Molecular Brain Research, 12 (1992) 215-223 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0169-328X/92/$03.50
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Changes in immediate early gene expression during postnatal development of cat cortex and cerebellum Matthew A. McCormack, Kenneth M. Rosen, Lydia Villa-Komaroff and George D. Mower Department of Neurology and Program in Neuroscience, Children's Hospital, Harvard Medical School, Boston, MA 02115 (U.S.A.) (Accepted 6 August 1991) Key word~: Immediate early gene; Proto-oncogene; Gene expression; Visual cortex: Cerebellum; mRNA
Postnatal brain development involves interactions between extracellular signals and preprogrammed genetic events. Immediate early genes (IEGs) are a group of genes that are induced by extracellular signals and their prote% products alter transcription by binding regulatory elements in other genes. Using Northern and slot blot analysis of total RNA isolated i,'om visual cortex, frontal cortex, and cerebellum of cats, we have determined the postnatal developmental patterns of mRNA expression for 5 of these genes, c-fos, egr-1, c-jun, ]un-B, and c-myc. Each gene had a distinct developmental pattern of mRNA expression, and for a given genie, these patterns were often different in different brain structures. These results suggest that temporal changes in the combinatorial interaction of different IEGs during early postnatal life are important for normal brain development. INTRODUCTION
Both the cerebral cortex and the cerebellum of cats continue to undergo substantial development during the early postnatal period 21. A number of anatomical changes occur, including neuronal proliferation and migration, dendritic elaboration, synaptogenesis, and gliogenesis. Physiological response properties mature in parallel with this anatomical development. Some of these developmental events are ~,~,,verned by preprogrammed genetic processes, while others involve a response to extracellular signals. A well characterized system where development is guided in part by externally driven neural activity is the cat visual cortex. Both the anatomy and physiology of the visual cortex can be dramatically altered by manipulation of the visual environment during a 'critical period', which includes the first several postnatal months t4. After this early postnatal period, the anatomy and physiology of the visual cortex is largely immutable. These characteristics of postnatal cortical development strongly suggest that an important mechanism involved in the developmental process must be changes in the pattern of gene expression in response to cues provided by cell-cell interactions an0 the environment. Over the last several years, a number of genes whose products mediate changes in gene expression have been identified 9'22'25'29'34. Expression of these genes, the immediate early genes (IEGs), has been studied exten-
sively in cultured cells derived from the nervous system and in the intact brain under conditions of electrical stimulation 3°'44"45'5°'51. Different groups of IEGs respond to different types of stimulation 2. The protein products of tl~ese genes alter transcription by binding regulatory elements in other genes 9'1~'23'25. These characteristics of IEG mRNA expression have led to the suggestion that their function is to couple extracellular signals at the plasma membrane with changes in the cells genetic expression 31. In the developing nervous system, the properties of IEGs are consistent with a possible role in a number of events, including, neuronal and glial proliferation, migration, and differentiation, synaptogenesis, and plasticity. Dete .training what role IEGs play in these processes requires systematic analysis of the expression of these genes during prenatal (e.g. refs. 6, 33, 46, 55) and postnatal (e.g. refs. 7, 17, 57) development. The present study had two major aims. One was to examine the expression of a l~anel of IEGs (egr-1, c-fos, c-jun, jun-B, and c-myc) in visual cortex of the cat to determine whether the temporal pattern of expression of these genes during postnatal life correlated with events occurring during the critical period in this species. A second aim was to determine whether the postnatal developmental pattern of expression of these genes differed in different brain structures by comparing visual cortex, frontal cortex, and cerebellum.
Correspondence: G.D. Mower, Department of Anatomical Sciences and Neurobiology, Health Sciences Center, University of Louisville School of Medicine, Louisville, KY 40292, U.S.A.
216 MATERIALS AND METHODS
Animals and tissue preparation A total of 17 cats at various postnatal ages (1 week, n = 2; 5 weeks, n = 2; 10 weeks, n - 2; 16 weeks, n -- 2; 20 weeks, n ffi 3; 35 weeks, n = 3, > 1 year, n = 3) were used in the study. All animals were housed and used under guidelines established by the Institutional Use and Care of Animals Committee of Children's Hospital. They were sacrificed by an overdose of sodium pentobarbital (7 mg/kg, i.p.). The visual cortical sample included nearly all of area 17 and part of area 18. It consisted of the entire lateral gyrus from lateral sulcus to cingulate sulcus. Anterior to posterior, it included from the ascending branch of the cingulate suicus to the posterior pole. Frontal cortical samples also included much of the motor cortex and part of the somatosensory cortex. These samples contained tissue anterior and medial of the central sulcus, the anterior and part of the posterior sigmoid gyms, and the frontal gy° rus. The cerebellum included the entire structure. It was obtained by cutting the peduncles and included both cortex and deep nuclei. After dissection, tissue was immediately frozen in liquid nitrogen and stored at -80 °C until used.
total RNA per lane) and then blotted onto charged nylon membranes by capillary transfer with 10x SSC. RNA was fixed on to the membranes by baking under vacuum at 80 °C for 2-4 h. Before hybridization, membranes were pre-washed in 0.5x SSC, 2% SDS at 60-65 °C for 1-2 h, and prehybridized for 4-20 h at 50 °C in prehybridization solution (50% formamide, 5x SSC, 10x Denhardt's solution, 50 mM sodium phosphate, pH 6.7, 0.5% SDS, 100 Fg/ml salmon sperm DNA). Hybridization was at 50 °C for approximately 20 h in hybridization solution (50% formamide, 5x SSC, l x Denhardt's, 20 mM sodium phosphate, pH 6.7, 100 /~g/ml salmon sperm DNA) which included the appropriate randomprimed 32p-labeled probe. After hybridization, membranes were washed to a final stringency of 0. l x SSC, 0.1% SDS at 50 °C for approximately 50 rain. Membranes were then exposed to Kodak XAR-5 film at -70 °C with intensifying screens. Densitometry of X-ray film was performed using an LKB Ultroscan laser de~tsi~vmeter. Membranes were stripped for reuse by heating in stripping solution (5 mM Tris, pH 8, 0.2 mM EDTA, 0.1x Denhardt's, 1 mM sodium pyrophosphate) at 60-65 °C for approximately 2 h. Stripped membranes were reprobed with32p-labeled glyceraldehyde-3-phospate dehydrogenase (GAPDH) cDNA following the same procedure as described above.
