Developmental Regulation of Neural Map Formation in the Mouse Olfactory System PDF

Developmental Regulation of Neural Map Formation in the Mouse Olfactory System Hirofumi Nishizumi,1 Hitoshi Sakano2 1 Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan 2 Department of Brain Function, Faculty of Medical Sciences,...

Developmental Regulation of Neural Map Formation in the Mouse Olfactory System Hirofumi Nishizumi,1 Hitoshi Sakano2 1 Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan 2 Department of Brain Function, Faculty of Medical Sciences, University of Fukui, Fukui 910-1193, Japan Received 26 October 2014; revised 6 January 2015; accepted 6 January 2015 ABSTRACT: In the mouse olfactory system, vari- location of OSNs within the olfactory epithelium (OE), ous odorants are detected by approximately 1000 differ- whereas A-P projection is instructed by expressed OR ent odorant receptors (ORs) expressed in the olfactory molecules using cyclic adenosine monophosphate (cAMP) sensory neurons (OSNs). It is well established that each signals. After birth, the map is further refined in an OSN expresses only one functional OR gene in a monoal- activity-dependent manner by its conversion from a con- lelic manner. Furthermore, OSN axons expressing the tinuous to a discrete map through segregation of glomer- same OR converge to a set of glomeruli in the olfactory ular structures. Here, we summarize recent progress bulb (OB). During embryonic development, a coarse map from our laboratory in understanding neural map is formed by the combination of two genetically pro- formation in the mouse olfactory system. VC 2015 Wiley grammed processes. One is OR-independent axonal pro- Periodicals, Inc. Develop Neurobiol 75: 594–607, 2015 jection along the dorsal-ventral (D-V) axis, and the other Keywords: neural map; olfactory sensory neurons; axo- is OR-dependent projection along the anterior-posterior nal projection; receptor baseline activity; odorant (A-P) axis. D-V projection is regulated by the anatomical receptors; cyclic AMP INTRODUCTION (Rodriguez, 2013). Furthermore, OSN axons express- ing the same type of OR converge on a set of glomer- In mammals, sensory information is spatially uli in the olfactory bulb (OB) (Mombaerts et al., encoded in the brain, forming neural maps that are 1996). Because each glomerulus represents a single fundamental for higher-order processing of sensory OR species, and a single odorant can interact with information. In the mouse olfactory system, there are multiple OR species (Malnic et al., 1999), odor sig- 1000 different odorant receptor (OR) species (Buck nals received in the olfactory epithelium (OE) are and Axel, 1991), and each olfactory sensory neuron converted into a topographic map of multiple glomer- (OSN) expressed only one functional OR gene uli activated with varying magnitudes (Mori and Sakano, 2011). Molecular mechanisms of sensory map formation in the visual system in particular have been exten- Correspondence to: H. Sakano; ([email protected]). sively studied. The topographic organization of the Contract grant sponsor: Grants in Aid and Specially Promoted Research Grants (Ministry of Education, Culture, Sports, Science projecting neurons in retina is maintained and pro- and Technology of Japan). jected onto the target preserving the nearest-neighbor Ó 2015 Wiley Periodicals, Inc. relationship (McLaughlin and O’Leary, 2005; Luo Published online 18 February 2015 in Wiley Online Library (wileyonlinelibrary.com). and Flanagan, 2007). In contrast, in the olfactory sys- DOI 10.1002/dneu.22268 tem, projecting axons of spatially dispersed cell 594  1932846x, 2015, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dneu.22268 by Emilie Gueguen - Universitätsbibliothek Zuerich , Wiley Online Library on [03/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Neural Map Formation in the Mouse Olfactory System 595 bodies with the same neuronal identity converge on ment in the OR genes (Eggan et al., 2004; Li et al., one glomerular location in each olfactory map in the 2004). Because the genetic translocation model OB (Mombaerts et al., 1996; Ressler et al., 1994; became unlikely, another possibility was explored, Vassar et al., 1994). Unlike the visual system, much namely a locus control region (LCR) that might reg- of olfactory map formation appears to occur autono- ulate the single OR gene choice. mously by axon–axon interaction of OSNs rather than by axon-target interaction (Sakano, 2010). Stochastic Activation of Single OR Gene A remarkable feature of axonal projection in the by cis-Acting LCR mouse olfactory system is that ORs play an instruc- tive role in projecting OSN axons to the OB (Mom- >LCR is defined as a cis-acting regulatory region that baerts, 2006). It was once thought that the OR protein controls multiple genes clustered at a specific genetic itself may act as an axon guidance receptor, detecting locus. It was assumed that transcription factors that the target cues in the OB, and that it mediates homo- physically interact with the remote promoter site, by philic interactions among “like” axons. Although looping out the intervening DNA, bind to the LCR. these models were attractive, our recent studies argue Two examples of such an LCR in the mouse olfactory against them. We have previously reported that both system, named H and P, have been identified (Seri- global targeting along the A-P axis and local