Visualizing in deceased COVID-19 patients how SARS-CoV-2 attacks the respiratory and olfactory mucosae but spares the olfactory bulb.

Anosmia, the loss of smell, is a common and often the sole symptom of COVID-19. The onset of the sequence of pathobiological events leading to olfactory dysfunction remains obscure. Here, we have developed a postmortem bedside surgical procedure to harvest endoscopically samples of respiratory and olfactory mucosae and whole olfactory bulbs. Our cohort of 85 cases included COVID-19 patients who died a few days after infection with SARS-CoV-2, enabling us to catch the virus while it was still replicating. We found that sustentacular cells are the major target cell type in the olfactory mucosa. We failed to find evidence for infection of olfactory sensory neurons, and the parenchyma of the olfactory bulb is spared as well. Thus, SARS-CoV-2 does not appear to be a neurotropic virus. We postulate that transient insufficient support from sustentacular cells triggers transient olfactory dysfunction in COVID-19. Olfactory sensory neurons would become affected without getting infected.

Regulation of the probability of mouse odorant receptor gene choice.

Each olfactory sensory neuron (OSN) in mouse chooses one of 1,200 odorant receptor (OR) genes for expression. OR genes are chosen for expression by greatly varying numbers of OSNs. The mechanisms that regulate the probability of OR gene choice remain unclear. Here, we have applied the NanoString platform of fluorescent barcodes and digital readout to measure RNA levels of 577 OR genes in a single reaction, with probes designed against coding sequences. In an inbred mouse strain with a targeted deletion in the P element, we find that this element regulates OR gene choice differentially across its cluster of 24 OR genes. Importantly, the fold changes of NanoString counts in ΔP or ΔH mice are in very close agreement with the fold changes of cell counts, determined by in situ hybridization. Thus, the P and H elements regulate the probability of OR gene choice, not OR transcript level per OSN.

Expert curation of the human and mouse olfactory receptor gene repertoires identifies conserved coding regions split across two exons.

BACKGROUND: Olfactory receptor (OR) genes are the largest multi-gene family in the mammalian genome, with 874 in human and 1483 loci in mouse (including pseudogenes). The expansion of the OR gene repertoire has occurred through numerous duplication events followed by diversification, resulting in a large number of highly similar paralogous genes. These characteristics have made the annotation of the complete OR gene repertoire a complex task. Most OR genes have been predicted in silico and are typically annotated as intronless coding sequences. RESULTS: Here we have developed an expert curation pipeline to analyse and annotate every OR gene in the human and mouse reference genomes. By combining evidence from structural features, evolutionary conservation and experimental data, we have unified the annotation of these gene families, and have systematically determined the protein-coding potential of each locus. We have defined the non-coding regions of many OR genes, enabling us to generate full-length transcript models. We found that 13 human and 41 mouse OR loci have coding sequences that are split across two exons. These split OR genes are conserved across mammals, and are expressed at the same level as protein-coding OR genes with an intronless coding region. Our findings challenge the long-standing and widespread notion that the coding region of a vertebrate OR gene is contained within a single exon. CONCLUSIONS: This work provides the most comprehensive curation effort of the human and mouse OR gene repertoires to date. The complete annotation has been integrated into the GENCODE reference gene set, for immediate availability to the research community.

The testicular soma of Tsc22d3 knockout mice supports spermatogenesis and germline transmission from spermatogonial stem cell lines upon transplantation.

Spermatogonial stem cells (SSCs) are adult stem cells that are slowly cycling and self-renewing. The pool of SSCs generates very large numbers of male gametes throughout the life of the individual. SSCs can be cultured in vitro for long periods of time, and established SSC lines can be manipulated genetically. Upon transplantation into the testes of infertile mice, long-term cultured mouse SSCs can differentiate into fertile spermatozoa, which can give rise to live offspring. Here, we show that the testicular soma of mice with a conditional knockout (conKO) in the X-linked gene Tsc22d3 supports spermatogenesis and germline transmission from cultured mouse SSCs upon transplantation. Infertile males were produced by crossing homozygous Tsc22d3 floxed females with homozygous ROSA26-Cre males. We obtained 96 live offspring from six long-term cultured SSC lines with the aid of intracytoplasmic sperm injection. We advocate the further optimization of Tsc22d3-conKO males as recipients for testis transplantation of SSC lines.

