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Expression analysis of RSK gene family members:the RSK2 gene, mutated in CofnLowry syndrome,is prominently expressed in brain structuresessential for cognitive function and learningMaria Zeniou 1 , Thomas Ding 1 , Elisabeth Trivier 2 and Andre Hanauer 1, *

1 Institut de Ge ne tique et de Biologie Mole culaire et Cellulaire, CNRS/INSERM/ULP, B.P. 10142, 67404 Illkirch Cedex,C.U. de Strasbourg, France and 2 Department of Medicine, University College London, London, WC1E 6JJ, UK

Received July 15, 2002; Revised and Accepted August 28, 2002

CofnLowry syndrome (CLS) is characterized by cognitive impairment, characteristic facial and digitalndings and skeletal anomalies. The gene implicated in CLS encodes RSK2, a serine/threonine kinase actingin the Ras/MAPK signalling pathway. In humans, RSK2 belongs to a family of four highly homologousproteins (RSK1RSK4), encoded by distinct genes. RSK2 mutations in CLS patients are extremelyheterogeneous. No consistent relationship between specic mutations and the severity of the disease orthe expression of uncommon features has been established. Together, the data suggest an inuence ofenvironmental and/or other genetic components on the presentation of the disease. Obvious modifyinggenes include those encoding other RSK family members. In this study we have determined the expression ofRSK1 , 2 and 3 genes in various human tissues, during mouse embryogenesis and in mouse brain. The threeRSK mRNAs were expressed in all human tissues and brain regions tested, supporting functionalredundancy. However, tissue specic variations in levels suggest that they may also serve specic roles.The mouse Rsk3 gene was prominently expressed in the developing neural and sensory tissues, whereasRsk1 gene expression was the strongest in various other tissues with high proliferative activity, suggestingdistinct 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. Thesestructures are essential components in cognitive function and learning. Based on the expression levels, ourresults suggest that in these areas, the Rsk1 and Rsk3 genes may not be able to fully compensate for a lack ofRsk2 function.

INTRODUCTIONCofnLowry syndrome (CLS), is an X-linked disorder (MIM303600) characterized by psychomotor and growth retardationand facial, hand and skeletal malformations (13). 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 ngers, are usually diagnostic features. Frequent skeletal anomalies are spinal kyphosis and/or scoliosis, whichshow progressive deterioration and often require surgicalcorrection in adulthood. The cognitive decit may be highlyvariable, 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 infancyhypotonia is observed. Some patients present with additionalfeatures not commonly associated with CLS, includingmicrocephaly, ventricular dilatation, seizures, sensorineuraldeafness and cardiac defects.

The gene mutated in CLS patients ( RPS6KA3) was identied

in 1996 by positional cloning in the Xp22.2 region (4). It encodes a protein of 740 amino acids, RSK2, which containstwo non-identical kinase catalytic domains (5,6). The RSK2gene belongs to a family comprising, in humans, four veryclosely related members, RSK1 RSK4, and homologues have been identied in vertebrate (mouse, chicken) and invertebrate(C. elegans, Drosophila melanogaster ) genomes (711).

RSKs are serine/threonine protein kinases, acting at the distalend of the RasMitogen-Activated Protein Kinase (MAPK)

*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: [email protected]

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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 mes-sengers, 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 agrowing list of nuclear substrates including histones, thetranscription 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 in uence geneexpression. In addition to their role in regulating transcription,RSKs have been shown to regulate apoptosis through phospho-rylation 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 familymember to the in vivo phosphorylation of most known substratesare currently not well de ned. Only two speci c physiologicalsubstrates have, for example, been de nitively identi ed for

RSK2; CREB, which appears essential for induction of theimmediate early gene c-fos (24) and histone H3 (25). Thus, it isstill unclear whether distinct cellular functions are regulated bythe four RSK proteins, or whether they perform unique plusoverlapping functions. Moreover, although it is clearly estab-lished that the RSK signalling pathway plays an important role incellular events such as growth and differentiation, the relevanceof these events to normal development and functioning of thewhole organism is unknown.

