Human Molecular Genetics, 2002, Vol. 11, No. 23 2929-2940
© 2002 Oxford University Press
Expression analysis of RSK gene family members: the RSK2 gene, mutated in Coffin–Lowry syndrome, is prominently expressed in brain structures essential for cognitive function and learning
1Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, B.P. 10142, 67404 Illkirch Cedex, C.U. de Strasbourg, France and 2Department of Medicine, University College London, London, WC1E 6JJ, UK
Received July 15, 2002; Accepted August 28, 2002
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ABSTRACT |
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Coffin–Lowry syndrome (CLS) is characterized by cognitive impairment, characteristic facial and digital findings and skeletal anomalies. The gene implicated in CLS encodes RSK2, a serine/threonine kinase acting in the Ras/MAPK signalling pathway. In humans, RSK2 belongs to a family of four highly homologous proteins (RSK1–RSK4), encoded by distinct genes. RSK2 mutations in CLS patients are extremely heterogeneous. No consistent relationship between specific mutations and the severity of the disease or the expression of uncommon features has been established. Together, the data suggest an influence of environmental and/or other genetic components on the presentation of the disease. Obvious modifying genes include those encoding other RSK family members. In this study we have determined the expression of RSK1, 2 and 3 genes in various human tissues, during mouse embryogenesis and in mouse brain. The three RSK mRNAs were expressed in all human tissues and brain regions tested, supporting functional redundancy. However, tissue specific variations in levels suggest that they may also serve specific roles. The mouse Rsk3 gene was prominently expressed in the developing neural and sensory tissues, whereas Rsk1 gene expression was the strongest in various other tissues with high proliferative activity, suggesting distinct roles during development. In adult mouse brain, the highest levels of Rsk2 expression were observed in regions with high synaptic activity, including the neocortex, the hippocampus and Purkinje cells. These structures are essential components in cognitive function and learning. Based on the expression levels, our results suggest that in these areas, the Rsk1 and Rsk3 genes may not be able to fully compensate for a lack of Rsk2 function.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Coffin–Lowry syndrome (CLS), is an X-linked disorder (MIM 303600) characterized by psychomotor and growth retardation and facial, hand and skeletal malformations (1–3). Typically, male patients are of short stature and exhibit a characteristic coarse face with prominent forehead, orbital hypertelorism, epicanthic folds, thick lips, a thick nasal septum, and irregular or missing teeth. Their large and soft hands, with lax skin and tapering fingers, are usually diagnostic features. Frequent skeletal anomalies are spinal kyphosis and/or scoliosis, which show progressive deterioration and often require surgical correction in adulthood. The cognitive deficit may be highly variable, but most male patients appear to be severely affected. Development of speech is always involved but also to variable degrees. Motor development is delayed and in infancy hypotonia is observed. Some patients present with additional features not commonly associated with CLS, including microcephaly, ventricular dilatation, seizures, sensorineural deafness and cardiac defects.
The gene mutated in CLS patients (RPS6KA3) was identified in 1996 by positional cloning in the Xp22.2 region (4). It encodes a protein of 740 amino acids, RSK2, which contains two non-identical kinase catalytic domains (5,6). The RSK2 gene belongs to a family comprising, in humans, four very closely related members, RSK1–RSK4, and homologues have been identified in vertebrate (mouse, chicken) and invertebrate (C. elegans, Drosophila melanogaster) genomes (7–11).
RSKs are serine/threonine protein kinases, acting at the distal end of the Ras–Mitogen-Activated Protein Kinase (MAPK) signalling pathway. They are directly phosphorylated and activated by ERK1/2 (Extracellular signal-Regulated Kinases) in response to many growth factors, peptide hormones and neurotransmitters (12–15). Importantly, multiple second messengers, such as cyclic adenosine monophosphate, calcium and diacylglycerol, can control ERK signalling via the small G proteins Ras and Rap1 (16).
