Development 102. 409-425 (1988) 409 Printed in Great Britain © The Company of Biologists Limited 1988 Development of macroglial cells in rat cerebellum II. An in situ immunohistochemical study of oligodendroglial lineage from precursor to mature myelinating cell RICHARD REYNOLDS and GRAHAM P. WILKIN Department of Biochemistry, Imperial College of Science and Technology, London SW7 2AZ, UK Summafy Using immunofluorescence with a panel of antibodies (WM) tracts of the folia, and then away from this that recognize antigens expressed by oligodendroglia, central white matter into the granule cell and Purkinje the myelin-producing cells of the CNS, at different cell layers, and finally the molecular layer. The time stages of differentiation from precursor to mature cell, delay between the expression of GC, CNP and MBP we have investigated the development of cells of this was the same for oligodendroglia in all of these layers, lineage in cryostat sections of rat cerebellum. Our suggesting the presence of an intrinsic clock control- results are consistent with the view that glial precur- ling the initial expression of these myelin components. sors, identified by their expression of the ganglioside The early appearance of CNP in oligodendroglia GD3, arise in the subependymal layers of the 4th ventricle and migrate to their final position in the suggests a role for this protein in the early stages of cerebellum via the superior medullary velum, and to myelinogenesis. some extent the peduncles. As the cells reach their final destination they make the transition to recogniz- able galactocerebroside (GC)-expressing oligoden- + + Abbreviations: GC, galactocerebroside; CNP, 2',3'-cyclic droglia, via a GD3 /GC intermediate. The myelin- nucleotide 3'-phosphohydrolase; MBP, myelin basic associated protein 2',3'-cyclic nucleotide 3'-phospho- protein; WM, white matter; CNS, central nervous hydrolase (CNP) appears at the same time as GC, system; SMV, superior medullary velum; GFAP, glial whereas myelin basic protein (MBP) is expressed 2-3 fibrillary acidic protein. days after GC and CNP, immediately prior to myelin formation. A very clear progression of oligodendro- Key words: oligodendroglia, astroglia, myelination, glial differentiation was observed from the SMV into cerebellum, glial cells, immunohistochemistry, rat, the base of the cerebellum, up into the white matter macroglia] cell. Introduction the oligodendroglial precursor cell into the mature myelin-producing oligodendroglia is crucial to the Although much is known about the morphological understanding of the relative failure of remyelination changes that occur during oligodendroglial differen- following the extensive demyelination seen in mul- tiation from earlier studies, both at the light and tiple sclerosis. Considerable debate still exists con- electron microscopic level (for review see Wood & Bunge, 1984), relatively little is known about the cerning the cell of origin and the mode of differen- sequential biochemical events that occur during the tiation of oligodendroglia in the developing CNS. The acquisition of oligodendroglial characteristics by morphological studies of Skoff et al. (1976) and immature cells, and during the assembly of the Imamoto et al. (1978) suggested that the immediate various myelin components. A knowledge of the precursors of astroglia and oligodendroglia are differ- sequence of events leading to the transformation of entiated enough to be quite distinct from each other. 410 R. Reynolds and G. P. Wilkin More recent studies have used dissociated CNS cell antibody was prepared and characterized as described cultures and immunocytochemistry with cell-specific previously (Reynolds & Herschkowitz, 1986). Rabbit anti- markers to extend these earlier morphological obser- CNP antibodies (provided by Dr E. M. Carey, Sheffield) vations. Using these techniques two groups have and anti-MBP were produced and characterized as de- independently reached the conclusion that a bipoten- scribed elsewhere (Reynolds et al. 1987b). Mouse mono- clonal antibody against MBP (clone 2C3) was provided by tial progenitor glial cell exists in both the optic nerve Dr R. Thompson (Cambridge) and has been characterized and cerebellum that is capable of differentiation into elsewhere (Elfman et al. 1986). Rabbit polyclonal anti- an oligodendroglia or a fibrous astroglia (Raff et al. bodies against the astrocytic marker glial fibrillary acidic 1983; Levi et al. 1986). The time period in early CNS protein (GFAP) were purchased from DAKO Ltd. development during which these multipotential gliob- lasts, identified in dissociated cell culture, can be found in vivo is not known. Even with the advent of Immunofluorescence antibodies as cell-specific markers the major difficulty The