Histological and Ultrastructural Characteristics ... - Wiley Online Library

THE ANATOMICAL RECORD 295:481–491 (2012)

Histological and Ultrastructural Characteristics of the Primordial Vomeronasal Organ in Lungfish SHOKO NAKAMUTA,1,2 NOBUAKI NAKAMUTA,1,2 KAZUMI TANIGUCHI,3 1,2 AND KAZUYUKI TANIGUCHI * 1 Laboratory of Veterinary Anatomy, Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan 2 United Graduate School of Veterinary Sciences, Gifu University, Gifu 501-1193, Japan 3 Laboratory of Veterinary Anatomy, School of Veterinary Medicine, Kitasato University, Towada, Aomori 034-8628, Japan

ABSTRACT Many vertebrates have two anatomically distinct olfactory organs—the olfactory epithelium and the vomeronasal organ—to detect chemicals such as general odorants and pheromones in their environment. The vomeronasal organ is not present in fish but is present in vertebrates of a higher order than amphibians. Among all extant fishes, the lungfish is considered to be genetically and phylogenetically closest to tetrapods. In this study, we examined the olfactory organs of African lungfish, Protopterus annectens, by lectin histochemistry, immunohistochemistry, and transmission electron microscopy. Two types of sensory epithelia were identified in the olfactory organ—the olfactory epithelium covering the surface of lamellae and the sensory epithelium lining the recesses both at the base of lamellae and in the wall of the nasal sac—and designated here as the lamellar olfactory epithelium and the recess epithelium, respectively. Based on analysis of G-protein expression and ultrastructure, the lamellar olfactory epithelium resembled the olfactory epithelium of ordinary teleosts and the recess epithelium resembled the vomeronasal organ of tetrapods. Furthermore, lectin histochemistry demonstrated that the axons from the recess epithelium converge and project to the ventrolateral part of the olfactory bulb, suggesting that lungfish possess a region homologous to the accessory olfactory bulb of tetrapods. Based on these results, it seems appropriate to refer to the recess epithelium as ‘‘a primordium of the vomeronasal organ.’’ This study may provide important clues to elucidate how the vomeronasal organ emerged during the evolution of vertebrates. C 2012 Wiley Periodicals, Inc. Anat Rec, 295:481–491, 2012. V

Key words: lungfish; vomeronasal organ; olfactory organ; ultrastructure; immunohistochemistry; lectin histochemistry

Many vertebrates have two distinct olfactory organs— the olfactory epithelium (OE) and the vomeronasal organ (VNO)—for detecting chemicals such as general

odorants and pheromones in their environment. Although it was previously believed that the OE detects general odorants and the VNO, pheromones, this view

Abbreviations used: OB ¼ olfactory bulb; OE ¼ olfactory epithelium; ORC ¼ olfactory receptor cell; TEM ¼ transmission electron microscopy; TRITC ¼ tetramethyl rhodamine isothiocyanate; VNO ¼ vomeronasal organ. *Correspondence to: Kazuyuki Taniguchi, Faculty of Agriculture, Laboratory of Veterinary Anatomy, Department of Veterinary Science, Iwate University, 3-18-8 Ueda, Morioka,

Iwate 020-8550, Japan. Fax: +81-19-621-6209. E-mail: [email protected] Received 29 May 2011; Accepted 28 December 2011. DOI 10.1002/ar.22415 Published online 23 January 2012 in Wiley Online Library (wileyonlinelibrary.com).

