Journal of Neuroscience Research 3 3 5 5 9 4 6 7 (1992)
Developmental Expression of Neuronal Calmodulin mRNA Species in the Rat Brain Analyzed by In Situ Hybridization B. Ni, C.F. Landry, and I.R. Brown Department of Zoology, University of Toronto, Scarborough Campus, West Hill, Ontario, Canada The temporal and spatial distribution of calmodulin mRNAs which are preferentially expressed in neurons was determined during postnatal development of rat central nervous system. Expression of these mRNAs was strongly detected in the developing neocortex, hippocampus, and cerebellum. Differences in the pattern of expression of a 1.8 and 4.0 kb neuronal calmodulin mRNA species were identified in the developing cerebellum. Expression of the smaller mRNA appeared to correlate with proliferating and developing cerebellar granule neurons while the larger mRNA was present in the mature granule neuron population. A transient elevation in the neuronal calmodulin mRNA species was observed in the superior and inferior colliculus and in the thalamus at postnatal days 5 and 10. 0 1992 Wiley-Liss, Inc. Key words: calmodulin mRNA, in situ hybridization, development INTRODUCTION Calmodulin is a calcium-binding protein of molecular weight 17,000, which is a multifunctional regulator of cellular processes such as cell growth and differentiation (Cheung, 1980; Klee and Vanaman, 1982; Means et al., 1982). Activation of calmodulin, through the binding of calcium, results in a cascade of regulatory events such as protein phosphorylation. Calmodulin has been implicated in several neural phenomena, including neurotransmitter release (Rubin, 1972; Rasanussen and Goodman, 1977; for review, see DeLorenzo, 1982), long-term potentiation (Malenka et al., 1989), neurite elongation and growth cone movement (Polak et al., 1991). Studies have suggested that this protein plays an important role in signal transduction in the CNS (Cheung, 1980; Means et al., 1982; Malenka et al., 1989; Siekevitz, 1991). Calmodulin is widely distributed and believed to be present in all eukaryotic species and tissues (Means et al., 1982). Although it appears to be enriched in the nervous system, its concentration varies markedly in dif0 1992 Wiley-Liss, Inc.
ferent brain regions (Zhou et al., 1985) and cell types (Egrie et al., 1977; Wood et al., 1980; Caceres et al., 1983). In addition, the protein is found in a number of subcellular fractions which may be involved in neurotransmission, such as the postsynaptic density (Grab et al., 1980; Lin et al., 1980; Caceres et al., 1983), postsynaptic membrane (Wood et al., 1980; Sobue et al., 1982), and synaptic vesicles (DeLorenzo, 1982). Calmodulin has been isolated and sequenced from a number of organisms that span the evolutionary spectrum from yeast to man including both lower and higher plants (Means et al., 1982). It has been shown that the protein sequence is highly conserved through evolution and is identical in mammals (Means et al., 1982). Multiple calmodulin mRNA species have been reported (SenGupta et a]., 1987; Fisher et al., 1988; Nojima, 1989). In rat, at least three calmodulin genes and four mRNA species have been found (Nojima et al., 1989; Ni et al., 1992) which show between 15 to 18% divergence in their coding regions and pronounced differences in their 3 ’ nontranslated sequences. We have previously cloned two rat calmodulin mRNA species, NGBl (4.0 kb) and NGB2 (1.8 kb), which are highly enriched in brain tissue and preferentially expressed in neurons of the rat brain (Ni et al., 1992). Both mRNA species are derived from the rat calmodulin gene CaM 1 by utilization of different polyadenylation addition sites (Ni et al., 1992). Sequence analysis has demonstrated that NGBl has a longer and unique 3‘ untranslated region containing an AU-rich “destabilizer-like” element. This sequence has been shown to influence mRNA half life in the cytoplasm (Shaw and Kamen, 1986) and is characteristic of many inducible mRNAs, such as c-fos (Morgan and Curran, 1989) and SCGlO (Stein et al., 1988). A recent study has shown that levels of the NGBl mRNA species are inReceived June 29, 1992; revised August 12, 1992; accepted August 14, 1992. Address reprint requests to Ian R. Brown, Dept. of Zoology, University of Toronto, Scarborough Campus, West Hill, Ontario, Canada M1C 1A4.
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creased in specific regions of the rat brain stem in response to the impulse activity evoked by reserpine (Ni and Brown, 1993). In addition, NGBl is induced in cells surrounding the site of wounding within a few hours following a cortical lesion in rats (Landry et al., 1992). Moreover, using Northern blotting, a fivefold increase in levels of the NGBl mRNA species has been detected during development of the rat cerebellum while a three fold increase was noted for NGB2 mRNA (Ni et al., 1992). In this report, we have applied in situ hybridization histochemistry to map out the pattern of the expression of neuronal calmodulin mRNAs during postnatal development of the rat brain.
MATERIALS AND METHODS Vectors and Probes We have previously described the molecular cloning of the calmodulin mRNA species, NGBl and NGB2, which are derived from the rat CaMl gene (Ni et al., 1992). For the production of antisense and sense riboprobes, two cDNA fragments, P1 and P2, were subcloned into the RNA synthesis vector Bluescript (Stratagene) and linearized for in vitro transcription with Pvu I1 (Ni et al., 1992). Synthesis of RNA Probes In vitro transcription reactions were carried out as described by Stratagene. ["SIUTP was utilized in the production of riboprobes for in situ hybridization. Preparation of Tissue for In Situ Hybridization Preparation of brain tissue was carried out as described by Landry et al. (1989). Briefly, animals were anaesthetized with 50 mg/kg pentobarbitol and perfused intracardially with 0.1 M phosphate-buffered saline (PBS), pH 7.2 followed by 4% paraformaldehyde in PBS. Brains were removed and mounted in OCT embedding compound. Cryostat sections (15 pm) were collected on glass microscope slides double-coated with a solution of 1% gelatin-0.05% chromium potassium sulfate. To facilitate probe penetration, tissue sections were briefly digested with proteinase K immediately before prehybridization (Landry et al., 1990). The concentration of proteinase K required for optimal tissue integrity and probe penetration was empirically determined for each age and ranged between 5 pg/mg for adult and 0.5 pg/mg for postnatal day I (Lewis and Cowan, 1985). Our previous Northern blot data which indicated a developmental increase in neuronal calmodulin mRNAs (Ni et al., 1992) are consistent with the present developmental in situ hybridization results. In Situ Hybridization In situ hybridization was carried out as described previously by Landry et al. (1989). Briefly, tissue sec-
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Fig. 1. Schematic illustration of probes utilized for in situ hybridization. Regions used for generating riboprobes for in situ hybridization are marked as follows: P1 delineates the region which was used for synthesizing a probe which recognizes both the 4.0 kb NGBl and the 1.8 kb NGB2 neuronal calmodulin mRNA species; P2 delineates the region used for generating a probe which is specific to the larger NGBl transcript (Ni et al., 1992).
tions were prehybridized in a solution of 50% formamide, 750 mM NaCl, 0.1% Ficol, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin, 0.1% SDS, 100 mM dithiothreitol and 50 mM PIPES, pH 6.9 at 42°C for 10 min. Hybridization was at 55°C for 14 hr with 2 X lo6 cpm "S-labelled antisense PI or P2 riboprobe added per slide. Slides were then incubated in a solution of 20 p,g/ml RNAse A in TES buffer (500 mM NaCl, 1 mM EDTA, 10 mM Tris, pH 7.4) at 37°C for 20 min. The slides were washed at 37°C for 1 hr in TES buffer containing 0.3 M P-mercaptoethanol and finally at 70°C for 1 hr in 0.1 X SSC and 0.3 M P-mercaptoethanol. The slides were then dehydrated through an ethanol series containing 0.33 M ammonium acetate, air-dried and processed for autoradiography with either Cronex sensitive X-ray film (Dupont) or NTB2 autoradiographic emulsion from Kodak (Landry et a]., 1989). Data shown are representative of three animals which were used at each developmental stage.