G-protein coupled receptors Mc4r and Drd1a can serve as surrogate odorant receptors in mouse olfactory sensory neurons.

In the mouse, most mature olfactory sensory neurons (OSNs) express one allele of one gene from the repertoire of ~1100 odorant receptor (OR) genes, which encode G-protein coupled receptors (GPCRs). Axons of OSNs that express a given OR coalesce into homogeneous glomeruli, which reside at conserved positions in the olfactory bulb. ORs are intimately involved in ensuring the expression of one OR per OSN and the coalescence of OSN axons into glomeruli. But the mechanisms whereby ORs accomplish these diverse functions remain poorly understood. An experimental approach that has been informative is to substitute an OR genetically with another GPCR that is normally not expressed in OSNs, in order to determine in which aspects this GPCR can serve as surrogate OR in mouse OSNs. Thus far only the ß2-adrenergic receptor (ß2AR, Ardb2) has been shown to be able to serve as surrogate OR in OSNs; the ß2AR could substitute for the M71 OR in all aspects examined. Can other non-olfactory GPCRs function equally well as surrogate ORs in OSNs? Here, we have generated and characterized two novel gene-targeted mouse strains in which the mouse melanocortin 4 receptor (Mc4r) or the mouse dopamine receptor D1 (Drd1a) is coexpressed with tauGFP in OSNs that express the OR locus M71. These alleles and strains are abbreviated as Mc4r → M71-GFP and Drd1a → M71-GFP. We detected strong Mc4r or Drd1a immunoreactivity in axons and dendritic knobs and cilia of OSNs that express Mc4r or Drd1a from the M71 locus. These OSNs responded physiologically to cognate agonists for Mc4r (Ro27-3225) or Drd1a (SKF81297), and not to the M71 ligand acetophenone. Axons of OSNs expressing Mc4r → M71-GFP coalesced into glomeruli. Axons of OSNs expressing Drd1a → M71-GFP converged onto restricted areas of the olfactory bulb but did not coalesce into glomeruli. Thus, OR functions in OSNs can be substituted by Mc4r or Drd1a, but not as well as by ß2AR. We attribute the weak performance of Drd1a as surrogate OR to poor OSN maturation.

Subpopulations of vomeronasal sensory neurons with coordinated coexpression of type 2 vomeronasal receptor genes are differentially dependent on Vmn2r1.

The mouse vomeronasal organ is specialized in the detection of pheromones. Vomeronasal sensory neurons (VSNs) express chemosensory receptors of two large gene repertoires, V1R and V2R, which encode G-protein-coupled receptors. Phylogenetically, four families of V2R genes can be discerned as follows: A, B, C, and D. VSNs located in the basal layer of the vomeronasal epithelium coordinately coexpress V2R genes from two families: Approximately half of basal VSNs coexpress Vmn2r1 of family C with a single V2R gene of family A8-10, B, or D ('C1 type of V2Rs'), and the other half coexpress Vmn2r2 through Vmn2r7 of family C with a single V2R gene of family A1-6 ('C2 type V2Rs'). The regulatory mechanisms of the coordinated coexpression of V2Rs from two families remain poorly understood. Here, we have generated two mouse strains carrying a knockout mutation in Vmn2r1 by gene targeting in embryonic stem cells. These mutations cause a differential decrease in the numbers of VSNs expressing a given C1 type of V2R. There is no compensatory expression of Vmn2r2 through Vmn2r7. VSN axons coalesce into glomeruli in the appropriate region of the accessory olfactory bulb in the absence of Vmn2r1. Gene expression profiling by NanoString reveals a differential and graded decrease in the expression levels across C1 type of V2Rs. There is no change in the expression levels of C2 type of V2Rs, with two exceptions that we reclassified as C1 type. Thus, there appears to be a fixed probability of gene choice for a given C2 type of V2R.

A ventral glomerular deficit in Parkinson's disease revealed by whole olfactory bulb reconstruction.

