Expression of the intermediate filament protein nestin by sustentacular cells in mature olfactory neuroepithelium

The intermediate filament protein nestin has been widely used as a marker for proliferating neural progenitor cells in the nervous system. The mammalian olfactory neuroepithelium is a region of the nervous system that robustly supports ongoing neurogenesis, yet where nestin has not been reported to mark proliferating progenitors. Using immunohistochemistry, we examined nestin expression in the mature olfactory neuroepithelium and found it to be tightly restricted to the basal compartment where the olfactory neuronal progenitor cell population resides. The pattern of nestin immunoreactivity was consistent with expression by the endfeet and inferior processes of sustentacular cells rather than basal cells. Using a bank of defined antibody markers, we confirmed nestin's pattern of distribution to be different from that of cytokeratin, vimentin, GBC-1, GAP43, and carnosine. It was highly similar to the pattern of SUS-4 immunoreactivity in the basal region of the neuroepithelium. Following surgical bulbectomy, nestin expression was up-regulated and became evident in the cell bodies of sustentacular cells situated more apically in the neuroepithelium. We have shown nestin to be present in the basal region of the adult olfactory neuroepithelium in the zone that supports ongoing neurogenesis in the adult, but its expression is restricted to the inferior parts of sustentacular cells rather than the neuronal progenitor cells. Nestin may play a potential role in the migration of recently proliferated olfactory neurons on the scaffolding of sustentacular cells in a manner analogous to its proposed role in radial glia during embryonic development of the central nervous system.     

B-50/GAP43 Gene Expression in the Rat Olfactory System During Postnatal Development and Aging

The olfactory neuroepithelium exhibits neurogenesis throughout adult life, and in response to lesions, a phenomenon that distinguishes this neural tissue from the rest of the mammalian brain. The newly formed primary olfactory neurons elaborate axons into the olfactory bulb. Thus, denervation and subsequent re-innervation of olfactory bulb neurons may occur throughout life. In this study the authors demonstrate the distribution of the growth-associated phosphoprotein B-50/GAP43 and its mRNA in the olfactory neuroepithelium and olfactory bulb during development and aging. In neonatal rats B-50/GAP43 mRNA was expressed in primary olfactory neurons throughout the olfactory epithelium and in their target neurons in the olfactory bulb, the mitral, juxtaglomerular and tufted cells. In contrast, in adult (7.5 weeks) and aging animals (6 - 18 months of age) B-50/GAP43 mRNA expression was progressively restricted to neurons in the basal region of the neuroepithelium and to some of their target mitral and juxtaglomerular cells in the olfactory bulb. The continuing expression of B-50/GAP43 mRNA in mitral- and juxtaglomerular cells in mature animals is thought to be related to their capacity to respond to continuously changing input from the primary olfactory neurons present in the olfactory neuroepithelium.     

Illuminating the terminal nerve: Uncovering the link between GnRH-1 neuron and olfactory development

During embryonic development, the olfactory placode (OP) generates migratory neurons, including olfactory pioneer neurons, cells of the terminal nerve (TN), gonadotropin-releasing hormone-1 (GnRH-1) neurons, and other uncharacterized neurons. Pioneer neurons from the OP induce olfactory bulb (OB) morphogenesis. In mice, GnRH-1 neurons appear in the olfactory system around mid-gestation and migrate via the TN axons to different brain regions. The GnRH-1 neurons are crucial in controlling the hypothalamic-pituitary-gonadal axis. Kallmann syndrome is characterized by impaired olfactory system development, defective OBs, secretion of GnRH-1, and infertility. The precise mechanistic link between the olfactory system and GnRH-1 development remains unclear. Studies in humans and mice highlight the importance of the prokineticin-2/prokineticin-receptor-2 (Prokr2) signaling pathway in OB morphogenesis and GnRH-1 neuronal migration. Prokr2 loss-of-function mutations can cause Kallmann syndrome (KS), and hence the Prokr2 signaling pathway represents a unique model to decipher the olfactory/GnRH-1 connection. We discovered that Prokr2 is expressed in the TN neurons during the critical period of GnRH-1 neuron formation, migration, and induction of OB morphogenesis. Single-cell RNA sequencing identified that the TN is formed by neurons distinct from the olfactory neurons. The TN neurons express multiple genes associated with KS. Our study suggests that the aberrant development of pioneer/TN neurons might cause the KS spectrum.     

