The chicken inner ear arises from a simple embryonic structure, the otic vesicle (Cohen and Fermin, 1978). During chicken embryonic development, the ventro-medial part of the otic epithelium differentiates into the cochlea and the latero-dorsal part into the vestibular organ (Torres and Giraldez, 1998). Many genes, e.g., bone morphogenetic protein-4 (BMP4), sensory organ homeobox-1 (SOHo-1), cognate of Drosophila orthodenticle gene-1 (Otx1), muscle segment homeobox gene-1 (Msx1) are temporally regulated in different domains of the otic vesicle and contribute to the identity of different anatomical structures of the ear (Brigande et al., 2000). Sonic hedgehog (Shh) secreted by the notochord and floor plate around the otic vesicle regulates the auditory cell fate of the inner ear by affecting several cell fate specification genes, e.g., paired-box gene Pax2, Otx1, and Otx2 (Riccomagno et al., 2002). In the developing chicken cochlea, Wnt ligands are transcribed predominantly in the non-sensory tissue domains, whereas Wnt receptors, e.g., Frizzled receptors, are expressed mainly in the sensory primordia, suggesting a role for paracrine Wnt signalling in developmental processes such as regionalization, cell fate specification, and synaptogenesis (Sienknecht and Fekete, 2008).
ADAMs are a family of zinc-dependent transmembrane metalloproteases with multiple functions, involving cell–cell and cell–matrix interactions, in proteolytic shedding of other membrane proteins, as well as in intracellular signal transduction (Wolfsberg et al, 1995; White, 2003; Blobel, 2005; Reiss and Saftig, 2009). Individual members of the ADAMs show variable expression patterns that are regulated spatiotemporally during embryonic development (Edwards et al., 2008; Lin et al., 2008; Alfandari et al., 2009). For example, ADAM13 is expressed in neural crest cell–derived structures, in digestive organs, and in the developing kidney during Xenopus and/or chicken development (Alfandari et al., 1997; Lin et al., 2007). ADAM9, ADAM10, ADAM12, ADAM22, and ADAM23 show spatiotemporal expression patterns in the developing brain and spinal cord (Lin et al., 2008, 2010). ADAM10 is found widely in the epidermis, somites, and in the gut as well as in cultured neural crest cells (Hall and Erickson, 2003). Finally, expressions of ADAM12 and ADAM19 are detected in the embryonic limb, the gut, and in the kidney (Lewis et al., 2004; Neuner et al., 2009).
ADAMs play an important role in morphogenesis and in tissue formation, and function as pivotal molecules in distinct developmental processes, such as fertilization, embryogenesis, neurogenesis, and cell migration (Yang et al., 2006; Edwards et al., 2008; Alfandari et al., 2009). For example, ADAM24 is expressed on the surface of the sperm and contributes to prevent polyspermic fertilization (Zhu et al., 2009). ADAM10 sheds Notch ligands and regulates Notch signaling, which plays a critical role in embryonic development (Muraguchi et al., 2007). ADAM28 regulates the differentiation of odontogenic mesenchymal cells and participates in tooth development (Zhao et al., 2006). ADAM21 may regulate neurogenesis and guide neuroblast migration by a cleavage-dependent activation of proteins and integrin binding (Yang et al., 2005). Finally, ADAM10 is essential for the correct projection of retinal ganglion cell axons to their target region in the tectum (Chen et al., 2007).
Our previous studies showed that the ADAMs are expressed in the developing chicken spinal cord and the brain, especially in auditory nuclei and their projections crossing the midline of the hindbrain (Lin et al., 2008, 2010). These findings raise the question of whether the ADAMs are also involved in the development of the cochlea and its nerve. Little is known about the expression and function of ADAMs during cochlear development. Therefore, in the present study, we continue to analyze the expression patterns of the ADAMs including ADAM9, ADAM10, ADAM17, ADAM22, and ADAM23 in the different developing cochlear structures at late chicken embryos. Our results show for the first time that each individual ADAM is expressed in distinct anatomical regions of the developing cochlea, but with a partial overlap.
