regional expression of the adams in developing chicken cochlea

a PATTERNS & PHENOTYPES

Regional Expression of the ADAMs inDeveloping Chicken CochleaXin Yan,1† Juntang Lin,2† Hong Wang,3‡ Annett Markus,1 Andreas Wree,4 Arndt Rolfs,1

and Jiankai Luo1,2*

The expression patterns of five members of the ADAM (a disintegrin and metalloprotease) family includingADAM9, ADAM10, ADAM17, ADAM22, and ADAM23 were analyzed in different anatomical structures of thedeveloping chicken cochlea by in situ hybridization and immunohistochemistry. Results show thatADAM9, ADAM10, and ADAM17 are widely expressed in the sensory epithelium of the basilar papilla, byhomogene cells, spindle-shaped cells, and acoustic ganglion cells, and in the tegmentum vasculosum, eachwith a different pattern. ADAM22 expression is restricted to spindle-shaped cells and acoustic ganglioncells, while ADAM23 is prominently expressed by hair cells and acoustic ganglion cells. Furthermore,ADAM10 protein is coexpressed with several members of the classic cadherins, including cadherin-7, N-cadherin, and R-cadherin in distinct anatomical regions of the cochlea except for acoustic ganglion cells.The expression of the ADAMs in the developing cochlea suggests a contribution of the ADAMs to the devel-opment of distinct cochlear structures. Developmental Dynamics 239:2256–2265, 2010. VC 2010 Wiley-Liss, Inc.

Key words: ADAM; cochlear development; chicken embryo

Accepted 1 June 2010

INTRODUCTION

The chicken inner ear arises from asimple embryonic structure, the oticvesicle (Cohen and Fermin, 1978).During chicken embryonic develop-ment, the ventro-medial part of theotic epithelium differentiates into thecochlea and the latero-dorsal part intothe vestibular organ (Torres andGiral-dez, 1998).Many genes, e.g., bonemor-phogenetic protein-4 (BMP4), sensoryorgan homeobox-1 (SOHo-1), cognateof Drosophila orthodenticle gene-1(Otx1), muscle segment homeoboxgene-1 (Msx1) are temporally regu-

lated in different domains of the oticvesicle and contribute to the identityof different anatomical structures ofthe ear (Brigande et al., 2000). Sonichedgehog (Shh) secreted by the noto-chord and floor plate around the oticvesicle regulates the auditory cell fateof the inner ear by affecting severalcell fate specification genes, e.g.,paired-box gene Pax2, Otx1, and Otx2(Riccomagno et al., 2002). In the devel-oping chicken cochlea, Wnt ligandsare transcribed predominantly in thenon-sensory tissue domains, whereasWnt receptors, e.g., Frizzled receptors,are expressed mainly in the sensory

primordia, suggesting a role for para-crine Wnt signalling in developmentalprocesses such as regionalization, cellfate specification, and synaptogenesis(Sienknecht and Fekete, 2008).ADAMs are a family of zinc-depend-

ent transmembrane metalloproteaseswith multiple functions, involvingcell–cell and cell–matrix interactions,in proteolytic shedding of other mem-brane proteins, as well as in intracellu-lar signal transduction (Wolfsberg etal, 1995; White, 2003; Blobel, 2005;Reiss and Saftig, 2009). Individualmembers of the ADAMs show variableexpression patterns that are regulated

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1Albrecht-Kossel-Institute for Neuroregeneration, School of Medicine, University of Rostock, Rostock, Germany2Institute of Anatomy I, School of Medicine, University of Jena, Jena, Germany3Department of Otorhinolaryngology, School of Medicine, University of Jena, Jena, Germany4Institute of Anatomy, School of Medicine, University of Rostock, Rostock, Germany†Xin Yan and Juntang Lin contributed equally to this work and should be considered co–first authors.‡Hong Wang’s present address is Offentlicher Gesundheitsdienst, Thuringer Landesverwaltungsamt, Weimar, Germany.Grant sponsor: German Research Foundation (DFG); Grant number: LU1455/1-1.*Correspondence to: Dr. Jiankai Luo, Albrecht-Kossel-Institute for Neuroregeneration, School of Medicine, University ofRostock, Gehlsheimer Strasse 20, D-18147 Rostock, Germany. E-mail: [email protected]

DOI 10.1002/dvdy.22360Published online 12 July 2010 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 239:2256–2265, 2010