RSK2 mutations in CLS patients are extremely heterogeneousand lead to premature termination of translation and/or to lossof phosphotransferase activity (6,26). No disorder associated with RSK1 or RSK3 mutations is known. Implication of RSK4in non-speci c X-linked mental retardation has been suspected,

but denitive evidence remains to be provided (9).The phenotype of CLS patients consists in a speci ccombination 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 mousedevelopment. Previous genetic studies in a population of CLS patients, have shown that there is no consistent relationship between speci c mutations and the severity of the disease or theexpression of particular features (6,26). In addition, investigationin 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 functionsas the RSK2 protein, may in uence the presentation of thedisease. Obvious modifying genes could be those encoding other RSK family members. Therefore, we have also analyzed theexpression patterns of two additional members of the RSK family, for which the murine homologues are available: RSK1and 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 centralnervous system. As a rst step to understand this role, we havealso investigated expression of RSK1, 2 and 3 in adult human and mouse brain.

RESULTS

RSK1 , 2 and 3 gene expression in adult human tissuesand 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)

RNAderived from a selection of human tissues. Two RSK2transcripts, of 3.5 and 8.5 kb, were detected in all tissuesanalyzed (Fig. 1A). Sequencing of cDNAs has demonstrated that alternative use of two different polyadenylation sites givesrise 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 wasobserved 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 highlyexpressed in skeletal muscle, heart and pancreas. The lowest levels of RSK3 mRNA were detected in the liver. RSK1 wasmainly expressed in kidney, lung and pancreas (Fig. 1A). A

northern blot containing poly(A)

RNA prepared fromdifferent structures of adult human brain was hybridized withthe same probes (Fig. 1B). The three RSK genes were againexpressed in all structures tested. The RSK1 gene was most abundantly expressed in the cerebellum, RSK2 in thecerebellum, the occipital pole and the frontal lobe, whereasthe RSK3 mRNA was primarily detected in the medulla.

Expression of Rsk genes during mouse embryogenesis

In order to investigate the expression patterns of Rsk1, 2 and 3genes during embryonic development, we have performed in situhybridization on sections of embryonic day (E)9.5 E16.5mouse 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. Rsk2and 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 wereobserved in Rsk2 expression whereas at E11.5, Rsk3 mRNAexpression increased to very high levels in the neural tube thedorsal root ganglia, the developing eye and the heart (Fig. 2Cand data not shown).

At later stages (E14.5 and onwards), low levels of Rsk2mRNA 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 genedecreased 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 moderateto high levels in various tissues including lung, liver, skeletalmuscle, thymus and kidney (Fig. 4B, E and H and data not shown). Additional sites of enhanced Rsk1 expression, such asthe pinna of the ear, the cochlea, the respiratory and olfactoryepithelia, the periphery of the tongue, the follicles of vibrissaeand the tooth buds are indicated in Figure 5B and D. Rsk1expression was also noticed in the inter-digital regions of the

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limb buds, con ned to the epithelial cells (data not shown). At E16.5, Rsk3 expression remained more restricted in the centraland the peripheral nervous system and was primarily limited toregions harbouring proliferating or differentiating cells. A high Rsk3 mRNA signal was observed in the roof of the neopallialcortex (which becomes the future cerebral cortex), thetelencephalic ventricular zone, the diencephalon (thalamus and hypothalamus), the olfactory lobes, the choroid plexus, themedulla oblongata and the pons. In the peripheral nervoussystem, Rsk3 was very strongly expressed in the trigeminal,vestibulocochlear and vagal ganglia, the dorsal root ganglia, the

sympathetic ganglia and the retina. Within retina, expressionwas 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 intestis cords, giving rise in the adult to seminiferous tubules.However, moderate to weak expression was also visible in someother tissues or organs including the proliferative regions of thedeveloping lens, the renal capsule, the myocardium and therespiratory epithelium (Fig. 4C, F and E, Fig. 5F and H and datanot shown). A more detailed description of the differential Rsk expression patterns at late stages (E14.5 E16.5) of mouseembryonic development is given in Table 1.

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 wasuniformly low (although the intensity was a little higher thanthe corresponding sense probe), except in the granular celllayer of the cerebellum, which displayed a relatively strongsignal (Fig. 6B). Rsk2 expression levels were also very low,except in some structures of the cerebellum, the hippocampusand the cerebral cortex. In the cerebellum, Rsk2 was stronglyexpressed in the Purkinje cells and in some cells of the deep

cerebellar nuclei (Fig. 6D). The highest Rsk2 mRNA levelswere detected in the CA3 region of the hippocampus. Stronglabelling was also seen in the CA1 CA2 areas (Fig. 6F).Finally, substantial Rsk2 expression was also detected through-out the neocortex, with the strongest signal in layers V and VI,and in the pyriform cortex (data not shown).