During chicken embryonic development, distinct anatomical structures and cell types of the cochlea can be distinguished morphologically by nuclear staining (Nu) with 4,6′-diamidino-2-phenylindole (DAPI) and by hematoxylin and eosin (HE) staining from E11, when hearing begins in chicken embryo (Saunders et al., 1973; Jackson and Rubel, 1978; Jones et al., 2006). For example, in transverse sections through the mid-region of the cochlea at E11 (stage 37), sensory hair cells and supporting cells located in the sensory epithelium (SE) of the basilar papilla (BP) are distinguished clearly (Fig. 1A), while homogene (columnar) cells are found between the BP and the tegmentum vasculosum (TV) neighboring the superior fibrocartilaginous plate. Non-neuronal spindle-shaped cells distribute between the superior edge of the BP and the neuronal acoustic ganglion cells (Fig. 1A, B) and along the nerve fibers projecting from acoustic ganglion cells to the SE. At this stage, spindle-shaped cells are easily distinguished from the round-shaped and large-sized acoustic ganglion cells by HE staining (Fig. 1B). At E17 (Fig. 1C), hair cells differentiated into one layer locate on the apical surface of the BP, while supporting cells are in place underneath the hair cells (Fig. 1C). At this stage, homogene cells are observed clearly.
The aim of this study was to analyze the expression patterns of the five members of the ADAM family in adjacent transverse sections through mid-regions of the cochlea from E11 (stage 37) to E18 (stage 44). Expression patterns are shown in Figure 1 for ADAM9 and ADAM17, in Figure 2 for ADAM10, and in Figure 3 for ADAM22 and ADAM23, and are organized according to the developmental stages (early to late). Furthermore, expression patterns of the ADAMs in adjacent longitudinal sections through the neural side of the BP at E11 are also shown in Figure 4. Sense RNA probes were used as negative controls (e.g., Fig. 1D). In general, each of the ADAMs investigated demonstrates a spatially restricted and temporally regulated expression pattern in the distinct anatomical structures and different cell types of the cochlea, with partial overlap between each other.
At E11, ADAM9 mRNA is abundantly expressed by the hair cells and supporting cells of the SE in the BP, and by the homogene cells, the cuboidal cells, and the acoustic ganglion cells, and in the TV (Fig. 1E–G). At E14, ADAM9 signals are strongly maintained in the cuboidal cells and the acoustic ganglion cells, and in the TV, moderately in the supporting cells and the homogene cells, but very weakly in the hair cells (Fig. 1H–J). At E16, strong expression remains in the acoustic ganglion cells and in the TV, but weak expression by the supporting cells (Fig. 1K–M). ADAM9 is no longer detectable in the hair cells and the homogene cells from E16 onward. Instead, the spindle-shaped cells start to strongly express ADAM9. At E18, strong expression remains in the acoustic ganglion cells, the spindle-shaped cells, and in the TV, but supporting cells only weakly express the ADAM (Fig. 1N–P).
At E11, ADAM17 mRNA is widely and strongly transcribed in the anatomical structures of the cochlea, e.g., in the SE of the BP, especially in the inferior part (arrow in Fig. 1R), by the cuboidal cells, the spindle-shaped cells and the acoustic ganglion cells, and in the TV, but only moderately by the homogene cells (Fig. 1Q–S). From E14 to E16, strong expression remains in the hair cells and supporting cells of the BP, in the spindle-shaped cells, the acoustic ganglion cells, and in the TV, but expression decreases in the homogene cells (Fig. 1T–Y). ADAM17 expression by the hair cells and supporting cells in the BP can be distinguished clearly. At E18, strong expression is found in the spindle-shaped cells and the acoustic ganglion cells, moderate expression in the supporting cells, and weak expression in the hair cells of the superior part of the BP and in the TV (Fig. 1Z–B′). At this stage, ADAM17 is no longer detectable in the homogene cells.
Similar to ADAM17, at E11, ADAM10 mRNA is abundantly and widely expressed in the SE of the BP, by the homogene cells, the cuboidal cells, the spindle-shaped cells, and the acoustic ganglion cells as well as in the TV (Fig. 2A–C). Remarkably, ADAM10 signals are also strong in the abneural mesenchymal cells of the inferior fibrocartilaginous plate (ifp in Fig. 2A). From E14 onward, strong expression is maintained in the supporting cells, the homogene cells, the spindle-shaped cells, and in the TV, but only moderate expression remains in the hair cells (Fig. 2D–H).
