ANeuronalIdentityCode for the Odorant Receptor-Specific and Activity-Dependent Axon Sorting

Shou Serizawa,1,2,4 Kazunari Miyamichi,1,2,4 Haruki Takeuchi,1,2,4 Yuya Yamagishi,1 Misao Suzuki,3 and Hitoshi Sakano1,2,* 1 Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0032, Japan 2 CREST Program of Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan 3 Center for Animal Resources and Development, Kumamoto University, Kumamoto 862-0976, Japan 4 These authors contributed equally to this work. *Contact: [email protected] DOI 10.1016/j.cell.2006.10.031

SUMMARY Feinstein et al., 2004; Lewcock and Reed, 2004; Shykind et al., 2004). It is well established that OSNs expressing In the mouse, olfactory sensory neurons (OSNs) the same OR converge their axons to a specific set of glo- expressing the same odorant receptor (OR) meruli in the olfactory bulb (OB) (Ressler et al., 1994; Vas- converge their axons to a specific set of glomer- sar et al., 1994; Mombaerts et al., 1996). Thus, the signals uli in the olfactory bulb. To study how OR- of odorant binding in the olfactory epithelium (OE) are con- instructed axonal fasciculation is controlled, verted to a topographic odor map of activated glomeruli in we searched for whose expression pro- the OB. For the positioning of glomeruli, previous studies demonstrated that the dorsal-ventral (D-V) arrangement files are correlated with the expressed ORs. Us- of glomeruli in the OB is correlated with the locations of ing the transgenic mouse in which the majority OSNs in the OE (Saucier and Astic, 1986; Ressler et al., of OSNs express a particular OR, we identified 1994; Vassar et al., 1994; Miyamichi et al., 2005). In con- such genes coding for the homophilic adhesive trast, the anterior/posterior arrangement of glomeruli ap- molecules Kirrel2/Kirrel3 and repulsive mole- pears to be independent of the epithelial locations of cules ephrin-A5/EphA5. In the CNGA2 knockout OSNs and more dependent on the expressed ORs (Miya- mouse, where the odor-evoked cation influx michi et al., 2005). In fasciculating axons, even a single is disrupted, Kirrel2 and EphA5 were down- amino acid difference in OR molecules can cause the seg- regulated, while Kirrel3 and ephrin-A5 were regation of axon termini (Ishii et al., 2001; Feinstein and upregulated, indicating that these genes are Mombaerts, 2004). transcribed in an activity-dependent manner. How is it, then, that the neuronal identity of each OSN is represented at axon termini during the process of glomer- Mosaic analysis demonstrated that gain of ular map formation? One possibility is that OR are function of these genes generates duplicated directly involved in converging axons to precise glomeru- glomeruli. We propose that a specific set of ad- lar locations (Mombaerts et al., 1996; Wang et al., 1998; hesive/repulsive molecules, whose expression Feinstein and Mombaerts, 2004). It is also proposed that levels are determined by OR molecules, regu- ORs, along with axon-guidance molecules, participate late the axonal fasciculation of OSNs during as guidance receptors in targeting axons to specific sites the process of glomerular map formation. (Cutforth et al., 2003). To address this issue, it was desir- able to obtain a homogeneous cell population that ex- presses the same OR. However, due to the absence of INTRODUCTION appropriate cell lines, it has been difficult to examine the question of neuronal identity of OSNs. To solve this prob- In the mouse olfactory system, each olfactory sensory lem, we employed a transgenic (Tg) mouse in which the neuron (OSN) expresses only one functional odorant re- majority of OSNs express a particular OR under ceptor (OR) gene in a monoallelic and mutually exclusive the control of a locus control region, H (Serizawa et al., manner (Buck and Axel, 1991; Chess et al., 1994; Malnic 2003). In the present study, we compared gene ex- et al., 1999; Serizawa et al., 2000). This one neuron-one re- pression profiles in the OE between Tg and non-Tg mice ceptor rule is maintained by negative feedback by the ex- and identified several cell-recognition molecules whose pressed OR molecules and forms the genetic basis for expression levels are uniquely regulated by ORs in an OR-instructed axonal projection (Serizawa et al., 2003; activity-dependent manner.

Cell 127, 1057–1069, December 1, 2006 ª2006 Elsevier Inc. 1057 Figure 1. The Transgenic System Where the Majority of OSNs Express a Particular OR Gene (A) Structure of the transgenic construct, H- MOR28. It contains the MOR28 minigene, the H region at its 50 end, and an IRES-tau-ECFP tag downstream of the MOR28 coding se- quence (lower line). A part of the genomic structure of the MOR28 cluster is also shown (upper line). (B) In situ hybridization of OE sections with the MOR28 gene probe. Two OE sections were an- alyzed: one from the nontransgenic mouse (non-Tg) and the other from the H-MOR28 transgenic mouse. Hybridization signals (dark grains) are much more intense and abundant in the H-MOR28 mouse OE. (C) In situ hybridization of OE sections with the S100A5 and Kirrel2 gene probes. Both non-Tg and H-MOR28 transgenic mice were analyzed. Expression of the two genes appears to be suppressed in the H-MOR28 mouse OE. (D) Numbers of tags per 100,000 in the SAGE analysis. The bar graph compares the numbers of tags between the non-Tg and H-MOR28

mice for OMP, S100A5, Kirrel2, Golf, and Kirrel3.