C 2012 WILEY PERIODICALS, INC. V

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has been challenged by recent studies indicating that they detect both types of chemicals (Ubeda-Ba~ non et al., 2011). Unlike the OE, which is present in all vertebrates, the VNO is not present in fish, birds, cetaceans, and humans (Døving and Trotier, 1998). The OE and VNO are anatomically and ultrastructurally distinct from each other (Eisthen, 1992). Although the OE of elasmobranches contains only microvillous olfactory receptor cells (ORCs) (Schluessel et al., 2008), the OE of most teleosts and some amphibians contains both ciliated ORCs and microvillous ORCs (Hansen and Zielinski, 2005). The OE of most terrestrial vertebrates contains only ciliated ORCs, although single ORCs possess both cilia and microvilli in the OE of most birds and some reptiles (Graziadei and Bannister, 1967; Kratzing, 1975; Wakabayashi and Ichikawa, 2008). In contrast to the ORC, which differs ultrastructurally between animal species, the vomeronasal receptor cells of most vertebrates possess only microvilli (Døving and Trotier, 1998). Thus, the clustering of microvillous receptor cells may be the definitive characteristic of the VNO of tetrapods. Two methods that have been used to differentiate between ORCs and vomeronasal receptor cells are lectinbinding patterns and G-protein localization. To date, distinct lectin-binding patterns between ORCs and vomeronasal receptor cells have been reported in amphibians (Franceschini et al., 2003; Saito et al., 2003), reptiles (Franceschini et al., 1996, 2001), and mammals (Lundh et al., 1989; Takami et al., 1994; Nakajima et al., 1998). These studies suggest that differences in the lectin-binding patterns may reflect the differences in function between ORCs and vomeronasal receptor cells. In addition, subsets of ORCs, which project their axons to distinct regions of the olfactory bulb (OB), have been distinguished by their unique lectinbinding patterns and are suggested to be different from each other biochemically (Riddle et al., 1993). In rodents, the ORCs express odorant receptors (ORs), whereas the vomeronasal receptor cells express type 1 vomeronasal receptors (V1R) or type 2 vomeronasal receptors (V2R) (Buck and Axel, 1991; Dulac and Axel, 1995; Herrada and Dulac, 1997). The OR, V1R, and V2R are coupled with different types of G-protein a subunits, that is, Gaolf, Gai2, and Gao, respectively (Buck and Axel, 1991; Dulac and Axel, 1995; Herrada and Dulac, 1997). The same couplings have also been demonstrated in teleosts (Hansen et al., 2003, 2004) and amphibians (Date-Ito et al., 2008). Segregation of ciliated ORCs and microvillous ORCs into two distinct epithelia may have occurred during the evolution from teleosts to tetrapods, leading to the appearance of two distinct olfactory organs: the OE and the VNO. Teleosts are classified into two groups: actinopterygians and sarcopterygians. Most of the general bony fishes are actinopterygians, whereas lungfishes, together with the coelacanths, are the only extant sarcopterygians. Lungfish diverged from the vertebrate lineage after the divergence of actinopterygians and prior to the divergence of amphibians (Diogo and Abdala, 2007). Genetic analyses suggest that the lungfish is the closest fish to tetrapods (Takezaki et al., 2004). Based on studies using immunohistochemistry, a VNO-specific marker, and DiI tracer, Gonz alez et al. (2010) proposed that the African lungfish Protopterus

TABLE 1. Binding specificities of the lectins Lectin DBA VVA BSL-I SBA SJA s-WGA

Concentration (mg/ml) 4.0 1.0 4.0 1.0 5.0 2.0

103 102 103 102 102 102

Binding specificity a-GalNAc a,b-GalNAc a-GalNAc, a-Gal a,b-GalNAc, Gal b-GalNAc, b-Gal b-GlcNAc

Gal: galactose; GalNAc: N-acetylgalactosamine; GlcNAc: Nacetylglucosamine.

dolloi possesses a vomeronasal system similar to that of tetrapods. In this study, we have further characterized the cells of this olfactory system in the African lungfish, Protopterus annectens, by lectin histochemistry, immunohistochemistry, and transmission electron microscopy (TEM). Our results suggest that the VNO had already started to diverge from the OE earlier in sarcopterygian phylogeny than has been previously believed.

MATERIALS AND METHODS Animals Nine juvenile or adult African lungfish, P. annectens (five males, four females), from 33 to 47 cm in length, were purchased from commercial suppliers. All procedures were approved by the local animal ethical committee of Iwate University. In all cases, the animals were anesthetized with ice and euthanized by decapitation.