RESULTS Generation of Specific Probes Used for Detection of Neuronal Calmodulin mRNA Species We have previously identified two rat calmodulin mRNA species, NGBl and NGB2, which are highly enriched in the rat brain and preferentially expressed in neurons (Ni et al., 1992). To further characterize the distribution of these mRNA species in the rat brain during postnatal development, two probes, designated P 1 and P2, were generated for in situ hybridization analysis (Ni et a]. , 1992). As shown in Figure 1, P1 is a sequence which lies within the coding regions of both NGBl and NGB2 and hence detects both mRNA species. P2, derived from the unique 3' noncoding region of NGB 1, is a probe which is specific to the larger 4.0 kb mRNA. As a control for non-specific probe binding, sagittal sections of the adult brain were hybridized with either sense or antisense P1 riboprobe. Binding was not observed in
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Fig. 2 . Distribution of neuronal calmodulin mRNA species in the adult rat brain. Sagittal rat brain sections were probed with 35S-labelledP1 riboprobe, exposed to NTB2 autoradiographic emulsion for 4 weeks and lightly stained with Cresyl violet. Signal was visualized by darkfield microscopy. A: Sagittal brain section probed with antisense riboprobe ( x 3). A neuronal enriched pattern of specific labelling is apparent in the cerebellum, hippocampus, cerebral cortex. B: Sagittal section probed with sense riboprobe as a control for nonspecific probe binding ( X 3).
tissue sections hybridized with sense riboprobe while signal was detected in neuronal enriched areas using antisense riboprobe (Fig. 2). We have previously described this pattern of expression in the adult rat brain (Ni et al., 1992; Ni and Brown, 1993). Our present efforts are directed towards the investigation of the developmental expression of the neuronal calmodulin mRNA species.
Distribution of the Neuronal Calmodulin mRNAs During Rat Brain Development In order to obtain an overall view of the developmental pattern of expression of the neuronal calmodulin mRNA species, 35S-labelled PI and P2 riboprobes were hybridized to sagittal brain sections and exposed directly to X-ray film. As shown in the Figure 3, five postnatal stages were included in this study: postnatal days (PD) 1, 5, 10, 15 and 20. Results with the P1 probe are shown in the left panels and results with the P2 probe in the right panels. The signal distribution suggests that the neuronal calmodulin mRNA species are expressed in various neuronal enriched regions of the hippocampus, cerebellum, neocortex, thalamus, superior and inferior colliculus and brain stem during postnatal development of the rat central nervous system. Although in situ hybridization with the two probes revealed a similar pattern in most regions of the rat brain, differences in the pattern of expression of the two calmodulin mRNA species were observed in the cerebellum during early postnatal development, as will be shown at higher magnification (Fig. 5). Cerebral cortex. As shown in Figure 3A,B, expression of the neuronal calmodulin mRNAs was detected in the neocortex (nc) and proliferative zone (pz) at PD 1. Expression was pronounced in the neocortex by
PD 5 (Fig. 5C,D) and the overall signal remained high in this brain region throughout the rest of postnatal development. Hippocampus. As shown in the overall X-ray film analysis (Fig. 3A,B) and at higher magnification using dark-field microscopy and emulsion autoradiography (Fig. 4A,B), a rather diffuse signal corresponding to the calmodulin mRNAs was apparent at postnatal day 1 in the hippocampus and dentate g y m . By PD 10 signal appeared to be localized over the pyramidal cell layer of the hippocampus (PLH) and the granule cell layer of the dentate gyrus (GLD) (Figs. 3E,F; 4C,D). This pattern became sharply defined by PD 15 and PD 20 (Figs. 3G-J; 4E,F). Overall a similar developmental pattern of expression was detected with the P1 and P2 probes during postnatal development of the hippocampus (compare Fig. 4A,C,E with Figure 4B,D,F). Superior and inferior colliculus, thalamus. A prominent and transient increase in levels of the neuronal calmodulin mRNAs was noted in the inferior colliculus (ic) and in the optic nerve layer (op) of superior colliculus (Fig. 3). With both the P1 and P2 probes, a diffuse signal was noted in these regions at PD 1 (panels A and B) followed by a strong, localized elevation in signal at PD 5 and 10 (panels C-F) and thereafter a greatly decreased signal by PD 15 and 20 (panels G-J). A similar transient increase in neuronal calmodulin mRNA levels was also observed in the thalamus (th). Cerebellum. As shown in Figure 3, levels of expression of the neuronal calmodulin mRNAs were lower in the cerebellum at PD 1 compared to other neuronenriched areas such as the hippocampus and neocortex (Fig. 3A,B). However, mRNA levels increased greatly during postnatal development of the cerebellum. AS will
Fig. 3. Expression of the neuronal calmodulin mRNA species during postnatal development of the rat brain. Brain tissue sections were hybridized with P1 o r P2 riboprobes described in Figure 1 and processed for in situ hybridization analysis with X-ray film. Signal was visualized by bright-field microscopy. Five postnatal stages were included: postnatal day (PD) 1
(A,B), 5 (C,D), 10 (E,F), 15 (G,H), and 20 (1,J). cb, cerebellum; hc, hippocampus; ic, inferior colliculus; nc, neocortex; pz, proliferative zone; op, optic nerve layer of superior colliculus; th, thalamus. P1 riboprobe was utilized in the panels shown on the left and P2 riboprobe in panels on the right.
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Fig. 4. Neuronal calmodulin mRNAs in the developing hippocampus. Tissue sections were hybridized with PI (A,C,E) and P2 (B,D,F) riboprobes as described in Figure 2. Signal was visuaIized by darkfield microscopy. Three postnatal stages were included: postnatal day (PD) 1 (A,B) ( X lo), 10 (C,D) ( X lo), 20 (E,F) ( X 13). GLD, granule cell layer of dentate gyrus; PLH, pyramidal cell layer of hippocampus.
be shown at higher magnification (Fig. 5 ) , in situ hybridization revealed different labelling patterns with the P1 and P2 probes during early development a€the cerebellum. This implies that 4.0 kb NGBl and 1.8 kb NGB2 neuronal calmodulin mRNA species may be differentially expressed in cellular layers of the developing cerebellum.
Expression of the Neuronal Calmodulin mRNAs in the Developing Cerebellum In order to investigate at higher resolution the pattern of expression of the neuronal calmodulin mRNA species in the developing rat cerebellum, in situ hybridization was carried out with the PI and P2 riboprobes
using autoradiographic emulsion (Fig. 5). Differences in the pattern of expression of the 4.0 kb NGBl and the 1.8 kb NGB2 neuronal calmadulin mRNA species were noted in the granule cell layers of the developing cerebellum. Granule neurons are generated postnatally by division of cells within the external granule cell layer (Burgoyne and Cambray-Deakin, 1988). The whole process of granule cell proliferation and migration to the internal granule cell layer occurs throughout the first 3 postnatal weeks in the rat cerebellum (Altman, 1972). Postnatal day 1. As shown in Figure 5A, at PD 1 the PI probe detected a diffuse signal in the Purkinje cell layer (PL) and in the external granule cell layer (EGL). The P2 probe, which is specific for the 4.0 kb NGBl
Fig. 5 .
Developmental Expression of Neuronal Calmodulin mRNAs
calmodulin mRNA, detected signal over the cells in the Purkinje cell layer (Fig. 5B). In contrast to the P1 probe, labelling was not observed with the P2 probe in external granule cell layer (Fig. 5B). Postnatal day 5. A laminar distribution of calmodulin mRNAs was apparent at PD 5 with the P1 probe which detects both calmodulin mRNA species (Fig. 5C). Signal was apparent in a thin line which clearly followed the external granule cell layer (EGL) of Cerebellum and was present from PD 5 until PD 15 (Fig. 5C,E,G). A punctate pattern of expression with the P1 probe was also observed in the internal granule neuron layer (GL) and the Purkinje cell layer (PL). With the P2 probe, which is specific for the 4.0 kb NGBl mRNA, a punctate pattern of expression was localized over the Purkinje cell layer (PL), however, signal was absent from the external granule cell layer (Fig. 5D). These results suggested that the 1.8 kb NGB2 mRNA species and not the 4.0 kb NGBl species is expressed in the proliferating and developing granule neuron population. A relatively strong signal was detected in neurons of the deep cerebellar nuclei (dwn) with both probes whereas no signal was observed in neuron-poor regions such as the deep white matter
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white matter (WM). The expression pattern of the neuronal calmodulin mRNAs observed at this stage (PD20) is identical to that observed in the adult rat brain.
