Olfactory Axon Targeting Research Articles

Insights into olfactory ensheathing cell development from a laser-microdissection and transcriptome-profiling approach.

Olfactory ensheathing cells (OECs) are neural crest-derived glia that ensheath bundles of olfactory axons from their peripheral origins in the olfactory epithelium to their central targets in the olfactory bulb. We took an unbiased laser microdissection and differential RNA-seq approach, validated by in situ hybridization, to identify candidate molecular mechanisms underlying mouse OEC development and differences with the neural crest-derived Schwann cells developing on other peripheral nerves. We identified 25 novel markers for developing OECs in the olfactory mucosa and/or the olfactory nerve layer surrounding the olfactory bulb, of which 15 were OEC-specific (that is, not expressed by Schwann cells). One pan-OEC-specific gene, Ptprz1, encodes a receptor-like tyrosine phosphatase that blocks oligodendrocyte differentiation. Mutant analysis suggests Ptprz1 may also act as a brake on OEC differentiation, and that its loss disrupts olfactory axon targeting. Overall, our results provide new insights into OEC development and the diversification of neural crest-derived glia.

Computationally Modeling Axonal Guidance Cue Expression in The Developing Olfactory Bulb

Olfactory sensory neurons (OSNs) in most mammals have cell bodies in the olfactory epithelium (OE). OSNs project axons to glomeruli in the olfactory bulb (OB) of the brain. Developmentally, signal guidance cues direct axons to their target glomeruli. We created a program that displays a model of the OB using a series of sagittal section images based on in situ hybridization (ISH) images from the Allen Brain Atlas (ABA). The ABA's program, Brain Explorer 2, allows for the limited 3D visualization of gene expression data. Our program adds functionality by allowing users to clearly visualize and quantify the expression of several genes simultaneously via superimposition.To optimize visualization of multiple ISH data sets simultaneously, we aimed to improve the signal to noise ration of those ISH data. We use color thresholding and density thresholding algorithms to computationally detect and filter guidance cue expression data. Color thresholds that establish gene expression from the background of the image were determined using ImageJ. The expression data were filtered using a density threshold in a 2D sliding window algorithm. The 2D sliding window algorithm is implemented for every pixel in the image. It distinguishes a window of the image around each pixel. The density of gene expression within that window is calculated. The expression for the current pixel is then decided using a density threshold. Methods for visualization of the superimposed genes were derived using the matplotlib and PIL libraries. Our program provides a tool for investigating olfactory axon targeting, glomeruli formation and glomeruli related pathologies in mammalian models. For example, users can examine the expression of different genes and investigate if guidance cue expression results in unique locations for mapping specific glomeruli. This program can also be adapted to examine guidance cue expression for other regions of nervous tissue.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Olfactory ensheathing cells abutting the embryonic olfactory bulb express Frzb, whose deletion disrupts olfactory axon targeting.

We and others previously showed that in mouse embryos lacking the transcription factor Sox10, olfactory ensheathing cell (OEC) differentiation is disrupted, resulting in defective olfactory axon targeting and fewer gonadotropin‐releasing hormone (GnRH) neurons entering the embryonic forebrain. The underlying mechanisms are unclear. Here, we report that OECs in the olfactory nerve layer express Frzb—encoding a secreted Wnt inhibitor with roles in axon targeting and basement membrane breakdown—from embryonic day (E)12.5, when GnRH neurons first enter the forebrain, until E16.5, the latest stage examined. The highest levels of Frzb expression are seen in OECs in the inner olfactory nerve layer, abutting the embryonic olfactory bulb. We find that Sox10 is required for Frzb expression in OECs, suggesting that loss of Frzb could explain the olfactory axon targeting and/or GnRH neuron migration defects seen in Sox10‐null mice. At E16.5, Frzb‐null embryos show significant reductions in both the volume of the olfactory nerve layer expressing the maturation marker Omp and the number of Omp‐positive olfactory receptor neurons in the olfactory epithelium. As Omp upregulation correlates with synapse formation, this suggests that Frzb deletion indeed disrupts olfactory axon targeting. In contrast, GnRH neuron entry into the forebrain is not significantly affected. Hence, loss of Frzb may contribute to the olfactory axon targeting phenotype, but not the GnRH neuron phenotype, of Sox10‐null mice. Overall, our results suggest that Frzb secreted from OECs in the olfactory nerve layer is important for olfactory axon targeting.

