Olfactory Receptor Accessory Proteins Play Crucial Roles in Receptor Function And

Olfactory Receptor Accessory Proteins Play Crucial Roles in Receptor Function and

Gene Choice

Ruchira Sharma

Department of Molecular Genetics and Microbiology Duke University

Date:______Approved:

______Hiroaki Matsunami, Supervisor

______Debra Silver

______Nina Sherwood

______Richard Mooney

______William Wetsel

Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Molecular Genetics and Microbiology in the Graduate School of Duke University

2017

ABSTRACT

Olfactory Receptor Accessory Proteins Play Crucial Roles in Receptor Function and

Gene Choice

Ruchira Sharma

Department of Department of Molecular Genetics and Microbiology Duke University

Date:______Approved:

______Hiroaki Matsunami, Supervisor

______Debra Silver

______Nina Sherwood

______Richard Mooney

______William Wetsel

An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Molecular Genetics and Microbiology in the Graduate School of Duke University

2017

Abstract

Understanding how we detect our environment is crucial to understanding how life evolved and now functions. Volatile chemicals from our surroundings are sensed by our olfactory system, a primitive sense that organisms have relied on for survival for millions of years. Mammals express a large family of odorant receptor (OR) genes in the sensory neurons in the nose that mediate this chemosensation. Each mature olfactory sensory neuron (OSN) expresses a single allele of a single OR gene at one time although in the absence of a functional gene OSNs can switch to another OR gene. A functional

OR can inhibit the expression of another OR by co-opting the unfolded protein response

(UPR). How OSNs make their initial OR gene choice and the mechanisms by which the

ORs interact with UPR factors remain unknown.

In this study, I make use of a mouse that has both RTP1 and RTP2 knocked out while keeping the intervening sequence intact. These proteins are required for the efficient surface trafficking of ORs in heterologous cells and the double knock-out mouse could be used to study the gene regulation of OSNs during a large-scale perturbation of the trafficking of ORs to the cell surface. We initially generate and validate the RTP1 and

RTP2 double knock out mouse (RTP1,2DKO) and show that consistent with our heterologous expression system, the mutant mice have OR trafficking defects. These OR trafficking defects give rise to higher rates of cell death and the mutant mice have fewer

mature OSNs. Surprisingly we identified a subset of ORs that were overrepresented in the RTP1,2DKO animals. Some of these ORs can target the cell surface in the absence of the RTPs. This finding gave rise to two cohorts of ORs, those that are underrepresented in the mutants and presumably dependent on the RTPs for cell surface trafficking and

ORs that are overrepresented in RTP1,2DKO. Previous studies have shown that OSNs co-opt the unfolded protein response (UPR) to stabilize OR gene choice of a functional

OR. We show that OSNs expressing underrepresented receptors were more likely to be unable to terminate UPR and had a higher tendency to switch the OR it was initially expressing. Using these two cohorts we showed that the trafficking of ORs to the cell surface is a crucial step in the stabilization of the expression of the OR. In the absence of this cell surface trafficking the OSN is unable to terminate the UPR pathway and either undergoes cell death or OR gene switching.

Dedication

To Amma for pushing and Daddy for worrying.

Contents

Abstract ...... iv

List of Tables ...... xii

List of Figures ...... xiii

Acknowledgements ...... xvi

1. Introduction ...... 1

1.1 Mammalian olfactory system ...... 2

1.2 Mammalian olfactory receptors ...... 4

1.2.1 Canonical odorant receptor signaling ...... 5

1.2.2 Multiple signaling pathways observed in OSNs ...... 7

1.2.3 Desensitization and adaptation ...... 8

1.3 OR gene expression ...... 9

1.3.1 All OR genes are silenced by default ...... 11

1.3.2 A single OR gene is chosen for expression ...... 11

1.3.3 Immature OSNs contain transcripts from multiple OR genes ...... 13

1.3.4 Negative feedback loop ...... 13

1.3.5 OSNs co-opt the unfolded protein response (UPR) pathway to stabilize OR gene choice ...... 14

1.4 Axon targeting ...... 17

1.4.1. ORs play a pivotal role in axon targeting ...... 17

1.4.2 The region where an OR is expressed in the OE determines the position of its glomerulus ...... 18

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1.4.3 The number of OSNs expressing an OR is correlated to the size of the glomerulus ...... 19

1.5 Odor coding ...... 20

1.6 Cell surface trafficking of ORs ...... 21

1.6.1 RTP1 and RTP2 ...... 22

2. RTP1 and RTP2 double knock-out mice show defects in their olfactory system ...... 24

2.1 Introduction ...... 24

2.2 Results ...... 25

2.2.1 Generation of RTP1,2DKO mice ...... 25

2.2.2 RTP1,2DKO OSNs show defects in OR trafficking ...... 27

2.2.3 RTP1,2DKO mice have fewer mature sensory neurons ...... 28

2.2.4 Odorant evoked electrophysiological responses in RTP1,2DKO mice are diminished ...... 32

2.2.5 RTP1,2DKO can detect odorants ...... 33

2.2.6 RTP1,2DKO show mating depression ...... 34

2.3 Conclusions ...... 35

3. RTP1,2DKO mice express a biased OR repertoire ...... 37

3.1 Introduction ...... 37

3.2 Results ...... 37

3.2.1 RNA-seq gene expression analysis on the RTP1,2DKO OE ...... 37

3.2.2 OR are one of the most differentially expressed gene families ...... 39

3.2.3 The proportion of OSNs expressing oORs increases in older RTP1,2DKO mice ...... 44

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3.2.4 Underrepresentation or overrepresentation cannot be predicted using sequence similarity ...... 46

3.2.5 Protein sequence determines whether an OR is underrepresented or overrepresented ...... 47

3.2.6 OSNs expressing oORs can mature ...... 49

3.2.7 The OSNs expressing oORs can detect odorants ...... 51

3.2.8 OSNs expressing uORs show increased rates of cell death in RTP1,2DKO OE52

3.3 Conclusions ...... 53

4. RTP1,2DKO mice expressing uORs show persistent UPR and unstable OR gene choice ...... 56

4.1 Introduction ...... 56

4.2 Results ...... 57

4.2.1 Ectopic expression of nATF5 in OSNs from RTP1,2DKO OE ...... 57

4.2.2 Protein sequence determines whether UPR persists in the OSN ...... 58

4.2.3 OR gene expression is unstable in OSNs expressing Olfr151 in RTP1,2DKO .. 61

4.2.4 RTP1,2DKO can form glomeruli for some ORs...... 64

4.3 Conclusions ...... 66

5. Effects of chronic stimulation on the representation of ORs ...... 68

5.1 Introduction ...... 68

5.2 Results ...... 69

5.2.1 There is no increase in Olfr151 gene choice stability with chronic odor exposure ...... 69

5.3 Conclusions ...... 70

6. Conclusions ...... 71

6.1 Differential control of OR representations by RTPs ...... 71

6.2 Prolonged UPRs in OSNs expressing uORs in RTP1,2DKO ...... 72

6.3 RTP1,2DKO mice show diminished but not abolished responses to odors ...... 76

6.4 Functional ORs expressed outside olfactory system ...... 78

6.5 Novel factors that promote OR trafficking to the cell surface ...... 79

7. Materials and Methods ...... 81

7.1 Media and Buffers ...... 81

7.1.1 Bacterial Culture ...... 81

7.1.2 Cell Culture ...... 83

7.1.3 Fluorescence-Activated Cell Sorting (FACS) ...... 84

7.1.4 In Situ Hybridization ...... 85

7.1.5 LacZ Staining ...... 86

7.2 Cell Culture ...... 87

7.2.1 Bacterial cell culture ...... 87

7.2.2 Mammalian Cell Culture ...... 96

7.3 Fluorescence-Activated Cell Sorting (FACS) ...... 97

7.4 Immunohistochemistry (IHC) ...... 99

7.5 In situ Hybridization ...... 101

7.6 RNA Extraction and Sequencing ...... 105

7.7 Whole mount LacZ Staining ...... 107

7.8 Phospho S6 Induction ...... 108

7.9 ImageJ Analysis ...... 108

7.9.1 Marker gene analysis ...... 109

7.9.2 Statistical analysis ...... 110

Appendix A ...... 111

References ...... 132

Biography ...... 154

List of Tables

Table 1: Number and genotype of viable offspring ...... 26

Table 2: Success rate of RTP1,2DKO crosses ...... 34

Table 3: Number of underrepresented, overrepresented and not significant ORs based on the data set used to calculate FDRs ...... 41

Table 4: Putative Chaperones for OR trafficking and regulation ...... 79

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List of Figures

Figure 1: A representation of the cellular organization of the olfactory system: ...... 4

Figure 2: The Olfactory Signaling Cascade: ...... 7

Figure 3: Loss of nATF5 leads to the stabilization of OR choice: ...... 16

Figure 4: Axon targeting to the OB ...... 19

Figure 5: Combinatorial odor coding: ...... 21

Figure 6: Knock out strategy for RTP1 and RTP2 ...... 26

Figure 7: RNA in situ hybridization to validate the RTP1,2DKO ...... 27

Figure 8: RTP1,2DKO OSNs show OR trafficking defects ...... 28

Figure 9: RTP1,2DKO OEs are thinner than wild-types...... 29

Figure 10: OMP positive layer is smaller in RTP1,2DKO ...... 30

Figure 11: RTP1,2DKO OE has a smaller OMP positive layer at day 1 and 21 ...... 30

Figure 12: ACIII positive layer is smaller in RTP1,2DKO ...... 31

Figure 13: The GAP43 positive layer is larger in 21 day old RTP1,2DKO OEs ...... 32

Figure 14: EOG responses of wild-types and RTP1,2DKO to 7 odorants ...... 33

Figure 15: Time spent freezing to 2MT in wild-types and RTP1,2DKO ...... 34

Figure 16: Comparison of expression level of all genes between wild-types and RTP1,2DKO ...... 38

Figure 17: OR expression is biased in RTP1,2DKO ...... 39

Figure 18: Volcano plot showing underrepresented and overrepresented ORs based on FDRs calculated from our OR only data set ...... 40

Figure 19: RTP1,2DKO OE has fewer OSNs expressing uORs ...... 42

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Figure 20: RTP1,2DKO OE has a larger number of OSNs expressing uORs ...... 42

Figure 21: Plot of mean abundance showing that oORs are more abundantly expressed43

Figure 22: Percent positive cells for a mix of uORs at 1 day, 21 day and 6 months ...... 45

Figure 23: Percent positive cells for a mix of oORs at 1 day, 21 day and 6 months ...... 46

Figure 24: Phylogenetic tree of ORs showing no clustering of either uORs or oORs...... 47

Figure 25: Representation of Olfr151 versus β2AR in RTP1,2DKO ...... 48

Figure 26: FACS analysis to determine cell surface targeting of ORs ...... 49

Figure 27: OSNs expressing oORs mature in RTP1,2DKO OE...... 50

Figure 28: OSNs expressing oORs in RTP1,2DKO can function ...... 51

Figure 29: A larger number of OSNs are undergoing cell death in RTP1,2DKO ...... 53

Figure 30: Expanded expression of nATF5 and LSD1 in RTP1,2DKO ...... 57

Figure 31: nATF5 persists in OSNs expressing Olfr151 but not β2AR in RTP1,2DKO OSNs ...... 58

Figure 32: nATF5 is more likely to persist in OSNs expressing uORs ...... 60

Figure 33: nATF5 is not co-expressed by OSNs expressing oORs ...... 60

Figure 34: Scheme for the lineage tracing experiment ...... 62

Figure 35: Destabilized gene choice is observed for OSNs expressing Olfr151 (uOR) in RTP1,2DKO ...... 63

Figure 36: Olfr151 does not switch to Olfr143, an oOR in the same gene locus ...... 64

Figure 37: OMP positive OSNs innervate the OB in RTP1,2DKO ...... 64

Figure 38: RTP1,2DKO can form glomeruli for some but not all ORs ...... 66

Figure 39: No increase in gene stabilization in response to chronic stimulation ...... 69

Figure 40: Olfr151 is significantly overrepresented in response to chronic stimulation .. 70

xiv

Figure 41: Model for the role played by the RTPs ...... 73

Figure 42: pCI expression vector (Promega) ...... 88

Acknowledgements

I would like to thank Dr. Hiroaki Matsunami for his support and guidance. None of this would be possible without Hiro, who patiently guided me through both the scientific thought process as well as how to properly express these ideas. Every figure, graph and paragraph has been influenced by what taught me, down to the alignment of the stars to represent significance in graphs.

The initial conception and the generation of these mice is all thanks to Dr.

Yoshiro Ishimaru. Kentaro Ikegami helped me with all the mapping and quantification of the RNA-seq experiments. This project would not have been successful without his quiet and friendly professionalism. Helena You helped with some of the FACS experiments as well as the challenges of compiling my manuscript.

My committee members Dr. Mooney, Dr Silver, Dr. Sherwood and Dr. Wetsel for believing in me and encouraging me finish this marathon as well as their timely scientific insight and advice that was indispensable to me. I would like to thank Dr

Sherwood for helping me express my ideas in my thesis by shaping both its language and construction and Dr Wetsel for his help with statistics. My lab members, both past and current especially Dr. Mainland who first taught me the ropes of scientific rigor and

Dr. Ho who helped me survive graduate student life. Dr. Dey, Dr. Roberts for helping a fledgeling Ph. D student. Dr. Adiepietro for all her help and guidance. Dr. Chein, Dr.

Zhou and Dr. Jiang for their friendship, scientific input and collaboration through the

xvi

years. Jessica Ni, Serene Hu for all their help with experiments and reagents, Dr Abaffy,

Dr. de March, Aashutosh Vihani, Kevin Zhu and Dr. Belser for reading my manuscript and helping me push through the editorial process. I would also like to thank all the undergraduate students I have worked with in the lab for infecting me with their enthusiasm and helping me see why I got into this in the first place.

xvii

1. Introduction

The quality of a human life depends upon the ability to perceive odors from the environment. The hedonistic pleasure of food and drink as well as the survival of dangers like a burning house are heavily derived from the information garnered by the olfactory sensory modality. The evolutionarily primitive sense is conserved throughout living organisms ranging from single celled bacteria to humans which indicates its underlying importance. Along with ability to decipher the environment, the sense of smell is also used for communication, for example, maternal behaviors are triggered by chemical cues from the offspring and sexual receptivity is often signaled by the release of pheromones. Lab bred mice that have never encountered predators show fear responses when subject their urine demonstrating that innate behaviors to certain odors are hardwired in the brain. The loss of olfactory ability is also associated with several neurodegenerative diseases like idiopathic Parkinson’s and Alzheimer’s and believed to be closely linked to the limbic system evoking memories and emotions. Therefore, the study of this subject is both fascinating as well as essential giving insight into how living organisms convert sensory input to behavioral responses and thus leading to a better understanding of human nature.

1.1 Mammalian olfactory system

The olfactory epithelium (OE), is primarily responsible for odorant detection in mammals. It consists of several different cells types including non-neuronal supporting cells like sustentacular cells, microvillar cells and the progenitor basal cells along with olfactory sensory neurons (OSNs) all organized in a pseudostratified columnar epithelium found in the upper portion of the nasal septum. The epithelium lies on top of a highly vascular lamina propia, which contains the Bowman’s gland. Each OSN expresses a single olfactory receptor (OR), members of the seven-transmembrane domain G-protein coupled receptor superfamily (Buck and Axel, 1991), in a monoallelic fashion (Chess et al., 1994; Ressler et al., 1994; Vassar et al., 1994). Basal cells are stem cells responsible for the replacement of the OSNs (Shipley et al., 2003) and the Bowman’s gland is responsible for secreting the serous component of the mucous layer covering the OE. OSNs in the OE project their dendrites to the nasal passage where the dendrite forms a knob like structure from which 5 to 20 non-motile cilia emerge giving larger surface areas to express the OR (Lancet, 1986). These cilia are bathed in the olfactory mucus which contains dissolved odorant molecules and this is where ORs first encounter their cognate ligands. OSNs expressing the same OR (within a range of 2500 to 37000, with a median number of 6,000 (Bressel et al., 2016)) project their axons to either one or two distinct locations in the olfactory bulb (OB) where they converge to

form an anatomical unit called the glomerulus (Mombaerts et al., 1996). The neuropil that comprise these glomeruli are surrounded by periglomerular cells and interact with mitral and tufted cells that act as OB output neurons and interneurons that connect neighboring glomeruli to co-ordinate response to odor mixtures (Hayar et al.,

2004)(Figure 1). Mitral cells project to five distinct regions of the brain: anterior olfactory nucleus, olfactory tubercle, amygdala, entorhinal cortex and piriform cortex. Granule cells are inhibitory interneurons that synapse with mitral tufted cells and inhibit them in response to glutamate released by neighboring Mitral/tufted cells. Hence, a single odorant is capable of both activating specific glomeruli as well as silencing others

(Economo et al., 2016; Murthy, 2011).

Figure 1: A representation of the cellular organization of the olfactory system: OSNs expressing the same receptors (represented here in the same color) project to the same glomeruli, which are innervated by afferent mitral and tufted cells. Interneurons connect neighboring glomeruli and coordinate their response. Supporting cells flank the OSNs while the underlying basal cell layer contains the stem cells from which OSNs are regularly replaced (Sharma and Matsunami, 2014).

1.2 Mammalian olfactory receptors

The number of OR genes is highly variable in different species with humans encoding 396 intact OR genes and mice 1130 (Niimura et al., 2014). ORs contain several

conserved motifs, some of which may be important for G-protein coupling and there are many variable sequences in transmembrane and extracellular domains of the protein that come together in the tertiary structure to form a ligand-binding site. The high degree of variability is thought to be important for activation by a structurally diverse set of volatile odor molecules (Kobilka, 1992). OSNs in the OE also express other minority chemoreceptors like GPCR trace amine-associated receptors (TAARs) which detect biogenic amines (Fleischer et al., 2007; Hashiguchi and Nishida, 2007; Liberles and

Buck, 2006) and four pass transmembrane MS4A receptors that detect pheromones and fatty acids (Greer et al., 2016).

1.2.1 Canonical odorant receptor signaling

The binding of a cognate ligand to its OR releases a specialized stimulatory G protein α subunit called Gαolf into the membrane from the βγ subunits (Belluscio et al.,

1998), into the membrane with the help of the guanine exchange factor Ric8b (Von

Dannecker et al., 2006; Yoshikawa and Touhara, 2009; Zhuang and Matsunami, 2007). Gαolf activates adenylate cyclase III (ACIII), and causes the conversion of ATP into cAMP (Imai and Sakano, 2008; Wong et al., 2000b). A surge in cAMP levels in the neurons leads to the activation of calcium permeable, tetrameric cyclic nucleotide gated (CNG) (Brunet et al.,

1996a) channels and an influx of Na+ and Ca2+(Firestein and Werblin, 1989), which in turn leads to an efflux of chloride via the Ano2/Tmem16b channel (Kleene and Gesteland, 1991;

Kleene, 1993; Rasche et al., 2010) resulting in the depolarization of the membrane potential

(Reisert et al., 2005). Once an action potential has been generated, the cell extrudes Ca2+ by

Na+/Ca2+ exchangers in order to return to its resting membrane potential (Noé et al., 1997;

Stephan et al., 2011). The major subunit of the Ca2+ channel was identified as CNGA2

(Nakamura and Gold, 1987) and the deletion of CNGA2 causes mice to become anosmic

(Brunet et al., 1996c). The deletion of Ano2 does not seem to abolish the sense of smell, although electrophysiological and cell culture experiments show that the OSNs maintain a high baseline of Cl- concentration and the opening of these channels leads to a low noise, nonlinear amplification of the signal (Kurahashi and Yau, 1993; Lowe and Gold, 1993;

Reuter et al., 1998). The unbound βγ subunit of GPCRs expressed outside the olfactory system have been shown to associate with G protein-regulated Inward Rectifying K

(GIRK) channels and kinases to signal via inositol tris-phosphate (IP3) (Daaka et al., 1997;

Dorsam and Gutkind, 2007; Huang et al., 1995; Menard and Mattingly, 2004) but have not been shown to act in the mouse olfactory system.

