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Olfactory receptor (OR) choice represents an example of genetically hardwired stochasticity, where every olfactory neuron expresses one out of ~2000 OR alleles in a probabilistic, yet stereotypic fashion. Here, we show that topographic restrictions in OR expression are established in neuronal progenitors by two opposing forces: polygenic transcription and genomic silencing, both of which are influenced by dorsoventral gradients of transcription factors NFIA, B, and X. Polygenic transcription defines spatially constrained OR repertoires, among which one OR allele may be selected for singular expression later in development. Heterochromatin assembly and genomic compartmentalization preferentially eliminate from this “privileged” repertoire ORs with more dorsal expression destinations, which are ectopically transcribed in neuronal progenitors throughout the olfactory epithelium. Our experiments identify early transcription as an “epigenetic” contributor to future developmental patterning and reveal how two spatially responsive probabilistic processes act in concert to establish deterministic, precise, and reproducible territories of stochastic gene expression.

Odorant receptor (OR) gene choice in mammals is a paradigmatic example of monogenic and monoallelic transcriptional selection, in which each olfactory sensory neuron (OSN) chooses to express one OR allele from over 1,000 encoded in the genome [1-3]. This process, critical for generation of the circuit from nose to brain [4-6], is thought to occur in two steps: a slow initial phase that randomly activates a single OR allele, followed by a rapid feedback that halts subsequent expression [7-14]. Inherent in this model is a finite failure rate wherein multiple OR alleles may be activated prior to feedback suppression [15, 16]. Confronted with more than one receptor, the neuron would need to activate a refinement mechanism to eliminate multigenic OR expression and resolve unique neuronal identity [16], critical to the generation of the circuit from nose to olfactory bulb. Here we used a genetic approach in mice to reveal a new facet of OR regulation that corrects adventitious activation of multiple OR alleles, restoring monogenic OR expression and unique neuronal identity. Using the tetM71tg model system, in which the M71 OR is expressed in >95% of mature OSNs and potently suppresses the expression of the endogenous OR repertoire [10], we provide clear evidence of a post-selection refinement (PSR) process that winnows down the number of ORs. We further demonstrate that PSR efficiency is linked to OR expression level, suggesting an underlying competitive process and shedding light on OR gene switching and the fundamental mechanism of singular OR choice.

Anxiety disorders are characterized by persistent fear in the absence of immediate threat and represent the most common psychiatric diseases, with an estimated 28% lifetime prevalence worldwide (Kessler et al., 2010). While symptoms of anxiety are typically evoked by sensory stimuli, it is unknown whether sensory deficits contribute to the development of anxiety disorders. Here we examine the effect of defined genetic mutations that compromise the function of the olfactory system on the development of anxiety-like behaviors in mice. We show that the functional inactivation of the main olfactory epithelium, but not the vomeronasal organ, causes elevated levels of anxiety. Anxiety-like behaviors are also observed in mice with a monoclonal nose, that are able to detect and discriminate odors but in which the patterns of odor-evoked neural activity are perturbed. In these mice, plasma corticosterone levels are elevated, suggesting that olfactory deficits can lead to chronic stress. These results demonstrate a central role for olfactory sensory cues in modulating anxiety in mice.

Histidine decarboxylase (HDC), the unique enzyme responsible for histamine generation, is highly expressed in myeloid cells, but its function in these cells is poorly understood. Here we show that Hdc-knockout mice show a high rate of colon and skin carcinogenesis. Using Hdc-EGFP bacterial artificial chromosome (BAC) transgenic mice in which EGFP expression is controlled by the Hdc promoter, we show that Hdc is expressed primarily in CD11b +Ly6G+ immature myeloid cells (IMCs) that are recruited early on in chemical carcinogenesis. Transplant of Hdc-deficient bone marrow to wild-type recipients results in increased CD11b + Ly6G + cell mobilization and reproduces the cancer susceptibility phenotype of Hdc-knockout mice. In addition, Hdc-deficient IMCs promote the growth of tumor allografts, whereas mouse CT26 colon cancer cells downregulate Hdc expression through promoter hypermethylation and inhibit myeloid cell maturation. Exogenous histamine induces the differentiation of IMCs and suppresses their ability to support the growth of tumor allografts. These data indicate key roles for Hdc and histamine in myeloid cell differentiation and CD11b+Ly6G+IMCs in early cancer development. © 2011 Nature America, Inc. All rights reserved.

