Regionspecific expression of cyclin-dependent kinase 5 (cdk5) and its activators, p35 and p39, in...

Region-Specific Expression of Cyclin-DependentKinase 5 (cdk5) and Its Activators, p35 and p39,in the Developing and Adult Rat CentralNervous System

Min Zheng,1 Conrad L. Leung,2 Ronald K. H. Liem1

1Department of Pathology, Anatomy and Cell Biology, Columbia University College of Physiciansand Surgeons, 630 West 168th St., New York, New York 10032

2Biochemistry and Molecular Biophysics, Columbia University College of Physicians and Surgeons,630 West 168th St., New York, New York 10032

Received 12 November 1997; accepted 15 December 1997

marginal zone, but are absent from the ventricularABSTRACT: The ubiquitously expressed cyclin-zone, which may restrict cdk5 activation to the postmi-dependent kinase 5 (cdk5) is essential for brain devel-totic neural cells in the developing brain. The expres-opment. Bioactivation of cdk5 in the brain requiression levels of p35 and p39 mRNAs in the marginalthe presence of one of two related regulatory subunits,zone increase by E15 and E17, paralleling the neuro-p35 and p39. Since either protein alone can activategenetic timetable. One exception is in the rostral fore-cdk5, the significance of their coexistence as cdk5 ki-brain, where p35 mRNA expression levels are high,nase activators is unclear. To determine whether thesuggesting that p35 may be the major activator fortwo activators are expressed in different cells through-cdk5 during telencephalic morphogenesis. A signifi-out the nervous system and during development, wecant level of p35 mRNA is present in the myotome atcompared the tissue distributions of cdk5, p35, andE12 and p35 expression persists in the premuscle massp39 mRNAs in the rat using in situ hybridization.and mature musculature at later stages, suggestingIn the adult rat, expression levels of p35 mRNA arethat p35 may also activate cdk5 during myogenesis.generally higher in the brain than in the spinal cord,q 1998 John Wiley & Sons, Inc. J Neurobiol 35: 141–159, 1998while the converse is observed for p39 mRNA. DuringKeywords: kinase activator; in situ hybridization; neu-neurogenesis, both p35 and p39 transcripts can berofilaments; muscle; cdk5detected as early as embryonic day 12 (E12) in the

INTRODUCTION brain and its cDNA was cloned based on homologyto cdc2 (Hellmich et al., 1992; Lew et al., 1992;Meyerson et al., 1992). Although cdk5 exhibits sig-In recent years, significant progress has been made

in elucidating the protein kinases, which regulate nificant sequence similarity to other cdks, its physi-ological role is fundamentally different from otherthe cell cycle. These kinases are activated by cyclins

and are therefore classified as cyclin-dependent ki- cdks. In contrast to the cell-cycle–related kinases,cdk5 kinase activity is detected only in terminallynases or cdks (reviewed in Morgan, 1995; Nurse,

1990; Pines, 1993). cdk5 was initially isolated from differentiated neural tissues (Tsai et al., 1993). Fur-thermore, cdk5 kinase activity is stimulated by ei-ther one of two proteins unrelated to cyclins, called

Correspondence to: R. Liem p35 and p39 (Lew et al., 1994; Tang et al., 1995;Contract grant sponsor: NIH; contract grant numbers:

Tsai et al., 1994).AG13185, EY07105, AG00189q 1998 John Wiley & Sons, Inc. CCC 0022-3034/98/020141-19 Among the known substrates of cdk5 are the neu-

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https://www.researchgate.net/publication/15661915_Molecular_Characterization_of_a_Neuronal-Specific_Protein_that_Stimulates_the_Activity_of_Cdk5?el=1_x_8&enrichId=rgreq-fb0110ba898d1d6b8f2e9c144c720726-XXX&enrichSource=Y292ZXJQYWdlOzI0Njg3MTg4MjtBUzoyMTg4MjAzNDMwMTMzNzZAMTQyOTE4MjI0NDkyNg==

142 Zheng, Leung, and Liem

decyl sulfate (SDS), 427C] with 32P-labeled rat p35rofilament (NF) proteins. NFs are composed of low-cDNA as a probe. A total of 24 positive plaques were(NF-L), medium- (NF-M), and high- (NF-H) mo-recovered and subcloned into the EcoRI site of pBlue-lecular-weight neurofilament proteins (Fliegner andscript II KS (Stratagene). By means of dideoxy sequenc-Liem, 1991; Liem, 1993; Lee and Cleveland, 1994).ing, six clones were identified as human p39. After EcoRINF-H is extensively phosphorylated in the axondigestion, the 1.2-kb fragment from the longest clone,

(Jones and Williams, 1982; Julien and Mushynski, containing an almost full-length p39, was isolated and1982), and this phosphorylation is important in used to screen a mouse genomic library. Briefly, the 1.2-modulating axonal caliber (de Waegh et al., 1992; kb EcoRI fragment containing the human p39 cDNA wasNixon and Sihag, 1991). cdk5 in the presence of purified, labeled with 32P by random primer synthesisp35 has been shown to phosphorylate NF-H both (Amersham), and used as a probe for screening an ampli-

fied lFIX II mouse genomic library (Stratagene). Ap-in vitro (Hisanaga et al., 1995; Lew et al., 1992;proximately 5 1 107 plaques were screened, and oneShetty et al., 1995) and in vivo (Guidato et al.,positive plaque was recovered and characterized.1996; Sun et al., 1996). Functional disruption of

