- Email: [email protected]
Developmental expression of myelin proteins by oligodendrocytes in the CNS of trout
Developmental Brain Research, 51 (1990) 27-34
27
Elsevier BRESD 50992
Developmental expression of myelin proteins by oligodendrocytes in the CNS of trout G. Jeserich 1, A. Mfiller I and C. Jacque 2 ZAbt. Zoophysiologie, Universitiit Osnabriick, Osnabrack (F.R. G.) and 2 Labatoire de Neurochimie, I.N.S.E.R.M.U. 134, HOpital de la Salpetri~re, Paris (France)
(Accepted 13 June 1989) Key words: Myelin protein; Development; Oligodendrocyte; Trout CNS
Using immunohistochemical techniques, the pattern of cytoplasmic staining and the temporal order of expression of 5 major myelin components of oligodendrocytes were studied in the developing central nervous system of trout. The two myelin glycoproteins, IP1 and IP2, in the cytoplasm of glial cells showed a granular pattern of immunostaining, whereas the 36K protein was homogeneously distributed. Analysis of freshly dissociated cells during early stages of myelinogenesis revealed a constant chronological sequence of expression of myelin proteins by the oligodendrocytes: glycoprotein IP2 was the first protein to appear during glial development together with the galactocerebroside GalC at stage 28 followed by the 36K at stage 30 and finally IP1 at stage 32. The deposition of myelin proteins into the nascent myelin sheath occurred in the same chronological order as their expression by oligodendrocytes. Moreover myelin basic protein, which was not detectable in glial cells, on tissue sections was found to appear in parallel with IP2. INTRODUCTION Myelin in the central nervous system (CNS) of vertebrates is produced by oligodendrocytes 3, which during late ontogenesis send out multiple branches of cell processes 25 to ensheath axons in their vicinity in a typical multilamellar fashion. During their cellular differentiation, oligodendrocytes undergo a characteristic sequence of cytoarchitectural and biochemical changes, which in cell culture can be conveniently monitored immunohistochemically by the expression of cell type and stage specific markers. Thus, it was recently shown that a bipotential progenitor cell, which can give rise to both astrocytes and oligodendrocytes 19, first develops into immature oligodendroblasts before maturing into functionally active oligodendrocytes capable of synthesizing a series of myelin specific components, especially proteins (for review see ref. 24). In the past, numerous studies, both in vivo 7'18'22'23 and under cell culture conditions 1"5'15A7'32, have been conducted to analyze the kinetics of appearance of the major myelin proteins in the developing oligodendrocytes and their deposition in the myelin sheath by means of immunohistochemistry. Thereby it was documented that the expression of specific myelinogenic functions by oligodendrocytes is not a synchronous event but typically
proceeds in a sequential manner: ensuing the initial expression of galactocerebrosides (GalC) on the cell surface, probably a key step in oligodendroglial differentiation, the emergence of Wolfgram proteins was noted, followed by the appearance of myelin basic proteins (MBP) and finally proteolipidprotein (PLP) ~s. Up to now, studies along this line have been performed almost exclusively with mammalian species, and to date very little is known about the biochemical differentiation of oligodendrocytes in the brain of lower vertebrates, in general. Recent comparative biochemical analyses of myelin proteins in different vertebrate classes, however, have revealed striking differences in the myelin protein composition of lower vertebrates as compared to those of higher classes3°'31. In the CNS of bony fishes, two or more hydrophobic myelin glycoproteins were encountered (intermediate proteins, IP1, IP2) that were immunologically related to the major peripheral nervous system (PNS) myelin glycoprotein Po but not to P L P 14'29. The CNS myelin of teleost fishes was additionally characterized by a novel protein component of about 36 kDa mol. wt., called 36K 1°, which does not cross-react with one of the known major myelin proteins of the mammalian brain. Taking advantage of the recent availability of specific antibodies against the major fish myelin proteins 12, the present investigation was made using freshly dissociated
Correspondence: G. Jeserich, Dept. Zoophysiology, University of Osnabrfick, Barbarastr. 11, 4500 Osnabriick, F.R.G.
0165-3806/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
28 brain cells to study the d e v e l o p m e n t a l
e x p r e s s i o n of
m y e l i n o g e n i c f u n c t i o n s by o l i g o d e n d r o c y t e s in the C N S o f trout.
MATERIALS AND METHODS
Animals Fertilized eggs and freshly hatched larvae of rainbow trout (Salmo gairdneri, Rich.) which were obtained from the Rosengarten hatchery at Georgsmarienhiitte, ER.G. were raised in constantly aerated water tanks at 10 °C. Antibodies All antibodies used in this study have been previously characterized in detail. Polyclonal antisera against the trout myelin proteins 36K, IP2 and MBP were raised in rabbits as described 12 and used at a 1:100 dilution. The mouse anti-IP1 antibody was a hybridoma IgG produced by spleen cells of BALB/c mice after fusion with hypoxanthine-, aminopterin-, thymidine-sensitive myeloma cells. As was revealed by electroimmunoblotting, this antibody selectively recognized the protein portion of the IP1 myelin glycoprotein (Jeserich and Strotmann, in preparation). Mouse ascitic fluid containing monoclonal antibodies against galactocerebroside was kindly provided by Dr. B. Ranscht and used in a 1:100 dilution 21. Frozen sections Entire heads of trout of larval stages 27, 28, 29, 30, 31, 33 as identified according to the criteria of Vernier et al. 28 were fixed overnight by immersion in 4% paraformaldehyde, dissolved in sodium cacodylate buffer (pH 7.4). After rinsing in phosphatebuffered saline (PBS, pH 7.4), the samples were soaked in 10% phosphate-buffered sucrose solution and rapidly frozen in liquid nitrogen-cooled isopentane. Serial sections (12/~m thick) through the brain were cut in a Frigocut freezing microtome (Reichert-Jung) and melted onto gelatinized microscopic slides. Prior to the immunostaining, sections were partially delipidated in a series of
graded ethanols (30-90%) and rehydrated in PBS containing 0.1 9; Triton X-100 and 1% bovine serum albumin (BSA).
Cell dissociation Dissociation of trout brain tissue was performed essentially as described 16. Developmental stages 27-37 were decapitated and the brains rapidly removed. For each experiment, the brains of 5-1(I animals were collected in 1 ml of Eagle's minimal essential medium (MEM), buffered with 0.02 M Hepes, pH 7.4. and cut into small fragments with fine scissors. For enzymatic digestion, the samples were successively incubated for 20 rain at 25 °C with 0.25% trypsin (type III, Sigma), 0.01% collagenase (Sigma) and DNase I (0.04 mg/ml). In some cases, trypsin was omitted from the incubation medium to preserve extracellular protein antigens. Subsequently, the tissue was mechanically dissociated by repeatedly aspirating through a 2-ml-glass pipette and syringing through a no. 26 gauge hypodermic needle. The resulting cell suspension was passed through a 85-#m-nylon screen and washed with MEM containing 5% BSA and 1% lamb serum. Approximately 7000 cells suspended in 30 ,ul MEM were transferred onto 13-mm-round-glass coverslips which had been precoated with 0.1 mg/ml poly-L-lysine and allowed to adhere for 30 min. For fixation, the coverslips were immersed in -20 °C cold methanol and rinsed with PBS. Immunofluorescence Fixed cells on coverslips were exposed for 30 rain at room temperature to 2% normal goat serum, then for 30 min to antibodies against the myelin-specific markers (anti-36K, 1:100; anti-IP2, 1:150; anti-BP, 1:50; anti-IP1 hybridoma supernatant, anti-GalC ascitic fluid). After thoroughly rinsing in MEM-Hepes containing 1% BSA, the cells were incubated for 20 min with either biotinylated anti-mouse lgG (1:100) or biotinylated anti-rabbit IgG (1 :100) followed by fluorescein-labeled streptavidin (1:100, 20 rain) with intervening washing in MEM-Hepes. Frozen tissue sections were essentially treated as described above, except for prolonged incubation times of 4 h for the first antibody and 1 h for the streptavidin-biotin system. In double-labeling experiments, the cells
Fig. 1. Immunohistochemical localization of myelin specific compounds in oligodendrocytes freshly dissociated from the CNS of trout. IP2 (a,b), 36K (c,d), GalC (e,0. In unfixed cells (a,c,e), IP2 and GalC show a distinct membrane labeling, whereas 36K remains unstained. In fixed cells (b,d,f), IP2 and 36K show a characteristic pattern of intracellular labeling whereas GalC is not labeled. Scale bars = 20/zm. In b and d, the same cells are shown after double labeling.
