Olfactory neurons expressing closely linked and homologous odorant receptor genes tend to project their axons to neighboring glomeruli on the olfactory bulb

Abstract

We have characterized two separate odorant receptor (OR) gene clusters to examine how olfactory neurons expressing closely linked and homologous OR genes project their axons to the olfactory bulb. Murine OR genes, MOR28, MOR10, and MOR83, share 75-95% similarities in the amino acid sequences and are tightly linked on chromosome 14. In situ hybridization has demonstrated that the three genes are expressed in the same zone, at the most dorsolateral and ventromedial portions of the olfactory epithelium, and are rarely expressed simultaneously in individual neurons. Furthermore, we have found that olfactory neurons expressing MOR28, MOR10, or MOR83 project their axons to very close but distinct subsets of glomeruli on the medial and lateral sides of the olfactory bulb. Similar results have been obtained with another murine OR gene cluster for A16 and MOR18 on chromosome 2, sharing 91% similarity in the amino acid sequences. These results may indicate an intriguing possibility that olfactory neurons expressing homologous OR genes within a cluster tend to converge their axons to proximal but distinct subsets of glomeruli. These lines of study will shed light on the molecular basis of topographical projection of olfactory neurons to the olfactory bulb.

Fig. 1.

Genomic structure of the MOR28, MOR10, and MOR83 genes. At the top, isolated phage and BAC clones are shown as horizontal lines. The bottomrepresents schematically the 5′-RACE analysis. The flanking regions of the MOR28, MOR10, and MOR83 genes are enlarged. Boxesindicate exons, and hatched portions depict coding regions. DCRs and MARs are shown by ovals andtriangles, respectively. The MAR sites are predicted by MAR finder according to the method of Singh et al. (1997).E, EcoRI; H,HindIII; P, PstI;Sf, SfiI; X,XbaI; Xh, XhoI.

Fig. 2.

Comparison of amino acid sequences of the MOR28, MOR10, and MOR83 proteins. The MOR28 sequence is shown withone-letter code, whereas only amino acid differences are indicated for the MOR10 and MOR83 sequences. Gray shading represents conserved amino acid residues among the three sequences. The predicted positions of the seven transmembrane domains (TM-I to TM-VII) are depicted below the sequences and the putative glycosylation and phosphorylation sites (blackand gray circles) above the sequences.

Fig. 3.

The 5′-noncoding sequences of the MOR10 and MOR28 transcripts. A, Nucleotide sequence comparison of the 5′-noncoding regions of the MOR10 and MOR28 genes. Identical nucleotides are connected by vertical bars, and both noncoding and coding sequences in exon 1 and exon 2 are depicted byuppercase letters. Upstream genomic sequences as well as intron sequences are noted by lowercase letters. The ATG initiation codons are boxed. Underlined sequences show regions used for antisense RNA probes in the RNase protection assay, and cross-hatched underlining sequences indicate protected regions. The major transcription start sites, identified by the 5′-RACE analysis and by the RNase protection assay, are marked bysmall and large arrowheads, respectively. Primers for the 5′-RACE and RT-PCR analyses are indicated byarrows along the sequences. B, The RNase protection assay. Antisense RNA probes labeled with ³²P were hybridized to poly(A)⁺ RNAs from the mouse liver or olfactory epithelium. After hybridization, samples were digested with RNase and separated on a denaturing polyacrylamide gel (6%). In olfactory poly(A)⁺ RNA, three protected fragments of 247, 252, and 258 nt were detected for the MOR10 probe, whereas those of 269, 271, and 276 nt were detected for the MOR28 probe. No protected products were detected in liver mRNA.

Fig. 4.

The MOR28, MOR10, and MOR83 genes are expressed in the same zone within the olfactory epithelium, but in different olfactory neurons. A–C, Coronal sections of the mouse olfactory epithelium were hybridized with a DIG-labeled antisense RNA probe from the MOR28, MOR10, or MOR83 gene. Sections were washed under a high stringency condition, followed by chromogenic reaction.A, The MOR28 probe hybridized to neurons that lie in some parts of ectoturbinates 1 and 2, according to the nomenclature ofAstic and Saucier (1986). The MOR10 probe (B) and the MOR83 probe (C) also hybridized to neurons that are located in the same zone as the MOR28 probe.D–F, Double-label in situ hybridization of olfactory epithelium sections with two differently labeled probes. Coronal sections were hybridized simultaneously with the following probes: D, MOR28 noncoding (FLU) and MOR10 noncoding (DIG); E, MOR28 noncoding (FLU) and MOR83 coding (DIG);F, MOR10 noncoding (FLU) and MOR83 coding (DIG). Positive cells are red in the first reaction (FLU), whereas positive cells are dark purple in the second reaction (DIG). Scale bars, 200 μm.

