Thesis
Reference
From elements of the olfactory transduction cascade to a precise glomerular map
DAL COL, Julien
Abstract
Animals benefit from a number of sensory tools that allow them to perceive the world, and thus to interact adequately with this latter. In a large number of mammalian species, one of these senses, olfaction, if particularly developed. This sense allows the perception of millions of different stimuli and to extract information from them, without prior experience of these molecules. This remarkable ability is based on the expression of specific odorant receptors by sensory neurons located in the nasal cavity. The genes coding for these receptors are numerous (over 1500 in the mouse) and variable. Each neuron expresses a single of these genes, providing to each sensory cell a defined and limited functionality. All axonal projections emanating from neurons that express the same odorant receptor gene converge in the olfactory bulb, forming spherical structures called glomeruli.
DAL COL, Julien. From elements of the olfactory transduction cascade to a precise glomerular map. Thèse de doctorat : Univ. Genève, 2010, no. Sc. 4279
URN : urn:nbn:ch:unige-142697
DOI : 10.13097/archive-ouverte/unige:14269
Available at:
http://archive-ouverte.unige.ch/unige:14269
Disclaimer: layout of this document may differ from the published version.
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Département de Zoologie Professeur Ivan Rodriguez et Biologie Animale
_____________________________________________________________________
From Elements of the Olfactory Transduction Cascade to a Precise Glomerular Map
THÈSE
Présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès Sciences, mention Biologie
par
Julien DAL COL de
Genève (GE) Suisse
Thèse n°4279
Genève
Atelier d’impression ReproMail 2010
Résumé
Les êtres vivants disposent d’une série d’outils sensoriels qui leurs permettent de percevoir le monde extérieur, et donc de pouvoir interagir adéquatement avec celui-ci. Chez beaucoup de mammifères, un de ces sens, l’olfaction, est particulièrement développé. Il permet de faire face à des millions de stimuli différents et d’en extraire une information sans expérience préalable de ces signaux.
Cette capacité est basée sur l’expression de récepteurs à odorants par des neurones sensoriels localisés dans la cavité nasale. Les gènes codant pour ces récepteurs, particulièrement nombreux et variés, sont au nombre de 1500 chez la souris. Chaque neurone exprime un seul de ces gènes, lui conférant donc une fonctionnalité définie et limitée. Les axones des neurones sensoriels exprimant le même gène de récepteur vont converger dans le bulbe olfactif, formant des structures sphériques, appelées glomérules. A chaque récepteur correspondent seulement deux glomérules par bulbe, un médial et un latéral, innervés par des neurones localisés respectivement dans la partie médiale (septale) ou latérale de la cavité nasale. La position de ces glomérules est également dépendante du récepteur exprimé. L’information sensorielle primaire est donc véhiculée par des lignes organisées en parallèle qui sont à la base du codage olfactif périphérique.
Ces lignes s’organisent en une carte topographique olfactive précise, constituée de milliers de glomérules dans le bulbe.
A ce jour, nous ne comprenons pas les mécanismes qui permettent ce câblage remarquable, mais nous disposons de pistes, en particulier celle d’un rôle potentiel joué par le récepteur olfactif lui même dans la guidance axonale. Ce travail s’inscrit dans cette réflexion.
Nous avons tenté, chez la souris, d’évaluer le rôle joué par l’activation des récepteurs olfactifs dans le guidage, en analysant des mutants nuls pour l’adénylyl cyclase 3 (un élément central de la cascade de transduction olfactive).
Nous avons observé chez ces animaux des altérations massives des projections olfactives, tant au niveau de la carte topographique (qui se rostralise), qu’au niveau de l’homogénéité fonctionnelle des glomérules (qui sont coinnervés par des populations distinctes). Un rôle majeur est donc joué par cette cyclase, et possiblement par la cascade olfactive, dans l’établissement de la carte topographique formée dans le bulbe.
Nous avons observé qu’une déficience de l’adénylyl cyclase 3 s’accompagnait de la modification transcriptionelle de nombreux autres gènes, notamment celui codant pour la Neuropiline-1. Nous avons donc examiné des souris mutantes conditionnelles pour cette protéine de guidance, et avons observé des altérations frappantes dans l’organisation des glomérules. Ces altérations incluent la modification des positions topographiques des glomérules, et de façon plus surprenante, l’apparition de liens directs entre glomérules médiaux et latéraux, résultant dans certains cas en une fusion des deux glomérules. La Neuropiline-1
joue donc un rôle critique dans la formation ou le maintien de la symétrie latéromédiale intrabulbaire.
Dans la deuxième partie de ce travail, nous nous sommes intéressés à une population de neurones sensoriels atypique que nous avons identifiée dans l’épithélium olfactif majeur. Cette population exprime TrpC2, un marqueur théoriquement spécifique et exclusif de la cascade induite par la détection de phéromones dans les neurones voméronasaux. Dans l’épithélium olfactif majeur, cette expression est principalement restreinte aux neurones jeunes, et diminue avec l’âge de ces derniers. Cette population neuronale a d’autres caractéristiques uniques : ils sont principalement localisés dans la partie apicale du neuroépithélium, et ils n’expriment pas les autres marqueurs connus des neurones olfactifs conventionnels, incluant le marqueur pan-olfactif OMP.
Nous avons poursuivi l’étude de ces neurones par une approche transgénique permettant de suivre les projections axonales des neurones exprimants TrpC2. Cette approche nous a révélé que ces derniers projettent dans le bulbe dans une région bien définie : leurs glomérules sont regroupés et se situent dans la partie rostromédiale du bulbe olfactif. Sachant qu’à certaines molécules partageant certains groupes chimiques correspondent des zones d’activation spécifiques dans le bulbe, le regroupement particulier des projections des neurones exprimant TrpC2 suggère naturellement que ces neurones expriment des récepteurs appartenant à une même classe. En conclusion, nous avons possiblement identifié une nouvelle sous population de neurones olfactifs.
Abstract
Animals benefit from a number of sensory tools that allow them to perceive the world, and thus to interact adequately with this latter. In a large number of mammalian species, one of these senses, olfaction, if particularly developed. This sense allows the perception of millions of different stimuli and to extract information from them, without prior experience of these molecules.
This remarkable ability is based on the expression of specific odorant receptors by sensory neurons located in the nasal cavity. The genes coding for these receptors are numerous (over 1500 in the mouse) and variable. Each neuron expresses a single of these genes, providing to each sensory cell a defined and limited functionality. All axonal projections emanating from neurons that express the same odorant receptor gene converge in the olfactory bulb, forming spherical structures called glomeruli. In each olfactory bulb, a pair of glomeruli (one medial and one lateral, innervated by neurons located respectively medially and laterally in the nasal cavity) corresponds to a given receptor. The position of these glomeruli is also dependent on the receptor identity. Primary sensory information is thus transmitted through parallel lines, which are at the base of the olfactory peripheral coding logic. These lines are organized into a precise olfactory topographical map, made of thousands of glomeruli.
To date, we do not understand the molecular mechanisms responsible for this remarkable wiring, although published and unpublished data support a role played by the odorant receptor itself in axonal guidance processes. This work builds on these observations.