When activated, RSKs have been shown to phosphorylate a growing list of nuclear substrates including histones, the transcription factors c-Fos, c-Jun, Nur77, SRF and CREB and to interact with the transcriptional co-activator CBP (17–20). Activation of RSKs is therefore thought to influence gene expression. In addition to their role in regulating transcription, RSKs have been shown to regulate apoptosis through phosphorylation and inactivation of the pro-apoptotic protein BAD, and cell cycle since the kinase Myt1 (a cell cycle regulator) is a RSK target (21–23). The respective contributions of each RSK family member to the in vivo phosphorylation of most known substrates are currently not well defined. Only two specific physiological substrates have, for example, been definitively identified for RSK2; CREB, which appears essential for induction of the immediate–early gene c-fos (24) and histone H3 (25). Thus, it is still unclear whether distinct cellular functions are regulated by the four RSK proteins, or whether they perform unique plus overlapping functions. Moreover, although it is clearly established that the RSK signalling pathway plays an important role in cellular events such as growth and differentiation, the relevance of these events to normal development and functioning of the whole organism is unknown.
RSK2 mutations in CLS patients are extremely heterogeneous and lead to premature termination of translation and/or to loss of phosphotransferase activity (6,26). No disorder associated with RSK1 or RSK3 mutations is known. Implication of RSK4 in non-specific X-linked mental retardation has been suspected, but definitive evidence remains to be provided (9).
The phenotype of CLS patients consists in a specific combination of symptoms, which are due to the lack of the pleiotropic effects of the RSK2 gene. To understand the origin of these pleiotropic effects, we have investigated, in the present study, the sites of RSK2 expression in humans and during mouse development. Previous genetic studies in a population of CLS patients, have shown that there is no consistent relationship between specific mutations and the severity of the disease or the expression of particular features (6,26). In addition, investigation in a few families with multiple affected individuals, revealed that there might be intra-familial variability for severity or for expression of uncommonly associated features. Together our current data suggest that environmental factors or other components that contribute to the same physiological functions as the RSK2 protein, may influence the presentation of the disease. Obvious modifying genes could be those encoding other RSK family members. Therefore, we have also analyzed the expression patterns of two additional members of the RSK family, for which the murine homologues are available: RSK1 and RSK3. The fact that cognitive impairment is a prominent feature in CLS indicates an important role of the MAPK–RSK signalling pathway in development and/or function of the central nervous system. As a first step to understand this role, we have also investigated expression of RSK1, 2 and 3 in adult human and mouse brain.
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RESULTS |
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RSK1, 2 and 3 gene expression in adult human tissues and in various structures of adult human brain
Probes corresponding to the coding regions of the RSK1, 2 and 3 cDNAs, were hybridized to northern blots of poly(A)+ RNA derived from a selection of human tissues. Two RSK2 transcripts, of
3.5 and 8.5 kb, were detected in all tissues analyzed (Fig. 1A). Sequencing of cDNAs has demonstrated that alternative use of two different polyadenylation sites gives rise to these two transcripts (unpublished data). The strongest expression of the RSK2 gene was found in skeletal muscle, heart and pancreas, whereas the weakest expression was observed in brain. Only one transcript, of 3.5 and 7 kb, was detected in all tissues tested with each of the RSK1 and RSK3 cDNA probes respectively. Like RSK2, RSK3 was highly expressed in skeletal muscle, heart and pancreas. The lowest levels of RSK3 mRNA were detected in the liver. RSK1 was mainly expressed in kidney, lung and pancreas (Fig. 1A). A northern blot containing poly(A)+ RNA prepared from different structures of adult human brain was hybridized with the same probes (Fig. 1B). The three RSK genes were again expressed in all structures tested. The RSK1 gene was most abundantly expressed in the cerebellum, RSK2 in the cerebellum, the occipital pole and the frontal lobe, whereas the RSK3 mRNA was primarily detected in the medulla.
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Expression of Rsk genes during mouse embryogenesis
In order to investigate the expression patterns of Rsk1, 2 and 3 genes during embryonic development, we have performed in situ hybridization on sections of embryonic day (E)9.5–E16.5 mouse embryos. At E9.5, Rsk1 was highly expressed in the neuroepithelium down the entire length of the neural tube, while only low expression was visible in all other tissues. Rsk2 and Rsk3 mRNAs were detectable at slightly above background levels in most tissues (data not shown). At E10.5 and E11.5, Rsk1 expression levels remained high in the neural tube and increased to moderate to high levels in hepatic primordium and to very high levels in midgut (Fig. 2B and data not shown). During the same period of time, only slight changes were observed in Rsk2 expression whereas at E11.5, Rsk3 mRNA expression increased to very high levels in the neural tube the dorsal root ganglia, the developing eye and the heart (Fig. 2C and data not shown).