Fig. 5.

The MOR10 and MOR28 genes are not expressed simultaneously in individual neurons. Double-label in situ hybridization of olfactory epithelium sections with two differently labeled probes from the MOR10- and MOR28-exon 1 regions. Coronal sections were hybridized simultaneously with the following probes: A,B, MOR10 (FLU) and MOR28 (DIG); C,D, MOR10 (FLU) and MOR10 (DIG). The photographs were taken sequentially after each color reaction: the first FLU-red reaction (A,C), the second DIG-purple reaction (B,D). Scale bars, 100 μm.

Fig. 6.

Localization of the MOR28, MOR10, and MOR83 transcripts to proximal glomeruli within the olfactory bulb. Serial coronal sections of the mouse olfactory bulb were hybridized with a³³P-labeled antisense RNA probe, washed under a high-stringency condition, and exposed to emulsion for 5 weeks. Dark-field micrographs of sections show two pairs of very close, but discrete glomeruli that hybridize to the MOR83 (A,D) or MOR10 (B,E) probe. It should be noted that there are almost neighboring sections exhibiting positive glomeruli in both the ventrolateral (A, B) and ventromedial (D,E) parts of the bulb. In addition, micrographs of sections indicate two pairs of proximal, but discrete glomeruli that hybridize to the MOR10 (B,E) or MOR28 (C,F) probe. These sections are ordered in an anteroposterior manner. Arrows depict positive glomeruli on the bulb. Scale bars, 500 μm.

Fig. 7.

Comparison of amino acid sequences of the A16 and MOR18 proteins. The A16 sequence is shown withone-letter code, whereas only amino acid differences are indicated for the MOR18 sequence. Gray shadingrepresents conserved amino acid residues between both sequences. The predicted positions of the seven transmembrane domains (TM-I to TM-VII) are depicted below the sequences and the putative glycosylation and phosphorylation sites (black and gray circles) above the sequences.

Fig. 8.

Localization of the A16 and MOR18 transcripts to proximal glomeruli within the olfactory bulb. Serial coronal sections of the olfactory bulb were hybridized with a ³³P-labeled antisense RNA probe, washed under a high-stringency condition, and exposed to emulsion for 6 weeks. Dark-field micrographs of sections indicate two pairs of proximal, but discrete glomeruli that hybridize to the A16 (A,C) or MOR18 (B,D) probe. These sections are ordered in an anteroposterior manner. Arrows depict positive glomeruli on the bulb. Scale bars, 500 μm.

Fig. 9.

Relative positions of MOR28, MOR10, MOR83, MOR18, and A16 glomeruli within the olfactory bulb. Consecutive coronal sections of the olfactory bulb in different mice were hybridized with five different ³³P-labeled antisense RNA probes, washed under a high stringency condition, and exposed to emulsion for 6 weeks. To summarize the results, signals of the ventrolateral (A,C) and ventromedial (B,D) glomeruli within the left half of the bulb are represented schematically. Relative positions of glomeruli for the OR genes in the MOR28 cluster (A, B) and the A16 cluster (C,D) are shown. Four sets of the coronal bulb sections from the four different mice were analyzed to determine the relative distances of glomeruli using the MOR10 or A16 glomerulus as a standard position. The relative distance between two glomeruli was calculated by a geometric mean of both distances of 20 μm coronal sections containing the center of each site and the two sites in the anteroposterior plane. The MOR10 and MOR83 glomeruli are arranged very closely at a distance of 80 ± 20 μm, and approximately in the same anteroposterior plane in both sides. The MOR10 and MOR28 glomeruli are arranged at a distance of 250 ± 50 μm and 190 ± 60 μm in the lateral and medial sides, respectively. The A16 and MOR18 glomeruli are arranged at a distance of 190 ± 50 μm and 210 ± 60 μm in the lateral and medial sides, respectively. Abbreviations are A (anterior),P (posterior), D (dorsal), andV (ventral).