DISCUSSION
The involvement of calmodulin and its target enzymes in the development of nervous system is an area of active study (Billingsley et al., 1990; Cimino et al., 1990; Weinman et al., 1991). Several lines of evidence have shown that calmodulin is involved during the course of development in regulation of its target enzymes which include cyclic nucleotide phosphodiesterase, protein phosphatase, calcineurin and protein kinase I1 which mediate important processes in neuronal activity (Hyman and Pfenninger, 1985; Hoskins and Ho, 1986; Ratan and Shelanski, 1986; Billingsley et al., 1990). In the present study, we have utilized in situ hybridization to determine the developmental pattern of expression of neuronal calmodulin mRNA species which we have recently characterized by molecular cloning (Ni et al., 1992). Particular effort was devoted to the investigation of the pattern of expression of these mRNA species in cellular layers of the rat cerebellum during postnatal development. In a WM). Postnatal days 10 and 15. An intense signal with previous study using Northern blotting we have reported P1 probe was apparent in the internal granule cell layer a developmental increase in a 1.8 kb (NGB2) and a 4.0 (GL) at PD 10 and persisted through to adulthood (Fig. kb (NGB 1) mRNA species in the cerebellum (Ni et al., 5E,G,I). Much lower levels of expression of the 4.0 kb 1992), an observation which is consistent with the NGBl calmodulin mRNA species were apparent in this present in situ hybridization results. The cerebellum has been frequently studied as an cellular layer from PD 5 through to PD 15 (Fig. ideal system for neuronal development because of its 5D,F,H). Postnatal day 20. Because the proliferation and laminated structure, the presence of a limited number of migration of granule neurons from the external granule neuronal cell types, and the detailed knowledge of the cell layer to the internal granule cell layer is completed morphological changes that occur during development of by the third postnatal week of development, no signal this brain region (Bernhardt and Matus, 1984). In the rat was evident at PD 20 with the P1 probe in the outermost cerebellum, the majority of granule cells are generated layer of cerebellum (Fig. 51). At PD 20, the two probes postnatally in the external granule cell layer (EGL) (Burdemonstrated a similar signal pattern (Fig. 51,J). A goyne and Cambray-Deakin, 1988). Immature granule strong signal was observed over the Purkinje layer (PL), cells in the EGL extend their axons and leading processes internal granule cell layer (GL), and deep cerebellar nu- down through the molecular layer and migrate towards clei. No signal was evident over glial cells in the deep the internal granular layer during early postnatal development, leaving their parallel fibres behind (Altman, 1972, 1975, 1982). The whole process of granule cell proliferation and migration occurs throughout the first 3 Fig. 5 . Distribution of neuronal calmodulin mRNA species in weeks of postnatal development in the rat cerebellum cellular layers of the developing cerebellum analyzed by in situ (Altman, 1972). hybridization. Tissue sections were hybridized with PI probe The present developmental study indicated that the (A,C,E,G,I) ( x 13) and NGBl specific probe P2 (B,D,F,H,J) two probes, P1 and P2, detected differences in signal ( x 13) as described in Figure 2 . Five postnatal stages were patterns during early postnatal development of the cereincluded: postnatal days (PD) 1 (A,B), 5 (C,D), 10 (E,F), 15 (G,H), and 20 (1,J). Signal was visualized by darkfield mi- bellum, suggesting that NGBl and NGB2 neuronal croscopy. dwn, deep white nuclei; EGL, external granule cell calmodulin mRNAs may be differentially expressed in layer; GL, internal granule cell layer; PL, Purkinje layer; WM, cellular layers of this brain region. In situ hybridization deep white matter. Longer exposures with the P2 probe did not revealed that relatively high levels of expression of the 1 .8 kb NGB2 mRNA were apparent in the external granchange the pattern of expression which is shown.
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ule cell layer during early postnatal development from PD 1 to 15, whereas the 4.0 kb NGBl mRNA was not. The strong PI signal and weak P2 signal in the internal granule cell layer from PD 5 to 15 suggest that the 1.8 kb NGB2 mRNA is preferentially expressed in developing granule neurons. Unlike the 1.8 kb NGB2 mRNA, the 4.0 kb NGBl mRNA species was not detected in the external granule cell layer, however it was expressed at abundant levels in the Purkinje cell layer from postnatal day 1 to 20 and also at PD 20 in the internal granule cell layer. In summary, our data support the notion that the 1.8 kb NGB2 mRNA species may be involved in cellular processes during the proliferation and maturation of granule neurons while the 4.0 kb neuronal calmodulin mRNA is present in mature granule neurons. Previous studies have noted that calmodulin immunoreactivity was present in proliferative regions of the cerebellum (Cumming et al., 1982; Caceres et al., 1983). At present it is not known why the 4.0 kb NGBl mRNA species, which contains an AU-rich “destabilizer-like” element, is expressed in mature granule neurons while the 1.8 kb NGB2 species, which does not contain this element, is expressed in developing granule neurons. The AU-rich ‘‘destabilizer-like” element may influence mRNA half life (Shaw and Kamen, 1986) and is characteristic of many inducible mRNAs such as c-fos (Morgan and Curran, 1989). Neuronal cells possessing an inducible calmodulin mRNA with a short half-life might utilize it in responsive mechanisms in calcium-dependent signalling events or in maintenance of calcium homeostasis. We have demonstrated that levels of the 4.0 kb mRNA species are rapidly increased in cells surrounding the site of a cortical lesion in rats (Landry et al., 1992) and that levels of this neuronal calmodulin mRNA species are increased in specific regions of the rat brain stem in response to impulse activity evoked by reserpine (Ni and Brown, 1993). Studies on staggerer (sg/sg) mutant mice, which have an intrinsic defect in Purkinje cells ( H e m p and Mullen, 1979a,b), have demonstrated that normal expression of a calmodulin gene is altered in Purkinje neurons (Messer et al., 1990). While the cause(s) of the degeneration of this population of neurons is not precisely known, the authors suggest that it may result from a defect in Purkinje-cell-specific regulation of a calmodulin gene. The protein coding region of the rat CaMl gene has a 94% homology with the mouse calmodulin gene whose expression is significantly altered in Purkinje neurons of the staggerer mutant (Messer et al., 1990; Ni et al., 1992). The study on the staggerer mutant mouse and our present observation of the presence of CaMl gene transcripts in developing Purkinje neurons may suggest an important role for CaMl gene transcripts in the development of this population of neurons.
In the cerebral cortex, neuronal calmodulin mRNA was detected at postnatal day 1 in the cortical plate and proliferative zone where the most recently formed neurons are localized. The distribution of the neuronal calmodulin mRNA species in the developing cerebral cortex correlates with the anatomical maturation of neurons in this region and is in agreement with the observation that calmodulin may be involved in the maturation and proliferation of neurons in the cerebral cortex (Rasmussen and Means, 1989). A transient and highly localized increase in neuronal calmodulin mRNA was observed in the inferior colliculus and optic nerve layer of the superior colliculus at PD 5 and 10. This may suggest that up-regulation of the CaMl gene occurs during steps involved in the formation of visional projection pathways. While the present study focuses on expression of neuronal calmodulin mRNAs during postnatal development of the rat brain, calmodulin immunoreactivity has been found in the marginal neural tube of developing mice embryos as early as day 9.5 of gestation (SetoOhshima et al., 1987). In addition, calmodulin mRNAs have been detected in the rat brain as early as embryonic day 14 by Northern blot analysis (Weinman et al., 1991) and by in situ hybridization (Landry, C.F., personal communication). Calmodulin may play an important role in growth cone function (Seto-Ohshima et al., 1983; Hyman and Pfenninger, 1985) and other processes involved in early development of the CNS (Hoskins and Ho, 1986; Seto-Ohshima et al., 1987).
ACKNOWLEDGMENTS This research was supported by grants from NSERC (Canada) to I.R.B.