Interestingly, it has been found that ADAM10 can shed the ectodomain of N-cadherin (N-Cad) and E-Cad, modulating the cell–cell adhesion and signal transduction (Maretzky et al., 2005; Reiss et al., 2005; Reiss and Saftig, 2009). Eight classic cadherins are expressed in distinct anatomical regions and cell types of the developing cochlea (Luo et al., 2007). Therefore, we further investigated the coexpression of ADAM10 together with the classic cadherins including Cad7, N-Cad, and R-Cad at protein level in the cochlea by double-immunohistochemistry at E11 (Fig. 2I–T). At E11, ADAM10 protein (green color in Fig. 2I–X) is expressed widely and strongly in the distinct anatomical structures of the cochlea, consistent with the expression of ADAM10 at the mRNA level (Fig. 2A–D). Cad7 is expressed strongly in the spindle-shaped cells (arrows in Fig. 2K, L), where ADAM10 is coexpressed (yellow color in Fig. 2L). In acoustic ganglia, Cad7 protein is expressed in the wraps around the cells expressing ADAM10 protein, but the Cad7-positive neural fibers do not coexpress ADAM10 (nf in Fig. 2K, L and the insert in K; Luo et al., 2007). N-Cad signals are strong in the hair cells and the supporting cells of the BP, where ADAM10 is also coexpressed (yellow color in Fig. 2P). Remarkably, the coexpression of ADAM10 and N-Cad in the BP is inversely related. For example, N-Cad expression is strong in the central and superior part of the SE, where ADAM10 expression is weaker. In contrast, N-Cad expression is weak in the inferior part of the SE, where ADAM10 is stronger (Fig. 2M–P). The different expression of ADAM10 or N-Cad in the SE of the BP forms a clear boundary (arrows in Fig. 2N–P). In the acoustic ganglion cells, N-Cad expression wraps around the cells expressing ADAM10 (Fig. 2O, P). Furthermore, the coexpression of ADAM10 and R-Cad by the homogene cells is also observed (Fig. 2Q–T). Similarly, the coexpression of ADAM10 and R-Cad by the homogene cells is also inversely related when comparing ADAM10 signals in the homogene cells to the cells of the adjacent tissues (Fig. 2R–T). In the acoustic ganglion cells, R-Cad protein is expressed in the wraps around the cells expressing ADAM10 (Fig. 2S, T).
Moreover, ADAM10 and neurofilament are coexpressed in the acoustic ganglion cells and their neurites (yellow color in Fig. 2X) as detected by immunostaining with antibody against neurofilament (red color in Fig. 2W, X), a specific marker for differentiated neurons and their processes (Hatta et al., 1987).
In contrast to the expression patterns of ADAM9, ADAM10, and ADAM17, mRNA signals of ADAM22 are prominently found in the restricted structures of the developing cochlea. From E11 to E18, ADAM22 is strongly and specifically expressed by the spindle-shaped cells and the acoustic ganglion cells (Fig. 3A–K).
At E11, ADAM23 mRNA is expressed strongly by the hair cells and supporting cells in the BP, by the acoustic ganglion cells, and in the TV, but weakly by the homogene cells (Fig. 3L–N). At E14, ADAM23 is gradually restricted to the hair cells in the superior part of the BP, and continuously strongly expressed in the acoustic ganglion cells and in the TV, but decreases in the supporting cells (Fig. 3O–Q). At this stage, the homogene cells show weak ADAM23 signals. From E16 onward, strong expression persists in the hair cells and the acoustic ganglion cells (Fig. 3R–W), but ADAM23 is no longer detectable in the supporting cells and the homogene cells (Fig. 3U–W).
Expression of the Five Members of the ADAMs in Longitudinal Sections of the Cochlea
In adjacent longitudinal sections through the neural side of the BP at E11, ADAM9 mRNA is strongly expressed by the hair cells, the supporting cells, the acoustic ganglion cells, and in the TV (Fig. 4A, B). ADAM10 signals are strong in the hair cells and the supporting cells of the SE, in the spindle-shaped cells and the acoustic ganglion cells, and also in the TV (Fig. 4C, D). ADAM17 expression is strong in the hair cells and the supporting cells of SE, in the spindle-shaped cells and the acoustic ganglion cells, and in the TV (Fig. 4E, F). ADAM22 mRNA transcription is restricted to the spindle-shaped cells and the acoustic ganglion cells (Fig. 4G, H), while ADAM23 mRNA is transcribed in the hair cells, the supporting cells, and in the acoustic ganglion cells (Fig. 4I, J).
