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2009 Ph.D. Thesis KyungHwa (Kirsten) Lee

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  • 1. 
 
 Sex Differences in a C. elegans Sensory Behavior by Kyung Hwa Lee Submitted in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Supervised by Professor Douglas S. Portman Interdepartmental Graduate Program in Neuroscience School of Medicine and Dentistry University of Rochester Rochester, New York 2009
  • 2. ii 
 To my family whose love and prayer has carried me through this endeavor
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 Curriculum Vitae The author was born in Seoul, Korea on August 5, 1980. She attended Handong Global University from March 1999 to December 2002, and graduated with a Bachelor of Science degree in February 2003. She came to the University of Rochester in the Summer of 2003 and began graduate studies in the Interdepartmental Graduate Program in Neuroscience. She pursued her research in “Sex differences in C. elegans olfactory behavior” under the direction of Professor Douglas S. Portman and received the Master of Science degree from the University of Rochester in March 2006. A part of the thesis is published in the journal: Current Biology 17, 1858– 1863, November 6, 2007.
  • 4. iv 
 Acknowledgements I am grateful to all members of the Portman lab for their challenging hard work, encouragements, and accountability to discuss science and to share life. In particular, I thank Dr. Renee Miller, William Mowrey and a former member, Dr. Adam Mason for insightful suggestions and discussions throughout years. Their valuable technical contributions to my thesis work are noted within the text. Foremost I offer my gratitude to my advisor, Dr. Douglas S. Portman, who has supported me throughout my graduate studies with his patience, knowledge, and encouragement. He was always available for discussions in person and online. One simply could not wish for a better advisor. I would like to thank my thesis committee members: Dr. Robert S. Freeman and Dr. Kathy W. Nordeen for their time and advice. I also appreciate Dr. Oliver Hobert for his time and kindness to serve on my committee and deliver valuable suggestions and insights. I thank Dr. White and Dr. Jorgensen for sharing oxEx862 and oxEx863 and for communicating unpublished data, Dr. Schwarz and Dr. Horvitz for generously providing ceh-30(n4289) mutants, and Dr. Bargmann for helpful suggestions. I cannot end without thanking my family, on whose constant love and prayer I have relied throughout my journey at the Academy.
  • 5. v 
 Abstract Sex differences in the structure and function of the nervous system exist throughout the animal kingdom. Together with sex-biases in neurological diseases, this highlights the importance of studying how sexual differentiation modifies neural circuits and function. Taking advantage of the unique strengths of the nematode C. elegans, we explore how “neural sex”, the sexual state of a given neuron established by cell-intrinsic sex determination, regulates the function of the “core” neural circuitry composed of neurons common to both sexes. To ask how neural sex influences behavior, we have examined olfaction, well-described in the C. elegans hermaphrodite but previously unstudied in the male. Using a novel assay involving the simultaneous presentation of two attractants, we have observed characteristic and distinct sex differences in olfactory preference behaviors. These sex differences were prominent before sexual maturation and did not require the gonad or germline, suggesting that core neural circuitry itself may be the cellular focus of sexually different shared behavior. To address this directly, we switched the sexual state of subsets of core neurons by cell-type specific expression of sexual regulators. We found that the neural sex of even a single sensory neuron, AWA, can determine the sexual phenotype of olfactory preference, indicating that AWA itself possesses sexually different functional properties. Moreover, at least some of these functional properties arise through sex differences in the expression of the odorant receptor ODR-10, providing a molecular mechanism for the generation of sexually different shared sensory function. This work has revealed a novel pathway for bringing about
  • 6. vi 
 sex differences in the function of shared neural circuitry, and may shed light on the nature of sexual dimorphisms in the vertebrate nervous system.
  • 7. vii 
 Table of Contents Chapter 1 Introduction 1 1 The general problem of sex differentiation in the nervous system 1 Sex, brain, and behavior 1 Sex differences are prominent in the neuroanatomy for sex-specific 2 behaviors Sex differences also occur in the areas of the brain not relevant to 2 reproductive behavior Sex differences are observed even in common behaviors non-relevant to 3 reproduction Sex-biases are prevalent in the nature and/or incidences of neurological 3 diseases Sex differences in behavioral symptoms of some neurological disorders 4 2 Sex hormones and the sex of the brain 5 Activity of sex hormones has been thought to regulate sex differences in 5 the brain Some sexually different behaviors are not explained by sex hormones 5 3 Chromosomal sex also control properties of neural structures and 6 behaviors Sex differences in neurological diseases are not all explained by the 6 activity of sex hormones Evidences of sex hormone-independent sexual differentiation in the 6 vertebrate system Cell-intrinsic sex regulators generate sex-specific behaviors in 7 invertebrate organisms The pathway of chromosomal sex regulation on the properties of the 8 neural circuit is largely unknown
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 4 Neural circuits and behaviors 8 Some gene expression differences change behaviors 8 Sex-specific behaviors are generated by sexually different 9 interpretations of the same sensory stimuli as a result of differences in gene expression Common behaviors between sexes or between species are modified 9 by gene expression differences to confer sex difference or species difference A complete diagram of the neural circuit for any complex behavior 10 is generally not described 5 C. elegans as a system to study sex differences in shared behaviors 10 C. elegans is an ideal model for neuroscience and for studying sexual 10 dimorphism in the nervous system The cell-intrinsic sex determination pathway regulates all known somatic 11 sex differences in C. elegans The C. elegans core neural circuitry has molecular sex difference 12 Sex differences in the C. elegans core neural function 12 6 C. elegans olfactory behaviors 17 The hermaphrodite olfactory system is well characterized in its structure 17 and function Olfactory neural circuit possess molecular properties for behavioral 21 plasticity Chapter 2 Neural sex modifies the function of a C. elegans sensory circuit 22 1 Introduction 22 2 Materials and Methods 23 3 Results 29 C. elegans exhibit significant sex difference in olfactory behaviors 29 Each sex displays distinct and characteristic olfactory preferences 30
  • 9. ix 
 Sexually different olfaction is not the secondary effect of male-specific 34 behaviors The male-specific CEM neurons do not have a primary role in the 37 sexually different shared sensory function Gonad signaling is not necessary for sex difference in olfaction 37 Sexual differences in olfaction are prominent before sexual maturation 38 Neural sex determines the sex phenotype of a common sensory function, 41 olfactory preference Pan-neural sex-transformation 45 Sex-transformation on sensory, interneuron and motor neurons 45 4 Discussion 49 Why are there sex differences in C. elegans olfaction? 49 Developmental regulation of sexually different olfactory behaviors 51 How does sex modify olfaction? 52 Neural Sex regulation on Behaviors 53 Chapter 3 Neural sex modifies the properties of a single sensory neuron to 55 generate sexually different olfactory behaviors 1 Introduction 55 2 Materials and Methods 56 3 Results 58 Sexually different olfaction arises through neural sex modification on 58 sensory neurons A single sensory neuron, AWA, generates sexually different olfactory 62 preference Neural sex regulation on sexually different olfaction is a property of the 65 C. elegans olfactory circuit Neural sex modifies a target gene in AWA neurons to bring about sex 69
  • 10. x 
 difference in olfaction 4 Discussion 80 Core sensory circuitry controls a sex difference in olfactory preference 80 A single sensory neural-switch between hermaphrodite and male olfaction 80 Neural sex regulates an effector gene critical for the function of a neural 82 circuit Appendix 1 Potential sex difference in AWA connectivity 85 Chapter 4 Discussion 87 The control of sex differences in a C. elegans sensory behavior 87 Insights on sexual dimorphisms in neurological diseases 89 References 91 Appendix 2 Strains 102
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 List of Tables Table A2.1 Nematode strains 102
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 List of Figures Figure 1.1 C. elegans Sexes 13 Figure 1.2 Somatic sex determination in C. elegans 14 Figure 1.3 The C. elegans core nervous system 15 Figure 1.4 Sexually dimorphic gene expressions in the C. elegans core 16 nervous syste Figure 1.5 Does the C. elegans core nervous system mediate sexually 19 dimorphic behaviors? Figure 1.6 The C. elegans olfactory neural circuit is a part of the core 20 nervous system Figure 2.1 Single odorant assay 25 Figure 2.2 Olfactory preference assay 27 Figure 2.3 Male olfaction is significantly different to hermaphrodite 31 olfaction Figure 2.4 Each sex has distinct and characteristic olfactory preferences 33 Figure 2.5 Sexually different olfactory preferences are generated neither 35 by sex-specific behaviors nor by structures Figure 2.6 Sex differences in olfaction precede sex-specific differentiation 39 Figure 2.7 The terminal sex regulator, tra-1, controls sex difference 43 in olfactory preference Figure 2.8 Sex-transformation of the nervous system 44 Figure 2.9 Neural sex-transformation 46 Figure 2.10 Neural sex determines the sex phenotype of olfactory preference 48 Figure 2.11 Sexually different properties of the sensory neurons control 50 the sex phenotype of olfactory preference Figure 3.1 Neural sex governs sex difference in olfactory preference 60
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 Figure 3.2 A neural sex mechanism modifies properties of the sensory 61 neurons to bring about sexually dimorphic olfactory preference Figure 3.3 The sexual state of a single sensory neuron, AWA, is 63 sufficient to impart sex differences in olfactory preference Figure 3.4 How AWA neurons are modified by neural sex? 66 Figure 3.5 Neural sex regulates sex differences in both AWA and AWC 67 olfactory preference behaviors Figure 3.6 Sexually different mechanisms underlying responses to 71 hermaphrodite AWA odorants Figure 3.7 How do male AWA neurons respond to hermaphrodite AWA 73 odorants? Figure 3.8 Male odr-10 expression is significantly reduced compared to that 75 of hermaphrodites Figure 3.9 ODR-10 expression is sex-specifically regulated: strong ODR-10 78 in hermaphrodites but weak ODR-10 in males Figure 3.10 Neural sex in AWA neurons regulates sexually dimorphic ODR-10 79 expression Figure 3.11 Neural sex generates sex difference in a target gene for sexually 84 different common sensory function Figure A1.1 AWA connectivity is similar between sexes 86
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 List of Symbols ANOVA Analysis of variance ATRX X-linked α thalassaemia/mental retardation syndrome AWA Amphid Wing type neuron A sense volatile attractants AWB Amphid Wing type neuron B sense repellents AWC Amphid Wing type neuron C sense volatile attractants bu 2-butanone (hermaphrodite AWC odorant) bz benzaldehyde (hermaphrodite AWC odorant) C. elegans Caenorhabditis elegans C.I. Chemotaxis Index CA Male-specific ventral cord motor neurons cAMP cyclic Adenosine MonoPhosphate CEM Male-specific cephalic sensory neurons CP Male-specific tail interneurons da diacetyl (hermaphrodite AWA odorant) DIC Differential Interference Contrast DM doublesex and mab-3 Ex Extrachromosomal array fem-3 FEMinization of XX and X0 animals
  • 15. xv 
 FMR1 fragile X mental retardation 1 GFP Green Fluorescence Protein glp-1 abnormal germlilne proliferation defective glr-1 glutamate receptor family (AMPA) H Hermaphrodite him-5 mutant strain for the high incidence of male progenies HSN Hermaphrodite-specific serotonergic motor neurons iaa isoamylalcohol (hermaphrodite AWC odorant) Is Integrated strain JARID1C Jumonji, AT rich interactive domain 1C M Male MECP2 methyl CpG binding protein 2 (Rett syndrome) odr odorant response abnormal OPI Olfactory Preference Index osm-5 OSMotic avoidance abnormal Pglr-1 promoter specific for interneurons, head motor neurons Podr-7 AWA neural specific promoter Posm-5 promoter specific for sensory neurons pkd-2 marks CEM neurons in the male head regions Prab-3 pan-neural promoter
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 PUFA Poly Unsaturated Fatty Acid py pyrazine (hermaphrodite AWA odorant) RFP Red Fluorescence Protein RnA Ray neuron type A RnB Ray neuron type B SEM Standard Errors of the Mean SNB-1 C. elegans Synaptobrevin srd-1 male specifically expressed 7-transmembrane receptor srj-54 male specifically expressed 7-transmembrane receptor tph-1 marks Herm.-specific HSNs, male-specific CP neurons tra transformer VC Hermaphrodite-specific cholinergic motor neurons
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 Chapter 1 Introduction 1 The general problem of sex differentiation in the nervous system Sex, brain, and behavior The sex of the brain has been historically discussed in terms of the relationships between sexual dimorphism in the hypothalamus, sex hormones, and sex behaviors [Levine, 1966]. Investigations in the past decade, however, have revealed abundant evidence that sex of the brain is not limited to the neural machinery of sexual activity. Rather, sex of the brain affects many different areas of brain and behaviors. Many areas of brain responsible for sensory processing, cognition, pain and stress response, and reward are sexually differentiated. Sex differences in a variety of behaviors common to both sexes, not related to reproductive behaviors, are well observed throughout the animal kingdom and in humans. Regarding these sex differences, the action of sex hormones during development and in adulthood has been thought to exclusively set up the neural substrates of sex differences in behavior. Any sex differences in a common behavior were suggested to result from sexual dimorphism in the structure of brain areas: significant sex differences in the size of nucleus of sub- cortical structures and/or in the connectivity of neurons (i.e., differences in cell numbers, thickness of cortical layers, numbers of spines, and electrophysiological properties). However, it has been revealed that anatomically common structures between sexes also give rise to sex differences in some behaviors and even in neurological diseases. Furthermore, some sexually different behaviors are not explained by sex hormones and recent studies reveal that cell-intrinsic sex differentiation is a significant regulator of the sex of the brain. Therefore, some behavioral sex differences may arise through sex differences in the molecular properties of common neural circuitry. A better understanding of these issues will shed light on how biological sex interacts with developmental control to impart plasticity to neural circuitry to bring about sex differences in behaviors.
  • 18. 
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 Sex differences are prominent in the neuroanatomy for sex-specific behaviors In mammals, reproductive behaviors are significantly different by sex. In some cases, neural substrates that may underlie these behaviors have been identified. For example, the rat hypothalamus was first described to have sexual dimorphism [Gorski et al., 1978]. The SDN-POA (sexually dimorphic nucleus of preoptic area) in the hypothalamus regulates male copulatory behaviors. It was revealed that lesions in the entire anterior preoptic area eliminate male courtship behavior and lesions restricted to SDN-POA significantly slow acquisition of male copulatory behavior. The volume of SDN-POA is about seven times larger in male rats than in female rats [Morris et al., 2004]. Another well-characterized sexually dimorphic area of the rat brain is the posterodorsal medial amygdala (MePd). This area receives pheromone stimuli and main olfactory inputs, which triggers male responses to female pheromones, male dominance social behavior, and female relations with litters. Lesions in MePd result in severe deficits in those behaviors. Consistent with obvious sex difference in the function of MePd, its volume is about 1.5 times larger in male rats than in female rats and its neurochemical characteristics are also sexually different [Cooke et al., 2005]. Together, SDN-POA and MePd are representative sexually dimorphic areas in the brain, offering good model systems to study the relationship between the brain sex and the sex-specific behaviors. Sex differences also occur in the areas of the brain not relevant to reproductive behavior Sex differences in the brain are not limited to areas dedicated to reproductive behavior. Sex differences also exist in many ‘cognitive’ regions such as hippocampus, amygdala, and neocortex [Juraska, 1991]. The hippocampus, the most well-known structure regulating learning and memory, is also sexually dimorphic in its structure and function [Juraska et al., 1985; McEwan et al, 2000]. Its normalized size compared to the whole brain is larger in women than in men. Specifically, the volume of the CA1 region, the number of pyramidal cells in CA1, and the neuronal
  • 19. 