CNS stem and progenitor cell differentiation into functional neuronal circuits in three-dimensional collagen gels

The mammalian central nervous system (CNS) has little capacity for self-repair after injury, and neurons are not capable of proliferating. Therefore, neural tissue engineering that combines neural stem and progenitor cells and biologically derived polymer scaffolds may revolutionize the medical approach to the treatment of damaged CNS tissues. Neural stem and progenitor cells isolated from embryonic rat cortical or subcortical neuroepithelium were dispersed within type I collagen, and the cell-collagen constructs were cultured in serum-free medium containing basic fibroblast growth factor. The collagen-entrapped stem and progenitors actively expanded and efficiently generated neurons, which developed neuronal polarity, neurotransmitters, ion channels/receptors, and excitability. Ca2+ imaging showed that differentiation from BrdU+/TuJ1- to BrdU-/TuJ1+ cells was accompanied by a shift in expression of functional receptors for neurotransmitters from cholinergic and purinergic to predominantly GABAergic and glutamatergic. Spontaneous postsynaptic currents were recorded by patch-clamping from precursor cell-derived neurons and these currents were partially blocked by 10-microM bicuculline, and completely blocked by additional 10 microM of the kainate receptor antagonist CNQX, indicating an appearance of both GABAergic and glutamatergic synaptic activities. Staining with endocytotic marker FM1-43 demonstrated active synaptic vesicle recycling occurring among collagen-entrapped neurons. These results show that neural stem and progenitor cells cultured in 3D collagen gels recapitulate CNS stem cell development; this is the first demonstration of CNS stem and progenitor cell-derived functional synapse and neuronal network formation in a 3D matrix. The proliferative capacity and neuronal differentiating potential of neural progenitors in 3D collagen gels suggest their potential use in attempts to promote neuronal regeneration in vivo.     

Neurons of the olfactory epithelium in adult rats contain vimentin

In the developing nervous system, the intermediate filament protein vimentin is found in the proliferating neuroepithelium and neural crest. As development proceeds, postmitotic neurons cease vimentin expression and neurofilament proteins begin to accumulate. We have shown that olfactory receptor neurons deviate from the general pattern of neuronal intermediate filament expression, in that they continue to express vimentin or a highly vimentin-like protein rather than neurofilament proteins in the adult rat. With light-microscopic immunohistochemistry, three independently derived antibodies to vimentin label all portions of the primary olfactory projection, including the sensory neuron cell bodies in the olfactory epithelium, the fascicles of the olfactory nerve, and their axonal arbors in the glomeruli of the olfactory bulb. In contrast, anti-neurofilament antisera stain only rare scattered receptor cells and a small number of axons in the olfactory nerve. Electron-microscopic immunohistochemistry shows dense staining of olfactory axons with anti-vimentin. The vimentin-like immunoreactive material in the olfactory nerve layer was characterized by SDS-PAGE and by immunoblotting. On immunoblots of homogenates of the olfactory nerve, the anti-vimentin monoclonal antibody SBV-21 (Blose et al., 1984) stains only a single protein of Mr = 55 kDa. This band comigrates with vimentin in crude cytoskeletal material from the neonatal rat brain prepared according to the method of Dahl et al. (1981). SBV-21 does not stain neurofilament triplet proteins or glial fibrillary acidic protein, which are also present in these blots. These results demonstrate that the vast majority of olfactory receptor neurons and their axons contain vimentin or a protein of similar immunological character and electrophoretic mobility, while identifiable expression of neurofilament proteins is confined to a very small subpopulation. Hence, the switch in intermediate filament proteins that normally accompanies neuronal maturation is arrested in most olfactory neurons, and a "juvenile" biochemical marker is retained. This population of neurons is also unique among mammalian neurons in several other respects, including that olfactory neurons die during normal adult life or following injury and then are replaced from a proliferating pool of stem cells.     

