Role of Membrane Bound G-Protein Coupled Estrogen Receptor

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ROLE OF MEMBRANE BOUND G-PROTEIN COUPLED ESTROGEN RECEPTOR

GPR30 AND Z-LINKED RIBOSOMAL GENE S6 (RPS6) IN SEXUALLY

DIMORPHIC DEVELOPMENT OF THE ZEBRA FINCH BRAIN

A dissertation submitted to

Kent State University in partial fulfillment

of the requirements for the degree of

Doctor of Philosophy

Kalpana D. Acharya

August 2012

Dissertation written by

Kalpana D. Acharya

B.A.M.S., Tribhuvan University, 2004

Ph.D., Kent State University, 2012

Approved by

Dr. Sean L. Veney , Chair, Doctoral Dissertation Committee

Dr. Gail C. Fraizer , Member, Doctoral Dissertation Committee

Dr. Heather K. Caldwell , Member, Doctoral Dissertation Committee

Dr. Eric M. Mintz , Member, Doctoral Dissertation Committee

Dr. Chun-Che Tsai , Member, Doctoral Dissertation Committee

Accepted by

Dr. Robert V. Dorman , Director, School of Biomedical Sciences

Dr. John R.D. Stalvey , Dean, College of Arts and Science

TABLE OF CONTENTS

LIST OFABBREVIATIONS…..…..……………………………………………………vi

LIST OF FIGURES……….………………………………………...…………...... x

LIST OF TABLES………………………………………………………...……………xii

ACKNOWLEDGEMENTS…….………………………………………………………xiii

DEDICATION……..…………………………………………………………………....xv

OVERALL ABSTRACT…….……………………………………………………...... 1

CHAPTER I. Introduction….………………………..…………………………………..2

Sexual differentiation of the vertebrate nervous system...... ……….…………....………2

Hormone mediated brain dimorphisms….…...……………..…………………...….…...3

Non-hormonal regulation of brain dimorphisms…..…………..………………...….…...9

Zebra finch: A model for sexual dimorphisms..………………….…………………...... 10

Hormonal regulation of zebra finch differentiation.…..……..……………..…………..11

Genetic regulation of zebra finch brain differentiation.………...……….……..…...... 15

Overall aims……………..………………….…………………………………..………..18

References…………….…………..……...…………………...…………..….….…….....19

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CHAPTER II. Characterization of the G-protein coupled membrane bound estrogen receptor GPR30 in the zebra finch brain reveals a sex difference in gene and protein expression (Published online in Journal of Developmental Neurobiology:

DOI: 10.1002/dneu.22004………………………………………………………….…36

Abstract………………...……………………………………..……………………….36

Introduction……………...………………………………………………..…………...37

Materials and methods………………………………………………………..…...…...40

Results……………………………………………………….………...……….……....48

Discussion……………………………………………….…………….…..…….……..59

Acknowledgements……………………………………...………………...….………..69

References…..………………………………………...…………………...….………..69

CHAPTER III. Use of differential display reverse transcription (DDRT) PCR to identify differentially expressed genes in the telencephalon of early post-hatching male and female zebra finches …………………………………………………………………..82

Abstract……….………………………………………………..…….……………...... 82

Introduction…………….……………………………………..…………………..…...83

Materials and methods……….…………………………………………...………...….84

Results………………….…………………………………….……………………..….86

Discussion…………………………………………...…………………………………88

References…..………………………………………………………….….………....…91

CHAPTER IV. Sexually dimorphic expression and estradiol mediated up-regulation of the Z-linked ribosomal gene rpS6 in the zebra finch brain…………………….....…..95

Abstract ………….……….…………………………………………….....…………..95

Introduction……………….…………………………………………...…..……...... 96

Materials and methods………………..………………………………...…..…………99

Results…………………..………………..………………….…………....….....……106

Discussion…………………..……...……………………….…...…………………...115

References.....………………………………………...…………..…...………..…….119

CHAPTER V. Global discussion…..……….……………..……………………...…129

Future directions…………..…………………..……………………………………...135

References………..…………………………………………………….…….……….137

LIST OF ABBREVIATIONS

A…………………………..……………………………………….….…….….arcopallium

AA………………………………………………….……….…anterior arcopallial nucleus

AVP………………….…………………………………………………..……..vasopressin

AVPV………………..…………anteroventral periventricular region of the hypothalamus

Bas………………………………………………….………….baso-rostral pallial nucleus

BDNF…………………….………………………..…… brain derived neurotrophic factor

IGF-II……………….……………………………..……………….insulin growth factor-II

BNSTp………………...…..……posterior region of the bed nucleus of the stria terminalis

Cb………………….…,,…………………………………………….……….…cerebellum

CoA……………...……………………………………..….……….…anterior commissure

DDRT-PCR………...………………………differential display reverse transcription PCR

DHT……………..…………………………..……………………….. dihydrotestosterone

DIG………………..…………………………..…………………………….…digoxigenin

DLM……………...………………..………...dorsolateral nucleus of the medial thalamus

E…….………..………………………..……………….………………entopallial nucleus

E………………..…………………………….………………….………………embryonic

E2……...…………………………………….…………………….……………….estradiol

EB……...……………………………………………………….……….estradiol benzoate

ERE…….…..…………………………………..…………….…estrogen response element

ERα….…..………………………………….……….….……..……..…estrogen receptor α

ERαKO…..………………………………….……...…estrogen receptor α knock-out mice

ERβ….…..…………………………………….……….…….………...estrogen receptor β

FA………………………………………………………..…..……..fronto-arcopallial tract

GP……………………………………………………………..…….……...globus pallidus

GPR30……………………………………………..…G-protein coupled estrogen receptor

Gs……….………………………………………….….…………..…stimulatory G-protein

HA……………………………………………..……..…….……….…apical hyperpallium

HD………….…………………………………..……….….……….…dorsal hyperpallium

HP……………………………………………………..………….……….….hippocampus

IEG…….……………………………………..……………..….…....immediate early gene

IHC…….…………………………………………….…………..…immunohistochemistry

IPTG…..…………………………………………….isopropyl-β-D-thiogalactopyranoside ir……………………………………………………….….…..………...…immunoreactive

ISH…..………………………………………..……………..……..…in situ hybridization

LMAN….…………….….….....lateral magnocellular nucleus of the anterior nidopallium

LSt…….……………………………………..…..….…….………….….…lateral striatum

M……….…………………………….……..……....…….………..……...….mesopallium

MePD…..……………………...………..…….…...postero-dorsal region of the amygdala

MSt……..………………….………………...…..….…….….……….…...medial striatum

N…………………………………..…………….…….….…….…..……….…nidopallium

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NC………..……………………….…………..……….………….……caudal nidopallium

NCM……...………………………….……….………..………...caudomedial nidopallium

Rt…………………………………….………..……………………...…...nucleus rotundus

Nif……...……………………………...……….…….nucleus interface of the nidopallium nXIIt…….………………………..…..tracheosyringeal branch of the hypoglossal nucleus

P…………………………………………..…………………………..……....post-hatching

PBF………………………………………..….…..…………..phosphate buffered formalin

PBS…………………………………………...…….…………...phosphate buffered saline

ORF……….…………………………………..….……..………..…opening reading frame

PI3K…….……………………………………..…..……………..phosphoinositol-3 kinase

RA……….……………………………….…..……….…….robust nucleus of arcopallium

RP……………………………………….…………………………….…ribosomal protein

RT………………………………………………………………….…reverse transcription

SDN-POA……………………………....sexually dimorphic nucleus of the pre-optic area

SNB…….………………………………………..…….spinal nucleus of bulbocavernosus

Sry…….………………………………….…………………...…sex-determining region Y

SSC….………………………………………….… sodium-chloride-sodium citrate buffer

T….………………………………………….…………………………………testosterone

TEA……….…………………………………..……….……………….… triethanolamine

TeO…….………………………………………………………….……….… optic tectum

Tfm…………………………………………………….…testicular feminization mutation

TH…….……………………………………………..………….……tyrosine hydroxylase

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Tn…….……………………………………..……..….… nucleus taeniae of the amygdala

TP……………………………………………………….………... testosterone propionate

Uva…….……………………………………………….…….………..uvaeformis nucleus

V…………………………………………..……..…...……………………..….….ventricle

LIST OF FIGURES

Page

CHAPTER I. Introduction

FIGURE 1.1. Flowchart representing the development of brain sex differences as a combined effect of genetic and hormonal sex that gives rise to phenotypic sex……...... 4

FIGURE 1.2. Schematic drawing of the song control regions (showing the posterior forebrain (motor) pathway and anterior forebrain (song learning) pathway)

……..……………………………………………………………………………..….…...12

FIGURE 1.3. A gynandromorphic zebra finch with male-specific plumage (zebra stripes and orange cheek patch) on the right side, and female specific plumage (no stripes or cheek patch) on the left side………………………………………...….…….……….….....17

CHAPTER II. Characterization of the G-protein coupled membrane bound estrogen receptor

GPR30 in the zebra finch brain reveals a sex difference in gene and protein expression.

FIGURE 2.1. Multiple alignments of GPR30 homologues in mouse, rat, human, zebra finch and chicken using clustalw2 program.………………..……...... …………….….….49

FIGURE 2.2. Relative expression of the GPR30 gene in the zebra finch telencephalon during development and adulthood…………………………….………………….……..51

FIGURE 2.3. Western blot confirming the specificity of the GPR30 polyclonal antibody

………..……………………………………………………………………………...….……52

FIGURE 2.4. Camera lucida drawings representing the relative intensity of GPR30 immunoreactivity at P21.………………………………..……………...…………....……..53

FIGURE 2.5. Numbers of GPR30- ir cells counted within a 0.01mm2 area of HVC...... 56

FIGURE 2.6. Photomicrographs of GPR30-ir cells in HVC……...……………....…...... 57

FIGURE 2.7. Numbers of GPR30- ir cells counted within a 0.01 mm2 area of RA..…..58

FIGURE 2.8. Photomicrographs of GPR30- ir cells in RA. ………...…………....…...... 60

FIGURE 2.9. Numbers of GPR30- ir cells counted within a 0.01mm2 area of LMAN...61

CHAPTER III. Use of differential display reverse transcription (DDRT) PCR to identify differentially expressed genes in the telencephalon of early post-hatching male and female zebra finches.

FIGURE 3.1. Gel showing amplified bands using DDRT- PCR………………...……....87

FIGURE 3.2. Bands representing ribosomal gene S6 (rpS6) cDNA amplified using

DDRT-PCR…………………………………………...…………………………..…….....89

CHAPTER IV. Sexually dimorphic expression and estradiol mediated up-regulation of the Z-linked ribosomal gene rpS6 in the zebra finch brain.

FIGURE 4.1. Relative expression of rpS6 gene in the zebra finch telencephalon…….108

FIGURE 4.2. RpS6 labeling in HVC of a P10 male and female zebra finch…...... …..109

FIGURE 4.3. RpS6 labeled cells counted within a 0.09mm2 area of HVC in P10 males and females………………………………………………………………………..…....110

FIGURE 4.4. RpS6 labeling in RA of a P10 male and female………………...………111

FIGURE 4.5. RpS6 labeled cells counted within a 0.09mm2 area of RA in P10 males and females……………………………………………………………………….....………112

FIGURE 4.6. Effect of E2 treatment on rpS6 gene expression in P21 male and female zebra finch telencephalon………………………………………………….…….……..114

CHAPTER V. Global Discussion.

Figure 5.1. Proposed mechanism of membrane bound estrogen receptor GPR30 and rpS6 gene in dimorphic development of the neural song system in zebra finch……………..130

LIST OF TABLES

Page

Table 2.1: Ages chosen for q-PCR and their significance……………….….…………..43

Table 3.1: Differentially expressed genes in the telencephalon of P3 and P8 animals as identified by DDRT-PCR, their chromosomal location and the direction of expression..90

Table 4.1: Ages chosen for q-PCR and their significance……………….….……...…..100

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ACKNOWLEDGEMENTS

First and foremost, I would like to thank my PhD advisor, Dr. Sean Veney for his tremendous guidance and support throughout my graduate life. Coming from a totally different academic background, I was very new to most of the research techniques and protocols when I joined his lab. On top of it, my thick accent surely wasn’t a pleasant addition to the situation. Despite these issues, throughout the years in his lab, he has been very motivating and constructive of my mistakes. His consistent positive feedback has been the main fuel for building confidence in me that I lacked a great deal. In addition to excellent guidance in research and academic work, his extremely understanding nature has further increased my respect towards him. Whether it were those difficult times when I had to make two or more emergency visits per week to the hospital, or my theme “last minute shocks”, he never showed any disappointment or frustration, but provided me whatever it took to cope with those situations. Had I not been blessed with such a PI, I wouldn’t have gotten the chance to be where I am now.

I am also very grateful to Dr. Eric Mintz for his immense help and guidance. I appreciate his answers for all types of problems from technical, research related to statistical ones, and for assurance through ups and downs of graduate life. I just hope every institution has someone like him who is there to deeply care about students. I would also like to thank Dr. Fraizer for her help with molecular biology techniques and protocols, and consistent feedback for my research. Additionally, I thank Dr. Caldwell for her guidance

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and her additional help outside of the research. I am also grateful to Dr. Tsai for his valuable feedback on my research.

I cannot imagine being able to finish my PhD degree without continuous support from my husband, Dr. Gyanu Acharya. He has always put me first and has provided me all opportunities I would wish for. Most importantly, he has sacrificed excellent career opportunities trying to make sure I am not deprived of any. He has been the best dad for our two boys and has given them love and care above and beyond any limits. I am thankful to two stars of my eyes, Aditya and Adwait. They are the main source of my energy and inspiration every day.