RNA isolation and Northern blot hybridization Total cellular RNA was isolated by the method of Chomcyznski and Sacchi ~°. Integrity of the RNA was checked after electrophoretic separation by ethidium bromide staining of ribosomal RNA. Northern blots were performed as described by Rosen et al.39. Briefly, total RNA was separated by electrophoresis in form° aldehyde-agarose gels (2.2 M formaldehyde, 1.2% agarose, 5/~g
egr-1
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jun-B
Slot blot hybridization Slot blots were prepared after the method of Sambrook et al. 42. Five/~g of total RNA was mixed with 20/d formamide, 8/A formaldehyde, and 2 / d 20 x SSC. This mixture was heated at 65 °C for 15 min, combined with 8 0 / d 20x SSC, and then applied to a charged nylon membrane. RNA was fixed on the membranes by baking under vacuum at 80 °C for 1-2 h. Membranes prepared in this way were used for hybridization as described above for Northern blots.
Probes c-los: 1.7 kb PstI fragment of a rat cDNA clone (pcfos3), kindly provided by M. Greenberg and J. Belasco; c-myc: 1.5 kb Sstl fragment derived from the human cDNA pHSR-1 (ATCC no. 41010); err-l: 700 bp Pstl fragment derived from a rat err.1 cDNA (kindly provided by V. Sukhatme); c-jun: 2.6 kb mouse cDNA pJAC.1 (ATCC no. 63026); jun-B: 1.9 kb mouse cDNA p465.20 (ATCC no. 63025); GAPDH: 500 Up Xbal-HindIIl fragment of a human GAPDH cDNA (ATCC no. 57090).
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RESULTS
To describe the postnatal developmental regulation of IEG mRNA levels in different brain structures, we iso-
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Fig. 1. Northern blot analysis of immediate early gene transcripts. For each gene analyzed, total RNA from two animals is presented. RNA was analyzed as described in Materials and Methods.
Fig. 2. Slot blot analysis of err-1 mRNA expression in visual cortex during postnatal development. Five ~g of total RNA was added to each well of a slot blot and the membrane was hybridized as described in Materials and Methods. After washing, filters were exposed to X-ray film in the presence of an intensifying screen at -70 °C. Filters were stripped and reprobed with GAPDH cDNA. RNA from 2 or 3 animals was analyzed at each of the ages indicated.
217
lated total RNA from visual cortex, frontal cortex, and cerebellum of cats at various ages (2-3 animals at each age). Total R N A from each sample was analyzed by Northern blots to establish the presence and integrity of the specific IEG transcripts (Fig. 1). In all cases, transcripts of the appropriate size were found: c-myc3;
c.fos15; c.jun41; jun.B41; egr152; G A P D H 53.
To obtain quantitative comparisons between multiple
c-fos/GAPDH
RNA samples, total RNA was applied to filters using a slot blot apparatus. These filters were then hybridized with radiolabeled IEG probes under the same conditions used for Northern blots. Each slot blot contained RNA samples from all three brain structures at each age studied, providing a direct comparison between age and structure. After hybridization, probed filters were exposed to X-ray film for various times to ensure an exposure whereby all samples were detectable and within the useful range of the film. Fig. 2 shows a representative autoradiogram of a portion of a slot blot. Autoradiograms were quantified by laser densitome-
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Age (weeks) Fig. 3. Expression of c-los mRNA during postnatal development of visual cortex, frontal cortex, and cerebellum. Autoradiograms of slot blot membranes probed with c-los cDNA, stripped, and reprobed with GAPDH cDNA were quantified with a scanning densitometer. The relative amount of hybridization for each sample was obtained by dividing the absorbancy for c-los signal by that of GAPDH signal, Points are data from individual animals to show the range at each age and open squares are the mean. The curve is the best-fit trend of the means.
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218 try. Ta correct densitometric values for variations in signal due to differences in the amounts of loaded RNA, filters were stripped and reprobed with cDNA encoding GAPDH. GAPDH mRNA levels were found to be essentially constant across development (see Fig. 2). All values were expressed as the ratio of the absorbance of the IEG probe to that of GAPDH. The resulting ratios of absorbances, when plotted with respect to age, provided a developmental pattern of expression for each IEG in each brain structure. Analysis of c-los mRNA demonstrated that levels of IEG mRNAs are developmentally regulated. Fig. 3 sum-
marizes the developmental changes in the level of expression of c-fos mRNA in visual cortex, frontal cortex, and cerebellum. The overall developmental pattern was similar in all three structures and was highlighted by a dramatic increase between the first and fifth postnatal week. After the sharp rise, c-fos mRNA levels remained high until approximately 20 weeks and then gradually declined into adulthood. The levels of c-fos were highest in visual cortex and cerebellum. These results are similar to those reported for postnatal cortex and cerebellum of rats 17. Analysis of egr-1 mRNA levels (Fig. 4) demonstrated
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Fig. 6. Expression of c-myc mRNA during postnatal development of visual cortex, frontal cortex, and cerebellum. Conventions as in Fig. 3.
