differential expression of trkc catalytic and noncatalytic isoforms suggests that they act...

Differential Expression of TrkC Catalyticand Noncatalytic Isoforms Suggests ThatThey Act Independently or in Association

BENEDICTE MENN,1 SERGE TIMSIT,2 GEORGES CALOTHY,1

AND FABIENNE LAMBALLE1*1CNRS UMR 146, Institut Curie, Centre Universitaire, 91405 Orsay Cedex, France

2INSERM U29, Hopital Port-Royal, 75674 Paris Cedex 14, France

ABSTRACTMembers of the trk gene family encode neurotrophin receptors. The trkC locus encodes

multiple neurotrophin-3 catalytic and noncatalytic receptor isoforms. We report the molecularcloning and characterization of mouse cDNAs encoding two noncatalytic TrkC receptors: novelisoforms designated as TrkC NC1 and TrkC NC2, the mouse homologue of the TrkC truncatedform previously identified in rat (Tsoulfas et al. [1993] Neuron 10:975–990; Valenzuela et al.[1993] Neuron 10:963–974). We extensively analyzed the transcription pattern of these twononcatalytic isoforms and that of the catalytic isoforms by Northern blotting and in situhybridization. We did not detect trkC NC1 transcripts in embryos, but we found that trkC NC1expression is restricted to specific areas in adult brain. In contrast, trkC NC2 transcripts arereadily detected early during embryogenesis and are expressed predominantly in adult brainand gonads. We also provide the first evidence for the existence of TrkC NC2 protein by usingpolyclonal antibodies that specifically recognize this isoform. By using in situ hybridization,we show for the first time that trkC NC2 transcripts are found in differentiating fields ofmaturing neurons and in mature neurons of laminar structures of adult brain. We also reporta similarity of localization between trkC NC2 transcripts and markers of oligodendrocyteprogenitors in the embryonic spinal cord. Furthermore, our results also show that trkC NC2and trkC catalytic transcripts could be either codistributed (in the central and peripheralnervous system) or independently expressed, especially outside the nervous system. Theseresults suggest that the TrkC NC2 isoform acts either independently or in association with itscatalytic counterpart. Finally, we show that TrkC NC2 is expressed in dendrites of pyramidalneurons of hippocampus and cerebral cortex. We propose that this receptor is involved inproliferation of oligodendrocyte progenitors, neuronal differentiation, and synaptic plasticity andthat it may also play a fundamental role in mediating neurotrophin-3 effects outside the nervoussystem. J. Comp. Neurol. 401:47–64, 1998. r 1998 Wiley-Liss, Inc.

Indexing terms: trk; growth factors; neurotrophin-3; neural development; tyrosine kinase

Neurotrophins play an essential role in regulating prolif-eration and differentiation of neuronal precursors duringembryonic development and in mediating survival of neu-rons in the adult nervous system (reviewed by Davies,1994; Snider, 1994). The neurotrophin family is composedof five members—nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3(NT-3), NT-4/5, and NT-6—and binds to two classes ofreceptors—p75NTR, the low affinity neurotrophin receptor,and receptor tyrosine kinases of the Trk family.

The TrkA, TrkB, and TrkC receptors are preferentiallyexpressed in the mammalian nervous system (Klein et al.,1989, 1990b; Martin-Zanca et al., 1990; Tessarolo et al.,1993; Lamballe et al., 1994). However, transcripts have

also been detected in nonneuronal tissues (Klein et al.,1989; Erhard et al., 1993; Tessarolo et al., 1993; Lamballeet al., 1994).

The complexity of the Trk family increases as additionalprotein isoforms, generated by alternative splicing, are

Grant sponsor: French Ministere de l’Education Nationale, del’Enseignement Superieur et de la Recherche; Grant sponsor: CentreNational de la Recherche Scientifique; Grant sponsor: Institut Curie; Grantsponsor: International Human Frontier Science Program Organization.

*Correspondence to: Dr. Fabienne Lamballe, CNRS UMR 146, InstitutCurie, Centre Universitaire, Laboratoire 110, 91405 Orsay Cedex, France.E-mail: [email protected]

Received 12 December 1997; Revised 2 July 1998; Accepted 7 July 1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 401:47–64 (1998)

r 1998 WILEY-LISS, INC.

identified. Splicing can occur in the extracellular domain,thus conferring different binding specificities to the cog-nate ligands of the different isoforms (Clary and Rei-chardt, 1994; Strohmaier et al., 1996). Alternative splicingalso occurs in the cytoplasmic domain. Thus, the trkB locusencodes at least three isoforms that differ in their intracel-lular regions: one catalytic isoform that exhibits tyrosinekinase activity (Klein et al., 1989) and two noncatalyticisoforms that lack the tyrosine kinase domain (Klein et al.,1990a; Middlemas et al., 1991). Expression of trkC is evenmore complex because this locus encodes at least eightisoforms. To date, four isoforms containing a tyrosinekinase domain, differing by the presence or absence of aninsert, have been described (Lamballe et al., 1993; Tsoul-fas et al., 1993; Valenzuela et al., 1993). These differentcatalytic isoforms exhibit different biological properties,suggesting that they are involved in different signalingpathways (Lamballe et al., 1993; Tsoulfas et al., 1996).

The trkC locus also encodes isoforms lacking the tyro-sine kinase domain. These isoforms have extracellular andtransmembrane domains identical to those of the catalyticisoforms. However, they contain unique and distinct cyto-plasmic sequences of different sizes, located downstreamof the juxtamembrane region shared by all TrkC proteins.To date, four cDNAs encoding different noncatalytic iso-forms have been identified in several species, namelyhuman, pig, mouse, rat, and chicken (Lamballe et al.,unpublished results; Tsoulfas et al., 1993; Valenzuela etal., 1993; Garner and Large, 1994; Shelton et al., 1995).However, neither the visualization of noncatalytic trkCtranscripts by Northern blot analysis nor that of theencoded TrkC proteins has been reported.

Although the existence of fibroblast growth factor (FGF)receptor isoforms truncated in their tyrosine kinase do-main has been reported (Shi et al., 1993), noncatalyticisoforms with unique cytoplasmic sequences appear to be aremarkable feature of the TrkB and TrkC receptors.Different hypotheses have been raised concerning thepotential roles of these noncatalytic receptors: (1) theymay establish gradients of neurotrophins and either pre-sent them to the catalytic receptors, thus leading to signaltransduction events, or mediate neurotrophin clearanceafter internalization; (2) they may also have a dominantnegative effect on signaling through the catalytic recep-tors; and (3) they may interact with cytoplasmic proteinsnot yet identified and be involved in specific signal trans-duction pathways (Lamballe, 1995).

The present work was undertaken as part of a functionalstudy of the noncatalytic TrkC receptors. We describe themolecular cloning and characterization of cDNAs encodingtwo mouse TrkC noncatalytic isoforms: one, designatedTrkC NC1, constitutes a novel isoform and the second,designated TrkC NC2, is the mouse homologue of the TrkCtruncated form previously isolated in rat (Tsoulfas et al.,1993; Valenzuela et al., 1993).1 We extensively analyzedthe transcription pattern of these two noncatalytic iso-forms during mouse embryogenesis and in adult tissues byNorthern blotting and in situ hybridization. Our resultsindicate that trkC NC1 expression appears to be restrictedto specific areas of the adult brain. The trkC NC2 tran-scripts are detected early during embryogenesis and are

found predominantly in the brain and the gonads. Byusing polyclonal antibodies directed against specific se-quences encoded by these two noncatalytic isoforms, weprovide the first evidence for the existence of TrkC NC2protein. Our observations demonstrate that the TrkC NC2isoform is expressed in mature neurons of laminar struc-tures in adult brain preferentially in apical dendrites andin differentiating fields of maturing neurons. Interestingly,we also observed a similarity of localization between trkCNC2 transcripts and markers of oligodendrocyte progeni-tors in the embryonic spinal cord. Our results also showthat trkC NC2 and trkC catalytic transcripts may be eithercodistributed in the central (CNS) and peripheral (PNS)nervous systems or differentially expressed in certainregions of the CNS–PNS and outside the nervous system.