I am extremely indebted to my father, Ram Mani Duwadi and my mother, Amrita

Duwadi for EVERYTHING they have done to me. No words will be sufficient to thank them enough! I am also grateful to my grandparents, especially to my grandmother, who came all the way here and stayed with me for almost a year to make sure that I could continue my research without any interruption. I also want to extend my thanks to my younger brothers Kapil and Kanchan for their support and love.

I would also like to extend my gratitude to past and present colleagues in Veney lab.

Specifically, Andie and Andrew have been true friends, and have been there for me through good and rough times. I would like to extend my thanks to Judy for her help

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with not only department and course related issues but being there for me as a great friend.

DEDICATION

I would like to dedicate this dissertation to my mother whom I miss every day and wish that I could share all my joys and sorrows with her!

OVERALL ABSTRACT

Neural dimorphisms in the songbird represent one of the most extreme examples of sex differences observed in vertebrates. Interestingly however, despite their identification more than four decades ago, information on the exact mechanisms related to how they arise is very limited. In this dissertation, I provide evidence supporting a potential role of the membrane bound G-protein coupled estrogen receptor GPR30, in estrogen- mediated dimorphic brain development of the zebra finch. In particular, this receptor may directly regulate sex differences within song nucleus HVC. Since brain dimorphisms are not completely dependent on sex steroids, the second part of my dissertation identified possible gene candidates that may also be involved. More specifically, I present evidence for a potential role of the Z-linked ribosomal gene rpS6 in the development and maintenance of dimorphic features in song nuclei HVC and RA where it is enhanced in males than in females. I sum up this dissertation by providing evidence that the function of rpS6 is likely modulated by estrogens, as evidenced by its up-regulation in the presence of estradiol. Taken together, my work provides a model for how genetic and hormonal factors may function to affect neural dimorphisms in the zebra finch.

CHAPTER I

INTRODUCTION

SEXUAL DIFFERENTIATION OF THE VERTEBRATE NERVOUS SYSTEM

Sex differences in the central nervous system are common features among vertebrates

(Cooke et al., 1998; Simerly, 2002; Morris et al., 2004). For example, humans, primates, rodents, and marsupials express dimorphisms in neuronal morphology, physiology and neurochemistry that directly correlate with sex-specific behaviors. Many of these neural differences are permanently organized by exposure to different levels of sex hormones during the perinatal period of development. The sex hormones are primarily secreted from the gonads, which initially develop based on which sex chromosomes are present.

In mammals, X and Y are the sex chromosomes; with males being the heterogametic sex

(XY) and females the homogametic sex (XX). Located on the Y chromosome is a sex- determining region Y (sry) that is necessary and sufficient for the formation of testes from an initially bi-potential gonad (Goodfellow and Lovell-Badge, 1993). In females, the absence of the Y chromosome, and thus the absence of the sry gene, initiates the development of ovaries. Soon after the testes are formed (around embryonic (E) day 14-

15 in mice (Karl and Capel, 1998) and rats (Tapanainen et al., 1984; Paranko et al.,

1986), and during the 7th-8th week of gestation in humans (Tapanainen et al., 1984) they

produce androgens; primarily testosterone (T) and its metabolite dihydrotestosterone

(DHT). Both hormones are released into the general circulation where they act to further amplify the masculinization of the partially differentiated gonad. Whereas formation of the testes and development of the associated male reproductive structures are completely masculinized by these hormones, the brain is not. In many mammals including rodents,

T can masculinize some parts of the brain, however the vast majority of masculinizing effects occur after its conversion into estradiol (E2); a reaction mediated by the aromatase enzyme. Aromatase is abundantly present in the brain and mediates the local production of E2 from T (Naftolin and MacLusky, 1984; Lephart, 1996). The resulting estradiol can then bind to estrogen receptors. While T serves as a primary precursor, some androgens, like DHT, cannot be converted into estrogens. Comparatively speaking, in females the ovaries are quiescent during this same period, so the lack of androgens and estrogens results in feminine brain and gonadal development (Figure 1.1).

HORMONE MEDIATED BRAIN DIMORPHISMS

There are several examples of sex differences in brain morphology and physiology that develop due to the actions of steroid hormones. One such nucleus in the rodent brain, for example, that is masculinized by estrogens is a specific region in the medial pre-optic area of the hypothalamus. This region contains more neurons, and is larger in males than in females (Raisman and Field, 1971, 1973). Later, Gorski and colleagues identified a similar region in rats that contained densely packed neurons and was many times larger in

Figure 1.1. Flowchart representing the development of brain sex differences in mammals as a combined effect of genetic and hormonal sex that gives rise to phenotypic sex.

males than in females, thus appropriately named it the sexually dimorphic nucleus of the the pre-optic area (SDN-POA), (Gorski et al., 1980). An analogous nucleus to the

SDN-POA was also identified in quails and was named as the medial preoptic nucleus

(POM) (Panzica et al., 1987). Since its initial discovery in rats and quails, this nucleus has additionally been described to be larger in other male vertebrates including ferrets

(Tobet et al., 1986), gerbils (Commins and Yahr, 1984), guinea pigs (Hines et al., 1985), and humans (Hofman and Swaab 1989; Allen et al., 1989). The SDN-POA is required for copulatory behaviors in male rats, gerbils and quails (De Jonge et al., 1989; Balthazart and Surlemont, 1990; Balthazart et al., 1990; Yahr and Gregory 1993), but its specific role in other mammals in which it has been identified is not clear (reviewed in Cooke et al., 1998). Administration of E2 or testosterone propionate (TP; an analog of T) shortly after birth masculinizes the volume and the size of neurons within the SDN-POA of females, whereas gonadectomy on the day of birth (Jacobson et al., 1981) or inhibition of aromatase in males (Ohe, 1994) feminizes the nucleus. The inability of the non- aromatizable androgen DHT to masculinize this region in either sex (Korenbrot et al.,

1975), combined with the fact that Tfm (testicular feminization mutation) male rats who do not express functional androgen receptors have a normal sized nucleus (Jacobson,

1980) indicate the exclusive role of estrogens, but not androgens in the masculinization of the SDN-POA.

Although estrogens are mainly responsible for masculinizing many regions of the brain, in some areas, these hormones actively feminize (make smaller) morphological features in other brain areas. For example, the anteroventral periventricular region of the hypothalamus (AVPV), which is larger and has more dopaminergic neurons (containing its precursor, tyrosine hydroxylase; TH) in females as compared to males (Simerly et al.,

1985), contains dense expression of estrogen receptors, but no androgen receptors.

Exposure to TP (acting on estrogen receptors) during the neonatal period reduces the number of TH immunoreactive (ir) neurons (Simerly et al., 1985), and the size of the nucleus (Davis et al., 1996) in female rats. A similar effect on TH immunoreactivity was observed in the AVPV of Prairie voles after the administration of TP or estradiol benzoate (EB) during the first week after birth (Lansing and Lonstein, 2006). The number of TH-ir neurons is significantly increased in estrogen receptor α knockout male mice (ERαKO) as compared to wild type males. Moreover, there is no difference in the number of TH-ir cells in the AVPV of Tfm males and wild type males (Simerly et al.,

1997), all suggesting that the hormonal effect in this region is purely estrogenic.

As it turns out, not all brain regions rely exclusively on E2 for masculinization or feminization. For example, the postero-dorsal region of the medial amygdala (MePD) and the posterior region of the bed nucleus of the stria terminalis (BNSTp) are both masculinized following exposure to androgens (aromatizable and non-aromatizable) or estrogens. The MePD is larger in volume, and contains more neurons with bigger soma sizes in male rodents than in females (Guillamon and Segovia, 1996; Cooke et al., 2003;

Morris et al., 2008a). This region has more androgen and estrogen receptors, as well as more aromatase containing neurons in male mice as compared to females (Simerly et al.,

1990; Wu et al., 2009). The MePD can be masculinized by androgens or estrogens

(Nishizuka and Arai, 1981), with the effect being larger when both hormones are used in combination (Cooke et al., 2003). Although a majority of the hormonal effects in this region occur during the organizational period of development (perinatally), either E2 or androgens can further masculinize the MePD later in life (Cooke et al., 1999), with the effect of E2 being larger as compared to non-aromatizable androgens (Cooke et al., 2003).

The size of MePD in adults changes with the fluctuation of sex hormones suggesting the activational effects to be transient (Morris et al., 2008b).

The BNSTp is another sexually dimorphic nucleus that is larger and contains more vasopressin (AVP) expressing neurons in male rats than in females (Del Abril et al.,

1987). Similar to the MePD, it also contains more androgen and estrogen receptors in males, and can be masculinized by the perinatal actions of androgens or estrogens (Hines et al., 1985; Guillamon et al., 1988; Han and De Vries, 2003). Whereas castration of male rats on the day of birth decreases the number of AVP-ir neurons to the levels typical of females, either TP or DHT administration during early postnatal period restores it to normal male levels (Wang et al., 1993). AVP neurons in BNSTp are also responsive to sex hormones in adulthood since gonadectomy reduces AVP expression and replacement of TP reinstates it during this period (De Vries et al., 1984). Taken together, these results indicate that androgens or estrogens can masculinize at least two regions of the brain, the

BNSTp and the MePD, with a larger masculinizing effect occurring when these regions are exposed to both hormones.

Thus far, I have discussed how E2 has both masculinizing and feminizing effects on the brain. I also talked about how some brain regions are masculinized by the actions of androgens or estrogens; or the additive effect of both. Under a third scenario, studies have demonstrated that there are nuclei in the CNS that are masculinized only by androgens and not estrogens. An example of such a case is observed in the sexually dimorphic spinal nucleus of bulbocavernosus (SNB). Motoneurons from this nucleus innervate perineal muscles bulbocavernosus (BC) and levator ani (LA), and are required for male copulatory behavior. The SNB is present in both sexes, and is monomorphic up until early postnatal ages. At that point, in females, the nucleus and target muscles significantly shrink in size due to the absence of androgens (Breedlove, 1992; Forger et al., 1992). In males, the nucleus and muscles grow. The SNB can be restored in females if they are treated with TP (which activate androgen receptors) or DHT within the first week after birth (Breedlove and Arnold, 1983). In Tfm males, the SNB is greatly reduced comparable to females, suggesting that the effect of hormones on the nucleus is exclusively mediated by androgens (Breedlove and Arnold, 1981). The dendrites of SNB neurons as well as target muscle of this nucleus in males can also be increased by androgens during adulthood (Rand and Breedlove, 1995).

To sum up, region-specific responses are observed in the nervous system that may be regulated by estrogens only, androgens only, or by the exposure to both groups of hormones. Collectively, they all provide evidences for the hormonal regulation of brain dimorphisms. However, some brain regions in mammals as well as some non- mammalian vertebrates display dimorphisms that do not appear to be regulated by sex hormones.

NON-HORMONAL REGULATION OF BRAIN DIMORPHISMS

Dimorphisms that arise and are maintained in the absence of the sex hormones, or ones that cannot be altered or reversed by pharmacological manipulation of hormones during the critical period of brain development, suggest the role of non-hormonal factors in the process. There are several dimorphisms that have been described in mammals which occur prior to the formation of gonads, and thus likely occur independent of sex hormones. For example, embryos of male rats, mice and cattle are bigger than those of females prior to hormone production from the gonads (Scott and Holson, 1977; Tsunoda et al., 1985; Gardner and Leese, 1987; Avery et al., 1991; Xu et al., 1992). Similarly,

TH-ir neurons in the diencephalon of male rats at E14 (before the brain is exposed to a sexually dimorphic level of sex steroids) are larger in males than in females, and are not affected by manipulations of either T or E2 (Beyer et al., 1991). Instead, TH expression in these neurons can be decreased by down-regulating the sry gene, suggesting the difference to be created by sex-linked gene(s) (Beyer et al., 1991; Dewing et al., 2006).

In general, sry and many other sex-linked genes are differentially expressed in the brain

of embryonic male and female mice before the formation of gonads, and thus may play an essential role in dimorphic brain development (Dewing et al., 2003).

ZEBRA FINCH: A MODEL FOR SEXUAL DIMORPHISM

In light of the possibility that hormones are not the only factors that are important for brain dimorphisms, the central question becomes what are the specific genes directly regulating this process? To address this question, zebra finches have become a very useful model. They exhibit one of the most extreme sex differences in brain morphology and behavior among all animals. In this species, there is a group of sexually dimorphic nuclei that are more robust in males, and are responsible for the male-exclusive singing behavior that females are not normally capable of producing (Gurney and Konishi, 1980;

Goldman and Nottebohm, 1983; Wade, 2001; Wade and Arnold, 2004). These nuclei are interconnected to form two functional circuits (Nottebohm and Arnold, 1976; Gurney,

1982; Konishi and Akutagawa, 1985). One circuit is responsible for song learning and the other for song production. The song learning pathway consists of telencephalic nucleus HVC (used as a proper name), which is connected indirectly to the lateral magnocellular nucleus of the anterior nidopallium (LMAN). The song production pathway consists of HVC connected to the robust nucleus of arcopallium (RA) that sends projections to the vocal organ (syrinx) via the tracheosyringeal branch of the hypoglossal nucleus (nXIIts). The motor nucleus HVC gets auditory feedback from two nuclei; uvaeformis nucleus (Uva) and the nucleus interface of the nidopallium (Nif) (Nottebohm

et al., 1976; Bottjer et al., 1985; Konishi and Akutagawa, 1985; Figure 1.2). There are dimorphisms at several levels within the song circuit. For example, the volumes of HVC,

RA, LMAN and nXIIts are bigger in males than females. This is partially due to a greater number, size and spacing between neurons within these nuclei. Area X can only be identified in males with a Nissl stain (Nottebohm and Arnold, 1976). And the synaptic connections between HVC and RA are more robust in males (Konishi and

Akutagawa, 1985).