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At later stages (E14.5 and onwards), low levels of Rsk2 mRNA were detected in dorsal root ganglia, trigeminal ganglia, skeletal muscle, kidney, lung and liver (Fig. 3B, D and F and data not shown). Interestingly, expression of the Rsk1 gene decreased dramatically in the developing nervous system and became undetectable at E16.5. However, very high levels of Rsk1 mRNA were detected in intestine (mucosa) and moderate to high levels in various tissues including lung, liver, skeletal muscle, thymus and kidney (Fig. 4B, E and H and data not shown). Additional sites of enhanced Rsk1 expression, such as the pinna of the ear, the cochlea, the respiratory and olfactory epithelia, the periphery of the tongue, the follicles of vibrissae and the tooth buds are indicated in Figure 5B and D. Rsk1 expression was also noticed in the inter-digital regions of the limb buds, confined to the epithelial cells (data not shown). At E16.5, Rsk3 expression remained more restricted in the central and the peripheral nervous system and was primarily limited to regions harbouring proliferating or differentiating cells. A high Rsk3 mRNA signal was observed in the roof of the neopallial cortex (which becomes the future cerebral cortex), the telencephalic ventricular zone, the diencephalon (thalamus and hypothalamus), the olfactory lobes, the choroid plexus, the medulla oblongata and the pons. In the peripheral nervous system, Rsk3 was very strongly expressed in the trigeminal, vestibulocochlear and vagal ganglia, the dorsal root ganglia, the sympathetic ganglia and the retina. Within retina, expression was strong in the inner nuclear layer and low in the outer nuclear and pigment layers. Outside the nervous system, strong Rsk3 expression was observed in the thyroid gland as well as in testis cords, giving rise in the adult to seminiferous tubules. However, moderate to weak expression was also visible in some other tissues or organs including the proliferative regions of the developing lens, the renal capsule, the myocardium and the respiratory epithelium (Fig. 4C, F and E, Fig. 5F and H and data not shown). A more detailed description of the differential Rsk expression patterns at late stages (E14.5–E16.5) of mouse embryonic development is given in Table 1.
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Expression of Rsk genes in adult mouse brain
We next investigated by in situ hybridization, the distribution of Rsk mRNAs in adult mouse brain. For Rsk1 the labelling was uniformly low (although the intensity was a little higher than the corresponding sense probe), except in the granular cell layer of the cerebellum, which displayed a relatively strong signal (Fig. 6B). Rsk2 expression levels were also very low, except in some structures of the cerebellum, the hippocampus and the cerebral cortex. In the cerebellum, Rsk2 was strongly expressed in the Purkinje cells and in some cells of the deep cerebellar nuclei (Fig. 6D). The highest Rsk2 mRNA levels were detected in the CA3 region of the hippocampus. Strong labelling was also seen in the CA1–CA2 areas (Fig. 6F). Finally, substantial Rsk2 expression was also detected throughout the neocortex, with the strongest signal in layers V and VI, and in the pyriform cortex (data not shown).
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Rsk3 mRNA was detected, to variable degrees, in a wide variety of regions of the adult mouse brain. Particularly strong expression was displayed by the lateral amygdaloid nucleus, the bed nucleus of the stria terminalis and the accumbens nucleus (Fig. 6H and J). In the hippocampus, high levels of expression were seen in the granule cell layer of the dentate gyrus (Fig. 6H). In the cerebral cortex, Rsk3 was expressed throughout the neocortex, but the highest mRNA levels were detected in layers II and III (Fig. 6H and G). Cells with relatively high Rsk3 expression were also observed in the pyriform cortex and the entorhinal cortex (Fig. 6H and J and data not shown). Finally, many thalamic and hypothalamic nuclei displayed also strong Rsk3 expression (Fig. 6L and data not shown).