Figure 2: The Olfactory Signaling Cascade: When activated, the OR catalyzes the release of the Golf subunit, which in turn activates ACIII, leading to an increase in the cAMP levels that activate CNGA2, the calcium channel responsible for the depolarization, and the chloride channel Tmem16b that opens after the surge of Ca2+ into the cell (Sharma and Matsunami, 2014).

1.2.2 Multiple signaling pathways observed in OSNs

OSNs in the OE utilize many signaling cascades other than the cAMP pathway.

Many OSNs that target their axons to the necklace glomeruli, glomeruli that lie on the border between the main olfactory bulb and the accessory olfactory bulb, express guanylate cyclase D (GC-D) (Fülle et al., 1995; Juilfs et al., 1997). These neurons utilize the cGMP signaling cascade where cGMP specific down-stream effectors like CNGA3

and PDE2 are activated expressed along with these receptors (Juilfs et al., 1997; Meyer et al., 2000). These neurons all express carbonic anhydrase II and show a concentration dependent Ca2+ response to CO2, indicating that they might be responsible for its detection. They also respond to certain natriuretic peptide hormones and seem to be responsible for the detection of carbon disulphide, which is a signal associated with food related social learning. Some OSNs also express Trpc2, a cation channel found largely in the vomeronasal organ, along with a soluble guanylate cyclase Gucy1b2 (Omura and

Mombaerts, 2015). TRPM5 is another calcium channel found in a subset of OSNs that also express CNGA2 (Lin et al., 2007).

1.2.3 Desensitization and adaptation

Constant exposure to a stimulus makes OSNs lose their responsiveness in a process called desensitization or adaptation, which can take place via various negative feedback pathways. CNG channels, ACIII and cAMP hydrolysis by phosphodiesterase are mediated by calcium through a calcium binding protein calmodulin (Munger et al.,

2001; Wayman et al., 1995; Yan et al., 1995)(Figure 2). ORs themselves may be phosphorylated or internalized to desensitize the cell (Dawson et al., 1993; Ferguson,

2001; Mashukova et al., 2006). GRK3 and b arrestin 2 mediate the uncoupling of the OR from its G protein. (Ferguson, 2001; Pitcher et al., 1998; Willets et al., 2003). Incubating

OSNs with antibodies to b arrestin 2 and GRK3 leads to elevation of cAMP response in

the presence of an odorant (Dawson et al., 1993) and b arrestin 2 has also been shown to be responsible for receptor internalization (Mashukova et al., 2006). b arrestin 2 competes with the G protein to bind to stimulated ORs and promotes OR endocytosis via the clathrin mediated pathway. Vesicles containing ORs can reside in cells for extended periods of time hence promoting the desensitization of the OSN to the odorant activating it (Dawson et al., 1993; Gainetdinov et al., 2004; Jiang et al., 2015b; Mashukova et al., 2006).

1.3 OR gene expression

Examination of OR mRNAs in the OE showed topographically distinct expression patterns that could be broadly divided into two zones along the dorso – ventral axis

(Vassar et al., 1994; Vassar et al., 1993; Yoshihara et al., 1997). Within a zone, it seems that OR expression is largely stochastic, although in the ventral zone there are further subdivisions into several overlapping zones (Miyamichi et al., 2005). OR genes are divided into class I and class II receptors based on their phylogeny with class I being expressed in the dorsal zone (Zhang and Firestein, 2002; Zhang et al., 2004).

Microdissection followed by microarray experiments showed about 300 class II receptors expressed in the dorsal epithelium while the remaining are expressed in the

ventral zone (Miyamichi et al., 2005). The mechanisms that control this semi-stochastic expression and regulate the one neuron-one receptor rule remain unclear.

Many ORs are ectopically expressed outside the olfactory epithelium in tissues like the heart, liver, lung, kidney and sperm (Zhang et al., 2007). The carotid body is a sensory organ vital in the detection of hypoxia. Olfr78 is highly expressed in this tissue and its loss has been shown to affect the ability of the mutant mouse to respond to oxygen deprivation (Chang et al., 2015). This evidence shows that at least one OR expressed outside the OE has a specific function in the tissue it is expressed.

The transcriptomic analysis of multiple mature OSNs shows the ubiquitous expression of a single OR allele in every cell, showing that the mechanisms responsible for the choice of this gene selection must be under tight regulation (Saraiva et al., 2015b).

Three major theories for how OSNs carry out this monogenic, monoallelic expression have been put forward: i. The OSNs undergo chromosomal recombination akin to immune cells to delete OR genes within the selected locus. Mice expressing a full OR repertoire have been cloned from a single post-mitotic OSN showing that there is no permanent DNA deletion in these cells (Eggan et al., 2004; Li et al., 2004). ii. Gene conversion where only the coding region of the active OR is kept in active euchromatin in the nucleus and the remaining genes are silenced. iii Regulation by a limiting concentration of trans elements on a single allele of one gene leading to only its

expression. Currently the evidence suggests that a mixture of gene conversion and limiting trans factors control the singular expression of an OR allele.

1.3.1 All OR genes are silenced by default

All OR genes bear the marks of constitutive heterochromatinization, H3K9me3 and H4K20me3 (Magklara et al., 2011). These genes congregate in multiple heterochromatin foci in the nucleus (Clowney et al., 2012) where the transient activity of a histone demethylase LSD1 de-silences a single OR gene (Lyons et al., 2013).

Presumably, context dependent cues like transcription factors and cis regulatory elements are responsible for the recruitment of LSD1 to a single OR gene. None of the cis regulatory factors important for the recruitment of LSDI to a specific gene locus have been discovered so far.

1.3.2 A single OR gene is chosen for expression

Upstream regions of certain OR loci (constituting minigenes), introduced into the

OE using transgenic lines, have been shown to drive OR expression in OSNs that do not express an endogenous gene (Vassalli et al., 2002). The discovery of a locus control region (LCR) like OR control element near the MOR28 receptor cluster first demonstrated a cis acting region responsible for initial gene choice (Serizawa et al.,

2003). This region lies more than 100 kbps upstream of the OR gene cluster and contains

a 2kbps homology sequence common to both humans and mice called the H-element, which acts like an enhancer (Nagawa et al., 2002). Insertion of the H-element closer to the MOR28 gene gives rise to an overrepresentation of this OR in its expression zone

(Serizawa et al., 2003). The core H-element sequence contains two homeodomain sequences and one O/E sequence which is frequently found in OR promoter regions

(Nishizumi et al., 2007). The H-element interacts with Lxh1, Emx2 and O/E family proteins (Hirota and Mombaerts, 2004; Hirota et al., 2007; Michaloski et al., 2006;

Rothman et al., 2005; Vassalli et al., 2002; Wang et al., 2004) as well as the promoters of multiple OR genes on different chromosomes (Lomvardas et al., 2006). Although the H- element has been shown to drive the expression of many ORs in transgenic lines, knocking out this sequence in vivo does not lead to a global loss of ORs (Fuss et al., 2007;

Khan et al., 2011; Nishizumi et al., 2007). The H-element is thus able to loop out and interact with OR gene loci, both on the same chromosome and on other chromosomes, but it is not necessary for all OR gene expression. Computational analysis has shown that OR promoters contain some unique epigenetic signatures and are AT rich (Clowney et al., 2011). Transcription factors like Bptf facilitate multiple enhancer interactions

(Markenscoff-Papadimitriou et al., 2014) giving rise to a model where an essential cis enhancer along with multiple trans enhancers and transcription factors are responsible

for the recruitment LSD1 to a single OR gene giving rise to its monogenic and monoallelic expression.

1.3.3 Immature OSNs contain transcripts from multiple OR genes

Transcriptomic analysis of single OSNs has revealed that immature OSNs express multiple OR genes at low levels (Hanchate et al., 2015b; Saraiva et al., 2015b; Tan et al., 2015). These ORs are on different chromosomes but are restricted by their expression in the same zone indicating that certain OR genes are silenced based on position in the OE (Hanchate et al., 2015b), however the factors regulating this process are unknown. Mechanisms governing the restriction of all but one OR that is expressed in the mature OSN remain unclear.

1.3.4 Negative feedback loop

Once the OSN starts expressing an OR, the factors that initiated the expression should be able to activate another OR’s expression. It has been shown that OSNs expressing pseudogenes or OR genes containing mutations or deletions go on to express a different OR. The expression of one functional OR on the surface of the OSN represses the expression of all other ORs (Feinstein et al., 2004b; Lewcock and Reed, 2004; Qasba and Reed, 1998; Serizawa et al., 2003; Shykind et al., 2004b; Wang et al., 1998). This OR mediated silencing does not require the activity of ACIII although the activity of this enzyme has been implicated in silencing LSD1 (Imai et al., 2006a; Lyons et al., 2013;

Nguyen et al., 2007b). In Zebrafish, the βγ subunit of the G-protein is responsible for OR mediated silencing (Ferreira et al., 2014).

1.3.5 OSNs co-opt the unfolded protein response (UPR) pathway to stabilize OR gene choice

The initial translation of the chosen OR leads to the accumulation of the OR protein in the endoplasmic reticulum (ER). This accumulation triggers the UPR pathway, possibly by the means of the PERK signaling pathway although no evidence has been put forward for this in the olfactory system. One of the downstream effects of the UPR is the phosphorylation of the initiation factor Eif2α which leads to the translation of an alternative open reading frame of the translation factor ATF5, which is an isoform expressed exclusively in the olfactory system. This alternate reading frame gives rise to a protein that can enter the nucleus (nATF5) (Dalton et al., 2013a; Hetz et al.,

2011). The deletion of ATF5 leads to neonatal lethality and a dramatic loss of mature

OSNs in the OE (Wang et al., 2012) indicating that the initial production of nATF5 is an important check point for the OSNs to assess successful translation of the chosen OR.

The expression of nATF5 is lost in mature neurons that have stabilized their OR choice and OSNs from ATF5 knockout mice show increased levels of OR gene switching i.e. these OSNs switch to another OR from their initial OR choice (Dalton et al., 2013a). nATF5 induces the expression of ACIII which in turn is implicated in the silencing of

LSD1 in an activity dependent manner, stabilizing the current OR choice made by the

OSN (Dalton et al., 2013a) (Figure 3). This has been inferred due to the fact that neurons expressing ACIII do not express LSD1. Hence, negative feedback regulation by an OR uses the UPR pathway for intracellular signaling.

In summary, during early development all ORs are methylated, a mark for heterochromatinization, and hence silenced. Enhancer elements like the H-element have been shown to drive the expression of ORs in transgenic lines. Knocking out the H- element does not lead to the global loss of OR expression indicating that additional regulatory elements control single OR expression. Recent studies have shown that immature OSNs express multiple receptors at low levels which hints at the possibility that a single OR wins in the “competition” to get established as the chosen OR. None of the factors that regulate this process have been elucidated. The initial translation of the

OR triggers the UPR leading to the production of nATF5, which enters the nucleus and acts as a transcription factor driving the expression of genes thought to be responsible for relieving UPR. Deletion of nATF5 leads to the expanded expression of LSD1 and an increase in OR gene switching but mature OSNs in wild-type mice stop expressing nATF5 showing that OSNs need to trigger and then terminate the UPR pathway for stable OR expression. Because of the loss of LSD1 expression observed in mature OSNs, it is thought that cAMP signaling via ACIII activity might be responsible for silencing its

expression. ACIII knockout-mice show expanded LSD1 and nATF5 expression further supporting this theory. How OSNs terminate UPR signaling is unknown.

Figure 3: Loss of nATF5 leads to the stabilization of OR choice: Accumulation of OR proteins in the ER triggers the UPR where receptors in the ER catalyze the phosphorylation of Eif2α, which in turn causes the translation of a downstream open reading frame of ATF5 which gives rise to nATF5, a version of the protein capable of entering the nucleus. nATF5 acts a translation factor inducing the expression of ACIII (ADCY3) (Li and Matsunami, 2013).

1.4 Axon targeting

Axons from OSNs expressing the same receptors converge and project to a few glomeruli in the OB. Axon targeting is primarily controlled by gradients of molecules divided into 2 major classes. Class I molecules include Neuropilin-1 and Plexin-A1, which establish the anterior-posterior axis, while class II molecules like Kirrel2 and

Kirrel3 aid in activity-dependent refined sorting. The deletion of BIG -2, which is only expressed in a subset of OSNs, leads to the erroneous innervation of glomeruli by those axons (Kaneko-Goto et al., 2008). More recently more molecules required for OR targeting have been identified (Assens et al., 2016; Hasegawa et al., 2016; Zapiec et al.,

2016).

1.4.1. ORs play a pivotal role in axon targeting

The expression of some axon targeting molecules depends on the cAMP levels, which are in turn modulated by a functioning OR (Bozza et al., 2002; Feinstein et al.,

2004b; Feinstein and Mombaerts, 2004; Imai et al., 2006b; Serizawa et al., 2006). (Figure 2)

In the case of a non-functioning OR, the cAMP levels are low and the axon is often unable to converge and find its position; such OSNs undergo apoptosis (Feinstein and

Mombaerts, 2004; Yu et al., 2004). Compromised cilia in the OE lead to aberrant axon targeting in the OB, suggesting proper OSN activation is also important in this process

(Tadenev et al., 2011). Studies have shown that the OR itself does not have a unique role

in axon targeting as replacing an OR with a functional G protein-like β adrenergic receptor leads to an ectopic but seemingly functional glomerulus (Feinstein et al., 2004b).

The initial targeting to the OB when the OSN is immature is independent of the OR, rather it is the refined targeting to the exact glomerulus that requires the expression of

OR in the OB (Rodriguez-Gil et al., 2015).

1.4.2 The region where an OR is expressed in the OE determines the position of its glomerulus

The organization of the glomeruli follows the logic of the epithelium where all the dorsal receptors project to the dorsal portion of the OB, while the class II ventral receptors project to the ventral portion of the bulb (Figure 4). The glomeruli in the OB are connected to one another by the dendro-dendritic connections of local inhibitory interneurons found in the glomerular layer (Adam and Mizrahi, 2010; Kosaka et al.,

1998; Pinching and Powell, 1971). It seems that these neurons are capable of silencing neighboring glomeruli responding with lower intensity to the same odor or in response to certain binary mixtures (Economo et al., 2016; Murthy, 2011).

Figure 4: Axon targeting to the OB The position of the glomerulus reflects the position in which the OR is expressed in the OE: The expression pattern of the OR in the OE (dorsal/ventral) is highly correlated to the position of that OR’s glomeruli. (Mori, 2009)

1.4.3 The number of OSNs expressing an OR is correlated to the size of the glomerulus

The total glomerular volume of one OR is linearly correlated to the number of

OSNs expressing it in the OE (Bressel et al., 2016; Morrison et al., 2015) and therefore dysregulation in the expression of ORs might lead to defects in the glomerulus formation.

1.5 Odor coding

An OR may be activated by a number of molecules and one odorant is capable of activating a number of ORs, enabling the olfactory system to detect and discriminate among tens of thousands of odorants using a combinatorial code of OR activation (Malnic et al., 1999) (Figure 5). The range of unique odorants able to elicit a response from the OR defines how broadly or narrowly it is tuned. There are ORs that are excited by a wide range of molecules and ORs that respond to only very specific cues. One example of a narrowly tuned OR is the human OR7D4, which is activated very selectively by androstenone and androstadienone. The receptor has common variants that change its function and alter the perception of these volatile steroids, showing an essential role of a single OR in odor perception (Keller et al., 2007; Saito et al., 2009; Sicard and Holley, 1984).

An odorant activates a specific set of ORs and hence excites a characteristic pattern of glomeruli which remains consistent across different individuals. Higher centers of the brain are capable of identifying odors based on these patterns of activation (Belluscio and

Katz, 2001). The olfactory system may also utilize temporally coded odor information because when different odorants stimulate the same OSN, the OSN depolarizes at different frequencies (Laurent and Davidowitz, 1994). The OB could also be generating complex temporal patterns encoding information about odors (Laurent et al., 2001; Spors and Grinvald, 2002).

Figure 5: Combinatorial odor coding:

Each odorant activates a unique set of receptors (row) and each OR is activated by multiple odorants (column). By using multiple receptors for the detection of an odorant the OE is theoretically capable of distinguishing between trillions of odorant molecules (Malnic et al., 1999).

1.6 Cell surface trafficking of ORs

One way to study OR-odor interaction would be to first express the OR in cell culture and screen a library of odorants to see which of them activate the receptor, and then to study common motifs in ORs activated by the same ligand. The main obstacle in this type of study is that ORs in cell culture accumulate in the ER and are not 21

transported to the cell surface (Gimelbrant et al., 1999; McClintock et al., 1997). Previous studies showed that co-expression of ORs with Receptor Transporting Protein1 (RTP1) and Receptor Transporting Protein2 (RTP2) increased the efficiency of the trafficking of receptors to the cell surface (Saito et al., 2004). Based on these findings, a heterologous cell assay system was developed for large scale screening with ligands in order to identify active ligands for many ORs (Zhuang and Matsunami, 2008). Careful deletion analysis has led to the discovery of RTP1S, a shorter version of RTP1 starting from an alternate start codon that is more efficient than RTP1 at promoting cell surface targeting of ORs (Zhuang and Matsunami, 2007). Multiple studies have been carried out to try and determine if there are any motifs or amino-acid residues that are necessary for the targeting of ORs to the cell surface. So far, no such residues have been identified

(Bubnell et al., 2015; Jamet et al., 2015).

1.6.1 RTP1 and RTP2

The RTP gene family consists of 4 proteins, 2 of which, RTP1 and RTP2, are exclusively and strongly expressed in the main olfactory epithelium and the vomeronasal organ (Saito et al., 2004). Both Rtp1 and Rtp2 are on the mouse chromosome 16 within ~500kb of each other. They have a single transmembrane domain and evidence suggests that they are on the same lipid raft as the OR proteins in the cell membrane and that their N terminal region is important to facilitate the OR proteins

escape from the ER (Wu et al., 2012). RTP3 is expressed in liver heart and testis and

RTP4 is expressed in many different tissues. Neither RTP3 nor RTP4 have been shown to increase the efficiency of trafficking any ORs and what they do in the tissue that they are expressed has not been studied (Mainland and Matsunami, 2012). The role played by

RTP1 and RTP2, henceforth referred to as “the RTPs”, in vivo is unknown. This study details the characterization of the RTP1 and RTP2 double knock-out mouse

(RTP1,2DKO) and the role played by these chaperone proteins in the olfactory system.