VC 2010 Wiley-Liss, Inc.

spatiotemporally during embryonicdevelopment (Edwards et al., 2008;Lin et al., 2008; Alfandari et al., 2009).For example, ADAM13 is expressed inneural crest cell–derived structures,in digestive organs, and in the devel-oping kidney during Xenopus and/orchicken development (Alfandari et al.,1997; Lin et al., 2007). ADAM9,ADAM10, ADAM12, ADAM22, andADAM23 show spatiotemporal expres-sion patterns in the developing brainand spinal cord (Lin et al., 2008, 2010).ADAM10 is found widely in the epider-mis, somites, and in the gut as well asin cultured neural crest cells (Hall andErickson, 2003). Finally, expressionsof ADAM12 and ADAM19 are detectedin the embryonic limb, the gut, and inthe kidney (Lewis et al., 2004; Neuneret al., 2009).

ADAMs play an important role inmorphogenesis and in tissue forma-tion, and function as pivotal moleculesin distinct developmental processes,such as fertilization, embryogenesis,neurogenesis, and cell migration(Yang et al., 2006; Edwards et al.,2008; Alfandari et al., 2009). Forexample, ADAM24 is expressed on thesurface of the sperm and contributesto prevent polyspermic fertilization(Zhu et al., 2009). ADAM10 shedsNotch ligands and regulates Notch sig-naling, which plays a critical role inembryonic development (Muraguchiet al., 2007). ADAM28 regulates thedifferentiation of odontogenic mesen-chymal cells and participates in toothdevelopment (Zhao et al., 2006).ADAM21 may regulate neurogenesisand guide neuroblast migration by acleavage-dependent activation of pro-teins and integrin binding (Yang et al.,2005). Finally, ADAM10 is essentialfor the correct projection of retinalganglion cell axons to their targetregion in the tectum (Chen et al.,2007).

Our previous studies showed thatthe ADAMs are expressed in the devel-oping chicken spinal cord and thebrain, especially in auditory nucleiand their projections crossing the mid-line of the hindbrain (Lin et al., 2008,2010). These findings raise the ques-tion of whether the ADAMs are alsoinvolved in the development of thecochlea and its nerve. Little is knownabout the expression and function ofADAMs during cochlear development.

Therefore, in the present study, wecontinue to analyze the expressionpatterns of the ADAMs includingADAM9, ADAM10, ADAM17,ADAM22, and ADAM23 in the differ-ent developing cochlear structures atlate chicken embryos. Our resultsshow for the first time that each indi-vidual ADAM is expressed in distinctanatomical regions of the developingcochlea, but with a partial overlap.

RESULTS

During chicken embryonic develop-ment, distinct anatomical structuresand cell types of the cochlea can be dis-tinguished morphologically by nuclearstaining (Nu) with 4,60-diamidino-2-phenylindole (DAPI) and by hematox-ylin and eosin (HE) staining from E11,when hearing begins in chickenembryo (Saunders et al., 1973; Jack-son and Rubel, 1978; Jones et al.,2006). For example, in transverse sec-tions through the mid-region of thecochlea at E11 (stage 37), sensory haircells and supporting cells located inthe sensory epithelium (SE) of the bas-ilar papilla (BP) are distinguishedclearly (Fig. 1A), while homogene (co-lumnar) cells are found between theBP and the tegmentum vasculosum(TV) neighboring the superior fibro-cartilaginous plate. Non-neuronalspindle-shaped cells distributebetween the superior edge of the BPand the neuronal acoustic ganglioncells (Fig. 1A, B) and along the nervefibers projecting from acoustic gan-glion cells to the SE. At this stage,spindle-shaped cells are easily distin-guished from the round-shaped andlarge-sized acoustic ganglion cells byHE staining (Fig. 1B). At E17 (Fig.1C), hair cells differentiated into onelayer locate on the apical surface of theBP, while supporting cells are in placeunderneath the hair cells (Fig. 1C). Atthis stage, homogene cells areobserved clearly.

The aim of this study was to analyzethe expression patterns of the fivemembers of the ADAM family in adja-cent transverse sections through mid-regions of the cochlea from E11 (stage37) to E18 (stage 44). Expression pat-terns are shown in Figure 1 forADAM9 and ADAM17, in Figure 2 forADAM10, and in Figure 3 forADAM22 and ADAM23, and are

organized according to the develop-mental stages (early to late). Further-more, expression patterns of theADAMs in adjacent longitudinal sec-tions through the neural side of the BPat E11 are also shown in Figure 4.Sense RNA probes were used as nega-tive controls (e.g., Fig. 1D). In general,each of the ADAMs investigated dem-onstrates a spatially restricted andtemporally regulated expression pat-tern in the distinct anatomical struc-tures and different cell types of thecochlea, with partial overlap betweeneach other.