cdk5 or p35 in primary neuronal cell cultures inhib-its neurite outgrowth (Nikolic et al., 1996). Tar- In Situ Hybridizationgeted ablation of the cdk5 gene in mice results in theloss of cortical lamination and cerebellar foliation, Tissue Preparation. All studies were conducted in accor-

dance with the principles and procedures outlined in theabnormal accumulation of NFs, and perinatal lethal-National Institute of Health Guide for the Care and Useity (Ohshima et al., 1996). In contrast, disruptionof Laboratory Animals. For in situ hybridization, embryosof p35 causes cortical lamination defects, but notfrom timed pregnant Sprague–Dawley female rats wereother defects observed in cdk5 knockout mice, andused. Adult brains were obtained from three female ratsthe mice survive for several weeks postnatally(250–300 g). For studies of developmental gene expres-(Chae et al., 1997).sion, at least one litter of rats for each age group (E12,

Although cdk5 transcripts are widely distributed E13, E15, and E17) was used, with two to four embryos(Meyerson et al., 1992; Tsai et al., 1993), cdk5-related examined for each age. The adult animals were sacrificedhistone H1 kinase activity has been isolated or immu- by CO2 suffocation, and whole embryos (E12–E15),noprecipitated only from neural tissue (Lew et al., whole fetuses or fetal head and trunk (E17), or brains1992; Shetty et al., 1995; Tsai et al., 1993). The re- were collected. Embryos and fetuses were staged ac-

cording to the morphology of their limb buds in referencequirement for the cdk5 kinase activators, p35 and/orwith the criteria established by Wanek et al. (1989). E12p39, may be the underlying mechanism responsibleembryos were fixed by immersion in 4% paraformalde-for this restriction of cdk5 kinase activity. Since eitherhyde for 6–8 h and equilibrated in 20% sucrose solutionp35 or p39 alone can activate cdk5 kinase activity,before embedding. Older embryos, fetuses, and adultthe significance of their coexistence as cdk5 kinasebrains were freshly frozen and embedded directly in OCTactivators is unclear. One possibility is that differencescompound without prior fixation. Both sagittal and trans-

in p35 and p39 distribution contribute to the in vivo verse cryostat sections (E12–E17) as well as frontal cra-activation of cdk5 in a region-specific or developmen- nial sections (E15–E17, adult brains) were prepared astally regulated manner. We compared the expressions described (Zheng and Pintar, 1995).of p35 and p39 mRNAs with each other and withcdk5 mRNA. Our results demonstrate distinct cellular Probe Preparation. 35S-labeled cRNA transcripts werepatterns of their expression both in the adult brain synthesized in vitro from the plasmid vectors harboringand in embryonic development. These observations the corresponding cDNA sequences using Riboprobesuggest that p35 and p39 may activate cdk5 kinase in Gemini Systems from Promega (Madison, WI). Plasmid

pBS-cdk5 contains a 876-bp rat cdk5 cDNA [generatedregion-specific and developmental stage-specific man-by reverse-transcriptase–polymerase chain reaction (RT-ners in neural tissues.PCR)] and corresponds to nucleotides 1–876 of the full-length coding region (Sun et al., 1996). This plasmidwas linearized with SalI (sense) or EcoRI (antisense)

MATERIALS AND METHODS to generate 876 nt cRNA sense or antisense transcripts.pGEM-p35 containing a 1158-bp rat p35 cDNA fragment(Sun et al., 1996) was linearized with HindIII (sense) orMolecular Cloning of the MouseEcoRI (antisense) . pBS-p39 contains a 1198-bp mousep39 Genep39 cDNA fragment (Fig. 1) that was linearized withEcoRI (sense) or BamHI (antisense) .Human p39 cDNA was isolated by screening a human

fetal brain cDNA library (Clontech) under low-strin- Both sense and antisense 35S-UTP-labeled cRNA wereprepared. cRNA probes were purified on Sephadex G-50gency hybridization conditions [11 SSC, 1% sodium do-




cdk5, p35, and p39 Expression in Rat CNS and Development 143



columns (Boehringer-Mannheim) and used for in situ for cdk5, the distributions of p35 and p39 are neuralhybridization experiments without hydrolysis. specific in the adult. Quantitative analyses of their

mRNA expression levels were not performed, al-In Situ Hybridization. In situ hybridization experiments though similar length of riboprobes and emulsionwere performed as described previously (Zheng and

exposure time were used for the three probes. Subre-Pintar, 1995). After nuclear emulsion, autoradiographygions of the brain and spinal cord were identifiedwas performed and slides were examined using a Leitzfrom a rat brain atlas (Paxinos and Watson, 1986).microscope under both bright-field and dark-field illu-

minations. Hybridization with control ( sense ) probesRhinencephalon. Moderate levels of cdk5, p35,yielded only low background staining in all cases.and p39 mRNAs were detected in the olfactory bulband anterior olfactory nuclei (data not shown). TheRESULTSpiriform cortex and tenia tecta were labeled heavilyby all three probes [Fig. 2(A–F)] .Molecular Cloning of the Mouse

p39 GeneTelencephalon.