sorting zawa et al., 2003; Khan et al., 2011). Deletion and of OSN axons for glomerular segregation are regu- mutation analyses of the H region revealed that the lated by OR-derived cyclic adenosine monophos- 124-bp core-H region, which contains two homeodo- phate (cAMP) signals (Imai et al., 2006; Serizawa main sequences and one O/E-like sequence, is suffi- et al., 2006). In the OB, A-P-targeting molecules are cient to achieve the enhancer activity (Nishizumi et al., detected on axon termini of OSNs, forming a comple- 2007). Homeodomain factors, Lhx2 and Emx2, and O/ mentary gradient in a glomerular map. OR-derived E family proteins are known to bind to their motifs in cAMP signals also regulate the expression of glomer- the OR gene promoter (Vassalli et al., 2002; Hirota and ular segregation molecules for map refinement. Mombaerts, 2004; Rothman et al., 2005; Michaloski Unlike A-P-targeting molecules, glomerular segrega- et al., 2006). It is possible that these nuclear factors tion molecules are regulated in an activity-dependent bind to the H region and form a complex that remodels manner and show mosaic distribution in the glomeru- the chromatin structure near the cluster, thereby acti- lar map. How is it, then, that A-P targeting and glo- vating one OR promoter site at a time by physical merular segregation are differentially regulated by interaction (Serizawa et al., 2003) (Fig. 1). the expressed ORs despite both using OR-derived Does the H region act on other OR gene clusters, cAMP as a second messenger? What is the source of not only in cis but also in trans, similar to the IL24 cAMP signals, and how are the signals generated? LCR in T cells (Spilianakis et al., 2005)? It was pro- posed that the single trans-acting enhancer may allow the stochastic activation of only one OR gene in each OR GENE CHOICE OSN (Lomvardas et al., 2006). However, knockout (KO) studies of H contradict the single trans-acting In the mouse olfactory system, each OSN chooses LCR model for the OR gene choice (Fuss et al., 2007; for expression only one functional OR gene in a Nishizumi et al., 2007). In heterozygous (H1/H-) KO monoallelic manner (Chess et al., 1994; Serizawa mice, the wild-type (WT) H1 allele could not rescue et al., 2000; Ishii et al., 2001), which is referred to the H- mutant allele in trans, indicating that H can act as the one neuron-one receptor rule. Such unique only in cis and not in trans (Nishizumi et al., 2007). expression forms the genetic basis for the OR- It has been reported that each OR gene possesses a instructed axonal projection of OSNs to the OB. unique expression area in the OE along the How is the singular OR gene choice regulated in dorsomedial-ventrolateral axis (Ressler et al., 1993; OSNs, and how is the one neuron-one receptor rule Vassar et al., 1993; Miyamichi et al., 2005). Thus, maintained? Irreversible DNA changes, that is, OR gene choice may not be totally stochastic, but DNA recombination and gene conversion, had been rather, it may be restricted by the OSN location in the attractive explanations for single OR gene expres- OE. How does this positional information within the sion because of the many parallels between the OE regulate OR gene choice? It is possible that this immune and olfactory systems. However, these the- regulation is determined by cell lineage, resulting in ories were dismissed after two groups cloned mice the use of zone-specific transcription factors, for from postmitotic OSN nuclei and determined that example, Msx1 and Foxg1 (Norlin et al., 2001; Dug- the mice showed no irreversible DNA rearrange- gan et al., 2008). Developmental Neurobiology  1932846x, 2015, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dneu.22268 by Emilie Gueguen - Universitätsbibliothek Zuerich , Wiley Online Library on [03/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 596 Nishizumi and Sakano Figure 1 Single OR genes expression in the mouse olfactory system. It is assumed that the activa- tion complex formed in the LCR stochastically chooses one promoter (P) site by random collision, activating one particular OR gene. However, the LCR-promoter interaction alone would not pre- clude the activation of a second OR gene located in the other allele or in other OR gene clusters. Once a functional gene is expressed, the OR molecules transmit inhibitory signals via ACIII and block the further activation of additional OR gene clusters. Stochastic activation of an OR gene by a cis-acting LCR and negative feedback regulation by the functional OR gene product, together, ensure the maintenance of the one neuron-one receptor rule. Negative Feedback Regulation of OR gene to be expressed (Serizawa et al., 2003; Feinstein Genes by Expressed OR Molecules et al., 2004; Lewcock and Reed, 2004; Shykind et al., 2004). Naturally-occurring frameshift mutants of In the OR gene system, the LCR-promoter interaction ORs also allow the coexpression of a functional OR alone would not preclude the activation of a second gene (Serizawa et al., 2003). Thus rate-limited activa- OR gene located in the alternative allele or in other tion of an OR gene by cis-acting LCRs and negative OR gene clusters. Therefore, it has been postulated feedback regulation by the OR gene product together that the functional OR proteins have an inhibitory appear to ensure the maintenance of the one neuron- role to prevent further activation of other OR genes one receptor