP and myelin basic protein (MBP) immunohistochemistry, cryostat-frozen sections were used for indirect immunofluorescence, and both Vibratome and paraffin sections were used for application of the unlabeled antibody enzyme technique. In selected samples, 1 #m Epon-embedded sections were de-Eponized, processed for immunohistochemistry and counterstained with toluidine blue for light microscopic (LM) examination. One micron sections stained with
toluidine blue were used primarily for survey purposes and adjacent sections were used for correlative immunohistochemical and EM analysis. Thin sections of selected regions were cut with a diamond knife on an LKB IV Uhratome, stained with uranyl acetate and lead citrate, and examined with Philips EM 201 and 400 electron microscopes.
j,
0
TABLE 1
Estimated ovulation age of ,wecimen based on crown-rump length No.
Specimen no.
C r o w nrump Estimated lenght (cm) ovulation age (week)
I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
R 718 873 681 664 693 833 691 844 675 725 625 832 764 828 745 830 848 682 701 709
0.84 1.20 2.25 2.67 2.82 3.57 4.35 4.77 4.85 5.32 6.46 8.35 10.27 10.40 10.42 11.24 12.80 13.27 14.35 15.12
6 6 7.5 8
8 9 10 10 10 10 I1 12 13.5 13.5 13.5 14 15 16 16.5 17
L
.
.
.
.
.
.
.
.
~._
_
Fig, I. Photomicrograph showing glial cells in the subpial region of the ventral column of the spinal cord of an I l-weekold human fetus. A mitotic figure (M) indicates proliferative activity of cells in this region. The majority of the nuclei show an evenly distributed, lightly staining chromatin pattern. Some nuclei contain prominent nucleoli. A pair of cells with dark nuclei and nucleoli and scanty amounts of dark cytoplasm probably represent recently divided oligodendroglia (O). Myelin (my) formation, though modest, is clearly recognizable. PM, pia mater. Epon-embedded I-,ttm section. Toluidine blue stain, x 400. Fig. 2. Photomicrograph showing exuberant myelin (my) formation and numerous oligodendroglia (O) within the subpial region of the ventral column of a 16-week-old human fetus. Only a few cells with nuclear features suggestive of astroglia (A) are present at this stage. PM, pia mater. Epon-embedded I-tLm section. Toluidine blue stain. X 400.
121 OBSERVATIONS
The subpial and marginal zones of the ventral columns of the spinal cords of embryos between the ages of 6 and 8 weeks are relatively cell-free and composed primarily of cell processes, including radial glial fibers. The radial glia during this period show characteristic conical swelling of the endfeet along the surface of the pia mater (PM) following Golgi impregnation and, by EM, the electron-lucent matrices of the shaft and endfeet are seen to contain glycogen granules and glial filaments 5. By 8- 10 weeks, there is a gradual accumulation of cells within the subpial region with evidence of mitotic activity. At this time, the majo-
rity of the cells in this region have ovoid to round nuclei with lightly stained nucleoplasm, evenly distributed chromatin and a prominent nuclear envelope. These are features typically observed in astroglia 9..3. As shown in Fig. 1, myelin formation is already beginning by i I weeks. Although at this stage the majority of the cells still show the nuclear characteristics of astroglia, there are also a few cells with dense nuclei, prominent nucleoli and scanty amounts of dense cytoplasm, features that are characteristic of oligodendroglia9.t4. A pair of such cells which appears to have just divided is shown in Figure 1. By 16 weeks, there is a notable increase in the population of subpial cells, the majority of which are oligodendroglia, as evidenced by their intimate associa-
Fig. 3. Electron micrograph showing an electron-dense glial-limiting membrane (glm) closely abutting the basal lamina of the PM. The astroglia (A) contains a typical nucleus with evenly distributed chromatin and cytoplasm filled with bundles ofglial filaments (arrowhead) and glycogen granules (small arrows). All scale bars of electron micrographs represent 1.0 ttm.
122
,71
-
Fig. 4. Electron micrograph showing oligodendroglia ((3) with typical nuclei and abundant cytoplasm The cytoplasmic extensions ofoligodendroglia are forming compact myelin. The cytoplasm is rich in organelles such as microtubules. Golgi complexes, mitochondria, ribosomes and rough endoplasmic reticulum. An astroglial process containing glycogen granules (small arrow) is closely associated with a myelin sheath; 16-week-old fetus. Fig. 5. Electron micrograph showing an oligodendroglial cell (O) with a long tapering process (thick arrow) myclinating at least 3 larger axons. The cytoplasmic matrix is relatively electron-dense with abundant organellcs such as mitochondria, microtubules, centriole, Golgi complex and ribosomes, etc. Human fetus. 16 weeks old
123 tion with well-developed myelin sheaths and by their dense nuclear and cytoplasmic staining (Fig. 2). Electron microscopy at about 10 weeks demonstrates a change in the appearance of the gliallimiting membrane (GLM) and of the processes of subpial astroglia (Fig. 3). The generally electron-lucent cytoplasmic matrix of the radial glia has become markedly electron-dense and is filled with bundles of glial filaments and scattered glycogen granules. Ultrastructural features of typical astroglia and oligodendroglia are relatively easy to distinguish and are illustrated in Figs. 3-6. Fig. 3 depicts a subpial astroglial cell at 11 weeks of ovulation age. The nuclear chromatin is evenly distributed except for a thin rim of condensation at the nuclear margin. Characteristically, the cytoplasm contains bundles of glial filaments, scattered glycogen granules, rough endoplasmic reticulum (RER), ribosomes
and mitochondria. Figs. 4-6 demonstrate typical oligodendroglia, with the associated formation of compact myelin, at 16 weeks of ovulation age. The nucleus shows clumping of the heterochromatin beneath the nuclear membrane and the formation of a distinct nucleolus. The cytoplasm is abundant and contains large numbers oforganelles, such as microtubules, cisternae of the Golgi apparatus, RER, ribosomes, mitochondria and dense bodies. Some oligodendroglia have long tapering processes apparently myelihating several axons, as shown in Figs. 5 and 6. Examination of many sections from specimens of different ages, however, revealed cells seemingly of transitional type that possessed features of both astroglia and oligodendroglia. Fig. 7 shows a subpial astroglial cell and a young oligodendroglial cell at 13 weeks. The astroglial cell has a long process extending up to the PM to become continuous with the GLM. The cyto-
Fig. 6. Electron micrograph of an oligodendroglial cell (O) extending a long process with formation of compact myelin around an axon (a). The nucleus is round with patchy and clumped heterochromatin throughout, particularly at the nuclear margin. The cytoplasm is electron-dense and contains stacks of RER, Golgi apparatus (G) and ribosomes. Note parallel arrays of microtubules (arrowsl in the oligodendroglial process.
