Ž .Molecular Brain Research 47 1997 1–10

Research report

Identification of preferentially expressed cochlear genes by systematicsequencing of a rat cochlea cDNA library

Ana Soto-Prior ), Mireille Lavigne-Rebillard, Marc Lenoir, Chantal Ripoll, Guy Rebillard,Philippe Vago, Remy Pujol, Christian P. Hamel´

INSERM U254 and UniÕersites de Montpellier 1 et 2, CHU Hopital Saint Charles, 34295 Montpellier Cedex 5, France´ ˆ ´

Accepted 26 November 1996

Abstract

Ž .107 expressed sequence tags ESTs from a rat cochlea cDNA library were identified by systematic sequencing coupled to databaseselection and RT-PCR analysis of novel sequences. This approach led us to select a clone, pCO8, showing no significant homology withany database sequence, that corresponds to a mRNA whose expression is restricted to the cochlea, except for traces detected in brain.Additional clones with novel sequences enriched in the cochlea were also found. ESTs bearing significant homologies with database

Ž .sequences 63 out of 107 were classified according to the putatively encoded protein. They include tissue-specific genes not previouslydescribed in the cochlea as well as known genes from other species. We performed in situ hybridization in cochlear tissues to localize thepCO8 mRNA and that of clone pCO6 which is 100% homologous to the delayed rectifier potassium channel drk1. We found that bothmRNAs were exclusively expressed in the cellular body of the primary auditory neurons from the spiral ganglion of the cochlea. Theseresults indicate that this approach is an efficient way to identify novel genes that could be of importance in cochlear function. q 1997Elsevier Science B.V. All rights reserved.

Keywords: Cochlea cDNA library; Systematic sequencing; RT-PCR; Hybridization, in situ; Novel gene; pCO8; drk1

1. Introduction

The mammalian auditory organ, the cochlea, is com-posed of highly specialized and precisely assembled tis-sues which provide proper sensitivity and selectivity to

Ž w x.stimulatory sounds see 36,43 . In the cochlea, the audi-tory message originates in the organ of Corti, a complexepithelial structure comprised of two types of sensory hair

Ž .cells inner and outer hair cells and various types ofsupporting cells. Inner and outer hair cells are connected totype I and type II primary auditory neurons, respectively,whose cellular bodies form the spiral ganglion of thecochlea. The mechano-electrical transduction occurring inhair cells is dependent on a high potassium concentrationin the endolymph, generated by another specialized epithe-lium, the stria vascularis. Given the unique features of

) Corresponding author. INSERM U254, Neurobiologie de l’Audition-Plasticite Synaptique, CHU Hopital Saint Charles, 34295 Montpellier´ ˆCedex 5, France. Fax: q33 6752-5601.´

these tissues and the complexity of the auditory mecha-nisms, it can be anticipated that many genes are preferen-tially or specifically expressed in the cochlea. Recentreports support this hypothesis. Several genes, includingsome homeobox genes, thought to play a role in cochleardevelopment, appear to be predominantly expressed in the

w xcochlear progenitor cells 17,37 . The relatively high inci-Ž .dence 1 out of a 1000 of children born with a hearing

impairment and the finding that mutations at many differ-w xent loci in both mice and human cause deafness 49 , are

also suggestive of genes exerting specific roles in thecochlea. However, very little is known of the molecularmechanisms responsible for the uniqueness of the cochlea,in part due to the limited amount of cochlear tissues. Infact, only a few studies have successfully addressed them olecu lar com position of coch lear tissuesw x12,22,32,38,39,41 .

Partial sequencing of randomly selected clones directlyfrom a cDNA library has been shown to be an excellentway of identifying new genes and describing the transcrip-

w xtional activity of a tissue or cell line 1,2,9,22,33 . Re-

0169-328Xr97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved.Ž .PII S0169-328X 97 00033-8

( )A. Soto-Prior et al.rMolecular Brain Research 47 1997 1–102

cently, cDNA libraries have been constructed either fromw xthe entire cochlea 24,32,39 or from the organ of Corti

w x55 , allowing the identification of cochlea-specific genes.In an attempt to gain rapid access to novel cochlear genesimplicated in auditory function and to simplify the processof isolating genes, we used the approach of systematicsequencing of a rat cochlea cDNA library. Randomlyisolated cDNA clones from the library were partially se-quenced and compared to database sequences. The expres-sion pattern of the unknown genes was analyzed by RT-PCR. We report the isolation of novel sequences preferen-tially expressed in the cochlea, as well as those of severalknown genes that we have classified, a number of whichencode structural proteins or enzymes previously associ-ated with hearing loss.

