Histochem Cell Biol (2008) 130:339–361 DOI 10.1007/s00418-008-0441-8

ORIGINAL PAPER

Molecular composition of tight and adherens junctions in the rat olfactory epithelium and Wla Axel Steinke · SoWa Meier-Stiegen · Detlev Drenckhahn · Esther Asan

Accepted: 6 May 2008 / Published online: 4 June 2008 © Springer-Verlag 2008

Abstract Tight and adherens junctions (TJs, AJs) between neurons, epithelial and glial cells provide barrier and adhesion properties in the olfactory epithelium (OE), and subserve functions such as compartmentalization and axon growth in the Wla olfactoria (FO). ImmunoXuorescence and immunoelectronmicroscopy were combined in sections of rat OE and FO to document the cellular and subcellular localization of TJ proteins occludin(Occl), claudins(Cl) 1–5 and zonula occludens(ZO) proteins 1–3, and of AJ proteins N-cadherin(cad), E-cad, and alpha-, betaand p120-catenin(cat). With the exception of Cl2, all TJ proteins were colocalized in OE junctions. DiVerences in relative immunolabeling intensities were noted between neuronal and epithelial TJs. In the FO, Cl5-reactivity was localized in olfactory ensheathing cell (OEC) junctions, Cl1-reactivity in the FO periphery, with diVerential colocalization with ZOs. Supporting cells formed N-cad-immunoreactive (ir) AJs with olfactory sensory neurons, E-cad-ir junctions with microvillar and gland duct cells, and both N-cad and E-cad-ir junctions in homotypic contacts. Alpha,
- and p120-cat were localized in all AJs of the OE. AJs were scarce in the globose basal cell layer. Immature and mature neurons formed numerous contacts. In the FO, AJs were documented between OECs, between OECs and axons, and between axons. Most AJs colocalized N-cad with catenins, occasionally E-cad-ir AJs were found in the FO periphery. Characteristics of molecular composition suggest diVerential properties of TJs formed by neuronal, epithelial and glial cells in the OE and FO. The presence and molecular composition of AJs are consistent with a role A. Steinke · S. Meier-Stiegen · D. Drenckhahn · E. Asan (&) Institute of Anatomy and Cell Biology, University of Wuerzburg, Koellikerstr. 6, 97070 Wuerzburg, Germany e-mail: [email protected]

of AJ proteins in neuroplastic processes in the peripheral olfactory pathway. Keywords Olfactory epithelium · Fila olfactoria · Claudins · Cadherins · Fluorescence immunohistochemistry · Immunoelectron microscopy Abbreviations AJ Adherens junction AMCA 7-Amino-4-methylcoumarin-3-acetic acid BC Basal cell BCL Basal cell layer BSA Bovine serum albumin
-TUB Beta-tubulin
III-TUB Neuron speciWc
-tubulin,
III isotype Cad Cadherin Cat Catenin Cl Claudin CK Cytokeratin EDC Excretory duct cell FO Fila olfactoria FI Fluorescence immunohistochemistry g Green Xuorescent GA Glutaraldehyde GAP43 Growth associated protein 43 GBC Globose basal cell GFAP Glial Wbrillary acidic protein HBC Horizontal basal cell -ir -immunoreactive LP Lamina propria m Monoclonal MC Microvillar cell NDS Normal donkey serum NGS Normal goat serum OB Olfactory bulb

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Occl OE OEC OG ONF ONL OMP OSN OSNL p p120 PE PFA PBS r RT SC SCL TJ V ZO ZO1m ZO1rp ZO1gp

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Occludin Olfactory epithelium Olfactory ensheathing cell Olfactory gland Olfactory nerve Wbroblast Olfactory nerve layer Olfactory marker protein Olfactory sensory neuron Olfactory sensory neuron layer Polyclonal p120-catenin Postembedding immunogold labeling Paraformaldehyde Phosphate buVered saline Red Xuorescent Room temperature Supporting cell Supporting cell layer Tight junction Vessel Zonula occludens protein Rat monoclonal anti-ZO1 Rabbit polyclonal anti-ZO1 Goat polyclonal anti-ZO1

Introduction The peripheral olfactory pathway is a unique part of the nervous system. Olfactory sensory neurons (OSN) are the only neurons which are situated in a surface epithelium, the olfactory epithelium (OE), and thus have to participate in establishing an epithelial barrier. They are directly exposed to toxic or infectious agents in the air, which may cause massive OSN degeneration. A loss of odor recognition is prevented by continuous generation of new OSN via asymmetric division of neuronal progenitor cells in the basal epithelium. Newly generated, immature OSN move apically in the epithelium, extend their dendrites towards the epithelial surface and their axons through the Wla olfactoria (FO) into the olfactory bulb (OB), where they target speciWc glomeruli and form synapses with OB projection neurons (for review see Schwob 2002). Intercellular junctions between diVerent cell types (stem cells, neurons, epithelial or glial cells) fulWl multiple functions in the peripheral olfactory system. Junctional complexes containing zonulae occludentes (tight junctions, TJs) and zonulae adherentes in the apical epithelium and in the olfactory gland (OG) and and excretory duct cells (EDC) presumably provide barrier and adhesion properties, and thus may be important for creating the olfactory mucus microenvironment necessary for odor signal transduction processes (Getchell and Getchell 1992; Menco 1994). As

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has been shown in other neurogenetic areas of the adult brain, formation and release of basolateral puncta adherentia may be instrumental in processes of progenitor division and neuronal diVerentiation (Chauvet et al. 2003; Seki et al. 2007; Gao et al. 2007). Intercellular contacts in the FO presumably subserve such varying functions as compartmentalization and axon growth (Mack and Wolburg 1986; Miragall et al. 1994). Both in development and in adulthood, axon growth and correct targeting appear to be inXuenced by intercellular contacts between growing OSN axons (Yoshihara et al. 1997; Alenius and Bohm 1997; Hummel and Zipursky 2004; Chung et al. 2004), and between OSN axons and the speciWc glial cells of the FO, the olfactory ensheathing cells (OEC; Chuah et al. 1991; Chuah and Au 1994). In all vertebrate species described to date, the OE is a pseudostratiWed epithelium containing supporting cells (SC), microvillar cells (MC), OSN of diVerent maturity, two types of basal cells (horizontal and globose; HBC and GBC), and cells lining the excretory ducts of olfactory glands (EDC) (e.g., Schwob 2002; Asan and Drenckhahn 2005). The lamina propria (LP) contains the olfactory glands (OG), and fascicles of the FO. Ultrastructural investigations have documented the presence of TJs and AJs in the OE and OG in various species (e.g., Menco 1980; Moran et al. 1982; Mendoza 1993; Herrera et al. 2005). In freeze-fracture studies, diVerential structural characteristics of TJs between the cell types were described and were suggested to be related to TJ dynamics (Menco 1980). Data on the molecular composition of OE TJs are rather scarce yet. In the rat OE, a peripheral TJ protein, Zonula occludens protein 1 (ZO1), and an integral membrane protein of TJs, occludin (Occl), were immunohistochemically localized (Miragall et al. 1994; Asan and Meyer-Stiegen 1998; Hussar et al. 2002). While immunoreaction intensities for ZO1 appeared to vary between diVerent types of epithelial TJs, similar observations were not reported for Occl (Miragall et al. 1994; Hussar et al. 2002). In freeze-fracture studies of the rat FO, Mack and Wolburg (1986) and Miragall et al. (1994) described TJs between glial cells. Ultrastructural analyses of FOs in diVerent species using transmission electron microscopy did not report the presence of conspicuous TJs between OEC processes (Field et al. 2003; Herrera et al. 2005). TJs between the olfactory nerve Wbroblasts (ONF), speciWc cells surrounding larger FO fascicles in the LP up to their passage through the pia into the olfactory nerve layer (ONL) of the olfactory bulb (OB), were described (Field et al. 2003; Herrera et al. 2005). ZO1-immunoreactivity was documented immunocytochemically in the FO periphery (Miragall et al. 1994). A detailed analysis of the contribution of other TJ proteins to the diVerent TJs on a cellular and subcellular level

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both in the OE and the FO is lacking. Especially, analysis of the expression and cellular localization of claudins, which have been suggested to be responsible for the ample variety in electrical resistance and in selective paracellular ionic permability displayed by diVerent TJs (e.g., GonzalesMariscal et al. 2003; Schneeberger and Lynch 2004; Hartsock and Nelson 2008), has not been carried out yet. In view of the fact that maintenance of the particular ionic composition of the olfactory mucus is of utmost importance for olfactory signal transduction, the exact molecular composition of epithelial TJs and a possible contribution of claudins to the diVerent types of TJs is of great interest. Additionally, a detailed analysis of TJs formed by OEC is of some importance. These unusual glial cells have been used in transplantation experiments to bridge central nervous lesions (e.g., Boyd et al. 2005; Ibrahim et al. 2006), and have been shown both to enhance supraspinal axon regrowth and to be able to myelinate the regrowing axons (e.g., Sasaki et al. 2006). TJs are components of central and peripheral myelin sheaths (Poliak et al. 2002), and of ensheathments of unmyelinated axons (Sugimoto et al. 2002) in the periphery. Data on the molecular composition of TJs formed by OEC would broaden the understanding of the neurobiology of these important glial cells, especially of their potency to aid axonal regeneration and remyelination in central and peripheral lesions. Adherens junctions (AJs) have been shown both ultrastructurally (e.g., Mendoza 1993; Herrera et al. 2005) and by immunocytochemical documentation of the localization of cadherins (cads), the integral membrane proteins of zonulae adherentes and puncta adherentia, both in the OE and the FO of diVerent species. N-cad has been reported to be present in the chick and developing mouse OE and olfactory nerve (Norgren and Brackenbury 1993; Akins et al. 2007). E-cad was detected in the rat and mouse OE (Whitesides and LaMantia 1996; Asan and Drenckhahn 2005; Akins et al. 2007), but not in mouse OSN axons (Whitesides and LaMantia 1996; Akins et al. 2007). Cadherin-mediated contact formation has been suggested to inXuence OSN axon growth and targeting. N-cad enhances mouse OSN axon growth (Chuah et al. 1991), and is instrumental in promoting sorting of OSN axon terminals into protoglomeruli in drosophila (Hummel and Zipursky 2004). In vitro studies have yielded controversial conclusions concerning the importance of OEC expression of N-cad for OSN axon elongation (Chuah et al. 1991; Chuah and Au 1994). Recent evidence indicates that catenins (cats), cytoskeletal linker proteins, play important roles as signal transduction molecules and regulators of actin dynamics (Noren et al. 2000; Hartsock and Nelson 2008). Therefore, cats have been proposed to integrate cell–cell junctions and cytoskeletal dynamics with signaling pathways governing morphogenesis, tissue homeostasis, and even intercellular