We tried to evaluate, in the mouse, the role played by the activation of odorant receptors in axon guidance processes. To this aim, we used null mutant of an essential element of the odorant receptor transduction cascade: adenylyl cyclase 3. We observed massive alterations of olfactory projections in these animals, both at the level of the topographical map (a map that is rostralized) and at the level of glomerular homogeneity (glomeruli are coinnervated by functionally distinct sensory neurons). A major role is thus played by this cyclase, and possibly by the olfactory cascade, in the establishment of the topographical map formed in the bulb.
We observed that the lack of adenylyl cyclase 3 leads to the transcriptional alteration of multiple genes, among which the one coding for Neuropilin-1. We therefore examined conditionally deficient mice for this guidance molecule, and observed striking alterations in the organization of glomeruli. These alterations include the modification of glomerular topographical positions, but more surprisingly, direct links between lateral and medial glomeruli, that culminate in some instances in the complete fusion of the medial and lateral glomeruli.
Neuropilin-1 plays thus a critical role in the formation or maintenance of the intrabulbar symmetry.
In the second part of this work, we investigated an atypical population of sensory neurons that we identified in the main olfactory neuroepithelium. This population expresses TrpC2, a marker specific of the pheromone-induced transduction cascade in vomeronasal neurons. This expression is mostly restricted to young sensory neurons, and decreases with the age of these latter.
These TrpC2-expressing neurons exhibit other unique characteristics: they are localized in the apical part of the neuroepithelium, and they do not express markers present in known olfactory populations, including OMP, the olfactory marker protein.
We further investigated these neurons by taking a transgenic approach allowing the visualization of the projections of TrpC2-expressing neurons. This revealed an interesting projection pattern: all labeled glomeruli are restricted to a rostromedial zone in the olfactory bulb. Considering that molecules sharing similar chemical characteristics activate specific zones of the bulb, the grouping of TRPC2 glomeruli in the bulb suggests that the TRPC2 population present in the main olfactory epithelium shares the expression of a class of olfactory receptors. Taken together, these observations support the existence of a novel population of sensory neurons in the main olfactory epithelium.
Résumé 5
Abstract 7
Introduction 11
The Mammalian Olfactory System(s) 11
The Main Olfactory System 12
The Vomeronasal System 17
The Grüneberg Ganglion 22
Axon Guidance… 23
… in the Olfactory System 23
Trp Channel Subunits 37
Objectives 39
Study 1: Olfactory Transduction and Axonal Guidance 39
Study 2: A TRPC2-expressing Sensory Population in the Main Olfactory Epithelium 39
Results 41
Study 1: Olfactory Transduction and Axonal Guidance 41
Expression of Adcy3 in the Olfactory System 41
Adcy3-deficient OSNs Target Aberrantly to the Olfactory Bulb 42
Anteroposterior Glomerular Shift in AC3-deficient OSNs 45
Choice of a Glomerulus 49
Lack of Nrp1 Expression in Adcy3-/- OSN projections 51
Transcriptome of Adcy3-/- OSNs 52
Adcy3 and OR Negative Feedback 55
Globally Unaltered Projections in Mice Lacking Nrp1 in Mature OSNs 56
Altered Glomerular Position in NRP1-deficient Mature OSNs 57
Early Lack of NRP1 Leads to the Fusion of the Lateral and Medial M71 Glomeruli 59 Study 2: A TrpC2-expressing Sensory Population in the Main Olfactory Epithelium 62 Transient Expression of TrpC2 in the Main Olfactory Epithelium 62
Permanent Labeling of TrpC2-expressing OSNs 64
Characterisation of the TrpC2 Expressors 64
Axonal Projections of TrpC2-expressing OSNs 66
Discussion 67
Study 1: Olfactory Transduction and Axonal Guidance 67
cAMP as a Major Player in OSN Axonal Guidance 67
AC3-Dependent and -Independent OSN Targeting 68
AC3 and Glomerular Formation 68
Adcy3 and Negative Feedback 69
AC3-dependent Genes 70
A Link Between AC3 and NRP1 70
NRP1-dependent OSN Targeting 71
Factor(s) Mediating Convergence of Like Fibers 72
Conclusion 73
Study 2: A TrpC2-expressing Sensory Population in the Main Olfactory Epithelium 74
A VSN Marker in the Main Olfactory Epithelium 74
Origin of the TrpC2-expressing OSN Population 74
The Zone-to-Zone Projection Rule Broken? 75
A Potential Substrate to Explain Behavioral Discrepancies Between TrpC2 Knockouts and
vomeronasal organ-ablated Animals 76
Conclusion 77
Materials and Methods 78
Mouse Lines 78
Cloning Strategy for TrpC2-IRES-Cre-IRES-GFP 78
Whole Mount Analyses 80
Microscopy 80
RT-PCRs 80
In Situ Hybridizations 80
Affymetrix Oligonucleotide Microarray Hybridization 81
Western Blots 81
Immunohistochemistry 82
Appendix 83
Abbreviations 83
Supplementary Figures 84
Acknowledgements 93
Bibliography 94
Introduction
Sensory systems allow us to extract visual, chemical, auditory and tactile information from our environment. Many higher organisms, including most mammals, rely heavily on olfaction. Chemosensory perception is probably the most ancient tool to evaluate the environment. It represents a highly valuable source of information concerning the surroundings of animals (and other living organisms). It serves both unidirectional (environment probing) as well as bidirectional (communication) purposes and is thus critical for survival and reproduction. However, the functioning of this sensory system is not fully understood.
Mice represent a very appropriate model system to study olfaction. This species developed a highly efficient sense of smell that is crucial to its survival. In addition, this species is very amenable to study this sense since its whole genome is sequenced and can be easily altered.
The Mammalian Olfactory System(s)
Mammals evolved a diversified toolbox to probe their environment. These tools are composed of peripheral sensory neurons that are in contact with the outside world. In the olfactory system, these sensory neurons are directly connected to the central nervous system. Several olfactory subsystems coexist in the mammalian nasal cavity. These include the main olfactory system, the vomeronasal system and the Grüneberg ganglion. These subsystems are composed of different anatomical structures (Figure 1) containing different types of sensory neurons. Each subsystem has thus specific molecular and functional properties. Each of them will be discussed here.
Figure 1: The different subsystems in the mouse olfactory system. The septal organ is roughly considered an extension of the main olfactory system and will not be discussed further. The insert shows a close-up of the Grüneberg ganglion. The white dashed line represents the border between the nasal cavity and the brain, and corresponds to the cribiform plate (see text). GG:
Grüneberg ganglion, VNO: vomeronasal organ, SO: septal organ, MOE: main olfactory epithelium, OB: olfactory bulb.
Inside each subsystem, sensory neurons are also divided into functionally different subpopulations. Each of them is defined by the expression of a given sensory receptor gene. This is an important notion in mammalian olfaction (and vision (Mazzoni et al., 2004)) called the “one neuron, one receptor” rule (Serizawa et al., 2004). It relies on a mechanism apparently shared among olfactory subsystems (Capello et al., 2009). It is noticeable that, in olfactory neurons, this monogenic expression extends to the allelic level (Chess et al., 1994; Rodriguez et al., 1999), i.e. a neuron expresses a given receptor gene from either the paternal or the maternal allele (monoallelic expression). This is at the base of the coding logic in the olfactory system. Subpopulations of sensory neurons, each expressing a given receptor, constitute parallel lines of information that are integrated in the brain. The combinatorial activation patterns are translated into meaningful signals such as what we perceive as odors, for instance.
