Neural crest and placode contributions to olfactory development.

Olfaction is the sense of smell that influences many primitive behaviors for survival, e.g., feeding, reproduction, social interaction, and fear response. The olfactory system is an evolutionarily ancient sensory system and composed of the olfactory epithelium (OE), the olfactory bulb (OB), and the olfactory cortex. The OE gives rise to olfactory receptor neurons (ORNs), i.e., primary sensory receptor cells whose axons project directly to the OB. The ORNs are unique in the way that they are continuously replaced during physiological turnover or following injury throughout life. In the OE, horizontal basal cells, i.e., flat and quiescent cells attached to the basal lamina, are now thought to be tissue stem cells. Although OE cells, especially ORNs, were hypothesized to be derived from the olfactory placode (OP), recent genetic fate-mapping studies using Cre reporter mice indicate a dual origin, i.e., the OP and neural crest (NC), of the olfactory system. The NC is a transient embryonic tissue that is formed between the dorsal neuroepithelium and epidermis. Neural crest cells (NCCs) are multipotent cells that migrate into various target tissues and differentiate into various cell types, including neurons and glia of the peripheral nervous system, cranial cartilage and bone, and melanocytes. Recent studies have revealed that neural crest-derived cells (NCDCs) are widely distributed in adult tissues, and that a subset of NCDCs still possesses NCC-like multipotency. Here, we review classical and recent studies of the olfactory system, especially focusing on the contribution of the NC and OP to the OE development.

Activation of α2A-containing nicotinic acetylcholine receptors mediates nicotine-induced motor output in embryonic zebrafish.

It is well established that cholinergic signaling has critical roles during central nervous system development. In physiological and behavioral studies, activation of nicotinic acetylcholine receptors (nAChRs) has been implicated in mediating cholinergic signaling. In developing spinal cord, cholinergic transmission is associated with neural circuits responsible for producing locomotor behaviors. In this study, we investigated the expression pattern of the α2A nAChR subunit as previous evidence suggested it could be expressed by spinal neurons. In situ hybridization and immunohistochemistry revealed that the α2A nAChR subunits are expressed in spinal Rohon-Beard (RB) neurons and olfactory sensory neurons in young embryos. To examine the functional role of the α2A nAChR subunit during embryogenesis, we blocked its expression using antisense modified oligonucleotides. Blocking the expression of α2A nAChR subunits had no effect on spontaneous motor activity. However, it did alter the embryonic nicotine-induced motor output. This reduction in motor activity was not accompanied by defects in neuronal and muscle elements associated with the motor output. Moreover, the anatomy and functionality of RB neurons was normal even in the absence of the α2A nAChR subunit. Thus, we propose that α2A-containing nAChRs are dispensable for normal RB development. However, in the context of nicotine-induced motor output, α2A-containing nAChRs on RB neurons provide the substrate that nicotine acts upon to induce the motor output. These findings also indicate that functional neuronal nAChRs are present within spinal cord at the time when locomotor output in zebrafish first begins to manifest itself.

Neural map formation in the mouse olfactory system.

In the mouse olfactory system, odorants are detected by ~1,000 different odorant receptors (ORs) produced by olfactory sensory neurons (OSNs). Each OSN expresses only one functional OR species, which is referred to as the "one neuron-one receptor" rule. Furthermore, OSN axons bearing the same OR converge to a specific projection site in the olfactory bulb (OB) forming a glomerular structure, i.e., the "one glomerulus-one receptor" rule. Based on these basic rules, binding signals of odorants detected by OSNs are converted to topographic information of activated glomeruli in the OB. During development, the glomerular map is formed by the combination of two genetically programmed processes: one is OR-independent projection along the dorsal-ventral axis, and the other is OR-dependent projection along the anterior-posterior axis. The map is further refined in an activity-dependent manner during the neonatal period. Here, we summarize recent progress of neural map formation in the mouse olfactory system.

Conditional ablation of Tbr2 results in abnormal development of the olfactory bulbs and subventricular zone-rostral migratory stream.