29 were simultaneously incubated with either anti-IP1 (raised in mouse) and anti-IP2 (raised in rabbits) or anti-IP1 (raised in mouse) and anti-36K (raised in rabbits), washed extensively with MEMHepes and subsequently treated with a mixture of fluoresceinconjugated anti-mouse IgG (1:100) and rhodamine-labeled goat anti-rabbit IgG (1:100) for 20 min in the dark. In other experiments aimed to visualize extracellular epitopes, fresbly dissociated cells were immunolabeled in suspension. After each incubation and washing step in this case, the cells were recollected by centrifugation (400 g, 5 min). Finally, the cells were left to adhere onto poly-L-lysine-coatedcoverslips and fixed in -20 °C methanol. In all cases, samples were mounted in gelvatol (Monsanto, Springfield, U.S.A.) to which N-propylgallate was added and examined under a Leitz epifluorescence microscope equipped with appropriate filters for viewing fluorescein and rhodamin. Specificity of the immunostaining was controlled by incubating cells in normal rabbit serum or non-immune ascitic fluid instead of polyclonal or monoclonal antibodies.
Quantitation The total number of cells was counted in randomly selected microscopic fields using a 40x objective and the proportion of oligodendrocytes was estimated after immunofluorescence labeling. To exclude possible error due to investigator bias, the cell preparations were coded and examined in a 'blind' manner. For each developmental stage, 3-8 cell preparations were analyzed separately. RESULTS
Pattern of immunostaining in dissociated cells In freshly dissociated cell p r e p a r a t i o n s from larval
trout brain, o l i g o d e n d r o c y t e s could be readily identified by their intense i m m u n o l a b e l i n g with one of the antibodies against myelin specific c o m p o u n d s . A s illustrated in Fig. 1, the cells exhibited a distinct p a t t e r n of immunofluorescence with each of the different myelin markers. G a l C staining was selectively localized on the surface of unfixed, living cells and was completely eliminated after m e t h a n o l fixation (Fig. le,f). By contrast, 36K, which could be visualized only after fixation of the cells, a p p e a r e d uniformly distributed throughout the cytoplasm, whereas the nuclei always r e m a i n e d unstained (Fig. l c , d ) . The myelin glycoproteins, IP1 and IP2, in unfixed cells that had been dissociated in the absence of trypsin showed a m e m b r a n e labeling similar to the G a l C staining, except that it d i s a p p e a r e d after trypsinization. Following fixation, the cells additionally exhibited a characteristic p a t t e r n of intracellular staining consisting of intensely fluorescent patches often located close to the nucleus (Fig. l b ) . This p a t t e r n of immunostaining was identical for both IP1 and IP2. Surprisingly, antibodies against M B P in most cases failed to stain oligodendrocytes from trout brain even in cell p r e p a r a tions that otherwise were distinctly l a b e l e d with one of the o t h e r myelin markers. H o w e v e r , when present, the M B P staining of o l i g o d e n d r o c y t e s was diffuse in the
O
Fig. 2. Immunostaining of IP2 glycoprotein in cell preparations freshly dissociated from the brain of trout at various developmental ages. There is a continous increase in IP2-positive cells during larval development (a, stage 27; b, stage 29; c, stage 32, d, stage 35). Scale bars = 50 ~m.
30
60 lz~0
cytoplasm as for 36K. Control preparations incubated with either normal rabbit serum or non-immune hybridoma supernatants did not show any significant immunolabeling.
IP2
o 20-
O (D
U,
27 28 29 30 31 32 33 34 35 d e v e l o p m e n t a l stage 60-
37
36 K
.> 40~ 20O 13_ O
c
27 28 29 30 31 32 33 34 35
E .E 60] ~S 40
37
IPl
t
C
30-
GalC
~ 2o10" 27 2B 29 30 31
developmental
32 33
stage
Fig. 3. The number of cells expressing IP2, 36K, IPI and GalC, respectively as determined immunohistochemically on freshly dissociated cell preparations of developing trout brain. Arrows indicate time of earliest detection. Results are the mean of at least 3 different experiments. The total number of cells counted for each stage are in the range of 6000 to 12,000.
Phenotypic expression of myelin markers by oligodendrocytes The first myelin protein that could be immunohistochemically detected during trout brain development in freshly dissociated cells was the IP2 glycoprotein. Appearing at stage 28 (8 days before hatching), this protein was expressed by at least one out of 2000 total cells (Figs. 2, 3). At the same time, the first GalC-positive cells could be seen, whereas the remaining major myelin proteins, 36K and 1P1, were not yet detected (Fig. 3). Later during brain development, the number of IP2-1abeled oligodendrocytes progressively increased from t4/200 at stage 29 to 39/2000 at stage 33 (8 days after hatching) to 53/2000 at stage 35 (20 days after hatching, Figs. 2, 3). The teleost-specific 36K protein first emerged at stage 30 (around hatching), when only one out 2000 cells was immunostained. During larval development (stages 3037), the number of oligodendrocytes expressing 36K continuously increased reaching 41/2000 cells at the end of the larval period (stage 37, 30 days after hatching). The third major myelin protein that appeared during oligodendrogliai differentiation was the IP1 gtycoprotein, which was first noted at stage 32 in one out of 2000 cells (Fig. 3). In order to allow a more direct comparison of the order of appearance of myelin proteins in oligodendrocytes during cell differentiation, double-labeling ex-
Fig. 4. Double-labeling immunostaining of IP1 (a) and IP2 (b) on freshly dissociated cells from trout brain at developmental stage 33. Two of the oligodendrocytes already express both myelin glycoproteins, whereas 3 others are IP2-positive but IPl-negative, c: interferential contrast photomicrograph. Scale bars = 25 pm,
31
h
Fig. 5. Double-labeling immunostaining of IP1 (a) and 36K (b) in freshly dissociated cells from trout brain at developmental stage 34. Two of the cells already express both myelin markers whereas a third one is selectively stained for 36K. c: interferential contrast photomicrograph. Scale bars = 20/~m. periments were performed using either mouse anti-trout IP1 and rabbit anti-trout IP2 antibodies (Figs. 4, 6a) or mouse anti-trout IP1 and rabbit anti-trout 36K antibodies (Figs. 5, 6b), respectively. In cell preparations derived from trout brains at stage 32, the great majority of immunopositive cells selectively stained with anti-IP2 antibodies (88%) and only 12% of the labeled cells were double-labeled for both IP1 and IP2, whereas cells staining for IP1 alone were not observed. At stage 34 the proportion of double-labeled cells
a
I
stage 32
stage
34
Irnmunostaining of tissue sections
u)
_-20 tO
%
~10 c~ E :D
b ~20] ~6 ,-10-
significantly increased to 59% of all labeled cells. None of them was IP1 positive and IP2 negative, emphasizing a delayed expression of IP1 compared to IP2. In double-labeling experiments with anti-36K and anti-IP1 antibodies (Figs. 5, 6b), at stage 32 the proportion of oligodendrocytes selectively stained with anti-36K antiserum was slightly higher than those which were stained for both 36K and IP1. Cells expressing IP1 but not 36K did not occur, emphasizing that 36K appears before IP1 during functional maturation of trout oligodendrocytes. Similar results were obtained at stage 34 except that nearly 68% of the oligodendrocytes were double-labeled for both 36K and IP1.