Olfactory dysfunction is common in Parkinson's disease and is an early symptom, but its pathogenesis remains poorly understood. Hindering progress in our mechanistic understanding of olfactory dysfunction in Parkinson's disease is the paucity of literature about the human olfactory bulb, both from normal and Parkinson's disease cases. Qualitatively it is well established that the neat arrangement of the glomerular array seen in the mouse olfactory bulb is missing in humans. But rigorous quantitative approaches to describe and compare the thousands of glomeruli in the human olfactory bulb are not available. Here we report a quantitative approach to describe the glomerular component of the human olfactory bulb, and its application to draw statistical comparisons between olfactory bulbs from normal and Parkinson's disease cases. We subjected horizontal 10 µm sections of olfactory bulbs from six normal and five Parkinson's disease cases to fluorescence immunohistochemistry with antibodies against vesicular glutamate transporter-2 and neural cell adhesion molecule. We scanned the immunostained sections with a fluorescence slide scanner, segmented the glomeruli, and generated 3D reconstructions of whole olfactory bulbs. We document the occurrence of atypical glomerular morphologies and glomerular-like structures deep in the olfactory bulb, both in normal and Parkinson's disease cases. We define a novel and objective parameter: the global glomerular voxel volume, which is the total volume of all voxels that are classified immunohistochemically as glomerular. We find that the global glomerular voxel volume in Parkinson's disease cases is half that of normal cases. The distribution of glomerular voxels along the dorsal-ventral dimension of the olfactory bulb in these series of horizontal sections is significantly altered in Parkinson's disease cases: whereas most glomerular voxels reside within the ventral half of olfactory bulbs from normal cases, glomerular voxels are more evenly spread among the ventral and dorsal halves of olfactory bulbs from Parkinson's disease cases. These quantitative whole-olfactory bulb analyses indicate a predominantly ventral deficit in the glomerular component in Parkinson's disease, consistent with the olfactory vector hypothesis for the pathogenesis of this neurodegenerative disease. The distribution of serine 129-phosphorylated α-synuclein immunoreactive voxels correlates with that of glomerular voxels. The higher the serine 129-phosphorylated α-synuclein load of an olfactory bulb from a Parkinson's disease case, the lower the global glomerular voxel volume. Our rigorous quantitative approach to the whole olfactory bulb will help understand the anatomy and histology of the normal human olfactory bulb and its pathological alterations in Parkinson's disease.

Multiplex assessment of the positions of odorant receptor-specific glomeruli in the mouse olfactory bulb by serial two-photon tomography.

In the mouse, axons of olfactory sensory neurons (OSNs) that express the same odorant receptor (OR) gene coalesce into one or a few glomeruli in the olfactory bulb. The positions of OR-specific glomeruli are traditionally described as stereotyped. Here, we have assessed quantitatively the positions of OR-specific glomeruli using serial two-photon tomography, an automated method for whole-organ fluorescence imaging that integrates two-photon microscopy with serial microtome sectioning. Our strategy is multiplexed. By repeated crossing, we generated two strains of mice with gene-targeted mutations at four or five OR loci for a total of six ORs: MOR23 (Olfr16), mOR37A (Olfr155), M72 (Olfr160), P2 (Olfr17), MOR256-17 (Olfr15), and MOR28 (Olfr1507). Glomerular imaging relied on intrinsic fluorescence of GFP or DsRed, or on whole-mount immunofluorescence with antibodies against GFP, DsRed, or ß-gal using the method of immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO). The high-resolution 3D-reconstructed datasets were segmented to identify the labeled glomeruli and to assess glomerular positional variability between the bulbs of one mouse (intraindividual) and among the bulbs of different mice (interindividual). In 26 mice aged 21 or 50 d or 10 wk, we made measurements of the positions of 352 glomeruli. We find that positional variability of glomeruli correlates with the OR: For instance, the medial MOR28 glomerular domain occupies a surface area that is an order of magnitude larger than the surface area of the medial MOR23 glomerular domain. Our results quantify the level of precision that is delivered by the mechanisms of OSN axon wiring, differentially for the various OSN populations expressing distinct OR genes.

Exclusive transmission of the embryonic stem cell-derived genome through the mouse germline.

