Proc. Nati. Acad. Sci. USAVol. 91, pp. 1819-1823, March 1994Neurobiology

Multiple tyrosine protein kinases in rat hippocampal neurons:Isolation of Ptk-3, a receptor expressed in proliferativezones of the developing brainMARINA P. SANCHEZ*, PETER TAPLEY*, SHAMSHER S. SAINI*, BIN HE*, DIEGO PULIDOt,AND MARIANO BARBACID*1*Department of Molecular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000; and tCentro de Biologia Molecular,Consejo Superior de Investigaciones Cientificas, Universidad Autonoma de Madrid, Canto Blanco, 28049 Madrid, Spain

Communicated by Leon E. Rosenberg, November 5, 1993 (receivedfor review September 10, 1993)

ABSTRACT Tyrosine protein kinases are likely to play animportant role in the maintenance and/or development of thenervous system. In this study we have used the PCR cloningtechnique to isolate sequences derived from tyrosine kinasegenes expressed in cultured hippocampal neurons obtainedfrom 17.5-day-old rat embryos. Nucleotide sequence analysisof 209 independent clones revealed sequences derived from 25tyrosine kinases, of which two corresponded to previouslyunreported genes. One of the PCR clones, ptk-2, belongs to theJak family of cytoplasmic tyrosine kinases. The second clone,ptk-3, was derived from a gene encoding an additional class oftyrosine kinase receptors whose extracellular domains containregions of homology with coagulation factors V and VM andcomplement component C1. Transcripts encoding the Ptk-3receptor are present in a variety ofembryonic and adult tissueswith highest levels observed in brain. During development,ptk-3 transcripts are most abundant in the proliferative neu-roepithelial cells of the ventricular zone, raising the possibilitythat this receptor may play an important role in the generationof the mammalian nervous system.

The complexity of the mammalian nervous system predictsthe existence of multiple signaling strategies to generateneuronal cells during embryonic development and to main-tain mature neurons during adult homeostasis. However, thesignal-transduction mechanisms that govern the growth andsurvival of neurons remain to be elucidated. The recentdiscovery that the nerve growth factor family of neurotro-phins mediate their trophic activities through the Trk tyrosineprotein kinase receptors (1-3) has raised the possibility thattyrosine phosphorylation may play a key role in the devel-opment and/or maintenance of neurons. The importance ofsignaling through the Trk receptors was recently illustratedby generating mice carrying a targeted trkB allele (4). Micehomozygous for this mutation have reduced numbers ofneurons in various structures of the central and peripheralnervous systems (4). Other growth factors such as acidicfibroblastic growth factor (FGF), basic FGF, insulin-likegrowth factor, epidermal growth factor, and platelet-derivedgrowth factor, known to signal through tyrosine kinasereceptors, also have neurotrophic activity (5, 6). More re-cently, the glial growth factor family of proteins has beenidentified as ligands for members of the epidermal growthfactor family of tyrosine protein kinase receptors (7, 8).These observations further support the concept that tyrosinekinases play a key role in signaling throughout the mamma-lian nervous system.Accumulating evidence indicates that neurons express a

large number of tyrosine kinase receptors (9). Lai and Lemke

(10) first used PCR-based amplification techniques to isolate aseries of tyrosine protein kinases (Tyro-1 to Tyro-13) fromenriched cDNA libraries derived from sciatic nerve andSchwann cells. We have used a similar strategy to identifythose tyrosine protein kinase receptors expressed in definedneuronal subpopulations ofthe central nervous system. In thisstudy, we report that at least 25 different tyrosine kinases,including 15 receptors, are present in rat embryonic hippo-campal neurons. Two of these tyrosine kinase sequences arederived from previously unreported genes, including a fourthmember ofthe Jak family of cytoplasmic tyrosine kinases anda tyrosine kinase receptor with distinctive structural motifs inits extracellular/ligand-binding domain.

MATERIALS AND METHODSPCR Amplification. Total RNA (5 pug) was isolated from

primary cultures of rat embryonic hippocampal neurons (11)and used to synthesize cDNA with oligo(dT) primers and theSuperscript cDNA synthesis system (GIBCO/BRL). Theresulting cDNA was submitted to PCR-aided amplificationwith degenerate primers corresponding to the conservedtyrosine protein kinase sequences HRDLAAR (5'-GQAAL-TCCAYCGNGAYYTNGCNGCNMCG-3') and DVWSFG(5'-GATGCGGCCGCNCCRAARSWCCANACRTC-3').These "amplimers" contain EcoRI and Not I cleavage sites(underlined) in their 5' regions for subcloning purposes.Amplified DNA was fractionated on a 2.2% agarose gel; themajor 220-bp band was then isolated by electroelution, di-gested with EcoRI and Not I, and subcloned into pBluescript.DNAs obtained from randomly selected bacterial transform-ants were submitted to nucleotide sequence analysis by usingthe Sequenase system (United States Biochemical).