2. RTP1 and RTP2 double knock-out mice show defects in their olfactory system

2.1 Introduction

In our study, we wanted to assess the in vivo role played by RTP1 and RTP2 and we therefore generated a double knockout mouse RTP1,2DKO. Knocking out genes responsible for olfaction often have negative consequences as evidenced by mice deficient in CNGA2, Gαolf and ACIII, all unable to survive longer than 1-2 days after birth. The same is observed for ATF5 knockout mice where ATF5 is a transcription factor expressed in the OE in response to UPR. These knockout mice are unable to suckle milk from the mother even in the absence of competing littermates leading to a 65-80% lethality rate depending on the gene that has been knocked out (Belluscio et al., 1998;

Brunet et al., 1996a; Wang et al., 2012; Wong et al., 2000b). In the sodium channel Nav1.7 knockout animal, even though the mice are anosmic they survive to adulthood, but they have reduced body weights and knockout mothers show maternal behavior defects because they do not retrieve their scattered pups back to their nest (Weiss et al., 2011).

Knocking out TMEM16b also known as ANO2 on the other hand, does not affect survival or mating even though this gene codes for an important Cl- channel required for olfactory signaling (Billig et al., 2011). Similarly mice with impaired Go subunit (no α subunit) are unable to mate and they are born 27% smaller than their littermates, but are 24

then able to grow to the same size (Luo et al., 2002). BBS2 is a protein required for OE cilia formation and its loss leads to deficient olfactory function. These Bbs2-/- mice are born smaller than their littermates but grow obese as they get older (Nishimura et al.,

2004; Shah et al., 2008). We studied the viability, morphology and mating in RTP1,2DKO to see if any of these aspects were affected. We expect that the deletion of the chaperone proteins would lead to defects in the olfactory system which could lead to early postnatal lethality or mating defects.

2.2 Results

2.2.1 Generation of RTP1,2DKO mice

In order to study the role played by RTP1 and RTP2 in regulating OR expression and trafficking in vivo, we consecutively knocked out these genes while the intervening

~500 kb genomic region was not disrupted in ES cells (Figure 6).

Following chimeric mice production and germline transmission, we established mouse lines with Rtp1 and Rtp2 double knock out alleles. We found no phenotypic difference between Rtp1(+/+);Rtp2(+/+) (wild-type) and Rtp1(+/-);Rtp2(+/-) (het) mice.

The Rtp1(-/-);Rtp2(-/-) homozygous mutants (RTP1,2DKO) showed no gross defects outside the olfactory system. Heterozygous crosses gave rise to wild-type, heterozygous and homozygous adults in roughly 1:2:1 ratio (Table 1), the expected numbers are shown in parenthesis below the observed values.

Figure 6: Knock out strategy for RTP1 and RTP2

Table 1: Number and genotype of viable offspring

Wild-types Hets RTP1,2DKO 19 34 10 (17) (34) (17) n=10 crosses 26

We validated the absence of RTP1 and RTP2 transcripts in RTP1,2DKO by performing RNA in situ hybridization (Figure 7).

Figure 7: RNA in situ hybridization to validate the RTP1,2DKO In situ hybridization with a probe mix against RTP1 and RTP2 showing that RTP1,2DKO does not express these genes. Scale bar =25µm

2.2.2 RTP1,2DKO OSNs show defects in OR trafficking

We used M71-IRES-tauGFP mice in which Olfr151 (also known as M71 and

MOR171-2) expressing OSNs co-express tauGFP to examine the OSNs for OR trafficking defects (Feinstein et al., 2004a) (Figure 8). In the RTP1,2DKO;M71-IRES-tauGFP OE, GFP staining was observed in the dendrites of Olfr151 positive OSNs, indicating that the morphology of their OSNs remains unchanged. In contrast, immunostaining against

Olfr151 (Barnea et al., 2004) was restricted to the cell body, indicating these OSNs are unable to traffic the OR to the dendrite (Figure 8).

Figure 8: RTP1,2DKO OSNs show OR trafficking defects

(Top) Cartoon depicting M71-IRES-GFP. Any neuron expressing Olfr151 will also express tauGFP. (Bottom) Antibody staining against Olfr151 (red) and GFP (green) showing that although in wild-type OSNs expressing Olfr151, the OR gets trafficked to the dendrite, in RTP1,2DKO Olfr151 does not. Scale bar =10µm

2.2.3 RTP1,2DKO mice have fewer mature sensory neurons

We compared sections from both wild-type and RTP1,2DKO littermates, matched using anatomical landmarks, and measured the thickness of the OE tissue. We found that the OE’s thickness was significantly reduced in RTP1,2DKO mice. (p=0.02 paired student t test) (Figure 9).

Figure 9: RTP1,2DKO OEs are thinner than wild-types. There is a reduction in thickness of the entire RTP1,2DKO OE compared to their wild-type littermates using sections matched using anatomical landmarks.

We examined the expression of various OSN developmental markers and signaling molecules in the OE to evaluate areas occupied by mature and immature OSNs in RTP1,2DKO. We compared OMP and adenylate cyclase 3 (ACIII), markers for mature neurons (Carter et al., 2004; Rogers et al., 1987), in 21 day old RTP1,2DKO mice and their littermates. We measured the area occupied by RNA in situ hybridization signals against OMP and found that mice showed an average of 22% reduction in RTP1,2DKO when compared to the wild-type (p<0.0001, paired student t test, wild-type mean area

71% +/- 5 (SD), RTP1,2DKO mean area 49% +/- 4 (SD)) (Figure 10).

Figure 10: OMP positive layer is smaller in RTP1,2DKO On comparing matched sections from 21 day old wild-type and RTP1,2DKO (example shown on the left) we find that in all cases, the OMP positive layer is thinner in the mutant OE.

Comparison of the OMP positive layer from wild-type and RTP1,2DKO OE collected at 1 day old, 21day old and 6 month old mice showed a significant reduction in

OMP expression at 1 day and 21 days (p<0.0001, unpaired student t test, p<0.0001, unpaired student t test) but not at 6 months (Figure 11).

Figure 11: RTP1,2DKO OE has a smaller OMP positive layer at day 1 and 21 (Left) RNA in situ hybridization against OMP in OEs collected at 1 day, 21 days and 6 months. Scale bar =25µm (Right) Percent area occupied by the OMP layer is smaller in the RTP1,2DKO at 1 day and 21 days.

Immunohistochemical analysis of expression of adenylate cyclase 3 (ACIII), a signaling molecule expressed in mature OSNs (Dal Col et al., 2007; Wei et al., 1998;

Wong et al., 2000a) showed a 17% decrease in the area occupied by the staining in 21 day old RTP1,2DKO OE (p=0.0004, unpaired student t test, wild-type mean area 44% +/- 9

(SD), RTP1,2DKO mean area 27%, +/- 3 (SD)). Consistent with OMP expression we observed a significant difference in ACIII expression at 1 day old (p=0.0165, unpaired student t test) but not at 6 months (Figure 12).

Figure 12: ACIII positive layer is smaller in RTP1,2DKO (Left) ACIII staining (red) in OEs collected at 1 day, 21 days and 6 months. Scale bar =25µm (Right) Percent area occupied by the ACIII layer is smaller in the RTP1,2DKO at 1 day and 21 days.

GAP43 is required by a number of neuronal populations for the formation and maintenance of a functional growth cone during axonal elongation (Meiri et al., 1986) and is used as a marker for immature olfactory neurons in the OE (Meiri et al., 1988;

Treloar et al., 1999; Verhaagen et al., 1990). The area occupied by GAP43 RNA in situ signal shows a 7% increase in the 21 day RTP1,2DKO OE (p=0.0267 unpaired student t test, wild-type mean area 20%, +/- 5 (SD), RTP1,2DKO mean area 27%, +/- 6 (SD)) and a

5% increase in 6 month old RTP1,2DKO (p=0.0441 unpaired student t test, wild-type mean area 13%, +/-2 (SD), RTP1,2DKO mean area 18%, +/- 1 (SD)). No significant difference in the GAP43 positive layer is observed between RTP1,2DKO and their littermates at 1 day (Figure 13).

Figure 13: The GAP43 positive layer is larger in 21 day old RTP1,2DKO OEs (Left) GAP43 positive layer in OEs collected at 1 day, 21 days and 6 months. Scale bar =25µm (Right) Percent area occupied by the GAP43 layer is larger in the RTP1,2DKO at 21 days and 6 months.

2.2.4 Odorant evoked electrophysiological responses in RTP1,2DKO mice are diminished

Upon observation of fewer OSNs in RTP1,2DKO mice and lack of OR trafficking to the cilia (Figure 8), we sought to test the olfactory ability by electroolfactogram (EOG).

We tested a diverse set of 7 odorants, in both wild-type and RTP1,2DKO littermates.

Wild-type mice show robust EOG responses to all odorants at concentrations as low as

0.01%. In contrast, RTP1,2DKO mice showed striking deficits in their response.

Responses to most odors were identical to the blank stimulus (air only), although some sensitivity was maintained for a subset of odorants (2-heptanone, amyl acetate, isomenthone) compared to the wild-types (Figure 14).

Figure 14: EOG responses of wild-types and RTP1,2DKO to 7 odorants EOG responses to 7 different odorants showed severely diminished or completely abolished responses from the RTP1,2DKO OE.

2.2.5 RTP1,2DKO can detect odorants

We decided to use behavior to determine the extent of the olfactory defect in these mutant mice. In a freezing assay with 2-methyl-2-thiazoline (2MT), a derivative of

2,5-dihydro-2,4,5-trimethylthiazoline (TMT) a predator odor known to elicit fear responses in naïve mice (Isosaka et al., 2015), we found that RTP1,2DKO froze in response to the odor showing that they were able to detect it (n=2)(Figure 15).

500 Wild type RTP1,2(-/-) 400

300

200

100 Freeze time (seconds)

0 No Odor 0.01% 2MT No Odor 0.01% 2MT

Figure 15: Time spent freezing to 2MT in wild-types and RTP1,2DKO RTP1,2DKO froze in response to 2MT, therefore they could detect it.

2.2.6 RTP1,2DKO show mating depression

RTP1,2DKO crosses are less likely to produce offspring than RTP1,2DKO crossed with hets or wild-type crosses. Table 2 shows the number of successful crosses amongst the various genotypes. Only 3/9 RTP1,2DKO x RTP1,2DKO crosses produced pups whereas 6/7 wild-type x wild-type crosses generated pups.

Table 2: Success rate of RTP1,2DKO crosses

Male Female Success Fail Total RTP1,2DKO RTP1,2DKO 3 6 9 Het RTP1,2DKO 4 2 6 RTP1,2DKO Het 2 1 3 Het Het 16 1 17 Wild-type Wild-type 6 1 7

2.3 Conclusions

Previous studies have shown that deleting vital components of the olfactory system leads to lethality. The RTP1,2DKO mouse we generated was not lethal. Although the overall morphology of these mice was not different from their wild-type littermates, there were significant differences in the OE. Consistent with our heterologous cell expression system, the absence of the RTPs led to a trafficking defect of ORs to the dendrites of the OSNs. The RTP1,2DKO mice had much thinner OEs than their wild- type littermates and thinner OMP and ACIII positive layers indicating that they suffered a loss of mature sensory neurons. In contrast, GAP43, a marker for immature neurons showed expanded expression in the mutant mice which indicates that these mice do not have any developmental defects in their OSNs. Although there is no significant difference between the number of mature OSNs in 6 month old OEs taken from wild- type and RTP1,2DKO littermates, there is a significant increase in the number of immature OSNs hinting at a compensatory mechanism for maintaining a steady number of OSNs in the OE.

The fact that RTP1,2DKO show a decrease in their mature OSNs but not a total loss could explain why they show some responses in their EOGs and are able to detect predator odors derivatives like 2MT. The loss of these OSNs is not completely compensated for by the remaining OSNs because the EOG response to certain odorants

is completely lost and RTP1,2DKO show a mating depression which might be linked to their lack of olfactory acuity as mating behaviors have been strongly associated with olfaction.

3. RTP1,2DKO mice express a biased OR repertoire

3.1 Introduction

Mouse OSNs express ORs in a monogenic and monoallelic fashion. The advent of cheap and large-scale sequencing has led to the study of the relative abundance of the cells expressing a given OR. The range of abundance in the OE of OSNs expressing different ORs covers a vast range with some ORs being abundantly expressed and some very rarely (Ibarra-Soria et al., 2014; Lyons et al., 2013). So far OR choice was believed to be made in a semi-stochastic manner but the abundance of an OR remains remarkably consistent within the same species showing that there must be regulatory mechanisms that control the number of OSNs expressing a given receptor. This regulation has far reaching impacts as the number of OSNs expressing an OR is co-related to the size of that OR’s glomerulus. We wanted to test whether the loss of the RTPs affects all ORs equally, in order to do so we collected the entire nasal mucosa, extracted RNA and sequenced it to study the gene expression patterns.

3.2 Results

3.2.1 RNA-seq gene expression analysis on the RTP1,2DKO OE

To obtain a comprehensive view of gene expression changes in RTP1,2DKO, we performed an RNA-Seq on isolated olfactory mucosa including OE and surrounding tissues. Differential expression analysis comparing RTP1,2DKO to wild-type littermates

revealed that 3.8% of all genes (926 / 24661) were differentially expressed between the 2 genotypes, among which 805 were downregulated and 121 were upregulated in

RTP1,2DKO (FDR corrected p<0.05) (Figure 16). Canonical signaling molecules known to be expressed in mature OSNs including Gnal (Gαolf), Adcy3 (ACIII), and Cnga2 were less abundant in the RTP1,2DKO consistent with a reduced number of mature OSNs in absence of RTP1 and RTP2. We found no significant difference in the expression levels of housekeeping genes like Gapdh and β actin (Kouadjo et al., 2007), neither did we see any compensatory increase in other RTP family members Rtp3 or Rtp4.

Figure 16: Comparison of expression level of all genes between wild-types and RTP1,2DKO

3.8% of all genes are differentially expressed between the 2 genotypes. ORs (green) are one of the top differentially expressed gene families. Housekeeping genes are not affected by the loss of the RTPs

3.2.2 OR are one of the most differentially expressed gene families

We then asked whether the loss of RTP1 and RTP2 equally affected all ORs. In a comparison between wild-type and RTP1,2DKO we found that 62% of ORs (678/1088) were significantly affected by the loss of RTP1 and RTP2 (Figure 17A). Close to half of the annotated intact ORs (562/1088) were downregulated in RTP1,2DKO (FDR corrected p<0.05), consistent with fewer OSNs in the mutant. Unexpectedly however, a small subset of OR transcripts (116/1088) were upregulated in RTP1,2DKO mice (FDR corrected p<0.05) (Figure 17B).

A B

Figure 17: OR expression is biased in RTP1,2DKO

(Left) A plot of only ORs where ORs differentially expressed (p<0.01, red; p<0.05 blue) are shown as colored dots. There are more ORs underrepresented in RTP1,2DKO mice. (Right) A volcano plot with log2(fold change) on the x-axis and false discovery rate (FDR) on the Y axis. There are some ORs that have a higher fold change in the RTP1,2DKO as compared to the wild-types.

The disparity in the abundance of transcripts for these ORs raised the possibility of a difference in probabilities of OSNs expressing each OR in RTP1,2DKO. To remove any possible confounding variables from non-OSN cells, we normalized our read counts using only reads mapped on intact ORs (no pseudogenes were included) and found that

531/1088 were underrepresented and 202/1088 were overrepresented (FDR corrected p<0.05) (Figure 18). The proportion of underrepresented ORs remain roughly the same but the number of overrepresented ORs increases. (Table 2)

Figure 18: Volcano plot showing underrepresented and overrepresented ORs based on FDRs calculated from our OR only data set The same volcano plot with colored dot representing significantly underrepresented or overrepresented ORs. Candidate ORs chosen for further validation

shown in yellow. Underrepresented candidate ORs are shown on the left and overrepresented candidates on the right.

Table 3: Number of underrepresented, overrepresented and not significant ORs based on the data set used to calculate FDRs

To further validate changes in numbers of OSNs expressing individual ORs in

RTP1,2DKO mice, we carried out RNA in situ hybridization with probes against individual underrepresented ORs (uORs) and counted the number of percent positive

OSNs. We found that for all our candidates fewer OSNs were positive in RTP1,2DKO

(Figure 19) (p<0.05 student t test).

Figure 19: RTP1,2DKO OE has fewer OSNs expressing uORs (Left) Representative images of RNA in situ hybridization against uOR Olfr522. RTP1,2DKO has fewer OSNs expressing this OR. Scale bar =25µm (Right) RTP1,2DKO had fewer OSNs expressing uORs for all candidates tested.

In stark contrast, the frequency of OSNs expressing tested oORs were greater in

RTP1,2DKO (p<0.05, student t test) (Figure 20).

Figure 20: RTP1,2DKO OE has a larger number of OSNs expressing uORs

(Left) Representative images of RNA in situ hybridization against oOR Olfr414. RTP1,2DKO has more OSNs expressing this OR. Scale bar =25µm (Right) RTP1,2DKO had a larger number of OSNs expressing oORs for all candidates tested.

Curiously, we found that oORs as a group are more abundantly expressed than uORs in the wild-type. The OR genes that were not classified as either underrepresented nor overrepresented (NS, not significant) exhibited wide range of changes in expression levels between the wild-type and RTP1,2DKO, but are expressed at significantly lower abundance levels than both oORs and uORs (Figure 21) (p<0.0001 one-way ANOVA,

Tukey’s post hoc test).

Figure 21: Plot of mean abundance showing that oORs are more abundantly expressed (Left) Each dot represents a single olfactory receptor classified as an uOR/ oOR/ NS based on normalization by ORs. The horizontal bars denote mean abundance (FPKM). oORs are significantly more abundant than uORs, NS are less abundant than both oORs and uORs (p<0.0001, one-way ANOVA, Tukey’s post hoc test) (right) zoomed in view of the plot showing uOR/oOR and NS abundance, horizontal bars denote mean abundance (FPKM).

3.2.3 The proportion of OSNs expressing oORs increases in older RTP1,2DKO mice

We wondered what happens to the proportion of OSNs expressing uORs and oORs in RTP1,2DKO mice at different ages. We performed RNA in situ hybridization with a 1) a probe mix containing 11 uORs and 2) a probe mix containing 25 oORs, all expressed in the dorsal region of the OE on 1 day, 21 day and 6 month old OE from both wild-type and RTP1,2DKO littermates. Although the efficacy of each probe is different and some probes have off target effects, we assumed these would equally affect both the wild-types and the mutants and therefore we could compare the number of OSNs between genotypes (but not between uOR and oOR groups). In the case of the uOR mix, wild-type showed an increase for OSNs expressing the uORs we tested both at 21 days and 6 months (Figure 21) consistent to the increasing proportion of mature OSNs in the

OE indicated by larger OMP and ACIII layers (Figure 11-12).

Figure 22: Percent positive cells for a mix of uORs at 1 day, 21 day and 6 months (Left) Representative images of the number of OSNs expressing our uOR probe mix. Scale bar =25µm (Right) Quantification of the percent positive OSNs for uOR probe mix showing that there is no change in RTP1,2DKO. An increase in the percentage is observed for the wild-types

However, RTP1,2DKO showed no obvious increase in the fraction of cells expressing these ORs with age. In RTP1,2DKO the number of OSNs expressing oORs showed a dramatic increase both from 1 day old to 21 days and from 21 days to 6 months (p<0.0001 one-way ANOVA, Tukey’s post hoc test) (Figure 23) demonstrating that the RTP1,2DKO OE is progressively populated by oORs.

