Olfactory receptor genes expressed in distinct lineages are sequestered in different nuclear compartments

Kyoung-hye Yoon; Tobias Ragoczy; Zhonghua Lu; Kunio Kondoh; Donghui Kuang; Mark Groudine; Linda B. Buck

doi:10.1073/pnas.1506058112

Contributed by Linda B. Buck, March 27, 2015 (sent for review January 6, 2015)

Significance

Odorants are detected in the mouse nose by 1,000 different odorant receptors (ORs) and 14 TAARs. Each olfactory sensory neuron (OSN) expresses one receptor allele. While ORs generate diverse odor perceptions, some TAARs appear to be involved in innate responses, raising questions about mechanisms that could segregate ORs and TAARs in functionally distinct OSN subsets. Here, we identify two OSN subsets with different epithelial expression patterns that express different subgroups of TAARs rather than ORs. Our studies show that Taar and Olfr genes localize in different nuclear compartments, suggesting a physical substrate for their differential regulation. We further find that activation of a Taar allele is accompanied by its escape from peripheral repressive heterochromatin to a permissive interior chromatin environment.

Abstract

The olfactory system translates a vast array of volatile chemicals into diverse odor perceptions and innate behaviors. Odor detection in the mouse nose is mediated by 1,000 different odorant receptors (ORs) and 14 trace amine-associated receptors (TAARs). ORs are used in a combinatorial manner to encode the unique identities of myriad odorants. However, some TAARs appear to be linked to innate responses, raising questions about regulatory mechanisms that might segregate OR and TAAR expression in appropriate subsets of olfactory sensory neurons (OSNs). Here, we report that OSNs that express TAARs comprise at least two subsets that are biased to express TAARs rather than ORs. The two subsets are further biased in Taar gene choice and their distribution within the sensory epithelium, with each subset preferentially expressing a subgroup of Taar genes within a particular spatial domain in the epithelium. Our studies reveal one mechanism that may regulate the segregation of Olfr (OR) and Taar expression in different OSNs: the sequestration of Olfr and Taar genes in different nuclear compartments. Although most Olfr genes colocalize near large central heterochromatin aggregates in the OSN nucleus, Taar genes are located primarily at the nuclear periphery, coincident with a thin rim of heterochromatin. Taar-expressing OSNs show a shift of one Taar allele away from the nuclear periphery. Furthermore, examination of hemizygous mice with a single Taar allele suggests that the activation of a Taar gene is accompanied by an escape from the peripheral repressive heterochromatin environment to a more permissive interior chromatin environment.

The mammalian olfactory system possesses enormous discriminatory power (1, 2). It can distinguish a multitude of volatile odorants as having specific odors as well as elicit innate behavioral or physiological responses (3 ⇓⇓–6).

Mice have 1,000 different odorant receptors (ORs), each expressed by a unique subset of olfactory sensory neurons (OSNs) scattered within one zone of the nasal olfactory epithelium (OE) (7 ⇓⇓⇓–11). ORs are used in a combinatorial manner to detect odorants, a strategy that explains how myriad odorants can be discriminated (12).

However, the OE also contains 14 trace amine-associated receptors (TAARs), whose expression patterns resemble those of ORs (13). Like ORs, TAARs are evolutionarily conserved in vertebrates, suggesting that they may serve a distinct function. Ligands found thus far for TAARs are volatile amines, including several in mouse or predator urine (5, 14, 15). Ligands for a few mouse, fish, and human TAARs elicit aversive or attractive behaviors in their respective species, hinting at a conserved ability of TAARs to stimulate innate responses of potentially adaptive significance (5, 6, 14, 16, 17).

If this is the case, one might envisage OSNs expressing TAARs as a distinct neuronal subgroup capable of conveying signals to the brain that elicit specific behaviors. Consistent with this idea, some OSNs appear biased to express TAARs. OR-expressing OSNs are thought to randomly select one odorant receptor (Olfr) allele for expression, but choose a second Olfr allele to express if the first one fails to produce a functional protein (18 ⇓–20). A high proportion of OSNs that express a mutated Taar gene also express a functional Taar allele, suggesting similar mechanisms for Olfr and Taar “gene choice,” but one tailored to Taars (21, 22).

What regulatory mechanisms dictate TAAR versus OR expression in OSNs? How do OSNs slated to express TAARs avoid expressing ORs when there are about 70 times as many Olfr as Taar alleles? Another question concerns the regulation of gene expression within the Taar family. Whereas Olfr genes are found in clusters at multiple loci on most chromosomes (11), Taar genes are clustered at a single chromosomal locus (23). Like Olfr genes, different Taar genes can be expressed in different spatial domains within the OE (13, 21). The differential expression suggests the existence of additional gene regulatory mechanisms that act at the level of the Taar gene locus, but remain to be elucidated.

Here, we investigated Taar gene choice within the Taar locus as well as at the level of nuclear architecture. At the level of the Taar locus, we find evidence for fine scale mechanisms that bias Taar gene choice to subsets of Taar genes expressed in different OE domains. At the level of the nucleus, we find a striking difference between the intranuclear compartments containing Taar versus Olfr genes. In sharp contrast to Olfr genes, which reside primarily in the nuclear interior near large heterochromatin aggregates (24, 25), Taar genes preferentially localize to a thin rim of heterochromatin adjacent to the nuclear envelope. Our studies further indicate that the activation of a Taar allele involves an escape from this peripheral heterochromatin rim to a more interior location, a shift likely to remove the Taar allele from a repressive heterochromatin environment to one permissive for Taar gene expression.

Results

Differential Regulation of Taar Gene Subsets.

Similar to Olfr genes, different Taar genes can be expressed in different OE spatial domains. Some are expressed in only the dorsal or ventral OE and others in both domains. In knockout (KO) mice with a single Taar gene replaced by a reporter gene, 50–60% of OSNs expressing the reporter also expressed a functional Taar gene (21, 22). Mutation of one dorsal Taar (Taar4) led to the secondary expression of mostly dorsal Taars (21), suggesting that there might be subsets of TAAR OSNs that differ in the Taar genes they can express.

To investigate this question, we compared KO mice with mutant alleles of Taar5 or Taar6, the first expressed in the dorsal OE and the second in both dorsal and ventral OE. In both mutant alleles, the Taar coding region was replaced by a lacZ gene encoding beta-galactosidase (β-gal).