Isolation of ptk-3 cDNA Clones. A AgtlO neonatal rat braincDNA library (Clontech) was screened with a 32P-labeled220-bp EcoRI-Not I probe derived from a plasmid (pPT9)containing the ptk-3 PCR-amplified clone. Three recombi-nant phages were isolated, and their respective inserts weresubcloned into pBluescript (pMS1, pMS2 and pMS3) andsubmitted to nucleotide sequence analysis. Because none ofthese cDNA clones contained the complete ptk-3 cDNAcoding sequences, a 510-bp EcoRI/Sac I probe derived fromthe 5' region of pMS3 was used to screen a Agt1O adult ratbrain cDNA library (Clontech). One clone (pMS25) con-tained the missing 5' cDNA coding sequences. A 729-bpEcoRI/Sac I fragment corresponding to the 5' region ofpMS25 was ligated to a 3014-bp Sac I/EcoRI DNA fragmentfrom pMS3 and inserted into pBluescript to generate afull-length ptk-3 cDNA clone pMS35.§

Abbreviations: FGF, fibroblastic growth factor; En, embryonicday n.*To whom reprint requests should be addressed.§The sequence reported in this paper has been deposited in theGenBank data base (accession no. L26525).

1819

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 15

, 202

1

1820 Neurobiology: Sanchez et al.

Immunoprecipitation and Tyrosine Kinase Assays. COS-7cells were transfected with pBH7 DNA, a pREX2N-derivedplasmid (12) that contains the ptk-3 cDNA insert of pMS35,using the DEAE-dextran/chloroquine technique as de-scribed (12). To assay for kinase activity, immunoprecipi-tates were incubated for 10 min at 300C in the presence of 10p.Ci of [y-32P]ATP (6000 Ci/mmol; NEN/DuPont; 1 Ci = 37GBq) and 10mM MnCl2. Samples were analyzed on SDS/8%PAGE gels as described (12).In situ Hybridization. A 290-bp PCR fragment correspond-

ing to nt 1105-1395 ofpMS35 was subcloned into pBluescriptand transcribed in the presence of either [a-35S]UTP (Amer-sham) or [y-33P]UTP (NEN/DuPont) with T3 and T7 poly-merases (Promega) to generate ptk-3 antisense and senseRNA probes, respectively. Frozen coronal sections fromadult mouse brain and sagittal sections from embryonic (E)day 12.5 and E17.5 embryos (10-pm thick) were hybridizedto the corresponding RNA probes as described (13).

RESULTS AND DISCUSSIONAmplification of Tyroumne Kinase Sequences from Embry-

onic Hippocampal Neurons. Primary cultures were preparedfrom E17.5 rat hippocampi as described by Scholz andPalfrey (11). At this time, >90%o of the neurons in thedeveloping hippocampus correspond to pyramidal neurons.Cells were plated onto poly(L-lysine)-coated plates and co-cultivated with feeder glial cell cultures isolated from thecerebral hemispheres of newborn rats. These glial cells wereplaced on inverted coverslips, so as not to directly contact theneuronal cultures. After 48 hr, neuritic processes were evi-dent in most (>90%o) of the cells in the embryonic hippocam-pal cultures. Moreover, <5% of these cells were of glialorigin, as determined by staining with anti-glial fibrillaryacidic protein antibodies.To amplify sequences related to tyrosine protein kinase

genes, we used a PCR cloning strategy based on the use oftwo degenerate oligonucleotide primers corresponding to thehighly conserved sequence motifs (HRDLAAR andDVWSFG) ofthe catalytic domain of tyrosine protein kinasereceptors (10). Of a total of 220 randomly selected plasmidDNAs, 209 contained sequences corresponding to 25 distincttyrosine protein kinase genes. Twenty-three of these geneswere either already known at that time or were subsequentlyreported by other laboratories. These genes included theubiquitously expressed Src regulatory kinase, Csk, the Src-related kinases Abl, Arg, Fps, and Fer, the focal adhesionkinase Fak, and the recently identified Jakl and Jak2 cyto-plasmic kinases. The remaining 15 genes were derived fromtyrosine kinase receptors. Nine of them were well charac-terized receptors for-FGFs (FGFR-1, FGFR-2, and FGFR-3),

Ptk- 2,. a.\._ V ./

Ptk- 3

I

_.