Figure 23: Percent positive cells for a mix of oORs at 1 day, 21 day and 6 months (Left) Representative images of the number of OSNs expressing our oOR probe mix. Scale bar =25µm (Right) Quantification of the percent positive OSNs for oOR probe mix showing a dramatic increase in RTP1,2DKO.

3.2.4 Underrepresentation or overrepresentation cannot be predicted using sequence similarity

We wondered whether the OR expression bias arose because of RTP on the regulatory elements of an OR’s gene locus or the protein. In our initial investigation, we did not find an obvious pattern or clustering for the genomic locations nor did we find obvious conserved residues or motifs amongst uORs or oORs (Figure 24, Appendix A).

Figure 24: Phylogenetic tree of ORs showing no clustering of either uORs or oORs. uORs (black) and oORs (red) plotted on the OR phylogenetic tree where sequence similarity is reflected by distance between ORs. No pattern or clustering based on evolution can be observed for uORs and oORs

3.2.5 Protein sequence determines whether an OR is underrepresented or overrepresented

In an attempt to causally identify the basis of the bias we used a mouse expressing β2AR-IRES-LacZ from the Olfr151 locus (Feinstein et al., 2004b) (Figure 25A) and asked whether the numbers of OSNs expressing Olfr151 or β2AR are similarly 47

affected in RTP1,2DKO. We saw that fewer Olfr151 expressing OSNs were present in

RTP1,2DKO (p<0.05, student t test), as expected for a uOR. Strikingly, more β2AR positive OSNs were present in RTP1,2DKO compared with wild-type (p<0.05, student t test) (Figure 25B-C), suggesting that it is the protein sequence and not the locus of the

OR that determines whether a given OR is underrepresented or overrepresented.

Figure 25: Representation of Olfr151 versus β2AR in RTP1,2DKO β2AR expressed from the Olfr151 locus (A) is overrepresented in the RTP1,2DKO where as Olfr151 itself is underrepresented. The only factor that could influence representation is the protein sequence. Representative images (B) Scale bar =25µm (C) Quantification of the percent positive cells

Given that β2AR is known to be efficiently trafficked to the cell surface when heterologously expressed in the absence of the RTPs (Omura et al., 2014), we asked whether uORs and oORs show differential capabilities in cell surface trafficking in heterologous cells. We carried out live cell surface staining of HEK293T cells transfected with either Rho tagged uORs or oORs in the absence of RTP1 and RTP2. In order to quantify the surface staining, we carried out FACS to measure the surface OR levels.

oORs as a group showed more OR surface expression than uORs (p<0.05, Mann-

Whitney U test) (Figure 26). Notably, the ORs that show most robust cell surface expression were all oORs.

Figure 26: FACS analysis to determine cell surface targeting of ORs FACS analysis of uORs (left) and oORs (middle) in HEK293T cells showing that uORs never go to the cell surface without the RTPs but it is possible for some oORs. Each color represents the histogram obtained from an individual OR (Right) Geometric mean of each ORs FACS histogram normalized by Olfr78 to remove variability arising from multiple experiments. oORs as a group have a significantly higher likelihood of reaching the cell surface in the absence of the RTPs.

3.2.6 OSNs expressing oORs can mature

Our results thus far suggest that OSNs expressing oORs can function despite the loss of the RTPs. To test this, we examined whether OSNs expressing uORs or oORs co- express OMP (Figure 27A-B). We found that the number of immature OMP-negative

OSNs expressing uORs are similar in RTP1,2DKO and het controls, whereas the number

of OMP-positive OSNs expressing uORs show a 69% decrease in RTP1,2DKO. (p=0.04, student t test) (Figure 27B).

Figure 27: OSNs expressing oORs mature in RTP1,2DKO OE. (A) Representative images of OMP, mature OSN marker, and a uOR and an oOR. OSNs expressing Olfr78 co-express OMP but not those expressing Olfr923 in RTP1,2DKO. Scale bar =25µm (B) Quantification of the number of OSNs expressing OMP and the 5 uORs/ oORs tested. There is no difference in the immature cells

expressing these ORs in the wild-type and RTP1,2DKO but the number of mature cells expressing underrepresented ORs falls dramatically in the mutant mice.

3.2.7 The OSNs expressing oORs can detect odorants

To evaluate function of OSNs expressing uORs or oORs, we chose a uOR and an oORs that have been previously deorphanized (Jiang et al., 2015a). Olfr1395 is an oOR found to respond to 2,5-dihydro-2,4,5-trimethylthiazoline (TMT) and Olfr923, a uOR, to acetophenone in vivo (Jiang et al., 2015a). Using the induction of phospho ribosomal protein S6 (pS6), a marker for neuronal activation(Knight et al., 2012), we found that

OSNs expressing Olfr1395 in both het and RTP1,2DKO were activated to its cognate ligand TMT (p<0.0001 one-way ANOVA, Tukey’s post hoc test) (Figure 28A&C). In contrast OSNs expressing Olfr923 in het, but not RTP1,2DKO were activated by their cognate ligand acetophenone (Figure 28A-B).

Figure 28: OSNs expressing oORs in RTP1,2DKO can function (A) Representative images for pS6 (red) and OR (green) double staining showing that oOR expressing OSNs and not uOR expressing OSNs are functional in RTP1,2DKO

mice. Scale bar =25µm (B) Quantification of pS6 co-expression with uOR Olfr923 with and without its cognate ligand in wild-type and RTP1,2DKO mice. There is no pS6 induction observed in the mutant. (C) Quantification of pS6 co-expression with oOR Olfr1395 with and without its cognate ligand in wild-type and RTP1,2DKO mice. There is robust pS6 induction observed in the mutant.

3.2.8 OSNs expressing uORs show increased rates of cell death in RTP1,2DKO OE

Immunostaining against active caspase 3, a cell death marker, suggested that

RTP1,2DKO mice have an increased number of OSNs undergoing cell death (p<0.001, student t test) (Cowan et al., 2001) (Figure 29). Active caspase 3 staining in conjunction with OMP suggested that more OSNs in immature and mature layers both undergo cell death in RTP1,2DKO (Figure 29A&C). Notably, immature OSNs in the OMP negative layer rarely undergo cell death in wild-type (Figure 29B) (Jia et al., 2010). Together, these observations are consistent with the idea that OSNs expressing uORs are more likely to undergo cell death in RTP1,2DKO.

Figure 29: A larger number of OSNs are undergoing cell death in RTP1,2DKO Co-expression analysis of OMP and active caspase 3 shows that a larger number of OSNs are undergoing cell death both inside and outside the OMP layer. (A) Representative images of caspase (red) and OMP (green) staining in 21 day OE. The white arrows indicate OMP positive neurons undergoing cell death, the blue ones show OMP negative OSNs that are caspase 3 positive. Scale bar =25µm (B) RTP1,2DKO has significantly higher percent OSNs undergoing cell death. (C) In RTP1,2DKO, both OMP positive and OMP negative cells show an increase in cell death.

3.3 Conclusions

3.9% of all genes are either upregulated or downregulated in response to the absence of the RTPs. Amongst the ORs, nearly 60% of the genes are affected and about

10% of these affected genes are upregulated in RTP1,2DKO. The remaining 40% is constituted by ORs that are expressed at low abundance and are likely to fall in either the upregulated or downregulated category if experiments of sufficient power were to be conducted. Upregulation and downregulation is reflected by an increase in the

number of OSNs expressing these ORs rather than the increase or decrease in the concentration of transcripts in each cell. Substituting the β2AR open reading frame (ORF) into the Olfr151 locus and studying the effect of the receptor representation in the

RTP1,2DKO shows that the protein sequence rather than the regulatory elements, drive the difference in representation. An interesting observation from this experiment is that even in wild-types, the representation of β2AR is significantly lower than Olfr151 meaning that the number of OSNs expressing a receptor is influenced by either the OR

ORF itself or the protein sequence.

Our experiments showed that an overrepresented ORs (oORs) are able to target the cell surface when heterologously expressed in the absence of the RTPs, an observation that was never made in the case of uOR. Our results show that as a group, oORs are more efficient at targeting the cell surface. There is a continuum where some oORs are trafficked up to 15 times more than the mean for all oORs tested. It is possible that some ORs can fold correctly in the absence of any chaperones and these are both oORs and expressed on the cell surface of HEK293T cells in the absence of the RTPs.

There might be other, as of yet unidentified, chaperone proteins that are required by a subset of oORs, present in the olfactory system but not in HEK293T cells that traffick oORs in vivo. Therefore, these oORs would not target the HEK293T cell surface but would still be overrepresented in the OE.

OSNs expressing uORs are presumably heavily reliant on the RTPs and in

RTP1,2DKO, the candidates we tested were unable to mature and express OMP. On testing OSNs expressing a candidate uOR for response to its cognate ligand, we found that although OSNs expressing this receptor are activated in Het animals, in RTP1,2DKO they fail to respond. The fact that OSNs expressing uORs do not mature or respond to odorants hints at the fact that these OSNs cannot survive and these are the OSNs that contribute to the higher caspase 3 activity in the mutants. The fact that some

RTP1,2DKO OSNs undergo cell death while they are still immature and some while they are in the OMP positive layer suggests that OSNs use a complex mechanism to assess whether the OR they have chosen is able to target the cell surface.

Older RTP1,2DKO mice OEs are largely populated by oORs. Two factors could contribute to this observation, the fact that there are more immature (GAP43 positive)

OSNs in RTP1,2DKO at 6 months, and that there are more (presumably uOR positive)

OSNs undergoing cell death in RTP1,2DKO mice. The fact that 6 month old RTP1,2DKO mice showed no morphological defects shows that this group of ORs (oORs) are sufficient for survival.

The discovery of oORs has given rise to a useful tool to understand how post differentiated OSNs regulate their life cycle and this group of ORs can be used to further study OSN development in the future.

4. RTP1,2DKO mice expressing uORs show persistent UPR and unstable OR gene choice

4.1 Introduction

Previous studies have shown that the expression of a functional OR is crucial for the survival of the OSN and this OR can elicit a negative feedback loop to inhibit the expression of another OR via the UPR (See Introduction 1.3.4 Negative feedback loop).

Our experiments have given rise to two groups of ORs and the study of the differences between OSNs expressing oORs and uORs would give insight into the mechanisms the

OSN uses to ensure that it is expressing a functional OR on its surface. We began by investigating markers for UPR (nATF5) and histone demethylase required for OR transcription (LSD1) to see if there were differences in the RTP1,2DKO and their wild- type littermates. We expect that OSNs expressing uORs in RTP1,2DKO mice would be unable to terminate UPR and hence show unstable OR gene choice. As uORs constitute about half of all ORs, we expect that RTP1,2DKO would show ectopic expression of UPR markers in their OE.

4.2 Results

4.2.1 Ectopic expression of nATF5 in OSNs from RTP1,2DKO OE

We observed that there were more nATF5 positive OSNs in RTP1,2DKO mice

(p<0.001, Mann-Whitney U Test) and some of these OSNs were located closer to the apical surface, a phenomenon that was not observed in the wild-types, suggesting that nATF5 expression persists during the OSN development in RTP1,2DKO animals (Figure

30). Similarly, expanded expression was observed for LSD1, a histone demethylase whose expression depends on nATF5 (Figure 30).

Figure 30: Expanded expression of nATF5 and LSD1 in RTP1,2DKO (A) Immuno-staining against nATF5 and LSD1 showing that RTP1,2DKO has a larger number of positive cells. Scale bar =25µm (B) A graph showing the number and position along the apical-basal axis of nATF5 positive cells. Not only does the mutant OE have more positive cells, but these positive cells reside in the portion of the OE

normally occupied by mature OSNs (which have stopped expressing nATF5 in the wild- types). (C) The number of LSD1 positive cells is significantly higher in RTP1,2DKO and similar to nATF5, LSD1 expression persists in more mature (apical cells) in the mutant.

4.2.2 Protein sequence determines whether UPR persists in the OSN

We first compared the co-expression of nATF5 and Olfr151 between the wild- type and RTP1,2DKO using M71-IRES-tauGFP mice, and then asked whether the same co-localization occurs for β2AR expressed from the Olfr151 gene locus using β2AR-IRES-

LacZ mice. We observed that the number of OSNs co-expressing Olfr151 and nATF5 was significantly higher in RTP1,2DKO than in the wild-type (p<0.05, Fisher’s exact test). In contrast, the number of OSNs co-expressing β2AR and nATF5 in the β2AR:IRES:tauLacZ mouse was not different from the wild-type (p=1, Fisher’s exact test)(Figure 31).

Figure 31: nATF5 persists in OSNs expressing Olfr151 but not β2AR in RTP1,2DKO OSNs

(A)Representative images for nATF5 co-localization with Olfr151 OSNs in RTP1,2DKO and wild-type. (B) Representative images for nATF5 co-localization with β2AR OSNs in RTP1,2DKO and wild-type. Scale bar =25µm (C) Quantification of the number of nATF5 positive and nATF5 negative OSNs expressing either Olfr151 or β2AR in RTP1,2DKO and wild-types. RTP1,2DKO has nearly twice as many nATF5 positive OSNs in the mutant whereas there is no significant difference in the number of nATF5 and β2AR expressing OSNs in the mutant.

We next asked whether the expression of nATF5 in RTP1,2DKO mice was different in OSNs expressing uORs or oORs. To answer this question, we carried out fluorescent in situ hybridization against 7 uORs and 6 oORs along with immunohistochemistry for nATF5 and quantified colocalization of the OR signal with nATF5 staining. As expected, higher numbers of OSNs expressing uORs colocalized with nATF5 in RTP1,2DKO as a group (p=0.007, Mann-Whitney U test) (Figure 32), whereas no significant difference was observed when oORs were tested (p = 0.937,

Mann-Whitney U test) (Figure 33).

Figure 32: nATF5 is more likely to persist in OSNs expressing uORs (Left) Representative images for nATF5 and uOR double staining. Scale bar =25µm (Right) Quantification showing that nATF5 has a higher tendency to co-localize with OSNs expressing uORs from RTP1,2DKO. Lighter points show individual ORs and the solid points and line show the average.

Figure 33: nATF5 is not co-expressed by OSNs expressing oORs

(Left) Representative images for nATF5 and oOR double staining. Scale bar =25µm (Right) Quantification showing that nATF5 co-localization with OSNs expressing oORs is no different between wild-type and RTP1,2DKO. 4.2.3 OR gene expression is unstable in OSNs expressing Olfr151 in RTP1,2DKO

Increased nATF5 levels in uOR-expressing OSNs of RTP1,2DKO mice suggests a lack of stable OR gene choice in these neurons. This led us to hypothesize that OSNs that initially express a uOR may later turn off the OR and stabilize the expression of another

OR. To directly address this, we used a lineage tracing strategy to study the stability of

OR gene choice in RTP1,2DKO (Abdus-Saboor et al., 2016; Dalton et al., 2013b; Shykind et al., 2004a). In our assay (Figure 34), we crossed a mouse that had one allele expressing

Cre recombinase under the Olfr151 promoter, M71-IRES-Cre, to a mouse that had the

Cre inducible fluorescent reporter Rosa26-lox-stop-lox-tdTomato (Madisen et al., 2010).

In this mouse, any OSN expressing the M71-IRES-Cre allele at any point in time, would give rise to the permanent expression of tdTomato even if the OSN went on to express a different OR gene. We counted the number of tdTomato positive neurons that were also

Olfr151 positive (double positive), which reflect the OSNs that initially chose as well as stably express Olfr151. OSNs that were tdTomato positive but not Olfr151 positive are the ones that switched their initial gene choice.

Figure 34: Scheme for the lineage tracing experiment

We found that only 17% of tdTomato positive cells from RTP1,2DKO OE

(n=24/140 neurons) also express Olfr151 in comparison to 31% OSNs from the wild-type

(n=79/258 neurons) (p<0.05, Fisher’s exact test) and 38% (n= 66/172 neurons) in the heterozygotes (p<0.05, Fisher’s exact test) (Figure 35).

Figure 35: Destabilized gene choice is observed for OSNs expressing Olfr151 (uOR) in RTP1,2DKO (Left) Representative images showing lineage tracing of Olfr151, double positive (white arrow heads) OSNs are those that show stable gene choice. OSNs that no longer express Olfr151, and hence have undergone gene switching are shown with white arrows. Scale bar =25µm (Right) Quantification of the number of OSNs that continue to express Olfr151 after initially choosing it. Each dot represents the average from one animal. Each dot represents the average for all neurons counted from an individual mouse.

To determine whether the Olfr151 gene switched to an oOR within the same locus we carried out a co-localization analysis between Olfr143, an oOR within the

Olfr151 locus and tdTomato under the control of M71-cre (Figure 36). We found no

Olfr143 and tdTomato double positive OSNs in both RTP1,2DKO or their wild-type littermates.

Figure 36: Olfr151 does not switch to Olfr143, an oOR in the same gene locus (A) Double staining by Fluorescent RNA in situ hybridization against Olfr143 and tdTomato to see if Olfr151 switches within the locus to an oOR. Scale bar =25µm (B) Number of neurons counted per genotype. (C) Representation of OSNs expressing Olfr151 and Olfr143 to confirm that they are a uOR and an oOR respectively. 4.2.4 RTP1,2DKO can form glomeruli for some ORs.

We investigated OSN axon targeting to the olfactory bulb (OB) in RTP1,2DKO.

Olfactory axons entered the OB and innervate to the glomerular layer based on our OMP immunostaining in RTP1,2DKO (Figure 37).

Figure 37: OMP positive OSNs innervate the OB in RTP1,2DKO

However, using M71-IRES-tau GFP mice we found that Olfr151 expressing OSNs were unable to converge in the OB in RTP1,2DKO while their wild-type littermates had two Olfr151 glomeruli in each of their OBs as expected (Figure 38). To investigate whether the axon targeting defect was ubiquitous to all receptors, we used β2AR-IRES- tauLacZ (Feinstein et al., 2004b) (Figure 25). Both the wild-type and RTP1,2DKO mice formed glomeruli, however the mutant mice had ectopic glomeruli for OSNs expressing this GPCR (Figure 38). These data suggest the RTP1,2DKO mice do not have a complete set of glomeruli, but retain the ability to form them. We observed tdTomato-positive axons forming small glomeruli in RTP1,2DKO;M71-IRES-cre; Rosa26-lox-stop-lox- tdTomato mice (2 out of 3 mice examined had 1 glomerulus each)(Figure 38).

Figure 38: RTP1,2DKO can form glomeruli for some but not all ORs (Left) A whole mount GFP fluorescence from axons expressing M71 from M71- IRES-tauGFP mice, tdTomato fluorescence from M71-IRES-Cre; Rosa26-lox-stop-lox- tdTomato and LacZ positive axons from β2AR-IRES-tauLacZ mice. RTP1,2DKO OBs lack Olfr151 (M71) glomeruli but have tdTomato and LacZ positive ones, while labeled glomeruli are observed in wild-type with M71-IRES-tauGFP, M71-IRES-Cre; Rosa26-lox- stop-lox-tdTomato and β2AR-IRES-tauLacZ. Only the dorso-lateral OB are visible for β2AR-IRES-LacZ in our preparation. Scale bar =25µm. (Right) Quantification of the total number of glomeruli observed in wild-type and RTP1,2DKO OBs. Each dot represents one mouse. 4.3 Conclusions

OSNs expressing oORs in RTP1,2DKO OSNs do not show an increase in nATF5 co-localization unlike those expressing uORs when compared to their wild-type littermates. This suggests that oOR, which are able to reach the cell surface, mature, function, and therefore stabilize their gene choice. OSNs that are unable to do so, like

those expressing uOR Olfr151, do not stabilize their initial gene choice and switch to another OR. These data show that surface trafficking is a crucial checkpoint for the OSN to ascertain that the initial OR choice is viable.