In summary, each individual ADAM is expressed in different anatomical structures of the cochlea during embryonic development (Fig. 3X). The expression patterns differ from each other but show partial overlap.
Expression of the ADAMs in Sensory Epithelium and Cell Differentiation
In the present study, ADAM9, ADAM10, ADAM17, and ADAM23 are observed to be expressed strongly by the hair cells and the supporting cells of the BP (Figs. 1–3), and as embryos develop, the expression of the individual ADAMs decreases. Hair cells and supporting cells share a common progenitor cell during chicken cochlear development (Fekete et al., 1998; Satoh and Fekete, 2005). However, little is known about how these cells differentiate into hair cells and supporting cells and which molecules regulate this process. One possible mechanism to explain such diversification of the sensory development is lateral inhibition, whereby signal interaction between neighboring cells induces the cells to adopt different developmental fates. For example, activation of Notch signaling promotes the differentiation of hair cells and supporting cells along distinct developmental pathways by mediating lateral inhibition (Daudet and Lewis, 2005; Kiernan et al., 2005), as detected in other sensory systems, e.g., in the retina (Henrique et al., 1997). Indeed, the chicken homologues of Notch receptor and Delta and Serrate ligands are found to be expressed in the developing cochlea, e.g., in the otic vesicle and the supporting cells (Warchol et al., 1993; Warchol, 2002). Of interest, it is known that the ADAMs, especially ADAM10 and ADAM17, participate in proteolytic cleavage of the Notch receptors, which are essential for controlling neural cell fate determination (Yang et al., 2006; Edwards et al., 2008). Therefore, the expression of ADAM10 and ADAM17 in the SE during cochlear development may contribute to the differentiation and cell fate determination of hair cells and supporting cells via proteolytic shedding of Notch.
Furthermore, several members of the cadherins are temporally regulated in the SE of the developing chicken cochlea, suggesting a role of cadherins in the differentiation of supporting cells and hair cells (Luo et al., 2007). N-Cad-mediated cell–cell interaction and β-catenin signalling regulate sensory cell proliferation in the chicken inner ear (Warchol, 2002). ADAM10 is a major protease shedding the extracellular domain of cadherins and modulating cell–cell adhesion and signal transduction (Maretzky et al., 2005; Reiss et al., 2005, 2006; Kohutek et al., 2009). In this study, expression of ADAM10 and N-Cad proteins was observed to be inversely related in the SE (Fig. 2N–P), suggesting that ADAM10 may also contribute to the proliferation and differentiation of hair cells and supporting cells by exclusively regulating the expression of N-Cad during cochlear development.
Moreover, that ADAM23 plays a role in neuronal differentiation has been suggested by an in vitro experiment with cultured P19 cells (Sun et al., 2007), and it also controls the differentiation of neural crest cells during embryonic development (Neuner et al., 2009). In the present study, ADAM23 is found to be expressed in the hair cells at different stages, especially in the superior edge of the BP (Fig. 3L–V). Therefore, it will be interesting to investigate whether ADAM23 contributes to the differentiation of hair cells, particularly the tall hair cells in the superior edge of the BP.