To analyze the coexpression of the mutant Taar alleles with other Taar genes, we costained OE tissue sections with anti–β-gal antibodies and a mix of Taar cRNA probes, or probes specific for individual Taars, or for the Taar7 or Taar8 subfamily, which have five and three members, respectively (Fig. S1). To distinguish expression in dorsal versus ventral OE domains, we used a probe for Nqo1 (NAD(P)H dehydrogenase, quinone 1), which is selectively expressed in the dorsal domain (26).

In both Taar5 and Taar6 heterozygous mutants, about 50–60% of β-gal+ OSNs were colabeled with Taar probes (Table S1). The percentage colabeled with the mixed Taar probe was similar to the summed percentages of those colabeled with single Taar probes, suggesting that individual β-gal+ OSNs expressed only one functional Taar gene.

Expression of the mutant Taar allele resembled that of the wild-type allele in both mutant strains (Fig. S2). Like Taar5, β-gal was expressed almost exclusively in the dorsal Nqo1+ domain in Taar5 mutants. Like Taar6, β-gal was expressed in both dorsal and ventral domains in Taar6 mutants, although the dorsal domain contained a higher percentage of the OSNs expressing β-gal (64.9%) than Taar6 (23.1%) (Table S2).

We observed a striking difference in the complement of Taars coexpressed with Taar5 versus Taar6 mutant alleles (Fig. 1). Relative to other OSNs, OSNs expressing the mutant Taar5 allele showed a similar or increased percentage of cells expressing Taars normally expressed in the dorsal OE domain (Taar2, 3, 4, 5, 8, 9) (21). However, the percentage expressing Taar6, which is normally expressed in both dorsal and ventral domains, was reduced ∼7.5-fold. In addition, there was an approximately threefold decrease in cells expressing Taar7 subfamily members, some of which are normally expressed in only the ventral OE (21). These results suggest that OSNs that express the Taar5 mutant allele are biased to express Taar genes normally expressed in only the dorsal OE domain.

Fig. 1.

Taar gene choice differs in OSNs expressing mutant alleles of Taar5 versus Taar6. (A and B) Histograms show the percentages of OSNs expressing different Taar genes (T2-T9) in Taar+/βgal+ and Taar+/βgal− OSNs in Taar5^lacZ/+ and Taar6^lacZ/+ mice. The profile of Taars coexpressed with the mutant allele differs in Taar5 versus Taar6 mutants. Average ± SEM, n = 4 per mutant. P values were calculated using two-way ANOVA followed by paired t test with Bonferroni correction, *P < 0.05, **P < 0.01, ***P < 0.001. (C–F) Images of the paired dorsal olfactory bulbs show β-gal+ OSN axons converging in a crescent of glomeruli in Taar5^lacZ/+ and Taar6^lacZ/+ mice. Taar6^lacZ/+, but not Taar6^lacZ/lacZ, mice, also show more posterior-lateral β-gal+ glomeruli. Anterior is at the top. (G–I) Darkfield images show rare glomeruli that hybridized to radioactive Taar2, Taar3, or Taar9 riboprobes in olfactory bulb sections (arrowheads). (Scale bar, 200 μm.) (J) A schematic of the dorsal bulb shows the locations of glomeruli that hybridized to four Taar probes in three animals.

In sharp contrast to Taar5 mutants, Taar6 mutants showed little or no bias for the coexpression of particular Taar genes with the mutant allele. The only significant difference was a slight decrease in the percentage of Taar+ OSNs that expressed Taar7 subfamily members in the β-gal+ population compared with other OSNs. These results suggest that OSNs expressing the Taar6 mutant allele either have little preference for the expression of different Taar genes or, alternatively, that they comprise at least two distinct subsets, one biased to dorsal Taars and the other to ventral Taars.

The biased coexpression of the Taar5 mutant allele with dorsal Taars in these studies is similar to that reported for a mutant allele of Taar4, another dorsal Taar (21). However, our findings differ from another study of the same Taar5 KO strain that failed to show a preference for coexpression of the mutant Taar5 allele with other dorsally expressed Taar genes (22). One possible explanation for this difference is that we removed the neomycin resistance gene selection cassette present in the original Taar5 KO allele whereas the other study did not. Previous studies have shown that the presence of such selection cassettes can lead to the abnormal expression of genes with which they are associated (27, 28). Consistent with this explanation, our preliminary experiments showed β-gal+ OSNs in the ventral OE domain of Taar5 mutant animals before, but not after, removal of the selection cassette (Fig. S3).

The differences we observed in Taar5 versus Taar6 mutant animals were reflected in the patterns of glomeruli innervated by β-gal+ OSN axons in the olfactory bulb (Fig. 1 C–F). Like OSNs expressing a particular Olfr, OSNs expressing a given Taar synapse in a few specific glomeruli in the bulb (22, 29). As reported previously for the axons of OSNs expressing Taar4 or Taar5 mutant alleles, β-gal+ OSN axons in both Taar5 and Taar6 heterozygous mutant mice converged in multiple glomeruli that formed a crescent in the dorsal bulb (Fig. 1 C and E). However, heterozygous Taar6 mutants showed additional strongly labeled glomeruli in the more posterior and lateral bulb. These additional glomeruli were absent in homozygous Taar6 mutants (Fig. 1F), suggesting that they were innervated by Taar6-expressing OSNs in the heterozygous mutant.

Using radioactive in situ hybridization to label OSN axons containing specific Taar mRNAs, we observed glomeruli for four different Taars in olfactory bulb sections (Fig. 1 G–J). These studies showed similarly located additional posterior-lateral glomeruli for Taar6, as also seen using Taar6 antibodies (22), whereas glomeruli labeled for three other Taars all had more anterior locations in the dorsal olfactory bulb.

Together, these studies suggest that there are subsets of Taar-expressing OSNs that are biased to express different Taar genes. Moreover, these subsets reflect spatial domain differences in the expression of Taar family members. The clear implication is that there are subgroups of Taar genes that are coregulated, with differences in Taar gene regulation linked to the domain in which the Taar-expressing OSN is located.

Taar and Olfr Genes Are Localized to Different Parts of the Nucleus.

