In situ hybridization analysis of olfactory receptor expression in the sea turtle olfactory organ.

The olfactory organ of turtles consists of an upper chamber epithelium (UCE) with associated glands, and a lower chamber epithelium (LCE) devoid of glands. The UCE and LCE are referred to as the air-nose and the water-nose, respectively, because the UCE is thought to detect airborne odorants, while the LCE detects waterborne odorants. However, it is not clear how the two are used in the olfactory organ. Odorant receptors (ORs) are the major olfactory receptors in turtles; they are classified as class I and II ORs, distinguished by their primary structure. Class I ORs are suggested to be receptive to water-soluble ligands and class II ORs to volatile ligands. This study analyzed the expression of class I and II ORs in hatchlings of the green sea turtle, Chelonia mydas, through in situ hybridization, to determine the localization of OR-expressing cells in the olfactory organ. Class I OR-expressing cells were distributed mainly in the LCE, implying that the LCE is receptive to waterborne odorants. Class II OR-expressing cells were distributed in both the UCE and LCE, implying that the entire olfactory organ is receptive to airborne odorants. The widespread expression of class II ORs may increase opportunities for sea turtles to sense airborne odorants.

Morphological Analysis of the Olfactory System of the Pig-Nosed Turtle, Carettochelys insculpta.

The turtle olfactory organ consists of the upper (UCE) and lower (LCE) chamber epithelium, projecting to the ventral and dorsal parts of the olfactory bulbs, respectively. The UCE is associated with glands, contains ciliated olfactory receptor neurons, and is assumed to detect odorants primarily in air, while the LCE is devoid of glands, contains microvillous olfactory receptor neurons, and is assumed to detect odorants primarily in water. Examining the olfactory system of the pig-nosed turtle, Carettochelys insculpta, this study found that both the upper and lower chambers of the nasal cavity were lined with sensory epithelium devoid of associated glands and contained ciliated olfactory receptor neurons. Moreover, the olfactory bulbs were not divided into dorsal and ventral parts. These results suggest that the olfactory system of the pig-nosed turtle is a single system specialized for detecting odorants in water.

Immunohistochemical distribution of proteins involved in glutamate release in subepithelial sensory nerve endings of rat epiglottis.

To elucidate the efferent functions of sensory nerve endings, the distribution of calretinin and vesicular glutamate transporter 1 (VGLUT1) in laryngeal laminar nerve endings and the immunohistochemical distribution of proteins associated with synaptic vesicle release, i.e., t-SNARE (SNAP25 and syntaxin 1), v-SNARE (VAMP1 and VAMP2), synaptotagmin 1 (Syt1), bassoon, and piccolo, were examined. Subepithelial laminar nerve endings immunoreactive for Na+-K+-ATPase α3-subunit (NKAα3) were largely distributed in the whole-mount preparation of the epiglottic mucosa, and several endings were also immunoreactive for calretinin. VGLUT1 immunoreactivity was observed within terminal part near the outline of the small processes of NKAα3-immunoreactive nerve ending. SNAP25, syntaxin 1, and VAMP1 immunoreactivities were detected in terminal parts of calretinin-immunoreactive endings, whereas VAMP2 immunoreactivity was only observed in a few terminals. Terminal parts immunoreactive for calretinin and/or VGLUT1 also exhibited immunoreactivities for Syt1, Ca2+ sensor for membrane trafficking, and for bassoon and piccolo, presynaptic scaffold proteins. The presence of vesicular release-related proteins, including SNARE proteins, in the terminals of laryngeal laminar endings indicate that intrinsic glutamate modulates their afferent activity in an autocrine-like manner.

Paternal knockout of Slc38a4/SNAT4 causes placental hypoplasia associated with intrauterine growth restriction in mice.