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that the developmental changes in the level of a specific IEG can be markedly different in different brain structures. In visual and frontal cortex, levels of egr-1 mRNA showed a developmental pattern similar to c-los with a dramatic rise between I and 5 weeks, relatively high levels until 20 weeks, and a gradual decline into adulthood. The absolute mRNA levels wer, highest in visual cortex. The developmental pattern in cerebellum was strikingly different from that in the neocortical structures. In the cerebellum, levels of egr-1 were very low throughout postnatal life. Analysis of ]un-B mRNA levels also revealed differences between brain structures (Fig. 5). In visual and frontal cortex, there was a marked rise between 1 and 10 weeks and a gradual decline into adulthood. Compared to c-fos and egr-1 mRNAs, peak levels of jun-B mRNA occurred later (10 vs. 5 weeks) and the relative decrease into adulthood was less. In cerebellum, there were no clear changes in the levels of ]un-B mRNA and rather high levels were maintained at all ages. The pattern of developmental regulation of c-myc mRNA was totally different from those of the other IEGs described thus far (Fig. 6). In visual and frontal cortex, there was a precipitous decline in mRNA levels between 1 and 5 weeks and levels remained extremely low into adulthood. It is possible that the decrease in c.myc mRNA expression was related to the increase in c-fos mRNA expression, since a dimer of fos and ]un has been demonstrated to bind to a negative regulator in the c.myc gene 19. At 1 week of age, the level of c-myc was considerably higher in visual than frontal cortex. Again, a markedly different developmental pattern was found in the cerebellum, where relatively constant levels were maintained throughout life (see also refs. 40, 57). After 5 weeks of age, levels of c-myc mRNA were higher in the cerebellum than in visual or frontal cortex. Northern blot analysis revealed the appearance of a second, larger transcript of c-myc n:RNA in the cerebellum be-
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tween 5 and 10 weeks of age (Fig. 7). This second transcript was not detected in visual or frontal cortex. It is possible that the two transcripts arise from alternate splicing or are the products of different promoters, as has been shown in the mouse cerebellum 4°, but our study did not analyze these possibilities and here we simply note their presence. The pattern of c-myc mRNA expression in the cat cerebellum is similar to that previously shown in the mouse cerebellum 4°, where the level of the second transcript appears correlated with the proliferation of granule cell precursors. c-]un was unique among the lEGs studied in that it
220 did not show any clear developmental alterations of its mRNA levels in any of the three structures (Fig. 8). Rather, c-jun mRNA levels were maintained at significant levels throughout life in visual cortex, fro,~tal cortex, and cerebellum. DISCUSSION The present study provides a systematic description of the pattern of IEG mRNA expression during postnatal development in visual cortex, frontal cortex, and cerebellum. One obvious conclusion is that different IEGs show different developmental patterns of mRNA expression. In visual and frontal cortex, three general developmental profiles were evident. Severe l IEG mRNAs (egr-1, c-fos, jun-B) showed dynamic changes, going from very low levels at birth, through a peak during the early postnatal months, and then a gradual deciine to adult levels, c-myc mRNA showed a precipitous drop from high levels immediately after birth to negligible levels from 5 weeks to adulthood. Levels of c-jun mRNA were essentially constant throughout life. The finding of distinct and systematic developmental patterns in basal levels of IEG expression is surprising since IEGs are generally thought to be quiescent until induced by appropriate extracellular stimulation s°. This finding is important for several reasons. One is that it provides strong evidence that the results are not due to stimulation received at the time of sacrifice. More importantly, it suggests that different IEGs are responding to different extracellular events occurring during development and that the differential induction of IEG mRNAs is an important step in normal brain development. A second conclusion from this study is that the developmental profile of specific IEG mRNAs is different in different brain structures. This is most evident in comparison of the cerebellum with the two neocortical regions. Visual and frontal cortex showed very similar developmental profiles for all IEGs tested. It is noteworthy, however, that the levels of the mRNAs were 2- to 3-fold higher in visual cortex for all IEGs except c-jun. In contrast, within the cerebellum, IEG mRNAs generally showed rather constant levels of expression from neonatal life to adulthood. The exception was c-fos, which showed a sharp rise at 5 weeks of age and then a gradual decrease to relatively high steady state levels in adulthood. Additionally, c-myc showed a marked change in the ratio of its two transcripts during development, a result that has also been reported in the mouse cerebellum4°. This change was not found in visual or frontal cortex. These results suggest that different regions of the brain require different patterns of IEG expression for normal development.