Haploinsufficiency of SIX3 Abolishes Male Reproductive Behavior Through Disrupted Olfactory Development, and Impairs Female Fertility Through Disrupted GnRH Neuron Migration.

Mating behavior in males and females is dependent on olfactory cues processed through both the main olfactory epithelium (MOE) and the vomeronasal organ (VNO). Signaling through the MOE is critical for the initiation of male mating behavior, and the loss of MOE signaling severely compromises this comportment. Here, we demonstrate that dosage of the homeodomain gene Six3 affects the degree of development of MOE but not the VNO. Anomalous MOE development in Six3 heterozygote mice leads to hyposmia, specifically disrupting male mounting behavior by impairing detection of volatile female estrus pheromones. Six3 is highly expressed in the MOE, main olfactory bulb (MOB), and hypothalamus; all regions essential in the proper migration of the gonadotropin-releasing hormone (GnRH) neurons, a key reproductive neuronal population that migrates along olfactory axons from the developing nose into the brain. Interestingly, we find that the reduction in Six3 expression in Six3 heterozygote mice compromises development of the MOE and MOB, resulting in mis-migration of GnRH neurons due to improper olfactory axon targeting. This reduction in the hypothalamic GnRH neuron population, by 45% in adulthood, leads to female subfertility, but does not impact male hormone levels, suggesting that male infertility is not related to GnRH neuron numbers, but exclusively linked to abnormal olfaction. We here determine that Six3 is haploinsufficient for MOE development, GnRH neuron migration, and fertility, and represents a novel candidate gene for Kallmann syndrome, a form of inherited infertility.

Reproductive endocrine phenotypes relating to CHD7 mutations in humans.

Mutations in the gene CHD7 cause CHARGE syndrome, a rare multi-organ syndromic disorder. Gonadal defects are common in individuals with CHARGE syndrome (seen in ∼60-80% of cases) and represent the letter "G" in the CHARGE syndrome acronym. The gonadal defect in CHARGE syndrome results from congenital deficiency of the hypothalamic hormone Gonadotropin-releasing hormone (GnRH), which manifests clinically as pubertal failure and infertility, and biochemically as hypogonadotropic hypogonadism (low sex steroid hormone levels with inappropriately normal or low gonadotropin levels). In addition to the gonadal endocrine abnormalities, in a small minority of individuals with CHARGE, additional endocrine defects including growth hormone deficiency, multiple pituitary hormone deficits and primary hypothyroidism may also be seen. CHD7 mutations disrupt the targeting of olfactory axons and the migration of GnRH-synthesizing neurons during embryonic development, resulting in congenital idiopathic hypogonadotropic hypogonadism (IHH) and anosmia (or hyposmia), two features that define human Kallmann syndrome. Since Kallmann syndrome is one of the constituent phenotypes within CHARGE, recent studies have investigated the role of CHD7 mutations in individuals with IHH and established that deleterious missense mutations in CHD7 are associated with Kallmann syndrome as well as normosmic form of IHH. These missense mutations affect the ATPase and nucleosome remodeling activities of the CHD7 protein. These observations suggest that CHD7 protein function is critical for the ontogeny of GnRH neurons and neuroendocrine regulation of GnRH secretion.

Distinct and Cooperative Functions for the Protocadherin-α, -β and -γ Clusters in Neuronal Survival and Axon Targeting.

The clustered protocadherin (Pcdh) genes are divided into the Pcdhα, Pcdhβ, and Pcdhγ clusters. Gene-disruption analyses in mice have revealed the in vivo functions of the Pcdhα and Pcdhγ clusters. However, all Pcdh protein isoforms form combinatorial cis-hetero dimers and enter trans-homophilic interactions. Here we addressed distinct and cooperative functions in the Pcdh clusters by generating six cluster-deletion mutants (Δα, Δβ, Δγ, Δαβ, Δβγ, and Δαβγ) and comparing their phenotypes: Δα, Δβ, and Δαβ mutants were viable and fertile; Δγ mutants lived less than 12 h; and Δβγ and Δαβγ mutants died shortly after birth. The Pcdhα, Pcdhβ, and Pcdhγ clusters were individually and cooperatively important in olfactory-axon targeting and spinal-cord neuron survival. Neurodegeneration was most severe in Δαβγ mutants, indicating that Pcdhα and Pcdhβ function cooperatively for neuronal survival. The Pcdhα, Pcdhβ, and Pcdhγ clusters share roles in olfactory-axon targeting and neuronal survival, although to different degrees.