NT LVESE-NLVK'ENNINVLLDNDR

SI LV

NCLVGE iIX'.i LVGK T IK

INCLVGE N|L VK iNCLVGAKL VK IiNC-TVGQG-i.7VVK '

NCLVGE KLI V VKNCMV HD FI T VKI

lNK.S WKIV!N2~LIC-E

{L,-~'JYiC NkE'K]

plateled-derived growth factors (PDGFRa and PDGFR(3),colony-stimulating factor 1 (CSF-1R), insulin (IR), insulin-like growth factor 1 (IGF-1R), brain-derived neurotrophicfactor (TrkB), and neurotrophin 4 (TrkB), all of which,except for the CSF-1R, are expressed in neuronal cells (4, 14,15). The remaining clones corresponded to six orphan recep-tors, including three members of the Eph subfamily oftyrosine kinases (Eck, Sek, and Eek) (16-18), Tyro-7 (10),Tyro-10 (10), and Vik (19). To date, there is no additionalinformation regarding the structure ofthe Tyro-7 and Tyro-10receptors outside their kinase catalytic domain (10). Vik is aputative tyrosine kinase receptor with a short extracellulardomain and unique residues in sequence motifs invariant toall other tyrosine protein kinases (19). Finally, two of the 25PCR-amplified cDNA clones, ptk-2 and ptk-3, were derivedfrom previously unreported tyrosine protein kinase genes(Fig. 1).

Ptk-2, an Addifional Member of the Jak Family of Cyto-plasmic Tyrosine Kinases. As illustrated in Fig. 1, the ptk-2sequence is highly related to the recently identified Jak familyof cytoplasmic tyrosine protein kinases. Known members ofthe Jak kinases include Jakl (20); Jak2 (21), a kinase recentlyshown to be associated with the erythropoietin and growthhormone receptors (22); and Tyk2, a kinase involved in thephosphorylation of the interferon-induced transcription fac-tor ISGF3a (23). These cytoplasmic kinases are character-ized by the presence of two catalytic domains, of which themost carboxyl-terminal region remains to be functionallydefined (20, 21). In addition, they share several regions ofhigh homology, one of which is closely related to the Srchomology 2 domains present in the Src kinase family. Thededuced amino acid sequence of ptk-2 is most closely relatedto Jak-2 (82% homology and 67% identity) and contains theWYAPE sequence motif characteristic of this group of ki-nases (Fig. 1). To establish whether the Ptk-2 kinase has theoverall structural features of this subfamily of cytoplasmictyrosine kinases it will be necessary to isolate full-lengthptk-2 cDNA clones.

Ptk-3 Is a Member of a Distinct Class of Tyrosine ProteinKinase Receptors. The ptk-3 PCR-derived clone exhibits thehighest level of homology to Tyro-10, another PCR-derivedsequence isolated from a sciatic nerve cDNA library (10)(Fig. 1). The ptk-3 kinase region is also highly related to othermammalian tyrosine kinases, such as the three members ofthe Trk family of neurotrophin receptors (24), the insulinreceptor (25), and to a lesser extent, Rorl and Ror2 (26) (Fig.1). ptk-3 also displays significant homology to the TorpedoRTK (27) and the Drosophila Dtrk gene (24).Molecular cloning of a full-length ptk-3 cDNA clone re-

vealed an open reading frame of 910 residues with a series ofstructural motifs distinct from those of previously known

KTAD-G~'G TGKDYYU~VVK EP QSP:FwYA.5:.; 4:I VKIOl;DFGLT. V.QDKEYYKVK EP ESP:FWYAPKS1w 7.VK I 1D " CLAAVpEGC;HEYYRVNR E G S PV W1Y A -|7ECv S-r."VKIGD7'GL ( H KAKEsTvW_7.-A?:-r

ADFGMSRL

CGDFIGMSR DVY7GFGMSRD'L Y

FGM SRC I YFGMDRDIY

SLrG "LI S IY

s EiFT

VGVR'<h P Y Y .S

_ATDYYR ir

"A.-rVYR.

LpTM I.P R - -E - |---x- 1

I '-JI KM~r-y,E'-. P. L-m.--A I RWiaA S ;[c

V 2 Rw DT-E .vxu-i

'-;' P RW-D 9.-Nt0-l 5h1' A-7

FIG. 1. Sequence homology of the deduced amino acid sequence of the PCR-amplified domain of Ptk-2 and Ptk-3 with other tyrosine proteinkinases. Sequences have been aligned using the PILEUP program (Genetics Computer Group). Percentage of homologous and identical aminoacid residues is indicated at right. Conserved residues are boxed and shaded. Torp, Torpedo; RTK, receptor tyrosine kinase; IR, insulin receptor.

