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Increased proliferation of oligodendrocytes in the hypomyelinated mouse mutant-jimpy
Brain Research, 248 (1982) 19-31 Elsevier Biomedical Press
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Increased Proliferation of Oligodendrocytes in the Hypomyelinated Mouse Mutant-Jimpy ROBERT P. SKOFF Department of Anatomy, Wayne State University, School of Medicine, 540 E. Canfield, Detroit, M I 48201 (U.S.A.)
(Accepted December 31st, 1981) Key words: jimpy - - myelin - - autoradiography - - neuroglia - - nervous system
Previous studies of the hypomyelinatedmouse mutant jimpy have shown that the number of oligodendrocytesare reduced about 50~. To determine the cause of the cellular reduction, light and electron microscopy were combined with thymidine autoradiographic techniques. The number of neuroglial cells which incorporate radioactive thymidine in the mutants is increased severalfold over control values. Electron microscopic autoradiograms indicate the majority of the labeled cells are oligodendroblasts. However, the total number of glia in the white matter of jimpy and control animals is the same during development and even up to the time of the animal's death. The presence of mitotic cells suggests that the oligodendrocytesundergo division but the abundance of dying cells suggests that they die sometime afterwards. The results of the quantitative autoradiographic studies in combination with our other data strongly suggest that the immediate failure of these cells to form myelin sheaths is due to a shortened life span and/or continued cell proliferation.
INTRODUCTION Numerous mouse mutants (e.g. dystrophic 6, jimpy zz, quaking z3, MSD 24, shiverer 4, trembler 20, twitcher 16) as well as several strains of other mammals (Landrace pig 5, Wistar rat 8, Spaniel dog 12) have been discovered which show abnormalities in the development of myelin. In some mutants, the abnormality is directly associated with the synthesis of myelin (twitcher) in. In others, the defect involves interactions between the myelin-forming cells and other cell types (dystrophic) 1. In the dystrophic mouse, the axons are apparently unable to signal the Schwann cells to make myelin 1. In another strain of mice zl,z2, a correlation exists between the amount of myelin in the CNS and serum levels of thyroxine, a hormone essential for adequate myelination in rodents 2. From the few examples mentioned here it is clear that myelination is regulated by many genes, some of which directly control the synthesis of myelin while others are involved in more remotely related events. With the exception of the twitcher mouse, the cell types directly affected by the genetic 0006-8993/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
mutation and the cause of the myelin deficiency are unknown. The jimpy mouse is one of the most interesting of the myelin mutants because myelin in the central nervous system (CNS) is severely reduced but is apparently normal 3a in the peripheral nervous system (PNS). This phenomenon suggests that the mutation is intrinsic to the CNS. Consequently, almost all studies of this mutant have been limited to the CNS. The cause of the deficiency has not been determined although biochemical 3,7,45 and morphologic11,22,a2 studies have documented that myelin is reduced as much as 95 ~. It is important to note here that other abnormalities in the jimpy mutants have been described which may be solely or partially responsible for the hypomyelination. The number of oligodendrocytes in these mutants is reduced about 50 ~19,~3,z4. Besides the oligodendrocyte abnormalities, the astrocytes show a striking hypertrophy of their processes 34. This hypertrophy begins when myelination begins and it has been postulated that this astrogliosis may interfere with the development of the oligodendrocyte line and subsequent myelina-
20 tion ~4. More recently, Hertz et al. 13 showed astrocytes grown in tissue culture from the jimpys exhibit abnormalities in their metabolism. The above observations suggest that the genetic defect in jimpy may not involve an abnormality in formation of myelin but rather interactions between the oligodendrocyte and other cells. They also suggest that the development of the oligodendrocyte cell line is defective. The present study was undertaken to determine the basis for the loss of oligodendrocytes. MATERIALSAND METHODS Jimpy mice (jpTa/Y) were bred in our laboratories by mating carrier females (jpTal+-[-) with Tabby males (Ta+/Y) obtained from Jackson Laboratories (Bar Harbor, ME). The jimpy gene is maintained in the heterozygous female along with another closely linked gene (Tabby). Because of the Tabby fur markings, it can be used to help identify the jimpy mutants before tremors appear at 12 days of age. Normal males ( + +/y) and females ( + +/ta+) including crossover littermates (Ta+/ta+) were used as controls. Crossover females for the jimpy gene (Tajp/Ta+ ;+jp/Ta+) which exhibited mosaicism in the optic nerve were not used~0. All mice were given a single intraperitoneal injection (7.5/zCi/g) of [methyl-ZH]thymidine (spec. act. 17-20 Ci/mM) (New England Nuclear) and sacrificed 1 h after the injection. Mice were injected at 4, 7, 9, 12, 16-18, 20 and 23 days after birth. The mice were sacrificed by intracardiac perfusion following anesthetization with chloral hydrate. The perfusate consisted of 2 ~ glutaraldehyde and 2 ~ formaldehyde in either a 0.1 M cacodylate or 0.1 M phosphate buffer at pH 7.2. The intracranial segment of the optic nerves and the upper brachial region of the spinal cord were dissected out, placed in fixative and refrigerated overnight. The next day, the tissue was osmicated, dehydrated in a graded series of alcohols, then absolute propylene oxide and finally embedded in Araldite plastic. Light microscopic autoradiograms of 1/~m thickness of optic nerves and spinal cords were prepared as previously described37. The number of animals used at each stage is presented in Figs. IA and lB. Sections were spaced at 10-I 5 #m intervals to avoid counting the same cells. Quantitation of labeled cells
was done under oil immersion at 1000 × magnification. The labeling index for the optic nerve was determined by counting the labeled cells and the unlabeled cells in complete transverse sections. Both sides of the nerve were analyzed; at least two different sections for each side were quantitated and averaged, and the values for both sides averaged. In the spinal cord, only the glial cells in the white matter on one side were quantitated; at least two different sections were counted and averaged for each animal. Electron microscopic autoradiograms were prepared from the 4, 7, 9, 16 and 20 day animals. The animals used for electron microscopy were chosen on the basis of the quality of fixation and the abundance of labeled cells. The procedure for preparing electron microscopic autoradiograms is detailed in Skoff et al. 37. Approximately 40 different cells were examined in the normal mice at different stages of development and more than 100 in the mutants. The majority of labeled cells examined are from the spinal cord as the area sampled is considerably greater than the nerve. RESULTS
Light microscopic autoradiography The number of glial cells incorporating radioactive thymidine was investigated in the spinal cord and optic nerve at 7 different stages of postnatal development. The earliest stage studied was at 4 days after birth and the last at 23 days postnatal which is close to the time of their death (25-30 days). The animals were sacrificed 1 h after the injection so that the identity of the proliferating cells could be ascertained. In normal animals the labeling index progressively declines from the time of birth until 20 days postnatally (Figs. 1A and 1B). The increase in the labeling index from 4 to 7 days in the nerve is possibly due to the later onset of myelination there. The mutants show a similar decline in their labeling pattern but it never decreases to the background levels seen for the normals. In the spinal cord, the mutants have a significantly higher percentage of labeled glia than controls at all stages of development except at 4 days postnatal. The difference in the labeling index between the jimpys and controls becomes more pronounced with age. At 9 days after birth, about a twofold difference exists in the cord;
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Fig. 1. A and B : the percentage of labeled glial cells in the spinal cord and optic nerve are shown at different stages of postnatal development. The mice were given a single i.p. injection of radioactive thymidine and sacrificed 1 h later. The number beside each point indicates the number of animals used at each stage of development. In the optic nerve, complete transverse sections of the intracranial segment were quantitated; the cell counts in the spinal cord were made from the white matter on one side of the cervical cord. Details of the cell counting are presented in Materials and Methods. C and D: the mean number of glial nuclei in the spinal cord and optic nerve are shown at the different stages of development. Glial nuclei in the spinal cord were counted from the white matter on one side and from complete transverse sections of the optic nerve. The same sections and the same number of animals were used as in A and B.