RESULTS from the non-Tg mouse. Five examples from the SAGE data are shown in Figure 1D. The genes that showed dif- Search for the Genes Whose Expression Levels ferences in the SAGE data were further screened by in Are Correlated with ORs situ hybridization. Among 150 genes with the greatest To study how OR-specific axonal segregation occurs, we numbers of SAGE tags from the non-Tg sample, Kirrel2 searched for genes whose expression profiles in the OE and S100A5 were the only genes that showed differences are uniquely correlated with the expressed OR species. in expression patterns between non-Tg and Tg OEs. Thus, For such an experiment, homogeneous cell samples that we chose these two genes for further analysis. Kirrel2 express a particular OR were needed. We previously re- belongs to the immunoglobulin (Ig) superfamily, whose ported a locus control region, H (homology), that activates homologs have been characterized in Drosophila and the MOR28 cluster for transcription and affects the choice C. elegans as adhesive cell-recognition molecules in the of the OR genes within the cluster (Serizawa et al., 2003). nervous system (Schneider et al., 1995; Ruiz-Gomez Using the H-region DNA, we were able to establish a Tg et al., 2000; Shen and Bargmann, 2003). S100A5 is a mem- mouse (H-MOR28) in which the majority of OSNs express ber of the low-molecular-weight and acidic calcium-bind- a particular OR gene, MOR28 (Serizawa et al., 2003). This ing proteins and has been reported to be expressed differ- Tg construct, the 13 kb H-MOR28, contains the H-region at entially in OSNs (Yu et al., 2005). Expression of both Kirrel2 the 50 end and an IRES-tau-ECFP tag downstream (Fig- and S100A5 in the OE was downregulated in the ure 1A). In the ventrolateral area of the OE in the homozy- H-MOR28 mouse (Figure 1C). gous Tg mouse, more than 90% of mature OSNs express Downregulation of Kirrel2 transcription in the MOR28- MOR28 (Figure 1B). It should be noted that the one neuron- expressing OSNs was observed not only for the H-MOR28 one receptor rule is maintained in the H-MOR28-express- transgene, but also for the endogenous MOR28.InFig- ing cells, even though the choice of the transgene over en- ure 2B, coexpression is examined by double in situ hybrid- dogenous ORs is greatly enhanced (Serizawa et al., 2003). ization of the OE sections isolated from a non-Tg mouse. In Using the OE of the H-MOR28 mouse, we performed OSNs expressing the endogenous MOR28 (stained green), serial analysis of gene expression (SAGE), searching for the level of Kirrel2 transcripts (stained red) was quite low. In genes whose expression levels differed among different the superimposed photos, MOR28-expressing OSNs did OSNs, but correlated with the particular OR gene ex- not show coexpression (yellow) with Kirrel2. We then ana- pressed. RNA profiles were compared between two OE lyzed two other subsets of OSNs expressing the endoge- samples: one from the H-MOR28 Tg mouse, and the other nous MOR10 and MOR83, which belong to the same

1058 Cell 127, 1057–1069, December 1, 2006 ª2006 Elsevier Inc. Figure 2. Complementary Expression of Kirrel2 and Kirrel3 Genes (A) Structures of the BAC transgenic constructs Tg-MOR28 and Tg-MOR28/83. MOR28 was tagged with IRES-tau-EYFP. The MOR28 cod- ing sequence is replaced with that of MOR83 in the Tg-MOR28/83 construct. (B) Double in situ hybridization of OE sections isolated from the 10-week-old nontransgenic mouse. The sections were hybridized with dif- ferently labeled RNA probes: OR gene probes (MOR28 and MOR83) were labeled with Flu (stained green), while the Kirrel2 probe was labeled with DIG (stained red). Coexpressing cells appear yellow in the merged photos. Kir- rel2 is coexpressed with MOR83 but is not with MOR28. (C) Double in situ hybridization of OE sections isolated from two different 10-week-old trans- genic (Tg) mice. One carries the 200 kb BAC Tg construct in which the MOR28 gene is tagged with IRES-tau-EYFP (Tg-MOR28). The other mouse essentially carries the same con- struct, but with the coding sequence replaced with that of MOR83 (Tg-MOR28/83). The OE sections were hybridized with differently la- beled RNA probes: the EYFP probe was la- beled with Flu (stained green) to detect the transgenic OR expression, while the Kirrel2 probe was labeled with DIG (stained red). (D and E) Quantifications of Kirrel2 and Kirrel3 transcripts. Signal intensities were measured in OSNs expressing either endogenous MOR28 or MOR83 OR in the non-Tg mouse (D). Kirrel2 and Kirrel3 transcripts were also quantified for the transgenic OR genes Tg- MOR28 and Tg-MOR28/83 (E). For each OR gene, signal intensities of in situ hybridization with Kirrel2 and Kirrel3 probes were measured in 50 dissociated OSN cells, using the Scion Image program. Expression of endogenous ORs was detected with OR gene probes, and expression of transgenic ORs was detected with the EYFP probe. Error bars indicate SEM; *p < 0.01, Student’s t test. (F) Double in situ hybridization of dissociated OSN cells with Kirrel2 (labeled red with Flu) and Kirrel3 (labeled green with DIG) probes. A large field of view of stained dissociated cells is shown on the left. Six examples of stained, individual cells are shown on the right. K2, Kirrel2; K3, Kirrel3; m, merge. (G) Scatter plot of OSNs doubly stained with the Kirrel2 and Kirrel3 probes (n = 78). For each dissociated OSN, hybridization signal intensities of Kirrel2 and Kirrel3 were measured using the Scion Image program. The straight line represents linear regression (boxed r value: Pearson product moment correlation coefficient, p < 0.001).

