Mechanisms underlying pre- and postnatal development of the vomeronasal organ.

The vomeronasal organ (VNO) is sensory organ located in the ventral region of the nasal cavity in rodents. The VNO develops from the olfactory placode during the secondary invagination of olfactory pit. The embryonic vomeronasal structure appears as a neurogenic area where migratory neuronal populations like endocrine gonadotropin-releasing hormone-1 (GnRH-1) neurons form. Even though embryonic vomeronasal structures are conserved across most vertebrate species, many species including humans do not have a functional VNO after birth. The vomeronasal epithelium (VNE) of rodents is composed of two major types of vomeronasal sensory neurons (VSNs): (1) VSNs distributed in the apical VNE regions that express vomeronasal type-1 receptors (V1Rs) and the G protein subunit Gαi2, and (2) VSNs in the basal territories of the VNE that express vomeronasal type-2 receptors (V2Rs) and the G subunit Gαo. Recent studies identified a third subclass of Gαi2 and Gαo VSNs that express the formyl peptide receptor family. VSNs expressing V1Rs or V2Rs send their axons to distinct regions of the accessory olfactory bulb (AOB). Together, VNO and AOB form the accessory olfactory system (AOS), an olfactory subsystem that coordinates the social and sexual behaviors of many vertebrate species. In this review, we summarize our current understanding of cellular and molecular mechanisms that underlie VNO development. We also discuss open questions for study, which we suggest will further enhance our understanding of VNO morphogenesis at embryonic and postnatal stages.

Immunolocalization of VEGF/VEGFR system in human fetal vomeronasal organ during early development.

The vomeronasal system (VNS) is an accessory olfactory structure present in most mammals adhibited to the detection of specific chemosignals implied in social and reproductive behavior. The VNS comprises the vomeronasal organ (VNO), vomeronasal nerve and accessory olfactory bulb. VNO is characterized by a neuroepithelium constituted by bipolar neurons and supporting and stem/progenitor cells. In humans, VNO is present during fetal life and is supposed to possess chemoreceptor activity and participate in gonadotropin-releasing hormone neuronal precursor migration toward the hypothalamus. Instead, the existence and functions of VNO in postnatal life is debated. Vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) have been demonstrated to play fundamental roles in various neurogenic events. However, there are no data regarding the localization and possible function of VEGF/VEGFRs in human fetal VNO. Therefore, this study was conceived to investigate the expression of VEGF/VEGFRs in human VNO in an early developmental period (9-12 weeks of gestation), when this organ appears well structured. Coronal sections of maxillofacial specimens were subjected to peroxidase-based immunohistochemistry for VEGF, VEGFR-1 and VEGFR-2. Double immunofluorescence for VEGF, VEGFR-1 or VEGFR-2 and the neuronal marker protein gene product 9.5 (PGP 9.5) was also performed. VEGF expression was evident in the entire VNO epithelium, with particularly strong reactivity in the middle layer. Strongly VEGF-immunostained cells with aspect similar to bipolar neurons and/or their presumable precursors were detected in the middle and basal layers. Cells detaching from the basal epithelial layer and detached cell groups in the surrounding lamina propria showed moderate/strong VEGF expression. The strongest VEGFR-1 and VEGFR-2 expression was detected in the apical epithelial layer. Cells with aspect similar to bipolar neurons and/or their presumable precursors located in the middle and basal layers and the detaching/detached cells displayed a VEGFR-1 and VEGFR-2 reactivity similar to that of VEGF. The basal epithelial layer exhibited stronger staining for VEGFRs than for VEGF. Cells with morphology and VEGF/VEGFR expression similar to those of the detaching/detached cells were also detected in the middle and basal VNO epithelial layers. Double immunofluorescence using anti-PGP 9.5 antibodies demonstrated that most of the VEGF/VEGFR-immunoreactive cells were neuronal cells. Collectively, our findings suggest that during early fetal development the VEGF/VEGFR system might be involved in the presumptive VNO chemoreceptor activity and neuronal precursor migration.