at 12 days the differential is 3; at 16, 6; and at 20, 8. In the optic nerve, the labeling index differs most strikingly between the mutants and the controls from 16 days onwards. As in the spinal cord, the difference increases with time. At 16 days, the difference is about threefold but at 20 and 23 days, when cell proliferation in normals is reduced to minimal levels, almost a tenfold difference is present. After the second postnatal week when cell proliferation has slowed considerably, light microscopic autoradiograms illustrate dramatically the difference in labeling between the two sets of animals (Fig. 2). The pictures shown here are a dramatic expression of the differences in the labeling index and they are typical of most of the animals. Almost without exception, the labeled cells are small and it is difficult to distinguish any cytoplasm surrounding the nucleus. After the first week a few labeled cells can be identified as microglial cells on the basis of cytoplasmic lipid inclusions.
The number of glial cells quantitated for determining the labeling index is shown in Fig. 1C and 1D. The results show that there is no major increase in the number of glial cells in the mutants in comparison to the controls, even at the later stages of development. The standard deviations for normal and mutants overlap at all stages of development and they generally show large fluctuations between animals. Such variability is not surprising because we have shown that even within a particular animal, the number of glial cells counted in serial transverse sections may change more than 20% within a 100 /~m distance 85,39.
Electron microscopic autoradiography The ultrastructure of cells incorporating [3H]thymidine was analyzed in both spinal cord and optic nerve at the different stages of development. The same types of glial cells are labeled in normal mice as in the rat 37. The vast majority of the cells labeled
Fig. 2. A and B: light microscopic autoradiograms showing the dorsal funiculi of spinal cords from 16-day-old mice. The dorsal funiculus of the normal mouse (A) is densely packed with myelinated fibers but the same area in the mutant has only a few scattered fibers. The control has one labeled cell (arrow) whereas the mutant has at least 9 labeled ceils (arrows). Many of the cells in the normal animals can be identified as astrocytes or oligodendrocytes on the basis of their staining but the cells in the mutants are small and difficult to identify. Degenerating cells (arrowheads) are numerous in the mutants, x 600.
23
Fig. 3. This electron microscopic autoradiogram is from the spinal cord of a 7-day-old normal mouse. The cytoplasmic and nuclear features of the labeled cell are characteristic of immature oligodendroglia. The density of the perikaryon and nucleus contrasts sharply with axoplasm and astrocytic processes. Free ribosomes, cisternae of the Golgi apparatus (G), numerous small mitochondria and thin strands of endoplasmic reticulum (ER) occupy the cytoplasm of immature oligodendroglia. The Golgi apparatus is adjacent to the nucleus and surrounded by mitochondria and endoplasmic reticulum. The nucleus and cytoplasm are usually located at opposite ends of the cell. x 20,000. during p o s t n a t a l d e v e l o p m e n t can be classified as a s t r o b l a s t s a n d o l i g o d e n d r o b l a s t s . The a s t r o b l a s t s are f o u n d p r i m a r i l y d u r i n g the first p o s t n a t a l week a n d the o l i g o d e n d r o b l a s t s afterwards. This is as expected since the time o f final cell division for astrocytes occurs p r e n a t a l l y a n d during t h e first
week o f p o s t n a t a l d e v e l o p m e n t 3s. T h e l a b e l e d astroblasts a n d o l i g o d e n d r o b l a s t s a p p e a r slightly m o r e differentiated in the m o u s e t h a n their c o u n t e r p a r t s in the rat. The a s t r o b l a s t s in mice usually have distinct clusters o f filaments within their c y t o p l a s m whereas filaments in the r a t are often lacking o r n o t
24 aggregated into prominent clusters. Oligodendroblasts in mice have a dark cytoplasmic matrix which is nearly identical in density to fully mature forms. A detailed description of the morphology of oligodeodroblasts and astroblasts has been presented previously 36,~7 and the emphasis in this paper will be placed upon the differences between oligodendroglia in normal and mutant mice. The morphology of the oligodendroblasts in normal mice is the same regardless of the stage of the animal's development (Fig. 3). The nucleus of the labeled oligodendroblast is characteristically located at one end of the cell. Immediately adjacent to the nucleus are many cisternae of the Golgi apparatus. Surrounding the Golgi and closer to the cell membrane are numerous mitochondria and short cisternae of granular endoplasmic reticulum. Microtubules and cisternae of rough endoplasmic reticulum are scattered throughout the cytoplasm but are not as prominent as in the mature forms. The nucleoplasm has a rather hazy appearance and a thin rim of chromatin surrounds the
nuclear membrane. The morphology of oligodendroglia can be compared and contrasted with that of a mononuclear cell which was observed to be labeled in a blood vessel of a normal mouse (Fig. 4). Chromatin in the monocyte forms large, dense patches which are scattered throughout the nucleoplasm. At the nuclear membrane, the chromatin is interrupted by lightly stained channels. These channels are lacking or at least not as prominent in macroglia. The morphology of labeled oligodendroblasts in the mutants (Figs. 5 and 6) is similar to their normal counterparts in most respects but there are several differences which should be mentioned. The electron density of their cytoplasm is less than that of normals, and while they contain the same types of organelles, their overall number, especially ribosomes and mitochondria, appears reduced in the oligodendroglia of jimpy (Figs. 5 and 6). At the later stages of development (16 and 20 days), the organization of the cytoplasm in some labeled oligoden-
Fig. 4. A labeled mononuclear cell in the lumen of a blood vessel of a normal 7-day-old mouse. The cytoplasm of this cell is packed with scattered ribosomes, a few cisternae of the Golgi apparatus (G) and mitochondria. The electron density of this cell is similar to that of oligodendroglia but the chromatin pattern is quite different from macroglia. × 16,000.
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Fig. 5. A labeled oligodendroblast from the spinal cord of a 16-day-old jimpy. The cytoplasmic density of oligodendroglia in the mutants is usually less than that of their normal counterparts but still considerably more dense than adjacent astroglial processes (AP) and axonal cytoplasm (A). The cytoplasm funnels down into a process packed with microtubules (M). Note the centriole (C) and the dense bodies (DB). Compare the cytoplasm of this oligodendrocyte with that of the adjacent microglial cell (Mg). The cytoplasm of microglia usually have a grainy appearance which contain a variety of different inclusions. The endoplasmic reticulum (Er) is of a narrow caliber and often branches (arrows) as it winds throughout the cytoplasm. × 18,000.