Proc. Natl. Acad. Sci. USA 91 (1994)

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 15

, 202

1

Proc. Nad. Acad. Sci. USA 91 (1994) 1821

tyrosine protein kinases (Fig. 2). By analogy with other signalpeptide sequences, residues 1-19 of the Ptk-3 receptor arelikely to correspond to its signal peptide. If so, the maturePtk-3 protein would be 891 amino acids long. The amino-terminal moiety of the putative extracellular/ligand-bindingdomain of Ptk-3 (residues 19-409) contains a region (residues32-185) of significant homology (49-61% homology and24-35% identity) to structural motifs present in solublemolecules known to associate with cellular membrane pro-teins, including the light chains of coagulation factors V andVIII and the milk fat globule membrane proteins MFG.E8and BA46 (28-30) (Fig. 2). These coagulation factor-likemotifs are also present in certain membrane proteins, includ-ing discoidin I, a Dictyostelium discoideum lectin involved incell aggregation (31), and AS, a Xenopus neural recognitionmolecule (32). The coagulation factor-like motif of Ptk-3 ismost related to those of the AS protein (61% homology) andcoagulation factor V (60% homology).The carboxyl-terminal half of the Ptk-3 extracellular do-

main also displays an internal repeat (residues 303-347 and349-388) with limited homology to domains I and III ofcomplement components Cir and Cls (33) (Fig. 2). Interest-ingly, the related Xenopus AS neural recognition protein alsodisplays these complement-like internal repeats (32). Theserepeats are contiguous in Ptk-3 and AS but not in thecomplement molecules in which they are separated by anepidermal growth factor-like domain (33). However, thePtk-3 repeats are considerably shorter (40 and 45 residues)than those present in the C1 and AS (114-135 residues)molecules (Fig. 2). The functional roles of these motifs in theC1 molecules remain to be firmly established. However, ithas been proposed that they might be involved in the inter-action between the dimeric Clr2Cls2 complex and the Clqcomponent (34). Whether these complement-like motifs playa role in the dimerization of the Ptk-3 receptors is aninteresting possibility that remains to be explored.The single transmembrane domain of Ptk-3 (residues 410-

436) is followed by a juxtamembrane region with unusualstructural features. In addition to its length (170 residues),this region contains a high percentage of glycine and prolineresidues, a feature that predicts a high degree of structuralflexibility (Fig. 2). As indicated above, the kinase catalytic

domain of Ptk-3 (residues 607-902) is most closely related toTyro-10 (85% homology and 71% identity), a putative tyro-sine kinase receptor primarily expressed in brain tissue (10).Ptk-3 and Tyro-10 also share an unusual 17-residue insertbetween kinase subdomains I and II. The presence of thisunique structural feature along with their high overall se-quence homology in their respective kinase domains raisesthe possibility that Tyro-10 and Ptk-3 may be members of thesame receptor subfamily.

Ptk-3 has an unusually short carboxyl-terminal tail ofjusteight amino acid residues (residues 903-910) (Fig. 2). Thisfeature also resembles the Trk family of neurotrophin recep-tors (24). However, the tail of the Trk receptors, but not ofPtk-3, contains a tyrosine residue, which is responsible fortheir interaction with certain substrates such as phospholi-pase Cy (35). While we were in the process of writing thismanuscript, Johnson et al. (36) have reported the isolation ofa tyrosine protein kinase receptor from a human breastcarcinoma cell line. This tyrosine protein kinase, designatedDDR, is 94% identical (96% homologous) to Ptk-3 andexhibits the same structural features (Fig. 2) (36). Theseobservations suggest thatDDR may be the human homologueof Ptk-3.

Biochemical Characterization of Ptk-3. COS-7 cells weretransfected with pBH7, a mammalian expression plasmidcontaining the full-length ptk-3 cDNA clone under the controlof a Rous sarcoma virus long terminal repeat. As shown inFig. 3A, pBH7, but not mock-transfected cells, expressed a120-kDa protein specifically immunoprecipitated with twodifferent antisera raised against a synthetic peptide corre-sponding to the predicted carboxyl terminus of Ptk-3 (resi-dues 897-910) and a bacterial glutathione S-transferase-Ptk-3fusion protein encompassing the entire Ptk-3 tyrosine kinasedomain (residues 599-910) (Fig. 2). Immunoprecipitation ofthe 120-kDa Ptk-3 protein by the antipeptide antibodies wascompletely abolished by preincubation with the immunizingpeptide (Fig. 3A). To determine whether this 120-kDa proteinhad tyrosine kinase activity, immunoprecipitates were incu-bated with [y-32P]ATP in the presence of Mn2+ ions. Asshown in Fig. 3B, the 120-kDa Ptk-3 protein became effi-ciently phosphorylated. Phosphoamino acid analysis of theresulting 32P-labeled Ptk-3 protein indicated that the majority