The placenta is critical in mammalian embryonic development because the embryo's supply of nutrients, including amino acids, depends solely on mother-to-embryo transport through it. However, the molecular mechanisms underlying this amino acid supply are poorly understood. In this study, we focused on system A amino acid transporters Slc38a1/SNAT1, Slc38a2/SNAT2, and Slc38a4/SNAT4, which carry neutral, short-side-chain amino acids, to determine their involvement in placental or embryonic development. A triple-target CRISPR screen identified Slc38a4/SNAT4 as the critical amino acid transporter for placental development in mice. We established mouse lines from the CRISPR founders with large deletions in Slc38a4 and found that, consistent with the imprinted paternal expression of Slc38a4/SNAT4 in the placenta, paternal knockout (KO) but not maternal KO of Slc38a4/SNAT4 caused placental hypoplasia associated with reduced fetal weight. Immunostaining revealed that SNAT4 was widely expressed in differentiating cytotrophoblasts and maturing trophoblasts at the maternal-fetal interface. A blood metabolome analysis revealed that amino acid concentrations were globally reduced in Slc38a4/SNAT4 mutant embryos. These results indicated that SNAT4-mediated amino acid transport in mice plays a major role in placental and embryonic development. Given that expression of Slc38a4 in the placenta is conserved in other species, our Slc38a4/SNAT4 mutant mice could be a promising model for the analysis of placental defects leading to intrauterine growth restriction in mammals.

Morphology of GNAT3-immunoreactive chemosensory cells in the nasal cavity and pharynx of the rat.

Solitary chemosensory cells and chemosensory cell clusters are distributed in the pharynx and larynx. In the present study, the morphology and reflexogenic function of solitary chemosensory cells and chemosensory cell clusters in the nasal cavity and pharynx were examined using immunofluorescence for GNAT3 and electrophysiology. In the nasal cavity, GNAT3-immunoreactive solitary chemosensory cells were widely distributed in the nasal mucosa, particularly in the cranial region near the nostrils. Solitary chemosensory cells were also observed in the nasopharynx. Solitary chemosensory cells in the nasopharyngeal cavity were barrel like or slender in shape with long lateral processes within the epithelial layer to attach surrounding ciliated epithelial cells. Chemosensory cell clusters containing GNAT3-immunoreactive cells were also detected in the pharynx. GNAT3-immunoreactive cells gathered with SNAP25-immunoreactive cells in chemosensory clusters. GNAT3-immunoreactive chemosensory cells were in close contact with a few SP- or CGRP-immunoreactive nerve endings. In the pharynx, GNAT3-immunoreactive chemosensory cells were also attached to P2X3-immunoreactive nerve endings. Physiologically, the perfusion of 10 mM quinine hydrochloride (QHCl) solution induced ventilatory depression. The QHCl-induced reflex was diminished by bilateral section of the glossopharyngeal nerve, suggesting autonomic reflex were evoked by chemosensory cells in pharynx but not in nasal mucosa. The present results indicate that complex shape of nasopharyngeal solitary chemosensory cells may contribute to intercellular communication, and pharyngeal chemosensory cells may play a role in respiratory depression.

Distribution and morphology of baroreceptors in the rat carotid sinus as revealed by immunohistochemistry for P2X3 purinoceptors.

The morphological characteristics of baroreceptors in the rat carotid sinus were reevaluated by whole-mount preparations with immunohistochemistry for P2X3 purinoceptors using confocal scanning laser microscopy. Immunoreactive nerve endings for P2X3 were distributed in the internal carotid artery proximal to the carotid bifurcation, particularly in the region opposite the carotid body. Some pre-terminal axons in nerve endings were ensheathed by myelin sheaths immunoreactive for myelin basic protein. Pre-terminal axons ramified into several branches that extended two-dimensionally in every direction. The axon terminals of P2X3-immunoreactive nerve endings were flat and leaf-like in shape, and extended hederiform- or knob-like protrusions in the adventitial layer. Some axons and axon terminals with P2X3 immunoreactivity were also immunoreactive for P2X2, and axon terminals were closely surrounded by terminal Schwann cells with S100 or S100B immunoreactivity. These results revealed the detailed morphology of P2X3-immunoreactive nerve endings and suggested that these endings respond to a mechanical deformation of the carotid sinus wall with their flat leaf-like terminals.

