Expression profiles of the connexin genes, Gjb1 and Gjb3, in thedeveloping mouse cochlea

Nuria Lopez-Bigasa,b, Maria L. Arbonesa,b,*, Xavier Estivilla,b, Lionel Simonneauc

aGenes and Disease Program, Centre de Regulacio Genomica, Barcelona, SpainbMedical and Molecular Genetics Center, Institut de Recerca Oncologica-IRO, L’Hospitalet de Llobregat, Barcelona, Spain

cLaboratoire de Neurobiologie de l’Audition-Plasticite synaptique, INSERM U254, Montpellier, France

Received 10 July 2002; received in revised form 12 August 2002; accepted 14 August 2002

Abstract

Several connexin genes (GJB1, GJB2, GJB3, GJB6 and GJA1) have been found mutated in patients with non-syndromic and/or syndromic

deafness indicating an important role of these proteins in the auditory system. In order to better understand the function of the connexins in

the inner ear we have analyzed the gene expression profiles of two connexin genes, Gjb1 (connexin 32) and Gjb3 (connexin 31), by in situ

hybridization during the mouse cochlea organogenesis, from early otocyst up to the mature organ in adult. In the developing otocyst

epithelium, some restricted domains expressed Gjb3 and Gjb1 whilst high levels of both transcripts were present in the surrounding

mesenchymal tissue. As development proceeds, expression of these two genes was found in various subtypes of fibrocytes, either within

the spiral limbus or along the spiral ligament, as well as in the basilar membrane cells, in the Reissner’s membrane cells, and in subsets of the

cellular elements of the cochlear ganglion. Gjb3 and Gjb1 expression was spatiotemporally modulated within the sensory hair cells and the

various supporting cells that compose the developing organ of Corti. A transitory expression of Gjb1 was found in the basal and intermediate

cells of the stria vascularis. In the adult cochlea Gjb1 transcripts disappeared while Gjb3 expression remained present in fibrocytes with

specific expression patterns. q 2002 Elsevier Science B.V. All rights reserved.

Keywords: Mouse development; Cochlea; Inner ear; Gap junctions; Deafness; Hearing loss; Organ of Corti; Beta-connexins; Connexin 31; Connexin 32;

Connexin 31.1; Gjb3; Gjb1; Gjb5

1. Results and discussion

Connexins, the subunits that form gap junctions, play an

important role in intercellular communication (Kumar and

Gilula, 1996). Five connexin genes, including GJB1

(Bergoffen et al., 1993) and GJB3 (Xia et al., 1998), cause

deafness when mutated (Rabionet et al., 2002). In order to

understand the role of connexins in the inner ear morpho-

genesis and in auditory function, we report here the spatio-

temporal expression patterns of two beta-connexin genes

(Gjb1 and Gjb3) during mouse cochlear development and

maturation.

1.1. Non-sensory-neural areas

At the first stage studied, embryonic day (E) 12, an

intense Gjb3 and Gjb1 expression was present in the

surrounding mesenchymal tissue and within some areas in

the epithelium of the otocyst (Figs. 1A and 2A). By E15,

when hair cells differentiation is just beginning, Gjb3 and

Gjb1 messenger RNAs (mRNAs) were still highly present in

the neighbouring mesenchyme, and were also detected in

the cochlear epithelium, Gjb3 as small puncta within the

presumptive cochlear sensory domain and Gjb1 within the

rostral wall of the cochlear duct from which the Reissner’s

membrane will form (Figs. 1B and 2B).

At neonatal stages, strong Gjb3 and Gjb1 gene expression

was detected in the Reissner’s membrane, in the basilar

membrane cells and in fibrocytes either neighbouring the

developing lateral wall and stria vascularis or, in the dark

and the light regions of the spiral limbus (Figs. 1C,D,F and

2C–F). At birth, the developing type II fibrocytes were

highly expressing Gjb3 and Gjb1 (Figs. 1F and 2F). With

a lesser intensity, the expression of both genes was main-

tained in fibrocytes, types II and IV, of the lateral wall and in

some fibrocytes within the spiral limbus, in the Reissner’s

membrane and in the basilar membrane cells up to postnatal

day (P) 13 (Figs. 1G,H,J,K and 2G,H,J,K). An intense Gjb1

expression persisted at P20 in type II fibrocytes (Fig. 2M).

Fibrocytes nomenclature was according to Kimura et al.

Gene Expression Patterns 2 (2002) 113–117

1567-133X/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved.

PII: S0925-4773(02)00299-X

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* Corresponding author. Tel.: 134-93-224-0900; fax: 134-93-224-0899.

E-mail address: mariona.arbones@crg.es (M.L. Arbones).