MOR28 gene cluster (Figure 1A), that have projection sites expressed OR species. Similar observations were made nearby those for MOR28 in the OB (Tsuboi et al., 1999). with the S100A5 gene (data not shown). Amino acid similarities of MOR28 to MOR10 and To examine whether such OR-correlated expression is MOR83 are 95% and 75%, respectively. In contrast to controlled by the OR proteins themselves or by the OR MOR28, the majority of MOR83-positive cells (stained gene promoters, via a common set of transcription factors, green) turned yellow in the merged pictures, indicating we performed a coding-swap experiment between the that there is coexpression of MOR83 with Kirrel2 (Figures MOR28 and MOR83 transgenes (Figure 2A). We first gen- 2B and 2D). MOR10-positive OSNs expressed Kirrel2, erated a control construct (Tg-MOR28) using a 200 kb bac- but at a lower level (data not shown). It appears that ex- terial artificial (BAC), in which the MOR28 pression levels of Kirrel2 are uniquely correlated with the gene was tagged with the IRES-tau-EYFP. We then

Cell 127, 1057–1069, December 1, 2006 ª2006 Elsevier Inc. 1059 replaced the MOR28 coding sequence with that of MOR83 at axon termini. When two consecutive sections of the (Tg-MOR28/83) in a BAC Tg construct. In the double OB were separately stained with anti-Kirrel2 and anti-Kir- in situ hybridization, the profile of Tg-MOR28/83 was rel3 antibodies, mosaic but complementary staining pat- more like MOR83 than MOR28 (Figures 2C and 2E), indi- terns were obtained for these molecules (Figure 3A). Stain- cating that the OR-correlated Kirrel2 expression is under ing intensities of Kirrel2 and Kirrel3 in each glomerulus the control of the coded product and not the promoter. were quantified and plotted on the scatter graph. As shown in Figure 3B, glomeruli with higher levels of Kirrel2 tended Complementary and OR-Specific Expression to show lower levels of Kirrel3, and vice versa. We also de- of Cell-Recognition Molecules termined the ranking of glomeruli for Kirrel2 and Kirrel3 ex- Encouraged by these initial results, we searched for addi- pression (Figure 3C). The staining intensities of glomeruli tional genes of cell-recognition molecules whose expres- were measured in the horizontal OB sections containing sion levels differed among subsets of OSNs expressing the MOR28 and MOR256-17 glomeruli (Figure 3D) (eight different ORs. Two OE sections, one from the H-MOR28 sections, n = 230 for Kirrel2; eight sections, n = 216 for Kir- mouse and the other from the non-Tg mouse, were hy- rel3). For Kirrel3, the MOR28-expressing glomeruli were al- bridized with RNA probes of the various cell-recognition ways stained darker than those expressing MOR256-17. and axon-guidance molecules, then immunostained For Kirrel2, the situation was the opposite: the MOR28- with anti-GFP antibodies to detect the expression of the positive glomeruli were fainter than those expressing ECFP-tagged H-MOR28. Among the genes examined, MOR256-17, when stained with anti-Kirrel2 antibodies. It Kirrel3, EphA5, EphA7, ephrin-A3, and ephrin-A5 showed should be noted that the relative order of glomerular plots differences between Tg and non-Tg OEs. Like Kirrel2, the for the Kirrel proteins (Figure 3C) is consistent with that for EphA genes were rarely expressed in the H-MOR28- the Kirrel gene transcripts shown in Figure S1. expressing OSNs. In contrast, ephrin-A genes and Kirrel3 To study the possible roles of Kirrel2 and Kirrel3 in con- were uniformly expressed in the OE of the H-MOR28 verging axons, we attempted to localize the putative mouse. ligands for the Kirrel proteins. The extracellular (ec) do- We then examined the OR-correlated expression of mains of Kirrel2 and Kirrel3 were each fused to alkaline Kirrel2 and Kirrel3 genes with various endogenous OR phosphatase (AP), and the resulting fusion proteins (ecK2- genes in non-Tg mice. Signal intensities of in situ hybrid- AP and ecK3-AP) were used as affinity probes (Figure 4A). ization with Kirrel2 and Kirrel3 probes were measured in When OB sections were treated with ecK2-AP and devel- 50 dissociated OSN cells expressing a particular OR oped with AP substrate, the staining patterns with ecK2- gene. The expressed OR species were identified by dou- AP (ligand) were basically the same as those with the ble in situ hybridization. As shown in Figure S1 in the Sup- anti-Kirrel2 antibodies (receptor) (Figure 4B). This was plemental Data available with this article online, different also true between ecK3-AP and anti-Kirrel3 antibodies. coexpression patterns were observed with different OR It should be noted that the removal of OSN axons with genes. For example, the MOR9-2 was coexpressed with Triton X resulted in the complete loss of ecK2-AP and Kirrel3, but not with Kirrel2. In contrast, I7-positive OSNs ecK3-AP signals, suggesting that the Kirrel ligands are lo- coexpressed Kirrel2, but not Kirrel3. It appears that Kirrel2 calized to the OSN axons (data not shown). What are the and Kirrel3 are expressed in a roughly complementary ligands for Kirrel2 and Kirrel3? One possibility is that the manner: when one is high, the other is low, and vice versa, receptor molecules act as ligands as well. We transfected in each subset of OSNs. Expression levels of Kirrel2 and COS cells with the Kirrel2 or Kirrel3 gene and treated them Kirrel3 were quantified in individual OSNs dissociated with the ecK2-AP or ecK3-AP probe (Figure 4C). It was from the non-Tg OE (Figure 2F). After in situ hybridization, found that the ecK2-AP probe detected the Kirrel2- staining intensities of the Kirrel2 (labeled red with Flu) and expressing COS cells but not the Kirrel3 cells, and vice Kirrel3 (labeled green with Dig) transcripts were mea- versa (Figure 4D). No interaction was observed between sured. The scatter plot demonstrates the inverse relation- Kirrel2 and Kirrel3. These results suggest that Kirrel pro- ship of the transcriptional levels between the Kirrel2 and teins act as ligands and receptors on OSN axons. Homo- Kirrel3 genes (Figure 2G). It should be noted that the signal philic and adhesive properties of Kirrel2 and Kirrel3 have intensities were variable to some extent even in individual also been implicated by Minaki et al. (2005) and Gerke OSNs with the same OR identity (Figures 2D and 2E and et al. (2005), respectively. Figure S1). Although this may be due in part to measure- ment accuracy, it is possible that there was sampling of Complementary Expression of the EphA5 immature OSNs that were OR positive but not yet fully ex- and ephrin-A5 Genes pressing the Kirrel genes. The variability might also reflect We also examined whether the expression of ephrin-A5 the temporal fluctuation of transcription for these genes. and EphA5 is regulated in an OR-correlated manner. As shown in Figure 5A, different coexpression patterns were The Homophilic Adhesion Molecules Kirrel2 observed with different OR genes. For example, the and Kirrel3 in the Axon Termini of OSNs MOR103-1 was coexpressed with EphA5, but not with We next sought to determine if the levels of transcripts in ephrin-A5. In contrast, cells expressing the MOR104-4 the OE had any relevance to the abundance of proteins coexpressed ephrin-A5, but not EphA5. It appears that