The Main Olfactory System
The main olfactory system allows the perception of odorant molecules. It is the substrate of what is commonly called the sense of smell. Olfactory signals emanating from food, smoke (useful for fire detection) or flowers, for instance, are perceived by this subsystem.
The main olfactory epithelium is a pseudostratified neuroepithelium located in the back of the nasal cavity. It lies on cartilaginous folds called turbinates (increasing its sensory surface), and is covered by mucus. The main olfactory epithelium contains olfactory sensory neurons (OSNs), which are bipolar cells extending their unique dendrite towards the lumen of the nasal cavity (the external world). Each dendrite ends up in 20-30 cilia, surrounded by mucus and harboring receptors. Each OSN projects its single axon towards a brain region called the main olfactory bulb. On their way towards the bulb, axons pass through the cribiform plate, a bony structure containing multiple microscopic holes, providing an entrance into the skull. At the surface of the main olfactory bulb, OSNs synapse with mitral and tufted cells. These relay information to higher brain centers. Inhibitory interneurons (periglomerular and granule cells) are also present in the main olfactory bulb. OSNs have a life span of about two months and are continuously renewed from a pool of basal self-renewing progenitors. Interneurons in the olfactory bulb are also renewed by progenitors coming from the subventricular zone via the rostral migratory stream.
Figure 2: Cellular populations of the olfactory bulb. White arrows denote excitatory synapses, and black arrows denote inhibitory synapses. OSN: olfactory sensory neurons, PG:
periglomerular cells, T: tufted cells, M: mitral cells, Gr: granule cells, GL: glomerulus (From (Mori et al., 1999)).
OSNs transcribe chemoreceptor genes belonging to different families: odorant receptors (ORs), trace amine-associated receptors (TAARs) or guanylyl cyclase 2D (GC-D). As previously mentioned, each neuron will express a single receptor gene (from one of these families). Monoallelic expression of TAAR genes in OSNs has not been tested yet, while both alleles of the gene encoding GC-D are expressed (Walz et al., 2007).
Odorant Receptors
ORs are seven transmembrane domain (7TM) G protein-coupled receptors (GPCRs) of the class A (rhodopsin-like1
1 Class A GPCRs are subdivided into 19 subfamilies but some receptors, like ORs, are
“unclassified”. Opsins belong to subfamily A16.
) family. They were discovered by Linda Buck and Richard Axel in 1991 (Buck and Axel, 1991) a finding recognized by the Nobel Prize in 2004. With over 1000 members in mammalian genomes (almost 900 and 400 functional OR genes in mice and humans, respectively (Glusman et al., 2001; Olender et al., 2008; Young et al., 2002; Zhang and Firestein, 2002)) they constitute the largest protein coding gene family. Mammalian OR genes are clustered in the genome (at multiple loci, on all chromosomes) and their coding sequences lie in a single exon. ORs are subdivided into two classes. Class I (or “fish-like”) OR genes are expressed in the dorsal zone of the main olfactory
epithelium, while class II (or “tetrapod-specific”) OR genes are expressed in the whole main olfactory epithelium. OSNs expressing a given OR gene are interspersed in a large but specific zones in the main olfactory epithelium. These zones are overlapping.
ORs are enriched on dendritic cilia, where signal detection takes place. Figure 3 shows the signal transduction cascade in OR-expressing OSNs. Upon ligand binding, the OR conformational modification induces the release of Gαolf, which can then stimulate adenylyl cyclase 3 (AC3) to produce cyclic adenosine monophosphate (cAMP) from adenosine triphosphate (ATP). As a 2nd messenger, cAMP triggers the opening of heteromeric cyclic nucleotide-gated channels (CNGCs) composed of CNGA2, CNGA4 and CNGB1b subunits. The resulting Ca2+
entry in the cell leads to the opening of chloride channels made of an anoctamin 22 splice variant (Stephan et al., 2009). Unlike other neurons, olfactory neurons actively maintain a high intracellular Cl- concentration (Kaneko et al., 2004; Reisert et al., 2005). The ligand-induced chloride efflux causes cell depolarization.
Figure 3: Schematics representing the signal transduction cascade in OR gene-expressing OSNs.
OR: odorant receptor, Gα, Gβ, Gγ: G protein subunits, GTP: guanosine triphosphate, GDP:
guanosine diphosphate, AC3: adenylyl cyclase 3, ATP: adenosine triphosphate, cAMP: cyclic adenosine monophosphate, CNGC: cyclic nucleotide-gated channel, ANO2: anoctamin 2.
The regulation mechanisms underlying gene choice and gene expression in the mammalian olfactory system are still unknown. A first model involves a negative feedback resulting from the expression of a functional3
2 Also known as TMEM16B.
receptor protein (Serizawa et al., 2003). If a nonfunctional receptor gene is first chosen, then another receptor gene can be expressed. This phenomenon, termed gene switching (Shykind et al., 2004), also occurs when a receptor gene coding sequence is replaced by a reporter coding sequence by genetic engineering, a situation where the production of the reporter protein replaces the one of a functional receptor. A second model was proposed, where negative selection would lead to the elimination of OSNs producing no, or more than one, functional OR (Mombaerts, 2004). However, artificial forced expression of two different functional OR genes indicated that this situation was compatible with cell survival (Nguyen et al., 2007).
3 The definition of “functional” is ambiguous. As described later in the text, an exception exists in the case of a mutant odorant receptor that cannot bind signal transduction elements but is still able to mediate negative feedback.
Figure 4: Out of the whole receptor gene repertoire, a single one is expressed per olfactory neuron. This is controlled by a negative feedback signal from the functional OR protein. b) When a nonfunctional receptor (or a reporter inserted into the locus and replacing the original coding sequence) is expressed, no negative feedback takes place and the cell expresses another receptor gene. (Modified from (Rodriguez, 2007))
The negative feedback, resulting from the expression of a functional OR gene and mediating monogenic expression, does apparently not involve signal transduction cascade elements since a mutant OR, unable to bind G proteins, is still able to prevent the expression of other OR genes (Imai et al., 2006; Nguyen et al., 2007).
Trace Amine-Associated Receptors
TAARs are 7TM GPCRs of the class A17 family. They are unrelated to ORs but share homologies with dopamine and serotonin receptors4 (Liberles and Buck, 2006). This group is less diversified than ORs, with 15 and 6 functional TAAR genes in the mouse and human genomes, repectively (Zucchi et al., 2006). TAAR genes form a cluster in the mouse genome and their coding sequences lie in a single exon (with the exception of TAAR2) (Lindemann et al., 2005). These receptors were first identified in the brain where TAAR1 binds trace amines (β-phenylethylamine, tyramine, tryptamine and octopamine) (Borowsky et al., 2001; Bunzow et al., 2001). In mice, all TAAR genes are expressed in the main olfactory epithelium with the exception of TAAR1 (Liberles and Buck, 2006).