26
Fig. 6. The nucleus of this labeled oligodendroblast has been grazed so that the chromatin forms a broad band beneath the nuclear envelope. This cell is from a jimpy animal at 16 days of age. The cytoplasm is packed with mitochondria, cisternae of the Golgi apparatus (G), and a few strands of endoplasmic reticulum. × 17,000. droblasts appears more differentiated than oligodendroblasts in the normal animal (Figs. 5 and 6). For example, the cluster of microtubules seen in the cell in Fig. 5 and the stacking of rough endoplasmic reticulum in the cell in Fig. 6 has never been observed in labeled oligodendroblasts of normal rats or mice. In the jimpy animals, labeled oligodendroblasts were observed in the spinal cord at all stages of development and in the optic nerve from 7 days on-
wards. At 16 and 20 days in the mutants, at least 50 ~o of the total number of labeled cells are oligodendroblasts. With the exception of one possible cell, astroblasts did not show label at 16 days and afterwards. In addition to labeled macroglia, microglial cells are abundantly labeled (Fig. 7) at the later stages. They account for 20-30 ~ of the labeled cells at 16 and 20 days. Microglial cells can be identified by their long, stringy endoplasmic reticulum, the
27
Fig. 7. A labeled microglial cell from a 16-day-old mutant. The cytoplasm contains long, stringy, endoplasmic reticulum (Er), mitochondria and a variety of different inclusions ranging from lipid droplets (L) to vacoules (V) containing floccular substance. Microtubules, filaments and glycogen are not found in microglia. Ribosomes are less densely packed than in oligodendroglia and they give a grainy appearance to the cytoplasm similar to that seen in the mononuclear cell (Fig. 4). × 18,000.
28 presence of numerous inclusions, moderate density and the absence of intermediate filaments and microtubules. The microglial cells in the mutants usually contain a variety of inclusions and are somewhat similar in appearance to reactive forms seen in experimental conditions such as Wallerian degeneration 41. The remainder of the labeled cells could not be positively identified because of the paucity of cytoplasm and lack of sufficient morphological features. Most of these cells appear to be oligodendrocytes based on the similarity of nuclear features to oligodendrocytes. DISCUSSION The severity of the myelin reduction in the CNS of the jimpy mouse has been documented in numerous biochemical 3,7,15,21,22,45 and morphological studies 19,2a,2s,3°. Biochemical analyses of myelin components, such as myelin basic protein, indicate reductions of as much as 95 ~3,7,45. Morphologic studies of different fiber tracts throughout the brain show that less than 5 ~ of the axons are myelinated (refs. 28, 30, 42). To date, however, the cell type directly affected by the genetic mutation has not been determined. As myelin in the peripheral nervous system (PNS) appears normal a~, it is reasonable to assume that the defect involves either the neuroglial cells or the CNS neurons. Consequently, virtually all studies of this mutant have been limited to the CNS. Several recent studies strongly suggest that the anon is not the site of the mutation. The female carriers of the jimpy gene exhibited mosaicism in their optic nerves with the result that some areas of the nerve are unmyelinated while others are myelinated 4°. As the patches of myelinated and unmyelinated areas occur along the nerve at different levels, this indicates that an individual axon is both myelinated and unmyelinated along its course. If the defect were in the anon, there should be a small population which are myelinated continuously along their length. Other evidence that the axon is not at fault comes from tissue culture studies of Wolff et al.4L In co-cultured explants containing mutant cerebella and normal optic nerve, the mutant cerebella showed an increase in the number of myelinated fibers. This increase is probably due to an influx of oligodendroglia from normal tissue
which then myelinate the axons 4a. Although biochemical studies have not focused directly upon membrane properties of the axons, markers for neurons appear normapO, 14. An additional argument against the axon as the site of the mutation relates to the presence of myelin in the PNS and its paucity in the CNS. If the anon is postulated to be the site, then it must be further postulated that the axonal membrane is abnormal in the CNS and normal in the PNS. While this is a possibility, it seems unlikely that the axonal membrane should change so abruptly. Although the evidence against the neuron is not yet conclusive, it seems more reasonable to focus our attention upon primary defects in the neuroglial cells. The astrocytes show an abnormal increase in their processes that begins around the same time as myelinogenesis 34,a5. We have postulated that the astrocytes may prevent interactions between the oligodendrocytes and axons either mechanically or biochemically. Whether the astrocytic hypertrophy is a primary defect or a secondary response has not been determined but a recent tissue culture study by Hertz et al.13 indicates that the astrocytes have an intrinsic abnormality in their oxygen metabolism. As the astrocytes were cultured shortly after birth and before myelination has progressed very far, it is unlikely that the metabolic abnormality represents a secondary response on the part of the astrocyte. The most likely candidate for the mutation site is the oligodendrocyte but evidence for a specific abnormality in this cell type is lacking. Numerous biochemical studies have documented reductions of the myelin components and the enzyme systems involved in the formation of myelin but no particular defect has yet been detected. The morphology of the oligodendrocyte is not particularly revealing either, and the cell does not appear strikingly abnormal except for the fact that it is less differentiated than its normal counterpart. They rarely contain inclusions and this may be significant because in experimental conditions in which myelination has been blocked, the oligodendrocytes contain inclusions of various sorts 9,29. In mice treated with inhibitors of cholesterol biosynthesis, the Schwann cells show a variety of inclusions 29. Administration of CNS antiserum in tissue culture also leads to an accumulation of debris in the oligodendrocytes9. In metachromatic leuko-
29 dystrophy, the absence of cerebroside sulfotransferase (arylsulfastase A), an enzyme which degrades cerebroside sulfate to cerebroside and sulfate, produces an accumulation of cytoplasmic inclusions within oligodendrocytes27. While these observations do not eliminate a defect in myelin synthesis or degradation, they suggest that the cause of the defect may affect more fundamental cellular processes such as glial metabolism, the development of the oligodendrocyte cell line or cellular interactions. The number of oligodendrocytes present in hypomyelinated mice is the most fundamental question that needs investigation. In jimpy mice the number of glial cells has been investigated at the light and electron mlcroscoplc levels in several studies• Light microscopic studies of the spinal cord and corpus callosum indicate the number of oligodendrocytes are significantly reducedtg,2a,28,a0. A quantitative electron microscopic study of the optic nerve indicates a reduction of about 50 7o84. A precise figure is difficult to come by because marry of the cells in jimpy are difficult to classify. These cells have sparse cytoplasm with few identifying features and classification as an oligodendrocyte or undifferentiated glia is often subjective. The cellular loss illustrates two peculiarities of the jimpy mutants. First, while oligodendrocytes are reduced in number, those present are not myelinating as many axons as possible and second, the cellular reduction is an indication that the cell line is affected. The present study was undertaken to determine the nature of the oligodendrocyte reduction. Using thymidine autoradiography in combination with light and electron microscopy, the extent of cell proliferation as well as the morphology of the proliferating cells was examined. Our results show that the labeling index between the normal animals and the mutants is quite different. In the spinal cord, the labeling index is significantly higher in the jimpys than in normals after the fourth day of development. In the nerve, significantly higher indices are present after the second week. The difference between jimpys and controls becomes most striking when cell proliferation slows to background levels in normal mice at 16 days. The differential is such that it should lead to a dramatic increase in the number of glial cells by the time of their death. It can be calculated that the number of glia should at least
double in cross-sections of the spinal cord if the length of their cell cycle is normal (about 20 h)17,18 and if it is further assumed that half of the cells exit from the cell cycle and the others continue to divide. In the optic nerve, the number of glia should almost double by 20 days postnatal. The quantitative data for both the spinal cord and optic nerves shows no sign of an increase at any time during the animal's life. However, mitotic figures are found at all stages of the mutant's life. The increase in cell proliferation and the presence of mitotic figures strongly suggest that the cells are capable of normal cell division. The fate of the dividing neuroglial cells is not certain but it appears that some apparently die prematurely while others continue to divide. Dying cells are especially common at the later stages of development. While dying glia are occasionally present in normal development, the mutants often have clusters of dying cells scattered throughout the white matter. Whether certain populations of cells are destined to die and others continue to divide or whether it is random is unknown at this stage of our investigation. The increased cell proliferation and death would not necessarily be significant were it not for the fact that the vast majority of the labeled cells are oligodendroblasts. Because of the astroglial hypertrophy, it might be expected that this cell type would be predominantly labeled. This finding is in agreement with our ultrastructural quantitative studies which show that astrocytes do not increase in number z4. It illustrates the fact that astroglial hypertrophy can occur without an accompanying hyperplasia. The proliferation of one cell type also suggests that there is a specific stimulus for the oligodendrocytes. In regards to the continued proliferation of oligodendroglia throughout the life of the jimpy, it cannot be argued that their time of origin is simply delayed as they are also labeled during the first week of postnatal development. This is in keeping with their time of origin in normal animalszS. A more important question is whether the increased proliferation of oligodendrocytes is a primary or secondary phenomenon. Arguments for a secondary effect come from studies of the PNS. The introduction of unmyelinated neurons into Schwann cell cultures stimulates the ensheathing cells to divide 44. In the dystrophic mouse25 and in Trembler26, proliferation
30 o f Schwann cells occurs t h r o u g h o u t the life o f the animal. The u n m y e l i n a t e d axons in t h e j i m p y m a y p r o v i d e a similar stimulus. Proliferation o f oligod e n d r o c y t e s might also be triggered b y other factors such as the extracellular a c c u m u l a t i o n o f myelin c o m p o n e n t s in j i m p y . Their d e a t h might also be explained in a similar manner. However, until a d d i t i o n a l evidence is a c c u m u l a t e d a b o u t cell proliferat i o n a n d d e a t h in the oligodendrocytes, it is p r e m a ture to conclude t h a t the o l i g o d e n d r o g l i a l changes are p r i m a r y o r secondary. A l t h o u g h the increased cell p r o l i f e r a t i o n a n d d e a t h is likely to be triggered b y s o m e o t h e r f a c t o r (either intrinsic or extrinsic to the oligodendrocyte), myelinogenesis c a n n o t occur in a system in which the o l i g o d e n d r o c y t e s are short lived o r are c o n t i n u a l l y dividing. I n this sense, the f a c t o r i m m e d i a t e l y responsible for the h y p o m y e l i o a t i o n is the a b n o r m a l cellular d e a t h a n d proliferation.