Confirmation of the in situ hybridization studies using antibodies directed against the Rsk1 and the Rsk2 proteins
In order to confirm the presence of Rsk proteins at the sites where the corresponding mRNA species were detected by in situ hybridization, we performed immunohistochemistry experiments on cryosections from E13.5 mouse embryos and adult mouse brain. To this end, we developed polyclonal antibodies directed against Rsk1, 2 and 3. However, only the antibodies recognizing Rsk1 and Rsk2 could be used for immunohistochemistry whereas the antibody directed against Rsk3 was not convenient for such analysis. At E16.5, the Rsk1 protein sites of expression included, as expected, liver, intestine, thymus, the submandibular gland, the respiratory and olfactory epithelia, the follicles of vibrissae and the periphery of the tongue (Fig. 7A, B and C). In the mouse brain, the Rsk1 predominant site of expression was the granular cell layer of the cerebellum (Fig. 7D and E). The Rsk2 protein was clearly detected in the Purkinje cell layer of the cerebellum, in the CA1–CA3 regions of the hippocampus and in neurons of the cerebral cortex (Fig. 7F and data not shown).
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DISCUSSION |
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To gain better understanding of the physiological roles of RSKs and whether they could have specific roles and/or overlapping functions in vivo, we have carried out an extensive analysis of their patterns of expression in human tissues, during mouse embryogenesis and in adult mouse brain. Northern blot analysis of a panel of eight human tissues demonstrated that the RSK1, 2 and 3 genes are all widely expressed. Interestingly, expression of both RSK2 and RSK3 is most abundant in the heart, the skeletal muscle and the pancreas, suggesting that they may have specific and overlapping functions in these organs. In the whole brain, the RSK3 gene shows the strongest expression, whereas expression of both RSK2 and RSK1 genes is hardly detectable. A more detailed analysis of the human adult brain by northern blotting, revealed a widespread expression of the three RSK genes in the eight regions explored (cerebellum, cerebral cortex, medulla, spinal cord, occipital pole, frontal lobe, temporal lobe and putamen). However, noticeably higher levels of RSK1 and RSK2 mRNA were observed in the cerebellum. The latter is also strongly expressed in the occipital and frontal cortex.
The expression of Rsk2 was subsequently compared to that of Rsk1 and 3 during development, using in situ hybridization on mouse embryo sections. This analysis revealed that the Rsk2 gene is expressed at very low levels throughout embryonic development. However, at late embryonic stages, its expression is weakly enhanced in some tissues including sensory ganglia, skeletal muscle and some peripheral organs, but not in the central nervous system. In contrast to Rsk2, the Rsk3 gene shows high levels of expression during development, almost exclusively restricted to the developing central and peripheral nervous system. Rsk3 expression is, in particular, detected in the ventricular zone bordering the lateral ventricle, a site of high proliferative activity, but also in differentiating cerebral fields such as the cortex, caudate putamen, thalamus, hypothalamus and brainstem. This result supports the hypothesis that during development, Rsk3 functions to regulate proliferation and differentiation of neuroepithelial cells. Although the most prominent site of Rsk3 expression during embryogenesis is the nervous system, significant expression is also detected in a few other tissues including testis cords and the thyroid gland.
Until late midgestation, Rsk1 is strongly expressed in the neuroepithelium of the neural tube, whereas at later stages it decreases dramatically and becomes undetectable in the nervous system. On the contrary, expression of Rsk3 becomes prominent in the central and peripheral nervous system from E11.5 and onwards. These results are consistent with a temporal regulation of the Rsk1 and Rsk3 genes and support the requirement of Rsk1 in early and Rsk3 in later development of the nervous system, respectively.
At late stages of development, Rsk1 is highly expressed in regions harbouring highly proliferating cells. These include liver, lung, thymus and olfactory and respiratory epithelia. Particularly intense Rsk1 expression was observed in the gut epithelium. From the expression patterns observed, Rsk1 seems to be more strictly linked to cellular proliferation and Rsk3 to cellular differentiation, in particular in the nervous system.
RSK1 and RSK3 have not yet been associated with a human disorder and no animal models have as yet been described. In adult mouse brain, Rsk1 was clearly detected only in the granular cell layer of the cerebellum. In contrast, high levels of Rsk3 expression were observed in various regions of the adult mouse brain, suggesting a specific function for Rsk3 in nervous system maintenance and/or in neural signal transmission. In particular, the strong expression observed in structures that can be related to cognitive function, such as the cerebral cortex, the dentate gyrus and the amygdala suggest that RSK3 is a good candidate for disorders displaying involvement of the central nervous system, including mental retardation.