5. Effects of chronic stimulation on the representation of ORs

5.1 Introduction

The RTPs significantly affect OR representation in the OE. Another possible way to perturb OR representation is chronic stimulation by a single volatile odorant.

Previous studies have shown that the density of OSNs expressing MOR23 decreases by

70% in response to chronic exposure to its cognate ligand lyral (Cadiou et al., 2014;

Grosmaitre et al., 2006). The forced expression of rat I7 odorant receptor followed by exposure to its cognate ligand octanal has shown an increase in OSN survival (Watt et al., 2004). On the other hand, a study using a transgenic mouse expressing the rat I7 from the M71 locus shows no difference in OR representation in response to octanal

(Kerr and Belluscio, 2006). Naris occlusion experiments have shown a dramatic reduction in M72 expressing OSNs compared to the unobstructed naris in wild-type mice (Cavallin et al., 2010). These data along with evidence suggesting that ACIII activity is required for gene stabilization (Dalton et al., 2013a; Lyons et al., 2013) all suggests that chronic exposure to a single volatile odorant might change the representation of the responding ORs. We hypothesize that chronic exposure to 2- hydroxyacetophenone (2HAph), a ligand known to stimulate Olfr151 (Zhang et al.,

2012), should increase the stabilization of Olfr151 gene choice in our lineage tracing experiment (Figure 34). In order to test this hypothesis, we carried out daily 16 hour

exposures to 2HAph on 0-21day M71-IRES-cre; Rosa26-lox-stop-lox-tdTomato mice and counted the number of tdTomato positive and tdTomato and Olfr151 positive OSNs.

5.2 Results

5.2.1 There is no increase in Olfr151 gene choice stability with chronic odor exposure

We quantified the number of ORs that initially chose Olfr151 marked by tdTomato and continued to express the OR. We found no difference in these OSNs between chronically stimulated and control mice. (p=0.9982, student t test; n=3)

Figure 39: No increase in gene stabilization in response to chronic stimulation (Top) Representative images of OSNs expressing Olfr151 and tdTomato in response to chronic stimulation. If persistent activity would increase gene stabilization, there would be an increase in the number of double positive OSNs after chronic stimulation. (Bottom) Quantification of gene stabilization.

Studies have shown that Olfr923 responds to acetophenone at low concentrations

(Jiang et al., 2015a). We quantified the percent positive OSNs expressing Olfr151, Olfr923 and Olfr78 as a control. We found that the percent positive OSNs expressing Olfr151 was significantly higher after chronic exposure to 2HAph (p=0301, student t test, n=3).

Neither of the other ORs were affected.

Figure 40: Olfr151 is significantly overrepresented in response to chronic stimulation

5.3 Conclusions

The number of M71 OSNs increase in response to chronic stimulation even though the number of tdTomato positive OSNs is the same. This implies that chronic stimulation increases the longevity of the activated OSN but does not change the rate of gene choice. Olfr923 responds to low concentrations of acetophenone but might not be as responsive to 2HAph. Further experiments to determine its response to 2HAph need to be carried out to determine why there is an increase in the number of Olfr151 but not

Olfr923 OSNs.

6. Conclusions

In the current study, we investigated the in vivo role of RTP1 and RTP2 in the mouse olfactory system by generating and analyzing RTP1,2DKO mice. Our results demonstrate a surprising link between receptor trafficking of ORs, the UPR and OR gene choice.

6.1 Differential control of OR representations by RTPs

The absence of RTP1 and RTP2 leads to the underrepresentation of nearly half of the ORs, while about 10% of the ORs are significantly overrepresented. This translates into a change in the numbers of OSNs choosing each OR. How do the RTPs regulate the probability of OR gene choice?

Our data from the β2AR→Olfr151 gene swapping experiment indicate that protein sequences of ORs differentially influence OR gene choice, which is linked with trafficking of ORs to the cell surface. Our attempts to identify protein motifs, domains, or features specific to either uORs or oORs were so far unsuccessful. This fits with recent reports where large-scale mutational analysis of Olfr151 in heterologous cells failed to identify any specific amino acids or domains that regulate its cell surface expression

(Hague et al., 2004; Jamet et al., 2015). Nevertheless, this study provides a large set of sequence information of ORs that will allow us to conduct future structure-function

studies by testing uOR/oOR chimeras and/or searching for common motifs or residues within uORs or oORs, which may in turn give us clues as to why nearly half of the ORs are retained in the ER in the absence of the RTPs.

6.2 Prolonged UPRs in OSNs expressing uORs in RTP1,2DKO

Previous studies have suggested the UPR protein nATF5 is induced once an OSN starts actively expressing an OR and is lost when OR expression is stabilized (Dalton et al., 2013b). Our results show an expanded expression of nATF5 in RTP1,2DKO OSNs, suggesting persistent UPR during OR gene choice. Importantly, the frequency of co- localization between nATF5 and uORs increases in RTP1,2DKO whereas this increase is not observed for oORs. This suggests that the persistent UPR phenotype observed in

RTP1,2DKO is due to the OSNs that express uORs resulting in unstable OR gene choice for these OSNs. This is reinforced by our observation that OSNs initially expressing

Olfr151, a uOR, are more likely to terminate their expression in RTP1,2DKO mice. We present a model for the role played by RTP1 and RTP2 in the gene choice made by an

OSN. In our model, the RTPs suppress UPR response by allowing ORs to exit the ER and be transported to the plasma membrane. uORs are not trafficked to the cell surface in the absence of the RTPs, giving rise to persistent UPR in the OSNs that express them.

Consequently, destabilization of the initial OR gene choice leads to cell death or to the

stabilization of another OR. In contrast, oORs are trafficked to the cell surface as functional proteins in the absence of the RTPs, allowing these OSNs to terminate UPR and stabilize gene expression.

Figure 41: Model for the role played by the RTPs

Although we cannot rule out that RTPs themselves play a role in the elimination of the UPR response, the lack of increase in nATF5 observed in OSNs expressing oORs in RTP1,2DKO suggests interaction between the RTPs and the UPR pathway through

ORs. Even for OSNs expressing oORs and β2AR, UPR is likely to be induced in the initial stage (Dalton et al., 2013b). Recent reports utilizing single cell RNA-Seq suggest that immature OSNs express as many as 12 ORs at low levels but mature OSNs only show the expression of one dominant OR (Hanchate et al., 2015a; Saraiva et al., 2015a; Tan et al., 2015). Low-level expression of uORs precedes the expression of the RTPs and may be

sufficient to trigger UPR in the developing OSNs. Alternatively, both oORs and uORs induce UPR at the initial stage.

RNA-Seq data in the wild-type suggests that oORs as a group tend to be more frequently expressed. It could be that initial expression of RTP1 and RTP2 in the developing OSNs is not stable or abundant enough, causing oORs to be stabilized.

Alternatively oORs tend to “win the competition” (Abdus-Saboor et al., 2016; Nguyen et al., 2007a) even in the presence of RTPs probably because of its ability to suppress UPR.

Both change in probability of OR gene choice and biased cell death could alter OR population representation both in the level of transcripts and in the number of OSNs.

The relative contributions of cell death and gene switching to the differential representation of ORs can be further clarified using Bax knockout mice where cell death in developing neurons is suppressed (Robinson et al., 2003).

OR genes that did not significantly change in RTP1,2DKO mice showed lower abundance in wild-type mice suggesting that these ORs are chosen less frequently.

Deeper sequencing and/or increased samples sizes will help classify these ORs as underrepresented, overrepresented, or not changed.

In our lineage tracing experiment, we were unable to distinguish tdTomato- positive, Olfr151-negative OSNs between those express Olfr151 as one of the low-level

ORs, and the others express Olfr151 as the dominant OR later switch to express another

OR. Irrespective of this our data show greater gene instability for Olfr151 expression without the RTPs. Consistent with multiple OR gene expression in developing OSNs, our lineage tracing results suggest that only 31% of OSNs that initially expressed Olfr151 went on to stabilize its expression in the wild-type. Curiously, the vast majority of OSNs that initially expressed Olfr1507 (MOR28) show stable expression in a similar experiment (Dalton et al., 2013b; Shykind et al., 2004a), suggesting that some ORs are more likely to be stabilized than other ORs.

It would be interesting to carry out a sequencing experiment to compare mature and immature OSNs choosing to express Olfr151 in our lineage tracing experiment. The low level expression seen in immature OSNs might be restricted to the same group of

ORs. The sequencing would also tell us if OR gene switching was deterministic or stochastic. Our initial efforts to identify the ORs that get expressed in OSNs initially expressing Olfr151 in RTP1,2DKO were unsuccessful. Sequencing the mature OSNs that initially chose to express Olfr151 would answer this question.

The frequency with which any OR is chosen is different and the underlying cause for this difference remain unknown. Our results show that the number of OSNs expressing β2AR expressed from the Olfr151 locus is lower than the number expressing

Olfr151. Our data calls for future experiments to test whether protein sequences of ORs differentially influence initial OR gene choice.

6.3 RTP1,2DKO mice show diminished but not abolished responses to odors

Our data suggest that mice without RTP1 and RTP2 had diminished but not abolished responses to odorants. Even though we see a clear reduction in the number of mature OSNs and dramatically diminished responses to odors in RTP1,2DKO mice, functional OSNs are not eliminated. Previous studies suggest that anosmic mice often die in the first few days after birth (Brunet et al., 1996b), but the RTP1,2DKO mice seem to have sufficient olfactory ability for postnatal development. Our data show that OSNs expressing oORs are likely to mature and be functional in RTP1,2DKO, explaining the residual responses observed in the mutant. These OSNs become more abundant in the mutant OE as the mouse ages indicating that these ORs help maintain the olfactory ability of these mice. It will be interesting to assess olfactory-mediated behaviors of the

RTP1,2DKO mice in which only a minor fraction of the ORs are functional, since this could address a fundamental question of why most mammals have so many ORs.

Further study into the ligands that activate oORs could indicate whether these

ORs are especially important for survival and hence multiple strategies to successfully express them have evolved. Conversely, it might be beneficial for OSNs to ensure the

expression of broadly tuned receptors to ensure a wide range of detection ability at the expense of discrimination.

No GFP-positive glomeruli were observed in RTP1,2DKO;M71-IRES-tauGFP indicating OSNs expressing Olfr151 are unable to converge their axons without RTP1 and RTP2. Yet we observed small tdTomato-positive glomeruli in RTP1,2DKO; M71-

IRES-cre; Rosa26-lox-stop-lox-tdTomato. These tdTomato-positive glomeruli could be formed by OSNs that initially chose Olfr151 and then switched and/or stabilized the expression of the same OR, presumably an oOR. Previous reports have shown that OR gene switching tend to take place within the same gene locus(Pacifico et al., 2012;

Roppolo et al., 2007) leading us to test the hypothesis that the tdTomato-positive axons forming these glomeruli in RTP1,2DKO mice could be stabilizing the expression of

Olfr143, an oOR near the Olfr151 gene locus. However, we found no tdTomato-positive

OSNs that also expressed Olfr143. This suggests that at least a portion of tdTomato positive OSNs stabilize the expression of the same OR, which is probably an oOR other than Olfr143. Further investigation of the identities of tdTomato-positive OSNs could further our understanding of the OR gene switching mechanism.

β2AR-IRES-LacZ expressing OSNs in RTP1,2DKO form multiple ectopic glomeruli. It has already been shown that the number of OSNs expressing a receptor determine the size of the glomerulus. It is possible that the global disruption of OR

representation changes the number of glomeruli formed. It would be interesting to see if reducing the number of OSNs expressing one receptor and increasing the number expressing another gives rise to an increase in the number of glomeruli for the other OR.

Another explanation of the multiple glomeruli is that OSNs expressing another receptor targeted to correctly to their glomerulus and then switched to β2AR semi-stochastically due to gene destabilization.

6.4 Functional ORs expressed outside olfactory system

A number of reports have shown that ORs are expressed in various organs outside the olfactory system (Feldmesser et al., 2006; Flegel et al., 2013; Griffin et al.,

2009; Kang and Koo, 2012), whereas expression of RTP1 and RTP2 appears to be confined to the peripheral olfactory tissues. One well-established example of a functional OR outside the olfactory system is Olfr78 (also known as MOR18-2) and its human ortholog OR51E2, which have been reported to function in the prostate, airway and kidney as well as the carotid body (Aisenberg et al., 2016; Chang et al., 2015;

Pluznick et al., 2013; Wang et al., 2006). This receptor is an oOR and is trafficked to the cell surface in heterologous cells without the RTPs (Neuhaus et al., 2009; Pluznick et al.,

2011; Zhou et al., 2016). It is also interesting to note that ORs expressed in the bladder and thyroid (Olfr544, Olfr558, and Olfr1386) are all oORs (Kang et al., 2015). Together, it

is tempting to speculate that oORs expressed outside the OE are more likely to play chemosensory roles (Feingold et al., 1999).

6.5 Novel factors that promote OR trafficking to the cell surface

Our data show that not all oORs are trafficked to the surface of the HEK293T cell surface in the absence of the RTPs. This hints that other factors, expressed in the OE but not in HEK293T cells, may be involved in promoting the trafficking of ORs to the cell surface. We can immediately discount other members of the RTP family from carrying out these functions because neither RTP3 nor RTP4 are expressed in the olfactory epithelium at detectable levels. We expect that some of these putative candidates would be expressed in high abundance in mature OSNs and be overrepresented in

RTP1,2DKO. We therefore picked the top candidates worth pursuing from our RNA-seq data (Table 4).

Table 4: Putative Chaperones for OR trafficking and regulation

Gene name Description log2(FoldChange) FDR Oligodendrocyte Transcription Olig1 1.00 1.41E-07 Factor 1 Pcdh11x Protocadherin 11 X-Linked 1.19 3.20E-07 Oligodendrocyte Transcription Olig2 0.86 0.00012 Factor 2 Myocardial Infarction Associated Miat 0.75 0.00023 Transcript (Non-Protein Coding) Cocaine And Amphetamine Cartpt 0.73 0.00075 Regulated Transcript Cellular Retinoic Acid Binding Crabp1 0.99 0.00179 Protein 1 79

Tuba1b Tubulin Alpha 1b 0.70 0.00240 Hematological And Neurological Hn1 0.59 0.00242 Expressed 1 Thrombospondin Type 1 Domain Thsd7b 0.75 0.00246 Containing 7B Coactosin Like F-Actin Binding Cotl1 0.68 0.00659 Protein 1 Pcdh8 Protocadherin 8 0.66 0.00730 Hes6 Hairy and enhancer of split 6 0.74 0.00797 Calb2 Calbindin 2 0.54 0.00845 secretoglobin family 2B member Scgb1b27 0.60 0.00918 27 SNF2 Histone Linker PHD RING Shprh 0.59 0.01206 Helicase Insm1 Insulinoma Associated 1 0.56 0.01343

In conclusion, our study suggests the importance of OR trafficking by the RTP family members in OSN function and probability of OR gene choice. Our studies contribute to a broader understanding of how cells discern the presence of a GPCR on their cell surface post translationally and link protein trafficking to epigenetic modifications that give rise to changes in the cell’s expression profile. In the future, it will be interesting to ask whether the UPR pathway that links the functional trafficking of receptors to the cell surface with epigenetic modifications is utilized by other tissue types (Sharma et al., 2017).

7. Materials and Methods

7.1 Media and Buffers

7.1.1 Bacterial Culture

2XYT + Amp bacterial growth media

Bacto-tryptone (BD 211705) 16g

Bacto-yeast extract (BD 212750) 10g

NaCl 5g

up to 1L volume with DW

100mg/mL Ampicillin

Transformation Buffer

PIPES 10mM

CaCl2·2H2O (American Bioanalytical AB00366) 15mM

KCl (Mallinckrodt Chemicals 6845-04) 250mM

Add 950ml Water

Adjust the pH up to 6.7 with KOH

MnCl2·4H2O 55mM

up to 1L volume with dH2O

Sterilize the solution by filtration through a 0.22 um filter unit.

Store at 4°C

SOB

bacto-tryptone 20.0g

bacto-yeast extract 5.0 g

NaCl 0.5g

KCl 250mM

Adjust the pH to7.0 with 10N NaOH

Autoclave the liquid for 20 min.

MgCl2(EMD MX0045-2, added just before use) 2M

LB-amp plate

Bacto-tryptone 10g

Yeast extract 5g

NaCl 10g

Add 950 ml of water

Adjust the pH to7.0 with NaOH

Agar (BD 214530) 15g

Autoclave for 20 min.

Leave the liquid until the temperature become at ~55°C

Add 1 ml of 60-100 mg/ml ampicillin and mix very well (final concentration: 60-

100ug/ml)

Pour 20ml/plate

7.1.2 Cell Culture

M10 cell maintenance media

Eagle’s Minimum Essential Medium (MEM) with L-glutamine, Earle’s salt and bicarbonate (Sigma M4655) 45ml

Fetal Bovine Serum 5ml

M10-PSF cell maintenance media with penicillin/streptomycin/amphotericin

MEM 45ml

Fetal Bovine Serum 5ml

Penicillin (10,000 units/mL)-Streptomycin (10mg/mL) (Sigma P4333) 250µl

Amphotericin B solution (250µg/mL, Gibco 15290) 250µl

Freezing Media

MEM 4ml

Fetal Bovine Serum 500µl

DMSO 500µl

1M HEPES (Gibco 15630) 50µl

F12Kserum cell maintenance media

Ham's F-12K (Kaighn's) Medium(F12K) (Gibco 21127) 41.25ml

Equine Serum 7.5ml

Fetal Bovine Serum 1.25ml

F12KserumPSF cell maintenance media with penicillin/streptomycin/amphotericin

F12K 41.25ml

Equine Serum 7.5ml

Fetal Bovine Serum 1.25ml

Penicillin (10,000 units/mL)-Streptomycin (10mg/mL) 250µl

Amphotericin B solution (250µg/mL) 250µl

7.1.3 Fluorescence-Activated Cell Sorting (FACS)

PBSss

Phosphate Buffered Saline (Corning Cellgro 21-040-CV) 500ml

Fetal Bovine Serum 10ml

1.5M NaN3 (EM Science SX0299-1) 200µl

7.1.4 In Situ Hybridization

Alkaline buffer

1M NaHCO3 24µl

1M Na2CO3 36µl

DW 240µl

Phosphate (PBS) Buffer

NaCl 137mM

KCl 2.7mM

Na2HPO4 (EM science SX0710-1) 10mM

KH2PO4 (Mallinckrodt 7088) 1.8mM

20 x Sodium citrate (SSC) buffer

DW 800ml

NaCl 3M

Trisodium Citrate 300mM

Adjust the pH to 7 with a few drops of 1M HCl make up the volume to 1L with DW

Sterilize by autoclaving

Prehybridization buffer

Formamide 50%

20xSSC 12.5ml

1M DTT 0.5ml

Yeast RNA (Sigma R-6750, 250µg/ml) (10mg/ml) 1.5ml

Heparin (Sigma H3393) (3000U/ml) 0.5ml

Herring Sperm DNA (Sigma)(10mg/ml) 0.5ml

DW 10ml

Developing Buffer:

Tris pH9 100mM

NaCl 100mM

MgCl2 50mM

7.1.5 LacZ Staining

Buffer A: PBS 18.96ml

1M MgCl2 40µl

100mM EGTA 1ml

Buffer B: PBS 19.9ml

1M MgCl2 40µl

10% Sodium deoxycholate 20µl

10% Nonidet P-40 (Roche 11754599001) 40µl

Buffer C: 100mM Potassium Ferricyanide 500µl

100mM Potassium Ferrocyanide 500µl

X-gal (Zymo research X1001) 1mg/ml

8.5ml Buffer B

7.2 Cell Culture

7.2.1 Bacterial cell culture

Plasmid:

pCI mammalian expression vector(Promega) containing the first 20 amino acids of bovine rhodopsin (N-MNGTEGPNFYVPFSNATGVVR-C), rho tag was digested with MluI(NEB R0198L) and NotI(NEB R0189L) and the insert was ligated in with T4 ligase (NEB M0202L).