One a p p r o a c h to solving this p r o b l e m is to study the female carrier o f the j i m p y gene. A s she lives to a d u l t h o o d , cell p r o l i f e r a t i o n a n d cell d e a t h can be studied over an extended p e r i o d o f time. T h e m o s a i cism in the optic nerve 40 p r o v i d e s a unique system to study the c a p a c i t y o f n o r m a l o l i g o d e n d r o c y t e s to invade a n d myelinate those areas affected b y the m u t a tion.
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optical study, Acta neuropath., 21 (1972) 272-281. 12 Griffiths, I. R., Duncan, I. D., McCullock, M. and Harvey, M. J. D., Shaking pups: a disorder of central myelination in the Spaniel dog, J. Neural Sci., 80 (1981) 423-433. 13 Hertz, L., Chaban, G. and Hertz, E., Abnormal metabolic response to excess potassium in astrocytes from the Jimpy mouse; a convulsing neurological mutant, Brain Research, 181 (1980) 482-487. 14 Jacque, M., Baumann, A. and Bock, E., Quantitative studies of the brain specific antigens GFA, 14-3-2, synaptin C1, D1, D2, D3, and D5 in jimpy mouse, Neurosci. Lett., 3 (1976) 41-44. 15 Kandutsch, A. A. and Saucier, S. E., Regulation of sterol synthesis in developing brains of normal and Jimpy mice, Arch. Biochem. Biophys., 135 (1969) 201-208. 16 Kobayashi, K., Yamanaka, Y., Jacobs, J. M., Teixeira, R. and Suzuki, K., The Twitcher mouse: an enzymatically authentic model of human globoid cell leukodystrophy (Krabble Disease), Brain Research, 202 (1980) 479-483. 17 Korr, H., Schultze, B. and Maurer, W., Autoradiographic investigations of glial proliferation in the brain of adult mice. I. The DNA synthesis phase of neuroglia and endothelial cells, J. comp. NeuroL, 150 (1973) 169-176. 18 Korr, H., Schultze, B. and Maurer, W., Autoradiographic investigations of glial proliferation in the brain of adult mice. I I. Cycle time and mode of proliferation of neuroglia and endothelial cells, J. comp. NeuroL, 160 (1975) 477490. 19 Kraus-Ruppert, R., Herschkowitz, N. and Furst, S., Morphological studies on neuroglial cells in the corpus callosum of the jimpy mutant mouse, J. Neuropath. exp. NeuroL, 32 (1973) 197-203. 20 Low, P. A. and McLeod, J. G., Hereditary demyelinating neuropathy in the Trembler mouse, J. neurol. Sci., 26 (1975) 565-574. 21 Neskovic, N. M., Sarlieve, L. L. and Mandel, P., Biosynthesis of glycolipids in myelin deficient mutants: brain
1 Aguayo, A. J., Bray, G. M. and Perkins, S. C., AxonSchwann cell relationships in neuropathies of mutant mice, Ann. N.Y. Acad. Sci., 317 (1979) 512-531. 2 Balazs, R., Brooksbank, B. W. L., Davison, A. N., Eayrs, J. T. and Wilson, D. A., The effect of neonatal thyroidectomy on myelination in the rat brain, Brain Research, 15 (1969) 219-232. 3 Barbarese, E., Carson, J. H. and Braun, P. E., Subcellular distribution and structural polymorphism of myelin basic protein in normal and Jimpy mouse brain, J. Neurochem., 32 (1979) 1437-1446. 4 Bird, T., Farrell, D. F. and Sumi, S. M., Brain lipid composition of the shiverer mouse: genetic defect in myelin development, at. Neurochem., 31 (1978) 387-391. 5 Blakemore, W. F., Harding, J. D. J. and Done, J. T., Ultrastructural observations on the spinal cord of a Landrace pig with congenital tremor type AIII, Res. Vet. Sci., 17 (1974) 174-178. 6 Bradley, W. G. and Jenkison, M., Abnormalities of peripheral nerves in murine muscular dystrophy, J. NeuroL Sci., 18 (1973) 227-247. 7 Campagnoni, A. T., Campagnoni, C. W., Huang, A. L. and Sapugna, J., Developmental changes in the basic proteins of normal and Jimpy mouse brain, Biochem. biophys. Res. Commun., 46 (1972) 700-707. 8 Csiza, C. K. and deLahunta, A., Myelin deficiency (md) a neurologic mutant in the Wistar rat, Amer. J. PathoL, 95 (1979) 215-219. 9 Diaz, M., Bornstein, M. B. and Raine, C. S., Disorganization of myelinogenesis in tissue culture by anti-CNS antiserum, Brain Research, 154 (1978) 231-239. 10 East, J. M. and Dutton, G. R., Muscarinic binding sites in developing normal and mutant mouse cerebellum, J. Neurochem., 34 (1980) 657-661. 11 Farkas-Bargeton, E., Robain, O. and Mandel, P., Abnormal glial maturation in the white matter in jimpy mice. An
ACKNOWLEDGEMENTS The a u t h o r extends his a p p r e c i a t i o n to Dr. K e n Liu for technical assistance a n d to Dr. Joyce Benjamins a n d A n n e Skoff for their critical review o f the manuscript. This research was s u p p o r t e d b y N I H G r a n t N S 15338.