Fig. 8. Electron micrograph showing a cell with typical nuclear and cytoplasmic features of an oligodendroglial cell with a typical nucleus and a prominent nucleolus. The cytoplasm is electron-dense and contains microtubules (large arrow), mitochondria, Goigi complexes and RER. Also scattered in the cytoplasm are glycogen granules (small arrows). Human fetus, 13 weeks old. Fig. 9. Electron micrograph showing a cell with nuclear morphology typical of an oligodendroglial cell. The cytoplasm, however, contains glial filaments (arrowhead) in addition to the usual organelles; lipid droplets (empty arrow) are also seen. Human fetus, ! 3 weeks old.
126 plasm is extremely electron-dense and contains bundles of glial filaments and scattered glycogen granules. The nucleus shows slight clumping of chromatin at the nuclear margin although no nucleolus is seen in the section. The pert-nuclear cytoplasm also contains scattered glycogen, Golgi complexes, a centriole, RER and ribosomes. Slender astroglial processes encircle and delineate adjacent large-diameter axons. The cell on the right side of Fig. 7 is a young oligodendroglial cell with clumping of heterochromatin and dense nucleoplasm. The cytoplasm is greatly expanded as compared to the neighboring astroglial cell and contains large numbers of organtiles, such as microtubules, Golgi complexes. mitochondria, RER and ribosomes. Many larger diameter axons are closely apposed to or encircled by its cytoplasmic extensions. In addition, this young oligodendroglial cell contains scattered glycogen granules and thin filaments within the cytoplasm. The presence of glycogen granules and microfilaments in the cytoplasm of otherwise typical oligodendroglia was also seen in many other cells, such as those shown in Figs. 8 and 9. immunohistochemical study of these 'transitional' types of cells was carried out on de-Eponized 1 ~m sections. Fig. I0 shows cells containing black G F A P immunoprecipitate within the cytoplasm and its processes. The cell on the left, which resembles an astroglial cell, contains a large nucleus with lightly stained chromatin and no nucleolus, and its cytoplasm is strongly immunoreactive for GFAP. The cell on the right, however, has a densely stained nucleus with a prominent nucleolus, features that are typical of oligodendroglia, despite the fact that its cytoplasm and processes are strongly GFAPpositive. Fig. 11 represents a section processed for immunohistochemistry to detect MBP. Scattered myelin sheaths can be seen which are rich in immunoprecipitate. There are cells with typical oligodendroglial nuclear features, but the cell in the center of the illustration, the nucleus of which resembles that of an astroglial cell, shows an intense reaction for MBP in the cytoplasm. Fig, 12, which represents composite camera iucida tracings of I ~m Epon-embedded sec-
i Fig. 10. Photomicrograph showing cells with black immunoprecipitates (arrowheads) of glial fibrillary acidic protein (GFAP) in the cytoplasm and its prtx:esses. The nucleus of a cell with a long tapering process shows margination of the chromatin and a prominent nucleolus, features typical ofoligodendroglia. The cell on the left contains a lightly stained nucleus with evenly distributed chromatin, a feature reminiscent of a typical astroglial cell. Both cells, however, contain G F A P immunoprecipitatc within their cytoplasm. Unlabeled antibody enzyme immunohistochemistry after dcEponization. post-stained with toluidine blue. × 20(0. Fig. 11. Photomicrograph ofde-Eponized 1/Lm section processed immunohistochemically for MBP and post-stained with toluidine blue. Note black immunoprecipitate (arrowheads) of M BP within the cytoplasm of a cell in the center of the picture. Its nucleus, however, resembles that of an astroglial cell. The cell on the right shows the typical features of an oligodendroglial cell. Scattered myelin sheaths (my) arc also seen tocontain MBP. x 2000.