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 density of the dentate gyrus are larger in males [Madeira et al., 1995]. The neurochemical systems within the hippocampus are also sexually different. Furthermore, the reactivity of hippocampus to stressful situations in both rats and humans are sexually different. In the amygdala of rat pups, the basomedial nucleus displays sexually different changes in serotonin receptor expressions upon separation from mother rodents [Ziabreva et al., 2003]. The amygdala of human brain also exhibits sex difference in the hemispheric lateralization of amygdala function for memory with emotional events [Cahill et al, 2004]. Together, these suggest that sexual dimorphisms in these ‘cognitive’ regions bring about sex differences even in behaviors common to both sexes. The mechanistic relationships between these anatomical and functional sex differences are largely unclear. Sex differences are observed even in common behaviors non-relevant to reproduction Recent findings reveal that sex differences in common behaviors are apparent between sexes. Behaviors fundamental to both sexes were different in mammalian model systems. Those behaviors encompass sensorimotor behaviors, hippocampal, striatal learning strategies, and drug-addiction [Dewing et al., 2006; Korol et al., 2004; Becker et al., 1999]. Furthermore, emotion, memory, vision, hearing, feeding, face processing, pain perception, navigation, and the effects of stress in the brain were also sexually differentiated in animals and humans. However, due to the complexity of the neural circuitry and the behavior itself, the mechanisms that give rise to sex differences even in common behaviors are not well understood. Sex-biases are prevalent in the nature and/or incidences of neurological diseases Clinical observations have reported that there are significant sex-biases in variety of aspects of diseases affecting the nervous system. These sex-biases affect the nature of disease, its incidence, and recovery ability [Cahill et al., 2006]. First, schizophrenia displays sex differences in both nature and incidences. For example,
  • 20. 
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 the morphology of the diseased brain areas is sexually different in that the normally sex-biased ratio of the size of amygdala to that of the orbitofrontal cortex is increased in men with psychosis but decreased in women with psychosis [Gur et al., 2004]. This disease occurs about 2.7 times more often in men than in women. Second, autism displays the extreme sex-bias in its incidences in that males get autism four times more often than females [Swaab et al., 2003]. Third, Parkinson’s disease (PD) reveals sex differences in its symptoms and drug responses. For example, male PD patients go through more serious rigidity than female PD patients and drug-related dyskinesias are more frequently observed in female PD patients [Brann et al., 2007]. These sex differences in the neurological diseases are suggested to result from sexual dimorphisms in the brain itself [Cahill, 2006; Wizemann et al., 2001; Swaab et al., 2003]. Substrates for sex-bias in brain diseases may be apparent sex differences in the structure and function of the affected brain regions. In addition, extensive sex differences in many neurotransmitter systems arise as important possible molecular substrates of sex-bias in neurological disorders. In particular, unipolar depression, which occurs more often in females than in males, may be understood better by studying the functional role of the higher mean rate of serotonin synthesis in males than in females [Nishizawa et al., 1997]. Sex differences in behavioral symptoms of some neurological disorders Certain neurological diseases such as mental retardation, neurodegenerative diseases, and neuropsychiatric disorders seriously impair a plethora of behavioral symptoms in humans. The affected behaviors in these diseases are influenced by gender, brining about sexually different behavioral symptoms in patients. For example, the APOE*E4 allele associated with increased risk of Alzheimer’s Disease (AD) is linked with significantly more serious memory disruption in female than in male AD patients [Fleisher et al., 2005]. In addition, the deficits in social behaviors of the valproic acid-induced rat autism model were similar to behavioral symptoms of autism patients and were present only in male rats [Schneider et al., 2008]. This
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 suggests that autism, with a strong (4:1) male-biased incidence, can at least in part be attributed to higher susceptibility of male behavioral substrates to disease risk factors. Therefore, investigations on how common behaviors between sexes are also sexually differentiated will improve our understanding of the pathology underlying behavioral symptoms of brain diseases and our chances of developing treatments. 2 Sex hormones and the sex of the brain Sex hormones regulate sex differences in the brain Scientists first demonstrated that testosterone masculinizes the brain by exposing female guinea pigs to testosterone in utero, which permanently hindered normal female reproductive behaviors in adulthood [Phoenix et al., 1959]. Organizing sexual differentiation of the brain by sex hormones during development was thought to be the major mechanism by which sex-specific behaviors are generated [Cooke et al., 1998]. In addition to the role of sex hormones during development, the activity of sex hormones in adulthood gives rise to sex-specific behaviors. For example, the treatment of the testosterone in the adult female canary brain at least temporarily generates male-specific courtship songs [Goldman et al., 1983]. Some sexually different behaviors are not explained by sex hormones The activity of circulating sex hormones, particularly testosterone and estrogen, has been thought to govern all known sexual differentiation of the brain and behaviors. However, findings over the past several decades have revealed that that is not the case. One major finding is that the rat dopaminergic neurons display sex differences even before sexual hormones are active [Reisert and Pilgrim, 1991]. In addition, many studies on sex differences in behaviors show that sex hormones cannot account for all those differences [Arnold, 2004]. For example, male aggression and female parental behaviors are also at least in part attributed to the role of sex chromosome
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 complement other than the testis-determining gene Sry on the Y chromosome [Gatewood et al., 2006]. Furthermore, social interactions are sexually differentiated by the sex chromosomal complement [McPhie-Lalmansingh, 2008]. 
 3 Chromosomal sex also control properties of neural structures and behaviors Sex differences in neurological diseases are not all explained by the activity of sex hormones Sex-biases in many aspects of brain diseases are thought to result from the activity of sex hormones during development and in adult. The simple notion was that sex hormones establish sexually different neuroanatomy and molecular substrates more susceptible to certain neurological diseases in one sex than in the other. Furthermore, sex hormones, especially estrogen, give rise to sex differences in post-injury brain damage and recovery. However, recent clinical reports reveal that sex differences exist in the developing brain, at times when the circulating sex hormonal activity is absent. For example, both post-ischemic brain damage and pattern of apoptosis were significantly different in two sexes of the postnatal day 7 rats at which sexual maturation is not established [Renolleau et al., 2008; Hurn et al., 2005; Edwards, 2004]. Thus, these findings highlight the importance of studying how the cell- intrinsic sex determination mechanisms give rise to sexual differentiation of the brain. Evidences of sex hormone-independent sexual differentiation in the vertebrate system Consistent with the insufficiency of sex hormonal activity to bring about sexual dimorphisms in the brain, behavior, and diseases, several lines of evidence reveal that the sex chromosomes themselves harbor information regulating neural sex differentiation and the generation of sexual dimorphisms. The ground-breaking finding of a rare gynandromorphic zebra finch that has two sex phenotypes throughout its body shows that sex chromosomal gene expression correlates with
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 sexually lateralized brain phenotype irrespective of the gonadal sex [Agate et al., 2003]. Furthermore, a series of experiments support the idea that the cell-intrinsic sex determination mechanisms control sexual differentiation in the brain in a variety of species [Arnold, 2004; Carruth et al., 2002; Dewing et al., 2006; Gahr et al., 2003]. In particular, Dewing et al., revealed that the Y chromosome-linked male- determining gene Sry may directly control the higher expression of tyrosine hydroxylase in dopaminergic neurons of the adult male substantia nigra system, imparting sex differences to the sensorimotor behaviors. Together, these studies reveal that cell-intrinsic sex regulators may directly regulate molecular sex differences to bring about behavioral sex differences. However, very little is known about the regulatory mechanisms that might link chromosomal sex to differences in neural development or function. Cell-intrinsic sex regulators generate sex-specific behaviors in invertebrate organisms Invertebrate systems have been an advantageous tool to study sex differences in neurobiology and behavior since their nervous systems and behaviors are relatively simple and tractable. Studies on sexual differentiation in the nervous system utilizing invertebrate systems, in particular, Drosophila, revealed how the cell-intrinsic mechanisms differentiate sex-specific neurons and generate sex-specific behaviors - i.e., behaviors present in one sex and unnecessary for the survival of the other sex - for reproduction. In Drosophila, a sex determination gene, fruitless (fru), has been shown to control male-specific behaviors by specifying masculine properties in multiple types of fly neurons, suggesting that fru might be a master regulator of male behavioral circuitry [Billeter et al., 2006; Manoli et al., 2006; Vrontou et al., 2006; Datta et al., 2008]. In addition, possibly in a smaller portion of male neurons, another sex determination gene, doublesex (dsx), also contributes to the development of masculine properties [Kimura et al., 2008; Rideout et al., 2007].
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 The pathway of chromosomal sex regulation on the properties of the neural circuit is largely unknown Altogether, in addition or in parallel to the sex hormonal control over all known sex differences in animals, the cell-intrinsic sex regulators are suggested to establish changes in the properties of the neural circuitry itself to generate sex differences in behaviors. However, due to the complexity of the neural circuitry for a given behavior, the lack of knowledge on downstream effectors of the sex chromosomes and target genes of those effectors, the specific mechanism through which the sex chromosomal signaling modifies the molecular/cellular/physiological properties of neural circuitry is not described. 
 4 Neural circuits and behaviors Some gene expression differences change behaviors Understanding how behaviors are generated by the neural circuitry is a major problem in neuroscience. In some cases, it has been suggested that behavior can be hard-wired into the corresponding neural circuitry. However, the surprising plasticity in behavior suggests that the neural circuitry can modify the behavior it generates to meet the needs of animals in survival and reproduction throughout generations. Consistent with this idea, studies in different systems have shown that some gene expression changes in neural circuitry give rise to differences in behaviors between sexes, species, and even in an individual animal. Sex-specific behaviors are generated by sexually different interpretations of the same sensory stimuli as a result of differences in gene expression Mouse pheromone responses require the vomeronasal organ (VNO) and the main olfactory epithelium (MOE). A recent study reveals that mouse TRPC2, an ion channel specific to the VNO, is responsible for sex-specific processing of the same
  • 25. 
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 sensory stimuli (mouse pheromones) to generate sex-specific responses according to the sex of the individual [Kimchi et al., 2007]. This study reveals that the same sensory stimuli trigger sexually different downstream signaling mediated by TRPC2 in the VNO sensory neurons to bring about activation of sex-specific behavior. In Drosophila, male flies execute male-specific courtship song mediated by the pattern generator. By light-activated channel expression in the motor circuits, song-like wing movement and sound were generated by both sexes. However, authentic male courtship song was only revealed in normal males and in females with expression of the male form of fruitless (fru, a master sex regulator of fly neurons). This indicates that fru sets up male characteristics to process the same stimuli in a male way and brings about the normal male singing [Clyne et al., 2008]. Together, these studies suggest that changes in gene expression in an otherwise common neural circuit between sexes can generate sexually different behaviors. Common behaviors between sexes or between species are modified by gene expression differences to confer sex difference or species difference Fiddler crabs display sexually different responses to food-related cues: the chemosensory neurons of male fiddler crabs are less sensitive to low concentrations of food cues than those of female fiddler crabs. Changes in the expression of cAMP signaling between male and female crabs underlie generation of sex differences in food-sensation [Weissburg et al., 2001]. In rodents, different expression levels of the oxytocin receptor (OTR) and vasopressin 1a receptor (V1aR) in the ventral forebrain reward circuitry results in species differences in social behaviors. High levels of these molecules contribute to monogamous social behavior of the prairie vole whereas low levels of them generate polygamous behavior of the montane and meadow voles [Hammock et al., 2006]. Therefore, these studies reveal that gene expression changes bring about modification even in common behaviors between sexes or between species. Since common behaviors are presumably generated by the same neural circuitry, these findings surprisingly suggest that the activity or
  • 26. 
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 physiology of the appropriate neural circuit for these behaviors may be made different by changes in gene expression. Furthermore, these findings indicate that the extent of plasticity in neural circuitry to bring about differences in behaviors must be quite broad. A complete diagram of the neural circuit for any complex behavior is generally not described Growing evidence, as described above, reveal that discrete changes in gene expression modify the properties of neural circuitry to generate sex differences or species differences in behaviors. However, due to the limited knowledge on the neural circuit sufficient or necessary for any given behavior, elucidation of the tight control of changes in molecular components on behaviors is challenging in vertebrate systems. By utilizing relatively simple model systems, this problem could be alleviated and understanding the relationship of gene, neural circuit, and behavior can be expedited. 
 5 C. elegans as a system to study sex differences in shared behaviors C. elegans is an ideal model for neuroscience and for studying sexual dimorphism in the nervous system C. elegans has a relatively simple nervous system, harboring about 300 neurons. The nervous system (Figure 1.3) mediates a plethora of behaviors that can be categorized into two classes: one, shared behaviors fundamental to both sexes mediated by the “core” nervous system (neural circuitry comprising common neurons between sexes) and the other, sex-specific behaviors for reproduction generated by sex-specific neural circuitry. With sophisticated genetic tools and the complete neuronal wiring diagram for adults of one sex (hermaphrodites), C. elegans provides a unique opportunity to study how neural circuits generate behavior at the resolution of single genes and single neurons.
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 The cell-intrinsic sex determination pathway regulates all known somatic sex differences in C. elegans C. elegans has two sexes: XX hermaphrodite and X0 male (Figure 1.1). Hermaphrodites are essentially somatic females except it can self-fertilize in the absence of males. As in most animals, C. elegans sex determination depends on sex chromosomes: XX or X0. However, in contrast to the vertebrate sex determination in which the early gonad primes tissues to adopt sex-specific characteristics through the influence of gonadal steroids [Morris et al., 2004], most sex differences in C. elegans extragonadal tissues do not depend on gonads. This has been experimentally demonstrated by laser ablation of early gonadal primordium cells, which had no effect on sex-specific somatic development [Kimble, 1981; Klass et al., 1976]. Furthermore, C. elegans does not have such sex hormones (e.g., estrogen) produced by its gonad, although the presence of potential gonad signaling is reported [Lipton et al., 2004; Kleemann et al., 2008]. C. elegans somatic sex determination mostly relies on the sexual state of each cell (Figure 1.2). By measuring the X chromosome dosage, an inhibitory genetic cascade converges onto the terminal sex regulator gene, tra-1 (transformer 1). tra-1 then directs sexual differentiation throughout the soma. Elegant genetic studies on the tra-1 null mutant revealed that tra-1 acts cell-autonomously in the specification of nearly all sexually dimorphic cell fates in the C. elegans soma [Hunter and Wood, 1990]. This indicates that TRA-1 activity imparts a sexual identity to each cell, either hermaphrodite (TRA-1 ON) or male (TRA-1 OFF) sexual fate. However, the downstream effectors of TRA-1 are not completely known. The C. elegans core neural circuitry has molecular sex difference In contrast to the conspicuous sex-specific neurons in each sex, it has been thought that the core nervous system possesses only minor, ultrastructural sex differences in connectivity. However, two recent findings revealed that the core nervous system is
  • 28. 