Lectin Histochemistry The nasal sac and brain were cut off from the heads of three fishes and fixed in Bouin’s solution at 4 C for 24 hr. The nasal septum, including olfactory nerve bundles, was fixed in Bouin’s solution and decalcified in 10% ethylenediamine tetraacetic acid in 0.1 M phosphate buffer (pH 7.4) at 4 C for 2 weeks. The specimens were routinely embedded in paraffin and cut sagittally at 5 lm in thickness. Sections were processed for lectin histochemistry or stained using the Klu¨ver-Barrera method for histological examination (Klu¨ver and Barrera, 1953). Lectin histochemistry with six biotinylated lectins (Vector Laboratories; listed in Table 1) was carried out using the avidin–biotin peroxidase complex method with a Vectastain ABC kit (Vector Laboratories). After deparaffinization, the sections were incubated in 0.3% H2O2 in methanol to inactivate endogenous peroxidase and then incubated with 1% bovine serum albumin in phosphate-buffered saline (PBS) at 37 C for 30 min to block nonspecific binding. The sections were then incubated with one of the biotinylated lectins at 4 C overnight, followed by Vectastain ABC reagent at 37 C for 30 min. The sections were colorized with 0.05 M Tris-HCl (pH 7.6) containing 0.01% 3-30 diaminobenzidine tetrahydrochloride and 0.003% H2O2 at 37 C for 15 min. Finally, the sections were counterstained with methyl green, being washed in PBS between each step. Control staining was performed by the use of PBS in place of biotinylated lectins. Concentrations and sugar specificity of lectins are listed in Table 1.

PRIMORDIUM OF VOMERONASAL ORGAN IN LUNGFISH

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TABLE 2. Primary antibodies and peptides

PGP 9.5 Acetylated tubulin Gao Gao Gas/olf Gas/olf Peptide Gas/olf Peptide Gao

Rabbit pAb Mouse mAb Rabbit pAb Rabbit pAb Mouse mAb Rabbit pAb

Dilution

Supplier

Article number

1:100 1:200 1:500 1:500 1:100 1:1000 1:100 1:100

UltraClone Sigma-Aldrich MBL Santa Cruz Santa Cruz Santa Cruz Santa Cruz Santa Cruz

RA95101 T7451 551 sc-387 sc-55545 sc-383 sc-383P sc-387P

Immunohistochemistry The nasal sac was removed from the heads of three fishes and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) overnight at 4 C. The specimens were cryoprotected and embedded in optimal cutting temperature compound (Sakura Finetek) and cut sagittally at 10–15 lm in thickness with a cryostat. Some of the sections were stained with hematoxylin–eosin for general histological examination. Immunohistochemistry was carried out using six primary antibodies (listed in Table 2). After washing in PBS and 0.1% Triton X-100 in PBS, the sections were treated with 2% normal donkey serum in PBS for 30 min at room temperature to block nonspecific binding, then incubated with one of the primary antibodies at 4 C overnight. After washing in PBS and 0.1% Triton X-100 in PBS, the sections were incubated for 2 hr at room temperature with one of two fluorescent-labeled secondary antibodies: tetramethyl rhodamine isothiocyanate (TRITC)-donkey anti-rabbit IgG (1:500, Jackson ImmunoResearch, 711-026-152) or TRITC-donkey antimouse IgG (1:500, Jackson ImmunoResearch, 715-026150). All antibodies were diluted in 1% bovine serum albumin in PBS. Control staining was performed by the use of PBS in place of primary antibodies. To examine the colocalization of two antigens in the same section, double-labeling immunohistochemistry was performed with a combination of two primary antibodies: Gao (MBL) and Gas/olf (sc-55545) or Gas/olf (sc383) and acetylated tubulin (Sigma-Aldrich). Primary antibodies were used in a cocktail at the same dilution as described above for each single labeling. As secondary antibodies, Alexa Fluor 488-donkey anti-rabbit IgG (Invitrogen, A21206) and TRITC-donkey anti-mouse IgG (Jackson ImmunoResearch, 715-026-150) were used in a cocktail (1:500 dilution for each).