ADAM9

At E11, ADAM9 mRNA is abundantlyexpressed by the hair cells and sup-porting cells of the SE in the BP, andby the homogene cells, the cuboidalcells, and the acoustic ganglion cells,and in the TV (Fig. 1E–G). At E14,ADAM9 signals are strongly main-tained in the cuboidal cells and theacoustic ganglion cells, and in the TV,moderately in the supporting cells andthe homogene cells, but very weakly inthe hair cells (Fig. 1H–J). At E16,strong expression remains in theacoustic ganglion cells and in the TV,but weak expression by the supportingcells (Fig. 1K–M). ADAM9 is no longerdetectable in the hair cells and thehomogene cells from E16 onward.Instead, the spindle-shaped cells startto strongly express ADAM9. At E18,strong expression remains in theacoustic ganglion cells, the spindle-shaped cells, and in the TV, but sup-porting cells only weakly express theADAM (Fig. 1N–P).

ADAM17

At E11, ADAM17 mRNA is widely andstrongly transcribed in the anatomicalstructures of the cochlea, e.g., in theSE of the BP, especially in the inferiorpart (arrow in Fig. 1R), by the cuboidalcells, the spindle-shaped cells and theacoustic ganglion cells, and in the TV,but only moderately by the homogenecells (Fig. 1Q–S). From E14 to E16,strong expression remains in the haircells and supporting cells of the BP, inthe spindle-shaped cells, the acousticganglion cells, and in the TV, butexpression decreases in the homogene

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ADAMS EXPRESSION IN DEVELOPING COCHLEA 2257

cells (Fig. 1T–Y). ADAM17 expressionby the hair cells and supporting cellsin the BP can be distinguished clearly.At E18, strong expression is found in

the spindle-shaped cells and theacoustic ganglion cells, moderateexpression in the supporting cells, andweak expression in the hair cells of the

superior part of the BP and in the TV(Fig. 1Z–B0). At this stage, ADAM17 isno longer detectable in the homogenecells.

Fig. 1. Expression of ADAM9 and ADAM17 in transverse sections through the mid-region of the developing chicken cochlea from incubation day11 (E11) to E18 (marked). Abneural part (ifp) is on the left side and neural part (sfp) on the right. A–C: Nuclear staining (Nu; A) and hematoxylinand eosin (HE) staining (B, C) show different structures of the cochlea. D: In situ hybridization using sense cRNA probe for ADAM9 as a negativecontrol. E–B0: In situ hybridization for ADAM9 (E–P) and ADAM17 (Q–B0) at different stages (marked). cu, cuboidal cells; hc, hair cells; ho, homo-gene cells; ifp, inferior fibrocartilaginous plate; se, sensory epithelium; sfp, superior fibrocartilaginous plate; sg, acoustic ganglion cells; sp, sup-porting cells; ssc, spindle-shaped cells; tv, tegmentum vasculosum. Scale bars ¼ 100 mm in B (applies to B and C) and in F (applies to F, G, I, J,L, M, O, P, R, S, U, V, X, Y, A0, B0); 200 mm in A (applies to A and D) and in E (applies to E, H, K, N, Q, T, W, Z).

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2258 YAN ET AL.

ADAM10

Similar to ADAM17, at E11, ADAM10mRNA is abundantly and widelyexpressed in the SE of the BP, by the

homogene cells, the cuboidal cells, thespindle-shaped cells, and the acousticganglion cells as well as in the TV(Fig. 2A–C). Remarkably, ADAM10

signals are also strong in the abneu-ral mesenchymal cells of the inferiorfibrocartilaginous plate (ifp in Fig.2A). From E14 onward, strong

