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

ii

To my family whose love and prayer has carried me through this endeavor

iii

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.

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communicating unpublished data, Dr.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.

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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

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sex differences in the function of shared neural circuitry, and may shed light on the

nature of sexual dimorphisms in the vertebrate nervous system.

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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

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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

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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

xiv

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

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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

1

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.

2

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

3

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,

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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

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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

10

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.

11

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

12

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.

13

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).

14

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.

15

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.

16

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.

17

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,

18

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].

19

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.

20

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.

21

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.

22

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

23

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].

24

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

25

Figure 2.1. Single odorant assay

“Single odorant assay” is essentially the same as the classical chemotaxis assay

(Bargmann et al., 1993)

26

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.

27

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.

28

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

29

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

30

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

31

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.

32

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.

33

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.

34

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.

35

36

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.

37

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

38

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

39

40

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.

41

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.

42

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.

43

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.

44

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.

45

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

46

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

47

(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.

48

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.

49

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

50

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.

51

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,

52

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

53

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

54

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.

55

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-

56

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

57

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

58

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

59

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.

62

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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64

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.

67

68

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.

69

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

70

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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72

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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76

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

83

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

88

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

89

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

90

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.

91

References

Agate, R.J., Grisham, W., Wade, J., Mann, S., Wingfield, J., Schanen, C., Palotie, A., and Arnold, A.P. (2003). Neural, not gonadal, origin of brain sex differences in a gynandromorphic finch. Proc Natl Acad Sci U S A 100, 4873-4878.

Arnold, A.P. (2004). Sex chromosomes and brain gender. Nat Rev Neurosci 5, 701-708.

Bany, I., Dong, M., and Koelle, M. (2003). Genetic and cellular basis for acetylcholine inhibition of Caenorhabditis elegans egg-laying behavior. J Neurosci 23, 8060-8069.

Bargmann, C.I. (2006). Chemosensation in C. elegans. WormBook.

Bargmann, C.I., Hartwieg, E., and Horvitz, H.R. (1993). Odorant-selective genes and neurons mediate olfaction in C. elegans. Cell 74, 515-527.

Barr, M.M., and Sternberg, P.W. (1999). A polycystic kidney-disease gene homologue required for male mating behaviour in C. elegans. Nature 401, 386-389.

Becker, J.B., Arnold, A.P., Berkley, K.J., Blaustein, J.D., Eckel, L.A., Hampson, E., Herman, J.P., Marts, S., Sadee, W., Steiner, M., et al. (2005). Strategies and methods for research on sex differences in brain and behavior. Endocrinology 146, 1650-1673.

Berube, N.G., Jagla, M., Smeenk, C., De Repentigny, Y., Kothary, R., and Picketts, D.J. (2002). Neurodevelopmental defects resulting from ATRX overexpression in transgenic mice. Hum Mol Genet 11, 253-261.

Billeter, J., Rideout, E., Dornan, A., and Goodwin, S. (2006). Control of male sexual behavior in Drosophila by the sex determination pathway. Curr Biol 16, R766-776.

Brann, D.W., Dhandapani, K., Wakade, C., Mahesh, V.B., and Khan, M.M. (2007). Neurotrophic and neuroprotective actions of estrogen: basic mechanisms and clinical implications. Steroids 72, 381-405.

Bratus, A., and Slota, E. (2006). DMRT1/Dmrt1, the sex determining or sex differentiating gene in Vertebrata. Folia Biol (Krakow) S 54, 81-86.

Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71-94.

Cahill, L. (2006). Why sex matters for neuroscience. Nat Rev Neurosci 7, 477-484.

Cahill, L., Uncapher, M., Kilpatrick, L., Alkire, M.T., and Turner, J. (2004). Sex-

92

related hemispheric lateralization of amygdala function in emotionally influenced memory: an FMRI investigation. Learn Mem 11, 261-266.

Carruth, L.L., Reisert, I., and Arnold, A.P. (2002). Sex chromosome genes directly affect brain sexual differentiation. Nat Neurosci 5, 933-934.

Certel, S., Savella, M., Schlegel, D., and Kravitz, E. (2007). Modulation of Drosophila male behavioral choice. Proc Natl Acad Sci U S A 104, 4706-4711.