The above results are consistent with the idea that there is a distinct OSN lineage that is biased to express Taar rather than Olfr genes. In most cells, heterochromatin containing inactive genes is located primarily at the nuclear periphery, adjacent to the nuclear envelope, or in multiple pericentromeric or perinucleolar foci in the nuclear interior. The interior foci are referred to as “chromocenters”, because they contain aggregates of DNA and are therefore strongly stained by DAPI (4′,6-diamidino-2-phenylindole dihydrochloride), which binds DNA. However, OSNs have an unusual nuclear architecture in which heterochromatin in the nuclear interior is aggregated at one or a few large chromocenters rather than in multiple smaller chromocenters, as seen in other cell types (24, 25). Previous studies indicate that Olfr genes are colocalized at these chromocenters (24, 25). In mutant mice lacking these large foci, Olfr genes do not cluster and are abnormally expressed, leading to the proposal that the aggregation of Olfr genes governs their monogenic expression (24).

Do Taar genes colocalize with Olfr genes in OSN chromocenters? To address this question, we used fluorescence in situ hybridization (FISH) with BAC (bacterial artificial chromosome) probes on OE tissue sections to visualize the nuclear locations of Taar and Olfr genes. Taar genes are all clustered at a single locus on chromosome 10 whereas Olfr genes are found on most chromosomes and at dozens of different loci (11). We examined five of these Olfr gene loci in addition to the Taar locus. To visualize the nuclear periphery, we used antibodies against lamin B, which marks the nuclear lamina, a filamentous protein network coating the inner nuclear membrane (30).

As previously reported, the nuclei of OSNs contained one or a few central aggregates of heterochromatin, as indicated by their intense staining with DAPI (Fig. 2 A–D and Movie S1). These large chromocenters were not seen to the same extent in “basal cells” at the base of the OE, the location of olfactory stem cells and progenitors, or in the most apical cell layer of supporting (sustentacular) cells adjacent to the nasal lumen. Instead, the basal and sustentacular cells tended to show multiple smaller chromocenters (Fig. 2A and Movie S1).

Fig. 2.

Taar and Olfr genes are sequestered in different nuclear compartments. DNA FISH using Taar and Olfr BAC probes (green) show that Taar alleles often overlap with lamin B (red), which marks the lamina at the nuclear periphery (A and E). Most Olfr alleles are instead located in the nuclear interior (B, C, and E), although one preferentially localizes to the lamina (D and E). The laminar localization of Taar alleles seen in OSNs is reduced or absent in OE basal cells and sustentacular (sus) cells (A and F). Arrowheads indicate Taar alleles colocalized with the lamina, and arrows indicate Taar alleles in the nuclear interior (A). Chromocenters are apparent from their intense DAPI staining (grayscale in A, blue in B–D). In histograms showing the locations of Taar and Olfr alleles (E and F), P+P+, P+P−, and P−P− indicate cells with, respectively, both, one, and no alleles at the nuclear periphery. Olfr BAC reference numbers are indicated (B–E). Average ± SEM based on three animals. n indicates total number of cells analyzed. (Scale bars, 5 μm.)

Each BAC probe labeled two small foci within the nucleus, marking the two alleles of a gene locus. Using Olfr probes, the labeled foci were mostly located within the nucleoplasm and often at or near central chromocenters, as previously reported (Fig. 2 B–E) (24, 25). Only one of the five Olfr BACs (155M18) labeled foci preferentially located at the nuclear periphery, with 60.7% of OSNs showing both alleles coincident with the nuclear lamina (Fig. 2 D and E), as determined by pixel overlap between the two color channels.

In sharp contrast, foci labeled with the Taar probe were predominantly located at the nuclear periphery, where they colocalized with lamin B (Fig. 2 A, E, and F). In 81.7% of OSNs, both Taar alleles were located at the nuclear lamina (Fig. 2E). Moreover, most of the remaining OSNs had one Taar allele at the lamina and one allele at a more interior location in the nucleus. Only 4.0% of the Taar foci abutted a large chromocenter, and none was seen inside a chromocenter.

Previous studies indicate that a gene can have a preferred location that correlates with its activity, but is not always found at that location, even in the same cell type. For example, the beta globin gene is silent in erythroid progenitors and highly expressed in mature erythroid cells, but only 70% of beta globin alleles localize to the nuclear periphery in the progenitors and 28% of alleles remain there in the mature cells (31). Our results indicate a strong preference for the localization of inactive Taar genes to the nuclear periphery.

Notably, other OE cell types did not show the same pattern of Taar gene localization as OSNs (Fig. 2 A and F). In sustentacular cells, the locations of Taar alleles were nearly the reverse of what was seen in OSNs, with 75.9% of cells showing both Taar alleles in the nuclear interior. Taar alleles were also located away from chromocenters, which were smaller and more numerous in sustentacular cells than OSNs. In basal cells, both Taar alleles were associated with the lamina in 38.9% of cells, an organization midway between OSNs and sustentacular cells. It is possible that some of the basal cells were stem or progenitor cells that give rise to both OSNs and sustentacular cells whereas others were “precursor” cells already destined to become OSNs.

These results indicate that Taar and Olfr genes are largely localized to different compartments within the OSN nucleus. They also indicate that the laminar localization of Taar genes seen in OSNs is present to a much lesser degree, or absent, in other OE cell types, and may therefore be specific for OSNs and important for Taar gene regulation in OSNs. Consistent with this idea are studies showing that genes can be targeted to different locations or compartments within the nucleus in a tissue-specific or cell-type specific manner that influences their expression (31 ⇓–33).

LAP2β May Tether Heterochromatin to the OSN Nuclear Periphery.

The vast majority of OSNs express ORs rather than TAARs. Thus, most of the Taar loci observed at the nuclear periphery in the above experiments were presumably silent and located in a repressive environment. To investigate whether there is repressive heterochromatin at the OSN nuclear periphery, we immunostained OE sections for two histone modifications characteristic of heterochromatin: H4K20me3 and H3K9me3 (34).

Antibodies against both histone modifications strongly labeled the large central chromocenters in OSNs (Fig. 3 A and B). However, they also showed faint yet distinct labeling at the nuclear periphery, which was costained for lamin B. This outline could also be faintly observed by DAPI staining, indicating that some condensed heterochromatin is present at the periphery.

Fig. 3.

The OSN nuclear periphery contains heterochromatin and LAP2β, but not lamin A. (A and B) Immunohistochemistry using antibodies against the heterochromatin marks H4K20me3 and H3K9me3 show a thin rim of staining along the nuclear lamina of OSNs, in addition to the intense staining of internal heterochromatin aggregates characteristic of OSN nuclei. DAPI-staining of heterochromatin is similar. Merged image shows the DAPI, heterochromatin marks, and lamin B pseudocolored in blue, green, and red, respectively. (C) Lamin A is seen at the nuclear periphery of OE basal cells, but not OSNs. (D) LAP2β, another lamina-associated protein, is present at the OSN nuclear lamina, where it colocalizes with lamin B. A, apical OE; B, basal OE. (Scale bars, 5 μm.)