Morphology of GNAT3-immunoreactive chemosensory cells in the rat larynx.

The upper airways play important roles in respiratory defensive reflexes. Although solitary chemosensory cells and chemosensory cell clusters have been reported in the laryngeal mucosa of mammalian species, the distribution and cellular morphology of chemosensory cells remain unclear. In the present study, the distribution and morphology of solitary chemosensory cells and chemosensory cell clusters were examined by immunofluorescence for GNAT3 on whole-mount preparations of the rat laryngeal mucosa. Electrophysiological experiments were performed to analyze the respiratory reflexes evoked by bitter stimuli to the laryngeal cavity. In the whole area of the laryngeal mucosa, the numbers of GNAT3-immunoreactive solitary chemosensory cells and chemosensory clusters were 421.0 ± 20.3 and 62.7 ± 6.9, respectively. GNAT3-immunoreactive solitary chemosensory cells were mainly distributed in the mucosa overlying epiglottic and arytenoid cartilage, and chemosensory clusters were mainly distributed on the edge of the epiglottis and aryepiglottic fold. GNAT3-immunoreactive solitary chemosensory cells were slender with elongated processes or had a flask-like/columnar shape. The number of GNAT3-immunoreactive cells in chemosensory clusters was 6.1 ± 0.4, ranging between 2 and 14 cells. GNAT3-immunoreactive cells in the cluster were variform and the tips of apical processes gathered at one point at the surface of the epithelium. The tips of apical cytoplasmic processes in solitary chemosensory cells and cells in the cluster were immunoreactive for espin, and faced the laryngeal cavity. Physiological experiments showed that the application of 10 mm quinine hydrochloride to the laryngeal cavity decreased respiratory frequency. The present results revealed the chemosensory field of the larynx and the morphological characteristics of the laryngeal chemosensory system for respiratory depression.

Morphology of P2X3-immunoreactive nerve endings in the rat laryngeal mucosa.

The morphological characteristics of P2X3-immunoreactive nerve endings in the laryngeal mucosa were herein examined using immunohistochemistry with confocal laser microscopy. Ramified intraepithelial nerve endings immunoreactive to P2X3 were distributed in the epiglottis and arytenoid region. The axon terminals of P2X3-immunoreactive ramified endings were beaded or flat in shape. These endings were also immunoreactive to P2X2 and not identical to the nerve endings immunoreactive to Na(+)-K(+)-ATPase α3-subunit, substance P (SP), and calcitonin gene-related peptide (CGRP). P2X3-immunoreactive axon terminals were also immunoreactive to vGLUT1, vGLUT2, and vGLUT3. In addition to ramified endings, P2X3-immunoreactive nerve endings were associated with α-gustducin-immunoreactive solitary chemosensory cells and/or SNAP25-immunoreactive neuroendocrine cells. Furthermore, P2X3-immunoreactive nerve endings were also observed in the taste bud-like chemosensory cell clusters of the stratified squamous epithelium covering epiglottic and arytenoid cartilage. The P2X3-immunoreactive nerve endings that associated with sensory and/or endocrine cells and chemosensory cell clusters were also immunoreactive to P2X2, vGLUT1, vGLUT2, and vGLUT3, but not to SP or CGRP. In conclusion, P2X3-immunoreactive nerve endings may be classified into two types, i.e., intraepithelial ramified nerve endings and nerve endings associated with chemosensory cells and neuroendocrine cells.

Three-dimensional architectures of P2X2-/P2X3-immunoreactive afferent nerve terminals in the rat carotid body as revealed by confocal laser scanning microscopy.