MATERIALS AND METHODS

Library screening and DNA sequencing

A lZAP oligo-(dT) cDNA library (1 3 106 phages) pre-pared from adult mouse brain (Stratagene, La Jolla, CA)was plated on a lawn of Escherichia coli LE392. Phageswere absorbed onto nitrocellulose filters and lysed, andtheir DNAs were hybridized overnight at 60°C understringent conditions (15% formamide, 0.5 M NaPO4, pH7.2, 7% sodium dodecyl sulfate [SDS], 0.1% EDTA, 1%bovine serum albumin [BSA]) with a 32P-labeled probe,referred to as probe EXT-TM (nucleotides 486–1530; Fig. 1).This probe corresponds to sequences encoding part of theextracellular domain and the transmembrane region ofpFL16, a mouse trkC cDNA clone (Lamballe et al., 1991).Filters were washed twice in 150 mM NaPO4, 0.1% SDSand once in 30 mM NaPO4, 0.1% SDS for 30 minutes at60°C. Positive phages were then hybridized with a cDNAprobe, designated as probe KIN, that encodes part of thetyrosine kinase domain of a catalytic isoform of mouseTrkC (mTrkC K), corresponding to nucleotides 1756–2142of pFL19 (Lamballe et al., 1991). Phages showing hybrid-ization signals to this catalytic probe were discarded.Those hybridizing with probe EXT-TM but not with thecatalytic probe were picked and plaque purified. Insertswere sequenced by the dideoxy chain termination method(Sanger et al., 1977), with synthetic oligonucleotides and amodified T7 DNA polymerase (T7 Sequencing Kit, Pharma-cia, Uppsala, Sweden).

Animals

Adult animals were killed by cervical dislocation accord-ing to procedures that conformed to NIH guidelines.Fetuses were removed from dams and immediately rinsed

1The trkC NC1 and trkC NC2 nucleotide sequence data reported in thepresent paper will appear in the Genbank under the accession numbersAF035399 and AF035400, respectively.

Fig. 1. Sequence analysis of mouse trkC NC1. A: Nucleotide anddeduced amino acid sequence of the pFL71 cDNA clone. The boxedamino acid sequence encodes the putative signal peptide. The consen-sus N-glycosylation sites are underlined by open bars. The cysteineresidues in the extracellular domain are circled. The transmembraneregion is underlined with a solid bar. The point of sequence divergence(nucleotide 1549) with cDNA clones encoding catalytic TrkC receptorsis indicated by an arrow. The inframe terminator codon TAA isindicated by asterisks. The polyadenylation motif in the 38 noncodingregion is underlined. B: Comparison of the intracellular amino acidsequences of mouse TrkC NC1 and mouse TrkB TK2 (Klein et al.,1990a). Identical amino acids are boxed. Arrow indicates the point ofdivergence of sequences with thoses of the respective catalytic recep-tors, TrkC K1 (Lamballe et al., 1991) and TrkB TK1 (Klein et al.,1989). Asterisks represent stop codons.

48 B. MENN ET AL.

Figure 1

in phosphate buffered saline (PBS) and immersed in 4%paraformaldehyde (PFA).

Northern blot analysis

Total cellular RNA was extracted from adult tissues ofICF mice according to the method described by Chomczyn-ski and Sacchi (1987). The poly(A)-containing fraction wasisolated by affinity chromatography on oligo-(dT) cellulosecolumns (Type 7, Pharmacia). Five micrograms of poly(A)-containing RNA were electrophoresed on 1% agarose-formaldehyde gels and transferred to nitrocellulose filters.Probes were generated by amplification of pFL71 (mtrkCNC1 subcloned into pBluescript) or pBM3 (mtrkC NC2subcloned into pBluescript) cDNA sequences by using thepolymerase chain reaction (PCR) method. Probe A is acDNA fragment that covers nucleotides 483–801 and corre-sponds to part of the extracellular domain (Figs. 1, 3).Probe NC1 (nucleotides 1574–1674; Figs. 1, 3) encom-passes specific sequences of mTrkC NC1. Probe NC2(nucleotides 1736–1977; Figs. 2, 3) is the entire cDNAfragment encoding specific sequences of mTrkC NC2.Amplified DNAs were purified by gel electrophoresis be-fore labeling. Probe KIN was used as described above.Hybridizations were performed under stringent conditions(18 hours at 42°C in 50% formamide, 53 saline sodiumcitrate buffer [13 SSC: 150 mM NaCl, 15 mM Na3 citrate,

pH 7.0], 1 3 Denhardt’s solution, 20 mM NaPO4, pH 7.0, 20µg/ml calf thymus DNA, 0.3% SDS, 10% dextran sulfate).Hybridized filters were washed 3 3 20 minutes at 50°C in0.13 SSC, 0.1% SDS for all probes except for probe KIN. Inthe case of KIN, filters were washed 3 3 15 minutes atroom temperature in 23 SSC, 0.1% SDS and 30 minutes at60°C in 0.13 SSC, 0.1% SDS. Filters were then exposed for15 days to Kodak X-OMAT film at 280°C with intensifyingscreens.

Fig. 3. Northern blot analysis of trkC transcripts in adult mousetissues. A: Schematic representation of the different trkC cDNAs usedto generate the various probes. Thick bars represent coding sequences.Thin bars represent 58 and 38 noncoding sequences. The putativesignal peptide (SP), extracellular (EXT), and transmembrane (TM)regions are indicated. The tyrosine kinase (TK) and the noncatalytic(NC1, NC2) domains are also shown. Position and length of thevarious probes used in this analysis are indicated. B: Northern blotanalysis. Five micrograms of poly(A)1 RNA isolated from the indicatedtissues were hybridized with probes specific for either the extracellu-lar domain (probe A), the kinase domain (KIN) of mTrkC K, orsequences encoding the specific intracellular region of mTrkC NC1 ormTrkC NC2. Autoradiograms shown were exposed for 15 days at270°C with the help of intensifying screens. The autoradiogramcorresponding to the hybridization with the NC1-specific probe is notshown because no hybridization signal was detected. Sizes of the trkCtranscripts are indicated.

Fig. 2. Sequence analysis of mouse trkC NC2. A: Schematicrepresentation of pBM3, a cDNA clone that encodes mTrkC NC2. Thethick bar represents coding sequences flanked by the initiating (ATG)and terminating (TAA) codons. The thin bars represent 58 and 38noncoding sequences. The predicted signal peptide (SP), extracellular(EXT), transmembrane (TM), and intracellular noncatalytic (NC2)domains are indicated. Points of sequence divergence with the mtrkCNC1 (Fig. 1) and catalytic (K) trkC cDNA clones are indicated by an

arrow and an arrowhead, respectively. B: Nucleotide and deducedamino acid sequence of the intracellular domain of mTrkC NC2. Thesite of the splice giving rise to the noncatalytic form of mTrkC NC1(Fig. 1) is indicated by an arrow. Arrowhead indicates the point ofsequence divergence with cDNA clones encoding catalytic TrkC.Amino acid residues that differ from the sequence of human TrkC NC2(called the truncated, or non-TK form, of TrkC; Shelton et al., 1995)are indicated in bold. Asterisks represent the stop codon.