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communication between diVerent cell types (Perez-Moreno and Fuchs 2006; Gao et al. 2007). DiVerential developmental distribution of cadherins and catenins was observed in the olfactory nerve layer of the OB (Akins and Greer 2006; Akins et al. 2007), leading to the suggestion that adhesion complexes may contribute to establishment and maintenance of olfactory networks (Akins and Greer 2006; Akins et al. 2007). Although, as described, the overall distribution of AJ proteins in the peripheral olfactory pathway has been studied, the molecular composition of contacts between the various cell types in the diVerent parts of the peripheral olfactory pathway is not clear yet. However, knowledge of the exact molecular composition is a necessary basis for further studies on the functions of these contacts in neuroplastic processes in the OE and FO. Therefore, in the present study, Xuorescence immunohistochemistry for various TJ and AJ proteins was carried out on 0.5–1
m semithin sections from freeze–dried, Eponembedded rat nasal mucosa and OB. This technique allows precise identiWcation of the immunoreactive cellular and subcellular compartments in single and double labelings (Asan and Drenckhahn 2005). The results were complemented by postembedding immunoelectron microscopy and Xuorescence immunohistochemistry on cryosections for selected proteins. The results signiWcantly broaden our knowledge of the molecular composition of diVerent types of TJs and AJs in the peripheral olfactory pathway.

Materials and methods Tissue preparation Investigations were carried out on adult Wistar rats of either sex. For preparation of material for Xuorescence immunohistochemistry, rats were anaesthetized and either immediately decapitated or perfusion Wxed via the left ventricle using, after a short prerinse with heparinized saline, 4% freshly polymerized paraformaldehyde (PFA) in 0.01 M phosphate buVered saline (PBS) pH 7.4 (Asan et al. 2003). The nasal mucosa including the distal FO and the OB were carefully dissected (Asan et al. 2003, Langenhan et al. 2005). Non-Wxed olfactory tissue was immediately frozen in liquid nitrogen-cooled isopentane, freeze dried and embedded in epoxy resin according to established methods (Asan and Drenckhahn 2005). Perfusion-Wxed tissue was postWxed for 2–3 h at room temperature (RT) or overnight at 4°C, rinsed in PBS, inWltrated successively with 10 and 20% sucrose in PBS, and frozen in liquid nitrogen-cooled isopentane. For conventional electron microscopic analysis, rats were perfusion-Wxed as described above with 3% GA in 0.1 M phosphate buVer pH 7.4, the nasal mucosa was dissected and postWxed for 3 h at RT, cut

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into small pieces, osmicated in 1% OsO4 in PBS for 1 h, dehydrated in ethanol and embedded in EPON (Serva, Heidelberg, Germany) according to established methods (Asan et al. 2003). For postembedding immunoelectron microscopy, anaesthetized rats were perfusion Wxed as described above using either 4% PFA in PBS, 4% PFA in 2% sodium acetate at pH 6.5 followed by 4% PFA and 0.02%GA in 0.1 M sodium carbonate/bicarbonate buVer at pH 11 (Berod et al. 1981), or 2% PFA and 0.1% glutaraldehyde (GA) in 0.1 M phosphate buVer pH 7.4. After postWxation as described above in the perfusion Wxative without glutaraldehyde, nasal mucosa was either snap-frozen in liquid nitrogen-cooled propane using a Leica CPC (Leica Systems, Bensheim, Germany), transferred to methanol at ¡90°C, cryosubstituted with Lowicryl HM 22 (Science Services, Munich, Germany) at ¡45°C and polymerized using UV light in a Leica AFS, or dehydrated in graded ethanol, transferred to LR White (Plano, Wetzlar, Germany) and polymerized using UV light at 4°C for 48 h. No signiWcant diVerences in ultrastructural preservation or antigen detectability were observed between the diVerent Wxation and embedding procedures used. Immunohistochemistry Fluorescence immunolabeling Preparation and etching of semithin (0.5–1
m) sections of freeze-dried, Epon-embedded material were carried out as described (Asan and Drenckhahn 2005). Cryostat sections (12
m) of perfusion-Wxed, frozen tissue were thawed onto Superfrost™ (Menzel, Braunschweig, Germany) objective slides and dried overnight. For immunoreactions, semithin sections were washed in PBS after etching while cryostat sections were preincubated in 0.01 M PBS containing 5% normal goat serum (NGS, Dako Systems, Hamburg, Germany) or normal donkey serum (NDS) and 1% Triton-X100 (Sigma, Taufkirchen, Germany) for 1.5–2 h at RT. The sections were covered with 20 or 100
l (semithin and cryostat sections, respectively), of the appropriate dilution of the primary antisera or combinations of antisera and antibodies (speciWcations and dilutions see Table 1) in incubation buVer (PBS containing 0.5% NGS or NDS and, for cryostat sections, 0.5% Triton-X-100), and were incubated overnight at 4°C in a humid chamber. After washing in PBS, the sections were incubated in the appropriate Cy3-, Cy2-, DichlorotriazinylaminoXuorescein (DTAF)- or 7Amino-4-methylcoumarin-3-acetic acid (AMCA)-labeled secondary antisera or combinations of antisera (goat antirabbit, goat anti-mouse, goat anti-rat, donkey anti-goat, donkey anti-rabbit, donkey anti-mouse IgG, all Dianova, Hamburg, Germany, dilution 1:200 or 1:600) in incubation buVer for 2 h. Sections were then washed in PBS and either

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immediately mounted in 60% glycerine in distilled water containing 1.5% n-propyl gallate (Serva, Heidelberg, Germany) as antifading agent, or dehydrated brieXy in graded ethanol and mounted in DePex (Serva). Each reaction was carried out repetitively on semithin sections of tissue from diVerent animals. Reactions on cryostat sections were done for selected TJ proteins as described in the results section. Controls were carried out by omitting from the reaction mixture the primary antiserum/antibody in single labeling, or either, two, or three of the primary antisera/antibodies in double and triple labelings. For Cl2-immunoreactions, which did not give staining in semithin sections from olfactory tissue, positive control immunoreactions were carried out on semithin sections from freeze–dried, Epon-embedded rat kidney, which has been documented to show Cl2staining in proximal tubule epithelia (Enck et al. 2001). Section preparation for conventional transmission electron microscopy, postembedding immunlabeling for electron microscopy Ultrathin (»70 nm) sections of the tissue embedded for electron microscopy were Xoated onto formvar-coated 200 mesh thin bar nickel grids (Plano). Grids were contrasted with uranyl acetate and lead citrate (Reynolds 1963), observed in a LEO 912 AB electron microscope, and documented using either a sheet Wlm or a slow scan CCD camera (Zeiss SMT, Oberkochen, Germany). For postembedding immunolabeling, sections were incubated with 0.05 M glycine in PBS for 15 min, transferred to 5% bovine serum albumin (BSA; BioTrend, Cologne, Germany), 0.1% cold water Wsh gelatine (BioTrend) and 5% NGS in PBS for 30 min, washed in 1%BSA in PBS (incubation buVer) 3 £ 5 min, incubated with the appropriate dilution of the primary antibody (see Table 1) in incubation buVer overnight at 4°C, washed in incubation buVer for 6 £ 5 min, incubated with 0.8 nm immunogold-labeled secondary antibodies of the appropriate species reactivity (BioTrend) for 2 h, washed again for 6 £ 5 min in incubation buVer and for 3 £ 5 min in PBS, postWxed in 2% GA in PBS for 5 min, washed in PBS and water and then silver-intensiWed according to the manufacturer’s instructions using the R-Gent SE-EM silver enhancement kit (BioTrend). For controls, primary antibodies were omitted from the reaction. Contrasting of the grids was done as described. Documentation of immunolabeling results ImmunoXuorescence was observed using an Olympus BHS microscope or a Zeiss axioskope equipped with appropriate Wlter systems. Microphotographs were taken with an Olympus analog or a Spot (Visitron Systems, Puchheim, Germany) digital camera. SpeciWc care was taken in

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Table 1 Labeling speciWcity, host species, source and dilution for Xuorescence immunohistochemistry (FI) and, if diVerent, for postembedding immunoelectron microscopy (PE; in parentheses) of antibodies used in the present study Marker proteins

Labeling speciWcity

Host

Source

Dilution FI (PE)

Olfactory marker protein (OMP)

Mature OSN

Goat (p)

Gift of Prof. F. Margolis or Wako Chemicals, Neuss, Germany

1:400

Growth associated protein (GAP) 43

Immature OSN

Rabbit (p)

Novus Biologicals, Acris antibodies, Hiddenhausen, Germany

1:500 1:100


III Tubulin (
III-TUB)

Newly committed neurons

Mouse (m)

R&D Systems Wiesbaden, Germany


-Tubulin (
-TUB)


-TUB isotypes

Mouse (m)

Sigma, Taufkirchen, Germany

1:200

Cytokeratin 18 (CK 18)

SC, MC and EDC

Mouse (m)

Progen, Heidelberg, Germany

1:100

CK14

HBC

Mouse (m)

Chemicon Schwalbach, Germany

1:100

Glial Wbrillary acidic protein (GFAP)

OEC

Mouse (m)

Sigma

1:500

Globose basal cell 1 (GBC 1)

Globose Basal Cells

Mouse (m)

Gift of Prof. J. Schwob

1:1

Junction proteins

Protein type

Occludin (Occl)

TJ, integral

(a) Mouse (m)

BD Transduction Laboratories Heidelberg, Germany

1:50

(b) Rabbit (p)

Zymed Laboratories Invitrogen, Karlsruhe, Germany

1:50 (1:200)

Zymed Laboratories

1:100 (1:50)

Claudin 1 (Cl-1)

TJ, integral

Rabbit (p)