rule (Serizawa et al., 2004; Rodriguez, (Serizawa et al., 2003; Lewcock and Reed, 2004) 2013). (Fig. 1). Transgenic experiments demonstrated that Lomvardas and colleagues reported that chromatin- the mutant OR genes lacking either the entire coding mediated silencing is important to prevent multiple sequence or the start codon can permit a second OR expression of OR genes in each OSN (Magklara Developmental Neurobiology  1932846x, 2015, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dneu.22268 by Emilie Gueguen - Universitätsbibliothek Zuerich , Wiley Online Library on [03/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Neural Map Formation in the Mouse Olfactory System 597 et al., 2011). They found that heterochromatic com- brain (Lemke and Reber, 2005; McLaughlin and paction of OR-gene clusters occurs before OR gene O’Leary, 2005; Petersen, 2007), there is no such transcription via two trimethyl marks of H3K9me3 correlation for the projection along the A-P axis in and H4K20me3. The enrichment for these silent the mouse olfactory system. Intriguingly, OR mole- marks is significantly reduced in an activated cules are detected in axon termini by tagging with OR-gene region, which is marked instead with green fluorescent protein (Feinstein and Mombaerts, H3K4me3. They assume that all OR-gene loci 2004) or by immunostaining with anti-OR antibod- become silenced before OR-gene transcription. At a ies (Barnea et al., 2004; Strotmann et al., 2004). On later stage, a limited enzymatic activity removes the basis of these observations, it was suggested that methylations of H3K9me3 and H4K20me3 from a the OR protein itself may recognize guidance cues stochastically chosen locus, activating its transcrip- in the target OB and also mediate homophilic inter- tion. Once a functional gene is expressed, negative action of similar axons (Mombaerts, 2006). How- feedback signals may prevent the enzyme/selector ever, recent studies indicate that instead of directly from activating other OR-gene loci. acting as guidance receptors or adhesion molecules, Recently, Lyons et al. (2013) reported that the ORs regulate transcription levels of axon-guidance transient expression of histone demethylase, LSD1, and sorting molecules using cAMP signals with lev- plays a key role in activation and suppression of OR els uniquely determined by the OR species (Imai genes to maintain the signal OR gene expression in et al., 2006; Serizawa et al., 2006) (Fig. 2). OSNs. They found that LSD1 is necessary as a tran- scriptional coactivator for desilencing and initiating A-P Targeting of OSN Axons is Regulated the transcription of OR genes. Once a functional OR by OR-Derived cAMP gene is activated and expressed, ACIII mediates the down-regulation of LSD1, which causes prevention In mature OSNs, the signals generated by odorants of further activation of other OR genes. Further- binding to ORs are converted into neuronal activ- more, Dalton et al. (2013) reported that the OR pro- ities via cAMP. The olfactory-specific G protein tein generates the feedback by activating the (Golf) activates adenylyl cyclase type III (ACIII), unfolded protein response. They found that OR generating cAMP, which opens cyclic nucleotide induces Perk-mediated phosphorylation of the trans- gated (CNG) channels (Wong et al., 2000). CNG lation initiation factor causing translation of a channels, together with chloride channels (Stephan transcription factor, ATF5, that induces ACIII et al., 2009), depolarize the plasma membrane, thus expression. Thus, ACIII down-regulates LSD1 pre- generating the action potential. Targeted KOs venting further activation of other OR genes. of Golf and CNG-A2 cause severe anosmia Recently, it was also reported that spatial compart- (Brunet et al., 1996). Despite their essential role in mentalization of OR genes and their enhancers in odor signal transduction, KOs of the genes did not OSN nuclei is important for the regulation of demonstrate major defects in axonal projection of OR gene expression (Clowney et al., 2012; OSNs (Belluscio et al., 1998; Lin et al., 2000; Zheng Markenscoff-Papadimitriou et al., 2014). et al., 2000). It was therefore assumed that OR- derived cAMP signals were not required for OSN projection. OR-INSTRUCTED OSN PROJECTION Despite these observations, it was possible that an alternate G protein mediates OR-instructed OSN projec- The olfactory map in the OB is comprised of dis- tion. To examine this possibility, Imai et al. (2006) gen- crete glomeruli, each representing a single OR spe- erated a mutant OR whose G protein coupling motif, cies (Ressler et al., 1994; Vassar et al., 1994; DRY, was mutated to RDY. They found that axons Mombaerts et al., 1996). Coding-swap experiments expressing the mutant OR remained in the anterior of OR genes demonstrated the instructive role of the region of the OB and failed to converge to a specific OR protein in OSN projection (Mombaerts et al., glomerulus in the OB. Interestingly, coexpression of the 1996; Wang et al., 1998; Feinstein and Mombaerts, constitutively active Gs restored axonal convergence and 2004). Because OSNs expressing the same OR are glomerular formation. Partial rescue was also observed scattered in the OE for A-P targeting, topographic with the constitutively active mutants of protein kinase organization must occur during the process of OSN PKA and transcription factor CREB. Thus, PKA- projection. Unlike