The recognition of odors is accomplished in the sensory epithelium where individual olfactory neurons express only one of 1,300 odorant receptor genes. Neurons expressing a given receptor project to two spatially invariant glomeruli in the olfactory bulb such that each odor elicits a distinct and sparse pattern of glomerular activity. We have altered the neural representation of odors in the brain by generating a mouse with a “monoclonal nose” in which greater than 95% of the sensory neurons express a single odorant receptor, M71. As a consequence, the frequency of sensory neurons expressing endogenous receptor genes is reduced twenty-fold. We observe that these mice can smell but odor discrimination and performance in associative olfactory learning tasks are impaired. However, these mice cannot detect the M71 ligand acetophenone despite the observation that virtually all sensory neurons and glomeruli are activated by this odor. The M71 transgenic mice readily detect other odors in the presence of acetophenone. These observations have implications for how receptor activation in the periphery is represented in the brain and how these representations encode odors.

The odorant receptors (ORs) make up the largest gene family in mammals. Each olfactory sensory neuron chooses just one OR from the more than 1000 possibilities encoded in the genome and transcribes it from just one allele. This process generates great neuronal diversity and forms the basis for the development and logic of the olfactory circuit between the nose and the brain. The mechanism behind this monoallelic regulation has been the subject of intense speculation and increasing experimental investigation, yet remains enigmatic. Recent genetic experiments have brought the outlines of the process into sharper relief, identifying a feedback mechanism in which the first odorant receptor expressed, generates a signal that stabilizes its choice, thus maintaining singular selection. In the absence of this signal, the olfactory neuron re-enters the selection process and switches to choose an alternate OR. Irreversible genetic changes in the nuclei of olfactory neurons do not accompany OR selection, which must therefore be initiated by an epigenetic process that may involve a stochastic mechanism.

A transgenic approach in mice allows the functional interrogation of an odorant receptor locus in vivo and reveals characteristics of its monogenic and monoallelic expression., The odorant receptor (OR) genes constitute the largest mammalian gene family and are expressed in a monogenic and monoallelic fashion, through an unknown mechanism that likely exploits positive and negative regulation. We devised a genetic strategy in mice to examine OR selection by determining the transcriptional activity of an exogenous promoter homologously integrated into an OR locus. Using the tetracycline-dependent transactivator responsive promoter (teto), we observed that the OR locus imposes spatial and temporal constraints on teto-driven transcription. Conditional expression experiments reveal a developmental change in the permissiveness of the locus. Further, expression of an OR transgene that suppresses endogenous ORs similarly represses the OR-integrated teto. Neurons homozygous for the teto-modified allele demonstrate predominantly monoallelic expression, despite their potential to express both copies. These data reveal multiple axes of regulation, and support a model of initiation of OR choice limited by nonpermissive chromatin and maintained by repression of nonselected alleles., Author Summary Odorant receptor (OR) gene choice is a paradigmatic example of transcriptional regulation in which each olfactory sensory neuron selects a single OR from a repertoire of over 1,000 genes. Two mechanistic models of OR choice have been proposed. One postulates the existence of a specialized transcriptional machinery that selects just one OR allele, while a second, kinetic model proposes that OR chromatin is intrinsically nonpermissive, such that inefficient activation during a critical window of time restricts expression to a single OR allele. Here, we used a transgenic approach in mice in which we inserted a conditionally regulated exogenous promoter into an OR locus by homologous recombination in embryonic stem cells. The resulting novel mouse lines allowed the functional interrogation of the OR locus in vivo during development of the olfactory epithelium, enabling us to directly test models of OR choice. Using this experimental strategy we found that OR loci are indeed slow to activate and that the subsequent phenomenon of spatial restriction of OR expression is accomplished by repression. We also observed a developmental shutdown of OR loci concomitant with expression of the OR repertoire. Together, these experiments provide prima facie evidence for a kinetic model of initiation of OR gene choice, coupled with repression of nonselected OR alleles.

