Six1 is indispensable for production of functional progenitor cells during olfactory epithelial development

The rodent olfactory epithelium (OE) is a good model system for studying the principles of stem and progenitor cell biology, because of its capacity for continuous neurogenesis throughout life and relatively well-characterized neuronal lineage. The development of mouse OE is divided into two stages, early and established neurogenesis. In established neurogenesis, which starts at embryonic day (E) 12.5, sustentacular cells and olfactory receptor neurons (ORNs) are produced from apical and basal progenitors, respectively. We previously reported that Six1(-/-) shows a lack of mature ORNs throughout development and disorganization of OE after E12.5. However, the molecular bases for these defects have not been addressed. Here, we show that Six1 is expressed in both apical and basal progenitors. In Six1(-/-) mice, apical proliferating cells were absent and no morphologically identifiable sustentacular cells were observed. Consistently, the expression of Notch2 and Jagged1 in the apical layer was absent in Six1(-/-) mice. On the other hand, basal proliferating cells were observed in Six1(-/-) animals, but the expression of Ngn1, NeuroD, Notch1, and Jagged2 in the basal layer was absent. The expression of Mash1, the determination gene for ORNs, and Hes genes was enhanced in Six1(-/-) mice. The present findings suggest that Six1 regulates production of functional apical and basal progenitors during OE development, through the regulation of various genes, such as neuronal basic helix-loop-helix (bHLH), neuronal repressor bHLH, and genes involved in the Notch signaling pathway.     

The Ginkgo biloba extract modulates the balance between proliferation and differentiation in the olfactory epithelium of adult mice following bulbectomy.

Abstract - The adult olfactory receptor neurons (ORNs), located in the olfactory epithelium (OE) are permanently renewed thanks to neuronal progenitors present in the deep part of the OE, the globose basal cells (GBCs). Following the ablation of their synaptic target, the olfactory bulb (OB), ORNs degenerate by apoptosis and a wave of neurogenesis, including proliferation of GBCs and neuronal differentiation of their progeny, restores the olfactory function. The Ginkgo biloba extract (EGb 761) (Beaufour Ipsen, France) was administered to adult mice at the doses of 50 or 100 mg/kg, following bilateral bulbectomy and its effects on the expression of PCNA, reflecting the number of proliferating GBCs and on growth associated protein 43 (GAP-43), expressed by differentiating neurons were measured by Western blotting. PCNA expression peaked 9 days post-bulbectomy in untreated animals, but 7 days post-lesion in EGb 761-treated animals. A simultaneous reduction in GAP-43 expression suggested that EGb 761 may temporarily favor the proliferation of GBCs rather than their entry into the differentiation pathway. Probably as a consequence of the earlier onset of the neurogenetic response to bulbectomy, neuronal differentiation was enhanced in the OE, 3 weeks post-bulbectomy. These data suggest that EGb 761 may have beneficial effects upon neurogenesis in the OE through changing the balance between proliferation and differentiation.     

Six1 is essential for early neurogenesis in the development of olfactory epithelium

The olfactory epithelium (OE) is derived from the olfactory placode (OP) during mouse development. At embryonic day (E) 10.0-E10.5, “early neurogenesis” occurs in the OE, which includes production of pioneer neurons that emigrate out of the OE and other early-differentiated neurons. Around E12.5, the OE becomes organized into mature pseudostratified epithelium and shows “established neurogenesis,” in which olfactory receptor neurons (ORNs) are differentiated from basal progenitors. Little is known about the molecular pathway of early neurogenesis. The homeodomain protein Six1 is expressed in all OP cells and neurogenic precursors in the OE. Here we show that early neurogenesis is severely disturbed despite the unaltered expression of Mash1 at E10.5 in the Six1-deficient mice (Six1^−/−). Expression levels of neurogenin1 (Ngn1) and NeuroD are reduced and those of Hes1 and Hes5 are augmented in the OE of Six1^−/− at E10.5. Pioneer neurons and cellular aggregates, which are derived from the OP/OE and situated in the mesenchyme between the OE and forebrain, are completely absent in Six1^−/−. Moreover, ORN axons and the gonadotropin-releasing hormone-positive neurons fail to extend and migrate to the forebrain, respectively. Our study indicates that Six1 plays critical roles in early neurogenesis by regulating Ngn1, NeuroD, Hes1, and Hes5.     