Rsk3 mRNA was detected, to variable degrees, in a widevariety of regions of the adult mouse brain. Particularly strongexpression 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 expressionwere seen in the granule cell layer of the dentate gyrus (Fig. 6H).

Figure 1. Northern blot analysis of RSK1, 2 and 3 expression. ( A) Multiple adult human tissue northern blot containing poly(A) RNA (Clontech laboratories, Inc.Palo Alto, California). For RSK1 and RSK3, one single transcript of 3.5 kb and 7 kb respectively, was observed. Two transcripts of 8.5kb and 3.5 kb weredetected for RSK2. Whereas RSK1 is mainly detected in kidney and pancreas, the highest RSK2 and RSK3 expression was observed in heart and skeletal muscle.(B) Northern blot containing poly(A) RNA from human adult brain tissues (Clontech laboratories, Inc. Palo Alto, California). RSK1 was mainly expressed in thecerebellum and RSK2 in the cerebellum, the occipital pole and the frontal lobe. The highest RSK3 expression was observed in the medulla.

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In the cerebral cortex, Rsk3 was expressed throughout theneocortex, but the highest mRNA levels were detected in layersII and III (Fig. 6H and G). Cells with relatively high Rsk3expression were also observed in the pyriform cortex and theentorhinal 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).

Conrmation of the in situ hybridization studiesusing antibodies directed against the Rsk1 andthe Rsk2 proteins

In order to con rm the presence of Rsk proteins at the siteswhere the corresponding mRNA species were detected byin situ hybridization, we performed immunohistochemistryexperiments on cryosections from E13.5 mouse embryos and adult mouse brain. To this end, we developed polyclonalantibodies 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 respiratoryand 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 celllayer of the cerebellum (Fig. 7D and E). The Rsk2 protein wasclearly detected in the Purkinje cell layer of the cerebellum, inthe CA1 CA3 regions of the hippocampus and in neurons of the cerebral cortex (Fig. 7F and data not shown).

DISCUSSIONTo gain better understanding of the physiological roles of RSKsand whether they could have speci c roles and/or overlapping

Table 1. Summary of the differential Rsk expression patterns at late stages of mouse embryonic development (E14.5 E16.5)

Rsk1 Rsk2 Rsk3

Brain Caudate putamen Choroid plexus Diencephalon Medulla oblongata

Neopallial cortex Olfactory bulb Telencephalic ventricular zone

Peripheral nervous system Dorsal root ganglia Sympathetic ganglia Trigeminal ganglia Vagal ganglia Vagal trunk

Spinal cord Mantle layer Eye Retina

Lens Ear Pinna

Cochlea Nose Respiratory epithelium

Olfactory epithelium Oral cavity Tongue Mucous membrane

Tooth buds Follicles of vibrissae Submandibular gland Thyroid gland Thymus Esophagus Trachea Epithelium Heart Myocardium Lung Mesenchyme Liver Kidney Cortical region

Renal capsule Adrenal gland Cortex Intestine Epithelium

Lamina propria Outer mesenchyme

Duodenum

Stomach Epithelium Testis Testis cords Skeletal muscle Interdigital space

, Very strong expression; , strong expression; , moderate expression; , weak expression; , no detectable expression.



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functions in vivo, we have carried out an extensive analysis of their patterns of expression in human tissues, during mouseembryogenesis and in adult mouse brain. Northern blot analysisof a panel of eight human tissues demonstrated that the RSK1, 2and 3 genes are all widely expressed. Interestingly, expression of both RSK2 and RSK3 is most abundant in the heart, the skeletalmuscle and the pancreas, suggesting that they may have speci cand overlapping functions in these organs. In the whole brain,the RSK3 gene shows the strongest expression, whereasexpression 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 lobeand putamen). However, noticeably higher levels of RSK1 and RSK2 mRNAwere observed in the cerebellum. The latter is alsostrongly 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 onmouse embryo sections. This analysis revealed that the Rsk2gene is expressed at very low levels throughout embryonicdevelopment. However, at late embryonic stages, its expressionis 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 geneshows high levels of expression during development, almost exclusively restricted to the developing central and peripheralnervous system. Rsk3 expression is, in particular, detected inthe ventricular zone bordering the lateral ventricle, a site of high proliferative activity, but also in differentiating cerebralelds such as the cortex, caudate putamen, thalamus,hypothalamus and brainstem. This result supports the hypo-thesis that during development, Rsk3 functions to regulate proliferation and differentiation of neuroepithelial cells.Although the most prominent site of Rsk3 expression duringembryogenesis is the nervous system, signi cant expression is

also detected in a few other tissues including testis cords and the thyroid gland.