2. Materials and methods

2.1. Construction of a rat cochlear cDNA library

100 cochleas from 24-day-old Wistar rats were dis-sected out. Cochleas were homogenized with a potterhomogenizer in a denaturing solution containing 4 M

Ž .guanidine thiocyanate Fluka, Switzerland , 25 mM sodiumcitrate pH 7.0, 0.5% N-lauryl sarcosine, 0.1 M 2-mercapto-ethanol and RNA extracted according to the single-step

w x Ž .qmethod of Chomczynski and Sacchi 13 . Poly A RNAsŽ .were selected from the total RNA obtained 440 mg and

used to construct a directional cDNA library in lZAP IIŽ .Stratagene, USA . The cDNA library, which contained-1% non-recombinant phages in 5.8=106 pfusrml, wasamplified to a titer of 3=1010 pfusrml.

2.2. Isolation of cDNA clones

XL1 Blue E. coli were infected with the library phagesand plated out at low density to allow for separation ofindividual plaques. To evaluate the size of inserts, 54plaques were randomly isolated, inserts were PCR-ampli-fied using T3 and T7 primers and the product from eachclone was run on a 1% agarose gel. Subsequently, 107individual plaques, including the 54 PCR-amplified ones,were randomly cored, the phagemid ‘in vivo excised’Ž .Stratagene and purified.

2.3. Sequence analysis

Sequences at the 5X-end of the coding strand of thecloned cDNAs were obtained by manual sequencing usingPUCrM13 reverse primer. Partial nucleotide sequences ofclones were compared to the nucleotide sequences de-posited in Genbank andror EMBL using the Fasta pro-

w xgramme 35 . Clones showing no significant homologywith known cDNAs were selected, and their tissue expres-

sion patterns evaluated by RT-PCR. The cellular localiza-tion of cDNAs expressed within cochlear tissues wassubsequently analyzed by in situ hybridization.

2.4. Tissue expression analysis by RT-PCR

Total RNAs from various tissues – brain, cerebellum,eye, lung, kidney, cochlea and liver – were isolated asdescribed above. The cDNA was synthesized by randompriming using 2 mg of total RNA from each tissue and 9.6

ŽU of a MuMLV reverse transcriptase Eurogentec, Bel-.gium in a 10-ml volume. 1r10 of the RT product volume

was amplified by PCR in a 20-ml volume, using 20-mer-specific primers for each clone, as follows: 958Cr2 min

Žfollowed by 35 cycles 958Cr30 s, T -38Cr30 s, 728Cr1m.min and 728Cr5 min. 35 PCR cycles are sufficient to

detect cDNA fragments from 20 fg of a specific low-abun-dance mRNA in 2 mg of total RNA on an ethidium

w xbromide-stained gel 30 . In these conditions, abundantmRNAs will lead to early saturated amplified cDNAspreventing proper relative estimate of the expression levelsof these mRNAs. However, an intense cDNA amplifica-tion from only one or a few tissues is enough to assigntissue-predominant expression to the highly amplified, cor-responding RNA, providing that cDNAs from ubiquitouslyexpressed mRNAs could be amplified in all tissues. Thislatter requirement was done by amplifying a 176-bp frag-

Žment from the rat ATPase subunit 6 Genbank accession. Ž Xnumber M27315 with forward 5 -AGAAGGGTGAA-

X . Ž XTACATAGGC-3 and reverse 5 -CGACTAA -X.CAGCAAACATTAC-3 primers using the same PCR

programme. For each clone, the absence of contaminatingcDNA in the buffers and reagents was verified in a PCRreaction omitting the cDNA. To ensure that RNA did notcontain any detectable genomic DNA, RNAs from eachtissue were subjected to PCR amplification without previ-ous reverse transcription. This latter control was performedfor each of the 5 clones exhibiting a differential tissuedistribution. The presence of the desired PCR product wasascertained by electrophoresis on an ethidium bromide-stained 4% agarose gel in 1=TAE.

2.5. Cochlear expression analysis by in situ hybridization

2.5.1. TissuesŽRats were deeply anesthesized with pentobarbital 60

.mgrkg and perfused via the aortic arch with 50 ml of 0.1M sodium phosphate buffer pH 7.4, followed by 200 ml of

Ž .cold 48C 4% paraformaldehyde in the same buffer. Thecochleas were dissected from the temporal bones, post-fixed at 48C for 1 h in 4% paraformaldehyde and im-mersed for 5 min in the decalcifying solution DC3Ž .Labonord, France . The eyes and brains were similarlyprocessed except for the decalcification step. Then, thetissues were incubated overnight in the phosphate buffer

( )A. Soto-Prior et al.rMolecular Brain Research 47 1997 1–10 3

Žcontaining 20% sucrose, placed in OCT compound Miles,.USA , frozen and sectioned at 15 mm on a Reichert-Jung

2800 cryostat. The sections were mounted on gelatin-coatedslides and either used immediately or stored at y708Cuntil use.