It is interesting to relate the developmental profiles of IEG mRNA expression to the various events occurring during postnatal brain development. Visual cortex is an ideal structure for such comparisons because much is known concerning its postnatal development and plasticity. A number of events occur, and here we can only briefly summarize some of the major ones. In the cat, the visual cortical mantle is very immature at birth in terms of both its anatomy and physiology. Cortical layers are not defined, considerable numbers of embryonic cells remain below the cortical mantle, and geniculocortical afferents have not yet assumed their adult position 2s. By 3 weeks of age, cellular migration has completed formation of the mature lamination pattern, embryonic cells have disappeared, and geniculocortical afferents have reached their final position and begun the process of segregating into ocular dominance columns 26' 4s. Correlated with these anatomical events is the appearance of adult-like physiological response properties in visual cortical neurons by about 4 weeks of age and little change in response properties occurs thereafter s'36. The initial phase of postnatal visual cortical development, therefore, can be characterized as a period of rapid change until 3-4 weeks of age when a relatively mature state is attained and subsequently maintained. Visual cortical development is not complete, however, at 3-4 weeks of age. A second, longer lasting phase ensues during which the loosely established anatomical and physiological characteristics are stabilized under the guidance of visual experience. One major event during this second phase is synaptic development. Synaptic density rises sharply from birth to a peak at 40-70 days and then declines, reaching adult levels at 3-4 months of age n's6. Maturation of both glial and neurotransmitter systems also occurs gradually over the first several postnatal months 4'~6'2~'32. The outstanding characteristic of this second developmental phase is plasticity, that is, the capacity for anatomical and physiological development to be altered by visual experience. The clearest demonstration of such environmental effects is to rear cats with one eye sutured closed, a condition that leads to dramatic anatomical and physiological alterations 49'54. Visual cortex is susceptible to monocular deprivation only during a 'critical period' of postnatal life. In cats, susceptibility to monocular deprivation is absent until about 3 weeks, peaks at 4-5 weeks, then gradually declines and disappears at about 6 months of age 2°'35. After 6 months of age, visual cortex has matured to adult levels and is largely immutable. The developmental profiles of IEG mRNA expression found here offer some intriguing parallels to the developmental events described above. Three of the IEGs studied (egr-1, c-fos, jun-B) showed dynamic develop-
221 mental patterns which were temporally similar to the second phase of visual cortical development and suggest a possible involvement of these IEGs in synaptic development and/or plasticity, c-myc m R N A showed a temporal pattern similar to the first phase of visual cortical development. The similarity of the IEG m R N A developmental profiles in visual and frontal cortex is also important in this context. Recent studies have shown that cortical lamination and synaptogenesis occur concurrently over diverse regions of the cerebral cortex, suggesting a common genetic signal 37. The present results provide direct evidence for such commonality in the development of gene expre:~sion in different cortical regions. Although the correlations between specific IEG developmental profiles and the developmentai events in neocortex are appealing, it is likely that any influences that IEGs exert on brain development are more complex. Increasing evidence indicates that the mechanism by which IEGs regulate target genes involves combinatorial interactions of different I E G protein products as well as changes ill the relative amounts of different IEG protein combinations 29'31'5°. This mechanism has been demonstrated most clearly for the interactions of the fos and jun families. The proteins formed by these gene families form heterodimeric complexes collectively termed AP-113aa'a4. The AP-1 complex then binds to target genes to alter their transcription rate 24'3s'4a. Genes involved in a wide array of cellular functions contain AP-1 binding sites in their regulatory elements t'24. Heterodimers of c-los and c-jun proteins activate many
genes, while heterodimers of c-fos and jun-B appear to be capable of activating certain genes and repressing others a'27'47. A second level of complexity is added by temporal differences in the time course of induction of different IEGs. For example, after seizure, different members of the fos and jun families are induced with different time courses s~. A single I E G inducing event can therefore produce a cascade of activation and repression of a number of different target genes. The present findings of dynamic developmental profiles for different IEGs suggests that similar mechanisms could be involved in brain development. Changing levels of a given I E G m R N A would produce different levels of specific heterodimers at different times in development when that gene product interacted with a second gene product. This would be true whether the second gene product showed dynamic or constant levels during development. The system appears designed to produce changing IEG combinations across development, presumably aimed at a critical sequence of indi~ction and repression of specific target genes. Undoubtedly, as more is learned about the targets and functions of IEG products and the cellular localization of those products in the developing and mature brain, the role of changes in gene expression in brain development will be clarified.
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Acknowledgements. The work described here was supported by grants from the National Institutes of Health to G.M.D. (NS25216), L.V.K. (NS27832) ~nd The Mental Retardation Research Center at Children's Host~itai P30-HD18655). M.A.M. was supported by NIH training Grant T32 NS07264,