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 also sexually differentiated at the molecular level (Figure 1.4). First, srd-1, a seven transmembrane receptor, is specifically expressed in the male ADF sensory neuron pair [Troemel et al., 1995]. Second, srj-54, a seven transmembrane receptor, is specifically expressed in the male AIM interneurons [D.S.P., unpublished data]. These ADF and AIM neurons are present in both sexes - i.e., both neurons are part of the core nervous system - and these genes exist in both sexes, however, gene expression was observed specifically in males. Despite their unknown functional significance, sex-specific gene expression in core neurons may give rise to sexual modifications in behavior since gene expression differences can result in functional differences. Together, these findings indicate that the core nervous system is also sexually differentiated at the molecular level. These molecular substrates of sexual dimorphism in the C. elegans core nervous system prompted us to initiate this investigation on potential sex difference in a shared sensory behavior, olfaction since chemosensation/olfaction may be affected by gene expression differences in neurons. Sex differences in the C. elegans core neural function The core nervous system mediates a variety of behaviors displayed by both hermaphrodites and males. In spite of sexually differentiated non-reproductive behaviors in the vertebrate systems, such as learning and memory, addiction, and stress responses, sex differences in the C. elegans shared behaviors of two sexes have not been understood well. Recently, some learning and memory functions of C. elegans, mediated by core neurons, have been reported to be sexually different [Vellai et al., 2006]. Furthermore, it has been found that the core nervous system mediates sexually different locomotion [W. Mowrey, unpublished data]. These results, along with molecular evidence of sex differences, suggest that other C. elegans core neural functions may be also sexually differentiated.
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 Figure 1.1. C. elegans Sexes C. elegans has two sexes: XX hermaphrodite and X0 male. An adult hermaphrodite and adult male is depicted above. In C. elegans, the hermaphrodite is essentially a female except for its ability to self-fertilize. In adults, the two sexes have significant sex differences in body size, tail morphology, and gonad structure. The adult hermaphrodite is larger, has a whip-like, tapered tail tip, a vulva, and a two-lobed gonad. The adult male is relatively smaller, has a rounded tail tip with several classes of specialized sensilla, and a one-armed gonad (Portman, 2007).
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 Figure 1.2. Somatic sex determination in C. elegans All known C. elegans sex differences are derived from the action of the cell-intrinsic sex determination pathway. The pathway originates from the ratio of sex chromosomes to autosomes signaled by the signal-element genes on X chromosomes and A autosomes (Rhind et al., 1995). Downstream of the sdc genes, dosage compensation (not shown) reduces gene expression from the X chromosomes in half and is controlled independently from the differentiation of somatic characteristics (Meyer, 2005). A repressive genetic cascade that ultimately regulates the global sex- determining gene tra-1 controls all known somatic sex characteristics. In XX animals, tra-1 is active, repressing male differentiation and promoting hermaphrodite differentiation. In X0 animals, tra-1 is inactive, allowing male differentiation.
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 Figure 1.3. The C. elegans nervous system The adult hermaphrodite (above) has 8 hermaphrodite-specific neurons (red): 6 VC neurons in the ventral cord and 2 HSN motor neurons. These neurons regulate hermaphrodite egg-laying behavior. The adult male (below) has 89 male-specific neurons (blue). The 4 CEM neurons, located in the head region contribute to male pheromone response (White et al., 2007). The CA and CP motor neurons in the male ventral code are implicated in specific steps of male mating behavior (Loer and Kenyon, 1993; Schindelman et al., 2006). The male tail contains a variety of sensory (RnA and RnB), motor, and interneurons. The sex-specific neurons overlay onto the 294 “core” neurons (green), common to both sexes. These core neurons generate common behavior between sexes.
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 Figure 1.4. Sexually dimorphic gene expressions in the C. elegans core nervous system (A) srd-1 and (B) srj-54, two seven transmembrane receptors, are male-specifically expressed in the (A) ADF sensory neurons (Troemel et al., 1995), (B) AIM interneurons (Portman, unpublished data) of the core nervous system, respectively.
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 6 C. elegans olfactory behaviors The hermaphrodite olfactory system is well characterized in its structure and function To address potential sex differences in the core nervous system, we systematically approached this question by studying behaviors (Figure 1.5). Among the many behaviors mediated by the core nervous system, we chose to study C. elegans olfactory behaviors, which are relatively simple, easily tractable, and well characterized in hermaphrodites. C. elegans must encounter thousands of chemicals in the wild environment (soil) and must be able to sort out massive information to find food sources, to locate mating partners, and to avoid harmful toxicants. As a result, worms execute attraction and repulsion behaviors in response to different sensory cues. In C. elegans, chemotaxis to volatile attractant sources is referred to as olfactory behavior and is mediated by the core nervous system. All data on the structure and function of C. elegans olfactory system are based on hermaphrodites [Bargmann et al., 1993]. In contrast, male olfaction has never been examined. The olfactory neuronal circuit (Figure 1.6) is composed of a set of sensory (olfactory) neurons, layers of interneurons, and motor neurons. These neurons are common between male and hermaphrodite. The connectivity between these neurons is completely identified in hermaphrodites. In addition, the characterization of the male neuronal wiring diagram is under way [S. Emmons, Albert Einstein College of Medicine; http://worms.aecom.yu.edu/pages/all%20male%20neurons.htm]. The olfactory sensory neurons (Figure 1.6) are AWA, AWB, and AWC, located in the amphid of head region. Each of these neurons is left and right paired, finger-shaped, contains branched ciliary endings, is located under the sheath cells, and is indirectly exposed to the environment [Bargmann et al., 1993; Bargmann, 2006]. AWA and AWC neurons sense attractive cues and AWB neurons detect aversive molecules. Olfactory neurons synapse onto intervening layers of interneurons, which are in turn connected to motor neurons. Through complex activity in the olfactory neural circuit,
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 C. elegans navigates a chemical gradient using temporal comparisons of encountered concentrations of chemical cues [Pierce-Shimomura et al., 1999; Dusenbery et al., 1980; Miller et al., 2005]. As with vertebrate and fly olfaction, C. elegans olfaction also conveys information through G-protein coupled receptor signaling [Colosimo et al., 2004; Komatsu et al., 1999; Troemel et al., 1995]. However, in contrast to vertebrate neurons that sense thousands of chemicals through thousands of olfactory neurons, each of them responding to single odorant, C. elegans can detect thousands of odorants with a few olfactory neurons. Only one chemical has been linked to its cognate receptor and the ligands for other receptors are as yet unknown. The only one de-orphanized odorant receptor in C. elegans is the diacetyl receptor, ODR-10 [Sengupta et al., 1996]. As in other chemosensory systems, interaction of an odorant with its cognate receptor is predicted to either activate or inhibit synaptic output of a chemosensory neuron [Wakabayashi et al., 2004; Gray, 2005; Chalasani et al., 2007].
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 Figure 1.5. Does the C. elegans core nervous system mediate sexually dimorphic behaviors? The C. elegans core nervous system may also be sexually differentiated and generate sexually different core neural functions.
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 Figure 1.6. The C. elegans olfactory neural circuit is a part of the core nervous system Shown is a simplified neuronal wiring cartoon based on information from WormAtlas (www. wormatlas.org). Olfactory sensory neurons (AWC, AWA, and AWB), layers of interneurons (AIY, AIZ, and RIA), and head motor neurons (SMD, RMD) consist of the olfactory neural circuit, which is a part of the core nervous system. Both sexes have these neuronal components. However, all known characteristics of structure and function of the olfactory system are based on the hermaphrodite characterization. Hermaphrodite AWA and AWC neurons sense volatile attractants: AWA (da, py and others) and AWC (bu, bz and others). Hermaphrodite AWB neurons respond to aversive stimuli.
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 Olfactory neural circuits possess molecular properties for behavioral plasticity The C. elegans chemosensory system is highly developed to mediate recognition, discrimination, and adaptation to chemical cues. Moreover, like sensory behaviors in other higher organisms, C. elegans can also modulate sensory behaviors according to its internal and external state such as memory of past experience, feeding state, and contextual cues. Furthermore, some genes involved in food/odor learning behaviors have been identified [Ishihara et al., 2002; Remy and Hobert, 2005]. In particular, cGMP and PKC signaling acts as a behavioral switch in a single neuron (AWC) to switch between odor preference and avoidance [Tsunozaki et al., 2008]. This behavioral switch is suggested as a mechanism underlying AWC olfactory sensitization/adaptation and provides an example of how changes in the molecular properties of a neuron can give rise to modification of behavior. Together with the fact that some chemosensory receptors are involved in the sensation of internal state modulating behaviors, modification of neural circuit function by gene expression changes may be an important property of behavioral plasticity in C. elegans.
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 Chapter 2 Neural sex modifies the function of a C. elegans sensory circuit 1 Introduction In spite of the importance of studying sex difference in the brain, it is not understood how chromosomal sex generates complex sex differences in the neural circuit and behavior. The relatively simple and well-characterized C. elegans nervous system and tractable innate behaviors make it feasible and interesting to study this complex issue. C. elegans has two sexes: XX hermaphrodites and X0 males. The cell- intrinsic sex determination pathway determines the sexual state of each somatic cell [Hodgkin, 1987; Hodgkin and Brenner, 1977; Hunter and Wood, 1990]. In the pathway, the TRA-1 (a terminal sex regulator) activity determines either hermaphrodite or male fate of a given cell. The C. elegans nervous system harbors extensive sexual dimorphism, including the structurally and functionally well- characterized sex-specific nervous system. The sex-specific nervous system is composed of distinct sets of sex-specific neurons, which mediate sex-specific behaviors for reproduction. This sex-specific nervous system connects to the “core” nervous system comprised of 294 common neurons between sexes. The core nervous system has been thought to exhibit only a few ultrastructural level sex differences. However, recent findings on two male-specific gene expressions in core neurons reveal that the core nervous system is also sexually differentiated in its molecular properties. Although the functional significance of these molecular sex differences in the core neurons is not understood, these could contribute to sex differences in neural function. Therefore, the core nervous system may also be sexually differentiated in its functions imparting sex differences to shared behaviors between the two sexes in C. elegans. To ask how core neural functions may differ between sexes, we choose to examine C. elegans olfactory behavior, a function of the core neural circuitry. Because male olfaction has never been carefully examined, we began characterizing male responses to simple canonical odorants for comparison to the well-characterized
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 hermaphrodite olfaction. My results reveal that the “neural sex”, the sexual state of a given neuron established by cell-intrinsic sex determination, determines the sexual phenotype of olfaction. Each sex revealed distinct and characteristic olfaction giving rise to significant sex differences. This suggests that neural sex establishes sexually different properties in core neural circuitry to bring about sex differences in a shared sensory behavior. 2 Materials and Methods Nematode Genetics, Strains, and Transgenes C. elegans cultures were grown on nematode growth medium (NGM) plates seeded with E. coli OP50 as described [Brenner et al., 1974]. him-5 (high incidence of males) was used as our lab stock for stable and abundant generation of male progenies in self-fertilizing population by him-5 hermaphrodites. him-5(e1490) mutation increases the chance of meiotic disjunction of chromosomes leading to the higher ratio of spontaneous male progenies in a given population. In all my experiments, him-5 genetic background was used except in strains containing tra-1. him-5 is noted as the wild-type group on figures and in the text. The following mutant alleles were used: tra-1(e1099) III, pha-1(e2123ts) III, him-5(e1490) V, lin-15(n765) X, and ceh-30(n4289) X. tra-1 XX pseudomales were obtained from the self progeny of tra-1(e1099)/pha-1(e2123) hermaphrodites. Sex-transformation constructs were generated using the GatewayTM cloning kit (Invitrogen). EG4391 and EG4392 strains containing the Prab-3::fem- 3(+)_mCherry::unc-54 3’UTR transgenes oxEx862 and oxEx863 were generously provided by J. White and E. Jorgensen [White et al., 2007]. To make Posm-5::fem- 3(+)_mCherry::unc-54 3’UTR and Pglr-1::fem-3(+)_mCherry::unc-54 3’UTR, we polymerase chain reaction (PCR) amplified the osm-5 [Qin et al., 2001; Haycraft et al., 2001] and glr-1 [Zheng et al., 1999] promoters and made 4-1 Entry clones for use in the Multisite Gateway System as described [White et al., 2007].
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 Extrachromosomal arrays were generated by the coinjection of the fem-3(+) construct at 50–75ng/ml with a coelomocyte::GFP marker (75ng/ml). UR226, UR227 strains containing the transgenes (fsEx160, fsEx161) and UR224, UR225 strains containing the transgenes (fsEx158, fsEx159) were cordially generated by D.A. Mason. The expression pattern of each transgene was verified by the observation of fluorescence from mCherry, encoded by the distal open reading frame (ORF) in these operon-based constructs. In behavioral experiments on worms with these transgenes, only those showing clear mCherry expression were assayed. The following transgenes were used for the marker-gene expression studies (fsIs6[srj-54::YFP + cc::GFP], bxIs14[pkd-2::GFP + pBX1], oxEx862[Prab-3::fem- 3(+)::mCherry + pkd-2::GFP + lin-15(+)], and zdIs13[tph-1::GFP]) (Figure 2.9). Behavioral assays Single odorant assay The single odorant assay (Figure 2.1) is essentially the same as the classical chemotaxis assay [Bargmann et al., 1993]. It measures worms’ responses to a single odorant diluted in 100% EtOH (1ul) placed on the left spot, 0.5cm apart from the edge of the assay plate. Opposite to the odorant spot, 100% EtOH as a control was placed on the right spot, 0.5cm apart from the edge of the plate. 100% EtOH is neutral to worms. It was used for a control spot to distinguish the specific response of worms to a single odorant presented simultaneously. To paralyze worms that get to the odorant source, 1 ul of 0.3 M NaN3 was placed on both EtOH and odorant spot. Populations of single sex worms were placed on the 2% agar assay plate without food and after 45 minutes the number of worms at both spots was counted for the quantification of olfactory behaviors. For quantification, the Chemotaxis Index (C.I.) which equals to (B-A)/Total was used. A and B represent the number of worms at odorant spots. The C.I. varies from +1 (complete attraction) to -1 (complete repulsion) [Bargmann et al., 1993]. An average of 50 animals were subjected to each
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 Figure 2.1. Single odorant assay “Single odorant assay” is essentially the same as the classical chemotaxis assay (Bargmann et al., 1993)
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 assay. Four to twelve assays of each sex at each dilution of an odorant were performed. For statistical significance, we have used a two-sample Student’s t-test assuming equal variances between sexes of each odorant at each dilution. To remove any possible effect of age variance in olfactory behavior, we used age-synchronized cultures for all behavior assays otherwise it is noted. This was carried out by synchronized egg-laying rather than hypochlorite treatment to avoid any potential side effects of larval starvation. Briefly, 20 gravid hermaphrodites were allowed to lay eggs on a seeded plate for 2 h and were then removed. The resulting progeny matured in a relatively synchronous manner. To avoid any potential influence of interaction between sexes onto olfaction, in all assays, animals were sex- segregated as L4 larvae, before male mating structures have not yet developed, and transferred to single-sex “holding plates” overnight before behavioral assays. Olfactory preference assays (Figure 2.2) The olfactory preference assay is done with basically the same set up and procedure as the single odorant assay [Chapter 2.1 Materials and Methods] except that another volatile attractant is placed on the left spot instead of 100% EtOH. Both odorants used for this assay are diluted into 100% EtOH and a 1/100 dilution of each odorant was used in all my olfactory preference assays. I quantified behaviors of single sex populations of worms in this assay using an Olfactory Preference Index (OPI), defined as (b-a)/(a+b), where a and b represent the numbers of animals migrating to odorants A and B, respectively. The OPI can vary from -1 (indicating a complete preference for odorant A) to +1 (a complete preference for odorant B). An OPI of 0 indicates that equal numbers of animals migrate to each of the two spots. Behaviors of wild-type animals were analyzed by one way analysis of variance (ANOVA) with equal variance with Bonferroni post- hoc test.