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glycosyl transferase in Jimpy and Quaking mice, Brain Research, 42 (1972) 147-157. Nussbaum, J. L., Neskovic, N. and Mandel, P., A study of lipid components in brains of the 'Jimpy' mouse, a mutant with myelin deficiency, J. Neurochem., 16 (1969) 927-934. Meier, C. and Bischoff, A., Oligodendroglial cell develop ment in Jimpy mice and controls, an electron-microscopic study in the optic nerve, J. Neurol. Sci., 26 (1975) 517-528. Meier, H. and MacPike, A. D., A neurological mutant (msd) of the mouse causing a deficiency of myelin synthesis, Exp. Brain Res., 10 (1970) 512-525. Perkins, C. S., Bray, G. M. and Aguayo, A. J., Persistant multiplication of axon-associated cells in the spinal roots of dystrophic mice, Neuropath. appL NeurobioL, 6 (1980) 83-91. Perkins, C. S., Aguayo, A. J. and Bray, G. M., Behavior of Schwann cells from Trembler mouse unmyelinated fibers transplanted into myelinated nerves, Exp. NeuroL, 71 (1981) 515-526. Poduslo, S. E., Tennekoon, G., Price, D., Miller, K. and McKhann, G. M., Fetal metachromatic leukodystrophy: pathology, biochemistry and a study of in vitro enzyme replacement in CNS tissue, J. Neuropath. exp. Neurol., 35 (1976) 622-632. Privat, A., Robain, O. and Mandel, P., Aspect ultrastructuraux du corps calleux chez la souris Jimpy, Acta neuropath., 21 (1972) 282-295. Rawlins, F. A. and Uzman, B. G., Retardation of peripheral nerve myelination in mice treated with inhibitors of cholesterol biosynthesis, J. Cell BioL, 46 (1970) 505-517. Robain, O. and Mandel, P., Etude quantitative de la myelinisation et de la croissance axonale dans le corps calleux et le cordon post6rieur de la moelle chez la souris Jimpy, Acta neuropath., 29 (1974) 293-309. Seyfried, T. N., Glaser, G. H. and Yu, R. K., Thyroid hormone influence on the susceptibility of mice to audiogenic seizures, Science, 205 (1979) 598-600. Seyfried, T. N. and Yu, R. K., Heterosis for brain myelin content in mice, Biochem. Genet., 18 (1980) 1229-1238. Sidman, R. L., Dickie, M. M. and Appel, S. H., Mutant mice (Quaking and Jimpy) with deficient myelination in the central nervous system, Science, 144 (1964) 309-311.
34 Skoff, R. P., Myelin deficit in the Jimpy mouse may be due to cellular abnormalities in astroglia, Nature (Lond.), 264 (1976) 560-562. 35 Skoff, R. P., Neuroglia: a reevaluation of their origin and development, Path. Res. Pract., 168 (1980) 279-300. 36 Skoff, R. P., Proliferation of oligodendroglial cells in normal animals and in a myelin deficient mutant-Jimpy. In E. Acosta Vidrio and S. Federoff (Eds.), Glial and Neuronal Cell Biology, Alan R. Liss, New York, N.Y., 1981, pp. 93-103. 37 Skoff, R. P., Price, D. L. and Stocks, A., Electron microscopic autoradiographic studies of gliogenesis in rat optic nerve. I. Cell proliferation, J. comp. Neurol., 169 (1976) 291-312. 38 Skoff, R. P., Price, D. L. and Stocks, A., Electron microscopic autoradiographic studies of gliogenesis in rat optic nerve. II. Time of origin, J. comp. NeuroL, 169 (1976) 313-333. 39 Skoff, R. P., Toland, D. and Nast, E., Pattern of myelination and the distribution of neuroglial cells along the developing optic system of the rat and rabbit, J. comp. NeuroL, 191 (1980) 237-253. 40 Skoff, R. and Montgomery, I. N., Expression of mosaicism in females heterozygous for Jimpy, Brain Research, 212 (1981) 175-181. 41 Vaughn, J. E. and Skoff, R. O., Neuroglia in experimentally altered central nervous system. In G. Bourne (Ed.), Structure and Function of Nervous Tissue, VoL 5, Academic Press, New York, 1972, pp. 39-72. 42 Webster, H. deF., Reier, P. J., Matthieu, J.-M. and Quarles, R. H., Axonal abnormalities in optic nerves of Jimpy mice, J. Neuropath. exp. NeuroL, 35 (1976) 309. 43 Wolf, M. K., Schwing, G. B., Adcock, L. H. and BillingsGagliardi, S., Hypomyelinated mutant mice. III. Increased myelination in mutant cerebellum co-cultured with normal optic nerve, Brain Research, 206 (1981) 193-197. 44 Wood, P. M. and Bunge, R. P., Evidence that sensory axons are mitogenic for Schwann cells, Nature (Lond.), 256 (1975) 662-664. 45 Zimmerman, J. R. and Cohen, S. R., The distribution of myelin basic protein in subcellular fractions of developing Jimpy mouse brain, J. Neurochem., 32 (1979) 817-821.
19
Increased Proliferation of Oligodendrocytes in the Hypomyelinated Mouse Mutant-Jimpy ROBERT P. SKOFF Department of Anatomy, Wayne State University, School of Medicine, 540 E. Canfield, Detroit, M I 48201 (U.S.A.)
(Accepted December 31st, 1981) Key words: jimpy - - myelin - - autoradiography - - neuroglia - - nervous system
Previous studies of the hypomyelinatedmouse mutant jimpy have shown that the number of oligodendrocytesare reduced about 50~. To determine the cause of the cellular reduction, light and electron microscopy were combined with thymidine autoradiographic techniques. The number of neuroglial cells which incorporate radioactive thymidine in the mutants is increased severalfold over control values. Electron microscopic autoradiograms indicate the majority of the labeled cells are oligodendroblasts. However, the total number of glia in the white matter of jimpy and control animals is the same during development and even up to the time of the animal's death. The presence of mitotic cells suggests that the oligodendrocytesundergo division but the abundance of dying cells suggests that they die sometime afterwards. The results of the quantitative autoradiographic studies in combination with our other data strongly suggest that the immediate failure of these cells to form myelin sheaths is due to a shortened life span and/or continued cell proliferation.