Fig. 2. Expression of ADAM10 in transverse sections through the mid-region of the developing chicken cochlea from incubation day 11 (E11) to E18(marked). Abneural part (ifp) is on left side and neural part (sfp) on right side. A–H: In situ hybridization for ADAM10 mRNA at different stages. Arrows inA–C indicate the sensory epithelium in the inferior part of the basilar papilla. I–X: Nuclear staining (Nu; I, M, Q, U), and double-labeled immunostainingfor ADAM10 (green in J, N, R, V), for Cad7 (red in K and the insert in K), N-Cad (red in O), R-Cad (red in S), and for neurofilament (NF; red in W) at E11,respectively. L shows the merged images displayed in J and K; P from N and O; T from R and S; and X from V and W. Arrows in K, L, V–X indicate theCad7-positive spindle-shaped cells (K, L) and the efferent neuronal fibers of the acoustic ganglia (V–X), and in N–P, R–T arrows show the differentexpression boundary of ADAM10 (N) and N-Cad (O) in the inferior part of the basilar papilla, and of ADAM10 (R) and R-Cad (S) by homogene clles andadjacent cells. cu, cuboidal cells; hc, hair cells; ho, homogene cells; ifp, inferior fibrocartilaginous plate; me, mesenchymal cells; nf, neural fiber; se,sensory epithelium; sfp, superior fibrocartilaginous plate; sg, acoustic ganglion cells; sp, supporting cells; ssc, spindle-shaped cells; tv, tegmentumvasculosum. Scale bars ¼ 50 mm in Q (applies to Q–T) and in U (applies to U–X); 100 mm in B (applies to B, C, E, G, H); 200 mm in A (applies to A, D, F).

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expression is maintained in the sup-porting cells, the homogene cells, thespindle-shaped cells, and in the TV,but only moderate expression remainsin the hair cells (Fig. 2D–H).

Interestingly, it has been found thatADAM10 can shed the ectodomain ofN-cadherin (N-Cad) and E-Cad, modu-lating the cell–cell adhesion and sig-nal transduction (Maretzky et al.,

2005; Reiss et al., 2005; Reiss andSaftig, 2009). Eight classic cadherinsare expressed in distinct anatomicalregions and cell types of the de-veloping cochlea (Luo et al., 2007).

Fig. 3. Expression of ADAM22 and ADAM23 in transverse sections through the mid-region of the developing chicken cochlea from incubationday 11 (E11) to E18 (marked). Abneural part (ifp) is on the left side and the neural part (sfp) on the right side. A–W: In situ hybridization forADAM22 (A–K) and ADAM23 (L–W) at different stages (marked). X: Schematic summary of the investigated ADAMs (represented by distinct colorcycles) expressed in different cochlear structures at E14. cu, cuboidal cells; hc, hair cells; ho, homogene cells; ifp, inferior fibrocartilaginous plate;me, mesenchymal cells; se, sensory epithelium; sfp, superior fibrocartilaginous plate; sg, acoustic ganglion cells; sp, supporting cells; ssc, spin-dle-shaped cells; tv, tegmentum vasculosum. Scale bars ¼ 100 mm in B (applies to B, D, E, G, H, J, K, M, N, P, Q, S, T, V, W); 200 mm in A(applies to A, C, F, I, L, O, R, U).

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Therefore, we further investigated thecoexpression of ADAM10 togetherwith the classic cadherins includingCad7, N-Cad, and R-Cad at proteinlevel in the cochlea by double-immuno-histochemistry at E11 (Fig. 2I–T). AtE11, ADAM10 protein (green color inFig. 2I–X) is expressed widely andstrongly in the distinct anatomicalstructures of the cochlea, consistentwith the expression of ADAM10 at themRNA level (Fig. 2A–D). Cad7 isexpressed strongly in the spindle-shaped cells (arrows in Fig. 2K, L),

where ADAM10 is coexpressed (yellowcolor in Fig. 2L). In acoustic ganglia,Cad7 protein is expressed in the wrapsaround the cells expressing ADAM10protein, but the Cad7-positive neuralfibers do not coexpress ADAM10 (nf inFig. 2K, L and the insert in K; Luoet al., 2007). N-Cad signals are strongin the hair cells and the supportingcells of the BP, where ADAM10 is alsocoexpressed (yellow color in Fig. 2P).Remarkably, the coexpression ofADAM10 and N-Cad in the BP isinversely related. For example, N-Cad