The expression of the growth associated protein B50/GAP43 in the olfactory system of neonatal and adult rats

B50/GAP43 is a neuron-specific phosphoprotein whose expression is associated with neural development and synaptic plasticity. Its postnatal ontogeny was investigated in the primary olfactory pathway of the rat using immunohistochemical methods. The unique ability of the olfactory neuroepithelium to generate new neurons from a population of precursor cells present in the basal cell layer of this tissue makes it a valuable model in the study of neural development. In newborn rats B50/GAP43 is present throughout the entire population of olfactory receptor neurons. These cells are stained throughout, from the ciliated dendritic knob to their axon terminals in the bulb. This appears to be the first example of unambiguous B50/GAP43 expression in dendritic processes. With increasing age the distribution of this protein becomes progressively restricted to a subpopulation of olfactory neurons. Comparison of the expression of B50/GAP43 and the olfactory marker protein (OMP), a polypeptide only present in mature olfactory neurons, revealed that during postnatal development of the olfactory system these 2 proteins are expressed in a nearly reciprocal fashion. In adult animals (3.5 months-6 months of age), B50/GAP43-positive cells are exclusively present adjacent to the basal cell layer of the neuroepithelium. Basal cells appear to be unstained. The region of the epithelium containing the B50/GAP43-positive cells is virtually devoid of OMP-positive neurons. A significant fraction of these B50/GAP43-containing cells bear dendritic and neuritic processes. However, these cells do not express olfactory cilia. It is probable that the olfactory neurons expressing the growth-associated B50/GAP43 protein may correspond to a particular subset of olfactory neurons at an intermediate state of maturation.     

Visualization of neurogenesis in the central nervous system using nestin promoter-GFP transgenic mice

Neurons are generated from neural progenitor cells not only during development but also in the mature brain. To develop an in vivo system for analyzing neurogenesis, we generated transgenic mice expressing green fluorescent protein (GFP) under the control of regulatory regions of the nestin gene. GFP fluorescence was observed in areas and during periods connected with neurogenesis, including embryonic neuroepithelium, neonatal cerebellum, and hippocampal dentate gyrus and rostral migratory pathway from the subventricular zone to the olfactory bulb in the adult. GFP-positive cells in the adult brain included immature neuronal cells expressing polysialylated NCAM. BrdU labeling experiments revealed that newly generated interneurons which migrated rostrally from the subventricular zone expressed GFP until they reached the olfactory bulb. These results indicate that nestin promoter-GFP transgenic mice can be utilized to visualize the regions of neurogenesis throughout the life of the animals and to follow the migration and differentiation of newly generated neurons.     

Transgenic expression of B-50/GAP-43 in mature olfactory neurons triggers downregulation of native B-50/GAP-43 expression in immature olfactory neurons

The adult mammalian olfactory neuroepithelium is an unusual neural tissue, since it maintains its capacity to form new neurons throughout life. Newly formed neurons differentiate in the basal layers of the olfactory neuroepithelium and express B-50/GAP-43, a protein implicated in neurite outgrowth. During maturation these neurons migrate into the upper portion of the epithelium, upregulate expression of olfactory marker protein (OMP) and concomitantly downregulate the expression of B-50/GAP-43. Transgenic mice that exhibit OMP-promoter directed expression of B-50/GAP-43 in mature olfactory neurons display an unexpected decrease in the complement of B-50/GAP-43-positive cells in the lower region of the olfactory epithelium [A.J.G.D. Holtmaat, P.A. Dijkhuizen, A.B. Oestreicher, H. J. Romijn, N.M.T. Van der Lugt, A. Berns, F.L. Margolis, W.H. Gispen, J. Verhaagen, Directed expression of the growth-associated protein B-50/GAP-43 to olfactory neurons in transgenic mice results in changes in axon morphology and extraglomerular growth, J. Neurosci. 15 (1995) 7953-7965]. We have investigated whether the decrement in B-50/GAP-43-positive cells in this region was due to a dislocation of the immature neurons to other regions of the olfactory epithelium or to a downregulation of B-50/GAP-43 synthesis in these immature neurons. In eight of nine independent transgenic mouse lines that express the transgene in different numbers of olfactory neurons, a decline in the number of B-50/GAP-43-expressing neurons in the basal portion of the olfactory neuroepithelium was observed, both at the protein level and the mRNA level. An alternative marker for immature cells, a juvenile form of tubulin, was normally expressed in this location, indicating that the olfactory epithelium of OMP-B-50/GAP-43 transgenic mice contains a normal complement of immature olfactory neurons and that most of these neurons display a downregulation of B-50/GAP-43 expression.     