Electron Microscopy The nasal sac was dissected out from the heads of three fishes and fixed in 2% glutaraldehyde plus 2.5% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) overnight at 4 C. Specimens were postfixed in 1% osmium tetroxide for 2 hr at 4 C, dehydrated in a graded series of ethanol, substituted with propylene oxide, and embedded in epoxy resin. Ultrathin sections were cut with a diamond knife, stained with uranyl acetate and lead citrate, and examined with a TEM H-800 (Hitachi).

RESULTS A pair of oval nasal sacs, approximately 15 mm in length, were located anterior to the eyes and opened into

the oral cavity by the anterior and posterior nostrils. Two rows of lamellae were suspended from the dorsal, medial, and lateral walls of the nasal sac and were distributed on each side of the dorsal midline raphe (Fig. 1A,B). The number of lamellae contained in each row varied from 15 to 20 (Fig. 1A,B). Small recesses were frequently observed, both along the wall and at the base of the lamellae (Fig. 1C). The recesses were separated from the surrounding tissue by nonsensory epithelium. The recesses were more numerous in the caudal part of the nasal sac than in the rostral part. The surface of the lamellae was covered with the OE, alternating with the nonsensory epithelium (Fig. 1C,D). We designated the OE of the lamella as ‘‘lamellar OE.’’ The lamellar OE, like the OE of the tetrapods, consisted of supporting cells, ORCs, and basal cells (Fig. 1D). The recesses were lined with an epithelium containing several layers of cells (Fig. 1E). For convenience, we designated this epithelium as the ‘‘recess epithelium.’’ The recess epithelium was arranged in an acinar structure of 100–150 lm in thickness (Fig. 1E). In most cases, the recess epithelium appeared to be associated with a gland (Fig. 1C,F), which was not present in the lamellar OE. The associated gland consisted of cylindrical cells with eosinophilic cytoplasm and round nuclei (Fig. 1F).

Lectin Histochemistry We performed lectin histochemistry with the six lectins listed in Fig. 2. The lectins showed different staining patterns between the lamellar OE and the recess epithelium: Dolichos biflous agglutinin (DBA) only weakly stained the ORCs in the lamellar OE, whereas DBA positively stained most cells in the recess epithelium. Vicia villosa agglutinin (VVA) only weakly stained the ORCs in the lamellar OE, whereas VVA positively stained the supranuclear cytoplasm of most cells in the recess epithelium. The ORCs in the lamellar OE were not stained with Bandeiraea simplicifolia lectin-I (BSL-I), soybean agglutinin (SBA), Sophora japonica agglutinin (SJA), and succinylated wheat germ agglutinin (s-WGA), whereas the supranuclear cytoplasm of most cells in the recess epithelium were positively stained with these lectins. No specific staining was observed in the control slides (data not shown). Next, we performed lectin histochemistry of the olfactory nerve bundles and the OB to elucidate the projection pathway from the recess epithelium to the OB (Fig. 3). In the olfactory nerve bundles, DBA and s-WGA stained a small subset of olfactory nerves more intensely than the rest (Fig. 3C,E). The olfactory nerve bundles showed no positive staining with the other four lectins (data not shown). The OB of lungfish was situated on

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Fig. 1. Structure of the olfactory organ of lungfish. (A) Schematic drawing and (B) dissection microscopic view from the medial aspect of the sagittally sectioned nasal sac. A number of lamellae are aligned perpendicularly to the dorsal midline raphe. Dorsal is top and caudal is right in A–C. (C) Low magnification view of the olfactory organ stained with hematoxylin–eosin. Cells of the recess epithelium (open arrows) and those of the associated gland (open arrowheads) are located both on the wall and at the base of lamellae. (D) High magnification view of the lamellar OE. The nonsensory epithelium (asterisks) and lamellar OE are alternately arranged. The lamellar OE consists of

supporting cell (Sp), ORC, and basal cell (BC). Nuclei of the supporting cells are located in the upper half, nuclei of the ORCs toward the lower half, and nuclei of the basal cells immediately above the basal membrane. (E, F) High magnification views of the recesses. The recesses are separated from the surrounding tissue by the nonsensory epithelium (asterisks). (E) The recess epithelium consists of several layers of epithelial cells. (F) The associated gland contains cylindrical cells with eosinophilic cytoplasm and round nuclei. Scale bars: 2 mm in B, 300 lm in C, and 50 lm in D–F.