expression is strong in the central andsuperior part of the SE, whereADAM10 expression is weaker. In con-trast, N-Cad expression is weak in theinferior part of the SE, where ADAM10is stronger (Fig. 2M–P). The differentexpression of ADAM10 or N-Cad in theSE of the BP forms a clear boundary(arrows in Fig. 2N–P). In the acousticganglion cells, N-Cad expressionwrapsaround the cells expressing ADAM10(Fig. 2O, P). Furthermore, the coex-pression of ADAM10 and R-Cad by thehomogene cells is also observed (Fig.2Q–T). Similarly, the coexpression ofADAM10 and R-Cad by the homogenecells is also inversely related whencomparing ADAM10 signals in thehomogene cells to the cells of the adja-cent tissues (Fig. 2R–T). In the acousticganglion cells, R-Cad protein isexpressed in thewraps around the cellsexpressing ADAM10 (Fig. 2S, T).Moreover, ADAM10 and neurofila-

ment are coexpressed in the acousticganglion cells and their neurites (yel-low color in Fig. 2X) as detected byimmunostaining with antibodyagainst neurofilament (red color inFig. 2W, X), a specific marker for dif-ferentiated neurons and their proc-esses (Hatta et al., 1987).

ADAM22

In contrast to the expression patternsof ADAM9, ADAM10, and ADAM17,mRNA signals of ADAM22 are promi-nently found in the restricted struc-tures of the developing cochlea. FromE11 to E18, ADAM22 is strongly andspecifically expressed by the spindle-shaped cells and the acoustic ganglioncells (Fig. 3A–K).

ADAM23

At E11, ADAM23 mRNA is expressedstrongly by the hair cells and support-ing cells in the BP, by the acoustic gan-glion cells, and in the TV, but weaklyby the homogene cells (Fig. 3L–N). AtE14, ADAM23 is gradually restrictedto the hair cells in the superior part ofthe BP, and continuously stronglyexpressed in the acoustic ganglioncells and in the TV, but decreases inthe supporting cells (Fig. 3O–Q). Atthis stage, the homogene cells showweak ADAM23 signals. From E16onward, strong expression persists in

Fig. 4. Expression of the ADAMs in adjacent longitudinal sections through the neural side ofthe BP at incubation day 11 (E11). Distal end is on the left side and proximal end on the rightside. In situ hybridization for ADAM9 (A, B), ADAM10 (C, D), ADAM17 (E, F), ADAM22 (G, H),and ADAM23 (I, J). hc, hair cells; sg, acoustic ganglion cells; sp, supporting cells; ssc, spindle-shaped cells; tv, tegmentum vasculosum. Scale bars ¼ 100 mm in B (applies to B, D, F, H, J);200 mm in A (applies to A, C, E, G, I).

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the hair cells and the acoustic ganglioncells (Fig. 3R–W), but ADAM23 isno longer detectable in the support-ing 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 sectionsthrough the neural side of the BP atE11, ADAM9 mRNA is stronglyexpressed by the hair cells, the sup-porting cells, the acoustic ganglioncells, and in the TV (Fig. 4A, B).ADAM10 signals are strong in the haircells and the supporting cells of theSE, in the spindle-shaped cells and theacoustic ganglion cells, and also in theTV (Fig. 4C, D). ADAM17 expressionis strong in the hair cells and the sup-porting cells of SE, in the spindle-shaped cells and the acoustic ganglioncells, and in the TV (Fig. 4E, F).ADAM22 mRNA transcription is re-stricted to the spindle-shaped cellsand the acoustic ganglion cells (Fig.4G, H), while ADAM23 mRNA is tran-scribed in the hair cells, the support-ing cells, and in the acoustic ganglioncells (Fig. 4I, J).

In summary, each individual ADAMis expressed in different anatomicalstructures of the cochlea during em-bryonic development (Fig. 3X). Theexpression patterns differ from eachother but show partial overlap.

DISCUSSION

Expression of the ADAMs in

Sensory Epithelium and Cell

Differentiation

In the present study, ADAM9,ADAM10, ADAM17, and ADAM23 areobserved to be expressed strongly bythe hair cells and the supporting cellsof the BP (Figs. 1–3), and as embryosdevelop, the expression of the individ-ual ADAMs decreases. Hair cells andsupporting cells share a common pro-genitor cell during chicken cochleardevelopment (Fekete et al., 1998;Satoh and Fekete, 2005). However, lit-tle is known about how these cells dif-ferentiate into hair cells and support-ing cells and which molecules regulatethis process. One possible mechanism