REFERENCES Altman J (1972): Postnatal development of the cerebellar cortex in the rat. 1. The external germinal layer and the transitional molecular layer. J Comp Neurol 145:353-398. Altman J (1975): Postnatal development of the cerebellar cortex in the rat. IV. Spatial organization of bipolar cells. Parallel fibres and glial pallisades. J Comp Neurol 163:427-448. Altman J (1982): Morphological development of the rat cerebellum and some of its mechanisms. Exp Brain Res Suppl 6:8-49. Bernhardt R, Matus A (1984): Initial phase of dendrite growth: Evidence for the involvement of high molecular weight rnicrotubule-associated proteins (HMWP) before the appearance of tubulin. J Cell Biol 92:589-593. Billingsley ML, Polli JW, Balaban CD, Kincaid L (1990): Developmental expression of calmodulin-dependent cyclic nucleotide phosphodiesterase in rat brain. Developmental Brain Res 53: 256-263. Burgoyne RD, Cambray-Deakin MA (1988): The cellular neurobiology of neuronal development: The cerebellar granule cell. Brain Res Rev 13:77-101. Caceres A, Bender P, Snavely L, Rebhun LI, Steward 0 (1983):
Developmental Expression of Neuronal Calmodulin mRNAs Distribution and subcellular localization of calmodulin in adult and developing brain tissue. Neuroscience 10:449-46 1. Cheung WY (1980): Calmodulin plays a pivotal role in cellular regulation. Science 207: 19-27. Cimino M, Chen JF, Weiss B (1990): Ontogenetic development of calmodulin mRNA in rat brain using in situ hybridization histochemistry. Developmental Brain Res 54:43-49. Cumming R, Krigman MR, Steine AL (1982): Perinatal ontogenesis of calmodulin and cyclic AMP-dependent protein kinase subunits in the Purkinje cell using immunofluorescence. Neurosci Lett 28:247-252. DeLorenzo RJ (1 982): Calmodulin in neurotransmitter release and synaptic function. Fed Proc 41:2265-2272. Egne JC, Campbell A, Flangas AL, Siege1 FL (1977): Regional, cellular and subcellular distribution of calcium-activated cyclic nucleotide phosphodiesterase and calcium-dependent regulator in porcine brain. J Neurochem 28:1207-1213. Fischer R, Koller M, Flura M, Mathews S , Strehler-Page, M-A, Krebs J, Penniston JT, Carafoli E, Strehler EE (1988): Multiple divergent mRNAs code for a single human calmodulin. J Biol Chem 263:17055-17062. Grab DJ, Carlin RK, Siekevitz P (1980): The presence of calmodulin in postsynaptic densities. Ann NY Acad Sci 35655-72. H e m p K, Mullen RJ (1979a): Staggerer chimeras: Intrinsic nature of Purkinje cell defects and implications for normal cerebellar development. Brain Res 178:443-457. H e m p K, Mullen RJ (1979b): Regional variation and absence of large neurons in the cerebellum of the staggerer mouse. Brain Res 172:l-12. Hoskins B, Ho 1K (1986): Effects of maturation and aging on calmodulin and calmodulin-regulated enzymes in various regions of mouse brain. Mech Aging Dev 36:137-186. Hyman C, Pfenninger KH (1985): Intracellular regulations of neuronal sprouting: Calmodulin-binding proteins of nerve growth cones. J Cell Biol 101:1153-1160. Klee CB, Vanaman TC (1982): Calmodulin. Adv Protein Chem 35: 213-231. Landry CF, Ivy GO, Dunn RJ, Marks A, Brown IR (1989): Expression of the gene encoding the f3-subunit of S-100 protein in the developing rat brain analyzed by in situ hybridization. Mol Brain Res 6:251-262. Landry CF, Ivy GO, Brown IR (1990): Developmental expression of glial fibrillary acidic protein mRNA in the rat brain analyzed by in situ hybridization. J Neurosci Res 25:194-203. Landry CF, Ivy GO, Brown IR (1992): Effect of a discrete dorsal forebrain lesion in the rat on the expression of neuronal and glial-specific genes: induction of calmodulin, NF-L, SCI and GFAP mRNAs. J Neurosci 32:280-289. Lewis SA, Cowan NJ (1985): Temporal expression of mouse glial fibrillary acidic protein studied by a rapid in situ hybridization procedure. J Neurochem 45:913-919. Lin CT, Dedman JR, Brinkley BR, Marcus AR (1980): Localization of calmodulin in rat cerebellum by irnmunoelectron microscopy. J Cell Biol 85:473-480. Malenka RC, Kauer JA, Perkel DJ, Mauk MD, Kelly PT, Nicoll RA, Waxham MN (1989): An essential role for postsynaptic calmodulin and protein kinase activity in long-term potentiation. Nature 34034-557. Means AR, Tash JS, Chafouleas JG (1982): Physiological implications of the presence, distribution and regulation of calmodulin in eukaryotic cells. Physiol Rev 62:l-39.
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Messer A , Plummer-Siegard J, Eisenberg B (1990): Staggerer mutant mouse Purkinje cells do not contain detectable calmodulin mRNA. J Neurochem 55:293-302. Morgan JI, Curran T (1989): Stimulus-transcription coupling in neurons: Role of cellular immediate-early genes. TINS 12:459462. Ni B, Rush S, Gurd JW, Brown IR (1992): Molecular cloning of calmodulin mRNA species which are preferentially expressed in neurons of the rat brain. Mol Brain Res 13:7-17. Ni B, Brown IR (1993): Modulation of a neuronal calmodulin mRNA species in the rat brain stem by reserpine. Neurochemical Res (in press). Nojima H (1989): Structural organization of multiple rat calmodulin genes. J Mol Biol 208:269-282. Polak KA, Edelman AM, Wasley JWF, Cohan CS (1991): A novel calmodulin antagonist, CGS 9343B, modulates calcium-dependent changes in neurite outgrowth and growth cone movements. J Neurosci 11534-542. Rasanussen H and Goodman DBP (1977): Relationship between calcium and cyclic nucleotides in cell activation. Physiol Rev 57:421-509. Rasmussen D, Means AR (1989): Calmodulin, cell growth and gene expression. TINS 12:433-438. Ratan RR, Shelanski ML (1986): Calcium and the regulation of mitotic events. Trends Biochem Sci 11:456-459. Rubin RP (1972): The role of calcium in the release of neurotransmitter substances and hormones. Pharmaco Rev 22:389-428. SenGupta B, Friedberg F, Betera-Wadleigh SD (1987): Molecular analysis of human and rat calmodulin complementary DNA clones. J Biol Chem 262:16663-16670, Seto-Ohshima A, Kitajima S , Sano M, Kato K, Mizutani A (1983): Immunohistochemical localization of calmodulin in mouse brain. Histochemistry 79:25 1-257. Seto-Ohshima A, Yamazaki Y, Kawamura N, Kitajima S, Sano M, Mizutani A (1987): The early expression of immunoreactivity for calmodulin in the nervous system of mouse embryos. Histochemistry 86337-343. Shaw G, Kamen R (1986): A conserved AU sequence from the 3’ untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46:659-667. Siekevitz P (1991): Possible role for calmodulin and the Ca+ +/calmodulin-dependent protein kinase I1 in postsynaptic neurotransmission. Proc Natl Acad Sci 885374-5378. Sobue K, Morimoto K, Kanda K, Kakiuchi S (1982): Ca+ -dependent binding of 3H-calmodulin to the microsomal fraction of brain. J Biochem 91:1313-1320. Stein R, Mori N, Matthews K, Lo L, Anderson D (1988): The NGFinducible SCGlO mRNA encodes a novel membrane-bound protein present in growth cones and abundant in developing neurons. Neuron 1:463-476. Weinman J, Gaspera BD, Dautigny A, Dinh DP, Wang J, Nojima H, Weinman S (1991): Developmental regulation of calmodulin gene expression in rat brain and skeletal muscle. Cell Regulation 2:819-826. Wood JG, Wallace RW, Whitaker JN, Cheung WY (1980): Immunocytochemical localization of calmodulin and a heat-labile calmodulin-binding protein (CaM-BP80) in basal ganglia of mouse brain. J Cell Biol 84:66-76. Zhou LW, Moyer JA, Muth EA, Clark B, Palkovits M, Weiss B (1985): Regional distribution of calmodulin activity in rat brain. J Neurochem 44:1657-1662. +
Developmental Expression of Neuronal Calmodulin mRNA Species in the Rat Brain Analyzed by In Situ Hybridization B. Ni, C.F. Landry, and I.R. Brown Department of Zoology, University of Toronto, Scarborough Campus, West Hill, Ontario, Canada The temporal and spatial distribution of calmodulin mRNAs which are preferentially expressed in neurons was determined during postnatal development of rat central nervous system. Expression of these mRNAs was strongly detected in the developing neocortex, hippocampus, and cerebellum. Differences in the pattern of expression of a 1.8 and 4.0 kb neuronal calmodulin mRNA species were identified in the developing cerebellum. Expression of the smaller mRNA appeared to correlate with proliferating and developing cerebellar granule neurons while the larger mRNA was present in the mature granule neuron population. A transient elevation in the neuronal calmodulin mRNA species was observed in the superior and inferior colliculus and in the thalamus at postnatal days 5 and 10. 0 1992 Wiley-Liss, Inc. Key words: calmodulin mRNA, in situ hybridization, development INTRODUCTION Calmodulin is a calcium-binding protein of molecular weight 17,000, which is a multifunctional regulator of cellular processes such as cell growth and differentiation (Cheung, 1980; Klee and Vanaman, 1982; Means et al., 1982). Activation of calmodulin, through the binding of calcium, results in a cascade of regulatory events such as protein phosphorylation. Calmodulin has been implicated in several neural phenomena, including neurotransmitter release (Rubin, 1972; Rasanussen and Goodman, 1977; for review, see DeLorenzo, 1982), long-term potentiation (Malenka et al., 1989), neurite elongation and growth cone movement (Polak et al., 1991). Studies have suggested that this protein plays an important role in signal transduction in the CNS (Cheung, 1980; Means et al., 1982; Malenka et al., 1989; Siekevitz, 1991). Calmodulin is widely distributed and believed to be present in all eukaryotic species and tissues (Means et al., 1982). Although it appears to be enriched in the nervous system, its concentration varies markedly in dif0 1992 Wiley-Liss, Inc.