Rsk2 expression was too low to be detected in most brain structures of adult mouse by in situ hybridization. It was, however, abundantly expressed in the pyramidal cell layer of the hippocampus, the neocortex, the pyriform cortex and in Purkinje cells and deep nuclei of the mature cerebellum. Neurons within all these structures are characterized by high synaptic activity, especially the Purkinje cells of the cerebellum and the pyramidal cells of the hippocampus. The latter ones are considered as the cells with the highest synaptic activity in the whole brain (27,28). The specificity of Rsk2 to neuronal regions with high synaptic activity supports the concept of a functional importance of RSK2 in neural transmission. The hippocampus and the neocortex are essential components in cognitive function and learning. Purkinje cells are commonly associated with motor and balance skills but are now increasingly thought to be involved in motor learning processes (29). The cells of the pyriform cortex are linked to amygdala formation, since the latter receives afferents from the pyriform cortex. The amygdala has been associated with a range of cognitive functions, including emotion, learning, memory, attention and perception (30). Noteworthy is also the strong expression of the RSK2 mRNA in the frontal lobe of the human cortex, as revealed by northern blot analysis (Fig. 1B). Recent advances have uncovered important roles for the frontal lobes in a multitude of cognitive processes, including executive function, attention and language. Interestingly, evidence has been provided that the prefrontal areas play also a crucial role in long-term memory and in integrating different types of information in working memory (31,32). These data do not demonstrate the association of RSK2 with learning and memory. However, they do provide a basis for analysis of the cell types in which RSK2 expression may play a key role. In addition, the expression of Rsk2 in adult mouse brain structures is very similar to the one observed for several genes implicated in mental retardation (33).
A mouse model for CLS, obtained by inactivation of the Rsk2 gene, was recently described (34). Mutant mice weigh 10% less and are 14% shorter than their wild-type littermates. This is in agreement with a role of RSK2 in growth. Very importantly, these mice exhibit impaired learning and poor co-ordination, providing evidence that RSK2 has similar roles in mental functioning both in mice and humans.
Recent reports have provided evidence that long-term memory formation requires the activity of the cAMP response element (CRE) binding protein (CREB) transcription factor and that CRE-regulated genes are expressed in the hippocampus in response to inhibitory avoidance training (35,36). CREB has been shown to be an in vivo target of RSK2 (19). Harum et al. (37) have demonstrated a direct relationship between the magnitude of in vitro RSK2-mediated CREB phosphorylation and intelligence level in CLS patients. Together these data suggest that the Ras/MAPK pathway, signalling through RSK2 to CREB, plays a prominent role in cognitive dysfunction in CLS patients. Our results, showing strong Rsk2 expression in the hippocampus, further support this hypothesis.
Interestingly, the pyramidal cells of the CA3 region of the hippocampus, intensively express the Rsk2 mRNA. Previous studies have indicated that fibroblast cell lines derived from CLS patients with RSK2 inactivating mutations are defective in EGF induced c-fos gene expression (24). There is now evidence that expression of Fos, the product of the immediate-early gene c-fos, in the CA3 region, may be necessary for spatial memory formation (38). Fos, participates in the formation of heterodimeric AP-1 transcription factors which are thought to activate the expression of late-effector genes. The expression of c-fos is induced by a variety of stimuli and after some forms of learning. Accordingly, Fos expression is considered as part of a mechanism by which brief stimuli can trigger long-term changes in gene expression and alter structural and functional properties of nerve cells.
The expression of RSK4, the most recently identified member of the RSK protein family (9), was not investigated in this study, since its mouse homologue was not available. However, it has been reported that in humans, the highest RSK4 expression was seen in kidney, brain and thyroid gland whereas in pancreas a low amount of RSK4 mRNA was detected. Thus, it seems that RSK4 displays a more restricted expression pattern than the other RSK proteins. However, a more detailed study in the mouse is required before a definitive conclusion can be drawn.
Intriguingly, given the skeletal abnormalities observed in CLS patients, no significant expression of any Rsk gene was detected in developing bones of mouse embryos. This result suggests that very low levels of Rsk expression are necessary for skeletal development. RT–PCR and immunocytochemistry studies in specific bone cell populations derived from wild type and Rsk2 deficient adult mice and embryos are underway, which should help to elucidate this point. It is worth noticing that mice carrying a Rsk2 null allele apparently do not have skeletal defects, which are among the major manifestations of the syndrome in humans (34). This latter observation suggests that there might be a greater degree of redundancy in mice, at least in the strain that has been used to generate the Rsk2 null mouse.