50 B. MENN ET AL.

Figure 3

DEVELOPMENTAL EXPRESSION OF CATALYTIC AND NONCATALYTIC TRKC ISOFORMS 51

In situ hybridization

The trkC NC1 riboprobes were generated from pFL61, apBluescript-derived plasmid that contains a 100-bp cDNAinsert that encodes specific sequences of mTrkC NC1(nucleotides 1574–1674; Fig. 1). To generate the antisenseriboprobe, pFL61 was linearized with HindIII and tran-scribed from the T7 promoter. The sense riboprobe used asa negative control was obtained by linearizing pFL61 withNotI, followed by transcription from the T3 promoter. A241-bp DNA fragment coding for specific sequences ofmTrkC NC2 (nucleotides 1736–1977; Fig. 2) was subclonedinto pBluescript to generate pFL62. To synthesize a single-stranded antisense cRNA probe, pFL62 was linearized bydigestion with NotI and in vitro transcribed with T3 RNApolymerase. The sense riboprobe was obtained by diges-tion of pFL62 with HindIII and transcription through theT7 promoter. The trkC catalytic riboprobes were generatedfrom pFL90, a plasmid containing a cDNA fragment(nucleotides 2074–2258; Lamballe et al., 1991) that en-codes part of the tyrosine kinase domain. The antisenseriboprobe was generated by linearizing pFL90 with EcoRI,followed by transcription from the T7 promoter. The senseriboprobe was obtained by linearizing pFL90 with XbaI,followed by transcription from the T3 promoter. Probeswere synthesized as described by Lamballe et al. (1991) byusing [35S]UTP (.1,000 Ci/mmol;Amersham, Buckingham-shire, UK).

Sections were obtained from either 4% PFA-fixed paraf-fin-embedded ICF mouse embryos or flash-frozen adultmouse brain. Paraffin-embedded sections (8 µm thick)were deparaffinized in xylene, rehydrated in graded (100–30%) ethanol solutions, and fixed in 4% PFA. Frozensections (15 µm thick) were directly fixed in 4% PFA.Paraffin-embedded tissues were treated with proteinase K(20 µg/ml). All sections were immersed in triethanolamine/acetic anhydride solution and dehydrated. Sections werethen hybridized with 5 3 107 cpm/ml of the respectiveriboprobes (see above). Hybridization was performed un-der stringent conditions (50% formamide, 0.3 M NaCl, 20mM Tris-HCl, pH 7.4, 5 mM EDTA, 10 mM Na2HPO4, 10%dextran sulfate, 1 3 Denhardt’s solution, 0.5 mg/ml yeasttRNA) for 17 hours at 55°C for the NC1 riboprobes and at50°C for the NC2 riboprobes. Hybridized sections werewashed in 53 SSC, 10 mM dithiotreitol (DTT) at 42°C for30 minutes and at 60°C for 20 minutes in a solutioncontaining 50% formamide, 23 SSC, and 10 mM DTT. Theslides were then incubated for 30 minutes at 37°C in 2 mMTris-HCl, pH 7.4, 0.4 M NaCl, 1 mM EDTA (20 µg/ml), andRNAse A. Sections were finally washed in the samesolution but without RNAse for 15 minutes at 37°C, in 23SSC for 15 minutes at 37°C, and in 0.13 SSC for 30minutes at 60°C for the NC1 riboprobes and at 50°C for theNC2 riboprobes. After dehydration, the slides were dippedinto NTB-2 emulsion (Kodak) and exposed for 10 days at4°C. They were then developed in Kodak D-19, fixed,dehydrated, and coverslipped. Darkfield photographs wereprinted on high-contrast paper. Sections used for histologywere stained with cresyl violet.

Preparation of antisera

The rabbit polyclonal anti-pan-TrkC antiserum wasraised against a synthetic peptide RESKIIHMDYYQEGEC,corresponding to amino acids 343–357 of mTrkC (Fig. 1).This high performance liquid chromatography (HPLC)–

purified peptide was conjugated to thyroglobulin (Sigma,St. Louis, MO) through the carboxy-terminal cysteineresidue by using maleimide benzoyl-N-hydroxysylfosuccin-imide ester (Pierce, Rockford, IL) as a coupling agent.

The cDNA fragment encoding specific sequences ofmTrkC NC2 (nucleotides 1726–1877; Fig. 2) was amplifiedby PCR with primers A26 (58-CCGGATCCGTGGGTCTT-TTC-38) and A27 (58-CTGAAAGCTTATGACCTTGG-38),which contain a BamHI and HindIII restriction site (italic),respectively. The resulting amplified DNA was digestedwith BamHI and HindIII, cloned in frame with sequencesof the pLC24 bacterial expression vector (Remaut et al.,1981), and sequenced. Recombinant plasmids were trans-ferred into Escherichia coli SG4044, and production of thefusion protein was induced by a 42°C temperature shift.The bacterial fusion protein was purified as described byGhysdael et al. (1986). It appeared as a single band ofappropriate molecular weight when stained with Coo-massie blue. This preparation was used for rabbit immuni-zation (Harlow and Lane, 1988).

Immunohistochemistry

Thirty-micrometer-thick floating sections of frozen adultmouse brain were incubated at room temperature, firstwith 0.1% hydrogen peroxide (H2O2) in 1 3 PBS for 10minutes and then in 1 3 PBS, 3% normal goat serum(Dako, Carpinteria, CA), 0.1% Triton X-100 for 30 minutes.Sections were then incubated overnight at 4°C with theaffinity-purified anti-NC2 antibody (dilution 1:250) inblocking buffer (1 3 PBS, 1% BSA, 0.5% gelatin, 3%normal goat serum, 0.1% Triton X-100). They were thenincubated for 90 minutes with the secondary antibody(goat anti-rabbit and biotinylated IgG, dilution 1:400;Vector Laboratories, Burlingame, CA) in blocking buffer.After rinsing, sections were incubated for 2 hours inavidin-biotin peroxydase complex (ABC, Vector Laborato-ries), and the reaction was developed with 0.6 mg/mldiaminobenzidine (Sigma) in 0.05 M Tris, pH 7.4, 0.3%H2O2.

Immunoprecipitation and Westernblot analysis

Mouse embryos and adult mouse tissues were homog-enized (0.5 g/ml) in RIPAE buffer (PBS containing 1%Triton X-100, 0.1% SDS, 5 mM EDTA, 1% aprotinin, and1% sodium deoxycholate). Clarified lysates were incubatedfor 2 hours in ice with anti-TrkC NC2 serum either inpresence or absence of 10 µg of the corresponding immuniz-ing fusion protein. The resulting immune complexes werecollected by precipitation with protein A Sepharose (Phar-macia), washed three times with RIPAE buffer, once with 1mM MgCl2 in 10 mM Tris-HCl, pH 7.5, and once withNP-40 buffer (0.5% NP-40, 50 mM NaCl in 20 mM Tris-HCl, pH 7.5). Immunoprecipitated proteins were eluted byboiling for 4 minutes in 30 µl of Laemmli sample buffer,fractionnated by 7.5% SDS-polyacrylamide gel electropho-resis, and transferred to polyvinylidene difluoride blottingmembranes (Immobilon-P, Millipore Corp., Bedford, MA).Membranes were saturated with blocking solution (5%nonfat milk in 20 mM Tris-HCl, pH 7.6, 0.9% NaCl, 0.2%Tween-20) and incubated with either anti-pan-TrkC oranti-TrkC NC2 antisera diluted 1:500 to 1:2,000 in block-ing solution overnight at 4°C. Immunostaining was per-formed with horseradish peroxidase-conjugated anti-rabbit IgG, with the enhance chemiluminescent (ECLy)

52 B. MENN ET AL.

Western blotting reagents (Amersham Corp.), according tothe manufacturer’s instructions.