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 Figure 2.2. Olfactory preference assay (A) The olfactory preference assay (Lee and Portman, 2007) is a modification of the single-odorant assay in which the control spot (“A”) is replaced with a second attractive odorant. All attractants were diluted to 1/100. A sex-segregated population of worms is placed 1 cm below the center of the plate. After 45 min, the number of animals within 2 cm of each spot is counted and used to calculate the olfactory preference index (OPI). This assay eliminates any potential confounding effects of other sexually different behaviors (e.g., movement rate or mating drive) or overall sensitivity on olfactory response, as it measures the relative difference in attraction to two different odorants. (B) OPI can range from -1 (strong preference for odorant A) and to +1 (strong preference for odorant B). OPI of +1 means more worms get to the odorant B spot than to the A spot. OPI of 0 indicates similar preference of both odorants as the assay plate reveals approximately equal number of worms gets to both spots.
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 Larvae cultures and assays I have used synchronized cultures set up from different time points to get simultaneous populations of L3, L4, and adult animals. L3 (36-40 hr), L4 (44-48 hr), and adult (~72 hr after laid egg) were used. L4 animals were separate-sexed and grown as adult for adult experimental group. To test statistical significance between sexes of each developmental stage, I used the Student t-test. Laser ablation and assays I followed a standard protocol to ablate gonad precursor cells [Bargmann et al., 1995]. Laser ablations were performed on gonad primordium cells (Z1, Z4) and germline precursor cells (Z2, Z3) in early larval stage 1 (L1) animals. Operated animals were rescued, grown up to larval stage 4 (L4) and separated by sex. Animals that did not undergo laser ablation (mock) were also separated by sex at L4 and assayed as adults. Animals were scored as responding to a particular odorant if, after 30 min on the assay plate, their distance from that odorant source was less than 40% of the distance to the other odorant source. (This constraint traces an arc around each odorant source, the radius of which varies from ~2.5 cm at the plate’s equator to ~2.8 cm at its edge.) Data from assays of laser-ablated animals were nonparametric and analyzed by logistic regression. Sexual mosaics Because hermaphrodites carrying Prab-3::fem-3(+)::mCherry::unc-54::3’UTR (oxEx862 and oxEx863) and Posm-5::fem-3(+)::mCherry::unc-54::3’UTR transgenes (fsEx160 and fsEx161) laid very late-stage eggs, these animals were manually staged as mid-L4s. In all assays, animals were sex-segregated as L4 larvae and transferred to single-sex “holding plates” overnight before behavioral assays. In behavioral experiments on worms with these transgenes, only those showing clear mCherry expression (or in the case of oxEx862 and oxEx863, the rescue of the lin-15 Muv (Multi-vulva) phenotype) were assayed. Comparisons of the behavior of wild-type
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 and transgenic animals were carried out with two way analysis of variance (ANOVA) with Bonferroni post-hoc tests. Statistical Analyses For all behavior assays, weighted means and standard errors of the mean (SEMs) were calculated with Stata 9 (StataCorp LP [College Station, TX]). I used the total number of worms in each single odorant assay or the number of responders in each olfactory preference assay to weigh the mean and SEM. Comparisons of the behavior of wild-type single sex population and mutant or other groups, depending on the experimental design, were carried out with one way or two way analysis of variance (ANOVA) with Bonferroni post-hoc tests. 3 Results C. elegans exhibit significant sex difference in olfactory behaviors Though hermaphrodite olfaction to a variety of odorants has been well characterized [Bargmann et al., 1993; Ward et al., 1975; Ware et al., 1975], male olfactory responses have never been systematically examined. To explore C. elegans male olfaction, we compared the responses of adults of each sex to four volatile attractants of hermaphrodites: diacetyl (da), benzaldehyde (bz), pyrazine (py), and 2-butanone (bu). I used a single odorant assay, previously described as the chemotaxis assay, for measuring olfactory behaviors to a single volatile attractant [Bargmann et al., 1993]. I have tested three serial dilutions of each odorant noted on the X axis. The response measured as the Chemotaxis Index (C.I.) is displayed as columns of each chart for each odorant. I examined male responses to three serial dilutions of all four volatile attractants of hermaphrodites. Male responses were all attractive (C.I. > 0) revealing that male olfactory responses are similar overall to the previously described
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 hermaphrodite olfactory responses [Bargmann et al., 1993]. However, male responses, in many cases, were significantly lower than those of hermaphrodites (Figure 2.3). Specifically for da (an AWA odorant in hermaphrodites) and bz (AWC), male responses were significantly reduced compared to hermaphrodites’ at all dilutions of both odorants except at bz 1/1000. In responses to py (AWA) and bu (AWC), male responses were similar to hermaphrodites at all dilutions of both odorants except at py 1/100 and bu 1/100. Together, these reveal that male responses to some, but not all, olfactory attractants are lower than hermaphrodite responses. Moreover, this suggests that C. elegans has odorant- and concentration-specific sex differences in olfaction. Each sex displays distinct and characteristic olfactory preferences To define sex differences in C. elegans olfaction more specifically, we have developed an olfactory preference assay in which two different odorants were simultaneously presented to single-sex population. Two attractants were placed on the opposing sides of an assay plate without food (Figure 2.2). Migration to one odorant source or the other should depend on an animal’s relative preference for each odorant. If male olfactory function is simply less efficient than that of hermaphrodites, males would be expected to distribute themselves among the two odorant spots in the same relative number as hermaphrodites do. If, however, there are more specific sex differences in olfactory behavior, males and hermaphrodites might exhibit differences in their relative attraction to the two odorants. Using the olfactory preference assay, we examined the behavior of wild-type adult hermaphrodites and males to four pairwise combinations of hermaphrodite attractants. I used 1/100 dilutions of each odorant, as this concentration usually results in peak responses under single-odorant conditions (Figure 2.3) [Bargmann et al., 1993]. Additionally, the response to each of these odorants at this concentration is known to be mediated predominantly by a hermaphrodite single sensory neuron
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 Figure 2.3. Male olfaction is significantly different to hermaphrodite olfaction (Lee and Portman, 2007) Young adult hermaphrodites (red) and males (blue) were assayed in sex-segregated populations in single odorant assays (Bargmann et al., 1993). Three dilutions of the odorants da and py (sensed in hermaphrodites by AWA) and bz and bu (sensed in hermaphrodites by AWC) were tested. Each data point represents the weighted mean of 4 to 12 assays each containing ~50 animals. Error bars show the weighted SEM. The statistical significance of sex differences in CI was determined using Student’s t- test. In all figures, statistical significance is indicated with asterisks as follows: ***, p < 0.001; **, p < 0.01 *, p < 0.05.
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 pair: hermaphrodite AWA neurons for da and py, hermaphrodite AWC neurons for bz and bu [Bargmann et al., 1993; Chou et al., 2001; Sengupta et al., 1994]. I first examined the responses of animals to opposing pairs of attractants sensed by two different sensory neurons of hermaphrodites (Figure 2.4). For both pairs tested, da-bz and py-bz, we found significant and robust sex differences in olfactory preference behavior. In the da-bz assay, males strongly preferred da although hermaphrodites displayed approximately equal preference for both odorants. This sex difference in OPIda-bz was statistically significant. Sex differences in the py- bz assay were also statistically significant; in this case hermaphrodites preferred bz while males preferred py. A model in which male olfactory responses are simply less efficient than those of hermaphrodites cannot easily account for these results. Instead, these pronounced disparities in OPI between males and hermaphrodites indicate that there are qualitative sex differences in C. elegans olfactory function. I also found significant sex differences in olfactory responses to two odorants sensed by the same sensory neuronal pair (Figure 2.4). In the da-py assay, in which both odorants are sensed primarily by hermaphrodite AWA neurons, hermaphrodites demonstrated a strong preference for da, while males showed a similarly strong preference for py. Again, this difference was statistically significant. I also observed sex differences in the bu-bz assay, in which both odorants are sensed by the hermaphrodite AWC neuron, and observed less pronounced sex difference. In another combination of hermaphrodite AWC odorants, bu and iaa (isoamylalcohol), male and hermaphrodite olfaction was significantly different (Figure 2.4). These results suggest that sex differences in olfactory behaviors can arise from olfactory signaling via a single sensory neuron pair and raise the possibility that sexual differentiation may influence the properties of sensory neurons themselves.
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 Figure 2.4. Each sex has distinct and characteristic olfactory preferences (A) The OPI of adult hermaphrodites (open red circles) and males (closed blue circles) is shown for each of the four odorant pairs (Lee and Portman, 2007) and the fifth odorant pair indicated at the left and right side of each cart. Four volatile attractants of hermaphrodites: diacetyl (da), benzaldehyde (bz), pyrazine (py), and 2- butanone (bu) were paired in various combinations for these olfactory preference assays. Each point represents the weighted mean of at least 7 olfactory preference assays each containing ~50 animals. Error bars indicate weighted SEM. The significance of sex differences in OPI to each odorant pair was determined using Student’s t-test.
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 Sexually different olfaction is not the secondary effect of male-specific behaviors To investigate how these highly characteristic and distinct sex differences in a shared sensory function of C. elegans come about, we tested the role of sex-specific behaviors and sex-specific structures. Sexually different olfactory preference might have resulted from the secondary effect of previously described sex-specific behaviors, particularly male- specific mating behaviors. My typical observations on male behaviors in a male only population were that males attempt to mate with neighboring worms when they ran into each other regardless of the sex of the neighbor. Together, male mating behaviors somehow might impart sex differences to olfaction. To examine the potential contribution of male-specific behaviors to male olfactory preference, we compared male behaviors in single-sex population, individuals, and mixed-sex population (Figure 2.5A). First, we tested the role of male mating behaviors. The single sex population behaviors were the control to which we compared the individual behaviors. In isolation, the mating behaviors should be less frequent than in population with neighbors since individual worm’s mechanosensation-mediated mating attempt is suppressed. Male mating attempt could be suppressed at least in part in isolation. I found that males in isolation exhibit the same olfactory preferences as males in male population, indicating that male olfactory preference is not a secondary effect of the male mating behavior. Second, the role of male mate-searching behaviors was examined by comparing male behaviors in male only population to that in mixed-sex population. Male mate- searching drive is a male-specific behavior in that males leave the food area, the bacterial lawn on the culture dish, in the absence of hermaphrodites. If male mate- searching drive is the primary contribution to sexual difference in olfactory preference, male behaviors in a mixed-sex population should be different from male behaviors in a male-only population.
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 Figure 2.5. Sexually different olfactory preferences are generated neither by sex- specific behaviors nor by structures (Lee and Portman, 2007) (A) Data are shown for animals assayed in standard single-sex population assays, mixed-sex population assays, and animals assayed individually. ceh-30 mutant males were assayed in standard, single sex population assays. Each data point represents the weighted mean and weighted SEM of at least ten assays, each containing ~50 animals, except for ceh-30, which represents six assays of ~40 animals each. Statistical significance was determined by ANOVA with Bonferroni post-hoc tests. Each experimental group revealed sex differences (***). ceh-30 males and wild-type males are significantly different (***). (B) Mock-ablated, germline and gonad (Z1-Z4)-ablated, and germline (Z2, Z3)- ablated animals were tested individually for da-py olfactory preference behavior as young adults. Behavioral responses were determined using a modified olfactory preference assay as described in Experimental Procedures. Each point represents odorant-preference behavior of single animal (open red circle, hermaphrodite; closed blue circle, male). Vertical bars indicate the median response of each group of animals. Logistic regression was used to determine the statistical significance of the sex difference in behavior in each of the three groups.
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 However, in the mixed-sex population, male behaviors were still significantly different from hermaphrodite behaviors. This indicates that male olfactory preference is not a secondary effect of the male mate-searching drive. Therefore, sexually different olfactory preferences might be attributed to specific modification in the machinery for olfactory behaviors rather than to an influence from the machinery for male-specific behaviors. The male-specific CEM neurons do not have a primary role in the sexually different shared sensory function Sex-specific neurons may contribute to sex differences in olfactory preferences through direct and/or indirect input to the core neurons. The male-specific CEM sensory neurons are located in the head region where the olfactory neural circuit resides. CEM neurons regulate male-specific responses to hermaphrodite conditioned medium, which contains hermaphrodite pheromone [White et al., 2007]. Therefore, I postulated that male-specific CEM neurons might be responsible for generating male olfactory preference. To address this, ceh-30 mutant males, in which all four CEM neurons are absent, were subjected to the da-py olfactory preference assay. Intriguingly, ceh-30 males behaved as the wild-type males indicating that male- specific CEM neurons are not required for the sexually different olfactory preference behavior (Figure 2.5A). This stands in contrast to the contribution of male-specific CEM neurons to the male-specific hermaphrodite pheromone response and suggests that the core nervous system itself may generate sex difference in a shared behavior, olfactory preference. Gonad signaling is not necessary for sex difference in olfaction It has been suggested that gonad signaling exists and regulates sex-specific behaviors in C. elegans, such as male mate-searching behaviors [Lipton et al., 2004; Kleemann et al., 2008]. I, therefore, asked whether gonad signaling influences olfactory preference. To address this idea, I first tested behaviors of glp-1 mutants, which lack
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 a germline. I found that adult glp-1 hermaphrodites have similar olfactory preference as wild-type hermaphrodites (data not shown). To directly test the role of gonad and germline, we generated worms without both gonad and germline or without germline only by laser ablation and tested their behaviors in the da-py olfactory preference assay. Adult worms without both gonad and germline or without germline maintained intact sex differences in their olfactory preference (Figure 2.5B). Therefore, gonad signaling is not necessary for sex difference in olfactory preference at least in the da-py assay and it is consistent with the possibility that the core nervous system itself imparts sexual dimorphism to olfactory preference. Sexual differences in olfaction are prominent before sexual maturation C. elegans life span (Figure 2.6B) begins with embryonic development and encompasses four larval stages and adulthood. Late in larval development, significant changes to the nervous system take place in both sexes. In L4 hermaphrodites, the egg-laying system matures [Schafer, 2006; Bany et al., 2003]; in L4 males, sensory circuits necessary for mating develop in the tail, neurons are added to the ventral cord, and the CEM head neurons undergo maturation [Sulston and Horvitz, 1977]. In addition, extensive gonad maturation occurs in both sexes at L4 stage [Kimble, 2005]. I therefore asked whether the appearance of sex differences in olfactory preference behavior coincided with any of these sex-specific developmental events. To address this, we compared the olfactory preferences of larvae to those of adults (Figure 2.6A). Since extensive sexual differentiation occurs at the late larval developmental stages, we asked whether the olfactory preference of larval stage 3 (L3) and larval stage 4 (L4) worms might be similar or different from that of adults. In the py-bz assay, both L3 and L4 larvae showed the same general trend in olfactory preference behavior exhibited by adults: males preferred py to bz more strongly than hermaphrodites did. This difference was marginally significant in L4
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 Figure 2.6. Sex differences in olfaction precede sex-specific differentiation L3, L4 and young adult animals of both sexes were tested in the py-bz (A, Left) and da-py (A, Right) assays. Hermaphrodite behavior is shown with open red circles; male behavior with closed blue circles. Each data point represents the weighted mean OPI and standard error of at least 10 assays each containing roughly 50 animals. The significance of sex differences in OPI at each developmental stage was determined using Student’s t-test and is indicated with asterisks: ***, p < 0.001; (*), p = 0.059. (B) Major sexual differentiation events in both sexes are noted including sexually different olfaction.