A near-full-length human p39 cDNA was recovered Neocortex. All three transcripts were expressedand subcloned into the EcoRI site of pBluescript II throughout the cerebral cortex, including the frontalKS (Stratagene). The isolated human p39 cDNA cortex, parietal cortex, temporal cortex, insular cor-was identical to the reported sequence (Tang et al., tex, and entorhinal cortex [Fig. 2(A–L)] (data not1995). This p39 cDNA was then used to screen a shown) and were nearly uniformly distributed in all129/SvJ mouse genomic library (Stratagene). A cortical layers except layer I [cdk5: Fig. 2(D,G,J);genomic clone was isolated which contained the p35: Figs. 2(E,H,K) and 3(B); p39: Figs. 2(F,I,L)entire coding region of the mouse p39 (Accession and 3(C)] .no. AF01639) (Fig. 1) . In addition, the genomic Hippocampal Formation. High levels of cdk5,clone contained 500 bp of 5 * flanking region and p35, and p39 transcripts were present in the hippo-13 kb of 3 * flanking region. The deduced amino campus. The pyramidal cells of subfields 1–4 ofacid sequence of mouse p39 is very similar to that Ammon’s horn, scattered hilar cells, and the granu-of human p39 (there are only 13 amino acid differ- lar cell layers of the dentate gyrus all displayed highences) . In addition, the p39 gene has no introns in levels of expression of cdk5 mRNA [Fig. 2(G,J)] ,the coding region similar to the reported genomic p35 mRNA [Figs. 2(H,K) and 3(E)] , and p39structure of mouse p35 (Chae et al., 1997; Ohshima mRNA [Figs. 2(I,L) and 3(F)] .et al., 1996). A 1198-base pair SmaI fragment con- Amygdala. All three genes were present at ataining the entire mouse p39 coding sequence was moderate level in the amygdaloid body, includingfurther subcloned into the SmaI site of pBluescript the anterior cortical nuclei, basolateral, and medialII KS and used for in situ hybridization studies. nuclei [Fig. 2(D–L)] (data not shown). Postero-

The mRNA distribution of cdk5 and its activators medial cortical amygdala nuclei displayed a slightlyp35 and p39 were mapped in the adult brain using higher level of p35 gene expression [Fig. 2(H)] .serial coronal sections (Fig. 2, and some highlights Basal Forebrain. Relatively similar levels ofin Fig. 3) . In addition, their expression was also cdk5, p35, and p39 gene expression were observedtraced back during prenatal development (E12– in the caudate-putamen and accumbens nuclei,E17), with emphasis placed on comparing the whereas their expressions in the globus pallidus andunique distribution of p35 and p39 mRNAs in the ventral pallidum were low [Fig. 2(A–F)] . Moder-developing nervous system (Figs. 3–9). ate levels of cdk5, p35, and p39 mRNAs were pres-

ent in the lateral septum, with slightly higher expres-Comparative Expression of cdk5, p35, sion in the intermediate lateral septal nuclei [Fig.and p39 in the Adult Central 2(D–F)] .Nervous System (CNS)Nearly ubiquitous expression of cdk5, p35, and p39 Diencephalon.

Thalamus. All three genes were expressed uni-mRNAs were observed in the adult CNS. Except

Figure 1 Nucleotide sequence and deduced amino acid sequence of mouse p39 gene. TheSmaI fragment was subcloned into pBluescript II KS vector (Stratagene), linearized, and usedas templates to synthesized both sense and antisense riboprobes.



Figure 2 Comparative mRNA distribution of cdk5, p35, and p39 in the adult brain. Adjacentcoronal sections in the anterior-posterior sequence are shown hybridized with cdk5(A,D,G,J,M,P), p35 (B,E,H,K,N,Q), and p39 (C,F,I,L,O,R). The following abbreviations ofnomenclature are based on Paxinos and Watson (1986): Acb Å accumbens nuclei; CA1–3



formly in various thalamic nuclei [Fig. 2(I)] (data 2(M,N) and 3(K)] were readily detected in thePurkinje cells, whereas p39 mRNA appeared to benot shown).

Epithalamus. Moderate levels of cdk5, p35, and less abundant in these cells [Figs. 2(O) and 3(L)] .Scattered positive cells labeled for cdk5 and p35p39 mRNAs were distributed in the medial and lat-

eral habenular nuclei (data not shown). mRNAs in the granular cell layer probably representGolgi II cells [Fig. 3(K)] (data not shown).Hypothalamus. Various hypothalamic nuclei

displayed low to moderate levels of cdk5, p35, and Deep Nuclei. cdk5, p35, and p39 gene expres-sions were noted in all deep nuclei of the cerebellump39 gene expression (data not shown).[Fig. 2(M–O)] (data not shown).

Mesencephalon. Ubiquitous expression of cdk5,p35, and p39 mRNAs were observed in most re- Choroid Plexus. A high level of cdk5 mRNA was

detected in nonneuronal structures of the brain, suchgions of the mesencephalon, such as deep mesence-phalic nuclei, substantia nigra, superior and inferior as the choroid plexus [see, for example, Fig.

2(D,M)]. Neither p35 nor p39 transcripts were de-colliculi, interstitial nuclei; raphe nuclei, and rednuclei [Fig. 2(J–L)] (data not shown). Heteroge- tected in the choroid plexus [compare Fig. 3(N)

and (O)].neity in cdk5 and p35 expression, however, wasobserved in several nuclei. While the cdk5 probeheavily labeled the oculomotor and red nuclei [Fig. Spinal Cord. In the spinal cord, cdk5, p35, and

p39 mRNAs were heterogeneously distributed. cdk52(J)] , the p35 probe only labeled the geniculatenuclei [Fig. 2(H)] , and the p39 probe did not sig- mRNA was expressed at a particularly high level

by the motor neurons of the anterior horn [Fig.nificantly label any of these nuclei [compare Fig.2(H) with (I) , Fig. 2(K) with (L), and Fig. 3(E) 2(P)] . Transcripts for p39 were more easily de-

tected in the spinal cord than for p35, especially inwith (F), and note their comparable level in theparietal lobe]. the superficial dorsal horn [compare Fig. 2(Q) and

(R)] . The spinal cord therefore represents the onlyCNS region where the expression level of p39Rhombencephalon.