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Fig. 2. Lectin staining patterns in the lamellar OE (LOE) and the recess epithelium (RecE). Although DBA and VVA stain the cytoplasm of the supporting cell (Sp) and the free border in the lamellar OE positively, they stain the ORC in the lamellar OE weakly. Most of the cells

in the RecE stained positively with DBA. The supranuclear cytoplasm of most cells in the RecE stained positively with VVA. BSL-I, SBA, SJA, and s-WGA do not stain the LOE but postively stain the supranuclear cytoplasm in most cells of the RecE. Scale bars: 50 lm.

the rostrodorsal part of the telencephalon (Fig. 3A) and had four layers: olfactory nerve layer, glomerular layer, mitral cell layer, and granule cell layer (Fig. 3B). A clearly distinguishable accessory OB was not found. However, the ventrolateral part of the OB was stained with DBA more intensely than the rest of the OB (Fig. 3D-D00 ), and the ventrolateral part of the OB was specifically stained with s-WGA (Fig. 3F-F00 ). In the OB, the staining patterns of VVA, BSL-I, SBA, and SJA were similar to that of s-WGA (data not shown). No specific staining was observed in the control slides (data not shown).

were immunopositive for Gas/olf (Fig. 6B-B00 ). The immunoreactivity for two Gao antibodies (MBL#551 and sc-387) was similar to each other, as was the immunoreactivity for two Gas/olf antibodies (sc-383 and sc-55545; data not shown). After preabsorption with the corresponding peptides (listed in Table 2), no immunoreactivity for either Gas/olf (sc-383) or Gao (sc-387) was observed (data not shown).

Immunohistochemistry We performed immunohistochemical analyses to further characterize the lamellar OE and the recess epithelium (Figs. 4–6). For neuronal marker Protein Gene Product 9.5 (PGP 9.5), the lamellar OE and the recess epithelium were both immunopositive whereas the adjacent nonsensory epithelium was immunonegative (Fig. 4A,C). For the cilia marker acetylated tubulin, cilia on the free border of the lamellar OE and the adjacent nonsensory epithelium were immunopositive, whereas the recess epithelium was immunonegative (Fig. 4B,D). In the lamellar OE, dendrites, somata, and axons of the ORCs were immunopositive for Gao (Fig. 5A), whereas the cilia on the free border and axons were immunopositive for Gas/olf (Fig. 5B). In the recess epithelium, somata and axons showed intensely positive immunoreactivity for Gao (Fig. 5C), whereas immunoreactivity for Gas/olf was not observed (Fig. 5D). Neither Gao nor Gas/olf immunoreactivity was observed in the nonsensory epithelium (asterisks in Fig. 5A–D). In the apical part of the lamellar OE, the microvillous layer and dendrites were immunopositive for Gao (Fig. 6A), whereas the cilia on the free border were immunopositive for Gas/olf (Fig. 6A0 ). The immunopositive regions for Gao did not overlap with those for Gas/olf (Fig. 6A00 ). In the apical part of the lamellar OE, some of the cilia

Electron Microscopy In addition, we examined the ultrastructure of the olfactory organ of lungfish by TEM. In the apical part of the lamellar OE, four types of cells were distinguished: ciliated ORC, microvillous ORC, ciliated supporting cells, and microvillous supporting cells (Fig. 7A). The ciliated ORC had both cilia and microvilli on the tip of the dendrite. The dendrite of ciliated ORC contained longitudinally arranged microtubules (Fig. 7B). The microvillous ORC had microvilli on the tip of the dendrite. The dendrite of microvillous ORC contained longitudinally arranged microtubules. In addition, a small number of centrioles were observed in the dendrite of microvillous ORC (Fig. 7C-C0 ). The centriole of lungfish appeared to be composed of nine singlet microtubules rather than the more common triplet microtubules (Fig. 7C0 ). The ciliated supporting cells had cilia and short microvilli, whereas the microvillous supporting cells had a number of microvilli on their free borders. Both ciliated and microvillous supporting cells contained numerous secretory granules in their cytoplasm (Fig. 7A). The ORCs and the supporting cells were easily distinguished because the former were more electron-lucent than the latter. In the apical part of the recess epithelium, the microvillous sensory cells alternated with the microvillous supporting cells (Fig. 8A). The microvillous sensory cells were 0.5–5 lm in diameter and had microvilli of 5–10 lm in length on the tip of the dendrite. The dendrites of