How might a thin rim of heterochromatin, and likely Taar genes, be tethered to the nuclear lamina? Previous studies indicate that OSNs lack the lamin B receptor (LBR), which can anchor heterochromatin to the nuclear lamina (24). Another protein implicated in heterochromatin anchoring to the lamina is lamin A. To investigate whether lamin A might serve this function in OSNs, we immunostained OE sections for lamin A.

These experiments revealed lamin A at the nuclear lamina of OE basal cells, but not OSNs (Fig. 3C). Thus, OSNs lack not only LBR, but also lamin A. Previous studies have shown that the loss of both LBR and lamin A can cause an inverted nuclear architecture in cell types with a conventional nuclear architecture, resulting in the appearance of a large central heterochromatin aggregate (35). It is conceivable that the related nuclear architecture of OSNs similarly derives from the lack of both proteins. Consistent with this idea, ectopic expression of LBR in OSNs leads to the disruption of their large central chromocenters and the altered expression of hundreds of genes, including Olfr genes (24).

Immunostaining for other candidate lamina-associated proteins showed that one, LAP2β, is reliably detected at the nuclear lamina of OSNs (Fig. 3D). LAP2β has previously been linked to transcriptional repression and the association of chromatin with the nuclear periphery (36, 37). Although we cannot exclude the involvement of other lamina-associated proteins, these results suggest that LAP2β may be responsible for tethering some heterochromatin, and Taar genes, to the nuclear lamina.

Taar Gene Expression Is Accompanied by a Shift in Nuclear Location.

The above studies indicated that Taar and Olfr genes largely localize to different nuclear compartments. However, because most OSNs express Olfrs, they could not exclude the possibility that the locations of Taar genes differ in OSNs expressing Taars.

To explore this question, we examined Taar4-IRES-mCherry (T4-mCh or “T4-RFP”) mice, which coexpress the fluorescent protein, mCherry with the intact Taar4 gene (21). OE sections were costained with antibodies against mCherry and a Taar BAC FISH probe.

These experiments revealed a marked difference between the locations of Taar genes in OSNs expressing a Taar gene versus other OSNs. DAPI staining indicated that the nuclear architecture of Taar-expressing OSNs resembles that of other OSNs. However, whereas both Taar alleles localized to the nuclear lamina in 81.7% of the total OSN population, this was the case for only 31.6% of mCherry+ OSNs, which express Taar4 (Fig. 4 A and B). Moreover, half of the mCherry+ OSNs had one Taar allele at the nuclear lamina and the other at a more interior location. Thus, there is a clear correlation between the status of Taar gene expression in OSNs and the location of Taar alleles within the nucleus.

Fig. 4.

Taar allele activation is accompanied by a shift in nuclear location. The locations of Taar alleles were examined in OSNs expressing TAAR4, using Taar4-IRES-mCherry mice. (A) Different confocal slices through three mCherry+ (red) OSNs show one Taar allele (green) located in the nuclear interior (above) and the other colocalized with lamin B (white) at the nuclear periphery (below) (arrows). (B) Compared with nearby mCherry- OSNs (mCh−), mCherry+ OSNs (mCh+) show a decrease in the percentage of cells with both Taar alleles at the periphery (P+P+) and increases in the percentages of cells with one (P+P−) or both (P−P−) Taar alleles in the nuclear interior. (C) Taar allele localization was scored in Taar4-IRES-mCherry × ΔT2-9 hemizygous mice, which only have one Taar locus allele. In mCh+ cells, the single Taar allele is always active. Compared with nearby mCh− OSNs, mCh+ OSNs show an increase in the percentage of cells with the Taar locus located in the nuclear interior (P−). Average ± SEM based on three animals. n indicates total number of cells analyzed. (Scale bar, 5 μm.)

Because Taar genes are monoallelically expressed, one question raised by these observations concerned the location of the expressed Taar allele. To address this question, we crossed the T4-mCh mice with mice in which all OE Taar genes are deleted (ΔT2-9 mice) (21). The resulting mice are hemizygous and express Taar genes from a single Taar locus allele.

In the hemizygous mice, the Taar BAC probe labeled a single focus in each cell. In mCherry− OSNs, 85.5% of Taar alleles were colocalized with the lamina. In contrast, the single active Taar allele colocalized with the lamina in only 41.3% of mCherry+ OSNs (Fig. 4C). In other words, 58.7% of OSNs expressing Taar4 had the active Taar allele located away from the lamina, whereas this was true for only 14.5% of other OSNs. Given that the nuclear location of a gene tends to vary somewhat even among cells of the same type, this degree of relocalization from the periphery to the nuclear interior is striking.

Together, these studies indicate that there is a dramatic difference between the nuclear locations of Taar versus Olfr genes in the general OSN population, with Olfr genes largely localized to large aggregates of heterochromatin in the nuclear interior and Taar genes primarily localized to a thin rim of heterochromatin at the nuclear periphery. They further indicate that the activation of a Taar gene is accompanied by a shift in its location from a repressive heterochromatin environment at the periphery to a more interior location that is likely to be permissive for Taar gene expression. This finding is reminiscent of previous observations showing the repositioning of genes away from repressive heterochromatin environments upon, or preceding, their activation (31, 33, 38).

Discussion

The evolutionary conservation of both ORs and TAARs as vertebrate odor detectors has been puzzling, particularly given the large number and combinatorial use of ORs in odor coding. Recent studies indicate that at least some TAARs can elicit innate behaviors (5, 6, 14). This finding suggests a model in which OSNs expressing TAARs versus ORs may be preprogrammed to express those receptor types and to transmit signals through different neural circuits in the brain. This model implies the involvement of distinct regulatory mechanisms in OSNs expressing TAARs versus ORs, but what those mechanisms might be is unknown.