The transcription factor Tfap2e/AP-2ε plays a pivotal role in maintaining the identity of basal vomeronasal sensory neurons.

The identity of individual neuronal cell types is defined and maintained by the expression of specific combinations of transcriptional regulators that control cell type-specific genetic programs. The epithelium of the vomeronasal organ of mice contains two major types of vomeronasal sensory neurons (VSNs): 1) the apical VSNs which express vomeronasal 1 receptors (V1r) and the G-protein subunit Gαi2 and; 2) the basal VSNs which express vomeronasal 2 receptors (V2r) and the G-protein subunit Gαo. Both cell types originate from a common pool of progenitors and eventually acquire apical or basal identity through largely unknown mechanisms. The transcription factor AP-2ε, encoded by the Tfap2e gene, plays a role in controlling the development of GABAergic interneurons in the main and accessory olfactory bulb (AOB), moreover AP-2ε has been previously described to be expressed in the basal VSNs. Here we show that AP-2ε is expressed in post-mitotic VSNs after they commit to the basal differentiation program. Loss of AP-2ε function resulted in reduced number of basal VSNs and in an increased number of neurons expressing markers of the apical lineage. Our work suggests that AP-2ε, which is expressed in late phases of differentiation, is not needed to initiate the apical-basal differentiation dichotomy but for maintaining the basal VSNs' identity. In AP-2ε mutants we observed a large number of cells that entered the basal program can express apical genes, our data suggest that differentiated VSNs of mice retain a notable level of plasticity.

Trpm5 expression in the olfactory epithelium.

The Ca2+-activated monovalent cation channel Trpm5 is a key element in chemotransduction of taste receptor cells of the tongue, but the extent to which Trpm5 channels are expressed in olfactory sensory neurons (OSNs) of the main olfactory epithelium (MOE) of adult mice as part of a specific pheromonal detection system is debated. Here, we used a novel Trpm5-IRES-Cre knockin strain to drive Cre recombinase expression, employed previously validated Trpm5 antibodies, performed in situ hybridization experiments to localize Trpm5 RNA, and searched extensively for Trpm5 splice variants in genetically-labeled, Trpm5-expressing MOE cells. In contrast to previous reports, we find no evidence for the existence in adult mouse OSNs of the classical Trpm5 channel known from taste cells. We show that Trpm5-expressing adult OSNs express a novel Trpm5 splice variant, Trpm5-9, that is unlikely to form a functional cation channel by itself. We also demonstrate that Trpm5 is transiently expressed in a subpopulation of mature OSNs in the embryonic olfactory epithelium, indicating that Trpm5 channels could play a specific role in utero during a narrow developmental time window. Ca2+ imaging with GCaMP3 under the control of the Trpm5-IRES-Cre allele using a newly developed MOE wholemount preparation of the adult olfactory epithelium reveals that Trpm5-GCaMP3 OSNs comprise a heterogeneous group of sensory neurons many of which can detect general odorants. Together, these studies are essential for understanding the role of transient receptor potential channels in mammalian olfaction.

Cell migration in the developing rodent olfactory system.

The components of the nervous system are assembled in development by the process of cell migration. Although the principles of cell migration are conserved throughout the brain, different subsystems may predominantly utilize specific migratory mechanisms, or may display unusual features during migration. Examining these subsystems offers not only the potential for insights into the development of the system, but may also help in understanding disorders arising from aberrant cell migration. The olfactory system is an ancient sensory circuit that is essential for the survival and reproduction of a species. The organization of this circuit displays many evolutionarily conserved features in vertebrates, including molecular mechanisms and complex migratory pathways. In this review, we describe the elaborate migrations that populate each component of the olfactory system in rodents and compare them with those described in the well-studied neocortex. Understanding how the components of the olfactory system are assembled will not only shed light on the etiology of olfactory and sexual disorders, but will also offer insights into how conserved migratory mechanisms may have shaped the evolution of the brain.