tions following the application of immunohistochemical procedures for MBP, depicts several forms of developing oligodendroglia in the early HFSC. In Fig. 12a a cell is seen with the cytoplasmic features of a subpiai astroglial cell but with the nuclear features of an oligodendroglial cell. Figure 12b demonstrates an oligodendroglial cell with a long tapering process, apparently myelinating an axon, that. together with its my-
127 r ~
,
f J
C Fig. 12. Camera lucida drawing of glial cells within the subpial region of the ventral column of the spinal cord of a 16-week-old human fetus, obtained from de-Eponized I/~m sections processed immunohistochemically for MBP and post-stained with toluidine blue. a: the cell on the right shows a typical astroglial cell (A) with a lightly stained nucleus, evenly distributed nuclear chromatin, and a slender cytoplasmic process. The cell on the left possesses a cytoplasmic process with features typical of those ofsubpial astroglia extending toward the PM. The branches of the process encircle several myelin sheaths, although they do not appear themselves to have formed myelin. The cell soma, however, possess features generally regarded as typical of oligodendroglia, namely, a small rim of dark cytoplasm and a dark nucleus containing a prominent nucleolus, b: the cell on the right is a neuron (N) which is shown for purposes of comparison. The cell on the left shows a typical oligodendroglial soma with a long extended process that has participated in the formation of a myelin sheath, c: the cells on the left represent typical dark oligodendroglia (O) some of which have formed myelin sheaths. The two larger cells on the right, though possessing lightly stained nuclei with a chromatin pattern similar to that ofastroglia, are also closely associated with myelin sheaths.
128 elin sheath, is strongly immunoreactive for MBP. Typical myelinating dark oligodendroglia with their dense nuclei and cytoplasm are shown in Fig. 12c. There are also cells with relatively large, light-staining nuclei, the cell bodies of which are closely applied to myelin sheaths. DISCUSSION
In the developing HFSC, radial glia are the first distinguishable neuroglial element among the population of cells within the ventricular zone, and can be identified by EM and following Golgi impregnation as early as 6 weeks of ovulation age. By 8 9 weeks the presence of G F A P in radial glia is demonstrable immunohistochemically~. With the aid of correlative EM, Golgi and immunohistochemical analysis, we have previously presented morphological evidence to suggest the transformation of radial glia into astroglia in the developing HFSC 6 and human fetal cerebrumL Similar findings have also been reported by Schmechel and Raki¢a4 in the monkey telencephalon. The possibility of direct transformation of ventricular cells to astroglia was also suggested by Skoff eta[. 26 in the developing rat optic nerve. Most of the progressive increase in cell density that occurs within the subpial and marginal zones of the ventral columns of the HFSC between the ages of 6 and 8 weeks is accounted for by cells with astroglial morphology, many of which possess radial processes extending toward the PM. Cells identifiable by EM as oligodendroglia make their appearance at about 11 weeks, just prior to the start of active myelin formation in this region. At this time only a few large-diameter axons are associated with the formation of compact myelin, the majority of neurites being unmyelinated. By 16 weeks, however, the population of subpial cells is primarily oligodendroglial, and many of the axons, particularly those of the ventral rootlets, are myelinated. The results of the present study, therefore, indicate that, within the ventral columns of the developing HFSC, gliogenesis is characterized early by the formation of astroglia and subsequently, when myelination is about to begin, by the for-
mation of oligodendroglia. Such a sequence has also been described by other investigators in studies of rat and human optic nerves 2~.3j". Following the application of well-established I.M and EM~4.,~.~,,.r: ~s ~,, : . ~ : ~ a ' , 4 ~, as well as immunohistochemical j:~:~'' criteria, typical astroglia and oligodendroglia were readily differentiated in this study. Many cells, however. could not be classified with either group; some. such as those illustrated in Figs. 7-9, though possessing many of the LM and EM characteristics of oligodendroglia, contained scattered glycogen and thin glial filaments in their cytoplasm. The presence of glycogen and glial filaments on the one hand and the presence of an electrondense cytoplasmic matrix on the other have been regarded as mutually exclusive identifying features of astrocytes and oligodendrocytes, respectively -~. The cells described above may, we believe, be interpreted as representing intermediate forms of oligodendroglia with residual astroglial features. Such an interpretation is further supported by the results obtained following the application of immunohistochemical procedures for G F A P and MBP. As shown in Fig. 10, cells with features typical of medium type oligodendroglia z4 demonstrated a strong immune reaction for G F A P within the cytoplasm and its processes. In addition, cells with the general morphological features of astroglia 9.'3 contained an abundance of MBP within their cytoplasm (Fig. 1 I). G F A P and MBP staining of adjacent de-Eponized I p.m sections would be valuable adjunctive means of identifying "transitional' forms, i.e. those cells containing both antigens. This would, in addition, allow us to provide, in support of our hypothesis, a quantitative evaluation of the various cell populations (including astroglial, oligodendroglial and 'transitional' cells) at different stages of development. Although we have made attempts to do this and are continuing to do so, because of technical problems we have not yet succeeded in demonstrating the presence of both antigens within the same cell. Efforts are also currently in progress to confirm the bivalent nature of such cells with the aid o f E M immunocytochemistry.
129 In recent years, uncertainty has been expressed regarding the nature of the cells that initiate myelin formation. Some investigators have suggested that astrocytes are either wholly or partly responsible 17,36.Our observations, however, like those of Hirano s, Nagashima ~6and Okado ~s, indicate that compact myelin is formed solely by oligodendroglial cells. The rather sudden appearance at around I 1 weeks of cells with the cytological, immunohistochemical and ultrastructural characteristics of oligodendroglia just prior to the onset of myelination is of considerable interest, particularly in view of our failure to find, at any earlier stage of development, cells that might be identified as 'oligodendroblasts'. We believe that this phenomenon could be accounted for by the presence of 'transitional' cells that possess features of both astroglial and oligodendroglial cells and, therefore, that the myelin-forming oligodendroglial cells develop from the radial glial cell-derived, axon-enveloping cells with astroglial characteristics that we have described in this report. Such a possibility has been addressed, albeit indirectly, by others who have also observed the close relationship between astroglial cells and axons prior to the onset of
myelination ~6:8,23. If this hypothesis is correct, it would imply that both astrocytes and oligodendrocytes ultimately originate from radial glial cells. Whether oligodendrocytes do so directly or through intermediate astroglial forms, however, cannot be determined at the present time. The signal for the hypothesized transformation of radial glia or of astroglial cells into oligodendroglia is not known, but it is difficult to escape the conclusion that nerve cells and their axons would have to play an important role. Perhaps, in accordance with the 'critical diameter' hypothesis, axonal size (or a change in diameter) is a major contributing factor; such a contention is supported by our observations and by those of others ~2,18that myelin formation appears to occur initially around axons of relatively large diameter.
