Plasminogen activator in the developing rat cerebellum: Biosynthesis and localization in granular neurons

149

Developmental Brain Research, 11 (1983) 149-158 Elsevier

Research Reports

Plasminogen Activator in the Developing Rat Cerebellum: Biosynthesis and Localization in Granular Neurons HERMONA SOREQ 1and RUTH MISKIN2 Departments o/1Neurobiology and 2Biochemistry, The Weizmann Institute of Science, Rehovot 76100 (Israel) (Accepted June 30th, 1983) Key words: plasminogen activator molecular forms - - agranular cerebellum - - granular neuron migration - - messenger RNA autoradiography - - X-irradiation

The histochemical localization of plasminogen activator (PA) and the level of the translatable mRNA species coding for active PA were analyzed during ontogenesis of normal and Of irradiation-agranulated rat cerebellum. Autoradiographic localization of PA activity was performed by plasminogen-dependent fixation of [125I]fibrindegradation products to frozen sections of developing rat cerebellum. Both the immature external and the adult internal granular layers were intensely labeled, in addition to labeling of meninges. In the irradiation-agranulated cerebellum, PA labeling could be observed in residual granular neurons which went through their final division prior to the irradiation protocol. The concentration of the mRNA species directing the synthesis of catalytically active PA (PAmRNA) was monitored by an in ovo bioassay, using Xenopus oocytes as a translation system. A major species of 80,000 and a minor species of 50,000 apparent molecular weight of active PA were translated by mRNA from either control or X-irradiated cerebellum throughout ontogenesis. These could be detected by electrophoretic analysis of extracts and incubation media of microinjected oocytes. Both the content and the concentration of PAmRNA were found to be the highest at the stage of cerebeUar development when granular neurons proliferate and migrate. These observations suggest that a major portion of the PA activity in the rat cerebellum is synthesized and localized in granular neurons through cerebellar ontogenesis, and that PA activity in the developing cerebellum is largely determined by the level of translatable mRNA coding for this enzyme. INTRODUCTION Proteolysis generated by the plasminogen activator system functions in thrombolysis. Plasminogen activator (PA) appears also to take part in cellular movement and tissue remodeling, both in adult and in developing organisms24,2s, 39. PAs, which are found as cell-associated and secreted enzymes, convert extraceUular plasminogen to a second protease, plasmin, by specific and limited proteolysis. Plasminogen comprises approximately 0 . 5 % of total plasma protein 31 and is also found in other extracellular fluids, including the cerebrospinal fluidS,26,40. Plasmin is a short-lived protease, due to autocatalytic degradation and, at least in the plasma, to a rapid block by protease inhibitors 7. Both the generation and continued presence of plasmin are, therefore, largely de-

pendent upon the synthesis and secretion of PA. A variety of modulators were shown to regulate the synthesis of P A in many cell types, and studies with inhibitors of macromolecular synthesis indicate that this regulation occurs at the level of transcription16,29,42. We have recently developed a direct bioassay to detect and quantify P A m R N A , by measuring its translation into an active protease in microinjected X e n o p u s oocytes 17. The possible involvement of the plasminogen activation system in brain development has recently been approached in several ways. The specific activity of P A in dissected brain regions was found to be higher during periods of development than at maturity32. In the developing cerebellum, high specific activity of P A was temporally correlated with neuronal proliferation and migration 33. Moreover, P A activity

Correspondence: Department of Neurobiology, The Weizmann Institute of Science, Rehovot 76100, Israel. 0165-3806/83/$03.00 © 1983 Elsevier Science Publishers B.V.

150 was detected in primary cultures of cerebellar cells 13 and in the internal granular layer of frozen sections of the mature cerebellum 33. However, none of these observations could clearly indicate whether migrating neurons actively produce PA throughout their in vivo development. To reveal whether this is the case, and to clarify the level of gene expression at which the specific activity of neuronal PA is regulated, we extended our studies of PA in the developing rat cerebellum using, in parallel, histochemical and molecular biology techniques. The rodent cerebellum is particularly suitable for studies of biochemical mechanisms underlying neuronal development for a number of reasons. The cell type complexity in the mature cerebellum is low relative to other brain regions 25. The spatial organization and intercellular connections are well-characterized and remarkably constant among species 27. Several genetic and experimental malformations of the rodent cerebellum, deficient in specific cell populations, are available 38. The main development of the rodent cerebellum occurs after birth, when new cell types proliferate, migrate and differentiate 1. Most intensive is the proliferation and movement of granular interneurons. These arise from germ cells in the external germinal layer (EGL) during the first two postnatal weeks and migrate from the surface of the cerebellum inward, through the developing molecular layer and the monolayer of Purkinje cells, to form the mature internal granular layer (IGL). In the mature cerebellum, granular neurons compose a major fraction of the population of cerebellar cells. The relatively simple composition of the rat cerebellum is also reflected in the properties of cerebellar mRNA. The complexity of cerebellar m R N A is lower than in other brain regions 4. Cerebellar development is accompanied by pronounced changes in the concentration of specific m R N A species 22,35 and some of these changes are altered in the cerebellum malformed by disease 9 or by irradiation-induced agranulationS. Experimental irradiation, which selectively eliminates mitotic granular neuronsl. 2.3s, also affects the level of PA in cerebellar homogenates 33. The normal and X-irradiated developing rat cerebellum can, therefore, serve as good model systems to examine the regulation of PA production. The present report deals with two specific aspects of PA in the developing cerebellum. The first is the