INTRODUCTION Numerous mouse mutants (e.g. dystrophic 6, jimpy zz, quaking z3, MSD 24, shiverer 4, trembler 20, twitcher 16) as well as several strains of other mammals (Landrace pig 5, Wistar rat 8, Spaniel dog 12) have been discovered which show abnormalities in the development of myelin. In some mutants, the abnormality is directly associated with the synthesis of myelin (twitcher) in. In others, the defect involves interactions between the myelin-forming cells and other cell types (dystrophic) 1. In the dystrophic mouse, the axons are apparently unable to signal the Schwann cells to make myelin 1. In another strain of mice zl,z2, a correlation exists between the amount of myelin in the CNS and serum levels of thyroxine, a hormone essential for adequate myelination in rodents 2. From the few examples mentioned here it is clear that myelination is regulated by many genes, some of which directly control the synthesis of myelin while others are involved in more remotely related events. With the exception of the twitcher mouse, the cell types directly affected by the genetic 0006-8993/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
mutation and the cause of the myelin deficiency are unknown. The jimpy mouse is one of the most interesting of the myelin mutants because myelin in the central nervous system (CNS) is severely reduced but is apparently normal 3a in the peripheral nervous system (PNS). This phenomenon suggests that the mutation is intrinsic to the CNS. Consequently, almost all studies of this mutant have been limited to the CNS. The cause of the deficiency has not been determined although biochemical 3,7,45 and morphologic11,22,a2 studies have documented that myelin is reduced as much as 95 ~. It is important to note here that other abnormalities in the jimpy mutants have been described which may be solely or partially responsible for the hypomyelination. The number of oligodendrocytes in these mutants is reduced about 50 ~19,~3,z4. Besides the oligodendrocyte abnormalities, the astrocytes show a striking hypertrophy of their processes 34. This hypertrophy begins when myelination begins and it has been postulated that this astrogliosis may interfere with the development of the oligodendrocyte line and subsequent myelina-
20 tion ~4. More recently, Hertz et al. 13 showed astrocytes grown in tissue culture from the jimpys exhibit abnormalities in their metabolism. The above observations suggest that the genetic defect in jimpy may not involve an abnormality in formation of myelin but rather interactions between the oligodendrocyte and other cells. They also suggest that the development of the oligodendrocyte cell line is defective. The present study was undertaken to determine the basis for the loss of oligodendrocytes. MATERIALSAND METHODS Jimpy mice (jpTa/Y) were bred in our laboratories by mating carrier females (jpTal+-[-) with Tabby males (Ta+/Y) obtained from Jackson Laboratories (Bar Harbor, ME). The jimpy gene is maintained in the heterozygous female along with another closely linked gene (Tabby). Because of the Tabby fur markings, it can be used to help identify the jimpy mutants before tremors appear at 12 days of age. Normal males ( + +/y) and females ( + +/ta+) including crossover littermates (Ta+/ta+) were used as controls. Crossover females for the jimpy gene (Tajp/Ta+ ;+jp/Ta+) which exhibited mosaicism in the optic nerve were not used~0. All mice were given a single intraperitoneal injection (7.5/zCi/g) of [methyl-ZH]thymidine (spec. act. 17-20 Ci/mM) (New England Nuclear) and sacrificed 1 h after the injection. Mice were injected at 4, 7, 9, 12, 16-18, 20 and 23 days after birth. The mice were sacrificed by intracardiac perfusion following anesthetization with chloral hydrate. The perfusate consisted of 2 ~ glutaraldehyde and 2 ~ formaldehyde in either a 0.1 M cacodylate or 0.1 M phosphate buffer at pH 7.2. The intracranial segment of the optic nerves and the upper brachial region of the spinal cord were dissected out, placed in fixative and refrigerated overnight. The next day, the tissue was osmicated, dehydrated in a graded series of alcohols, then absolute propylene oxide and finally embedded in Araldite plastic. Light microscopic autoradiograms of 1/~m thickness of optic nerves and spinal cords were prepared as previously described37. The number of animals used at each stage is presented in Figs. IA and lB. Sections were spaced at 10-I 5 #m intervals to avoid counting the same cells. Quantitation of labeled cells
was done under oil immersion at 1000 × magnification. The labeling index for the optic nerve was determined by counting the labeled cells and the unlabeled cells in complete transverse sections. Both sides of the nerve were analyzed; at least two different sections for each side were quantitated and averaged, and the values for both sides averaged. In the spinal cord, only the glial cells in the white matter on one side were quantitated; at least two different sections were counted and averaged for each animal. Electron microscopic autoradiograms were prepared from the 4, 7, 9, 16 and 20 day animals. The animals used for electron microscopy were chosen on the basis of the quality of fixation and the abundance of labeled cells. The procedure for preparing electron microscopic autoradiograms is detailed in Skoff et al. 37. Approximately 40 different cells were examined in the normal mice at different stages of development and more than 100 in the mutants. The majority of labeled cells examined are from the spinal cord as the area sampled is considerably greater than the nerve. RESULTS
Light microscopic autoradiography The number of glial cells incorporating radioactive thymidine was investigated in the spinal cord and optic nerve at 7 different stages of postnatal development. The earliest stage studied was at 4 days after birth and the last at 23 days postnatal which is close to the time of their death (25-30 days). The animals were sacrificed 1 h after the injection so that the identity of the proliferating cells could be ascertained. In normal animals the labeling index progressively declines from the time of birth until 20 days postnatally (Figs. 1A and 1B). The increase in the labeling index from 4 to 7 days in the nerve is possibly due to the later onset of myelination there. The mutants show a similar decline in their labeling pattern but it never decreases to the background levels seen for the normals. In the spinal cord, the mutants have a significantly higher percentage of labeled glia than controls at all stages of development except at 4 days postnatal. The difference in the labeling index between the jimpys and controls becomes more pronounced with age. At 9 days after birth, about a twofold difference exists in the cord;
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Fig. 1. A and B : the percentage of labeled glial cells in the spinal cord and optic nerve are shown at different stages of postnatal development. The mice were given a single i.p. injection of radioactive thymidine and sacrificed 1 h later. The number beside each point indicates the number of animals used at each stage of development. In the optic nerve, complete transverse sections of the intracranial segment were quantitated; the cell counts in the spinal cord were made from the white matter on one side of the cervical cord. Details of the cell counting are presented in Materials and Methods. C and D: the mean number of glial nuclei in the spinal cord and optic nerve are shown at the different stages of development. Glial nuclei in the spinal cord were counted from the white matter on one side and from complete transverse sections of the optic nerve. The same sections and the same number of animals were used as in A and B.
at 12 days the differential is 3; at 16, 6; and at 20, 8. In the optic nerve, the labeling index differs most strikingly between the mutants and the controls from 16 days onwards. As in the spinal cord, the difference increases with time. At 16 days, the difference is about threefold but at 20 and 23 days, when cell proliferation in normals is reduced to minimal levels, almost a tenfold difference is present. After the second postnatal week when cell proliferation has slowed considerably, light microscopic autoradiograms illustrate dramatically the difference in labeling between the two sets of animals (Fig. 2). The pictures shown here are a dramatic expression of the differences in the labeling index and they are typical of most of the animals. Almost without exception, the labeled cells are small and it is difficult to distinguish any cytoplasm surrounding the nucleus. After the first week a few labeled cells can be identified as microglial cells on the basis of cytoplasmic lipid inclusions.
The number of glial cells quantitated for determining the labeling index is shown in Fig. 1C and 1D. The results show that there is no major increase in the number of glial cells in the mutants in comparison to the controls, even at the later stages of development. The standard deviations for normal and mutants overlap at all stages of development and they generally show large fluctuations between animals. Such variability is not surprising because we have shown that even within a particular animal, the number of glial cells counted in serial transverse sections may change more than 20% within a 100 /~m distance 85,39.
Electron microscopic autoradiography The ultrastructure of cells incorporating [3H]thymidine was analyzed in both spinal cord and optic nerve at the different stages of development. The same types of glial cells are labeled in normal mice as in the rat 37. The vast majority of the cells labeled
Fig. 2. A and B: light microscopic autoradiograms showing the dorsal funiculi of spinal cords from 16-day-old mice. The dorsal funiculus of the normal mouse (A) is densely packed with myelinated fibers but the same area in the mutant has only a few scattered fibers. The control has one labeled cell (arrow) whereas the mutant has at least 9 labeled ceils (arrows). Many of the cells in the normal animals can be identified as astrocytes or oligodendrocytes on the basis of their staining but the cells in the mutants are small and difficult to identify. Degenerating cells (arrowheads) are numerous in the mutants, x 600.