Chalasani, S., Chronis, N., Tsunozaki, M., Gray, J., Ramot, D., Goodman, M., and Bargmann, C. (2007). Dissecting a circuit for olfactory behaviour in Caenorhabditis elegans. Nature 450, 63-70.

Chalfie, M., Horvitz, H.R., and Sulston, J.E. (1981). Mutations that lead to reiterations in the cell lineages of C. elegans. Cell 24, 59-69.

Chou, J., Bargmann, C., and Sengupta, P. (2001). The Caenorhabditis elegans odr-2 gene encodes a novel Ly-6-related protein required for olfaction. Genetics 157, 211-224.

Colosimo, M.E., Brown, A., Mukhopadhyay, S., Gabel, C., Lanjuin, A.E., Samuel, A.D., and Sengupta, P. (2004). Identification of Thermosensory and Olfactory Neuron-Specific Genes via Expression Profiling of Single Neuron Types. Curr Biol 14, 2245-2251.

Colosimo, M.E., Tran, S., and Sengupta, P. (2003). The divergent orphan nuclear receptor ODR-7 regulates olfactory neuron gene expression via multiple mechanisms in Caenorhabditis elegans. Genetics 165, 1779-1791.

Conradt, B., and Horvitz, H.R. (1998). The C. elegans protein EGL-1 is required for programmed cell death and interacts with the Bcl-2-like protein CED-9. Cell 93, 519-529.

Cooke, B., Hegstrom, C.D., Villeneuve, L.S., and Breedlove, S.M. (1998). Sexual differentiation of the vertebrate brain: principles and mechanisms. Front Neuroendocrinol 19, 323-362.

Cooke, B.M., and Woolley, C.S. (2005). Sexually dimorphic synaptic organization of the medial amygdala. J Neurosci 25, 10759-10767.

Cornelius, M.L., Duan, J.J., and Messing, R.H. (2000). Volatile host fruit odors as attractants for the oriental fruit fly (Diptera: Tephritidae). J Econ Entomol 93, 93-100.

Datta, S.R., Vasconcelos, M.L., Ruta, V., Luo, S., Wong, A., Demir, E., Flores, J., Balonze, K., Dickson, B.J., and Axel, R. (2008). The Drosophila pheromone cVA

93

activates a sexually dimorphic neural circuit. Nature 452, 473-477.

de Bono, M., Zarkower, D., and Hodgkin, J. (1995). Dominant feminizing mutations implicate protein-protein interactions as the main mode of regulation of the nematode sex-determining gene tra-1. Genes Dev 9, 155-167.

Dewing, P., Chiang, C.W., Sinchak, K., Sim, H., Fernagut, P.O., Kelly, S., Chesselet, M.F., Micevych, P.E., Albrecht, K.H., Harley, V.R., et al. (2006). Direct Regulation of Adult Brain Function by the Male-Specific Factor SRY. Curr Biol 16, 415-420.

Dewing, P., Shi, T., Horvath, S., and Vilain, E. (2003). Sexually dimorphic gene expression in mouse brain precedes gonadal differentiation. Brain Res Mol Brain Res 118, 82-90.

Doniach, T., and Hodgkin, J. (1984). A sex-determining gene, fem-1, required for both male and hermaphrodite development in Caenorhabditis elegans. Dev Biol 106, 223-235.

Dwyer, N.D., Troemel, E.R., Sengupta, P., and Bargmann, C.I. (1998). Odorant receptor localization to olfactory cilia is mediated by ODR-4, a novel membrane-associated protein. Cell 93, 455-466.

Edwards, D. (2004). Brain protection for girls and boys. J Pediatr 145, 723-724.

Ember, C.R., and Ember, M. (2003). Encyclopedia of sex and gender : men and women in the world's cultures, (New York: Kluwer Academic/Plenum Publishers).

Erdman, S., and Burtis, K. (1993). The Drosophila doublesex proteins share a novel zinc finger related DNA binding domain. EMBO J 12, 527-535.

Fleisher, A., Grundman, M., Jack, C.R., Jr., Petersen, R.C., Taylor, C., Kim, H.T., Schiller, D.H., Bagwell, V., Sencakova, D., Weiner, M.F., et al. (2005). Sex, apolipoprotein E epsilon 4 status, and hippocampal volume in mild cognitive impairment. Arch Neurol 62, 953-957.

