Matrix Gla protein deficiency impairs nasal septum growth, causing midface hypoplasia.

Genetic and environmental factors may lead to abnormal growth of the orofacial skeleton, affecting the overall structure of the face. In this study, we investigated the craniofacial abnormalities in a mouse model for Keutel syndrome, a rare genetic disease caused by loss-of-function mutations in the matrix Gla protein (MGP) gene. Keutel syndrome patients show diffuse ectopic calcification of cartilaginous tissues and impaired midface development. Our comparative cephalometric analyses of micro-computed tomography images revealed a severe midface hypoplasia in Mgp-/- mice. In vivo reporter studies demonstrated that the Mgp promoter is highly active at the cranial sutures, cranial base synchondroses, and nasal septum. Interestingly, the cranial sutures of the mutant mice showed normal anatomical features. Although we observed a mild increase in mineralization of the spheno-occipital synchondrosis, it did not reduce the relative length of the cranial base in comparison with total skull length. Contrary to this, we found the nasal septum to be abnormally mineralized and shortened in Mgp-/- mice. Transgenic restoration of Mgp expression in chondrocytes fully corrected the craniofacial anomalies caused by MGP deficiency, suggesting a local role for MGP in the developing nasal septum. Although there was no up-regulation of markers for hypertrophic chondrocytes, a TUNEL assay showed a marked increase in apoptotic chondrocytes in the calcified nasal septum. Transmission electron microscopy confirmed unusual mineral deposits in the septal extracellular matrix of the mutant mice. Of note, the systemic reduction of the inorganic phosphate level was sufficient to prevent abnormal mineralization of the nasal septum in Mgp-/-;Hyp compound mutants. Our work provides evidence that modulation of local and systemic factors regulating extracellular matrix mineralization can be possible therapeutic strategies to prevent ectopic cartilage calcification and some forms of congenital craniofacial anomalies in humans.

Quantitation of nasal development in the early prenatal period using geometric morphometrics and MRI: a new insight into the critical period of Binder phenotype.

OBJECTIVES: Disturbance of the development of the nasal septum in the early prenatal period causes congenital facial anomalies characterized by a flat nose and defects of the anterior nasal spine (ANS), such as Binder phenotype. The present research aimed to assess the development of the nasal septum and the ANS with growth in the early prenatal period. METHODS: Magnetic resonance images were obtained from 56 specimens. Mid-sagittal images were analyzed by using geometric morphometrics for the development of the nasal septum, and angle analysis was performed for the development of the ANS. Additionally, we calculated and visualized the ontogenetic allometry of the nasal septum. RESULTS: Our results showed that the nasal septum changed shape in the anteroposterior direction in smaller specimens, while it maintained an almost isometric shape in larger specimens. Furthermore, mathematical evidence revealed that the maturation periods of the shapes of the ANS and the nasal septum were around 12 and 14 weeks of gestation, respectively. CONCLUSION: The anteroposterior development of the nasal septum is specific until 14 weeks of gestation, and it is important for nasal protrusion and the development of the ANS. Therefore, the disturbance of such development could induce low nasal deformity, including Binder phenotype. © 2017 John Wiley & Sons, Ltd.

[Immunohistochemical study of human fetal vomerona structures the nasal septum, by applying neuron-specific beta3-tubulin antibodies].

The functioning of Jacobson's or vomeronasal organ (VNO) in man is the subject-matter of discussion today. It is generally taken that VNO as an anatomic structure also remains in the adult; however, its receptor apparatus still degenerates in the fetal stage of ontogenesis. Nevertheless, the data available in the literature on the time and specific features of degenerative changes in the human fetal VNO are conflicting and ambiguous. The authors examined the human fetal nasal septum from the 8th week of development to birth, by applying the traditional histological procedures and neuron-specific beta3-tubulin antibodies. An immunohistochemical study could first show the receptor apparatus of the human fetal VNO at weeks 8-26 of development. The immunohistochemical study on a series of sections could reveal the regularities of spatial receptor distribution depending on the time of fetal development. In addition, the developed human fetal vomeronasal nerve and ganglion at weeks 8-26 were described, in human fetuses at weeks 8-26. The neuron-specific marker test has shown the nerve fibers departing directly from the VNO wall, which is inconsistent with the data available in the literature on vomeronasal nerve degeneration in this sign just after the 18th week of development.