Olfactory neuron-specific expression of NeuroD in mouse and human nasal mucosa

Human olfactory neuroepithelium (OE) is situated within the olfactory cleft of the nasal cavity and has the characteristic property of continually regenerating neurons during the lifetime of the individual. This regenerative ability of OE provides a unique model for neuronal differentiation, but little is known about the structure and biology of human olfactory mucosa. Thus, to better understand neurogenesis in human OE, we studied the expression of olfactory marker protein (OMP), TrkB and NeuroD in human nasal biopsies and autopsy specimens and compared these data with those obtained from normal and regenerating mouse OE. We show that NeuroD and TrkB are coordinately expressed in human OE. Thus, by using these markers we have been able to extend the known boundaries of the human OE to include the inferior middle turbinate. In normal mouse OE, TrkB and OMP expression overlap in cells closest to the superficial layer, but TrkB is expressed more strongly in the lower region of this layer. In contrast, NeuroD expression is more basally restricted in a region just above the globose basal cells. These characteristic expression patterns of OMP, TrkB and NeuroD were also observed in the regenerating mouse OE induced by axotomy. These results support a role of NeuroD and brain-derived neurotrophic actor (BDNF), the preferred ligand for TrkB, in the maintenance of the olfactory neuroepithelium in humans and mice.     

Olfactory epithelia differentially express neuronal markers

All three olfactory epithelia, the olfactory epithelium proper (OE), the septal organ of Masera (SO), and the vomeronasal organ of Jacobson (VNO) originate from the olfactory placode. Here, their diverse neurochemical phenotypes were analyzed using the immunohistochemical expression pattern of different neuronal markers. The olfactory bulb (OB) served as neuronal control. Neuronal Nuclei Marker (NeuN) is neither expressed in sensory neurons in any of the three olfactory epithelia, nor in relay neurons (mitral/tufted cells) of the OB. However, OB interneurons (periglomerular/granule cells) labeled, as did supranuclear structures of VNO supporting cells and VNO glands. Protein Gene Product 9.5 (PGP9.5 = C-terminal ubiquitin hydrolase L1 = UCHL1) expression is exactly the opposite: all olfactory sensory neurons express PGP9.5 as do OB mitral/tufted cells but not interneurons. Neuron Specific Enolase (NSE) expression is highest in the most apically located OE and SO sensory neurons and patchy in VNO. In contrast, the cytoplasm of the most basally located neurons of OE and SO immunoreacted for Growth Associated Protein 43 (GAP-43/B50). In VNO neurons GAP-43 labeling is also nuclear. In the cytoplasm, Olfactory Marker Protein (OMP) is most intensely expressed in SO, followed by OE and least in VNO neurons; further, OMP is also expressed in the nucleus of basally located VNO neurons. OB mitral/tufted cells express OMP at low levels. Neurons closer to respiratory epithelium often expressed a higher level of neuronal markers, suggesting a role of those markers for neuronal protection against take-over. Within the VNO the neurons show clear apical–basal expression diversity, as they do for factors of the signal transduction cascade. Overall, expression patterns of the investigated neuronal markers suggest that OE and SO are more similar to each other than to VNO.     

Expression patterns of homeobox genes in the mouse vomeronasal organ at postnatal stages

Homeodomain proteins are encoded by homeobox genes and regulate development and differentiation in many neuronal systems. The mouse vomeronasal organ (VNO) generates in situ mature chemosensory neurons from stem cells. The roles of homeodomain proteins in neuronal differentiation in the VNO are poorly understood. Here we have characterized the expression patterns of 28 homeobox genes in the VNO of C57BL/6 mice at postnatal stages using multicolor fluorescent in situ hybridization. We identified 11 homeobox genes (Dlx3, Dlx4, Emx2, Lhx2, Meis1, Pbx3, Pknox2, Pou6f1, Tshz2, Zhx1, Zhx3) that were expressed exclusively in neurons; 4 homeobox genes (Pax6, Six1, Tgif1, Zfhx3) that were expressed in all non-neuronal cell populations, with Pax6, Six1 and Tgif1 also expressed in some neuronal progenitors and precursors; 12 homeobox genes (Adnp, Cux1, Dlx5, Dlx6, Meis2, Pbx2, Pknox1, Pou2f1, Satb1, Tshz1, Tshz3, Zhx2) with expression in both neuronal and non-neuronal cell populations; and one homeobox gene (Hopx) that was exclusively expressed in the non-sensory epithelium. We studied further in detail the expression of Emx2, Lhx2, Meis1, and Meis2. We found that expression of Emx2 and Lhx2 initiated between neuronal progenitor and neuronal precursor stages. As far as the sensory neurons of the VNO are concerned, Meis1 and Meis2 were only expressed in the apical layer, together with Gnai2, but not in the basal layer.     