Despite several differences, a considerable overlap in the expression patterns of the three RSK genes was seen. Together with the fact that the various RSK proteins share high homology, our results support the hypothesis that other RSK genes may be able to partially compensate for one missing RSK molecule. Their expression levels may be an important factor in the variable phenotypic severity observed in different individuals with RSK2 mutations.
Indeed, this comparative analysis of RSK expression begins to explain the phenotype observed in CLS patients. RSK1 and 3 should be able to compensate for the lack of the Rsk2 protein in most tissues, in which they are expressed at higher levels than Rsk2. Interestingly, in the pyramidal cells of the hippocampus, in the Purkinje cells of the cerebellum and in deep layers of the neocortex of the adult mouse brain, the Rsk2 gene shows very high levels of expression, whereas Rsk1 and Rsk3 mRNA expressions are not detectable. Preliminary data in Rsk2 knockout mice provide evidence that expression of at least Rsk1 and Rsk3 are not increased in response to Rsk2 deficiency (unpublished results, H.A.). These data support the hypothesis that in these areas RSK1 and RSK3 might not be able to fully compensate for RSK2 deficiency in Rsk2 null mice and in CLS patients, and thus may provide an additional clue to the understanding of the cognitive dysfunction.
To explore the full range of physiological functions performed by members of the RSK family and to further define redundant and specific functions, especially for RSK2, it will now be important to mutate all the members of the Rsk gene family and to make different combinations of Rsk deficient mice.
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MATERIALS AND METHODS |
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Northern blot analysis
Two northern blots, the first containing human poly(A)+ RNA from heart, total brain, placenta, lung, liver, skeletal muscle, kidney and pancreas (Clontech, Palo Alto, CA, USA, Human MTNTM Blot, 7760-1), and the second human poly(A)+ RNA from cerebellum, cerebral cortex, medulla, spinal cord, occipital pole, frontal lobe, temporal lobe and putamen (Clontech Human Brain MTNTM Blot II, 7755-1) were hybridized with radioactively labelled cDNA probes (2x106 cpm/ml) corresponding to the rat RSK1, the human RSK2 and the human RSK3 coding sequences respectively, following the Clontech protocol. The probes were obtained by random primer extension following standard protocols.
Preparation of mouse embryos
Pregnant CD-1 females from natural overnight matings (morning of vaginal plug was considered as 0.5 dpc) were sacrificed by cervical dislocation and embryos were collected, embedded in Tissue-Tek® O.C.TTM medium (SAKURA, Zoeterwoude, The Netherlands) and frozen on the surface of dry ice. Serial cryosections (10 µm thick) were obtained and subsequently analyzed.
Preparation of adult mouse brains
Adult (8 week old) CD-1 mice were sacrificed and brains were processed as described above.
In situ hybridization
To avoid cross-reaction, sequences corresponding to parts of the 3' untranslated regions of Rsk1, 2 and 3 mRNAs were used as riboprobes. 719 and 990 bp Rsk1 and Rsk3 EcoRI–BamHI cDNA fragments, respectively, were cloned in pBluescriptSK+ (Stratagene, Amsterdam-Zuidoost, The Netherlands). A 276 bp Rsk2 cDNA fragment was cloned between the SacI and EcoRI sites of the same vector. 1 µg of the linearized vector was used in T3 (17 units) and T7 (15 units) polymerase (Promega, Charbonnières, France) reactions including 3 µl [35S]-CTP (Amersham Biosciences Europe GmbH, Orsay, France), to produce sense and antisense riboprobes. The reactions were carried out for 2 h at 37°C in the presence of 5 mM of each of the nucleotides ATP, GTP and UTP, 0.25 mM CTP, 100 mM dithiothreitol and 50 units RNase inhibitor (Promega). The reaction volume was 20 µl. After a 20 min treatment with 10 units of RNase-free DNase I (Boehringer, Ingelheim, Germany) at 37°C, the length of the probes was reduced by alkaline hydrolysis with 0.1 M NaOH.