Until late midgestation, Rsk1 is strongly expressed in theneuroepithelium of the neural tube, whereas at later stages it decreases dramatically and becomes undetectable in thenervous system. On the contrary, expression of Rsk3 becomes prominent in the central and peripheral nervous system fromE11.5 and onwards. These results are consistent with atemporal 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 inregions harbouring highly proliferating cells. These includeliver, lung, thymus and olfactory and respiratory epithelia.Particularly intense Rsk1 expression was observed in the gut epithelium. From the expression patterns observed, Rsk1 seemsto be more strictly linked to cellular proliferation and Rsk3 tocellular differentiation, in particular in the nervous system.

RSK1 and RSK3 have not yet been associated with a humandisorder and no animal models have as yet been described.In adult mouse brain, Rsk1 was clearly detected only in thegranular cell layer of the cerebellum. In contrast, high levels of Rsk3 expression were observed in various regions of the adult mouse brain, suggesting a speci c 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, thedentate gyrus and the amygdala suggest that RSK3 is a good candidate for disorders displaying involvement of the centralnervous system, including mental retardation.

Rsk2 expression was too low to be detected in most brainstructures 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 inPurkinje cells and deep nuclei of the mature cerebellum. Neurons within all these structures are characterized by high

Figure 2. Rsk1 and Rsk3 expression at early stages of mouse embryonic development (E11.5). ( A) Bright eld view of an E11.5 mouse embryo sagittal section, toshow the histology. ( B and C ) Dark eld views of the same or neighbouring sections, showing the hybridization signal grain as white dots. All subsequent Figuressimilarly show dark eld views, as well as the corresponding bright eld view for histology. ( B) At this stage, a wide distribution of the Rsk1 mRNAwas observed.However, the strongest Rsk1 expression was observed in the midgut. ( C ) Rsk3 expression was very strong in the neural tube, the dorsal root ganglia, the heart and

the developing eye. drg, Dorsal root ganglia; ey, developing eye (optic cup); hp, hepatic primordium; ht, heart; mg, midgut; nt, neural tube.



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Figure 3. Rsk2 expression at E14.5 and E15.5. ( A and B) Sagittal sections of an E15.5 embryo. Low Rsk2 expression was observed in dorsal root ganglia (arrows),lung, skeletal muscle, and kidney. ( C and D) Sagittal sections of an E14.5 mouse embryo. The arrow in (D) shows Rsk2 expression in the trigeminal ganglion.(E and F ) Dot-like expression pattern of Rsk2 in liver at E15.5. drg, Dorsal root ganglia; ki, kidney; li, liver; lu, lung; mu, muscle; tg, trigeminal ganglion.



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synaptic activity, especially the Purkinje cells of the cerebellumand the pyramidal cells of the hippocampus. The latter ones areconsidered as the cells with the highest synaptic activity in thewhole brain (27,28). The speci city of Rsk2 to neuronalregions with high synaptic activity supports the concept of afunctional importance of RSK2 in neural transmission. Thehippocampus and the neocortex are essential components incognitive function and learning. Purkinje cells are commonlyassociated with motor and balance skills but are nowincreasingly 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 pyriformcortex. The amygdala has been associated with a range of cognitive functions, including emotion, learning, memory,attention and perception (30). Noteworthy is also the strongexpression of the RSK2 mRNA in the frontal lobe of the humancortex, as revealed by northern blot analysis (Fig. 1B). Recent advances have uncovered important roles for the frontal lobesin a multitude of cognitive processes, including executivefunction, attention and language. Interestingly, evidence has been provided that the prefrontal areas play also a crucial rolein 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 thecell types in which RSK2 expression may play a key role.In addition, the expression of Rsk2 in adult mouse brainstructures is very similar to the one observed for several genesimplicated in mental retardation (33).