Gahr, M. (2003). Male Japanese quails with female brains do not show male sexual behaviors. Proc Natl Acad Sci U S A 100, 7959-7964.

Gatewood, J.D., Wills, A., Shetty, S., Xu, J., Arnold, A.P., Burgoyne, P.S., and Rissman, E.F. (2006). Sex chromosome complement and gonadal sex influence aggressive and parental behaviors in mice. J Neurosci 26, 2335-2342.

Goldman, S.A., and Nottebohm, F. (1983). Neuronal production, migration, and differentiation in a vocal control nucleus of the adult female canary brain. Proc Natl Acad Sci U S A 80, 2390-2394.

94

Goodman, M.B. (2006). Mechanosensation. WormBook.

Gorski, R.A., Gordon, J.H., Shryne, J.E., and Southam, A.M. (1978). Evidence for a morphological sex difference within the medial preoptic area of the rat brain. Brain Res 148, 333-346.

Grady, D.a.o. (1992). Hormone therpay to prevent disease and prolong life in postmenopausal women. Ann Intern Med 117, 1016-1037.

Gray, J.M., Hill, J.J., and Bargmann, C.I. (2005). A circuit for navigation in Caenorhabditis elegans. Proc Natl Acad Sci U S A 102, 3184-3191.

Gruninger, T., Gualberto, D., LeBoeuf, B., and Garcia, L. (2006). Integration of male mating and feeding behaviors in Caenorhabditis elegans. J Neurosci S 26, 169-179.

Hagerman, R.J. (2006). Lessons from fragile X regarding neurobiology, autism, and neurodegeneration. J Dev Behav Pediatr 27, 63-74.

Hall, D., Lints, R., and Altun, Z. (2006). Nematode neurons: anatomy and anatomical methods in Caenorhabditis elegans. Int Rev Neurobiol S 69, 1-35.

Hall, J.C. (1977). Portions of the central nervous system controlling reproductive behavior in Drosophila melanogaster. Behav Genet 7, 291-312.

Hammock, E.A., and Young, L.J. (2006). Oxytocin, vasopressin and pair bonding: implications for autism. Philos Trans R Soc Lond B Biol Sci 361, 2187-2198.

Hartley, J.L., Temple, G.F., and Brasch, M.A. (2000). DNA cloning using in vitro site-specific recombination. Genome Res 10, 1788-1795.

Haycraft, C.J., Swoboda, P., Taulman, P.D., Thomas, J.H., and Yoder, B.K. (2001). The C. elegans homolog of the murine cystic kidney disease gene Tg737 functions in a ciliogenic pathway and is disrupted in osm-5 mutant worms. Development 128, 1493-1505.

Hodgkin, J. (1987). A genetic analysis of the sex-determining gene, tra-1, in the nematode Caenorhabditis elegans. Genes Dev 1, 731-745.

Hodgkin, J., and Brenner, S. (1977). Mutations causing transformation of sexual phenotype in the nematode Caenorhabditis elegans. Genetics 86, 275-287.

Hong, C.S., Park, B.Y., and Saint-Jeannet, J.P. (2007). The function of Dmrt genes in vertebrate development: it is not just about sex. Dev Biol 310, 1-9.

Horvitz, H.R., and Sulston, J.E. (1980). Isolation and genetic characterization of cell-lineage mutants of the nematode Caenorhabditis elegans. Genetics 96, 435-454.

95

Hunter, C.P., and Wood, W.B. (1990). The tra-1 gene determines sexual phenotype cell-autonomously in C. elegans. Cell 63, 1193-1204.

Hurn, P.D., Vannucci, S.J., and Hagberg, H. (2005). Adult or perinatal brain injury: does sex matter? Stroke 36, 193-195.

Ishihara, T., Iino, Y., Mohri, A., Mori, I., Gengyo-Ando, K., Mitani, S., and Katsura, I. (2002). HEN-1, a secretory protein with an LDL receptor motif, regulates sensory integration and learning in Caenorhabditis elegans. Cell 109, 639-649.