Gene targeting in embryonic stem (ES) cells remains best practice for introducing complex mutations into the mouse germline. One aspect in this multistep process that has not been streamlined with regard to the logistics and ethics of mouse breeding is the efficiency of germline transmission: the transmission of the ES cell-derived genome through the germline of chimeras to their offspring. A method whereby male chimeras transmit exclusively the genome of the injected ES cells to their offspring has been developed. The new technology, referred to as goGermline, entails injecting ES cells into blastocysts produced by superovulated homozygous Tsc22d3 floxed females mated with homozygous ROSA26-Cre males. This cross produces males that are sterile due to a complete cell-autonomous defect in spermatogenesis. The resulting male chimeras can be sterile but when fertile, they transmit the ES cell-derived genome to 100% of their offspring. The method was validated extensively and in two laboratories for gene-targeted ES clones that were derived from the commonly used parental ES cell lines Bruce4, E14, and JM8A3. The complete elimination of the collateral birth of undesired, non-ES cell-derived offspring in goGermline technology fulfills the reduction imperative of the 3R principle of humane experimental technique with animals. genesis 54:326-333, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.

The extremely broad odorant response profile of mouse olfactory sensory neurons expressing the odorant receptor MOR256-17 includes trace amine-associated receptor ligands.

The mouse olfactory system employs ~1100 G-protein-coupled odorant receptors (ORs). Each mature olfactory sensory neuron (OSN) is thought to express just one OR gene, and the expressed OR determines the odorant response properties of the OSN. The broadest odorant response profile thus far demonstrated in native mouse OSNs is for OSNs that express the OR gene SR1 (also known as Olfr124 and MOR256-3). Here we showed that the odorant responsiveness of native mouse OSNs expressing the OR gene MOR256-17 (also known as Olfr15 and OR3) is even broader than that of OSNs expressing SR1. We investigated the electrophysiological properties of green fluorescent protein (GFP)+ OSNs in a MOR256-17-IRES-tauGFP gene-targeted mouse strain, in parallel with GFP+ OSNs in the SR1-IRES-tauGFP gene-targeted mouse strain that we previously reported. Of 35 single chemical compounds belonging to distinct structural classes, MOR256-17+ OSNs responded to 31 chemicals, compared with 10 for SR1+ OSNs. The 10 compounds that activated SR1+ OSNs also activated MOR256-17+ OSNs. Interestingly, MOR256-17+ OSNs were activated by three amines (cyclohexylamine, isopenthylamine, and phenylethylamine) that are typically viewed as ligands for chemosensory neurons in the main olfactory epithelium that express trace amine-associated receptor genes, a family of 15 genes encoding G-protein-coupled receptors unrelated in sequence to ORs. We did not observe differences in membrane properties, indicating that the differences in odorant response profiles between the two OSN populations were due to the expressed OR. MOR256-17+ OSNs appear to be at one extreme of odorant responsiveness among populations of OSNs expressing distinct OR genes in the mouse.

Trpc2-expressing sensory neurons in the mouse main olfactory epithelium of type B express the soluble guanylate cyclase Gucy1b2.

Chemoreception in the mouse olfactory system occurs primarily at two chemosensory epithelia in the nasal cavity: the main olfactory epithelium (MOE) and the vomeronasal epithelium. The canonical chemosensory neurons in the MOE, the olfactory sensory neurons (OSNs), express the odorant receptor (OR) gene repertoire, and depend on Adcy3 and Cnga2 for chemosensory signal transduction. The canonical chemosensory neurons in the vomeronasal epithelium, the vomeronasal sensory neurons (VSNs), express two unrelated vomeronasal receptor (VR) gene repertoires, and involve Trpc2 for chemosensory signal transduction. Recently we reported the discovery of two types of neurons in the mouse MOE that express Trcp2 in addition to Cnga2. These cell types can be distinguished at the single-cell level by expression of Adcy3: positive, type A and negative, type B. Some type A cells express OR genes. Thus far there is no specific gene or marker for type B cells, hampering further analyses such as physiological recordings. Here, we show that among MOE cells, type B cells are unique in their expression of the soluble guanylate cyclase Gucy1b2. We came across Gucy1b2 in an explorative approach based on Long Serial Analysis of Gene Expression (LongSAGE) that we applied to single red-fluorescent cells isolated from whole olfactory mucosa and vomeronasal organ of mice of a novel Trcp2-IRES-taumCherry gene-targeted strain. The generation of a novel Gucy1b2-IRES-tauGFP gene-targeted strain enabled us to visualize coalescence of axons of type B cells into glomeruli in the main olfactory bulb. Our molecular and anatomical analyses define Gucy1b2 as a marker for type B cells within the MOE. The Gucy1b2-IRES-tauGFP strain will be useful for physiological, molecular, cellular, and anatomical studies of this newly described chemosensory subsystem.