Figure 42: pCI expression vector (Promega)

Insert:

Mouse OR sequences were PCR amplified with primers containing MluI and

NotI sites from the C57BL6 strain. All PCR reactions were carried out using the general protocol:

DW 5µl

5xPhusion buffer 2µl

2mM dNTP 1µl

5’ primer (MluI site AAACGCGT) 1µl

3’ primer (NotI site AAGCGGCCGC) 1µl

Phusion polymerase 0.1µl

Template (5-50ng/µl) 1µl

Thermocycler Program

98°C 30sec

98°C 5sec

55°C 15sec

72°C 1min

34 cycles

72°C 5min

4°C Soak

1µl of the PCR product (amplicon) was run on 1.1% agarose (RPI A20090-500.0) gel to verify the success of the PCR and the concentration by looking at the band size and intensity.

Qiagen PB purification:

200µl of PB (Qiagen 19066) was added and the solution was spun for 30 seconds in elution columns (Qiagen 28604) (The flow through was discarded after every spin step). 750µl of buffer PE (Qiagen 28604) was added and the column was spun again for

30 seconds and then for 2 minutes. 10µl of buffer EB (Qiagen 28604) was added making

sure to completely cover the membrane and the columns was spun for 2 minutes and the eluted DNA was collected in a microfuge tube.

Restriction digestion:

The amplicon purified above was restricted for 2 hours at 37°C with the following:

Amplicon/pCI Vector(100ng/µl) 9µl

Buffer 3 (NEB) 2µl

MluI 0.5µl

NotI 0.5µl

BSA (NEB) 0.2µl

DW 8µl

Gel Purification:

4xloading dye was added and 18µl was run on 1.1% agarose gel and the appropriate band was cut out, added to 500µl of bufferQG (Qiagen 28604) at 50°C for 10 minutes and was mixed occasionally. If the volume was less than 750µl, 150µl isopropanol was added and this solution was added to the same elution column used

during the PB purification step and spun for 30 seconds. 500µl of QG was added and spun for 30 seconds, followed by 750µl of PE which was spun for 30 seconds and then for 2 minutes. Add 10µl of EB (30µl for vector) and spun for 2 minutes and collected for ligation.

Ligation:

Ligation was carried out for more than 1 hour at room temperature using the following protocol:

Insert 3µl

Vector 0.5µl

T4 ligase (NEB M0202L) 0.5µl

T4 ligase buffer (NEB) 0.5µl

DW 0.5µl

Transformation:

DH5α cells grown in SOB were treated with transformation buffer on ice for 10 minutes and DMSO was added to achieve a final concentration of 7% before small aliquots were flash frozen in liquid nitrogen and stored at -80°C. 25µl – 50µl of

competent cells and 2.5µl of the ligation product were kept on ice for more than 10 minutes and plated on LB amp plates. The plates were incubated at 37°C overnight.

Colony PCR:

8-16 colonies were picked using a 10µl pipette tip and soaked in 20µl of DW.

10µl of this solution was used as a template to carry out colony PCR using the following protocol in order to verify that the vector in the cell contains the insert.

DW 5µl

10x HotStar Taq buffer 1µl

2mM dNTP 1µl

HotStarTaq polymerase (Qiagen 203205) 0.05µl

5mM 5’ pCI primer 1µl

5mM 3’ pCI primer 1µl

Bacteria soaked DW 10µl

Thermocycler Program

95°C 15min

95°C 15sec

55°C 15sec

72°C 1min

24 cycles

72°C 1min

4°C Soak

1µl of the PCR product was run on a 1.1% agarose gel and 10µl of those colonies that contained the insert was used to inoculate 4.5ml of 2xYT-amp and grown at 37°C overnight with shaking at 300 rpm.

Plasmid Preparation:

The plasmid was extracted using a modified mini prep kit (Denville Scientific) with an added phenol chloroform step. The following protocol was followed:

1. Bacteria grown overnight in 5mL 2xYT-Amp was eluted in a 2mL Eppendorf

tube

2. It was suspended in 250µl resuspension solution (P1)

3. 250µl lysis buffer (P2) was added and mixed gently by inversion >10 times until solution became viscous and slightly clear

4. 525µl of the neutralization solution (P3) was added within 5 minutes of the addition lysis buffer and mixed gently by inversion >20 times. Visible precipitate was observed in the tube.

5. The tube was centrifuged for 5 min to pellet the precipitate

6. The supernatant was transferred to a fresh Eppendorf tube containing 500µl of

phenol(Sigma P4557)/chloroform/isoamyl alcohol (25:24:1 ratio)

7. The tube was centrifuged for 5 min to separate the phases

8. The upper aqueous phase was carefully transferred to a miniprep column

9. The tube was centrifuged for 30 seconds

10. 750µl wash solution was added and the tube centrifuged for 30 seconds.

11. The tube was centrifuged for an additional 2 min to dry the column

12. The column was transferred to a 1.5ml Eppendorf tube and 104µl of elution buffer was added

13. The tube was centrifuged for 2 min to elute plasmid.

14. OD was measured from 4ul of plasmid DNA.

15. The concentration was adjusted to 100ng/µl and stored at 4°C or lower

Sequencing:

Sequencing was carried out using 3100 Genetic Analyzer (ABI Biosystems). 40µl of Sephadex G50 was added to a 96-well Whatman filter plate with 300µl DW per well and soaked for 3 hours. The sequencing reaction with the following protocol was set during this time:

DNA template (100ng/ul) 2µl

Primer (5uM 1µl

BigDye v1.1 0.5µl

ABI 5X Sequencing buffer 2µl

dW 5.5µl

Total 11µl

96°C 10sec

50°C 5sec

60°C 2min

25 cycles

The 96-well plate was spun at 2000rpm for 5 minutes and then 10µl of DW was added to the plate before the sample was added and the sample was spun at 2000rpm for 5 minutes and collected into 96-well semi skirted plate and read with a sequencer.

7.2.2 Mammalian Cell Culture

All cells were grown in a 37°C incubator containing 5% CO2.

HEK293T cells were cultured in M10PSF and PC12 cells in F12KserumPSF.

Thawing:

Cells frozen at -80°C were thawed in 37°C water and then resuspended in 9ml of media before they were spun down. The supernatant was discarded and then the cells resuspended in the appropriate volume of media (determined by the dilution factor) and plated in 10 mm plates.

Maintenance:

HEK293T:

When cells reached 90-100% confluence they were washed in PBS (Corning

Cellgro 21-040-CV), 3ml of trypsin (0.05%, Sigma) was added and the plate was gently shaken until the cells were floating in the solution in chunks. 5ml of M10 was added to the trypsin and the cells were triturated and spun down for 5 minutes for 1000 rpm and the supernatant is aspirated. The cells are suspended in an appropriate volume of

M10PSF added to new 100mm plates and incubated at a 37°C, 5% CO2 and saturating humidity.

Freezing:

Cells were grown to 100% confluence and spun down as described above. They were resuspended in 1ml freezing media and kept overnight in an isopropanol bath before storing for the long term under -80°C

7.3 Fluorescence-Activated Cell Sorting (FACS)

HEK293T cells were transfected with Rho tagged ORs in the absence of RTP1 and

RTP2 to study the efficiency with which they were trafficked to the surface of the cell.

Plating:

HEK293T cells were suspended at 25% confluence in 2ml M10-PSF in a 35mm plate and incubated overnight at 37°C, 5% CO2 and saturating humidity.

Transfection:

1.2µg OR, 30ng GFP were added to 100µl MEM followed by 4µl Lipofectamine

2000 Transfection Reagent (Invitrogen 11668) in 100µl MEM and incubated for 15 minutes at room temperature. 1.8ml of M10 was added and used to replace the media in the 35mm plates which were incubated overnight at 37°C, 5% CO2 and saturating humidity.

Live Cell Staining:

The plates were washed in 1ml PBSss. 1ml Cellstripper (Corning Cellgro 25-056-

CI) was added and the cells were triturated and added to a 5ml round bottomed polystyrene tubes (Falcon 2052) kept on ice and 1ml PBS was added to the plates to remove the last of the cells and transferred to the tube. The tubes were centrifuged at low speeds for 2 minutes at 4°C, the supernatant discarded and the cells were resuspended in 50µl PBSss containing 1:50 dilution of anti-Rho 4D2 primary antibody and incubated on ice for 30 minutes. 2ml PBS was added to the tubes and they were centrifuged at low speed at 4°C for 2 minutes. The supernatant was discarded and the cells were now incubated 50µl PBSss staining buffer containing 1:100 dilution of PE- conjugated anti-mouse secondary antibody (Jackson) for 30 mins on ice. 2ml PBSss was added and the cells centrifuged as described above. The cells were resuspended in

500µL PBS staining buffer containing 1µl 7-Aminoactinomycin D (7AAD) and vortexed before being read with BD FACSCanto II FACS. FACsdivo software was used to ensure that only viable spherical single cells were counted and the cap on the maximum number counted was kept at 10,000 GFP positive cells. This data was then exported to the FloJo program where histograms were plotted and the geometric mean obtained for comparison between different ORs.

7.4 Immunohistochemistry (IHC)

PC12 cells: PC12 cells of a low passage number were grown to 70 to 80% confluence on collagen-coated plates, collected by pipetting 10ml F12Kserum up and down with a 5ml pipette and triturated with a 1ml pipette before spinning and resuspending in 8ml/plate of F12KserumPSF. 2mls of cell suspension was used to grow the culture in a new collagen coated plate and 300µl of the cell suspension was used to seed 35mm plates with collagen (sigma C5533, 200µl 0.1mg/ml) coated coverslips. These plates were allowed to sit in the incubator for 60 to 90 minutes after which 1.7mls of F12Kserum was added and the plates were incubated overnight.

Transfection was carried out in F12K with no serum using Lipofectamine LTX

(8µl, Invitrogen 15338-100) and plus agent (1:1 DNA, Invitrogen 15338-100). 1.2 µg OR,

30ng GFP and plus agent were incubated for 10 mins before adding LTX diluted in F12K and this mixture was kept for 25 mins before bringing up the volume to 2mls and replacing the media in the 35mm plates. The plates were incubated for 24 hours in 37°C,

5% CO2 and saturating humidity.

The coverslips were fixed with 4% Paraformaldehyde (PFA, EMS 19208) in PBS for 20 mins and then blocked with 4% donkey serum (Jackson Immunoresearch, no glycerol) and stained with anti-ATF5 goat antibody (Table #) overnight at 4°C. The cells were simultaneously stained with anti-Rho mouse antibody and this double staining

was visualized using cy3 and 488 Alexa fluor secondary antibodies (Table #). The coverslips were stained with 0.0001% bisBenzimide H 33258 (Sigma, B2883) to visualize the nucleus, mounted in Molwiol (Calbiochem 475904) and pictures were taken under a

40x oil immersion lens (Carl Zeiss, Axioskop). 6 images were taken from different parts of each cover slip and the number of cells both ATF5 and Rho positive were counted as well as only Rho positive cells using ImageJ software. The average percentage of double stained cells was used to compare the ability of different ORs to induce ATF5

Mouse sections: Mice were sacrificed and either the whole snout and olfactory bulb region or the

OE and OB separately were placed in OCT (Tissue-Tek 4583), flash frozen in liquid nitrogen and stored at -80°C. The blocks were sectioned in a cryostat, each section was cut at a thickness between 18µm - 25µm and placed on SuperFrost Plus slides (VWR

71869-11). Each slide contained 7-14 sections of both wildtype and RTP1,2DKO tissue and were dried thoroughly before storing at -80°C.

Prefixing: Mice were sacrificed and the dorsal OE was removed and placed in 4% PFA for

30 mins after which the PFA was vacuumed out and the tissue was rinsed twice with

PBS for 30 mins each. The tissue was placed in 30%sucrose containing PBS overnight. All

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steps were carried out at 4°C with constant gentle shaking. The tissue was frozen and sectioned as described above.

Immunostaining: The slides were dried using a hair dryer and fixed with 4% PFA in PBS for 20 mins, rinsed twice with PBS and then blocked with 4% donkey serum (no glycerol) and stained with the antibody (Table #) overnight at 4°C. In the case of the prefixed tissue, the slides were kept overnight at room temperature. The antibodies were visualized using cy3 and/or 488 Alexa fluor secondary antibodies (Table #). The slides were stained with 0.0001% bisBenzimide H 33258 to visualize the nucleus, mounted in Molwiol and pictures were taken and analyzed.

7.5 In situ Hybridization

RNA probes were prepared using the following protocol:

Plasmids (pCI) containing the gene of interest were amplified using a reverse plasmid containing a T3 promoter site.

Plasmid DNA (1-10ng/ul) 1µl

10X HotStar Taq Buffer 1µl

2mM dNTP 1µl

5µM primer (pCI 5'A) 1µl

5µM primer (T3-pCI3'A) 1µl 101

HotStar Taq 0.05µl

dW 6µl

Thermocycler program:

95°C 15min

95°C 15sec

55°C 15sec

72°C 1min

24 cycles

72°C 5min

Qiagen PB purification

30 seconds and then for 2 minutes. 10µl of buffer EB (Qiagen 28604) was added making sure to completely cover the membrane and the columns was spun for 2 minutes and the eluted DNA was collected in a microfuge tube.

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RNA synthesis:

Anti sense RNA labeled by DIG was synthesized by the following protocol:

5x RNA pol buffer (Promega P2083) 2µl

Dig or FITC RNA labeling mix (Roche 11277073910) 1µl

Template DNA (purified amplicon) 1µl

RNase inhibitor (Promega AM2682) 0.5µl

RNA pol (Helix P2083) 0.5µl

DTT (100mM) 1µl

DW 4µl

Incubate at 37°C for 2 hours

Alkaline Hydrolysis:

25µl of alkaline buffer was added and the solution was incubated at 60°C for 15 mins. Column purification was carried out using Micro Bio-Spin 30 (Bio-Rad). The columns were thoroughly shaken, spun at 3400 rpm for 2 min, the 35µl of alkaline hydrolysis solution was added and spun for 4 mins and 35µl of formamide was added to the labeled probe collected. This was stored at -80°C and used as the probe.

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Hybridization with sections

Slides were dried using a hair dryer and immersed in 4% PFA for 15 minutes followed by 2x 5 min PBS washes. Slides were submerged in triethanolamine solution

(VWR JT9468-1, 800mlDW + 8.2ml triethanolamine). Adding 1.75ml acetic acid dropwise carried out acetylation and the slides were rinsed once in PBS before being placed on railings in plates containing paper towels saturated with 5xSSC, 50% formamide and incubated for at least 1 hour in prehybridization buffer. 1µl probe was added to 200µl prehybridization buffer and added to the slides and they were covered using a strip of parafilm and kept at 58°C overnight.

Antibody staining

Slides were briefly dipped in a bucket containing 5xSSC equilibriated at 72°C while the parafilm strip was removed using a pair of forceps. The slides were transferred to another bucket containing 5xSSC at 72°C, transferred to 02.xSSC at 72°C breifly and to 02.xSSC at 72°C for 30 minutes. The slides were moved to a new bucket containing 02.xSSC at 72°C for 30 minutes and then washed in PBS and moved to slide mailer boxes. Here they were subject to at least 1 hour of blocking with 0.5% blocking agent (Roche 11096176001) in Maleic acid buffer and then anti-DIGAP antibody (1:5000) in 0.5% blocking agent for 1 hour. Slides were rinsed twice and then washed in PBS 3

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times for 20 mins each. Slides were kept for 5 minutes in in developing buffer and then in NBT/BCIP solution (NBT333µg/ml, BCIP167µg/ml, Promega S3771) in developing buffer for 1 day. Slides were examined under a dissection microscope and once the color had developed satisfactorily, the slides were stained with 0.0001% bisBenzimide H 33258 to visualize the nucleus, mounted in Molwiol and pictures were taken and analyzed using ImageJ.

7.6 RNA Extraction and Sequencing

The entire OE was extracted along with the VNO (Vomeronasal organ) and placed in a 5ml tube containing 1 ml TRIzol (Ambion 15596) and the tissue was homogenized using a polytron homogenizer. The homogenized solution was transferred to a sterile 2ml centrifuge tube and spun for 10 mins at 15000rpm and the supernatant from this was added to a new tube containing 200µl Chloroform (EMD CX1055-6) and thoroughly shaken for 3 minutes. The new tube was centrifuged for 15 minutes at

15000rpm and 500µl of the aqueous phase was removed and added to a tube containing

500µl of isopropanol mixed well and incubated for 5 minutes before centrifuging for 10 mins at 15000 rpm. The RNA pellet thus obtained was washed twice, once in 150µl of

75% ethanol and then with 180µl of 75% ethanol and was briefly air-dried by kepping in an open tube covered in plastic wrap for 10 minutes. The pellet was dissolved in 50µl

105

DW (at 55ºC if necessary) and a 1:10 dilution of the above was used to measure the concentration of RNA and the concentration adjusted to 100µg/ml

DNase treatment was carried out using the following protocol:

10X Buffer (M6101) 10µl

RNase free DNaseI (M6101) 2µl

RNA (prepped above) 100µg

DW Up to 100µl

Keep for 37C 20 minutes

350µl Buffer RLT (Qiagen 79216) was added to the DNase treated RNA and mixed and centrifuged for 1 second. 250µl ethanol (99%) was added, mixed and immediately transferred to the RNeasy mini column (Qiagen 74134) in a 2ml collection tube. The tube was centrifuged and the flow-through along with the tube was discarded.

500µl Buffer RPE (Qiagen 1018013) was added to the column and centrifuged twice. The column was placed in a new collection tube and centrifuged for 2 mins at 15000rpm. The column was placed in a 1.5ml centrifuge tube and 50µl RNase free water on the silica gel membrane and centrifuged for 2 mins. 1:10 dilution of the solution was used to measure

RNA.

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50µl of the RNA extracted was sent to the Duke Center for Genomic and

Computational Biology for library formation and sequencing by Illumina HiSeq.