RESULTS

Identification and characterization of cDNAsencoding noncatalytic TrkC receptors

To isolate noncatalytic TrkC receptors, we screened anadult mouse brain cDNA library with a probe correspond-ing to a part of the extracellular and transmembranedomains of the mouse trkC gene product (see Materialsand Methods). More than 100 recombinant phages werefound to be positive. Filters containing these phages wererehybridized with a probe that encodes the catalytictyrosine kinase domain of mouse TrkC to eliminate clonesthat encode the catalytic isoforms. Because we used anoligo-(dT)-primed cDNA library, the residual clones wereexpected to encode TrkC receptors lacking the tyrosinekinase domain. We selected these phages and analyzedtheir inserts by restriction enzyme mapping. Thus, weobtained several independent clones that could be classi-fied into two groups according to the restriction enzymepattern: one group contained two clones and the othercontained 47 clones. We subcloned the longest inserts ofeach group into pBluescript and determined the entirenucleotide sequence of plasmids pFL71 and pBM3 repre-senting each group. The results are presented in Figures 1and 2, respectively.

Analysis of pFL71 nucleotide sequence (Fig. 1A) showedthat this cDNA consists of 2813 bp, of which only the first1,548 nucleotides are identical to those present in pFL16, acDNA clone that encodes gp145trkC, a catalytic isoform ofmouse TrkC (mTrkC K; Lamballe et al., 1991). Nucleotides1–1548 correspond to sequences that encode the putativesignal peptide, the extracellular and transmembrane re-gions, and the first 13 amino acids of mTrkC K intracellu-lar domain. The similarity between pFL16 and pFL71ends at amino acid residue 466. The point of sequencedivergence is indicated by an arrow in Figure 1A,B. ThepFL71 open reading frame extends 36 amino acids down-stream before encountering a stop codon. These 36 aminoacid residues do not share significant similarity withsequences deposited in databanks. Therefore, pFL71 en-codes a 502-amino-acid polypeptide with a calculatedrelative molecular mass of 56,358. These results indicatethe existence of a novel noncatalytic TrkC receptor iso-form, designated TrkC NC1 (for noncatalytic-1). Interest-ingly, the TrkC NC1 receptor is spliced at the same site asTrkB TK2 (Klein et al., 1990a; Fig. 1B), which suggeststhat the genomic organization of the trkB and the trkC lociis similar in their juxtamembrane region.

Analysis of pBM3 nucleotide and deduced amino acidsequence (Fig. 2B) showed that this cDNA encodes anoncatalytic isoform of TrkC similar to that previouslydescribed in pig (Lamballe and Barbacid, unpublishedobservations), in rat as TrkC TK2 (Tsoulfas et al., 1993) orTrkC(ic158) (Valenzuela et al., 1993), and in human(Shelton et al., 1995). This receptor has been named mouseTrkC NC2 (for noncatalytic-2). It encodes a 612-amino-acid-long polypeptide with a calculated relative molecular massof 68,522. Sequences encoding the signal peptide, theextracellular and transmembrane domains, and the first75 amino acids of the intracellular region are identical tothose of pFL16 that encode mTrkC K. The point ofsequence divergence (nucleotide 1735), located in the

cytoplasmic region, is indicated by an arrowhead in Figure2A,B. The TrkC NC2 sequence diverges from that of TrkCNC1 at nucleotide 1548, indicated by an arrow in Figure2A,B. The specific sequence of mTrkC NC2 consists of 84amino acid residues followed by a stop codon. These 84amino acids are highly conserved in mouse, rat, pig, andhuman, with only two differences (indicated in bold lettersin Fig. 2B) with the human sequence.

Expression of trkC catalytic- andnoncatalytic-specific transcripts as

determined by Northern blot analysis

Previous studies of the expression pattern of trkC tran-scripts in different adult tissues were performed only withprobes corresponding to either the extracellular or thecatalytic domain of TrkC. They have shown that trkCmRNAs are abundantly expressed in brain and moreweakly in testis and ovary (Lamballe et al., 1991; Tsoulfaset al., 1993; Valenzuela et al., 1993; Shelton et al., 1995).To investigate the relative expression of transcripts encod-ing each of the catalytic and noncatalytic TrkC isoforms,we analyzed poly(A)1 RNA from several adult tissues byNorthern blotting and hybridization with specific probes.Hybridization with probe A, an extracellular probe (Fig.3A; see Materials and Methods), demonstrated at leastthree transcripts of about 15.0 kb, 4.7 kb, and 4.4 kb inbrain (Fig. 3B). We detected the 4.7-kb mRNA in ovaryafter a long exposure of the Northern blot (data notshown). Although none of these transcripts was observedin testis, a band corresponding to a 2.1-kb mRNA speciescould be visualized in this tissue. We did not detect trkCexpression in spleen (Fig. 3B).

A duplicate RNA blot was then hybridized with the KINprobe encompassing most of the sequences encoding thetyrosine kinase domain (Fig. 3A). This probe detected onlythe 15.0-kb transcript in brain. No hybridization signalswere observed in the other tissues analyzed (Fig. 3B).These results identify the 15.0-kb transcript as that encod-ing the TrkC catalytic isoform(s).

We next investigated the presence of transcripts encod-ing TrkC NC1 and TrkC NC2 by using probes containingsequences specific for each of the noncatalytic receptors(Fig. 3A). The NC2-specific probe hybridized with the4.7-kb and the 4.4-kb transcripts in brain and with the2.1-kb transcript in testis (Fig. 3B). This NC2 probe alsodetected the 4.7-kb transcript in ovary. No transcriptswere detected when the same RNA blot was hybridizedwith the NC1-specific probe (data not shown).

These results show that transcription of the trkC locusproduces several mRNA species: the 15.0-kb transcriptappears to encode the catalytic isoforms, whereas the4.7-kb and the 4.4-kb mRNAs appear to encode the noncata-lytic TrkC NC2 receptor. Interestingly, the 2.1-kb tran-script was detected only in testis. The structure of theTrkC NC2 receptor encoded by this transcript remains tobe determined. Our inability to detect trkC NC1-specifictranscript by Northern blot analysis indicates that it isexpressed at a low level.

In situ hybridization analysis of trkCcatalytic and noncatalytic transcriptsduring mouse embryonic development

and in adult brain

To determine more precisely the spatial and temporalexpression of trkC NC1, trkC NC2, and trkC catalytic


transcripts, we performed in situ hybridization with 35S-labeled NC1, NC2, or catalytic riboprobes during mousedevelopment and in adult mouse brain.

trkC NC1 expression during embryonic development.

We investigated the localization of trkC NC1 transcript byin situ hybridization on paraffin-embedded sections ofembryos of different ages (E9.5, E11.5, E13.5, E15.5,E17.5) with an antisense NC1 riboprobe. We did not detecthybridization signals with the control sense NC1 cRNAprobe. We could not detect hybridization signals with theantisense NC1 riboprobe in embryos at different stages,despite the reduced stringency conditions used in theseexperiments. This result indicates that, in agreement withNorthern blot analysis, the levels of trkC NC1 expressionare low. They can be detected only by the sensitive PCRtechnique, which shows that trkC NC1 transcript is ex-pressed early in embryogenesis until late stages of develop-ment (data not shown).

trkC NC2 expression during embryonic development.

In situ hybridization analyses were then performed withNC2-specific riboprobes. No hybridization signals weredetected with the sense probe. When we hybridized adja-cent sections with the antisense NC2 riboprobe, we did notdetect a signal in 9.5-day-old embryos. The trkC NC2expression was first observed at E11.5 and remainedconstant until late stages of embryogenesis. Expression in

the CNS extended grossly to the whole brain with a doublegradient, one rostrocaudal and the other lateromedial.Transcripts were also detected in the PNS, mainly inneural crest derivatives, and in additional regions outsidethe nervous system.