Chemokine receptor expression by neural progenitor cells in neurogenic regions of mouse brain

We previously demonstrated that chemokine receptors are expressed by neural progenitors grown as cultured neurospheres. To examine the significance of these findings for neural progenitor function in vivo, we investigated whether chemokine receptors were expressed by cells having the characteristics of neural progenitors in neurogenic regions of the postnatal brain. Using in situ hybridization we demonstrated the expression of CCR1, CCR2, CCR5, CXCR3, and CXCR4 chemokine receptors by cells in the dentate gyrus (DG), subventricular zone of the lateral ventricle, and olfactory bulb. The pattern of expression for all of these receptors was similar, including regions where neural progenitors normally reside. In addition, we attempted to colocalize chemokine receptors with markers for neural progenitors. In order to do this we used nestin-EGFP and TLX-LacZ transgenic mice, as well as labeling for Ki67, a marker for dividing cells. In all three areas of the brain we demonstrated colocalization of chemokine receptors with these three markers in populations of cells. Expression of chemokine receptors by neural progenitors was further confirmed using CXCR4-EGFP BAC transgenic mice. Expression of CXCR4 in the DG included cells that expressed nestin and GFAP as well as cells that appeared to be immature granule neurons expressing PSA-NCAM, calretinin, and Prox-1. CXCR4-expressing cells in the DG were found in close proximity to immature granule neurons that expressed the chemokine SDF-1/CXCL12. Cells expressing CXCR4 frequently coexpressed CCR2 receptors. These data support the hypothesis that chemokine receptors are important in regulating the migration of progenitor cells in postnatal brain.     

Expression of calmodulin mRNA in rat olfactory neuroepithelium.

A calmodulin (CaM) cDNA was isolated by differential hybridization screening of a lambda gt10 library prepared from rat olfactory mucosa. This cDNA fragment, containing most of the open reading frame of the rat CaMI gene, was subcloned and used to characterize steady-state expression of CaM mRNA in rat olfactory neuroepithelium and bulb. Within the bulb mitral cells are the primary neuronal population expressing CaM mRNA. The major CaM mRNA expressed in the olfactory mucosa is 1.7 kb with smaller contributions from mRNAs of 4.0 and 1.4 kb. CaM mRNA was primarily associated with the olfactory neurons and, despite the cellular complexity of the tissue and the known involvement of CaM in diverse cellular processes, was only minimally evident in sustentacular cells, gland cells or respiratory epithelium. Following bulbectomy CaM mRNA declines in the olfactory neuroepithelium as does olfactory marker protein (OMP) mRNA. In contrast to the latter, CaM mRNA makes a partial recovery by one month after surgery. These results, coupled with those from in situ hybridization, indicate that CaM mRNA is expressed in both mature and immature olfactory neurons. The program regulating CaM gene expression in olfactory neurons is distinct from those controlling expression of B50/GAP43 in immature, or OMP in mature, neurons respectively.     

Expression of collapsin response mediator proteins 1, 2 and 5 is differentially regulated in newly generated and mature neurons of the adult olfactory system

Collapsin-response mediator proteins (CRMPs) are highly expressed in the developing brain where they take part in several aspects of neuronal differentiation. CRMPs are still present postnatally, but their function remains speculative in the adult brain. We studied the expression and localization of CRMP1, CRMP2 and CRMP5 in two areas of the nervous system with persistent neurogenesis in adult mice, the olfactory mucosa and the olfactory bulb. In the olfactory mucosa, we have established that CRMP expression is restricted to postmitotic cells of the olfactory neurons lineage. CRMP5 is coexpressed with growth associated protein of 43 kDa (GAP43) in immature olfactory neurons and is down-regulated in olfactory marker protein-positive mature neurons. In contrast, CRMP1 and CRMP2 persist at all stages of differentiation from immature GAP43-positive to fully mature olfactory neurons. In the olfactory bulb, CRMP1, CRMP2 and CRMP5 are abundant in neuronal progenitors of the subependymal layer and in differentiating interneurons. In both areas, the subcellular distribution of CRMP1 or CRMP2 is different in mature vs. immature neurons, suggesting that these proteins are sequentially involved in various cellular events during neuronal lifetime. The variations of CRMP expression following axotomy are consistent with their differential localization and functional involvement in immature vs. mature neurons of the olfactory system. Our data bring new insight to the putative functions of CRMPs within areas of the adult nervous system with permanent neurogenesis, some related to differentiation of newly generated neurons but others occurring in mature neurons with a limited lifespan.     