Expression of the ADAMs in Homogene Cells
In this study, we show that the ADAMs, including ADAM9, ADAM10, ADAM17, and ADAM23, are transcribed in the homogene cells, where their expressions gradually decrease from E11 onward and become weak at E16 to E18 (Figs. 1–3). The homogene cells locate in the superior fibrocartilaginous plates between the TV and SE with a columnar shape from E11. The adjacent homogene cells are connected laterally with well-developed tight junctions (Hirokawa, 1980). During chicken cochlear development, the upper lumenal side of the tectorial membrane appears as a smooth amorphous layer and is produced by homogene cells, which secrete the extracellular matrix (ECM) directly into the lumen of the cochlea (Tanaka and Smith, 1975; Cohen and Fermin, 1985; Runhaar, 1989; Shiel and Cotanche, 1990). During the different phases of the secretory activity, the homogene cells show some differences in their size, shape, and density of the cytoplasmic contents, contributing to the production of the tectorial membrane (Cohen and Fermin, 1985; Shiel and Cotanche, 1990). Furthermore, homogene cells may be involved in a mechanical process adjusting the tension of the tectorial membrane due to their high expression of homogenin and filamentous actin (Heller et al., 1998). The ADAM proteins contain a metalloprotease domain and a disintegrin domain, which are involved in cell–cell and cell–ECM interaction by cleaving and releasing cell-surface proteins and by remodelling the ECM (White, 2003; Yang et al., 2006; Edwards et al., 2008). The changes of ADAM mRNA levels in the homogene cells coincide with alterations of the cytoplasmic organelles of the homogene cells during their secretory phase. For example, at the beginning of secretion (e.g., E11–14), the mRNA levels of ADAMs are higher in the cells (Figs. 1–3) that may be responsible for the synthesis and secretion of the respective proteins. At later stages of development (e.g., E18), when the formation of the tectorial membrane is almost completed (Cohen and Fermin, 1985; Shiel and Cotanche, 1990), the transcript levels of ADAMs decline gradually (Figs. 1–3). Whether the investigated ADAMs participate in the formation of the tectorial membrane via proteolytic shedding and/or cell–ECM interaction remains to be elucidated.
Expression of the ADAMs in Spindle-Shaped Cells and Acoustic Ganglion Cells
Spindle-shaped cells are located in the superior fibrocartilaginous plates between the SE and the acoustic ganglia and support the signal transfer of the sensory dendrites from hair cells to the acoustic ganglia (Heller et al., 1998). In the present study, ADAM10, ADAM17, and ADAM22 are strongly expressed in the spindle-shaped cells from E11 onward, and ADAM9 is strongly expressed from E16 onward (Figs. 1–3). What role these ADAMs play in the spindle-shaped cells during cochlear development is unclear.
In the cochlea, projections of acoustic ganglion cells to targeted cochlear nucleus undergo refinement to form precise cochleotopic terminals and provide the basis for tonotopic mapping throughout the central auditory system. The guidance of the nerve fibers mediating this tonotopic projection is controlled by different molecules (Richardson et al., 1987; Kajikawa et al., 1997; Lee et al., 2004). The present study reveals that the five ADAM mRNAs are strongly expressed by the acoustic ganglion cells during development (Figs. 1–3) and ADAM10 protein is also expressed in both the acoustic ganglion cells and their neurites, where neurofilament is coexpressed (Fig. 2U–X). Previous studies have shown that the ADAMs are involved in axon outgrowth and guidance. For example, ADAM8, ADAM10, and ADAM17 can shed the ectodomain of neural cell adhesion molecule (NCAM), modulating the neurite outgrowth and/or branching (Hinkle et al., 2006; Kalus et al., 2006). ADAM21 is present in growing axonal tracts and participates in the final axonal outgrowth and/or synapse formation (Yang et al., 2005). ADAM10 plays a pivotal role in the correct projection of retinal ganglion cell axons to their target tectum (Chen et al., 2007). Finally, ADAM22 and ADAM23 are also expressed in the auditory nuclei of the hindbrain (Lin et al., 2008). Therefore, expressions of the investigated ADAMs in the acoustic ganglion cells and the auditory nuclei during cochlear development may suggest a role of the ADAMs in the guidance of acoustic ganglion neurites to their target nuclei in the hindbrain.
Furthermore, Neuner and his colleagues (2009) demonstrated in Xenopus that ADAM23 could regulate the differentiation of neural crest cells during embryonic development. The ADAMs including ADAM9, ADAM10, ADAM22, and ADAM23 have been identified to be spatiotemporally regulated in the dorsal root ganglion and play a role in the neuronal differentiation of the sensory neurons (Lin et al., 2010). Therefore, the five members of the ADAMs studied here may also be involved in the differentiation of the acoustic ganglion cells during cochlear development.