OSNs expressing a given TAAR gene are interspersed in overlapping zones in the main olfactory epithelium. In these cells, TAARs are expressed monogenically and in a mutually exclusive manner with OR genes.
The signal transduction cascade downstream TAARs is unknown. Since TAAR gene-expressing OSNs are positive for Gαolf (Liberles and Buck, 2006) and TAAR1 stimulate cAMP synthesis upon stimulation in vitro (Borowsky et al., 2001; Bunzow et al., 2001), it is possible that TAAR- and OR gene-expressing OSNs share other transduction elements.
4 TAAR1 has only 16% identity to its closest OR relative. It is 33% identical to serotonin receptor 4. TAAR1 is the only member of its family that is not expressed in the main olfactory epithelium (Liberles and Buck, 2006). The homology between other TAARs and ORs has not been reported.
Guanylyl Cyclase 2D
GC-D is a transmembrane guanylyl cyclase5 coded by the Gucy2d gene. Among the seven membrane-bound guanylyl cyclase genes in the mouse genome, it is the only one known to be expressed in the main olfactory epithelium by a subset of OSNs (Fulle et al., 1995). These neurons coexpress a specific phosphodiesterase (PDE2), distinct from those found in OR gene-expressing OSNs (PDE1C2 and PDE4A), and vice-versa (Juilfs et al., 1997). Concerning CNGC subunits, the situation is similar. Gucy2d-expressing OSNs express Cnga36 (a subunit typically found in cone photoreceptors), while OR gene-expressing OSNs express Cnga2, Cnga4 and Cngb1. In addition, Gucy2d-expressing neurons do not express GnaI or Adcy3(genes coding for Gαolf and AC3, respectively) (Meyer et al., 2000).
The role of GC-D in signal transduction is still controversial, possibly reflecting multiple signaling pathways. Gucy2d-expressing OSNs respond to CS2 (Munger et al., 2010) (a social stimulus found in rodent breath) and CO2 (Hu et al., 2007) (sensitivity to CS2 is several orders of magnitude greater than that to CO2). CO2
response is mediated by the conversion of atmospheric carbon dioxide to bicarbonate by intracellular carbonic anhydrase type 2 in Gucy2d-expressing OSNs. Bicarbonate then intracellularly activates G-CD (Guo et al., 2009; Sun et al., 2009), leading to the production of cyclic guanosine monophosphate (cGMP) and the opening of CNGCs composed of CNGA3 subunit isoforms. CS2 response is not fully understood but also involves GC-D, carbonic anhydrase type 2 and CNGA3 (Munger et al., 2010). On the other hand, guanylin and uroguanylin7 can also stimulate Gucy2d-expressing OSNs (Leinders-Zufall et al., 2007) at even lower concentrations compared to CS2 (Munger et al., 2010). This response is lost in either Gucy2d or Cnga3 deficient mice. Car2 knockout mice lacking functional carbonic anhydrase type 2 cannot perceive CO2 (Hu et al., 2007). Interestingly, CO2 is odorless to humans and the Gucy2d orthologue8 is a pseudogene in primates (Young et al., 2007).
5 Membrane-bound and soluble guanylyl cyclases convert guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP).
6 The nomenclature concerning CNGC subunits changed multiple times and is source of confusion. For instance CNGA2 is also known as CNGα3, while CNGA3 is also known as CNGα2.
For more details browse:
http://www.iuphar-db.org/DATABASE/FamilyMenuForward?familyId=71
7 Guanylin and uroguanylin are peptide hormones secreted by cells in the colon and duodenum, repectively. Uroguanylin (but not guanylin) is present in urine as well.
8 Here again, nomenclature is suboptimal: the human GUCY2D gene codes for GC-E, a retinal specific guanylyl cyclase.
The Vomeronasal System
In amphibians, reptiles and mammals, the vomeronasal system allows the perception of pheromones. These are molecules emitted by an individual eliciting physiological and/or behavioral responses when perceived by conspecifics. Pheromones carry information such as identity, gender, reproductive status and social status.
The vomeronasal organ9 is located above the palate10. It is a dead-end tubular-shaped bony structure containing a sensory neuroepithelium and filled with mucus. The vomeronasal epithelium contains vomeronasal sensory neurons (VSNs). These are bipolar neurons extending their unique dendrite towards the vomeronasal organ lumen i.e. the external world. Microvillous processes at the tip of each dendrite harbor vomeronasal receptors (VRs). VSNs project their axons towards a particular region of the olfactory bulb, called the accessory olfactory bulb. At the surface of the accessory olfactory bulb, VSNs synapse with mitral/tufted cells. These relay information to higher brain centers. Interneurons in the accessory olfactory bulb are similar to those in the main olfactory bulb.
VSNs transcribe receptor genes belonging to different families: vomeronasal type 1 receptors (V1Rs), vomeronasal type 2 receptors (V2Rs) and formyl peptide receptors (FPRs). These gene families are unrelated to each other or to ORs.
Again, the genetic expression of these receptor genes is mutually exclusive.
Vomeronasal Type 1 Receptors
V1Rs are 7TM GPCRs of the class A family. The first members of this gene family were discovered in 1995 (Dulac and Axel, 1995). Figure 5 shows both that the size of V1R gene repertoires in different mammalian species is surprisingly variable (with almost 200 functional V1R genes in mice, but less than ten in dogs and humans) and that the ratio between functional genes and pseudogenes is low (Grus et al., 2005; Young et al., 2005). Figure 6 illustrates how V1R genes are further classified into sometimes species-specific families (Grus and Zhang, 2004; Young et al., 2005; Young et al., 2010). V1R genes are clustered in the genome (at multiple loci) and their coding sequences lie in a single exon (Dulac and Axel, 1995). The somata of V1R gene-expressing VSNs are located in the apical part of the vomeronasal epithelium. These neurons coexpress Gnai2, the gene coding for Gαi2. VSNs expressing a given V1R gene are interspersed in the vomeronasal epithelium. The genetic regulation of V1Rs shows a remarkable feature called “gene cluster lock” (Roppolo et al., 2007). Briefly, if a non- functional receptor gene (or allele) is first chosen, the lack of negative feedback
9 Also known as the Jacobson’s organ
10 In primates, this organ is vestigial. Human fetuses have a vomeronasal organ that partially or totally regresses after birth. In addition, TrpC2, a crucial signal transduction element (see further) is a pseudogene in humans (Wes et al., 1995) and many other primates (Liman and Innan, 2003).
allows for the expression of another receptor gene. This switch cannot occur between genes located in cis on the same cluster. The region thus seems to be transcriptionally “locked”. The mechanism mediating this “cluster lock” is unknown.
