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Research ArticleNew Research, Sensory and Motor Systems

Lhx2 Determines Odorant Receptor Expression Frequency in Mature Olfactory Sensory Neurons

Guangfan Zhang, William B. Titlow, Stephanie M. Biecker, Arnold J. Stromberg and Timothy S. McClintock
eNeuro 17 October 2016, 3 (5) ENEURO.0230-16.2016; DOI: https://doi.org/10.1523/ENEURO.0230-16.2016
Guangfan Zhang
1Department of Physiology, University of Kentucky, Lexington, Kentucky 40536-0298
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William B. Titlow
1Department of Physiology, University of Kentucky, Lexington, Kentucky 40536-0298
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Stephanie M. Biecker
1Department of Physiology, University of Kentucky, Lexington, Kentucky 40536-0298
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Arnold J. Stromberg
2Department of Statistics, University of Kentucky, Lexington, Kentucky 40536-0082
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Timothy S. McClintock
1Department of Physiology, University of Kentucky, Lexington, Kentucky 40536-0298
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  • Figure 1.
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    Figure 1.

    Most OSNs in 123Cre:Lhx2fl/fl mice continue to express Lhx2. A, In situ hybridization for Lhx2 mRNA in a wild type (WT) mouse. B, In situ hybridization for Lhx2 mRNA in a 123Cre:Lhx2fl/fl mouse. Sus, Sustentacular cell layer; mOSN, mature OSN cell layer; iOSN, immature OSN cell layer; Basal, basal cell layer. Scale bars, 40 µm.

  • Figure 2.
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    Figure 2.

    Conditional deletion of Lhx2 in immature OSNs decreases the abundance of most OR mRNAs. A, In 123Cre:Z/EG mice, recombination-dependent GFP expression marks OSNs in the immature and mature OSN layers of the olfactory epithelium. Dashed line, location of the basal lamina separating the olfactory epithelium and lamina propria. Scale bar, 20 µm. B–E, Gap43 immunoreactivity (B, D) and in situ hybridization for Gap43 mRNA (C, E) identify immature OSN cell bodies in 123Cre:Lhx2fl/fl mice (D, E) and littermate controls (B, C). F–I, Omp immunoreactivity (F, H) and in situ hybridization for Omp mRNA (G, I) identify mature OSN cell bodies in 123Cre:Lhx2fl/fl mice (H, I) and littermate controls (F, G). Scale bars, 20 µm. J, In 123Cre:Lhx2fl/fl mice, the abundance of 676 OR mRNAs decreases (p < 0.01; n = 4; FDR <1%). •, Significant decrease; +, no difference from littermate controls. K, The percentage of class I ORs affected by Lhx2 deletion in immature OSNs is very similar to the percentage of Class II ORs affected, and the magnitude of the decreases are similar for both classes of ORs. Error bars represent SDs.

  • Figure 3.
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    Figure 3.

    Conditional deletion of Lhx2 in immature OSNs reduces OR gene expression frequencies. A, In situ hybridization for Olfr615 labels scattered OSNs in the dorsal olfactory epithelium. B, In 123Cre:Lhx2fl/fl mice, the frequency of OSNs expressing Olfr615 appears to be reduced. C, Normalized cell counts show that ORs are consistently less frequently expressed in 123Cre:Lhx2fl/fl mice compared with littermate controls. Numbers in control bars are the numbers of littermate pairs tested. Diff., Normalized fold differences from the microarray experiment; *Significant difference (p < 0.01; n = 4; FDR <1%). NC, Air space of the nasal cavity; S, nasal septum; arrows, blood vessels in the lamina propria. Scale bars, 100 µm.

  • Figure 4.
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    Figure 4.