Reads were mapped using a method established in (Jiang et al., 2015a), using kallisto (Bray et al., 2016), to the mouse transcripts which were downloaded from UCSC

Genome Browser (https://genome.ucsc.edu) and whose OR genes were replaced with extended OR gene annotations modified from previously published definitions (Ibarra-

Soria et al., 2014). Reads assigned on each gene were counted by kallisto. The read count table thus generated was analyzed using EdgeR. DESeq was used to calculate the size factors of individual libraries, FDR (False Discovery Rate) was used to adjust multiple comparisons between the ORs.

7.7 Whole mount LacZ Staining

3 week old mice were sacrificed and their OE and OB was dissected out and kept in 4% PFA for 30 minutes on ice. It was then transferred to a plate containing buffer A for 30 mins and then the buffer was replaced with new Buffer A and kept for 5 minutes.

Buffer A was replaced with Buffer B twice for 5 minutes and then with Buffer C overnight at room temperature.

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7.8 Phospho S6 Induction

3 week old mice were kept in a white paper bucket (capacity of approximately

3L) for an hour with an empty tissue tek cassette in order to remove any baseline effects.

They were then moved to a new bucket containing a tissue tek cassette with a small filter paper soaked in either 10µl water or 10µ 1% acetophenone for an hour and then sacrificed and their OBs and their OEs were frozen as described above. Each stimulation was carried out in parallel with a wildtype and RTP1,2DKO littermates.

7.9 ImageJ Analysis

All images were captured on Zeiss Axioskop 2 fluorescent microscope using Q capture pro. Images were then analyzed using ImageJ. For OR bias, Phosphorylated S6 and caspase 3 experiments, nuclear staining was quantified by selecting the OSN layer in the OE and using the maxima function in ImageJ to select and count all cells followed by quantification of OR or Caspase 3 positive OSNs by hand scoring using the cell counter function. Percent positive cells were calculated as (Positive cells/ total number of cells)*100. ATF5 was quantified by selecting the OSN layer and digitally straightening it using ImageJ followed by manually selecting the ATF5 positive cells and using the measure function to get the X and Y co-ordinates to measure the height from the basal end of the epithelium. All colocalization experiments were manually scored by selecting

108

the OR positive cell’s nucleus and measuring the pixel intensity for the same selection.

The pixel intensity of the neighboring area was subtracted to remove background and determine positive cells.

7.9.1 Marker gene analysis

The area occupied by OSNs in the OE was hand selected using nuclear staining in image J. The thickness of the sections was measured by straightening the OE followed by measuring the height of the straightened section in 4 places. The average height for every position was compared between the two genotypes. The OMP or ACIII or GAP43 positive area was selected using imageJ thresholding and these areas were measured and expressed as percentages in the figures. Images were taken at 5 roughly equivalent positions in the OE using the VNO and OB as landmarks (anterior VNO, middle VNO, posterior VNO, anterior OB and middle OB, Supplementary figure S1 shows an example of matched anterior OB sections). We further analyzed all the sections from RTP1,2DKO mice at 1 day, 21 day and 6 months and compared the difference in percent area occupied by the OMP positive layer, ACIII positive layer and GAP43 positive layer with that of their littermates. Each data point is obtained from one image from one matched section. The data sets were compared using paired student t test or Mann Whitney U test as indicated in the figure legend.

109

7.9.2 Statistical analysis

Percent positive cells were calculated by hand scoring positive cells and calculating percentage based on the total number of OSNs in the image counted using nuclear staining. Each individual section was counted as an individual data point.

Height of positive cells were calculated by straightening OE sections and obtaining the Y co-ordinates of hand scored ATF5 positive cells. Multiple comparison data from our

ANOVA analysis is included in supplementary table 3.

110

Appendix A id Class DV Chr Olfr1000 II Ventral chr2 uOR Olfr1002 II Dorsal chr2 uOR Olfr1006 II Ventral chr2 uOR Olfr1009 II Dorsal chr2 uOR Olfr1014 II Ventral chr2 uOR Olfr1015 II Ventral chr2 uOR Olfr1018 II Ventral chr2 uOR Olfr1026 II Dorsal chr2 uOR Olfr1029 II Dorsal chr2 uOR Olfr1030 II Dorsal chr2 uOR Olfr1032 II Dorsal chr2 uOR Olfr1034 II Dorsal chr2 uOR Olfr1036 II Dorsal chr2 uOR Olfr1037 II Dorsal chr2 uOR Olfr1039 II Dorsal chr2 uOR Olfr1042 II Dorsal chr2 uOR Olfr1043 II Dorsal chr2 uOR Olfr1044 II Dorsal chr2 uOR Olfr1045 II Dorsal chr2 uOR Olfr1046 II Dorsal chr2 uOR Olfr1047 II Dorsal chr2 uOR Olfr1048 II Dorsal chr2 uOR Olfr1049 II Dorsal chr2 uOR Olfr1051 II Dorsal chr2 uOR Olfr1052 II Dorsal chr2 uOR Olfr1053 II Dorsal chr2 uOR Olfr1065 II Dorsal chr2 uOR Olfr107 II Dorsal chr17 uOR Olfr1085 II Ventral chr2 uOR Olfr1087 II Ventral chr2 uOR Olfr1090 II Ventral chr2 uOR Olfr1093 II Ventral chr2 uOR Olfr1094 II Dorsal chr2 uOR

111

Olfr1097 II Ventral chr2 uOR Olfr1098 II Ventral chr2 uOR Olfr1099 II Ventral chr2 uOR Olfr11 II Ventral chr13 uOR Olfr1100 II Ventral chr2 uOR Olfr1102 II Dorsal chr2 uOR Olfr1104 II Dorsal chr2 uOR Olfr1106 II Dorsal chr2 uOR Olfr1111 II Dorsal chr2 uOR Olfr1112 II Ventral chr2 uOR Olfr1115 II Ventral chr2 uOR Olfr1118 II Ventral chr2 uOR Olfr112 II Ventral chr17 uOR Olfr1121 II Ventral chr2 uOR Olfr1123 II Ventral chr2 uOR Olfr1129 II Ventral chr2 uOR Olfr1130 II Ventral chr2 uOR Olfr1131 II Dorsal chr2 uOR Olfr1133 II Dorsal chr2 uOR Olfr1134 II Dorsal chr2 uOR Olfr1135 II Ventral chr2 uOR Olfr1137 II Dorsal chr2 uOR Olfr114 II Ventral chr17 uOR Olfr1140 II Dorsal chr2 uOR Olfr1143 II Ventral chr2 uOR Olfr1148 II Ventral chr2 uOR Olfr1151 II Ventral chr2 uOR Olfr1152 II Ventral chr2 uOR Olfr1154 II Dorsal chr2 uOR Olfr1155 II Dorsal chr2 uOR Olfr1157 II Dorsal chr2 uOR Olfr116 II Ventral chr17 uOR Olfr1160 II Dorsal chr2 uOR Olfr1161 II Dorsal chr2 uOR Olfr1162 II Dorsal chr2 uOR

112

Olfr1170 II Dorsal chr2 uOR Olfr1176 II Dorsal chr2 uOR Olfr1178 II Ventral chr2 uOR Olfr1182 II Ventral chr2 uOR Olfr1183 II Ventral chr2 uOR Olfr1184 II Ventral chr2 uOR Olfr1196 II Ventral chr2 uOR Olfr1198 II Ventral chr2 uOR Olfr12 II Ventral chr1 uOR Olfr1200 II Ventral chr2 uOR Olfr1202 II Ventral chr2 uOR Olfr1205 II Ventral chr2 uOR Olfr1206 II Ventral chr2 uOR Olfr1208 II Ventral chr2 uOR Olfr121 II Ventral chr17 uOR Olfr1211 II Ventral chr2 uOR Olfr1212 II Ventral chr2 uOR Olfr1215 II Ventral chr2 uOR Olfr1217 II Ventral chr2 uOR Olfr122 II Ventral chr17 uOR Olfr1221 II Ventral chr2 uOR Olfr1222 II Ventral chr2 uOR Olfr1225 II Ventral chr2 uOR Olfr1228 II Ventral chr2 uOR Olfr1229 II Ventral chr2 uOR Olfr123 II Ventral chr17 uOR Olfr1233 II Ventral chr2 uOR Olfr1234 II Ventral chr2 uOR Olfr1238 II Ventral chr2 uOR Olfr124 II Ventral chr17 uOR Olfr1240 II Ventral chr2 uOR Olfr1241 II Ventral chr2 uOR Olfr1242 II Ventral chr2 uOR Olfr1246 II Ventral chr2 uOR Olfr125 II Ventral chr17 uOR

113

Olfr1251 II Ventral chr2 uOR Olfr1252 II Ventral chr2 uOR Olfr1253 II Ventral chr2 uOR Olfr1254 II Ventral chr2 uOR Olfr1255 II Ventral chr2 uOR Olfr1256 II Ventral chr2 uOR Olfr1258 II Ventral chr2 uOR Olfr1259 II Ventral chr2 uOR Olfr1260 II Ventral chr2 uOR Olfr1261 II Ventral chr2 uOR Olfr1262 II Ventral chr2 uOR Olfr1263 II Ventral chr2 uOR Olfr1264 II Ventral chr2 uOR Olfr127 II Ventral chr17 uOR Olfr1270 II Ventral chr2 uOR Olfr1290 II Ventral chr2 uOR Olfr1294 II Ventral chr2 uOR Olfr13 II Ventral chr6 uOR Olfr130 II Ventral chr17 uOR Olfr1301 II Ventral chr2 uOR Olfr1302 II Ventral chr2 uOR Olfr1307 II Ventral chr2 uOR Olfr1308 II Ventral chr2 uOR Olfr1309 II Ventral chr2 uOR Olfr1311 II Ventral chr2 uOR Olfr1312 II Ventral chr2 uOR Olfr1313 II Ventral chr2 uOR Olfr1316 II Ventral chr2 uOR Olfr132 II Ventral chr17 uOR Olfr1320 II Ventral chrX uOR Olfr1321 II Ventral chrX uOR Olfr1322 II Ventral chrX uOR Olfr1323 II Ventral chrX uOR Olfr1324 II Ventral chrX uOR Olfr1325 II Ventral chrX uOR

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Olfr133 II Dorsal chr17 uOR Olfr1330 II Ventral chr4 uOR Olfr1331 II Ventral chr4 uOR Olfr1339 II Dorsal chr4 uOR Olfr1340 II Dorsal chr4 uOR Olfr1341 II Ventral chr4 uOR Olfr1342 II Ventral chr4 uOR Olfr1344 II Ventral chr7 uOR Olfr1349 II Ventral chr7 uOR Olfr135 II Ventral chr17 uOR Olfr1350 II Ventral chr7 uOR Olfr1352 II Ventral chr10 uOR Olfr1354 II Ventral chr10 uOR Olfr1356 II Ventral chr10 uOR Olfr136 II Ventral chr17 uOR Olfr1361 II Dorsal chr13 uOR Olfr1364 II Ventral chr13 uOR Olfr1366 II Dorsal chr13 uOR Olfr1368 II Ventral chr13 uOR Olfr138 II Ventral chr17 uOR Olfr1380 II Ventral chr11 uOR Olfr1384 II Ventral chr11 uOR Olfr1389 II Dorsal chr11 uOR Olfr139 II Dorsal chr11 uOR Olfr1392 II Ventral chr11 uOR Olfr1393 II Ventral chr11 uOR Olfr1406 II Dorsal chr1 uOR Olfr1408 II Dorsal chr1 uOR Olfr141 II Dorsal chr2 uOR Olfr1410 II Ventral chr1 uOR Olfr1412 II Ventral chr1 uOR Olfr1414 II Ventral chr1 uOR Olfr1417 II Ventral chr19 uOR Olfr1419 II Ventral chr19 uOR Olfr142 II Ventral chr2 uOR

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Olfr1424 II Ventral chr19 uOR Olfr1426 II Ventral chr19 uOR Olfr1427 II Ventral chr19 uOR Olfr1428 II Ventral chr19 uOR Olfr1431 II Ventral chr19 uOR Olfr1437 II Ventral chr19 uOR Olfr144 II uOR Olfr1440 II Ventral chr19 uOR Olfr1443 II Ventral chr19 uOR Olfr1444 II Dorsal chr19 uOR Olfr1446 II Ventral chr19 uOR Olfr1447 II Ventral chr19 uOR Olfr1451 II Dorsal chr19 uOR Olfr1454 II Ventral chr19 uOR Olfr146 II Dorsal chr9 uOR Olfr1461 II Ventral chr19 uOR Olfr1463 II Ventral chr19 uOR Olfr1467 II Dorsal chr19 uOR Olfr147 II Dorsal chr9 uOR Olfr1471 II Ventral chr19 uOR Olfr1472 II Ventral chr19 uOR Olfr1477 II Ventral chr19 uOR Olfr148 II Dorsal chr9 uOR Olfr1484 II Ventral chr19 uOR Olfr1487 II Ventral chr19 uOR Olfr149 II Dorsal chr9 uOR Olfr1490 II Ventral chr19 uOR Olfr1491 II Ventral chr19 uOR Olfr1494 II Ventral chr19 uOR Olfr1495 II Dorsal chr19 uOR Olfr1497 II Dorsal chr19 uOR Olfr150 II Dorsal chr9 uOR Olfr1500 II Dorsal chr19 uOR Olfr1510 II Dorsal chr14 uOR Olfr1511 II Dorsal chr14 uOR

116

Olfr1513 II Dorsal chr14 uOR Olfr155 II Ventral chr4 uOR Olfr156 II Ventral chr4 uOR Olfr167 II Ventral chr16 uOR Olfr17 II Ventral chr7 uOR Olfr170 II Ventral chr16 uOR Olfr177 II Ventral chr16 uOR Olfr178 II Ventral chr16 uOR Olfr18 II Ventral chr9 uOR Olfr181 II Ventral chr16 uOR Olfr183 II Ventral chr16 uOR Olfr19 II Dorsal chr16 uOR Olfr190 II Ventral chr16 uOR Olfr195 II Ventral chr16 uOR Olfr198 II Ventral chr16 uOR Olfr199 II Ventral chr16 uOR Olfr2 II Ventral chr7 uOR Olfr206 II Ventral chr16 uOR Olfr211 II Ventral chr6 uOR Olfr212 II Ventral chr6 uOR Olfr215 II Ventral chr6 uOR Olfr221 II Dorsal chr14 uOR Olfr222 II Ventral chr11 uOR Olfr224 II Ventral chr11 uOR Olfr228 II Dorsal chr2 uOR Olfr23 II Dorsal chr11 uOR Olfr231 II Dorsal chr1 uOR Olfr235 II Ventral chr19 uOR Olfr239 II Ventral chr17 uOR Olfr247 II Dorsal chr10 uOR Olfr250 II Dorsal chr9 uOR Olfr259 II Ventral chr2 uOR Olfr263 II Ventral chr13 uOR Olfr267 II Ventral chr4 uOR Olfr270 II Ventral chr4 uOR

117

Olfr272 II Ventral chr4 uOR Olfr273 II Ventral chr4 uOR Olfr275 II Ventral chr4 uOR Olfr279 II Ventral chr15 uOR Olfr281 II Ventral chr15 uOR Olfr283 II Ventral chr15 uOR Olfr284 II Ventral chr15 uOR Olfr285 II Ventral chr15 uOR Olfr297 II Ventral chr7 uOR Olfr298 II Ventral chr7 uOR Olfr3 II Dorsal chr2 uOR Olfr30 II Dorsal chr11 uOR Olfr301 II Ventral chr7 uOR Olfr303 II Ventral chr7 uOR Olfr305 II Ventral chr7 uOR Olfr307 II Ventral chr7 uOR Olfr308 II Ventral chr7 uOR Olfr309 II Ventral chr7 uOR Olfr311 II Ventral chr11 uOR Olfr312 II Ventral chr11 uOR Olfr313 II Dorsal chr11 uOR Olfr314 II Ventral chr11 uOR Olfr316 II Ventral chr11 uOR Olfr317 II Dorsal chr11 uOR Olfr318 II Ventral chr11 uOR Olfr319 II Ventral chr11 uOR Olfr32 II Ventral chr2 uOR Olfr324 II Ventral chr11 uOR Olfr325 II Ventral chr11 uOR Olfr328 II Ventral chr11 uOR Olfr330 II Ventral chr11 uOR Olfr332 II Ventral chr11 uOR Olfr338 II Dorsal chr2 uOR Olfr341 II Dorsal chr2 uOR Olfr346 II Ventral chr2 uOR

118

Olfr347 II Ventral chr2 uOR Olfr348 II Dorsal chr2 uOR Olfr350 II Dorsal chr2 uOR Olfr354 II Ventral chr2 uOR Olfr357 II Ventral chr2 uOR Olfr358 II Dorsal chr2 uOR Olfr360 II Ventral chr2 uOR Olfr372 II Dorsal chr8 uOR Olfr373 II Dorsal chr8 uOR Olfr376 II Dorsal chr11 uOR Olfr38 II Dorsal chr6 uOR Olfr389 II Ventral chr11 uOR Olfr39 II Ventral chr9 uOR Olfr390 II Ventral chr11 uOR Olfr392 II Ventral chr11 uOR Olfr394 II Ventral chr11 uOR Olfr397 II Ventral chr11 uOR Olfr399 II Dorsal chr11 uOR Olfr410 II Dorsal chr11 uOR Olfr411 II Dorsal chr11 uOR Olfr417 II Ventral chr1 uOR Olfr419 II Dorsal chr1 uOR Olfr424 II Dorsal chr1 uOR Olfr430 II Dorsal chr1 uOR Olfr433 II Dorsal chr1 uOR Olfr434 II Ventral chr6 uOR Olfr435 II Dorsal chr6 uOR Olfr437 II Dorsal chr6 uOR Olfr44 II Ventral chr9 uOR Olfr448 II Dorsal chr6 uOR Olfr45 II Ventral chr7 uOR Olfr450 II Ventral chr6 uOR Olfr452 II Ventral chr6 uOR Olfr453 II Dorsal chr6 uOR Olfr455 I Ventral chr6 uOR

119

Olfr457 II Ventral chr6 uOR Olfr459 II Ventral chr6 uOR Olfr46 II Ventral chr7 uOR Olfr460 II Ventral chr6 uOR Olfr461 II Ventral chr6 uOR Olfr462 II Ventral chr11 uOR Olfr463 II Ventral chr11 uOR Olfr464 II Ventral chr11 uOR Olfr466 II Ventral chr13 uOR Olfr467 II Dorsal chr7 uOR Olfr469 II Ventral chr7 uOR Olfr47 II Ventral chr6 uOR Olfr473 II Ventral chr7 uOR Olfr476 II Ventral chr7 uOR Olfr478 II Dorsal chr7 uOR Olfr48 II Ventral chr2 uOR Olfr481 II Dorsal chr7 uOR Olfr482 II Dorsal chr7 uOR Olfr483 II Dorsal chr7 uOR Olfr484 II Dorsal chr7 uOR Olfr487 II Ventral chr7 uOR Olfr488 II Dorsal chr7 uOR Olfr491 II Ventral chr7 uOR Olfr497 II Ventral chr7 uOR Olfr50 II Dorsal chr2 uOR Olfr502 II Ventral chr7 uOR Olfr506 II Dorsal chr7 uOR Olfr508 II Dorsal chr7 uOR Olfr51 II Dorsal chr11 uOR Olfr510 II Dorsal chr7 uOR Olfr516 II Dorsal chr7 uOR Olfr517 II Dorsal chr7 uOR Olfr518 II Dorsal chr7 uOR Olfr52 II Dorsal chr2 uOR Olfr521 II Dorsal chr7 uOR