Maternal Folic Acid Deficiency Is Associated to Developing Nasal and Palate Malformations in Mice.

Craniofacial development requires extremely fine-tuned developmental coordination of multiple specialized tissues. It has been evidenced that a folate deficiency (vitamin B9), or its synthetic form, folic acid (FA), in maternal diet could trigger multiple craniofacial malformations as oral clefts, tongue, or mandible abnormalities. In this study, a folic acid-deficient (FAD) diet was administered to eight-week-old C57/BL/6J female mouse for 2-16 weeks. The head symmetry, palate and nasal region were studied in 24 control and 260 experimental fetuses. Our results showed a significant reduction in the mean number of fetuses per litter according to maternal weeks on FAD diet (p < 0.01). Fetuses were affected by cleft palate (3.8%) as well as other severe congenital abnormalities, for the first time related to maternal FAD diet, as head asymmetries (4.6%), high arched palate (3.5%), nasal septum malformed (7.3%), nasopharynx duct shape (15%), and cilia and epithelium abnormalities (11.2% and 5.8%). Dysmorphologies of the nasal region were the most frequent, appearing at just four weeks following a maternal FAD diet. This is the first time that nasal region development is experimentally related to this vitamin deficiency. In conclusion, our report offers novel discoveries about the importance of maternal folate intake on midface craniofacial development of the embryos. Moreover, the longer the deficit lasts, the more serious the consequent effects appear to be.

The acid-sensing ion channel 2 (ASIC2) of ciliated cells in the developing rat nasal septum.

The airway epithelium is exposed to an acidic environment in certain conditions. The acid-sensing ion channel 2 (ASIC2) belongs to the epithelial amiloride-sensitive sodium channel and degenerin (ENaC/DEG) family and is expressed on cilia of the respiratory epithelium. The aim of this study was to detect the expression of ASIC2 in the nasal septum in the embryonic stage of the rat. ASIC2 expression was not observed in the primary cilium but was found in some cilia on embryonic day 17 (E17). After E18, all cilia showed ASIC2 immunoreactivity. RT-PCR analysis revealed that ASIC2b, a subtype of ASIC2, was expressed in the nasal septum while ASIC2a was not. Quantitative Real-time RT-PCR studies indicated that the expression level of ASIC2 mRNA was highest on E21, just before birth. These results imply that ASIC2 plays little part in the development of the nasal septum epithelium. On the other hand, ASIC2, especially ASIC2b, may function for the survival and retention of ciliated cells of the nasal septum against dynamic changes in the pH environment at birth.

Amygdaloid nucleus: new afferent input from the vomeronasal organ.

Terminal degeneration stained by the Fink-Heimer technique was found in the medial and cortical amygdaloid nuclei in a discrete zone after lesions were inflicted in the accessory olfactory bulb but not after lesions were made in the main olfactory bulb in the rabbit. Since the accessory olfactory bulb receives the endings of the vomeronasal nerve, the mediocortical complex of the amygdala is the central projection area for the vomeronasal sensory organ. The vomeronasal organ is seen as having new potential significance in sexual and feeding behavior because the cortical amygdaloid nucleus projects to the anterior, medial hypothalamus and the ventromedial nucleus.

Tbx22 expressions during palatal development in fetuses with glucocorticoid-/alcohol-induced C57BL/6N cleft palates.