BACKGROUND: Development of the olfactory bulb (OB) is a complex process that requires contributions from several progenitor cell niches to generate neuronal diversity. Previous studies showed that Tbr2 is expressed during the generation of glutamatergic OB neurons in rodents. However, relatively little is known about the role of Tbr2 in the developing OB or in the subventricular zone-rostral migratory stream (SVZ-RMS) germinal niche that gives rise to many OB neurons. RESULTS: Here, we use conditional gene ablation strategies to knockout Tbr2 during embryonic mouse olfactory bulb morphogenesis, as well as during perinatal and adult neurogenesis from the SVZ-RMS niche, and describe the resulting phenotypes. We find that Tbr2 is important for the generation of mitral cells in the OB, and that the olfactory bulbs themselves are hypoplastic and disorganized in Tbr2 mutant mice. Furthermore, we show that the SVZ-RMS niche is expanded and disordered following loss of Tbr2, which leads to ectopic accumulation of neuroblasts in the RMS. Lastly, we show that adult glutamatergic neurogenesis from the SVZ is impaired by loss of Tbr2. CONCLUSIONS: Tbr2 is essential for proper morphogenesis of the OB and SVZ-RMS, and is important for the generation of multiple lineages of glutamatergic olfactory bulb neurons.

Asymmetric neural development in the Caenorhabditis elegans olfactory system.

Asymmetries in the nervous system have been observed throughout the animal kingdom. Deviations of brain asymmetries are associated with a variety of neurodevelopmental disorders; however, there has been limited progress in determining how normal asymmetry is established in vertebrates. In the Caenorhabditis elegans chemosensory system, two pairs of morphologically symmetrical neurons exhibit molecular and functional asymmetries. This review focuses on the development of antisymmetry of the pair of amphid wing "C" (AWC) olfactory neurons, from transcriptional regulation of general cell identity, establishment of asymmetry through neural network formation and calcium signaling, to the maintenance of asymmetry throughout the life of the animal. Many of the factors that are involved in AWC development have homologs in vertebrates, which may potentially function in the development of vertebrate brain asymmetry.

Developmental expression of the calcium-activated chloride channels TMEM16A and TMEM16B in the mouse olfactory epithelium.

Calcium-activated chloride channels are involved in several physiological processes including olfactory perception. TMEM16A and TMEM16B, members of the transmembrane protein 16 family (TMEM16), are responsible for calcium-activated chloride currents in several cells. Both are present in the olfactory epithelium of adult mice, but little is known about their expression during embryonic development. Using immunohistochemistry we studied their expression in the mouse olfactory epithelium at various stages of prenatal development from embryonic day (E) 12.5 to E18.5 as well as in postnatal mice. At E12.5, TMEM16A immunoreactivity was present at the apical surface of the entire olfactory epithelium, but from E16.5 became restricted to a region near the transition zone with the respiratory epithelium, where localized at the apical part of supporting cells and in their microvilli. In contrast, TMEM16B immunoreactivity was present at E14.5 at the apical surface of the entire olfactory epithelium, increased in subsequent days, and localized to the cilia of mature olfactory sensory neurons. These data suggest different functional roles for TMEM16A and TMEM16B in the developing as well as in the postnatal olfactory epithelium. The presence of TMEM16A at the apical part and in microvilli of supporting cells is consistent with a role in the regulation of the chloride ionic composition of the mucus covering the apical surface of the olfactory epithelium, whereas the localization of TMEM16B to the cilia of mature olfactory sensory neurons is consistent with a role in olfactory signal transduction.

Olfactory wiring logic in amphibians challenges the basic assumptions of the unbranched axon concept.

Olfactory receptor neurons extend axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. The consensus view is that in vertebrates individual receptor neurons project unbranched axons into one specific glomerulus of the olfactory bulb. We report here that, strikingly different from the generally assumed wiring principle in vertebrate olfactory systems, axons of single receptor neurons of Xenopus laevis regularly bifurcate and project into more than one glomerulus. Specifically, the innervation of multiple glomeruli is present in all ontogenetic stages of this species, from the larva to the postmetamorphic frog. Also, we show that this unexpected wiring pattern is not restricted to axons of immature receptor neurons, but that it is also a feature of mature neurons of both the main and accessory olfactory system. This glomerular innervation pattern is unique among vertebrates investigated so far and represents a new olfactory wiring strategy.

Possible roles of Robo1+ ensheathing cells in guiding dorsal-zone olfactory sensory neurons in mouse.

In the mouse olfactory system, the anatomical locations of olfactory sensory neurons (OSNs) correlate with their axonal projection sites along the dorsoventral axis of the olfactory bulb (OB). We have previously reported that Neuropilin-2 expressed by ventral-zone OSNs contributes to the segregation of dorsal and ventral OSN axons, and that Slit is acting as a negative land mark to restrict the projection of Robo2+, early-arriving OSN axons to the embryonic OB. Here, we report that another guidance receptor, Robo1, also plays an important role in guiding OSN axons. Knockout mice for Robo1 demonstrated defects in targeting of OSN axons to the OB. Although Robo1 is colocalized with dorsal-zone OSN axons, it is not produced by OSNs, but instead by olfactory ensheathing cells. These findings indicate a novel strategy of axon guidance in the mouse olfactory system during development.