axonal projection in other sen- mediated transcriptional regulation appears to be sory systems in which relative positional informa- involved in OSN projection. Furthermore, constitutively tion is preserved between the periphery and the active Gs results in a posterior shift of glomeruli when Developmental Neurobiology  1932846x, 2015, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dneu.22268 by Emilie Gueguen - Universitätsbibliothek Zuerich , Wiley Online Library on [03/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 598 Nishizumi and Sakano Figure 2 Global targeting and local sorting of OSN axons in olfactory map formation. (A) OSN axons are guided to approximate destinations in the OB by a combination of D-V patterning and A- P patterning. D-V projection is regulated by the anatomical locations of OSNs within the OE. A-P projection is achieved through cAMP signals induced by OR baseline activities. These processes, forming a coarse map topography, are genetically programmed and are independent of neuronal activity. (B) During the neonatal period, the map is further refined in an activity-dependent manner. Glomerular segregation occurs via adhesive and repulsive interactions of neighboring axons. OE, olfactory epithelium; OB, olfactory bulb. expressed with the WT OR. In contrast, dominant- into the central compartment of the bundle, whereas negative PKA results in an anterior shift of glomeruli Nrp1high/Sema3Alow axons are confined to the outer- (Imai et al., 2006). These findings suggest that it is the lateral compartment. OSN-specific KOs of Nrp1 not OR-derived cAMP signals, rather than the direct action only perturbed axon sorting within the bundle, but of OR molecules themselves, that determine the target also caused an anterior shift of glomeruli in the OB. destination of OSNs along the A-P axis in the OB. These results indicate that pretarget axon sorting within the bundle contributes to olfactory map forma- tion along the A-P axis (Fig. 3). Pretarget Axon Sorting Establishes the A-P Topography As forementioned, OSNs producing high levels of OR-INDEPENDENT OSN PROJECTION cAMP project their axons to the posterior OB, whereas those producing low levels target the ante- In contrast to A-P projection, D-V projection is not rior OB. When protein levels of Neuropilin 1 (Nrp1) regulated by OR molecules. For OSN projection along were measured in axon termini of OSNs, Nrp1 was the D-V axis, there is a close correlation between the found in an anterior-low/posterior-high gradient in anatomical locations of OSNs in the OE and their the OB (Imai et al., 2006). Increases or decreases of axonal projection sites in the OB (Astic et al., 1987; Nrp1 expression in OSNs caused posterior or anterior Miyamichi et al., 2005). The preservation of the spa- glomerular shifts, respectively. How then do guid- tial relationship of neuronal cell bodies and their pro- ance molecules regulate olfactory map formation? jection sites is widely seen in other brain regions, Map order emerges in axon bundles, well before they including the visual system (McLaughlin and reach the target (Satoda et al., 1995). It has been O’Leary, 2005; Luo and Flanagan, 2007; Petersen, reported that pretarget axon sorting plays an impor- 2007). On the basis of the expression patterns of tant role in the organization of the olfactory map zone-specific markers, the OE can be divided into two (Imai et al., 2009). Nrp1 and its repulsive ligand nonoverlapping zones: dorsal and ventral. Two sets of Semaphorin 3A (Sema3A) are both expressed in repulsive ligands/receptors, Slits/Robo2 and Sema3F/ OSNs, but in a complementary manner. Within the Nrp2, have been shown to participate in OSN projec- axon bundles, Nrp1low/Sema3Ahigh axons are sorted tion along the D-V axis (Norlin et al., 2001; Developmental Neurobiology  1932846x, 2015, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dneu.22268 by Emilie Gueguen - Universitätsbibliothek Zuerich , Wiley Online Library on [03/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Neural Map Formation in the Mouse Olfactory System 599 the D-V axis. Loss-of-function and gain-of-function experiments demonstrated that Nrp2 indeed regu- lates the axonal projection of OSNs along this axis (Takeuchi et al., 2010). Based on the visual system, the repulsive ligand Sema3F was expected to show a gradient in the target OB. Curiously, however, the Sema3F transcript was not detected in the OB. In the olfactory system, an axon-guidance receptor, Nrp2, and its repulsive ligand, Sema3F, are both expressed by OSN axons in a complementary man- ner to regulate D-V projection. These observations indicate that axon–axon interactions of OSNs using the repulsive signaling of Nrp2/Sema3F are essen- tial to the segregation of dorsal-zone and ventral- zone axons. Expression levels of D-V guidance molecules, Figure 3 Two different strategies for neural map forma- such as Nrp2 and Sema3F, are closely correlated with tion. Axon-target versus axon–axon interactions in neural the expressed OR species. However, unlike Nrp1 and map formation. Preservation of the relative spatial relation- Sema3A, which are involved in A-P positioning, the ships of projecting axons between origin and target sites is transcription of Nrp2 and Sema3F gene is not down- a general feature of neural map formation. In the vertebrate stream of OR signaling. If D-V guidance molecules visual system, graded