Expression of glycoproteins in the vomeronasal organ reveals a novel spatiotemporal pattern of sensory neurone maturation

The main olfactory and the accessory olfactory systems are both anatomically and functionally distinct chemosensory systems. The primary sensory neurones of the accessory olfactory system are sequestered in the vomeronasal organ (VNO), where they express pheromone receptors, which are unrelated to the odorant receptors expressed in the principal nasal cavity. We have identified a 240 kDa glycoprotein (VNO(240)) that is selectively expressed by sensory neurones in the VNO but not in the main olfactory neuroepithelium of mouse. VNO(240) is first expressed at embryonic day 20.5 by a small subpopulation of sensory neurones residing within the central region of the crescent-shaped VNO. Although VNO(240) was detected in neuronal perikarya at this age, it was not observed in the axons in the accessory olfactory bulb until postnatal day 3.5. This delayed appearance in the accessory olfactory bulb suggests that VNO(240) is involved in the functional maturation of VNO neurones rather than in axon growth and targeting to the bulb. During the first 2 postnatal weeks, the population of neurones expressing VNO(240) spread peripherally, and by adulthood all primary sensory neurones in the VNO appeared to be expressing this molecule. Similar patterns of expression were also observed for NOC-1, a previously characterized glycoform of the neural cell adhesion molecule NCAM. To date, differential expression of VNO-specific molecules has only been reported along the rostrocaudal axis or at different apical-basal levels in the neuroepithelium. This is the first demonstration of a centroperipheral wave of expression of molecules in the VNO. These results indicate that mechanisms controlling the molecular differentiation of VNO neurones must involve spatial cues organised, not only about orthogonal axes, but also about a centroperipheral axis. Moreover, expression about this centroperipheral axis also involves a temporal component because the subpopulation of neurones expressing VNO(240) and NOC-1 increases during postnatal maturation.     

Cytochrome oxidase staining reveals functionally important activity bands in the olfactory epithelium of newborn rat

We used cytochrome oxidase (CytOx) staining intensity, which is correlated with neuronal functional activity, to evaluate maturity and functionality of newborn rat olfactory epithelium (OE) and olfactory receptor neurons (ORNs). Nasal olfactory tissue of neonatal rats was stained with CytOx and analyzed qualitatively and quantitatively. Results revealed that newborn OE shows six differentially stained horizontal bands. Bands run parallel to the OE surface and were categorized as very light, medium or darkly stained. A narrow and pale Band 1 overlapped with horizontal basal cells. Next, a wide and lightly stained Band 2 was observed that coincides with the globose basal cell layer and immature ORNs, deep in OE. Next apically, a medium-staining Band 3 overlapped with ORN perikarya. Closer to the surface, a medium to light Band 4 was discerned where dendrites of mature ORNs normally occur. This band was interrupted with lighter areas due to the presence of supporting cells nuclei. Next, a superficial but dark Band 5 occurred, populated by the apical portions of ORN dendrites and their ciliated knobs and by supporting cell apices; mitochondria in apices of supporting cells contribute predominantly to dense staining of this Band 5. Apical to Band 5, a thin and fairly light Band 6 was observed which overlaps with the mucus layer that contains part of the ORN knobs, their cilia and supporting cell microvilli. Along the length of ORN dendrites, apical segments just below the ORN knobs, and wide basal segments showed a darker staining than the middle segments implying “microzones” of higher neural activity within the most apical and basal regions of dendrites. Our findings agree with ultrastructural studies showing a presence of mitochondria in knobs, basal portions of ORN dendrites and in OE supporting cell apices, suggesting that apical regions of both olfactory and supporting cells near the surfaces are metabolically most active, in odorant detection, signal processing, and detoxification, the latter for supporting cells.     