Evidence of newly generated neurons in the human olfactory bulb

The subventricular zone (SVZ) is known to be the major source of neural stem cells in the adult brain. In rodents and nonhuman primates, many neuroblasts generated in the SVZ migrate in chains along the rostral migratory stream (RMS) to populate the olfactory bulb (OB) with new granular and periglomerular interneurons. In order to know if such a phenomenon exists in the adult human brain, we applied single and double immunostaining procedures to olfactory bulbs obtained following brain necropsy in normal adult human subjects. Double immunofluorescence labelling with a confocal microscope served to visualize cells that express markers of proliferation and immature neuronal state as well as markers that are specific to olfactory interneurons. Newborn cells that express cell cycle proteins [Ki-67, proliferating cell nuclear antigen (PCNA)] were detected in the granular and glomerular layers (GLs) of the human olfactory bulb; these cells coexpressed markers of immature neuronal state, such as Doublecortin (DCX), NeuroD and Nestin. Numerous differentiating cells expressed molecular markers of early committed neurons [beta-tubulin class III (TuJ1)] and were also immunoreactive for glutamic acid decarboxylase (GAD), a marker of GABAergic neurons, or tyrosine hydroxylase (TH), a marker of dopaminergic neurons. Other early committed neurons expressed the calcium-binding proteins calretinin (CR) or parvalbumin (PV). These results provide strong evidence for the existence of adult neurogenesis in the human olfactory system. Despite its relatively small size compared to that in rodents and nonhuman primates, the olfactory bulb in humans appears to be populated, throughout life, by new granular and periglomerular neurons that express a wide variety of chemical phenotypes.     

Phosphatidyl-inositide signalling proteins in a novel class of sensory cells in the mammalian olfactory epithelium

Ciliated sensory neurons, supporting cells and basal stem cells represent major cellular components of the main olfactory epithelium in mammals. Here we describe a novel class of sensory cells in the olfactory neuroepithelium. The cells express phospholipase C beta-2 (PLC beta2), transient receptor potential channels 6 (TRPC6) and inositol 3, 4, 5-trisphosphate receptors type III (InsP3R-III). Unlike ciliated olfactory neurons, they express neither olfactory marker protein nor centrin, adenylyl cyclase or cyclic nucleotide-gated cation channels. Typical components of the cytoskeleton of microvilli, ezrin and actin are found co-localized with PLC beta2 and TRPC6 in apical protrusions of the cells. In Ca2+-imaging experiments, the cells responded to odours. They express neuronal marker proteins and possess an axon-like process, but following bulbectomy the cells do not degenerate. Our results suggest a novel class of microvillous secondary chemosensory cells in the mammalian olfactory system. These cells, which utilize phosphatidyl-inositides in signal transduction, represent about 5% of all olfactory cells. Their abundance indicates that they play an important role in stimulus-dependent functions and/or the regeneration of the olfactory system.     

Gap junctions: multifaceted regulators of embryonic cortical development

The morphological development of the cerebral cortex from a primitive neuroepithelium into a complex laminar structure underlying higher cognition must rely on a network of intercellular signaling. Gap junctions are widely expressed during embryonic development and provide a means of cell-cell contact and communication. We review the roles of gap junctions in regulating the proliferation of neural progenitors as well as the migration and differentiation of young neurons in the embryonic cerebral cortex. There is substantial evidence that although gap junctions act in the classical manner coupling neural progenitors, they also act as hemichannels mediating the spread of calcium waves across progenitor cell populations and as adhesive molecules aiding neuronal migration. Gap junctions are thus emerging as multifaceted regulators of cortical development playing diverse roles in intercellular communication.     

