The inner ear is a complex sensory organ that is responsible for sound detection and body balance. It arises from a simple embryonic structure, the otic vesicle, which originates in a transient epithelial thickening of cranial ectoderm at the level of the hindbrain. During embryonic development, the epithelium of the otic vesicle differentiates into distinct anatomical domains of the mature inner ear (Cohen and Fermin,1978). The ventromedial part of the otic epithelium gives rise to the cochlea and the laterodorsal region to the vestibular organ (Torres and Giraldez,1998). The formation of the mature inner ear is regulated by a combinatorial action of genes (Brigande et al.,2000) and a balanced interplay between different signaling mechanisms acting from inside and outside the otic vesicle (Riccomagno et al.,2002,2005; Bok et al.,2003; Ozaki et al.,2004). Remarkably, the mutation of many genes (at least 35 known genes) can lead to hearing loss in hereditary deafness (Holme and Steel,1999).
Cell adhesion molecules, including cadherins, are suggested to be involved in the genesis of the cochlea (Richardson et al.,1987; Raphael et al.,1988; Kelley,2003; Novince et al.,2003). Cadherins are Ca2+-dependent adhesion molecules and play important roles in the regulation of morphogenesis and tissue formation during embryo development (Redies,1995,2000; Takeichi,1995). Several cadherins were found to be expressed during the development of the cochlea. In the zebrafish, cadherin-2 (Cad2 or N-Cad) and cadherin-4 (Cad4 or R-Cad) are transcribed only in the sensory patches and in the statoacoustic ganglion, while cadherin-11 (Cad11) mRNA is expressed widely in the early otic placode and restricted later to a subset of cells, including hair cells (Novince et al.,2003). In the chicken, N-Cad and E-cadherin (E-Cad) are coexpressed first in the epithelium of the otic vesicle and subsequently, N-Cad is maintained in the sensory epithelia of the basilar papilla, whereas E-Cad is detected in the nonsensory epithelia (Richardson et al.,1987; Raphael et al.,1988). During rat cochlear ontogeny, truncated-cadherin (T-Cad) is present in fibrocytes and in the subdomains of the pillar cells. E-Cad is expressed in cochlear epithelial cells except for the inner hair cells (IHCs). N-Cad appears first in the developing IHCs and then in the neighboring Kölliker's organ (Simonneau et al.,2003). Furthermore, protocadherin-15 (Pcdh15) and cadherin-23 (Cad23) are involved in the formation of the stereociliary bundles of the hair cells during mouse cochlear development and may regulate the mechanoelectrical transduction in the hair cells (Alagramam et al.,2001a,b; Siemens et al.,2004). Mutation of the Pcdh15 and Cad23 genes results in defects of the hair cells and causes a recessive deafness in mice (Hampton et al.,2003; Noben-Trauth et al.,2003; Adato et al.,2005; El-Amraoui and Petit,2005).
The distribution and function of cadherins in the developing cochlea have still remained largely unexplored. In the present study, we investigated the relationship between cadherin expression patterns and developing cochlear structures at late embryonic stages in the chicken. Our results show that the expression of each of eight different classic cadherins is restricted to distinct parts and cell types of the cochlea, although the expression patterns overlap partially.