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37 Rakic, E, Bourgeois, J.-P., Eckenhoff, M.F., Zecevic, N. and Goldman-Rakic, P., Concurrent overproduction of synapses in diverse regions of the primate cerebral cortex, Science, 232 (1986) 232-236. 38 Rauscher, F.J. Ill, Sambucetti, L.C., Curtan, T., Distel, R.J. and Spiegelman, B.M., Common DNA binding site for Fos protein complexes and transcriptioa factor AP-1, Cell, 52 (1988) 471-480. 39 Rosen, K.M., Lamperti, E.D. and Villa-Komaroff, L., Optimizing the northern blot procedure, BioTechniques, 8 (1990) 398-403. 40 Ruppert, C., Goldowitz, D. and Wille, W., Proto-oncogene c-myc is expressed in cerebellar neurons at different develop. mental stages, EMBO/., 5 (1986) 1897-1901. 41 Ryder, K. and Nathans, D., Induction of protooncogene c-jun by serum growth factors, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 8464-8467. 42 Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Clon. ing. A Laboratory Manual., 2nd edn., Cold Spring Harbor Press, New York, 1989, 753 pp. 43 Sassone-Corsi, E, Lamph, W.W., Kamps, M. and Verma, I.M., Fos-associated cellular p39 is related to nuclear transcription factor AP-1, Cell, 54 (1988) 553-560. 44 Saffen, D.W., Cole, A.J., Worley, EE, Christy, B.A., Ryder, K. and Baraban, J.M., Convulsant-induced increase in transcription factor messenger RNAs in rat brain, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 775-779. 45 Sagar, S.M., Sharp, E R. and Curran, T., Expression of c-los protein in brain: metabolic mapping at the cellular level, Science, 240 (1988) 1328-1331. 46 Schmid, P., Schulz, W.A. and Hameister, H., Dynamic expression pattern of the myc protooncogene in midgestation mouse embryos, Science, 243 (1989) 226-229. 47 Schutte, J., ViaUet, J., Nau, M., Segal, S., Fedorko, J. and Minna, J., jun-B inhibits and c-fos stimulates the transforming and trans-activating activities of c-jun, Ceil, 59 (1989) 987-997. 48 Shatz, C.J. and Luskin, M.B., The relationship between the geniculocortical afferents and their cortical target cells during development of the cat's primary visual cortex, J. Neurosci., 6 (1986) 3655-3668. 49 Shatz, C.J. and Stryker, M.P., Ocular dominance in layer IV of the cat's visual cortex and the effects of monocular deprivation, J. Physiol., 281 (1978) 267-283. 50 Sheng, M. and Greenberg, M.E., The regulation of c-fos and other immediate early genes in the nervous system, Neuron, 4 (1990) 477-485. 51 Sonnenberg, J.L., Macgregor-Leon, P.E, Curran, T. and Morgan, J.I., Dynamic alterations occur in the levels and composition of transcription factor AP-1 complexes after seizure, Neuron, 3 (1989) 359-369. 52 Sukhatme, V.E, Cao, X., Chang, L.C., Tsai-Morris, C.-H., Stamenkovich, D., Ferreira, EC.E, Cohen, D.R., Edwards, S.A., Shows, T.B., Curran, T., LaBeau, M.M. and Adamson, E.D., A zinc finger-encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization, Cell, 53 (1988) 37-43. 53 Tso, J.Y., Sun, X.-H., Kao, T.-h., Reece, K.S. and Wu, R., Isolation and characterization of rat and human glyceraldehyde3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene, ?Cud. Acids Res., 13 (1985) 2485-2502. 54 Wiesel, T.N. and Hubel, D.H., Single cell responses in striate cortex of kittens deprived of vision in one eye, J. Neurophysiol., 26 (1963) 1003-1017. 55 °i¢iikinson, D.G., Bhatt, S., Ryseck. R.E and Bravo, R., Tissue specific expression of c-jun and ,un-B during organogenesis in the mouse. Development, 106 (1989) 465-471. 56 Winfield, D.A., The postnatal development of synapses in the different laminae of the visual cortex in the normal kitten and in kittens with eyelid suture, Dev. Brain Res., 9 (i983) i55-169.
223 57 Zimmerman, K.A., Yancopoulos, G.D., Collum, R.G., Smith, R.K., Kohl, N.E., Denis, K.A., Nau, M.M., Witte, O.N., Toran-Allerand, D., Gu, C.E., Minna, J.D. and Alt, F.W., Dif-
ferential expression of myc family genes during murine development, Nature, 319 (1986) 780-783.
215
BRESM 70373
Changes in immediate early gene expression during postnatal development of cat cortex and cerebellum Matthew A. McCormack, Kenneth M. Rosen, Lydia Villa-Komaroff and George D. Mower Department of Neurology and Program in Neuroscience, Children's Hospital, Harvard Medical School, Boston, MA 02115 (U.S.A.) (Accepted 6 August 1991) Key word~: Immediate early gene; Proto-oncogene; Gene expression; Visual cortex: Cerebellum; mRNA
Postnatal brain development involves interactions between extracellular signals and preprogrammed genetic events. Immediate early genes (IEGs) are a group of genes that are induced by extracellular signals and their prote% products alter transcription by binding regulatory elements in other genes. Using Northern and slot blot analysis of total RNA isolated i,'om visual cortex, frontal cortex, and cerebellum of cats, we have determined the postnatal developmental patterns of mRNA expression for 5 of these genes, c-fos, egr-1, c-jun, ]un-B, and c-myc. Each gene had a distinct developmental pattern of mRNA expression, and for a given genie, these patterns were often different in different brain structures. These results suggest that temporal changes in the combinatorial interaction of different IEGs during early postnatal life are important for normal brain development. INTRODUCTION
Both the cerebral cortex and the cerebellum of cats continue to undergo substantial development during the early postnatal period 21. A number of anatomical changes occur, including neuronal proliferation and migration, dendritic elaboration, synaptogenesis, and gliogenesis. Physiological response properties mature in parallel with this anatomical development. Some of these developmental events are ~,~,,verned by preprogrammed genetic processes, while others involve a response to extracellular signals. A well characterized system where development is guided in part by externally driven neural activity is the cat visual cortex. Both the anatomy and physiology of the visual cortex can be dramatically altered by manipulation of the visual environment during a 'critical period', which includes the first several postnatal months t4. After this early postnatal period, the anatomy and physiology of the visual cortex is largely immutable. These characteristics of postnatal cortical development strongly suggest that an important mechanism involved in the developmental process must be changes in the pattern of gene expression in response to cues provided by cell-cell interactions an0 the environment. Over the last several years, a number of genes whose products mediate changes in gene expression have been identified 9'22'25'29'34. Expression of these genes, the immediate early genes (IEGs), has been studied exten-
sively in cultured cells derived from the nervous system and in the intact brain under conditions of electrical stimulation 3°'44"45'5°'51. Different groups of IEGs respond to different types of stimulation 2. The protein products of tl~ese genes alter transcription by binding regulatory elements in other genes 9'1~'23'25. These characteristics of IEG mRNA expression have led to the suggestion that their function is to couple extracellular signals at the plasma membrane with changes in the cells genetic expression 31. In the developing nervous system, the properties of IEGs are consistent with a possible role in a number of events, including, neuronal and glial proliferation, migration, and differentiation, synaptogenesis, and plasticity. Dete .training what role IEGs play in these processes requires systematic analysis of the expression of these genes during prenatal (e.g. refs. 6, 33, 46, 55) and postnatal (e.g. refs. 7, 17, 57) development. The present study had two major aims. One was to examine the expression of a l~anel of IEGs (egr-1, c-fos, c-jun, jun-B, and c-myc) in visual cortex of the cat to determine whether the temporal pattern of expression of these genes during postnatal life correlated with events occurring during the critical period in this species. A second aim was to determine whether the postnatal developmental pattern of expression of these genes differed in different brain structures by comparing visual cortex, frontal cortex, and cerebellum.