2.5.2. ProbesLinearized drk1 and pCO8 cDNA templates were used

Žto synthesize digoxigenin-labeled sense linearized with. Ž .XhoI and antisense linearized with EcoRI riboprobes

Žaccording to the manufacturer’s protocol Boehringer.Mannheim, Germany . The length of drk1 and pCO8

riboprobes was 2900 and 426 nucleotides, respectively.The drk1 riboprobe was reduced to 400-nucleotide frag-

w xments by alkaline hydrolysis 4 . The probes were finallydenatured at 858C for 2 min immediately before hybridiza-tion.

2.5.3. Hybridization procedureThe tissue sections were permeabilized with 15 mgrml

Ž .of Proteinase K Bioprobe Systems, France in 0.1 MTris–HCl pH 8.0 and 50 mM EDTA at 378C for 15 min,post-fixed with 4% paraformaldehyde, acetylated, soaked

Žat room temperature in the pre-hybridization buffer 4=

SSC, 1=Denhart’s solution, 1% sodium N-lauryl sarco-. w xsine for 1 h, dehydrated and air-dried 42 . They were

Žthen incubated in 20 ml of hybridization buffer 50%deionized formamide, 4 mM EDTA, 0.2% sodium N-laurylsarcosine, 600 mM NaCl, 0.05% disodiumpyrophosphate,0.05% tetrasodiumpyrophosphate, 80 mM Tris–HCl pH

.7.5 containing 4 to 20 ngrml of the labeled riboprobe.Cochlear sections were hybridized overnight at 508C in

a moist chamber, washed at room temperature in 4=SSCŽfor 15 min and then treated with ribonuclease A 100

.mgrml, 30 min at 378C in 2=SSC. They were thensuccessively washed in 2=SSC for 1 h at room tempera-ture, in 0.1=SSC for 15 min at 508C and again in thesame buffer for 10 min at room temperature. They werefinally processed for digoxigenin immunodetection using

Žthe DIG nucleic acid detection kit Boehringer Mannheim,.Germany according to the manufacturer’s protocol. Sec-

tions were examined with a Reichert Polyvar microscope.

3. Results

In order to evaluate the insert sizes of our amplified ratcochlea cDNA library, we initiated a pilot study by ran-dom selection of 54 clones. The sizes of the inserts werebetween 400 and 4300 nucleotides, among which 1.8%were -0.5 kb, 27.7% were 0.5–1 kb and 70.4% were)1 kb.

3.1. Classification and database selection of sequencedclones

107 clones were randomly isolated. On average, 130nucleotides of sequence were obtained from each clone. Acomprehensive view of the strategy used and of the resultsobtained is illustrated in Fig. 1. The nucleotide sequenceswere analyzed and compared to those in Genbank andEMBL databases. Clones showing either nucleotide identi-ties to database sequences )85% or FASTA scores )150were considered as identified genes; those showing nu-cleotide identities -85% or scores -150 were classifiedas unknown genes. Following this first screening, 63 clonesŽ .58.8% were classified as identified genes and 40 clonesŽ .37.4% represented unknown rat genes. Four clones werenot interpretable due to a poly-T tail at both the 3X- and5X-ends of the insert.

Among the 63 identified clones, 58 corresponded topreviously characterized genes and 5 to sequence tagsfrom various tissues that we classified as uncharacterized

Ž .genes see Table 1 for summary of results . Among the 58characterized clones, 37 corresponded to previously de-scribed rat genes and 21 were homologous to genes fromother species, including mouse, bovine, monkey and hu-man, therefore, probably representing their rat counterpart

Fig. 1. Distribution and expression analysis of clones obtained from the systematic sequencing of the rat cochlea cDNA library.