expression of Eph receptors on reti- are not regulated by OR-derived signals, how are nal axons and their repulsive ligands, ephrins, from the their expression levels determined and correlated superior colliculus regulates axonal projection of retinal with the expressed OR species? OR gene choice is ganglion cells (upper). In the mouse olfactory system topo- not purely stochastic and is affected by location in graphic order emerges in axon bundles well before they reach the target, suggesting pretarget axon–axon interac- the OE (Miyamichi et al., 2005). It appears that both tions. An axon guidance receptor, Nrp1, and its repulsive OR gene choice and Nrp2 expression levels are com- ligand, Sema3A, are both expressed by OSNs in the OE in monly regulated by positional information within the a complementary manner and mediate pretarget axon sort- OE. This regulation is likely determined by cell line- ing within the bundles (lower). age, resulting in the use of specific sets of transcrip- tion factors, which can explain the anatomical Cho et al., 2007; Nguyen-Ba-Charvet et al., 2008; correlation along the dorsomedial-ventrolateral axis Takeuchi et al., 2010). of the OE. Temporal Regulation of D-V Targeting Positional Information of OSNs in the OE of OSN Axons Regulates D-V Projection Complementary and graded expression of Nrp2 and OR genes expressed by OSNs that project to the dor- Sema3F in OSNs suggested that repulsive interac- sal OB are distributed throughout the dorsal zone in tions occur among OSN axons to establish map order. the OE (Tsuboi et al., 2006). However, ventral-zone It has been shown that OSNs in the dorsal zone OR genes exhibit spatially limited expression along mature earlier than those in the ventral zone during the dorsomedial-ventrolateral axis of the OE (Miya- embryonic development. Glomerular structures first michi et al., 2005). The relationship between the D- emerge in the anterodorsal domain of the OB (Bailey V positioning of glomeruli and the locations of et al., 1999). The olfactory map appears to expand OSNs in the OE has been demonstrated by retro- ventrally as the OB grows during development. grade DiI staining of OSN axons (Astic et al., 1987; Where and how, then, does Sema3F interact with Miyamichi et al., 2005). These observations suggest Nrp21 OSN axons? Although Sema3F is not pro- that the anatomical locations of OSNs in the OE duced by the cells in the OB, it is detected in the contribute to the D-V positioning of glomeruli in the outer olfactory nerve layer of the anterodorsal OB, OB. How is this positional information of OSNs in which serves as an axon-sorting area before converg- the OE translated to their target site during olfactory ing at its final destination. These observations point map formation? Nrp2 is expressed on OSN axons in toward an intriguing possibility that a repulsive such a way that it forms a gradient in the OB along ligand, Sema3F, is secreted by early arriving dorsal- Developmental Neurobiology  1932846x, 2015, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dneu.22268 by Emilie Gueguen - Universitätsbibliothek Zuerich , Wiley Online Library on [03/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 600 Nishizumi and Sakano Figure 4 Axonal projection of OSNs along the D-V axis. In the OE, D-zone OSNs mature first and extend their axons to the OB. These axons express Robo2 and project to the embryonic OB with the aid of repulsive interactions with Slit1 (left). Axonal extension of OSNs occurs sequen- tially along the D-V axis of the OE as the OB grows ventrally during embryonic development. In OSNs, an axon guidance receptor, Nrp2, and its repulsive ligand, Sema3F, are expressed in a com- plementary and graded manner. Sema3F is secreted in the anterodorsal region of the OB by early- arriving D-zone axons (middle). Sequential projection of OSN axons in the OB prevents the late- arriving Nrp21 axons from invading the dorsal region of the OB (right). ED, embryonic day. zone axons and is deposited in the anterodorsal OB to destinations in the OB, further refinement of the glo- serve as a guidance cue to repel late-arriving ventral- merular map needs to occur through fasciculation zone axons that express the Nrp2 receptor (Takeuchi and segregation of axon termini in an activity- et al., 2010). dependent manner (Fig. 2). Then, what guides pioneer dorsal-OSN axons to the anterodorsal area in the OB? It has been reported that Robo21 dorsal-OSN axons project to the dorsal Conversion of the Olfactory Map from region of the OB by repulsive interactions with Continuous to Discrete secreted ligands (Cho et al., 2007; Nguyen-Ba- Charvet et al., 2008). One of the repulsive ligands of To study how OR-specific glomerular segregation is Robo2, Slit1, is detected in the septum and ventral regulated, Serizawa et al. (2006) searched to find a OB during early developmental stages. These obser- group of genes with expression profiles that correlate vations suggest that Robo2/Slit1 signaling plays an with the expressed OR species. Using a transgenic important role in D-V projection of OSNs. In the total mouse in which the majority of OSNs express a par- KO for the Robo/Slit system, OSN axons can mistar- ticular OR, they were able to identify such genes. get to surrounding non-OB tissues (Nguyen-Ba-Char- They include ones that code for homophilic adhesive vet et al., 2008). Repulsive interactions between