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 animals (p = 0.059); however, it was not significant in L3 animals. Additionally, L4 animals of both sexes showed a temporary but marked positive shift in OPIpy-bz, the significance of which is unclear. In the da-py assay, developmental changes were also apparent. Both L3 and L4 animals showed significant sex differences in OPIda-py, indicating that clear sex differences are present well before the majority of the sex- specific nervous system develops. Interestingly, hermaphrodites, but not males, undergo a significant change in da-py preference behavior as they mature from L4s to adults, such that the magnitude of the sex difference in OPIda-py is much greater in adults than in larvae. This suggests that an alteration of hermaphrodite behavior coinciding with maturation of the reproductive system accounts for the full extent of sex differences in adult da-py preference. Together, these data demonstrate that developmental changes in larvae influence sex differences in olfaction, but that some sex differences in behavior clearly precede the maturation of sex-specific neurons indicating that the core nervous system possesses properties for sexually different olfaction even before construction of the sex-specific nervous system. Neural sex determines the sex phenotype of a common sensory function, olfactory preference All known somatic sex differences in C. elegans are regulated by the terminal sex regulator gene, tra-1. tra-1 loss in XX hermaphrodites results in development of tra- 1 pseudomales, which are virtually identical to wild-type X0 males. Since tra-1 regulates sex-specific development in the C. elegans soma, we postulated that tra-1 also controls sex differences in olfactory preference. In both da-py and py-bz assays, the behavior of tra-1 XX pseudomales was statistically indistinguishable from that of the wild-type X0 males (Figure 2.7A). tra-1 XX pseudomales also exhibited male- like responses in da, py single odorant assays (Figure 2.7B). Therefore, tra-1 also determines the sex phenotype of a shared behavior, olfactory preference. Furthermore, it confirms that the cell-intrinsic sex determination pathway controls sex differences in both structure and function of the nervous system.
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 Next, we explored directly whether the sexual state of the core nervous system governs sex differences in olfactory preferences. If it does, switching the sexual state of the nervous system should reverse the sex phenotype of an olfactory behavior. Studies on both naturally occurring gynandromorph animals and artificial gynandromorph animals have shown that behavioral sex is determined by the sexual state of neural substrates for the behavior [Agate et al., 2003; Hall et al., 1977; Weissburg et al., 2001]. To investigate directly whether the sexual state of neurons regulates sexually different behaviors, we reprogrammed the sexual state of neurons by taking advantage of the cell-intrinsic sex determination in C. elegans. By generation of sexual mosaics – i.e., animals possessing neurons of the opposite sex in the body of one sex – we could test if changing in sexual state of certain neurons also changes the sex phenotype of its neural functions (Figure 2.8B). To reprogram neural sex, we modified the tra-1 activity by manipulation of the somatic sex determination pathway. In this pathway, FEM-3, a ubiquitin-ligase cofactor, degrades TRA-1 and promotes male development. Overexpression of FEM- 3 is sufficient to masculinize XX animals [Mehra et al., 1999]. By overexpression of FEM-3 specifically in the nervous system using a pan-neural promoter (Figure 2.8A), Prab-3, we switched the sexual state of the nervous system from hermaphrodite to male. To confirm that sex-transformation on the core neurons was successful, we asked if male-specific gene expression in core neurons came on in the hermaphrodites with masculinized neurons. The male-specific expression of a putative seven transmembrane receptor, srj-54 in the AIM core interneuron of wild-type animals was used as a reporter for the sex-transformation of core neurons (Figure 1.4). I first crossed the srj-54::yfp reporter strain with the strain carrying masculinizing transgene (Prab-3::fem-3) and found that the male-specific srj-54::yfp expression came on in the AIM interneuron of hermaphrodites by the Prab-3::fem-3 transgene activity (Figure 2.9). This indicates that the core nervous system is sexually transformed by the masculinizing transgene even at the gene expression level.
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 Figure 2.7. The terminal sex regulator, tra-1, controls sex difference in olfactory preference (Lee and Portman, 2007) (A) tra-1(e1099) XX pseudomales were assayed for py-bz and da-py olfactory preference. In both cases, the loss of tra-1 function led to complete masculinization of the behavior of XX animals. (B) Wild-type animals and tra-1 XX pseudomales were tested for attraction to da and py at 1/100 with standard single odorant assays. Each point represents the weighted mean and weighted SEM of (A) at least eleven assays each containing ~56 animals, (B) at least four assays each containing ~50 animals. tra-1 XX pseudomales displayed significant olfactory attraction to both odorants, similar olfaction as wild-type males. Statistical significance was determined by ANOVA with Bonferroni post-hoc tests.
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 Figure 2.8. Sex-transformation of the nervous system (Lee and Portman, 2007) (A) A simplified diagram of the genetic cascade of hermaphrodite and male sex determination: In XX animals, tra-2 represses fem-3 and tra-1 is allowed to promote hermaphrodite development. In X0 animals, fem-3 represses tra-1 and male differentiation is initiated. (B) Two sexual mosaics: (above) a hermaphrodite with masculinized nervous system by overexpression of a masculinizing gene, fem-3(+) driven by the pan-neural promoter Prab-3 and (below) a male with feminized nervous system by overexpression of a feminizing gene, tra-2(ic) (tra-2 intracellular domain) driven by Prab-3.
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 I additionally checked if the pan-neural masculinizing transgene could generate male-specific neurons in hermaphrodites. As pkd-2::gfp reporter of male- specific neurons was not revealed in hermaphrodites carrying the pan-neural masculinizing transgene (Figure 2.9), we, therefore, concluded that masculinizing the nervous system is sufficient to switch the sexual state of the core neurons only. Male- specific neurons were not generated by the masculinizing transgene (Prab-3::fem-3) due to the later expression of the transgene than the sex determination of sexually dimorphic cell lineages [Portman, 2007]. This technique ensured me to examine the sole role of the core neurons in sexually different-shared behaviors and to exclude potential role of the sex-specific neurons. Pan-neural sex-transformation Using these sexual mosaics, hermaphrodites with masculinized neurons and males with feminized neurons, we looked for sex reversal in olfactory preference due to the sex-transformation on core neurons. In the da-py assay, the response of hermaphrodites with masculinized neurons was statistically different from the response of the wild-type hermaphrodites (Figure 2.10). Unlike the wild-type hermaphrodites, hermaphrodites from two different lines strongly preferred py, similar to the behavior of wild-type males. Thus, switching the neural sex is sufficient to transform the sexual phenotype of behavior. This indicates that the sexual state of the core neural circuit represents the sex phenotype of olfactory preference. Sex-transformation on sensory, interneuron and motor neuron I have identified the core neurons as the generator of sex differences in olfactory preference. Among various types of neurons in the core neural circuit, we have selectively sex-transformed most of the sensory neurons or subsets of interneurons and motorneurons. To target neuronal subtypes for sex-transformation, we have used cell-type specific promoters: for sensory neurons, we have used the Posm-5 [Qin et
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 Figure 2.9. Neural sex-transformation (Lee and Portman, 2007) (A) Whole-animal views of young adults showing a wild-type hermaphrodite, an oxEx862 hermaphrodite, and a wild-type male. The transgenic hermaphrodite has a normal soma but retains extra eggs (bracketed area), indicating that the masculinization of the nervous system disrupts egg-laying behavior. A similar phenotype was seen in the Posm-5::fem-3(+) hermaphrodites (data not shown), indicating that this phenotype might stem from defects in hermaphrodite-specific sensory control of egg laying. (B) Roughly half of oxEx862 adult hermaphrodites express srj-54::GFP in the head AIM neuron (arrowhead). This expression is never seen in wild-type hermaphrodites but is observed in nearly all wild-type adult males. (C) The pkd-2::GFP transgene marks the CEM neurons in the head of adult males
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 (arrowheads). Expression is only very rarely observed in wild-type or oxEx862 hermaphrodites, indicating that oxEx862 does not result in the generation of CEM neurons in hermaphrodites. (D) In the male tail, pkd-2::GFP is expressed in the male-specific RnB and HOB neurons (bracket). No expression was observed in the tails of wild-type or oxEx862 hermaphrodites, indicating that these male-specific neurons do not form in oxEx862 hermaphrodites. (E) tph-1::GFP is expressed in the hermaphrodite-specific neuron HSN in wild-type and oxEx862 hermaphrodites (arrowheads). Interestingly, tph-1::GFP expression is usually reduced in the HSNs of oxEx862 hermaphrodites, and the HSN neurons in these animals are sometimes mispositioned (data not shown), indicating that fem-3(+) expression might disrupt HSN differentiation. I also sometimes observed tph-1::GFP expression in two cells flanking the vulva in oxEx862 hermaphrodites; this might indicate that the hermaphrodite-specific cells VC4 and VC5 can adopt a CP-like fate in these animals. In adult males, tph-1::GFP marks these male-specific CP ventral cord neurons. Four of the CP neurons (open arrowheads) are visible in this view of a wild-type adult male.
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 Figure 2.10. Neural sex determines the sex phenotype of olfactory preference Animals carrying fem-3(+) overexpression transgene were assayed in the da-py olfactory preference assay. Two different transgenic lines are shown. Prab-3::fem- 3(+) (oxEx862 and oxEx863) expresses fem-3(+) throughout the nervous system. Each point represents the weighted mean and standard error of at least 6 assays with an average of 56 animals per assay (wild-type), 25 animals per assay (Prab-3::fem- 3(+)). Statistical significance was determined by ANOVA with Bonferroni post-hoc tests.
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 al., 2001; Haycraft et al., 2001] and for many interneurons (and a small set of head motor neurons), we have used Pglr-1 [Maricq et al., 1995]. In the da-py assay, the response of hermaphrodites with masculinized sensory neurons was statistically different from the response of the wild-type hermaphrodites (Figure 2.11). Unlike the wild-type hermaphrodites, hermaphrodites from two different lines strongly preferred py, similar to the behavior of the wild-type males in the da-py assay. This indicates that the sensory neural circuit itself is sufficient to bring about sexually different olfactory preference. Furthermore, it suggests that the sensory neural circuit must have sexually different properties giving rise to two different forms of the shared sensory function. In contrast, the response of hermaphrodites with masculinized glr-1- expressing cells was not different from the response of the wild-type hermaphrodites (Figure 2.11). As the wild-type hermaphrodites, hermaphrodites from two different lines strongly preferred da in the da-py assay. This indicates that the properties of glr-1-expressing interneurons and motor neurons are not modified by neural sex. 4 Discussion Why are there sex differences in C. elegans olfaction? I have found for the first time that there is a great deal of sex difference in C. elegans olfactory behaviors. Unlike sex-specific chemosensory behaviors utilized for the reproduction, the olfactory behaviors we characterized here are exhibited by both sexes. In the sense that behaviors are naturally selected and evolve according to beneficiary effects, the sex difference in olfaction we observed may have adaptive value in worm survival. One possible reason for this sex difference in olfaction is males’ necessity for finding mates. In spite of incomplete identification of the hermaphrodite pheromone components, canonical attractants we have examined are
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 Figure 2.11. Sexually different properties of the sensory neurons control the sex phenotype of olfactory preference Animals carrying three different fem-3(+) overexpression transgenes were assayed in the da-py olfactory preference assay. Two different transgenic lines are shown for each construct. Prab-3::fem-3(+) (oxEx862 and oxEx863) expresses fem-3(+) throughout the nervous system, Posm-5::fem-3(+) (fsEx160 and fsEx161) expresses fem-3(+) in ~60 sensory neurons (Qin et al., 2001; Haycraft et al., 2001), and Pglr- 1::fem-3(+) (fsEx158 and fsEx159) expresses fem-3(+) in a large set of interneurons and motor neurons (Maricq et al., 1995). Each point represents the weighted mean and standard error of at least 6 assays with an average of 56 animals per assay (wild-type), 25 animals per assay (Prab-3::fem- 3(+)), 43 animals per assay (Posm-5::fem-3(+)), and 38 animals per assay (Pglr- 1::fem-3(+)). Statistical significance was determined by ANOVA with Bonferroni post-hoc tests.
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 not thought to be pheromones in C. elegans. Rather, it is more probable that sex difference in the necessity of nutrition results in sex difference in olfactory behaviors. Together with the fact that many olfactory attractants are bacterial metabolites, we prefer this speculation that C. elegans olfaction is sex-specifically modified for biased nutrition necessity. Males may need more carbohydrates for their high motility and hermaphrodites may need more proteins for their large volume of oocyte cytoplasms. For most insects, adult food preferences were dependent on their nutrition necessity for the reproduction [Cornelius et al., 2000]. Sex differences in non mating-related chemosensory behaviors were also revealed in Fiddler crabs (Uca spp.). Their feeding behaviors were sexually different in that male fiddler crabs confine their feeding in a more food rich regions and display less sensitivity to blends of food-related chemicals than females having higher sensitivity to low concentrations of foods [Weissburg and Derby, 1995]. It is suggested that these sex differences may also arise from sex difference in the necessity of sufficient food. Therefore, it is a property of evolution of animal behaviors to adopt discrete modification even in a common behavior between sexes for the advantage of each sex. Developmental regulation of sexually different olfactory behaviors Sexually different olfactory preference was found in developing worms in which no sex-specific neural circuitry and gonads were established (Figure 2.6). This shows that the core neural circuitry itself rather than the sex-specific systems generates sexually different olfaction. However, the diverging hermaphrodite olfactory preference during the development suggests some influence of the sex-specific systems on the olfactory preference. The single odorant responses of L4 hermaphrodites to da 1/100 are significantly smaller than that of adult hermaphrodites (data not shown). L4 males however do not have any difference from adult males in their single odorant response to da 1/100 (data not shown). Consistent with the diverging responses of hermaphrodite responses in the da-py assay throughout the L3,
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 L4, and adult stages (Figure 2.6A, Right), the single odorant response of L4 animals suggest that some non cell-autonomous influences from the development of sex- specific structures on the core neural circuitry may also contribute to the complete extent of sexually different olfaction in the da-py assay. Furthermore, in the bz-py assay, the marked positive shift in the larvae OPI (Figure 2.6A, Left) may be either revealing unstable olfaction at the sexually differentiating developmental stages or displaying variability in the larvae olfaction more prominently in the py-bz assay than in other assays. Altogether, sexually different olfactory preferences seem to be certainly controlled throughout development. Analysis of the behavior of animals with temporal manipulation of the sex determinants will give a better understanding of how sex and development factors coordinate together to establish the complete set of sex differences in a sensory behavior. How does sex modify olfaction? Opposing the dogma that sexual hormones regulate all known sex differences in the structure and function of the nervous system, my findings reveal that the cell-intrinsic sex regulators establish sexually different properties in the common neurons between sexes. In particular, sensory neurons are the generator of sexually different olfactory preference. Sensory neurons themselves should produce sexually different neural output giving rise to actual display of sex difference in behavior. Assuming the connectivity of olfactory neurons to others is the same between sexes, we suggest that sensory neurons may send sexually different neurotransmission to the postsynaptic neurons. The neurotransmission may be either qualitatively and/or quantitatively different between sexes. As the chemosensory neurons of fiddler crabs display sexually different neural response by modulation of the same cAMP-signaling pathway [Weissburg, 2001], sensory neurons in C. elegans may also convey two different outputs by differential regulation on the same signaling components. At one extreme, however, qualitative and/or quantitative sex difference in critical signaling molecules may bring about behavioral sex difference. Since the sufficiency of
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 sensory neurons for sexually different responses were revealed in the da-py assay in which AWA odorants are tested, it is likely that AWA neurons itself possess sex differences in substrates such as a critical AWA signaling component: da receptor ODR-10. Although sensory neurons were sufficient for determining the sex phenotype of olfactory preference, other neurons may also be required to display the complete extent of sex difference in olfactory preference. Two lines of hermaphrodites with masculinized sensory neurons display intermediate OPIs between the OPI of wild-type hermaphrodites and males (Figure 2.11). It is possible that other subsets of core neurons together with sensory neurons may bring about the complete sex difference in olfactory preference. Major postsynaptic neurons of olfactory sensory neurons (AWA and AWC) are AIY and AIZ interneurons. Despite no contribution of glr-1-expressing neurons, interneurons and head neurons in connectivity to olfactory sensory neurons may contribute to sexual difference in olfaction. I suggest that sex-transforming both olfactory neurons and the AIY and AIZ interneurons might fully reverse the sex phenotype of olfactory preference. Alternatively, male-specific sensory neurons in addition to core sensory neurons may be required for the full extent of sex difference in olfactory preference. Since the sensory neural masculinizing transgene in the hermaphrodites does not establish male-specific sensory neurons, these sensory neural masculinized hermaphrodites could have displayed the incomplete sex-switch in behaviors (Figure 2.11). Neural Sex regulation on Behaviors Together with recent work in Drosophila [Billeter et al., 2006; Manoli et al., 2006; Vrontou et al., 2006; Certel et al., 2007; Datta et al, 2008], my work emphasizes advantages of utilizing the invertebrate systems to elucidate the mechanisms by which neural sex establishes sexual dimorphisms in the nervous system. I investigate how neural sex generates sexually different-shared behaviors mediated by the common neural circuit and this study is distinct from studies on sex-specific neurons and behaviors. My findings reveal that the cell-intrinsic sex determinants establish
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 sex difference in the properties of the core nervous system and generate sexually different-shared behaviors. Together with recent evidences that the sex chromosomal signaling determines sex of the brain, independently from sex hormones [Arnold et al., 2004; Dewing et al., 2006], my findings may shed light on understanding how brain is modified by neural sex to accommodate substrates for sex-biases in the nature and incidences of neurological diseases. Last, studying sex differences in the common neurons will help unravel the mechanisms by which neural circuitry is plastic and brings about a variety of behaviors that enhance animals’ survival and adaptation to the environment. 