( )A. Soto-Prior et al.rMolecular Brain Research 47 1997 1–104

Table 1Rat cochlea ESTs matched to known genes in GenbankrEMBL databases

Clone Accession Putative identified protein and its accession number Species Score % Ntnumber

MetabolismŽ .pCO3 AA108320 ATP synthase g-subunit gbL19927 Rat 272 97.3 72

Ž .pCO9 AA108321 Mitochondrial genome, ORF 41 gbJ01435 Rat 453 99.1 115Ž .pCO21 AA108322 UV-damaged DNA-binding protein emL20216 Mon 365 93.5 106

Ž .pCO24 AA108323 Mitochondrial genome, ORFa6 gbM27315 Rat 453 93.5 132Ž .pCO30 AA108262 Protective protein gbJ05261 Mou 303 87.9 115

Ž .pCO32 AA108263 Cytochrome oxidase subunit II gbJ01435 Rat 543 97.2 114Ž .pCO38 AA108264 Stearyl-CoA desaturase gbJ02585 Rat 222 84.1 83

q q Ž . Ž .pCO41 AA108265 Na , K -ATPase a q gbM14512 Rat 274 97.2 76q Ž .pCO44 AA108266 NAD -dependent isocitratedehydrogenase emU07980 Bov 358 83.8 134

Ž .pCO46 AA108267 Peroxisomal 3-ketoacyl-CoA thiolase gbD90055 Rat 301 97.4 79Ž .pCO55 AA108268 Prostaglandin-H-2 D-isomerase gbM94134 Rat 614 98.7 157

Ž .pCO62 AA108269 Histone-1 gbM29260 Mou 233 79.1 103Ž .pCO75 AA108270 Ubiquinone oxidoreductase emT07882 Hum 302 76.8 127

Ž .pCO78 AA108271 L-Arg: Gly amidinotransferase gbU07971 Rat 454 91.6 149Ž .pCO86 AA108272 Cytochrome oxydase III gbJ01435 Rat 862 98.2 225

Ž .pCO89 AA108273 Aldolase A gbM12919 Rat 685 93.5 213Ž .pCO91 AA108274 Carbonyl reductase gbX84348 Rat 613 100 183

Ž .pCO92 AA108275 a-Globin gbM17083 Rat 176 96.1 50Ž .pCO96 AA108276 a-Globin gbM17083 Rat 729 99.5 185

Ž . Ž .pCO100 AA108277 Heat-shock protein hsp-E7I gbL40406 Mou 446 86.8 158Ž .pCO102 AA108278 Cytochrome b gbJ01436 Rat 448 98.3 118Ž .pCO107 AA108279 Cytochrome b gbX14848 Rat 125 91.1 42Ž .pCO113 AA108280 Cytochrome b gbX14848 Rat 733 96.0 200

Ž .pCO123 AA108281 Phosphatase 2A-b protein emM23591 Rat 117 96.8 38qŽ . Ž .pCO130 AA108282 S -Malate NADP oxidoreductase emM26594 Rat 641 98.2 168

Ž .pCO133 AA108283 b-Globin emX67613 Rat 421 95.9 123Ž .pCO136 AA108284 Carbonic anhydrase III emM22413 Rat 476 89.6 205

Cell signaling and transportersŽ .pCO6 AA108285 Drk1 potassium channel gbX16476 Rat 296 100 74

Ž .pCO42 AA108286 Guanine nucleotide regulatory protein emU01147 Hum 342 93.8 96Ž .pCO49 AA108287 Chloride channel RCL1 gbD13985 Rat 153 80.0 61

Ž .pCO51 AA108288 Ras-related protein gbJ02998 Rat 323 89.5 130Ž .pCO52 AA108289 Stathmin gbJ04979 Rat 371 94.2 122Ž .pCO63 AA108290 Calretinin gbX66974 Rat 320 92.5 137

q Ž .pCO87 AA108291 Mitochondrial H rphosphate symporter gbM23984 Rat 884 96.7 241Ž . Ž .pCO93 AA108292 ADP ribosylation factor ARF -like protein gbX16476 Rat 468 99.2 215

Ž .pCO112 AA108293 a-Platelet-derived growth factor receptor gbM63837 Rat 620 100 155Ž .pCO120 AA108294 14-3-3 Protein b-subtype emD17446 Rat 526 96.6 145Ž .pCO121 AA108295 GDP-dissociation inhibitor emX79353 Hum 246 85.1 142

StructuralŽ .pCO43 AA108296 Schwann cell peripheral myelin protein P0 gbK03242 Rat 316 100 97

Ž .pCO53 AA108297 Myelin proteolipid protein gbM11185 Rat 364 98.9 97Ž .pCO70 AA108298 Microtubule-associated protein 1A gbM83196 Rat 664 100 166

Ž .pCO82 AA108299 b-Tubulin gbM28730 Mou 116 87.5 39Ž .pCO85 AA108300 a-Collagen gbU16789 Mou 178 95.8 55

Ž .pCO90 AA108301 b-Tropomyosin gbL00372 Rat 673 98.9 173Ž . Ž .pCO116 AA108302 Peripheral myelin protein P0 emM62857 Hum 404 95.5 127

Ž .pCO129 AA108303 Cell-binding bone sialoprotein emJ04215 Rat 290 88.9 100Transcription factors and translation machinery