Claudin 2 (Cl-2)

TJ, integral

Mouse (m)

Zymed Laboratories

1:100

Claudin 3 (Cl-3)

TJ, integral

Rabbit (p)

Zymed Laboratories

1:100

Claudin 4 (Cl-4)

TJ, integral

a) Mouse (m)

Zymed Laboratories

1:100

b) Rabbit (p)

Zymed Laboratories

1:100

Claudin 5 (Cl-5)

TJ, integral

Rabbit (p)

Zymed Laboratories

1:50

Zonula occludens protein (ZO)1

TJ, peripheral

(a) Rat (m)

Chemicon

1:100

(b) Goat (p)

Santa Cruz Biotechnology, Heidelberg, Germany

1:100

ZO2

TJ, peripheral

ZO3 N-cadherin (N-cad)

(c) Rabbit (p)

Zymed Laboratories

1:100

Goat (p)

Santa Cruz Biotechnology

1:100

TJ, peripheral

Goat (p)

Santa Cruz Biotechnology

1:100

AJ, integral

Rabbit (p)

Calbiochem Merck, Darmstadt, Germany

1:200 (1:500)

E-cadherin (E-cad)

AJ, integral

Mouse (m)

BD Transduction Laboratories

1:100 (1:25)

Alpha-catenin (-cat)

AJ, peripheral

Mouse (m)

BD Transduction Laboratories

1:100

Beta-catenin (
-cat)

AJ, peripheral

(a) Mouse (m)

BD Transduction Laboratories

1:100 (1:50)

(b) Rabbit (p)

Sigma

1:100

Mouse (m)

BD Transduction Laboratories

1:100 (1:50)

p120catenin (p120)

AJ, peripheral

m monoclonal, p polyclonal

observation and documentation of double labelings to block bleeding of Xuorescence emitted from one Xuorescent marker into the detection system of the second Xuorescent marker, and in preparing and adjusting digital photographs to represent exactly the visual image. Electron microscopic analysis was carried out as described above. No speciWc immunolabeling was detected in any control sections.

Results OE and FO morphology and identiWcation of cell types Immunoreactions using antibodies against established markers for diVerent cell types (Table 1; Schwob et al. 1992; Roskams et al. 1998; Schwob 2002) were carried out

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Fig. 1 a–f Triple immunolabeling in the OE for OMP(blue), GAP43(red) and
III-TUB (green). OMP-ir mature OSN are localized in the upper OSN layer (a, e, f) and display weak
III-TUB-immunoreactivity (c, f). GAP43-ir immature OSN (b) are strongly
III-TUB-ir (c, d). Occasionally, a GAP43/OMP-double labeled OSN is observed which displays weak
III-TUB-labeling (a–f, large arrows). Colocalization of strong
III-TUB- with OMP-immunoreactivity is only occasionally observed in GAP43-negative dendrites (small arrows in b, c, e, f). g Labeling of CK18-immunoreactive Wlaments in SCs and EDCs

demonstrates longitudinally oriented position of EDC cell bodies (arrows). h–k OMP- (h) and GAP43-ir axons (i) are found in distinct bundles in the FO (k). GAP43-immunoreactivity is stronger in the FO than in OSN cell bodies of the OE (cf. i, b, e). Strong
III-TUB-immunoreactivity is colocalized in GAP43-ir axons, OMP-ir axons are weakly
III-TUB-ir (j, k). l OMP(blue)/GFAP(red)-double labeling marks the localization of OEC in the lamina propria FOs arising from the OE. Bar in f for a–f 10
m, in g and in l for h–l 5
m

on freeze–dried, Epon-embedded material (Fig. 1) in order to test whether these markers yielded suitable labeling in our material to enable cell type identiWcation. Additionally, detection of markers and transmission electron microscopy of OE and FO were combined to achieve a clear idea of the OE and FO cellular and contact architecture, and thus to enable unequivocal identiWcation of contacts between identiWed cells using Xuorescence immunolabeling. OMPimmunoreactions yielded intense labeling of mature OSN cell bodies and their dendrites in the OE and of OSN axons in the LP FO fascicles (Fig. 1a, e, f, h, k, l). GAP 43-immunoreactivity was restricted to cell bodies, dendrites and axons of neurons in the lower OSN layer (Fig. 1b, d, e), presumably representing immature neurons (e.g., Schwob

2002).
III-TUB-immunoreactivity was also particularly strong in cell bodies and dendrites of OSN in the immature neuron layer, while only faint labeling was detected in mature, OMP-ir OSN (Fig. 1a, c, d, f). Double labelings showed colocalization of GAP 43- and
III-TUB-immunoreactivity in OSN cell bodies (Fig. 1d). In the majority of GAP43-ir OSN cell bodies,
III-TUB-immunoreaction intensity was strong. In individual GAP43-ir OSN cell bodies,
III-TUB-immunoreactivity was faint, and some of these OSN displayed OMP-immunoreactivity (Fig. 1a–f, large arrows). Colocalization of OMP-immunoreactivity in strongly
III-TUB-immunoreactive(ir) OSN cell bodies was never observed. However, some moderate to strongly
III-TUB-ir dendrites lacked GAP43-immunoreactivity but

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displayed weak OMP-immunoreactivity (Fig. 1b, c, e, f, small arrows). Using an antibody against all
-TUB isotypes, labeling was weak in all OSN cell bodies and intense in dendrites and axons (not shown; cf. Asan and Drenckhahn 2005). The Wndings and comparison with literature data (e.g., Verhaagen et al. 1989; Schwob et al. 1992; Roskams et al. 1998; Schwob 2002), suggest that cell bodies and developing dendrites of newly born OSN can be identiWed by GAP43/strong
III-TUB-immunoreactivity. It appears that, in the course of maturation, OSNs lose
III-TUB-immunoreactivity from the cell body and start synthesizing OMP probably before GAP43 is completely lost from the entire neuron.
III-TUB-immunoreactivity in dendrites is only slowly reduced to the level present in the majority of OMPir mature neurons. Immunoreactions using antisera against GBC marker GBC1 or HBC marker CK14 did not yield speciWc staining in the semithin sections. CK18-immunoreactivity was present in SC and particularly strongly in EDC, as has been shown before (Fig. 1g; Asan and Drenckhahn 2005), and documented that EDC span the entire OE with their long axis oriented parallel to the excretory duct. In the FO fascicles of the LP, GAP43-immunoreactivity was localized in axon bundles which lacked OMP-immunoreactivity (Fig. 1h, i, k). GAP43-immunoXuorescence in axons was considerably more intense than in cell bodies and dendrites (Fig. 1b, i). Strong
III-TUB-immunoreactivity was colocalized in GAP43-ir axons, faint
III-TUB-immunoreactivity in OMP-ir axons (Fig. 1h–k). GFAP- and OMP-immunolabelings clearly demonstrated the localization of OECs and OSN axons, respectively, in the FO (Fig. 1l). The antibody against all
-TUB-isotypes labeled all axons in FO (see below). Electron microscopically, diVerent cell types (Fig. 2a) and apical junctional complexes consisting of TJs and AJs (Fig. 2b) were easily recognizable in longitudinal (Fig. 2a, b) and tangential/oblique OE sections (Fig. 2c, d). The latter showed that SCs completely surrounded and formed junctional complexes with apical OSN dendrites (D) recognizable by their content of numerous basal bodies (Fig. 2c, arrowhead). Dendrites were either situated between two or several adjacent SCs, or were embedded into one SC which formed a self-associating contact (Fig. 2c, large arrow). Thus, dendrites formed bicellular D/SC contacts and one, two, or more tripartite bi- and tricellular D/SC contacts (Fig. 2c, small arrows). The cross sectional diameters of the dendrites at the level of D/SC junctions were of a fairly uniform size (»1.2
m; Fig. 2c, d). SCs also completely surrounded and formed junctional complexes with apical parts of MCs, which often possessed larger diameters than dendrites and contained only few organelles (Fig. 2a, d).

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Occasionally, a basal body was observed in the apical cytoplasm of microvillar cells (Fig. 2d, arrowhead). Junctional complexes between SCs formed polygonal proWles in transverse sections (Fig. 2c, d). Apical contacts between dendrites without interposition of SCs were observed, but were extremely rare. In longitudinal sections, small lateral puncta adherentia were localized between all cell types in the OSN layer (not shown). Although adherens contacts were observed in the basal cell layer, it was not unequivocally possible to identify the cell types or processes of cells (OSN axons, SC or MC basal processes) forming them. FO ultrastructural morphology in the LP was as has been previously described, with OEC cell bodies and larger processes surrounding bundles of OSN axons, covered on the outer surface by a basal lamina and collagen Wbrils, small OEC processes separating axonal territories within the bundles, and ONF forming a narrow sheath surrounding the bundles, separated from the basal lamina by a small space containing collagen Wbrils (not shown; cf. Field et al. 2003). In our material, numerous contacts with ultrastructural features of AJs were found between OEC processes (Fig. 2e, inset, small arrow), between OEC processes and axons (Fig. 2h, arrow, i), and between axons (Fig. 2h, inset). Occasionally, triaxonal contacts were observed (Fig. 2h, inset). As has been shown before (Blinder et al. 2003), OEC processes also formed conspicuous gap junction-like contacts (Fig. 2f, g). Fusion of plasma membrane outer leaXets, suggesting the presence of TJs, was observed between OEC processes in the FO periphery (Fig. 2e, inset, large arrow) and between small OEC processes within the FO (Fig. 2g), often immediately adjacent to gap junctionlike contacts (Fig. 2g, inset). The morphological observations led us to propose a scheme of contact form and localization in longitudinal and transverse sections of the OE shown in Fig. 3. In longitudinal sections (Fig. 3a), apical junctional complexes (SC/SC, D/SC and MC/SC zonulae occludentes and adherentes) are represented by spots or small longitudinal lines just below the epithelial surface, puncta adherentia by spots within the epithelium, and EDC contacts by longitudinal lines spanning the epithelium. The presence and localization of contacts in the BCL layer is not clear from the morphological investigations. OG contacts are localized in the lamina propria (LP; Fig. 3a). In tangential/ oblique sections (Fig. 3b, c), D/SC contacts are represented by small round proWles. Larger round proWles represent MC/SC contacts, SC/SC contacts are characteristic polygonal proWles. Tripartite bi- and tricellular D/SC contacts are recognizable only in tangential/oblique sections (Fig. 3b, arrowheads). Luminar EDC contacts appear as spots or short lines oriented radially around the duct lumen (Fig. 3c). SC/EDC AJs are large punctate circles