Hormonal regulation of zebra finch brain differentiation

When zebra finches were first used in experiments related to brain development, they were investigated to determine if hormones played a similar role in brain dimorphisms as was observed in mammals. To test this, estrogens as well as androgens were manipulated.

Based on the studies in which DHT or TP administration to post-hatching females did not significantly masculinize the song system, the role of androgens was arguably determined to be less important for masculinization of the song system (Gurney ME, 1981; Schlinger and Arnold, 1991).

In contrast, E2 does significantly masculinize the brain of female zebra finches. When females are treated with elevated pharmacological doses within the first few weeks after hatching, they develop a partially masculinized song circuit. This is marked by an increase in the volume of song regions, an increase in the number and size of neurons within these areas as well as the formation of Area X, which is normally not detectable in

Figure 1.2. Schematic drawing of the song control regions. The black arrows connect the posterior forebrain (motor) pathway, the white arrows show connections within nuclei of anterior forebrain (song learning) pathway and the dotted lines connect the two pathways. HVC: acronym used as the proper name; RA: robust nucleus of the arcopallium; X: Area X; DLM: dorsolateral nucleus of the medial thalamus; LMAN: lateral magnocellular nucleus of anterior nidopallium; nXIIts: the tracheosyringeal portion of the hypoglossal nucleus; Uva: nucleus uvaeiformis; NIf: nucleus interface of the nidopallium. (Reproduced with some modifications from Reiner etal., 2004).

females (Gurney and Konishi, 1980; Gurney, 1981; Simpson and Vicario, 1991; Grisham et al., 1994; Adkins-Regan et al., 1994). Interestingly, even at the highest doses, the morphological effects in treated females are still less than what appears in normal males, suggesting that other factors are likely involved. The effects of E2 in females are organizational, and are limited to early development. The efficacy of this hormone on the song system is diminished by post-hatching day (P) 30, and has even less effect on the brain after P45 (Konishi and Akutagawa, 1988; Simpson and Vicario, 1991).

Based on the masculinizing effects of E2 in females, it can be predicted that the male brain would be demasculinized when deprived of the hormone, similar to what occurs in many regions of the mammalian brain. Curiously, most studies have reported that preventing the synthesis or action of gonadal E2 is not sufficient to demasculinize the song system (Wade and Arnold, 1994; Balthazart et al., 1994; Wade et al., 1999). In particular, neither castration at or shortly after birth, nor the exposure to aromatase inhibitors (fadrozole or vorozole) were sufficient to cause a significant demasculinization

(Wade and Arnold 1994, Foidart and Balthazart, 1995). Similarly, attempts to block E2 action by using antagonists such as tamoxifen, LY117018 or CI628 also have been unsuccessful (reviewed in Wade and Arnold, 2004). In contrast, results from our lab have demonstrated demasculinization in some features of the song system when the brain was deprived of E2. In one study, intracranial injections of the estrogen receptor (ERα) antagonist, ICI 182,780 for 25 days after hatching resulted in a reduction of neuron soma sizes in both sexes (Bender and Veney, 2008). In an additional study, intracranial

administration of the aromatase inhibitor fadrozole decreased neuron soma size as well as the nuclear volumes of HVC and RA in both males and females (Bender and Veney, personal communication). Although the reasons for the discrepancies between studies are not clear, they may be related to the type of compound used, the dosage, or the route of drug administration. Regardless, even in work from our lab where limiting estrogens did result in significant demasculinzation of the brain, the effects were not detected globally throughout all song control regions, suggesting that other factors are likely involved. And generally speaking, the large number of inconsistencies across studies from other labs makes it difficult to even understand how hormones normally act in this system.

The observed sex differences in response to E2 can be expected to arise as a result of differences in the amount of hormone in the system, or differences in the number of its receptors. In zebra finches, there have been no detectable sex differences in plasma levels of this hormone during early post-hatching development when the brain is in the process of differentiation (Hutchison et al., 1984; Adkins- Regan et al., 1990; Schlinger and Arnold, 1992). Additionally, the available information on estrogen receptors is not conclusive. In particular, the nuclear receptors for estrogens (ERα and ERβ) are the only ones that have been investigated in the zebra finch brain to date. The distribution of ERα mRNA has been described at various embryonic, post-hatching and adult ages (Jacobs et al., 1996; Jacobs et al., 1999; Perlman and Arnold, 2003; Saldanha and Coomaralingam,

2005). In general, labeling was detected in several telencephalic regions. More

specifically, within the song circuit, HVC contained abundant ERα positive cells from

P10- P25. RA contained a substantially fewer number, but only at P25 (Jacobs et al.,

1999). At embryonic ages, song control nuclei are not yet formed. However, areas that are destined to develop into song regions were devoid of labeling (Perlman and Arnold,

2003). In adults (greater than 100 days of age), HVC was the only song control nucleus that contained labeling. Interestingly, at no age was a sex difference in ERα reported, and no study has detected ERα positive cells in LMAN or Area X.

In comparison to ERα, ERβ mRNA has not been as extensively investigated in zebra finches. At late embryonic (E30, E34) and an early post-hatching age (P1) ERβ mRNA was detected in several telencephalic regions, but without a sex difference. There was no indication that areas destined to develop into song regions contained labeling.

Preliminary studies from our lab revealed that there is no sex difference in ERβ gene in the telencephalic extract of post-hatching zebra finches. To our knowledge, ERβ has not been described in detail at later ages in the zebra finch, but in the closely related

European Starling, its mRNA is not found in song regions of adults (Bernard et al.,

1999). Thus, a lack of a sex difference in ERα or ERβ suggests the possibility of the presence of additional estrogen receptor(s) in the brain.

Genetic regulation of zebra finch brain differentiation

The partial role of E2 in the masculinization of the female brain, and the lack of clear sex difference related to plasma hormone levels, receptor availability and distribution, all additionally suggest the possibility of other mechanisms in the regulation of differentiation of the zebra finch song system. One possibility is that sex-linked genes may direct the earliest occurring dimorphisms that may be further enhanced by the action of hormones. Support for the genetic regulation of song system development is provided by a naturally occurring gynandromorphic zebra finch that displayed male typical features on the right half and female typical features on the left half (Agate et al., 2003;

Figure 1.3). Avians have Z and W sex chromosomes, and unlike mammals, females are heterogametic (ZW) and males, homogametic (ZZ). The hermaphrodite brain displayed a split genotype as verified by twice the expression of a Z-linked gene pkc-iz in the right half of the brain as compared to the left. The expression of W-linked genes chd1w and asw was restricted to the left half of the brain (Figure 1.3B and 1.3C). This finding is consistent with a lack of dosage compensation in the avian Z chromosome, which has been further verified by examination of their regulatory regions (Itoh et al., 2007; Warren et al., 2010). In addition to the laterality in genotype, the right brain contained a significantly larger HVC than on the left side, suggesting a clear effect of Z-linked genes on masculine morphology. Although sex-linked genes seemed to have the major affect on the song nucleus development, the presence of slightly larger song nuclei in the female half (left) as compared to normal females, suggest some hormonal influence on the dimorphism.

Figure 1.3. A gynandromorphic zebra finch with the male-specific plumage, zebra stripes, and orange cheek patch on the right side, and female specific plumage without the cheek patch and the stripes on the left side (A). Hybridization signal for a W-gene asw (B), and for a Z-gene pkci-z (C) representing a split genotype on two halves of the brain (obtained from Agate et al., 2003).

The recent availability of the zebra finch genome has provided additional insights into genetic regulation of brain dimorphisms by providing information about the chromosomal location of genes, their promoters, and regulatory sequences. In avians, unlike in mammals, a homologue for the mammalian sry gene has not been identified.

Instead, a completely different mechanism than in mammals seems to be responsible for the genetic regulation of dimorphisms. Specifically, a lack of dosage compensation (Itoh et al., 2007; Warren et al., 2010) suggests that Z-linked genes are expressed in a double dose, and therefore may directly contribute to the masculinization of males. Recent works have identified some candidate genes. Some examples include trkB (neurotrophin receptor; Chen et al., 2005), ribosomal proteins (rp) L17 and L37 (Tang and Wade,

2006), and mitochondrial rpS27, which other labs are currently following up on. In each case, expression of these genes is enhanced within specific song nuclei of developing males as compared to females. Given the likelihood that these may not be the only candidates required for zebra finch brain masculinization, as part of this dissertation, I have investigated additional sex-linked genes that may be required for sex-specific neuronal development.

OVERALL AIMS

The existing evidence in zebra finches collectively suggests a combined role of genes and hormones in the regulation of differential brain development. My dissertation work focused on identifying hormonal and genetic components that are actively involved in this process during various stages of early development, and examined if there is an

interaction between them. I have put forth following aims to better understand the regulation of sexual dimorphism in the brain.

1. To determine if GPR30 gene and protein expression are dimorphic in the zebra

finch brain.

2. To use differential display reverse transcription PCR as a tool to identify

differentially expressed genes in the telencephalon of early post-hatching male

and female zebra finches.

3. To examine the expression and distribution of rpS6 in the zebra finch brain and

investigate if the gene expression is affected by estrogens.

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CHAPTER II

CHARACTERIZATION OF THE G-PROTEIN COUPLED MEMBRANE

BOUND ESTROGEN RECEPTOR GPR30 IN THE ZEBRA FINCH BRAIN

REVEALS A SEX DIFFERENCE IN GENE AND PROTEIN EXPRESSION**

HYPOTHESIS: GPR30 GENE AND PROTEIN ARE DIFFERENTIALLY

EXPRESSED IN THE DEVELOPING BRAIN

ABSTRACT

Estrogen induced structural dimorphisms exist in the songbird brain and correlate with sex differences in singing behavior. How they arise, however, is not clear since there is a scarce distribution of ERα and lack of ERβ in song control nuclei. This suggests that other receptors are involved. The G-protein coupled membrane bound estrogen receptor,

GPR30, is a candidate but has never been investigated in songbirds. In this study, we characterized its gene and protein in the zebra finch brain. Analysis of the putative

GPR30 protein sequence revealed a strong similarity to avian and mammalian homologues. Quantitative PCR indicated that the gene was elevated in the telencephalon

** Reproduced with permission of John Wiley and Sons Publishing, USA, as it appears in Journal of Developmental Neurobiology (Epub: DOI: 10.1002/dneu.22004) by K.D. Acharya & S.L. Veney.

of both sexes from posthatching day (P) 15 to P45, with a male biased sex difference at

P21 and P30. In comparison, expression at younger post-hatching ages and in adults was significantly less. At P21, GRP30 protein was widespread, non-uniform, and overlapped with song control nuclei. Of particular interest, the number of immunoreactive cells was greatest in HVC and RA, but less in LMAN and Area X. Labeling in HVC was also dimorphic; with more cells present in males than in females. In parallel with the gene, by adulthood, protein expression was also reduced across most brain regions. Taken together, these data suggest that GPR30 may contribute to differences in song system development by mediating dimorphic estrogen responses. In addition, the extensive protein distribution indicates that it may also have a role in general brain development in both sexes.

INTRODUCTION

Zebra finches are widely studied as a model for understanding how sex differences in brain anatomy and physiology develop and control reproductive behaviors (Gurney and

Konishi, 1980; Goldman and Nottebohm, 1983; Wade, 2001; Wade and Arnold, 2004).

In this species males produce a courtship vocalization (song) that females are not normally capable of producing. This singing behavior is controlled by a sexually dimorphic group of interconnected nuclei within the telencephalon that are larger and contain more neurons in males as compared to females (Nottebohm and Arnold, 1976;

Gurney, 1982; Konishi and Akutagawa, 1985). The factor(s) that direct these brain

regions to become dimorphic are not known, but may in part depend on steroid hormones. In particular, estradiol (E2) has been most effective at masculinizing the song system in females (Gurney and Konishi, 1980; Gurney, 1981; Simpson and Vicario,

1991; Grisham et al., 1994; Adkins-Regan et al., 1994). Interestingly, most attempts to demasculinize the male brain by blocking the formation or action of E2 have not been successful (Arnold, 1980; Mathews et al., 1988; Mathews and Arnold, 1990; Balthazart et al., 1994; Wade and Arnold, 1994). However, in our hands, intra-cranial administration of the estrogen receptor antagonist ICI 182,780 did result in a significant reduction of neuron soma size in both sexes (Bender and Veney, 2008). Although the reason(s) for the discrepancy across studies is not clear, one cannot ignore the E2 manipulations that were effective. The major question, however, is how do estrogens normally act to influence brain sex differences?

In songbirds the nuclear receptors for estrogens (ERα and ERβ) are the only ones that have been investigated to date. The distribution of ERα mRNA and protein has been described in zebra finches at various embryonic (E), post-hatching (P) and adult ages

(Gahr and Konishi, 1988; Jacobs and Arnold, 1996; Jacobs et al., 1999; Perlman and

Arnold, 2003). In general, signal was detected throughout several telencephalic regions.