T-box transcription factor 22 (Tbx22) belongs to the T-box family of transcription factors and was originally found using an in silico approach to identify new genes in the human Xq12-Xq21 region. Mutations in Tbx22 have been reported in families with X-linked cleft palate and ankyloglossia, but the underlying pathogenetic mechanism remains unknown. The aim of this study was to evaluate the expression of Tbx22 messenger RNA (mRNA) during palatogenesis in glucocorticoid-/alcohol-induced cleft palate in a C57BL/6N mouse model. Palatal development was monitored by histomorphologic and immunohistochemical studies and by in situ hybridization. Thirty pregnant C57BL/6N mice at 8 weeks of age, weighing 20 to 25 g, were used in this study. In the experimental group, 12 mice were exposed to alcohol for 7 days before mating, and 12 mice in the control group were not exposed. Six mice in a sham group were exposed to neither alcohol nor glucocorticoids. A total of 18 fetuses with induced cleft palates each from 102 fetuses in the experimental group, 109 in the control group, and 58 in the sham group were used. In both the experimental and the control groups, glucocorticoids were injected subcutaneously on gestational days (GD) 9.5, 10.5, and 11.5, and each mouse was killed on GDs 10.5 to 15.5. Histomorphologic findings were studied using hematoxylin and eosin staining, and antibodies against proliferation cell nuclear antigen, matrix metallopeptidase 9, zinc finger protein 422 (Krox25) heat shock protein 70, and Tbx22 were used in immunohistochemical analysis. Mouse Tbx22 mRNA was identified, and its expression was analyzed during embryogenesis by polymerase chain reaction and in situ hybridization. Coronal sections of the cleft maxilla of the embryos with induced cleft palates had a gap between the palatal shelves, where 2 palatal shelves had fused as in normal development but failed to meet and fuse to each other. By in situ hybridization, Tbx22 mRNA was found to be expressed in distinct areas of the head, such as the mesenchyme of the inferior nasal septum, the posterior palatal shelf before fusion, and the attachment of the tongue during normal development of the palate and maxilla from GD 11.5. Localization in the tongue frenum correlated with the ankyloglossia phenotype in the induced cleft palate animal model.

Septal organ of Grüneberg is part of the olfactory system.

The olfactory system in rodents and many other mammals is classically divided into two anatomically separate, and morphologically distinct, sensory systems: the main olfactory system and the accessory olfactory system. We have now identified a novel third population of olfactory marker protein-expressing sensory neurons that is located in a discrete pocket of the rostral nasal septum, which we refer to as the septal organ of Grüneberg (SOG). Neurons in this region of the septum are located in the submucosa, in small grape-like clusters, rather than in a pseudostratified neuroepithelium, as seen in both the olfactory and vomeronasal neuroepithelia. Despite their unusual location, axons projecting from the SOG neurons fasciculate into several discrete bundles and terminate in a subset of main olfactory bulb glomeruli. These glomeruli most likely represent a subset of atypical glomeruli that are spatially restricted to the caudal main olfactory bulb. The unique rostral position of the SOG suggests that the SOG may be functionally specialized for the early detection of biologically relevant odorants.

Incorporation of 3H-thymidine in the embryonic vomeronasal and olfactory epithelial of garter snakes.

Previous studies have shown that the vomeronasal and olfactory epithelia of adult vertebrates provide good models for studying normal neuronal turnover and regeneration in response to axotomy. However, little is known about the cell dynamics in the embryonic vomeronasal and olfactory epithelia or the origins of different cell types in these structures. By using 3H-thymidine autoradiography, both in vivo and in vitro, the origins of receptor and supporting cells and the survival of labelled cells in the embryonic vomeronasal and olfactory epithelial of garter snakes were examined. The results of this study suggest that the receptor and supporting cells of both epithelial arise from separate stem cells and that two subpopulations of stem cells exist for receptor cells in the embryonic vomeronasal epithelium. One subpopulation generates cells that migrate through the receptor cell columns, while another subpopulation remains at the base of the epithelium for approximately 50 days. Although it is unclear how long receptor cells in the embryonic olfactory epithelium survive, the results of this study suggest that they survive at least 37 days and may survive over 56 days. In addition, the development of these sensory epithelia appears different in early versus late embryos, and regeneration in the vomeronasal and olfactory epithelia of adult garter snakes appears similar to development during late gestation. Cells in the developing receptor cell layer of the olfactory epithelium lose their ability to incorporate 3H-thymidine before those in the vomeronasal epithelium, suggesting that the onset of neuronal maturation occurs earlier in the olfactory epithelium than in the vomeronasal epithelium.