In vivo identification of eugenol-responsive and muscone-responsive mouse odorant receptors.

Our understanding of mammalian olfactory coding has been impeded by the paucity of information about the odorant receptors (ORs) that respond to a given odorant ligand in awake, freely behaving animals. Identifying the ORs that respond in vivo to a given odorant ligand from among the ∼1100 ORs in mice is intrinsically challenging but critical for our understanding of olfactory coding at the periphery. Here, we report an in vivo assay that is based on a novel gene-targeted mouse strain, S100a5-tauGFP, in which a fluorescent reporter selectively marks olfactory sensory neurons that have been activated recently in vivo. Because each olfactory sensory neuron expresses a single OR gene, multiple ORs responding to a given odorant ligand can be identified simultaneously by capturing the population of activated olfactory sensory neurons and using expression profiling methods to screen the repertoire of mouse OR genes. We used this in vivo assay to re-identify known eugenol- and muscone-responsive mouse ORs. We identified additional ORs responsive to eugenol or muscone. Heterologous expression assays confirmed nine eugenol-responsive ORs (Olfr73, Olfr178, Olfr432, Olfr610, Olfr958, Olfr960, Olfr961, Olfr913, and Olfr1234) and four muscone-responsive ORs (Olfr74, Olfr235, Olfr816, and Olfr1440). We found that the human ortholog of Olfr235 and Olfr1440 responds to macrocyclic ketone and lactone musk odorants but not to polycyclic musk odorants or a macrocyclic diester musk odorant. This novel assay, called the Kentucky in vivo odorant ligand-receptor assay, should facilitate the in vivo identification of mouse ORs for a given odorant ligand of interest.

A family of nonclassical class I MHC genes contributes to ultrasensitive chemodetection by mouse vomeronasal sensory neurons.

The mouse vomeronasal organ (VNO) has a pivotal role in chemical communication. The vomeronasal sensory neuroepithelium consists of distinct populations of vomeronasal sensory neurons (VSNs). A subset of VSNs, with cell bodies in the basal part of the basal layer, coexpress Vmn2r G-protein-coupled receptor genes with H2-Mv genes, a family of nine nonclassical class I major histocompatibility complex genes. The in vivo, physiological roles of the H2-Mv gene family remain mysterious more than a decade after the discovery of combinatorial H2-Mv gene expression in VSNs. Here, we have taken a genetic approach and have deleted the 530 kb cluster of H2-Mv genes in the mouse germline by chromosome engineering. Homozygous mutant mice (ΔH2Mv mice) are viable and fertile. There are no major anatomical defects in their VNO and accessory olfactory bulb (AOB). Their VSNs can be stimulated with chemostimuli (peptides and proteins) to the same maximum responses as VSNs of wild-type mice, but require much higher concentrations. This physiological phenotype is displayed at the single-cell level and is cell autonomous: single V2rf2-expressing VSNs, which normally coexpress H2-Mv genes, display a decreased sensitivity to a peptide ligand in ΔH2Mv mice, whereas single V2r1b-expressing VSNs, which do not coexpress H2-Mv genes, show normal sensitivity to a peptide ligand in ΔH2Mv mice. Consistent with the greatly decreased VSN sensitivity, ΔH2Mv mice display pronounced deficits in aggressive and sexual behaviors. Thus, H2-Mv genes are not absolutely essential for the generation of physiological responses, but are required for ultrasensitive chemodetection by a subset of VSNs.

Promoter architecture of mouse olfactory receptor genes.