detection and identification of cells expressing PA in situ. This was carried out by histochemical autoradiography of PA activity in frozen sections of the developing cerebellum. The second question was whether the regulation of cerebellar PA occurs at the level of mRNA. This was approached by translation of cerebellar m R N A into active PA in microinjected Xenopus oocytes. The results indicate that at all developmental stages, a major fraction of cerebellar P.A can be ascribed to granular neurons, and that the synthesis of cerebellar PA is largely, although not exclusively, controlled at the m R N A level. MATERIALS AND METHODS

Animals Sprague-Dawley rats were obtained from the Weizmann Institute breeding Center. The proliferation and migration of granular neurons in the developing cerebellum of these rats, as revealed from the width of the E G L and the I G L layers in cresyl violetstained cerebellar sections 30, appear to occur similarly to the same morphological changes as reported for Wistar rats 2. X-irradiation of the right side only of the cerebellum of developing rats was according to Ben-Barak 6 and as described previouslyS,33,36. Adult Xenopus laevis females were obtained from the South African Snake Farm (Fish Hoek, South Africa).

Preparation and microinjection of cerebeUar mRNA Poly(A)-containing m R N A was prepared from normal and irradiated rat cerebellum at various developmental stages8, 35. The level of translatable P A m R N A was determined by bioassay of PA activity induced in Xenopus laevis oocytes microinjected with cerebellar m R N A 17. Fifty nanograms of nonfractionated cerebellar poly(A)-containing RNA, which is a subsaturating amount for expression of PAmRNA, were injected per oocyte. Incubation was f o r 8 h at 21 °C.

Analysis of PA activity In all of the quantitative PA assays used, the plasminogen-dependent fibrinolytic activity of plasmin, which was produced from plasminogen by PA, was determined. Preparation of cerebellar homogenates and the fibrin plate assay were as described 33. PA ac-

151 tivity in oocyte homogenates and incubation medium was determined by the fibrin plate assay 17. The molecular forms of active PA in tissue homogenates and in oocyte samples were determined by miniscale gel electrophoresis in casein-containing gels 18. Autoradiography of [125I]fibrin degradation products, produced by PA and cross-linked to creostat sections of developing rat cerebellum, was carried out as described 33. RESULTS

Granular neurons display PA activity throughout cerebellar ontogenesis In situ localization of PA activity can be carried out in frozen cerebellar sections, by emulsion autoradio-

graphy of fixed [125I]fibrin degradation products produced in Agar overlays. In the 17-day cerebellum, such analysis localized a major part of cerebellar PA activity to the I G L 33. The autoradiographic analysis has now been extended to the immature cerebellum, and is presented in the photographs assembled in Figure 1. At 10 days after birth, PA labeling was found to be most intense in the surface region of the cerebellar lobules, which includes the meninges and the embryonic EGL, containing stem cells and premigratory neurons. A similar degree of labeling has also been detected in the developing IGL, which is mostly composed at this stage of differentiating postmigratory granular neurotis (Fig. 1, lower left). Less intense labeling has been observed in the growing molecular

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Fig. 1. PA autoradiography in cerebellar frozen sections. Autoradiography of PA activity was carried out as described33on 10/xmthick frozen sections of cerebella from 10-day and 17-day-oldrats. Magnification: x 30 (low) and x 200 (high). Exposure was for 3 weeks with Ilford K5 emulsion. Note intense labeling of EGL at 10 days (left) and IGL at 17 days (right), and weak labeling of Purkinje cell layer (10 days and 17 days, up).