In the present studies, we explored this question by investigating receptor gene choice in mice carrying mutant Taar alleles and by examining the nuclear organization of Taar versus Olfr genes. Our findings are consistent with the idea that Taar OSNs comprise a distinct OSN lineage. They further suggest that there are subsets of Taar OSNs that are biased to express different subgroups of Taar genes, and that those subsets underlie the differential expression patterns of Taars within the OE. Our studies of the OSN nucleus reveal a dramatic difference in the nuclear organization of Taar versus Olfr genes. Although Olfr genes primarily localize to central heterochromatin aggregates, Taar genes localize predominantly to a thin rim of repressive heterochromatin at the nuclear periphery. This finding suggests that the differential regulation of Taar versus Olfr genes involves their sequestration in distinct chromatin compartments within the nucleus. We further find that Taar gene activation is accompanied by a movement away from the periphery, a mechanism that may permit an escape from a repressive environment to one permissive for Taar expression.

TAAR OSNs Comprise a Distinct Lineage Containing Subsets.

Previous studies of Olfr gene expression have suggested that the developing OSN selects a single Olfr allele for expression, but will select a second Olfr allele to express if the first one chosen fails to produce a functional OR protein (18 ⇓–20). Our studies are consistent with the operation of similar mechanisms of receptor gene choice in OSNs that express TAARs and ORs. By examining Taar gene expression in mice containing mutated Taar alleles, we find that about 50–60% of OSNs expressing a mutated Taar allele also express another, functional Taar allele, as previously reported. Whether the remaining 40–50% of these OSNs instead express an Olfr gene is unknown. Previous studies indicate that OSNs expressing a mutant Taar allele can coexpress an Olfr gene, but a degenerate Olfr probe labeled only about 1% of the cells, suggesting that this may be a relatively rare event (21, 22).

Similar studies of Olfr genes could not assess whether OSNs that first choose a mutant Olfr allele subsequently choose another single Olfr allele or multiple Olfr alleles for expression. However, due to the biased secondary expression of Taars in our studies and the relatively small number of Taar versus Olfr genes, it was possible to address this question. By examining OSNs that express a mutant Taar allele for hybridization to different Taar probes versus a mixed Taar probe, we found evidence that only one functional Taar allele is coexpressed with the mutant allele per OSN. These results are consistent with a model in which an OSN that selects a nonfunctional Taar allele for expression can go on to secondarily express only one additional Taar allele.

The above findings clearly indicate that TAAR-expressing OSNs comprise a distinct lineage. However, our results further indicate that there are also TAAR OSN subsets, which are biased to express different subgroups of Taar genes. Moreover, these subsets reflect the spatial domains within which individual TAARs are expressed in the OE. OSNs that select a mutated allele of Taar5, which is normally expressed in the dorsal domain, preferentially coexpress another Taar allele expressed in the same domain. Studies of mice with another mutant dorsal Taar (Taar4) gave similar results (21). In sharp contrast, we find that OSNs that select a mutated Taar gene (Taar6) expressed in both the dorsal and ventral domain can go on to secondarily select a Taar expressed in either domain. One possible explanation for these findings is that Taar6 belongs to two different subsets, one dorsal and one ventral, with each subset restricted to select among Taar genes appropriate for a single domain. This scenario would suggest the existence of at least two Taar OSN subsets: one dorsal and one ventral.

The gene regulatory mechanisms that control the expression of individual Taar genes within specific OE spatial domains are unknown, as are those that control the expression of Olfr genes in different OE zones. Although there are dozens of Olfr gene loci in the genome, Olfr genes expressed in the same zone are found at different loci and those found at the same locus can be expressed in different OE zones (39). The clustering of Taar genes at a single chromosomal locus should facilitate future studies to explore cis regulatory elements and/or epigenetic processes that exert spatial restrictions on Taar gene expression that differ across the Taar gene locus.

Taar and Olfr Genes Are Sequestered in Different Nuclear Compartments.

How is the expression of Taar and Olfr genes segregated in different OSNs? Taar genes resemble Olfr genes in their singular monoallelic expression in individual neurons as well as the expression of different receptor genes in different OE spatial domains. These similarities suggest, at the very least, the existence of similar molecular mechanisms underlying Olfr and Taar gene choice and the maintenance of expression of a single receptor gene in the mature OSN. Although one can imagine how an OSN might randomly pick one receptor allele from the total 2,000 Olfr and 28 Taar alleles, it is more difficult to envision how an OSN that initially chooses a mutated Taar allele is subsequently restricted to secondarily choose one of the other 27 Taar alleles and not any of the 2,000 Olfr alleles.

Our studies reveal one mechanism that may contribute to the segregated expression of Taar versus Olfr genes in the OSN population: the sequestration of Olfr versus Taar genes in different compartments within the nucleus.

OSN nuclei show an unusual organization of heterochromatin. In most cell types, silenced genes are found primarily in heterochromatin compartments located at the nuclear periphery or at multiple foci in the nuclear interior. In OSNs, however, heterochromatin is largely concentrated in one or a few large central heterochromatin aggregates. It has been reported that most Olfr genes colocalize in or near these large chromocenters (24, 25). Ectopic OSN expression of the lamina binding protein LBR prevents the formation of the large chromocenters and Olfr gene clustering, and causes the misexpression of hundreds of genes, including Olfr genes.

We find that the nuclear organization of Taar genes is strikingly different from that of Olfr genes. Using DNA FISH to visualize the nuclear locations of Taar and Olfr gene loci, Olfr gene loci were often found at or near the large central aggregates of heterochromatin. In sharp contrast, Taar alleles localized primarily to the nuclear lamina. In 81.7% of OSNs, both Taar alleles were found at the periphery and, in the total OSN population, ∼90% of Taar alleles were localized to the periphery.

Our studies indicate that OSNs lack not only LBR, but also lamin A, which is thought to be important for the maintenance of a peripheral heterochromatin compartment in the absence of LBR (35). However, we find that OSNs do express LAP2β, another lamina-associated protein that may tether heterochromatin, and Taar genes, to the nuclear periphery.

Although it was conceivable that Taar alleles might have a different location in Taar-expressing neurons, this possibility was excluded by examination of OSNs that coexpress a fluorescent reporter protein (mCherry) with a specific Taar gene. Like other OSNs, Taar-expressing OSNs had large central chromocenters, but Taar alleles were rarely located near the chromocenters.

Taar Gene Expression Is Marked by a Shift in Nuclear Localization.

Our studies indicate that the expression of a Taar gene is generally accompanied by a movement of the encoding Taar locus from the nuclear periphery to a more interior nuclear location. Using mice that coexpress a reporter (mCherry) with a specific Taar gene, it was possible to determine the locations of Taar genes in Taar-expressing OSNs. Strikingly, half of these OSNs showed one Taar allele located at the nuclear periphery and the other allele at a more interior location. This disparate localization was only observed in 16.3% of other OSNs, the majority of which showed both Taar alleles at the periphery.