Ptk-3DDR

Ptk-3DDR

Ptk-3DDR

Ptk-3DDR

1 MGTGTLSSLL LLLLLVTIGD ADMKGHFDPA N YALGMQD RTIPDSDISV SSSWSDSTAA RHSRLESSDG DGAPAGPV FPKEEEYLQV DLRRLHLVALPEA - AS A S Q

101 GTQGRHAGG LGKEFSRSYR LRYSRDGRRW MDWKDRWGQE VISGNEDPGG VVLKDLGPPM VARLVRFYPR ADRVMSR VELY WRD GLLSYTAPVG100 G E

201 QTMQLSEMVY LNDSTYDGYT AGGLQYGGLG QLADGVVGLD DFRQSQELRV WPGYDYVGWS NHSFPSGYVE MEFEFDRLRS FQTMQVHODN MHTLGARLPG200 Y A lH V K 1S A A....... ~~~ ~~~ ~~~~~~~~~~~~~~.

.. ... .........301 GVE~.FKRGP AMAWEGEPVHEALGGSLGDP RAPAISVPLG GHVGRFLt: FLFAGPWLLF -SEISFISDVV NDSS. . .:.D......G.TTFSL300 R : N N N Y:A:.: GG

Ptk-3 397 LEPRGQ.QPV AKAEGSPTAI LIGCLVAIIL LLLLIIALML WRLHWRRLLS KAERRVLEEE LTVHLSVBGD TILINNR REBEYQEER iPRTkTHSA2DDR 400 PR <_ N e

Ptk-3 496 CVkNfSALLL SNRAYRLLLA TYAR2jRG £BTLAWAKkT NTQACSGDYM EP-EKP..AFLL PPPQNSVjH YAEADIVTLQ fVTfNTYAV PALEMAVGDDDR 500 Y

Ptk-3 596 gPPRVDFPRS R4KEKLGE GQFGEVHLCE VEDPQDLVTS DFPISVQKGH PLLVAVKILR PDATKNARND FLKEVKIMSR LKDLNIIRLL GVCVQDDPLCDDR 600 I> . DS SL LN R P

Ptk-3 696 MITDXMENGD LNQFLSAIQL ENKVTQGLPG DRESDQGPTI S-PMLLHVGA QIASGMRfLA TLNFVHRDLA TRNCLVGENF TIKIADFGMS RNLYAGDYYRDDR 700 D AAEA GQAA A

Ptk-3 796 VQGRAVLPIR WMAWECILMG KFTTASDVWA FGVTLWEVLM LCRSQPFGQL TDEQVIENAG EFFRDQGRQV -YLSRPPACPQ TLYELMLRCW SREPEQRPPFDDR 800 V A G S

Ptk-3 896 SQLHRFIAD D ALNTVDDR 900 .J E

FIG. 2. Deduced amino acid sequence of the rat Ptk-3 tyrosine kinase receptor. The predicted signal peptide is underlined by a stippled bar.A region homologous to coagulation factors is boxed. Two partial internal repeats showing limited homology to complement proteins Clr andCls are highlighted by stippled boxes. Putative N-glycosylation sites in the extracellular region are underlined by open bars. Extracellularcysteine residues are circled. The single transmembrane domain is underlined by a solid bar. A proline/glycine-rich domain in thejuxtamembraneregion is indicated by open arrows. Each of the proline and glycine residues in this region are underlined for easier identification. The catalytictyrosine kinase domain is flanked by solid arrows. Tyrosine residues in the kinase catalytic domain are shaded. Nonidentical residues of thehighly related human DDR receptor (36) are shown for comparative purposes. Residues absent in Ptk-3 but present in DDR are indicated bydots. A Ptk-3 residue not present in DDR tyrosine protein kinase is indicated by a dash.

Neurobiology: Sdnchez et al.