Pons. Higher levels of cdk5 mRNA were de- mRNA is apparently higher than that of p35 mRNA.tected in the pontine nuclei than in other pontinestructures, while p35 and p39 mRNAs were ex- Comparative Expression of cdk5, p35,pressed more uniformly [Fig. 2(J–L)] . and p39 mRNAs in the Embryonic

Medulla. Transcripts of cdk5, p35, and p39 Developmentwere broadly distributed in various medullary struc-tures [Fig. 2(M–O)] (data not shown). All three The distribution of cdk5, p35, and p39 mRNAs was

analyzed in rat embryos and fetuses ranging fromgenes were expressed at a significantly high levelin the rostroventrolateral reticular nuclei and in the E12 to E17, which represent a critical period when

the nervous system and other major organ systemsgigantocellular nuclei [Fig. 2(M–O)]. The expres-sion of p35 mRNA in the adjacent inferior olive are forming. Anatomic structures or cell types ex-

pressing cdk5, p35, and p39 mRNAs were identifiedwas also more readily apparent than p39 mRNA[compare Fig. 3(H) and (I)] . by comparing them with several atlases (Altman

and Bayer, 1995; Kaufman, 1992; Sidman et al.,1971). Below, we summarize their distribution atCerebellum.

Cerebellar cortex. cdk5 and p35 mRNAs [Figs. different stages.

Å fields Ca1–3 of Ammon’s horn; ccÅ corpus callosum; CgÅ cingulate cortex; ChPÅ choroidplexus; CPu Å caudate-putamen; DA Å dorsal hypothalamic area; DC Å deep cerebellar nuclei;DG Å dentate gyrus; DLG Å dorsal lateral geniculate nuclei; DpMe Å deep mesencephalicnuclei; Ent Å entorhinal cortex; Fr Å frontal cortex; Gi Å gigantocellular regicular nuclei; GPÅ globus pallidus; IC Å insular cortex; IO Å inferior olive; DLG Å dorsal lateral geniculatenuclei; LS Å lateral septal nuclei; MG Å medial geniculate nuclei; MVe Å medial vestibularnuclei; Par Å parietal cortex; Pir Å piriform cortex; PMCo Å posteromedial cortical amygdalanuclei; Pn Å pontine nuclei; RVL Å rostroventrolateral reticular nuclei; SNR Å substantianigra; SuC Å superior colliculus; Th Å thalamic nuclei; TT Å tenia tecta; VA Å ventralhypothalamic area; VP Å ventral pallidum.



Figure 3 Localization of cdk5, p35, and p39 mRNAs in various adult brain regions. Leftcolumn shows bright-field micrographs of Cresyl violet–stained brain sections, with the adja-cent sections hybridized with cdk5 (N), p35 (B,E,H,K), and p39 (C,F,I,L). See Results fordetails. AA Å Amygdaloid area; CA2–3 Å fields CA2–3 of Ammon’s horn; ChP Å choroidplexus; CPu Å caudate-putamen; Fr Å frontal cortex; GCL Å granular cell layer; Gi Å giganto-cellular regicular nuclei; Layer I–VI Å cerebral cortex layers I–VI; Lat Å lateral cerebellarnuclei; MG Å medial geniculate nuclei; ML Å molecular layer; IO Å inferior olive; ParÅ parietal cortex; PCL Å Purkinje cell layer.

E12. The neural tube has already closed and the zone or geminal layer) and reside in the outer rimof the neural tube, called the marginal zone. A lownervous system is in an active phase of expansion.

As neural progenitor cells exit the cell cycle, they level of cdk5 mRNA expression was observed inall embryonic structures [Fig. 4(A,D)] . In the de-migrate out of the neuroepithelial layer (ventricular



Figure 4 At E12 embryo, p35 and p39 mRNAs were expressed in the marginal zones ofthe developing nervous system. Sagittal sections (A–C,G,H) and frontal sections (D–F,I,J) ,hybridized with cdk5 (A,D), p35 (B,E,H,J) , and p39 (C,F) riboprobes. Relative position ofsections shown in (D–F) is illustrated on a schematic drawing adjacent to them. A regionsimilar to the rectangal one in (E) is enlarged and shown in (I) (bright field) and (J) (darkfield) . Note the expressions of p35 and p39 are confined to the intermediate zones of the CNS(B,C,E,F,H), whereas cdk5 is expressed at a low level by all embryonic structures (A,D). BrSÅ Brain stem; Cb Å cerebellum; CC Å cingulate cortex; CP Å cardiac primordium; DRGÅ dorsal root ganglia; Fp Å floorplate; LB Å (hind)limb bud; M Å medulla; MA Å madibulararch; Ht Å hypothalamus; Pd Å pallidum; Sm Å somite; SC Å spinal cord; Tt Å tectum. Scalebars Å 1 mm (A–F), 0.5 mm (G–J).