152 layer, and may, perhaps, be ascribed to granular neurons migrating through this layer from the E G L to the IGLL In cerebellar sections from 17-day rats (Fig. 1, right) PA labeling was most intense in the IGL, as previously reported 33. Labeling of the I G L was equally intense in the various cerebellar lobules, with no regional differences. The surface labeling was reduced to a thin line, representing PA in the cerebellar meninges. This observation is compatible with the finding that migration of granular neurons is already completed at this age in the cerebellum of SpragueDawley rats. The difference in intensity of staining between the internal granular layer and the molecular layer ap-

pears to be more pronounced at 17 days than at 10 days, when part of the cell bodies of granular neurons are still migrating through the developing molecular layer, and before all of the granular neurons are located in the IGL. A monolayer of Purkinje cells, separating between the molecular layer and IGL, can be clearly detected in larger magnification photographs of both stages (Fig. 1, upper part). However, it is difficult to determine from these photographs whether specific labeling can be assigned to the surface membrane of the Purkinje cells. Autoradiography of PA activity in sections of 30-day cerebellum agranulated by X-irradiation revealed a great reduction in labeling of the agranulated lobules, indicating that granular neurons are re-

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Fig. 2. Elimination of IGL labeling from agranulated cerebellar sections. PA autoradiography in individual lobules from frozen sections of X-irradiated cerebellum of a 30-day-old rat is presented, x, right side (X-irradiated) cerebellar hemisphere; c, left side (control) cerebellar hemisphere. Note disappearance of labeling from internal region of X-irradiated Iobules.

153 sponsible for the labeling. In contrast to the reduction in IGL labeling, labeling of the meninges appeared not to be affected by irradiation. Within the irradiated lobules, labeling could be seen in the defective IGL, which is composed of granular neurons that were post-mitotic at the time of irradiation. The reduction in size of each lobule and the residual amount of labeling depend, therefore, on the time at which these lobules develop. Indeed, the reduction in size and in PA labeling is more pronounced in the late developing Ansiformis lobule II than it is in the early developing Ansiformis lobule I (Fig. 2). Thus, the histochemical analysis shows that the immature EGL, the mature IGL and the residual neurons in the irradiated cerebellum contain a large fraction of cerebellar PA, indicating that ganular neurons display PA activity throughout cerebellar ontogenesis.

Cerebellar mRNA directs the synthesis of PA Two forms of active PA, a major one of 80,000 and a minor one of 50,000 apparent molecular weight were detected in cerebellar homogenates33, in bovine cortex synaptosomes 46 and in various lines of mouse neuroblastoma cells34. However, a large fraction of the cerebellar mass is contributed by incoming fibers, extending from cell bodies in other brain regions. It could not, therefore, be established whether cerebellar neurons actively produce either of these molecular forms of PA or whether this activity is largely contributed by incoming fibers. To resolve this question, an analysis at the mRNA level is necessary, since mRNA is found exclusively in cell bodies and missing from processes 3. The biosynthesis of mammalian PA is directed by poly(A)-containing mRNA species, capable of inducing the production and secretion of active PA in microinjected Xenopus oocytes. The amount of PA produced in oocytes increases linearly with the amount of nonfractionated poly(A)-containing RNA injected, up to 100 ng/oocyte 17. Within this range, the amount of PA synthesized in the oocytes reflects, therefore, the abundance of PAmRNA in the injected mRNA population. The in ovo bioassay for the expression of PAmRNA was employed to detect and quantify the level of PAmRNA in the developing rat cerebellum. Poly(A)-containing RNA was extracted from rat cerebellum at different developmental stages and in-

jected into Xenopus oocytes in double groups of 10. Oocytes were incubated for 8 h, the optimal time for accumulation of newly synthesized PA 17. PA activity in oocyte homogenates and incubation medium was determined by the quantitative fibrin plate assay33 and in miniscale polyacrylamide gels containing casein TM. Cerebellar mRNA from all developmental stages examined reproducibly induced the synthesis of significant amounts of PA, well above the endogenous activity of control oocytes, injected with Barth medium. The newly synthesized PA, which was secreted into the incubation medium of oocytes injected with cerebellar mRNA, was identified as rat PA by its electrophoretic migration. Two molecular forms of active PA were detected by gel analysis in samples of the incubation medium of oocytes injected with mRNA. Similar patterns were obtained using both control and irradiation-agranulated cerebellum at 3 developmental stages. The major and heavier PA form had an apparent molecular weight of 80,000, whereas the faint, faster migrating PA form comigrated with the 50,000 major PA from mouse urine (Fig. 3A). Neither of the proteolytic forms could be detected when plasminogen was omitted from the gel, indicating that proteolysis was due to PA and not to a plasminogen-independent protease. No comigrating PA forms could be seen in the gel slot loaded with medium of control oocytes, proving that the newly synthesized PA is not an endogenous amphibian protease. Similarly migrating PA forms, with a similar ratio of intensities between the 80,000 and the 50,000 forms, could be detected in cerebellar extracts (Fig. 3B). Therefore, the analysis of PA forms produced in microinjected oocytes indicates that endogenous cerebellar cell bodies contain the mRNA species directing the production of both of the cerebellar PA forms, throughout development. Equal amounts of mRNA from control and irradiated cerebellum were injected into the oocytes. The fraction of mRNA contributed by granular neurons is drastically reduced in the irradiated cerebellum. Thus, the fact that similar patterns are obtained when mRNA from irradiated cerebellum is injected implies that these mRNA species are not confined to granular neurons. The capacity of equal amounts of mRNA to produce PA in microinjected oocytes, as measured by