Incorporation of 3H-thymidine in telencephalic structures of the vomeronasal and olfactory systems of embryonic garter snakes.

Previous studies have shown that the vomeronasal and, possibly, olfactory systems are functional in newborn garter snakes. However, little is known about neurogenesis in these chemosensory pathways. In the companion paper, we describe the embryonic growth of the sensory epithelia for both the vomeronasal and olfactory systems. In the present study, we examine neurogenesis in the telencephalic structures of these chemosensory systems by using 3H-thymidine autoradiography (ARG). The majority of neurogenesis appears to occur before birth in the accessory and main olfactory bulbs and their principal projection sites, the nucleus sphericus and lateral cortex, respectively. The data suggest that some postnatal neurogenesis may occur in the accessory and main olfactory bulbs and in the nucleus sphericus. Although the neuronal constituents of the accessory and main olfactory bulbs appear to mature concurrently, those of the lateral cortex appear to mature before those of the nucleus sphericus. Along with previous findings, this latter result supports the hypothesis that the olfactory system develops before the vomeronasal system in garter snakes. There appears to be a rostral to caudal gradient of neurogenesis within the mural layer of the nucleus sphericus. However, an "outside to inside" gradient of neurogenesis was not observed in the mantle layer of the lateral cortex, as described for other reptiles. Similarities and differences observed by other investigators in other reptilian species and mammals are discussed.

Immunocytochemical demonstration of neural cell adhesion molecule (NCAM) along the migration route of luteinizing hormone-releasing hormone (LHRH) neurons in mice.

Contact between the developing forebrain and the ingrowing central processes of the olfactory, vomeronasal and terminal nerves is preceded by a migration of neural cell adhesion molecule (NCAM)-immunoreactive cells from the epithelium of the olfactory pit and the formation of an NCAM-immunoreactive cellular aggregate in the mesenchyme between the olfactory pit and the forebrain. The axons of the olfactory, vomeronasal, and terminal nerves, also NCAM-immunoreactive, grow into the cellular aggregate, which as development proceeds, becomes continuous with the rostral tip of the forebrain. The lateral and more rostral part of the cellular aggregate receives the ingrowing axons of the olfactory nerves and becomes the olfactory nerve layer of the olfactory bulb. The medial, more caudal part receives the central processes of the vomeronasal and terminal nerves. The vomeronasal nerve ends in the accessory olfactory bulb. The central processes of the terminal nerve end in the medial forebrain. Luteinizing hormone-releasing hormone (LHRH)-immunoreactive neurons, like the vomeronasal and terminal nerves, originate from the medial part of the olfactory pit. These LHRH cells migrate into the brain along and within a scaffolding formed by the NCAM-immunoreactive axons of the vomeronasal and terminal nerves, and they are never seen independent of this NCAM scaffold as they cross the nasal lamina propria. The results suggest that: (1) NCAM is likely to be necessary for scaffold formation, and (2) the scaffold may be essential for the subsequent migration of LHRH neurons into the brain. Because they aggregate, migrating LHRH-immunoreactive neurons, on which we did not detect NCAM immunoreactivity, may interact via other cell adhesion molecules (CAM). Inasmuch as the interaction between the LHRH-immunoreactive neurons and the NCAM-immunoreactive scaffold is heterotypic, the possibility of a heterophilic (NCAM to other CAM) interaction is not ruled out. These findings focus our attention on the functional role of NCAM in this migratory system.

Identification of a new non-neuronal cell type in rat olfactory epithelium.