Human Neural Cells Transiently Express Reelin during Olfactory Placode Development.

Reelin, an extracellular glycoprotein is essential for migration and correct positioning of neurons during development. Since the olfactory system is known as a source of various migrating neuronal cells, we studied Reelin expression in the two chemosensory olfactory systems, main and accessory, during early developmental stages of human foetuses/embryos from Carnegie Stage (CS) 15 to gestational week (GW) 14. From CS 15 to CS 18, but not at later stages, a transient expression of Reelin was detected first in the presumptive olfactory and then in the presumptive vomeronasal epithelium. During the same period, Reelin-positive cells detach from the olfactory/vomeronasal epithelium and migrate through the mesenchyme beneath the telencephalon. Dab 1, an adaptor protein of the Reelin pathway, was simultaneously expressed in the migratory mass from CS16 to CS17 and, at later stages, in the presumptive olfactory ensheathing cells. Possible involvements of Reelin and Dab 1 in the peripheral migrating stream are discussed.

Human vomeronasal epithelium development: An immunohistochemical overview.

The vomeronasal organ (VNO) is the receptor structure of the vomeronasal system (VNS) in vertebrates. It is found bilaterally in the submucosa of the inferior part of the nasal septum. There are ongoing controversies regarding the functionality of this organ in humans. In this study we propose the immunohistochemical evaluation of changes in components of the human vomeronasal epithelium during foetal development. We used 45 foetuses of different age, which were included in three age groups. After VNO identification immunohistochemical reactions were performed using primary antibodies against the following: neuron specific enolase, calretinin, neurofilament, chromogranin, synaptophysin, cytokeratin 7, pan-cytokeratin and S100 protein. Digital slides were obtained and following colorimetric segmentation, surface area measurements were performed. The VNO was found in less than half of the studied specimens (42.2%). Neuron specific enolase and calretinin immunoexpression showed a decreasing trend with foetal age, while the other neural/neuroendocrine markers were negative in all specimens. Cytokeratin 7 expression increased with age, while Pan-Ctk had no significant variations. S100 protein immunoexpression also decreased around the VNO. The results of the present work uphold the theory of regression of the neuroepithelium that is present during initial stages of foetal development.

Immunohistochemical and lectin histochemical studies on the developing olfactory organs of fetal camel.

Little is known about the development of the olfactory organs of camel. In this study, prenatal development and neuronal differentiation of the vomeronasal organ (VNO) and the olfactory epithelium (OE) of the one-humped camel were studied by immunohistochemistry and lectin histochemistry. A neuronal marker, protein gene product (PGP) 9.5, but not a marker of fully differentiated olfactory receptor cells, olfactory marker protein, intensely labeled the olfactory receptor cells of the VNO and OE at 395 mm, 510 mm, and 530 mm fetal ages, indicating that the olfactory receptor cells are differentiated, but not fully matured both in the VNO and the OE. In 187 mm and 190 mm fetuses, PGP 9.5 yielded faint immunoreactive signals in the VNO, but not in the OE, although the presence of olfactory receptor cells were demonstrated in both tissues by intense WGA and LEL stainings. We conclude that the camel VNO and OE bear differentiated, but still immature receptor cells; in addition, the onset of neuronal differentiation seems to be somewhat earlier in the VNO than in the OE till half of the prenatal life.

Transient appearance of the epithelial invagination in the olfactory pit of chick embryos.

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.

Progressive effects of N-myc deficiency on proliferation, neurogenesis, and morphogenesis in the olfactory epithelium.