Using hemizygous mice with only one functional Taar locus, it was further possible to examine the nuclear location of an active Taar allele in Taar-expressing OSNs. The single active allele had an interior location in 58.7% of Taar-expressing OSNs versus only 14.5% of other OSNs. Importantly, these experiments indicate that it is the active Taar allele that transitions from the nuclear lamina to a more interior nuclear location in Taar-expressing neurons.

These findings suggest that the activation of a Taar gene is accompanied, or perhaps preceded, by a shift of the gene away from a repressive heterochromatin environment. Activated Olfr genes also show a locational shift, but in this instance away from the large central heterochromatin aggregates (24). Movement of activated genes away from heterochromatin has also been observed in many other instances, often concomitant with differentiation. For example, as the murine β-globin locus becomes activated during erythroid differentiation, it moves away from the periphery (31), as does the Mash1 locus during neural differentiation of mouse embryonic stem cells (33). In these cases, silenced genes relocate away from a repressive compartment as they are activated and gain access to a transcriptionally permissive environment.

An additional level of spatial regulation is the sequestration of crucial transcription factors in one nuclear compartment and the corresponding gene target in another. For example, during erythroid differentiation, the β-globin locus moves away from the periphery, where it associates with corepressors, into a euchromatic nuclear compartment that contains a transcriptional activator essential for its expression (40). During myogenesis, the relocation of MyoD away from the nuclear periphery is accompanied by a switch in core factors from TFIID to the alternate, and exclusively nucleoplasmic, TAF3 (41). It is conceivable that such mechanisms regulate the expression of numerous genes, including Taar and Olfr genes. By escaping a repressive heterochromatin environment, individual Taar or Olfr genes may gain access to key transcription factors crucial to their activation.

Materials and Methods

Mice.

All procedures using animals were approved by the Fred Hutchinson Cancer Research Center Institutional Animal Care and Use Committee. Adult male and female C57BL/6J mice were obtained from The Jackson Laboratory. Taar5 and Taar6 KO mice (Taar5^{tm1(KOMP)Vlcg} and Taar6^{tm1(KOMP)Vlcg} mice) were generated by the trans-NIH Knockout Mouse Project and obtained from the KOMP Repository (www.komp.org). In these mice, the Taar coding region is replaced by a lacZ sequence followed by a loxP-flanked selection cassette containing a neomycin resistance gene (neo). To remove the selection cassette, we crossed the mice with EIIa-Cre mice, which carry a Cre recombinase transgene under the control of the adenovirus EIIa promoter [EIIa-cre strain (B6.FVB-Tg(EIIa-cre)C5379Lmgd/J) (The Jackson Laboratory (JAX 003724)]. The mice were then backcrossed to C57BL/6J mice, intercrossed, and genotyped to verify the removal of the neo sequence, the presence of the lacZ sequence, and absence of the Taar5 or Taar6 coding sequence. Taar4-IRES-mCherry and ΔT2-9 mice were kindly provided by Thomas Bozza (21).

RNA In Situ Hybridization with Immunohistochemistry.

Fluorescence RNA in situ hybridization was performed as described (13) with minor modifications listed in SI Materials and Methods. For β-gal immunohistochemistry, antibodies used were chicken anti–β-gal (Abcam, ab9361) followed by Alexa-488–conjugated goat anti-chicken Ig (Life Technologies).

Radioactive RNA in Situ Hybridization.

Radioactive in situ hybridization was performed as described with slight modifications (42). See SI Materials and Methods for details.

Whole-Mount X-Gal Staining.

Wholemount staining of β-gal+ OSN axons in the olfactory bulb was performed as described (43).

Fluorescence DNA in Situ Hybridization (FISH) and Immuno-FISH.

3D Immuno-FISH was largely performed as published (24, 31, 44) with some modifications. See SI Materials and Methods for details.

Image Acquisition.

For FISH images, a Zeiss LSM780 confocal microscope running Zeiss Zen 2011 SP2 v.8 software and equipped with a Zeiss Plan-Apochromat 63x/1.40 oil immersion objective was used to collect image stacks (up to 7–10 μm) at 0.25-μm intervals. Image stacks were viewed and scored using either the Zeiss software or ImageJ. FISH spots were deemed to colocalize with the lamina (or chromocenters) if there was pixel overlap between the two channels after appropriate thresholding.

Acknowledgments

We thank Julio Vazquez and the staff of the Scientific Imaging facility at Fred Hutchinson Cancer Research Center for their assistance with the acquisition of microscope images. We would also like to thank the members of the Buck and Groudine labs for helpful discussions and comments throughout the study. This work was supported by the Howard Hughes Medical Institute (L.B.B.) and National Institutes of Health Grants F32 DC011699 (to K.-h.Y.), R01 DC009324 (to L.B.B.), and R01 HL065440 and R37 DK044746 (to M.G.).

Footnotes

↵¹K.-h.Y. and T.R. contributed equally to this work.
↵²Present address: Division of Biological Science and Technology, Yonsei University, Wonju, 220-710, South Korea.
↵³To whom correspondence should be addressed. Email: lbuck{at}fhcrc.org.

Author contributions: K.-h.Y., T.R., Z.L., M.G., and L.B.B. designed research; K.-h.Y., T.R., Z.L., and K.K. performed research; K.-h.Y., T.R., Z.L., K.K., D.K., and M.G. analyzed data; and K.-h.Y., T.R., and L.B.B. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1506058112/-/DCSupplemental.

Freely available online through the PNAS open access option.

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Proceedings of the National Academy of Sciences: 112 (18)

Table of Contents

Submit

[1] ↵
Kandel E,
Schwartz J,
Jessell T,
Siegelbaum S,
Hudspeth AJ
Buck LB,
Bargmann C
(2012) Smell and Taste: The Chemical Senses. Principles of Neuroscience, eds Kandel E, Schwartz J, Jessell T, Siegelbaum S, Hudspeth AJ (McGraw-Hill, New York), pp 712–742
.

[2] Kandel E,

[3] Schwartz J,

[4] Jessell T,

[5] Siegelbaum S,

[6] Hudspeth AJ

[7] Buck LB,

[8] Bargmann C

[9] ↵
Munger SD,
Leinders-Zufall T,
Zufall F
(2009) Subsystem organization of the mammalian sense of smell. Annu Rev Physiol 71:115–140
.
OpenUrl CrossRef PubMed

[10] Munger SD,

[11] Leinders-Zufall T,

[12] Zufall F

[13] ↵
Kobayakawa K, et al.
(2007) Innate versus learned odour processing in the mouse olfactory bulb. Nature 450(7169):503–508
.
OpenUrl CrossRef PubMed

[14] Kobayakawa K, et al.