Figure 5: Variability of the V1R gene repertoire size among six mammalian species. Pies represent the complete V1R gene repertoires corresponding to each species, and their surface the number of V1R genes. The surface of the disk on the lower left corner of the figure corresponds to one gene. Black and gray indicate potentially functional V1R genes and pseudogenes, respectively. (From (Rodriguez, 2005))
Figure 6: Phylogenetic tree of the mouse, rat and dog V1R gene repertoires. This tree was obtained by maximum likelihood anaysis with 1000 bootstraps (the most significant values are shown). Families h and i are specific to the mouse, while family m is exclusively present in rat. All other families contain members in both species. (From (Capello, 2009))
Most signal transduction components present in OSNs are absent in VSNs11 (Berghard et al., 1996). The signal transduction cascade of V1R gene-expressing VSNs is unknown but elements such as phospholipase C β2 and the transient receptor potential cation channel C2 (TrpC2) as well as 2nd messengers like inositol triphosphate (IP3), diacylglycerol (DAG) and arachidonic acid possibly play a role in signal transduction. Pharmacological inhibitors of phospholipase C β2, IP3 or DAG lipase abolish VSN response to urine while arachidonic acid induces transient intracellular calcium peaks (Holy et al., 2000;
Inamura et al., 1997; Spehr et al., 2002). TrpC2-deficient mice also show highly decreased responses to urine or known pheromones (Leypold et al., 2002; Lucas et al., 2003; Stowers et al., 2002). Gnai2 knockout mice have only half of V1R gene-expressing VSNs compared to controls (which leads to several anatomical and behavioral phenotypes). Although it is believed that signal transduction cascade in VSNs rather involves Gβ and Gγ subunits, mutants lacking Gαi2 also show a lack of signal in the accessory olfactory bulb in standard c-Fos staining assays (Norlin et al., 2003). Figure 7 illustrates the current consensus concerning V1R signaling, although a study refutes the involvement of IP3, DAG lipase and arachidonic acid in activating the DAG-gated cation channel present in VSNs (Lucas et al., 2003). Although many synthetic compounds or natural blends (such as urine) induce responses by V1R gene-expressing VSNs, only one receptor- agonist pair is known: V1RB2 binds 2-heptanone12 (Boschat et al., 2002).
Figure 7: Putative signal transduction cascade in VSNs, based on analogies with signal transduction in Drosophila photoreceptors. VR: vomeronasal receptor, Gα, Gβ, Gγ: G protein subunits, GTP: guanosine triphosphate, GDP: guanosine diphosphate, PIP2: phosphatidyl inositol biphosphate, PLCβ2: phospholipase C β2, IP3: inositol triphosphate, DAG: diacylglycerol, AA:
arachidonic acid, TrpC2: transient receptor potential cation channel C2.
11 The gene coding for CNGA4 is expressed in VSNs but its function in these cells is not known.
Similarly, AC2 (another adenylyl cyclase) is present in VSNs but its implication in signal transduction has not been demonstrated (Berghard and Buck, 1996; Berghard et al., 1996).
12 2-heptanone is a pheromone extending the duration of the female estrus period (Novotny et al., 1986).
Vomeronasal Type 2 Receptors
V2Rs are 7TM GPCRs of the class C family harboring a large extracellular N-terminus. Three different groups identified V2R genes in 1997 (Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997). V2R gene repertoires are highly variable among species (a situation similar to that of V1R genes), mice having around 70 functional V2R genes (and as many pseudogenes), while only a few pseudogenes remain in the human genome (Shi and Zhang, 2007). Figure 8 illustrates how V2R genes are further classified into families.
V2R genes are clustered in the genome (at multiple loci). Their coding sequences span multiple exons although all or most of the 7TM domain coding sequence is intronless (Herrada and Dulac, 1997; Matsunami and Buck, 1997). The somata of V2R gene-expressing VSNs are located in the basal part of the vomeronasal epithelium. These neurons coexpress Gnao1, the gene coding for Gαo. VSNs expressing a given V2R gene are interspersed in the vomeronasal epithelium.
The “one neuron, one receptor” rule is not strictly respected in V2R gene-expressing VSNs since a given member of the V2Rc gene family is always coexpressed with a V2R gene from another family (a, b or d) (Martini et al., 2001). However, this form of coexpression is not random (Silvotti et al., 2007) and V2Rc gene expression is mutually exclusive. In V2R gene-expressing VSNs the rule seems to have changed to “one neuron, one receptor, plus its c-family partner”. It is noteworthy that V2Rc genes are distantly related to other V2R genes (Yang et al., 2005).
Figure 8: An unrooted phylogenetic tree of the mouse V2R genes. V2Rc family is also known as V2R2 family, which was previously mistaken for a single gene. Numbers in red indicate the amount of members in each (sub)family. Numbers in black indicate bootstrap values (From (Silvotti et al., 2007)).
Concerning V2Rs, the signal transduction cascade is thought to be similar to what is known for V1Rs. However, as previously mentioned, V2R genes are coexpressed with the gene coding for Gαo (instead of Gαi2, in V1R gene-expressing VSNs), but, again, the involvment of these α subunits in signal transduction is not demonstrated. Although many synthetic compounds or natural blends (such as urine) induce responses by V2R gene-expressing VSNs,
only one receptor-agonist pair is known: V2RP513 binds exocrine-gland- secreting peptide 1(ESP114) (Haga et al., 2010).
V2R gene-expressing VSNs produce other proteins of interest, notably members of the major histocompatibility complex (MHC) class 1 and class 1b families.
Genes coding for proteins of the M1 and M10 families15 are coexpressed with V2R genes in a complex but non-random fashion (Ishii et al., 2003; Loconto et al., 2003). The presence of β2 microglobulin16 in all V2R gene-expressing VSNs (Ishii et al., 2003), and the fact that some V2Rs do not reach the membrane in β2 microglobulin-deficient animals (Loconto et al., 2003), led to the hypothesis that this molecule was required as a chaperone for proper trafficking of V2Rs to the membrane, a role potentially shared with proteins of the M10 family (Loconto et al., 2003). However other authors contest these observations since they were unable to repeat these results. These authors further showed that V2R1B17 trafficking to the membrane occurs normally in β2 microglobulin- deficient mice (Ishii and Mombaerts, 2008). Moreover they showed that proteins of the M1 or M10 families are absent in V2r1b-expressing VSNs.
Formyl Peptide Receptors
FPRs are 7TM GPCRs of the class A8 family. Originally known as players in the immune response, it was very recently shown that many FPR genes are expressed in VSNs (Liberles et al., 2009; Riviere et al., 2009). This gene family has seven functional members (and a few pseudogenes) in the mouse genome.
These form a cluster, intermingled with, or in close proximity to V1R, V2R and other genes. Five FPR genes are expressed in the vomeronasal epithelium (four in the apical and one in the basal zone, respectively), while the remaining two are expressed in the immune system. Humans have only three functional FPR genes, all expressed in the immune system. FPR coding sequences are intronless (Gao et al., 1998; Wang and Ye, 2002). The role of these receptors in the vomeronasal organ may be to mediate the avoidance of sick conspecifics observed in mice and other mammals (Riviere et al., 2009).
FPR signal transduction cascade in VSNs is still unexplored but is probably similar to V1R or V2R signaling. FPR gene-expressing VSNs respond with variable affinities to a broad range of disease-related compounds (known to bind FPRs in the immune system) that are present in urine of sick animals (Riviere et al., 2009).