    Deletion of Lhx2 in mature OSNs decreases the abundance of most OR mRNAs. A, In OmpCre:Z/EG mice, recombination-dependent GFP expression marks OSNs in the mature OSN layer. Dashed line, Location of the basal lamina separating the olfactory epithelium and lamina propria. Scale bar, 20 µm. B, In OmpCre:Lhx2fl/fl mice, the abundance of 765 OR mRNAs is decreased relative to littermate controls (p < 0.01; n = 4; FDR <1%). •, Significant decrease; +, no difference from littermate controls; ▴, increased abundance. C, Deletion of Lhx2 in immature OSNs (123Cre:Lhx2) and mature OSNs (OmpCre:Lhx2) produced similar effects on the abundance of most OR mRNAs relative to littermate controls. Line, Pearson linear correlation fit; •, significant decrease in at least one of the two datasets; +, no decrease compared with littermate controls.

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    Figure 5.

    Conditional deletion of Lhx2 in mature OSNs reduces OR expression frequencies. A, In situ hybridization shows that Olfr156 is expressed in scattered OSNs in the ventral olfactory epithelium. B, In OmpCre:Lhx2fl/fl mice, the frequency of OSNs expressing Olfr156 is reduced. C, Normalized cell counts from in situ hybridization images show that the ORs tested are less frequently expressed in OmpCre:Lhx2fl/fl mice compared with littermate controls, which is in agreement with normalized fold differences (Diff.) measured in the microarray experiment. *Significant difference (p < 0.01; n = 4; FDR < 1%). T, Turbinate; S, nasal septum. Scale bars, 100 µm.

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    Figure 6.

    Conditional deletion of both Lhx2 and Emx2 in immature OSNs decreases the abundance of 755 OR mRNAs and increases the abundance of 10 OR mRNAs. •, Significant decrease; +, no difference from littermate controls; ▴, increased abundance.

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    Figure 7.

    Conditional deletion of both Lhx2 and Emx2 in immature OSNs reduces OR expression frequencies. A, In situ hybridization shows that Olfr308 is expressed in scattered OSNs in the ventral olfactory epithelium. B, In 123Cre:Lhx2fl/fl:Emx2fl/fl mice, the frequency of OSNs expressing Olfr308 appears to be reduced. NC, Air space of the nasal cavity; S, nasal septum; T, turbinate. Scale bars, 100 µm. C, Normalized cell counts from in situ hybridization images show that ORs are less frequently expressed in 123Cre:Lhx2fl/fl:Emx2fl/fl mice compared with littermate controls. Diff., Normalized fold differences from the microarray experiment. *Significant difference (p 0.01; n 4; FDR 1%).

  • Figure 8.
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    Figure 8.

    The absence of Lhx2 and the absence of Emx2 have distinctly different effects on Olfr15 expression frequency. A, In situ hybridization image showing normal Olfr15 expression in the ventral region of the olfactory epithelium in a littermate control. B, When Lhx2 is deleted in OSNs, Olfr15 expression frequency decreases. C, An image of the left half of a more caudal section from a littermate control shows the normal expression of Olfr15 in the ventral olfactory epithelium. This image shows one of the rare instances of the misexpression of Olfr15 in the dorsal olfactory epithelium in control genotypes (arrow). D, When both Lhx2 and Emx2 are deleted, Olfr15 expression frequency decreases in the ventral region, where it is normally expressed, but misexpression increases in the dorsal region. The approximate boundaries between dorsal and ventral olfactory epithelium are marked in C and D. NC, Air space of the nasal cavity; S, nasal septum; T, turbinate. Scale bars, 100 µm.

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    Figure 9.

    OSN turnover in Lhx2 and Emx2 mutant mice is indistinguishable from that of littermate controls. A, Image of active caspase 3 immunoreactivity showing a cell undergoing apoptosis in the OSN layer of the olfactory epithelium (arrow). B, Counts of the number of active caspase 3 immunoreactive OSNs reveal no significant differences between mutant mice and control littermates (n = 3 mice). C, Phosphorylated histone 3 (phospho-H3) immunoreactivity in the basal cell layer identifies basal cells undergoing mitosis (arrows). D, Quantification of phosphorylated histone 3 immunoreactive basal cells shows no significant differences between mutant mice and control littermates (n = 3 mice). E, CD68 immunoreactivity identifies activated macrophages (arrows). F, Quantification of CD68-immunoreactive macrophages in the olfactory epithelium reveals no significant differences between mutant mice and control littermates (n = 3 mice). Dashed lines, Location of the basal lamina. Scale bars, 50 µm.