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Histochem Cell Biol (2008) 130:339–361

Fig. 2 Electron microscopy of intercellular contacts in the OE (a–d) and FO (e–i). a In longitudinal sections, diVerent cell types are easily recognized (for details, see text). Arrows denote the apical junctional complexes. b At high magniWcation, TJs (large arrow) and AJs (small arrow) are seen between SC and D. c, d Tangential sections through the apical junctional complex in a preparation used for postembedding immunolabeling. D and MC apical processes are completely surrounded by SC, which form typical D/SC, SC/SC, and MC/SC junctions. Large arrow in c shows a self-associating contact where an SC completely surrounds a D, small arrows in c denote tripartite bi- and tricellular D/SC junctions. Arrowheads show cilia in D (c) and MC (d).

e–i Contacts with ultrastructural features of typical AJs are formed between OEC processes (small arrow in e, inset), between OSN axons and large (arrow in h) and small (arrow in i) OEC processes, and between axons, occasionally in a triaxonal arrangement (box and inset in h). Fusion of outer membrane leaXets indicative of TJs is found between large OEC processes (large arrow in e, inset). Small OEC processes surround axon bundles (arrowheads in f) and form conspicuous contacts (box in f, higher magniWcation in g) which show morphological characteristics of gap junctions (arrowheads in g), but occasionally also display small regions of membrane fusion (inset in g). Bar in a and c, for c, d 1
m, in b, g and i 0.1
m, in f for e, f and in h 0.2
m

surrounding the duct with its radially oriented luminar contacts.

7) were in accordance with the scheme shown in Fig. 3. Of all claudins analyzed in the present study, only Cl2-immunoreactivity was not detected in our material. Since immunoreactions for Cl2 carried out on semithin sections from freeze–dried, Epon-embedded rat kidney, which has been shown to display Cl2-immunoreactivity in proximal tubule epithelia (Enck et al. 2001), gave positive staining in apical junctions of proximal tubules (not shown), this Wnding indicated that Cl2 does not contribute to TJ formation in the

Immunolabeling for TJ proteins Olfactory epithelium and glands Labeling patterns for TJ proteins in longitudinal and tangential/oblique semithin sections of the OE (Figs. 4, 5, 6,

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Histochem Cell Biol (2008) 130:339–361

Fig. 3 Scheme of contact form and localization in longitudinal sections (a) and in tangential/oblique sections through the apical junctional complexes (b) and the upper epithelium including olfactory gland excretory ducts (c). TJs are black, AJs (zonulae adherentes in the apical epithelium, puncta adherentia in the lower epithelial layers) are gray. Localization of AJs in the BCL in a is putative. Arrowheads in b point to tripartite bi- and tricellular D/SC junctions

peripheral olfactory pathway. Strong reactivity was found for Cls 1 and 5 in all apical epithelial and in EDC/EDC junctions (Fig. 4). Double labeling with OMP (Fig. 4a, b) documented that Cl-immunoreactive(ir) small round proWles represented D/SC junctions in oblique/transverse sections (Fig. 4b, inset), and thus veriWed the attribution of the form of immunolabeled proWles to speciWc intercellular junctions shown in Fig. 3. Fluorescence immunolabelings for Cls 1 and 5 on cryostat sections gave similar results, albeit with higher background and lower morphological resolution (not shown). In Cl5/
III-TUB double labelings, TJs were found also on strongly
III-TUB-ir dendrites (Fig. 4c, arrows). The cell bodies extending these dendrites were localized more apically than most other strongly
III-TUB-ir immature neurons, and some displayed weak
III-TUB-immunoreactivity (Fig. 4c, arrowheads). Silver-intensiWed postembedding immunogold labeling for Cls 1 and 5 proved localization of the two claudins

Fig. 4 a, b Cl1 r(red)/OMP g (green) and Cl5 r/OMP g double labeling shows Cl-immunoreactivity in D/SC and other apical TJs (arrows) and in EDC junctions. b inset Tangential/oblique sections document small round Cl5-ir proWles surrounding OMP-ir dendrites validating the scheme shown in Fig. 3. c Cl5 r/
III-TUB g double labeling

347

speciWcally in those parts of D/SC, SC/SC and MC/SC junctional complexes in which the membranes appeared fused (Fig. 5). Labeling for occludin and ZO1 was found in the same localization (not shown). Labeling for Cls 3 and 4 (using both the mouse monoclonal and the rabbit polyclonal anti-Cl4) was somewhat less intense with higher background than for Cl1 and 5, but identically localized in apical epithelial junctions (Fig. 6a, b, d). However, while EDC/EDC TJs were strongly reactive for Cls 3 (Fig. 6a, inset, arrow) and 5 (Fig. 6d, arrow), as were OG TJs (not shown), labeling for Cl4 was weak to not detectable in EDC/EDC (Fig. 6d, arrow) and completely lacking in OG junctions (not shown). Occl-immunoreactivity in the apical OE showed the same labeling pattern as immunoreactivity for Cls 1, 3, 4 and 5 (Fig. 6e–g). For all TJ integral membrane proteins studied, Xuorescence intensity varied between diVerent TJs. SC/SC-junctions showed moderate to strong immunoXuorescence, D/SC-Xuorescence ranged from equally intense to somewhat less intense compared to SC/SC junctions. Conspicuously high Xuorescence was observed at tripartite bi- and tricellular junctions between dendrites and their surrounding SCs (arrowheads in Fig. 6a–c, e–g). Tripartite bi- and tricellular SC/SC junctions only occasionally showed increased Xuorescence. EDC/EDC and OG junctions were strongly labeled for Cls 1, 3 and 5, weakly to not detectably labeled for Cl4, and displayed a very intense reactivity for Occl (Fig. 4a, b, arrows; Fig. 6a, inset, arrow, d, arrow, Fig. 7a, c, arrows). ZO1-immunoreactivity detected with a monoclonal rat anti-ZO1 (ZO1m; Table 1) colocalized completely with Cl1-, 3-, 5- and Occl-immunoreactivity in OE and OG junctions (Fig. 7a–h). The same labeling pattern was achieved using polyglonal goat and polyclonal rabbit anti-ZO1 (ZO1gp, ZO1rp), however, background labeling was higher (Fig. 7h, i). Fluorescence intensity in preparations both using the monoclonal and the polyclonal anti-ZO1s was particularly strong in D/SC junctions, less intense in SC/SC and rather faint in EDC/EDC junctions (Fig. 7b, e). This

indicates that strongly
III-TUB-ir dendrites of also form tight junctions (arrows). The cell bodies of these neurons (arrowheads) display diVerential
III-TUB-immunoreaction intensities. Bar in a for a–c 5
m, in b (inset) 2
m

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348

Histochem Cell Biol (2008) 130:339–361

Fig. 5 Postembedding immunoelectron microscopy for Cls 1 and 5 in tangential apical OE sections. SpeciWc labeling is restricted to junctions with fused membranes in all types of intercellular contacts. AJs (arrows) are not labeled. Bar in c for a–c 1
m

Fig. 6 a–c Tangential/oblique sections show localization of Cl3-, 4and 5-immunoreactivity in all types of apical junctions and Cl3-reactivity in EDC junctions (arrow in a, inset). d Cl4 r (red)/Cl5 g (green) double labeling documents lack of Cl4-reactivity in EDC junctions (arrow). e–g Cl4/Occl double labeling shows colocalization in all

apical junctions. Arrowheads in a–c and e–g point to tripartite bi- and tri-cellular D/SC TJs which display particularly strong immunoXuorescence for all detected proteins. Fluorescence intensity in bicellular D/ SC junctions is as high or somewhat lower than in bicellular SC/SC TJs. Bar in a and in c for b, c, g–h 2
m, in d 5
m

was most obvious in merged images of Occl/ZO1 and Cl5/ ZO1 double labelings (Fig. 7c, f). Tripartite bi- and tricellular D/SC junctions did not show enhanced ZO1-immunoXuorescence compared to bicellular D/SC junctions. Immunoreaction patterns for ZO2 (Fig. 7k, l, small arrows) and ZO3 (not shown) in the OE and OG were largely identical to those described for ZO1. However, background labeling for both antigens was rather high, and diVerences in immunoreaction intensities between diVerent types of epithelial TJs were not as clearly discernible. Taken together, the Wndings provide evidence that all TJs in the OE contain Occl, speciWc claudins, and ZO1-3. However, the exact molecular composition of TJs varies according to the cell types forming them. The results are summarized in Table 2.

layer (ONL) of the olfactory bulb (OB, Fig. 8a–c). Inner layers of the ONL showed less Cl5 immunoreactivity (Fig. 8a). Double labeling with GFAP indicated that Cl5-immunoreactivity was closely associated with GFAP-ir OEC processes (Fig. 8d). In FO cross sections double labeled for OMP, Cl5ir spots were particularly frequently localized at the periphery of OMP-negative, presumed OEC proWles, but were absent from OMP-ir axon bundles (Fig. 8e). Cl1-immunoreactivity was found in spots and lines adjacent to the outer circumference of FOs close to but not intermingled with GFAP- and OMPir proWles (Fig. 8h, i). Reactivity for Cl3- and Cl4 was not detected in the FO (not shown), even in sections in which apical epithelial labeling for the diVerent antigens was strong. In semithin sections, there was only faint diVuse labeling in the FOs in Occlimmunoreactions (using both the poly- and the monoclonal anti-Occl). Cryosections displayed high background labeling, and, in sections labeled with the polyclonal anti-Occl, inconsistent but apparently speciWc weak Occl-immunoreactivity was observed in proWles within and surrounding the FOs (not shown). ZO1-immunoreactivity was consistently localized in proWles surrounding larger FOs in the LP, and in FO fascicles at the junction with the ONL of the OB (Fig. 8b, f). It

Fila olfactoria Intense Cl5-immunoreactivity was present within FOs of the LP in semithin and cryosections. In longitudinal FO sections, Cl5-immunoreactivity appeared as longitudinal lines (Figs. 7g, j, 8d, j), in transverse sections as small spots (Fig. 8a, d inset, e). Cl5-ir proWles were found throughout the Wla, and also in FOs at the junction with the olfactory nerve