I P 2 IP1
I P 2 IP1
stage 32
stage 34
36K IPI
3 6 K IP1
Q; 4~
E C
Fig. 6. Quantitative evaluation of double-labeling experiments using anti-36K and anti-IP1 antibodies (b) or anti-IP1 and anti-IP2 antibodies (a), respectively. White bars represent the number of IP2+/IP1 cells in a, and of 36K+/IP1 cells in b. Gray bars indicate double-labeled cells.
In order to follow the sequence of deposition of myelin proteins into the nascent myelin sheath, frozen tissue sections of early developmental stages of trout brain were immunohistochemically analyzed. The first myelinating fibers, identified by their immunostaining with anti-IP2 antibodies, were initially detected at stage 29 in the spinal cord and brainstem, whereas in the remaining brain regions, including tectum, cerebellum and forebrain, no immunofluorescent fibers could be detected at that age. As shown in Figs. 7 and 8, on coronal sections these immunofluorescent fibers were typically arranged in two major fiber tracts that were longitudinally aligned on either side of the central canal. On longitudinal sections, these fiber bundles could be further traced into more rostral regions of the brainstem by their immunostaining (not shown here). On adjacent coronal sections through a selected region of the medulla oblongata, it was possible to immunohistochemically analyze the same fiber bundles with antisera against different myelin proteins. As shown in Fig. 7, at stage 30 these fiber bundles were brightly stained with both anti-IP2 as well
32
~ ~ . . . . . . Fig. 7. Localization of IP2 (a), 36K (b), IP1 (c) and MBP (d) on adjacent parasagittal sections through the medulla oblongata of trout brain at developmental stage 30. Note bundles of myetinating nerve fibers which are brightly staining for IP2 and MBP, more weakly staining for 36K and unlabeled with anti-IP1 antibodies. Scale bars = 50/~m.
as anti-MBP antibodies, but were only weakly labeled for 36K, whilst the IP1 glycoprotein was still undetectable. A b o u t one week later at stage 33, in the same region of the medulla oblongata the myelinated fiber bundles had distinctly expanded in size and their immunostaining was now equally strong for IP2, MBP and 36K (Fig. 8). Staining for IP1 was now visibie as well, but it was still significantly weaker than for the other myelin antigens. As was.expected from earlier studies 1~, glial cells in tissue sections of trout brain did not immunostain at any age. Hence a direct comparison of the appearance of myelin markers in the oligodendroglial cells and their deposition in the myelin sheath was not possible. Nevertheless, the developmental stage of intial expression of myelin proteins in dissociated cell preparations correlated well with the earliest occurrence of myelinated fibers staining for both M B P and IP2.
DISCUSSION In the present study, by means of immunocytochemistry, the pattern of intracellular distribution and the order of expression of 4 major myelin-specific compounds were analyzed during oligodendroglial differentiation in the brain of a lower vertebrate species. In dissociated cell preparations of trout brain, a distinct pattern of immunolabeling was observed for 36K on the one hand and the two myelin glycoproteins 1P1 and IP2 on the other. Whereas IP1 and IP2 both typically exhibited a granular pattern of staining, suggesting a processing of these proteins in the endoplasmic reticulum and/or Golgi compartment, 36K appeared uniformly distributed in the cytoplasm, closely resembling the pattern of staining described for MBP in mammalian glial cells 6'8'24. Since MBP in mammalian species has been convincingly shown to be synthesized on free polyri-
Fig. 8. Localization of IP2 (a), 36K (b), IP1 (c) and MBP (d) on adjacent parasagittal sections through the medulla oblongata of trout brain at developmental stage 33. Bundles of myelinating nerve fibers have considerably expanded in size and are labeled equally strong for IP2, 36K and MBP, whereas staining for IP1 is still significantly weaker. Scale bars = 50 pro.
33 bosomes 4, a similar site of synthesis could be suggested for 36K from its pattern of staining described here. In fact, results from recent in vitro metabolic labeling experiments performed with trout optic nerve (Jeserich et al., unpublished) revealed similar kinetics of entry into myelin membranes for both MBP and 36K, lending support to the above interpretation. Clearly, however. additional immunocytochemical work at the electronmicroscopical level will be required to elucidate this question. The first myelin components that could be detected in trout oligodendrocytes during early stages of myelination were GalC and IP2, followed by the appearance of 36K and finally IP1. This order of expression during oligodendroglial differentiation in trout brain was further corroborated by the results of double-labeling experiments, emphasizing a delayed expression of IP1 as compared to 36K or IP2. Immunolabeling of MBP, in contrast, was only rarely detectable in trout oligodendrocytes. This was, however, not unexpected, since in a previous study 1~ it was noted that MBP during trout brain development did not accumulate in oligodendroglial cell bodies beyond the level of immunocytochemical detectability. In general, the temporal order of expression of the major myelin proteins by trout oligodendrocytes correlated well with their order of detection in the nascent myelin sheath as revealed in frozen tissue sections. Similarly, the earliest expression of myelin-related antigens in dissociated cell populations of trout CNS coincided well with the first appearance of myelinated nerve fibers in defined brain regions. From the present data it is concluded that in the CNS of trout, myelinogenesis is initiated in the spinal cord and hindbrain well before hatching and long before myelination starts in the tectum and cerebellum. The latter fully agrees also with the results of earlier biochemical and electronmicroscopical investigations on the developmental course of myelinogenesis in the different parts of trout brain 9"1°. Interestingly, MBP was expressed in parallel with IP2 by fibers which in immediately adjacent sections were only weakly stained for 36K and completely unlabeled for IP1. Accordingly, in the trout CNS, MBP can be regarded as an early marker of myelination. Considering that by means of immunochemical and proteolysis peptide comparison IP1 and IP2 have previously been identified as homologous proteins 12'29, the strongly delayed expression of IP1 as compared to IP2 described here was surprising. This finding, however, is fully consistent with the results of earlier electrophoretic analyses w, demonstrating that the proportion of IP1 is low in myelin isolated from early stages of brain development and is continuously increasing during sub-
sequent myelin maturation. Therefore, it is tempting to speculate that the IP1 glycoprotein might play a role in the stabilization of the myelin sheath, e.g. during myelin compaction. In cell culture experiments 1"5't5 as well as from in vivo studies 18'22'23, it was consistently noted that GalC and Wolfgram protein were the first myelin markers to be expressed by mammalian oligodendrocytes during early myelinogenesis, followed by MBP and PLP 7 after a certain delay. These data, however, are difficult to compare with ours on trout, because in the myelin of fish, neither Wolfgram protein nor PLP nor any immunologically related protein constituents are expressed 29'3°. The two IP components of trout myelin, on the other hand, have been shown to share common antigenic sites with the major myelin glycoprotein of the mammalian PNS, Po 14, hence suggesting a comparison with the biochemical differentiation of Schwann cells. Interestingly enough, Po in Schwann cells, like IP2 in trout oligodendrocytes, obviously represents an early marker of cell differentiation 17, which under in vitro conditions seems to appear slightly before MBP and shortly after GalC. It is interesting to note that during phylogenetic development, too, MBP and Po-like glycoproteins are the first major myelin protein constituents that occur during evolution of the myelin sheath in the vertebrate CNS 3t. Taking into account the striking capacity for remyelination of both Schwann cells and the oligodendroglia of fish CNS, it appears likely that apart from the biosynthesis of Po-like proteins these two types of myelinforming glia do have further biochemical properties in common. Therefore, it will be of particular future interest to analyze the regulation of biochemical differentiation of oligodendrocytes from the CNS of fish in comparison with those of Schwann cells in an in vitro approach. In this regard, it should be first established if trout oligodendrocytes require a persistent neuronal signal to maintain their differentiated phenotype as it is known for Schwann cells 2,17'19 or if they are able to express their myelin-related cell functions in the total absence of neurons over longer periods of time as mammalian oligodendrocytes do 17"26"27. Studies are now underway to isolate and culture the myelin-forming cells from the CNS of trout and to immunohistochemically monitor the expression of myelinogenic functions under controlled cell culture conditions.