REFERENCES
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1 Antanitus, D. S., Choi, B. H. and Lapham, L. W., lmmunofluorescence staining of astrocytes in vitro using antiserum to glial fibrillary acidic protein, Brain Res., 89 (1975) 363- 367. 2 Bignami, A. and Dahl, D.,Specificity of the glial fibrillary acidic protein for astroglia, J. Histochem. Cytochem., 25 (1977) 466- 469. 3 Bunge, R. P., Glial cells and the central myelin sheath, J. Physiol. (Lond.), 48 (1968) 197-251. 4 Caley, D. W. and Maxwell, D. S., An electron microscopic study of the neuroglia during postnatal development of the rat cerebrum, J. comp. Neurol., 133 (1968) 56- 70. 5 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. 6 Choi, B. H., Radial glia of developing human fetal spinal cord: Golgi, immunohistochemical and electron microscopic study, Develop. Brain Res., I ( 1981 ) 249- 267. 7 Eng, L. F., Vanderhaeghen, J. J., Bignami, A. and Gerstl, B., An acidic protein isolated from fibrous astrocytes, Brain Res., 28 (1971) 351-354. 8 Hirano, A., A confirmation of the oligodendroglial origin
ACKNOWLEDGEMENTS
This study is partly supported by NIEHS Grants R01-ES 02928, ES 01722 and ES 01247. The superb technical assistance of Ms. Teresa Espinosa and excellent secretarial support of Ms. Lucia Wisdom are gratefully acknowledged.
130 16 Nagashima. K., Ultrastructural study of myelinating cells and subpial astrocytes in developing rat spinal cord. J. neurol. Sci., 44(1979) I- 12. 17 Narang, H. K.. Electron microscopic development of neuroglia in epiretinal portion of post-natal rabbits, J. neurol. Sck, 34 (1977) 39 I- 406. 18 Okado, N., Early myelin formation and glial cell development in the human spinal cord, Anat. Rec.. 202 (1982) 483- 490. 19 Penfield, W., Oligodendroglia and its relation to classical neuroglia, Brain. 47 (1924) 430--452. 20 Peters, A.. Palay, S. L. and Webster, H. de F., The Fine Structure of the Nervous System. The Neurons and Supporting Cells, W. B. Saunders, Philadelphia, 1976. 21 Privat, A., Postnatal gliogenesis in the mammalian brain, Int. Rev. C vtol.. 40 (1975) 281- 323. 22 Ramon-Moliner, E., A study on neuroglia. The problem of transitional forms. J. comp. Neurol.. 110 (1958) 157 171. 23 Reier, P. J. and Webster. H. de F., Regeneration and remyelination of Xenopus tadpole optic nerve fibers following transection or crush, J. Neurocvtol.. 3 (1974) 591618. 24 Schmechel, D. E. and Rakic, P., A Golgi study of radial glial cells in developing monkey telencephalon: morphogenesis and transformation into astrocytes, Anat. Embrvo1., 156(1979)115- 152. 25 Skoff, R. P., Neuroglia: a reevaluation of their origin and development, Path. Res. Pract., 168 (1980) 279- 300. 26 Skoff, R. P., Price. D. L. and Stocks, A., Electron microscopic autoradiographic studies ofgliogenesis in rat optic nerve. I. Cell proliferation, J. comp. Neurol.. 169 (1976) 291 312. 27 Skoff, R. P., Price. D. L. and Stocks, A., Electron microscopic autoradiographic studies ofgliogenesis in rat optic
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nerve. I1. Time of origin, J. comp. Neurol., 169 (19761 313 334. Stensaas, L. J., The development of hippocampal and dorsolateral pallial regions of the cerebral hemisphere in fetal rabbits, I. Fifteen millimeter stage, spongioblast morphology. J. comp. Neurol., 129 (1967) 59 70. Stensaas, k. J., The ultrastructure ofastrocytes, oligodendrocytes, and microglia in the optic nerve of urodele amphibians (A. punctatum, 7~ pyrrhogaster, T. viridescens). J. Neuroc)'tol., 6 (1977) 269 286. Sternberger, N. H., Itoyama. Y., Kies, M. W. and Webster, H. F., Immunocytochemical method to identify basic protein in myelin-forming oligodendrocytes of newborn rat CNS. J. Neuro~Ttol.. 7 (1978) 25 I-263. Sturrock, R. R., A light and electron microscopic study of proliferation and maturation of fibrous astrocytes in the optic nerve of the human emb .ryo, J. Anat.. 119 (1975) 223- 234. Tennekoon, G. I.. Cohen, S. R., Price, D. L. and McKhann, G. M.. Myelinogenesis in optic nerve. A morphological autoradiographic and biochemical analysis. J. CellBiol.. 72(1977)604 616. Van der Loos, H., Une combinaison de deux vieilles methodes histologiques pour le systeme nerveux central, Mschr. Psychiat. NeuroL. 132 (1956)330-334. Vaughn, J. E., An electron microscopic analysis ofgliogenesis in rat optic nerves, Z. Zellforsch., 94 (1969) 292 324. Vaughn, J. E. and Peters, A.. Electron microscopy of early postnatal development of fibrous astrocytes, Amer. J. Anat., 121 (1967) 131 152. Wendell-Smith, C. P., Blunt, M. J. and Baldwin, F., The ultrastructural characterization of macroglial cell types. J. comp. NeuroL. 127(1966) 219- 240.