1060 Cell 127, 1057–1069, December 1, 2006 ª2006 Elsevier Inc. Figure 3. Complementary and Mosaic Staining of Glomeruli in the OB with Anti-Kirrel2 and -Kirrel3 Antibodies (A) Two consecutive OB sections were immu- nostained either red with anti-Kirrel2 or green with anti-Kirrel3. A merged picture of the two photos is also shown. (B) Scatter plot of staining intensities of glomer- uli. Intensities of each glomerulus stained with anti-Kirrel2 and anti-Kirrel3 antibodies were measured using the Scion Image program (n = 157). The straight line represents linear re- gression (boxed r value: Pearson product mo- ment correlation coefficient, p < 0.01). (C) Ranking of glomeruli for Kirrel2 and Kirrel3 expression. Intensities of immunostaining with anti-Kirrel2 and anti-Kirrel3 antibodies were determined for each glomerulus and plotted in order. Plots for the MOR28-positive glomer- uli are colored red, and those for the MOR256- 17 are green. Eight OB sections were analyzed for each. Glomeruli for MOR28 and MOR256- 17 were identified by immunostaining adjacent OB sections with respective anti-OR anti- bodies. *p < 0.01, Student’s t test. (D) Immunostaining of OB sections with anti- Kirrel2 and anti-Kirrel3 antibodies. Glomeruli for MOR28 and MOR256-17 were identified with anti-MOR28 and anti-MOR256-17 anti- bodies, respectively. Adjacent OB sections were immunostained with anti-Kirrel2 and anti-Kirrel3 antibodies. MOR28- and MOR256- 17-positive glomeruli are indicated by arrows. Note that, in the MOR28-positive glomerulus, the level of Kirrel3 is high, but that of Kirrel2 is low. The situation is the inverse in the 256-17 glomerulus.

EphA5 and ephrin-A5 are expressed in a complementary Because the EphA5 and ephrin-A5 genes were shown manner in each subset of OSNs: when one is high, the to be transcribed in a complementary manner in the OE, other is low, and vice versa. It should be noted that the we expected that EphA5 and ephrin-A5 proteins are expression levels of ephrin-A5/EphA5 were relatively low also expressed in a complementary manner at axon ter- compared with those of Kirrel2/Kirrel3. For this reason, it mini in individual glomeruli. This was demonstrated to was difficult to quantify ephrin-A5/EphA5 in individual be the case by using the extracellular domains of OSNs, particularly when their expression levels were inter- EphA5 and ephrin-A5 fused to AP (Figure 5C). Cutforth mediary. To solve this problem, complementary expres- et al. (2003) reported that OSNs expressing different sion was examined by double in situ hybridization of OE ORs express different levels of ephrin-A proteins at sections with differently labeled ephrin-A5 and EphA5 axon termini. They assumed that different ORs may in- probes (Figure 5B). The ephrin-A5-positive OSNs (green) teract differently with ephrin-A proteins, thereby regulat- were mainly found on the apical side of the OE, while the ing the ephrin-A levels at axon termini (Cutforth et al., EphA5-positive cells (red) were found on the more basal 2003). Our present studies demonstrate that the OR-cor- side. In the merged picture, most green cells remained related expression can be found not only for ephrin-A5, green, and red cells stayed red. It should be noted that but also for EphA5. Furthermore, the OR-specific eph- both types of cells were OMP positive and appeared to rin-A5/EphA5 expression is regulated at the transcrip- be mature OSNs (data not shown). tional level.

Cell 127, 1057–1069, December 1, 2006 ª2006 Elsevier Inc. 1061 Figure 4. Homophilic and Adhesive Inter- action of Kirrel Proteins (A) Schematic diagrams of Kirrel2 and Kirrel3, and their AP-tagged fusion proteins, ecK2-AP and ecK3-AP. The extracellular domains of Kirrel2 and Kirrel3 were fused to alkaline phos- phatase (AP). (B) Detection of Kirrel proteins and their ligands in the OB. Two consecutive OB sections were stained with the AP fusion proteins and Kirrel antibodies, in various combinations. K2, Kir- rel2; K3, Kirrel3. (C) Binding of AP fusion proteins to the Kirrel gene-transfected COS cells. Binding was de- tected by AP activity using the NBT/BCIP sub- strate. Expressions of Kirrel2 and Kirrel3 in transfected cells were detected with anti-Kir- rel2 and anti-Kirrel3 antibodies, respectively. (D) Measurements of binding affinities of ecK2- AP and ecK3-AP to the COS cells transfected with Kirrel2 (K2), Kirrel3 (K3), or vector plasmid alone (V). Binding was measured by the AP ac- tivity using p-nitrophenyl phosphate as the substrate. Significant binding was detected only in the homologous combinations, Kirrel2- transfected COS cells with ecK2-AP, or Kir- rel3-transfected COS cells with ecK3-AP, indi- cating homophilic and adhesive interactions. Error bars indicate SEM; *p < 0.01, Student’s t test.