Ž .pCO27 AA108304 Ribosomal protein L7 gbM17422 Rat 230 94.2 93Ž .pCO29 AA108305 Primase large subunit gbD13545 Mou 198 85.1 118

Ž .pCO50 AA108306 Transcription factor P45 NF-E2 gbL09600 Mou 309 87.6 107Ž .pCO94 AA108307 Ribosomal protein L7 gbM17422 Rat 557 98.0 147

Ž .pCO97 AA108308 DNA-binding protein mdm2 gbX58876 Mou 320 78.9 186Ž .pCO104 AA10830 9 Ribosomal protein S10 gbX13549 Rat 609 96.9 161

Ž .pCO109 AA108310 Testis-specific transcription elongation factor emD12927 Rat 482 94.0 145Neuron-specific

Ž .pCO16 AA108311 Neuron-specific MAP kinase gbL35236 Mou 409 91.8 125Ž .pCO71 AA108312 Latexin gbX76985 Rat 441 97.5 118

( )A. Soto-Prior et al.rMolecular Brain Research 47 1997 1–10 5

Ž .Table 1 continued

Clone Accession Putative identified protein and its accession number Species Score % Ntnumber

Secreted factorsŽ .pCO80 AA108313 Chromogranin B gbX53028 Mou 280 87.5 110Ž .pCO95 AA108314 Apolipoprotein D gbX55572 Rat 526 90.3 165

Cell surfaceŽ .pCO106 AA108315 Synaptophysin gbX06388 Rat 425 97.4 121

Not characterizedpCO4 AA108316 EST from embryonal carcinoma F9 cell cDNA, 82E07 Mou 184 85.9 85

.emD21724X Ž .pCO79 AA108317 cDNA clone 69812 5 EST emT49922 Hum 300 80.6 117X Ž .pCO81 AA108318 cDNA clone 74208 3 EST emT48384 Hum 306 93.8 126

Ž .pCO118 AA108319 mRNA for ORF emD26068 Hum 347 82.3 133Ž .pCO122 AA109476 cDNA clone HEA35T EST emZ36286 Hum 410 91.7 166

Ž .Classification of the 63 identified sequences out of 107 randomly sequenced clones from the rat cochlea cDNA library. Database searches were done inŽ . Ž .Genbank gb and EMBL em . Species are mok for monkey, bov for bovine, mou for mouse and hum for human. Scores were established using the

Ž . Ž . Ž .FASTA programme see Materials and methods . Percentages of identity % and nucleotide overlap nt between clone and identified sequences areindicated.

genes. Among the 63 identified clones, three bore the samemitochondrial cytochrome b sequences, two coded for theribosomal protein L7 and two for a-globin.

From our 58 clones homologous to characterized genes,Ž .46.5% ns27 are related to metabolism. Of these, 17

clones participate in energy metabolism, 2 are related toDNA metabolism and 8 participate to other metabolisms.Less numerous are the clones coding for proteins ranged in

Ž .the categories of cell signaling and transporters 18.9% ,Ž .structural proteins 13.8% and transcription factors and

Ž .translation machinery proteins 12.0% . A few number ofŽ .clones 8.6% belong to scarcely represented categories,

including neuron-specific, secretory and cell surface pro-teins.

3.2. Expression analysis of selected clones

The 40 clones showing no significant identity with anysequence in the databases were selected for tissue expres-sion analysis. RT-PCR from seven different rat tissuesŽ .cochlea, eye, brain, cerebellum, lung, kidney and liverwas performed with each selected clone. The expression of

Žthe ATPase subunit 6 was equivalent in the 7 tissues Fig..2A , indicating proper quality of RNAs. As a result of this

expression screening, 35 clones exhibited ubiquitous tissueexpression whereas 5 showed a differential tissue distribu-tion. These five latter clones, namely pCO101, pCO115,pCO119, pCO135 and pCO8, received particular attention.For each of these 5 clones, the absence of detectable

ŽcDNA fragments when RNA was directly amplified see.material and methods indicated that RNAs were not con-

Ž .taminated with genomic DNA not shown .Ž .Clones pCO115 and pCO119 Fig. 2A showed low

expression levels in the cochlea compared to those in someŽother tissues brain, cerebellum, eye and liver in the case

.of pCO115 and brain, eye and lung in the case of pCO119 .Ž .In contrast, clones pCO101, pCO135 Fig. 2A and pCO8

Ž .Fig. 2B showed their highest level of expression in thecochlea indicating a preferential expression in this tissue.Clones pCO101 and pCO135 exhibited additional strong