N-myc belongs to the myc proto-oncogene family, which is involved in numerous cellular processes such as proliferation, growth, apoptosis, and differentiation. Conditional deletion of N-myc in the mouse nervous system disrupted brain development, indicating that N-myc plays an essential role during neural development. How the development of the olfactory epithelium and neurogenesis within are affected by the loss of N-myc has, however, not been determined. To address these issues, we examined an N-myc(Foxg1Cre) conditional mouse line, in which N-myc is depleted in the olfactory epithelium. First changes in N-myc mutants were detected at E11.5, with reduced proliferation and neurogenesis in a slightly smaller olfactory epithelium. The phenotype was more pronounced at E13.5, with a complete lack of Hes5-positive progenitor cells, decreased proliferation, and neurogenesis. In addition, stereological analyses revealed reduced cell size of post-mitotic neurons in the olfactory epithelium, which contributed to a smaller olfactory pit. Furthermore, we observed diminished proliferation and neurogenesis also in the vomeronasal organ, which likewise was reduced in size. In addition, the generation of gonadotropin-releasing hormone neurons was severely reduced in N-myc mutants. Thus, diminished neurogenesis and proliferation in combination with smaller neurons might explain the morphological defects in the N-myc depleted olfactory structures. Moreover, our results suggest an important role for N-myc in regulating ongoing neurogenesis, in part by maintaining the Hes5-positive progenitor pool. In summary, our results provide evidence that N-myc deficiency in the olfactory epithelium progressively diminishes proliferation and neurogenesis with negative consequences at structural and cellular levels.

Morphological characterization of the nasopalatine region in human fetuses and its association to pathologies.

UNLABELLED: The nasopalatine region is composed of structures such as the vomeronasal organ and nasopalatine duct. The nasopalatine duct may provide the communication of the mouth to the nasal cavity in human fetuses and can be obliterated in an adult human. Knowledge on the development of the nasopalatine region and nasopalatine duct in humans is necessary for understanding the morphology and etiopathogenesis of lesions that occur in this region. OBJECTIVE: The aim of the present study was to describe the morphological aspects of the nasopalatine region in human fetuses and correlate these aspects with the development of pathologies in this region. MATERIAL AND METHODS: Five human fetuses with no facial or palatine abnormalities were used for the acquisition of specimens from the nasopalatine region. After demineralization, the specimens were histologically processed. Histological cuts were stained with methylene blue to orient the cutting plane and hematoxylin-eosin for the descriptive histological analysis. RESULTS: The age of the fetuses was 8.00, 8.25, 9.00 and 9.25 weeks, and it was not possible to determine the age in the last one. The incisive canal was observed in all specimens as an opening delimited laterally by the periosteum and connecting oral and nasal cavity. The nasopalatine duct is an epithelial structure with the greatest morphological variation, with either unilateral or bilateral occurrence and total patent, partial patent and islet forms. The vomeronasal organ is a bilateral epithelized structure located alongside the nasal septum above the incisive canal in all the fetuses. CONCLUSIONS: The incisive canal, nasopalatine duct and vomeronasal organ are distinct anatomic structures. The development of nasopalatine duct cysts may occur in all forms of the nasopalatine duct.

Morphological characterization of the nasopalatine region in human fetuses and its association to pathologies

The nasopalatine region is composed of structures such as the vomeronasal organ and nasopalatine duct. The nasopalatine duct may provide the communication of the mouth to the nasal cavity in human fetuses and can be obliterated in an adult human. Knowledge on the development of the nasopalatine region and nasopalatine duct in humans is necessary for understanding the morphology and etiopathogenesis of lesions that occur in this region. Objective The aim of the present study was to describe the morphological aspects of the nasopalatine region in human fetuses and correlate these aspects with the development of pathologies in this region. Material and Methods Five human fetuses with no facial or palatine abnormalities were used for the acquisition of specimens from the nasopalatine region. After demineralization, the specimens were histologically processed. Histological cuts were stained with methylene blue to orient the cutting plane and hematoxylin-eosin for the descriptive histological analysis. Results The age of the fetuses was 8.00, 8.25, 9.00 and 9.25 weeks, and it was not possible to determine the age in the last one. The incisive canal was observed in all specimens as an opening delimited laterally by the periosteum and connecting oral and nasal cavity. The nasopalatine duct is an epithelial structure with the greatest morphological variation, with either unilateral or bilateral occurrence and total patent, partial patent and islet forms. The vomeronasal organ is a bilateral epithelized structure located alongside the nasal septum above the incisive canal in all the fetuses. Conclusions The incisive canal, nasopalatine duct and vomeronasal organ are distinct anatomic structures. The development of nasopalatine duct cysts may occur in all forms of the nasopalatine duct. .