We have examined adult and embryonic rat olfactory epithelia by immunohistochemical techniques using the monoclonal antibody 1A-6, which was raised against embryonic rat olfactory epithelia. A heretofore unidentified cell type, reactive with the monoclonal antibody 1A-6, was observed scattered within the epithelium. The 1A-6 reactivity of these cells is most intense on the microvilli projecting from the luminal cell surfaces. For several reasons, we believe these cells are not neurons but a distinct subpopulation of supporting cells or some other sort of non-neuronal cells. (1) They have no identifiable axonal process, are not reactive with an antibody against olfactory marker protein, and are not in juxtaposition with trigeminal axons. (2) They survive ablation of the olfactory bulb. (3) Their nuclei lie within the supporting cell layer, and they resemble supporting cells morphologically and in their [3H]thymidine birthdating and turnover characteristics. However, the 1A-6-positive cells fail to react with the general supporting cell-specific monoclonal antibody SUS-1 [see Hempstead J. L. and Morgan J. I. (1983) Brain Res. 188, 289-295] a finding which suggests that they are not typical supporting cells. Immunoreactivity to 1A-6 is developmentally regulated. Immunohistochemical preparations of almost all tissues we examined showed widespread reactivity in the embryo but a much more restricted pattern in the adult. In the olfactory epithelium of the fetus, the luminal surfaces of all cells, including supporting cells and olfactory receptor cells and cilia, are reactive, while in the adult only the non-neuronal cell subpopulation shows this reactivity. We also found that during the reconstitution of olfactory epithelium which occurs in response to olfactory bulbectomy-induced neuronal degeneration, fetal patterns of 1A-6 reactivity are not re-expressed, i.e. the only 1A-6-positive cells are the non-neuronal cells seen in unperturbed adult olfactory epithelium. Preliminary biochemical analyses of membrane fractions from E19 brain and from adult olfactory mucosa indicate that the 1A-6 reactivity is associated with two bands, having molecular weights of 42,000 and 46,000 on Western blots.

Stimulus access to the accessory olfactory system in the prenatal and perinatal rat.

The fetal rat can detect odors in its amniotic fluid. Indirect evidence suggests that the accessory olfactory system may be mediating chemoreception prenatally. The primary goal of this study was to determine if stimuli in the amniotic fluid gain access to the receptor neurons of the accessory olfactory system that are sequestered inside the vomeronasal organ. Other goals of the study were to compare the access of stimuli to the vomeronasal organ in the prenatal and young postnatal rat and to examine the role of the autonomic nervous system in this process. On the day before birth fluorescent beads (0.95 microns diameter) were injected into the amniotic sacs of rat fetuses from seven dams. After 4-6 h one group of mothers received an i.p. injection of epinephrine (either 60 or 75 micrograms) and another group received no injection. One hour later pups were collected and processed for histological examination using fluorescence microscopy. A portion of the fetuses from both treatment groups had significant numbers (< 50) of beads in their nasal passages. Some of these subjects also had beads in the vomeronasal organ. There was no significant difference in the proportion of subjects with beads in the nasal cavities or vomeronasal organ across the two treatments. In a second experiment, five- to seven-day-old rat pups had beads infused into one naris. After 3-4 h one group was injected i.p. with epinephrine (2-4 micrograms), while a second group received no injection. As expected, virtually all the subjects had large numbers of beads in their nasal cavities upon post mortem examination.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructure of the human vomeronasal organ.

Virtually all vertebrates have a vomeronasal system whose involvement in pheromone detection plays a crucial role in reproduction. In humans, the vomeronasal organ has been assumed to be vestigial or absent and without functional significance. In the present study involving over 400 subjects, vomeronasal pits were observed in all individuals except those with pathological conditions affecting the septum. Electron microscopy of the adult human vomeronasal organ indicates the presence of two potential receptor elements in the pseudostratified epithelial lining: microvillar cells, and unmyelinated, intraepithelial axons. In addition, unmyelinated axons are common in the lamina propria surrounding the organ. They appear to constitute the components essential for a functional chemosensory system, and may thus provide the basis for a pheromone detection system as in other animals.

Sensory receptor-like cells in the human foetal vomeronasal organ.

The vomeronasal organ (VNO) was studied in ten human foetuses, 12 to 36 weeks old. At 12 to 23 weeks, it was lined by a smooth pseudostratified epithelium, with neurone-specific enolase (NSE) positive cells looking like olfactory receptors. Clusters of NSE-positive cells were seen in relation with the posterosuperior end of the organ and along nerve fascicles in the nasal septum. At 36 weeks, the organ was lined by a respiratory epithelium and did not show any receptor-like cells; some pear-shaped NSE-positive cells of unknown significance were seen at the upper part of the respiratory epithelium. Our results suggest that during the early foetal period, the VNO could have some, as yet unknown, sensory function.