A mouse model for CLS, obtained by inactivation of the Rsk2gene, was recently described (34). Mutant mice weigh 10% lessand are 14% shorter than their wild-type littermates. This is inagreement 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 mentalfunctioning both in mice and humans.Recent reports have provided evidence that long-term

memory formation requires the activity of the cAMP responseelement (CRE) binding protein (CREB) transcription factor and that CRE-regulated genes are expressed in the hippocampus inresponse 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 themagnitude of in vitro RSK2-mediated CREB phosphorylationand intelligence level in CLS patients. Together these datasuggest that the Ras/MAPK pathway, signalling through RSK2

Figure 5. Rsk1 and Rsk3 expression at E16.5. ( A D) Rsk1 expression on frontal sections of an E16.5 mouse embryo. Arrows in (B) and (D) indicate Rsk1expression in the pinna of the ear, the respiratory and olfactory epithelia, the follicles of vibrissae, the cochlea, the periphery of the tongue and the tooth budsrespectively. ( E H ) Rsk3 expression on frontal sections. ( F ) Rsk3 expression was detected in many parts of the brain, including the olfactory bulb, the choroid plexus (arrow), the cortical plate of the neopallial cortex, the ventricular zone of the telencephalon, the sulcus (indicated respectively by arrows) and the striatum.(H) Strong Rsk3 expression was seen in the inner nuclear layer of the retina and the lens. The thalamus and the pons were also stained by the Rsk3 riboprobe. Like Rsk1, Rsk3 was detected in the respiratory epithelium. 3 V, Third ventricle; 4 V, fourth ventricle; co, cochlea; cop, cortical plate of the neopallial cortex; cp, choroid plexus; ea, ear; ls, lens; ob, olfactory bulb; oe, olfactory epithelium; po, pons; re, respiratory epithelium; ret, retina; st, striatum; su, sulcus; tb, tooth buds;th, thalamus; to, tongue; vi; follicles of vibrissae; vz, ventricular zone of the telencephalon.



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to CREB, plays a prominent role in cognitive dysfunction inCLS patients. Our results, showing strong Rsk2 expression inthe hippocampus, further support this hypothesis.

Interestingly, the pyramidal cells of the CA3 region of thehippocampus, intensively express the Rsk2 mRNA. Previousstudies have indicated that broblast cell lines derived fromCLS patients with RSK2 inactivating mutations are defective inEGF induced c-fos gene expression (24). There is now evidencethat expression of Fos, the product of the immediate-early gene

c-fos , in the CA3 region, may be necessary for spatial memoryformation (38). Fos, participates in the formation of hetero-dimeric AP-1 transcription factors which are thought to activatethe expression of late-effector genes. The expression of c-fos isinduced by a variety of stimuli and after some forms of learning. Accordingly, Fos expression is considered as part of amechanism by which brief stimuli can trigger long-termchanges in gene expression and alter structural and functional properties of nerve cells.

Figure 6. Rsk expression in adult mouse brain. ( A F ) Sagittal sections of adult brain. (B) Rsk1 expression was primarily observed in the granular cell layer of thecerebellum. (D and F) Rsk2 expression in the Purkinje cell layer and the deep nuclei of the cerebellum (arrows in D) and the hippocampus (arrows in F), respec-tively. The weak staining observed in the dentate gyrus was not speci c. (G L ) Frontal sections of adult brain. (H) Arrows show Rsk3 expression in the dentategyrus, the amygdala and the pyriform cortex. Layers II and III of the cerebral cortex were also stained by the Rsk3 riboprobe. (J) Strong Rsk3 expression wasobserved in the bed nucleus of the stria terminalis and moderate expression in the accumbens nucleus (indicated by arrows). (L) Rsk3 expression in the arcuatenucleus of the hypothalamus. an, Lateral amygdaloid nucleus; acn, accumbens nucleus; arn, arcuate nucleus; bn, bed nucleus of the stria terminalis; ce, cerebellum;cx, cerebral cortex; dg, dentate gyrus; dn, deep cerebellar nuclei; gcl, granular cell layer of the cerebellum; hi, hippocampus; ht, hypothalamus; pc, pyriform cortex;

pcl, Purkinje cell layer of the cerebellum; th, thalamus.



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The expression of RSK4, the most recently identi ed 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 wasseen in kidney, brain and thyroid gland whereas in pancreas a lowamount of RSK4 mRNA was detected. Thus, it seems that RSK4displays a more restricted expression pattern than the other RSK proteins. However, a more detailed study in the mouse is required before a denitive conclusion can be drawn.