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 15

, 202

1

1822 Neurobiology: Sanchez et al.

A BMock pBH7 Mock pBH7 MW Mock pBH7

a b b a b b c d IC x103 cd c d

- 200

Ptk-3 -_.0

*E

2."iV

- 97 -

69

- 46 -

COMPETING _ + - +

PEPTIDE:

FIG. 3. (A) Expression of Ptk-3 in COS-7 cells. COS-7 cells weretransfected either with no DNA (Mock) or with pBH7 DNA, apMEX-neo-derived expression plasmid containing the entire cDNAcoding sequences of ptk-3. Transfected cells were labeled withTran35S-label, lysed, and immunoprecipitated with either preimmune(lanes a and c) or immune (lanes b and d) sera elicited against a

peptide corresponding to the carboxyl-terminal sequence ofthe Ptk-3protein in the absence (-) or presence (+) of 10 pLg of competingpeptide (lanes b) and against a bacterial glutathione S-transferasePtk-3 fusion protein (lane d). (B) Immunoprecipitates were alsoassayed for in vitro tyrosine kinase activity as described. Sampleswere loaded onto SDS/8% PAGE gels. Electrophoresed gels wereeither fluorographed (A) or submitted to alkali treatment, dried, andexposed to Kodak X-Omat film (B) for either 30 (A) or 48 (B) hr.Migration of the 120-kDa Ptk-3 protein is indicated by arrows.Molecular-weight markers include myosin (200,000), phosphorylaseb (97,000), bovine serum albumin (69,000), and ovalbumin (46,000).

(>90%) of the radioactivity was incorporated as phosphoty-rosine (data not shown).

of ptk-3 In Adult and Embryonic Mouse Tissues.

The ptk-3-encoding gene directs the synthesis of a singletranscript of 4.3 kb in all the tissues analyzed (data notshown). ptk-3 transcripts are also present during mousedevelopment, particularly after E10.5. To better define itspattern of expression, we performed in situ hybridizationanalysis of tissue sections derived from mouse embryos atvarious stages of development, as well as from adult mousebrain. In E12.5 embryos, ptk-3 is preferentially expressed inthe developing nervous system, including the forebrain,midbrain, hindbrain, and the spinal cord (Fig. 4A). ptk-3expression outside the central nervous system appears lim-ited to the olfactory epithelium lining the nasal cavity and theyolk sac (Fig. 4A). With the onset of organogenesis, ptk-3

FIG. 4. In situ hybridization analysis of ptk-3 expression duringmouse embryonic development. Sagittal sections of 12.5-day-old (A)and 17.5-day-old (B) mouse embryos. cb, Cerebellum; d, dienceph-alon; f, forebrain; h, hindbrain; he, heart; in, intestine; k, kidney; li,liver; Mc, Meckel's cartilage; m, midbrain; me, mesencephalon; oe,olfactory epithelium; sc, spinal cord; t, telencephalon; th, thymus;to, tongue; v, vibrissae; ys, yolk sac; 4V, fourth ventricle. (A, x4.5;B, x2.2).

expression expands to most developing organs of the em-bryo. However, at E15.5, the strongest ptk-3 hybridizationsignal was still detected in the ventricular proliferative zonesof the brain (data not shown).

In E17.5 embryos, ptk-3 is widely expressed in nonneuraltissues. High levels of ptk-3 expression can be observed inolfactory and nasal structures, vibrissae, ossifying Meckel'scartilage, tooth primordia, tongue, thyroid cartilage, thymus,bone, heart, intestine, pancreas, and kidney (Fig. 4B). Skin,adipose, and liver tissue also display significant levels ofptk-3 transcripts (Fig. 4B). In the nervous system, ptk-3expression is highly localized to well-defined structures. Inthe telencephalon, there are abundant ptk-3 transcripts in thecaudate putamen, as well as in the ventricular zone of thecerebral cortex, where those mitoses giving rise to theyounger cortical neurons are still in progress. These obser-vations indicate that ptk-3 is preferentially expressed inproliferative zones of the developing cortex, whereas there islittle or no ptk-3 expression in regions of neuronal migrationand differentiation. In the diencephalon, low levels of ptk-3transcripts can be detected in the dorsal thalamus. However,none of the other diencephalic structures displays detectablelevels of ptk-3 expression. In the midbrain, only the inferiorcolliculus shows a high density of ptk-3 hybridization. In thehindbrain, ptk-3 transcripts can be found in the cerebellum aswell as in some dorsal structures of the medulla. The choroidplexus of the lateral and fourth ventricles, a region ofcontinuous cellular turnover, also shows a high density ofptk-3 transcripts. Finally, moderate levels of ptk-3 expres-sion could be observed in the pituitary gland and in the spinalcord (Fig. 4B), as well as in the peripheral nervous system,where we observed homogeneous distribution of ptk-3 hy-bridization signals in all the ganglia analyzed (data notshown).