We investigated the three-dimensional architectures of P2X2-/P2X3-immunoreactive nerve terminals in the rat carotid body using immunohistochemistry with confocal laser microscopy. Nerve endings immunoreactive for P2X2 and P2X3 were associated with clusters of type I cells, whereas some nerve endings were sparsely distributed in a few clusters. Most nerve endings surrounding type I cells were hederiform in shape and extended several flattened axon terminals, which were polygonal or pleomorphic in shape and contained P2X2-/P2X3-immunoreactive products. Three-dimensional reconstruction views revealed that some flattened nerve endings with P2X3 immunoreactivity formed arborized, sac- or goblet-like terminal structures and were attached to type I cells immunoreactive for tyrosine hydroxylase (TH). However, P2X3-immunoreactive axon terminals were sparsely distributed in type I cells immunoreactive for dopamine beta-hydroxylase. Multi-immunolabeling for P2X2, S100, and TH revealed that P2X2-immunoreactive axon terminals were attached to TH-immunoreactive type I cells on the inside of type II cells with S100 immunoreactivity. These results revealed the detailed morphology of P2X2-/P2X3-immunoreactive nerve terminals and suggest that sensory nerve endings may integrate chemosensory signals from clustered type I cells with their variform nerve terminals.

Sympathetic and sensory innervation of small intensely fluorescent (SIF) cells in rat superior cervical ganglion.

The sympathetic ganglion contains small intensely fluorescent (SIF) cells derived from the neural crest. We morphologically characterize SIF cells and focus on their relationship with ganglionic cells, preganglionic nerve fibers and sensory nerve endings. SIF cells stained intensely for tyrosine hydroxylase (TH), with a few cells also being immunoreactive for dopamine ß-hydroxylase (DBH). Vesicular acetylcholine transporter (VAChT)-immunoreactive puncta were distributed around some clusters of SIF cells, whereas some SIF cells closely abutted DBH-immunoreactive ganglionic cells. SIF cells contained bassoon-immunoreactive products beneath the cell membrane at the attachments and on opposite sites to the ganglionic cells. Ganglion neurons and SIF cells were immunoreactive to dopamine D2 receptors. Immunohistochemistry for P2X3 revealed ramified nerve endings with P2X3 immunoreactivity around SIF cells. Triple-labeling for P2X3, TH and VAChT allowed the classification of SIF cells into three types based on their innervation: (1) with only VAChT-immunoreactive puncta, (2) with only P2X3-immunoreactive nerve endings, (3) with both P2X3-immunoreactive nerve endings and VAChT-immunoreactive puncta. The results of retrograde tracing with fast blue dye indicated that most of these nerve endings originated from the petrosal ganglion. Thus, SIF cells in the superior cervical ganglion are innervated by preganglionic fibers and glossopharyngeal sensory nerve endings and can be classified into three types. SIF cells might modulate sympathetic activity in the superior cervical ganglion.

Vesicular glutamate transporter 2-immunoreactive afferent nerve terminals in the carotid body of the rat.

The carotid body is a peripheral chemoreceptor that detects decreases in arterial pO2 and subsequently activates the carotid sinus nerve. The hypoxia-evoked activity of the carotid sinus nerve has been suggested to be modulated by glutamate. In the present study, we investigate the immunohistochemical localization of vesicular glutamate transporters in the carotid body of the rat. Vesicular glutamate transporter 2 (VGLUT2) labeling was closely associated with glomus cells immunoreactive to tyrosine hydroxylase but was not in the cytoplasm of these cells. The VGLUT2 immunoreactivity was observed within nerve endings that were immunoreactive to P2X3 and densely localized inside P2X3-immunoreactive axon terminals. These results suggest that VGLUT2 is localized in the afferent nerve terminals of the carotid body. Glutamate may be released from afferent nerve terminals to modulate the chemosensory activity of the carotid body.

Immunohistochemical localization of P2Y12 purinoceptors in the rat carotid body.