Correspondence: G.D. Mower, Department of Anatomical Sciences and Neurobiology, Health Sciences Center, University of Louisville School of Medicine, Louisville, KY 40292, U.S.A.
216 MATERIALS AND METHODS
Animals and tissue preparation A total of 17 cats at various postnatal ages (1 week, n = 2; 5 weeks, n = 2; 10 weeks, n - 2; 16 weeks, n -- 2; 20 weeks, n ffi 3; 35 weeks, n = 3, > 1 year, n = 3) were used in the study. All animals were housed and used under guidelines established by the Institutional Use and Care of Animals Committee of Children's Hospital. They were sacrificed by an overdose of sodium pentobarbital (7 mg/kg, i.p.). The visual cortical sample included nearly all of area 17 and part of area 18. It consisted of the entire lateral gyrus from lateral sulcus to cingulate sulcus. Anterior to posterior, it included from the ascending branch of the cingulate suicus to the posterior pole. Frontal cortical samples also included much of the motor cortex and part of the somatosensory cortex. These samples contained tissue anterior and medial of the central sulcus, the anterior and part of the posterior sigmoid gyms, and the frontal gy° rus. The cerebellum included the entire structure. It was obtained by cutting the peduncles and included both cortex and deep nuclei. After dissection, tissue was immediately frozen in liquid nitrogen and stored at -80 °C until used.
total RNA per lane) and then blotted onto charged nylon membranes by capillary transfer with 10x SSC. RNA was fixed on to the membranes by baking under vacuum at 80 °C for 2-4 h. Before hybridization, membranes were pre-washed in 0.5x SSC, 2% SDS at 60-65 °C for 1-2 h, and prehybridized for 4-20 h at 50 °C in prehybridization solution (50% formamide, 5x SSC, 10x Denhardt's solution, 50 mM sodium phosphate, pH 6.7, 0.5% SDS, 100 Fg/ml salmon sperm DNA). Hybridization was at 50 °C for approximately 20 h in hybridization solution (50% formamide, 5x SSC, l x Denhardt's, 20 mM sodium phosphate, pH 6.7, 100 /~g/ml salmon sperm DNA) which included the appropriate randomprimed 32p-labeled probe. After hybridization, membranes were washed to a final stringency of 0. l x SSC, 0.1% SDS at 50 °C for approximately 50 rain. Membranes were then exposed to Kodak XAR-5 film at -70 °C with intensifying screens. Densitometry of X-ray film was performed using an LKB Ultroscan laser de~tsi~vmeter. Membranes were stripped for reuse by heating in stripping solution (5 mM Tris, pH 8, 0.2 mM EDTA, 0.1x Denhardt's, 1 mM sodium pyrophosphate) at 60-65 °C for approximately 2 h. Stripped membranes were reprobed with32p-labeled glyceraldehyde-3-phospate dehydrogenase (GAPDH) cDNA following the same procedure as described above.
RNA isolation and Northern blot hybridization Total cellular RNA was isolated by the method of Chomcyznski and Sacchi ~°. Integrity of the RNA was checked after electrophoretic separation by ethidium bromide staining of ribosomal RNA. Northern blots were performed as described by Rosen et al.39. Briefly, total RNA was separated by electrophoresis in form° aldehyde-agarose gels (2.2 M formaldehyde, 1.2% agarose, 5/~g
egr-1
c-fos
jun-B
Slot blot hybridization Slot blots were prepared after the method of Sambrook et al. 42. Five/~g of total RNA was mixed with 20/d formamide, 8/A formaldehyde, and 2 / d 20 x SSC. This mixture was heated at 65 °C for 15 min, combined with 8 0 / d 20x SSC, and then applied to a charged nylon membrane. RNA was fixed on the membranes by baking under vacuum at 80 °C for 1-2 h. Membranes prepared in this way were used for hybridization as described above for Northern blots.
Probes c-los: 1.7 kb PstI fragment of a rat cDNA clone (pcfos3), kindly provided by M. Greenberg and J. Belasco; c-myc: 1.5 kb Sstl fragment derived from the human cDNA pHSR-1 (ATCC no. 41010); err-l: 700 bp Pstl fragment derived from a rat err.1 cDNA (kindly provided by V. Sukhatme); c-jun: 2.6 kb mouse cDNA pJAC.1 (ATCC no. 63026); jun-B: 1.9 kb mouse cDNA p465.20 (ATCC no. 63025); GAPDH: 500 Up Xbal-HindIIl fragment of a human GAPDH cDNA (ATCC no. 57090).
~t
RESULTS
To describe the postnatal developmental regulation of IEG mRNA levels in different brain structures, we iso-
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Fig. 2. Slot blot analysis of err-1 mRNA expression in visual cortex during postnatal development. Five ~g of total RNA was added to each well of a slot blot and the membrane was hybridized as described in Materials and Methods. After washing, filters were exposed to X-ray film in the presence of an intensifying screen at -70 °C. Filters were stripped and reprobed with GAPDH cDNA. RNA from 2 or 3 animals was analyzed at each of the ages indicated.