Developmental Brain Research, 8 (1983) 119 130 Elsevier Biomedical Press
D o R a d i a l G l i a G i v e Rise to B o t h A s t r o g l i a l a n d O l i g o d e n d r o g l i a l Cells?* BEN H. CHOI, RONALD C. KIM and LOWELL W. LAPHAM
Division of Neuropathology, Department of Pathology, University of California lrvine, California College of Medicine, Irvine, CA 92 717 and (1.. W. I.. ) Universi(v of Rochester Medical Center. Rochester. N Y 14642 ( U. S. A. ) (Accepted November 9th. 19821
Key words: gliogenesis - human fetus - spinal cord - radial glia - astroglia - oligodendroglia - myelinogenesis
The development and differentiation of gliai cells in the human fetal spinal cord was studied by correlative electron microscopic and immunohistochemical analysis in 20 embryos and fetuses between 6 and 17 weeks of ovulation age. Gliogenesis is characterized initially by the formation of radial glia and astroglia and subsequently, when myelination is about to begin, by the formation of oligodendroglia. The radial glial origin of astroglial cells has previously been demonstrated. The finding of'transitional' ceils with cytological, ultrastructurai and immunohistochemical features intermediate between those of astroglial and oligodendroglial cells andthe close relationship that develops between astroglial cells and axons just prior to the onset of mvelination suggest that oligodendroglia may also be derived from radial glial cells, either directly or through intermediate astroglial forms. INTRODUCTION
With the advent of immunohistochemical methods using specific markers for glial cells and with the correlative use of Golgi methods, radioautography and electron microscopy (EM), our understanding of the genesis and differentiation ofneuroglial ceils in the developing central nervous system (CNS) has improved greatly in recent years. For example, it has been demonstrated that radial glial cells are identifiable in the developing human fetal spinal cord (HFSC) as early as 6 weeks of ovulation age, at which time they exhibit the Golgi, immunohistochemical and EM features of astroglial cells6. It has also been shown, by EM and by immunohistochemistry (using antiserum to the astrocyte-specific glial fibrillary acidic protein (GFAP)), that neuronal and glial precursor cells coexist in the cerebral ventricular zone of the fetal monkey Iz. Thus it has become apparent that neurons and glial cells are generated concomitantly in the ventricular zone of the early devel-
oping CNS. This view is contrary to the previously held belief that gliogenesis takes place much later in ontogenetic development, i.e. after neurons have been generated from germinal matrix cells. Considerable debate exists still regarding the cell of origin and the mode of differentiation of oligodendroglial cells in the developing CNS. Evidence has been presented to indicate early divergence of astroglial and oligodendroglial cell lines25-27.32in the developing rat optic nerve; however, the timing of this event and the cells from which the oligodendroglia originated were not clearly defined. The difficulty is due primarily to limitations in our ability to classify embryonic and fetal CNS cells accurately. The purpose of this study was to investigate the development and differentiation of neuroglial cells in the early HFSC, particularly during the period of myelinogenesis, with the combined use of EM and immunohistochemistry, and to clarify the relationships between radial glia, astroglia and oligodendroglia.
* Presented in part at the 57th Annual Meeting of the American Association of Neuropathologists in Vancouver, Canada, June, 1981. 0165-3806/83/0000 0000/$03.00 '-~1983 Elsevier Science Publishers
120 MATERIALS AND METHODS
Twenty HFSC obtained from aborted embryos and fetuses received as surgical specimens were used for this study. The ovulation ages of the specimens ranged from 6 to 17 weeks (Table !). The spinal cords were removed by the dorsal approach as soon as the specimens were received. Spinal cord sections obtained from various levels with the aid of a dissecting microscope were placed into appropriate fixatives for EM 5, rapid Golgi 2~ and Goigi-Cox 33 staining and paraffin embedding. Younger embryos were serially sectioned in toto in the transverse plane and processed in a similar manner. For GFAP and myelin basic protein (MBP) immunohistochemistry, cryostat-frozen sections were used for indirect immunofluorescence, and both Vibratome and paraffin sections were used for application of the unlabeled antibody enzyme technique. In selected samples, 1 #m Epon-embedded sections were de-Eponized, processed for immunohistochemistry and counterstained with toluidine blue for light microscopic (LM) examination. One micron sections stained with
toluidine blue were used primarily for survey purposes and adjacent sections were used for correlative immunohistochemical and EM analysis. Thin sections of selected regions were cut with a diamond knife on an LKB IV Uhratome, stained with uranyl acetate and lead citrate, and examined with Philips EM 201 and 400 electron microscopes.
j,
0
TABLE 1
Estimated ovulation age of ,wecimen based on crown-rump length No.
Specimen no.