23
Fig. 3. This electron microscopic autoradiogram is from the spinal cord of a 7-day-old normal mouse. The cytoplasmic and nuclear features of the labeled cell are characteristic of immature oligodendroglia. The density of the perikaryon and nucleus contrasts sharply with axoplasm and astrocytic processes. Free ribosomes, cisternae of the Golgi apparatus (G), numerous small mitochondria and thin strands of endoplasmic reticulum (ER) occupy the cytoplasm of immature oligodendroglia. The Golgi apparatus is adjacent to the nucleus and surrounded by mitochondria and endoplasmic reticulum. The nucleus and cytoplasm are usually located at opposite ends of the cell. x 20,000. during p o s t n a t a l d e v e l o p m e n t can be classified as a s t r o b l a s t s a n d o l i g o d e n d r o b l a s t s . The a s t r o b l a s t s are f o u n d p r i m a r i l y d u r i n g the first p o s t n a t a l week a n d the o l i g o d e n d r o b l a s t s afterwards. This is as expected since the time o f final cell division for astrocytes occurs p r e n a t a l l y a n d during t h e first
week o f p o s t n a t a l d e v e l o p m e n t 3s. T h e l a b e l e d astroblasts a n d o l i g o d e n d r o b l a s t s a p p e a r slightly m o r e differentiated in the m o u s e t h a n their c o u n t e r p a r t s in the rat. The a s t r o b l a s t s in mice usually have distinct clusters o f filaments within their c y t o p l a s m whereas filaments in the r a t are often lacking o r n o t
24 aggregated into prominent clusters. Oligodendroblasts in mice have a dark cytoplasmic matrix which is nearly identical in density to fully mature forms. A detailed description of the morphology of oligodeodroblasts and astroblasts has been presented previously 36,~7 and the emphasis in this paper will be placed upon the differences between oligodendroglia in normal and mutant mice. The morphology of the oligodendroblasts in normal mice is the same regardless of the stage of the animal's development (Fig. 3). The nucleus of the labeled oligodendroblast is characteristically located at one end of the cell. Immediately adjacent to the nucleus are many cisternae of the Golgi apparatus. Surrounding the Golgi and closer to the cell membrane are numerous mitochondria and short cisternae of granular endoplasmic reticulum. Microtubules and cisternae of rough endoplasmic reticulum are scattered throughout the cytoplasm but are not as prominent as in the mature forms. The nucleoplasm has a rather hazy appearance and a thin rim of chromatin surrounds the
nuclear membrane. The morphology of oligodendroglia can be compared and contrasted with that of a mononuclear cell which was observed to be labeled in a blood vessel of a normal mouse (Fig. 4). Chromatin in the monocyte forms large, dense patches which are scattered throughout the nucleoplasm. At the nuclear membrane, the chromatin is interrupted by lightly stained channels. These channels are lacking or at least not as prominent in macroglia. The morphology of labeled oligodendroblasts in the mutants (Figs. 5 and 6) is similar to their normal counterparts in most respects but there are several differences which should be mentioned. The electron density of their cytoplasm is less than that of normals, and while they contain the same types of organelles, their overall number, especially ribosomes and mitochondria, appears reduced in the oligodendroglia of jimpy (Figs. 5 and 6). At the later stages of development (16 and 20 days), the organization of the cytoplasm in some labeled oligoden-
Fig. 4. A labeled mononuclear cell in the lumen of a blood vessel of a normal 7-day-old mouse. The cytoplasm of this cell is packed with scattered ribosomes, a few cisternae of the Golgi apparatus (G) and mitochondria. The electron density of this cell is similar to that of oligodendroglia but the chromatin pattern is quite different from macroglia. × 16,000.
25
Fig. 5. A labeled oligodendroblast from the spinal cord of a 16-day-old jimpy. The cytoplasmic density of oligodendroglia in the mutants is usually less than that of their normal counterparts but still considerably more dense than adjacent astroglial processes (AP) and axonal cytoplasm (A). The cytoplasm funnels down into a process packed with microtubules (M). Note the centriole (C) and the dense bodies (DB). Compare the cytoplasm of this oligodendrocyte with that of the adjacent microglial cell (Mg). The cytoplasm of microglia usually have a grainy appearance which contain a variety of different inclusions. The endoplasmic reticulum (Er) is of a narrow caliber and often branches (arrows) as it winds throughout the cytoplasm. × 18,000.
26
Fig. 6. The nucleus of this labeled oligodendroblast has been grazed so that the chromatin forms a broad band beneath the nuclear envelope. This cell is from a jimpy animal at 16 days of age. The cytoplasm is packed with mitochondria, cisternae of the Golgi apparatus (G), and a few strands of endoplasmic reticulum. × 17,000. droblasts appears more differentiated than oligodendroblasts in the normal animal (Figs. 5 and 6). For example, the cluster of microtubules seen in the cell in Fig. 5 and the stacking of rough endoplasmic reticulum in the cell in Fig. 6 has never been observed in labeled oligodendroblasts of normal rats or mice. In the jimpy animals, labeled oligodendroblasts were observed in the spinal cord at all stages of development and in the optic nerve from 7 days on-
wards. At 16 and 20 days in the mutants, at least 50 ~o of the total number of labeled cells are oligodendroblasts. With the exception of one possible cell, astroblasts did not show label at 16 days and afterwards. In addition to labeled macroglia, microglial cells are abundantly labeled (Fig. 7) at the later stages. They account for 20-30 ~ of the labeled cells at 16 and 20 days. Microglial cells can be identified by their long, stringy endoplasmic reticulum, the
27
Fig. 7. A labeled microglial cell from a 16-day-old mutant. The cytoplasm contains long, stringy, endoplasmic reticulum (Er), mitochondria and a variety of different inclusions ranging from lipid droplets (L) to vacoules (V) containing floccular substance. Microtubules, filaments and glycogen are not found in microglia. Ribosomes are less densely packed than in oligodendroglia and they give a grainy appearance to the cytoplasm similar to that seen in the mononuclear cell (Fig. 4). × 18,000.