The zinc finger transcription factor Jing is required for dendrite/axonal targeting in Drosophila antennal lobe development.

An important role in olfactory system development is played by transcription factors which act in sensory neurons or in their interneuron targets as cell autonomous regulators of downstream effectors such as cell surface molecules and signalling systems that control neuronal identity and process guidance. Some of these transcriptional regulators have been characterized in detail in the development of the neural elements that innervate the antennal lobe in the olfactory system of Drosophila. Here we identify the zinc finger transcription factor Jing as a cell autonomously acting transcriptional regulator that is required both for dendrite targeting of projection neurons and local interneurons as well as for axonal targeting of olfactory sensory neurons in Drosophila olfactory system development. Immunocytochemical analysis shows that Jing is widely expressed in the neural cells during postembryonic development. MARCM-based clonal analysis of projection neuron and local interneuron lineages reveals a requirement for Jing in dendrite targeting; Jing loss-of-function results in loss of innervation in specific glomeruli, ectopic innervation of inappropriate glomeruli, aberrant profuse dendrite arborisation throughout the antennal lobe, as well as mistargeting to other parts of the CNS. ey-FLP-based MARCM analysis of olfactory sensory neurons reveals an additional requirement for Jing in axonal targeting; mutational inactivation of Jing causes specific mistargeting of some olfactory sensory neuron axons to the DA1 glomerulus, reduction of targeting to other glomeruli, as well as aberrant stalling of axons in the antennal lobe. Taken together, these findings indicate that Jing acts as a key transcriptional control element in wiring of the circuitry in the developing olfactory sensory system in Drosophila.

Odorant responsiveness of embryonic mouse olfactory sensory neurons expressing the odorant receptors S1 or MOR23.

The mammalian olfactory system has developed some functionality by the time of birth. There is behavioral and limited electrophysiological evidence for prenatal olfaction in various mammalian species. However, there have been no reports, in any mammalian species, of recordings from prenatal olfactory sensory neurons (OSNs) that express a given odorant receptor (OR) gene. Here we have performed patch-clamp recordings from mouse OSNs that express the OR gene S1 or MOR23, using the odorous ligands 2-phenylethyl alcohol or lyral, respectively. We found that, out of a combined total of 20 OSNs from embryos of these two strains at embryonic day (E)16.5 or later, all responded to a cognate odorous ligand. By contrast, none of six OSNs responded to the ligand at E14.5 or E15.5. The kinetics of the odorant-evoked electrophysiological responses of prenatal OSNs are similar to those of postnatal OSNs. The S1 and MOR23 glomeruli in the olfactory bulb are formed postnatally, but the axon terminals of OSNs expressing these OR genes may be synaptically active in the olfactory bulb at embryonic stages. The upper limit of the acquisition of odorant responsiveness for S1 and MOR23 OSNs at E16.5 is consistent with the developmental expression patterns of components of the olfactory signaling pathway.

The microRNA mir-71 inhibits calcium signaling by targeting the TIR-1/Sarm1 adaptor protein to control stochastic L/R neuronal asymmetry in C. elegans.

The Caenorhabditis elegans left and right AWC olfactory neurons communicate to establish stochastic asymmetric identities, AWC(ON) and AWC(OFF), by inhibiting a calcium-mediated signaling pathway in the future AWC(ON) cell. NSY-4/claudin-like protein and NSY-5/innexin gap junction protein are the two parallel signals that antagonize the calcium signaling pathway to induce the AWC(ON) fate. However, it is not known how the calcium signaling pathway is downregulated by nsy-4 and nsy-5 in the AWC(ON) cell. Here we identify a microRNA, mir-71, that represses the TIR-1/Sarm1 adaptor protein in the calcium signaling pathway to promote the AWC(ON) identity. Similar to tir-1 loss-of-function mutants, overexpression of mir-71 generates two AWC(ON) neurons. tir-1 expression is downregulated through its 3' UTR in AWC(ON), in which mir-71 is expressed at a higher level than in AWC(OFF). In addition, mir-71 is sufficient to inhibit tir-1 expression in AWC through the mir-71 complementary site in the tir-1 3' UTR. Our genetic studies suggest that mir-71 acts downstream of nsy-4 and nsy-5 to promote the AWC(ON) identity in a cell autonomous manner. Furthermore, the stability of mature mir-71 is dependent on nsy-4 and nsy-5. Together, these results provide insight into the mechanism by which nsy-4 and nsy-5 inhibit calcium signaling to establish stochastic asymmetric AWC differentiation.