Cloning and Sequence Analysis of Chicken cDNAs Encoding Cadherins
To obtain cDNAs encoding chicken Cad8, Cad11, Cad19, and Cad20, reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using pairs of specific or degenerate primers under optimal parameters (see Table 1). The PCR fragments were subcloned into pBluescript-SK(+) or pCRII-TOPO vectors, and the clones were sequenced. A search of the NCBI GenBank database (http://www.ncbi.nlm.nih.gov) using the BLAST program revealed that two fragments of 2,850 bp and 2,055 bp, respectively, had the same nucleotide sequences as the published chicken Cad11 (GenBank accession no. NM_001004371) and Cad20 (GenBank accession no. NM_204134). Two other fragments of 1,759 bp and 1,407 bp, respectively, shared highest similarity with the predicted sequence Cad8 (GenBank accession no. XM_425125) and Cad19 (GenBank accession no. XM_419115) of chicken. To identify whether these two fragments were chicken Cad8 and Cad19, rapid amplification of cDNA ends (RACE) was carried out using different primers (see Table 1). Two full-length sequences encoding 799 amino acids (see Supplementary Figure S1, which can be viewed at http://www.interscience.wiley.com/jpages/1058-8388/suppmat) and 776 amino acids (see Supplementary Figure S2) were obtained, respectively. On the nucleotide level, these two full-length sequences share highest similarity with the predicted chicken Cad8 and Cad19 sequences, respectively. On the amino acid level, the proteins deduced from the two full-length sequences show highest similarity to human Cad8 (90% identity; GenBank accession no. BC113416) and to human Cad19 (57% identity; GenBank accession no. AJ007607), respectively. Alignment of the amino acid sequences between the obtained chicken molecules and known cadherins revealed that the deduced chicken proteins possess the domains characteristic of the classic cadherin family (see Supplementary Figures S1 and S2). Taken together, the two molecules obtained were identified as chicken Cad8 and Cad19, respectively. The complete sequences of chicken Cad8 (accession no: EF608154) and Cad19 (accession no: EF608155) have been submitted to NCBI GenBank.
Table 1. Primers of PCR or RACE and the PCR Parameters for Different Cadherinsa
Primer (PCR or RACE)
PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends.
30 sec at 94°C, 30 sec at 50°C
3 min at 68°C
15 sec at 94°C, 30 sec at 55°C
3.5 min at 68°C
30 sec at 94°C, 30 sec at 54°C
2 min at 68°C
15 sec at 94°C, 30 sec at 51°C
2.5 min at 68°C
Expression of Cadherins in the Developing Cochlea
We investigated the gene expression patterns of eight classic cadherins in transverse sections through midregions of the cochlea from 11 days of incubation (E11; stage 37), when hearing in chicken embryo begins (Saunders et al.,1973; Jackson and Rubel,1978; Jones et al.,2006), to the prehatching stage (E18). At E11, different types of cells in distinct anatomical regions can be recognized in the embryonic cochlea (Cohen and Fermin,1978; Tanaka and Smith,1978). For example, sensory hair cells and supporting cells are located in the basilar papilla, and ganglion cells in the acoustic ganglion (Fig. 1A,B). The three cell types can be visualized by their Islet-1 expression (Li et al.,2004) (Fig. 1C). Homogene cells are found adjacent to the superior fibrocartilaginous plate and spindle-shaped cells are distributed between the acoustic ganglion cells and the sensory epithelium (Fig. 1A). In the following paragraphs, we will describe the expression of each cadherin in the different cell types and tissues associated with the cochlea.
Cad6B mRNA was detectable at E11 and E14 (Fig. 1D,E) in the spindle-shaped cells and the acoustic ganglion cells, but disappeared at E18 (Fig. 1F). From E14, Cad6B began to be expressed in the tall hair cells (Fig. 1E–G). Cad8 mRNA was extremely abundant in the supporting cells at the inferior edge of the basilar papilla at E11 and E14 (Fig. 1H,I) and was distributed homogeneously at E18 (Fig. 1J).
For Cad11, previous studies have indicated that this molecule is expressed widely by neural cells as well as by mesenchymal cells. Cad11 is involved in various morphogenetic events, e.g., neurogenesis and chondrogenesis, during embryonic development (Simonneau et al.,1995; Simonneau and Thiery,1998; Vallin et al.,1998). In our study, strong expression of Cad11 was found in the chondroblast cells and the mesenchymal cells around the cochlea. Furthermore, Cad11 was found in the supporting cells and the homogene cells from E11 to E18 (Fig. 1K–M). N-Cad was expressed abundantly in the hair cells, in the supporting cells of the sensory epithelium, and in acoustic ganglion cells at E11 and E14 (Figs. 1N,O, 3F,J) and was retained at E18 (Fig. 1P).
