Typically, ∼0.1% of the total number of olfactory sensory neurons (OSNs) in the main olfactory epithelium express the same odorant receptor (OR) in a singular fashion and their axons coalesce into homotypic glomeruli in the olfactory bulb. Here, we have dramatically increased the total number of OSNs expressing specific cloned OR coding sequences by multimerizing a 21-bp sequence encompassing the predicted homeodomain binding site sequence, TAATGA, known to be essential in OR gene choice. Singular gene choice is maintained in these “MouSensors.” In vivo synaptopHluorin imaging of odor-induced responses by known M71 ligands shows functional glomerular activation in an M71 MouSensor. Moreover, a behavioral avoidance task demonstrates that specific odor detection thresholds are significantly decreased in multiple transgenic lines, expressing mouse or human ORs. We have developed a versatile platform to study gene choice and axon identity, to create biosensors with great translational potential, and to finally decode human olfaction.

Introduction

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Mombaerts P. Axon guidance of mouse olfactory sensory neurons by odorant receptors and the beta2 adrenergic receptor. Feinstein et al., 2004 Feinstein P.

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Mombaerts P. Axon guidance of mouse olfactory sensory neurons by odorant receptors and the beta2 adrenergic receptor. Feinstein and Mombaerts, 2004 Feinstein P.

Mombaerts P. A contextual model for axonal sorting into glomeruli in the mouse olfactory system. Odorant receptor (OR) genes form the largest multigene family in mammals, with about 1,200 members in the mouse and 350 in humans (). The main olfactory epithelium (MOE) expresses ORs through a poorly understood singular gene choice mechanism, whereby only one allele of any OR gene is selected for specific expression in a given neuron (). Axons from olfactory sensory neurons (OSNs) that express identical ORs coalesce into 2 out of the roughly 1,800 glomeruli per olfactory bulb (OB). The OR coding sequence (CDS) plays a role in the maintenance of gene choice; that is, if the OR is not capable of this maintenance, then a second OR allele is tested for functionality (). Hence, deletion of an OR CDS precludes the convergence of axons into a specific glomerulus and results in OSNs choosing to express one of the other OR genes and concomitantly projecting to a variety of glomeruli in the OB. In addition, OR proteins are necessary for promoting axon guidance, axon identity, and stabilizing neurons that have chosen to express those ORs (). Finally, the OR protein needs to be targeted to the olfactory cilia where it will function in odor signal transduction.

There has been limited success in odor profiling of ORs expressed in heterologous cells in vitro. Part of this limitation is due to the inability of OR proteins to traffic to the plasma membrane. In addition, given the biological properties of the olfactory system, many OR alleles characterized in vitro may not be functional in an in vivo setting and thus could be intact pseudogenes. The major drawback, however, has been the ability to rapidly contrast how odors presented to the OR in liquid phase (in vitro) correspond to odors presented in vapor phase within their mucosal environment (in vivo). Even ex vivo patching of dendritic knobs from transgenic and gene-targeted mice suffers from an absence of vapor phase delivery of odors. Finally, the study of both OR gene choice and OR coding in vivo is hampered by the low representation of a given OR, which, on average, is only expressed in 0.1% of the total neuronal population of a wild-type mouse.

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Mombaerts P. The promoter of the mouse odorant receptor gene M71. Vassalli et al., 2011 Vassalli A.

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Mombaerts P. Homeodomain binding motifs modulate the probability of odorant receptor gene choice in transgenic mice. Vassalli et al., 2011 Vassalli A.

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Mombaerts P. Homeodomain binding motifs modulate the probability of odorant receptor gene choice in transgenic mice. To increase specific OR representation by modulating OR gene choice, we have been characterizing OR minigenes for the past decade. We have identified highly conserved motifs in the promoter sequences of several mouse OR genes that are necessary and sufficient for singular gene choice (). In the case of an M71 OR minigene, a 7.5-kb DNA fragment accurately recapitulates the functionality of the gene-targeted M71 locus and imparts an expression pattern paralleling that of endogenous genes. We have previously observed the following two highly conserved sequences: a single candidate Olfactory-1/Early B Cell Factor (Olf1/EBF or O/E) binding site and two candidate-LIM homeobox 2 (Lhx2) binding sites (TAATXX or HD) within a 161-bp region. Our experiments suggested that an HD sequence is critical for regulating the probability for any OR gene to be expressed ().

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Mombaerts P. Mapping of class I and class II odorant receptors to glomerular domains by two distinct types of olfactory sensory neurons in the mouse. TAATGA between them. When a 19-mer containing this 13-mer sequence from the P element was multimerized nine times (9x19) and placed upstream of the MOR23 transgene backbone, modest increases in cell numbers were observed in 3/17 transgenic founders. Because analysis of chimeric P/P3 promoter transgenes suggested that DNA spacing of the HD might influence OR gene choice (data not shown), we designed a gene choice enhancer consisting of various multimers of a 21-bp sequence from the H element (ACATAACTTTTTAATGAGTCT), each covering two DNA turns of 10.5 bp and thereby allowing for maximum cooperativity of transcription factors, resulting in a radical increase in expression of any cloned OR CDS, which was never obtained with the 9x19 transgenic approach. We refer to these designer mice as “MouSensors.” Analysis of sequences known to strongly influence OR gene choice such as the mouse H (the homology region that activates the MOR28 cluster []) and P (a sequence with high homology to the P3 minimal promoter []) elements have revealed a set of three HD binding sites (TAATGA) in close proximity to each other, and an associated O/E site, with one of the HD sites sharing the same 13-mer AACTTTTbetween them. When a 19-mer containing this 13-mer sequence from the P element was multimerized nine times (9x19) and placed upstream of the MOR23 transgene backbone, modest increases in cell numbers were observed in 3/17 transgenic founders. Because analysis of chimeric P/P3 promoter transgenes suggested that DNA spacing of the HD might influence OR gene choice (data not shown), we designed a gene choice enhancer consisting of various multimers of a 21-bp sequence from the H element (ACATAACTTTTGTCT), each covering two DNA turns of 10.5 bp and thereby allowing for maximum cooperativity of transcription factors, resulting in a radical increase in expression of any cloned OR CDS, which was never obtained with the 9x19 transgenic approach. We refer to these designer mice as “MouSensors.”

We provide a genetic platform, which increases the total population of OSNs expressing a specific OR, enabling us to robustly study OR gene choice, axon identity, and odor coding simultaneously in its intact in vivo environment. Importantly, we show that we can also express human ORs in large numbers of mouse OSNs in our MouSensors, providing a breakthrough technology to crack the human olfactory code.