Fig. 4. Expression of trkC NC2 transcripts in the developing mousebrain. Darkfield photomicrographs of sagittal (A,C,E,G,I–L), coronal(B,D), and transverse (F,H) sections of 11.5- (A,B), 13.5- (C,D), 15.5-(E–H), and 17.5- (I–L) day-old embryos. Sections were hybridized withantisense trkC NC2 cRNA probes. Arrowheads in A–D indicate thebasal telencephalon. cb, cerebellum; cp, caudatoputamen complex; cpl,

cortical plate; fc, frontal cortex; ibo, inferior bulbar olive; lv, lateralventricle; mv, mesencephalic vesicle; p, pons; sc, spinal cord; tc,temporal cortex; tel, telencephalon; vz, ventricular zone; Vg, trigemi-nal ganglion; 3v, third ventricle; 4v, fourth ventricle. Scale bars 52,000 µm in I, 1,000 µm in C,E,F,H, 500 µm in A,B,D,G,J–L.

Fig. 5. In situ localization of trkC NC2 transcripts in 11.5- and13.5-day-old mouse embryos. A–D: E11.5 embryo. Darkfield photomi-crographs of sagittal (A) and coronal (B–D) sections. E–K: E13.5embryo. Darkfield (E,F,H,I) and brightfield (G,J,K) photomicrographsof sagittal (E,H) and coronal semioblique (F,G,I–K) sections. Sectionswere hybridized with antisense trkC NC2 cRNA probes. Magnifiedviews of the dorsal aorta and the abdominal wall (C), the sympathetictrunk indicated by arrows (D), and the genital tubercle (H). G:Brightfield photomicrograph of an adjacent section to that shown in F.I,J: Magnified views show the upper parts of F and G. K: Highmagnification view of the inferior bulbar olive indicated in J. Arrows inB and F indicate the trkC NC2 expression around the central canal ofthe spinal cord. Note also the strong signal in the inferior bulbar olivein a 13.5-day-old embryo (F,I). The black arrows in G indicate theventral part of the central canal of the spinal cord. See elongated cellsprobably representing neurons in the differentiating field (arrowsin K). a, dorsal aorta; aw, abdominal wall; dia, diaphragm; drg, dorsalroot ganglia indicated by arrowheads; gt, genital tubercule; ibo,inferior bulbar olive; p, pons; pc, pericardial cavity; sc, spinal cord; t,tongue; tec, tectum; tel, telencephalon; ugr, urogenital ridge; Vg,trigeminal ganglion; 4v, fourth ventricle. Scale bars 5 1,000 µm in E,500 µm in A–D,F–J, 5 µm in K.

54 B. MENN ET AL.

Figure 5


trkC NC2 mRNA is differentially expressed in brainduring development. At E11.5, trkC NC2 expression wasvery weak and was detected only in the basal telencepha-lon (Fig. 4A,B).

At E13.5, the expression pattern became more complex.Transcripts detected previously in the basal telencephalonextended medially, as shown on sagittal (Fig. 4C) andcoronal (Fig. 4D) sections. At this stage, trkC NC2 mRNAswere also present in the dorsal telencephalon (Fig. 4C,D),where the labeling was homogeneous in the whole thick-ness of the neural tube from the lateral ventricle to themarginal layer (Fig. 4C). The diencephalon did not showhybridization signals. In contrast, trkC NC2 transcriptswere also expressed, although less intensely than in thetelencephalon, in the mesencephalon, particularly in thetectum (Fig. 5E) and rhombencephalon (Figs. 4C, 5E). Asignal was also detected in the differentiating field of theinferior bulbar olive (Fig. 5I). Interestingly, the ependymallayer of the fourth ventricle, which expresses high levels oftrkB noncatalytic transcripts (Klein et al., 1990a), alsoexhibited high levels of trkC NC2 mRNAs (Figs. 4C, 5E).We could also detect trkC NC2 expression in the cerebel-lum primordium (data not shown).

At E15.5, the basal telencephalon, which gives rise tothe caudate-putamen complex, was intensely labeled(Fig. 4F). In the dorsal telencephalon, the pattern ofexpression differentiated concomitantly with CNS differen-tiation and was no longer homogeneous. An intense signalwas detected in the cortical plate (Fig. 4G,H) and theventricular zone (Fig. 4G). At this stage, no signal wasdetected in the diencephalon, whereas the rhombencepha-lon exhibited low levels of trkC NC2 expression. In con-trast, the tectum (Fig. 6A) and the primordium of thecerebellum expressed high levels of trkC NC2 transcriptsat a distance from the fourth ventricle, in the differenti-ated field (Fig. 4E,F). The intense signal in the ependymallayer of the third (Fig. 4F,H) and fourth (Fig. 4H) ven-tricles persisted.

At later stages of development (E17.5), trkC NC2 tran-scripts were present throughout all levels of the neuraxisexcept the diencephalon. Although the caudate-putamencomplex was intensely stained in the basal telencephalon(Fig. 4I), the heterogeneity of labeling was even morepronounced in the dorsal telencephalon. The cortical plateexhibited high levels of expression (Fig. 4I,L). A homoge-neous signal, although weaker than in the cortical plate,was observed in the ventricular zone (Fig. 4I,L). Further-more, labeling of the more caudal regions of the dorsaltelencephalon seemed to be stronger than that of the morerostral counterpart (Fig. 4L). In contrast to earlier stages,at E17.5 the trkC NC2 transcripts were abundant in themesencephalon and the rhombencephalon, including brain-stem and cerebellum. The signal in the ependymal layer ofthe lateral (Fig. 4L) and third (Fig. 4J) ventricles and themesencephalic vesicle (Fig. 4I,K) persisted.

trkC NC2 is expressed in the spinal cord. At E11.5, trkCNC2 transcripts were detected in the entire spinal cord(Fig. 5A). However, hybridization signals were rather lowand heterogeneous, with a stronger intensity located in theventral part around the central canal (Fig. 5B). A similarexpression pattern was seen at E13.5 (Fig. 5F). At laterstages in development (E15.5 and E17.5), trkC NC2 expres-sion was detected in the entire spinal cord (data notshown).

trkC NC2 is expressed in neural crest derivatives. ThetrkC NC2 expression in the dorsal root ganglia (DRGs)

reflects that detected with a trkC extracellular probe(Lamballe et al., 1994). At E11.5, the DRGs displayed highlevels of trkC NC2 expression (Fig. 5A,B). This homoge-neously distributed hybridization signal persisted untilE13.5 (Fig. 5F). At E17.5, a change in pattern was ob-served, i.e., trkC NC2 transcripts were located in a subsetof neurons at the periphery of the DRGs (Fig. 6F,G). ThetrkC NC2 transcripts were also detected in other struc-tures of the PNS including the trigeminal ganglion (Figs.4E, 5A) and other cranial nerve nuclei, namely IX and X(data not shown). Transcripts were also present in thesympathetic trunk (Fig. 5D) and were strongly expressed,at all stages analyzed, in the wall of the aorta (Figs. 5C,6F,G), particularly in smooth muscular cells, as detectedby brightfield analysis (data not shown).

trkC NC2 expression outside the nervous system. ThetrkC NC2 transcripts were strongly expressed outside thenervous system in structures such as the urogenital tu-bercle, which exhibited a strong expression as early asE11.5 (Figs. 5A,H, 6A,C,H). In addition, we observed trkCNC2 transcripts in the pigmentary epithelium of the eye(Fig. 6E), the vibrissae (Fig. 6F,J), the olfactory epithelium(Fig. 6I), the acini of the submandibular gland (Fig.6A,B,F), the adipose tissue (Fig. 6F), and the thyroid (Fig.6F). The trkC NC2 mRNAs were also detected in mesoder-mal derivatives, namely muscles in the process of differen-tiation such as the tongue (Figs. 5E, 6A), cervical muscles(Fig. 6F), diaphragm (Fig. 5A), body wall overlying thepericardial cavity, and the abdomen (Fig. 5A,C). A hybrid-ization signal was also seen in the interdigital spaces atE15.5 (Fig. 6D), concomitantly with the necrosis/apoptosisprocess that occurs in the intervening webbing (Saunderset al., 1962).

trkC catalytic transcripts expression during embry-

onic development. We next compared the localization oftrkC catalytic transcripts with the transcription pattern oftrkC NC2. In situ hybridization demonstrated high levelsof catalytic transcripts in the CNS and PNS. These tran-scripts were not significantly detectable outside the ner-vous system.