Cad7 was expressed in the spindle-shaped cells and the acoustic ganglion cells at both the mRNA and protein level from E11 to E18 (Figs. 2A–D, 3C,D). Cad19 mRNA was found in the spindle-shaped cells (Fig. 2E–G), while Cad20 transcript was present in the acoustic ganglion cells (Fig. 2H–J).
R-Cad expression was present in the homogene cells and the acoustic ganglion cells at E11 and E14 (Figs. 2K,L, 3I) and was reduced in the homogene cells and disappeared in the acoustic ganglion cells at E18 (Fig. 2M).
In summary, the expression of each of the eight cadherins was restricted to different types of cells and distinct anatomic structures of the cochlea, with partial overlap (Fig. 2N).
Furthermore, the neurites of the acoustic ganglion were also studied. By immunostaining against neurofilament (NF), a specific marker for differentiated neurons and their processes (Hatta et al.,1987), and β-tubulin III (Tuj-1), a marker for neurons, axons, and chicken hair cells (Cheng et al.,2003), the neurites of the acoustic ganglion cells (nf in Fig. 3B,E) and their terminals that contact the hair cells (arrows in Fig. 3B,E and insert in 3E) were visualized. The neurites of the acoustic ganglion cells expressed Cad7 (nf in Figs. 2D, 3C,D), N-Cad (nf in Fig. 3F), and R-Cad (nf in Fig. 3I). Moreover, N-Cad and R-Cad were coexpressed in the contact areas between the neurite terminals and the hair cells (arrows in Fig. 3F,G,I,J).
Cadherins in the Sensory Epithelia and the Differentiation of Hair Cells and Supporting Cells
In the present study, expression of Cad6B and N-Cad was demonstrated in the hair cells (Fig. 1E–G,N–P), while Cad8, Cad11, and N-Cad mRNAs were present in the supporting cells of the basilar papilla (Fig. 1H–P). During chicken cochlear development, hair cells and supporting cells share a common progenitor cell (Weisleder et al.,1995; Fekete et al.,1998). Supporting cells can differentiate into hair cells when hair cells are destroyed by acoustic noise or ototoxic drugs (Girod et al.,1989; Adler and Raphael,1996; Roberson et al.,2004; Yamagata et al.,2006). It is remarkable that N-Cad signaling can induce cell proliferation of both hair cells and supporting cells. Blocking the function of N-Cad results in an inhibition of the proliferation of both hair cells and supporting cells (Warchol,2002). Therefore, the expression of cadherins in the supporting cells and the hair cells may have an effect on the differentiation of the supporting cells and the hair cells during normal chicken cochlear development.
Cadherins in Homogene Cells, Spindle-Shaped Cells, and Acoustic Ganglion Cells
Cad11 and R-Cad were exclusively expressed by the homogene cells of the chicken cochlea (Figs. 1K–M, 2K–M), which are involved in the formation of the tectorial membrane during chicken cochlear development (Tanaka and Smith,1975; Cohen and Fermin,1985). The temporal profile of Cad11 and R-Cad expression in the homogene cells may relate to the generation of the tectorial membrane.
The human homologues of Cad7, Cad19, and Cad20 are closely related and found at the same chromosomal location of the human genome (Kools et al.,2000). Of interest, they were expressed in an overlapping manner during cochlear development in the chicken. Cad19 was transcribed by the spindle-shaped cells, Cad20 in the acoustic ganglion cells, and Cad7 in both of them (Fig. 2A–J). This result suggests that the regulation of gene expression for the three cadherins is only partially diverged in the cochlea and may coregulate the development of the spindle-shaped cells and the acoustic ganglion cells.