154 the fibrin plate assay, was found to change with development. A representative microinjection experiment, utilizing 50 ng of m R N A extracted from the cerebellum of rats at different ages, is displayed in Fig. 4. Maximal biosynthetic capacity is detected at day 10, and gradually decreases thereafter. This pattern was reproduced with different m R N A preparations and in several independent microinjection experiments.

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Fig. 3. Detection of active PA in cerebellar homogenates and in medium of microinjected oocytes following electrophoretic separation, A: Xenopusoocytes were injected with 50 ng each of poly(A)-containing RNA from irradiated (x) and control (c) cerebellar hemispheres at the indicated ages. Irradiation was according to Eliyahu and Sore@ Injected oocytes were incubated 8 h at 21 °C. Six/d samples of incubation medium were electrophoretically separated in casein-containing gels according to Miskin and Soreq TM. Clear zones represent PA activity. UK, human urokinase (2 UKmu); MU, mouse urine (2 UKmu); (-), incubation medium from control oocytes, microinjected with 50 nl of Barth medium. B: cerebellar homogenates containing PA activity equivalent to 2 and 4 milliunits urokinase, from rats of 1 and 47 days old, were applied to each slot, in addition to 1.5 milliunit of human urokinase and to mouse urine. Separation was as in A.

The level of total poly(A)-containing R N A , expressed as/~g/g tissue, changes with cerebellar development 35 (see dashed line in Fig. 4). The level of PAmRNA/g cerebellar tissue in each of the stages examined depends, therefore, both on the fraction of P A m R N A within the injected m R N A population and on the level of total poly(A)-containing RNA/g cerebellum at that stage. Similarly, the total content of P A m R N A in the cerebellum can be derived, at each stage, from the fraction of P A m R N A and the total amount of poly(A)-containing R N A in the cerebellum. It is evident from Fig. 5 that the amount of P A m R N A / g cerebellar tissue increases by about 2-fold from day 1 to day 10 after birth, when it reaches a maximal value. It then falls sharply between day 10 and day 30, parallel to the decrease in the width of the E G L , and continues to decrease at a slow rate with cerebellar aging (Fig. 5). However, the total content of P A m R N A increases in the developing rat cerebellum during the first 10 postnatal days by about 10-fold, parallel to the increase in total cerebellar protein and to the width of the IGL, which comprises a large fraction of the cerebellar section area. Following cerebellar maturation, P A m R N A content decreases continuously (Fig. 5). DISCUSSION Histochemical autoradiography of plasminogendependent fibrinolysis in frozen cerebellar sections localized P A activity at all developmental stages examined to cellular layers rich in granular neurons. These include the E G L and I G L at 10 days after birth, and at 17 days, the well-defined I G L which is mostly composed of densely packed cell bodies of fully differentiated granular neurons. Thus, it appears that P A is found in cell bodies of granular neurons in situ, prior to, during, and after migration. Localization of P A to granular neurons is also compatible with the observation that primary cells, obtained from 7 to 9-day-old mouse cerebellum, display P A activity in culture 13. The molecular layer in the mature cerebellum displays PA activity with much lower intensity than that observed in the IGL. This layer is mainly composed of the axons of granular neurons, forming parallel fibers, and of the dendritic trees of Purkinje cells 2s. The relatively low labeling in the molecular layer