Odorous chemicals are detected by the mouse main olfactory epithelium (MOE) by about 1100 types of olfactory receptors (OR) expressed by olfactory sensory neurons (OSNs). Each mature OSN is thought to express only one allele of a single OR gene. Major impediments to understand the transcriptional control of OR gene expression are the lack of a proper characterization of OR transcription start sites (TSSs) and promoters, and of regulatory transcripts at OR loci. We have applied the nanoCAGE technology to profile the transcriptome and the active promoters in the MOE. nanoCAGE analysis revealed the map and architecture of promoters for 87.5% of the mouse OR genes, as well as the expression of many novel noncoding RNAs including antisense transcripts. We identified candidate transcription factors for OR gene expression and among them confirmed by chromatin immunoprecipitation the binding of TBP, EBF1 (OLF1), and MEF2A to OR promoters. Finally, we showed that a short genomic fragment flanking the major TSS of the OR gene Olfr160 (M72) can drive OSN-specific expression in transgenic mice.

The ß2-adrenergic receptor as a surrogate odorant receptor in mouse olfactory sensory neurons.

In the mouse, mature olfactory sensory neurons (OSNs) express one allele of one of the ~1200 odorant receptor (OR) genes, which encode G-protein coupled receptors (GPCRs). Axons of OSNs that express the same OR coalesce into homogeneous glomeruli at conserved positions in the olfactory bulb. ORs are involved in OR gene choice and OSN axonal wiring, but the mechanisms remain poorly understood. One approach is to substitute an OR genetically with another GPCR, and to determine in which aspects this GPCR can serve as a surrogate OR under experimental conditions. Here, we characterize a novel gene-targeted mouse strain in which the mouse ß2-adrenergic receptor (ß2AR) is coexpressed with tauGFP in OSNs that choose the OR locus M71 for expression (ß2AR→M71-GFP). By crossing these mice with ß2AR→M71-lacZ gene-targeted mice, we find that differentially tagged ß2AR→M71 alleles are expressed monoallelically. The OR coding sequence is thus not required for monoallelic expression - the expression of one of the two alleles of a given OR gene in an OSN. We detect strong ß2AR immunoreactivity in dendritic cilia of ß2AR→M71-GFP OSNs. These OSNs respond to the ß2AR agonist isoproterenol in a dose-dependent manner. Axons of ß2AR→M71-GFP OSNs coalesce into homogeneous glomeruli, and ß2AR immunoreactivity is detectable within these glomeruli. We do not find evidence for expression of endogenous ß2AR in OSNs of wild-type mice, also not in M71-expressing OSNs, and we do not observe overt differences in the olfactory system of ß2AR and ß1AR knockout mice. Our findings corroborate the experimental value of the ß2AR as a surrogate OR, including for the study of the mechanisms of monoallelic expression.

Statistical analysis of differential gene expression relative to a fold change threshold on NanoString data of mouse odorant receptor genes.

BACKGROUND: A challenge in gene expression studies is the reliable identification of differentially expressed genes. In many high-throughput studies, genes are accepted as differentially expressed only if they satisfy simultaneously a p value criterion and a fold change criterion. A statistical method, TREAT, has been developed for microarray data to assess formally if fold changes are significantly higher than a predefined threshold. We have recently applied the NanoString digital platform to study expression of mouse odorant receptor genes, which form with 1,200 members the largest gene family in the mouse genome. Our objectives are, on these data, to decrease false discoveries when formally assessing the genes relative to a fold change threshold, and to provide a guided selection in the choice of this threshold. RESULTS: Statistical tests have been developed for microarray data to identify genes that are differentially expressed relative to a fold change threshold. Here we report that another approach, which we refer to as tTREAT, is more appropriate for our NanoString data, where false discoveries lead to costly and time-consuming follow-up experiments. Methods that we refer to as tTREAT2 and the running fold change model improve the performance of the statistical tests by protecting or selecting the fold change threshold more objectively. We show the benefits on simulated and real data. CONCLUSIONS: Gene-wise statistical analyses of gene expression data, for which the significance relative to a fold change threshold is important, give reproducible and reliable results on NanoString data of mouse odorant receptor genes. Because it can be difficult to set in advance a fold change threshold that is meaningful for the available data, we developed methods that enable a better choice (thus reducing false discoveries and/or missed genes) or avoid this choice altogether. This set of tools may be useful for the analysis of other types of gene expression data.

Temporal patterns of odorant receptor gene expression in adult and aged mice.