Figure 5 At E13, p39 expression level was significantly lower in the developing forebraincompared to p35. Sagittal sections (A–C) and transverse sections (D–I) hybridized with cdk5(A,D,G), p35 (B,E,H), and p39 (C,F,I) . Relative planes of sectioning for sections shown in(D–I) are illustrated on a schematic drawing of the embryo. Compared to p35, the expressionof p39 was barely detectable in the telencephalon in the sagital sections (B,C) and transversesections (H,I) . BrS Å Brain stem; Cb Å cerebellum; Di Å diencephalon; DRG Å dorsal rootganglia; H Å heart; Ht Å hypothalamus; LB Å (fore)limb bud; Lv Å liver; Me Å mesencepha-lon; Mg Å midgut; Nc Å neocortex; NP Å nasal process; PrM Å premuscle mass; SC Å spinalcord; Tt Å tectum; Te Å telencephalon; Tm Å tegmentum; To Å tongue. Scale bars Å 1 mm.



Figure 6 At E15, distinction between p35 and p39 gene expression in the developing nervoussystem maintained, while the expression of p35 in the muscle continued to expand. Sagittalsections (A–C), frontal sections (D–F), and oblique-transverse sections (G–I) were hybrid-ized with cdk5 (A,D,G), p35 (B,E,H), and p39 (C,F,I) . Note a continued lack of p39 expres-sion in the forebrain compared to p35 [compare (C) and (B)] . A moderate to high level ofp35 expression was observed in the muscular structures in the cranial region (E) and trunk(H). Cb Å cerebellum; CC Å cerebral cortex; Hi Å hippocampus; Ht Å hypothalamus; LvÅ liver; Mb Å midbrain; Md Å medulla; Nc Å neocortex; NVZ Å neocortical ventricularzone; OB Å olfactory bulb; OE Å olfactory epithelium; Pn Å pons; Pir Å piriform cortex; PdÅ pallidum; PIZ Å pallidal intermediate zone; St Å septum; SC Å spinal cord; SM Å skeletalmuscle masses; Th Å thalamus; To Å tongue. Scale bars Å 1 mm.



Figure 7 Comparison of cdk5, p35, and p39 gene expression in E17 embryos, especially inthe cerebral cortex. Sagittal sections hybridized with cdk5 (A,D), p35 (B,E), and p39 (C,F).(D–F) Enlargement of rostral brain regions shown in (A–C). Note that cdk5 mRNA wasexpressed in all layers of neocortex, including cortical plate, neocortical intermediate zone, andneocortical ventricular zone (D), whereas p35 mRNA was expressed in cortical plate andneocortical intermediate zone (E) and p39 mRNA only in the cortical plate (F). CrD Å Crusof diaphragm; CP Å cortical plate; H Å heart; Ht Å hypothalamus; InM Å intercostal muscle;Lu Å lung; Lv Å liver; Mg Å midgut; Nc Å neocortex; NIZ Å neocortical intermediate zone;NVZ Å neocortical ventricular zone; OB Å olfactory bulb; OE Å olfactory epithelium; PdÅ pallidum; PeM Å pectoralis muscle; Pit Å pituitary primordium; PrM Å prevertebral muscle;PsM Å psoas muscle; To Å tongue; Th Å thalamus. Scale bars Å 1 mm.

veloping nervous system, cdk5 mRNA was ex- in these regions [Fig. 4(C)] (data not shown).Other brain regions expressing predominantly p35pressed in both the ventricular and marginal zones,

although the latter displayed a higher level of cdk5 mRNA included tectum, hypothalamus, cerebellum,and brain stem [Fig. 4(B,E)] . Both p35 and p39gene expression [Fig. 4(A,D)] . In contrast, mid to

high levels of p35 mRNA expression and low levels mRNAs were expressed in the spinal cord, withventral horn accumulating more transcripts thanof p39 gene expression were confined exclusively

to the marginal zone in the developing CNS [Fig. dorsal horn [Fig. 4(J)] (data not shown). Neitherp35 nor p39 mRNAs were detected in the floorplate4(B,C)] . In general, the signal for p39 mRNA was

very weak in the brain and spinal cord at this stage, per se [Fig. 4(J)] , although at the hindbrain level,neural cells lateral to the floorplate contained p35whereas p35 mRNA could readily be observed. This

difference was particularly evident in the rostral and p39 mRNAs [Fig. 4(E,F)] . The developingperipheral nervous system (PNS), such as the dor-forebrain, where p35 mRNA labeled a rim of post-

mitotic neural cells in the cingulate cortex and pal- sal root ganglia, also expressed p35 and p39 tran-scripts [Fig. 4(C,J)] . Surprisingly, p35 mRNA waslidum [Fig. 4(B); see also enlargement in Fig.

4(H)] , whereas p39 mRNA was barely detectable also detected at low to moderate levels in skeletal



Figure 8 Frontal sections of E17 embryos comparing cdk5 (A,D,G), p35 (B,E,H), and p39(C,F,I) gene expression in the cranial region, especially their differential expressions in theneocortex. Relative planes of sectioning for sections are illustrated on a schematic drawing ofthe embryo. Am Å Amygdala; Apn Å anterior pons; ChP Å choroid plexus; Cb Å cerebellum;CP Å cortical plate; ETh Å epithalamus; Hi Å hippocampus; Ht Å hypothalamus; Md Å me-dulla; NIZ Å neocortical intermediate zone; NR Å neural retina; NVZ Å neocortical ventricularzone; OE Å olfactory epithelium; Pir Å piriform cortex; PPn Å posterior pons; Str Å striatum;Th Å thalamus; Tg Å trigeminal ganglia; Tm Å tegmentum; To Å tongue; Tt Å tectum. Scalebar Å 1 mm.