Iwase, S., Lan, F., Bayliss, P., de la Torre-Ubieta, L., Huarte, M., Qi, H.H., Whetstine, J.R., Bonni, A., Roberts, T.M., and Shi, Y. (2007). The X-linked mental retardation gene SMCX/JARID1C defines a family of histone H3 lysine 4 demethylases. Cell 128, 1077-1088.

Juraska, J.M. (1991). Sex differences in "cognitive" regions of the rat brain. Psychoneuroendochrinology 16, 105-109.

Juraska, J.M. (1991). Sex differences in "cognitive" regions of the rat brain. Psychoneuroendocrinology 16, 105-109.

Juraska, J.M., Fitch, J.M., Henderson, C., and Rivers, N. (1985). Sex differences in the dendritic branching of dentate granule cells following differential experience. Brain Res 333, 73-80.

Kahn-Kirby, A., Dantzker, J., Apicella, A., Schafer, W., Browse, J., Bargmann, C., and Watts, J. (2004). Specific polyunsaturated fatty acids drive TRPV-dependent sensory signaling in vivo. Cell 119, 889-900.

Kimble, J., and Crittenden, S.L. (2005). Germline proliferation and its control. WormBook, 1-14.

Kimble, J., Edgar, L., and Hirsh, D. (1984). Specification of male development in Caenorhabditis elegans: the fem genes. Dev Biol 105, 234-239.

Kimble, J.E., and White, J.G. (1981). On the control of germ cell development in Caenorhabditis elegans. Dev Biol 81, 208-219.

Kimchi, T., Xu, J., and Dulac, C. (2007). A functional circuit underlying male sexual behaviour in the female mouse brain. Nature 448, 1009-1014.

Kimura, K., Hachiya, T., Koganezawa, M., Tazawa, T., and Yamamoto, D. (2008). Fruitless and doublesex coordinate to generate male-specific neurons that can initiate courtship. Neuron 59, 759-769.

96

Klass, M., Wolf, N., and Hirsh, D. (1976). Development of the male reproductive system and sexual transformation in the nematode Caenorhabditis elegans. Dev Biol 52, 1-18.

Kleemann, G., Jia, L., and Emmons, S. (2008). Regulation of Caenorhabditis elegans Male Mate Searching Behavior by the Nuclear Receptor DAF-12. Genetics.

Komatsu, H., Jin, Y.H., L'Etoile, N., Mori, I., Bargmann, C.I., Akaike, N., and Ohshima, Y. (1999). Functional reconstitution of a heteromeric cyclic nucleotide-gated channel of Caenorhabditis elegans in cultured cells. Brain Res 821, 160-168.

Komatsu, H., Mori, I., Rhee, J., Akaike, N., and Ohshima, Y. (1996). Mutations in a cyclic nucleotide-gated channel lead to abnormal thermosensation and chemosensation in C. elegans. Neuron 17, 707-718.

Korol, D.L. (2004). Role of estrogen in balancing contributions from multiple memory systems. Neurobiol Learn Mem 82, 309-323.

Kuwabara, P.E., and Kimble, J. (1995). A predicted membrane protein, TRA-2A, directs hermaphrodite development in Caenorhabditis elegans. Development 121, 2995-3004.

Lee, K., and Portman, D. (2007). Neural sex modifies the function of a C. elegans sensory circuit. Curr Biol 17, 1858-1863.

Levine, S. (1966). Sex-role identification and parental perceptions of social competence. Am J Ment Defic 70, 822-824.

Lints, R., and Emmons, S.W. (2002). Regulation of sex-specific differentiation and mating behavior in C. elegans by a new member of the DM domain transcription factor family. Genes Dev 16, 2390-2402.

Madeira, M.D., and Lieberman, A.R. (1995). Sexual dimorphism in the mammalian limbic system. Prog Neurobiol 45, 275-333.

Madl, J., and Herman, R. (1979). Polyploids and sex determination in Caenorhabditis elegans. Genetics 93, 393-402.

Manoli, D., Meissner, G., and Baker, B. (2006). Blueprints for behavior: genetic specification of neural circuitry for innate behaviors. Trends Neurosci 29, 444-451.

Maricq, A.V., Peckol, E., Driscoll, M., and Bargmann, C.I. (1995). Mechanosensory signalling in C. elegans mediated by the GLR-1 glutamate receptor. Nature 378, 78-81.