to explain such diversification of thesensory development is lateral inhibi-tion, whereby signal interactionbetween neighboring cells induces thecells to adopt different developmentalfates. For example, activation of Notchsignaling promotes the differentiationof hair cells and supporting cells alongdistinct developmental pathways bymediating lateral inhibition (Daudetand Lewis, 2005; Kiernan et al., 2005),as detected in other sensory systems,e.g., in the retina (Henrique et al.,1997). Indeed, the chicken homologuesof Notch receptor and Delta and Ser-rate ligands are found to be expressedin the developing cochlea, e.g., in theotic vesicle and the supporting cells(Warchol et al., 1993; Warchol, 2002).Of interest, it is known that theADAMs, especially ADAM10 andADAM17, participate in proteolyticcleavage of the Notch receptors, whichare essential for controlling neural cellfate determination (Yang et al., 2006;Edwards et al., 2008). Therefore, theexpression of ADAM10 and ADAM17in the SE during cochlear developmentmay contribute to the differentiationand cell fate determination of haircells and supporting cells via proteo-lytic shedding of Notch.

Furthermore, several members ofthe cadherins are temporally regu-lated in the SE of the developingchicken cochlea, suggesting a role ofcadherins in the differentiation ofsupporting cells and hair cells (Luoet al., 2007). N-Cad-mediated cell–cellinteraction and b-catenin signallingregulate sensory cell proliferation inthe chicken inner ear (Warchol, 2002).ADAM10 is a major protease shed-ding the extracellular domain of cad-herins and modulating cell–cell adhe-sion and signal transduction(Maretzky et al., 2005; Reiss et al.,2005, 2006; Kohutek et al., 2009). Inthis study, expression of ADAM10and N-Cad proteins was observed tobe inversely related in the SE (Fig.2N–P), suggesting that ADAM10 mayalso contribute to the proliferationand differentiation of hair cells andsupporting cells by exclusively regu-lating the expression of N-Cad duringcochlear development.

Moreover, that ADAM23 plays a rolein neuronal differentiation has beensuggested by an in vitro experimentwith cultured P19 cells (Sun et al.,

2007), and it also controls the differen-tiation of neural crest cells during em-bryonic development (Neuner et al.,2009). In the present study, ADAM23is found to be expressed in the haircells at different stages, especially inthe superior edge of the BP (Fig. 3L–V). Therefore, it will be interesting toinvestigate whether ADAM23 contrib-utes to the differentiation of hair cells,particularly the tall hair cells in thesuperior edge of the BP.


Homogene Cells

In this study, we show that theADAMs, including ADAM9, ADAM10,ADAM17, and ADAM23, are tran-scribed in the homogene cells, wheretheir expressions gradually decreasefrom E11 onward and become weak atE16 to E18 (Figs. 1–3). The homogenecells locate in the superior fibrocartila-ginous plates between the TV and SEwith a columnar shape from E11. Theadjacent homogene cells are connectedlaterally with well-developed tightjunctions (Hirokawa, 1980). Duringchicken cochlear development, theupper lumenal side of the tectorialmembrane appears as a smooth amor-phous layer and is produced by homo-gene cells, which secrete the extracel-lular matrix (ECM) directly into thelumen of the cochlea (Tanaka andSmith, 1975; Cohen and Fermin, 1985;Runhaar, 1989; Shiel and Cotanche,1990). During the different phases ofthe secretory activity, the homogenecells show some differences in theirsize, shape, and density of the cyto-plasmic contents, contributing to theproduction of the tectorial membrane(Cohen and Fermin, 1985; Shiel andCotanche, 1990). Furthermore, homo-gene cells may be involved in a me-chanical process adjusting the tensionof the tectorial membrane due to theirhigh expression of homogenin and fila-mentous actin (Heller et al., 1998).The ADAM proteins contain a metallo-protease domain and a disintegrin do-main, which are involved in cell–celland cell–ECM interaction by cleavingand releasing cell-surface proteinsand by remodelling the ECM (White,2003; Yang et al., 2006; Edwards et al.,2008). The changes of ADAM mRNAlevels in the homogene cells coincidewith alterations of the cytoplasmic

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organelles of the homogene cells dur-ing their secretory phase. For exam-ple, at the beginning of secretion (e.g.,E11–14), the mRNA levels of ADAMsare higher in the cells (Figs. 1–3) thatmay be responsible for the synthesisand secretion of the respective pro-teins. At later stages of development(e.g., E18), when the formation of thetectorial membrane is almost com-pleted (Cohen and Fermin, 1985; Shieland Cotanche, 1990), the transcriptlevels of ADAMs decline gradually(Figs. 1–3). Whether the investigatedADAMs participate in the formation ofthe tectorial membrane via proteolyticshedding and/or cell–ECM interactionremains to be elucidated.