A critical role for the cholesterol-associated proteolipids PLP and M6B in myelination of the central nervous system.

The formation of central nervous system myelin by oligodendrocytes requires sterol synthesis and is associated with a significant enrichment of cholesterol in the myelin membrane. However, it is unknown how oligodendrocytes concentrate cholesterol above the level found in nonmyelin membranes. Here, we demonstrate a critical role for proteolipids in cholesterol accumulation. Mice lacking the most abundant myelin protein, proteolipid protein (PLP), are fully myelinated, but PLP-deficient myelin exhibits a reduced cholesterol content. We therefore hypothesized that "high cholesterol" is not essential in the myelin sheath itself but is required for an earlier step of myelin biogenesis that is fully compensated for in the absence of PLP. We also found that a PLP-homolog, glycoprotein M6B, is a myelin component of low abundance. By targeting the Gpm6b-gene and crossbreeding, we found that single-mutant mice lacking either PLP or M6B are fully myelinated, while double mutants remain severely hypomyelinated, with enhanced neurodegeneration and premature death. As both PLP and M6B bind membrane cholesterol and associate with the same cholesterol-rich oligodendroglial membrane microdomains, we suggest a model in which proteolipids facilitate myelination by sequestering cholesterol. While either proteolipid can maintain a threshold level of cholesterol in the secretory pathway that allows myelin biogenesis, lack of both proteolipids results in a severe molecular imbalance of prospective myelin membrane. However, M6B is not efficiently sorted into mature myelin, in which it is 200-fold less abundant than PLP. Thus, only PLP contributes to the high cholesterol content of myelin by association and co-transport.

Dual origins of the mammalian accessory olfactory bulb revealed by an evolutionarily conserved migratory stream.

The accessory olfactory bulb (AOB) is a critical olfactory structure that has been implicated in mediating social behavior. It receives input from the vomeronasal organ and projects to targets in the amygdaloid complex. Its anterior and posterior components (aAOB and pAOB) display molecular, connectional and functional segregation in processing reproductive and defensive and aggressive behaviors, respectively. We observed a dichotomy in the development of the projection neurons of the aAOB and pAOB in mice. We found that they had distinct sites of origin and that different regulatory molecules were required for their specification and migration. aAOB neurons arose locally in the rostral telencephalon, similar to main olfactory bulb neurons. In contrast, pAOB neurons arose caudally, from the neuroepithelium of the diencephalic-telencephalic boundary, from which they migrated rostrally to reach their destination. This unusual origin and migration is conserved in Xenopus, providing an insight into the origin of a key component of this system in evolution.

The wiring of Grueneberg ganglion axons is dependent on neuropilin 1.

The Grueneberg ganglion is a specialized olfactory sensor. In mice, its activation induces freezing behavior. The topographical map corresponding to the central projections of its sensory axons is poorly defined, as well as the guidance molecules involved in its establishment. We took a transgenic approach to label exclusively Grueneberg sensory neurons and their axonal projections. We observed that a stereotyped convergence map in a series of coalescent neuropil-rich structures is already present at birth. These structures are part of a peculiar and complex neuronal circuit, composed of a chain of glomeruli organized in a necklace pattern that entirely surrounds the trunk of the olfactory bulb. We found that the necklace chain is composed of two different sets of glomeruli: one exclusively innervated by Grueneberg ganglion neurons, the other by axonal inputs from the main olfactory neuroepithelium. Combining the transgenic Grueneberg reporter mouse with a conditional null genetic approach, we then show that the axonal wiring of Grueneberg neurons is dependent on neuropilin 1 expression. Neuropilin 1-deficient Grueneberg axonal projections lose their strict and characteristic avoidance of vomeronasal glomeruli, glomeruli that are innervated by secondary neurons expressing the repulsive guidance cue and main neuropilin 1 ligand Sema3a. Taken together, our observations represent a first step in the understanding of the circuitry and the coding strategy used by the Grueneberg system.