155 may therefore imply that PA activity is unevenly distributed within the granular neurons, being mainly concentrated in cell bodies and in dendritic arborizations in the IGL but not in the axonal processes in the molecular layer. This possibility is in agreement with our recent observation of uneven distribution of PA activity in processes of neuroblastoma cells in culture 34. It is also possible that Purkinje cell dendrites do not express PA activity, which reduces the labeling in the molecular layer. The assignment of PA activity to granular neurons is supported by the reduction in PA labeling that has been observed using sections of the irradiation-agranulated cerebellum. It is also possible that at least part of PA activity in the irradiated cerebellum is directly induced in the residual granular neurons by DNA damage caused by the irradiation. Such a possibility is suggested by the finding, that a variety of DNA damaging agents induce PA synthesis in embryonic fibroblasts 16. Furthermore, enhancement of PA levels by ultraviolet light irradiation occurs in

cells deficient in DNA repair mechanisms 19. In addition, post-mitotic granular neurons were reported to be deficient in DNA repair 11, a deficiency which is probably responsible for the high sensitivity of these cells to ionizing irradiation 12. The histochemical staining of acetylcholinesterase in cerebellar sections reveals changes in the intensity of staining of individual lobules during cerebellar development 23. Developmental differences in quantities and distribution of neuronal PA activity have recently been observed, using the fibrin plate assay, in neuroblastoma cells undergoing differentiation in culture. PA labeling in these cells has been localized to perikarya, neurites and growth cones 34. Fibrinolytic activity of neuronal growth cones of neuroblastoma cells has also been detected by the fibrin plaque assay TM. However, the histochemical autoradiography of PA activity in frozen cerebellar sections revealed a similar extent of labeling in all of the cerebellar lobules at all the developmental stages examI

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Fig. 4. Developmental variations in the ability of cerebeltar mRNA to induce the synthesis of PA in microinjected oocytes. Poly(A)-containing RNA was extracted from cerebella of rats at the indicated ages as described 35. Fifty nanograms of 1 mg/ml cerebellar poly(A)-containing RNA from the various preparations were microinjected into 2 groups of 10 single, stage 6 mature oocytes 17. Following 8 h incubation at 21 °C, PA activity was assayed in oocyte extracts and incubation medium in the standard fibrin plate assay 33. Control oocytes were injected with 50 nl of Barth medium and incubated under standard conditions. Each point represents average cumulative activity in the extract and incubation medium of a single oocyte. Data are presented as newly synthesized urokinase Ploug units per oocyte (O), and the endogenous PA activity in control oocytes (0.19 UKmu/oocyte) was subtracted. The total content of poly(A)-containing RNA/cerebellum at the different developmental stages (O) is according to 35.

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Fig. 5. Developmental modifications in the level and content of cerebellar PAmRNA. A: The content of PAmRNA, in UKmu per cerebellum (O) was derived from PAmRNA concentration/50 ng of poly(A)-containing RNA and the total content of poly(A)-containing RNA in the cerebellum at the examined developmental stages (see Fig. 4). The dashed line presents the width of internal granular layer (IGL) after Altman and Anderson 2. B: the level of PAmRNA, in UKmu/g cerebellar tissue (O) was derived from PAmRNA concentration and the level of cerebellar poly(A)-containing RNA 35, (see text for details). The dashed line presents the width of external granular layer (EGL), after Altman and Anderson 2.

156 ined. PA autoradiography is preceded by prolonged incubation under Agar overlays, when the fibrinolytic reaction takes place. Therefore, the autoradiography is a qualitative, rather than a quantitative technique, and at present, it cannot resolve whether granular neurons display different levels of PA activity at different developmental stages. The I G L region, in which PA labeling is most intense, is mainly composed of post-migratory granular neurons and of numerous incoming fibers, the cell bodies of which reside in other brain areas 25. The resolution power of the histochemical analysis, carried out with light microscopy, is too low to distinguish which of these elements is responsible for PA labeling. Thus, both the histochemical detection of in situ PA activity and the biochemical measurements of PA in cerebellar homogenates also include the contribution of incoming fibers. However, protein synthesis occurs exclusively in neuronal cell bodies and not in processes 3. The level of P A m R N A in the cerebellum should, therefore, provide the true measure of the biosynthetic potential of endogenous cerebellar cell bodies. Quantification of P A m R N A at different developmental stages reproducibly showed a 2- to 3-fold increase from day 1 to day 10 after birth, whether calculated as a fraction of the total cerebellar m R N A or as units of PA activity/g of cerebellar tissue which can be produced by P A m R N A molecules under the experimental conditions utilized. Between day 10 and 30, we observed a sharp decline in P A m R N A content/g cerebellum, but only a minor decrease when calculated as a fraction of total mRNA. Indeed, the content of total m R N A decreases sharply in the maturing cerebellar tissue 35. This decrease might account for the reduction in P A m R N A content, and our results thus indicate that endogenous cerebellar cell bodies continuously produce PA throughout cerebellar ontogenesis. The ontogenetic pattern of PA production is not a general one. The levels of the translatable m R N A for Sn-Glycerol 3-phosphate dehydrogenase increase by 6-fold during development of mouse cerebellum, when calculated as the fraction of total poly(A)-containing m R N A 15. The mRNAs coding for tubulin and actin gradually decrease in concentration during cerebellar development 22, and the concentration of cerebellar acetylcholinesterase m R N A is highest at day