Acknowledgements. This study was supported by the Deutsche Forschungsgemeinschaft (Grant Je 114/2-4) and a travel grant from the DAAD (A.M.). The authors are indebted to Dr. B. Ranscht who kindly provided monoclonal anti-GalC antibodies. We also wish to thank M. Monge and I. Suard for expert technical help. Prof. Lueken is thanked for general support and Mrs. Knehans for typing the manuscript.
34 REFERENCES 1 Bologa-Sandru, L., Siegrist, H.P.Z., Graggen, A., Hofmann,
K., Wiesmann, U., Dahl, D. and Herschkowitz, N., Expression of antigenic markers during the development of oligodendroeytes in mouse brain cell cultures, Brain Res., 210 (1981) 217-229. 2 Brockes, J.P., Raft, M.C., Nishiguchi, D.J. and Winter, J., Studies on cultured rat Schwann cells. III. Assays for peripheral myelin proteins, J. Neurocytol., 9 (1979) 66-77. 3 Bunge, R.P., Glial cells in the central myelin sheath, Physiol. Rev., 48 (1968) 197-251. 4 Colman, D.R., Kreibich, G., Frey, A.B. and Sabatini, D.D., Synthesis and incorporation of myelin polypeptides into CNS myelin, J. Cell BioL, 95 (1982) 598-608. 5 Dubois-Dalcq, M., Behar, T., Hudson, L. and Lazzarini, R.A., Emergence of three myelin proteins in oligodendrocytes cultured without neurons, J. Cell Biol., 102 (1986) 384-392. 6 Hartman, B.K., Agrawal, H.C., Kalmbach, S. and Shearer, W.T., A comparative study of the immunohistochemical localization of basic protein in myelin and oligodendrocytes in rat and chicken brain, J. Comp. Neurol., 188 (1979) 273-290. 7 Hartman, B.K., Agrawal, H.C., Agrawal, D. and Kalmbach, S., Development and maturation of central nervous system myelin: comparison of immunohistochemical localization of proteolipid protein and basic protein in myelin and oligodendrocytes, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 4217-4220. 8 Jacque, C.M., Collet, A., Raoul, M., Monge, M. and Gumpel, M., Functional maturation of the oligodendrocytes and myelin basic protein expression in the olfactory bulb of the mouse, Dev. Brain Res., 21 (1985) 277-282. 9 Jeserich, G., A morphological and biochemical study of myelinogenesis in fish brain, Dev. Neurosci., 4 (1981) 373-381. 10 Jeserich, G., Protein analysis of myelin isolated from the CNS of fish: developmental and species comparisons, Neurochem. Res., 8 (1983) 957-969. 11 Jeserich, G. and Jacque, C., Immunohistochemical localization of myelin basic protein in the developing optic system of trout, J. Neurosci. Res., 13 (1985) 529-538. 12 Jeserich, G. and Waehneldt, T.V., Bony fish myelin: evidence for common major structural glycoproteins in central and peripheral myelin of trout, J. Neurochem., 46 (1986) 525-533. 13 Jeserich, G. and Waehneldt, T.V., Characterization of antibodies against major fish CNS myelin proteins: immunoblot analysis and immunohistoehemical localization of 36K and IP2 proteins in trout nerve tissue, J. Neurosci. Res., 15 (1986) 147-158. 14 Jeserich, G. and Waehneldt, T.V., Antigenic sites common to major fish myelin glycoproteins (IP) and to major tetrapod PNS myelin glycoprotein (Po) reside in the amino acid chains, Neurochem. Res., 12 (1987) 821-825. 15 Knapp, EE., Bartlett, W.P. and Skoff, R.P., Cultured oligodendrocytes mimic in vivo phenotypic characteristics: cell shape, expression of myelin-specific antigens, and membrane production, Dev. Biol., 120 (1987) 356-365. 16 Miller, R.H., David, S., Patel, R., Abney, E.R. and Raft, M.C., A quantitative immunohistochemical study of macroglial cell development in the rat optic nerve: in vivo evidence for two distinct astrocyte lineages, Dev. Biol., 111 (1985) 35-41. 17 Mirsky, E., Winter, J., Abney, E.R., Pruss, R.M., Gavrilovic,
18
19
20
21
22
23 24
25
26 27
28 29 30
31 32
J. and Raft, M.C., Myelin specific proteins and glycolipids in rat Schwann cells and oligodendrocytes in cultures, J. Cell Biol., 84 (1980) 483-494. Monge, M., Kadiiski, D., Jacque, C. and Zalc, B., Oligodendroglial expression and deposition of four major myelin consituents in the myelin sheath during development. An in vivo study, Dev. Neurosci., 8 (1986) 222-235. Politis, M.J., Sternberger, N., Ederle, K. and Spencer, P.S.. Studies on the control of myelinogenesis. IV. Neuronal induction of Schwann cell myelin-specific protein synthesis during nerve fiber regeneration, J. Neurosci., 2 (1982) 1252-1266. Raft, M.C., Miller, R.H. and Noble, M., A glial progenitor cell that develops in vitro into an astrocyte or an oligodendroeyte depending on culture medium, Nature (Lond.), 303 (1983) 390-396. Ranscht, B., Clapshaw, P.A., Price, J., Noble, M. and Seifert, W., Development of oligodendrocytes and Schwann cells studied with a monoclonal antibody against galactocerebroside, Proc. Nat. Acad. Sci. U.S.A., 79 (1982) 2709-2713. Reynolds, R. and Wilkin, G.E, Development of macroglial cells in rat cerebellum. II. An in situ immunohistochemical study of oligodendroglial lineage from precursor to mature myelinating cell, Development, 102 (1988) 409-425. Roussel, G. and Nussbaum, J.L., Comparative localization of Wolfgram W1 and myelin basic protein in the rat brain during ontogenesis, Histochem. J., 13 (1981) 1029-1047. Sternberger, N.H., Patterns of oligodendrocyte function seen by immunocytochemistry. In W.T. Norton (Ed.), Advances in Neurochemistry, Vol. 5, Oligodendroglia, Plenum. New York, 1984, pp. 125-173. Sternberger, N.H., Itoyama, Y., Kies, M.W. and Webster, H.E, Immunohistochemical method to identify basic protein in myelin-forming oligodendrocytes of newborn rat CNS, J. Neurocytol., 7 (1978) 251-263. Szuchet, S., Stefansson, K., Wollman, R.L., Dawson, G. and Arnason, B.G., Maintenance of isolated oligodendrocytes in long-term culture, Brain Res., 200 (1980) 151-164. Szuchet, S., Myelin palingenesis: the reformation of myelin by mature oligodendrocytes in the absence of neurons. In H.H. Althaus and W. Seifert (Eds.), Glial-Neuronal Communication in Development and Regeneration, Springer, Heidelberg, 1987, pp. 756-777. Vernier, J.M., Table chronologique du drveloppement embryonaire de la truite arc-en-ciel Salmo gairdneri, Rich. 1836, Ann. Embryol. Morphog., 2 (1969) 495-520. Waehneldt, T.V. and Jeserich, G., Biochemical characterization of the central nervous system myelin proteins of the rainbow trout, Salmo gairdneri, Brain Res., 309 (1984) 127-134. Waehneldt, T.V., Malotka, J., Karin, N.J. and Matthieu, J.-M., Phylogenetic examination of vertebrate central nervous system myelin proteins by electro-immunoblotting, Neurosci. Lett., 57 (1985) 97-102. Waehneldt, T.V., Matthieu, J.-M. and Jeserich, G., Appearance of myelin proteins during vertebrate evolution, Neurochem. Int., 9 (1986) 463-474. Zubriggen, A., Vandevelde, M., Steck, A. and Angst, B., Myelin-associated glycoprotein is produced before myelin basic protein in cultured oligodendrocytes, J. Neuroimmunol., 6 (1984) 41-49.
