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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.

Generally, the olfactory organ of vertebrates consists of the olfactory epithelium (OE) and the vomeronasal organ (VNO). The OE contains ciliated olfactory receptor neurons (ORNs), while the VNO contains microvillous ORNs. The ORNs in the OE express odorant receptors (ORs), while those in the VNO express type 1 and type 2 vomeronasal receptors (V1Rs and V2Rs). In turtles, the olfactory organ consists of the upper (UCE) and lower chamber epithelia (LCE). The UCE contains ciliated ORNs, while the LCE contains microvillous ORNs. Here we investigated the distribution of cells expressing vomeronasal receptors in the olfactory organ of turtles. The turtle vomeronasal receptors were encoded by two V1R genes and two V2R genes. Among them, V2R1 and V2R26 were mainly expressed in the LCE, while V1R3 was expressed both in the UCE and LCE. Notably, vomeronasal receptors were expressed by a limited number of ORNs, which was confirmed by the expression of the gene encoding TRPC2, an ion channel involved in the signal transduction of vomeronasal receptors. Furthermore, expression of ORs by the majority of ORNs was suggested by the expression of the gene encoding CNGA2, an ion channel involved in the signal transduction of ORs. Thus, olfaction of turtle seems to be mediated mainly by the ORs rather than the vomeronasal receptors. More importantly, the relationship between the fine structure of ORNs and the expression of olfactory receptors are not conserved among turtles and other vertebrates.

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.

The nasal cavity of turtles is composed of the upper and lower chambers, lined by the upper and lower chamber epithelia, respectively. In many turtles including the Reeve's turtle Mauremys reevesii, the upper chamber epithelium contains ciliated olfactory receptor neurons (ORNs) and the lower chamber epithelium contains microvillous ORNs. However, in the olfactory organ of the Chinese soft-shelled turtle Pelodiscus sinensis, both the upper and lower chamber epithelia contain ciliated ORNs. In the present study, we immunohistochemically examined the developmental process of olfactory organs in soft-shelled turtle and the Reeve's turtle to clarify the developmental origins of the lower chamber epithelium in these turtles. Obtained data indicate that olfactory organs of these turtles have identical origin and follow similar process of development, suggesting that, in the lower chamber epithelium of the nasal cavity, ciliated ORNs differentiate in soft-shelled turtle whereas microvillous ORNs differentiate in the Reeve's turtle.

The olfactory organ of turtle consists of the upper chamber epithelium (UCE) and the lower chamber epithelium (LCE), detecting air-borne odorants and water-borne odorants, respectively. In this study, we investigated the number of olfactory receptor neurons (ORNs) in the UCE and LCE of soft-shelled turtle in order to find their possible differences among terrestrial, semi-aquatic and highly-aquatic turtles. The number of ORNs in the soft-shelled turtle was higher in the LCE than in the UCE, suggesting its close relationship to the environment the turtle lives. In addition, relative abundance of the ORNs in the LCE to the UCE varied in accordance with the size of individuals, although its functional significance remains elusive.

In general, the nasal cavity of turtles is divided into two chambers: the upper chamber, lined with the olfactory epithelium containing ciliated olfactory receptor cells, and the lower chamber, lined with the vomeronasal epithelium containing microvillous receptor cells. In the nasal cavity of soft-shelled turtles, however, differences between the upper and lower chamber epithelia are unclear due to the presence of ciliated receptor cells in both epithelia. In the olfactory organ of vertebrates, the surface of sensory epithelium is covered with secretory products of associated glands and supporting cells, playing important roles in the olfaction by dissolving odorants and transporting them to the olfactory receptors. Here, the associated glands and supporting cells in the olfactory organ of soft-shelled turtles were analyzed histochemically and ultrastructurally. The upper chamber epithelium possessed associated glands, constituted by cells containing serous secretory granules; whereas, the lower chamber epithelium did not. In the upper chamber epithelium, secretory granules filled the supranuclear region of supporting cells, while most of the granules were distributed near the free border of supporting cells in the lower chamber epithelium. The secretory granules in the supporting cells of both epithelia were seromucous, but alcian blue stained them differently from each other. In addition, distinct expression of carbohydrates was suggested by the differences in lectin binding. These data indicate the quantitative and qualitative differences in the secretory properties between the upper and lower chamber epithelia, suggesting their distinct roles in the olfaction.