Activity-Dependent Expression of Cell-Adhesion of Kirrel2 and Kirrel3 were compared between the two Molecules types of glomeruli: those that were CNGA2 positive and How, then, is the OR-correlated expression of cell- those that were CNGA2 negative. As shown in Figure 6B, adhesion molecules regulated? We assumed that the CNGA2-negative glomeruli contained reduced levels of OR-evoked signaling cascade may be involved in the Kirrel2, but increased levels of Kirrel3. We performed OR-correlated regulation. To examine the possibility, we a similar experiment with the anti-ephrin-A5 antibodies analyzed the expressions of Kirrel2 and Kirrel3 in the het- and found that CNGA2-negative glomeruli contained in- erozygous mutant for the cyclic nucleotide-gated (CNG) creased levels of ephrin-A5 (Figure S3B). Downregulated channel gene (Brunet et al., 1996), CNGA2, located on expression of EphA5 was observed in the CNGA2-defi- the mouse chromosome X (Lin et al., 2000). Random X in- cient mouse (Figure S3A). The CNG channel-dependent activation in the heterozygous female mouse allows the expression of Kirrel2 and Kirrel3 was also examined for mosaic analysis of the CNGA2 function (Zhao and Reed, glomeruli with a known OR, 256-17 (Figure 6C). In the het- 2001)(Figure 6A). Segregation of glomeruli for the erozygous female mouse, two adjacent glomeruli were CNGA2-positive and -negative OSNs has been reported detected for 256-17: one was CNGA2 positive, and the for M71 (Zheng et al., 2000). A similar observation was other was CNGA2 negative. Here again, CNG-negative made for two additional OR genes, 256-17 (Figure 6C) glomeruli contained reduced levels of Kirrel2, but in- and MOR28 (Figure S2). We examined the expression creased levels of Kirrel3. Basically, the same results levels of Kirrel2 and Kirrel3 in the CNGA2-positive were obtained with the ligand of Kirrel3, i.e., ecK3-AP and -negative glomeruli by immunostaining. Intensities fusion protein (Figure 6C).

1062 Cell 127, 1057–1069, December 1, 2006 ª2006 Elsevier Inc. Figure 5. Complementary Expression of the ephrin-A5 and EphA5 Genes (A) Double in situ hybridization of OE sections. OE sections of the 10-week-old nontransgenic mouse were hybridized with Flu-labeled OR gene probe (103-1 or 104-4) (stained green) and DIG-labeled ephrin-A5 or EphA5 probe (stained red). Right panels show quantifications of ephrin-A5 or EphA5 transcripts in OSN sub- populations expressing 103-1 or 104-4. Fifty green-stained OSN images were collected and measured for mean signal intensities of ephrin-A5 or EphA5 expression (red). Error bars indicate SEM; *p < 0.01, Student’s t test. (B) Double in situ hybridization of an OE section isolated from a nontransgenic mouse. The sec- tions were hybridized with DIG-labeled EphA5 (stained red) and Flu-labeled ephrin-A5 (stained green) probes. (C) Affinity labeling of OB sections using AP fu- sion proteins. Two consecutive OB sections were stained with either EphA5-AP or ephrin- A5-AP to detect ephrin-A or EphA, respec- tively. Note that the two staining patterns are complementary to each other.

Activity-dependent expression was further demon- Mosaic Analysis of the Tg Kirrel2 and Kirrel3 Genes strated by naris occlusion, which is supposed to reduce We then attempted to determine whether the OR-coordi- the neuronal activity in OSNs (Figure S4). A group of genes nated genes are indeed involved in the convergence of (e.g., ephrin-A5 and Kirrel3) expressed in the MOR28-pos- OSN axons. The gain-of-function experiment has been re- itive OSNs were upregulated by naris occlusion. In con- ported for ephrin-A5 using the OR-IRES-ephrin-A5 con- trast, the other group of genes (e.g., EphA5 and Kirrel2) struct (Cutforth et al., 2003). However, it is difficult to eval- whose expression levels were low in the MOR28-express- uate the result without an appropriate negative control. ing OSNs became downregulated when the signaling was Manipulation of the OR gene locus itself could cause the blocked. Downregulation of Kirrel2 was seen as soon as alteration in glomerular targeting. Using the H-region 6 hr of naris occlusion for RNA in the OE and 24 hr for pro- DNA, we devised a Tg system that generates an additional tein in the OB. These results further support the notion that repertoire of OSNs expressing the particular gene to be the OR-correlated expression is regulated in an activity- analyzed at an elevated level. Figure 7A shows the dependent manner. EYFP-tagged H-MOR28 construct in which the MOR28

Cell 127, 1057–1069, December 1, 2006 ª2006 Elsevier Inc. 1063 Figure 6. Activity-Dependent and Com- plementary Expression of Kirrel2 and Kirrel3 (A) Mosaic analysis of the CNGA2 mutant mouse. Due to the stochastic inactivation of X in the female mouse, two types of OSN populations are generated in the het- erozygous mutant for the CNGA2 gene: one that is CNG+ and the other that is CNG. (B) Immunostaining of OB sections. Three con- secutive OB sections from a heterozygous fe- male mouse (CNGA2+/) were subjected to im- munostaining with anti-Kirrel2, anti-CNG, and anti-Kirrel3 antibodies. CNG-negative glomer- uli are indicated by dotted circles. Mean signal intensities of anti-Kirrel2 and anti-Kirrel3 immu- nostainings were measured for each glomeru- lus with the Scion Image program. On the right, CNG-positive (+) and CNG-negative () glo- meruli were plotted by signal intensities for Kir- rel2 or Kirrel3. *p < 0.001, Mann-Whitney U test and Student’s t test. Red circles and bars indi- cate means and ±SEMs, respectively. (C) Immunostaining of MOR256-17 glomeruli. Three consecutive sections of the OB dis- sected from the heterozygous female mouse (CNG+/) were subjected to staining with anti- MOR256-17, anti-Kirrel2, anti-Kirrel3, anti- CNG, and ecK3-AP. Two MOR256-17 glomer- uli are circumscribed with broken lines, one that is CNG positive and one that is CNG neg- ative. Note that Kirrel2 expression is downre- gulated in the CNG glomerulus, while Kirrel3 expression is upregulated. OB sections were counterstained with DAPI (blue).