Figure 9 Transverse sections of E17 embryos comparing cdk5 (A,D,G), p35 (B,E,H), andp39 (C,F,I) gene expression in the cranial region (A–F) and trunk (G–I) . Relative planes ofsectioning for sections are illustrated on a schematic drawing of the embryo. Note the wide-spread expression of p35 mRNA in the musculatures (E,H). Ad Å Adrenal gland; AwMÅ abdominal wall musculature; Cb Å cerebellum; ChP Å choroid plexus; DRG Å dorsal rootganglia; Hi Å hippocampus; K Å kidney; Lv Å liver; Md Å medulla; Mg Å midgut; NcÅ neocortex; NR Å neural retina; Pn Å pons; PsM Å psoas muscle; S Å stomach; SC Å spinalcord; Th Å thalamus. Scale bar Å 1 mm.



muscle progenitors, such as the paraxial myotomes cells) , and preplate (or cortical plate) [Fig.6(A,D)] . In contrast, p35 mRNA was confined to[Fig. 4(J)] .the neocortex and intermediate zone [Fig. 6(B,E)] ,whereas p39 mRNA was nearly undetectable [Fig.E13. Expressions of cdk5, p35, and p39 mRNAs

were compared in adjacent sagittal sections [Fig. 6(C)] . In other regions of the forebrain, p39 mRNAwas nearly undetectable in the hippocampus and5(A–C)] and transverse sections [Fig. 5(D–I)] .

cdk5 mRNA was expressed in a ubiquitous fashion pallidal subventricular zone, which was in sharpcontrast to p35 mRNA [compare Fig. 6(C) andin all embryonic structures, with particularly high

levels in the marginal zones of the developing (B)] . The neural layer of the retina displayed stronglabeling for both p35 and p39 mRNAs (data notnervous system [Fig. 5(A,D,G)]. In comparison,

p35 labeling highlighted the marginal zones of shown). Both p35 and p39 mRNAs were expressedin the PNS, including cranial ganglia, dorsal rootvarious CNS structures representing neural cells,

which have withdrawn from the cell cycle [Fig. ganglia, and enteric ganglionic structures (data notshown). In regions outside of the nervous system,5(B,E,H)] . In the developing forebrain, for in-

stance, cdk5 mRNA was detected in both neocorti- both p35 and p39 mRNAs were found in the olfac-tory epithelium [Fig. 6(B,C)] . Expression of p35cal ventricular zone and preplate, which will give

rise to the cortical plate after E15 in the rat (Bayer mRNA in the skeletal muscle became more promi-nent as more premuscle mass differentiated into in-and Altman, 1990, 1991; Valverde et al., 1989),

whereas p35 mRNA was present only in the preplate dividual muscular structures, such as the paraxialmuscles and muscular shaft in the limb [Fig. 6(E)][compare Fig. 5(D) and (E)] . Similar to p35

mRNA, p39 mRNA was expressed in the marginal (data not shown).zones of the CNS [Fig. 5(C,F,I)] , although thesignal for p39 mRNA was generally lower than that E17. Similar to the previous stages, cdk5 mRNA

was widely distributed in all embryonic tissues,of p35 mRNA [compare Fig. 5(B) and (C)] . Inaddition, p39 mRNA expression in the forebrain with a particularly high level in the developing

nervous system, olfactory epithelium, and adrenalwas barely detectable, which was particularly evi-dent in the transverse sections [compare Fig. 5(E) cortical primordium [Figs. 7 (A,D) , 8 (A,D,G) ,

and 9(A,D,G)]. Compared with the previous stage,and (F)] . As is apparent, p39 mRNA in the telen-cephalon was nearly undetectable, whereas the adja- both p35 mRNA and p39 mRNA were more widely

distributed in the brain, reflecting the fact that mostcent diencephalon and mesencephalon were labelednearly as strongly for p39 mRNA as p35 mRNA. neural cells have withdrawn from the cell cycle

[Fig. 7(B,C)] . Prominent differences in their ex-Both p35 and p39 transcripts continued to be presentin the PNS [Fig. 5(B,C)] . In the trunk, p35 mRNA pression in the cerebral cortex remained. At this

stage, the neocortex is subdivided into several well-continued to be present in the skeletal muscle pro-genitors, such as the cervical myotome premuscle defined layers. These include the neocortical ven-

tricular zone, which is composed of undifferentiatedmass [Fig. 5(H)] . In contrast, no positive signalfor p39 mRNA was present in any structure outside neural cell progenitors and cells undergoing mitosis;

the cortical plate, which consists of differentiatedof the nervous system [Fig. 5(C,F,I)] .neural cells; the deep subplate; and the intermediatezone which lies in between. cdk5 mRNA was ex-E15. Similar to the previous stage (E13), cdk5

mRNA was expressed in all embryonic structures pressed in all four layers of the neocortex [Fig.7(D)] , whereas p35 mRNA was present in the outer[Fig. 6(A)] . Higher cdk5 mRNA expression was

observed in differentiated neural structures and the cortical plate, deep subplate, and intermediate zone[Fig. 7(E)] . In contrast, p39 mRNA expressionolfactory epithelium. Expression of p35 mRNA had

expanded in the nervous system, keeping up with a was missing from the ventricular zone, intermediatezone, and deep subplate, with only a low level ofhigher proportion of mature neural structures com-

pared to the previous stage [Fig. 6(B)] . Expression expression detected in the cortical plate [Fig.7(F)] . Significant differences were also observedof p39 mRNA had also expanded [Fig. 6(C)] . Ma-

jor differences in the expression of cdk5, p35, and in the hippocampus, where the expression level ofp35 mRNA was high, while p39 mRNA was almostp39 mRNAs were observed in the cerebral cortex.