Immunohistochemical distribution of galectin-1, galectin-3, and olfactory marker protein in human olfactory epithelium

The expression pattern of galectin-1 and galectin-3 in the human olfactory epithelium was investigated in relation to olfactory marker protein (OMP) using confocal laser immunofluorescence in human specimens and postmortem biopsies. OMP expression was found in olfactory receptor neurons (ORNs) in the olfactory mucosa and in fibers of the olfactory nerve crossing the submucous connective tissue. Galectin-1 was expressed in both the connective tissue of the nasal cavity and in the basal layer of the olfactory epithelium. In contrast, galectin-3 expression was limited to cells of the upper one-third of the olfactory epithelium. Expression of galectin-3 occurred in a subset of OMP-positive cells. However, between areas of galectin-1 and galectin-3 expression in the lower and upper portion of the epithelium, OMP-positive ORNs did not stain for both galectins. Considering the potential role of galectin-1 and galectin-3 in cell differentiation and maturation, the differential localization of galectins in the olfactory epithelium appears to be consistent with a significant role of these molecules in the physiological turnover of ORNs. Accepted: 20 December 1999     

Notch1 expression and ligand interactions in progenitor cells of the mouse olfactory epithelium

Despite the relatively simplified organization of the olfactory epithelium (OE), our understanding of the factors that regulate its cellular diversity is limited. Genetic and localization studies suggest that Notch signaling may be important in this process. We characterize here a population of Notch1 ⁺ olfactory basal cells in embryonic mice that coordinately express both the Notch effector Hes5 and the glycosyltransferase Lfng. These cells are distinct from Mash1 ⁺ neuronal precursors, but give rise to sensory neurons, suggesting that Notch1 signals may in part function to maintain a neurogenic progenitor pool. Furthermore, Lfng ⁺ cells also generate a population of cells in the migratory mass that appear to be ensheathing glial precursors, indicating potential multipotency in these progenitors. The Notch ligand Dll4 is expressed by basal OE cells that are interspersed with Notch1 ⁺ progenitors during later OE neurogenesis. In contrast, mice deficient in Dll1 exhibit a smaller OE and a loss of Hes5 expression, indicating an earlier function in olfactory progenitor cell development. Taken together, these results further support a role for Notch signaling in the regulation of olfactory neurogenesis and cell diversity.     

FGF2 suppresses neuronogenesis of a cell line derived from rat olfactory epithelium

Neurogenesis continues throughout adulthood in the mammalian olfactory epithelium (OE), and both neurons as well as nonneuronal cells are reconstituted following experimental injury. Underlying the capacity of the OE to replenish its mature elements is a population of progenitor basal cells. Although the precise lineage relationships among progenitor and mature cell types are incompletely understood, the population of globose basal cells (GBCs) contains immediate precursors to neurons as well as amplifying progenitors, and retroviral lineage analyses suggest that multipotential GBCs are activated following direct injury to the OE. To assess the controls on the process of epithelial regeneration, we have characterized a cell line derived from rat OE and studied the effects of serum and tissue extracts, fibroblast growth factor-2 (FGF2) and transforming growth factor-α (TGFα) on the cells. Using a panel of cell type-specific markers whose patterns of labeling in the OE are well defined, including recently developed markers for GBCs, we characterized the phenotype of the cell line under differing culture conditions. In complete medium, which contains serum and tissue extracts, the cell line displayed characteristics of GBCs that are prominent during regeneration. Serum and extract withdrawal induced the cells to differentiate into neurons. In contrast, FGF2 prevented neuronal differentiation and maintained a GBC phenotype. TGFα had a mitogenic or differentiative effect that was context dependent. Finally, we demonstrate here that FGF2 is contained in mature olfactory neurons and sustentacular cells in vivo, suggesting a physiologic role for this growth factor in OE cell regulation. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 411–428, 1997     

Macro- and micromorphological remodeling of olfactory organs throughout the ontogeny of the fire salamander Salamandra salamandra (Linnaeus, 1758)