1 and decreases sharply thereafter 37. Unlike these cases, the highest abundance of P A m R N A coincides with extensive neuronal migration and differentiation. The specific activity of the PA protein in the developing rat cerebellum was found to be highest during the first two postnatal weeks and to decline sharply to about one-third of the maximal level thereafter (not shown), similar to the pattern we previously reported for PA in the developing mouse cerebellum 32. The developmental decrease in the specific activity of PA which occurs at the second week after birth parallels the decrease in P A m R N A concentration, when both are calculated as units of activity/g cerebellum. This similarity suggests that most of the PA activity in the cerebellum is actively produced within cerebellar cell bodies and is largely determined by the level of available functional mRNA. In the cerebellum of newborn rats, however, the specific activity of the enzyme is as high as that observed in 10-day cerebellum, whereas the concentration of P A m R N A is relatively low. This difference between the level of PAmRNA and that of the PA protein in the newborn rat cerebellum may be due to a difference between the rates of degradation of these molecules. Alternatively, or in addition, it may imply that early in development, part of the PA activity is not produced by endogenous cerebellar cells, but is brought into the cerebellum by incoming fibers. All of our findings indicate that in the developing rat cerebellum, PA is mostly produced and contained in granular neurons, in addition to meninges, ependymas and choroid plexus cells. This conclusion is compatible with observations suggesting that glial cells in culture are rather poor producers of the enzyme 14,34,41. However, it is impossible to exclude the possibility that less abundant neuronal populations in the cerebellum, as well as specific types of incoming fibers, also contribute to the specific activity of cerebellar PA. Two major species of PA, of apparent molecular weights of 80,000 and 50,000, were detected in homogenates of the developing cerebellum and of Xenopus oocytes microinjected with cerebellar mRNA. Similarly migrating species of PA were shown to comprise distinct immunological types 7,21, analogous to the human tissue activator and urokinase 43,45. Both species were found in several lines of neuroblas-

157 t o m a cells 34. The m a j o r form of P A in the rat cerebellum is the heavier one, whereas in o t h e r tissues, such as muscle, most of the P A activity comigrates with the 50,000 molecular weight form. The P A forms p r o d u c e d in microinjected oocytes a p p e a r to reflect accurately the quantitative distribution between the two P A forms in each tissue 20, indicating that the two enzyme forms are c o d e d by different m R N A species and do not result from posttranslational processing of a c o m m o n protein precursor. It is not known, however, whether the m R N A s for the enzymatic forms are coded by different genes, or whether they are transcribed from a single gene into two distinct m R N A species. A c D N A clone to rat P A m R N A would be required to clarify this issue. Several u n r e l a t e d functions might be envisioned for P A in the developing and the m a t u r e cerebellum. It is possible that P A is required for the migration of granular neurons towards the internal granular layer. This suggestion is reinforced by the sudden increase in the level of P A m R N A which occurs at the time of granular neuron migration. In m a t u r e neurons, P A may play a role in the turnover of m e m b r a n e components 10 and in the processing of p e p t i d e h o r m o n e s REFERENCES 1 Altman, J., Autoradiographic and histological studies of postnatal neurogenesis. In N. A. Buchwald and M. A. B. Brazier (Eds.), Brain Mechanisms in Mental Retardation, Academic Press, NY, 1978, pp. 41-91. 2 Altman, J. and Anderson, W. J., Experimental reorganization of the cerebellar cortex. I. Morphological effects of elimination of all microneurons with prolonged X-irradiation started at birth, J. comp. Neurol., 146 (1972) 355--405. 3 Barondes, S. H., Protein synthesis in the nervous system. In G. J. Siegel et al. (Eds.), Basic Neurochemistry, Little, Brown, Boston, 1980, pp. 329-341. 4 Beckman, S. L., Chikaraishi, D. K., Deeb, S. S. and Seuka, N., Sequence complexity of nuclear and cytoplasmic ribonucleic acids from cional neurotumor cell lines and brain section of the rat, Biochemistry, 20 (1981) 2684-2692. 5 Beers, W. H., Follicular plasminogen and plasminogen activator and the effect of plasmin on ovarian follicle wall, Cell, 6 (1975) 379-386. 6 Ben-Barak, J., The development of the cholinergic system in rat hippocampus following postnatal X-irradiation, Brain Research, 227 (1981) 171-186. 7 Christman, J. K., Silverstein, S. C. and Acs, G., Plasminogen activators. In Proteinases in Mammalian Cells and Tissues, Elsevier/North-Holland, Amsterdam, 1977, pp. 91-149. 8 Eliyahu, D. and Soreq, H., Degranulation of rat cerebellum induces selective variations in gene expression, J. Neurochem., 38 (1982) 313-321.