Within song control nuclei, HVC (proper name) contained a high amount of labeling from P10 into adulthood. Labeling in the robust nucleus of arcopallium (RA) was detectable only at P25, and was considerably less than that in HVC (Jacobs et al., 1996;

Jacobs et al., 1999). In neither of these regions was a sex difference reported. The lateral

magnocellular nucleus of the anterior nidopallium (LMAN) and Area X did not contain any ERα positive cells. Compared to ERα, ERβ mRNA has not been as extensively investigated in zebra finches. At a late embryonic stage (E34) and an early post-hatching age (P1) ERβ mRNA was detected in a number of telencephalic regions, however, song control regions are not yet formed and there was no indication that areas destined to develop into song regions contained labeling. To our knowledge ERβ has never been described at later ages in the zebra finch, but in the closely related European Starling, its mRNA is not found in song regions (Bernard et al., 1999).

Based on this information, it does not seem obvious that ERα or ERβ would be primarily responsible for dimorphic estrogen responses in song nuclei. Therefore it is likely that other estrogen receptor(s) are involved. One possibility is GPR30, a newly characterized

G-protein coupled membrane receptor that binds to a stimulatory G protein (Gs) and activates several downstream pathways. It was first identified in breast cancer cells, and is known to play a role in estrogen mediated responses (Filardo et al., 2002; Thomas et al., 2005; Brailoiu et al., 2007; Hazell et al., 2009; Lebesgue et al., 2009). It has also been reported in estrogen-responsive regions of the rat, mouse, and human brain, several of which contain limited ERα and ERβ (Brailoiu et al., 2007; Hazell et al., 2009;

Lebesgue et al., 2009). Unfortunately, due to the lack of information on membrane receptors in zebra finches, it is unclear how/if GPR30 contributes to dimorphic brain development. As a first step towards exploring this, we first identified the gene and

examined its expression at select ages from hatching through adulthood. We also characterized the protein distribution in the juvenile and adult brain.

MATERIALS AND METHODS

Animals

Subjects used in this study were obtained from our animal facility at Kent State

University. Zebra finches were housed in communal aviaries, each containing 6-7 pairs of adult breeders held on a 14:10 light: dark cycle. Their diet consisted of finch bird seed and weekly supplements of hard boiled chicken eggs mixed with bread, fresh oranges or spinach. Food and water were available ad libitum. Adequate measures were taken to minimize pain and discomfort. All procedures were in accordance with Kent State

University’s Institutional Animal Care and Use Committee and conformed to NIH national guidelines.

RT- PCR

Total RNA was extracted from the telencephalon of P21 zebra finches (n=1 each sex) using the RNeasy Mini kit (Qiagen). This age was chosen because it represents a time when the song control nuclei are visible and show a robust response to E2 (Gurney and

Konishi 1980; Bottjer et al., 1985; Nordeen et al., 1986). We reasoned that if GPR30 is relevant to this system its expression should be abundant during this age. The quantity and quality of RNA was checked on a spectrophotometer and visualized on an RNA gel.

It was reverse transcribed (RT) into cDNA with the High Capacity cDNA Reverse

Transcription kit (ABI), and then amplified by performing PCR (GPR30 forward primer:

5’-GTGAGAAGATGACTATCCCAGACCT-3’and GPR30 reverse primer: 5’-

AGACCTCTGATTGCTCATTGCT-3’). At the time this work was started, the zebra finch genome was not available and no information existed about GPR30 in this species.

Thus the primers we used were designed based on the chicken GPR30 sequence. The

PCR product was cleaned using the Qiaquick PCR Purification kit (Qiagen).

Cloning and sequence analysis

The purified product was ligated into the pGEM- T Easy vector overnight at 4°C. The vector was then transfected into JM109 competent cells and grown on agar plates containing 100µg/ml of ampicillin, 40µg/ml X-gal, and 0.5mM isopropyl-β-D- thiogalactopyranoside (IPTG). Colonies that had incorporated the insert were picked and grown overnight at 37°C in Luria broth containing 100µg/ml of ampicillin. The plasmids were isolated (Wizard plus Miniprep DNA purification system; Promega), digested using a restriction enzyme, and visualized on an agarose gel to verify the correct size of insert prior to sequencing (Ohio State University, Plant Microbes Genomic Sequencing

Facility). The zebra finch GPR30 gene was translated into its amino acid sequence using

Translate tool (Swiss Institute of Bioinformatics). The protein sequences for human, mice, rat, and chicken GPR30 were aligned with zebra finch GPR30 protein using clustalw2 alignment. Pair wise alignment was performed using the blast program bl2seq to calculate the similarity between examined species.

Quantitative PCR

Total RNA was extracted from the telencephalon of P3, P8, P15, P21, P30, P45, and adult (n=6 males, n=6 females per group) animals using the RNeasy Mini kit protocol

(Qiagen). These ages were chosen as “snap shot” periods during development; ages when significant changes are occurring in the brain as described in Table 2.1. The cDNA synthesis was performed as previously explained. Negative RT controls were made for each sample by omitting the reverse transcriptase from the reaction. Quantitative PCR analysis was performed (ABI Prism 7000) using the SYBR Green PCR master mix kit with 20 ng of cDNA and 200 nM final primer concentration, in a total volume of 20 μl.

IDT primer tool, Primer Quest, was used to design the GPR30 forward (5'-

GTGAGAAGATGACTATCCCAGACCT-3') and the GPR30 reverse (5’-

ACCTCAATGAGAGAATCAGCGACT-3’) primers. The reactions were run in triplicate for each sample and primer combination. Amplification of GAPDH gene

(forward primer: 5’-TGTGGACCTGACCTGCCGTCTG-3’and reverse primer: 5’-

TGAAGTCACAGGAGACAACCTG-3’) was used as a loading control. Cycling conditions were 50˚C for 2 min, 95˚C for 10 min, and 40 cycles at 95˚C for 15 sec, and

60˚C for 1 min.

To confirm specific amplification of the GPR30 gene, negative RT controls and no template controls were used. Negative RT controls were synthesized by incubating RNA

Table 2.1: Ages chosen for q-PCR and their significance

with the reverse transcription reagents and omitting the reverse transcriptase enzyme. No template controls were run with only the primers. Dissociation curves were examined for both (the controls, neither of which showed any peaks, suggesting no genomic DNA contamination (negative RT) and no primer-dimer formation (no template controls).

Dissociation curves were also examined for the samples in which cDNA had been synthesized (RT positive) and showed a single peak of the expected size (85 bp long with a Tm of 75 ˚C) indicating the absence of non-specific amplification. Moreover, amplification of the GAPDH gene occurred 20 or more cycles later in the negative RT controls as compared to the RT positive samples further confirming the absence of genomic DNA.

The average δCt was obtained by subtracting the Ct value of GAPDH from the Ct value of GPR30. The relative expressions were calculated using the equation 2^- δCt values and were compared against P3 males whose expression was taken as 100%. Statistical analyses were performed using Sigma Stat. A Two Way ANOVA was conducted on relative expression values to examine the effect of age and sex on the gene expression.

The level of significance was determined at P<0.05.

Western blot analysis

To verify the specificity of the GPR30 primary antibody, Western analysis was conducted on telencephalic tissue from P21 animals (n=3; 2 males and 1 female). Brains were collected after rapid decapitation, homogenized using a glass mortar and pestle, and

placed in ice-cold RIPA lysis buffer containing protease inhibitor. The lysate was kept on ice for half an hour to allow complete lysis followed by centrifugation at 10,000 rpm for 10 minutes at 4˚C. The supernatant was collected and protein concentrations were quantified. 50µg of protein were electrophoresed on a 4-15% gradient Tris HCl gel (Bio-

Rad) and transferred onto a PVDF membrane, both for 1 hr at 100V. The membrane was exposed to 5% non-fat milk for half an hour at room temperature to minimize non- specific binding. The membrane was then incubated overnight at 4˚C with an anti-

GPR30 rabbit polyclonal antibody (LS-A4268; 1:2000; MBL International). It was then treated with a horseradish peroxidase conjugated goat anti-rabbit secondary antibody

(1:2500; Vector Labs) at room temperature for 1 hr. Immunoreactivity was detected by chemiluminescence (ECL, LAS-3000, Intelligent Dark Box, FujiFilm) after adding luminol mixed with H2O2 (10:1) to the membrane for 1 min. To further confirm the specificity of the antibody, a pre-adsorption control with 10-fold blocking peptide was also used (LS-P4268; MBL International). The control blot did not reveal any protein bands.

Immunohistochemistry (IHC)

P21 and adult zebra finches (n=5 males, n=5 females for each group) were used. We chose P21 for three main reasons. First, this was the age at which we initially identified the GPR30 gene. Secondly, song nuclei could be visualized so it would be clearly evident if GPR30 was expressed within each nucleus. Third, preliminary work indicated a sex difference in gene expression at this age and we wanted to verify if this was likely

to correspond to a functional difference at the protein level. Animals were deeply anesthetized with equithesin and perfused with 0.75% saline followed by 10% phosphate buffered formalin (PBF). The brains were post-fixed for 24 hrs in 10% PBF and cryo- protected overnight in 20% sucrose at 4˚C. Tissue was then instant frozen on dry ice and stored at -80˚C until processing. Brains were coronally cryo-sectioned into 30 µm slices, mounted onto gelatin-coated slides and once again stored at -80˚C until antibody labeling. Adjacent sections were mounted onto alternate slides. On the first day of IHC, tissue was warmed to room temperature followed by fixation in 4% formalin in 0.1M phosphate buffered saline (PBS) for 15 min. The slides were then rinsed 6X in 0.1 M

PBS for 5 min each, followed by a 15 min rinse in 0.5% H2O2 to inactivate endogenous peroxidases. To block non-specific binding, 10% donkey serum in PBS with 0.3% Triton

X-100 (PBS-T) was added to the sections for 1 hr. They were then rinsed 3X in 0.1 M

PBS, each for 5 min. The tissue was incubated with the GPR30 primary antibody (same as used for Western; 1:2500) for 48 hrs at 4°C. After exposure to the primary, sections were rinsed in PBS-T for 5 min, incubated with a biotinylated goat-anti rabbit secondary antibody (1:2000; Vector labs) for 1 hr, followed by another 1 hr exposure to avidin- biotin complex. The immunoreactive product was detected with diaminobenzidine. To confirm the specificity of the labeling, we omitted the primary antibody and used the pre- adsorbed primary with GPR30 blocking peptide (MBL International). The control IHC sections did not show any signal.

IHC analysis

Sections containing immunoreactive (ir)-GPR30 cells were viewed and digital images were captured at 20X. Since one major goal was to analyze GPR30 protein expression, we utilized thionin stained sections along with the canary atlas (nomenclature updated) to identify brain regions outside of the song control nuclei where labeling was present

(Stokes et al., 1974; Reiner et al., 2004). Because ir-cells were widespread, we opted to semi-quantitatively analyze the relative expression based on visual inspection (Veney et al., 2003; Hazell et al., 2009). To get an impression of the overall distribution, an observer blind to the sex of the animals ranked the intensity of labeling on a 4 point scale

(represented by different shades of gray). These ratings were designed to be approximately evenly divided over the intensity range. Within song nuclei, a quantitative analysis of labeled cells was also performed. A 0.01mm2 box was placed within each nucleus. This sized object fit equally within all song control nuclei of both sexes without extending beyond their borders. The analysis was performed bilaterally, using a single representative section at approximately the middle of each nucleus where the area was the greatest. Two way ANOVAs followed by Tukey-Kramer post-hoc tests were conducted on cell counts from HVC, RA and LMAN to test the effect of age and sex on

GPR30 immunoreactivity. Cell count data for Area X is reported, however it was not statistically analyzed because this region is not present in females (no sex effect) and in adults, this region is completely devoid of ir-labeling (no age effect).

RESULTS

Zebra finch GPR30 gene sequence

An 846 bp cDNA coding the partial open reading frame (ORF) of zebra finch GPR30 gene was originally identified (available in NCBI database under accession number

GU292793). It shared 90% homology to the chicken GPR30 gene and translated into a

281 amino acid long protein sequence. A few months after this work was completed, the zebra finch genome was published allowing access to the full GPR30 sequence (Warren et al., 2010). Alignment of the complete sequence with our partial sequence revealed a

99% similarity. This verified that the product we originally identified and used to make primers was in fact, GPR30. Pair wise comparisons of the 281 amino acid partial sequence revealed 93% identity and 96% similarity with chicken GPR30 protein. It also shared 80% identity and 88% similarity to human, and 81% identity and 87% similarity to both mouse and rat GPR30 protein (Figure 2.1).

GPR30 gene expression

Quantitative PCR revealed a significant effect of sex (F= 6.76, P=0.01), age (F= 52.48,

P<0.001) and an interaction (F=3.53, P=0.007) on GPR30 gene expression. At P21 and

P30, the gene expression was significantly greater in the male telencephalon as compared to that of females. Across the examined ages, GPR30 was elevated from P15- P45, with males showing the highest expression at P30. The lowest levels were detected in P3 and

Figure 2.1. Multiple alignments of GPR30 homologues of mouse, rat, human, zebra finch and chicken using clustalw2 program. Identical amino acids are denoted by “*”, conserved substitutions by “:” and semi-conserved substitutions by “.”

adult animals. At P8, the gene expression was greater than P3 and adulthood, but less than P15-P45 (Figure 2.2).

GPR30 protein expression

Western blot analysis revealed a 37 kDa band that corresponded to the expected molecular weight for GPR30 (Figure 2.3). We also detected a faint band of approximately 31 kDa. Blasting the antibody sequence to the protein database suggested that the 31 kDa band most likely represented nucleolar protein 9. Since this is a nuclear product and GPR30 is cytoplasmic, there were no issues with our ability to correctly identify positively labeled cells. Cells without a clear nucleus, which were rarely noticed, were excluded from the analyses.