In the CNS, at E11.5, the signal was barely detectable inthe developing telencephalon (Fig. 7A). As the CNS struc-tures differentiated, trkC catalytic expression increasedconsiderably. At E13.5, a signal was observed in thetelencephalon, mesencephalon, diencephalon, the inferiorbulbar olive, and the spinal cord (Fig. 7B). At E15.5, allthese structures remained labeled, with a higher expres-sion level in the external layer and the caudal part of thetelencephalon (Fig. 7C). At E17.5, the intense signalremained more pronounced.

High levels of expression were observed in the PNS,especially in the DRGs (at the periphery and in the centerof the ganglia, in contrast to NC2 transcripts; Fig. 7A,C,E)and in cranial ganglia such as the trigeminal ganglion,which exhibited a punctated signal (Fig. 7A,B,D).

trkC NC1 expression in adult mouse brain. We alsoinvestigated trkC NC1 expression by in situ hybridizationin frozen sections of adult mouse brain. The sense trkCNC1 riboprobe produced hybridization signals in brainregions such as the pyramidal layers of the hippocampusand the dentate gyrus (Fig. 8C) and in the white matterand the internal granular layer of the cerebellum (Fig. 8E).The intensity of this signal was considered to be back-ground. Adjacent sections were hybridized with the anti-sense NC1 riboprobe. Sense and antisense cRNA probeswere identical in length and had the same specific activity.

56 B. MENN ET AL.

The antisense NC1 cRNA probe produced a signal abovebackground level and showed expression of trkC NC1transcripts in the dentate gyrus of the hippocampus (Fig.

8A,B) and the internal granular layer of the cerebellum(Fig. 8A,D). Whether Purkinje cells express this transcriptremains an open question because observations of cresyl

Fig. 6. In situ localization of trkC NC2 transcripts in 15.5- and17.5-day-old mouse embryos. A–E: E15.5 embryo. Darkfield photomi-crographs of sagittal sections. F–J: E17.5 embryo. Darkfield photomi-crographs of sagittal sections. Sections were hybridized with antisensetrkC NC2 cRNA probes. Magnified views show the submaxillary gland(B), the genital tubercle (C,H), the paw (D), the eye (E), the dorsal rootganglia and the aorta (G), the olfactory epithelium (I), and thevibrissae (J). Note the signal in the interdigital space in the 15.5-day-old embryo indicated by arrows (D). In E17.5, note the distribution of

trkC NC2 transcripts in the periphery of the drg (G). a, aorta; at,adipose tissue; cb, cerebellum; cm, cervical muscle; cp, caudatoputa-men complex; drg, dorsal root ganglia indicated by arrowheads; el,eyelid; gt, genital tubercle; is, interdigital space; mv, mesencephalicvesicle; oe, olfactory epithelium; p, pons; pe, pigmentary epithelium;sc, spinal cord; smg, submaxillary gland; tec, tectum; tel, telencepha-lon; tj, tongue–jaw; th, thyroid; vi, vibrissae. Scale bars 5 2,000 µm inA,F, 500 µm in B,D,E,G–J, 100 µm in C.


Fig. 7. In situ localization of trkC catalytic transcripts in mouseembryos. Darkfield photomicrographs of sagittal sections of 11.5- (A),13.5- (B), 15.5- (C), and 17.5- (D,E) day-old embryos. Magnified viewshows the dorsal root ganglia of a 17.5-day-old embryo (E). die,

diencephalon; drg, dorsal root ganglia; ibo, inferior bulbar olive; p,pons; sc, spinal cord; tec, tectum; tel, telencephalon; Vg, trigeminalganglion. Scale bars 5 2,000 µm in D, 1,000 µm in B,C, 500 µm in A,E.

58 B. MENN ET AL.

violet–stained sections at the microscopic level showedonly a few silver grains in these cells (Fig. 8F).

trkC NC2 expression in adult mouse brain. As men-tioned above (Fig. 3B), Northern blot analysis indicatedthat trkC NC2 transcripts are expressed in adult mousebrain. In situ hybridization with a sense trkC NC2 cRNAprobe did not demonstrate any signal (data not shown).The trkC NC2 transcripts were widely detected with anantisense trkC NC2 cRNA probe in different structures ofthe brain. In the cerebral cortex, we observed a strongsignal in layers 2 and 3 (Fig. 8G,H). The hippocampusexhibited high levels of expression in the pyramidal layers(ca1, ca2, ca3) and even stronger signals in the dentategyrus (Fig. 8G). In the cerebellum, the internal granular

layer was intensely stained (Fig. 8G,I). However, micro-scopic observations of cresyl violet–stained sections showed,on the one hand, that granule cells express heteroge-neously trkC NC2 mRNAs and, on the other hand, thatonly some Purkinje cells express these transcripts (Fig.8K). In the olfactory bulb, trkC NC2 transcripts werehighly expressed in the internal granular layer and in themitral cells (Fig. 8H,J). The trkC NC2 transcripts werealso detected, although less intensely, in other structuressuch as the caudate putamen (Fig. 8G). However, we didnot detect expression in the thalamus, hypothalamus, orbrainstem, all of which exhibited hybridization signalswith a trkC extracellular probe in previous studies (Tess-arollo et al., 1993; Lamballe et al., 1994). These results

Fig. 8. Expression of trkC NC1, trkC NC2, and trkC catalytictranscripts in adult mouse brain. A–F: trkC NC1. G–K: trkC NC2.L–P: trkC catalytic. Darkfield (A–E,G–I,L–N) and brightfield(F,J,K,O,P) photomicrographs of sagittal (A,G,L) and coronal (B–F,H–K,M–P) sections of adult mouse brain. Sections were hybridized witheither antisense (A,B,D,F–P) or sense (C,E) cRNA-specific probes.Magnified views show the hippocampus (B,C), the cerebellum (D,E),the olfactory bulb (H,M), and the internal granular layer of thecerebellum (I,N). High magnifications show the internal granular

layer (F,K,P) and the olfactory bulb (J,O). Arrows in F,I,K,N, and Pindicate Purkinje cells. Closed arrowheads in H,J,M, and O indicatemitral cells. The open arrowhead in M indicates the lateral olfactorytract. c, cortex; ca1, ca3, pyramidal layers; cb, cerebellum; cp, cau-datoputamen complex; dg, dentate gyrus; h, hippocampus; igl, inter-nal granular layer; lot, lateral olfactory tract; ob, olfactory bulb. Scalebars 5 2,000 µm in A,D,E,G,H,L,M, 750 µm in B,C,N, 5 µm inF,J,K,O,P.


show that trkC NC2 expression is maintained in maturebrain and is in agreement with embryogenesis data.

trkC catalytic transcripts expression in adult mouse

brain. The sense trkC catalytic cRNA probe did notproduce any signal. Hybridization with the correspondingantisense riboprobe showed that trkC catalytic transcriptswere present in the same structures as trkC NC2 RNAs,i.e., the cerebellum, the hippocampus (Ammon’s horn anddentate gyrus), caudate putamen, and the cerebral cortex(Fig. 8L). However, catalytic transcripts were also observed inthalamus and hypothalamus that were not highlighted withthe NC2 riboprobe. In the cerebellum, labeling was ob-served in granule cells of the internal granular layer (Fig.8L,N) and heterogeneously in the Purkinje cells (Fig. 8P).In the olfactory bulb, trkC catalytic RNAs were visualizedin the mitral cells, in the internal granular layer, and inthe lateral olfactory tract (Fig. 8M,O).