123

Histochem Cell Biol (2008) 130:339–361

349

Fig. 7 Occl/ZO1m (a–c) and Cl5/ZO1m (d–f) double labeling documents colocalization of Occl and Cl5 with ZO1 in apical, EDC and OG junctions. Arrows in a–c and small arrows in d–f point to D/SC junctions which display particularly intense ZO1 immunoXuorescence, especially obvious in the reddish color of small round proWles compared to the yellow color of SC/SC polygonal proWles in merged double labeling images (inset 1 in c, arrow in f). ZO1-Xuorescence intensity is weak in EDC junctions (large arrows in a–c), most obvious by the greenish color in the merged Occl/ZO1m image in c. ZO1mimmunoreactivity in OG junctions (insets 2 in a–c) is stronger than in EDC junctions. g Overviews of Cl5 g(green)/ZO1m r(red)-double

labeled sections show intense green Xuorescent Cl5-immunoreactivity in FOs of the LP (large arrows) in addition to apical TJs, which are additionally ZO1-ir (yellow color; small arrows). h ZO1m-immunoreactivity is not detectable in the LP (large arrows) in sections which display intense ZO1-immunoreactivity in apical OE TJs (small arrows). i ZO1rp-immunreactions label both apical epithelial junctions (small arrow) and proWles in FOs (large arrows). j–l ZO2-immunoreactions show comparatively high background labeling, but speciWc reactivity is detected both in OE junctions (small arrows) and in the LP (large arrows). Bar in c for a–c and insets 2 5
m, in inset 1 in c for insets 1 in a–c 1
m, in f for d–f 1
m, in l for g–l 10
m

was colocalized with Cl1 (not shown). Inner layers of the ONL lacked ZO1-ir proWles surrounding FO fascicles (Fig. 8b, c). Using ZO1m, there was practically no immunoreactivity detectable within the FOs in both semithin and cryosections, even in sections which showed intensely labeled epithelial TJs (Fig. 7h). Labeling of endothelial TJs with this antibody was relatively faint compared to labeling of epithelial and pial TJs (Fig. 8b, c). When ZO1gp was used, background labeling was generally high, and a faint Xuorescence was observed in FOs, which, however, was not consistently colocalized with Cl5-immunoreactivity (not shown). On the other hand, immunolabeling using ZO1rp showed equally strong labeling of endothelial and

epithelial TJs in the OE (Figs. 7i, 8f), and additionally clear labeling of proWles resembling Cl5-ir proWles in FOs both in semithin and cryosections (Figs. 7i, 8f). ZO1m/ZO1rp double labeling showed colocalization of immunoreactivities in endothelial TJs and at the FO periphery (Fig. 8f, g, V and small arrows), but not within the FOs (Fig. 8f, g, large arrows). Faint to moderate immunoreactivity was observed for ZO2 in Cl5-ir proWles in FOs (Fig. 7j–l, large arrows, Fig. 8 j–l, large arrows). ZO2-immunoreactivity also colocalized with immunolabeling using anti-ZO1rp within the FOs and with Cl1 and ZO1 in junctions surrounding the FO (not shown), which were not reactive for Cl5 (Fig. 8j–l, small arrows).

123

350 Table 2 Immunolabeling intensities of diVerent TJs in the OE and LP with (¡): no, (+) weak or inconsistent, (++) moderate, and (+++) strong immunoXuorescence

* DiVerential reactivity of OEC TJs for diVerent ZO-antibodies (details see text and Figs. 7, 8)

Histochem Cell Biol (2008) 130:339–361

Localization

Olfactory epithelium

Lamina propria Fila olfactoria

TJ protein

SC/SC

D/SC bicellular

D/SC tripartite

EDC/EDC

OG

OEC

Periphery

Occludin

+++

++

++++

+++

+++

(+)

(+)

Claudin 1

++

++

+++

++

++

¡

++

Claudin 3

++

++

+++

++

+++

¡

¡

Claudin 4

++

++

+++

(+)

¡

¡

¡

Claudin 5

++

++

+++

++

++

+++

¡

ZO1

++

+++

+++

+

++

(¡)/++*

+

ZO2

++

+++

+++

++

++

+

+

ZO3

+++

+++

+++

++

++

¡

¡

Fig. 8 a–c Cl5-immunoreactivity is found in FOs at the junction with the ONL of the OB. ZO1m-immunoreactivity is strong in pial tissue and rather faint in pial vessel endothelia (V), where it is colocalized with Cl5-labeling. Small spots of ZO1m-immunoreactivity are found surrounding FO bundles only at their passage through the pial surface of the OB (large arrows). Cl5-ir proWles of the FO (e.g., small arrows) are not detectably ZO1m-ir. d Cl5 (r-red)-immunoreactivity is overlapping with GFAP (g-green)-ir peripheral processes of OEC and closely associated with GFAP-ir proWles within the FOs in longitudinal and transverse (inset) sections. e Small Cl5-ir red puncta within FOs are localized between OMP-ir green axon bundles and non-OMP-ir

proWles, but are absent from the center of OMP-ir axon bundles. f–g ZO1m/ZO1rp-double labeling shows colocalization of immunoreactivities in vessel endothelia (V) and in the FO periphery (small arrows), but ZO1rp-ir proWles within the FO lack ZO1m-reactivity (large arrows). h, i Red Cl1-immunoreactivity is found in spots and lines (arrows) adjacent to the outer FO circumference, close to but not between GFAP-ir (h, arrows) and OMP-ir (i) proWles. (j–l) High magniWcation shows ZO2-immunoreactivity colocalized with Cl5-ir within FOs (large arrows). ZO2-immunoreactive proWles in the FO periphery (small arrows) lack Cl5-immunoreactivity. Bars 10
m

Immunoelectron microscopy corroborated localization of Cl5-immunoreaction product in OEC junctions both at the FO periphery (Fig. 9a, inset) and within the FOs

between axon bundles (large arrows in Fig. 9a, b), although clear TJs could not be distinguished in the material used. Immunogold labeling for Cl1, ZO1 (using all antibodies)

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Histochem Cell Biol (2008) 130:339–361

Fig. 9 Postembedding immunoelectron microscopy shows immunogold Cl5-labeling restricted to OEC processes at the FO periphery (box and inset in a) and between axon bundles (AB; a, large arrows, b). Small arrows in a denote basal lamina at FO surface. Bar in a, c; for b, c 0.5
m

and Occl did not yield conclusive results. Background labeling was higher than in Cl5-immunoreactions and speciWc labeling was not recognizable in the FO or in junctions of olfactory nerve Wbroblasts surrounding larger FO fascicles (not shown). The results provide evidence that OEC form Cl5-mediated TJs, in which ZOs appear to be colocalized. Cl1immunolabeling is restricted to the outer circumference of large FO fascicles up to their junction with the ONL. The data are summarized in Table 2. Immunolabeling for AJ proteins Olfactory epithelium Strong, continuous apical and punctate basolateral labeling was found for E- and N-cad and for -,
- and p120-cats in longitudinal sections of the OE (Fig. 10). In merged images of N-cad/E-cad-double labeling it was obvious that the two cadherins were diVerentially distributed. Thus, the overview showed that EDC/EDC contacts and individual contacts in the apical OE (Fig. 10a–c, large and small arrows, respectively) were exclusively E-cad-ir. The same was true for OG AJs in the LP (not shown). N-cad-single labeling was observed in apical junctions, and N-cad-single labeled puncta were frequent in the OSN layer (Fig. 10a–c, large

351

arrowheads). In some epithelial contacts, E- and N-cadimmunoreactivities appeared intermingled. In the basal layer, N-cad-immunoreactivity was weaker than in the other layers. Individual cells localized just above the basal lamina were outlined by numerous E-cad-ir puncta (Fig. 10a–c, small arrowheads). Immunoreaction patterns for the diVerent cats were largely identical in the OE. Cat-staining was particularly strong apically and in the basal cell layer, somewhat less intense in the regions just above the basal layer, and strong again in OSN and SC layers (Fig. 10d–f). Apparently all diVerent types of AJs showed labeling for the cats tested. Double labeling of
-cat with N-cad or E-cad indicated that there were only very few
-cat-ir contacts, mainly in the basal cell layer, which did not appear to colocalize either of the cads (see below). N-cad/E-cad-double labeled tangential/oblique sections of the apical OE showing the junctional complex region and the region of apical OE cell processes documented that the small round D/SC zonulae adherentes were exclusively N-cad-ir (Fig. 11a, b). Strongly and exclusively E-cad-ir zonulae adherentes (Fig. 11a, arrow) were found between strong CK18-ir cells and supporting cells (not shown) and, based on earlier investigations (Asan and Drenckhahn 2005), could be identiWed as MC/SC junctions (Fig. 11a, MC with arrow). EDC/EDC and SC/EDC contacts were also exclusively E-cad-ir. In the polygonal zonulae adherentes between SCs, both proteins appeared colocalized, presenting yellow Xuorescence in double labelings (Fig. 11a ,b). In SC/SC puncta adherentia, E-cad-Xuorescence predominated, but often it appeared that E-cad- and N-cad-ir puncta were intermingled (Fig. 11b, c, arrows). D/ SC puncta adherentia (Fig. 11c, arrowhead) were exclusively N-cad-ir. Triple labeling of N-cad,
-cat and OMP (Fig. 12a, b) showed that the cat was localized in D/SC AJs identiWed by dendritic OMP-immunoreactivity (Fig. 12a, b, small arrowheads) as well as in MC/SC, SC/SC (Fig. 12a, b, large arrowheads), EDC/EDC and EDC/SC AJs. The Xuorescence intensity appeared relatively weak in N-cad-ir neuronal junctions (Fig. 12a, inset, small arrowhead). The same was true for - and p120-cat (not shown). Electron microscopy of the apical epithelium provided further proof of the speciWc localization of N-cad, E-cad,
-cat (Fig. 13) and p120-cat (not shown) to AJs between the diVerent cell types. Presence and cellular localization of E-cad-, N-cad- and cat-ir AJs in the basal OE were analyzed in detail in double labelings for N-cad and
III-TUB (Fig. 14a, c), in triple labelings for N-cad,
-cat and OMP (Fig. 14b), and in double labelings for E-cad and
-cat (Fig. 14d, e). Similar reactions were done for - and p120-catenin, yielding results comparable to those described for
-cat.