27
Elsevier BRESD 50992
Developmental expression of myelin proteins by oligodendrocytes in the CNS of trout G. Jeserich 1, A. Mfiller I and C. Jacque 2 ZAbt. Zoophysiologie, Universitiit Osnabriick, Osnabrack (F.R. G.) and 2 Labatoire de Neurochimie, I.N.S.E.R.M.U. 134, HOpital de la Salpetri~re, Paris (France)
(Accepted 13 June 1989) Key words: Myelin protein; Development; Oligodendrocyte; Trout CNS
Using immunohistochemical techniques, the pattern of cytoplasmic staining and the temporal order of expression of 5 major myelin components of oligodendrocytes were studied in the developing central nervous system of trout. The two myelin glycoproteins, IP1 and IP2, in the cytoplasm of glial cells showed a granular pattern of immunostaining, whereas the 36K protein was homogeneously distributed. Analysis of freshly dissociated cells during early stages of myelinogenesis revealed a constant chronological sequence of expression of myelin proteins by the oligodendrocytes: glycoprotein IP2 was the first protein to appear during glial development together with the galactocerebroside GalC at stage 28 followed by the 36K at stage 30 and finally IP1 at stage 32. The deposition of myelin proteins into the nascent myelin sheath occurred in the same chronological order as their expression by oligodendrocytes. Moreover myelin basic protein, which was not detectable in glial cells, on tissue sections was found to appear in parallel with IP2. INTRODUCTION Myelin in the central nervous system (CNS) of vertebrates is produced by oligodendrocytes 3, which during late ontogenesis send out multiple branches of cell processes 25 to ensheath axons in their vicinity in a typical multilamellar fashion. During their cellular differentiation, oligodendrocytes undergo a characteristic sequence of cytoarchitectural and biochemical changes, which in cell culture can be conveniently monitored immunohistochemically by the expression of cell type and stage specific markers. Thus, it was recently shown that a bipotential progenitor cell, which can give rise to both astrocytes and oligodendrocytes 19, first develops into immature oligodendroblasts before maturing into functionally active oligodendrocytes capable of synthesizing a series of myelin specific components, especially proteins (for review see ref. 24). In the past, numerous studies, both in vivo 7'18'22'23 and under cell culture conditions 1"5'15A7'32, have been conducted to analyze the kinetics of appearance of the major myelin proteins in the developing oligodendrocytes and their deposition in the myelin sheath by means of immunohistochemistry. Thereby it was documented that the expression of specific myelinogenic functions by oligodendrocytes is not a synchronous event but typically
proceeds in a sequential manner: ensuing the initial expression of galactocerebrosides (GalC) on the cell surface, probably a key step in oligodendroglial differentiation, the emergence of Wolfgram proteins was noted, followed by the appearance of myelin basic proteins (MBP) and finally proteolipidprotein (PLP) ~s. Up to now, studies along this line have been performed almost exclusively with mammalian species, and to date very little is known about the biochemical differentiation of oligodendrocytes in the brain of lower vertebrates, in general. Recent comparative biochemical analyses of myelin proteins in different vertebrate classes, however, have revealed striking differences in the myelin protein composition of lower vertebrates as compared to those of higher classes3°'31. In the CNS of bony fishes, two or more hydrophobic myelin glycoproteins were encountered (intermediate proteins, IP1, IP2) that were immunologically related to the major peripheral nervous system (PNS) myelin glycoprotein Po but not to P L P 14'29. The CNS myelin of teleost fishes was additionally characterized by a novel protein component of about 36 kDa mol. wt., called 36K 1°, which does not cross-react with one of the known major myelin proteins of the mammalian brain. Taking advantage of the recent availability of specific antibodies against the major fish myelin proteins 12, the present investigation was made using freshly dissociated
Correspondence: G. Jeserich, Dept. Zoophysiology, University of Osnabrfick, Barbarastr. 11, 4500 Osnabriick, F.R.G.
0165-3806/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
28 brain cells to study the d e v e l o p m e n t a l
e x p r e s s i o n of
m y e l i n o g e n i c f u n c t i o n s by o l i g o d e n d r o c y t e s in the C N S o f trout.
MATERIALS AND METHODS
Animals Fertilized eggs and freshly hatched larvae of rainbow trout (Salmo gairdneri, Rich.) which were obtained from the Rosengarten hatchery at Georgsmarienhiitte, ER.G. were raised in constantly aerated water tanks at 10 °C. Antibodies All antibodies used in this study have been previously characterized in detail. Polyclonal antisera against the trout myelin proteins 36K, IP2 and MBP were raised in rabbits as described 12 and used at a 1:100 dilution. The mouse anti-IP1 antibody was a hybridoma IgG produced by spleen cells of BALB/c mice after fusion with hypoxanthine-, aminopterin-, thymidine-sensitive myeloma cells. As was revealed by electroimmunoblotting, this antibody selectively recognized the protein portion of the IP1 myelin glycoprotein (Jeserich and Strotmann, in preparation). Mouse ascitic fluid containing monoclonal antibodies against galactocerebroside was kindly provided by Dr. B. Ranscht and used in a 1:100 dilution 21. Frozen sections Entire heads of trout of larval stages 27, 28, 29, 30, 31, 33 as identified according to the criteria of Vernier et al. 28 were fixed overnight by immersion in 4% paraformaldehyde, dissolved in sodium cacodylate buffer (pH 7.4). After rinsing in phosphatebuffered saline (PBS, pH 7.4), the samples were soaked in 10% phosphate-buffered sucrose solution and rapidly frozen in liquid nitrogen-cooled isopentane. Serial sections (12/~m thick) through the brain were cut in a Frigocut freezing microtome (Reichert-Jung) and melted onto gelatinized microscopic slides. Prior to the immunostaining, sections were partially delipidated in a series of
graded ethanols (30-90%) and rehydrated in PBS containing 0.1 9; Triton X-100 and 1% bovine serum albumin (BSA).
Cell dissociation Dissociation of trout brain tissue was performed essentially as described 16. Developmental stages 27-37 were decapitated and the brains rapidly removed. For each experiment, the brains of 5-1(I animals were collected in 1 ml of Eagle's minimal essential medium (MEM), buffered with 0.02 M Hepes, pH 7.4. and cut into small fragments with fine scissors. For enzymatic digestion, the samples were successively incubated for 20 rain at 25 °C with 0.25% trypsin (type III, Sigma), 0.01% collagenase (Sigma) and DNase I (0.04 mg/ml). In some cases, trypsin was omitted from the incubation medium to preserve extracellular protein antigens. Subsequently, the tissue was mechanically dissociated by repeatedly aspirating through a 2-ml-glass pipette and syringing through a no. 26 gauge hypodermic needle. The resulting cell suspension was passed through a 85-#m-nylon screen and washed with MEM containing 5% BSA and 1% lamb serum. Approximately 7000 cells suspended in 30 ,ul MEM were transferred onto 13-mm-round-glass coverslips which had been precoated with 0.1 mg/ml poly-L-lysine and allowed to adhere for 30 min. For fixation, the coverslips were immersed in -20 °C cold methanol and rinsed with PBS. Immunofluorescence Fixed cells on coverslips were exposed for 30 rain at room temperature to 2% normal goat serum, then for 30 min to antibodies against the myelin-specific markers (anti-36K, 1:100; anti-IP2, 1:150; anti-BP, 1:50; anti-IP1 hybridoma supernatant, anti-GalC ascitic fluid). After thoroughly rinsing in MEM-Hepes containing 1% BSA, the cells were incubated for 20 min with either biotinylated anti-mouse lgG (1:100) or biotinylated anti-rabbit IgG (1 :100) followed by fluorescein-labeled streptavidin (1:100, 20 rain) with intervening washing in MEM-Hepes. Frozen tissue sections were essentially treated as described above, except for prolonged incubation times of 4 h for the first antibody and 1 h for the streptavidin-biotin system. In double-labeling experiments, the cells
Fig. 1. Immunohistochemical localization of myelin specific compounds in oligodendrocytes freshly dissociated from the CNS of trout. IP2 (a,b), 36K (c,d), GalC (e,0. In unfixed cells (a,c,e), IP2 and GalC show a distinct membrane labeling, whereas 36K remains unstained. In fixed cells (b,d,f), IP2 and 36K show a characteristic pattern of intracellular labeling whereas GalC is not labeled. Scale bars = 20/zm. In b and d, the same cells are shown after double labeling.