97

Mason, D., Rabinowitz, J., and Portman, D. (2008). dmd-3, a doublesex-related gene regulated by tra-1, governs sex-specific morphogenesis in C. elegans. Development 135, 2373-2382.

McEwen, B.S. (2000). The neurobiology of stress: from serendipity to clinical relevance. Brain Res 886, 172-189.

McPhie-Lalmansingh, A.A., Tejada, L.D., Weaver, J.L., and Rissman, E.F. (2008). Sex chromosome complement affects social interactions in mice. Horm Behav 54, 565-570.

Mehra, A., Gaudet, J., Heck, L., Kuwabara, P.E., and Spence, A.M. (1999). Negative regulation of male development in Caenorhabditis elegans by a protein-protein interaction between TRA-2A and FEM-3. Genes Dev 13, 1453-1463.

Miller, A., Thiele, T., Faumont, S., Moravec, M., and Lockery, S. (2005). Step-response analysis of chemotaxis in Caenorhabditis elegans. J Neurosci 25, 3369-3378.

Morris, J.A., Jordan, C.L., and Breedlove, S.M. (2004). Sexual differentiation of the vertebrate nervous system. Nat Neurosci 7, 1034-1039.

Nelson, G., Lew, K., and Ward, S. (1978). Intersex, a temperature-sensitive mutant of the nematode Caenorhabditis elegans. Dev Biol 66, 386-409.

Nishizawa, S., Benkelfat, C., Young, S.N., Leyton, M., Mzengeza, S., de Montigny, C., Blier, P., and Diksic, M. (1997). Differences between males and females in rates of serotonin synthesis in human brain. Proc Natl Acad Sci U S A 94, 5308-5313.

Nonet, M., Staunton, J., Kilgard, M., Fergestad, T., Hartwieg, E., Horvitz, H., Jorgensen, E., and Meyer, B. (1997). Caenorhabditis elegans rab-3 mutant synapses exhibit impaired function and are partially depleted of vesicles. J Neurosci 17, 8061-8073.

Nopoulos, P., Flaum, M., and Andreasen, N.C. (1997). Sex differences in brain morphology in schizophrenia. Am J Psychiatry 154, 1648-1654.

O'Hagan, R., and Chalfie, M. (2006). Mechanosensation in Caenorhabditis elegans. Int Rev Neurobiol 69, 169-203.

Phoenix, C.H., Goy, R.W., Gerall, A.A., and Young, W.C. (1959). Organizing action of prenatally administered testosterone propionate on the tissues mediating mating behavior in the female guinea pig. Endocrinology 65, 369-382.

Pierce-Shimomura, J.T., Dores, M., and Lockery, S.R. (2005). Analysis of the effects

98

of turning bias on chemotaxis in C. elegans. J Exp Biol 208, 4727-4733.

Pierce-Shimomura, J.T., Morse, T.M., and Lockery, S.R. (1999). The fundamental role of pirouettes in Caenorhabditis elegans chemotaxis. J Neurosci 19, 9557-9569.

Portman, D. (2007). Genetic Control of Sex Differences in C. elegans Neurobiology and Behavior. Adv Genet 59, 1-37.

Qin, H., Rosenbaum, J.L., and Barr, M.M. (2001). An autosomal recessive polycystic kidney disease gene homolog is involved in intraflagellar transport in C. elegans ciliated sensory neurons. Curr Biol 11, 457-461.

Raymond, C.S., Shamu, C.E., Shen, M.M., Seifert, K.J., Hirsch, B., Hodgkin, J., and Zarkower, D. (1998). Evidence for evolutionary conservation of sex-determining genes. Nature 391, 691-695.

Reisert, I., and Pilgrim, C. (1991). Sexual differentiation of monoaminergic neurons--genetic or epigenetic? Trends Neurosci 14, 468-473.

Remy, J.J., and Hobert, O. (2005). An interneuronal chemoreceptor required for olfactory imprinting in C. elegans. Science 309, 787-790.

Renolleau, S., Fau, S., and Charriaut-Marlangue, C. (2008). Gender-related differences in apoptotic pathways after neonatal cerebral ischemia. Neuroscientist 14, 46-52.