Intriguingly, given the skeletal abnormalities observed inCLS patients, no signi cant expression of any Rsk gene wasdetected in developing bones of mouse embryos. This result suggests that very low levels of Rsk expression are necessaryfor skeletal development. RT PCR and immunocytochemistry

studies in speci c bone cell populations derived from wild typeand Rsk2 decient adult mice and embryos are underway,which should help to elucidate this point. It is worth noticingthat mice carrying a Rsk2 null allele apparently do not haveskeletal defects, which are among the major manifestations of the syndrome in humans (34). This latter observation suggeststhat there might be a greater degree of redundancy in mice, at least in the strain that has been used to generate the Rsk2 nullmouse.

Despite several differences, a considerable overlap in theexpression 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 missingRSK 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 beginsto explain the phenotype observed in CLS patients. RSK1 and 3 should be able to compensate for the lack of the Rsk2 proteinin most tissues, in which they are expressed at higher levelsthan Rsk2. Interestingly, in the pyramidal cells of thehippocampus, in the Purkinje cells of the cerebellum and indeep layers of the neocortex of the adult mouse brain, the Rsk2gene 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 Rsk2deciency (unpublished results, H.A.). These data support thehypothesis that in these areas RSK1 and RSK3 might not beable to fully compensate for RSK2 de ciency in Rsk2 null miceand in CLS patients, and thus may provide an additional clue tothe understanding of the cognitive dysfunction.

To explore the full range of physiological functions performed by members of the RSK family and to further de ne redundant and speci c functions, especially for RSK2, it will now beimportant to mutate all the members of the Rsk gene family and to make different combinations of Rsk decient mice.

Figure 7. Immunohistochemical analysis of Rsk1 and Rsk2 on E13.5 mouse embryo sections and on adult mouse brain sections. ( A , B and C ) The Rsk1 proteinwas detected in liver, intestine, thymus, the submandibular gland, the olfactory epithelium, the periphery of the tongue and the follicles of vibrissae. ( D, E and F )Rsk1 and Rsk2 expression in adult mouse brain. (D) The strongest Rsk1 expression was seen in the granular cell layer of the cerebellum. (E) Higher magni cationof the granular cell layer of the cerebellum, stained with the anti-Rsk1 antibody. (F) The presence of the Rsk2 protein was detected in Purkinje cells and in some of the deep nuclei of the cerebellum. dn, Deep cerebellar nuclei; gcl, granular cell layer of the cerebellum; in, intestine; li, liver; oe, olfactory epithelium; pcl, Purkinjecell layer of the cerebellum; sg, submandibular gland; th, thymus; to, tongue.



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MATERIALS AND METHODS

Northern blot analysis

Two northern blots, the rst containing human poly(A) RNAfrom heart, total brain, placenta, lung, liver, skeletal muscle,

kidney and pancreas (Clontech, Palo Alto, CA, USA, HumanMTN TM Blot, 7760-1), and the second human poly(A) RNAfrom cerebellum, cerebral cortex, medulla, spinal cord,occipital pole, frontal lobe, temporal lobe and putamen(Clontech Human Brain MTN TM Blot II, 7755-1) werehybridized with radioactively labelled cDNA probes(2 106 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 byrandom primer extension following standard protocols.

Preparation of mouse embryos

Pregnant CD-1 females from natural overnight matings

(morning of vaginal plug was considered as 0.5dpc) weresacriced by cervical dislocation and embryos were collected,embedded in Tissue-Tek 1 O.C.T TM medium (SAKURA,Zoeterwoude, The Netherlands) and frozen on the surface of dry ice. Serial cryosections (10 mm thick) were obtained and subsequently analyzed.

Preparation of adult mouse brains

Adult (8 week old) CD-1 mice were sacri ced and brains were processed as described above.