29 were simultaneously incubated with either anti-IP1 (raised in mouse) and anti-IP2 (raised in rabbits) or anti-IP1 (raised in mouse) and anti-36K (raised in rabbits), washed extensively with MEMHepes and subsequently treated with a mixture of fluoresceinconjugated anti-mouse IgG (1:100) and rhodamine-labeled goat anti-rabbit IgG (1:100) for 20 min in the dark. In other experiments aimed to visualize extracellular epitopes, fresbly dissociated cells were immunolabeled in suspension. After each incubation and washing step in this case, the cells were recollected by centrifugation (400 g, 5 min). Finally, the cells were left to adhere onto poly-L-lysine-coatedcoverslips and fixed in -20 °C methanol. In all cases, samples were mounted in gelvatol (Monsanto, Springfield, U.S.A.) to which N-propylgallate was added and examined under a Leitz epifluorescence microscope equipped with appropriate filters for viewing fluorescein and rhodamin. Specificity of the immunostaining was controlled by incubating cells in normal rabbit serum or non-immune ascitic fluid instead of polyclonal or monoclonal antibodies.
Quantitation The total number of cells was counted in randomly selected microscopic fields using a 40x objective and the proportion of oligodendrocytes was estimated after immunofluorescence labeling. To exclude possible error due to investigator bias, the cell preparations were coded and examined in a 'blind' manner. For each developmental stage, 3-8 cell preparations were analyzed separately. RESULTS
Pattern of immunostaining in dissociated cells In freshly dissociated cell p r e p a r a t i o n s from larval
trout brain, o l i g o d e n d r o c y t e s could be readily identified by their intense i m m u n o l a b e l i n g with one of the antibodies against myelin specific c o m p o u n d s . A s illustrated in Fig. 1, the cells exhibited a distinct p a t t e r n of immunofluorescence with each of the different myelin markers. G a l C staining was selectively localized on the surface of unfixed, living cells and was completely eliminated after m e t h a n o l fixation (Fig. le,f). By contrast, 36K, which could be visualized only after fixation of the cells, a p p e a r e d uniformly distributed throughout the cytoplasm, whereas the nuclei always r e m a i n e d unstained (Fig. l c , d ) . The myelin glycoproteins, IP1 and IP2, in unfixed cells that had been dissociated in the absence of trypsin showed a m e m b r a n e labeling similar to the G a l C staining, except that it d i s a p p e a r e d after trypsinization. Following fixation, the cells additionally exhibited a characteristic p a t t e r n of intracellular staining consisting of intensely fluorescent patches often located close to the nucleus (Fig. l b ) . This p a t t e r n of immunostaining was identical for both IP1 and IP2. Surprisingly, antibodies against M B P in most cases failed to stain oligodendrocytes from trout brain even in cell p r e p a r a tions that otherwise were distinctly l a b e l e d with one of the o t h e r myelin markers. H o w e v e r , when present, the M B P staining of o l i g o d e n d r o c y t e s was diffuse in the
O
Fig. 2. Immunostaining of IP2 glycoprotein in cell preparations freshly dissociated from the brain of trout at various developmental ages. There is a continous increase in IP2-positive cells during larval development (a, stage 27; b, stage 29; c, stage 32, d, stage 35). Scale bars = 50 ~m.
30
60 lz~0
cytoplasm as for 36K. Control preparations incubated with either normal rabbit serum or non-immune hybridoma supernatants did not show any significant immunolabeling.
IP2
o 20-
O (D
U,
27 28 29 30 31 32 33 34 35 d e v e l o p m e n t a l stage 60-
37
36 K
.> 40~ 20O 13_ O
c
27 28 29 30 31 32 33 34 35
E .E 60] ~S 40
37
IPl
t
C
30-
GalC
~ 2o10" 27 2B 29 30 31
developmental
32 33
stage
Fig. 3. The number of cells expressing IP2, 36K, IPI and GalC, respectively as determined immunohistochemically on freshly dissociated cell preparations of developing trout brain. Arrows indicate time of earliest detection. Results are the mean of at least 3 different experiments. The total number of cells counted for each stage are in the range of 6000 to 12,000.
Phenotypic expression of myelin markers by oligodendrocytes The first myelin protein that could be immunohistochemically detected during trout brain development in freshly dissociated cells was the IP2 glycoprotein. Appearing at stage 28 (8 days before hatching), this protein was expressed by at least one out of 2000 total cells (Figs. 2, 3). At the same time, the first GalC-positive cells could be seen, whereas the remaining major myelin proteins, 36K and 1P1, were not yet detected (Fig. 3). Later during brain development, the number of IP2-1abeled oligodendrocytes progressively increased from t4/200 at stage 29 to 39/2000 at stage 33 (8 days after hatching) to 53/2000 at stage 35 (20 days after hatching, Figs. 2, 3). The teleost-specific 36K protein first emerged at stage 30 (around hatching), when only one out 2000 cells was immunostained. During larval development (stages 3037), the number of oligodendrocytes expressing 36K continuously increased reaching 41/2000 cells at the end of the larval period (stage 37, 30 days after hatching). The third major myelin protein that appeared during oligodendrogliai differentiation was the IP1 gtycoprotein, which was first noted at stage 32 in one out of 2000 cells (Fig. 3). In order to allow a more direct comparison of the order of appearance of myelin proteins in oligodendrocytes during cell differentiation, double-labeling ex-
Fig. 4. Double-labeling immunostaining of IP1 (a) and IP2 (b) on freshly dissociated cells from trout brain at developmental stage 33. Two of the oligodendrocytes already express both myelin glycoproteins, whereas 3 others are IP2-positive but IPl-negative, c: interferential contrast photomicrograph. Scale bars = 25 pm,
31
h
Fig. 5. Double-labeling immunostaining of IP1 (a) and 36K (b) in freshly dissociated cells from trout brain at developmental stage 34. Two of the cells already express both myelin markers whereas a third one is selectively stained for 36K. c: interferential contrast photomicrograph. Scale bars = 20/~m. periments were performed using either mouse anti-trout IP1 and rabbit anti-trout IP2 antibodies (Figs. 4, 6a) or mouse anti-trout IP1 and rabbit anti-trout 36K antibodies (Figs. 5, 6b), respectively. In cell preparations derived from trout brains at stage 32, the great majority of immunopositive cells selectively stained with anti-IP2 antibodies (88%) and only 12% of the labeled cells were double-labeled for both IP1 and IP2, whereas cells staining for IP1 alone were not observed. At stage 34 the proportion of double-labeled cells
a
I
stage 32
stage
34
Irnmunostaining of tissue sections
u)
_-20 tO
%
~10 c~ E :D
b ~20] ~6 ,-10-
significantly increased to 59% of all labeled cells. None of them was IP1 positive and IP2 negative, emphasizing a delayed expression of IP1 compared to IP2. In double-labeling experiments with anti-36K and anti-IP1 antibodies (Figs. 5, 6b), at stage 32 the proportion of oligodendrocytes selectively stained with anti-36K antiserum was slightly higher than those which were stained for both 36K and IP1. Cells expressing IP1 but not 36K did not occur, emphasizing that 36K appears before IP1 during functional maturation of trout oligodendrocytes. Similar results were obtained at stage 34 except that nearly 68% of the oligodendrocytes were double-labeled for both 36K and IP1.