IHC demonstrated that GPR30 protein was widely distributed throughout the zebra finch brain in both males and females. Based on the morphology of cells that were labeled by antibody, and reports indicating that a majority of cells in song control nuclei are neurons

(Kirn and DeVoogd, 1989) we strongly believe that GPR30 is primarily expressed in neurons. It is, however, not exclusive to neurons since some irregularly shaped cells

(characteristic of glia) were also positive for the antibody. For all analyses, to aid in the localization of signal, labeled tissue was compared against Nissl stained sections.

Because immunoreactivity was variable and detected in some regions that did not contain clearly defined borders, we opted to semi-quantitatively analyze the relative intensity and pattern of labeling (Figure 2.4).

Figure 2.2. Relative expression of the GPR30 gene in the zebra finch telencephalon during development and adulthood. The expression in P3 males was designated as100% and gene expression for the other groups was compared to this. Bars with different letters indicate significant differences across groups. Asterisks denote sex differences within an age. Error bars represent the standard error of mean.

Figure 2.3. Western blot confirming the specificity of the GPR30 polyclonal antibody. Arrow points to a 37kDa band corresponding to zebra finch GPR30. The faint band below GPR30, approximately 31kDa, is most likely nucleolar protein 9.

Figure 2.4. Camera lucida drawings representing the relative intensity of GPR30 immunoreactivity at P21. Darker shades of gray correspond to a greater intensity of labeling. Selected coronal sections are arranged rostral to caudal (top left to bottom right). The distribution pattern was nearly identical in both sexes with the exception of HVC where a sex difference was detected in protein labeling that was significantly less in females. Abbreviations: A, arcopallium; AA, anterior arcopallial nucleus; Bas, baso- rostral pallial nucleus; Cb, cerebellum; CoA, anterior commissure; E, entopallial nucleus; FA, fronto-arcopallial tract; GP, globus pallidus; HA, apical hyperpallium; HD, dorsal hyperpallium; HP, hippocampus; LMAN, lateral magnocellular nucleus of the anterior nidopallium; LSt, lateral striatum; M, mesopallium; MSt, medial striatum; N, nidopallium; NC, caudal nidopallium; RA, robust nucleus of the arcopallium; Rt, nucleus rotundus; TeO, optic tectum; Tn, nucleus taeniae of the amygdala; V, ventricle.

The densest GPR30 immunoreactivity was detected in HVC (Figure 2.4E), nucleus taeniae (Figure 2.4F) and the entopallial nucleus (Figures 2.4C and D). Additionally, the rostro-caudal extent of the hippocampus (Figures 2.4D and E), ventro-lateral portion of the caudal regions of the nidopallium and the fronto-arcopallial fiber tract (Figures 2.4E and F) also revealed dense labeling.

A moderate intensity of labeling was observed spanning the lateral border of the telencephalon, rostral to caudal, until approximately the level of the entopallial nucleus

(Figures 2.4A-C). Moreover, the rostro-caudal extent of the apical hyperpallium (Figures

2.4A-C), lateral striatum (Figures 2.4C and D), ventro-medial border of the medial striatum (Figure 2.4A and B), lateral and medial preoptic areas (Figure 2.4C), caudomedial nidopallium (NCM), RA, ventral border of arcopallium (Figures 2.4E and F), and the anterior commissure (Figure 2.4D) all contained a moderate intensity of ir-GPR30 cells. In addition, we also noticed a continuous band of moderately labeled cells outlining the lateral ventricles that was distinct from the surrounding telencephalon

(Figure 2.4A-C).

A low intensity of labeling was identified in the mesopallium, dorsal hyperpallium

(Figures 2.4A-C), globus pallidus (Figure 2.4C), optic tectum, a majority of the arcopallium (Figures 2.4E and F), dorso-medial portion of the nidopallium surrounding

HVC, and the caudal region of the lateral striatum (Figure 2.4E). The lowest intensity of

immunoreactivity was detected in LMAN, lobus parolfactoris including Area X (Figure

2.4A), cerebellum (Figures 2.4E and F), and a majority of the nidopallium.

Overall, the pattern of labeling in adults was similar to that at P21. However, there was a proportional decrease in the intensity as they aged, such that the regions containing dense labeling at P21 had moderate to low labeling in adults. Regions with moderate to low

GPR30 labeling at P21, expressed little to no labeling at the later age. Exceptions were noted in the hippocampus, entopallial nucleus and ventro-lateral nidopallium where the intensity of labeling did not differ between P21 and adults.

Song control nuclei

Quantitative analysis from a single representative section of HVC indicated a significant effect of sex (F=5.16, p =0.03) and age (F=77.73, p<0.001) but no interaction on the number of GPR30-ir cells (Figure 2.5). At P21, ir cells were distributed throughout this nucleus, appeared to define its borders (Figure 2.6A), and were significantly higher in males than in females (Figures 2.6B and 2.6C). In adults, protein expression was less as compared to the juvenile age and no sex difference was detected (Figures 2.6D and 2.6E).

Quantitative analysis from a single representative section of RA indicated a significant effect of age (F=123.07, p<0.001), but no effect of sex (F=0.14, p=0.71) (Figure 2.7).

Compared to HVC, the numbers of ir-cells in RA were fewer and the nuclear border was

Figure 2.5. Numbers of GPR30- ir cells counted within a 0.01mm2 area of HVC. Bars with different letters signify age differences across groups, whereas an asterisk denotes a sex difference within a group.

Figure 2.6. Photomicrographs of GPR30-ir cells in HVC. (A) represents protein labeling in a P21 male. (B and C) are magnified views in a P21 male and female, respectively, where a sex difference is observed. (D and E) are magnified views in an adult male and female, respectively, where no sex difference is evident. Arrows outline the border of HVC in (A) and in (B) they depict neurons with cytoplasmic labeling and a clear nucleus, which is consistent with GPR30 specific localization.

Figure 2.7. Numbers of GPR30- ir cells counted within a 0.01 mm2 area of RA. Bars with different letters signify age differences across groups. There is no sex difference detected in this region at this age.

not as distinctly defined, so Nissl stained sections were used to verify its extent (Figure

2.8A). There was a similar intensity of labeling in males and females at P21 (Figures

2.8B and 2.8C). In adults very few labeled cells were present in RA and these numbers were significantly less than animals at P21.

Similar to RA, GPR30 ir-cells in LMAN and Area X also did not define the borders of these regions, but instead overlapped with them. Using an overlay of Nissl stained tissue to identify the boundaries, an analysis of the number of ir-cells from the single representative section of LMAN revealed that there was an effect of age (F=40.8, p<0.001), but no effect of sex (F=0.09, p=0.76) or an interaction (Figure 2.9). Juvenile tissue contained more labeled cells than adults. Overall, there were fewer GPR30-ir cells in LMAN as compared to HVC and RA. In contrast to all other song control regions,

Area X contained the fewest number of ir-cells. At P21, males had an average of (3 + 1) cells counted from the single representative section and area, and in adults this nucleus was completely devoid of immunoreactivity.

DISCUSSION

This study verified and described the presence of GPR30 in the songbird brain. This membrane bound estrogen receptor is present at various post-hatching ages, as well as in adults. Our findings revealed that gene expression continuously increased from P3 to

Figure 2.8. Photomicrographs of GPR30- ir cells in RA. (A) represents protein labeling in a P21 male. Arrows outline the border of this nucleus. (B and C) are magnified views in a P21 male and female, respectively.

Figure 2.9. Numbers of GPR30- ir cells counted within a 0.01mm2 area of LMAN. Bars with different letters signify age differences across groups.

P15 in both sexes, and remained elevated until P45, before falling back to minimal levels at adulthood. Expression was greatest in P30 males as compared to all other groups. We also report a sex difference (greater in males than in females) that first becomes detectable around P21 and continues through at least P30.

Analysis of GPR30 protein at P21 demonstrated that receptor labeling was in agreement with gene expression. Labeled cells were widely dispersed throughout the brain and overlapped with several song control regions. Closer examination of song nuclei revealed that HVC had the greatest amount of labeling, and that the number of GPR30 positive cells was sexually dimorphic (greater in males than in females). The extensive distribution of GPR30 is consistent with the idea that it may be required for common aspects of general brain development and/or function in both sexes. However, the sex difference in gene and protein suggests that it may also have an increased functionality in males during a restricted period of development. Given the timing of the dimorphic expression, this receptor is most likely not the trigger of neural sex differences in zebra finches (since the first morphological differences appear around P12), but instead may regulate or direct mechanisms of this process once they have started (Nixdorf-Bergweiler

BE, 1996).

By adulthood, GPR30 gene and protein were significantly reduced as compared to animals at P21; but in a few regions (e.g. hippocampus, entopallial nucleus and the ventro-lateral portion of the caudal nidopallium) the intensity of expression remained the

same. Although it has not been investigated as extensively in male zebra finches, in females, the reduction in GPR30 parallels a decreased responsiveness to E2 as they age.

More specifically, masculinizing effects of E2 begin to diminish in females around P35, and have an even lesser effect on altering brain morphology after P45 (Konishi and

Akutagawa, 1988). Coincidentally, GPR30 gene expression also declines after P45.

Because there have been no reported changes in other estrogen receptors to account for this, we propose that the decreasing effects of E2 may be directly related to the number of

GPR30 receptors available.

To our knowledge this study is the first to document a sex difference in an estrogen receptor within the song system at any age. The male biased expression of GPR30 overlaps a period of accelerated brain differentiation marked by an increased amount of apoptosis that occurs in females (Nixdorf-Bergweiler BE, 1996; Kirn and DeVoogd,

1989). Because there is no sex difference in ERα (Jacobs et al., 1996; 1999), and ERβ is not present in HVC (Bernard et al., 1999), we hypothesize that the increased GPR30 protein in males contributes to dimorphic neural development by mediating E2 induced neuroprotection to maintain a greater number of neurons and a larger nuclear volume.

Support for the role of GPR30 in neuroprotection comes from a study where the agonist

G1, administered shortly after insult, was sufficient to protect CA1 pyramidal neurons in ovariectomized rats against global ischemia (Lebesgue at al., 2009). Complementary to this, pre-treatment with the GPR30 antagonist G15, abolished the neuroprotective effects

of E2 in cultured rat hippocampal neurons (Gingerich et al., 2010). In a more recent study, it was demonstrated that G15 was also able to block the neuroprotection of primary rat neurons by raloxifene (a selective estrogen receptor modulator) (Abdelhamid et al., 2011). The specific cellular mechanisms that occur in response to GPR30 mediated neuroprotection are not known in songbirds. However, in mammals, binding of

GPR30 by estrogens activates several signaling molecules and cascades. In particular,

GPR30 activation of phosphoinositol-3 kinase (PI3K) affects the cell proliferative kinase

Akt, and induces neuroprotection (Abdelhamid et al., 2011). Akt can induce cell survival by inhibiting phosphorylation of the pro-apoptotic protein BAD (Datta et al., 1997) or by the induction of pro-survival genes such as NF-κB (Romashkova and Makarov, 1999).

Additionally, GPR30 is known to up-regulate pro-survival genes bcl-2 and pro-caspase 3 in cortical neurons (Liu et al., 2011). Thus, by stimulating these cascades more in males,

GPR30 in the songbird brain may be directly contributing to neuron survival and dimorphic development of HVC.

Although the number of receptors counted in the representative section of RA at P21 was high, no sex difference was detected. However, we still believe that GPR30 may contribute to dimorphic development of this nucleus. Neurons from HVC make axonal connections with those in RA (Konishi and Akutagawa, 1985). A significance of HVC-

RA synapses was revealed by a study demonstrating masculinization of RA when estrogen receptors were activated exclusively in HVC (Meitzen et al., 2007). Such activation up-regulates brain derived neurotrophic factor (BDNF; Dittrich et al., 1999).

BDNF has a masculinizing effect on the songbird brain as confirmed by its infusion close to RA that increased soma size and neuronal density (Wissman and Brenowitz, 2009).

Blocking the activity of BDNF abolished this effect. Thus, we propose that an increased response to estrogens in the male HVC induces a greater production of BDNF that contributes to masculine development of RA.

In contrast to HVC and RA, LMAN and Area X contained the fewest number of GPR30- ir neurons at P21. Although these nuclei respond morphologically to E2 at this age, ERα and ERβ are absent (Jacobs et al., 1996; 1999; Bernard et al., 1999; Metzdorf et al., 1999) and GPR30 is minimal. Thus, dimorphic development of these regions most likely occurs through alternate mechanisms. One possibility is the presence of an unidentified estrogen receptor in these nuclei. Alternatively the effects of E2 on Area X may be indirect. Similar to that in RA, activation of GPR30 in HVC may also regulate development of Area X. In males, HVC sends projections to Area X and those neurons contain growth promoters such as insulin growth factor (IGF) II that enhance neuronal survival (Holzenberger et al., 1997). Additionally, regulation through cofactors is a third possibility, where the number of receptors does not have to be dimorphic but the response to hormones can be. An example is the co-activator RPL7/SPA, which has a greater expression in the song nuclei of males as compared to females (Duncan and Carruth,

2007). Knocking down this co-activator during early development decreased the volume of Area X (Duncan et al., 2009). Curiously, RPL7/SPA is reported as an ERα specific co-activator, but why and how it affects Area X is not clear since this nucleus is devoid of

this estrogen receptor. Although we can offer testable hypotheses about how E2 induced changes in Area X occur, it is less clear how the latter two possibilities, in particular, explain sex differences in LMAN. This nucleus does not receive a direct connection from HVC, and knocking down rpL7/SPA does not affect the volume of this nucleus

(Duncan et al., 2009). Thus it is not clear how/if GPR30 contributes to dimorphic development of LMAN.