 
 
 
 
 
 
 
 
 
 
 
 
 

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 Chapter 3 Neural sex modifies the properties of a single sensory neuron to generate sex differences in olfactory behaviors 5 Introduction Understanding the neurobiological substrates of behavior is one of the main problems in neuroscience. Due to the complexity of the nervous system, it has been challenging to elucidate mechanisms that innate behaviors, prevalent and conserved across species, are generated. Sex differences in behaviors have provided a unique opportunity to study how behaviors are encoded in neural circuitry. Most studies of sex differences in behavior have focused on sex-specific behaviors, mostly for the reproduction, present in one sex but not in the other. Extensive studies in C. elegnas, Drosophila, and mouse have shown that sex-specific neural circuits generate sex- specific behaviors [Portman, 2007; Villella and Hall, 2008]. The relatively simple C. elegans nervous system allows me to directly address how common neural circuit is modified to impart sex differences to a shared sensory behavior. The absence of sex hormones and sex differences even in common behaviors [Lee and Portman, 2007] are the advantages for utilizing this system to explore the cell-intrinsic sex regulator, properties of common neural circuit, and shared behavior. I previously revealed that neural sex, the sexual state of a given neuron established by cell-intrinsic sex determination, determines sex phenotype of a shared sensory behavior. Furthermore, sensory neurons are the sufficient core neurons for sex differences in olfaction [Lee and Portman, 2007]. However, the mechanisms that neural sex modifies the properties of sensory neurons to generate sexually different olfaction were not understood. A pair of left and right AWA olfactory neurons has its cell bodies positioned in the amphid and its long dendrites project to the tip of nose at which branched ciliated endings reside [Bargmann et al., 1993]. In hermaphrodites, the AWA neurons sense volatile odorants: diacetyl (da), pyrazine (py), and 2,4,5-
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 trimethylthiazole (tmt). Responses to these canonical attractants generally peak at the 1/100 dilution of each odorant. The identity of AWA neurons is specified by AWA- exclusive expression of ODR-7, the only member of the large divergent nuclear receptor family, which activates the expression of AWA-specific signaling genes to establish AWA function [Colosimo et al., 2003]. Hermaphrodites carrying the null mutation odr-7(ky4) fail to express AWA-specific signaling genes [Sengupta et al., 1996] and fail to respond to all odorants sensed by the AWA neurons [Sengupta et al., 1994] despite intact AWA neural morphology. odr-7 controls expression of the single ligand-identified odorant receptor, odr-10 (the diacetyl receptor), in AWA neurons. Here, I examine how neural sex generates sexually different olfactory behaviors and reveal that neural sex modifies target gene expression in the AWA neuron to bring about sexually different olfactory behaviors. 2 Materials and Methods Nematode Genetics, Strains, and Transgenes C. elegans strains were cultured as described in Chapter 2. Sex-transformation constructs were generated by the GatewayTM cloning kit from Invitrogen. To make Prab-3::tra-2(ic)_mCherry::unc-54 3’UTR and Posm- 5::tra-2(ic)_mCherry::unc-54_3’UTR, I polymerase chain reaction (PCR) amplified the rab-3 [Nonet et al., 1997] and the osm-5 [Qin et al., 2001; Haycraft et al., 2001] promoters and made 4-1 Entry clones for use in the Multisite Gateway System as described [Hartley et al., 2000]. Extrachromosomal arrays were generated by the coinjection of the tra-2(ic) (75ng/ul) construct with a coelomocyte::GFP marker (75ng/ul) into him-5. UR249, UR250 strains containing the Prab-3::tra-2(ic)_mCherry::unc-54
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 3’UTR transgenes (fsEx187, fsEx188) and UR247, UR248 strains containing the Posm-5::tra-2(ic)_mCherry::unc-54_3’UTR transgenes (fsEx185, fsEx186) were generated by W. Mowrey. To make Podr-7::fem-3(+)_mCherry::unc-54 3’UTR and Podr-7::tra- 2(ic)_mCherry::unc-54 3’UTR, I polymerase chain reaction (PCR) amplified the odr- 7 [Sengupta et al., 1994, Colosimo et al., 2003] promoter and made 4-1 Entry clones for use in the Multisite Gateway System as described [Hartley et al., 2000]. Extrachromosomal arrays were generated by the coinjection of the fem-3(+) construct (75ng/ml) with a coelomocyte::GFP marker (75ng/ul). UR245, UR246 strains containing the Podr-7::fem-3(+)_mCherry::unc-54 3’UTR transgenes (fsEx202, fsEx203) were generated by myself and D.A. Mason, respectively. UR462, UR463 containing the Podr-7::tra-2(ic)_mCherry::unc-54 3’UTR transgenes (fsEx204, fsEx205) were generated by W. Mowrey. The expression pattern of each transgene was verified by the observation of fluorescence from mCherry, encoded by the distal open reading frame (ORF) in these operon- based constructs. In behavioral experiments on worms with these transgenes, only those showing clear mCherry expression were assayed. UR223 (odr-7 (ky4); him-5), UR463 (odr-10 (ky32); him-5), UR458 (kyIs37; him-5), UR460 (kyIs53; him-5) and UR488 (kyIs53; him-5; fsEx204[Podr-7::tra- 2(ic)_mCherry::unc-54 3’UTR]) were generated by crosses, utilizing the following mutant strains obtained from the Caenorhabditis Genetics Center: CX4 (ky4), CX32 (ky32), CX3260 (kyIs37), and CX3344 (kyIs53). The following reporters were used for the odr-10 expression studies: transcriptional reporter (kyIs37[Podr-10::GFP]) and translational reporter (kyIs53[Podr-10::ODR-10::GFP]). The pENTRY[SNB-1_eGFP] construct containing the sequence of SNB-1 and eGFP translational fusion was generated by performing the B-P recombination
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 reaction utilizing the GatewayTM cloning kit from Invitrogen (W. Mowrey). Podr- 7::SNB-1_eGFP::unc-54 3’UTR, an expression clone encoding SNB-1 only in AWA neurons, was generated by performing the multi-site gateway L-R recombination reaction. To locate AWA neurons relevant to adjacent neurons, I selected the AIY neural-specific reporter strain OH1098 carrying otIs133[Pttx-3::rfp]. The fsEx201[Podr-7:: SNB-1_eGFP::unc-54 3’UTR] was generated in the OH1098 strain by W. Mowrey. To examine male expression of SNB-1 compared to hermaphrodites, the transgenic animals were crossed with fsIs14; him-5 and obtained the UR459 (otIs133; him-5; fsEx201). Behavioral assays Single odorant assays and olfactory preference assays were carried out as described in Chapter 2. 3 Results Sexually different olfaction arise through neural sex modification on sensory neurons In chapter 2, I demonstrated that the masculinization of hermaphrodite sensory neurons is sufficient to generate masculinized sensory function, namely male olfactory preference in hermaphrodites [Lee and Portman, 2007]. To more thoroughly examine the role of neural sex in sexually different sensory function, I asked if the feminization of male neurons could feminize male olfactory preference. To feminize all neurons, I overexpressed TRA-2(ic), the constituitively active intracellular domain of TRA-2, in the nervous system using the pan-neural promoter Prab-3 [Nonet et al., 1997]. In the sex determination pathway, TRA-2 inhibits FEM- 3 allowing the activation of TRA-1 to result in hermaphrodite development (Figure 2.8). Expression of TRA-2(ic), the predicted intracellular carboxy-terminal domain
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 of TRA-2A, can feminize X0 males [Kuwabara and Kimble 1995]. Utilizing this technique, I could generate sexual mosaic worms in which the male nervous system is feminized. This provides mosaics that are complementary to hermaphrodites with the masculinized nervous system [Lee and Portman, 2007]. I then examined whether change in the sexual state of neurons reverses the sex phenotype of neural function. Two lines of males with feminized nervous system revealed hermaphrodite-like olfactory preference in da-py assay (Figure 3.1). This indicates that feminization of all neurons in males is sufficient to generate hermaphrodite olfactory preference. Furthermore, sexual properties in the neural circuitry are plastic enough to be modified in both masculinization and feminization directions to generate sexually different neural function. Together, this confirms that neural sex governs sex difference in olfactory preference. To feminize only sensory neurons, I overexpressed TRA-2(ic) in the ~60 sensory neurons driven by a sensory neural promoter, Posm-5. osm-5 encodes cilia protein, which regulates cilia development and osm-5 is expressed in all ciliary neurons including both core neurons and male-specific neurons in the male tail [Qin et al., 2001; Haycraft et al., 2001]. Utilizing this technique, I could generate males with the feminized sensory neurons, in addition to hermaphrodites with masculinized sensory neurons [Lee and Portman, 2007]. Then I examined whether change in the sexual state of sensory neurons reverses the sex phenotype of sensory function. Two lines of males with feminized sensory neurons behaved significantly different from wild-type males and rather revealed similar response as wild-type hermaphrodite olfactory preference in da-py assay (Figure 3.2). This indicates that the sexual state of sensory neurons determines sex phenotype of olfactory preference in da-py assay. Since the osm-5 promoter affects even male-specific sensory neurons in addition to common sensory neurons, this result supports the idea that the male-specific sensory neurons are not required for the olfactory behavioral sex. Together with the sufficiency of masculinized sensory neurons for male olfactory preference [Lee and
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 Figure 3.1. Neural sex governs sex difference in olfactory preference Animals carrying tra-2(ic) overexpression transgene were assayed in the da-py olfactory preference assay. Two different transgenic lines are shown. Prab-3::tra- 2(ic) (fsEx187 and fsEx188) expresses tra-2(ic) throughout the nervous system. Each point represents the weighted mean and standard error of at least 4 assays with an average of 55 animals per assay (wild-type), or 30 animals per assay (Prab-3::tra- 2(ic)). Statistical significance was determined by ANOVA with Bonferroni post-hoc tests.
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 Figure 3.2. A neural sex mechanism modifies properties of the sensory neurons to bring about sexually different olfactory preference Animals carrying tra-2(ic) overexpression transgene were assayed in the da-py olfactory preference assay. Two different transgenic lines are shown for each construct. Posm-5::tra-2(ic) (fsEx185 and fsEx186) expresses tra-2(ic) in all sensory neurons (Qin et al., 2001; Haycraft et al., 2001). Each point represents the weighted mean and standard error of at least 3 assays with an average of 46 animals per assay (wild-type), or 29 animals per assay (Posm-5::tra- 2(ic)). Statistical significance was determined by ANOVA with Bonferroni post-hoc tests.
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 Portman, 2007], this indicates that the neural sex mechanism modify properties of the sensory neural circuit to bring about sexually different olfactory preference. A single sensory neuron, AWA, generates sexually different olfactory preference Since both da and py odorants are sensed by hermaphrodite AWA olfactory neurons and sensory neurons are sufficient to mediate sexually different AWA-mediated olfactory preference, I speculated that AWA neurons themselves may contribute to sexually different olfactory preference. To explore if AWA olfactory neurons possess sexual dimorphism, I tested olfaction in animals in which only AWA was sex- reversed. To switch the sexual state of AWA neurons, I overexpressed sex factors utilizing the AWA-specific promoter Podr-7 [Sengupta et al., 1994]. odr-7 is a member of the nuclear receptor family that regulates the AWA neural fate. The timing of the odr-7 promoter activity, therefore, must be early enough for modification of the sexual state of AWA neurons. I found that hermaphrodites with masculinized AWA neurons behave significantly different from the wild-type hermaphrodites (Figure 3.3A). Two lines of hermaphrodites with masculinized AWA neurons strongly preferred py as the wild-type males in da-py assay. This indicates that masculinization of AWA neurons is sufficient to bring about male olfactory preference. It is fascinating that a single sensory neuron can determine behavioral sex of the AWA-mediated olfactory preference. Conversely, I examined olfactory preference of males with feminized AWA neurons. I found that two lines of males with feminized AWA neurons exhibit wild-type hermaphrodite-like olfactory preference (Figure 3.3B). This strengthens the idea that neural sex of AWA neurons is sufficient to generate sexually different olfactory preference at least in da-py (AWA/AWA) olfactory preference assay. Altogether, this indicates that neural sex regulates the molecular properties of AWA neurons to bring about sexually different olfactory preference. I speculate that masculinization or feminization of the molecular properties of the AWA neurons must be the basis of the sexually different olfactory preference in
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 Figure 3.3. The sexual state of a single sensory neuron, AWA, is sufficient to impart sex differences in olfactory preference (A) Animals carrying fem-3(+) overexpression transgene and (B) tra-2(ic) overexpression transgene were assayed in the da-py olfactory preference assay. Two different transgenic lines are shown for each construct. (A) Podr-7::fem-3(+) (fsEx202 and fsEx203) expresses fem-3(+), (B) Podr-7::tra-2(ic) (fsEx204 and fsEx205) expresses tra-2(ic) only in AWA single neurons. Each point represents the weighted mean and standard error of (A) at least 6 assays with an average of 36 animals per assay (wild-type), or 9 animals per assay (Prab- 3::fem-3(+)), (B) 4 assays with an average of 25 animals per assay (wild-type), or 17 animals per assay (Prab-3::tra-2(ic)). Statistical significance was determined by ANOVA with Bonferroni post-hoc tests. (C) In my model, neural sex, defined as cell-autonomous signals originated from sex chromosomes in the neuron itself, feminizes or masculinizes the AWA neuron, modifies the function of the olfactory circuit, and generates sex differences in olfactory preference.