The olfactory receptors (ORs), which are G-protein coupled receptors, number more than 1,000 and comprise the largest gene family in the mammalian genome. ORs are expressed both monogenically and monoallelically in olfactory sensory neurons (OSNs) and the mechanism that controls their regulation is largely unknown. ORs reside in constitutive heterochromatin and selection of one OR and from one allele occurs stochastically. To ensure singular monoallelic OR expression, a negative feedback mechanism is elicited by the first selected OR to suppress non-selected ORs in a given neuron. Here we describe results for mice with a 'monoclonal' nose that express one OR, M71,in 95% of all mature OSNs. The M71 transgene suppresses expression of endogenous ORs, and remaining endogenous expression is mostly restricted to immature neurons in the olfactory epithelium. We show that the endogenous OR repertoire are expressed prior to suppression by M71 transgene expression, contrary to current models. When we introduced a second transgene into M71 mice that expressed another OR in most mature OSNs, OSNs uncharacteristically expressed both of the ORs. We hypothesize that unresolved OR competition compromised the neuron's ability to express only one receptor. We further show that suppression of endogenous ORs by M71 is not reversible, and that M71 does not need to be continuously expressed for endogenous ORs to remain suppressed. In these experiments, we have engineered OSNs to express more than one OR in an OSN at a time, which is normally a low probability event. We believe that these experiments reveal the existence of a backup pathway that ensures only one OR will be expressed per neuron. It is possible that mammals have evolved similar mechanisms in other examples of stochastic, monoallelic gene expression, such as in random X-chromosome inactivation or lymphocyte receptor expression.

Background and Objective: The Arabian oryx (Oryx leucoryx) was hunted to near extinction in from the 1950s to the 1970s. Thus, the oryx ranks among the rarest mammals in the world. Qatar currently has ~800 oryx and has donated numerous oryx to breeding programs around the world. As such, Qatar may be a significant source of genetic diversity to the worldwide oryx population. The primary purpose of this project was to provide a genome sequence and Single Nucleotide Polymorphism (SNP) data in hopes it can be used for improving breading strategies by maintaining as much genetic diversity as possible. Methods: A DNA sample was provided from an oryx male in the Wabra Wildlife Preservation in Qatar. The animal's whole genome was sequenced using next-generation sequencing approach. After assembling the contigs, we utilized a 67bp kmer and ~2.4B paired 100bp (~80X coverage) reads from the Illumina HiSeq. These reads were distributed across libraries ranging in size from 300-1200bp for paired-end and 2000-5000bp for mate-pair libraries. Genome physical coverage by mate-pair libraries was approximately 15X. De novo gene prediction was conducted on scaffolds >500bp. SNPs were also detected. Results: Predicted genome size of ~3Gb (similar to other Mammals), a scaffold N50 of ~300kb, an assembly spanning ~2.5Gb of the genome which is likely >90% of euchromatic sequence. The sequence is distributed across 36,964 scaffolds greater than 500bp. We detected ~1M SNPs between parental alleles, which is significantly fewer than in other "rare" animals such as the giant panda. Conclusion: The initial analysis of polymorphisms suggests a relatively high level of inbreeding, and further study will be needed to clarify whether this is only in certain herds or a worldwide issue.

Each olfactory sensory neuron in the mammalian nose selects just a single member of the large odorant receptor (OR) gene family. The core processes regulating OR selection may involve mechanisms that limit initial OR transcription and once a single OR is chosen, repress subsequent activations. We have used a genetic strategy to monitor the transcriptional permissiveness of the OR gene P2 by inserting an exogenous promoter, the tetracycline-dependent transactivator responsive promoter (teto), into its start site through homologous recombination. We observe that the OR locus limits the expression of the teto: repressing it outside of the wild type P2 zone while allowing sporadic activation from within its zone. Staged conditional expression experiments reveal that the receptor locus becomes fully repressed over time and that this repression does not require the receptor's open reading frame. Further, the exogenous promoter is inhibited by an OR transgene that similarly suppresses the endogenous rec...