ferent brain regions (Zhou et al., 1985) and cell types (Egrie et al., 1977; Wood et al., 1980; Caceres et al., 1983). In addition, the protein is found in a number of subcellular fractions which may be involved in neurotransmission, such as the postsynaptic density (Grab et al., 1980; Lin et al., 1980; Caceres et al., 1983), postsynaptic membrane (Wood et al., 1980; Sobue et al., 1982), and synaptic vesicles (DeLorenzo, 1982). Calmodulin has been isolated and sequenced from a number of organisms that span the evolutionary spectrum from yeast to man including both lower and higher plants (Means et al., 1982). It has been shown that the protein sequence is highly conserved through evolution and is identical in mammals (Means et al., 1982). Multiple calmodulin mRNA species have been reported (SenGupta et a]., 1987; Fisher et al., 1988; Nojima, 1989). In rat, at least three calmodulin genes and four mRNA species have been found (Nojima et al., 1989; Ni et al., 1992) which show between 15 to 18% divergence in their coding regions and pronounced differences in their 3 ’ nontranslated sequences. We have previously cloned two rat calmodulin mRNA species, NGBl (4.0 kb) and NGB2 (1.8 kb), which are highly enriched in brain tissue and preferentially expressed in neurons of the rat brain (Ni et al., 1992). Both mRNA species are derived from the rat calmodulin gene CaM 1 by utilization of different polyadenylation addition sites (Ni et al., 1992). Sequence analysis has demonstrated that NGBl has a longer and unique 3‘ untranslated region containing an AU-rich “destabilizer-like” element. This sequence has been shown to influence mRNA half life in the cytoplasm (Shaw and Kamen, 1986) and is characteristic of many inducible mRNAs, such as c-fos (Morgan and Curran, 1989) and SCGlO (Stein et al., 1988). A recent study has shown that levels of the NGBl mRNA species are inReceived June 29, 1992; revised August 12, 1992; accepted August 14, 1992. Address reprint requests to Ian R. Brown, Dept. of Zoology, University of Toronto, Scarborough Campus, West Hill, Ontario, Canada M1C 1A4.
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creased in specific regions of the rat brain stem in response to the impulse activity evoked by reserpine (Ni and Brown, 1993). In addition, NGBl is induced in cells surrounding the site of wounding within a few hours following a cortical lesion in rats (Landry et al., 1992). Moreover, using Northern blotting, a fivefold increase in levels of the NGBl mRNA species has been detected during development of the rat cerebellum while a three fold increase was noted for NGB2 mRNA (Ni et al., 1992). In this report, we have applied in situ hybridization histochemistry to map out the pattern of the expression of neuronal calmodulin mRNAs during postnatal development of the rat brain.
MATERIALS AND METHODS Vectors and Probes We have previously described the molecular cloning of the calmodulin mRNA species, NGBl and NGB2, which are derived from the rat CaMl gene (Ni et al., 1992). For the production of antisense and sense riboprobes, two cDNA fragments, P1 and P2, were subcloned into the RNA synthesis vector Bluescript (Stratagene) and linearized for in vitro transcription with Pvu I1 (Ni et al., 1992). Synthesis of RNA Probes In vitro transcription reactions were carried out as described by Stratagene. ["SIUTP was utilized in the production of riboprobes for in situ hybridization. Preparation of Tissue for In Situ Hybridization Preparation of brain tissue was carried out as described by Landry et al. (1989). Briefly, animals were anaesthetized with 50 mg/kg pentobarbitol and perfused intracardially with 0.1 M phosphate-buffered saline (PBS), pH 7.2 followed by 4% paraformaldehyde in PBS. Brains were removed and mounted in OCT embedding compound. Cryostat sections (15 pm) were collected on glass microscope slides double-coated with a solution of 1% gelatin-0.05% chromium potassium sulfate. To facilitate probe penetration, tissue sections were briefly digested with proteinase K immediately before prehybridization (Landry et al., 1990). The concentration of proteinase K required for optimal tissue integrity and probe penetration was empirically determined for each age and ranged between 5 pg/mg for adult and 0.5 pg/mg for postnatal day I (Lewis and Cowan, 1985). Our previous Northern blot data which indicated a developmental increase in neuronal calmodulin mRNAs (Ni et al., 1992) are consistent with the present developmental in situ hybridization results. In Situ Hybridization In situ hybridization was carried out as described previously by Landry et al. (1989). Briefly, tissue sec-
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Fig. 1. Schematic illustration of probes utilized for in situ hybridization. Regions used for generating riboprobes for in situ hybridization are marked as follows: P1 delineates the region which was used for synthesizing a probe which recognizes both the 4.0 kb NGBl and the 1.8 kb NGB2 neuronal calmodulin mRNA species; P2 delineates the region used for generating a probe which is specific to the larger NGBl transcript (Ni et al., 1992).
tions were prehybridized in a solution of 50% formamide, 750 mM NaCl, 0.1% Ficol, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin, 0.1% SDS, 100 mM dithiothreitol and 50 mM PIPES, pH 6.9 at 42°C for 10 min. Hybridization was at 55°C for 14 hr with 2 X lo6 cpm "S-labelled antisense PI or P2 riboprobe added per slide. Slides were then incubated in a solution of 20 p,g/ml RNAse A in TES buffer (500 mM NaCl, 1 mM EDTA, 10 mM Tris, pH 7.4) at 37°C for 20 min. The slides were washed at 37°C for 1 hr in TES buffer containing 0.3 M P-mercaptoethanol and finally at 70°C for 1 hr in 0.1 X SSC and 0.3 M P-mercaptoethanol. The slides were then dehydrated through an ethanol series containing 0.33 M ammonium acetate, air-dried and processed for autoradiography with either Cronex sensitive X-ray film (Dupont) or NTB2 autoradiographic emulsion from Kodak (Landry et a]., 1989). Data shown are representative of three animals which were used at each developmental stage.