Fig. 2. Expression of selected unkown genes using RT-PCR analysis invarious tissues. Total RNAs were reverse transcribed, amplified by PCR

Ž .using clone-specific primers see Materials and methods and the PCRproducts analyzed on a 4% agarose gel stained with Ethidium bromide:Br, brain; Cb, cerebellum; Ey, eye; Lu, lung; Kd, kidney; Co, cochlea; Li,

Ž .liver; No, no cDNA. A: pCO115 124 bp , for this clone, the lane 4 is) Ž . Ž . Ž .heart , pCO119 111 bp , clones: pCO101 153 bp , pCO135 142 bp ,

Ž .ubiquitously expressed ATPase subunit 6 176 bp . B: expression ofpCO8 clone. Same legends as in A and Mu, muscle; Te, testis; He, heart.A 124-bp band corresponding to a pCO8 cDNA fragment is present in thecochlea and is hardly visible in brain.

( )A. Soto-Prior et al.rMolecular Brain Research 47 1997 1–106

Ž . Ž .expression in brain both clones and eye pCO101 , andŽ .lower expression levels in lung both clones , cerebellum

Ž . Ž .pCO101 and eye pCO135 .Clone pCO8 was the sole one to be noticeably ex-

Ž .pressed in only one tissue, that is the cochlea Fig. 2B . ItŽwas not found in eight other tissues cerebellum, eye,

.kidney, liver, skeletal muscle, testis, heart, lung . How-ever, a slight band was apparent in brain although it wasnot consistently present in repeated experiments. Onlytraces in other tissues were detected when a 40-cycle PCR

Ž .was performed not shown . Consequently, pCO8 wasconsidered as a cochlear-restricted cDNA.

The cellular expression of the unknown pCO8 cloneŽand of the identified pCO6 clone 100% identical to

.nucleotides 823–890 of the rat drk1 potassium channelwere examined by in situ hybridization. Clone pCO8 waschosen because of its predominant expression in the cochleaand clone pCO6 because of the importance of potassiumchannels in cochlear physiology. We observed that both

Ž . Ž .pCO8 Fig. 3A and drk1 Fig. 4A mRNAs were stronglyexpressed in the type I spiral ganglion neurons. They werelocalized only in the cytoplasm of these neurons, the nucleiand the axons were not stained. The intensity of the signalwas uniform all along the cochlear turns. Although thedistribution of the two mRNAs was similar, the signalintensity observed with the pCO8 riboprobe was higherthan that observed with the drk1 riboprobe. A minorpopulation of type I spiral ganglion neurons remainedweakly stained or unstained. The glial cells surroundingthe type I neuronal cell bodies appeared unstained with

Ž . Ž .pCO8 Fig. 3B as well as with pCO6 Fig. 4B . The typeII spiral ganglion neurons, characterized by a small cellbody and by the absence of myelin are difficult to identifyon cryostat sections. Putatively identified type II spiralganglion neurons were not stained with the drk1 and pCO8riboprobes. Border effects could be seen on some sections

Ž .with the stria vascularis and the organ of Corti Fig. 3A ;they do not represent authentic hybridization signals. No

Žspecific staining was observed with the sense probe Fig..4C .

Fig. 3. In situ hybridization analysis of the pCO8 clone in an adult ratŽ .cochlea using digoxigenin-labeled antisense riboprobes A,B . A: general

Ž . Ž .view of the first 1 and second 2 turn of the cochlea showing thelocalization of the mRNA of this clone in the spiral ganglion neuronsŽ . Ž .SG . No staining is observed in the organ of Corti asterisks at the

Ž . Ž .levels of the inner hair cells I and outer hair cells O . Other cochlearŽstructures SV; stria vascularis, sl, spiral limbus, osl; osseous spiral

.lamina are also not stained. B: high magnification of the spiral ganglionin the first turn of the cochlea showing the localization of the pCO8mRNA in the cytoplasm of the primary auditory neurons. Stained type I

Ž .auditory neurons large arrows are surrounded by unstained glial cellsŽ . Ž .small arrows . Some type I auditory neurons are not stained arrowheads

Ž .as well as possible type II auditory neurons double arrows , character-ized by a small cellular body and by the absence of myelin. Scale

Ž . Ž .barss100 in A and s20 mm in B .

Because of a slight expression of pCO8 mRNA in theŽ .brain see results of RT-PCR analysis , we performed in

situ hybridization in several parts of brain using the sametissue treatment and hybridization procedure than those forthe cochlea. As a positive control, we used the pCO6riboprobe since drk1 is highly expressed in the CNSw x16,19,21,52 . All the brain structures examined, includinghippocampus, cortex, thalamus, hypothalamus and those of

( )A. Soto-Prior et al.rMolecular Brain Research 47 1997 1–10 7

Žthe auditory pathway superior olivary complex, cochlear.nuclei, inferior colliculus and lateral lemniscus , remained

negative with pCO8 whereas various neuronal structuresŽ .were labeled with pCO6 not shown .