I P 2 IP1
I P 2 IP1
stage 32
stage 34
36K IPI
3 6 K IP1
Q; 4~
E C
Fig. 6. Quantitative evaluation of double-labeling experiments using anti-36K and anti-IP1 antibodies (b) or anti-IP1 and anti-IP2 antibodies (a), respectively. White bars represent the number of IP2+/IP1 cells in a, and of 36K+/IP1 cells in b. Gray bars indicate double-labeled cells.
In order to follow the sequence of deposition of myelin proteins into the nascent myelin sheath, frozen tissue sections of early developmental stages of trout brain were immunohistochemically analyzed. The first myelinating fibers, identified by their immunostaining with anti-IP2 antibodies, were initially detected at stage 29 in the spinal cord and brainstem, whereas in the remaining brain regions, including tectum, cerebellum and forebrain, no immunofluorescent fibers could be detected at that age. As shown in Figs. 7 and 8, on coronal sections these immunofluorescent fibers were typically arranged in two major fiber tracts that were longitudinally aligned on either side of the central canal. On longitudinal sections, these fiber bundles could be further traced into more rostral regions of the brainstem by their immunostaining (not shown here). On adjacent coronal sections through a selected region of the medulla oblongata, it was possible to immunohistochemically analyze the same fiber bundles with antisera against different myelin proteins. As shown in Fig. 7, at stage 30 these fiber bundles were brightly stained with both anti-IP2 as well
32
~ ~ . . . . . . Fig. 7. Localization of IP2 (a), 36K (b), IP1 (c) and MBP (d) on adjacent parasagittal sections through the medulla oblongata of trout brain at developmental stage 30. Note bundles of myetinating nerve fibers which are brightly staining for IP2 and MBP, more weakly staining for 36K and unlabeled with anti-IP1 antibodies. Scale bars = 50/~m.
as anti-MBP antibodies, but were only weakly labeled for 36K, whilst the IP1 glycoprotein was still undetectable. A b o u t one week later at stage 33, in the same region of the medulla oblongata the myelinated fiber bundles had distinctly expanded in size and their immunostaining was now equally strong for IP2, MBP and 36K (Fig. 8). Staining for IP1 was now visibie as well, but it was still significantly weaker than for the other myelin antigens. As was.expected from earlier studies 1~, glial cells in tissue sections of trout brain did not immunostain at any age. Hence a direct comparison of the appearance of myelin markers in the oligodendroglial cells and their deposition in the myelin sheath was not possible. Nevertheless, the developmental stage of intial expression of myelin proteins in dissociated cell preparations correlated well with the earliest occurrence of myelinated fibers staining for both M B P and IP2.
DISCUSSION In the present study, by means of immunocytochemistry, the pattern of intracellular distribution and the order of expression of 4 major myelin-specific compounds were analyzed during oligodendroglial differentiation in the brain of a lower vertebrate species. In dissociated cell preparations of trout brain, a distinct pattern of immunolabeling was observed for 36K on the one hand and the two myelin glycoproteins 1P1 and IP2 on the other. Whereas IP1 and IP2 both typically exhibited a granular pattern of staining, suggesting a processing of these proteins in the endoplasmic reticulum and/or Golgi compartment, 36K appeared uniformly distributed in the cytoplasm, closely resembling the pattern of staining described for MBP in mammalian glial cells 6'8'24. Since MBP in mammalian species has been convincingly shown to be synthesized on free polyri-
Fig. 8. Localization of IP2 (a), 36K (b), IP1 (c) and MBP (d) on adjacent parasagittal sections through the medulla oblongata of trout brain at developmental stage 33. Bundles of myelinating nerve fibers have considerably expanded in size and are labeled equally strong for IP2, 36K and MBP, whereas staining for IP1 is still significantly weaker. Scale bars = 50 pro.
33 bosomes 4, a similar site of synthesis could be suggested for 36K from its pattern of staining described here. In fact, results from recent in vitro metabolic labeling experiments performed with trout optic nerve (Jeserich et al., unpublished) revealed similar kinetics of entry into myelin membranes for both MBP and 36K, lending support to the above interpretation. Clearly, however. additional immunocytochemical work at the electronmicroscopical level will be required to elucidate this question. The first myelin components that could be detected in trout oligodendrocytes during early stages of myelination were GalC and IP2, followed by the appearance of 36K and finally IP1. This order of expression during oligodendroglial differentiation in trout brain was further corroborated by the results of double-labeling experiments, emphasizing a delayed expression of IP1 as compared to 36K or IP2. Immunolabeling of MBP, in contrast, was only rarely detectable in trout oligodendrocytes. This was, however, not unexpected, since in a previous study 1~ it was noted that MBP during trout brain development did not accumulate in oligodendroglial cell bodies beyond the level of immunocytochemical detectability. In general, the temporal order of expression of the major myelin proteins by trout oligodendrocytes correlated well with their order of detection in the nascent myelin sheath as revealed in frozen tissue sections. Similarly, the earliest expression of myelin-related antigens in dissociated cell populations of trout CNS coincided well with the first appearance of myelinated nerve fibers in defined brain regions. From the present data it is concluded that in the CNS of trout, myelinogenesis is initiated in the spinal cord and hindbrain well before hatching and long before myelination starts in the tectum and cerebellum. The latter fully agrees also with the results of earlier biochemical and electronmicroscopical investigations on the developmental course of myelinogenesis in the different parts of trout brain 9"1°. Interestingly, MBP was expressed in parallel with IP2 by fibers which in immediately adjacent sections were only weakly stained for 36K and completely unlabeled for IP1. Accordingly, in the trout CNS, MBP can be regarded as an early marker of myelination. Considering that by means of immunochemical and proteolysis peptide comparison IP1 and IP2 have previously been identified as homologous proteins 12'29, the strongly delayed expression of IP1 as compared to IP2 described here was surprising. This finding, however, is fully consistent with the results of earlier electrophoretic analyses w, demonstrating that the proportion of IP1 is low in myelin isolated from early stages of brain development and is continuously increasing during sub-
sequent myelin maturation. Therefore, it is tempting to speculate that the IP1 glycoprotein might play a role in the stabilization of the myelin sheath, e.g. during myelin compaction. In cell culture experiments 1"5't5 as well as from in vivo studies 18'22'23, it was consistently noted that GalC and Wolfgram protein were the first myelin markers to be expressed by mammalian oligodendrocytes during early myelinogenesis, followed by MBP and PLP 7 after a certain delay. These data, however, are difficult to compare with ours on trout, because in the myelin of fish, neither Wolfgram protein nor PLP nor any immunologically related protein constituents are expressed 29'3°. The two IP components of trout myelin, on the other hand, have been shown to share common antigenic sites with the major myelin glycoprotein of the mammalian PNS, Po 14, hence suggesting a comparison with the biochemical differentiation of Schwann cells. Interestingly enough, Po in Schwann cells, like IP2 in trout oligodendrocytes, obviously represents an early marker of cell differentiation 17, which under in vitro conditions seems to appear slightly before MBP and shortly after GalC. It is interesting to note that during phylogenetic development, too, MBP and Po-like glycoproteins are the first major myelin protein constituents that occur during evolution of the myelin sheath in the vertebrate CNS 3t. Taking into account the striking capacity for remyelination of both Schwann cells and the oligodendroglia of fish CNS, it appears likely that apart from the biosynthesis of Po-like proteins these two types of myelinforming glia do have further biochemical properties in common. Therefore, it will be of particular future interest to analyze the regulation of biochemical differentiation of oligodendrocytes from the CNS of fish in comparison with those of Schwann cells in an in vitro approach. In this regard, it should be first established if trout oligodendrocytes require a persistent neuronal signal to maintain their differentiated phenotype as it is known for Schwann cells 2,17'19 or if they are able to express their myelin-related cell functions in the total absence of neurons over longer periods of time as mammalian oligodendrocytes do 17"26"27. Studies are now underway to isolate and culture the myelin-forming cells from the CNS of trout and to immunohistochemically monitor the expression of myelinogenic functions under controlled cell culture conditions.