The present study investigated the localization of the adenosine 5'-diphosphate (ADP)-selective P2Y12 purinoceptors in the rat carotid body using multilabeling immunofluorescence. Punctate immunoreactive products for P2Y12 were distributed in chemoreceptive type I cells immunoreactive to vesicular nucleotide transporter (VNUT) or dopamine beta-hydroxylase, but not in S100B-immunoreactive glial-like type II cells. P2Y12 immunoreactivity was localized in cell clusters containing VNUT-immunoreactive type I cells surrounded by the perinuclear cytoplasm and cytoplasmic processes of type II cells immunoreactive for ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) and NTPDase3, which hydrolyze extracellular nucleotide tri- and/or di-phosphates. In ATP bioluminescence assays using carotid bodies, the degradation of extracellular ATP was attenuated in the presence of the selective NTPDases inhibitor ARL67156, suggesting ATP-degrading activity by NTPDases in the tissue. These results suggest that ATP released from type I cells is degraded into ADP and adenosine 5'-monophosphate by NTPDases expressed in type II cells, and that ADP modulates type I cells via P2Y12 purinoceptors.

Localization of the primordial vomeronasal organ and its relationship to the associated gland in lungfish.

The lungfish, the closest fish to tetrapods, has two types of sensory epithelia in the olfactory organ: the lamellar olfactory epithelium and the recess epithelium. The former resembles the olfactory epithelium of ordinary teleosts and the latter resembles the vomeronasal organ of tetrapods with respect to the G-protein expressions and the morphological properties of olfactory receptor cells. In contrast to the lamellar olfactory epithelium covering the surface of olfactory lamella, the recess epithelium, together with the glandular epithelium, lines the recesses at the base of olfactory lamellae and is separated from the surrounding tissues by nonsensory epithelium. In the present study, we examined the distribution of these recesses and the relationship between the recess epithelium and the associated gland in the nasal sac of lungfish. We found that the posterior part of the nasal sac contained more recesses than the anterior one, and the medial one contained more recesses than the lateral one. In addition, virtually all recesses consisted of both the recess epithelium and the glandular epithelium. Furthermore, the glandular epithelium was invariably situated proximal to the midline raphe of the nasal sac, and the recess epithelium distal to it. Possible roles of the recess epithelium and the glandular epithelium are discussed.

Plastic loss of motile cilia in the gills of Polypterus in response to high CO2 or terrestrial environments.

The evolutionary transition of vertebrates from water to land during the Devonian period was accompanied by major changes in animal respiratory systems in terms of physiology and morphology. Indeed, the fossil record of the early tetrapods has revealed the existence of internal gills, which are vestigial fish-like traits used underwater. However, the fossil record provides only limited data on the process of the evolutionary transition of gills from fish to early tetrapods. This study investigated the gills of Polypterus senegalus, a basal ray-finned/amphibious fish which shows many ancestral features of stem Osteichthyes. Based on scanning electron microscopy observations and transcriptome analysis, the existence of motile cilia in the gills was revealed which may create a flow on the gill surface leading to efficient ventilation or remove particles from the surface. Interestingly, these cilia were observed to disappear after rearing in terrestrial or high CO2 environments, which mimics the environmental changes in the Devonian period. The cilia re-appeared after being returned to the original aquatic environment. The ability of plastic changes of gills in Polypterus revealed in this study may allow them to survive in fluctuating environments, such as shallow swamps. The ancestor of Osteichthyes is expected to have possessed such plasticity in the gills, which may be one of the driving forces behind the transition of vertebrates from water to land.

Immunohistochemical distribution of Ca2+/calmodulin-dependent protein kinase II subunits in the rat carotid body.