Rideout, E.J., Billeter, J.C., and Goodwin, S.F. (2007). The sex-determination genes fruitless and doublesex specify a neural substrate required for courtship song. Curr Biol 17, 1473-1478.

Roayaie, K., Crump, J.G., Sagasti, A., and Bargmann, C.I. (1998). The G alpha protein ODR-3 mediates olfactory and nociceptive function and controls cilium morphogenesis in C. elegans olfactory neurons. Neuron 20, 55-67.

Schafer, W. (2006). Genetics of egg-laying in worms. Annu Rev Genet 40, 487-509.

Schneider, T., Roman, A., Basta-Kaim, A., Kubera, M., Budziszewska, B., Schneider, K., and Przewlocki, R. (2008). Gender-specific behavioral and immunological alterations in an animal model of autism induced by prenatal exposure to valproic acid. Psychoneuroendocrinology 33, 728-740.

Segal, S.P., Graves, L.E., Verheyden, J., and Goodwin, E.B. (2001). RNA-Regulated TRA-1 nuclear export controls sexual fate. Dev Cell 1, 539-551.

Sengupta, P. (2007). Generation and modulation of chemosensory behaviors in C.

99

elegans. Pflugers Arch 454, 721-734.

Sengupta, P., Chou, J.H., and Bargmann, C.I. (1996). odr-10 encodes a seven transmembrane domain olfactory receptor required for responses to the odorant diacetyl. Cell 84, 899-909.

Sengupta, P., Colbert, H.A., and Bargmann, C.I. (1994). The C. elegans gene odr-7 encodes an olfactory-specific member of the nuclear receptor superfamily. Cell 79, 971-980.

Shen, M.M., and Hodgkin, J. (1988). mab-3, a gene required for sex-specific yolk protein expression and a male-specific lineage in C. elegans. Cell 54, 1019-1031.

Shibayama, A., Cook, E.H., Jr., Feng, J., Glanzmann, C., Yan, J., Craddock, N., Jones, I.R., Goldman, D., Heston, L.L., and Sommer, S.S. (2004). MECP2 structural and 3'-UTR variants in schizophrenia, autism and other psychiatric diseases: a possible association with autism. Am J Med Genet B Neuropsychiatr Genet 128B, 50-53.

Sulston, J.E., and Horvitz, H.R. (1977). Postembryonic cell lineages of the nematode Caenorhabditis elegans. Dev Biol 56, 110-156.

Sulston, J.E., Schierenberg, E., White, J.G., and Thomson, J.N. (1983). The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev Biol 100, 64-119.

Swaab, D.F., Chung, W.C., Kruijver, F.P., Hofman, M.A., and Hestiantoro, A. (2003). Sex differences in the hypothalamus in the different stages of human life. Neurobiol Aging 24 Suppl 1, S1-16; discussion S17-19.

Swaab, D.F., and Hofman, M.A. (1988). Sexual differentiation of the human hypothalamus: ontogeny of the sexually dimorphic nucleus of the preoptic area. Brain Res Dev Brain Res 44, 314-318.

Thorne, N., Chromey, C., Bray, S., and Amrein, H. (2004). Taste perception and coding in Drosophila. Curr Biol 14, 1065-1079.

Trent, C., Tsuing, N., and Horvitz, H. (1983). Egg-laying defective mutants of the nematode Caenorhabditis elegans. Genetics 104, 619-647.

Troemel, E.R., Chou, J.H., Dwyer, N.D., Colbert, H.A., and Bargmann, C.I. (1995). Divergent seven transmembrane receptors are candidate chemosensory receptors in C. elegans. Cell 83, 207-218.

Tsai, H.W., Grant, P.A., and Rissman, E.F. (2009). Sex differences in histone

100

modifications in the neonatal mouse brain. Epigenetics 4.

Tsunozaki, M., Chalasani, S.H., and Bargmann, C.I. (2008). A behavioral switch: cGMP and PKC signaling in olfactory neurons reverses odor preference in C. elegans. Neuron 59, 959-971.

Vellai, T., McCulloch, D., Gems, D., and Kovacs, A. (2006). Effects of sex and insulin/insulin-like growth factor-1 signaling on performance in an associative learning paradigm in Caenorhabditis elegans. Genetics S 174, 309-316.