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.

IgSF8: a developmentally and functionally regulated cell adhesion molecule in olfactory sensory neuron axons and synapses.

Here, we investigated an Immunoglobulin (Ig) superfamily protein IgSF8 which is abundantly expressed in olfactory sensory neuron (OSN) axons and their developing synapses. We demonstrate that expression of IgSF8 within synaptic neuropil is transitory, limited to the period of glomerular formation. Glomerular expression decreases after synaptic maturation and compartmental glomerular organization is achieved, although expression is maintained at high levels within the olfactory nerve layer (ONL). Immunoprecipitations indicate that IgSF8 interacts with tetraspanin CD9 in the olfactory bulb (OB). CD9 is a component of tetraspanin-enriched microdomains (TEMs), specialized microdomains of the plasma membrane known to regulate cell morphology, motility, invasion, fusion and signaling, in both the nervous and immune systems, as well as in tumors. In vitro, both IgSF8 and CD9 localize to puncta within axons and growth cones of OSNs, consistent with TEM localization. When the olfactory epithelium (OE) was lesioned, forcing OSN regeneration en masse, IgSF8 was once again able to be detected in OSN axon terminals as synapses were reestablished. Finally, we halted synaptic maturation within glomeruli by unilaterally blocking functional activity and found that IgSF8 did not undergo exclusion from this subcellular compartment and instead continued to be detected in adult glomeruli. These data support the hypothesis that IgSF8 facilitates OSN synapse formation.

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.

Protein tyrosine phosphatase σ regulates the synapse number of zebrafish olfactory sensory neurons.

The formation and refinement of synaptic connections are key steps of neural development to establish elaborate brain networks. To investigate the functional role of protein tyrosine phosphatase (PTP) σ, we employed an olfactory sensory neuron (OSN)-specific gene manipulation system in combination with in vivo imaging of transparent zebrafish embryos. Knockdown of PTPσ enhanced the accumulation of synaptic vesicles in the axon terminals of OSNs. The exaggerated accumulation of synaptic vesicles was restored to the normal level by the OSN-specific expression of PTPσ, indicating that presynaptic PTPσ is responsible for the regulation of synaptic vesicle accumulation. Consistently, transient expression of a dominant-negative form of PTPσ in OSNs enhanced the accumulation of synaptic vesicles. The exaggerated accumulation of synaptic vesicles was reproduced in transgenic zebrafish lines carrying an OSN-specific expression vector of the dominant-negative PTPσ. By electron microscopic analysis of the transgenic line, we found the significant increase of the number of OSN-mitral cell synapses in the central zone of the olfactory bulb. The density of docked vesicles at the active zone was also increased significantly. Our results suggest that presynaptic PTPσ controls the number of OSN-mitral cell synapses by suppressing their excessive increase.

Fas-associated factor 1 as a regulator of olfactory axon guidance.

Axon guidance is a crucial part of neural circuit formation. While precise axonal targeting forms the basis of accurate information delivery, the mechanisms that regulate this process are still unclear. Apoptotic signaling molecules have been identified in the axon terminal, but their specific role in axon guidance is not well understood. Here we use the mouse olfactory system as an in vivo model to demonstrate that by modulating Fas-associated factor 1 (FAF1), an apoptosis regulatory molecule, we can rewire axonal projections. Interestingly, FAF1 is highly expressed in the developing mouse olfactory system, but its expression is downregulated postnatally. Using a tetracycline-inducible promoter Tet-Off system, we generated transgenic mice in which FAF1 is specifically expressed in immature olfactory sensory neurons (OSNs) and show that overexpression of FAF1 not only misroutes OSN axons to deep layers of the olfactory bulb but also leads to widespread disruption of the glomerular layer. In addition, we also demonstrate that the specific convergence of P2 receptor OSN axons is completely distorted in the FAF1 mice. Strikingly, all of the mutant phenotypes can be recovered by shutting down FAF1 expression through the administration of doxycycline. Together, our study provides clear in vivo evidence that an apoptotic molecule can indeed regulate axon targeting and that OSNs can restore their organization even after broad disruption.

Composition of the migratory mass during development of the olfactory nerve.