123

352

Histochem Cell Biol (2008) 130:339–361

Fig. 11 a–c N-cad (g-green)/E-cad (r-red) double labeling clearly shows that D/SC zonulae adherentes (small round green proWles in a, b) and puncta adherentia (arrowhead in c) are only N-cad-ir while MC/ SC, EDC/EDC and SC/EDC AJs are exclusively E-cad-ir. E-cad- and N-cad-immunoXuorescences appear colocalized in polygonal SC/SC zonulae adherentes (yellow color of polygonal proWles in a, b) and are intermingled in basolateral SC/SC puncta adherentia (arrows in b, c). Bars in a and in c for b, c 2
m

Fig. 10 a–c E-cad/N-cad double labeling shows intense but distinct reactivity for both antigens in apical and basolateral OE junctions. Some apical (small arrows) and all EDC-junctions (large arrows) are solely E-cad-ir. N-cad single labeling is frequent in the apical junctions, and numerous N-cad-single labeled puncta are apparent in the OSNL (large arrowheads). Some basal cells (small arrowheads) display predominantly E-cad-ir puncta. d–f The three catenins detected show more or less identical and ubiquitous distribution in the OE with particularly strong apical, basal and EDC/OG labeling. Bar in c for a–c 5
m, in f for d–f 10
m

BetaIII-TUB-ir immature cells in the lower OSN layer formed numerous N-cad-ir contacts with surrounding cells (Fig. 14a, c, small arrows). These closely spaced AJs at the cell body level allowed identiWcation of non-labeled immature neurons also in N-cad/
-cat/OMP triple immunoreactions (Fig. 14b, small arrow). OMP-ir mature neurons also formed numerous N-cad-ir contacts (Fig. 14b, large arrows), and thus their N-cad-ir AJ-covered outline could be identiWed also in N-cad/
III-TUBimmunoreactions (Fig. 14a, large arrow). The reddish color of AJs formed by immature and mature neuronal cell bodies in N-cad (red)/
-cat (green)-colocalizations (Fig. 14b) indicated that
-cat-reactivity in these AJs was relatively low, just as has been described for D/SC AJs (see above). N-cad-ir puncta were rarely found between cells adjacent to the basal lamina, which possessed oblong, horizontally oriented nuclei, and cells localized just apically to them with round to oval nuclei (Fig. 14a, c, large and small

123

arrowheads, respectively). On the other hand, the presence of green puncta on the outlines of Xat and roundish cells attached to the basal lamina in N-cad (red)/
-cat (green)immunoreactions (Fig. 14b, large arrowheads) suggested that AJs were formed at least by some of the diVerent basal cells with surrounding structures. In some large, basally localized cells,
-cat/E-cad-colocalization was occasionally observed (Fig. 14d, e, arrowheads). Especially in epithelial regions of high thickness, many round to oval cells localized just apical to the basalmost cells were non-ir for
III-TUB or OMP and possessed only a very scarce covering with puncta reactive for any of the AJ components tested (Fig. 14a–c, small arrowheads). Fila olfactoria In both transverse and longitudinal sections of FO, N-cadand
-cat-ir puncta were extremely numerous and were found both between non-labeled proWles of presumed OECs and axons (Fig. 15a, b, arrows) and within
-TUB- or OMPir OSN axon bundles (Fig. 15a, b, arrowheads). In the vast majority of puncta within FOs, N-cad- and
-cat-Xuorescence appeared colocalized (Fig. 15c). Some exclusively
cat-ir puncta were observed at the periphery of FO fascicles (Fig. 15c, arrows). Comparable results were found for - and p120-cat in the Wla (not shown). In N-cad/Ecad-double labelings, a few E-cad-ir puncta were present in the FO periphery (Fig. 15d–f, arrows). Colocalization of
-cat in these puncta was observed in E-cad/
cat-reactions (not shown).

Histochem Cell Biol (2008) 130:339–361

353

Fig. 12 a N-cad (r-red)/
-cat (g-green) double and b N-cad (r-red)/
cat (g-green)/OMP (b-blue) triple labeling shows that
-cat is colocalized with N-cad in D/SC and SC/SC zonulae adherentes (reddish and yellow-orange color, respectively) while MC/SC zonulae adherentes lack N-cad-Xuorescence (green color, M plus arrow in a, b). In basolateral puncta adherentia between SCs (large arrowheads in a, b), both proteins are colocalized (yellow color). EDC/EDC and SC/EDC junctions lack N-cad-Xuorescence (green color). In puncta adherentia between SCs and OMP-ir Ds (small arrowheads), N-cad-Xuorescence is strong (reddish color), while
-cat-reactivity is weaker than in SC/SC contacts (inset in a: single
-cat-labeling of boxed area). Bar in b for a, b 5
m

Postembedding immunogold labeling of N-cad and E-cad in FOs showed high background and was diYcult to attribute to speciWc AJs in the FOs or in the ONF sheath (not shown). Immunogold reactions for
-cat in longitudinally sectioned FOs, on the other hand, clearly supported the observation of numerous AJs between OECs and OSN axons and between axons (Fig. 16). Data of labeling characteristics for diVerent AJs in the apical OE and in FOs are summarized in Table 3.

Discussion The present study provides novel data contributing to a detailed knowledge of the molecular composition of interTable 3 Immunolabeling for cadherins and catenins in identiWed zonulae adherentes/puncta adherentia in the apical OE and the OG with (¡) no, (+) weak, (++) moderate, and (+++) strong immunoXuorescence and for puncta adherentia in the FOs with (¡) no, (+) scarce and (++) numerous puncta

Localization

Fig. 13 Postembedding immunogold labeling of the OE (a–d) shows E-cad-immunoreactivity restricted to SC/SC AJs (a, b). N-cad- (c) and
-cat-(d) labeling is localized in both D/SC and SC/SC AJs. The labeling is restricted to AJs and absent from contacts with fused membranes (e.g., inset in c). Bars in a, b 0.25
m, in c, d 0.5
m

cellular contacts in the rat OE and FO. A diVerential contribution of integral and peripheral membrane proteins to TJs between identiWed cells was documented in the OE: in addition to occludin and ZO1, claudins 1, 3, 4 and 5 were found in apical TJs, Cl 1, 3, and 5 in TJs of olfactory glands and ducts, and ZOs 2 and 3 in all TJs. Cl2 was not detected in the OE. The relative immunoreaction intensity for speciWc proteins varied between the identiWed TJs. The diVerential molecular composition may be related to structural dynamics of the various TJs, and may be of importance for paracellular permeability and thus for maintaining the particular ionic microenvironment required for olfactory signal transduction. In the FOs, TJs intensely reactive for Cl5 but lacking all other studied claudins were documented between OECs. Furthermore, the formation of cell-type speciWc AJs in the OE was analyzed, and the contribution of E-cad, N-cad and catenins to these AJs was documented. The results showed that AJs between neurons and SCs were exclusively mediated by N-cad. AJs between SCs and cells of presumably epithelial origin (MCs, EDCs) and homo-

Apical olfactory epithelium

Lamina propria Fila olfactoria

AJ protein

SC/SC

D/SC

MC/SC

SC/EDC

EDC/EDC

N-cad

++

+++

¡

¡

¡

E-cad

++

¡

+++

+++

+++

Alpha-catenin

++

+

+++

+++

+++

Beta-catenin

++

+

+++

+++

+++

p120 catenin

++

+

+++

+++

+++

OG

Center

Periphery

¡

++

++

+++

¡

+

+++

++

++

+++

++

++

+++

++

++

123

354

Fig. 14 a N-cad (g-green)/
III-TUB (r-red) double, b N-cad (r-red)/
-cat (g-green)/OMP (b-blue) triple and c N-cad (r-red)/
III-TUB (g-green)/DAPI (blue) triple labelings show that
III-TUB-ir OMPnon-ir immature neurons (small arrows in a–c) are surrounded by numerous N-cad-ir puncta which display weak
-cat Xuorescence (reddish color in b). OMP-ir mature neurons (large arrows in a, b) also possess many N-cad-ir puncta with only moderate
-cat-reactivity.

Histochem Cell Biol (2008) 130:339–361

Cells localized adjacent to the basal lamina (large arrowheads in a–c) show few N-cad-ir and more numerous
-cat-ir puncta adherentia. Immediately above these cells, cells non-ir for both
III-TUB and OMP-immunoreactivity (small arrowheads in a–c) lack both N-cadand
-cat-ir puncta. d, e E-cad and
-cat are colocalized in AJs of some basal cells. Bar in a for a, b and in e for c, d 5
m

Fig. 15 In N-cad (r-red)/
-TUB (g-green) (a) and
-cat (g-green)/OMP (b-blue) double labelings immunoreactive puncta are observed between non-labeled presumed OEC proWles and
-TUB- or OMP-r axon bundles (arrows) and within axon bundles (arrowheads).
cat (g-green)/ N-cad (r-red)/OMP (b-blue) triple labeling (c) shows complete colocalization of
-cat with N-cad within FOs and some exclusively
cat-ir puncta at the outer FO surface (arrows). In N-cad/Ecad double labelings (d–f), E-cad-ir puncta are observed at the periphery of FOs (arrows). Bar in c for a–c and in f for d–f 5
m

typic contacts between EDCs and between OG cells were exclusively mediated by E-cad. In homotypic SC contacts, AJs formed by N-cad and by E-cad appeared intermingled. The formation of AJs among the neuronal population in the OE appeared to vary with maturation state. In the FOs, the

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presence of cadherin/catenin mediated AJs between OECs, OECs and OSN axons, and between axons was documented. The Wndings are consistent with a role of AJ proteins in the neuroplastic processes constantly occurring in the peripheral olfactory system.