In the adult brain, the highest density of ptk-3 transcriptsare present in the white matter (Fig. 5 A and C), suggestingthat the Ptk-3 receptor is expressed in glial cells. Significantlevels of ptk-3 mRNA can also be observed in the pyramidallayer of the hippocampus (Fig. 5 A and C), as well as in thechoroid plexus and ependymal cell layer surrounding thecerebral ventricles (Fig. 5D). Interestingly, the expression ofptk-3 in the choroid plexus and the ependymal layers isreminiscent of the pattern of expression of those trkB tran-scripts encoding the noncatalytic receptor isoform gp95rB(37). However, upon close examination, ptk-3 expressionappears to be uniformly distributed throughout the entireventricle, whereas trkB transcripts have been found only inthe ventral region of the ependymal layer of the third ven-tricle (37). Although the functional significance of theseobservations remains to be established, they reveal thepresence of at least two classes of neuroepithelial cells in theventricular lining. Finally, the specificity of these in situhybridization studies is illustrated by the low levels ofhybridization detected when adjacent sections were hybrid-ized with the corresponding ptk-3 sense probe (Fig. 5B).The present studies illustrate that during development, a

specific subpopulation of neurons can express multiple ty-rosine protein kinases including 15 different receptors. Themajority of these kinases, including the two isolates Ptk2 andPtk-3, are also expressed in nonneuronal cells. Yet, thesekinases may play distinct roles in neuronal cells through theuse of specific signaling pathways not present in proliferatingcells. This scenario was recently illustrated with the Trkfamily of neurotrophin receptors, which can induce theneuronal differentiation of PC-12 and neuroblastoma cells, aswell as the malignant transformation of mouse fibroblasts. Asimilar situation may occur with the Ptk-3 receptor, whichhas been recently found to be overexpressed in human breastcarcinoma cells (36). The high levels of expression of ptk-3transcripts in the proliferating structures of the developing

Proc. Natl. Acad Sci. USA 91 (1994)

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 15

, 202

1

Proc. Natl. Acad. Sci. USA 91 (1994) 1823

FIG. 5. In situ hybridization analysis of ptk-3 expression in adult mouse brain. Frozen coronal sections (10 pim) were hybridized with eitherantisense (A, C, and D) or sense (B) ptk-3 probes. (C) Expanded areas indicated by an open square in upper left corner of A. (D) Expandedarea indicated by an open square in lower right part of A. CC, cerebral cortex; ChP, choroid plexus; EpL, ependymal cell layer; Hip,hippocampus; LV, lateral ventricle; Or, stratum oriens of the hippocampus; Py, pyramidal cell layer of the hippocampus; Tha, thalamus; WM,white matter; 3V, third ventricle; VI, cortical layer VI. (A, x5.5; B, x5.5; C, X85; D, x45).

nervous system compared with those in the adult brain raisesthe possibility that Ptk-3 may play a role during the prolif-erative stages of the developing brain. Identification of itscognate ligand should provide important information regard-ing the role of the Ptk-3 receptor in the ontogeny of themammalian nervous system.

We thank F. Lamballe for the neuronal cultures; N. Barclay andS. Davis for their assistance in the analysis ofthe Ptk-3 sequence; andX. R. Bustelo, D. Carrasco, R. Klein, S. Jing, and R. Smeyne forhelpful discussions. We are also grateful to S. Bryant and A. Lewinfor their excellent technical assistance. M.P.S. was partially sup-ported by a Fellowship from the Spanish Ministerio de Educacion yCiencia. D.P. was supported by grants from Direccion General deInvestigacion Cientifica y Tecnica and from Fundacion RamonAreces.

1. Kaplan, D. R., Martin-Zanca, D. & Parada, L. F. (1991) Nature(London) 350, 158-160.

2. Klein, R., Jing, S., Nanduri, V., O'Rourke, E. & Barbacid, M.(1991) Cell 65, 189-197.

3. Barbacid, M. (1993) Oncogene 8, 2033-2042.4. Klein, F. R., Smeyne, R. J., Wurst, W., Long, L. K., Auerbach,

B. A., Joyner, A. L. & Barbacid, M. (1993) Cell 75, 113-122.5. Barde, Y.-A. (1989) Neuron 2, 1525-1534.6. Walicke, P. A. (1989) Annu. Rev. Neurosci. 12, 103-126.7. Falls, D. L., Rosen, K. M., Corfas, G., Lane, W. S. & Fischbach,

G. D. (1993) Cell 72, 801-815.8. Marchionni, M. A., Goodearl, A. D. J., Su Chen, M., Bermingham-

McDonogh, O., Kirk, C., Hendricks, M., Danehy, F., Misumi, D.,Sudhalter, J., Kobayashi, K., Wroblewski, D., Lynch, C., Baldas-sare, M., Hiles, I., Davis, J. B., Hsuan, J. J., Totty, N. F., Otsu,M., McBurney, R. N., Waterfield, M. D., Stroobant, P. & Gwynne,D. (1993) Nature (London) 362, 312-318.