Expression of the ADAMs in Tegmentum Vasculosum
The TV is a pleated epithelial layer consisting of a mosaic of light and dark staining cells, which are involved in the water and ion homeostasis of the endolymph (Cotanche et al., 1987; Ryals et al., 1995). The TV in chicken separates the endolymphatic fluid in the scala media from the perilymph of the scala vestibuli and functions like the stria vascularis in mammals (Ryals et al., 1995; Manley, 2000). Both inner ear fluids and cerebrospinal fluid show remarkably stable ionic concentrations, and the stria vascularis and choroid plexus, which are responsible for production of the respective fluids, share similar characteristics, e.g., they contain the same proteins (Lecain et al., 2000; Saito et al., 2001). Of interest, in the developing chicken brain, ADAM9, ADAM10, ADAM12, and ADAM23 are temporally expressed in the choroid plexus (Lin et al., 2008). In the present study, we also found that ADAM9, ADAM10, ADAM17, and ADAM23 are strongly expressed in the TV. Therefore, whether the expression of the ADAMs in both the TV and choroid plexus plays a general role in the development of them should be further elucidated.
Chicken Embryos, RNA Probes, and Antibodies
Fertilized eggs from white Leghorn chicken (Gallus domesticus) were incubated in a forced-draft egg incubator (BSS160, Ehret, Germany) at 37°C with 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 PBS buffer (13 mM NaCl, 7 mM Na2HPO4, 3 mM NaH2PO4; pH 7.4). Cochleae were then separated and collected at embryonic incubation day 11 (E11), E14, E16, E17, and E18 (stages 37, 40, 42, 43, and 44, respectively; at least 5 cochleae for each stage).
For in situ hybridization, digoxigenin-labeled sense and antisense cRNA probes were synthesized in vitro using plasmids containing previously cloned ADAM sequences (Lin et al., 2008) as cDNA templates according to the manufacturer's instructions (Roche, Mannheim, Germany). Sense cRNA probes were used as a negative control.
For immunohistochemistry, primary rabbit polyclonal antibody against ADAM10 (Chemicon, Hampshire, UK; Hall and Erickson, 2003), and primary mouse and rat monoclonal antibodies against Cad7 (CCD7-1; Nakagawa and Takeichi, 1998), N-Cad (NCD-2; Hatta et al., 1987), R-Cad (RCD-2; Redies et al., 1992), and neurofilament (NF; Hatta et al., 1987) were used. CCD7-1, NCD-2, RCD-2, and NF antibodies were kind gifts of Dr. S. Nakagawa and Dr. M. Takeichi (RIKEN Center for Developmental Biology, Kobe, Japan). Alexa 488-labeled (Molecular Probes, Eugene, OR) and Cy3-labeled (Dianova, Hamburg, Germany) secondary antibodies against rabbit, mouse, or rat IgG were used.
In Situ Hybridization
In situ hybridization on sections through mid-regions of the cochlea was performed according to the protocol described previously (Luo et al., 2004). In brief, after post-fixation with 4% formaldehyde in PBS, cryostat sections were pretreated with proteinase K and acetic anhydride. Then sections were hybridized with cRNA probe at a concentration of about 1–5 ng/μl overnight at 70°C in hybridization solution (50% formamide, 3× SSC, 1× Denhardt's solution, 250 μg/ml yeast 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, Mannheim, Germany) 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 colour reaction on sections was viewed and photographed under a transmission microscope (BX40; Olympus, Hamburg, Germany) equipped with a digital camera (DP70; Olympus). Photographs were adjusted in contrast and brightness with the Photoshop software (Adobe System, Mountain View, CA).
Fluorescent immunostaining was performed on sections through mid-regions of the cochlea using the method described previously (Luo and Redies, 2004). In brief, after post-fixation in 4% formaldehyde, cryostat sections of 20 μm thickness were pre-incubated with a blocking solution (5% skimmed milk and 0.3% Triton X-100 in TBS) at room temperature for 60 min. Then sections were incubated overnight at 4°C with the primary antibody, followed by the secondary antibody at room temperature for 1 hr. Finally, cell nuclei were stained with 4′-6-Diamidino-2-phenylindole (DAPI; Sigma, Munich, Germany). Fluorescence was imaged under a fluorescent microscope (BZ-8000; Keyence Deutschland GmbH, Neu-Isenburg, Germany). Digital images were adjusted in contrast and brightness with the Photoshop software (Adobe Systems).
For double-label fluorescent immunohistochemistry, sections were first immunostained with an antibody against ADAM10 using the method described above. Subsequently, immunostainings for cadherin or neurofilament were performed.
We thank Dr. S. Nakagawa and Dr. M. Takeichi for their kind gifts of the antibodies, Dr. C. Redies for support of this study, and Dr. E. Mix for the critical reading of this manuscript.