Spindle-Shaped Cells and

Acoustic Ganglion Cells

Spindle-shaped cells are located inthe superior fibrocartilaginous platesbetween the SE and the acoustic gan-glia and support the signal transfer ofthe sensory dendrites from hair cellsto the acoustic ganglia (Heller et al.,1998). In the present study, ADAM10,ADAM17, and ADAM22 are stronglyexpressed in the spindle-shaped cellsfrom E11 onward, and ADAM9 isstrongly expressed from E16 onward(Figs. 1–3). What role these ADAMsplay in the spindle-shaped cells dur-ing cochlear development is unclear.

In the cochlea, projections of acous-tic ganglion cells to targeted cochlearnucleus undergo refinement to formprecise cochleotopic terminals andprovide the basis for tonotopic map-ping throughout the central auditorysystem. The guidance of the nervefibers mediating this tonotopic projec-tion is controlled by different mole-cules (Richardson et al., 1987; Kaji-kawa et al., 1997; Lee et al., 2004).The present study reveals that thefive ADAM mRNAs are stronglyexpressed by the acoustic ganglioncells during development (Figs. 1–3)and ADAM10 protein is alsoexpressed in both the acoustic gan-glion cells and their neurites, whereneurofilament is coexpressed (Fig.2U–X). Previous studies have shownthat the ADAMs are involved in axonoutgrowth and guidance. For exam-ple, ADAM8, ADAM10, and ADAM17

can shed the ectodomain of neural celladhesion molecule (NCAM), modulat-ing the neurite outgrowth and/orbranching (Hinkle et al., 2006; Kaluset al., 2006). ADAM21 is present ingrowing axonal tracts and partici-pates in the final axonal outgrowthand/or synapse formation (Yang et al.,2005). ADAM10 plays a pivotal role inthe correct projection of retinal gan-glion cell axons to their target tectum(Chen et al., 2007). Finally, ADAM22and ADAM23 are also expressed inthe auditory nuclei of the hindbrain(Lin et al., 2008). Therefore, expres-sions of the investigated ADAMs inthe acoustic ganglion cells and the au-ditory nuclei during cochlear develop-ment may suggest a role of theADAMs in the guidance of acousticganglion neurites to their targetnuclei in the hindbrain.

Furthermore, Neuner and his col-leagues (2009) demonstrated in Xeno-pus that ADAM23 could regulate thedifferentiation of neural crest cellsduring embryonic development. TheADAMs including ADAM9, ADAM10,ADAM22, and ADAM23 have beenidentified to be spatiotemporally regu-lated in the dorsal root ganglion andplay a role in the neuronal differen-tiation of the sensory neurons (Linet al., 2010). Therefore, the five mem-bers of the ADAMs studied here mayalso be involved in the differentiationof the acoustic ganglion cells duringcochlear development.


Tegmentum Vasculosum

The TV is a pleated epithelial layerconsisting of a mosaic of light and darkstaining cells, which are involved inthe water and ion homeostasis of theendolymph (Cotanche et al., 1987;Ryals et al., 1995). The TV in chickenseparates the endolymphatic fluid inthe scala media from the perilymph ofthe scala vestibuli and functions likethe stria vascularis in mammals(Ryals et al., 1995; Manley, 2000).Both inner ear fluids and cerebrospi-nal fluid show remarkably stable ionicconcentrations, and the stria vascula-ris and choroid plexus, which areresponsible for production of the re-spective fluids, share similar charac-teristics, e.g., they contain the sameproteins (Lecain et al., 2000; Saito

et al., 2001). Of interest, in the devel-oping chicken brain, ADAM9,ADAM10, ADAM12, and ADAM23 aretemporally expressed in the choroidplexus (Lin et al., 2008). In the presentstudy, we also found that ADAM9,ADAM10, ADAM17, and ADAM23 arestrongly expressed in the TV. There-fore, whether the expression of theADAMs in both the TV and choroidplexus plays a general role in the de-velopment of them should be furtherelucidated.