C r o w nrump Estimated lenght (cm) ovulation age (week)
I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
R 718 873 681 664 693 833 691 844 675 725 625 832 764 828 745 830 848 682 701 709
0.84 1.20 2.25 2.67 2.82 3.57 4.35 4.77 4.85 5.32 6.46 8.35 10.27 10.40 10.42 11.24 12.80 13.27 14.35 15.12
6 6 7.5 8
8 9 10 10 10 10 I1 12 13.5 13.5 13.5 14 15 16 16.5 17
L
.
.
.
.
.
.
.
.
~._
_
Fig, I. Photomicrograph showing glial cells in the subpial region of the ventral column of the spinal cord of an I l-weekold human fetus. A mitotic figure (M) indicates proliferative activity of cells in this region. The majority of the nuclei show an evenly distributed, lightly staining chromatin pattern. Some nuclei contain prominent nucleoli. A pair of cells with dark nuclei and nucleoli and scanty amounts of dark cytoplasm probably represent recently divided oligodendroglia (O). Myelin (my) formation, though modest, is clearly recognizable. PM, pia mater. Epon-embedded I-,ttm section. Toluidine blue stain, x 400. Fig. 2. Photomicrograph showing exuberant myelin (my) formation and numerous oligodendroglia (O) within the subpial region of the ventral column of a 16-week-old human fetus. Only a few cells with nuclear features suggestive of astroglia (A) are present at this stage. PM, pia mater. Epon-embedded I-tLm section. Toluidine blue stain. X 400.
121 OBSERVATIONS
The subpial and marginal zones of the ventral columns of the spinal cords of embryos between the ages of 6 and 8 weeks are relatively cell-free and composed primarily of cell processes, including radial glial fibers. The radial glia during this period show characteristic conical swelling of the endfeet along the surface of the pia mater (PM) following Golgi impregnation and, by EM, the electron-lucent matrices of the shaft and endfeet are seen to contain glycogen granules and glial filaments 5. By 8- 10 weeks, there is a gradual accumulation of cells within the subpial region with evidence of mitotic activity. At this time, the majo-
rity of the cells in this region have ovoid to round nuclei with lightly stained nucleoplasm, evenly distributed chromatin and a prominent nuclear envelope. These are features typically observed in astroglia 9..3. As shown in Fig. 1, myelin formation is already beginning by i I weeks. Although at this stage the majority of the cells still show the nuclear characteristics of astroglia, there are also a few cells with dense nuclei, prominent nucleoli and scanty amounts of dense cytoplasm, features that are characteristic of oligodendroglia9.t4. A pair of such cells which appears to have just divided is shown in Figure 1. By 16 weeks, there is a notable increase in the population of subpial cells, the majority of which are oligodendroglia, as evidenced by their intimate associa-
Fig. 3. Electron micrograph showing an electron-dense glial-limiting membrane (glm) closely abutting the basal lamina of the PM. The astroglia (A) contains a typical nucleus with evenly distributed chromatin and cytoplasm filled with bundles ofglial filaments (arrowhead) and glycogen granules (small arrows). All scale bars of electron micrographs represent 1.0 ttm.
122
,71
-
Fig. 4. Electron micrograph showing oligodendroglia ((3) with typical nuclei and abundant cytoplasm The cytoplasmic extensions ofoligodendroglia are forming compact myelin. The cytoplasm is rich in organelles such as microtubules. Golgi complexes, mitochondria, ribosomes and rough endoplasmic reticulum. An astroglial process containing glycogen granules (small arrow) is closely associated with a myelin sheath; 16-week-old fetus. Fig. 5. Electron micrograph showing an oligodendroglial cell (O) with a long tapering process (thick arrow) myclinating at least 3 larger axons. The cytoplasmic matrix is relatively electron-dense with abundant organellcs such as mitochondria, microtubules, centriole, Golgi complex and ribosomes, etc. Human fetus. 16 weeks old
123 tion with well-developed myelin sheaths and by their dense nuclear and cytoplasmic staining (Fig. 2). Electron microscopy at about 10 weeks demonstrates a change in the appearance of the gliallimiting membrane (GLM) and of the processes of subpial astroglia (Fig. 3). The generally electron-lucent cytoplasmic matrix of the radial glia has become markedly electron-dense and is filled with bundles of glial filaments and scattered glycogen granules. Ultrastructural features of typical astroglia and oligodendroglia are relatively easy to distinguish and are illustrated in Figs. 3-6. Fig. 3 depicts a subpial astroglial cell at 11 weeks of ovulation age. The nuclear chromatin is evenly distributed except for a thin rim of condensation at the nuclear margin. Characteristically, the cytoplasm contains bundles of glial filaments, scattered glycogen granules, rough endoplasmic reticulum (RER), ribosomes
and mitochondria. Figs. 4-6 demonstrate typical oligodendroglia, with the associated formation of compact myelin, at 16 weeks of ovulation age. The nucleus shows clumping of the heterochromatin beneath the nuclear membrane and the formation of a distinct nucleolus. The cytoplasm is abundant and contains large numbers oforganelles, such as microtubules, cisternae of the Golgi apparatus, RER, ribosomes, mitochondria and dense bodies. Some oligodendroglia have long tapering processes apparently myelihating several axons, as shown in Figs. 5 and 6. Examination of many sections from specimens of different ages, however, revealed cells seemingly of transitional type that possessed features of both astroglia and oligodendroglia. Fig. 7 shows a subpial astroglial cell and a young oligodendroglial cell at 13 weeks. The astroglial cell has a long process extending up to the PM to become continuous with the GLM. The cyto-
Fig. 6. Electron micrograph of an oligodendroglial cell (O) extending a long process with formation of compact myelin around an axon (a). The nucleus is round with patchy and clumped heterochromatin throughout, particularly at the nuclear margin. The cytoplasm is electron-dense and contains stacks of RER, Golgi apparatus (G) and ribosomes. Note parallel arrays of microtubules (arrowsl in the oligodendroglial process.