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Fig. 3. Lectin staining patterns in the olfactory nerve bundles (ONB) and the OB. (A) Schema of dorsal view of the brain. Rostral is top. The gray areas indicate the OB and the ONBs. The OB is situated on the rostrodorsal part of the telencephalon (Tel). The sagittal sections of the OB cut at line M (medial part), I (intermediate part), and L (lateral part) are shown as M-OB (D and F), I-OB (D0 and F0 ), and LOB (D00 and F00 ). (B) Sagittal section of the OB stained with the Klu¨verBarrera method. The OB demonstrates four layers: olfactory nerve

layer (ONL), glomerular layer (GL), mitral cell layer (MCL), and granule cell layer (GRL). Dorsal is top and rostral is left in B–F00. (C, E) Staining patterns of DBA (C) and s-WGA (E) in the ONB. A small subset of axons of the ONBs (arrows) is stained more intensely. (D-D00 ) DBA stains the ventrolateral part of the OB (encircled by dotted line) more intensely than the rest. (F-F00 ) s-WGA specifically stains the ventrolateral part of the OB (encircled by dotted line). Scale bars: 250 lm.

these cells contained numerous microtubules (Fig. 8A,B). The microvillous supporting cells had microvilli of 2–3 lm in length on their free border and contained numerous secretory granules of 200 nm in diameter in the

apical cytoplasm (Fig. 8A,B). Desmosomes were observed between the two cell types (Fig. 8B). A small number of centrioles were observed in the dendrites of the microvillous sensory cells (Fig. 8C). Nuclei of both cell types


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Fig. 4. Immunohistochemistry for PGP 9.5 and acetylated tubulin (AcT) in the lamellar OE (LOE) and the recess epithelium (RecE). (A) ORCs in the LOE are immunopositive for PGP 9.5. The nonsensory epithelium (asterisk) is immunonegative for PGP 9.5. (B) Cilia on the free border of the LOE are immunopositive for AcT. (C) Cells in the RecE are immunopositive for PGP 9.5. The nonsensory epithelium (asterisks) is immunonegative for PGP 9.5. (D) Free border of the RecE is immunonegative for AcT. Cilia on the free border of the adjacent nonsensory epithelium (asterisks) are immunopositive for AcT. Scale bars: 100 lm.

Fig. 5. Immunohistochemistry for Gao and Gas/olf in the lamellar OE (LOE) and the recess epithelium (RecE). (A) The free border, dendrites, somata, and axons of the ORCs in the LOE are immunopositive for Gao. (B) The free border of the LOE is immunopositive for Gas/olf. (C) Cells in the RecE are immunopositive for Gao. (D) The RecE is immunonegative for Gas/olf. The nonsensory epithelium (asterisks in A–D) is immunonegative for both Gao and Gas/olf. Scale bars: 100 lm.

Fig. 6. Double-labeling immunohistochemistry in the lamellar OE (LOE). (A–A00 ) The LOE double-labeled with Gao and Gas/olf. The microvillous layer on the free border and dendrites are immunopositive for Gao (A, green). Cilia on the free border are immunopositive for Gas/olf (A0 , red). There is no overlap between the immunopositive

regions for Gao and those for Gas/olf (A00 ). (B–B00 ) The LOE double-labeled with acetylated tubulin (AcT) and Gas/olf. Cilia on the free border are immunopositive for AcT (B, red). Some, but not all of the cilia on the free border are also immunopositive for Gas/olf (B0 , green and B00 , yellow). Scale bars: 20 lm.