In the cochlea, the projection of peripheral receptive regions onto auditory structures of the central nervous system is tonotopically organized. The guidance of the nerve fibers mediating this tonotopic projection is therefore of special interest. Several cell adhesion molecules have been found to be involved in the guidance of the extension of the neurites from the acoustic ganglion to their target hair cells (Richardson et al.,1987; Kajikawa et al.,1997). The present study reveals that Cad7, N-Cad, and R-Cad are coexpressed by the neurites of the acoustic ganglion axons (nf in Figs. 2D, 3F,I) and that N-Cad and R-Cad are also coexpressed in the areas of contact between the neurite terminals and the hair cells (arrows in Fig. 3I–K). Previous studies have demonstrated that Cad7, N-Cad, and R-Cad play an important role in neurite outgrowth and fasciculation, in synaptogenesis and/or in synaptic plasticity during embryonic development (Redies et al.,1992, Redies,1995; Fannon and Colman,1996; Riehl et al.,1996; Tanaka et al.,2000; Treubert-Zimmermann et al.,2002). Therefore, the three cadherins may be involved in the guidance of neurites from the acoustic ganglion to the sensory epithelium and in synaptogenesis with the target cells.
Animals, Antibodies, and cRNA Probes
Eggs from White Leghorn chicken (Gallus domesticus) were incubated in a forced-draft incubator (BSS160, Ehret, Germany) at 37°C under 60% humidity. Chicken embryos were staged according to Hamburger and Hamilton (1951). After the embryos were deeply anesthetized by cooling on ice, they were removed from the shell and perfused through the heart with 4% formaldehyde in 0.1 M cacodylate buffer (Roth, Karlsruhe, Germany; containing 1 mM Ca2+ and 1mM Mg2+). Cochleae were separated and collected for this study at E11, E14, and E18 (stages 37, 40, and 44, respectively; at least six cochleae for each stage). The experiments were carried out under an approved institutional protocol according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
For immunohistochemistry, primary mouse and rat monoclonal antibodies raised against Cad6B (CCD6B-1) and Cad7 (CCD7-1; Nakagawa and Takeichi,1998), N-Cad (NCD-2; Hatta and Takeichi,1986), R-Cad (RCD-2; Redies et al.,1992), Islet-1 (Li et al.,2004), neurofilament (Hatta et al.,1987), and β-tubulin III (Tuj-1; Sigma) were used. CCD6-1, CCD7-1, NCD-2, RCD-2, and neurofilament antibodies were kind gifts of Dr. S. Nakagawa and Dr. M. Takeichi (RIKEN Center for Developmental Biology, Kobe, Japan). Antibodies against Islet-1 were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences (Iowa City, IA 52242).
For in situ hybridization, purified plasmids containing the full-length sequences encoding chicken Cad6B, Cad7, N-Cad, and R-Cad (kind gifts of Dr. S. Nakagawa and Dr. M. Takeichi), and the RT-PCR fragments encoding chicken Cad8, Cad11, Cad19, and Cad20 were used as templates for the in vitro synthesis of cRNA probes. Digoxigenin-labeled sense and antisense cRNA probes were transcribed according to the manufacturer's instructions (Roche, Mannheim, Germany). Sense cRNA probes were applied as a negative control for in situ hybridization.
RT-PCR and RACE Reaction
To obtain chicken cDNAs encoding cadherins (Cad8, Cad11, Cad19, and Cad20), mRNA from E14 (stage 40) chicken brain was purified using Micro-FastTrack 2.0 mRNA Isolation Kit (Invitrogen, Karlsruhe, Germany) and first-strain cDNA was synthesized in vitro with the SuperScript First-Strand Synthesis System (Invitrogen). Then PCR was performed with a pair of specific or degenerate primers (see Table 1). The PCR templates were denatured for 2 min at 94°C, followed by 35 cycles of amplification with optimal denaturing, annealing, and extension conditions for each cadherin (see Table 1). After the last cycle, the reaction was extended finally for 15 min at 72°C. The PCR fragments obtained were cloned into pBluescript-SK(+) vector (for Cad8, Cad11, and Cad20) or into pCR II-TOPO vector using the TA-cloning kit (Invitrogen; for Cad19). The plasmids were purified using the Plasmid Maxi Kit (Qiagen, Hilden, Germany) and sequenced by a commercial company (MWG, Ebersberg, Germany).