N. Lopez-Bigas et al. / Gene Expression Patterns 2 (2002) 113–117114

Fig. 1. Gjb3 expression in the developing mouse cochlea. The stage is indicated at the upper right corner of the pictures. (A, B) Gjb3 expression at E12 and E15

in the mesenchyme surrounding the otocyst (asterisk) and in subdomains of the otocyst (arrowhead). (C) The expression at E18 was detected in fibrocytes (Fc)

of the lateral wall (lw) and basilar membrane cells (bmc) but not in the organ of Corti (oc). (D) Expression in Reissner’s membrane cells (Rm), Claudius cells

(Cl) and bmc at P0. (E) Micrograph of the spiral ganglion (g) at P0 showing Gjb3 expression in the outer cellular elements (arrowhead). (F) Expression in Fc

type II (FcII) neighboring the stria vascularis (sv) and the lw. (G) Micrograph of a cochlea turn at P7 showing expression in cells of the inner sulcus (isc), of the

regressing Kolliker organ (k), in the Rm, in FcII and in Fc type IV (FcIV). (H) Higher magnification of the micrograph in panel G at the organ of Corti showing

expression in the outer hair cells (ohc), pillar cells (Pc) and bmc but not in the inner hair cells (ihc). (I) Expression at P7 was also found in Fc but not in the sv.

(J) Micrograph showing two turns of a P13 cochlea, the oc of the apical and basal turns are pointed with arrowheads. (K) Higher magnification of panel J

showing high expression in Deiter cells (Dc), Hensen cells (Hc) and bmc. (L) At P13 the expression was present in FcII but not in the Rm or in the sv. In the

adult cochlea expression was absent in the oc (M), but was observed in FcII, Fc type III (FcIII), FcIV, tension fibroblasts (TFb), stellate limbal cells (slc) of the

spiral limbus and, in some neurons in the cochlear g (N). Bars represent 100 mm (J), 50 mm (A, B, G, N), 25 mm (C, D, E, F, I, L, M) and 10 mm (H, K).

N. Lopez-Bigas et al. / Gene Expression Patterns 2 (2002) 113–117 115

Fig. 2. Gjb1 expression in the developing mouse cochlea. The stage is indicated at the upper right corner of the pictures. (A, B) Gjb1 expression at E12 and E15

in the mesenchyme (asterisk) surrounding the otocyst and in subdomains of the otocyst (arrowheads). (C) Gjb1 transcripts at E18 were detected in Fc, Rm cells,

bmc and in Hensen cells (Hc) and Pc of the organ of Corti. (D) Expression, at P0, was detected in the limbal dark cells (ldc) and limbal light cells (llc) of the

spiral limbus, in the Rm and in the spiral g cells. (E) Higher magnification of panel D at the organ of Corti showing strong expression in Hc, Pc and bmc. (F)

Intense Gjb1 expression in FcII, in the Rm and in intermediate and basal cells (arrow) of the sv. (G) Micrograph of a cochlea turn at P7, showing expression in

ganglion cells, in the isc, in the regressing Kolliker (k) and in the Rm. (H) Magnification of panel G at the organ of Corti showing expression in the ohc, ihc, Pc,

Hc and bmc. (I) Magnification of the sv at P7 showing intense expression in FcII and the basal and intermediate cells of the sv (arrow). (J) Micrograph of a

cochlea turn at P13 showing uneven expression within the g cells. (K) Magnification of panel J at the organ of Corti showing negative or very weak Gjb1

expression. (L) Magnification of a cochlea section at P13 showing FcII expression in the suprastial zone close to the sv. (M) Gjb1 expression in FcII at P20. (N)

Micrograph of an adult cochlea turn showing absence of expression. Bars represent 100 mm (J), 50 mm (A, B, D, G, N), 25 mm (C, L, M) and 10 mm (E, F, H, I,

K).

(1990) and Kikuchi et al. (1995). Note that, at P0 and P7, no

Gjb3 mRNA was detected within the whole stria vascularis

but Gjb1 transcripts were observed in its deeper cellular

layers, basal and intermediate cells (Figs. 1F,I and 2F,I).

The stria vascularis cellular layer terms were according to

Lavigne-Rebillard and Bagger-Sjoback (1992).

In adult, while no Gjb1 transcript was observed in any

part of the cochlea (Fig. 2N), intense Gjb3 expression was

restricted to types I, III and IV fibrocytes, in areas of the

lateral wall that also contain tension fibroblasts, and to stel-

late type fibrocytes of the spiral limbus (Fig. 1N). At neona-

tal stages, the Kolliker’s organ was devoid of Gjb3 and Gjb1

expression but, interestingly, at P7, when its regression is

committed, gradually leading to the inner sulcus cellular

layer, both tissues contained Gjb3 and Gjb1 transcripts

(Figs. 1G and 2G).

Another beta-connexin gene analyzed, (Gjb5, coding for

connexin 31.1) was nearly undetectable at every develop-

mental stage except at birth, where Gjb5 transcripts were

observed in the Reissner’s membrane and in fibrocytes of

the lateral wall and of the spiral limbus (data not shown).

1.2. Organ of Corti

At E18 or P0, no Gjb3 transcripts were detected but Gjb1

mRNA was observed in the developing pillar and Hensen

cells (Figs. 1C,D and 2C–E). Gjb1 expression remained in

these cells at P7 but extended to the inner hair cells (IHCs)

and, more strongly, to the outer hair cells (OHCs) (Fig. 2H).