9. Maness, P. F. & Cox, M. E. (1992) Semin. Cell Biol. 3, 117-126.10 Lai, C. & Lemke, G. (1991) Neuron 6, 691-704.11. Scholz, W. K. & Palfrey, H. C. (1991) J. Neurosci. 11, 2422-2432.12. Jing, S., Tapley, P. & Barbacid, M. (1992) Neuron 9, 1067-1069.13. Bustelo, X. R., Rubin, S. D., Suen, K.-L., Carrasco, D. & Barb-

acid, M. (1993) Cell Growth Differ. 4, 297-308.14. Fantl, W. J., Johnson, D. E. & Williams, L. T. (1993) Annu. Rev.

Biochem. 62, 453-481.15. Schlessinger, J. & Ullrich, A. (1992) Neuron 9, 383-391.

16. Lindberg, R. A. & Hunter, T. (1990) Mol. Cell. Biol. 10, 6316-6324.17. Chan, J. & Watt, V. M. (1991) Oncogene 6, 1057-1061.18. Gilardi-Hebenstreit, P., Nieto, M. A., Frain, M., Mattei,

M.-G., Chestier, A., Wilkinson, D. G. & Charnay, P. (1992) On-cogene 7, 2499-2506.

19. Kelman, Z., Simon-Chazottes, D., Guenet, J. L. & Yarden, Y.(1993) Oncogene 8, 37-44.

20. Wilks, A. F., Harpur, A. G., Kurban, R. R., Ralph, S. J., Zurcher,G. & Ziemiecki, A. (1991) Mol. Cell. Biol. 11, 2057-2065.

21. Harpur, A. G., Andres, A.-C., Ziemiecki, A., Aston, R. R. &Wilks, A. F. (1992) Oncogene 7, 1347-1353.

22. Witthuhn, B. A., Quelle, F. W., Silvennoinen, O., Yi, T., Tang, B.,Miura, 0. & Ihle, J. N. (1993) Cell 74, 227-236.

23. Velazquez, L., Fellous, M., Stark, G. R. & Pellegrini, P. (1992) Cell70, 313-322.

24. Barbacid, M., Lamballe, F., Pulido, D. & Klein, R. (1991) Biochim.Biophys. Acta Rev. Cancer 1072, 115-127.

25. Ullrich, A., Bell, J. R., Chen, E. Y., Herrera, R., Petruzzelli,L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y.-C., Tsu-bokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C., Rosen,0. M. & Ramachandran, J. (1985) Nature (London) 313, 756-761.

26. Masiakowski, P. & Carroll, R. D. (1992) J. Biol. Chem. 267,26181-26190.

27. Jennings, C. G. B., Dyer, S. M. & Burden, S. J. (1993) Proc. Nati.Acad. Sci. USA 90, 2895-2899.

28. Kane, W. H. & Davie, E. W. (1986) Proc. Natl. Acad. Sci. USA 83,6800-6804.

29. Stubbs, J. D., Lekutis, C., Singer, K. L., Bui, A., Yuzuki, D.,Srinivasan, U. & Parry, G. (1990) Proc. Nat!. Acad. Sci. USA 87,8417-8421.

30. Larocca, D., Peterson, J. A., Urrea, R., Kuniyoshi, J., Bistrain,A. M. & Ceriani, R. L. (1991) Cancer Res. 51, 4994-4998.

31. Poole, S., Firtel, R. A. & Lamer, E. (1981) J. Mol. Biol. 153,273-289.

32. Takagi, S., Hirata, T., Agata, K., Mochii, M., Eguchi, G. &Fujisawa, H. (1991) Neuron 7, 295-307.

33. Tosi, M., Duponchel, C., Meo, T. & Julier, C. (1987) Biochemistry26, 8516-8524.

34. Arlaud, G. J., Colomb, M. G. & Cagnon, G. (1987) Immunol. Today8, 106-111.

35. Obermeier, A., Halfter, H., Weismuller, K. H., Jung, G., Schles-singer, J. & Ullrich, A. (1993) EMBO J. 12, 933-941.

36. Johnson, J. D., Edman, J. C. & Rutter, W. J. (1993) Proc. Nat!.Acad. Sci. USA 90, 5677-5681.

37. Klein, R., Conway, D., Parada, L. F. & Barbacid, M. (1990) Cell61,647-656.

Neurobiology: Sdnchez et al.

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 15

, 202

1