Many animals have evolved mechanisms to withstand the harsh desert environment, characterized by extreme high temperatures and scarce water supplies. The Arabian Camel, and the Arabian Oryx are valued economically and culturally. These animals can survive for several days without food or water. As the Arabian peninsula undergoes rapid and vast industrial changes, it is increasingly important to understand the biological aspects of these animals. From the unicellular microbes to the sophisticated multi-cellular animals, sensing the chemical composition of the surrounding environment is essential for survival. The vertebrate chemosensory receptors genes, which are members of the seven transmembrane -helical G-protein coupled receptors (GPCRs), are encoded by six different multigene families. Four of these genes encode receptor proteins for sensing odors. The olfactory (odorant) receptors (ORs) are predominately expressed in the sensory neurons of the main olfactory epithelium, and can sense either water-soluble (class I) or volatile (class II) molecules. Furthermore, certain OR genes are expressed in non-olfactory tissues, such as brain, kidney, testis, and placenta. Being adapted to very harsh conditions with elevated temperature, scarce water supply and limited vegetation, we hypothesize that desert animals have evolved the ability to detect water either from volatiles liberated by water in the environment or through the blooming of short-lived vegetation, via their olfactory systems. To explore this possibility we have undertaken the study of the Camel OR genes. We identified approximately one hundred candidate OR genes, all of which are ortholgos to OR genes in other mammals and most closely related to those of the Equus caballus. Preliminary analysis revealed an enrichment in OR gene family 2/13, found in the highest proportion in aquatic animals, as compared to other mammals. This finding provides the intriguing suggestion that desert animals have evolved specific OR genes to adapt to the desert ecosystem. We are currently working to identify the complete OR gene repertoire in the Camel, and to identify and characterize the OR subgenome in other desert animals such as the Arabian Oryx.

Smell is an essential sense that allows animals to find food and mates while avoiding predators. In humans smell is considered an aesthetic sensory modality, but olfactory disorders may presage neurological disease including Parkinson's, Alzheimer's, and schizophrenia. The odorant receptors (ORs) comprise the largest gene family in mammals and endow an animal with the ability to smell. Critical to the development and function of olfaction is the regulation of OR gene transcription, with each sensory neuron selecting just one OR for expression, at random, from only one allele. While recent experiments have brought the outlines of this remarkable process into focus,the core mechanism has remained obscure. Using a genetic approach in mice we present data that supports a model of single OR gene choice initiated by infrequent, stochastic transcriptional activation and governed by feedback suppression mechanism.In the face extreme requirements for diversity, cells and organisms have evolved stochastic p...

Background/aim: The anterior and posterior vagal trunks supply the each side of the gastric mucosa separately, and the denervation of either side does not impair the functional capacity of the other, leaving gastric acid output and circulating gastrin levels unchanged. In this study, we investigated the importance of the neural innervation and the brain-gut axis in cancer initiation by performing vagotomy in hypergastrinemic transgenic (INS-GAS) mice with a phenotype of spontaneous gastric cancer. Methods: Age/sex matched wild-type (WT) and INS-GAS mice were killed at 1, 2, 4, 6, 8, 10 and 12 months of age (at least 10 mice/ group) for histological examination of gastric cancer. INS-GAS mice at 6 months of age were randomized into 4 groups of surgery: unilateral anterior truncal vagotomy (29 mice), bilateral truncal vagotomy with pyloroplasty (24), pyloroplasty (27), and sham operation (25). Both the anterior and posterior sides of the stomachs were removed at 6 months postoperatively (i.e., 12 months of age) for pathological and immunohistochemical analyses. Results: Gastric cancer was found neither in WT mice regardless of age nor in INS-GAS mice that were

The mouse major transplantation antigens H–2K, H–2D and H–2L are highly polymorphic cell-surface glycoproteins which may serve as recognition elements in cell–cell interactions1. Each antigen possesses a number of alloantigenic determinants defined by antisera of various specificities. Recently, monoclonal antibodies have been produced which redefine and extend our knowledge of these determinants2,3, but structural information has not yet been correlated with the serological definition of the antigens. We have previously reported the molecular cloning of genes for H–2Ld and H–2Dd transplantation antigens from the BALB/c mouse and the expression of these genes in mouse L cells4,5. To localize the serological determinants to discrete regions of the H–2 protein, we have now constructed new H–2 antigen genes by joining together fragments of the H–2Ld and H–2Dd genes. In L cells, these genes direct the synthesis of hybrid H–2 proteins and by using monoclonal antibodies of defined specificities, we have mapped classically defined serological specificities to structurally defined domains of the transplantation antigen protein. We conclude that polymorphic determinants recognized by monoclonal antibodies are located in functionally distinct portions of the protein.