cdk5 mRNA was expressed by all layers of the undetectable [compare Fig. 8(F) and (E) and Fig.9(C) and (B)] . The zona incerta of hypothalamus,cerebrum, including the neocortical neuroepithel-

ium (ventricular zone), neocortex (or differentiat- which is located ventrolateral to the thalamus, alsodisplayed a low level of p39 mRNA, whereas p35ing field) , intermediate zone (migrating neural



mRNA was readily observed [compare Fig. 8(F) reported detection of p35 mRNA in the E12 ratforebrain by Northern blot analysis (Tomizawa etand (E)] . Intense expressions of cdk5, p35, and

p39 mRNAs were observed in the neural layer of the al., 1996), although in the mouse p35 transcriptshave been reported to be present as early as E10retina [Fig. 8(A–C)]. Expression of p39 mRNA in

the pituitary primordium and the olfactory epithe- (Delalle et al., 1997). At E12 in the telencephalon,weak levels of cdk5 and p35 gene expression arelium was also more readily apparent than p35

mRNA [compare Fig. 7(C) and (B)] . All three detected, concurrent with the initiation of neurogen-esis in this structure (Bayer and Altman, 1990;genes remained expressed in the PNS, including the

cranial ganglia [Fig. 8(D,E)] , dorsal root ganglia Bayer et al., 1993). Therefore, the cdk5/p35 com-plex may be present at the earliest time of cortico-[Fig. 9(D–I)] , sympathetic ganglia (data not

shown), and the enteric ganglionic population of genesis. In other regions of the brain, concurrentexpressions of cdk5, p35, and p39 mRNAs are ob-midgut [Figs. 7(A–C) and 9(G–I)] . All three

genes continued to be expressed in the olfactory served. The dramatic increase in their levels of ex-pression from E12 to E17 is in general parallel toepithelium [Figs. 7(A–C) and 8(A–C)]. Similar

to the previous stage, cdk5 mRNA, but not p35 the neurogenetic timetable (for a review, see Bayeret al., 1993). The presence of cdk5, p35, and p39mRNA or p39 mRNA, was expressed in the choroid

plexus [Figs. 8(D) and 9(A)]. Transcripts for p35 mRNAs during this period suggests that the acti-vated cdk5 kinase complex is available early toremained abundantly expressed in the skeletal mus-

culature throughout the embryo [Fig. 9(E,H)] (data phosphorylate various protein substrates throughoutneurogenesis.not shown).

p35 and p39 share weak sequence similarity inthe cyclin box region with other cyclins (Tang etal., 1995), and the activation domain of p35 isDISCUSSIONthought to adopt a conformation similar to that ofcyclin A (Brown et al., 1995; Tang et al., 1997).In this report, we have provided the first comprehen-

sive, comparative analysis of the cellular localiza- Although cyclin D1 is expressed during brain matu-ration and binds to cdk5, it does not stimulate cdk5tion of mRNAs encoding cdk5 and its two activa-

tors, p35 and p39, in the adult and developing rat kinase activity (Xiong et al., 1997). Therefore, p35and p39 are the only proteins that have been shownbrain. Our results clearly show a heterogeneous dis-

tribution of cdk5 and p35 mRNA in the brain, with to associate with, as well as to stimulate, cdk5 ki-nase activity (Lew et al., 1994; Tang et al., 1995;particularly high levels of expression of these two

mRNAs in the cerebral cortex, hippocampus, pons, Tsai et al., 1994). The significance of the coexis-tence of these two structurally and functionally ho-and medullary nuclei. We have extensively com-

pared the expression of p35 and p39 mRNAs in mologous proteins in the CNS is unclear. One possi-bility is that the two kinase activators, p35 and p39,the adult rat CNS using adjacent section analysis.

Although both genes are expressed ubiquitously in convey different substrate specificities for cdk5phosphorylation, although thus far no differencesthe brain and spinal cord, prominent region speci-

ficity in their levels of expression is observed. Both have been identified. Among the known substratesof cdk5/p35 are the middle- and high-molecular-p35 and p39 mRNAs are expressed at high levels

in the piriform cortex, tenia tectum, and hippocam- weight neurofilament proteins (NF-M and NF-H),which are extensively phosphorylated with as manypus. A significantly higher level of p35 gene expres-

sion, however, is observed in several brain regions, as 50 phosphorylation sites on NF-H (Jones andWilliams, 1982; Julien and Mushynski, 1982). Thisincluding cortical layers II and III, geniculate nuclei,

inferior olive, and cerebellar Purkinje cells. In con- post-translational modification is believed to be im-portant in stabilizing axonal caliber and modulatingtrast, the expression of p39 mRNA is more readily

apparent than p35 mRNA in the spinal cord. axonal transport (de Waegh et al., 1992; Nixon andSihag, 1991). In the rat, NF-L and NF-M can beOur results also show that cdk5 expression is

initiated at least as early as E12. This is in agreement detected as early as E12, but they are limited tothe hindbrain, spinal cord, and peripheral gangliawith the reported initial detection of cdk5 transcripts

at E11–E12.5 in the mouse (Ino et al., 1994; Tsai (Fliegner et al., 1994; Kaplan et al., 1990). Phos-phorylation of NF-M is already initiated at this stageet al., 1993). Similar to cdk5, p35 mRNA can be

detected at E12, in a broad area of the developing (Carden et al., 1987). The low, but significant levelof cdk5, p35, and p39 gene expression detected atCNS including forebrain, midbrain, hindbrain, and

spinal cord. This is consistent with the previously this stage may be responsible for the observed NF-