The embryonic development of the olfactory nerve includes the differentiation of cells within the olfactory placode, migration of cells into the mesenchyme from the placode, and extension of axons by the olfactory sensory neurons (OSNs). The coalition of both placode-derived migratory cells and OSN axons within the mesenchyme is collectively termed the "migratory mass." Here we address the sequence and coordination of the events that give rise to the migratory mass. Using neuronal and developmental markers, we show subpopulations of neurons emerging from the placode by embryonic day (E)10, a time at which the migratory mass is largely cellular and only a few isolated OSN axons are seen, prior to the first appearance of OSN axon fascicles at E11. These neurons also precede the emergence of the gonadotropin-releasing hormone neurons and ensheathing glia which are also resident in the mesenchyme as part of the migratory mass beginning at about E11. The data reported here begin to establish a spatiotemporal framework for the migration of molecularly heterogeneous placode-derived cells in the mesenchyme. The precocious emigration of the early arriving neurons in the mesenchyme suggests they may serve as "guidepost cells" that contribute to the establishment of a scaffold for the extension and coalescence of the OSN axons.

Expression and function of the empty spiracles gene in olfactory sense organ development of Drosophila melanogaster.

In Drosophila, the cephalic gap gene empty spiracles plays key roles in embryonic patterning of the peripheral and central nervous system. During postembryonic development, it is involved in the development of central olfactory circuitry in the antennal lobe of the adult. However, its possible role in the postembryonic development of peripheral olfactory sense organs has not been investigated. Here, we show that empty spiracles acts in a subset of precursors that generate the olfactory sense organs of the adult antenna. All empty spiracles-expressing precursor cells co-express the proneural gene amos and the early patterning gene lozenge. Moreover, the expression of empty spiracles in these precursor cells is dependent on both amos and lozenge. Functional analysis reveals two distinct roles of empty spiracles in the development of olfactory sense organs. Genetic interaction studies in a lozenge-sensitized background uncover a requirement of empty spiracles in the formation of trichoid and basiconic olfactory sensilla. MARCM-based clonal mutant analysis reveals an additional role during axonal targeting of olfactory sensory neurons to glomeruli within the antennal lobe. Our findings on empty spiracles action in olfactory sense organ development complement previous studies that demonstrate its requirement in olfactory interneurons and, taken together with studies on the murine homologs of empty spiracles, suggest that conserved molecular genetic programs might be responsible for the formation of both peripheral and central olfactory circuitry in insects and mammals.

Embryonic Pax7-expressing progenitors contribute multiple cell types to the postnatal olfactory epithelium.

Prolonged neurogenesis driven by stem/progenitor cells is a hallmark of the olfactory epithelium (OE), beginning at the placodal stages in the embryo and continuing throughout adult life. Despite the progress made to identify and study the regulation of adult OE progenitors, our knowledge of embryonic OE precursors and their cellular contributions to the adult OE has been stalled by the lack of markers able to distinguish individual candidate progenitors. Here we identify embryonic OE Pax7+ progenitors, detected at embryonic day 10.5 (E10.5) in the olfactory pit with an antigen profile and location previously assigned to presumptive OE stem cells. Using Cre-loxP technology (Pax7-cre/ROSA YFP mice), we expose a wide range of derivatives, including CNS and olfactory neurons, non-neuronal cells, and olfactory ensheathing glia, all made from embryonic Pax7+ cells. Importantly, the expression of Pax7 in the embryonic OE is downregulated from E15.5, such that after birth, no Pax7+ cells are found in the OE, and thus the progenitor population here identified is restricted to embryonic stages. Our results provide the first evidence for a population of Pax7-expressing embryonic progenitors that contribute to multiple OE lineages and demonstrate novel insights into the unique spatiotemporal patterning of the postnatal OE.

Cis-regulatory mechanisms of gene expression in an olfactory neuron type in Caenorhabditis elegans.

The generation of cellular diversity is dependent on the precise spatiotemporal regulation of gene expression by both cis- and trans-acting mechanisms. The developmental principles regulating expression of specific gene subsets in individual cell types are not fully understood. Here we define the cis-regulatory mechanisms driving expression of cell-selective and broadly expressed genes in vivo in the AWB olfactory neuron subtype in C. elegans. We identify an element that is necessary to drive expression of neuron-selective chemoreceptor genes in the AWB neurons, and show that this element functions in a context-dependent manner. We find that the expression of broadly expressed sensory neuronal genes in the AWB neurons is regulated by diverse cis- and trans-regulatory mechanisms that act partly in parallel to the pathways governing expression of AWB-selective genes. We further demonstrate that cis-acting mechanisms driving gene expression in the AWB neurons appear to have diverged in related nematode species. Our results provide insights into the cis-regulatory logic driving cell-specific gene expression, and suggest that variations in this logic contribute to the generation of functional diversity.