Tables

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    Table 1.

    GeneChip Mouse Gene 1.0 ST array probe set OR transcript clusters

    TypeTranscript clusters (n)OR genes (n)
    Single functional gene996994
    Single pseudogene3434
    Multiple gene112104
    No functional gene2
    • View popup
    Table 2.

    In situ hybridization probe cDNAs

    Gene symbol NCBI accession no. Region used
    OmpNM_011010569-1064
    Gap43NM_008083369-835
    Lhx2NM_001290646532-1251
    Olfr6NM_206897237-741
    Olfr15NM_008762172-658; 477-932
    Olfr17NM_020598208-684
    Olfr90NM_146477371-866
    Olfr129NM_146327692-1139
    Olfr156NM_019474125-598
    Olfr308NM_14662125-882
    Olfr545NM_1468401-827
    Olfr615NM_1470801-942
    Olfr963NM_001011827138-851
    Olfr1440NM_146684229-726
    Olfr1465NM_00101184135-454
    • Probes from the two fragments of Olfr15 give identical results. The region of Lhx2 used is contained within exons 1–3, the floxed region of the mutated Lhx2 gene.

    • View popup
    Table 3.

    OR mRNAs significantly affected in conditional Emx2 deletion mutants

    Gene symbol123Cre:Emx2 fold differenceOmpCre:Emx2 fold difference
    Olfr10870.5583*0.7984
    Olfr110.74440.7893*
    Olfr1110.6708*0.7826
    Olfr121.05220.8621*
    Olfr12490.74370.7533*
    Olfr12771.13810.6814*
    Olfr1300.97350.8090*
    Olfr13090.96570.7151*
    Olfr13250.48730.6895*
    Olfr13550.6195*0.7902
    Olfr13590.55820.4949*
    Olfr13640.78810.7082*
    Olfr14190.6804*0.7846*
    Olfr14240.5759*0.6292*
    Olfr14250.84550.7678
    Olfr1441.8484*0.9650
    Olfr14570.82570.7559*
    Olfr14950.6704*1.0229
    Olfr151.6782*1.1469
    Olfr2141.7787*0.8971
    Olfr2220.6071*0.6777*
    Olfr2700.4249*0.8188
    Olfr2830.8051*0.8693
    Olfr2910.6210*0.8153
    Olfr2980.38930.5313*
    Olfr3351.1136*1.0004
    Olfr3610.79650.7296*
    Olfr420.86770.5843*
    Olfr5230.8668*0.8304
    Olfr710.74720.8246*
    Olfr8010.4410*0.8148
    Olfr901.6394*1.0124
    Olfr911.3067*0.8880
    Olfr990.6356*0.8504
    • ↵* Significant difference.

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Lhx2 Determines Odorant Receptor Expression Frequency in Mature Olfactory Sensory Neurons
Guangfan Zhang, William B. Titlow, Stephanie M. Biecker, Arnold J. Stromberg, Timothy S. McClintock
eNeuro 17 October 2016, 3 (5) ENEURO.0230-16.2016; DOI: 10.1523/ENEURO.0230-16.2016

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Lhx2 Determines Odorant Receptor Expression Frequency in Mature Olfactory Sensory Neurons
Guangfan Zhang, William B. Titlow, Stephanie M. Biecker, Arnold J. Stromberg, Timothy S. McClintock
eNeuro 17 October 2016, 3 (5) ENEURO.0230-16.2016; DOI: 10.1523/ENEURO.0230-16.2016
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Keywords

  • enhancer
  • gene expression
  • olfaction
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