M phosphorylation. At E15–E16, up-regulation of lamination defects similar to that observed in thecdk5 disruption (Chae et al., 1997). Taken togetherNF-L and NF-M gene expression is observed, and

their expression patterns have expanded to several with the observed high level of p35 expression andlack of significant p39 expression in the cortex inrostral structures, including basal ganglia and thala-

mus (Fliegner et al., 1994). The expression of NF- brain development, these observations suggest thatthe cdk5/p35 kinase complex is necessary for theH is first detected at E15 in the ventral spinal cord

and peripheral ganglia, existing in either nonphos- general organization of the cortex during develop-ment. In contrast, the widespread expression of p39phorylated to variable phosphorylated states (Car-

den et al., 1987). This is matched by an increased in neural structures caudal to the telencephalon maycompensate for p35 in the p35 knockout mouse,expression of cdk5, p35, and p39 mRNAs detected

in this study and could account for the phosphoryla- which lacks the multiple brain defects observed inthe cdk5 knockout mouse (Chae et al., 1997). It willtion of NF-H at this stage. A recent study has shown

that p39 expression levels were most prominent in be interesting to know whether double knockouts ofp35 and p39 will result in all the multiple defects1-, 4-week-old rat brain (Cai et al., 1997). The up-

regulation of p39 during this period may account for observed for cdk5 ablation.In adult animals, cdk5 mRNA is detected bythe concurrent high levels of NF-H phosphorylation.

Indeed, p39-activated cdk5 appears to be more ro- Northern blot analysis in all tissues and cell typesexamined, with the highest levels observed in thebust in phosphorylating NF-H than p35-activated

cdk5 (Sun, Leung, and Liem, unpublished observa- brain (Meyerson et al., 1992). Paradoxically, cdk5-associated kinase activity toward histone H1 hastions) .

Another substrate of the cdk5 kinase is tau, a been isolated or immunoprecipitated only frombrain (Lew et al., 1992; Tsai et al., 1993), whichmicrotubule-associated protein (MAP) (Paudel et

al., 1993; Segnupta et al., 1997). MAPs help to is thought to be due to the tissue-specific distribu-tion of the cdk5 activators p35 and p39 (Lew et al.,maintain the balance between stability and plasticity

of the neuronal cytoskeleton by modulating the as- 1994; Tang et al., 1995; Tsai et al., 1994). It ispossible that cdk5 expressed in nonneural tissuessembly of microtubules (Kosik, 1993; Maccioni and

Cambiazo, 1995). Overlap between cdk5, p35, and may be inactive due to the lack of p35 or p39, orthat cdk5 activated by other activators present inp39 gene expression and tau is observed in neuro-

genesis. At E15, for instance, cdk5 and its activators these tissues does not possess kinase activity againsthistone H1. Furthermore, existing cdk5 antibodiesare coexpressed with low-molecular-weight tau iso-

forms in the dorsal root ganglia (Nothias et al., may not be able to immunoprecipitate active cdk5kinase from these tissues.1995). However, it should be noted that throughout

prenatal development, a broad range of brain struc- In contrast to previous reports (Delalle et al.,1997; Tsai et al., 1994), we also detected p35tures express significant levels of cdk5, p35, and

p39 mRNAs, where no significant levels of NFTPs mRNA expression in muscle development. p35mRNA is detectable in the myotome at E12 andor tau gene expression have been observed. It is

likely, therefore, that the cdk5 kinase complex is continues to be expressed in the premuscle massand mature musculatures at later stages, although atinvolved in the phosphorylation of additional sub-

strates throughout the developing CNS. lower levels than in the developing nervous system.The presence of both cdk5 and p35 during muscleWidespread expression of both p35 and p39

mRNAs are observed in the developing nervous sys- development suggests that cdk5 is active and playsa role in myogenesis. In fact, cdk5-associated H1tem, with the exception of the forebrain. In the cor-

tex, p35 mRNA is expressed at high levels in all kinase activity has been detected in cultured mousemyoblasts, and its level increases when myoblastslayers except the ventricular zone, whereas p39

mRNA is barely detectable in the marginal zone are induced to differentiate (Lazaro et al., 1997).Furthermore, a dominant-negative mutant form ofand later in the cortical plate. In cdk5 knockout

mice, prominent defects in cortical lamination are cdk5 disrupts myogenic differentiation (Lazaro etal., 1997). Similar results have been obtained inobserved. Other abnormalities in the CNS are also

observed, such as abnormal stratification in the hip- Xenopus laevis, where expression of a Xenopuscdk5 dominant-negative mutant disrupted somiticpocampus, lack of cerebellar foliation, and certain

pathological features in the brain stem, as well as muscle patterning (Philpott et al., 1997). Indeed,an activator of Xenopus cdk5, named Xp35.1, hasperinatal lethality (Ohshima et al., 1996). In con-

trast, defects observed in p35 gene ablation seem been cloned and exhibits a significant sequence ho-mology to mammalian p35. Similar to rat p35,to be confined to the telencephalon, with cortical



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DELALLE, I., BHIDE, P. G., CAVINESS, V. S. J., and TSAI,with the coexpression of cdk5 and p35 demonstratedL.-H. (1997). Temporal and spatial patterns of expres-in our study, these results suggest that p35, but notsion of p35, a regulatory subunit of cyclin-dependent

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