Histochem Cell Biol (2008) 130:339–361

355

enabling us to identify the diVerent types of contacts in junctional complexes of the apical OE and in the lower OE. Additionally, we found transmission electron microscopical evidence of the presence of TJs between OECs and of AJs between OECs, OECs and axons, and between axons. Axonal contacts were comparatively frequent and were sometimes found in a triaxonal arrangement. Tight junctions in the OE Fig 16 In the FO,
-cat-ir AJs are found between OECs and axons (arrow) and between axons (arrowheads). Bar 0.1
m

Methodological considerations Immunohistochemistry on semithin sections has numerous advantages over other light microscopical immunohistochemical methods for identiWcation of the cellular localization of antigens. The fact that quick-frozen, freeze–dried and Epon-embedded material is used preserves the antigenicity of some proteins and the precise localization better than other methods. Additionally, the 0.5 to 1-
m thick sections provide a better resolution of Xuorescence signals even than virtual sections of comparable thickness generated by confocal microscopy, and the results can be observed by eye, without the aid of a computerized detection system. On the other hand, some antibodies which yield good speciWc labeling on conventionally prepared material occasionally fail to detect the same antigens in freeze–dried, Epon-embedded material. Therefore, it was essential in our investigations to Wrst check whether established markers for diVerent OE cell types showed speciWc reactivity in our system. We found that immunoreactions for OMP, the classical marker of mature OSNs (e.g., Margolis 1980), detect both OSNs and their processes very eYciently. The same was true for immature neuron markers, GAP43 and
III-TUB (e.g., Roskams et al. 1998). Our colocalization experiments indicated that strong
III-TUBimmunoreactivity was a more speciWc marker for immature OSN cell bodies, since GAP43-immunoreactivity was occasionally observed in OMP-ir cell bodies, while strong
III-TUB-immunoreactivity was found exclusively in nonOMP-ir cell bodies. Additionally, our investigations indicated that strong dendritic
III-TUB-immunoreactivity is only lost after OSNs have integrated their dendrites into the epithelial surface and have started to produce OMP. Markers for other cell types also worked in our material, but, unfortunately, basal cell markers tested did not. Thus, further work is required to speciWcally identify the diVerent stem cells and neuronal precursors in our system. Our morphological investigations provided us with a clear idea of the form of intercellular contacts in the OE

Our results provide conclusive evidence that, in addition to Occl, claudins 1, 3, 4 and 5 contribute to the formation of apical (D/SC, MC/SC and SC/SC) TJs, that Cl 1, 3, and 5 are present in OG and EDC TJs, and that, in addition to ZO1 (Miragall et al. 1994), ZOs 2 and 3 participate in the formation of all OE and OG TJs. Furthermore, we have noted diVerences in the relative contribution of the integral and peripheral proteins to speciWc TJs between identiWed cells. In the light of earlier Wndings on TJ ultrastructure in the OE (Menco 1980) and of recent observations concerning TJ dynamics and permability, the diVerences in TJ molecular composition documented in the present study suggest some interesting hypotheses about the possible relevance of the TJ proteins for morphological and functional parameters. Menco (1980) described that, in the rat, bicellular D/SC, SC/SC and gland (presumably corresponding to our EDC/ EDC) TJs were composed of similar numbers of strands, while tricellular D/SC TJs showed signiWcantly higher strand numbers. This correlates with our Wnding of consistently more intense immunoXuorescence for all integral TJ proteins in tripartite bi- and tri-cellular D/SC junctions than in other epithelial TJs, indicating that in these junctions immunolabeling intensity for integral TJ proteins is a measure for strand numbers. Since strand numbers in gland (EDC/EDC) TJs were only marginally lower than in D/SC and SC/SC TJs (Menco 1980), the presence or absence of Cl4 apparently has little inXuence on this TJ feature. In accordance with the observations reported by Miragall et al. (1994), we consistently found ZO1-immunoreactivity to be much stronger in D/SC than in SC/SC TJs, and extremely weak in EDC/EDC TJs, suggesting that ZO1presence in TJs is not correlated with strand numbers. Menco (1980) reported other conspicuous diVerences between D/SC and SC/SC or EDC/EDC TJs: while neuronal TJs were characterized by strands which mainly consisted of particles in the P-faces, epithelial TJs showed P-face strands composed of “bars of diVerent lengths”. Based on very early studies showing that maturation of TJs involves a transition from particles to continuous bars in the P-face (Luciano et al. 1979; Suzuki and Nagano 1979), Menco suggested that morphological diVerences may be related to the TJ maturation state. Under normal environ-

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356

mental conditions, the turnover rate of OSN, and thus the need to integrate dendrites into the epithelial barrier and form new TJs, is higher than that of SCs and EDCs (e.g., Graziadei and Graziadei 1979; Calof et al. 1998; Schwob 2002). Indeed, we were able to document that dendrites of strongly
III-TUB-ir neurons formed TJs, indicating that a considerable number of new OSNs are constantly integrated into the apical OE barrier. Recent studies have suggested that ZO1 is required for dynamic processes of junction assembly and remodeling (Fanning et al. 2007). Thus, removal of ZO1 from cultured epithelial cells by homologous recombination (Umeda et al. 2004) or siRNA (McNeil et al. 2006) leads to marked slowing of TJ assembly, and ZO1 and 2 can independently determine whether and where claudins are polymerized into TJ strands (Umeda et al. 2006). Taken together, results and interpretations suggest that a high content of ZO1 may indicate high TJ dynamics in D/SC junctions, and possibly in other barriers, and may be related to speciWc morphological features of TJs, a hypothesis which should be easily testable in the future. While the precise function of Occl for barrier properties of TJs has not been completely clariWed, claudins have been conclusively shown to diVerentially determine (ion-selective) TJ permeability (e.g., Schneeberger and Lynch 2004; Gonzales-Mariscal et al. 2003; Hartsock and Nelson 2008). The family of claudins consists of at least 24 members, which are diVerentially expressed in mammalian tissues and convey heterogeneous barrier functions to diVerent epithelia and endothelia (Turksen and Troy 2004). Thus, while Cls1 and 3 appear to be essential for high-resistance epithelia and Cl5 reduces permeability for low molecular substances in brain endothelia, the transepithelial resistance is decreased by the presence of Cl2 in TJs, and the absence of Cl4 from TJs increases Na+ permeability without inXuencing Cl¡-permeability (for reviews, see Schneeberger and Lynch 2004; Gonzales-Mariscal et al. 2003). The ionic composition of the olfactory mucus is of utmost importance for the perception of odorants, since inwardly directed monovalent cation- and Ca2+-currents and outwardly directed Cl¡-currents driven by a concentration gradient between olfactory cilia and mucus are essential for signal propagation to the OSN cell body (Reuter et al. 1998; Reisert et al. 2005; Nickell et al. 2006, 2007). Maintenance of the mucus ionic composition is the result of secretion processes of the olfactory glands and of secretion, reabsorption and trans- and paracellular transport/permeation processes in the olfactory excretory ducts and the apical epithelial surface (Getchell and Getchell 1992). Although the exact complement of all claudins in OE TJs needs further analysis, the present Wndings documenting the contribution of Cls 1, 3, 4 and 5 to all apical epithelial TJs, and the absence of Cl2 indicate that the apical barrier is comparatively tight,

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so that here transmembraneous ion transport/Xuxes can become eVective, a prerequisite for the signal transduction processes. Since Cl4 is lacking in OGs and ducts, paracellular permeation processes, possibly processes linked to Na+transport or Xuxes, appear to contribute to the primary mucus composition. Tight junctions of the FOs The presence of TJs between OECs was suggested by freeze-fracturing and immunocytochemical studies (Mack and Wolburg 1986; Miragall et al. 1994). Although TJs between OECs and axons or between axons have not been described and were not apparent in our morphological analyses, the observation that Schwann cells form TJs with regenerating axons (Dezawa and Adachi-Usami 2000) raised the question of whether OEC/axon TJs might also occur in the constantly regenerating peripheral olfactory pathway. In the present study, we found very intense Cl5-, moderate ZO2- and weak Occl-immunoreactivity within the FOs. TJ proteins appeared to be exclusively localized to OEC contacts. Moderate ZO1-immunoreactivity in OEC TJs was documented using a rabbit polyclonal anti-ZO1, but these junctions were not detectable using a rat monoclonal anti-ZO1. The monoclonal antibody, in the same sections, labeled epithelial TJs with high intensity, endothelial ones less eYciently. The pre-ZO1 mRNA possesses several alternative splicing domains in rodents and humans (Underwood et al. 1999; Gonzales-Mariscal et al. 1999, 2003). Two isoforms of ZO1, ZO1+ and ZO1¡, the latter lacking the 80-amino-acids long -motif resulting from the alternative splicing of the 240-nt -exon (Willott et al. 1992), are synthesized both in epithelia and endothelia. While the +isoform is much more abundant than the ¡-isoform in epithelial TJs, the opposite relation is found in endothelial TJs (Willott et al. 1992; Balda and Anderson 1993; Underwood et al. 1999; Gonzales-Mariscal et al. 2003). The Wnding that the rat monoclonal anti-ZO1 detected endothelial TJs less eYciently than epithelial ones, while the rabbit polyclonal ZO1 antibody showed no diVerence in detection eYciency, indicates that ZO1m does not detect all ZO1 isoforms. Thus, a possible explanation for the diVerential Wndings in the FOs in our material could be that a ZO1 isoform other than ZO1+, possibly ZO1¡, is the predominant ZO1 isoform in OEC TJs. This could have functional implications, since the +-isoform appears to confer high transepithelial resistance to TJs while the ¡-isoform appears to be preferentially localized in immature, highly dynamic and leaky TJs (Balda and Anderson 1993; Underwood et al. 1999; Gonzales-Mariscal et al. 2003; Ghassemifar et al. 2006). Experiments using isoform-speciWc ZO1 antibodies will solve this question. Cl1/ZO1/ZO2-colocalization was consistently detected in proWles at the outer circumference