Acknowledgements. This study was supported by the Deutsche Forschungsgemeinschaft (Grant Je 114/2-4) and a travel grant from the DAAD (A.M.). The authors are indebted to Dr. B. Ranscht who kindly provided monoclonal anti-GalC antibodies. We also wish to thank M. Monge and I. Suard for expert technical help. Prof. Lueken is thanked for general support and Mrs. Knehans for typing the manuscript.
34 REFERENCES 1 Bologa-Sandru, L., Siegrist, H.P.Z., Graggen, A., Hofmann,
K., Wiesmann, U., Dahl, D. and Herschkowitz, N., Expression of antigenic markers during the development of oligodendroeytes in mouse brain cell cultures, Brain Res., 210 (1981) 217-229. 2 Brockes, J.P., Raft, M.C., Nishiguchi, D.J. and Winter, J., Studies on cultured rat Schwann cells. III. Assays for peripheral myelin proteins, J. Neurocytol., 9 (1979) 66-77. 3 Bunge, R.P., Glial cells in the central myelin sheath, Physiol. Rev., 48 (1968) 197-251. 4 Colman, D.R., Kreibich, G., Frey, A.B. and Sabatini, D.D., Synthesis and incorporation of myelin polypeptides into CNS myelin, J. Cell BioL, 95 (1982) 598-608. 5 Dubois-Dalcq, M., Behar, T., Hudson, L. and Lazzarini, R.A., Emergence of three myelin proteins in oligodendrocytes cultured without neurons, J. Cell Biol., 102 (1986) 384-392. 6 Hartman, B.K., Agrawal, H.C., Kalmbach, S. and Shearer, W.T., A comparative study of the immunohistochemical localization of basic protein in myelin and oligodendrocytes in rat and chicken brain, J. Comp. Neurol., 188 (1979) 273-290. 7 Hartman, B.K., Agrawal, H.C., Agrawal, D. and Kalmbach, S., Development and maturation of central nervous system myelin: comparison of immunohistochemical localization of proteolipid protein and basic protein in myelin and oligodendrocytes, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 4217-4220. 8 Jacque, C.M., Collet, A., Raoul, M., Monge, M. and Gumpel, M., Functional maturation of the oligodendrocytes and myelin basic protein expression in the olfactory bulb of the mouse, Dev. Brain Res., 21 (1985) 277-282. 9 Jeserich, G., A morphological and biochemical study of myelinogenesis in fish brain, Dev. Neurosci., 4 (1981) 373-381. 10 Jeserich, G., Protein analysis of myelin isolated from the CNS of fish: developmental and species comparisons, Neurochem. Res., 8 (1983) 957-969. 11 Jeserich, G. and Jacque, C., Immunohistochemical localization of myelin basic protein in the developing optic system of trout, J. Neurosci. Res., 13 (1985) 529-538. 12 Jeserich, G. and Waehneldt, T.V., Bony fish myelin: evidence for common major structural glycoproteins in central and peripheral myelin of trout, J. Neurochem., 46 (1986) 525-533. 13 Jeserich, G. and Waehneldt, T.V., Characterization of antibodies against major fish CNS myelin proteins: immunoblot analysis and immunohistoehemical localization of 36K and IP2 proteins in trout nerve tissue, J. Neurosci. Res., 15 (1986) 147-158. 14 Jeserich, G. and Waehneldt, T.V., Antigenic sites common to major fish myelin glycoproteins (IP) and to major tetrapod PNS myelin glycoprotein (Po) reside in the amino acid chains, Neurochem. Res., 12 (1987) 821-825. 15 Knapp, EE., Bartlett, W.P. and Skoff, R.P., Cultured oligodendrocytes mimic in vivo phenotypic characteristics: cell shape, expression of myelin-specific antigens, and membrane production, Dev. Biol., 120 (1987) 356-365. 16 Miller, R.H., David, S., Patel, R., Abney, E.R. and Raft, M.C., A quantitative immunohistochemical study of macroglial cell development in the rat optic nerve: in vivo evidence for two distinct astrocyte lineages, Dev. Biol., 111 (1985) 35-41. 17 Mirsky, E., Winter, J., Abney, E.R., Pruss, R.M., Gavrilovic,
18
19
20
21
22
23 24
25
26 27
28 29 30
31 32
J. and Raft, M.C., Myelin specific proteins and glycolipids in rat Schwann cells and oligodendrocytes in cultures, J. Cell Biol., 84 (1980) 483-494. Monge, M., Kadiiski, D., Jacque, C. and Zalc, B., Oligodendroglial expression and deposition of four major myelin consituents in the myelin sheath during development. An in vivo study, Dev. Neurosci., 8 (1986) 222-235. Politis, M.J., Sternberger, N., Ederle, K. and Spencer, P.S.. Studies on the control of myelinogenesis. IV. Neuronal induction of Schwann cell myelin-specific protein synthesis during nerve fiber regeneration, J. Neurosci., 2 (1982) 1252-1266. Raft, M.C., Miller, R.H. and Noble, M., A glial progenitor cell that develops in vitro into an astrocyte or an oligodendroeyte depending on culture medium, Nature (Lond.), 303 (1983) 390-396. Ranscht, B., Clapshaw, P.A., Price, J., Noble, M. and Seifert, W., Development of oligodendrocytes and Schwann cells studied with a monoclonal antibody against galactocerebroside, Proc. Nat. Acad. Sci. U.S.A., 79 (1982) 2709-2713. Reynolds, R. and Wilkin, G.E, Development of macroglial cells in rat cerebellum. II. An in situ immunohistochemical study of oligodendroglial lineage from precursor to mature myelinating cell, Development, 102 (1988) 409-425. Roussel, G. and Nussbaum, J.L., Comparative localization of Wolfgram W1 and myelin basic protein in the rat brain during ontogenesis, Histochem. J., 13 (1981) 1029-1047. Sternberger, N.H., Patterns of oligodendrocyte function seen by immunocytochemistry. In W.T. Norton (Ed.), Advances in Neurochemistry, Vol. 5, Oligodendroglia, Plenum. New York, 1984, pp. 125-173. Sternberger, N.H., Itoyama, Y., Kies, M.W. and Webster, H.E, Immunohistochemical method to identify basic protein in myelin-forming oligodendrocytes of newborn rat CNS, J. Neurocytol., 7 (1978) 251-263. Szuchet, S., Stefansson, K., Wollman, R.L., Dawson, G. and Arnason, B.G., Maintenance of isolated oligodendrocytes in long-term culture, Brain Res., 200 (1980) 151-164. Szuchet, S., Myelin palingenesis: the reformation of myelin by mature oligodendrocytes in the absence of neurons. In H.H. Althaus and W. Seifert (Eds.), Glial-Neuronal Communication in Development and Regeneration, Springer, Heidelberg, 1987, pp. 756-777. Vernier, J.M., Table chronologique du drveloppement embryonaire de la truite arc-en-ciel Salmo gairdneri, Rich. 1836, Ann. Embryol. Morphog., 2 (1969) 495-520. Waehneldt, T.V. and Jeserich, G., Biochemical characterization of the central nervous system myelin proteins of the rainbow trout, Salmo gairdneri, Brain Res., 309 (1984) 127-134. Waehneldt, T.V., Malotka, J., Karin, N.J. and Matthieu, J.-M., Phylogenetic examination of vertebrate central nervous system myelin proteins by electro-immunoblotting, Neurosci. Lett., 57 (1985) 97-102. Waehneldt, T.V., Matthieu, J.-M. and Jeserich, G., Appearance of myelin proteins during vertebrate evolution, Neurochem. Int., 9 (1986) 463-474. Zubriggen, A., Vandevelde, M., Steck, A. and Angst, B., Myelin-associated glycoprotein is produced before myelin basic protein in cultured oligodendrocytes, J. Neuroimmunol., 6 (1984) 41-49.