retracted: expression profiles of the connexin genes, gjb1 and gjb3, in the developing mouse cochlea
TRANSCRIPT
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.
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* Corresponding author. Tel.: 134-93-224-0900; fax: 134-93-224-0899.
E-mail address: [email protected] (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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