coding sequence (exon 2) was deleted and the Kirrel2 We utilized this technique to create mosaic mice as an coding sequence was inserted into the noncoding exon alternative to the traditional knockin method, which re- 1. Due to the H-region, the Tg Kirrel2, now under the con- quires a promoter on the X chromosome like the CNGA2 trol of the MOR28 promoter (H-Kirrel2), is frequently cho- gene (Zhao and Reed, 2001). In the mouse containing sen and activated for transcription in OSNs. Furthermore, the H-Tg construct, two types of MOR28-positive OSNs since the MOR28 coding sequence has been deleted, (stained red) were indeed identified (Figure 7B): those OSNs expressing the H-Kirrel2 transgene stochastically that were transgene-positive (which turned yellow) and activate any one of the endogenous OR genes (Serizawa those that were transgene negative (which remained et al., 2003). In such cells, the level of Kirrel2 should be- red). We then analyzed the H-Kirrel2 Tg mouse for the pro- come uniformly elevated because the H-Kirrel2 is under jection of OSNs expressing the endogenous MOR28. the control of the strong MOR28 promoter in the Tg con- Figure 7C shows immunostaining of OB sections with struct. In contrast, if OSNs did not choose the MOR28 pro- antibodies against MOR28 (red). The sections also moter first in the H-Kirrel2 construct, they should express fluoresced green for EYFP to detect the Tg H-Kirrel2- the endogenous OR genes with normal levels of Kirrel2. In expressing axons. With anti-MOR28 antibodies, two these OSNs, the H-Kirrel2 should be silent due to the neg- neighboring glomeruli were detected as the projection ative feedback regulation by the expressed ORs (Serizawa sites for the endogenous MOR28. However, only one of et al., 2003; Lewcock and Reed, 2004). In this way, we can the two glomeruli was fluorescent for EYFP, suggesting generate two sets of OSNs in the same Tg animal: one ex- that one is H-Kirrel2 positive and the other is H-Kirrel2 pressing normal levels of Kirrel2 correlated with ORs, and negative. Immunostaining with anti-Kirrel2 antibodies the other containing elevated levels. confirmed this notion (data not shown). With the control

1064 Cell 127, 1057–1069, December 1, 2006 ª2006 Elsevier Inc. Figure 7. Mosaic Analysis of the Kirrel2 and Kirrel3 Transgenes (A) Structure of the transgenic H-Kirrel con- struct. The MOR28 coding sequence was de- leted from the EYFP-tagged H-MOR28 con- struct, and the Kirrel2 coding sequence was inserted into the noncoding exon 1 under the control of the MOR28 promoter. In the H-Kirrel transgenic animal, two different repertoires of OSNs are generated: one expressing and the other not expressing the H-Kirrel transgene. Due to the negative feedback regulation by functional OR molecules, OSNs that have cho- sen the endogenous OR genes first cannot ac- tivate the Tg-MOR28 promoter in the H-Kirrel construct. In contrast, OSNs that have chosen the Tg-MOR28 promoter first to activate the H-Kirrel can still choose the endogenous OR genes for expression in a stochastic manner because the coding sequence of the Tg- MOR28 is deleted (no feedback). Two types of MOR28-expressing OSNs are schematically shown: one that expresses the Tg H-Kirrel (shaded green) and one that does not. (B) Staining of OE sections isolated from the H- Kirrel transgenic mouse. MOR28-expressing OSNs were detected by in situ hybridization (red). On the same section, OSNs expressing the H-Kirrel transgene were immunostained with anti-EYFP antibodies (green). In the merged picture, two types of MOR28-positive OSNs (red) are seen: one is transgene positive (turned yellow), and the other is negative (re- mained red). (C) Staining of OB sections isolated from the H- Kirrel2 transgenic mouse. The sections were stained red with MOR28 antibodies and fluo- resced green for EYFP to detect transgene expression. Note that anti-MOR28 antibodies detect two neighboring glomeruli, but only one of them is EYFP positive. Basically the same results were obtained with three other transgenic lines analyzed (lines 7, 51, and 59). (D) Analysis of the H-Kirrel3 transgenic mouse (line 3). The results obtained with the H-Kirrel3 transgenic mouse were similar to those obtained with the H-Kirrel2 mice. Another H-Kirrel3 transgenic line (line 1) was analyzed and gave the same result. (E) The H-(Kirrel)del transgenic mouse. As a control, we also analyzed the H-(Kirrel)del construct where the Kirrel coding sequence is absent. The result from one line (line 1) is shown, but basically the same results were obtained with two other lines (line 4 and line 5).

construct, H-(Kirrel)del, in which the Kirrel2 coding se- differences may reflect the spectrum of relative ratios of quence is absent, only one glomerulus was detected Kirrel2 produced by the transgene over the OR-dependent with anti-MOR28 antibodies, which was also EYFP posi- endogenous levels. It is interesting that, in the CNGA2- tive (Figure 7E). These results demonstrated that the deficient hemizygous mutant background, most glomeruli new population of OSNs expressing elevated levels of demonstrated full segregation by forced expression of the Kirrel2 project their axons to a new set of glomeruli, indi- H-Kirrel2 transgene (data not shown). This is probably be- cating that Kirrel2 may be involved in the axonal fascicula- cause the endogenous Kirrel2 expression was downregu- tion of OSNs. Basically the same result was obtained for lated in the hemizygous male animal. the Kirrel3 gene, using the H-expression system as de- scribed for Kirrel2 (Figure 7D). DISCUSSION In the H-Kirrel2 Tg mouse, not every glomerulus was segregated into Tg and non-Tg glomeruli. Some showed OR-Correlated Expression of Kirrel2 and Kirrel3 segregation within a single glomerular structure: one por- Our present study demonstrated that the Kirrel2 and tion being Tg positive and the other Tg negative. In some Kirrel3 genes, whose expression levels are uniquely corre- glomeruli, both Tg-positive and -negative axon termini lated with the expressed ORs, are transcribed in an activ- were intermingled. Therefore, the extent of segregation ity-dependent and complementary manner. In Drosophila, appears to depend upon the expressed OR species: the the Rst/IrreC protein (fly homolog of Kirrel2/3) is known to