[15] ↵
Mandiyan VS,
Coats JK,
Shah NM
(2005) Deficits in sexual and aggressive behaviors in Cnga2 mutant mice. Nat Neurosci 8(12):1660–1662
.
OpenUrl CrossRef PubMed

[16] Mandiyan VS,

[17] Coats JK,

[18] Shah NM

[19] ↵
Ferrero DM, et al.
(2011) Detection and avoidance of a carnivore odor by prey. Proc Natl Acad Sci USA 108(27):11235–11240
.
OpenUrl Abstract/FREE Full Text

[20] Ferrero DM, et al.

[21] ↵
Dewan A,
Pacifico R,
Zhan R,
Rinberg D,
Bozza T
(2013) Non-redundant coding of aversive odours in the main olfactory pathway. Nature 497(7450):486–489
.
OpenUrl CrossRef PubMed

[22] Dewan A,

[23] Pacifico R,

[24] Zhan R,

[25] Rinberg D,

[26] Bozza T

[27] ↵
Buck L,
Axel R
(1991) A novel multigene family may encode odorant receptors: A molecular basis for odor recognition. Cell 65(1):175–187
.
OpenUrl CrossRef PubMed

[28] Buck L,

[29] Axel R

[30] ↵
Ressler KJ,
Sullivan SL,
Buck LB
(1993) A zonal organization of odorant receptor gene expression in the olfactory epithelium. Cell 73(3):597–609
.
OpenUrl CrossRef PubMed

[31] Ressler KJ,

[32] Sullivan SL,

[33] Buck LB

[34] ↵
Vassar R,
Ngai J,
Axel R
(1993) Spatial segregation of odorant receptor expression in the mammalian olfactory epithelium. Cell 74(2):309–318
.
OpenUrl CrossRef PubMed

[35] Vassar R,

[36] Ngai J,

[37] Axel R

[38] ↵
Zhang X,
Firestein S
(2002) The olfactory receptor gene superfamily of the mouse. Nat Neurosci 5(2):124–133
.
OpenUrl CrossRef PubMed

[39] Zhang X,

[40] Firestein S

[41] ↵
Godfrey PA,
Malnic B,
Buck LB
(2004) The mouse olfactory receptor gene family. Proc Natl Acad Sci USA 101(7):2156–2161
.
OpenUrl Abstract/FREE Full Text

[42] Godfrey PA,

[43] Malnic B,

[44] Buck LB

[45] ↵
Malnic B,
Hirono J,
Sato T,
Buck LB
(1999) Combinatorial receptor codes for odors. Cell 96(5):713–723
.
OpenUrl CrossRef PubMed

[46] Malnic B,

[47] Hirono J,

[48] Sato T,

[49] Buck LB

[50] ↵
Liberles SD,
Buck LB
(2006) A second class of chemosensory receptors in the olfactory epithelium. Nature 442(7103):645–650
.
OpenUrl CrossRef PubMed

[51] Liberles SD,

[52] Buck LB

[53] ↵
Li Q, et al.
(2013) Synchronous evolution of an odor biosynthesis pathway and behavioral response. Curr Biol 23(1):11–20
.
OpenUrl CrossRef PubMed

[54] Li Q, et al.

[55] ↵
Zhang J,
Pacifico R,
Cawley D,
Feinstein P,
Bozza T
(2013) Ultrasensitive detection of amines by a trace amine-associated receptor. J Neurosci 33(7):3228–3239
.
OpenUrl Abstract/FREE Full Text

[56] Zhang J,

[57] Pacifico R,

[58] Cawley D,

[59] Feinstein P,

[60] Bozza T

[61] ↵
Hussain A, et al.
(2013) High-affinity olfactory receptor for the death-associated odor cadaverine. Proc Natl Acad Sci USA 110(48):19579–19584
.
OpenUrl Abstract/FREE Full Text

[62] Hussain A, et al.

[63] ↵
Horowitz LF,
Saraiva LR,
Kuang D,
Yoon KH,
Buck LB
(2014) Olfactory receptor patterning in a higher primate. J Neurosci 34(37):12241–12252
.
OpenUrl Abstract/FREE Full Text

[64] Horowitz LF,

[65] Saraiva LR,

[66] Kuang D,

[67] Yoon KH,

[68] Buck LB

[69] ↵
Serizawa S, et al.
(2003) Negative feedback regulation ensures the one receptor-one olfactory neuron rule in mouse. Science 302(5653):2088–2094
.
OpenUrl Abstract/FREE Full Text

[70] Serizawa S, et al.

[71] ↵
Lewcock JW,
Reed RR
(2004) A feedback mechanism regulates monoallelic odorant receptor expression. Proc Natl Acad Sci USA 101(4):1069–1074
.
OpenUrl Abstract/FREE Full Text

[72] Lewcock JW,

[73] Reed RR

[74] ↵
Shykind BM, et al.
(2004) Gene switching and the stability of odorant receptor gene choice. Cell 117(6):801–815
.
OpenUrl CrossRef PubMed

[75] Shykind BM, et al.

[76] ↵
Pacifico R,
Dewan A,
Cawley D,
Guo C,
Bozza T
(2012) An olfactory subsystem that mediates high-sensitivity detection of volatile amines. Cell Reports 2(1):76–88
.
OpenUrl CrossRef PubMed

[77] Pacifico R,

[78] Dewan A,

[79] Cawley D,

[80] Guo C,

[81] Bozza T

[82] ↵
Johnson MA, et al.
(2012) Neurons expressing trace amine-associated receptors project to discrete glomeruli and constitute an olfactory subsystem. Proc Natl Acad Sci USA 109(33):13410–13415
.
OpenUrl Abstract/FREE Full Text

[83] Johnson MA, et al.

[84] ↵
Lindemann L,
Hoener MC
(2005) A renaissance in trace amines inspired by a novel GPCR family. Trends Pharmacol Sci 26(5):274–281
.
OpenUrl CrossRef PubMed

[85] Lindemann L,

[86] Hoener MC

[87] ↵
Clowney EJ, et al.
(2012) Nuclear aggregation of olfactory receptor genes governs their monogenic expression. Cell 151(4):724–737
.
OpenUrl CrossRef PubMed

[88] Clowney EJ, et al.