A new model for olfactory placode development

The peripheral nervous system of vertebrate animals arises primarily from the interaction of cranial neural crest and sensory placodes. Placodes are described as thickenings of ectoderm that arise through cell division during neural tube formation. The olfactory sensory system is one component of the peripheral nervous system that arises from paired sensory placodes during development. The olfactory placodes give rise to the primary sensory neurons, support cells and basal cells of the olfactory epithelium. Recent evidence from work in zebrafish and chick suggests that the olfactory and auditory placodes arise from large fields of cells that converge to form the sensory placode. The olfactory placodes arise from within the neural plate, and cell division is apparent only after the sensory placodes are morphologically distinct. As the olfactory placode is forming, its precursors must segregate from their neighboring fields which will give rise to the adenohypophyseal placode, cranial neural crest, and telencephalon. Analysis has shown that the endocrine cells thought to arise from the olfactory placode originate in the neighboring adenohypophyseal and cranial neural crest domains. The borders separating the domains are plastic, where cells sort as they move, and cell fate is dependent on the identity of neighbors once the cells have converged to form the sensory placode. Thus there is degeneracy built into the system such that cells accommodate changes in the environment until cell migrations controlling the formation of the sensory placodes are complete.     

Distribution of neuronal intermediate filament proteins in the developing mouse olfactory system

The distribution of neuronal intermediate filament proteins in the developing mouse olfactory bulb and olfactory epithelium was characterized by immunocytochemical approach. Antibodies against α-internexin, neurofilament triplet proteins (NFTPs; NF-L, NF-M, and NF-H) and peripherin were used to determine their expression at different developmental stages. α-Internexin and peripherin were first found to be co-localized in the olfactory neuroepithelium during early development. At the perinatal stage, expression patterns of α-internexin and peripherin are distinguishable by spatial and temporal manner: peripherin is predominantly expressed in the olfactory nerves; whereas α-internexin is expressed in both olfactory nerves and olfactory bulb. Our observation suggests that peripherin as well as α-internexin may play some roles in the process formation of olfactory nerves during development. In the developing olfactory periglomerulus, α-internexin was found around postnatal Day 3, whereas NFTPs were not observed until postnatal Day 7. Our data showed that the expression of α-internexin preceded those of the NFTPs in most neurons of the developing olfactory bulb. Some small neurons in the adult olfactory bulb were uniquely labeled with antibody to α-internexin. Our results suggest that α-internexin may play a functional role in the neuronal cytoarchitecture of developing olfactory system, and can be a neuronal marker for detecting postmitotic migrating neurons in the adult olfactory bulb. J. Neurosci. Res. 54:353–363, 1998. © 1998 Wiley-Liss, Inc.     

MicroRNA-124 is a subventricular zone neuronal fate determinant

New neurons are continuously generated from neural stem cells with astrocyte properties, which reside in close proximity to the ventricle in the postnatal and adult brain. In this study we found that microRNA-124 (miR-124) dictates postnatal neurogenesis in the mouse subventricular zone. Using a transgenic reporter mouse we show that miR-124 expression is initiated in the rapid amplifying progenitors and remains expressed in the resulting neurons. When we stably inhibited miR-124 in vivo, neurogenesis was blocked, leading to the appearance of ectopic cells with astrocyte characteristics in the olfactory bulb. Conversely, when we overexpressed miR-124, neural stem cells were not maintained in the subventricular zone and neurogenesis was lost. In summary, our results demonstrate that miR-124 is a neuronal fate determinant in the subventricular zone.     

GnRH,anosmia and hypogonadotropic hypogonadism – Where are we?

Gonadotropin releasing hormone (GnRH) neurons originate the nasal placode and migrate into the brain during prenatal development. Once within the brain, these cells become integral components of the hypothalamic–pituitary–gonadal axis, essential for reproductive function. Disruption of this system causes hypogonadotropic hypogonadism (HH). HH associated with anosmia is clinically defined as Kallman syndrome (KS). Recent work examining the developing nasal region has shed new light on cellular composition, cell interactions and molecular cues responsible for the development of this system in different species. This review discusses some developmental aspects, animal models and current advancements in our understanding of pathologies affecting GnRH. In addition we discuss how development of neural crest derivatives such as the glia of the olfactory system and craniofacial structures control GnRH development and reproductive function.