Detection of apoptosis by electron microscopy and in situ labelling in the rat olfactory pit.

The purpose of this study was to provide further information upon the cell death process by apoptosis occurring in the olfactory pit during the primary palate formation and the vomeronasal organ detachment. Apoptotic cells were detected by coupling ultrastructural observations and in situ end-labelling of DNA breaks (TUNEL labelling) in E12-E15 rat embryos. During the primary palate formation and the vomeronasal organ closure, a strong apoptotic cell death process was observed along the midline epithelial seam after the epithelial fusion. The topographical distribution of labelled nuclei was in agreement with the morphological distribution of dying cells. One day before the nasal swellings fused, numerous degenerating cells were also detected in the regions of prospective contact which thus appeared as regions of programmed cell death.

Developmental studies on the rat vomeronasal organ: vascular pattern and neuroepithelial differentiation. I. Light microscopy.

The origin and the developmental sequence of the rat vomeronasal organ and its vascular supply are followed by means of India ink injection in serial sections of celloidin-embedded embryos from the eleventh day of gestation up to birth. The anlage of the vomeronasal organ has been established by the twelfth day of gestation (E 12). It appears as a shallow longitudinal impression of the medial wall of the nasal pit. At day E 14, it separates from the epithelium of the primary nasal cavity, forming a tube. The lumen of the organ remains continuous with the nasal cavity frontally, but ends blindly at the edge of the primary palate dorsally. From day E 16 to E 18 the lateral surface of the tubular vomeronasal organ invaginates toward the lumen forming a wide longitudinal furrow. The lumen is bordered by the developing neuroepithelium and receptor-free epithelium by this time. The vomeronasal organ receives a separate arterial blood supply arising from septal tributaries of the olfactory artery, a branch of the anterior cerebral artery from the earliest stage of development. Blood from the vomeronasal complex is collected in the vomeronasal vein lying in the longitudinal furrow next to the receptor-free epithelium. The typical vascular pattern of the vomeronasal organ is established by the eighteenth day of gestation. At this time, the first capillary loops appear within the neuroepithelium and the vomeronasal vein can already be seen to extend along the long axis of the organ.

Developmental studies on the rat vomeronasal organ: vascular pattern and neuroepithelial differentiation. II. Electron microscopy.

The present electron microscopical study demonstrates that the establishment of the typical vascular pattern of the rat vomeronasal organ by the eighteenth day of gestation is accompanied by neither complete maturation of the wall of the blood vessels nor of the vomeronasal neuroepithelium. In the newborn rat, however, the vasculature and the neuroepithelium of the vomeronasal organ present morphological elements suggesting functional capability at birth.

Is nestin a marker for chemosensory precursor cells?

The vomeronasal and olfactory systems are unique in that their chemosensory neurons undergo continuous neurogenesis after development. Immunoreactivity to nestin, a neuronal precursor marker protein, was investigated in the developing rat vomeronasal organ to determine its potential as a cell marker. From postnatal day 1 (P1) to P22, the distribution of nestin positive cells became progressively restricted to the area adjacent to the basement membrane. By P29, the vomeronasal organ reached structural maturity and only a few nestin positive cells were observed. Results suggest that nestin may be a useful marker for neuronal precursor cells in studies of neurogenesis and development of chemosensory systems.

Luteinizing hormone-releasing hormone (LHRH)-expressing cells in the nasal septum of human fetuses.

In 12-19 weeks old human fetuses, LHRH-immunoreactive (LHRH-Ir) cells were detected in the nerve fascicles arising from the vomeronasal organ (VNO), from their origin to their end point at the medial aspect of the olfactory bulb (vomeronasal and/or terminal nerves). In one 22-weeks-old fetus, LHRH-Ir cells were present only in the upper part of the nasal septum, in the nerve fascicles crossing the cribriform plate. No LHRH cells could be detected in two fetuses, 23 and 36 weeks old. Our results are discussed with regard to the migratory hypothesis of LHRH cells from the medial olfactory placode to the brain.