RESULTS Generation of Specific Probes Used for Detection of Neuronal Calmodulin mRNA Species We have previously identified two rat calmodulin mRNA species, NGBl and NGB2, which are highly enriched in the rat brain and preferentially expressed in neurons (Ni et al., 1992). To further characterize the distribution of these mRNA species in the rat brain during postnatal development, two probes, designated P 1 and P2, were generated for in situ hybridization analysis (Ni et a]. , 1992). As shown in Figure 1, P1 is a sequence which lies within the coding regions of both NGBl and NGB2 and hence detects both mRNA species. P2, derived from the unique 3' noncoding region of NGB 1, is a probe which is specific to the larger 4.0 kb mRNA. As a control for non-specific probe binding, sagittal sections of the adult brain were hybridized with either sense or antisense P1 riboprobe. Binding was not observed in
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Fig. 2 . Distribution of neuronal calmodulin mRNA species in the adult rat brain. Sagittal rat brain sections were probed with 35S-labelledP1 riboprobe, exposed to NTB2 autoradiographic emulsion for 4 weeks and lightly stained with Cresyl violet. Signal was visualized by darkfield microscopy. A: Sagittal brain section probed with antisense riboprobe ( x 3). A neuronal enriched pattern of specific labelling is apparent in the cerebellum, hippocampus, cerebral cortex. B: Sagittal section probed with sense riboprobe as a control for nonspecific probe binding ( X 3).
tissue sections hybridized with sense riboprobe while signal was detected in neuronal enriched areas using antisense riboprobe (Fig. 2). We have previously described this pattern of expression in the adult rat brain (Ni et al., 1992; Ni and Brown, 1993). Our present efforts are directed towards the investigation of the developmental expression of the neuronal calmodulin mRNA species.
Distribution of the Neuronal Calmodulin mRNAs During Rat Brain Development In order to obtain an overall view of the developmental pattern of expression of the neuronal calmodulin mRNA species, 35S-labelled PI and P2 riboprobes were hybridized to sagittal brain sections and exposed directly to X-ray film. As shown in the Figure 3, five postnatal stages were included in this study: postnatal days (PD) 1, 5, 10, 15 and 20. Results with the P1 probe are shown in the left panels and results with the P2 probe in the right panels. The signal distribution suggests that the neuronal calmodulin mRNA species are expressed in various neuronal enriched regions of the hippocampus, cerebellum, neocortex, thalamus, superior and inferior colliculus and brain stem during postnatal development of the rat central nervous system. Although in situ hybridization with the two probes revealed a similar pattern in most regions of the rat brain, differences in the pattern of expression of the two calmodulin mRNA species were observed in the cerebellum during early postnatal development, as will be shown at higher magnification (Fig. 5). Cerebral cortex. As shown in Figure 3A,B, expression of the neuronal calmodulin mRNAs was detected in the neocortex (nc) and proliferative zone (pz) at PD 1. Expression was pronounced in the neocortex by
PD 5 (Fig. 5C,D) and the overall signal remained high in this brain region throughout the rest of postnatal development. Hippocampus. As shown in the overall X-ray film analysis (Fig. 3A,B) and at higher magnification using dark-field microscopy and emulsion autoradiography (Fig. 4A,B), a rather diffuse signal corresponding to the calmodulin mRNAs was apparent at postnatal day 1 in the hippocampus and dentate g y m . By PD 10 signal appeared to be localized over the pyramidal cell layer of the hippocampus (PLH) and the granule cell layer of the dentate gyrus (GLD) (Figs. 3E,F; 4C,D). This pattern became sharply defined by PD 15 and PD 20 (Figs. 3G-J; 4E,F). Overall a similar developmental pattern of expression was detected with the P1 and P2 probes during postnatal development of the hippocampus (compare Fig. 4A,C,E with Figure 4B,D,F). Superior and inferior colliculus, thalamus. A prominent and transient increase in levels of the neuronal calmodulin mRNAs was noted in the inferior colliculus (ic) and in the optic nerve layer (op) of superior colliculus (Fig. 3). With both the P1 and P2 probes, a diffuse signal was noted in these regions at PD 1 (panels A and B) followed by a strong, localized elevation in signal at PD 5 and 10 (panels C-F) and thereafter a greatly decreased signal by PD 15 and 20 (panels G-J). A similar transient increase in neuronal calmodulin mRNA levels was also observed in the thalamus (th). Cerebellum. As shown in Figure 3, levels of expression of the neuronal calmodulin mRNAs were lower in the cerebellum at PD 1 compared to other neuronenriched areas such as the hippocampus and neocortex (Fig. 3A,B). However, mRNA levels increased greatly during postnatal development of the cerebellum. AS will
Fig. 3. Expression of the neuronal calmodulin mRNA species during postnatal development of the rat brain. Brain tissue sections were hybridized with P1 o r P2 riboprobes described in Figure 1 and processed for in situ hybridization analysis with X-ray film. Signal was visualized by bright-field microscopy. Five postnatal stages were included: postnatal day (PD) 1
(A,B), 5 (C,D), 10 (E,F), 15 (G,H), and 20 (1,J). cb, cerebellum; hc, hippocampus; ic, inferior colliculus; nc, neocortex; pz, proliferative zone; op, optic nerve layer of superior colliculus; th, thalamus. P1 riboprobe was utilized in the panels shown on the left and P2 riboprobe in panels on the right.
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Fig. 4. Neuronal calmodulin mRNAs in the developing hippocampus. Tissue sections were hybridized with PI (A,C,E) and P2 (B,D,F) riboprobes as described in Figure 2. Signal was visuaIized by darkfield microscopy. Three postnatal stages were included: postnatal day (PD) 1 (A,B) ( X lo), 10 (C,D) ( X lo), 20 (E,F) ( X 13). GLD, granule cell layer of dentate gyrus; PLH, pyramidal cell layer of hippocampus.
be shown at higher magnification (Fig. 5 ) , in situ hybridization revealed different labelling patterns with the P1 and P2 probes during early development a€the cerebellum. This implies that 4.0 kb NGBl and 1.8 kb NGB2 neuronal calmodulin mRNA species may be differentially expressed in cellular layers of the developing cerebellum.
Expression of the Neuronal Calmodulin mRNAs in the Developing Cerebellum In order to investigate at higher resolution the pattern of expression of the neuronal calmodulin mRNA species in the developing rat cerebellum, in situ hybridization was carried out with the PI and P2 riboprobes
using autoradiographic emulsion (Fig. 5). Differences in the pattern of expression of the 4.0 kb NGBl and the 1.8 kb NGB2 neuronal calmadulin mRNA species were noted in the granule cell layers of the developing cerebellum. Granule neurons are generated postnatally by division of cells within the external granule cell layer (Burgoyne and Cambray-Deakin, 1988). The whole process of granule cell proliferation and migration to the internal granule cell layer occurs throughout the first 3 postnatal weeks in the rat cerebellum (Altman, 1972). Postnatal day 1. As shown in Figure 5A, at PD 1 the PI probe detected a diffuse signal in the Purkinje cell layer (PL) and in the external granule cell layer (EGL). The P2 probe, which is specific for the 4.0 kb NGBl
Fig. 5 .
Developmental Expression of Neuronal Calmodulin mRNAs
calmodulin mRNA, detected signal over the cells in the Purkinje cell layer (Fig. 5B). In contrast to the P1 probe, labelling was not observed with the P2 probe in external granule cell layer (Fig. 5B). Postnatal day 5. A laminar distribution of calmodulin mRNAs was apparent at PD 5 with the P1 probe which detects both calmodulin mRNA species (Fig. 5C). Signal was apparent in a thin line which clearly followed the external granule cell layer (EGL) of Cerebellum and was present from PD 5 until PD 15 (Fig. 5C,E,G). A punctate pattern of expression with the P1 probe was also observed in the internal granule neuron layer (GL) and the Purkinje cell layer (PL). With the P2 probe, which is specific for the 4.0 kb NGBl mRNA, a punctate pattern of expression was localized over the Purkinje cell layer (PL), however, signal was absent from the external granule cell layer (Fig. 5D). These results suggested that the 1.8 kb NGB2 mRNA species and not the 4.0 kb NGBl species is expressed in the proliferating and developing granule neuron population. A relatively strong signal was detected in neurons of the deep cerebellar nuclei (dwn) with both probes whereas no signal was observed in neuron-poor regions such as the deep white matter
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white matter (WM). The expression pattern of the neuronal calmodulin mRNAs observed at this stage (PD20) is identical to that observed in the adult rat brain.