Lhx2-dependent specification of olfactory sensory neurons is required for successful integration of olfactory, vomeronasal, and GnRH neurons.

Inactivation of the LIM-homeodomain 2 gene (Lhx2) results in a severe defect in specification of olfactory sensory neurons (OSNs). However, the ramifications of lack of Lhx2-dependent OSN specification for formation of the primary olfactory pathway have not been addressed, since mutant mice die in utero. We have analyzed prenatal and postnatal consequences of conditionally inactivating Lhx2 selectively in OSNs. A cell-autonomous effect is that OSN axons cannot innervate their target, the olfactory bulb. Moreover, the lack of Lhx2 in OSNs causes unpredicted, non-cell-autonomous phenotypes. First, the olfactory bulb shows pronounced hypoplasia in adults, and the data suggest that innervation by correctly specified OSNs is necessary for adult bulb size and organization. Second, absence of an olfactory nerve in the conditional mutant reveals that the vomeronasal nerve is dependent on olfactory nerve formation. Third, the lack of a proper vomeronasal nerve prevents migration of gonadotropin-releasing hormone (GnRH) cells the whole distance to their final positions in the hypothalamus during embryo development. As adults, the conditional mutants do not pass puberty, and these findings support the view of an exclusive nasal origin of GnRH neurons in the mouse. Thus, Lhx2 in OSNs is required for functional development of three separate systems.

Developmental changes affecting lectin binding in the vomeronasal organ of domestic pigs, Sus scrofa.

This study investigated the developmental changes of glycoconjugate patterns in the porcine vomeronasal organs (VNOs) and associated glands (Jacobson's glands) from prenatal (9 weeks of gestation) and postnatal (2 days after birth) to the sexually mature stage (6 months old). The VNO of pigs (Sus scrofa) was examined using the following: Dolichos biflorus agglutinin (DBA), Bandeiraea simplicifolia agglutinin isolectin B4 (BSI-B4), Triticum vulgaris agglutinin (WGA), Ulex europaeus agglutinin I (UEA-I), and soybean agglutinin (SBA). At the fetal stage, all lectins examined were detected mainly in the free border of the vomeronasal epithelium, but few (WGA and UEA-I) and or absent in the VNO cell bodies. At the postnatal and sexually mature stages, the reactivity of some lectins, including WGA, UEA-I, DBA and SBA, were shown to increase in the VNO sensory epithelium as well as the free border. The increased reactivity of lectins as development progressed was also observed in Jacobson's gland acini. These findings suggest that binding sites of lectins, including those of WGA, UEA-I, DBA, and SBA, increase during development from fetal to postnatal growth, possibly contributing to the increased ability of chemoreception in the pig.

The vomeronasal organ of New World monkeys (platyrrhini).

Although all platyrrhine primates possess a vomeronasal organ (VNO), few species have been studied in detail. Here, we revisit the microanatomy of the VNO and related features in serially sectioned samples from 41 platyrrhine cadavers (14 species) of mixed age. Procedures to identify terminally differentiated vomeronasal sensory neurons (VSNs) via immunolabeling of olfactory marker protein (OMP) were used on selected specimens. The VNO varies from an elongated epithelial tube (e.g., Ateles fusciceps) to a dorsoventrally expanded sac (e.g., Saguinus spp.). The cartilage that surrounds the VNO is J-shaped or U-shaped in most species, and articulates with a groove on the bony palate. Preliminary results indicate a significant correlation between the length of this groove and length of the VNO neuroepithelium, indicating this feature may serve as a skeletal correlate. The VNO neuroepithelium could be identified in all adult primates except Alouatta, in which poor preservation prevented determination. The VNO of Ateles, described in detail for the first time, had several rows of VSNs and nerves in the surrounding lamina propria. Patterns of OMP-reactivity in the VNO of perinatal platyrrhines indicate that few or no terminally differentiated VSNs are present at birth, thus supporting the hypothesis that some platyrrhines may have delayed maturation of the VNO. From a functional perspective, all platyrrhines studied possess structures required for chemoreception (VSNs, vomeronasal nerves). However, some microanatomical findings, such as limited reactivity to OMP in some species, indicate that some lineages of New World monkeys may have a reduced or vestigial vomeronasal system.