Cell 127, 1057–1069, December 1, 2006 ª2006 Elsevier Inc. 1065 Figure 8. A Model for the Segregation of OSN Axons (Left) OR-specific and activity-dependent ex- pression of cell-adhesion molecules. Suppose that the neuronal activity is higher in OSNs ex- pressing OR-A than in those expressing OR-B. OSNs expressing OR-A would produce higher levels of Kirrel2 and EphA, but lower levels of Kirrel3 and ephrin-A. In contrast, OSNs ex- pressing OR-B would express higher levels of Kirrel3 and ephrin-A, but lower levels of Kirrel2 and EphA. (Right) Schematic diagrams depict- ing how Kirrel2, Kirrel3, EphA, and ephrin-A contribute to the OR-specific segregation of axon termini. OSNs expressing the same type of OR fasciculate their axons by homophilic ad- hesive interactions of Kirrel2 or Kirrel3. Axon termini of OSNs expressing different types of ORs are separated by the repulsive interaction between EphA and ephrin-A.

function as an adhesive axon-guidance molecule in a correlate with the expressed OR species. Among them, homophilic manner (Schneider et al., 1995). Also in the ephrin-A/EphA family genes are particularly interest- C. elegans, the Kirrel2/3 homolog SYG-1 plays a role in ing. In other tissues, ephrin-As and EphAs are known to in- determining the location of specific HSNL synapses teract with each other, causing repulsion of the interacting (Shen and Bargmann, 2003). In the present study, affinity cells (Wilkinson, 2001). Since different types of cells pro- probe in situ and COS cell experiments confirmed the ho- duce different levels of ephrin-As and EphAs in a comple- mophilic, adhesive properties of Kirrel2 and Kirrel3 pro- mentary manner, the ephrin-A/EphA system has been teins. Homophilic interactions of these molecules at proposed to prevent cells from interacting with different axon termini were also confirmed with the H-Kirrel Tg types or from migrating into inappropriate territories in mice (Figure S5). Mosaic analyses using the H-Tg mice the tissue (Xu et al., 1999). Axonal competition for the tar- demonstrated that forced expression of either Kirrel2 or get sites with differential repulsive interactions seems to Kirrel3 generates a separate set of glomeruli. These ob- be mediated by the relative, and not the absolute, levels servations indicate potential roles for Kirrel2 and Kirrel3 of ephrin-A and EphA molecules (Brown et al., 2000). in segregating like axons via homophilic, adhesive interac- In the mouse olfactory system, expression of ephrin and tions (Figure 8). Eph proteins has been analyzed with various antibodies Protein levels of Kirrel2 and Kirrel3 are not all-or-none in (St John et al., 2002). It has been reported that OSNs ex- each OSN but are found as a range with levels that are de- pressing different ORs express different levels of ephrin-A termined by the coexpressed OR species. What would proteins on their axons (Cutforth et al., 2003). Here, we happen to the axons whose expression levels of both found that EphAs are also differentially expressed in differ- Kirrel2 and Kirrel3 are intermediary? One possibility is ent subsets of OSNs. Since ephrin-As and EphAs are ex- that relatively small differences in the expression levels pressed in a complementary manner in each subset of of Kirrel2 and Kirrel3 are sufficient to segregate axon OSNs, the repulsive interaction between two different termini. Alternatively, other sets of adhesion molecules sets of axons, one that is ephrin-Ahigh/EphAlow and the may be involved in fasciculating axons, whose expression other that is ephrin-Alow/EphAhigh, may be important in levels are also correlated with the expressed ORs but in the fasciculation of OSN axons (Figure 8). a different manner from that for Kirrel proteins. It is not clear at this point what the relative difference of Kirrel pro- Neuronal Activity Evoked by the CNG Channel tein levels is that is needed to segregate two sets of OSN Regulates the Axonal Fasciculation of OSNs axons. In our experiment with the H-Kirrel3 transgene We demonstrated that the expression levels of OR-corre- (Figure 7D), the added expression of Kirrel3 protein by lated cell-recognition molecules are affected by the the transgene to the endogenous Kirrel3 was sufficient CNGA2 mutation in OSNs. Since the CNG channel con- to cause the separation of MOR28 glomeruli. verts the OR activity to a membrane potential and Ca2+ entry, an intriguing possibility is that the OR-mediated Complementary Expression of ephrin-A and EphA neuronal activity regulates the expression of the cell- In addition to the Kirrel2 and Kirrel3 genes, several other recognition molecules. In the Drosophila OSNs, it has been genes are also transcribed in OSNs at various levels that reported that spontaneous firing rates are determined