This article studies the morphological remodeling of olfactory organs in the fire salamander (Salamandridae, Caudata), from the larval stages of ontogeny to the adult and throughout the course of the annual cycle. The fire salamander exhibits adaptations to the aquatic environment during premetamorphic life and terrestrial adaptations after metamorphosis. During adulthood, the annual activity of this species is divided into three seasonal periods: a breeding period, a nonbreeding period, and hibernation. We observed significant differences in morphology of olfactory organs between developmental stages as well as between each period within the annual cycle. For the first time in caudates, we examined the morphology of olfactory organs during the winter period (wintering larvae, hibernating adults). The results show that the remodeling of olfactory organs during the life of the fire salamander occurs both on macro- and micromorphological levels. Macromorphological ontogenetic variability includes the shape of the main olfactory chamber (MOC) and the distribution of olfactory epithelium (OE) in the MOC and in the vomeronasal organ (VNO). In larvae, the vomeronasal epithelium (VNE) is in a separate cavity, while in the post-metamorphic stages of ontogeny, the VNE occurs in the diverticulum of the MOC. In adult fire salamanders, both olfactory organs are most developed during the breeding season and reduced during hibernation. The VNE and OE in the MOC are also reduced during hibernation. Micro-morphological changes included different types/subtypes of olfactory receptor neurons (ORNs) in the OE in particular stages of ontogeny and periods within the annual cycle, for example, ciliate ORNs are present in the VNE only in the larval stages and giant ORNs occur only in nonbreeding adults. Also, there was a variable set of types of olfactory supporting cells in the VNO of the fire salamander during pre- and postmetamorphic life stages.     

Fgf8 expression defines a morphogenetic center required for olfactory neurogenesis and nasal cavity development in the mouse

In vertebrate olfactory epithelium (OE), neurogenesis proceeds continuously, suggesting that endogenous signals support survival and proliferation of stem and progenitor cells. We used a genetic approach to test the hypothesis that Fgf8 plays such a role in developing OE. In young embryos, Fgf8 RNA is expressed in the rim of the invaginating nasal pit (NP), in a small domain of cells that overlaps partially with that of putative OE neural stem cells later in gestation. In mutant mice in which the Fgf8 gene is inactivated in anterior neural structures, FGF-mediated signaling is strongly downregulated in both OE proper and underlying mesenchyme by day 10 of gestation. Mutants survive gestation but die at birth, lacking OE, vomeronasal organ (VNO), nasal cavity, forebrain, lower jaw, eyelids and pinnae. Analysis of mutants indicates that although initial NP formation is grossly normal, cells in the Fgf8-expressing domain undergo high levels of apoptosis, resulting in cessation of nasal cavity invagination and loss of virtually all OE neuronal cell types. These findings demonstrate that Fgf8 is crucial for proper development of the OE, nasal cavity and VNO, as well as maintenance of OE neurogenesis during prenatal development. The data suggest a model in which Fgf8 expression defines an anterior morphogenetic center, which is required not only for the sustenance and continued production of primary olfactory (OE and VNO) neural stem and progenitor cells, but also for proper morphogenesis of the entire nasal cavity.     

Neuronal degeneration and regeneration induced by axotomy in the olfactory epithelium of Xenopus laevis

The olfactory epithelium (OE) has the remarkable capability to constantly replace olfactory receptor neurons (ORNs) due to the presence of neural stem cells (NSCs). For this reason, the OE provides an excellent model to study neurogenesis and neuronal differentiation. In the present work, we induced neuronal degeneration in the OE of Xenopus laevis larvae by bilateral axotomy of the olfactory nerves. We found that axotomy induces specific‐ neuronal death through apoptosis between 24 and 48h post‐injury. In concordance, there was a progressive decrease of the mature‐ORN marker OMP until it was completely absent 72h post‐injury. On the other hand, neurogenesis was evident 48h post‐injury by an increase in the number of proliferating basal cells as well as NCAM‐180– GAP‐43+ immature neurons. Mature ORNs were replenished 21 days post‐injury and the olfactory function was partially recovered, indicating that new ORNs were integrated into the olfactory bulb glomeruli. Throughout the regenerative process no changes in the expression pattern of the neurotrophin Brain Derivate Neurotrophic Factor were observed. Taken together, this work provides a sequential analysis of the neurodegenerative and subsequent regenerative processes that take place in the OE following axotomy. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1308–1320, 2017