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 response to AWA odorants (Figure 3.3C). The potential mechanisms underlying sexual differentiation of AWA neurons by sex determinants may be through modification on AWA olfactory signaling molecules, synaptic changes, and/or6 differential activity of the circuit (Figure 3.4). Neural sex regulation on sexually different olfaction is a property of the C. elegans olfactory circuit Neural sex generates sexually different olfactory preference in response to simultaneous presentation of AWA odorants. The extent to which neural sex establishes sex differences in neural functions is not fully understood. In other words, I asked if neural sex controls sexually different olfactory preference in response to other combinations of odorants. I therefore tested sex-transformed animals in response to two AWC odorants, bu and iaa. By spontaneous chromosomal integration of an extrachromosomal array, oxEx862[Prab-3::fem-3_mCherry::unc-54 3’UTR], I obtained fsIs15[Prab-3::fem-3_mCherry::unc-54 3’UTR]; him-5 animals. I first tested fsIs15; him-5 in the da-py assay in which both odorants are detected by AWA neurons and confirmed that the integrated masculinizing gene works as the transgene (Figure 3.5A). Then I explored whether sexually different olfactory preference is revealed in another olfactory preference assay. Among the different combinations of odorants sensed by AWC neurons, I selected bu-iaa assay in which the most sex difference between wild-type male and hermaphrodite was observed (Figure 2.4). Hermaphrodites with pan-neural masculinized neurons (fsIs15; him-5 Herm.) behaved as wild-type males with marginal statistical difference (p-value = 0.059) (Figure 3.5B). This indicates that neural sex is sufficient to determine sexually different olfactory preference in the bu-iaa assay in which both odorants are detected by AWC. Therefore, neural sex regulation of behavior is not a specific property of AWA neurons. Furthermore, this suggests that neural sex control of core neural functions is a property of C. elegans sexual differentiation.
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 Figure 3.4. How AWA neurons are modified by neural sex? ODR-10 is the only receptor with an identified ligand, the AWA-sensed odorant diacetyl (da). SNB-1 (synaptobrevin) is an integral protein of the synaptic vesicle and marks the pre-synapse. In addition to odorant receptor and synaptic organization, AWA signaling components could also be targets of neural sex.
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 Figure 3.5. Neural sex regulates sex differences in both AWA and AWC olfactory preference behaviors Animals with the integrated transgene fsIs15 (Prab-3::fem-3(+)) were tested in the da-py (AWA/AWA) assay (A) and in the bu-iaa (AWC/AWC) assay (B) (Top). Animals carrying the fem-3(+) overexpression transgene: Podr-3::fem-3(+) (fsEx206) in a small number of neurons including AWC neurons were assayed in the bu-iaa (AWC/AWC) assay (B) (Bottom). Each point represents the weighted mean and standard error of (A) 5 assays with an average of 40 animals per assay (wild-type), or 37 animals per assay (fsIs15), (B) (Top) 13 assays with an average of 42 animals per assay (wild-type), or 40 animals per assay (fsIs15) (Bottom) 4 assays with an average of 22 animals per assay (wild- type), or 16 animals per assay (Podr-3::fem-3(+)). Statistical significance was determined by ANOVA with Bonferroni post-hoc tests.
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 Since both bu and iaa odorants are sensed by hermaphrodite AWC olfactory neurons, I speculated that AWC neurons themselves may contribute to sexually different olfactory preference. To explore if AWA olfactory neurons possess sex differences that generate sexually different preferences in the bu-iaa (AWC/AWC) assay, I tested the olfaction of animals in which AWC was sex-reversed. To switch the sexual state of the AWC neurons, I overexpressed the masculinizing gene fem-3(+) utilizing the Podr-3 expressed majorly in AWC neurons [Roayaie et al., 1998]. odr-3 encodes a Gi/Go-like Ga protein that regulates at least in part of the AWC neural fate. I found that hermaphrodites with masculinized AWC neurons display similar behavior as the wild-type hermaphrodites (Figure 3.5B). This indicates that masculinization of AWC neurons is not sufficient to bring about male olfactory preference. Together with the sufficiency of pan-neural masculinization to masculinize behaviors in the bu-iaa (AWC/AWC) assay, this suggests that core neurons other than AWC and/or non-cell autonomous influence from the sex-specific structures are responsible for sexually different olfactory preference in response to AWC-odorants. Neural sex modifies a target gene in AWA neurons to bring about sex difference in olfaction Although I know that AWA neurons are sufficient for sex-specific AWA-mediated olfactory preference and AWA neurons must have sexual dimorphism, I do not know how AWA neurons are sexually different. Therefore, I investigated how AWA neurons are sexually different by examining how male AWA neurons respond to hermaphrodite AWA neurons. I already know that males also display attraction behaviors to the hermaphrodite AWA odorants (Figure 2.3). However, how males sense those odorants have never been carefully examined. To ask whether male AWA neurons also detect da and py, as they do in hermaphrodites, I used odr-7 (ky4) mutants in which AWA function is absent despite intact AWA morphology. odr-7 hermaphrodites fail to respond to da and py [Sengupta et al., 1994]. If male AWA
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 neurons also sense da and py, odr-7 males should also fail to respond to da and py as odr-7 hermaphrodites do. I tested male and hermaphrodite responses to each of py 1/100, da 1/100 in single odorant assays. According to the previous work [Sengupta et al., 1994], odr-7 hermaphrodite response to py 1/100 and da 1/100 is greatly reduced down to C.I. = 0. I found that both odr-7 hermaphrodites and males exhibited significantly reduced response to py 1/100 (p < 0.001, p < 0.001) (Figure 3.6A). This indicates that male AWA neurons are required for py 1/100 response, just as hermaphrodite AWA neurons are (Figure 3.7). odr-7 hermaphrodites respond to da 1/100 significantly lower than the wild-type hermaphrodites with statistical significance (p < 0.05) (Figure 3.6B). However, da 1/100 response of odr-7 males was similar to that of the wild-type males (Figure 3.6B). It reveals that male AWA neurons are not required for da 1/100 response and suggests that male response to da 1/100 is mediated by other neurons (Figure 3.7). Together, it suggests that the ability of AWA neurons to mediate da response is a property of sexual dimorphism in AWA neurons. The odr-7 hermaphrodite response (C.I. = ~0.5) is AWC neuron-mediated response to da at this concentration (Figure 3.7) (Chou et al., 2001). Together with the previous finding that hermaphrodite AWC neurons sense da at high concentrations ( >= da 1/100), the odr-7 male response (C.I. = ~0.5) may result from male AWC neurons detecting da at 1/100 (Figure 3.7). Since male responses to da 1/100 do not require AWA neurons, I asked how male AWA neurons do not mediate da response. One simple potential mechanism is that the da receptor ODR-10 is either absent or weakly expressed in male AWA neurons giving rise to its inability to detect da. Therefore, I began to examine ODR- 10 expression by comparing both odr-10 transcriptional reporter and ODR-10 translational reporter between the sexes.
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 Figure 3.6. Sexually different mechanisms underlying responses to hermaphrodite AWA odorants Young adult wild-type hermaphrodites (red), wild-type males (blue), odr-7 (ky4); him-5 hermaphrodites (light pink), and odr-7(ky4); him-5 males (light blue) were assayed in sex-segregated populations in the (A) py 1/100 single odorant assay and the (B) da 1/100 single odorant assay. Each data point represents the weighted mean of 4 to 7 assays each containing ~43 animals. Error bars show the weighted SEM. Statistical significance was determined by ANOVA with Bonferroni post-hoc tests.
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 Figure 3.7. How do male AWA neurons respond to hermaphrodite AWA odorants? (Top) Male AWA neurons detect py as they do in hermaphrodites. (Bottom) In contrast to hermaphrodite AWA neurons, male AWA neurons are not required for response to da. This sex difference in AWA neurons to respond to da could be based on a sex difference in the expression of the da receptor ODR-10.
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 odr-10 transcriptional reporter gene expression was examined in kyIs37[Podr- 10::GFP]; him-5 animals. I first compared Podr-10::GFP (transcriptional reporter) expression of ~20 adult worms of each sex. I visually scaled the intensity of GFP signal of AWA neurons in each worm from 1 (dimmest) to 5 (brightest) and determined the number of worms at each intensity level. This preliminary study was performed under the upright compound microscope. Despite bright Podr-10::GFP expression in hermaphrodites (15/16 had an expression level of 5), male Podr- 10::GFP expressions was mostly dim (9/13 had an expression level of 2) (Figure 3.8B). A representative picture of the Podr-10::GFP expression of both sexes is shown in Figure 3.8A. This significant sex difference suggests that the promoter activity of the odr-10 may be sexually different, such that males have lower transcription of odr-10. In spite of this significant sex difference, I cannot yet eliminate the possibility that males in general exhibit lower intensities of fluorescence reporter genes. The ODR-10 protein expression was examined in the kyIs53[Podr- 10::ODR-10::GFP]; him-5 animals under the upright compound microscope. I first compared Podr-10::ODR-10::GFP (translational reporter) expression between sexes. In hermaphrodites, bright ODR-10 receptor expression was observed in the ciliary endings of the AWA dendrites at the tip of the nose with relatively consistent intensity and was rarely observed in the cell body of AWA neurons as previous studies have shown [Dwyer et al., 1998]. In males, however, ODR-10 protein expression was either absent or very weak at the ciliary endings of the AWA neurons and anywhere else in the amphid (Figure 3.9A).
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 Figure 3.8. Male odr-10 expression is significantly reduced compared to that of hermaphrodites (A) Expression of an integrated Podr-10::GFP transcriptional reporter in wild-type animals (lateral views). (Top) DIC of an adult hermaphrodite (Left) and an adult male (Right). (Bottom) GFP of an adult hermaphrodite and an adult male. The solid arrow indicates the position of the AWA cell body showing Podr-10::GFP expression. (B) Visually measured qualitative sex difference in kyIs37[Podr-10::GFP] intensity is displayed on the X axis as a scale from 1 (dimmest) to 5 (brightest). The number of worms in each category of brightness is noted on the Y axis. ~20 age-matched adult worms of each sex were examined.
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 To verify the absence of ODR-10 expression in males with higher magnification, I imaged males and hermaphrodites using LEICA confocal microscopy. Preliminary evidence confirmed that males do not express ODR-10 [4/4 males] although hermaphrodites have strong ODR-10 expression [4/4 hermaphrodites]. To visually measure the qualitative sex difference in ODR-10 expression, I again scaled the intensity of the GFP signal of each worm from 1 (dimmest) to 5 (brightest) and charted the number of worms at each intensity level. Despite bright ODR-10 expression in hermaphrodites [26/33 had signal intensities of 4 or 6], male ODR-10 expression was mostly dim [33/34 showed no signal] (Figure 3.9B). This indicates that there is a significant sex difference in the expression of ODR-10, the da receptor. Next, I asked how this dramatic sex difference in ODR-10 expression came about. I postulated that the neural sex controls ODR-10 expression giving rise to sexually different level of ODR-10. To test this, I generated the kyIs53; him-5; fsEx204[Podr-7::tra-2(ic)_mCherry::unc-54 3’UTR] which have both the ODR-10 translational reporter (kyIs53) and the AWA-feminization construct (fsEx204). If neural sex regulates the ODR-10 expression, males with feminized AWA neurons should reveal increased ODR-10 level similar to the ODR-10 expression in the wild- type hermaphrodites. Intriguingly, AWA-feminized males revealed ODR-10 expression not as bright as but similar to the wild-type hermaphrodite ODR-10 expression (Figure 3.10). This indicates that neural sex controls the sexually different ODR-10 expression. Moreover, this identifies ODR-10 expression as the first identified molecular property that may regulate sex differences in olfaction. Together, this work reveals that neural sex controls sexually different expression of a target gene that may directly give rise to sexually different sensory function.
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 Figure 3.9. ODR-10 expression is sex-specifically regulated: strong ODR-10 in hermaphrodites but weak ODR-10 in males (A) Expression of an integrated Podr-10::GFP translational reporter in wild-type animals (lateral views). DIC and GFP of an adult hermaphrodite (Left) and an adult male (Right). (B) Visually measured qualitative sex difference in the kyIs53[Podr-10:: ODR- 10::GFP] brightness is displayed on the X axis as a scale from 1 (no expression or very weak) to 5 (the brightest). The number of worms in each category of the kyIs53 brightness is noted on the Y axis. ~35 age-matched adult worms of each sex were examined.
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 Figure 3.10. Neural sex in AWA neurons regulates sexually different ODR-10 expression Expression of an Podr-10::ODR-10::GFP integrated fusion gene in wild-type animals (lateral views). DIC (Left) and GFP (Right) of an adult hermaphrodite (Top), an adult male (Middle), and an adult male with feminized AWA neurons (Bottom). The AWA neuron specific expression of the feminizing gene tra-2(ic) is displayed as mCherry signal.
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 4 Discussion Core sensory circuitry controls a sex difference in olfactory preference The feasibility of behavioral sex-switching experiments in both directions by sex- transformation of subsets of neurons reveals two important points. First, both masculinization and feminization transgenes identified the same sufficient sets of neurons for sexually different olfactory preference. If any neurons absolutely required for sex difference in a sensory function were sexually reversed by masculinization but not by feminization, the sensory function would be sex- transformed by masculinization but not by feminization. Second, the core neural circuitry is sufficiently plastic to be modified by neural sex in both directions for behavioral specialization. Together, sexually different olfactory preference may be attributed to sex differences in molecular and/or physiological properties of the sensory neural circuit rather than to sexually different neural connectivity. Furthermore, it reveals that the cell-intrinsic sex regulators must modify the properties of core neurons to impart sex differences in the shared sensory function. I have examined the cellular focus of sex difference in the da-py assay in which the most significant sex difference in olfactory preference is observed among five preference assays with different odorant combinations [Lee and Portman, 2007]. Sex differences in olfactory preferences upon exposure to other combinations of odorants may also be regulated by neural sex. I found that hermaphrodites with masculinized neurons could also reverse hermaphrodite behavior to males’ in the bu- iaa (AWC/AWC) assay (Figure 3.5). This indicates that neural sex regulation of sex differences in olfaction is not limited to the single AWA pair neurons and is a property of the C. elegans olfactory circuit. A single sensory neural-switch between hermaphrodite and male olfaction Sex-transformation of the single sensory neuron pair AWA was sufficient to reverse the sex phenotype of AWA-mediated olfactory preference in both directions.