In the mouse, the sense of smell relies predominantly on the expression of ~1200 odorant receptor (OR) genes in the main olfactory epithelium (MOE). Each mature olfactory sensory neuron (OSN) in the MOE is thought to express just one of these OR genes; conversely, an OR gene is expressed in thousands to tens of thousands of OSNs per mouse. Here, we have characterized temporal patterns of OR gene expression in a cohort of inbred C57BL6/N mice from the Aged Rodent Colonies of the National Institute on Aging. We applied the NanoString multiplex platform to quantify RNA abundance for 531 OR genes in whole olfactory mucosa (WOM) tissue samples. The five study groups were females aged 2, 6, 12, 18, and 31 months (mo). We classified the 531 temporal patterns using a step-down quadratic regression method for time course analysis. The majority of OR genes (58.4%) are classified as flat: there is no significant difference from a horizontal line within this time window. There are 32.8% of OR genes with a downward profile, 7.2% with an upward profile, and 1.7% with a convex or concave profile. But the magnitude of these decreases and increases tends to be small: only 4.3% of OR genes are differentially expressed (DE) at 31 mo compared to 2 mo. Interestingly, the variances of NanoString counts for individual OR genes are homogeneous among the age groups. Our analyses of these 15,930 OR gene expression data of C57BL6/N mice that were raised and housed under well-controlled conditions indicate that OR gene expression at the MOE level is intrinsically stable.

Odorant receptor gene choice and axonal wiring in mice with deletion mutations in the odorant receptor gene SR1.

In the mouse, a mature olfactory sensory neuron (OSN) of the main olfactory epithelium (MOE) expresses one allele of one of the 1200 odorant receptor (OR) genes in the genome. The mechanisms that underlie the one receptor-one neuron rule remain poorly understood. A popular experimental paradigm for OR gene choice is to delete an OR coding region by gene targeting or in a transgene. Here we have applied this ∆OR paradigm to SR1, also known as MOR256-3 or Olfr124. This gene is expressed in OSNs of the MOE, and in ~50% of the OSNs of the septal organ. In heterozygous ∆SR1 mice, we observe an unprecedented biallelic expression rate of 30% at the SR1 locus. In homozygous ∆SR1 mice, we find a significant increase in the number of septal organ OSNs that undergo apoptosis. As a population, ∆SR1 OSNs project their axons to 81-85 glomeruli in each half of the OB, and coexpress at least 77 OR genes as evaluated by single-cell molecular analysis. There are no obvious or simple rules for the set of OR genes that are coexpressed with the ∆SR1 allele. The frequencies of coexpression are different for ∆SR1 OSNs in the septal organ compared to those in the MOE. We propose that there are as many as five scenarios for the fate of individual ∆SR1 OSNs.

Carotid Body Function in Tyrosine Hydroxylase Conditional Olfr78 Knockout Mice.

The Olfr78 gene encodes a G-protein-coupled olfactory receptor that is expressed in several ectopic sites. Olfr78 is one of the most abundant mRNA species in carotid body (CB) glomus cells. These cells are the prototypical oxygen (O2) sensitive arterial chemoreceptors, which, in response to lowered O2 tension (hypoxia), activate the respiratory centers to induce hyperventilation. It has been proposed that Olfr78 is a lactate receptor and that glomus cell activation by the increase in blood lactate mediates the hypoxic ventilatory response (HVR). However, this proposal has been challenged by several groups showing that Olfr78 is not a physiologically relevant lactate receptor and that the O2-based regulation of breathing is not affected in constitutive Olfr78 knockout mice. In another study, constitutive Olfr78 knockout mice were reported to have altered systemic and CB responses to mild hypoxia. To further characterize the functional role of Olfr78 in CB glomus cells, we here generated a conditional Olfr78 knockout mouse strain and then restricted the knockout to glomus cells and other catecholaminergic cells by crossing with a tyrosine hydroxylase-specific Cre driver strain (TH-Olfr78 KO mice). We find that TH-Olfr78 KO mice have a normal HVR. Interestingly, glomus cells of TH-Olfr78 KO mice exhibit molecular and electrophysiological alterations as well as a reduced dopamine content in secretory vesicles and neurosecretory activity. These functional characteristics resemble those of CB neuroblasts in wild-type mice. We suggest that, although Olfr78 is not essential for CB O2 sensing, activation of Olfr78-dependent pathways is required for maturation of glomus cells.