and neurotransmitters, similar to the conversion of proinsulin to insulin 44. In conclusion, o u r observations indicate that granular neurons are the m a j o r p r o d u c e r s of P A in the cerebellum throughout its ontogenesis. This conclusion is based on several arguments: (a) histochemical labeling reveals in situ P A activity both in the E G L and in the I G L ; (b) the I G L labeling is greatly reduced in irradiation-agranulated cerebellum; and (c) cerebellar m R N A directs the biosynthesis of active P A in microinjected oocytes, and the ontogenetic pattern of P A m R N A parallels that of granular neuron development. ACKNOWLEDGEMENTS We thank Mr. Daniel Eliyahu for his assistance throughout this work, and Prof. Israel Silman for critical reading of the manuscript. This work was partially s u p p o r t e d by grants from the Israeli Commission for Basic Research (to H . S . ) and from the U n i t e d States-Israel Binational Science F o u n d a t i o n (BSF) Jerusalem (to R . M . ) .

9 Griffin, W. S. T., Head, J. R., Pardue, S. and Morrison, M. R., Changes in RNA synthesis and messenger RNA content in the cerebella of rats with graft versus host disease, J. Neurochem., 35 (1980) 880-888. 10 Hatzfeld, J., Miskin, R. and Reich, E., Acetylcholine receptor: effects of proteolysis on receptor metabolism, J. Cell Biol., 62 (1982) 176-182. 11 Heyting, C. and Vant Veer, L., Repair of ethylnitrosoureainduced DNA damage in the newborn rat. II. Localization of unschedule DNA synthesis in the developing rat brain, Carcinogenesis, 2 (1981) 1173-1180. 12 Hicks, S. and D'Amato, C., Effects of ionizing radiations on mammalian development. In D. H. M. Woodlam (Ed.), Advances of Tetratology, Logos Press, London, 1966, pp. i95--250. 13 Krystosek, A. and Seeds, N. W., Plasminogen activator secretion by granule neurons in cultures of developing cerebellum, Proc. nat. Acad. Sci. U.S.A., 78 (1981) 7~10-7814. 14 Krystosek, A. and Seeds, N. W., Plasminogen activator release at the neuronal growth cone, Science, 213 (1981) 1532-1533. 15 Kozak, L. P. and Ratner, P. L., Genetic regulation of translatable mRNA levels for mouse sn-glycerol-e-phosphate dehydrogenase during development of the cerebellum, J. biol. Chem., 255 (1980) 7589-7594. 16 Miskin, R. and Reich, E., Plasminogen activator: induction of synthesis by DNA damage, Cell, 19 (1980) 217-224. 17 Miskin, R. and Soreq, H., Microinjected Xenopus oocytes synthesize active human plasminogen activator, Nucl. Acids Res., 9 (1981) 3355-3364.

158 18 Miskin, R. and Soreq, H., Sensitive autoradiographic quantification of electrophoretically separated proteases, Analyt. Biochem., 118 (1981) 252-258. 19 Miskin, R. and Ben-Ishai, R., Induction of plasminogen activator by UV light in normal and Xeroderma pigrnentosurn fibroblasts, Proc. nat. Acad. Sci. U.S.A., 78 (1981) 6236-6240. 20 Miskin, R. and Soreq, H., Human and rat mRNAs are translated into urokinase-type and tissue plasminogen activators in microinjected Xenopus oocytes. In C. McLeod (Ed.), Progress in Fibrinolysis -- 6th International Congress in Fibrinolysis, Lausanne, 1982,Churchill Livingstone, Edinburgh, in press. 21 Marotti, K. R., Belin, D. and Strickland, S., The production of distinct forms of plasminogen activator by mouse embryonic cells, Develop. Biol., 90 (1982) 154-159. 22 Morrison, M. R., Pardue, S. and Griffin, S. T., Developmental alterations in the levels of translationally active messenger RNAs in the postnatal rat cerebellum, J. biol. Chem., 257 (1981) 3550-3556. 23 Olschowka, J. A. and Vijayan, V. K., Postnatal development of cholinergic neurotransmitter enzymes in the mouse cerebellum, biochemical, light microscopic and electron microscopic cytochemical investigations, J. comp. Neurol., 191 (1980) 77-101. 24 Ossowski, L., Biegel, D. and Reich, E., Mammary plasminogen activator: correlation with involution, hormonal modulation and comparison between normal and neoplastic tissue, Cell, 16 (1979) 929-940. 25 Palay, S. L. and Chan-Palay, V., Cerebellar cortex, Cytology and Organization, Springer, Berlin, 1974. 26 Porter, J. M., Acinapura, A. J., Kapp, J. P. and Donald, S., Plasminogen activator in the cerebrospinal fluid, Neurology, 19 (1969) 47-52. 27 Ramon y Cajal, S., Histologie du Systeme Nerveux de l'Homme et des Vertebres, Translated from Spanish by Dr. L. L. Asoulay Consejo, Superior de Investigation Cientificas, Madrid, 1972, pp. 107-149. 28 Reich, E., Activation of plasminogen: a widespread mechanism for generating localized extracellular proteolysis. In E. Rudden (Ed.), Biological Markers of Neoplasia: Basic and Applied Aspects, Elsevier/North-Holland, Amsterdam, 1978, pp. 491-500. 29 Rifkin, D. B., Beal, L. P. and Reich, E., Properties of plasminogen activators formed by neoplastic human cell cultures. In E. Reich et al. (Eds.), Proteases and Biological Control, Cold Spring Harbor Laboratory, New York, 1975, pp. 841-847. 30 Safran, A., Variations in translatable mRNAs During Development of the Rat Cerebellum: Selection of Ontogenetically Regulated Protein Markers. M. Sc. Thesis, Weizmann Institute, 1982. 31 Schultze, H. E. and Herman, J. F., Molecular Biology of