molecules, for example, Kirrel2 and Kirrel3. Repul- Robo2 and Slit1 are probably needed to restrict sive molecules, such as ephrin-As and EphAs, were the first wave of OSN projection to the anterodorsal also identified, which are expressed in a complemen- OB (Fig. 4). tary manner in each subset of OSNs. Therefore, inter- actions between two subsets of axons, one that is ephrin-Ahigh/EphAlow and another that is ephrin- Alow/EphAhigh, may be important for the segregation ACTIVITY-DEPENDENT MAP of dissimilar OSN axons. We assume that a specific REFINEMENT set of adhesive and repulsive molecules, whose expression levels are determined by OR molecules, During embryonic development, a coarse olfactory regulates the axonal fasciculation of OSNs (Serizawa map topography is established by a combination of et al., 2006) (Fig. 5). It is not clear at this point how D-V patterning based on anatomical locations of many sets of sorting molecules are involved in glo- OSNs, and A-P patterning based on OR-derived merular segregation. However, several sets of adhe- cAMP signals. It has been shown that neighboring sion/repulsion molecules should be enough to sort glomerular structures are intermingled before birth, out neighboring glomerular structures. and discrete glomeruli emerge during the neonatal It should be noted that a coarse map generated dur- period. After OSN axons reach their approximate ing embryonic development is a continuous map Developmental Neurobiology  1932846x, 2015, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dneu.22268 by Emilie Gueguen - Universitätsbibliothek Zuerich , Wiley Online Library on [03/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Neural Map Formation in the Mouse Olfactory System 601 Figure 5 Activity-dependent local sorting of OSN axons for glomerular segregation. OR-specific and activity-dependent expression of adhesion and repulsive molecules (left). OSNs expressing activity-high OR (OR-A) would produce higher levels of Kirrel2 and EphA but lower levels of Kir- rel3 and ephrin-A. In contrast, OSNs expressing activity-low OR (OR-B) would express higher levels of Kirrel3 and ephrin-A but lower levels of Kirrel2 and EphA. Schematic diagrams depict how Kir- rel2, Kirrel3, EphA, and ephrin-A contribute to the OR-specific axon sorting for glomerular segrega- tion (right). OSNs expressing the same type of OR fasciculate their axons by hemophilic adhesive interactions of Kirrel2 or Kirrel3. Axon termini of OSNs expressing different types of ORs are sepa- rated by the repulsive interaction between EphA and ephrin-A. Modified from Serizawa et al., 2006. whose topography is established by graded distribu- molecules were affected by unilateral naris occlusion. tion of axon guidance molecules. This map is then In the occluded naris, Kirrel2 expression was down- converted to the discrete glomerular map during the regulated and Kirrel3 expression was up-regulated, neonatal period. Activity-dependent glomerular seg- whereas expression of the A-P guidance receptors regation is important not only for map refinement, by Nrp1 and PlxnA1 was not affected (Serizawa et al., eliminating satellite glomerular structures, but also 2006). These results indicate that stimulus-driven OR for the conversion of the map from one that is contin- activity contributes to the local sorting of OSN axons uous to one that is discrete (Zhao and Reed, 2001; Yu but does not affect global targeting along the A-P et al., 2004). axis. It is interesting to study whether odorous stimuli could change the expression profile of glomerular Activity-Dependent Glomerular segregation molecules (Nakashima et al., 2013). Segregation Transgenic mice in which the MOR29B gene is Unlike the global targeting of OSN axons in tagged with IRES-gapEYFP were housed in the pres- embryos, local sorting occurs in an activity- ence of vanillin, a ligand for MOR29B. When the dependent manner after birth. Blocking neuronal mice were exposed to vanillin, Kirrel2 expression activity through the expression of an inward rectify- was dramatically increased. However, Nrp1 expres- ing potassium channel, Kir2.1, severely affects axo- sion was not affected by vanillin exposure in the nal convergence (Yu et al., 2004). Mice that are MOR29B-positive OSNs. These results indicate that mosaic KOs for CNG-A2 reveal the segregation of the expression of glomerular segregation molecules CNG-positive and -negative glomeruli for the same is regulated by ligand-induced OR signals, whereas OR (Zheng et al., 2000; Serizawa et al., 2006). Inter- the expression of A-P-targeting molecules is likely to estingly, expression levels of glomerular segregation be driven by ligand-independent OR activity. Developmental Neurobiology  1932846x, 2015, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dneu.22268 by Emilie Gueguen - Universitätsbibliothek Zuerich , Wiley Online Library on [03/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 602 Nishizumi and Sakano BASELINE ACTIVITY OF GPCR Ballesteros et al., 2001). Recently, the 3D structures of b2-AR in its active state and in a complex with a As described above, OR-derived signals that regulate stimulatory G protein, Gs, have been determined A-P targeting molecules are not affected by extrinsic (Rasmussen et al., 2011a, 2011b). As a result of these stimuli or odor ligands. Furthermore, neuronal activ- favorable features, we used b2-AR for the transgenic ity is