Characterization and distributionof the TrkC NC2 protein

To assess the presence of TrkC NC2 protein, we raisedpolyclonal antibodies against amino acids encoded byspecific sequence of the NC2 isoform (see Materials andMethods). Expression of TrkC NC2 protein was investi-gated by immunoprecipitation and Western blot analysesat different stages of embryonic development and in differ-ent adult tissues.

Anti-TrkC NC2 antibodies specifically recognized a pro-tein with an apparent molecular weight of 100,000 inmouse embryos (Fig. 9A). The TrkC NC2 protein can bedetected as early as E9.5 and throughout embryonicdevelopment. Starting from stage E11.5, the presence ofTrkC NC2 protein was investigated separately in theheads and trunks. Expression of TrkC NC2 seemed to bemore abundant in the head than in the rest of the body. Inaccordance with in situ hybridization data, this findingsuggests that the TrkC NC2 protein is strongly expressedin the CNS. In adult mouse, the TrkC NC2 protein wasdetected only in brain (Fig. 9B), with higher amounts incortex and hippocampus (Fig. 9C). Immunoprecipitation ofTrkC NC2 was completely inhibited by preincubating theantiserum with the immunizing antigen (Fig. 9C). More-over, we did not detect a TrkC protein by immunoprecipita-tion with the preimmune serum (data not shown). Inconclusion, we show for the first time the existence of aTrkC noncatalytic protein, the 100-kDa TrkC NC2 protein,in mouse embryos and adult brain.

We next performed immunohistochemistry on adultmouse brain sections with the affinity-purified anti-TrkCNC2 serum. A specific staining was clearly detected in thecerebellum in the dendrites of first and second order butnot in the axons of the Purkinje cells (Fig. 10B,C). Whetherdendrites of third order and spines are labeled remains tobe determined. In the hippocampus, only apical dendritesof pyramidal cells were heavily stained, especially in theca1 region (Fig. 10E,F). No axonal staining was evident,especially in the mossy fibers and in the Schaffer collater-als. Dendrites of the dentate gyrus were less intensilystained (data not shown). Similarly, in the cerebral cortex,apical dendrites of the pyramidal neurons of layer 5 werestained (Fig. 10H,I), but proximal basal dendrites werealso labeled (Fig. 10H,I). In the olfactory bulb, cells of theinternal granular layer were stained (data not shown). Aweak signal was also observed in dendrites of mitral cells(data not shown). Competition with the immunizing fusion

protein completely blocked the staining in the dendrites(Fig. 10A,D,G), with the exception of the cellular bodies ofthe Purkinje cells (Fig. 10A).

Fig. 9. Expression of the TrkC NC2 receptor throughout mouseembryogenesis (A) and in adult mouse tissues (B,C). Embryonic heads(H) and trunks (T) and adult mouse tissues were homogenized inRIPAE buffer (see Materials and Methods) and immunoprecipitatedwith rabbit polyclonal antisera raised against unique sequences of theTrkC NC2 receptor (TrkC NC2 antiserum). C: Different brain struc-tures, i.e., cortex, midbrain (Midbr.), hippocampus (Hipp.), and cerebel-lum (Cb), were homogenized in RIPAE buffer and immunoprecipitatedwith anti-TrkC NC2-specific antibodies in the presence (1) or absence(2) of 10 µg of competing antigen. The immunoprecipitates wereresolved on 7.5% sodium dodecyl sulfate–polyacrylamide gel electro-phoresis and immunoblotted with anti-pan-TrkC antibodies. Theywere visualized by chemiluminescence. Migration of the TrkC NC2protein is indicated by a black arrowhead. The white arrowhead pointsto nonspecific bands, and the black arrow indicates the immunoglobu-lins. Coelectrophoresed molecular weight markers are indicated onthe right.

60 B. MENN ET AL.

To address the question of colocalization of catalytic andnoncatalytic isoforms, we performed in situ hybridizationwith the trkC kinase riboprobe followed by immunohisto-chemistry with the anti-TrkC NC2 antibody on adultmouse brain sections. TrkC NC2 protein was detected inapical dendrites of the ca1 pyramidal neurons of thehippocampus, and catalytic transcripts were found incellular bodies of the same cells (data not shown). Thesepreliminary results show that catalytic and noncatalyticreceptors can be expressed in the same cells.

DISCUSSION

The TrkC receptor displays multiple catalytic or noncata-lytic isoforms generated by alternative splicing (Lamballeet al., 1993; Tsoulfas et al., 1993; Valenzuela et al., 1993).Although the role of the catalytic isoforms is not wellunderstood, they exhibit distinct signaling capabilities(Lamballe et al., 1993; Tsoulfas et al., 1996). Moreover,function of the TrkC noncatalytic receptors is unknown. Asa prerequisite for understanding the physiological roles ofthese noncatalytic isoforms, we extensively analyzed their

expression pattern. We have shown that trkC NC2 tran-scripts are either expressed independently or coexpressedwith trkC catalytic transcripts. Moreover, the presence ofTrkC NC2 in dendrites suggests that this receptor isinvolved in neuronal differentiation and synaptic plastic-ity.

We have reported the identification of a novel noncata-lytic isoform, designated TrkC NC1. The trkC NC1 tran-scripts were detected by reverse transcriptase–PCR inalmost all tissues analyzed but at a very low level (data notshown). In situ hybridization results show that trkC NC1expression in adult brain is restricted to the pyramidalcells of the hippocampus and the granular layer of thecerebellum, two structures that express high levels of NT-3transcripts (Ernfors et al., 1992). The functional relevanceof these observations remains to be determined.

We also cloned a second noncatalytic isoform, designatedTrkC NC2, that is the mouse homologue of the previouslydescribed truncated TrkC receptor (Tsoulfas et al., 1993;Valenzuela et al., 1993 [TrkC (ic 158)]; Garner and Large,1994). The cDNAs encoding the TrkC NC2 receptor exhib-ited high conservation at the amino acid level (98–100%) in

Fig. 10. Distribution of TrkC NC2 in adult mouse brain. Immuno-histochemistry was performed with the affinity-purified TrkC NC2antibody. Competition with the immunizing fusion protein is shown(A,D,G). A–C: Purkinje cells. D–F: Pyramidal neurons of the ca1region of the hippocampus. G–I: Pyramidal neurons of the cerebral

cortex. Arrowheads indicate labeled dendrites corresponding to apicaldendrites in B,E,H, and I. Arrows show basal dendrites (H,I). cx,cortex; igl, internal granular layer; pc, Purkinje cells; so, stratumoriens; sp, stratum pyramidale; sr, stratum radiatum. Scale bar 5 2.7µm in A,B,D,E,G,H, 1.8 µm in C,F,I.


the specific cytoplasmic region, suggesting an importantrole in the cellular response to NT-3.

Analysis of its transcriptional pattern indicated that theTrkC NC2 receptor is highly expressed in the CNS andPNS of mouse embryos but also outside the nervoussystem and in adult mouse brain and gonads.

Our data suggest three interesting possibilities that willbe discussed in more detail: TrkC NC2 may (1) be involvedin proliferation of oligodendrocyte progenitors, (2) act bothindependently and in association with TrkC catalyticreceptors, and (3) be involved in neuronal differentiationand plasticity.

In the CNS, TrkC NC2 may be expressedin oligodendrocyte progenitors

In situ hybridization demonstrated a similarity of local-ization of trkC NC2 mRNA at the contact of the centralcanal all along the spinal cord with that of the PDGF alphareceptor (Pringle and Richardson, 1993) and the DM-20transcripts (Timsit et al., 1995), two molecules supposed tobe expressed in oligodendrocytes progenitors. NT-3 hasbeen shown to control proliferation and survival of oligoden-drocytes precursors (Barres et al., 1993; Barres and Raff,1994; Cohen et al., 1996). Furthermore, a synergistic effectof NT-3 and PDGF on oligodendrocyte progenitor cellsproliferation has been reported (Barres et al., 1993). Thisobservation suggests that TrkC NC2 controls proliferationof these cells.