Frozen cryosections were immediately placed in ice cold acetone (Merck, Fontenay-sons-Bois, France) for 4 min and then allowed to air-dry for 40 min at room temperature. They were subsequently fixed for 15 min in a 4% formaldehyde in PBS (Dulbecco's phosphate buffered saline, Sigma-Aldrich, Saint Quentin Fallavier, France) solution, at room temperature and washed 2x5 min in 1x PBS. A 5 min treatment in 0.1 M triethanolamine, was followed by a 10 min treatment in 0.1 M triethanolamine, 0.25% acetic anhydride, 2x2 min washings in 1xPBS at room temperature and a 10 min treatment in 50% formamide (in PBS) at 60°C. The slides were then placed for 1 min subsequently in 50%, 70% and 100% pre-cooled at -20°C ethanol solutions and allowed to dry for at least 1 h.
The riborobes (25x106 cpm/ml of pre-hybridization mix (0.3 M NaCl, 20 mM Tris-HCl pH 6.8, 5 mM EDTA, 1 mM NaPO4 buffer pH 6.8, 0.1% Ficoll, 0.1% polyvinyl pyrrolidon 25, 0.1% bovine serum albumin, 50% formamide, 10% dextran sulfate, 10 mM dithiothreitol and yeast tRNA (0.5 µg/ml))), were incubated for 5 min at 65°C, placed 5 min on ice and then added to the slides. The hybridization was carried out overnight at 52°C in a humid chamber. Subsequent washings were as follows: 1 h at 55°C in 5x SSC (1x SSC: 150 mM NaCl, 15 mM C6H5O7
Na32H2O), 1 h at 55°C in 2x SSC, 15 min at 37°C in 4x SSC, 30 min at 37°C in 4x SSC with RNaseA (Sigma-Aldrich) at 20 µg/ml, 10 min at 37°C in 4x SSC, 1 h at 55°C in 2x SSC/50% formamide, 15 min at 55°C in 2x SSC and 15 min at 55°C in 0.1x SSC. The slides were then placed for 1 min in each of the solutions: 30% ethanol and 0.4 M AcNH4, 60% ethanol and 0.4 M AcNH4, 85% ethanol and 0.4 M AcNH4, 95% ethanol and 0.4 M AcNH4 and 100% ethanol and they were then allowed to dry. The times for emulsion autoradiography were 3–5 weeks. As expected, control sense riboprobes only gave uniform background labelling (data not shown).
Antibodies
Rabbit polyclonal 1793 antibody was raised and affinity purified against the synthetic peptide LMEDDGKPRAPQAPL corresponding to human RSK1 amino acids 386–400. Rabbit polyclonal 1801 antibody was raised and affinity purified against the synthetic peptide MDEPMGEEEINPQTEEVS corresponding to human RSK2 amino acids 29–46.
Immunohistochemistry
Frozen brain cryosections obtained as described above were placed in ice cold solutions of 50% acetone, 100% acetone and 50% acetone, for 5 min, 2 min and 5 min successively. They were subsequently washed once with 1x PBS and fixed with 4% paraformaldehyde for 10 min. After fixation and washing with 1x PBS for 10 min, endogenous peroxidase was inhibited by a 20 min treatment with 0.5% H2O2 solution in PBS. Blocking of non-specific sites was carried out by incubating the sections in 10% normal goat serum in PBS for 1 h at room temperature. The primary antibodies were added to the sections in 1% normal goat serum, 0.5% Tween-20, in PBS, and incubated overnight at 4°C. Antibody dilutions were as follows: rabbit anti-RSK1 (1793, 1:10) and rabbit anti-RSK2 (1801, 1:100). The sections were subsequently washed four times for 10 min in 1x PBS and incubated for 2 h at room temperature with an Alexa Fluor® 488 goat anti-rabbit IgG (H+L) secondary antibody (Molecular Probes, Leiden, The Netherlands), (dilution 1:200). After four washes with 1x PBS samples were mounted with KAISER's glycerol gelatin (Merck).
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ACKNOWLEDGEMENTS |
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We would like to thank G. Duval for production of polyclonal antibodies, J.L. Vonesch, D. Hentsch and M. Boeglin for help with imaging and S. Pannetier for excellent technical assistance. The work was supported by grants from the Association Française pour la Recherche contre le Cancer, the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale and the Hôpital Universitaire de Strasbourg.
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FOOTNOTES |
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* To whom correspondence should be addressed at: I.G.B.M.C., B.P. 10142, 67404 Illkirch Cedex, C.U. de Strasbourg, France. Tel: +33 388653400; Fax: +33 388653246; Email: andre.hanauer{at}titus.u-strasbg.fr
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REFERENCES |
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