A rather similar but faint pattern of expression was observed

with Gjb3 probes except that the IHCs were negative (Fig.

1H). Note that the underlying basilar membrane cells were

intensively expressing both genes (Figs. 1H and 2H). At

P13, mainly at the apical cochlear turn, Gjb3 transcripts

were strongly present in Deiter’s and Hensen’s cells (Figs.

1J,K), whereas Gjb1 expression was nearly at background

level (Fig. 2K). Neither of these genes was expressed in the

organ of Corti in adult mice (Figs. 1M,N and 2N).

1.3. Ganglion

In mouse embryos, Gjb3 and Gjb1 expression in early

statoacoustic ganglion and in the developing cochlear gang-

N. Lopez-Bigas et al. / Gene Expression Patterns 2 (2002) 113–117116

Table 1

expression patterns in the developing mouse cochleaa

Gjb3 (Connexin 31) Gjb1 (Connexin 32)

E12 Neighbouring mesenchyme (111) Neighbouring mesenchyme (111)

Otocyst subdomains (1) Otocyst subdomains (1)

E15 Neighbouring mesenchyme (111) Neighbouring mesenchyme (111)

Small puncta within presumptive sensory domains (1) Future ectodermal Reissner’s membrane (11 )

E18–P0 FC neighbouring the stria vascularis (111) FC the developing stria vascularis (111)

FC in the future dark region of the spiral limbus (11 ) FC in the future dark region of the spiral limbus (11 )

Reissner’s membrane (111) Reissner’s membrane (111)

Basilar membrane cells (1) Basilar membrane cells (111)

Outer neuronal elements of the ganglion (1) Uneven expression within the whole ganglion

Organ of Corti: pillar cells (11 ), Hensen cells (111)

P7 All FC of the spiral ligament (11 ) All FC of the spiral ligament (111)

Developing supralimbal dark cells (1) Developing supralimbal dark cells (11 )

Reissner’s membrane (1) Reissner’s membrane (111)

Basilar membrane cells (111) Basilar membrane cells (111)

Regressing Kolliker’s organ (11 ) Regressing Kolliker’s organ (11 )

Developing inner sulcus cells (11 ) Developing inner sulcus cells (11 )

Organ of Corti: OHC (1), pilar cells (1), Hensen cells (1) Organ of Corti: IHC (1), pillar cells (1), OHC (11 ) Hensen

cells (11 )

Basal cells of the stria vascularis (11 )

P13 FC II and IV of the spiral ligament (11 ) FC II and IV of the spiral ligament (11 )

Basilar membrane cells (111) Basilar membrane cells (1)

Uneven expression within the spiral ganglion Uneven expression within the spiral ganglion

Organ of Corti: Deiter cells (111) and Hensen cells (111)

mostly in the apical turn

P20 FC II and IV of the spiral ligament (111)

Adult FC II, III, IV and tension fibroblasts (111)

Uneven expression within the spiral ganglion

a (1), (11 ) and (111) designate low, medium and high levels of expression. FC, fibrocytes; OHC, outer hair cells; IHC, inner hair cells.

lion was dispersed and almost undetectable (Figs. 1A,B and

2A). Expression of both genes started, at neonatal stages, in

the outer cellular elements of the ganglion for Gjb3 (Fig.

1E) or as a dispersed and uneven labeling for Gjb1 (Fig.

2D). During the maturation of the cochlea, the expression of

both genes within the ganglion was always unequal with

highly positive neurons neighboring with faintly or nega-

tively expressing neurons (Figs. 1J,N and 2G,J). In adult,

some expressing Gjb3 neurons were present while no Gjb1

mRNA was detected (Figs. 1N and 2N).

The above results, summarized in Table 1, show that the

expression pattern of Gjb1 and Gjb3 during mouse cochlea

development is complex and highly regulated.

2. Materials and methods

DNA fragments corresponding to the 3 0untranslated

region of the mouse Gjb3, Gjb1 and Gjb5 gene were cloned

into the pGEM-T Easy Vector (Promega) and used to gener-

ate sense and antisense riboprobes by in vitro transcription

with the Digoxigenin labeling kit (Roche Molecular

Biochemicals).

Cochleas from C57BL/6 mice at several stages of the

development (E18, P0, P7, P13, P20 and adult) and, E12

and E15 embryos were obtained and fixed in 4% parafor-

maldehyde in phosphate-buffered saline. In situ hybridiza-

tions were performed on paraffin tissue sections, essentially

as described previously (Nieto et al., 1996), using two

different probes for each gene analyzed. Sections were

examined with a light microscope Leica DMRB equipped

with a Nikon digital camera Dxm 1200 (software Nikon

ACT-1).

Acknowledgements

We thank Dr Marc Lenoir for helpful advices and sugges-

tions and Mireille Gallego for technical assistance. This

work was supported by grants from the Marato de TV3

(98/1710) and the Generalitat de Catalunya (ACI2001-8).

N.L.-B. is supported by a BEFI/FISS fellowship (00/9379).

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