Dysregulation of Semaphorin7A/ß1-integrin signaling leads to defective GnRH-1 cell migration, abnormal gonadal development and altered fertility.

Reproduction in mammals is dependent on the function of specific neurons that secrete gonadotropin-releasing hormone-1 (GnRH-1). These neurons originate prenatally in the nasal placode and migrate into the forebrain along the olfactory-vomeronasal nerves. Alterations in this migratory process lead to defective GnRH-1 secretion, resulting in heterogeneous genetic disorders such as idiopathic hypogonadotropic hypogonadism (IHH), and other reproductive diseases characterized by the reduction or failure of sexual competence. Combining mouse genetics with in vitro models, we demonstrate that Semaphorin 7A (Sema7A) is essential for the development of the GnRH-1 neuronal system. Loss of Sema7A signaling alters the migration of GnRH-1 neurons, resulting in significantly reduced numbers of these neurons in the adult brain as well as in reduced gonadal size and subfertility. We also show that GnRH-1 cells differentially express the Sema7 receptors ß1-integrin and Plexin C1 as a function of their migratory stage, whereas the ligand is robustly expressed along developing olfactory/vomeronasal fibers. Disruption of Sema7A function in vitro inhibits ß1-integrin-mediated migration. Analysis of Plexin C1(-/-) mice did not reveal any difference in the migratory process of GnRH-1 neurons, indicating that Sema7A mainly signals through ß1-integrin to regulate GnRH-1 cell motility. In conclusion, we have identified Sema7A as a gene implicated in the normal development of the GnRH-1 system in mice and as a genetic marker for the elucidation of some forms of GnRH-1 deficiency in humans.

Epithelial membrane transporters expression in the developing to adult mouse vomeronasal organ and olfactory mucosa.

To contribute clarifying mechanisms operating in nose chemosensory epithelia and their developmental patterns, we analyzed the expression of different epithelial membrane transporters as well as the Clara cell secretory protein, CC26 in the olfactory, vomeronasal organ (VNO), and respiratory epithelia of embryonic (E13-E19) and postnatal (P1-P60) mice by means of immunohistochemistry and reverse transcriptase-polymerase chain reaction. Results showed that CC26, cAMP-activated chloride channel (CFTR), and the water channel protein aquaporin 2, 3, 4, and 5 (AQP2, AQP3, AQP4, and AQP5) are expressed in developing to adult chemosensory epithelia with differential timing; moreover, their pattern of expression is not identical in VNO and olfactory epithelia as well as the corresponding associated glands; co-localization experiments using olfactory marker protein showed that CFTR, CC26, and AQP4 are not expressed in olfactory neurones. CFTR is expressed in sustentacular cells of the VNO and olfactory epithelium as well as blood vessels of the underlying mucosa, and VNO (but not Bowman's) glands; a similar pattern (excluding blood vessels) is present for AQP2; AQP4 is found in the two chemosensory epithelia and in Bowman's glands. AQP3 is expressed in the olfactory epithelium and the associated Bowman's glands, but not in the VNO chemosensory epithelium and glands. AQP5 is expressed in the olfactory epithelium and both Bowman's and VNO glands. These results indicate that water/ions handling as well as antioxidant mechanisms operating at the surface and/or inside the nose chemosensory epithelia start developing in utero and are maintained up to sexual maturity.