13 Also known as VMN2R116, a member of family V2Ra cladeIII (Tirindelli et al., 2009).
14 ESP1 is a peptidic pheromone enhancing female sexual receptive behavior (Haga et al., 2010).
15 MHC class 1b molecules.
16 An MHC class 1 molecule.
17 Also known as VMN2R26, a member of V2Rb family.
The Grüneberg Ganglion
The membership of the Grüneberg ganglion to the olfactory system was only recently determined in mice (Fleischer et al., 2006a; Fuss et al., 2005; Koos and Fraser, 2005; Roppolo et al., 2006; Storan and Key, 2006) and few solid evidences concerning its role or its functioning are available. The original identification of this neuronal population was made in mice, and similar structures were found in other mammals including human embryos (Gruneberg, 1973). However, closely related mammals seem to differ concerning the presence of the Grüneberg ganglion, and adult humans have not been investigated.
Like every other mature sensory neuron in the system, Grüneberg ganglion neurons are positive for the olfactory marker protein (OMP), a cytoplasmic protein that has no known function. On the other hand, Grüneberg ganglion neurons constitute the only sensory neuronal population in the olfactory system that is not self-renewed. The Grüneberg ganglion is apparently mediating perception of alarm pheromones18 (Brechbuhl et al., 2008) and/or cold temperatures by pups19 (Mamasuew et al., 2008; Schmid et al., 2010), also possibly playing an alarm role upon separation from the parents. Grüneberg ganglion neurons are clustered at the tip of the nose, close to the nostrils. They lie underneath the nasal epithelium and have no apparent contact with the nasal cavity, since they lack dendrites. Each Grüneberg ganglion neuron projects a single axon towards a caudal region of the main olfactory bulb. Most Grüneberg ganglion neurons coexpress the vomeronasal receptor gene V2r83 (Fleischer et al., 2006b), Gucy2g, the gene coding for guanylyl cyclase 2G20 (GC-G), Cnga3 and Pde2a (Fleischer et al., 2009; Liu et al., 2009; Mamasuew et al., 2010), while other Grüneberg ganglion neurons express TAAR genes (Fleischer et al., 2007).
Most if not all Grüneberg ganglion neurons coexpress Gnai2 and Gnao1, the genes coding for Gαi2 and Gαo subunits (expressed in V1R- and V2R gene-expressing VSNs dendrites respectively; Gαo is also present in OSNs axons) (Fleischer et al., 2006b). It is noteworthy that in Grüneberg ganglion neurons, TAARs are apparently not coexpressed with GnaI, the gene coding for Gαolf, or other transduction elements present in OSNs.
18 The authors termed “alarm pheromones” a mix of rapidly degrading unidentified compounds that were collected during the euthanasia of mice with CO2.
19 These coolness-induced responses were only observed in a distinct subset of Grüneberg ganglion neurons expressing the gene coding for V2R83, a member of the V2Rc family broadly expressed in basal VSNs. Surprisingly, classical cold sensor proteins are not present in Grüneberg ganglion neurons (Mamasuew et al., 2008).
20 GC-G is the only other guanylyl cyclase present in the olfactory system (in addition to GC-D in the main olfactory epithelium)
Axon Guidance…
The wiring of the brain is an extraordinarily complex process involving multiple levels of regulation. Projection neurons send fibers to remote target(s), their growth cone acting as homing heads. Various studies revealed some basic principles in this process (Chedotal and Richards, 2010). First, pioneering axons seem to pave the way for subsequent axonal projections. Accurate initial targeting is thus a critical step. A palette of receptors allows axons to recognize guidance cues present along the path. Pioneering axons progress using intermediate targets such as transient glial populations as well as “corridor cells”, both key players in the development of appropriate neuronal connections.
Both chemoattractive and chemorepulsive signals are involved and many different families of proteins have been identified as receptors or guidance cues.
Among them, various cell adhesion molecules and homophilic interaction molecules seem to be extensively recruited in different situations. In addition, activity-dependent mechanisms control final synaptical refinements at the target site.
Sensory systems are often used as models of axon guidance. Topographical maps representing the outside world are generated in the brain to integrate signals coming from sensory neurons. These maps can be continuous, like in the visual system where neighboring projections reflect neighboring photoreceptors, or discrete, like in the olfactory system where neighboring projections reflect neuronal identity (see further). The generation, refinement and maintenance of such maps are all tightly regulated processes, possibly involving distinct factors.
… in the Olfactory System
Despite the scattering of neurons expressing a given receptor gene in olfactory sensory epithelia, functionally identical OSNs send their axons towards a specific location in the olfactory bulb. It is thus a neuron’s identity (defined by the receptor gene it expresses) that specifies its target, rather than its position in the sensory epithelium. Thus, in the olfactory bulb, fibers from neurons expressing the same receptor gene converge and form neuropil-rich structures called glomeruli. These glomeruli are the site where sensory neurons synapse with second order neurons. Although subtle local permutations can occur (Strotmann et al., 2000), the topographical maps formed in the olfactory bulb are conserved between right and left bulbs and among individuals.
Figure 9: A) OSNs expressing the same receptor gene (represented using the same color) target the same glomeruli in the main olfactory bulb. B) VSNs expressing the same receptor gene target the same set of glomeruli in the accessory olfactory bulb. V1R gene-expressing VSNs target the rostral accessory olfactory bulb, while V2R gene-expressing VSNs target the caudal accessory olfactory bulb. (MOB: main olfactory bulb, MOE: main olfactory epithelium, VNO: vomeronasal organ, AOB: accessory olfactory bulb) (Modified from (Dulac and Torello, 2003))
Axonal projection characteristic of each olfactory subsytem will be now discussed.
OSNs and ORs
In the main olfactory epithelium, OSNs project to glomeruli located in the main olfactory bulb. Since the main olfactory epithelium and the olfactory bulbs are bilateral structures that can be divided into their medial and lateral parts, all OSNs expressing a given OR gene thus form one medial and one lateral glomerulus in each bulb (Figure 10). These four glomeruli are exclusively innervated by fibers coming from neurons expressing this receptor gene.
Figure 10: OSNs located in the lateral part of the left nasal cavity project to the lateral region of the left olfactory bulb. The left bulb mirrors the right one and the medial region mirrors the lateral one in each bulb. Axons of OSNs expressing a given OR gene (visualized here by the coexpression of lacZ) thus converge into four glomeruli in the main olfactory bulbs, one in each half bulb. (L-L: lateral left, M-L: medial left, M-R, medial right, L-R: lateral right)
The establishment of a topographical map first requires the innervation by OSN axonal projections of a defined area in the bulb and then an ability of like fibers to recognize each other and converge.
An attractive global model is proposed in a review by Hitoshi Sakano (Sakano, 2010). According to this model, the position of an OSN in the main olfactory epithelium first restricts OR gene choice and defines a first set of guidance elements21 involved in the regulation of projections along the dorsoventral axis.
Second, a neuronal identity code is provided by the OR gene choice. This translates into specific cAMP levels driving the expression of another set of guidance elements22, involved in early axon sorting and in the regulation of projections along the anteroposterior axis. Finally, activity regulates the expression of a third set of guidance elements23, involved in convergence.
To uncouple gene expression of the second and third set of guidance molecules, the model proposes that spontaneous activity (and associated cAMP levels) regulate expression of the second set, while ligand-induced activity (and associated Ca2+ levels) are determinant for the regulation of the third set. A temporal uncoupling is also necessary; otherwise activity-dependant cAMP levels would affect the regulation of molecules of the second set.