4. Discussion

In order to identify genes important for inner ear func-tion and potentially involved in hearing loss, we haveexamined a rat cochlea cDNA library. This library containsa sufficient number of clones to include representation ofrelatively scarce mRNAs, even with the diversity of tissuesthat make up the cochlea. 107 sequence tags were ran-domly collected and classified according to the type of theencoded protein. Our approach allowed us to successfullyidentify several new mRNAs highly expressed in thecochlea, including one, pCO8, almost exclusively ex-pressed in this tissue.

Our primary goal was to identify cochlea-specific mR-NAs, based on the assumption that they must code forproteins important in cochlear function, rather than to edit

Ž .an expressed sequence tag EST catalogue of the cochlea.Other approaches using cochlear cDNA libraries for theidentification of cochlea-specific mRNAs have been re-

w xported 39,41 . In these works, specific mRNAs wereselected by various techniques used in combination, in-

w x w xcluding subtraction of libraries 38 , Northern blotting 39w xand in situ hybridization 41,8 . To avoid the drawbacks of

the subtraction procedure, we chose a systematic sequenc-ing approach driven by a primary database selection ofunknown sequences followed by a secondary selectionbased on the expression analysis by RT-PCR. Although theresults of RT-PCR analysis should be interpreted withcaution due to variations in the efficiency of amplificationin different tissues, we believe that great differences inamounts of an amplified cDNA are the reflection of au-thentic differences in amounts of mRNAs. We consideredthat RT-PCR was the easiest and fastest technique toexamine the expression of many clones. In fact, large-scalescreening of expression obviates the use of time consum-ing Northern blots or in situ hybridization at this step.Only for those mRNAs which were selected for theirhighest interest was in situ hybridization applied as thefinal step.

One of the most noticeable features of our ESTs is thatŽmitochondrial transcripts are fairly abundant 15.9% of the

.total of the characterized clones compared to percentagesw xfound in other tissues such as 12% in heart 29 , 1.7% in

Fig. 4. In situ hybridization analysis of drk1 expression in the basal turnŽ .of the adult rat cochlea. Digoxigenin-labeled antisense A,B and sense

Ž .C riboprobes were hybridized to cryostat sections. A: primary auditoryŽ .neurons of the spiral ganglion SG display a high hybridization signal

Ž . Ž .while inner I and outer hair cells O remain negative. B: stained type IŽ .auditory neurons large arrows are surrounded by unstained glial cells

Ž .small arrows . Some auditory neurons are unstained: they included a fewŽ .type I auditory neurons arrowheads and possible type II auditory

Ž .neurons double arrows . C: with the sense riboprobe, no staining isŽ . Ž .visible in primary neurons arrowheads . Scale barss100 in A and

Ž .s10 mm in B,C .

( )A. Soto-Prior et al.rMolecular Brain Research 47 1997 1–108

w x w xfetal brain 1 , 10.3% in hippocampus 1 , 1% in pancreaticw x w xislet cells 50 and -0.2% in HepG2 cells 33 . However,

w xin EST catalogs from adult brain and skeletal muscle 26higher frequencies of mitochondrial sequences, 50 and24.8%, respectively, were observed. The finding of rela-tively high levels of mitochondrial messages in our cochlearlibrary may be a reflection of the abundance of mitochon-

w xdria in hair cells and stria vascularis 48 . This observationand recent work reporting mutations of mitochondrial genesin multisystemic disorders that often include hearing lossw x6,54 indicate the importance of mitochondrial metabolism

Ž .in the cochlea. The percentage 37.4% of unknown genesfound in the present study is in accordance with that

w xreported in recent large-scale EST studies 5,28 . Only 1out of 40 clones showed a highly predominant cochlearexpression; if we assume that the other 63 identified clonesare all expressed in the cochlea and in other tissues, thiswould indicate that -1% of the clones in our samplerepresent mRNAs predominantly expressed in cochlea.