28 presence of numerous inclusions, moderate density and the absence of intermediate filaments and microtubules. The microglial cells in the mutants usually contain a variety of inclusions and are somewhat similar in appearance to reactive forms seen in experimental conditions such as Wallerian degeneration 41. The remainder of the labeled cells could not be positively identified because of the paucity of cytoplasm and lack of sufficient morphological features. Most of these cells appear to be oligodendrocytes based on the similarity of nuclear features to oligodendrocytes. DISCUSSION The severity of the myelin reduction in the CNS of the jimpy mouse has been documented in numerous biochemical 3,7,15,21,22,45 and morphological studies 19,2a,2s,3°. Biochemical analyses of myelin components, such as myelin basic protein, indicate reductions of as much as 95 ~3,7,45. Morphologic studies of different fiber tracts throughout the brain show that less than 5 ~ of the axons are myelinated (refs. 28, 30, 42). To date, however, the cell type directly affected by the genetic mutation has not been determined. As myelin in the peripheral nervous system (PNS) appears normal a~, it is reasonable to assume that the defect involves either the neuroglial cells or the CNS neurons. Consequently, virtually all studies of this mutant have been limited to the CNS. Several recent studies strongly suggest that the anon is not the site of the mutation. The female carriers of the jimpy gene exhibited mosaicism in their optic nerves with the result that some areas of the nerve are unmyelinated while others are myelinated 4°. As the patches of myelinated and unmyelinated areas occur along the nerve at different levels, this indicates that an individual axon is both myelinated and unmyelinated along its course. If the defect were in the anon, there should be a small population which are myelinated continuously along their length. Other evidence that the axon is not at fault comes from tissue culture studies of Wolff et al.4L In co-cultured explants containing mutant cerebella and normal optic nerve, the mutant cerebella showed an increase in the number of myelinated fibers. This increase is probably due to an influx of oligodendroglia from normal tissue
which then myelinate the axons 4a. Although biochemical studies have not focused directly upon membrane properties of the axons, markers for neurons appear normapO, 14. An additional argument against the axon as the site of the mutation relates to the presence of myelin in the PNS and its paucity in the CNS. If the anon is postulated to be the site, then it must be further postulated that the axonal membrane is abnormal in the CNS and normal in the PNS. While this is a possibility, it seems unlikely that the axonal membrane should change so abruptly. Although the evidence against the neuron is not yet conclusive, it seems more reasonable to focus our attention upon primary defects in the neuroglial cells. The astrocytes show an abnormal increase in their processes that begins around the same time as myelinogenesis 34,a5. We have postulated that the astrocytes may prevent interactions between the oligodendrocytes and axons either mechanically or biochemically. Whether the astrocytic hypertrophy is a primary defect or a secondary response has not been determined but a recent tissue culture study by Hertz et al.13 indicates that the astrocytes have an intrinsic abnormality in their oxygen metabolism. As the astrocytes were cultured shortly after birth and before myelination has progressed very far, it is unlikely that the metabolic abnormality represents a secondary response on the part of the astrocyte. The most likely candidate for the mutation site is the oligodendrocyte but evidence for a specific abnormality in this cell type is lacking. Numerous biochemical studies have documented reductions of the myelin components and the enzyme systems involved in the formation of myelin but no particular defect has yet been detected. The morphology of the oligodendrocyte is not particularly revealing either, and the cell does not appear strikingly abnormal except for the fact that it is less differentiated than its normal counterpart. They rarely contain inclusions and this may be significant because in experimental conditions in which myelination has been blocked, the oligodendrocytes contain inclusions of various sorts 9,29. In mice treated with inhibitors of cholesterol biosynthesis, the Schwann cells show a variety of inclusions 29. Administration of CNS antiserum in tissue culture also leads to an accumulation of debris in the oligodendrocytes9. In metachromatic leuko-
29 dystrophy, the absence of cerebroside sulfotransferase (arylsulfastase A), an enzyme which degrades cerebroside sulfate to cerebroside and sulfate, produces an accumulation of cytoplasmic inclusions within oligodendrocytes27. While these observations do not eliminate a defect in myelin synthesis or degradation, they suggest that the cause of the defect may affect more fundamental cellular processes such as glial metabolism, the development of the oligodendrocyte cell line or cellular interactions. The number of oligodendrocytes present in hypomyelinated mice is the most fundamental question that needs investigation. In jimpy mice the number of glial cells has been investigated at the light and electron mlcroscoplc levels in several studies• Light microscopic studies of the spinal cord and corpus callosum indicate the number of oligodendrocytes are significantly reducedtg,2a,28,a0. A quantitative electron microscopic study of the optic nerve indicates a reduction of about 50 7o84. A precise figure is difficult to come by because marry of the cells in jimpy are difficult to classify. These cells have sparse cytoplasm with few identifying features and classification as an oligodendrocyte or undifferentiated glia is often subjective. The cellular loss illustrates two peculiarities of the jimpy mutants. First, while oligodendrocytes are reduced in number, those present are not myelinating as many axons as possible and second, the cellular reduction is an indication that the cell line is affected. The present study was undertaken to determine the nature of the oligodendrocyte reduction. Using thymidine autoradiography in combination with light and electron microscopy, the extent of cell proliferation as well as the morphology of the proliferating cells was examined. Our results show that the labeling index between the normal animals and the mutants is quite different. In the spinal cord, the labeling index is significantly higher in the jimpys than in normals after the fourth day of development. In the nerve, significantly higher indices are present after the second week. The difference between jimpys and controls becomes most striking when cell proliferation slows to background levels in normal mice at 16 days. The differential is such that it should lead to a dramatic increase in the number of glial cells by the time of their death. It can be calculated that the number of glia should at least
double in cross-sections of the spinal cord if the length of their cell cycle is normal (about 20 h)17,18 and if it is further assumed that half of the cells exit from the cell cycle and the others continue to divide. In the optic nerve, the number of glia should almost double by 20 days postnatal. The quantitative data for both the spinal cord and optic nerves shows no sign of an increase at any time during the animal's life. However, mitotic figures are found at all stages of the mutant's life. The increase in cell proliferation and the presence of mitotic figures strongly suggest that the cells are capable of normal cell division. The fate of the dividing neuroglial cells is not certain but it appears that some apparently die prematurely while others continue to divide. Dying cells are especially common at the later stages of development. While dying glia are occasionally present in normal development, the mutants often have clusters of dying cells scattered throughout the white matter. Whether certain populations of cells are destined to die and others continue to divide or whether it is random is unknown at this stage of our investigation. The increased cell proliferation and death would not necessarily be significant were it not for the fact that the vast majority of the labeled cells are oligodendroblasts. Because of the astroglial hypertrophy, it might be expected that this cell type would be predominantly labeled. This finding is in agreement with our ultrastructural quantitative studies which show that astrocytes do not increase in number z4. It illustrates the fact that astroglial hypertrophy can occur without an accompanying hyperplasia. The proliferation of one cell type also suggests that there is a specific stimulus for the oligodendrocytes. In regards to the continued proliferation of oligodendroglia throughout the life of the jimpy, it cannot be argued that their time of origin is simply delayed as they are also labeled during the first week of postnatal development. This is in keeping with their time of origin in normal animalszS. A more important question is whether the increased proliferation of oligodendrocytes is a primary or secondary phenomenon. Arguments for a secondary effect come from studies of the PNS. The introduction of unmyelinated neurons into Schwann cell cultures stimulates the ensheathing cells to divide 44. In the dystrophic mouse25 and in Trembler26, proliferation
30 o f Schwann cells occurs t h r o u g h o u t the life o f the animal. The u n m y e l i n a t e d axons in t h e j i m p y m a y p r o v i d e a similar stimulus. Proliferation o f oligod e n d r o c y t e s might also be triggered b y other factors such as the extracellular a c c u m u l a t i o n o f myelin c o m p o n e n t s in j i m p y . Their d e a t h might also be explained in a similar manner. However, until a d d i t i o n a l evidence is a c c u m u l a t e d a b o u t cell proliferat i o n a n d d e a t h in the oligodendrocytes, it is p r e m a ture to conclude t h a t the o l i g o d e n d r o g l i a l changes are p r i m a r y o r secondary. A l t h o u g h the increased cell p r o l i f e r a t i o n a n d d e a t h is likely to be triggered b y s o m e o t h e r f a c t o r (either intrinsic or extrinsic to the oligodendrocyte), myelinogenesis c a n n o t occur in a system in which the o l i g o d e n d r o c y t e s are short lived o r are c o n t i n u a l l y dividing. I n this sense, the f a c t o r i m m e d i a t e l y responsible for the h y p o m y e l i o a t i o n is the a b n o r m a l cellular d e a t h a n d proliferation.
One a p p r o a c h to solving this p r o b l e m is to study the female carrier o f the j i m p y gene. A s she lives to a d u l t h o o d , cell p r o l i f e r a t i o n a n d cell d e a t h can be studied over an extended p e r i o d o f time. T h e m o s a i cism in the optic nerve 40 p r o v i d e s a unique system to study the c a p a c i t y o f n o r m a l o l i g o d e n d r o c y t e s to invade a n d myelinate those areas affected b y the m u t a tion.
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ACKNOWLEDGEMENTS The a u t h o r extends his a p p r e c i a t i o n to Dr. K e n Liu for technical assistance a n d to Dr. Joyce Benjamins a n d A n n e Skoff for their critical review o f the manuscript. This research was s u p p o r t e d b y N I H G r a n t N S 15338.
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