DISCUSSION
The involvement of calmodulin and its target enzymes in the development of nervous system is an area of active study (Billingsley et al., 1990; Cimino et al., 1990; Weinman et al., 1991). Several lines of evidence have shown that calmodulin is involved during the course of development in regulation of its target enzymes which include cyclic nucleotide phosphodiesterase, protein phosphatase, calcineurin and protein kinase I1 which mediate important processes in neuronal activity (Hyman and Pfenninger, 1985; Hoskins and Ho, 1986; Ratan and Shelanski, 1986; Billingsley et al., 1990). In the present study, we have utilized in situ hybridization to determine the developmental pattern of expression of neuronal calmodulin mRNA species which we have recently characterized by molecular cloning (Ni et al., 1992). Particular effort was devoted to the investigation of the pattern of expression of these mRNA species in cellular layers of the rat cerebellum during postnatal development. In a WM). Postnatal days 10 and 15. An intense signal with previous study using Northern blotting we have reported P1 probe was apparent in the internal granule cell layer a developmental increase in a 1.8 kb (NGB2) and a 4.0 (GL) at PD 10 and persisted through to adulthood (Fig. kb (NGB 1) mRNA species in the cerebellum (Ni et al., 5E,G,I). Much lower levels of expression of the 4.0 kb 1992), an observation which is consistent with the NGBl calmodulin mRNA species were apparent in this present in situ hybridization results. The cerebellum has been frequently studied as an cellular layer from PD 5 through to PD 15 (Fig. ideal system for neuronal development because of its 5D,F,H). Postnatal day 20. Because the proliferation and laminated structure, the presence of a limited number of migration of granule neurons from the external granule neuronal cell types, and the detailed knowledge of the cell layer to the internal granule cell layer is completed morphological changes that occur during development of by the third postnatal week of development, no signal this brain region (Bernhardt and Matus, 1984). In the rat was evident at PD 20 with the P1 probe in the outermost cerebellum, the majority of granule cells are generated layer of cerebellum (Fig. 51). At PD 20, the two probes postnatally in the external granule cell layer (EGL) (Burdemonstrated a similar signal pattern (Fig. 51,J). A goyne and Cambray-Deakin, 1988). Immature granule strong signal was observed over the Purkinje layer (PL), cells in the EGL extend their axons and leading processes internal granule cell layer (GL), and deep cerebellar nu- down through the molecular layer and migrate towards clei. No signal was evident over glial cells in the deep the internal granular layer during early postnatal development, leaving their parallel fibres behind (Altman, 1972, 1975, 1982). The whole process of granule cell proliferation and migration occurs throughout the first 3 Fig. 5 . Distribution of neuronal calmodulin mRNA species in weeks of postnatal development in the rat cerebellum cellular layers of the developing cerebellum analyzed by in situ (Altman, 1972). hybridization. Tissue sections were hybridized with PI probe The present developmental study indicated that the (A,C,E,G,I) ( x 13) and NGBl specific probe P2 (B,D,F,H,J) two probes, P1 and P2, detected differences in signal ( x 13) as described in Figure 2 . Five postnatal stages were patterns during early postnatal development of the cereincluded: postnatal days (PD) 1 (A,B), 5 (C,D), 10 (E,F), 15 (G,H), and 20 (1,J). Signal was visualized by darkfield mi- bellum, suggesting that NGBl and NGB2 neuronal croscopy. dwn, deep white nuclei; EGL, external granule cell calmodulin mRNAs may be differentially expressed in layer; GL, internal granule cell layer; PL, Purkinje layer; WM, cellular layers of this brain region. In situ hybridization deep white matter. Longer exposures with the P2 probe did not revealed that relatively high levels of expression of the 1 .8 kb NGB2 mRNA were apparent in the external granchange the pattern of expression which is shown.
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ule cell layer during early postnatal development from PD 1 to 15, whereas the 4.0 kb NGBl mRNA was not. The strong PI signal and weak P2 signal in the internal granule cell layer from PD 5 to 15 suggest that the 1.8 kb NGB2 mRNA is preferentially expressed in developing granule neurons. Unlike the 1.8 kb NGB2 mRNA, the 4.0 kb NGBl mRNA species was not detected in the external granule cell layer, however it was expressed at abundant levels in the Purkinje cell layer from postnatal day 1 to 20 and also at PD 20 in the internal granule cell layer. In summary, our data support the notion that the 1.8 kb NGB2 mRNA species may be involved in cellular processes during the proliferation and maturation of granule neurons while the 4.0 kb neuronal calmodulin mRNA is present in mature granule neurons. Previous studies have noted that calmodulin immunoreactivity was present in proliferative regions of the cerebellum (Cumming et al., 1982; Caceres et al., 1983). At present it is not known why the 4.0 kb NGBl mRNA species, which contains an AU-rich “destabilizer-like” element, is expressed in mature granule neurons while the 1.8 kb NGB2 species, which does not contain this element, is expressed in developing granule neurons. The AU-rich ‘‘destabilizer-like” element may influence mRNA half life (Shaw and Kamen, 1986) and is characteristic of many inducible mRNAs such as c-fos (Morgan and Curran, 1989). Neuronal cells possessing an inducible calmodulin mRNA with a short half-life might utilize it in responsive mechanisms in calcium-dependent signalling events or in maintenance of calcium homeostasis. We have demonstrated that levels of the 4.0 kb mRNA species are rapidly increased in cells surrounding the site of a cortical lesion in rats (Landry et al., 1992) and that levels of this neuronal calmodulin mRNA species are increased in specific regions of the rat brain stem in response to impulse activity evoked by reserpine (Ni and Brown, 1993). Studies on staggerer (sg/sg) mutant mice, which have an intrinsic defect in Purkinje cells ( H e m p and Mullen, 1979a,b), have demonstrated that normal expression of a calmodulin gene is altered in Purkinje neurons (Messer et al., 1990). While the cause(s) of the degeneration of this population of neurons is not precisely known, the authors suggest that it may result from a defect in Purkinje-cell-specific regulation of a calmodulin gene. The protein coding region of the rat CaMl gene has a 94% homology with the mouse calmodulin gene whose expression is significantly altered in Purkinje neurons of the staggerer mutant (Messer et al., 1990; Ni et al., 1992). The study on the staggerer mutant mouse and our present observation of the presence of CaMl gene transcripts in developing Purkinje neurons may suggest an important role for CaMl gene transcripts in the development of this population of neurons.
In the cerebral cortex, neuronal calmodulin mRNA was detected at postnatal day 1 in the cortical plate and proliferative zone where the most recently formed neurons are localized. The distribution of the neuronal calmodulin mRNA species in the developing cerebral cortex correlates with the anatomical maturation of neurons in this region and is in agreement with the observation that calmodulin may be involved in the maturation and proliferation of neurons in the cerebral cortex (Rasmussen and Means, 1989). A transient and highly localized increase in neuronal calmodulin mRNA was observed in the inferior colliculus and optic nerve layer of the superior colliculus at PD 5 and 10. This may suggest that up-regulation of the CaMl gene occurs during steps involved in the formation of visional projection pathways. While the present study focuses on expression of neuronal calmodulin mRNAs during postnatal development of the rat brain, calmodulin immunoreactivity has been found in the marginal neural tube of developing mice embryos as early as day 9.5 of gestation (SetoOhshima et al., 1987). In addition, calmodulin mRNAs have been detected in the rat brain as early as embryonic day 14 by Northern blot analysis (Weinman et al., 1991) and by in situ hybridization (Landry, C.F., personal communication). Calmodulin may play an important role in growth cone function (Seto-Ohshima et al., 1983; Hyman and Pfenninger, 1985) and other processes involved in early development of the CNS (Hoskins and Ho, 1986; Seto-Ohshima et al., 1987).
ACKNOWLEDGMENTS This research was supported by grants from NSERC (Canada) to I.R.B.