Olfactory ensheathing glia are required for embryonic olfactory axon targeting and the migration of gonadotropin-releasing hormone neurons.

SummaryKallmann's syndrome is caused by the failure of olfactory axons and gonadotropin-releasing hormone (GnRH) neurons to enter the embryonic forebrain, resulting in anosmia and sterility. Sox10 mutations have been associated with Kallmann's syndrome phenotypes, but their effect on olfactory system development is unknown. We recently showed that Sox10 is expressed by neural crest-derived olfactory ensheathing cells (OECs). Here, we demonstrate that in homozygous Sox10lacZ/lacZ mouse embryos, OEC differentiation is disrupted; olfactory axons accumulate in the ventromedial olfactory nerve layer and fewer olfactory receptor neurons express the maturation marker OMP (most likely owing to the failure of axonal targeting). Furthermore, GnRH neurons clump together in the periphery and a smaller proportion enters the forebrain. Our data suggest that human Sox10 mutations cause Kallmann's syndrome by disrupting the differentiation of OECs, which promote embryonic olfactory axon targeting and hence olfactory receptor neuron maturation, and GnRH neuron migration to the forebrain.

Linking Cell Fate, Trajectory Choice, and Target Selection: Genetic Analysis of Sema-2b in Olfactory Axon Targeting

Neural circuit assembly requires selection of specific cell fates, axonal trajectories, and synaptic targets. By analyzing the function of a secreted semaphorin, Sema-2b, in Drosophila olfactory receptor neuron (ORN) development, we identified multiple molecular and cellular mechanisms that link these events. Notch signaling limits Sema-2b expression to ventromedial ORN classes, within which Sema-2b cell-autonomously sensitizes ORN axons to external semaphorins. Central-brain-derived Sema-2a and Sema-2b attract Sema-2b-expressing axons to the ventromedial trajectory. In addition, Sema-2b/PlexB-mediated axon-axon interactions consolidate this trajectory choice and promote ventromedial axon-bundle formation. Selecting the correct developmental trajectory is ultimately essential for proper target choice. These findings demonstrate that Sema-2b couples ORN axon guidance to postsynaptic target neuron dendrite patterning well before the final target selection phase, and exemplify how a single guidance molecule can drive consecutive stages of neural circuit assembly with the help of sophisticated spatial and temporal regulation.

Odorant receptor proteins in olfactory axons and in cells of the cribriform mesenchyme may contribute to fasciculation and sorting of nerve fibers

Odorant receptors (ORs) have been shown to be present not only in the chemosensory cilia of the olfactory sensory neurons, but also in their axon terminals. This observation has emphasized the notion that the receptor protein may contribute to the precise receptor-specific targeting of olfactory axons in the olfactory bulb. This concept implies a particularly important role for the axonal receptor protein during the onset and early phase of the wiring process during development. In the present study, we have demonstrated, by means of specific antibodies, that, as early as mouse embryonic day E12, the OR protein can be visualized in outgrowing axonal processes of the olfactory epithelium and in cells located in the cribriform mesenchyme. On their trajectory from the olfactory epithelium through the cribriform mesenchyme toward the forebrain, axons with strong OR immunoreactivity have only been seen in the dorsal part of the mesenchyme where they traverse the region of OR-positive cells. Upon visualization by specific antibodies, these cells have been revealed to have long protrusions extending along the surface of nerve fascicles. They are often located at bifurcations where two small axon fascicles merge to form a stronger bundle. Within this region, fascicles coalesce forming a coherent nerve. Moreover, within the now compact nerve bundle, axons visualized by the OR-specific antibody are no longer distributed evenly but are segregated from other axonal populations within the nerve. These findings suggest that OR proteins in the membrane of axonal processes and of cells in the cribriform mesenchyme are involved in crucial processes such as fasciculation and the sorting of outgrowing axons, both of which are fundamental for the initiation and establishment of the precise wiring of the olfactory system during early development.

Olfactory receptor proteins in axonal processes of chemosensory neurons.