In turtles, the epithelia lining the upper and lower chambers of the nasal cavity project axons to the ventral and dorsal parts of the olfactory bulbs, respectively. In a semi-aquatic soft-shelled turtle, Pelodiscus sinensis, more than 1,000 odorant receptor genes have been found, but it is not known where they are expressed. In this study, we aimed to clarify the distribution of cells expressing these genes in the olfactory organs of soft-shelled turtles. Immunoreactions for the Gαolf, the α subunit of G protein coupled to the odorant receptors, were detected on the surface of epithelia lining both the upper and lower chambers of the nasal cavity. The receptor cells in the epithelium of both chambers possessed cilia on the tip of their dendrites, whereas microvillous, non-ciliated, receptor cells were not found. These data suggest that the odorant receptor genes are expressed by the ciliated receptor cells in the upper and lower chamber epithelia. Precise location of the vomeronasal epithelium is not known at present.

In this study, immunohistochemical analysis has been performed using neuronal markers (GAP43, NCAM and PGP 9.5) to characterize the epithelial invagination in the medial wall of the olfactory pit in the chick embryos. At stages 26–27, the epithelial invagination was primarily composed of characteristic round-shaped cells, which were negative for neuronal markers. These cells were also found in the medial wall of the olfactory pit at stage 24, whereas the epithelial invagination was not observed at any stages other than stages 26–27. The possible relationship between the round-shaped cells and the migratory cells is discussed.

In this study, the olfactory system of a semi-aquatic turtle, the snapping turtle, has been morphologically investigated by electron microscopy, immunohistochemistry, and lectin histochemistry. The nasal cavity of snapping turtle was divided into the upper and lower chambers, lined by the sensory epithelium containing ciliated and non-ciliated olfactory receptor neurons, respectively. Each neuron expressed both Gαolf, the α-subunit of G-proteins coupling to the odorant receptors, and Gαo, the α-subunit of G-proteins coupling to the type 2 vomeronasal receptors. The axons originating from the upper chamber epithelium projected to the ventral part of the olfactory bulb, while those from the lower chamber epithelium to the dorsal part of the olfactory bulb. Despite the identical expression of G-protein α-subunits in the olfactory receptor neurons, these two projections were clearly distinguished from each other by the differential expression of glycoconjugates. In conclusion, these data indicate the presence of two types of olfactory systems in the snapping turtle. Topographic arrangement of the upper and lower chambers and lack of the associated glands in the lower chamber epithelium suggest their possible involvement in the detection of odorants: upper chamber epithelium in the air and the lower chamber epithelium in the water.

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.

Most vertebrates have two olfactory organs, the olfactory epithelium (OE) and the vomeronasal organ. African clawed frog, Xenopus laevis, which spends their entire life in water, have three types of olfactory sensory epithelia: the OE, the middle chamber epithelium (MCE) and the vomeronasal epithelium (VNE). The axons from these epithelia project to the dorsal part of the main olfactory bulb (d-MOB), the ventral part of the MOB (v-MOB) and the accessory olfactory bulb, respectively. In the MCE, which is thought to function in water, two types of receptor neurons (RNs) are intermingled and express one of two types of G-proteins, Golf and Go, respectively. However, axonal projections from these RNs to the v-MOB are not fully understood. In this study, we examined the expression of G-proteins by immunohistochemistry to reveal the projection pattern of olfactory RNs of Xenopus laevis, especially those in the MCE. The somata of Golf- and Go-positive RNs were separately situated in the upper and lower layers of the MCE. The former were equipped with cilia and the latter with microvilli on their apical surface. These RNs are suggested to project to the rostromedial and the caudolateral regions of the v-MOB, respectively. Such segregation patterns observed in the MCE and v-MOB are also present in the OE and olfactory bulbs of most bony fish. Thus, Xenopus laevis is a very interesting model to understand the evolution of vertebrate olfactory systems because they have a primitive, fish-type olfactory system in addition to the mammalian-type olfactory system.

In this study, we examined the olfactory epithelium (OE) of the barfin flounder by transmission electron microscopy. As in the case of the ordinary teleost, the OE of the barfin flounder had 3 types of olfactory receptor cells (ciliated olfactory receptor cell, microvillous olfactory receptor cell and crypt cell), 3 types of supporting cells (ciliated, microvillous and crypt supporting cells) and basal cells. Each type of OE cells in the barfin flounder had similar ultrastructure to that of the ordinary teleost. Crypt cell is the third type of olfactory receptor cell unique to fish, whose function is unclear. The barfin flounder may be a suitable material to study crypt cells because it has relatively abundant crypt cells in the OE.

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.