In addition to song control regions, high but monomorphic expression of GPR30 protein was detected in many other areas of the brain including the hippocampus, entopallial nucleus and the NCM. Songbirds such as canaries express ERα in the hippocampus, whereas others such as the great tit have both ERα and ERβ in this region (Gahr and

Metzdorf, 1997; Hodgson et al., 2008). In contrast, there is some inconsistency in the literature about the expression of estrogen receptors in the zebra finch hippocampus.

With the exception of an early study from Gahr et al., (1993) who reported few ER-ir cells in this region (presumably ERα) others do not report the presence of ERα or ERβ

(Bernard et al., 1999; Jacobs et al., 1999; Metzdorf et al., 1999). Thus, the majority of the effects of E2 in the zebra finch hippocampus may depend upon GPR30. For example,

E2 enlarges the size of hippocampal neurons in zebra finches which is thought to be essential for facilitating spatial memory (Oberlander et al., 2004). And fadrozole treatment (estrogen synthase inhibitor) results in a reduced volume of the zebra finch hippocampus (Schlinger and Saldanha, 2005).

The entopallial nucleus is another brain area where we detected intense GPR30 labeling but to our knowledge does not contain ERα or ERβ. This nucleus is part of the visual processing pathways in birds and lesioning this nucleus abolishes their capacity to discriminate between conspecific individuals and spatial patterns (Karten and Hodos,

1970; Watanabe 1996; 2003). The fact that GPR30 expression in this region varied little between P21animals and adults suggests that estrogens play a major role in this sensory ability throughout life.

GPR30 may also play an important functional role in NCM. It is a major auditory processing center (Mello et al; 1992; Mello and Clayton, 1994; Pinaud and Terleph,

2008) that expresses abundant GPR30 as well as ERβ, but minimal ERα (Shen et al.,

1995; Saldanha and Coomaralingam, 2005; Jeong et al., 2011). NCM is essential for the recognition of and discrimination between the bird’s own song, tutor song and other conspecific songs (Gobes and Bolhuis, 2007; Pinaud and Terleph, 2008; Remage-Healey et al., 2010; Tremere and Pinaud, 2011). The selective responses to these songs are expressed either by increased neuronal firing in NCM (Stripling et al., 1997; Remage-

Healey et al., 2010) or by the increased expression of immediate early genes (IEGs) such as zenk, fos, and arc (Bailey et al., 2002; Mello et al., 2002; Tremere et al., 2009).

Neuronal activity in NCM can be regulated by E2. In response to this hormone, firing rate is amplified in both sexes. E2 can also regulate IEGs in NCM since blocking the hormone decreases their expression (Tremere et al., 2009). These physiological responses occur very rapidly, and although both ERα and ERβ can mediate rapid

responses in cells (reviewed in Raz et al., 2008), they are primarily regulated by membrane bound receptors. For that reason, we speculate that GPR30 plays an important role in these activities.

Interestingly, we also noted that GPR30 protein labeling was not uniform throughout each brain region. For example, moderate labeling was observed along the lateral borders of approximately the rostral half of the telencephalon whereas a decreased intensity of labeling was seen medially. Similarly, densely labeled cells were present in the caudal sections of the ventro-lateral nidopallium compared to the remaining nidopallium, which contained less labeling. Although it is not clear why this pattern exists in either area, the ventro-lateral nidopallium has been reported to contain dense aromatase immunoreactivity in male zebra finches (Balthazart et al., 1996), further supporting the functionality of GPR30 in this region.

In summary, GPR30 may contribute to dimorphisms in morphology by influencing structural differences in HVC, RA, and Area X through greater neuroprotection in males.

Although its function(s) in behavioral regulation is not known, it is tempting to speculate that it plays a role in song production. Early vocalizations of song begin in males around

P25 (Immelmann, 1969; Arnold, 1992). The extensive presence of GPR30 in HVC and

RA (a few days earlier at P21), is possibly required for the organization of the circuits necessary for song production. In contrast, minimal expression in LMAN and Area X indicate a lesser or non-existent role in song learning. In addition, GPR30 may also be

necessary for memories, including that of song, by acting within the NCM and hippocampus. The plentiful expression of this receptor in regions outside of the song system indicate that common estrogenic processes occurring during brain development may also be associated with GPR30. As a next step, follow up studies using gene knock- down or specific antagonists should be conducted to better understand the role of this receptor in sex-specific brain development and behavior.

ACKNOWLEDGEMENTS

We would like to thank Dr. Eric Mintz for statistical assistance. This work was supported by Kent State University laboratory start-up fund.

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CHAPTER III

USE OF DIFFERENTIAL DISPLAY REVERSE TRANSCRIPTION (DDRT) PCR

TO IDENTIFY DIFFERENTIALLY EXPRESSED GENES IN THE

TELENCEPHALON OF EARLY POST-HATCHING MALE AND FEMALE ZEBRA

FINCHES

HYPOTHESIS: SEX-LINKED GENES ARE DIFFERENTIALLY EXPRESSED

IN THE BRAIN DURING THE FIRST WEEK POST-HATCHING

ABSTRACT

Differential display reverse transcription (DDRT) PCR is a technique that allows for the identification of differentially expressed genes when microarray analysis is not available.

Random primers are used to amplify genes from a cDNA sample. The samples are run on a gel and bands of interest are excised, cloned, sequenced, and compared against known products in a gene database. Since the zebra finch genome was not published and microarray chips were not publicly available at the time this study began, I utilized this method to identify differentially expressed genes at two select ages during the first week

post-hatching. Based on telencephalic tissue from P3 and P8 males and females, 15 genes were found to have a sex-biased expression. Following cloning and sequencing, 12 of these differentially expressed products matched with known homologues in chicken

(the closest avian species in the database). The remaining three matched to hypothetical proteins, and thus are not reported here. Interestingly, approximately 50% of the genes identified were Z-linked suggesting not that these products may have roles in sex-specific processes.

INTRODUCTION

Zebra finches exhibit remarkable sex differences in brain and behavior

(Nottebohm and Arnold, 1976; Wade, 2001; Wade and Arnold, 2004). However, little is understood about the exact mechanisms responsible for such differences. As explained in the first chapter of this dissertation, sex-linked genes have been of great interest as one of the earliest factors that may be important for the initiation of neural sex differences. In support of this, several genes have been identified as being differentially expressed during the first few weeks after hatching; a time just prior to, or at the initial stages when dimorphic features are emerging (Veney et al., 2003; Duncan et al., 2009; Tomaszycki et al., 2009). Unfortunately, however, their exact function(s) are not known. Given the possibility that these are not the only products important for this process, the goal of this study was to identify additional differently expressed genes that may also have a role on this process. Since microarray chips were not an option (not commercially available for

zebra finches) at the time this study began, DDRT-PCR was used to identify genes that may be involved in dimorphic brain development.

DDRT-PCR is a technique that can be used for the detection of differentially expressed cDNAs from two or more samples (Liang and Pardee, 1992). One disadvantage is that it can give rise to the amplification of a large number of that do not represent true differentially expressed genes. However, modifications such as the use of longer primers or increasing the primer binding specificity by adding specific regulator sequences within primers have been helpful towards improving specificity of the technique.

MATERIALS AND METHODS

Animals

Subjects used in this study were obtained from our animal facility at Kent State

University. Zebra finches were housed in communal aviaries, each containing 6-7 pairs of adult breeders held on a 14:10 light: dark cycle. Their diet consisted of finch birdseed and weekly supplements of hard boiled chicken eggs mixed with bread, fresh oranges or spinach. Food and water were available ad libitum. Adequate measures were taken to minimize pain and discomfort. All procedures were in accordance with Kent State

University’s Institutional Animal Care and Use Committee and conformed to NIH national guidelines.

DDRT-PCR

Total RNA was extracted from the telencephalon of P3 and P8 juveniles (n=2 males, n=2 females per age) using the RNeasy Mini kit (Qiagen). We chose these ages because they represent a period prior to visible detection of song regions (P3), and when the nuclei are first identifiable with a Nissl stain (P8) (Bottjer et al, 1985; Gahr and Metzdorf, 1999).

The quantity and quality of RNA was verified. GeneFishing DEG Premix kit was used to synthesize cDNA and identify differentially expressed genes during the first week and early second week that may be necessary for the initiation of zebra finch brain dimorphisms. cDNA was synthesized by combining 3 µg (1 µg/µl concentration) of

RNA with 20 µM of oligo dT arbitrary primer (dT-ACP1; GeneFishing DEG Premix kit) in a total volume of 10 µl. The mixture was incubated at 80°C for 3 min in a thermal cycler. Next, 4 µl of 5X RT buffer, 2 µl of 5 mM dNTPs, 0.5 µl of 40 U/µl RNAse, 1 µl of 200 U/µl M-MLV reverse transcriptase, and water were added to make a total volume of 20 µl. Reverse transcription occurred at 42°C for 90 min, 94°C for 2 min and 4°C for 2 min. The obtained cDNA was stored at -20°C until use.

50 ng of cDNA, 2 μl of 5 μM arbitrary ACP (1-80), 1 μl of 10 μM dT-ACP2, and 10 μl of

2X SeeAmp master mix were combined in a total reaction volume of 20 µl (Gene fishing

DEG premix kit) and amplified under following conditions: 94°C for 5 min, 50°C for 3 min, 72°C for 1 min; 40 cycles of 94°C for 40 sec, 65°C for 40 sec, 72°C for 40 sec; followed by a final extension of 72°C for 5 min. The PCR product was combined with

DNA loading buffer and loaded onto a 3% agarose gel to run for 3 hours. The gel was

placed under UV light and bands of interest were excised. Genes that were expressed exclusively in one sex, or more strongly in one sex compared to the other, were of greatest interest. The chosen bands were cleaned using the PCR Cleanup Kit (Qiagen).

Cloning and sequencing

Purified products were ligated overnight at 4°C into the pGEM-T Easy vector. The vector was then transfected into JM109 competent cells and grown on agar plates containing 100µg/ml of ampicillin, 40µg/ml X-gal and 0.5mM isopropyl-β-D- thiogalactopyranoside (IPTG). Colonies with the insert were identified and grown overnight at 37°C in Luria broth containing 100µg/ml of ampicillin. The plasmids were isolated (Wizard plus Miniprep DNA purification system; Promega), digested using a restriction enzyme, and visualized on an agarose gel to verify the correct size of the insert. The insert was then sequenced using T7 and SP6 vector primers (Ohio State

University, Plant Microbes Genomic Sequencing Facility).

RESULTS

A large number of products were amplified by DDRT-PCR as detected on the gel.

Characteristically, some bands showed a difference in the intensity between the sexes and ages, whereas others were expressed similarly (Figure 3.1). This is very common with

DDRT-PCR since degenerate primers used for the amplification identify many more

Figure 3.1. Gel showing amplified bands using differential display reverse transcription (DDRT) PCR in P3 (n=2 per sex; first six lanes) and P8 (remaining four lanes; n=2 per sex). Females (F) and males (M) of same age were run together to allow accurate comparison. In this specific gel, none of the bands showed a consistent difference between sexes, thus, were not taken for further analysis.

products than genes with true differences. Such bands were not potentially dimorphic between sexes, and thus, were not examined further. Only those band products where a difference was consistent among samples were chosen for further examination. Based on the animals that were run, a total of 15 cDNA bands were expressed in a greater intensity in one sex as compared to the other, although the direction of expression was not same for all samples. Some of the amplified genes were expressed more in males, and others were expressed more in females such that the direction of the sex difference and the age at which a sex difference was detected, varied among genes identified. For example, bands corresponding to rpS6 gene were more intense in males than in females at both ages examined (Figure 3.2). These bands were extracted from the gel, cloned and sequenced for their identification. Of them, 12 matched to the genes that had high homology to chicken homologues I compared these products to the chicken genome because during the time of this study, zebra finch genome was not yet available. The genes identified to be expressed differently between the sexes are presented in table 3.1.

DISCUSSION

DDRT-PCR has been widely used to compare gene expression. The use of degenerate primers where different nucleotide combinations have been used to amplify genes is responsible for non-specific amplification of several products all of which may not be truly differentially expressed among tissues. In this study, I have identified 12 gene

Figure 3.2. Bands representing ribosomal gene S6 (rpS6) cDNA amplified using differential display reverse transcription (DDRT) PCR in P3 (n=2 per sex; first four lanes) and P8 (n=1 per sex; 5th and 6th lanes). Boxed area contains rpS6 bands (were identified after sequencing) in males (M) which are either faint or absent in females (F). Since degenerate primers were used to amplify these bands, extra bands are also seen in the gel.

Table 3.1: Differently expressed genes in the telencephalon of P3 and P8 males as identified by DDRT-PCR, their location on the chromosome and the direction of expression.

products that showed a sex biased expression between males and females at P3 and P8.

Although false positive rates are high with this technique, 50% (6) of the total examined genes are Z-linked suggesting that the products were specific and real. Nonetheless, further comparison using q-PCR will be necessary to verify the sex differences of these genes.

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Grisham W, Mathews,GA, Arnold AP. 1994. Local intracerebral implants of estrogen masculinize some aspects of the zebra finch song system. Dev Neurobio 25:185-196.

Gurney ME, Konishi M. 1980. Hormone-induced sexual differentiation of brain and behavior in zebra finches. Science 208:1380-1383.