EXPERIMENTAL

PROCEDURES

Chicken Embryos, RNA

Probes, and Antibodies

Fertilized eggs from white Leghornchicken (Gallus domesticus) wereincubated in a forced-draft egg incuba-tor (BSS160, Ehret, Germany) at 37�Cwith 60% humidity. Chicken embryoswere staged according to Hamburgerand Hamilton (1951). After theembryos were deeply anesthetized bycooling on ice, they were removed fromthe shell and perfused through theheart with 4% formaldehyde in PBSbuffer (13 mM NaCl, 7 mM Na2HPO4,3 mM NaH2PO4; pH 7.4). Cochleaewere then separated and collected atembryonic incubation day 11 (E11),E14, E16, E17, and E18 (stages 37, 40,42, 43, and 44, respectively; at least 5cochleae for each stage).For in situ hybridization, digoxige-

nin-labeled sense and antisense cRNAprobes were synthesized in vitro usingplasmids containing previously clonedADAM sequences (Lin et al., 2008) ascDNA templates according to theman-ufacturer’s instructions (Roche,Mannheim, Germany). Sense cRNAprobes were used as a negativecontrol.For immunohistochemistry, pri-

mary rabbit polyclonal antibodyagainst ADAM10 (Chemicon, Hamp-shire, UK; Hall and Erickson, 2003),and primary mouse and rat monoclo-nal 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 neu-rofilament (NF; Hatta et al., 1987)were used. CCD7-1, NCD-2, RCD-2,and NF antibodies were kind gifts ofDr. S. Nakagawa and Dr. M. Takeichi

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(RIKEN Center for DevelopmentalBiology, Kobe, Japan). Alexa 488-la-beled (Molecular Probes, Eugene, OR)and Cy3-labeled (Dianova, Hamburg,Germany) secondary antibodiesagainst rabbit, mouse, or rat IgG wereused.

In Situ Hybridization

In situ hybridization on sectionsthrough mid-regions of the cochleawas performed according to the proto-col described previously (Luo et al.,2004). In brief, after post-fixation with4% formaldehyde in PBS, cryostat sec-tions were pretreated with proteinaseK and acetic anhydride. Then sectionswere hybridized with cRNA probe at aconcentration of about 1–5 ng/ml over-night at 70�C in hybridization solution(50% formamide, 3� SSC, 1� Den-hardt’s solution, 250 mg/ml yeast RNA,and 250 mg/ml salmon sperm DNA).After the unbound cRNAwas removedby RNAse, the sections were incubatedwith alkaline phosphatase-conjugat-ed anti-digoxigenin Fab fragments(Roche, Mannheim, Germany) at 4�Covernight. For visualization of the la-beled mRNA, a substrate solution ofnitroblue tetrazolium salt (NBT) and5-bromo-4-chloro-3-indoyl phosphate(BCIP) was added. The colour reactionon sections was viewed and photo-graphed under a transmission micro-scope (BX40; Olympus, Hamburg,Germany) equipped with a digitalcamera (DP70; Olympus). Photo-graphs were adjusted in contrast andbrightness with the Photoshop soft-ware (Adobe System, Mountain View,CA).

Immunohistochemistry

Fluorescent immunostaining was per-formed on sections through mid-regions of the cochlea using themethoddescribed previously (Luo and Redies,2004). In brief, after post-fixation in4% formaldehyde, cryostat sections of20 mm thickness were pre-incubatedwith a blocking solution (5% skimmedmilk and 0.3% Triton X-100 in TBS) atroom temperature for 60 min. Thensections were incubated overnight at4�C with the primary antibody, fol-lowed by the secondary antibody atroom temperature for 1 hr. Finally, cellnuclei were stained with 40-6-Diami-

dino-2-phenylindole (DAPI; Sigma,Munich, Germany). Fluorescence wasimaged under a fluorescentmicroscope(BZ-8000; Keyence DeutschlandGmbH, Neu-Isenburg, Germany). Dig-ital images were adjusted in contrastand brightness with the Photoshopsoftware (Adobe Systems).

For double-label fluorescent immu-nohistochemistry, sections were firstimmunostained with an antibodyagainst ADAM10 using the methoddescribed above. Subsequently, immu-nostainings for cadherin or neurofila-ment were performed.

ACKNOWLEDGMENTSWe thank Dr. S. Nakagawa and Dr. M.Takeichi for their kind gifts of the anti-bodies, Dr. C. Redies for support of thisstudy, and Dr. E. Mix for the criticalreading of this manuscript.

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regional expression of the adams in developing chicken cochlea

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