Human Proteins, Elsevier, New York, 1966. o. 219. 32 Soreq, H. and Miskin, R., Screening of the protease plasminogen activator in the developing mouse brain. In U.Z. Littauer et al. (Eds.), Neurotransmitters and Their Receptors, John Wiley, Chichester, London, 1980, pp. 55%563. 33 Soreq, H. and Miskin, R., Plasminogen activator in the rodent brain, Brain Res., 216 ( 1981) 361-374. 34 Soreq, H., Miskin, R., Zutra, A. and Littauer, U. Z., Modulation in the levels and localization of plasminogen activator in differentiating neuroblastoma cells, Develop. Brain Res., (1983) 257-269. 35 Soreq, H., Safran, A. and Zisling, R., Ontogenetic variations in cerebellar gene expression. Develop. Brain Res., 3 (1982) 65-79. 36 Soreq, H., Gurwitz, D., Eliyahu, D. and Sokolovsky, M., Altered ontogenesis of muscarinic receptors in agranular cerebellar cortex, J. Neurochem.. 39 (1982) 756-763. 37 Soreq, H., Parvari, R. and Silman, l., Ontogenic changes in acetylcholinesterase and in its translatable mRNA in normal and irradiation agranulated rat cerebellum, Proceedings of the Society for Neuroscience 12th Annual Meeting, 1982. 38 Sotelo, C., Mutant mice and the formation of cerebellar circuitry, Trends Neurochem. Sci., 3 (!980) 33-36. 39 Strickland, S., Plasminogen activator in early development. In M. H. Johnson (Ed.), Development of Mammals, Vol. 4. Elsevier, North-Holland, 1980, pp. 81. 40 Takashima, S., Koga, M. and Tanaka, K., Fibrinolytic activity of human brain and cerebrospinal fluid, Brit. J. exp. Path., 50 (1969) 533-539. 41 Tucker, W. S., Kitsch, W. M., Martinez-Hernanez, A. and Find, L. M., In vitro plasminogen activator activity in human brain tumors, Cancer Res.. 38 (1978) 297-302. 42 Vassalli, J. D., Hamilton, J. and Reich, E., Macrophage plasminogen activator: modulation of enzyme production by anti-inflammatory steroids, mitotic inhibitors and cyclic nucleotides, Cell, 8 (1976) 271-280. 43 Vetterlein, D., Bell, T. E., Young, P. L. and Roblin, R., Immunological quantitation and immunoadsorption of urokinase-like plasminogen activator secreted from human cells, J. biol. Chem., 255 (1980) 3665-3672. 44 Virgi, M. A. G., Vassalli, J. D., Estensen, R. D. and Reich, E., Plasminogen activator of islets of Langerhans: modulation by glucose and correlation with insulin production, Proc. nat. Acad. Sci. U.S.A., 77 (1980) 875-879. 45 Wilson, E. L., Becker, M. K. B., Hoal, E. G. and Dowdle, E. B., Molecular species of plasminogen activators secreted by normal and neoplastic human cells, Cancer Res., 40 (1980) 933-938, 46 Zisapel, N., Miskin, R., Laudon, M. and Soreq, H., Plasminogen activator is enriched in the synaptosomal plasma membranes, Brain Res., 248 (1982) 12%139.