Fig. 8. Electron micrograph showing a cell with typical nuclear and cytoplasmic features of an oligodendroglial cell with a typical nucleus and a prominent nucleolus. The cytoplasm is electron-dense and contains microtubules (large arrow), mitochondria, Goigi complexes and RER. Also scattered in the cytoplasm are glycogen granules (small arrows). Human fetus, 13 weeks old. Fig. 9. Electron micrograph showing a cell with nuclear morphology typical of an oligodendroglial cell. The cytoplasm, however, contains glial filaments (arrowhead) in addition to the usual organelles; lipid droplets (empty arrow) are also seen. Human fetus, ! 3 weeks old.
126 plasm is extremely electron-dense and contains bundles of glial filaments and scattered glycogen granules. The nucleus shows slight clumping of chromatin at the nuclear margin although no nucleolus is seen in the section. The pert-nuclear cytoplasm also contains scattered glycogen, Golgi complexes, a centriole, RER and ribosomes. Slender astroglial processes encircle and delineate adjacent large-diameter axons. The cell on the right side of Fig. 7 is a young oligodendroglial cell with clumping of heterochromatin and dense nucleoplasm. The cytoplasm is greatly expanded as compared to the neighboring astroglial cell and contains large numbers of organtiles, such as microtubules, Golgi complexes. mitochondria, RER and ribosomes. Many larger diameter axons are closely apposed to or encircled by its cytoplasmic extensions. In addition, this young oligodendroglial cell contains scattered glycogen granules and thin filaments within the cytoplasm. The presence of glycogen granules and microfilaments in the cytoplasm of otherwise typical oligodendroglia was also seen in many other cells, such as those shown in Figs. 8 and 9. immunohistochemical study of these 'transitional' types of cells was carried out on de-Eponized 1 ~m sections. Fig. I0 shows cells containing black G F A P immunoprecipitate within the cytoplasm and its processes. The cell on the left, which resembles an astroglial cell, contains a large nucleus with lightly stained chromatin and no nucleolus, and its cytoplasm is strongly immunoreactive for GFAP. The cell on the right, however, has a densely stained nucleus with a prominent nucleolus, features that are typical of oligodendroglia, despite the fact that its cytoplasm and processes are strongly GFAPpositive. Fig. 11 represents a section processed for immunohistochemistry to detect MBP. Scattered myelin sheaths can be seen which are rich in immunoprecipitate. There are cells with typical oligodendroglial nuclear features, but the cell in the center of the illustration, the nucleus of which resembles that of an astroglial cell, shows an intense reaction for MBP in the cytoplasm. Fig, 12, which represents composite camera iucida tracings of I ~m Epon-embedded sec-
i Fig. 10. Photomicrograph showing cells with black immunoprecipitates (arrowheads) of glial fibrillary acidic protein (GFAP) in the cytoplasm and its prtx:esses. The nucleus of a cell with a long tapering process shows margination of the chromatin and a prominent nucleolus, features typical ofoligodendroglia. The cell on the left contains a lightly stained nucleus with evenly distributed chromatin, a feature reminiscent of a typical astroglial cell. Both cells, however, contain G F A P immunoprecipitatc within their cytoplasm. Unlabeled antibody enzyme immunohistochemistry after dcEponization. post-stained with toluidine blue. × 20(0. Fig. 11. Photomicrograph ofde-Eponized 1/Lm section processed immunohistochemically for MBP and post-stained with toluidine blue. Note black immunoprecipitate (arrowheads) of M BP within the cytoplasm of a cell in the center of the picture. Its nucleus, however, resembles that of an astroglial cell. The cell on the right shows the typical features of an oligodendroglial cell. Scattered myelin sheaths (my) arc also seen tocontain MBP. x 2000.
tions following the application of immunohistochemical procedures for MBP, depicts several forms of developing oligodendroglia in the early HFSC. In Fig. 12a a cell is seen with the cytoplasmic features of a subpiai astroglial cell but with the nuclear features of an oligodendroglial cell. Figure 12b demonstrates an oligodendroglial cell with a long tapering process, apparently myelinating an axon, that. together with its my-
127 r ~
,
f J
C Fig. 12. Camera lucida drawing of glial cells within the subpial region of the ventral column of the spinal cord of a 16-week-old human fetus, obtained from de-Eponized I/~m sections processed immunohistochemically for MBP and post-stained with toluidine blue. a: the cell on the right shows a typical astroglial cell (A) with a lightly stained nucleus, evenly distributed nuclear chromatin, and a slender cytoplasmic process. The cell on the left possesses a cytoplasmic process with features typical of those ofsubpial astroglia extending toward the PM. The branches of the process encircle several myelin sheaths, although they do not appear themselves to have formed myelin. The cell soma, however, possess features generally regarded as typical of oligodendroglia, namely, a small rim of dark cytoplasm and a dark nucleus containing a prominent nucleolus, b: the cell on the right is a neuron (N) which is shown for purposes of comparison. The cell on the left shows a typical oligodendroglial soma with a long extended process that has participated in the formation of a myelin sheath, c: the cells on the left represent typical dark oligodendroglia (O) some of which have formed myelin sheaths. The two larger cells on the right, though possessing lightly stained nuclei with a chromatin pattern similar to that ofastroglia, are also closely associated with myelin sheaths.