not involved in the regulation. Then, what kind analysis of the agonist-independent GPCR activity in of OR activity could be responsible for A-P targeting, OSN axonal projections. and how is it generated? We hypothesized that OR baseline activity may be regulating A-P targeting. G- Transgenic Analysis of Activity Mutants protein-coupled receptors (GPCRs), including ORs, are known to possess two different conformation Among the b2-AR mutations analyzed, some affected states, active and inactive (Kobilka and Deupi, 2007). both agonist-independent and dependent activities, Agonists stabilize the receptor in an active form, whereas others affected only one or the other (Naka- whereas inverse agonists lock it in an inactive form. shima et al., 2013). It is expected that mutations In the absence of agonists and inverse agonists, affecting G-protein activation would alter both GPCRs produce a baseline level of cAMP by sponta- agonist-dependent and -independent activities, neously flipping between active and inactive confor- whereas those altering ligand interactions would mations [Fig. 6(A)]. change agonist-dependent activity. Mutations that affect conformational transitions should alter the agonist-independent receptor activity. For further Agonist-Independent Activity that transgenic studies, we selected three b2-AR mutants Regulates A-P Targeting from the collection that significantly altered agonist- independent receptor activity, but not agonist- For different OR species, variable but specific levels dependent activity. of baseline activities can be detected (Reisert, 2010; Nakashima et al. (2013) generated transgenic mice Nakashima et al., 2013). This agonist-independent expressing the mutant or WT b2-AR using an OR activity had long been considered to be noise created gene promoter. This was performed by replacing the by GPCRs, and its functional role was not fully MOR23 coding sequence with that of b2-AR in the appreciated. Since naris occlusion did not affect the MOR23 minigene cassette. The glomerular locations expression of A-P targeting molecules, the ligand- were studied for the WT and mutant b2-ARs tagged independent OR activity was assumed to regulate A- with different fluorescent markers. The activity-low P targeting of OSN axons. To examine this possibil- b2-AR mutants generated glomeruli anterior to that ity, Nakashima et al. (2013) attempted to generate of the WT. In contrast, the activity-high mutation, OR activity mutants. The initial experiment with ORs caused a posterior shift of glomeruli. This finding was not successful due to the challenges of achieving showed a good correlation between the agonist- adequate membrane expression in the heterologous independent activities of b2-ARs and their corre- system. Additionally, the vast diversity of OR family sponding glomerular locations in the OB along the proteins and the lack of three-dimensional (3D) struc- A-P axis. The expression levels of A-P-targeting mol- tural information made prediction and screening of ecules were examined in the b2-AR glomeruli (Naka- activity mutants difficult for OR molecules. shima et al., 2013). Nrp1 expression levels in b2-AR- In contrast, the b2-adrenergic receptor (b2-AR), a expressing OSNs were increased by the activity-high GPCR with the highest sequence homology to ORs, mutation, but lowered by the activity-low mutations is much easier to express in transfected cells and [Fig. 6(B)]. The results were inverse for PlxnA1 com- shares many functional similarities with ORs. When pared with those of Nrp1. It is notable that expression expressed in OSNs with the OR gene promoter, the levels of glomerular segregation molecules, for b2-AR maintains the “one neuron-one receptor rule,” example, Kirrel2 and Kirrel3, were not affected at all couples with the a subunit of Gs or Golf, and substi- by the baseline-activity mutations, indicating that tutes ORs for receptor-instructed axonal projection glomerular segregation molecules are regulated by (Feinstein et al., 2004). Furthermore, b2-AR has distinct OR signals. advantages of being well-characterized for distinct receptor functions. Based on mutational studies, the key amino acid residues in the b2-AR required for G Differential Usages of Gs and Golf in OSNs protein coupling, ligand binding, and generation of agonist-independent activity are well characterized As aforementioned, OR-instructed A-P targeting and (O’Dowd et al., 1988; Savarese and Fraser, 1992; glomerular segregation are differentially regulated by Developmental Neurobiology  1932846x, 2015, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dneu.22268 by Emilie Gueguen - Universitätsbibliothek Zuerich , Wiley Online Library on [03/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Neural Map Formation in the Mouse Olfactory System 603 Figure 6 Agonist-independent GPCR activity that regulates A-P targeting of OSN axons. (A) Generation of baseline activity of GPCRs. GPCRs possess two different conformations, active and inactive. 