Carotid body (CB) activity stimulated by a lower partial oxygen pressure in rats is enhanced by exposure to chronic intermittent hypoxia. However, the mechanisms that modulate CB activity remain unclear. In the present study, the expression and distribution of one of the candidate molecules to modulate reactivity, Ca2+/calmodulin-dependent protein kinase II (CaMKII) were examined in the rat CB using reverse transcriptional polymerase chain reaction and immunofluorescence with isoform-specific antibodies. CaMKIIγ and CaMKIIδ were distributed in CB chemoreceptor cells, and exhibited intense immunoreactivity in dopamine ß-hydroxylase-positive chemoreceptor cells. CaMKIIß and CaMKIIγ were distributed in sensory nerve endings attached to chemoreceptor cells of the CB. In the petrosal ganglion, immunoreactivities for CaMKIIα, CaMKIIß, CaMKIIγ, and CaMKIIδ were detected in the perinuclear region of ganglion cells. The present results indicate that CaMKIIγ and CaMKIIδ in chemoreceptor cells and CaMKIIß and CaMKIIγ in sensory nerve endings enhanced reciprocal synaptic transmission, i.e., noradrenaline and ATP for cells to neurons and glutamate for neurons to cells.

Type 1 vomeronasal receptors expressed in the olfactory organs of two African lungfish, Protopterus annectens and Protopterus amphibius.

Lungfish are the fish related most closely to tetrapods. The olfactory organ of lungfish contains two distinct sensory epithelia: the lamellar olfactory epithelium (OE) and the recess epithelium (RecE). Based on their ultrastructural and histological characteristics, the lamellar OE and the RecE are considered to correspond respectively to the teleost OE and a primitive vomeronasal organ (VNO). In tetrapods, the OE and VNO have been shown to express different families of olfactory receptors; for example, in mammals, the OE expresses odorant receptors and trace amine-associated receptors, while the VNO expresses type 1 (V1Rs) and type 2 (V2Rs) vomeronasal receptors. In the present study, we examined the expression of V1Rs in the olfactory organs of two African lungfish, Protopterus annectens and Protopterus amphibius. RNA sequencing and phylogenetic analyses identified 29 V1R genes in P. annectens and 50 V1R genes in P. amphibius. Most V1Rs identified in these lungfish were classified as the tetrapod-type V1Rs initially found in tetrapods and distinct from fish-type V1Rs. In teleost, which all lack a VNO, all olfactory receptors are expressed in the OE, while in Xenopus V1Rs are expressed exclusively in the OE, and not in the VNO. In situ hybridization analysis indicated that lungfish V1Rs were expressed mainly in the lamellar OE and rarely in the RecE. These results imply that V1R expression in lungfish represents an intermediate step toward the complete segregation of V1R expression between the OE and VNO, reflecting the phylogenetic position of lungfish between teleosts and amphibians.

Distribution of proteins for synaptic release in nerve endings associated with the trachealis muscle of rats.

The immunohistochemical localization of proteins for synaptic release was examined in smooth muscle-associated sensory nerve endings using whole-mount preparations of the rat trachea. Plant-like smooth muscle-associated nerve endings with immunoreactivity for Na+-K+-ATPase, α3-subunit were identified in the trachealis muscle. VGLUT1, synapsin1, t-SNARE proteins (SNAP25 and syntaxin1), v-SNARE proteins (VAMP1 and VAMP2), and a presynaptic active zone-related protein (piccolo) were detected in the terminal parts of these endings. These results suggest that smooth muscle-associated nerve endings secrete glutamate to modulate sensorimotor functions in the lung deflation reflex.

Immunohistochemical analysis of glutamatergic and serotonergic signaling pathways in chemosensory cell clusters in the pharynx and larynx of rats.

The present study examined cellular components and the localization of vesicular glutamate transporter (VGLUT) 1 and 2 and serotonin (5-HT) in chemosensory cell clusters in the rat pharynx and larynx. Triple immunolabeling for guanine nucleotide-binding protein G (t), subunit ⍺3 (GNAT3) and nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) with synaptotagmin-1 (Syt1) revealed NTPDase2-immunoreactive type I-like cells in addition to GNAT3-immunoreactive type II-like and Syt1-immunoreactive type III-like cells in pharyngolaryngeal chemosensory cell clusters. Therefore, these clusters appear to comprise similar cell types to those in the lingual taste buds with slight morphological modifications. An immunofluorescence analysis of VGLUT1 or VGLUT2 and GNAT3 with P2X3 purinoceptors revealed that VGLUTs co-localized to P2X3-immunoreactive spherical nerve terminals closely associated with GNAT3-immunoreactive type II-like cells. Moreover, triple immunolabeling for Syt1/synaptosomal-associated protein, 25 kDa (SNAP25) and P2X3 with VGLUT1 or VGLUT2 revealed punctate immunoreactive products for VGLUT1 and VGLUT2 within P2X3-immunoreactive flat axon terminals wrapped around Syt1/SNAP25-immunoreactive type III-like cells. The afferent nerve fibers innervating cell clusters may contain glutamate and release it by exocytosis. On the other hand, immunoreactive products for 5-HT and dopa decarboxylase were detected in Syt1-immunoreactive cells, indicating the release of 5-HT by these cells. The present results suggest that chemosensory cell clusters in the pharynx and larynx may be modulated by intrinsic glutamate and 5-HT.