Differential expression of TrkC isoformssuggests that the TrkC NC2 receptor acts

both independently and in associationwith its catalytic counterpart

Regions that coexpress catalytic and noncatalytic

isoforms. In the CNS of the embryo, trkC catalytic andnoncatalytic NC2 transcripts were coexpressed in thetelencephalon, mesencephalon, rhombencephalon, and thespinal cord. Coexpression was also detected in neural crestderivatives in the PNS, but the pattern of distributionseemed to be distinct in dorsal ganglion.

In adult, trkC catalytic and noncatalytic NC2 tran-scripts appeared to be codistributed in different struc-tures. The distribution of the signals obtained with specificriboprobes suggests that expression of these isoformsoverlap. Coexpression was indeed demonstrated in pyrami-dal neurons of the hippocampus by double labeling thatdetected catalytic transcripts and NC2 protein in the samecells. This finding suggests that catalytic and noncatalyticisoforms can generate heterodimers and that the noncata-lytic receptor modulates neurotrophin response.

Similarly, the trkB locus also encodes noncatalytic recep-tors. Recent functional studies have shown that ‘‘trun-cated’’ TrkB receptors may modulate neuronal responsesto neurotrophins (Armanini et al., 1995) by acting asdominant negative (Eide et al., 1996). Furthermore, it hasbeen shown that catalytic trkB transcripts precede theexpression of their noncatalytic counterparts (Cabelli etal., 1994; Fryer et al., 1996). In contrast, we did notobserve a temporal delay in the expression of trkC cata-lytic and noncatalytic isoforms. Thus, the function ofnoncatalytic TrkB and TrkC receptors may be different.

Regions that exclusively express the catalytic or the

noncatalytic isoform. In the CNS of the embryo, onlythe catalytic trkC transcript was expressed in the dien-

cephalon. Similarly, in adults, thalamus and hypothala-mus expressed only the tyrosine kinase transcripts. Thesedata are in agreement with previous results concerningthe expression of trkC transcripts demonstrated by anextracellular probe in these structures (Tessarolo et al.,1993; Lamballe et al., 1994). The distinct expression ofcatalytic and noncatalytic transcripts suggests that theyare under the control of regulatory genes involved in theneuromeric organization of the forebrain (Puelles andRubenstein, 1993). Furthermore, it has recently beenshown that the TrkB catalytic and noncatalytic isoformsare involved in the segmentation in the anterior–posteriorpatterning of the Xenopus CNS (Islam et al., 1996).

Outside the nervous system, only the NC2 isoform wasdetected in different structures. Thus, it was detected inthe genital tubercles in the embryo. The absence of expres-sion of the catalytic form and the strong expression of trkCNC2 in this region suggest that this noncatalytic receptorby itself mediates NT-3 signaling. Interestingly, it hasrecently been reported that, in nonneuronal cells, thetruncated TrkB receptor could transduce a signal in re-sponse to BDNF (Baxter et al., 1997).

Possible role of TrkC NC2 during neuronaldifferentiation and plasticity

Analysis of trkC NC2 expression during embryonicdevelopment showed that this isoform was expressed inregions of neuronal maturation. Expression appeared firstin the lateral part of the basal telencephalon, a region ofearly differentiation, and then extended medially. In thedorsal telencephalon, the signal gradually increasedthroughout development, with modulation of the expres-sion concomitant to corticogenesis. Interestingly, we ob-served a contrast of labeling in different regions: the signalwas intense in the cortical plate, where neurons havemigrated, and was low in the proliferative ventricular zoneat E17.5. Moreover, in the cortical plate, trkC NC2 expres-sion exhibited a rostrocaudal gradient concomitant to theneuronal differentiation gradient. Similar data were alsoobtained in the cerebellum primordium, where the label-ing was observed at distance from the ventricle and in theinferior bulbar olive.

NT-3 expression parallels critical periods of neural devel-opment. It is expressed early in development, and then itsexpression decreases with maturation (Ernfors et al.,1992). The trkC NC2 expression correlates well with thepresence of NT-3 transcripts. It has recently been shownthat NT-3 is a differentiating factor for calbindin-express-ing hippocampal neurons but does not affect their prolifera-tion (Vicario-Abejon et al., 1995). Similarly, other studieshave indicated that, as far as cortical neurogenesis isconcerned, NT-3 antagonizes the proliferative effect ofbFGF and enhances neuronal differentiation (Ghosh andGreenberg, 1995). Therefore, TrkC NC2 may play a role,during either the withdrawal from the cell cycle in theventricular zone or the differentiation phase itself, in thecortical plate. During these two phases, neuronal death isprominent in the proliferative zone and in regions ofpostmitotic neurons (Blaschke et al., 1996). Furthermore,recent studies have shown that neuronal death can also beinduced by an interaction between ligands and specificreceptors, as described in the immune system (Ju et al.,1995). Frade et al. (1996) reported that, during develop-ment, NGF can induce death of retinal neurons expressingthe nontyrosine kinase p75NTR receptor. Similarly, TrkC

62 B. MENN ET AL.

NC2 may be involved in neuronal cell death by activatingpathways leading to apoptosis or by a mechanism relatedto its interaction with its cognate catalytic isoform. Thus,the TrkC NC2 receptor may decrease the tyrosine kinaseactivity of the catalytic isoform and, therefore, woulddiminish the ability of NT-3 to act as a survival factor.Furthermore, trkC NC2 is expressed outside the nervoussystem. In the interdigital space, it is expressed exactlywhen apoptosis occurs (Saunders et al., 1962). These datastrengthen the possible involvement of TrkC NC2 inprogrammed cell death, assuming that similar mecha-nisms of apoptosis take place inside and outside thenervous system.

Concerning the neuronal differentiation phase itself,McAllister et al. (1995) showed that exogeneous neuro-trophins regulate dendritic growth in the developing vi-sual cortex. However, apical dendrites of pyramidal neu-rons responded more modestly to exogenous NT-3 than didbasal dendrites. This polarity response was even morepronounced for endogenous NT-3. Thus, only basal den-dritic growth was modulated by NT-3, positively or nega-tively, depending on the layers of the visual cortex consid-ered (McAllister et al., 1997). Interestingly, ourimmunohistochemistry results showed that TrkC NC2protein was preferentially expressed in apical dendrites ofpyramidal neurons of the cortex and hippocampus. Thisresult suggests that the TrkC NC2 receptor plays a role inthe asymetry of dendritic response to NT-3. As for BDNF,this neurotrophin was able to modulate dendritic growthresponse (McAllister et al., 1995). The TrkB receptors arepresent on the cell bodies and proximal apical and basaldendrites (Cabelli et al., 1996; Fryer et al., 1996). However,no asymetry (basal vs. apical) of truncated TrkB dendriticimmunostaining has been reported.

These data suggest that the noncatalytic TrkC NC2receptor modulates neuronal synaptic plasticity on bothshort-term and long-term signaling (Lo, 1995). NT-3 isinvolved in short-term plasticity in the CNS because itexerts acute effects on synaptic transmission (Lohof et al.,1993; Kang and Schuman, 1995). In addition, synapticactivity is required to allow neurotrophin regulation ofcortical dendritic growth (McAllister et al., 1996). Theconsolidation of these short-term effects into long-termchanges likely requires changes of neuronal morphologyand more specifically of dendritic arborization.

ACKNOWLEDGMENTS

We thank I. Mestres for excellent technical assistanceand C. Guillou for providing the animals. We are gratefulto Drs. Y. Ben-Ari, M. Khrestchatisky, and M. Wassef forcritical reading of the manuscript and for helpful com-ments. B. Menn was supported by a fellowship from theFrench Ministere de l’Education Nationale, del’Enseignement Superieur et de la Recherche.

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64 B. MENN ET AL.

differential expression of trkc catalytic and noncatalytic isoforms suggests that they act...

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