3D structures of b2-AR (left) are modified from Rasmussen et al. (2011a, 2011b). In the absence of ligand, GPCRs spontaneously interchange between the active and inactive conforma- tions, generating agonist-independent baseline activity (right). (B) A-P targeting regulated by base- line activity. Each OR possesses a unique level of baseline activity and generates a specific amount of cAMP using Gs, but not Golf. The levels of cAMP signals are converted to transcription levels of A-P targeting molecules, for example, Nrp1 and PlxnA1. Activity-high axons project to the poste- rior OB, whereas activity-low axons project to the anterior OB. TM, transmembrane domain; OR, odorant receptor; GPCR, G protein-coupled receptor. two distinct OR-derived cAMP signals. To address activate ACIII in OSNs. However, their functional how these two types of regulation are separately con- differences in the cellular context were not fully rec- trolled during development, the onset of expression ognized. What could be the reason that Gs and Golf was studied for various genes involved in axon guid- are differentially expressed in OSNs during develop- ance and signal transduction in OSNs (Nakashima ment? Imai et al. generated Gs and Golf fusion pro- et al., 2013). At embryonic day (ED) 13.5, hybridiza- teins for different ORs, whose agonists have been tion signals were detected for A-P-targeting mole- established (Nakashima et al., 2013). Both agonist- cules (e.g., Nrp1), but not for glomerular segregation independent and -dependent signals were measured molecules (e.g., Kirrel2). Kirrel2 expression became in vitro by the dual luciferase assay for cAMP pro- prominent only at the late stage of embryonic devel- duction (Saito et al., 2009). Much higher agonist- opment. We also analyzed the onset of Gs, Golf, and independent cAMP signals were detected with Gs other signal transduction molecules. Hybridization than with Golf, whereas ligand response properties signals were detected for Gs at ED13.5. In contrast, were similar between Gs and Golf. It was concluded Golf was expressed at ED17.5, but not ED13.5, indi- that Gs mediates agonist-independent activity more cating that Golf is not required for the expression of efficiently than Golf. This notion was further con- A-P-targeting molecules. firmed by the loss-of-function experiments using KO Gs and Golf are structurally similar, sharing 88% mice of Gs and Golf. Expression of A-P targeting mol- amino acid identity, and both mediate OR signals and ecules was affected by the Gs conditional KO in Developmental Neurobiology  1932846x, 2015, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dneu.22268 by Emilie Gueguen - Universitätsbibliothek Zuerich , Wiley Online Library on [03/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 604 Nishizumi and Sakano Figure 7 Two distinct signal transduction pathways in OSNs. (A) A-P targeting is regulated by agonist-independent OR activity using a noncanonical signaling pathway. In immature OSNs, each OR generates a unique level of cAMP by agonist-independent baseline activity via Gs and ACIII. The level of cAMP signals is converted to the transcription level of A-P targeting molecules, for example, Nrp1 and PlxnA1, through the cAMP-activated PKA pathway, phosphorylating the transcription fac- tor CREB. (B) Glomerular segregation is regulated by stimulus-driven neuronal activity using a canonical signal transduction pathway. In mature OSNs, different ORs generate different levels of neuronal activity using extrinsic stimuli, which ultimately determine the transcription levels of glo- merular segregation molecules, for example, Kirrel2 and Kirrel3. OR, odorant receptor; AC, adenylyl cyclase; PKA, protein kinase A; CREB, cAMP responsive element-binding protein. OSNs, whereas the glomerular segregation molecules 2013). Glomerular segregation is regulated by remained unaffected. In contrast, the Golf KO stimulus-driven neuronal activity, whereas A-P tar- affected glomerular segregation, but not A-P target- geting is regulated by agonist-independent baseline ing. Taken together, these results indicate that Gs activity of ORs (Fig. 7). Different OR signals are plays a major role in regulating A-P targeting in processed for cAMP production at different stages of immature OSNs, followed by the role of Golf for glo- olfactory development. Differences between the two merular segregation in mature OSNs (Fig. 7). types of regulation may also be due to the subcellular localization of ORs, namely, cilia in mature OSNs versus axon termini in immature OSNs. DISCUSSION Although spatial and temporal insulation of two distinct OR signals may be important for differential In the mouse olfactory system, a neural map is regulation, the difference in the source of cAMP sig- formed by the combination of OR-dependent (A-P nals is the major basis for the observed distribution of targeting) and OR-independent (D-V targeting) proc- A-P targeting (graded) and glomerular segregation esses during embryonic development [Fig. 2(A)]. (mosaic) molecules in the glomerular map. Our study These processes are genetically programmed and demonstrated that the equilibrium of conformational independent from the neuronal activity. The map is transition of GPCRs determines the steady-state lev- further refined in an activity-dependent manner dur- els of cAMP in immature OSNs, which ultimately ing the neonatal period [Fig. 2(B)]. Recent studies determine the expression levels of A-P-targeting mol- revealed that A-P targeting and glomerular segrega- ecules (Fig. 6). In contrast, expression of glomerular tion molecules are separately regulated by distinct segregation molecules is regulated by the stimulus- signals of ORs, even though both utilize OR-derived driven neuronal activity in mature OSNs. 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