The identities of our randomly picked sample of cochlearclones appears as a good molecular signature of the cochlea.In fact, we found proteins largely distributed in the organof Corti such as the extracellular matrix protein a collagenw x w x23 , the cytoskeletal proteins b tubulin 45,53 and b

w xtropomyosin 7,46 . Proteins present in many parts of thecochlea were also identified. For example, theNaqrKqATPase, previously described in the stria vascu-laris, spiral limbus, spiral ligament and subpopulations of

w xcochlear neurons of the spiral ganglion 40,57 and calre-tinin, a calcium-binding protein which has been detected inthe inner hair cells, supporting cells and spiral ganglion

w xneurons 14,15 , were found. Another structural protein,w xthe Schwann cell peripheral myelin protein, PMP 0, 27

obviously involved in myelination and nerve conduction ofthe type I auditory neurons of the spiral ganglion, wasidentified. Recent assignment of mutations to genes coding

w x q q w xfor a collagen 11,25 and Na rK ATPase 44 thatcauses hearing loss indicate that many cochlear genescould be involved in genetic deafness.

Even though many of the clones identified largelyrepresent housekeeping genes, some others can be classi-fied as tissue-specific genes. We did not examine theexpression of these clones at the cellular level in thecochlea since our study was aimed at isolating newcochlea-specific genes. Yet, in situ hybridization studies ofsuch clones might reveal interesting cell-specific expres-sion in the cochlea. For example, a clone named pCO109was found to code for the SII transcription factor. Thisgene has been reported to be specifically transcribed in

w xtestis 56 . Clone pCO16 codes for a neuron-specific MAPŽ .kinase accession number gbL35236 . Clones pCO50 and

pCO129 code for proteins homologous to the transcriptionfactor P45 NF-E2 and a cell-binding bone sialoprotein,respectively, which show tissue-specific expression. Theformer one is restricted to haematopoietic tissues and cell

w xlines 3 while the latter one is restricted to bone matrix

w x34 . Clone pCO42 codes for a guanine nucleotide regula-w xtory protein weakly present in all tissues except brain 51 .

One of our characterized clones corresponds to theq w xdelayed rectifier K channel drk1 18 . This channel was

not identified before in the cochlea. Since potassium is thephysiological charge transporter of the mechano-electricaltransduction in hair cells and because it creates the hyper-polarization current induced by acetylcholine in outer hair

w xcells of the organ of Corti 10 , we analyzed its cellularlocalization in the cochlea by in situ hybridization. Wehave not found drk1 mRNA in the organ of Corti; instead,it was exclusively expressed in the cell body of most of thetype I auditory neurons. This is in accordance with previ-ous works reporting its localization in neuronal cell bodies

w x w xof the brain 16,19,21,52 and of the retina 20 in adult rat.The cDNA of another delayed rectifier potassium channel,NGK2, was recently isolated from the chick auditorysensory epithelium. It is predominantly expressed at the

w xapical end of the basilar papilla 31 . This latter observa-tion suggests that certain delayed rectifier Kq channelsmay be involved in hair cell physiology.

Most importantly, pCO8, one of the clones that did notshow significant identity with any database sequence wasexpressed in the type I auditory neurons of the cochlea. Noexpression was found in any other tissues examined, in-cluding other neurons of the auditory pathway, although alow level of expression was detected in brain by RT-PCR.This implies that primary auditory neurons could haveparticular features. In fact, 60% of the type I neurons havea high spontaneous rate of activity which correlates with alow threshold to a stimulatory sound. They are highlyactive neurons, both metabolically and electrically and

w xshow specific calcium buffer systems 47,14 . However,very little is known about the molecular mechanisms im-plicated in the physiology of the auditory response and theelements involved in the regulation of the metabolic path-way of these neurons. Isolation and characterization ofmolecules predominantly expressed in neurons of the spi-ral ganglion, as is pCO8, should allow better understand-ing of their functional characteristics.

5. Conclusions

Identification of ESTs using a systematic sequencingapproach coupled to RT-PCR analysis is a rapid and usefulway to characterize novel genes expressed in the cochlea.Using this strategy we have identified and characterizedseveral novel cDNA clones from a rat cochlea cDNAlibrary that exhibit differential tissue expression, including3 clones preferentially expressed in the cochlea. One ofthem, pCO8, almost exclusively found in the primaryauditory neurons of the cochlea except for low levels ofexpression in the brain, is currently under investigation.Additional known cDNAs not previously reported in thecochlea were also isolated, including the drk1 potassium

( )A. Soto-Prior et al.rMolecular Brain Research 47 1997 1–10 9

channel which is highly expressed in the type I auditoryneurons of the cochlear spiral ganglion. Our resultsdemonstrate the utility of this methodology and its abilityto identify preferentially expressed cochlear genes.

Acknowledgements

We want to gratefully thank M. Eybalin and J.L. Puelfor comments on the manuscript and J.L. Pasquier forphotographic work. EST sequences from clones corre-sponding to unknown genes are available in GenbankŽunder accession numbers: U74018 to U74052 and U75317

.to U75320 .

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