Type 1 vomeronasal receptor expression in juvenile and adult lungfish olfactory organ.

Lungfish are the most closely related fish to tetrapods. The olfactory organ of lungfish contains lamellae and abundant recesses at the base of lamellae. Based on the ultrastructural and histochemical characteristics, the lamellar olfactory epithelium (OE), covering the surface of lamellae, and the recess epithelium, contained in the recesses, are thought to correspond to the OE of teleosts and the vomeronasal organ (VNO) of tetrapods. With increasing body size, the recesses increase in number and distribution range in the olfactory organ. In tetrapods, the expression of olfactory receptors is different between the OE and VNO; for instance, the type 1 vomeronasal receptor (V1R) is expressed only in the OE in amphibians and mainly in the VNO in mammals. We recently reported that V1R-expressing cells are contained mainly in the lamellar OE but also rarely in the recess epithelium in the olfactory organ of lungfish of approximately 30 cm body length. However, it is unclear whether the distribution of V1R-expressing cells in the olfactory organ varies during development. In this study, we compared the expression of V1Rs in the olfactory organs between juveniles and adults of the African lungfish Protopterus aethiopicus and South American lungfish, Lepidosiren paradoxa. The density of V1R-expressing cells was higher in the lamellae than in the recesses in all specimens evaluated, and this pattern was more pronounced in juveniles than adults. In addition, the juveniles showed a higher density of V1R-expressing cells in the lamellae compared with the adults. Our results imply that differences in lifestyle between juveniles and adults are related to differences in the density of V1R-expressing cells in the lamellae of lungfish.

Expression patterns of the transcription factors Fezf1, Fezf2, and Bcl11b in the olfactory organs of turtle embryos.

Many tetrapod vertebrates have two distinct olfactory organs, the olfactory epithelium (OE) and vomeronasal organ (VNO). In turtles, the olfactory organ consists of two types of sensory epithelia, the upper chamber epithelium (UCE; corresponding to the OE) and the lower chamber epithelium (LCE; corresponding to the VNO). In many turtle species, the UCE contains ciliated olfactory receptor cells (ORCs) and the LCE contains microvillous ORCs. To date, several transcription factors involved in the development of the OE and VNO have been identified in mammals. Fez family zinc-finger protein 1 and 2 (Fezf1 and 2) are expressed in the OE and VNO, respectively, of mouse embryos, and are involved in the development and maintenance of ORCs. B-cell lymphoma/leukemia 11B (Bcl11b) is expressed in the mouse embryo OE except the dorsomedial parts of the nasal cavity, and regulates the expression of odorant receptors in the ORCs. In this study, we examined the expression of Fezf1, Fezf2, and Bcl11b in the olfactory organs of embryos in three turtle species, Pelodiscus sinensis, Trachemys scripta elegans, and Centrochelys sulcata, to evaluate their involvement in the development of reptile olfactory organs. In all three turtle species, Bcl11b was expressed in the UCE, Fezf2 in the LCE, and Fezf1 in both the UCE and LCE. These results imply that the roles of the transcription factors Fezf1, Fezf2, and Bcl11b in olfactory organ development are conserved among mammals and turtles.