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Fig. 7. Ultrastructure of the lamellar OE. (A) At the apical part, two types of ORC and two types of supporting cell (Sp) are depicted: cORC, ciliated ORC; mORC, microvillous ORC; cSp, ciliated Sp; mSp, microvillous Sp. Both the ciliated Sp and the microvillous Sp contain numerous secretory granules (SG). (B) High magnification view of the cORC. The cORC has cilia (Ci) and short microvilli (m) on the tip of the dendrite. In the dendrite, microtubules (arrows) are observed. (C) High

magnification view of the mORC and the ciliated Sp. The mORC contains a few centrioles (white arrowheads) in the dendrite. The ciliated Sp is equipped with cilia (Ci) and short microvilli (m). Arrows indicate microtubules. Higher magnification view of the centriole indicated by rectangle is shown in C0 . (C0 ). This centriole appears to be composed of nine singlet microtubules. Scale bars: 2 lm in A, 1 lm in B and C, 0.1 lm in C0 .


Fig. 8. Ultrastructure of the RecE. (A) At the apical part, the microvillous sensory cells (mSCs; white arrowheads) and the microvillous supporting cells (mSps; black arrowheads) are alternately arranged. (B) High magnification view of the mSCs and the mSps. The mSC contains numerous microtubules in the dendrite (black arrows). The mSp contains numerous secretory granules (SG). A desmosome (white arrow) is observed between the mSC and the mSP. (C) Higher magnification view of the

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apical part of the mSC. In the dendrite, a few centrioles (white arrowheads) are observed. (D, E) The middle layer. The Sp contains numerous SGs and the SC contains well-developed smooth endoplasmic reticulum (sER). The axon bundle (AX) is observed between cells. (F) The basal layer. The basal cell (BC) contains rough ER (rER). The axon is observed between basal cells. Double arrowheads indicate the basal membrane. Scale bars: 1 lm in A, 0.5 lm in B, 0.25 lm in C, and 2 lm in D–F.

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were situated in the middle layer of the recess epithelium (Fig. 8D). The microvillous sensory cell contained welldeveloped smooth endoplasmic reticulum in the perinuclear cytoplasm (Fig. 8D). The axon bundles were seen in the middle and basal layers of the recess epithelium (Fig. 8E,F). The basal cells of the recess epithelium contained well-developed rough endoplasmic reticulum (Fig. 8F).

DISCUSSION The recess epithelium we have described here is likely to represent the same cells as the epithelial crypt described by Gonz alez et al. (2010). Although they described the crypt epithelium as being only at the base of lamellae, we found this epithelium not only at the base but also in the wall of the nasal sac. Thus, we have intentionally used another name for these cells—recess epithelium. The two sensory epithelia in the olfactory organ of lungfish, the lamellar OE and the recess epithelium, were found to differ from each other. First, the neuronal cells in the recess epithelium and the ORCs in the lamellar OE were distinguishable by the difference in their lectin-binding patterns, implying that the recess epithelium and the lamellar OE have separate functions. However, whether the neuronal cells in the recess epithelium are actually involved in olfaction has yet to be demonstrated, although the results of our lectin histochemistry suggested that they project to the OB in the same manner as the ORCs in the lamellar OE. Second, our immunohistochemical analysis of G-proteins in the olfactory organ of lungfish revealed that the lamellar OE contains Gaolf-expressing ciliated ORCs and Gao-expressing microvillous ORCs, whereas the recess epithelium contains Gao-expressing microvillous sensory cells but no Gaolf-expressing cells. In general, Gaolf is coupled with OR and Gao with V2R, and OR and V2R localize to cilia (Gaolf) and microvilli (Gao) on the tip of the ORC dendrites, respectively (Buck and Axel, 1991; Herrada and Dulac, 1997). In the olfactory organ of lungfish, apical cilia and microvilli showed intense immunoreactivity for Gaolf and Gao, suggesting the presence of OR and V2R in the olfactory organ of lungfish. In the ordinary teleosts, the OE contains Gaolf-expressing ciliated ORCs and Gao-expressing microvillous ORCs (Hansen and Zielinski, 2005; Hamdani and Døving, 2007), whereas the VNO of amphibians contains Gao-expressing microvillous vomeronasal receptor cells (Date-Ito et al., 2008). Based on G-protein expression, the lamellar OE of lungfish resembles the OE of ordinary teleosts and the recess epithelium resembles the VNO of amphibians. Although the ultrastructure of the OE has previously been reported in several species of lungfish (Theisen, 1972; Derivot et al., 1979; Hansen et al., 1994), the ultrastructure of the recess epithelium has not been previously described. In accordance with the earlier reports by Derivot et al. (1979) and Hansen et al. (1994), the ultrastructure of the lamellar OE shared characteristics with the OE of ordinary teleosts (Hansen and Zielinski, 2005). On the other hand, our studies have shown that the ultrastructure of the recess epithelium is considerably different from that of the lamellar OE in that the recess epithelium contained microvillous sensory cells but not ciliated sensory cells. Ciliated cells were also demonstrated in the lamellar OE but not in the recess epithelium by immunohistochemistry with acetylated