In situ hybridization

To avoid cross-reaction, sequences corresponding to parts of the 30 untranslated regions of Rsk1, 2 and 3 mRNAs were used as riboprobes. 719 and 990 bp Rsk1 and Rsk3 EcoRI BamHIcDNA fragments, respectively, were cloned in pBluescriptSK (Stratagene, Amsterdam-Zuidoost, The Netherlands). A 276 bp Rsk2 cDNA fragment was cloned between the Sac I and Eco RIsites of the same vector. 1 mg of the linearized vector was used in T3 (17 units) and T7 (15 units) polymerase (Promega,Charbonnie res, France) reactions including 3 ml [

35 S]-CTP(Amersham Biosciences Europe GmbH, Orsay, France), to produce sense and antisense riboprobes. The reactions werecarried out for 2 h at 37 C in the presence of 5 m M of each of the nucleotides ATP, GTP and UTP, 0.25m M CTP, 100 m Mdithiothreitol and 50 units RNase inhibitor (Promega). The

reaction volume was 20 ml. After a 20 min treatment with 10units of RNase-free DNase I (Boehringer, Ingelheim, Germany)at 37 C, the length of the probes was reduced by alkalinehydrolysis 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. Theywere subsequently xed for 15 min in a 4% formaldehyde inPBS (Dulbecco s phosphate buffered saline, Sigma-Aldrich,Saint Quentin Fallavier, France) solution, at room temperatureand washed 2 5 min in 1 PBS. A 5 min treatment in 0.1 Mtriethanolamine, was followed by a 10 min treatment in 0.1 M

triethanolamine, 0.25% acetic anhydride, 2 2 min washingsin 1 PBS 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 (25 106 cpm/ml of pre-hybridization mix

(0.3M

NaCl, 20mM

Tris-HCl pH 6.8, 5 mM

EDTA, 1 mM

NaPO 4 buffer pH 6.8, 0.1% Ficoll, 0.1% polyvinyl pyrrolidon25, 0.1% bovine serum albumin, 50% formamide, 10% dextransulfate, 10 m M dithiothreitol and yeast tRNA (0.5 mg/ml))),were incubated for 5 min at 65 C, placed 5 min on ice and thenadded to the slides. The hybridization was carried out overnight at 52 C in a humid chamber. Subsequent washings were asfollows: 1 h at 55 C i n 5 SSC (1 SSC: 150 m M NaCl,15 m M C6 H5 O7 Na3 2H 2 O), 1 h at 55 C in 2 SSC, 15 min at 37 C in 4 SSC, 30 min at 37 C in 4 SSC with RNaseA(Sigma-Aldrich) at 20 mg/ml, 10 min at 37 C in 4 SSC, 1 h at 55 C in 2 SSC/50% formamide, 15 min at 55 C in 2 SSCand 15min at 55 C in 0.1 SSC. The slides were then placed for 1 min in each of the solutions: 30% ethanol and 0.4 M

AcNH 4 , 60% ethanol and 0.4 M AcNH 4 , 85% ethanol and 0.4 MAcNH 4 , 95% ethanol and 0.4 M AcNH 4 and 100% ethanol and they were then allowed to dry. The times for emulsionautoradiography were 3 5 weeks. As expected, control senseriboprobes only gave uniform background labelling (data not shown).

Antibodies

Rabbit polyclonal 1793 antibody was raised and af nity puried against the synthetic peptide LMEDDGKPRAPQAPLcorresponding to human RSK1 amino acids 386 400. Rabbit polyclonal 1801 antibody was raised and af nity puri ed

against the synthetic peptide MDEPMGEEEINPQTEEVScorresponding 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. Theywere subsequently washed once with 1 PBS and xed with4% paraformaldehyde for 10 min. After xation and washingwith 1 PBS for 10 min, endogenous peroxidase was inhibited by a 20 min treatment with 0.5% H 2 O2 solution in PBS.Blocking of non-speci c sites was carried out by incubating the

sections in 10% normal goat serum in PBS for 1 h at roomtemperature. The primary antibodies were added to the sectionsin 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 10min in 1 PBS and incubated for 2 h at room temperaturewith an Alexa Fluor 1 488 goat anti-rabbit IgG (H L)secondary antibody (Molecular Probes, Leiden, The Netherlands), (dilution 1 : 200). After four washes with 1PBS samples were mounted with KAISER s glycerol gelatin(Merck).



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ACKNOWLEDGEMENTSWe would like to thank G. Duval for production of polyclonalantibodies, J.L.Vonesch,D. Hentsch and M. Boeglin for help withimaging and S. Pannetier for excellent technical assistance. Thework was supported by grants from the Association Franc aise pour la Recherche contre le Cancer, the Centre National de laRecherche Scienti que, the Institut National de la Sante et de laRecherche Me dicale and the Ho pital Universitaire de Strasbourg.

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