21 This set of molecules would include the slit receptor Robo2 and the Semaphorin receptor Neuropilin 2 (see further).
22 “Type-1” molecules would include the Semaphorin receptor Neuropilin1 (see further) and mediate axon guidance.
23 “Type-2” molecules would include Kirrels, ephrinAs and their EphA receptors (see further) and mediate convergence of like axons.
A - Guidance
Factors involved in axon guidance will first be discussed and will be followed by those involved in convergence.
Swap experiments where an OR coding sequence was replaced by another one showed that the expressed receptor plays an instructive role in axonal targeting:
1) Replacing an OR coding sequence by the one of a different OR leads to the formation of a new distinct glomerulus (Feinstein et al., 2004; Mombaerts et al., 1996; Wang et al., 1998), 2) Replacing an OR coding sequence by the one of another 7TM GPCR24 leads to the formation of a new glomerulus, indicating that other GPCRs can substitute for ORs in axon guidance (Feinstein et al., 2004), 3) Replacing an OR coding sequence by the one of an unrelated reporter25 leads to fibers targeting many glomeruli together with other axons not expressing the reporter (Feinstein et al., 2004; Wang et al., 1998). This likely reflects gene switching (and therefore rewiring) subsequent to the lack of feedback from the reporter protein, a situation similar to the expression of an OR pseudogene.
However, these data do not indicate if the instructive signal, emanating from the OR, originates in dendrites or in the growth cone. This is a fair question, since ORs (both their corresponding mRNA and protein (Barnea et al., 2004;
Strotmann et al., 2004)), as well as many other signal-transduction elements are present in OSN axons and growth cones (in addition to the dendritic endings).
These elements could have a dual function and be used to direct axon guidance in this context. However, mice that lack Gαolf and CNGA2 show no or very subtle alterations in their axonal projections to the main olfactory bulb (Belluscio et al., 1998; Lin et al., 2000; Zheng et al., 2000).
An interesting experiment would be to analyze the topographical projections in the previously mentioned transgenic animals in which a subpopulation of OSNs expressing two functional OR genes are present in the main olfactory epithelium (Nguyen et al., 2007) (such neurons perceive both ligands known to selectively activate each OR but the projections of these OSNs were unfortunately not reported).
In addition, the level of expression of a given OR gene as well as subtle modifications in its coding sequence affect the position of glomeruli (Feinstein et al., 2004; Feinstein and Mombaerts, 2004). Axon sorting by homotypic interactions based on the expressed OR gene was proposed (Feinstein and Mombaerts, 2004; Vassalli et al., 2002). However, in most cases, swap experiments result in axons of OSNs expressing an OR gene from its endogenous locus targeting different glomeruli relative to axons of OSNs expressing the same OR gene from a different locus. This suggests that other guidance cues are involved and that the OR does not determine these.
As previously mentioned, OSNs expressing a given receptor gene are scattered in a defined zone in the main olfactory epithelium. OR-specific zones seem to be
24 β2 adrenergic receptor, a member of class A17 family (like TAARs, for instance).
25 The green fluorescent protein-coding gene (GFP) or IRES-Tau:lacZ, for instance.
relevant regarding axonal targeting. In the main olfactory bulb, the dorsoventral position of a glomerulus formed by OSNs expressing a given OR gene correlates with the zone (or expression domain) of this OR in the main olfactory epithelium (Miyamichi et al., 2005; Mori and Yoshihara, 1995; Tsuboi et al., 2006). This reflects zone-specific expression of cell adhesion molecules (Yoshihara et al., 1997) and other guidance factors (Norlin et al., 2001) (see further).
As previously mentioned, mutants lacking signal transduction cascade elements such as Gαolf or CNGA2 (also present in OSN axons) show no or limited alterations in OSN axon wiring. On the other hand, glomeruli in Adcy3 knockout mice were reported as being somehow disorganized (Trinh and Storm, 2003).
The possibility that spontaneous OSN activity could play a role in axon guidance was further tested in experiments where OSN depolarization was prevented by the inducible expression of a transgenic potassium channel (Yu et al., 2004).
When the transgene expression is induced in a subpopulation of OSNs expressing a given OR gene, these cells disappear. However, in a non-competitive environment (when all OSNs express the transgene), OSNs expressing a given OR gene project to multiple glomeruli. This is similar to the situation observed in experiments involving nostril occlusion at birth, where map refinements are prevented and inappropriate neighboring glomeruli (or OSNs lacking CNGA2 in mosaic female mice26) persist on the occluded side (Nakatani et al., 2003; Zhao and Reed, 2001; Zou et al., 2004). Activity is thus important for the refinement of the olfactory map by elimination of inappropriate connections.
Signal transduction elements present on OSN axons are unlikely to mediate activity-driven refinements of the olfactory map. However, as previously mentioned, glomeruli are disorganized in Adcy327 knockouts but not in Cnga2 mutants. The role of cAMP in axon guidance was recently investigated by us and others (Chesler et al., 2007; Dal Col et al., 2007; Imai et al., 2006; Zou et al., 2007). OSNs expressing a given OR gene target multiple regions in the main olfactory bulb of Adcy3 knockout mice. Their axons do not converge properly and innervate multiple loosely defined glomeruli. The expression of guidance cues is dependant on signaling involving cAMP, protein kinase A and cAMP responsive element-binding protein. Modulation of cAMP signaling using mutant G protein α subunits, protein kinase A, or cAMP responsive element-binding protein leads to modifications of glomerular position. Apparently, specific levels of intracellular cAMP correlate with the expression of specific molecules involved in guidance.
26Cnga2 is located on mouse chromosome X. Random X inactivation allows thus the generation of mosaic females if they carry a single functional Cnga2 allele.
27 Like many knockouts of an olfactory signal transduction element, AC3 deficient mice are anosmic, a condition that impairs the feeding of pups. In addition, since AC3 is expressed in other tissues (brain, heart, lungs, pancreas…) and is probably involved in many other developmental processes, most of the homozygous mutant pups die soon after birth and survivors are much weaker than control littermates until adulthood. The role of AC3 in the acrosomal reaction during fertilization causes the sterility observed in the few surviving homozygous males (Livera et al., 2005).
Figure 11: Schematics illustrating a simplified version of gene activation via cAMP. Upon cAMP production, protein kinase A regulatory subunits release the catalytic subunits (pink rounded squares), which can then enter the nucleus to phosphorylate cAMP responsive element-binding protein. Phosphorylated cAMP responsive element-binding protein can bind cAMP responsive element sequences and activate gene transcription, together with other cofactors. OR: odorant receptor, Gα, Gβ, Gγ: G protein subunits, AC3: adenylyl cyclase 3, ATP: adenosine triphosphate, cAMP: cyclic adenosine monophosphate, PKA: protein kinase A, CREB: cAMP responsive element- binding protein.