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 Masculinization of AWA neurons generated male-like olfactory preference of otherwise wild-type hermaphrodites and feminization of AWA neurons brought about hermaphrodite-like olfactory preference of otherwise wild-type males. These results clearly reveal that neural sex of a single sensory neuron type modifies a shared sensory behavior. A decision on the sex phenotype of olfactory preference at the single sensory neural level may contribute to regulation of innate behaviors across generations. Since a few sensory neurons are exposed to thousands of other kinds of sensory stimuli and also informed with internal states, it could be advantageous for a system to segregate behavioral substrates for two different behaviors at the level of sensory neurons. This discovery of the sensory neural segregation of two different behaviors is also observed in behaviors of other systems. This sensory neural role for turning on a behavior of either sex resembles the mouse pheromone activation of sex-specific behavior by VNO (vomeronasal organ). In mouse, VNO of each sex determines sex difference in response to pheromones. Male pheromone increases the females’ receptivity to male sexual behavior although it induces male-male aggression in males. These different responses are controlled by VNO sensory neural input to the downstream neural circuits and independent of disruption in estrous cycle or sex hormones [Kimchi et al., 2007]. Drosophila male courtship song generation also resembles this mechanism in which sexually different sensory neural input (this case, either presence or absence of the male-specific input) modifies common postsynaptic effector machinery to bring about male singing only in male flies [Clyne et al, 2008]. Taken together, my findings that sex differences in sensory neurons presynaptic to common neural circuitry generates sex differences in a shared behavior may be a general mechanism for behavioral plasticity at the circuit level and for regulation of behaviors across generations. The analogy between systems above suggests that the properties of sensory receptor neurons responding to sensory stimuli should be sexually different to bring
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 about sex difference in a sensory behavior. The dramatic sex difference in ODR-10 expression may be the direct molecular substrate for sex difference in AWA neural function including AWA-mediated olfactory preference. The degree of ODR-10 function corresponding to the ODR-10 expression level could affect ODR-10 downstream signaling upon exposure to da. I suggest that the differences in ODR-10 expression lead to changes in PUFA (Poly Unsaturated Fatty Acids)-mediated AWA olfactory signaling which in turn results in different AWA neural activity. Sex difference in the AWA neural output to postsynaptic neurons modulating locomotion would be ultimately displayed as sex difference in the AWA-mediated olfaction. Together with preliminary results from the measurements that synaptic properties (connectivity) of AWA neurons are not sexually different (Figure A1.1), I speculate that sex differences in AWA-mediated olfactory behaviors may rely on the sex difference in the AWA neurons itself which may be expressed as differential modulation of AWA synaptic transmission. A recent study reveals an ability of another olfactory neuron, AWC, to switch between attraction and repulsion by the activity of olfactory signaling components [Tsunozaki et al., 2008]. Together with my speculation that a difference in ODR-10 expression level may generate sexually different behaviors, the modulation of critical sensory neural genes may be a common principle for imparting plasticity to a sensory behavior. Neural sex regulates an effector gene critical for the function of a neural circuit Neural sex controls sex difference in ODR-10 expression: strong ODR-10 expression in hermaphrodites and weak expression/absence of ODR-10 in males. Feminization of AWA male neurons could switch very faint male ODR-10 expression to significantly brighter ODR-10 expression in males similar to the level of the ODR-10 expression in wild-type hermaphrodites. I suggest that the increased ODR-10 expression in feminized male AWA neurons brings about hermaphrodite AWA functions. Cell-intrinsic sex regulators, tra-1 downstream effectors, must therefore
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 regulate ODR-10 to confer sex differences in AWA neurons. Although the link between the terminal sex regulator tra-1 and target gene odr-10 is not understood yet (Figure 3.11), my findings give insights on how chromosomal sex may establish sex difference in molecular/cellular substrates. Together, I have found that neural sex modifies the expression of a target gene in a single sensory neuron to confer sex differences in a shared sensory behavior. Our study differs from other studies describing mechanisms underlying sex-specific neuron-regulated sex-specific behaviors. I suggest that cell-intrinsic sex regulators in a sensory neuron, acting independently from external factors (e.g., sex-specific structures/functions in the system), modify the activity of sensory neural circuit to generate either feminized or masculinized shared sensory function. Further work in this area will shed light on understanding how brain is sexually differentiated to impart sex differences to common behaviors of the vertebrate system.
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 Figure 3.11. Neural sex generates sex difference in a target gene for sexually different common sensory function In hermaphrodites, active TRA-1 initiates hermaphrodite development and, through an unknown downstream mechanism, establishes strong ODR-10 expression. In contrast, inactive TRA-1 in males gives rise to very weak or no ODR-10 expression.
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 Appendix 1 Potential sex difference in AWA connectivity A simple mechanism underlying AWA-mediated sexually different olfactory preference may be the sex difference in AWA synaptic properties. I chose to use the synaptobrevin-1 localization as a read-out of synapse properties. I generated worms, which specifically express snb-1, synaptobrevin in C. elegans, driven by the AWA- specific promoter Podr-7. SNB-1 is a synaptic vesicle integral membrane protein and marks the presynaptic region. In vivo, it shows up as multiple punctate structures. As a guide to the localization of AWA synapses, the AIY interneuron is displayed with the otIs133(Pttx-3::rfp). Overall, I found that the qualitative properties of synapses, namely their number, location, and intensity of puncta (Podr-7::SNB-1_eGFP), were similar between sexes (Figure A1.1). This indicates that sex difference in AWA- mediated olfactory preference is not attributed to sex difference in the AWA connectivity.
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 Figure A1.1. AWA connectivity may not differ between sexes (A) AWA-specific SNB-1 expression (green puncta) is shown by fsEx201(Podr- 7::SNB-1_eGFP) and AIY neurons are marked with the otIs133(Pttx-3::RFP) (B) The number of AWA synapses along the AWA axons was visually compared on the age-matched 34 adult hermaphrodites and 32 adult males. The average number of AWA synapses in hermaphrodites and males were 9.4 and 8.6, respectively.

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 Chapter 4 Discussion The control of sex differences in a C. elegans sensory behavior I have, for the first time, characterized the significant sex differences in a C. elegans common sensory behavior, olfaction. These sex differences are controlled by neural sex, the sexual state of a neuron determined by the cell-intrinsic sex regulators. I identified that the single sensory neuron pair AWA is sufficient cellular foci to bring about sexually different AWA-mediated olfactory preference. Furthermore, I revealed the significant sex difference in the ODR-10 (the da receptor, critical for AWA function). Finally, I elucidated that the neural sex controls the sex difference in ODR-10 expression in AWA. I have found that neural sex controls the expression of a specific target gene, ODR-10, in the sensory neuron pair AWA (Figure 3.10). However, the functional role of the dramatic sex difference in ODR-10 expression has yet to be examined. Increasing the level of ODR-10 expression in male AWAs to the level seen in wild- type hermaphrodites may bring hermaphrodite-like behavior in the da-py assay (AWA/AWA). This would indicate that neural sex brings about sexually different olfactory behaviors through differential regulation on the common sensory genes. Furthermore, it would reveal that changes in gene expression is directly associated with modification in a sensory behavior. Second, the missing link between the cell-intrinsic sex regulators and the sexually different gene expressions must be addressed (Figure 3.11). Since odr-10 upstream region does not seem to contain a consensus binding site for the terminal sex regulator TRA-1, it is unlikely that sexually different ODR-10 expression is directly controlled by TRA-1 activity. TRA-1 downstream effectors, the conserved DM domain genes, are the likely upstream regulators of ODR-10. These DM (doublesex and mab-3)-domain genes specify partially overlapping subsets of male characteristics in C. elegans [Lints and Emmons, 2002; Shen and Hodgkin, 1988; Yi
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 et al., 2000; Mason et al., 2008]. By epistatic analysis (genetic interaction tests) on DM domain genes and ODR-10 expression, the role of DM domain genes in the sexually different target gene expressions may be revealed. If these conserved DM domain genes, known to regulate sex differentiation in other metazoans [Zarkower, 2006], modulate ODR-10 expression, it would suggest that neural sex regulation on a critical signaling gene is the common principle to generate sexually different sensory behaviors in different animal systems. Last, the full extent of neural sex regulation on behavior has not yet been thoroughly explored. I defined the AWA neurons as a sufficient cellular focus of the sex difference in AWA-mediated olfactory preference behavior. Our results in four other olfactory preference settings (Figure 2.4) show significant sex differences, suggesting that other neurons may also have sexually different properties established by neural sex. The neural sex control on the behavioral sex in AWC/AWC olfactory preference (Figure 3.5) supports this idea. Testing sex-transformed animals in different kinds of olfactory preference assays will widen the spatial map of neural sex control on sensory behaviors. Sex-transformation on combinations of different single neurons or subsets of sensory neurons and interneurons will reveal the complete neural components for the full extent of sex difference in olfactory preferences. The temporal activity of neural sex is another intriguing question (Figure 2.6B). Understanding when neural sex is established will tell me if the determination on the sex phenotype of a behavior is based on adult behavioral plasticity or developmental control. If switching neural sex in adult worms is sufficient to reverse behavioral sex, it indicates that neural circuit plasticity in the adult (i.e., including changes in the molecular properties of already established neural circuits) may give rise to sexually different behaviors. Otherwise it suggests that some of developmental sexual differentiation is critical for sexually different behaviors. Furthermore, it is possible that many other target genes critical for neural function are regulated by neural sex. The AWA neurons may have sex differences in the
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 expression of other olfactory signaling genes in addition to ODR-10. To elucidate the spectrum of molecular sex differences in any neural focus of behavior, systemic comparisons of mRNA profiles of single neurons or subsets of sensory neurons could be useful. This approach may identify discrete sex differences in gene expressions. Insights on sexual dimorphisms in neurological diseases Increasing attention on the significant sex differences in many aspects of neurological diseases has captured the interest of both physicians and neurobiologists. However, with the complexity of brain diseases and the nervous system itself, the etiology of many neurological diseases is poorly understood. Studying how the nervous system is sexually differentiated utilizing simple invertebrate animal models will yield insights on the sex-biases in multiple brain diseases of the vertebrates and human and on the development of better treatments for neurological diseases. Cell-intrinsic regulation on sexually different gene expressions may be substrates of the sex-biases in diseases Historically, epidemiological findings reported that sex hormones control sex differences in the pathologies of diseases and responses to therapeutics [Grady et al., 1992]. However, recent clinical reports highlight the role of cell-intrinsic sex regulators of sex differences in brain diseases, independently from the role of sex hormones. Sex differences in post-ischemic brain damages and the recovery were observed in the developing brains of child patients before sexual maturation. Interestingly, the cell death pathways acting upon the ischemic injury in the developing rat brain (postnatal day 7) exhibited significant sex differences. These indicate that prominent sex differences exist in the developing brain that contribute to sex differences in the pathology of brain diseases [Renolleau et al., 2008; Hurn et al., 2005; Edwards, 2004]. However, the exact mechanism of sex-specific regulation on cell death pathways has not been understood. Our finding on neural sex-regulated gene expression (ODR-10, critical for sensory function) suggests that chromosomal
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 sex may somehow modify conventional cell death pathways to give rise to sexually different pathogenesis upon brain injury. Sex differences in gene expressions may give rise to sex differences in behavioral symptoms of diseases Many neurological diseases that occurr with sex-biased incidence are associated with a variety of disrupted behaviors. As I discussed in Chapter 1, for example, autism patients and autism mouse models both display a plethora of disrupted social behaviors only in males [Schneider et al., 2008]. In addition, epigenetic control on sexually different gene expression (i.e., MECP2, FMR1, ATRX and JARID1C) in both human and mouse models is suggested as a mechanism for sex-biases in various aspects of neurobehavioral disease [Tsai et al., 2009; Shibayama et al., 2004; Iwase et al, 2007; Hagerman et al., 2006; Berube et al, 2002; Weaving et al., 2004]. Our studies on how sex differences in gene expressions modify behaviors give insights on the mechanisms underlying sex-biased behavioral symptoms of multiple neuropsychiatric disorders. 

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  • 118. 102 Appendix 2 Strains Table A2.1. Nematode strains Genotype Strain Name Note him-5; kyIs37 UR458 kyIs37 (Podr-10::GFP) him-5; kyIs53 UR460 kyIs53 (Podr-10::ODR-10_GFP) odr-7(ky4); him-5 UR223 odr-10(ky32); him-5 UR463 glp-1(e2141ts)III CB4037 nIs331; him-5(e1467ts)V; ceh- 30(n4289) MT13570 him-5; kyIs53; fsEx204 UR488 fsIs6; oxEx862 fsIs6[srj-54::YFP + cc::GFP] him-5; bxIs14; oxEx862 bxIs14[pkd-2::GFP + pBX1] him-5; zdIs13[tph-1::GFP]; oxEx862 UR222 zdIs13; him-5 was isolated from OH4134 strain no lin-15/lin-15 background fsIs15; him-5 UR236 fsIs15 is the oxEx862 Integration
  • 119. 103 Genotype Strain Name Construct him-5; fsEx158 UR224 Pglr-1::fem-3_mCherry::unc-54 3’UTR + coelomocyte::GFP him-5; fsEx159 UR225 Pglr-1::fem-3_mCherry::unc-54 3’UTR + coelomocyte::GFP him-5; fsEx160 UR226 Posm-5::fem-3_mCherry::unc-54 3’UTR + coelomocyte::GFP him-5; fsEx161 UR227 Posm-5::fem-3_mCherry::unc-54 3’UTR + coelomocyte::GFP him-5; fsEx185 UR247 Posm-5::tra-2(ic)_mCherry::unc-54 3’UTR + coelomocyte::GFP Line #1 him-5; fsEx186 UR248 Posm-5::tra-2(ic)_mCherry::unc-54 3’UTR + coelomocyte::GFP Line #3 him-5; fsEx187 UR249 Prab-3::tra-2(ic)_mCherry::unc-54 3’UTR + coelomocyte::GFP Line #4 him-5; fsEx188 UR250 Prab-3::tra-2(ic)_mCherry::unc-54 3’UTR + coelomocyte::GFP Line #8 otIs133[Pttx-3::rfp]; fsEx200 UR 456 Podr-7::SNB-1_eGFP::unc-54 3’UTR + coelomocyte::GFP Line #1 otIs133; fsEx201 UR 457 Podr-7::SNB-1_eGFP::unc-54 3’UTR + coelomocyte::GFP Line #3 him-5; otIs133; fsEx201 UR459 Podr-7::SNB-1_eGFP::unc-54 3’UTR + coelomocyte::GFP Line #3 him-5; fsEx202 UR245 Podr-7::fem-3_mCherry::unc-54 3’UTR + coelomocyte::GFP Line #3 him-5; fsEx203 UR246 Podr-7::fem-3_mCherry::unc-54 3’UTR + coelomocyte::GFP Line #6 him-5; fsEx204 UR461 Podr-7::tra-2(ic)_mCherry::unc-54 3’UTR + coelomocyte::GFP Line #4 him-5; fsEx205 UR462 Podr-7::tra-2(ic)_mCherry::unc-54 3’UTR + coelomocyte::GFP Line #1 him-5; fsEx206 UR454 Podr-3::fem-3_mCherry::unc-54 3’UTR + coelomocyte::GFP Line #1 him-5; lin-5; oxEx862 EG4391 Prab-3::fem-3_mCherry::unc-54 3’UTR + pkd-2::GFP + lin-15(+) him-5; lin-5; oxEx863 EG4392 Prab-3::fem-3_mCherry::unc-54 3’UTR + pkd-2::GFP + lin-15(+)
  • 120. 104