Olfactory receptors are supposed to act not only as molecular sensors for odorants but also as cell recognition molecules guiding the axons of olfactory neurons to their appropriate glomerulus in the olfactory bulb. This concept implies that olfactory receptor proteins are located in sensory cilia and in the axons. To approach this critical issue, antibodies were generated against two peptides, one derived from olfactory receptor mOR256-17, one derived from the "mOR37" subfamily. By means of immunohistochemistry and double-labeling studies using transgenic mouse lines as well as Western blot analyses, it was demonstrated that the newly generated antibodies specifically recognized the receptor proteins. To scrutinize the hypothesis that olfactory receptor proteins may also be present in the axonal processes and the nerve terminals, serial sections through the olfactory bulb were probed with the antibodies. Two glomeruli in each bulb were stained by anti-mOR256-17, one positioned in the medial, one in the lateral hemisphere. Fiber bundles approaching the glomeruli through the outer nerve layer also displayed intense immunofluorescence. A similar picture emerged for the antibody anti-mOR37, a small number of glomeruli in the ventral domain of the bulb was stained. On serial sections through the olfactory bulb of mOR37-transgenic mouse lines, double-labeling experiments demonstrated that distinct immunoreactive glomeruli corresponded to glomeruli that were targeted by neurons expressing a particular member of the mOR37 receptor subfamily. These data indicate that olfactory receptor (OR) proteins are indeed present in the axonal processes and nerve terminals of olfactory sensory neurons, thus supporting the notion that ORs may participate in the molecular processes underlying the fasciculation and targeting of olfactory axons.

Theoretical consideration of olfactory axon projection with an activity-dependent neural network model

Olfactory sensory neurons (OSNs) that express a given odorant receptor (OR) target their axons onto a few specific glomeruli in the olfactory bulb. Although the odorant receptor plays an indispensable role in olfactory axon targeting, the mechanisms underlying this guidance are largely unknown. In particular, there is much controversy regarding the involvement of activity-dependent mechanism in the targeting process. In this study, we developed an activity-dependent self-organization model of the glomerular layer in the olfactory bulb and simulated the targeting of olfactory axons onto the layer. Our model successfully constructed discrete glomeruli that received olfactory axons expressing a common odorant receptor through odorant-evoked neural activities. Furthermore, our model explained the perplexing experimental results that have been reported in olfactory axon targeting. For example, dispersion of olfactory axons in knockout mice for the odorant receptor gene was reasonably reproduced in the simulation. The segregated projection of the axons that express the same odorant receptor transcribed from the different alleles was also successfully simulated if the genetically modified allele was assumed to express a smaller amount of the receptor protein. The activity-dependent model even explained the inconsistent effects of disruption of the activity-evoking ion channel on axons expressing different odorant receptors, although some of these results were regarded as evidence for activity-independency of the olfactory targeting. Taken together, the activity-dependent targeting of olfactory axons seems to be a simple probable mechanism that can provide a unified explanation of glomerular formation.

Targeting of olfactory neurons.

Olfactory sensory neurons detect an enormous variety of small volatile molecules with extremely high sensitivity and specificity. The actual recognition and discrimination of odorous compounds is accomplished by specific receptor proteins located in the ciliary membrane of the sensory neurons. Axonal connections into the olfactory bulb, the first relay station for odor processing in the brain, are organized such that all neurons expressing the same odorant receptor converge their axons onto common glomeruli which are located at similar positions in all individuals from one species. For the establishment of this precise targeting of olfactory axons to their appropriate glomeruli, combinatorial functions of axon-associated cell adhesion molecules and odorant receptor proteins appear to be required. Odorants that stimulate distinct receptor cell populations will thereby activate a specific combination of glomeruli in the bulb; this characteristic activity pattern may be used by the system to encode the quality of a particular odorant.

Basic principles and molecular mechanisms of olfactory axon pathfinding.

The present review describes several lines of recent evidence providing new insights into the basic principles and mechanisms of axon projection from the olfactory epithelium to the olfactory bulb. Olfactory sensory neurons are classified into approximately 1000 subtypes according to the expression of specific odorant receptors. Olfactory sensory neurons expressing a given odorant receptor are distributed within one zone out of the four circumscribed zones of the olfactory epithelium and send their axons to the corresponding zone of the olfactory bulb: the principle of zone-to-zone projection. We discuss possible functions of a novel cell adhesion molecule, viz., OCAM, in the formation and maintenance of zone-to-zone projection of both olfactory and vomeronasal axons. Furthermore, olfactory sensory neurons expressing a given odorant receptor converge their axons onto only two topographically fixed glomeruli among the 1500-3000 glomeruli in the olfactory bulb: the principle of glomerular convergence. These axonal connection patterns give rise to the response specificity of the second-order neurons, viz., the mitral/tufted cells, to a particular range of odor molecules. In the process of glomerular convergence, combinatorial functions of axon-associated cell adhesion molecules and odorant receptor proteins may be required for the establishment of the precise targeting of olfactory axons to the appropriate glomeruli.