217
lated total RNA from visual cortex, frontal cortex, and cerebellum of cats at various ages (2-3 animals at each age). Total R N A from each sample was analyzed by Northern blots to establish the presence and integrity of the specific IEG transcripts (Fig. 1). In all cases, transcripts of the appropriate size were found: c-myc3;
c.fos15; c.jun41; jun.B41; egr152; G A P D H 53.
To obtain quantitative comparisons between multiple
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RNA samples, total RNA was applied to filters using a slot blot apparatus. These filters were then hybridized with radiolabeled IEG probes under the same conditions used for Northern blots. Each slot blot contained RNA samples from all three brain structures at each age studied, providing a direct comparison between age and structure. After hybridization, probed filters were exposed to X-ray film for various times to ensure an exposure whereby all samples were detectable and within the useful range of the film. Fig. 2 shows a representative autoradiogram of a portion of a slot blot. Autoradiograms were quantified by laser densitome-
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Age (weeks) Fig. 3. Expression of c-los mRNA during postnatal development of visual cortex, frontal cortex, and cerebellum. Autoradiograms of slot blot membranes probed with c-los cDNA, stripped, and reprobed with GAPDH cDNA were quantified with a scanning densitometer. The relative amount of hybridization for each sample was obtained by dividing the absorbancy for c-los signal by that of GAPDH signal, Points are data from individual animals to show the range at each age and open squares are the mean. The curve is the best-fit trend of the means.
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218 try. Ta correct densitometric values for variations in signal due to differences in the amounts of loaded RNA, filters were stripped and reprobed with cDNA encoding GAPDH. GAPDH mRNA levels were found to be essentially constant across development (see Fig. 2). All values were expressed as the ratio of the absorbance of the IEG probe to that of GAPDH. The resulting ratios of absorbances, when plotted with respect to age, provided a developmental pattern of expression for each IEG in each brain structure. Analysis of c-los mRNA demonstrated that levels of IEG mRNAs are developmentally regulated. Fig. 3 sum-
marizes the developmental changes in the level of expression of c-fos mRNA in visual cortex, frontal cortex, and cerebellum. The overall developmental pattern was similar in all three structures and was highlighted by a dramatic increase between the first and fifth postnatal week. After the sharp rise, c-fos mRNA levels remained high until approximately 20 weeks and then gradually declined into adulthood. The levels of c-fos were highest in visual cortex and cerebellum. These results are similar to those reported for postnatal cortex and cerebellum of rats 17. Analysis of egr-1 mRNA levels (Fig. 4) demonstrated
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that the developmental changes in the level of a specific IEG can be markedly different in different brain structures. In visual and frontal cortex, levels of egr-1 mRNA showed a developmental pattern similar to c-los with a dramatic rise between I and 5 weeks, relatively high levels until 20 weeks, and a gradual decline into adulthood. The absolute mRNA levels wer, highest in visual cortex. The developmental pattern in cerebellum was strikingly different from that in the neocortical structures. In the cerebellum, levels of egr-1 were very low throughout postnatal life. Analysis of ]un-B mRNA levels also revealed differences between brain structures (Fig. 5). In visual and frontal cortex, there was a marked rise between 1 and 10 weeks and a gradual decline into adulthood. Compared to c-fos and egr-1 mRNAs, peak levels of jun-B mRNA occurred later (10 vs. 5 weeks) and the relative decrease into adulthood was less. In cerebellum, there were no clear changes in the levels of ]un-B mRNA and rather high levels were maintained at all ages. The pattern of developmental regulation of c-myc mRNA was totally different from those of the other IEGs described thus far (Fig. 6). In visual and frontal cortex, there was a precipitous decline in mRNA levels between 1 and 5 weeks and levels remained extremely low into adulthood. It is possible that the decrease in c.myc mRNA expression was related to the increase in c-fos mRNA expression, since a dimer of fos and ]un has been demonstrated to bind to a negative regulator in the c.myc gene 19. At 1 week of age, the level of c-myc was considerably higher in visual than frontal cortex. Again, a markedly different developmental pattern was found in the cerebellum, where relatively constant levels were maintained throughout life (see also refs. 40, 57). After 5 weeks of age, levels of c-myc mRNA were higher in the cerebellum than in visual or frontal cortex. Northern blot analysis revealed the appearance of a second, larger transcript of c-myc n:RNA in the cerebellum be-
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tween 5 and 10 weeks of age (Fig. 7). This second transcript was not detected in visual or frontal cortex. It is possible that the two transcripts arise from alternate splicing or are the products of different promoters, as has been shown in the mouse cerebellum 4°, but our study did not analyze these possibilities and here we simply note their presence. The pattern of c-myc mRNA expression in the cat cerebellum is similar to that previously shown in the mouse cerebellum 4°, where the level of the second transcript appears correlated with the proliferation of granule cell precursors. c-]un was unique among the lEGs studied in that it
220 did not show any clear developmental alterations of its mRNA levels in any of the three structures (Fig. 8). Rather, c-jun mRNA levels were maintained at significant levels throughout life in visual cortex, fro,~tal cortex, and cerebellum. DISCUSSION The present study provides a systematic description of the pattern of IEG mRNA expression during postnatal development in visual cortex, frontal cortex, and cerebellum. One obvious conclusion is that different IEGs show different developmental patterns of mRNA expression. In visual and frontal cortex, three general developmental profiles were evident. Severe l IEG mRNAs (egr-1, c-fos, jun-B) showed dynamic changes, going from very low levels at birth, through a peak during the early postnatal months, and then a gradual deciine to adult levels, c-myc mRNA showed a precipitous drop from high levels immediately after birth to negligible levels from 5 weeks to adulthood. Levels of c-jun mRNA were essentially constant throughout life. The finding of distinct and systematic developmental patterns in basal levels of IEG expression is surprising since IEGs are generally thought to be quiescent until induced by appropriate extracellular stimulation s°. This finding is important for several reasons. One is that it provides strong evidence that the results are not due to stimulation received at the time of sacrifice. More importantly, it suggests that different IEGs are responding to different extracellular events occurring during development and that the differential induction of IEG mRNAs is an important step in normal brain development. A second conclusion from this study is that the developmental profile of specific IEG mRNAs is different in different brain structures. This is most evident in comparison of the cerebellum with the two neocortical regions. Visual and frontal cortex showed very similar developmental profiles for all IEGs tested. It is noteworthy, however, that the levels of the mRNAs were 2- to 3-fold higher in visual cortex for all IEGs except c-jun. In contrast, within the cerebellum, IEG mRNAs generally showed rather constant levels of expression from neonatal life to adulthood. The exception was c-fos, which showed a sharp rise at 5 weeks of age and then a gradual decrease to relatively high steady state levels in adulthood. Additionally, c-myc showed a marked change in the ratio of its two transcripts during development, a result that has also been reported in the mouse cerebellum4°. This change was not found in visual or frontal cortex. These results suggest that different regions of the brain require different patterns of IEG expression for normal development.