Gurney ME. 1981. Hormonal control of cell form and number in the zebra finch song system. J Neurosci 1:658-673.

Jacobs EC, Arnold AP, Campagnoni AT. 1996. Zebra finch estrogen receptor cDNA:

Cloning and mRNA expression. J Ster Biochem Mol Biol 59:135-145.

Jacobs EC, Arnold AP, Campagnoni AT. 1999. Developmental regulation of the distribution of aromatase and estrogen receptor mRNA expressing cells in the zebra finch brain. Dev Neurosci 21:453- 472.

Liang P, Pardee AB. 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257:967-971.

Mathews GA, Arnold AP. 1990. Antiestrogens fail to prevent the masculine ontogeny of the zebra finch song system. Gen Comp Endocrinol 80: 48-58.

Nottebohm F, Arnold AP. 1976. Sexual dimorphism in vocal control areas of the songbird brain. Science 194: 211-213.

Perlman WR, Arnold AP. 2003. Expression of estrogen receptor and aromatase mRNAs in embryonic and posthatch zebra finch brain. J Neurobiol 55: 204-219.

Simpson HB, Vicario DS. 1991. Early estrogen treatment alone causes female zebra finches to produce learned, male-like vocalizations. J Neurobiol 22:755–776.

Tomaszycki ML, Peabody C, Replogle K, Clayton DF, Tempelman RJ, Wade J. 2009.

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Wade J. 2001. Zebra finch sexual differentiation: The aromatization hypothesis revisited. Microsco Res Tech 54:354–363.

Wade J, Arnold AP. 1994. Post-hatching inhibition of aromatase activity does not alter sexual differentiation of the zebra finch song system. Brain Res 639:347-50.

Wade J, Arnold AP. 2004. Sexual differentiation of the zebra finch song system. Behav

Neurobiol Birdsong 1016:540-559.

Warren WC, Clayton DF, Ellegren H, Arnold AP, Hillier LW, Künstner A, Searle S,

White S, Vilella AJ, Fairley S, Heger A, Kong L, Ponting CP, Jarvis ED et al., 2010. The genome of a songbird. Nature 464:757-6.

CHAPTER IV

SEXUALLY DIMORPHIC EXPRESSION AND ESTRADIOL MEDIATED UP-

REGULATION OF A SEX-LINKED RIBOSOMAL GENE RPS6 IN THE ZEBRA

FINCH BRAIN

HYPOTHESIS: THE Z-LINKED RPS6 IS EXPRESSED GREATER IN MALES

AND THE GENE EXPRESSION IS UPREGULATED IN RESPONSE TO

ESTROGENS EXPOSURE.

ABSTRACT

Sex-linked genes have been considered as the primary regulators of sex differences among vertebrates. In mammals, sex-linked genes that are expressed in a dimorphic manner prior to an exposure of the brain to gonadal steroids initiate brain dimorphisms.

In this study, I have characterized a Z-linked gene rpS6 in the zebra finch brain that is enhanced in males throughout life. The gene expression is approximately twice in males as compared to females at all ages examined, consistent with incomplete dosage compensation in avians. The expression also differed among ages such that the gene expression was highest at P3 and P8, started to decrease by P15, and reached at the lowest level in both sexes by adulthood. Analysis of the mRNA distribution at P10, an age when the gene is not only up-regulated in both sexes, but also when song control

nuclei begin to be visible, revealed a widespread distribution throughout the brain.

Specifically, more numbers of rpS6 positive cells were present in song nuclei HVC and

RA of males as compared to females. To investigate if rpS6 interacts with estrogens to possibly influence dimorphic development of these regions, the gene expression was investigated at different time intervals following a single systemic injection of estradiol

(E2). A significant up-regulation of the gene was observed in both males and females as an effect of the hormone treatment, and E2 mediated up-regulation was approximately twice in males as compared to females. Thus, these pieces of information suggest that rpS6 may be a possible candidate in the sexual differentiation of the zebra finch brain, and its function may be facilitated by E2.

INTRODUCTION

Zebra finches serve as a useful vertebrate model to study brain sex differences. Within the telencephalon of this songbird, there are dimorphic nuclei involved in song learning and production collectively referred to as the neural song system (Nottebohm and Arnold,

1976; Wade and Arnold, 2004). This interconnected circuit consists of nuclei HVC

(proper name), RA (robust nucleus of the arcopallium), LMAN (lateral magnocellular nucleus of the anterior nidopallium) and Area X. The volumes of HVC and RA are greater in males than in females, in part, due to larger and more numerous neurons

(Bottjer et al., 1985; Konishi and Akutagawa, 1985). LMAN is monomorphic in volume; however, the soma sizes of neurons within this nucleus are larger in males (Nixdorf-

Bergweiler, 1996). Area X, which is easily identifiable in males with a Nissl stain, is not present in females (Nottebohm and Arnold, 1976). These male-biased morphological differences are responsible for observed behavioral differences; only males normally sing

(Bottjer et al., 2002).

The current hypothesis is that a combined effect of hormonal and genetic factors account for sex differences within the song system (reviewed in Wade and Arnold, 2004; London et al., 2009). Although the exact connection is not entirely clear, the contribution of hormones in this system has historically gained the most attention. Only more recently has focus shifted towards understanding the genetic influences. In particular, one family of proteins that has gained increasing interest in the past few years is ribosomal proteins

(RP). Although they are widely accepted as housekeeping components necessary for general cellular processes that occur in both sexes, studies in zebra finches have challenged this idea since some are dimorphically expressed in the brain during early development or are located on sex chromosomes. For example, rpL17, rpL37, and mitochondrial (M) rpS27 are present on the Z sex chromosome (Tang and Wade, 2006;

Qi et al., 2012). In avians, males are homogametic (ZZ sex chromosomes) and females heterogametic (ZW). Because sex chromosome dosage compensation is not as efficient in zebra finches as in mammals (Warren et al., 2010), these genes have enhanced expression in males as compared to females. More specifically, at post-hatching (P) day

25, when song nuclei are rapidly differentiating to give rise to masculine morphology, the ribosomal genes L17 and L37 are detected in Area X (which is present in males only)

and expressed more greatly in male RA. In addition, the gene for M rpS27 is expressed more significantly in male HVC. Although the specific role of these ribosomal genes as it relates to dimorphic brain development has not yet been identified, the timing and differential expression suggests that they may promote processes associated with masculine brain development. In addition, another ribosomal protein, L7/SPA is also dimorphically expressed during an early posthatching period when some of the earliest morphological differences in the song circuit begin to appear (Duncan and Carruth, 2007,

2009). Although rpL7/SPA is not sex-linked, both its gene and protein show a greater expression in the developing male brain, implying its potential role in sex-specific brain development.

In the current study we report on the identification of an additional novel rp (rpS6; also known as phosphoprotein NP33), that may also be important for brain dimorphisms. As reported in the previous chapter, this gene showed a male-biased expression in P3 and P8 males through DDRT-PCR. The goal of the current study was to characterize rpS6 by examining its gene expression in the telencephalon of both sexes at select ages during the first month after hatching and in adults. We also localized its mRNA within specific anatomical brain regions at an early developmental age. In addition, because dimorphisms in the brain are believed to result from a combination of hormonal and non- hormonal factors, and rps L17 and L37 are reported to have a possible interaction with estradiol (Tang and Wade, 2009), we wanted to investigate if a similar regulatory relationship existed with rpS6.

MATERIALS AND METHODS

Animals

Subjects used in this study were obtained from our animal facility at Kent State

University. Zebra finches were housed in communal aviaries, each containing 6-7 pairs of adult breeders held on a 14:10 light: dark cycle. Their diet consisted of finch birdseed and weekly supplements of hard boiled chicken eggs mixed with bread, fresh oranges or spinach. Food and water were available ad libitum. Adequate measures were taken to minimize pain and discomfort. All procedures were in accordance with Kent State

University’s Institutional Animal Care and Use Committee and conformed to NIH national guidelines.

Quantitative PCR

To confirm the differential expression of rpS6 and extend the ages for gene investigation, additional animals were used. Non-perfused telencephalic tissue was collected from animals at P3, P8, P15, P21, P30 and adults over 100 days old (n= 6 males, n=6 females per group). These ages represent “snap-shot” developmental periods when significant morphological changes are occurring in the brain. Significances of these ages are summarized on table 4.1.

RNA was extracted using the RNeasy mini kit (Qiagen). After verifying the quality, it was then reverse transcribed into cDNA with the High Capacity cDNA Reverse

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Table 4.1 Ages chosen for q-PCR and their significance are summarized.

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Transcription kit (ABI). Negative RT controls were made for each sample by omitting the reverse transcriptase from the reaction. Quantitative (q) PCR analysis was performed

(ABI Prism 7500) using the SYBR Green PCR master mix kit with 20 ng of cDNA and

200 nM final primer concentration, in a total volume of 20 μl. IDT primer design tool

“primer quest” was used to design the primers for the rpS6 and GAPDH gene (used as a normalizer). The partial sequence of rpS6 gene amplified using ddPCR was used as the template to design the primers. The primers for GAPDH were designed based on the conserved regions of chicken and mammalian homologues since the zebra finch sequences were not published when this work was started. RpS6 forward primer (5’-

TTGGATCTTGGGAGCTTTCG -3’), rpS6 reverse primer (5’-

AACTGTGCCCCGTCGTCT-3’), GAPDH forward primer (5’-

TGTGGACCTGACCTGCCGTCTG-3’), and GAPDH reverse primer: 5’-

TGAAGTCACAGGAGACAACCTG-3’) were used to amplify the respective zebra finch genes. The absence of primer-dimer formation for both primer pairs was verified by running control reactions with water instead of cDNA template. The PCR reactions were run in triplicate for each sample and primer combination. Cycling conditions were 50˚C for 2 min, 95˚C for 10 min, 40 cycles at 95˚C for 15 sec, and 60˚C for 1 min. The dissociation curves of the amplified PCR products were examined to confirm the absence of DNA contamination and any unwanted products. The average δCt was obtained by subtracting the Ct value of GAPDH from the Ct value of rpS6. Relative expression values were calculated using the equation 2-δδCt. Statistical analysis was performed using

Sigma Stat. A Two Way ANOVA was conducted on relative expression values to

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examine the effect of age and sex on gene expression. The level of significance was determined at P<0.05.

Riboprobe synthesis for in situ hybridization (ISH)

The plasmid containing the rpS6 insert that was amplified from DDPCR and confirmed to be rpS6 gene after sequencing was used as the template for in vitro transcription. The transcription was carried out using the Megascript protocol (Ambion) with SP6 and T7 primers. Digoxigenin (DIG) labeled UTP was added to the transcription reaction and incorporated into the probe, which at the end of the reaction, was cleaned by precipitating in a sodium acetate solution. The probe quality was confirmed prior to its use by running it on an agarose gel and checking absorbance values on a spectrophotometer. The incorporation of the DIG UTP was confirmed by dot blot (Roche).

In situ hybridization

P10 animals (n=6 males, n=6 females) were used for this study. Preliminary gene expression of rpS6 revealed an overall increase at P8 that decreased by P15. Since song control nuclei are minimally visible at P8, we opted to use an age greater than this when the nuclei would be more discernible and gene expression would likely be abundant, but not as late as P15 when expression decreased. Animals were anesthetized with CO2 and trans-cardially perfused with 0.75% saline followed by 10% phosphate buffer formalin

(PBF). The brain was removed and stored overnight in 10% PBF at 4°C to complete the fixation. The next day it was transferred to 20% sucrose, held at 4oC overnight, quickly

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frozen, and stored at -80°C. Each brain was coronally cryo-sectioned into 15µm thick sections onto alternate slides and stored at -80°C until further use. One set of slides was used for Nissl staining and utilized to aid in in identifying the brain regions. The remaining two sets were used for in situ labeling with one each for antisense and sense labeling. On the first day of in situ, tissue was removed from the freezer and air dried to room temperature (RT) for 20 min. The slides were then fixed in 4% formaldehyde at

RT for another 20 min. Sections were acetylated with 0.25% acetic anhydride in 0.1% triethanolamine (TEA) followed by two washes with phosphate buffered saline (PBS) for

5 min each. Tissue was then covered with 300 µL of pre-hybridization buffer

(containing 4X SSC, 10% dextran sulphate, Denhardt’s solution, 2mM EDTA, 50% deionized formamide, herring sperm DNA) for 90 min. To prepare the probe, it was first denatured at 80°C for 5 min and mixed with hybridization buffer (same as pre- hybridization buffer) to make a final concentration of 200ng/ml. Two hundred µL was dispensed onto each slide, covered by parafilm strips, and allowed to hybridize with the tissue in a moist chamber for 16-20 hours at 55°C.

On the second day, slides were removed from the oven and dipped in 1X SSC (sodium chloride- sodium citrate buffer) for 5 min at RT. They were placed in 50% formamide and 50% 2X SSC for 5 min at 50oC. Tissue was then washed in 1X SSC followed by

0.5X SSC, each for 5 min, at 37°C. Slides were placed in wash buffer (100mM Tris-HCl and 150mM NaCl; pH 7.5) for 5 min. Tissue was then blocked in 1% sheep serum in wash buffer for 1 hr, followed by incubation in anti-Dig-AP diluted in 1% sheep serum in

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wash buffer (1:2500) for 2 hrs. Brain sections were dipped in wash buffer twice for 5 min at RT, and placed in Tris buffer (100mM Tris-HCl and 100mM NaCl, pH 9.5) for 10 min. Detection buffer (NBT/BCIP and Levamisole dissolved in Tris buffer) was added and slides were stored in a dark humid chamber overnight (16-24 hrs) at RT. The reaction was stopped by rinsing the tissue in TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.0) for 5 min. The sections were then washed, mounted with Aquamount and coverslipped.