1066 Cell 127, 1057–1069, December 1, 2006 ª2006 Elsevier Inc. by the expressed ORs (Hallem et al., 2004). Also in the instead establish the neuronal identity by a set of cell- mouse, OSNs may vary the rate of spontaneous activity, recognition molecules expressed at axon termini. It has depending upon the expressed OR species. Since the been reported that the D-V arrangement of glomeruli is pattern of the neuronal activity and calcium influx regulate roughly determined by the locations of OSNs in the OE the expression of a particular set of genes in other systems (Saucier and Astic, 1986; Ressler et al., 1994; Vassar (Itoh et al., 1995; Dolmetsch et al., 1997; Chang and Berg, et al., 1994; Miyamichi et al., 2005). This positional infor- 2001; Borodinsky et al., 2004; Hanson and Landmesser, mation may be represented by the expression level of 2004), the OR-specific activity rate may also be converted guidance molecules forming a gradient along the D-V to the expression pattern of cell-recognition molecules. axis. Along the anterior-posterior axis, a different set of If we assume that the OR-specific activity is primarily axon-guidance molecules appear to be involved, whose spontaneous, why does naris occlusion demonstrate an expression levels are correlated with the OR species but effect on Kirrel2/Kirrel3 and ephrin-A5/EphA5 expres- regulated in an activity-independent manner (our unpub- sion? It is possible that every neuronal activity prevented lished data). After axons are guided to approximate desti- by naris occlusion is elicited by odorants. In such a sce- nations in the OB, we assume that the axon termini are nario, we have to assume that most ORs are continuously sorted based on the expressed OR species. Our present activated by odorants in the opened naris. Alternatively, results demonstrated that the levels of transcription of one can assume that the naris occlusion result was inde- a set of cell adhesive and repulsive molecules are uniquely pendent of odorants. It is possible that changes in the mu- correlated with the expressed OR species and are regu- cosal fluid caused by the occlusion resulted in the effect. lated in an activity-dependent manner (Figure 8). We Such changes may affect the membrane potential of propose that a unique combination of cell-recognition/ OSNs and the activity rates in an odorant-independent axon-guidance molecules, whose expression levels are manner. The changes may also affect the intrinsic activity determined by OR molecules, constitute the neuronal level of OR, and thus affect the spontaneous activity rate in identity code for individual OSNs and contribute to the an OR-specific manner. axonal projection during the process of glomerular map In other sensory systems, correlated neuronal activity is formation. known to refine synaptic connections during development (Katz and Shatz, 1996). Axonal inputs simultaneously acti- EXPERIMENTAL PROCEDURES vated with postsynaptic cells are thought to be reinforced by retrograde signals (Cramer and Sur, 1995; Fitzsimonds Tg Mice Details of the plasmid construction are described in the Supplemental and Poo, 1998). In the mouse olfactory system, it has been Experimental Procedures. Eight Tg mouse lines were obtained for the assumed that the correlated presynaptic neuronal activity H-(Kirrel)del, and three lines gave rise to transgene expression. Copy is not required for the OSN projection, since the glomeru- numbers of the Tg constructs in each line were quantified by Southern lar formation is not severely affected by the CNGA2 muta- hybridization and are as follows: one copy for line 1, one copy for line 4, tion (Lin et al., 2000; Zheng et al., 2000). If the neuronal and two copies for line 5. Eight lines were obtained for the H-Kirrel2, activity is essential for sorting OSN axons, why is the fas- and four lines gave expression. Copy numbers are as follows: two cop- ies for line 5, seven copies for line 7, twelve copies for line 51, and six ciculation not severely affected in the CNGA2-deficient copies for line 59. Five lines were obtained for the H-Kirrel3, and two mouse? One possibility is that other channels or signaling lines gave expression. Copy numbers are as follows: one copy for pathways can deliver sufficient levels of activity. It should line 1 and five copies for line 3. Two lines were established for both be noted that residual activity is observed in the CNGA2 Tg-MOR28 and Tg-MOR28/83. mutant (Zhao and Reed, 2001). If this is the case, further The CNGA2 mutant mouse was obtained from The Jackson reduction of the activity should result in the deficient sort- Laboratory. ing of OSN axons. In this connection, inhibition of the spik- Antibodies ing activity of OSNs by overexpressing Kir2.1 disrupted We generated antibodies against Kirrel2 using a unique synthetic pep- the convergence of like axons of OSNs. Thus, spontane- tide, CQASQAGLRSRPAQ. The KLH-conjugated peptide was injected ous neuronal activity appears to be required for the estab- into rabbits (Tanpakushitu-Seisei-Kougyo, Japan). Antibodies against lishment and maintenance of the olfactory sensory map MOR28 were generated with the KLH-conjugated peptide (CKKLIRR (Yu et al., 2004). KEGKEK) in rabbits (Sigma). Antibodies against MOR256-17 and Kirrel3 were generous gifts from Dr. H. Breer (Strotmann et al., 2004) and Dr. H. Ueno (Ueno et al., 2003), respectively. Antibodies against Glomerular Map Formation ephrin-A5 were purchased from Santa Cruz Biotechnology, Inc. Anti- While it has been thought that OR molecules play an in- bodies against CNGA2 were purchased from Alomone Labs. structive role in forming the glomerular map, it has re- mained entirely unclear how this occurs at the molecular In Situ Hybridization and Immunostaining level. OR molecules at axon termini have been assumed OE and OB sections were prepared as described (Tsuboi et al., 1999). to recognize guidance cues on the OB and also mediate To isolate dissociated OSNs, mice were anesthetized with sodium pentobarbital (2.5 mg/animal) and perfused intracardially with 4% the homophilic and adhesive interactions of like axons paraformaldehyde (PFA) in PBS. The OE was dissected out, and (Mombaerts et al., 1996; Wang et al., 1998; Feinstein OSNs were dissociated with a scraper. Cells were filtrated with a nylon and Mombaerts, 2004). However, ORs may not directly mesh and incubated with 4% PFA in PBS on ice for 1 hr. After washing act as guidance receptors or adhesion molecules, but with PBS, dissociated OSNs were mounted on slide glasses.

Cell 127, 1057–1069, December 1, 2006 ª2006 Elsevier Inc. 1067 In situ hybridization and immunostaining were performed essentially T. Yamamori, and members of our laboratory for valuable comments, as described (Tsuboi et al., 1999; Serizawa et al., 2003). For details, and T. Tsubokawa and A. Morinaka for expert technical assistance. also see the Supplemental Experimental Procedures. Received: August 4, 2006 In Situ Binding with AP Fusion Proteins Revised: September 14, 2006 AP stainings were performed essentially as described (Flanagan et al., Accepted: October 18, 2006 2000; Cutforth et al., 2003). Fresh-frozen OB sections were postfixed Published: November 30, 2006 for 20 s at 20C in 100% methanol before continuing with the pub- lished protocol (Flanagan et al., 2000). For EphA5-AP staining, sec- REFERENCES tions were counterstained with 0.3% neutral red. Plasmid construction is described in the Supplemental Experimental Procedures. 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