Receptors of the Roundabout (Robo) family bind secreted repellants of the Slit family. In vertebrates, each of these families has three members. These factors are involved in axon guidance, notably during midline crossing by commissural axons. The expression of Slits and Robos in the main olfactory system, as well as the phenotype of mutants, is not completely clear (Cho et al., 2007; Li et al., 1999;
Nguyen-Ba-Charvet et al., 2008). However, a recent study proposed a model based on the discovery of a ROBO2 gradient in the main olfactory epithelium:
dorsomedial OSNs expressing more Robo2 than ventrolateral ones (Cho et al., 2007). On the other hand, SLIT3 is expressed in the ventral olfactory bulb (SLIT1 is also transiently expressed in the embryonic ventral olfactory bulb). Robo2 knockout is lethal during the perinatal period in mice but analysis of these pups showed that some OSN fibers inappropriately target the ventral olfactory bulb.
This phenotype is also present in adult Slit1 (but not Slit3) knockouts (Cho et al., 2007). Thus, SLIT1 and ROBO2 are involved in the appropriate targeting of dorsal OSNs.
Figure 12: Slit1 and Robo2 are required for the segregation of dorsal OSN axons to the dorsal region of the main olfactory bulb. (From (Cho et al., 2007))
Neuropilins (NRPs) are coreceptors (together with plexins) for secreted class-3 semaphorins (playing roles in axon guidance) and vascular endothelial growth factor (playing roles in angiogenesis). NRP1 binds preferentially SEMA3A28, while NRP2 binds preferentially SEMA3F. These interactions result in repulsion.
In the olfactory system, Nrp1-expressing OSNs project to the anterolateral and caudomedial regions of the main olfactory bulb (Schwarting et al., 2000;
Schwarting et al., 2004). NRP1-positive fibers are repelled by SEMA3A, which is secreted in the anteromedial, ventral and caudolateral regions of the main olfactory bulb, apparently by ensheating cells, second order neurons and other cell types (Crandall et al., 2000; Pasterkamp et al., 1998; Schwarting et al., 2000).
An NRP2 gradient is present in the main olfactory epithelium (low dorsomedial to high ventrolateral expression by a subset of OSNs) (Norlin et al., 2001;
Takahashi et al., 2010; Walz et al., 2002). Sema3f expression by OSNs in the main olfactory epithelium also forms a gradient, complementary to that of NRP2 (Takeuchi et al., 2010).
Knocking out Nrp1 results in embryonic lethality. Sema3a knockout animals display defects in axon guidance with NRP1-positive OSNs targeting the whole main olfactory bulb (Schwarting et al., 2000). In such animals, OSNs expressing a given OR gene project to multiple glomeruli. This situation seems true even for OSNs that do not express Nrp1 (Schwarting et al., 2004) although it was not observed in another study (Taniguchi et al., 2003).
Nrp2 knockout mice are viable. In such mice, some OSNs sometimes target multiple glomeruli in the ventrolateral main olfactory bulb and the position of these glomeruli is sometimes altered (Takahashi et al., 2010). In addition, a
28 Originally found in chick brain and termed collapsin-1 (Luo et al., 1993). Also known as SemD or Semaphorin D in mice (Puschel et al., 1995).
recent study showed that complementary SEMA3F and NRP2 gradients are present in the main olfactory epithelium. Figure 13 depicts how OSNs expressing high levels of Sema3f and low levels of Nrp2 project dorsally in the main olfactory bulb, where secreted SEMA3F acts as a repellent for axons of OSNs expressing low levels of Sema3f and high levels of Nrp2. These latter neurons project thus ventrally in the main olfactory bulb29. Blocking SEMA3F signaling, by selectively knocking out Nrp2 (or the gene coding for its coreceptor PlxnA3) in a specific subpopulation of OSNs, results in a dorsal shift of its ventral glomerulus (Takeuchi et al., 2010). This study illustrates how OSN subpopulations can generate guidance cues used by other OSN subpopulations.
Another important aspect of this study comes from the generation of animals where OSNs expressing a given OR gene are subdivided into NRP2-positive and NRP2-negative subpopulations (or the mirror experiment with PlexinA3), depending on the sequential activation of an OR gene and an OR promoter-driven Cre transgene (in this construct, due to the negative feedback, activation of Cre is possible only if it occurs prior to the activation of another functional OR gene). These subpopulations of OSNs, expressing the same OR gene, project to distinct glomeruli.
Figure 13: A model for the axonal projection of OSNs along the dorsoventral axis. SEMA3F secreted at the anterodorsal region of the main olfactory bulb by early-arriving axons prevents the late-arriving NRP2-positive axons from invading the dorsal region of the main olfactory bulb.
(Modified from (Takeuchi et al., 2010))
29 A similar mechanism was previously described in Drosophila (Sweeney et al., 2007).
Eph receptors30 bind transmembrane or membrane-bound ligands called ephrins. These bidirectional cell-signaling components form families composed of 16 and 9 members for Eph receptors and ephrins, respectively31. They play multiple roles in development (such as in segmentation, cell migration and axon guidance, for instance) and are famous for their participation in the formation of the visual system topographic map. Interaction of ephrins with their receptor can result in attraction or repulsion. In the olfactory system, both EphAs and ephrinAs genes are expressed by OSNs32, in a complementary manner that correlates with the selected OR gene (Cutforth et al., 2003; Serizawa et al., 2006).
This means that OSNs expressing high levels of EphA will express low levels of ephrinA and that this ratio will be similar in OSNs expressing the same OR gene.
EphAs and ephrinAs are found on OSN axons, where they apparently mediate repulsion (axons expressing high levels of EphAs repel axons expressing high levels of ephrinAs). Modulating ephrin genes in OSNs expressing a given OR gene results in glomerular anteroposterior shifts reflecting ephrin levels (Cutforth et al., 2003).
BIG-2 is a GPI-anchored cell adhesion protein of the contactin family. It is widely present on OSN axons33 and its expression level correlates with OR gene choice (Kaneko-Goto et al., 2008). Mice lacking BIG-2 show OSNs expressing a given OR gene targeting multiple glomeruli in the main olfactory bulb. BIG-2 receptor is not known, but it is distinct from BIG-2 itself.
B - Convergence
As previously mentioned, the formation of a glomerulus is dependent on convergence of like fibers that were previously guided to a specific location in the main olfactory bulb. Now factors involved in axonal convergence will be presented and the different models will be discussed.
Axons of OSNs producing a mutant OR, unable to bind G proteins (the RDY34 mutant) fail to converge (Imai et al., 2006). AC3 deficient mice are also heavily impaired in OSN axonal convergence. Constitutively active Gαs35
30 Eph receptors are receptor tyrosine kinases.
and protein kinase A restore axonal convergence in OSNs producing the RDY mutant OR.
Constitutively active Gαs expressed from an OR locus also induces convergence of
31 These families are subdivided into EphAs (10 members) and EphBs (6 members), for receptors, and glycosylphosphatidylinositol (GPI) anchored ephrinAs (5 members) and transmembrane ephrinBs (3 members) are their corresponding ligands.
32 ephrinA3, ephrinA5 (Cutforth et al., 2003) and EphA5 (Serizawa et al., 2006).
33 The BIG-2 coding gene is also highly expressed in VSNs, but its role in the vomeronasal system has not been investigated.
34 Many GPCRs contain a DRY amino acid sequence in the cytoplasmic side of the 3rd transmembrane domain. These three amino acids are essential for the binding of G proteins.
When the first two amino acid are inverted, the mutant receptor can not bind G proteins anymore.
35 Gαs is a distinct α subunit present in immature OSNs.