indirect immunofluorescent staining technique with in all of these studies remains the identification of frozen sections was used. Sprague-Dawley rats ranging dividing and immature glial cells and the extrapol- from immediately postnatal to adult were anaesthetized ation from development in culture to development in with sodium pentobarbitone (Sagatal, May and Baker) and vivo. In a recent study, we used a monoclonal perfused through the left ventricle for 2min with PBS followed by 5 min with 4 % paraformaldehyde in PBS. The antibody LBj, which binds to G ganglioside (Curtis D3 cerebella were removed and placed in the same fixative for et al. 1987), to show that GD3 ganglioside expression a further 4-5 h and then in 30 % sucrose in PBS overnight. is characteristic of immature neuroectodermal cells in The tissue was frozen in isopentane cooled in solid CO2 and postnatal rat cerebellar cultures and, in particular, a 3-5/im parasagittal sections cut on a cryostat (Reichert- glial progenitor cell similar to that found in the rat Jung) at — 25°C. The sections were mounted on gelatin- optic nerve (Raff et al. 1983). GD3 was also shown to coated slides and placed in a humid box for 1-2 h before be expressed by cells of the germinal subventricular use. Sections were incubated twice with sodium boro- zones of the IVth ventricle, in agreement with Gold- hydride in PBS (0-5mgmr') and then washed extensively. man et al. (1984), and the immediate precursors of For single immunofluorescent staining with anti-CNP and anti-MBP antibodies, the sections were incubated in meth- oligodendrocytes in the rat cerebellum in vivo (Curtis anol (-20°C) for 5 min to improve antibody accessibility to et al. 1987). Thus it has proved possible to investigate the myelin antigens. the early stages of oligodendroglial differentiation in For single immunofluorescent staining of cryostat sec- vivo. tions, incubation with primary antibody was carried out During the period of rapid CNS myelination, a overnight followed by two rinses in PBS and incubation for number of specific proteins and lipids are synthesized 1 h with the appropriate fluorochrome-conjugated immuno- by developing oligodendroglia and, although the in globulins. For double-label immunofluorescence, the sec- vivo distribution and development of a number of tions were incubated overnight with anti-GD3 (ascites fluid these have been individually investigated (Stern- diluted 1:200-1:1000), monoclonal anti-GC (ascites fluid berger et al. 1978; Zalc et al. 1981), very little is diluted 1:200), or rabbit anti-CNP (diluted 1:200) anti- known about the sequential appearance of these bodies, washed twice with PBS and then incubated for 1 h with TRITC-conjugated sheep anti-mouse IgM (Serotec, oligodendroglial components. In the present study, 1:100 for GD3), FTTC-conjugated goat anti-mouse IgG3 we have investigated the development of cells of the (Nordic, 1:100 for GC) or FITC-conjugated swine anti- oligodendroglial lineage in the rat cerebellum using rabbit IgG (Nordic, 1:200 for CNP). After two 10 min PBS immunofluorescence with antibodies that recognize washes, the sections were incubated for 3h with mono- antigens expressed by these cells at different stages of clonal anti-GC antibodies (for GD3/GC colabelling), differentiation, from bipotential precursor to the monoclonal anti-MBP antibodies (ascites diluted 1:500, for mature myelinating cell. CNP/MBP colabelling) or with rabbit anti-CNP, anti- MBP, anti-GFAP or anti-GC antibodies (all diluted 1:200), followed by FITC-conjugated goat anti-mouse IgG3 (for Materials and methods GD3/GC colabelling), TRITC-conjugated goat anti-mouse IgG (for CNP/MBP colabelling), FITC-conjugated swine Antibodies anti-rabbit IgG (Nordic, 1:200, for GD3/CNP, GD3/MBP and G /GFAP colabelling), or TRITC-conjugated goat Mouse monoclonal antibody LB which binds to G D3 1; D3 anti-rabbit IgG (Cappel Labs, 1:400, for GC/CNP, ganglioside, has been described previously (Curtis et al. GC/MBP and GC/GFAP colabelling). Two 10 min PBS 1987) and was provided by Dr J. Cohen (University washes followed each incubation step. In some exper- College, London). Monoclonal antibodies, O3 and O4, iments, to allow anti-CNP and anti-MBP antibodies greater against sulphatide (Singh & Pfeiffer, 1985) were a gift from access to antigens in the myelin sheath, sections were Professor M. Schachner (Heidelberg). Monoclonal anti- incubated for 2 min in methanol at -20°C after the first galactocerebroside (anti-GC) antibody was provided by Dr primary antibody and fluorescence conjugate incubations. B. Ranscht (London; Ranscht et al. 1982). Rabbit anti-GC Oligodendroglial lineage in the cerebellum 411 Control stainings were carried out with either fluorescence- 1985). Although the monoclonal antibodies