Histochem Cell Biol (2008) 130:339–361

of FOs. These proWles accompanied the FOs up to the point of passage of FO fascicles through the pial surface of the OB, but were absent from inner ONL layers. Similar ZO1ir proWles were detected by Miragall et al. (1994) at the margins of FOs. Our Wndings render it likely that the Cl1/ ZO1/ZO2-reactive TJs are formed by ONF processes which closely surround the FO fascicles (Field et al. 2003; Herrera et al. 2005). Morphologically and functionally, ONFs appear closely related to perineurial Wbroblasts (Herrera et al. 2005), which express Cl1 and ZO1 (Pummi et al. 2004). Thus, OECs clearly form extensive TJs which are characterized by their intense Cl5-immunoreactivity, but apparently lack the other claudins studied. In addition to Cl5, these TJs contain moderate amounts of ZO1 (possibly the ¡-isoform) and ZO2, and little if any Occl, a Wnding in accordance with earlier studies (Hussar et al. 2002). Although the presence of further claudins is yet to be analyzed, these OEC TJs appear to be rather unusual. Their function, also, is not readily apparent. OEC barrier formation towards the FO environment in the LP may provide a necessary isolation of the axon bundles from external inXuences. TJs are also found in mesaxons of non-myelinating Schwann cells (Sugimoto et al. 2002), sealing the adaxonal from the abaxonal interstitium. The signiWcance of TJs between small OEC processes surrounding FO fascicles is more diYcult to assess. Myelinating Schwann cells localize Cl5 to TJs in Schmidt-Lantermann incisures, and are suggested to contribute to maintaining the radial pathway formed by gap junctions which links the outer Schwann cell compartment to the adaxonal cytoplasm (Poliak et al. 2002). Since electron microscopy indicated that TJs in OECs are localized in the vicinity of gap junctions, a supportive function for gap junction communication between small OEC processes appears feasible. Another possible function may be a compartmentalization of FO fascicles; however, since the separation of axon territories by OEC processes does not appear to be complete (Field et al. 2003), the possible functional signiWcance of this is not clear. In any case, expression of Cl5 is common to OECs and myelinating Schwann cells. Although Schwann cells additionally express Cl 1 (Poliak et al. 2002), and also Cl19 (Miyamoto et al. 2005) this Wnding may be of relevance for the still controversial capacity of OECs to remyelinate regenerating central axons after transplantation into spinal cord lesions (Boyd et al. 2005; Ibrahim et al. 2006). Adherens junctions in the OE The present results are the Wrst to comprehensively document cell-type speciWc formation of AJs in the OE. SCs are distinguished from other OE cell types in that they express both E-cad and N-cad, while OSN express only N-cad, and

357

epithelial cells (MCs, EDCs, OG cells), and possibly some basal cells synthesize only E-cad. The observation of Ecad-immunoreactivity in the developing mouse OSN layer reported by Akins et al. (2007) is thus most likely attributable to AJs of SC and MC processes rather than of OSNs. E- and N-cad only interact homotypically (Marrs and Nelson 1996). Thus, diVerential localization of cads to speciWc membrane domains of the SCs is a consequence of the localization of the corresponding cads on the partner cell membrane. In basolateral SC/SC puncta adherentia, interactions of both types of cads could theoretically occur, and indeed high-magniWcation images conWrmed a distinct formation of separate E-cad and N-cad contacts. This principle appears not to apply to SC/SC apical zonulae adherentes, however, it is likely that the apparent merging of E-cad and N-cad Xuorescence labeling in these contacts was a consequence of the high content of cads, obscuring a separate localization of E- and N-cad interaction in the contact zones. Colocalization experiments of cads with catenins indicated that all epithelial AJs contain the three catenins analyzed, and that there are only few AJs present mainly in the basal epithelium which do not colocalize either E- or N-cad. Tangential/oblique sections in the apical OE showed that direct contacts between dendrites are extremely rare. At the level of the OSN cell bodies, interneuronal contacts appeared to be more frequent. Both mature and immature OSNs form numerous N-cad-ir AJs on their cell bodies and dendrites. The Xuorescence for cats in these AJs was lower than in epithelial contacts. Since cats proposedly participate not only in linking the cads to the actin cytoskeleton but also in regulating the organization of the cytoskeleton (e.g., Baumgartner and Drenckhahn 2002; Baumgartner et al. 2003; Hartsock and Nelson 2008), this observation may indicate that neuronal AJs and their anchoring are more instable/dynamic than epithelial AJs. Recently, it has been shown that newborn neurons in the postnatal mouse hippocampus, just like immature OSN, localize
-cat in their cell bodies and dendrites, and that targeted knockout of this expression results in dendritic malformation (Gao et al. 2007). In these newborn neurons,
-cat was absent from the nuclei, but was colocalized with cadherin. Based on this observation, the authors suggested that the function of
-cat in dendritic morphogenesis more likely relates to a regulation of the dynamics of actin formation than to the wellknown function of the cat as a nuclear signaling molecule in the canonical Wnt pathway (Hartsock and Nelson 2008; Gao et al. 2007). Thus, the formation and release of N-cad/
-cat-containing AJs may be of relevance for the elaboration of dendrites and possibly also axons of immature OSN, and for their integration into the OE cell context. It has long been known that when OSN death is induced by surgical or chemical manipulations, the production of new OSN is

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markedly upregulated until the original state of the OE is restored (Schwob 2002; Kawauchi et al. 2003). Consequently, it was proposed that dying OSN either release factor(s) stimulating neurogenesis, or alternatively, cease to release factors inhibiting it. Growth factors such as TGF-, FGF2, BMPs, TGF-
, GDF11, Follistatin, and others have been proposed to mediate the neurogenetic events (e.g., Schwob 2002; Kawauchi et al. 2003; Nicolay et al. 2006). It does not appear to be evident what the initial step is in inducing the critical alteration of neurogenetic factor production in the microenvironment of the OE. In this context, a further hypothesis for OSN AJ function may be put forward: release of the junctions by dying neurons may initiate intracellular, catenin-mediated signaling cascades in the dying OSNs and/or in the surrounding cells, contributing to the production and release of factors and/or inducing neurogenetic/diVerentiation processes. In the basal epithelium, some cells formed E-cad-ir contacts. Although they morphologically resembled HBCs, they could also represent OG cells that protrude into the OE. Roundish cells above the basal lamina displayed
cat-ir puncta, but lacked apparent E- or N-cad-immunoreactivity. It appears to be generally accepted that a slowly dividing, multipotent stem cell situated close to the basal lamina gives rise to both neuronal and non-neuronal progeny; however, it is still a matter of debate whether this stem cells is found among the HBCs or the GBCs (Mackay-Sim and Kittel 1991; Schwob 2002; Chen et al. 2004; Carter et al. 2004, Beites et al. 2005). Since this cell is proposedly “non-migrating” (Mackay-Sim and Kittel 1991), it appears possible that it is integrated into the epithelial context via AJs during the time period between divisions. Cells with morphological characteristics of GBCs, which, according to Kawauchi et al. (2003) encompass stem cells, progenitors and immediate neuronal precursors, display particularly few AJs. This may be related to the fact that they possess a comparatively high proliferative activity, and thus do not engage in extensive interactions with neighboring cells (Beites et al. 2005). On the other hand, recent evidence indicates that N-cad and
-cat expression mediates neuronal diVerentiation and neurite outgrowth (Chen et al. 2006). Further investigations are necessary using the battery of markers for the diVerent basal cell types in order to analyze contact formation in the basal OE in more detail. Adherens junctions in the FOs Recent studies have shown the expression of various cads and cats in the developing mouse peripheral olfactory system (Akins and Greer 2006; Akins et al. 2007), and have concluded that the AJ proteins contribute to the establishment and relative stability of olfactory

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Histochem Cell Biol (2008) 130:339–361

networks. The methods used were not suited to precisely localize the proteins to AJs between identiWed cells. Our morphological and immunohistochemical Wndings provide, for the Wrst time, conclusive evidence for the formation of numerous N-cad- and cat-mediated AJs between OECs, OECs and OSN axons, and between OSN axons in the adult rat. We also found some E-cad-ir puncta at the FO periphery, but it was not absolutely clear whether these were localized between peripheral OEC processes or in AJs adjacent to the outer FO surface. ClariWcation of this question is of interest since myelinating Schwann cells have been shown to form E-cad-mediated junctions (Menichella et al. 2001). Further electron microscopic investigations will serve to clarify this point. The signiWcance of AJs in the highly neuroplastic system remains to be determined. Recently, it was shown that N-cad expression in embryonic rat peripheral nerves is observed in Schwann cell precursors and is restricted to the period of axon outgrowth (Wanner et al. 2006). Ncad-negative Schwann cells are a less favorable substrate for axon growth, but reinduction of N-cad expression restores their ability to support axon elongation. Axongrowth promoting properties of OECs are well established (e.g., KaWtz and Greer 1999; Chung et al. 2004, Pastrana et al. 2007), and are presumably mediated both by direct cellular contact and by secretion of soluble factors (Chung et al. 2004). Early in vitro-Wndings addressed at the question of whether N-cad in OECs supports OSN axon growth have not yielded conclusive results (Chuah et al. 1991; Chuah and Au 1994). However, the suggestion that N-cad may not be eVective in this respect relied on the fact that only a minority of cultured OECs expressed the protein. Our studies show that, in vivo, there are virtually no parts of the FOs where N-cad-ir puncta are not found, and electron microscopically, AJs between OECs and axons were very numerous, indicating that a growth-supporting function may be possible. Another interesting feature is the interaxonal contacts. DiVerential adhesiveness mediated by cads has been proposed to be important for the development of axon tracts and in axonal targeting (Treubert-Zimmermann et al. 2002; Hirano et al. 2003). Targeting of the continuously outgrowing axons of newly formed OSN is a complicated process depending on expression of odorant receptors and adhesion molecules (Alenius and Bohm 1997; Yoshihara et al. 1997; Feinstein and Mombaerts 2004; Feinstein et al. 2004). Whether AJs between speciWc axon types are relevant in this context remains to be determined. The necessary next step, of course, is to study whether AJs between OECs and axons and between axons are speciWcally formed with axons of immature neurons and/or between axons expressing speciWc receptors.

Histochem Cell Biol (2008) 130:339–361

Conclusion Our detailed analyses have shed light on the highly diverse and speciWc molecular composition of intercellular contacts in the rat peripheral olfactory system. The Wndings suggested a number of new hypotheses concerning the functions of these contacts, and thus open various interesting lines of further research. Acknowledgments The authors are deeply indebted to Rita Hermann, Sieglinde Schenk and Karin Reinfurt for technical support, and to Prof. Frank Margolis, Baltimore, and Prof. James Schwob, Boston, for their generous gift of antibodies. The studies were supported by the Deutsche Forschungsgemeinschaft, SFB 581, TPZ3.

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