Villella, A., and Hall, J. (1996). Courtship anomalies caused by doublesex mutations in Drosophila melanogaster. Genetics 143, 331-344.

Villella, A., and Hall, J.C. (2008). Neurogenetics of courtship and mating in Drosophila. Adv Genet 62, 67-184.

Von Stetina, S., Treinin, M., and Miller, D.r. (2006). The motor circuit. Int Rev Neurobiol S 69, 125-167.

Vrontou, E., Nilsen, S., Demir, E., Kravitz, E., and Dickson, B. (2006). fruitless regulates aggression and dominance in Drosophila. Nat Neurosci 9, 1469-1471.

Wakabayashi, T., Kitagawa, I., and Shingai, R. (2004). Neurons regulating the duration of forward locomotion in Caenorhabditis elegans. Neurosci Res 50, 103-111.

Wang, Z., Singhvi, A., Kong, P., and Scott, K. (2004). Taste representations in the Drosophila brain. Cell 117, 981-991.

Weaving, L.S., Christodoulou, J., Williamson, S.L., Friend, K.L., McKenzie, O.L., Archer, H., Evans, J., Clarke, A., Pelka, G.J., Tam, P.P., et al. (2004). Mutations of CDKL5 cause a severe neurodevelopmental disorder with infantile spasms and mental retardation. Am J Hum Genet 75, 1079-1093.

Weissburg, M.J. (2001). Sex, sensitivity, and second messengers: differential effect of cyclic nucleotide mediated inhibition in the chemosensory system of fiddler crabs. J Comp Physiol [A] 187, 489-498.

Weissburg, M.J., and Derby, C.D. (1995). Regulation of sex-specific feeding behavior in fiddler crabs: physiological properties of chemoreceptor neurons in claws and legs of males and females. J Comp Physiol [A] 176, 513-526.

Weissburg, M.J., Derby, C.D., Johnson, O., McAlvin, B., and Moffett, J.M., Jr. (2001). Transsexual limb transplants in fiddler crabs and expression of novel sensory capabilities. J Comp Neurol 440, 311-320.

101

White, J., Nicholas, T., Gritton, J., Truong, L., Davidson, E., and Jorgensen, E. (2007). The sensory circuitry for sexual attraction in C. elegans males. Curr Biol 17, 1847-1857.

White, J.G. (1988). The anatomy. In The nematode Caenorhabditis elegans, W.B. Wood, ed. (Cold Spring Harbor Laboratory Press).

White, J.G., Southgate, E., Thomson, J.N., and Brenner, S. (1986). The structure of the nervous system of the nematode Caenorhabditis elegans. Phil Trans R Soc Lond B 314, 1-340.

Wizemann, T.M., and Pardue, M.L. (2001). Exploring the biological contributions to human health: Does sex matter?, (Washington, D.C: National Academy Press).

Yi, W., Ross, J.M., and Zarkower, D. (2000). mab-3 is a direct tra-1 target gene regulating diverse aspects of C. elegans male sexual development and behavior. Development 127, 4469-4480.

Zarkower, D. (2001). Establishing sexual dimorphism: conservation amidst diversity? Nat Rev Genet 2, 175-185.

Zarkower, D. (2006). Somatic sex determination. WormBook, 1-12.

Zarkower, D., and Hodgkin, J. (1992). Molecular analysis of the C. elegans sex-determining gene tra-1: a gene encoding two zinc finger proteins. Cell 70, 237-249.

Zarkower, D., and Hodgkin, J. (1993). Zinc fingers in sex determination: only one of the two C. elegans Tra-1 proteins binds DNA in vitro. Nucleic Acids Res 21, 3691-3698.

Zheng, Y., Brockie, P., Mellem, J., Madsen, D., and Maricq, A. (1999). Neuronal control of locomotion in C. elegans is modified by a dominant mutation in the GLR-1 ionotropic glutamate receptor. Neuron 24, 347-361.

Ziabreva, I., Poeggel, G., Schnabel, R., and Braun, K. (2003). Separation-induced receptor changes in the hippocampus and amygdala of Octodon degus: influence of maternal vocalizations. J Neurosci 23, 5329-5336.

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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

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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(+)

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