REFERENCES Altman J (1972): Postnatal development of the cerebellar cortex in the rat. 1. The external germinal layer and the transitional molecular layer. J Comp Neurol 145:353-398. Altman J (1975): Postnatal development of the cerebellar cortex in the rat. IV. Spatial organization of bipolar cells. Parallel fibres and glial pallisades. J Comp Neurol 163:427-448. Altman J (1982): Morphological development of the rat cerebellum and some of its mechanisms. Exp Brain Res Suppl 6:8-49. Bernhardt R, Matus A (1984): Initial phase of dendrite growth: Evidence for the involvement of high molecular weight rnicrotubule-associated proteins (HMWP) before the appearance of tubulin. J Cell Biol 92:589-593. Billingsley ML, Polli JW, Balaban CD, Kincaid L (1990): Developmental expression of calmodulin-dependent cyclic nucleotide phosphodiesterase in rat brain. Developmental Brain Res 53: 256-263. Burgoyne RD, Cambray-Deakin MA (1988): The cellular neurobiology of neuronal development: The cerebellar granule cell. Brain Res Rev 13:77-101. Caceres A, Bender P, Snavely L, Rebhun LI, Steward 0 (1983):
Developmental Expression of Neuronal Calmodulin mRNAs Distribution and subcellular localization of calmodulin in adult and developing brain tissue. Neuroscience 10:449-46 1. Cheung WY (1980): Calmodulin plays a pivotal role in cellular regulation. Science 207: 19-27. Cimino M, Chen JF, Weiss B (1990): Ontogenetic development of calmodulin mRNA in rat brain using in situ hybridization histochemistry. Developmental Brain Res 54:43-49. Cumming R, Krigman MR, Steine AL (1982): Perinatal ontogenesis of calmodulin and cyclic AMP-dependent protein kinase subunits in the Purkinje cell using immunofluorescence. Neurosci Lett 28:247-252. DeLorenzo RJ (1 982): Calmodulin in neurotransmitter release and synaptic function. Fed Proc 41:2265-2272. Egne JC, Campbell A, Flangas AL, Siege1 FL (1977): Regional, cellular and subcellular distribution of calcium-activated cyclic nucleotide phosphodiesterase and calcium-dependent regulator in porcine brain. J Neurochem 28:1207-1213. Fischer R, Koller M, Flura M, Mathews S , Strehler-Page, M-A, Krebs J, Penniston JT, Carafoli E, Strehler EE (1988): Multiple divergent mRNAs code for a single human calmodulin. J Biol Chem 263:17055-17062. Grab DJ, Carlin RK, Siekevitz P (1980): The presence of calmodulin in postsynaptic densities. Ann NY Acad Sci 35655-72. H e m p K, Mullen RJ (1979a): Staggerer chimeras: Intrinsic nature of Purkinje cell defects and implications for normal cerebellar development. Brain Res 178:443-457. H e m p K, Mullen RJ (1979b): Regional variation and absence of large neurons in the cerebellum of the staggerer mouse. Brain Res 172:l-12. Hoskins B, Ho 1K (1986): Effects of maturation and aging on calmodulin and calmodulin-regulated enzymes in various regions of mouse brain. Mech Aging Dev 36:137-186. Hyman C, Pfenninger KH (1985): Intracellular regulations of neuronal sprouting: Calmodulin-binding proteins of nerve growth cones. J Cell Biol 101:1153-1160. Klee CB, Vanaman TC (1982): Calmodulin. Adv Protein Chem 35: 213-231. Landry CF, Ivy GO, Dunn RJ, Marks A, Brown IR (1989): Expression of the gene encoding the f3-subunit of S-100 protein in the developing rat brain analyzed by in situ hybridization. Mol Brain Res 6:251-262. Landry CF, Ivy GO, Brown IR (1990): Developmental expression of glial fibrillary acidic protein mRNA in the rat brain analyzed by in situ hybridization. J Neurosci Res 25:194-203. Landry CF, Ivy GO, Brown IR (1992): Effect of a discrete dorsal forebrain lesion in the rat on the expression of neuronal and glial-specific genes: induction of calmodulin, NF-L, SCI and GFAP mRNAs. J Neurosci 32:280-289. Lewis SA, Cowan NJ (1985): Temporal expression of mouse glial fibrillary acidic protein studied by a rapid in situ hybridization procedure. J Neurochem 45:913-919. Lin CT, Dedman JR, Brinkley BR, Marcus AR (1980): Localization of calmodulin in rat cerebellum by irnmunoelectron microscopy. J Cell Biol 85:473-480. Malenka RC, Kauer JA, Perkel DJ, Mauk MD, Kelly PT, Nicoll RA, Waxham MN (1989): An essential role for postsynaptic calmodulin and protein kinase activity in long-term potentiation. Nature 34034-557. Means AR, Tash JS, Chafouleas JG (1982): Physiological implications of the presence, distribution and regulation of calmodulin in eukaryotic cells. Physiol Rev 62:l-39.
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Messer A , Plummer-Siegard J, Eisenberg B (1990): Staggerer mutant mouse Purkinje cells do not contain detectable calmodulin mRNA. J Neurochem 55:293-302. Morgan JI, Curran T (1989): Stimulus-transcription coupling in neurons: Role of cellular immediate-early genes. TINS 12:459462. Ni B, Rush S, Gurd JW, Brown IR (1992): Molecular cloning of calmodulin mRNA species which are preferentially expressed in neurons of the rat brain. Mol Brain Res 13:7-17. Ni B, Brown IR (1993): Modulation of a neuronal calmodulin mRNA species in the rat brain stem by reserpine. Neurochemical Res (in press). Nojima H (1989): Structural organization of multiple rat calmodulin genes. J Mol Biol 208:269-282. Polak KA, Edelman AM, Wasley JWF, Cohan CS (1991): A novel calmodulin antagonist, CGS 9343B, modulates calcium-dependent changes in neurite outgrowth and growth cone movements. J Neurosci 11534-542. Rasanussen H and Goodman DBP (1977): Relationship between calcium and cyclic nucleotides in cell activation. Physiol Rev 57:421-509. Rasmussen D, Means AR (1989): Calmodulin, cell growth and gene expression. TINS 12:433-438. Ratan RR, Shelanski ML (1986): Calcium and the regulation of mitotic events. Trends Biochem Sci 11:456-459. Rubin RP (1972): The role of calcium in the release of neurotransmitter substances and hormones. Pharmaco Rev 22:389-428. SenGupta B, Friedberg F, Betera-Wadleigh SD (1987): Molecular analysis of human and rat calmodulin complementary DNA clones. J Biol Chem 262:16663-16670, Seto-Ohshima A, Kitajima S , Sano M, Kato K, Mizutani A (1983): Immunohistochemical localization of calmodulin in mouse brain. Histochemistry 79:25 1-257. Seto-Ohshima A, Yamazaki Y, Kawamura N, Kitajima S, Sano M, Mizutani A (1987): The early expression of immunoreactivity for calmodulin in the nervous system of mouse embryos. Histochemistry 86337-343. Shaw G, Kamen R (1986): A conserved AU sequence from the 3’ untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46:659-667. Siekevitz P (1991): Possible role for calmodulin and the Ca+ +/calmodulin-dependent protein kinase I1 in postsynaptic neurotransmission. Proc Natl Acad Sci 885374-5378. Sobue K, Morimoto K, Kanda K, Kakiuchi S (1982): Ca+ -dependent binding of 3H-calmodulin to the microsomal fraction of brain. J Biochem 91:1313-1320. Stein R, Mori N, Matthews K, Lo L, Anderson D (1988): The NGFinducible SCGlO mRNA encodes a novel membrane-bound protein present in growth cones and abundant in developing neurons. Neuron 1:463-476. Weinman J, Gaspera BD, Dautigny A, Dinh DP, Wang J, Nojima H, Weinman S (1991): Developmental regulation of calmodulin gene expression in rat brain and skeletal muscle. Cell Regulation 2:819-826. Wood JG, Wallace RW, Whitaker JN, Cheung WY (1980): Immunocytochemical localization of calmodulin and a heat-labile calmodulin-binding protein (CaM-BP80) in basal ganglia of mouse brain. J Cell Biol 84:66-76. Zhou LW, Moyer JA, Muth EA, Clark B, Palkovits M, Weiss B (1985): Regional distribution of calmodulin activity in rat brain. J Neurochem 44:1657-1662. +