It is interesting to relate the developmental profiles of IEG mRNA expression to the various events occurring during postnatal brain development. Visual cortex is an ideal structure for such comparisons because much is known concerning its postnatal development and plasticity. A number of events occur, and here we can only briefly summarize some of the major ones. In the cat, the visual cortical mantle is very immature at birth in terms of both its anatomy and physiology. Cortical layers are not defined, considerable numbers of embryonic cells remain below the cortical mantle, and geniculocortical afferents have not yet assumed their adult position 2s. By 3 weeks of age, cellular migration has completed formation of the mature lamination pattern, embryonic cells have disappeared, and geniculocortical afferents have reached their final position and begun the process of segregating into ocular dominance columns 26' 4s. Correlated with these anatomical events is the appearance of adult-like physiological response properties in visual cortical neurons by about 4 weeks of age and little change in response properties occurs thereafter s'36. The initial phase of postnatal visual cortical development, therefore, can be characterized as a period of rapid change until 3-4 weeks of age when a relatively mature state is attained and subsequently maintained. Visual cortical development is not complete, however, at 3-4 weeks of age. A second, longer lasting phase ensues during which the loosely established anatomical and physiological characteristics are stabilized under the guidance of visual experience. One major event during this second phase is synaptic development. Synaptic density rises sharply from birth to a peak at 40-70 days and then declines, reaching adult levels at 3-4 months of age n's6. Maturation of both glial and neurotransmitter systems also occurs gradually over the first several postnatal months 4'~6'2~'32. The outstanding characteristic of this second developmental phase is plasticity, that is, the capacity for anatomical and physiological development to be altered by visual experience. The clearest demonstration of such environmental effects is to rear cats with one eye sutured closed, a condition that leads to dramatic anatomical and physiological alterations 49'54. Visual cortex is susceptible to monocular deprivation only during a 'critical period' of postnatal life. In cats, susceptibility to monocular deprivation is absent until about 3 weeks, peaks at 4-5 weeks, then gradually declines and disappears at about 6 months of age 2°'35. After 6 months of age, visual cortex has matured to adult levels and is largely immutable. The developmental profiles of IEG mRNA expression found here offer some intriguing parallels to the developmental events described above. Three of the IEGs studied (egr-1, c-fos, jun-B) showed dynamic develop-
221 mental patterns which were temporally similar to the second phase of visual cortical development and suggest a possible involvement of these IEGs in synaptic development and/or plasticity, c-myc m R N A showed a temporal pattern similar to the first phase of visual cortical development. The similarity of the IEG m R N A developmental profiles in visual and frontal cortex is also important in this context. Recent studies have shown that cortical lamination and synaptogenesis occur concurrently over diverse regions of the cerebral cortex, suggesting a common genetic signal 37. The present results provide direct evidence for such commonality in the development of gene expre:~sion in different cortical regions. Although the correlations between specific IEG developmental profiles and the developmentai events in neocortex are appealing, it is likely that any influences that IEGs exert on brain development are more complex. Increasing evidence indicates that the mechanism by which IEGs regulate target genes involves combinatorial interactions of different I E G protein products as well as changes ill the relative amounts of different IEG protein combinations 29'31'5°. This mechanism has been demonstrated most clearly for the interactions of the fos and jun families. The proteins formed by these gene families form heterodimeric complexes collectively termed AP-113aa'a4. The AP-1 complex then binds to target genes to alter their transcription rate 24'3s'4a. Genes involved in a wide array of cellular functions contain AP-1 binding sites in their regulatory elements t'24. Heterodimers of c-los and c-jun proteins activate many
genes, while heterodimers of c-fos and jun-B appear to be capable of activating certain genes and repressing others a'27'47. A second level of complexity is added by temporal differences in the time course of induction of different IEGs. For example, after seizure, different members of the fos and jun families are induced with different time courses s~. A single I E G inducing event can therefore produce a cascade of activation and repression of a number of different target genes. The present findings of dynamic developmental profiles for different IEGs suggests that similar mechanisms could be involved in brain development. Changing levels of a given I E G m R N A would produce different levels of specific heterodimers at different times in development when that gene product interacted with a second gene product. This would be true whether the second gene product showed dynamic or constant levels during development. The system appears designed to produce changing IEG combinations across development, presumably aimed at a critical sequence of indi~ction and repression of specific target genes. Undoubtedly, as more is learned about the targets and functions of IEG products and the cellular localization of those products in the developing and mature brain, the role of changes in gene expression in brain development will be clarified.
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