128 elin sheath, is strongly immunoreactive for MBP. Typical myelinating dark oligodendroglia with their dense nuclei and cytoplasm are shown in Fig. 12c. There are also cells with relatively large, light-staining nuclei, the cell bodies of which are closely applied to myelin sheaths. DISCUSSION
In the developing HFSC, radial glia are the first distinguishable neuroglial element among the population of cells within the ventricular zone, and can be identified by EM and following Golgi impregnation as early as 6 weeks of ovulation age. By 8 9 weeks the presence of G F A P in radial glia is demonstrable immunohistochemically~. With the aid of correlative EM, Golgi and immunohistochemical analysis, we have previously presented morphological evidence to suggest the transformation of radial glia into astroglia in the developing HFSC 6 and human fetal cerebrumL Similar findings have also been reported by Schmechel and Raki¢a4 in the monkey telencephalon. The possibility of direct transformation of ventricular cells to astroglia was also suggested by Skoff eta[. 26 in the developing rat optic nerve. Most of the progressive increase in cell density that occurs within the subpial and marginal zones of the ventral columns of the HFSC between the ages of 6 and 8 weeks is accounted for by cells with astroglial morphology, many of which possess radial processes extending toward the PM. Cells identifiable by EM as oligodendroglia make their appearance at about 11 weeks, just prior to the start of active myelin formation in this region. At this time only a few large-diameter axons are associated with the formation of compact myelin, the majority of neurites being unmyelinated. By 16 weeks, however, the population of subpial cells is primarily oligodendroglial, and many of the axons, particularly those of the ventral rootlets, are myelinated. The results of the present study, therefore, indicate that, within the ventral columns of the developing HFSC, gliogenesis is characterized early by the formation of astroglia and subsequently, when myelination is about to begin, by the for-
mation of oligodendroglia. Such a sequence has also been described by other investigators in studies of rat and human optic nerves 2~.3j". Following the application of well-established I.M and EM~4.,~.~,,.r: ~s ~,, : . ~ : ~ a ' , 4 ~, as well as immunohistochemical j:~:~'' criteria, typical astroglia and oligodendroglia were readily differentiated in this study. Many cells, however. could not be classified with either group; some. such as those illustrated in Figs. 7-9, though possessing many of the LM and EM characteristics of oligodendroglia, contained scattered glycogen and thin glial filaments in their cytoplasm. The presence of glycogen and glial filaments on the one hand and the presence of an electrondense cytoplasmic matrix on the other have been regarded as mutually exclusive identifying features of astrocytes and oligodendrocytes, respectively -~. The cells described above may, we believe, be interpreted as representing intermediate forms of oligodendroglia with residual astroglial features. Such an interpretation is further supported by the results obtained following the application of immunohistochemical procedures for G F A P and MBP. As shown in Fig. 10, cells with features typical of medium type oligodendroglia z4 demonstrated a strong immune reaction for G F A P within the cytoplasm and its processes. In addition, cells with the general morphological features of astroglia 9.'3 contained an abundance of MBP within their cytoplasm (Fig. 1 I). G F A P and MBP staining of adjacent de-Eponized I p.m sections would be valuable adjunctive means of identifying "transitional' forms, i.e. those cells containing both antigens. This would, in addition, allow us to provide, in support of our hypothesis, a quantitative evaluation of the various cell populations (including astroglial, oligodendroglial and 'transitional' cells) at different stages of development. Although we have made attempts to do this and are continuing to do so, because of technical problems we have not yet succeeded in demonstrating the presence of both antigens within the same cell. Efforts are also currently in progress to confirm the bivalent nature of such cells with the aid o f E M immunocytochemistry.
129 In recent years, uncertainty has been expressed regarding the nature of the cells that initiate myelin formation. Some investigators have suggested that astrocytes are either wholly or partly responsible 17,36.Our observations, however, like those of Hirano s, Nagashima ~6and Okado ~s, indicate that compact myelin is formed solely by oligodendroglial cells. The rather sudden appearance at around I 1 weeks of cells with the cytological, immunohistochemical and ultrastructural characteristics of oligodendroglia just prior to the onset of myelination is of considerable interest, particularly in view of our failure to find, at any earlier stage of development, cells that might be identified as 'oligodendroblasts'. We believe that this phenomenon could be accounted for by the presence of 'transitional' cells that possess features of both astroglial and oligodendroglial cells and, therefore, that the myelin-forming oligodendroglial cells develop from the radial glial cell-derived, axon-enveloping cells with astroglial characteristics that we have described in this report. Such a possibility has been addressed, albeit indirectly, by others who have also observed the close relationship between astroglial cells and axons prior to the onset of
myelination ~6:8,23. If this hypothesis is correct, it would imply that both astrocytes and oligodendrocytes ultimately originate from radial glial cells. Whether oligodendrocytes do so directly or through intermediate astroglial forms, however, cannot be determined at the present time. The signal for the hypothesized transformation of radial glia or of astroglial cells into oligodendroglia is not known, but it is difficult to escape the conclusion that nerve cells and their axons would have to play an important role. Perhaps, in accordance with the 'critical diameter' hypothesis, axonal size (or a change in diameter) is a major contributing factor; such a contention is supported by our observations and by those of others ~2,18that myelin formation appears to occur initially around axons of relatively large diameter.
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ACKNOWLEDGEMENTS
This study is partly supported by NIEHS Grants R01-ES 02928, ES 01722 and ES 01247. The superb technical assistance of Ms. Teresa Espinosa and excellent secretarial support of Ms. Lucia Wisdom are gratefully acknowledged.
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