tubulin. With regard to the ultrastructure, the lamellar OE resembles the OE of ordinary teleosts and the recess epithelium resembles the VNO of tetrapods. Differential distribution of the ciliated ORCs and microvillous ORCs in a single OE has been described in several teleosts (Thommesen, 1982; Hansen et al., 2003, 2004). Hansen et al. (2003, 2004) suggested that such segregation may be the presage of the segregation of their ORCs into distinct epithelial chambers, such as the OE and the VNO. In catfish and goldfish, microvillous ORCs are preferentially located on the dorsal side and near the midline raphe (Hansen et al., 2003, 2004). However, the accumulation of microvillous ORCs, as in the recess epithelium of lungfish, is not found in these fishes. This all indicates that a higher degree of segregation between ciliated and microvillous ORCs has been achieved in lungfish than in the case of general actinopterygians. Our lectin histochemical analysis suggested that axons from the recess epithelium converge and project to the ventrolateral part of the OB. This axonal projection suggested by our data agrees with the previous result demonstrated by DiI tracer injection (Gonz alez et al., 2010). The nerve systems projecting from the olfactory organ of lungfish include the olfactory nerve, the nervus terminalis, and extrabulbar olfactory system (von Bartheld, 2004). The recess epithelium is not likely to belong to the latter two, because it projects to the OB. Several previous studies have suggested the presence of VNO in lungfish (Rudebeck, 1944; Bertmar, 1965, 1969, 1981; Schnitzlein and Crosby, 1967), whereas other studies have failed to identify the VNO, the vomeronasal nerves, and the accessory OB in lungfish (Derivot, 1984; Eisthen, 1992; Reiner and Northcutt, 1987; von Bartheld et al., 1988). Francescini et al. (2000) used lectin histochemistry to demonstrate that the ventrolateral part of the lungfish OB corresponds to the accessory OB of tetrapods, although they failed to find an epithelium homologous to the VNO in the olfactory organ of lungfish. Our study, using additional immunohistochemistry, as well as lectin histochemistry and TEM, has revealed detailed differences in the histological and ultrastructural characteristics between the lamellar OE and the recess epithelium. As it is possible that the recess epithelium could be a differentiating lamellar OE or a sensory epithelium other than the VNO, further studies would be required for clarification. In general, the VNO of tetrapods is situated on the ventral side of the nasal cavity as a distinct organ, whereas that of the urodela, that is, newt and salamander, arises in the lateral part of the nasal cavity as a lateral diverticulum (Taniguchi et al., 2011). Unlike the VNO of tetrapods, the recess epithelium was not anatomically distinct from the OE but rather scattered at the base of lamellae and unrecognized by gross morphological examination. Therefore, the recess epithelium should be referred to as ‘‘a primordium of VNO.’’ If sufficient data were obtained about the distribution of recess epithelium, it would be possible to discuss the change in localization of the VNO during phylogeny.

ACKNOWLEDGEMENTS This work was supported by Grant-in-Aid for Graduate Students from The United Graduate School of Veterinary Sciences, Gifu University (S.N.). We would like to


thank Shuichiro Hayashi, the Center for Regional Collaboration in Research and Education, Iwate University and Kuniaki Sasaki, the Technical Division, Iwate University for use of the TEM.

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