120

Olfr522 II Dorsal chr7 uOR Olfr523 II Ventral chr7 uOR Olfr524 II Ventral chr7 uOR Olfr525 II Ventral chr7 uOR Olfr530 II Ventral chr7 uOR Olfr531 II Ventral chr7 uOR Olfr532 II Ventral chr7 uOR Olfr533 II Ventral chr7 uOR Olfr535 II Ventral chr7 uOR Olfr536 II Ventral chr7 uOR Olfr538 II Ventral chr7 uOR Olfr54 II Dorsal chr11 uOR Olfr541 II Ventral chr7 uOR Olfr55 II Ventral chr17 uOR Olfr553 I Dorsal chr7 uOR Olfr556 I Ventral chr7 uOR Olfr566 I Dorsal chr7 uOR Olfr569 I Dorsal chr7 uOR Olfr570 I Dorsal chr7 uOR Olfr582 I Dorsal chr7 uOR Olfr584 I Dorsal chr7 uOR Olfr586 I Dorsal chr7 uOR Olfr59 II Ventral chr11 uOR Olfr592 I Dorsal chr7 uOR Olfr594 I Dorsal chr7 uOR Olfr596 II Dorsal chr7 uOR Olfr597 I Dorsal chr7 uOR Olfr598 I Dorsal chr7 uOR Olfr599 I Dorsal chr7 uOR Olfr6 II Ventral chr7 uOR Olfr60 II Ventral chr7 uOR Olfr601 I Dorsal chr7 uOR Olfr605 I Dorsal chr7 uOR Olfr606 I Dorsal chr7 uOR Olfr608 I Dorsal chr7 uOR

121

Olfr61 II Ventral chr7 uOR Olfr610 I Dorsal chr7 uOR Olfr618 I Dorsal chr7 uOR Olfr62 II Dorsal chr4 uOR Olfr622 I Dorsal chr7 uOR Olfr623 I Dorsal chr7 uOR Olfr629 I Dorsal chr7 uOR Olfr630 I Dorsal chr7 uOR Olfr633 I Dorsal chr7 uOR Olfr635 I Dorsal chr7 uOR Olfr640 I Dorsal chr7 uOR Olfr646 I Dorsal chr7 uOR Olfr652 I Dorsal chr7 uOR Olfr655 I Dorsal chr7 uOR Olfr657 I Dorsal chr7 uOR Olfr658 I Dorsal chr7 uOR Olfr659 I Dorsal chr7 uOR Olfr66 I Dorsal chr7 uOR Olfr665 I Dorsal chr7 uOR Olfr668 I Dorsal chr7 uOR Olfr67 I Dorsal chr7 uOR Olfr670 I Dorsal chr7 uOR Olfr671 I Dorsal chr7 uOR Olfr675 I Dorsal chr7 uOR Olfr676 I Dorsal chr7 uOR Olfr677 I Dorsal chr7 uOR Olfr68 I Dorsal chr7 uOR Olfr681 I Dorsal chr7 uOR Olfr683 I Dorsal chr7 uOR Olfr684 I Dorsal chr7 uOR Olfr686 I Ventral chr7 uOR Olfr689 I Dorsal chr7 uOR Olfr69 I Dorsal chr7 uOR Olfr692 I Dorsal chr7 uOR Olfr693 II Ventral chr7 uOR

122

Olfr694 II Ventral chr7 uOR Olfr695 II Ventral chr7 uOR Olfr698 II Ventral chr7 uOR Olfr699 II Ventral chr7 uOR Olfr70 II Ventral chr4 uOR Olfr703 II Ventral chr7 uOR Olfr704 II Ventral chr7 uOR Olfr705 II Ventral chr7 uOR Olfr706 II Ventral chr7 uOR Olfr713 II Ventral chr7 uOR Olfr714 II Ventral chr7 uOR Olfr716 II Ventral chr7 uOR Olfr720 II Ventral chr14 uOR Olfr722 II Ventral chr14 uOR Olfr725 II Ventral chr14 uOR Olfr726 II Ventral chr14 uOR Olfr728 II Ventral chr14 uOR Olfr736 II Ventral chr14 uOR Olfr741 II Ventral chr14 uOR Olfr742 II Ventral chr14 uOR Olfr744 II Dorsal chr14 uOR Olfr745 II Dorsal chr14 uOR Olfr746 II Dorsal chr14 uOR Olfr749 II Dorsal chr14 uOR Olfr763 II Dorsal chr10 uOR Olfr765 II Dorsal chr10 uOR Olfr768 II Dorsal chr10 uOR Olfr77 II Dorsal chr9 uOR Olfr772 II Ventral chr10 uOR Olfr786 II Ventral chr10 uOR Olfr787 II Ventral chr10 uOR Olfr788 II Ventral chr10 uOR Olfr791 II Dorsal chr10 uOR Olfr792 II Dorsal chr10 uOR Olfr796 II Ventral chr10 uOR

123

Olfr8 II Ventral chr10 uOR Olfr801 II Ventral chr10 uOR Olfr807 II Dorsal chr10 uOR Olfr809 II Dorsal chr10 uOR Olfr813 II Dorsal chr10 uOR Olfr815 II Dorsal chr10 uOR Olfr818 II Ventral chr10 uOR Olfr819 II Dorsal chr10 uOR Olfr820 II Ventral chr10 uOR Olfr824 II Ventral chr10 uOR Olfr825 II Ventral chr10 uOR Olfr826 II Dorsal chr10 uOR Olfr827 II Ventral chr10 uOR Olfr828 II Ventral chr9 uOR Olfr835 II Ventral chr9 uOR Olfr836 II Ventral chr9 uOR Olfr843 II Ventral chr9 uOR Olfr844 II uOR Olfr845 II Ventral chr9 uOR Olfr846 II Dorsal chr9 uOR Olfr847 II Dorsal chr9 uOR Olfr849 II Ventral chr9 uOR Olfr851 II Ventral chr9 uOR Olfr857 II Ventral chr9 uOR Olfr860 II Ventral chr9 uOR Olfr867 II Dorsal chr9 uOR Olfr868 II Ventral chr9 uOR Olfr869 II Ventral chr9 uOR Olfr870 II Ventral chr9 uOR Olfr872 II Ventral chr9 uOR Olfr874 II Dorsal chr9 uOR Olfr878 II Dorsal chr9 uOR Olfr881 II Dorsal chr9 uOR Olfr884 II Dorsal chr9 uOR Olfr887 II Ventral chr9 uOR

124

Olfr890 II Ventral chr9 uOR Olfr894 II Dorsal chr9 uOR Olfr898 II Ventral chr9 uOR Olfr91 II Ventral chr17 uOR Olfr910 II Ventral chr9 uOR Olfr912 II Ventral chr9 uOR Olfr913 II Ventral chr9 uOR Olfr916 II Dorsal chr9 uOR Olfr918 II Dorsal chr9 uOR Olfr919 II Dorsal chr9 uOR Olfr920 II Ventral chr9 uOR Olfr921 II Ventral chr9 uOR Olfr922 II Dorsal chr9 uOR Olfr923 II Dorsal chr9 uOR Olfr924 II Dorsal chr9 uOR Olfr93 II Ventral chr17 uOR Olfr933 II Dorsal chr9 uOR Olfr937 II Dorsal chr9 uOR Olfr938 II Dorsal chr9 uOR Olfr943 II Ventral chr9 uOR Olfr948 II Ventral chr9 uOR Olfr95 II Ventral chr17 uOR Olfr957 II Dorsal chr9 uOR Olfr958 II Dorsal chr9 uOR Olfr96 II Ventral chr17 uOR Olfr961 II Dorsal chr9 uOR Olfr963 II Dorsal chr9 uOR Olfr965 II Dorsal chr9 uOR Olfr967 II Dorsal chr9 uOR Olfr969 II Dorsal chr9 uOR Olfr97 II Ventral chr17 uOR Olfr971 II Dorsal chr9 uOR Olfr974 II Ventral chr9 uOR Olfr976 II Dorsal chr9 uOR Olfr981 II Dorsal chr9 uOR

125

Olfr982 II Dorsal chr9 uOR Olfr984 II Ventral chr9 uOR Olfr985 II Dorsal chr9 uOR Olfr986 II Ventral chr9 uOR Olfr988 II Dorsal chr2 uOR Olfr99 II Ventral chr17 uOR Olfr992 II Dorsal chr2 uOR Olfr994 II Dorsal chr2 uOR Olfr1370 II Ventral chr13 oOR Olfr799 II Ventral chr10 oOR Olfr220 II Ventral chr1 oOR Olfr959 II Dorsal chr9 oOR Olfr1297 II Ventral chr2 oOR Olfr1396 II Ventral chr11 oOR Olfr204 II Ventral chr16 oOR Olfr129 II Ventral chr17 oOR Olfr370 II Ventral chr8 oOR Olfr800 II Ventral chr10 oOR Olfr1395 II Ventral chr11 oOR Olfr653 I Dorsal chr7 oOR Olfr145 II Dorsal chr9 oOR Olfr1448 II Dorsal chr19 oOR Olfr544 I Dorsal chr7 oOR Olfr794 II Ventral chr10 oOR Olfr743 II Ventral chr14 oOR Olfr557 I Dorsal chr7 oOR Olfr449 II Ventral chr6 oOR Olfr43 II Ventral chr11 oOR Olfr168 II Ventral chr16 oOR Olfr290 II Ventral chr7 oOR Olfr286 II Ventral chr15 oOR Olfr1056 II Dorsal chr2 oOR Olfr1209 II Ventral chr2 oOR Olfr571 I Dorsal chr7 oOR Olfr790 II Ventral chr10 oOR

126

Olfr558 I Dorsal chr7 oOR Olfr1193 II Ventral chr2 oOR Olfr1110 II Ventral chr2 oOR Olfr1195 II Ventral chr2 oOR Olfr65 I Dorsal chr7 oOR Olfr194 II Ventral chr16 oOR Olfr727 II Ventral chr14 oOR Olfr169 II Ventral chr16 oOR Olfr365 II Ventral chr2 oOR Olfr339 II Ventral chr2 oOR Olfr1197 II Ventral chr2 oOR Olfr1509 II Ventral chr14 oOR Olfr101 II Ventral chr17 oOR Olfr111 II Ventral chr17 oOR Olfr1347 II Ventral chr7 oOR Olfr56 II Ventral chr11 oOR Olfr1362 II Dorsal chr13 oOR Olfr715 II Ventral chr7 oOR Olfr769 II Ventral chr10 oOR Olfr78 I Dorsal chr7 oOR Olfr16 II Dorsal chr1 oOR Olfr447 II Dorsal chr6 oOR Olfr1239 II Ventral chr2 oOR Olfr479 II Ventral chr7 oOR Olfr1079 II Dorsal chr2 oOR Olfr1508 II Ventral chr14 oOR Olfr748 II Dorsal chr14 oOR Olfr1386 II Ventral chr11 oOR Olfr1168 II Dorsal chr2 oOR Olfr64 I Dorsal chr7 oOR Olfr351 II Dorsal chr2 oOR Olfr218 II Dorsal chr1 oOR Olfr577 I Dorsal chr7 oOR Olfr166 II Ventral chr16 oOR Olfr545 I Dorsal chr7 oOR

127

Olfr1404 II Dorsal chr1 oOR Olfr414 II Ventral chr1 oOR Olfr729 II Ventral chr14 oOR Olfr203 II Ventral chr16 oOR Olfr173 II Ventral chr16 oOR Olfr1303 II Ventral chr2 oOR Olfr1057 II Dorsal chr2 oOR Olfr291 II Ventral chr7 oOR Olfr1107 II Ventral chr2 oOR Olfr1318 II Ventral chr2 oOR Olfr432 II Dorsal chr1 oOR Olfr733 II Ventral chr14 oOR Olfr970 II Dorsal chr9 oOR Olfr740 II Ventral chr14 oOR Olfr810 II Dorsal chr10 oOR Olfr33 I Dorsal chr7 oOR Olfr632 I Dorsal chr7 oOR Olfr119 II Ventral chr17 oOR Olfr15 II Ventral chr16 oOR Olfr527 II Ventral chr7 oOR Olfr143 II Dorsal chr9 oOR Olfr781 II Ventral chr10 oOR Olfr1226 II Ventral chr2 oOR Olfr288 II Ventral chr15 oOR Olfr20 II Dorsal chr11 oOR Olfr666 I Dorsal chr7 oOR Olfr131 II Ventral chr17 oOR Olfr1505 II Dorsal chr19 oOR Olfr873 II Ventral chr9 oOR Olfr1348 II Ventral chr7 oOR Olfr1080 II Ventral chr2 oOR Olfr53 II Ventral chr7 oOR Olfr73 II Dorsal chr2 oOR Olfr812 II Dorsal chr10 oOR Olfr214 II Ventral chr6 oOR

128

Olfr1336 II Ventral chr7 oOR Olfr1391 II Ventral chr11 oOR Olfr611 I Dorsal chr7 oOR Olfr644 I Dorsal chr7 oOR Olfr120 II Ventral chr17 oOR Olfr353 II Ventral chr2 oOR Olfr806 II Ventral chr10 oOR Olfr1250 II Ventral chr2 oOR Olfr798 II Dorsal chr10 oOR Olfr1387 II Ventral chr11 oOR Olfr1289 II Ventral chr2 oOR Olfr192 II Ventral chr16 oOR Olfr1359 II Ventral chr13 oOR Olfr1442 II Ventral chr19 oOR Olfr578 I Dorsal chr7 oOR Olfr1381 II Ventral chr11 oOR Olfr1257 II Ventral chr2 oOR Olfr205 II Ventral chr16 oOR Olfr616 I Dorsal chr7 oOR Olfr1219 II Ventral chr2 oOR Olfr152 II Dorsal chr2 oOR Olfr1535 II Ventral chr13 oOR Olfr672 I Dorsal chr7 oOR Olfr1013 II Ventral chr2 oOR Olfr1346 II Ventral chr7 oOR Olfr609 I Dorsal chr7 oOR Olfr403 II Ventral chr11 oOR Olfr620 I Dorsal chr7 oOR Olfr1269 II Ventral chr2 oOR Olfr197 II Ventral chr16 oOR Olfr612 I Dorsal chr7 oOR Olfr1382 II Ventral chr11 oOR Olfr617 I Dorsal chr7 oOR Olfr987 II Dorsal chr2 oOR Olfr209 II Ventral chr16 oOR

129

Olfr968 II Dorsal chr9 oOR Olfr1507 II Ventral chr14 oOR Olfr191 II Ventral chr16 oOR Olfr901 II Ventral chr9 oOR Olfr1295 II Ventral chr2 oOR Olfr543 I Dorsal chr7 oOR Olfr691 I Dorsal chr7 oOR Olfr1243 II Ventral chr2 oOR Olfr1288 II Ventral chr2 oOR Olfr366 II Ventral chr2 oOR Olfr1278 II Ventral chr2 oOR Olfr57 II Dorsal chr10 oOR Olfr134 II Dorsal chr17 oOR Olfr723 II Ventral chr14 oOR Olfr102 II Dorsal chr17 oOR Olfr24 II Dorsal chr9 oOR Olfr735 II Ventral chr14 oOR Olfr550 I Dorsal chr7 oOR Olfr613 I Dorsal chr7 oOR Olfr229 II Dorsal chr9 oOR Olfr1213 II Ventral chr2 oOR Olfr519 II Dorsal chr7 oOR Olfr1299 II Ventral chr2 oOR Olfr340 II Ventral chr2 oOR Olfr109 II Ventral chr17 oOR Olfr642 I Dorsal chr7 oOR Olfr103 II Ventral chr17 oOR Olfr1475 II Ventral chr19 oOR Olfr1105 II Dorsal chr2 oOR Olfr1012 II Dorsal chr2 oOR Olfr651 I Dorsal chr7 oOR Olfr110 II Ventral chr17 oOR Olfr690 I Dorsal chr7 oOR Olfr1089 II Ventral chr2 oOR Olfr398 II Ventral chr11 oOR

130

Olfr1231 II Ventral chr2 oOR Olfr732 II Ventral chr14 oOR Olfr1281 II Ventral chr2 oOR Olfr734 II Ventral chr14 oOR Olfr1132 II Dorsal chr2 oOR Olfr724 II Ventral chr14 oOR Olfr251 II Dorsal chr9 oOR Olfr935 II Dorsal chr9 oOR Olfr172 II Ventral chr16 oOR Olfr1357 II Dorsal chr10 oOR Olfr679 I Dorsal chr7 oOR Olfr118 II Ventral chr17 oOR Olfr1275 II Ventral chr2 oOR Olfr1040 II Dorsal chr2 oOR Olfr1061 II Ventral chr2 oOR Olfr1388 II Ventral chr11 oOR Olfr1425 II Ventral chr19 oOR Olfr1283 II Ventral chr2 oOR Olfr907 II Ventral chr9 oOR Olfr1466 II Ventral chr19 oOR Olfr1280 II Ventral chr2 oOR Olfr1286 II Ventral chr2 oOR Olfr1416 II Ventral chr1 oOR Olfr678 I Dorsal chr7 oOR Olfr92 II Ventral chr17 oOR Olfr1328 II Dorsal chr4 oOR Olfr1390 II Ventral chr11 oOR Olfr310 II Dorsal chr7 oOR Olfr161 II Dorsal chr16 oOR Olfr711 II Ventral chr7 oOR Olfr1008 II Ventral chr2 oOR Olfr157 II Ventral chr4 oOR Olfr600 I Dorsal chr7 oOR Olfr368 II Dorsal chr2 oOR Olfr1019 II Dorsal chr2 oOR

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Biography

Ruchira Sharma DOB October 18th, 1986 Place of birth Durham USA Education & Training PhD Thesis: Olfactory receptor accessory proteins play crucial roles in receptor function and gene choice, Duke University, Durham, USA

Masters in Science (Biochemistry), Maharaja Sayajirao University of Baroda, Department of Biochemistry

Bachelor of Science (Life Sciences,Honors), St. Xavier’s College Mumbai, Department of Life Sciences Fellowships/Awards 2016 Young Investigator Award, 17th International Symposium on Olfaction and Taste (ISOT2016) 2016 Chairman’s Travel Award, Dept. of MGM, Duke University 2014-2016 Conference Travel Awards, Duke University Graduate School 2014 Association for Chemoreception Studies Housing Award, Duke University, 2013 Commendation for Animal Handling, Duke-IACUC 2009 Council of Scientific and Industrial Research Fellowship (CSIR India) Publications Ruchira Sharma, Yoshiro Ishimaru, Ian Davison, Kentaro Ikegami, Ming-Shan Chien, Helena You, Quiyi Chi, Momoka Kubota, Masafumi Yohda, Michael Ehlers, and Hiroaki Matsunami. Olfactory receptor accessory proteins play crucial roles in receptor function and gene choice (Accepted eLife, 2017)

Ruchira Sharma and Hiroaki Matsunami. Mechanisms of Olfaction Bioelectronic Nose. Springer Netherlands, 2014. 23-45.

2005 Ruchira Sharma, Sonia Sen, Gautam Dey, and Veronica Rodrigues. A New Olfactory Trap Assay. Drosophila Information Service, 88:115–118, 2005

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