Date of Degree

9-2020

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Paul Feinstein

Carmen Melendez

Mitchell Goldfarb

Mark Emerson

Thomas Bozza

Ivan Rodriguez

Subject Categories

Biology | Molecular and Cellular Neuroscience | Molecular Genetics

Keywords

Gene regulation, olfaction, odorant receptors

Abstract

The ability to detect odorous chemicals in the environment is the oldest of the senses necessary for survival, from escaping danger, finding mates, to locating food. It is said that humans can identify and discriminate up to a trillion different odor mixtures. For chemoreception to have such a high discriminatory power, would require a diverse population of cells dedicated for odor detection. These detector cells are the olfactory sensory neurons (OSNs), which express odorant receptors (ORs) that bind to chemical odors in the environment. In order to increase specificity and sensitivity, an essential property in olfaction is for each OSN to express an OR in a monogenic and monoallelic fashion. Out of a large repertoire of OR genes, it is curious how a single OSN will choose not just one OR gene but only one allele of that gene to be expressed. The mechanisms establishing how one OR is expressed per OSN, referred to as singular gene choice, remain to be fully resolved. Current models stipulate OR co-expression is possible, but limited to immature OSNs due to competition for transcription and negative feedback mechanisms that ensure only one OR is expressed in a mature cell. This work aims to understand how singular gene choice may be regulated by employing a unique platform that can test whether singularity can be broken. We make use of a high probability (HP) gene choice element to test whether it is possible to express two ORs in a single OSN so we can directly test both the competition for transcription and negative feedback models. We have previously shown that this element results in a dramatic increase in the population of OSNs expressing a defined cloned OR coding sequence (CDS) in transgenic mice, while seemingly using the same endogenous expression and choice mechanism for wild type ORs. These HP transgenes range in representation in the main olfactory epithelium (MOE); from ~2% to about ~50% of OSNs expressing the defined OR. We crossed several HP transgenes to study the competition of two high probability OR loci in the same mouse. Through a combination of confocal imaging and immunofluorescence, as well as fluorescence activated cell sorting (FACS) we analyzed labeled OSNs in the MOE and glomeruli in the olfactory bulb (OB). We observed that in several transgenic crosses, a population of OSNs are double positive for both HP expressed ORs. The number of these double positive OSNs is sufficient for the axons to converge and form stable projections in the olfactory bulb. Interestingly double positive OSNs are not equal in fluorescence intensity for both reporter proteins as shown through FACS, suggesting a difference in the timing of protein translation. This observation was reflected in the OB, where double labeled axons converge to a defined OR glomerulus but within segregated regions, forming a mosaic pattern of various levels of the amount of protein co-expression. In order to evaluate timing of expression, we performed single molecule fluorescent RNA in situ hybridization and found an obvious site of high level signal in the nucleus, indicative of active transcriptional bursting. Surprisingly, although cytoplasmic RNA signal from both transgenic loci were simultaneously detected in the same OSN, more often, active transcriptional bursts in the nucleus only showed signal from one of the two transgenes. We propose a new model identifying that OR transcriptional machinery is singular and exclusive, but where two high probability loci can cycle through it for expression. We suspect that the number of OSNs expressing the HP transgene in the MOE is indicative of how stably maintained that locus is for expression. In competitive HP transgenic crosses, loci can bully each other out from access to transcriptional machinery and result in exclusive active RNA expression despite high levels of protein co-expression and even cytoplasmic RNA co-expression. Some HP loci may not be as stably maintained with transcriptional machinery, depending on context of the other HP locus. Even HP deletions can compete for transcriptional machinery and tend to have high rates of cycling between loci with intact OR CDS, yet we still witness exclusive active RNA expression. The ability for HP deletions to compete with intact OR loci in an exclusive manner signifies a singular dedicated transcriptional machinery for OR choice. We found the only opportunity for transcriptional machinery to simultaneously express multiple ORs at a higher rate is when these are genomically linked and active RNA transcriptional bursts were localized to a singular spot in the nucleus . These results demonstrate that OR protein co-expression is possible and can be stably maintained in a mature OSN, disputing negative feedback models as a means for singularity. However RNA transcription remains singular and exclusive, implying a dedicated transcriptional machinery that can only accept one locus at a time.

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