[89] ↵
Armelin-Correa LM,
Gutiyama LM,
Brandt DY,
Malnic B
(2014) Nuclear compartmentalization of odorant receptor genes. Proc Natl Acad Sci USA 111(7):2782–2787
.
OpenUrl Abstract/FREE Full Text

[90] Armelin-Correa LM,

[91] Gutiyama LM,

[92] Brandt DY,

[93] Malnic B

[94] ↵
Gussing F,
Bohm S
(2004) NQO1 activity in the main and the accessory olfactory systems correlates with the zonal topography of projection maps. Eur J Neurosci 19(9):2511–2518
.
OpenUrl CrossRef PubMed

[95] Gussing F,

[96] Bohm S

[97] ↵
Kim CG,
Epner EM,
Forrester WC,
Groudine M
(1992) Inactivation of the human beta-globin gene by targeted insertion into the beta-globin locus control region. Genes Dev 6(6):928–938
.
OpenUrl Abstract/FREE Full Text

[98] Kim CG,

[99] Epner EM,

[100] Forrester WC,

[101] Groudine M

[102] ↵
Fiering S,
Kim CG,
Epner EM,
Groudine M
(1993) An “in-out” strategy using gene targeting and FLP recombinase for the functional dissection of complex DNA regulatory elements: Analysis of the beta-globin locus control region. Proc Natl Acad Sci USA 90(18):8469–8473
.
OpenUrl Abstract/FREE Full Text

[103] Fiering S,

[104] Kim CG,

[105] Epner EM,

[106] Groudine M

[107] ↵
Bozza T, et al.
(2009) Mapping of class I and class II odorant receptors to glomerular domains by two distinct types of olfactory sensory neurons in the mouse. Neuron 61(2):220–233
.
OpenUrl CrossRef PubMed

[108] Bozza T, et al.

[109] ↵
Dechat T, et al.
(2008) Nuclear lamins: Major factors in the structural organization and function of the nucleus and chromatin. Genes Dev 22(7):832–853
.
OpenUrl Abstract/FREE Full Text

[110] Dechat T, et al.

[111] ↵
Ragoczy T,
Bender MA,
Telling A,
Byron R,
Groudine M
(2006) The locus control region is required for association of the murine beta-globin locus with engaged transcription factories during erythroid maturation. Genes Dev 20(11):1447–1457
.
OpenUrl Abstract/FREE Full Text

[112] Ragoczy T,

[113] Bender MA,

[114] Telling A,

[115] Byron R,

[116] Groudine M

[117] ↵
Peric-Hupkes D, et al.
(2010) Molecular maps of the reorganization of genome-nuclear lamina interactions during differentiation. Mol Cell 38(4):603–613
.
OpenUrl CrossRef PubMed

[118] Peric-Hupkes D, et al.

[119] ↵
Williams RR, et al.
(2006) Neural induction promotes large-scale chromatin reorganisation of the Mash1 locus. J Cell Sci 119(Pt 1):132–140
.
OpenUrl Abstract/FREE Full Text

[120] Williams RR, et al.

[121] ↵
Martens JH, et al.
(2005) The profile of repeat-associated histone lysine methylation states in the mouse epigenome. EMBO J 24(4):800–812
.
OpenUrl Abstract/FREE Full Text

[122] Martens JH, et al.

[123] ↵
Solovei I, et al.
(2013) LBR and lamin A/C sequentially tether peripheral heterochromatin and inversely regulate differentiation. Cell 152(3):584–598
.
OpenUrl CrossRef PubMed

[124] Solovei I, et al.

[125] ↵
Furukawa K,
Fritze CE,
Gerace L
(1998) The major nuclear envelope targeting domain of LAP2 coincides with its lamin binding region but is distinct from its chromatin interaction domain. J Biol Chem 273(7):4213–4219
.
OpenUrl Abstract/FREE Full Text

[126] Furukawa K,

[127] Fritze CE,

[128] Gerace L

[129] ↵
Somech R, et al.
(2005) The nuclear-envelope protein and transcriptional repressor LAP2beta interacts with HDAC3 at the nuclear periphery, and induces histone H4 deacetylation. J Cell Sci 118(Pt 17):4017–4025
.
OpenUrl Abstract/FREE Full Text

[130] Somech R, et al.

[131] ↵
Zink D, et al.
(2004) Transcription-dependent spatial arrangements of CFTR and adjacent genes in human cell nuclei. J Cell Biol 166(6):815–825
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Olfactory receptor genes expressed in distinct lineages are sequestered in different nuclear compartments

Significance

Abstract

Results

Differential Regulation of Taar Gene Subsets.

Taar and Olfr Genes Are Localized to Different Parts of the Nucleus.

LAP2β May Tether Heterochromatin to the OSN Nuclear Periphery.

Taar Gene Expression Is Accompanied by a Shift in Nuclear Location.

Discussion

TAAR OSNs Comprise a Distinct Lineage Containing Subsets.

Taar and Olfr Genes Are Sequestered in Different Nuclear Compartments.

Taar Gene Expression Is Marked by a Shift in Nuclear Localization.

Materials and Methods

Mice.

RNA In Situ Hybridization with Immunohistochemistry.

Radioactive RNA in Situ Hybridization.

Whole-Mount X-Gal Staining.

Fluorescence DNA in Situ Hybridization (FISH) and Immuno-FISH.

Image Acquisition.

Acknowledgments

Footnotes

References

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Main menu

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Search

Significance

Abstract

Results

Differential Regulation of Taar Gene Subsets.

Taar and Olfr Genes Are Localized to Different Parts of the Nucleus.

LAP2β May Tether Heterochromatin to the OSN Nuclear Periphery.

Taar Gene Expression Is Accompanied by a Shift in Nuclear Location.

Discussion

TAAR OSNs Comprise a Distinct Lineage Containing Subsets.

Taar and Olfr Genes Are Sequestered in Different Nuclear Compartments.

Taar Gene Expression Is Marked by a Shift in Nuclear Localization.

Materials and Methods

Mice.

RNA In Situ Hybridization with Immunohistochemistry.

Radioactive RNA in Situ Hybridization.

Whole-Mount X-Gal Staining.

Fluorescence DNA in Situ Hybridization (FISH) and Immuno-FISH.

Image Acquisition.

Acknowledgments

Footnotes

References

Citation Manager Formats

Article Classifications

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