To obtain the full-length sequences of chicken Cad8 and Cad19, 5- and 3-end RACE reactions were performed with the SMART RACE cDNA Amplification Kit (Takara Bio Europe/Clontech, Saint-Germain-en-Laye, France). The specific primers for RACE were chosen from the obtained PCR fragments according to the instructions of the RACE Kit User Manual. The primers for 5-end RACE PCR (Cad8-anti-1 and Cad8-anti-2 for chicken Cad8; Cad19-anti-1 and Cad19-anti-2 for chicken Cad19) and 3-end RACE PCR (Cad8-sense-1 and Cad8-sense-2 for Cad8; Cad19-sense-1 and Cad19-sense-2 for Cad19) are shown in Table 1. The touch-down PCR for 5- and 3-end RACE amplification was performed using the 5- and 3-end RACE PCR primers (Cad8-anti-1 and Cad19-anti-1 for 5-end RACE; Cad8-sense-1 and Cad19-sense-1 for 3-end RACE, respectively) together with the Universal Primer Mix. For the first 5 cycles, the touch-down PCR was performed at 94°C for 30 sec and at 72°C for 3 min. The next 5 cycles were performed at 94°C for 30 sec, 70°C for 30 sec, and 72°C for 3 min. At last, another 27 cycles were performed at 94°C for 30 sec, at 68°C for 30 sec, and at 72°C for 3 min. To obtain more specific bands, nested PCR was performed following the touch-down PCR for 5- and 3-end cDNA amplification with the primers (Cad8-anti-2 and Cad19-anti-2 for 5-end RACE; Cad8-sense-2 and Cad19-sense-2 for 3-end RACE) together with the Nested Universal Primer, respectively. The PCR templates were denatured for 1 min at 95°C, followed by 20 cycles of amplification (denaturing for 30 sec at 95°C, and annealing and extension for 3 min at 68°C). The PCR products were cloned into pCR II-TOPO vector (Invitrogen) and sequenced by a commercial company (MWG). After ligating the RACE sequences with the obtained RT-PCR fragment sequences, two molecules with the full-length nucleotide sequences were obtained.
To detect the expression of cadherins in the cochlea, fluorescent immunostaining was performed on sections of chicken cochleae according to the protocol described previously (Luo and Redies,2004). In brief, after post-fixation with 4% formaldehyde, cryostat sections of 20 μm thickness were preincubated with blocking solution (5% skimmed milk, 0.3% Triton X-100 in TBS) at room temperature for 60 min. Then the sections were incubated overnight at 4°C with the primary antibody against cadherin, followed by Alexa 488-labeled or Cy3-labeled secondary antibody against mouse or rat IgG (Dianova, Hamburg, Germany) at room temperature for 1 hr. Finally, cell nuclei were counterstained with dye Hoechst 33258 (Molecular Probes, Eugene, OR). Fluorescence was imaged under a fluorescence microscope (BX40, Olympus, Hamburg, Germany) equipped with a digital camera (DP70, Olympus).
For double-label fluorescent immunohistochemistry, sections were first immunostained with antibodies against R-Cad or β-tubulin III using the method described above. Subsequently, immunostaining for N-Cad was performed.
In Situ Hybridization
In situ hybridization on cryosections was performed according to the protocol described previously (Redies and Takeichi,1993). In brief, after post-fixation with 4% formaldehyde in phosphate-buffered saline, cryostat sections of 20 μm thickness were pretreated with proteinase K and acetic anhydride and hybridized overnight with cRNA probe at a concentration of approximately 1–5 ng/μl at 70°C in hybridization solution (50% formamide, 3× SSC, 1× Denhardt's solution, 250 μg/ml yeast transfer RNA, and 250 μg/ml salmon sperm DNA). After the unbound cRNA was removed by RNAse, the sections were incubated with alkaline phosphatase-conjugated anti-digoxigenin Fab fragments (Roche) at 4°C overnight. For visualization of the labeled mRNA, a substrate solution of nitroblue tetrazolium salt (NBT) and 5-bromo-4-chloro-3-indoyl phosphate (BCIP) was added. The color reactions were analyzed under a microscope (BX40, Olympus) and photographed with a digital camera (DP70, Olympus).
We thank Dr. S. Nakagawa and Dr. M. Takeichi for their kind gifts of plasmids and antibodies, and Ms. S. Hänβgen for technical assistance.