Quantitative analysis of rpS6 labeled cells within song control nuclei

Images were captured under bright field microscopy at 40X using an Olympus BX51 microscope. Since one major goal was to analyze rpS6 mRNA distribution, we utilized thionin stained sections and a zebra finch atlas (Nixdorf-Bergweiler and Bischof, 2007) to identify where labeling was present. HVC and RA were most clearly identifiable in

Nissl stained tissue at P10 so a quantitative analysis was performed within these nuclei.

In contrast, LMAN and Area X were not consistently identifiable among all animals studied, and thus, was not analyzed. For the analysis, a 0.09mm2 box, which covered the maximum cross-sectional area of RA and HVC without extending outside the borders, was placed at approximately the middle of each nucleus. In all cases, Nissl stained sections were used to identify the borders of song nuclei. RpS6 labeled cells in RA and

HVC were counted bilaterally in two sections on each side and added together. A two- tailed unpaired t-test was conducted to examine the effect of sex on the number of labeled

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cells in HVC and RA in (n=6) males, (n=6) females. The sections hybridized to the sense probe were examined to confirm the absence of any non-specific labeling.

E2 injections and gene quantification

P21 animals (n=6 males, n= 6 females per injection group) were collected for this study.

At this age, the zebra finch brain is highly responsive to estrogens, thus, we reasoned that if rpS6 is affected by E2, it would likely occur at this age (Gurney and Konishi 1980;

Nordeen et al., 1986). Animals were subcutaneously injected with a single dose of 50μg of E2 and collected 2, 6 and 18 hrs post-injection. This dose has been previously used and shown to be effective in partially masculinizing the song system in females (Konishi and Akutagawa, 1988; Grisham et al., 2008). We chose these time points to represent a range when changes in gene expression, as a result of treatment, were expected to occur.

Controls were injected with vehicle (propylene glycol) and animals for this group were also collected 2, 6 and 18 hrs post-injection. However, since relative expression on rpS6 gene did not differ among controls collected at different time points, these data were pooled and presented as a single data point.

To examine the effect of E2 on rpS6 gene expression, telencephalon from control and E2 treated animals were used. Since song control nuclei are located in the telencephalon, we looked at the change in gene expression in this brain region. Non-perfused telencephalic tissue was collected and rapidly frozen in ice-cold methylbutane from P21 animals at 2, 6 and 18 hrs post-injection.

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RNA extraction, cDNA synthesis and q-PCR were performed as explained previously.

Similarly, δCt and relative expressions were calculated as described above. A Two-Way

ANOVA (sex x time) was performed on relative expression values to examine the effect of E2 treatment on rpS6 gene expression. The level of significance was determined at

P<0.05.

RESULTS

RpS6 gene sequence

Blasting the 820 bp long sequence obtained from DDRT-PCR against the chicken genome (the zebra finch genome was not published when this work was started) confirmed that the gene was rpS6, since it shared 98% identity to the chicken homologue.

Months later when the zebra finch genome became available, blasting the sequence (that was initially identified) against the full length zebra finch rpS6 sequence (which was 833 bp in length) indicated 100% identity, further confirming that we had isolated rpS6. The full length gene translated into a 247 amino acid long sequence. A 2kb long promoter sequence was obtained from NCBI, and examined it for possible hormone responsive regions. Analysis using the MatInspector software revealed the presence of a potential estrogen response element (ERE) 393 bp upstream of the transcription start site, which indicated that this gene may directly respond to estrogens.

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Gene expression

Statistical analysis of rpS6 gene expression at P3, P8, P15, P21, P30 and adults indicated a significant effect of sex (F=57.155, p<0.001) and age (F=20.002, p<0.001). The sex difference persisted through adulthood with males having a greater expression at all ages as compared to females. The dimorphism was greatest at P3 and P8. From P15 through adulthood, although gene expression was still greater in males, overall expression was less compared to the younger ages (Figure 4.1).

Messenger RNA distribution

At P10, expression of rpS6 mRNA was ubiquitous throughout the brain as would be expected for a ribosomal gene (Tang and Wade, 2006; Duncan and Carruth, 2011).

Based on a visual inspection, no qualitative sex difference was observed in its distribution outside the song control nuclei. As previously explained, labeling in HVC and RA were quantitatively analyzed since only these two nuclei were clearly identified in Nissl stained tissue. In HVC, the labeled neurons clearly defined the border of the nucleus

(Figure 4.2). In addition, a clear sex difference was detected with a significantly higher number of rpS6 labeled neurons in HVC of males as compared to females (t=5.76, p<0.001; Figure 4.3). Similar to HVC, rpS6 labeling also defined the border of RA

(Figure 4.4). Within the defined region, the number of rpS6 labeled cells was significantly higher in males as compared to females (t = 6.11, p<0.001; Figure 4.5).

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Figure 4.1. Relative expression of rpS6 gene in the zebra finch telencephalon (n=6 males, 6 females per group). Two Way ANOVA was conducted on the relative expression values to examine the effect of age and sex on the gene. The expression in P3 females was designated as 100% and gene expression for the other groups was compared to this. Across ages, bars with different letters are significantly different from each other. Asterisks denote a difference within the group. Error bars represent the standard error of mean.

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A C

B D

Figure 4.2. RpS6 labeling in the song control nucleus HVC of a P10 male (A) and a female zebra finch (B). White arrows outline the border of the song nucleus (confirmed with Nissl). (C) Magnified view of labeling in (A) where arrowheads point to the neurons with cytoplasmic labeling. The sections hybridized to the sense probe (D) did not contain labeling indicating the specificity of the probe.

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Figure 4.3. Counts + SEM of rpS6 labeled cells in the song control nucleus HVC of P10 male and female zebra finches (n=6 males, 6 females). Measurements were performed bilaterally from two adjacent sections, within a 0.09mm2 sized box placed in the middle of the nucleus where the area was greatest. A significant effect of sex on the number of labeled cells was observed (t=5.76, p<0.001).

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Figure 4.4. RpS6 labeling in the song control nucleus RA of a P10 male (A) and female zebra finch (B). White arrows outline the border of the song nucleus.

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Figure 4.5. Counts + SEM of rpS6 labeled cells in RA (n=6 males, 6 females). Measurements were performed bilaterally from two adjacent sections, within a 0.09mm2 sized box placed in the middle of the nucleus where the area was greatest. A significant effect of sex on the number of labeled cells was observed (t=6.11, p<0.001).

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Apart from the song nuclei HVC and RA, at the age of investigation (P10), borders of

LMAN and Area X could not be clearly identified in all animals even in the Nissl stained sections. However, labeling in the presumptive regions appeared to be of a similar intensity as in other song control nuclei. In addition, the labeling was continuous in the surrounding region from corresponding LMAN and Area X making it very difficult to choose a region within the nuclei with certainty. Therefore, counting was not performed in regions representing LMAN and Area X.

Effect of E2 on rpS6 gene expression

Quantitative analysis of gene expression following E2 injection revealed a significant effect of sex (F=84.84, p<0.001) and treatment (F=30.64, p<0.001). In particular, males and females exhibited a significant increase in the rpS6 gene 2 hrs after a single E2 injection. Although the effect of treatment was significant in both sexes, it was approximately twice as robust in males as compared to females. Six hours post-injection, there was a significant drop in both sexes as compared to the 2 hrs-post treatment level.

The gene expression did not differ between controls and 6 hrs post-treatment. By 18 hrs following the injection, the gene had decreased significantly in both males and females as compared to the controls (Figure 4.6).

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Figure 4.6. Effect of E2 treatment (a single subcutaneous injection of 50 µg) on rpS6 gene expression in P21 males and females (n=6 males, 6 females per group) examined by using a two way ANOVA on relative expression values. Gene expression in controls at different time points did not differ from each other, thus these data were pooled and presented as a single data point. The expression in control P3 females was designated as 100% and gene expression for the other groups was compared to this. Within each sex and at the same time point across sexes, bars with different letters are significantly different from each other. A significant effect of sex (F=84.84, p<0.001) and treatment (F=30.64, p<0.001) was revealed.

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DISCUSSION

In the current study, we present evidence for a potential role of the Z-linked gene rpS6 in dimorphic development of the zebra finch brain. From an early post-hatching age through adulthood, rpS6 expression is more enhanced in males as compared to females.

Moreover, expression in males is approximately twice the level than in females, and is greatest in both sexes during the first and early half of the second week post-hatching.

Analysis of the mRNA indicated a widespread distribution throughout the brain that appeared monomorphic. However, within song nuclei HVC and RA, quantitatively there were more rpS6 positive cells in males than in females. Consistent with existing studies in this and other vertebrate models in which the effects of a gene can be modulated by hormones (Dittrich et al., 1999; Peterson et al., 2001; Carrer and Cambiasso, 2002;

Sanford et al., 2010), a single systemic injection of E2 at P21 resulted in an up-regulation of rpS6 in both sexes. Collectively, these results suggest that rpS6 plays an important role in dimorphic development of the brain, and that E2 may have a facilitative effect on rpS6 function.

The specific mechanisms by which rpS6 may affect brain sex differences is not exactly known. However, one hypothesis can be made based on its pattern and timing of expression. For example, one possibility is that it may be essential for the incorporation of new neurons into HVC. In males, during the first and second week after hatching, more neurons are added into HVC from the proliferative VZ that borders this nucleus,

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than at any other age (Alvarez-Buylla et al., 1990; Burek et al., 1994; DeWulf and

Bottjer, 2005; Scott and Lois, 2007).

Since rpS6 labeling was greater in male HVC compared to female at least at one age during this period, the gene may affect the dimorphism in neuronal addition within the nucleus. Interestingly, our data revealed that rpS6 displayed dimorphic expression even at ages earlier than when HVC (and all other song control nuclei) have morphologically developed. Thus, this suggests that rpS6 may additionally be involved in early processes that direct the initial formation of song nuclei.

In addition to HVC, rpS6 is also dimorphic in RA. Existing data suggest that a similar number of neurons are added to RA in males and females beginning at an early embryonic period and lasting through P6 (Kirn and DeVoogd, 1989). However, glia are added throughout the first month at a greater rate in males than in females (Nordeen et al., 1996). Although we examined mRNA distribution at P10 only when labeling was greater in this song nucleus, the telencephalic gene expression profile revealed dimorphism throughout life, suggesting that rpS6 may mediate dimorphism in recruitment of glia in RA.

In mammals, sex-chromosome inactivation is a process that allows females to have the same complement of X-linked genes as males. Interestingly, sex-chromosome inactivation is not common in avians, and this may result in dimorphic effects on a

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system (Itoh et al., 2007; Ellegren et al., 2007; Warren et al., 2010). Consistent with this idea, approximately twice the expression of rpS6 in males from the earliest age examined

(P3) through adulthood implies that the transcription of both copies of this Z-linked gene exert a double effect in males (ZZ) as compared to females (ZW). This, along with male- biased expression of its mRNA in HVC and RA, all suggest a dimorphic effect of rpS6 on brain development.

In vertebrates, the effects of genes can be amplified by their interaction with hormones

(Dittrich et al., 1999; Peterson et al., 2001; Sanford et al., 2010). In the current study, a significant up-regulation of rpS6 gene was observed following E2 treatment. RpS6 may primarily act to enhance addition of neurons and glia into HVC and RA respectively, and

E2 by itself is also known to increase the recruitment of both of these cell types (Nordeen and Nordeen, 1989; Burek et al., 1997; Peterson et al., 2004; Lee et al., 2007). Thus, we propose that these two components acting together may lead to a greater enhancement of neuronal and glial recruitment into these song control nuclei. The ERE identified in the zebra finch rpS6 promoter through in silico analysis suggests that its interaction with E2 is mediated by nuclear estrogen receptors. The most common way of E2 mediated up- regulation of target genes containing an ERE is through nuclear receptors (ERα and ERβ) that act as transcription factors (reviewed in Tsai and O’Malley, 1994; Nilsson et al.,

2001). The activated receptors form dimers and translocate to the nucleus, bind to ERE, and mediate gene transcription (reviewed in Tetel et al., 2009). In zebra finches, ERβ has not been shown to be present in song control nuclei (Bernard et al., 1999; Perlman and

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Arnold, 1999). In contrast, ERα is present, but in limited amounts and only within specific song control nuclei. For example, it is present in HVC (Jacobs et al., 1996;

1999; Gahr and Metzdorf, 1998), which also contains male-biased rpS6 expression.

Thus, there may be a direct interaction of these two components at this site to induce masculinization. In RA, ERα expression is very minimal (Gahr and Metzdorf, 1998;

Jacobs et al., 1999) making it less likely to have a significant effect on rpS6. Instead, dimorphic effect of rpS6 within RA may be independent of local estrogenic effects within the nucleus. Alternatively, neuronal projections from HVC possibly exert dimorphic effects of E2 on rpS6 function within RA since E2 induced masculinization has been previously reported in RA (Dittrich et al., 1999).

In song nuclei LMAN and Area X, the role of rpS6 is less clear. Dense rpS6 labeling was observed in brain regions that corresponded to where LMAN and Area X would be located. However, we were unable to specifically measure gene expression in these nuclei due to difficulty in consistently identifying borders in all animals. Although both of these nuclei can be masculinized by E2 in females, the interaction of hormone with rpS6 is unlikely since they do not express nuclear estrogen receptors (Jacobs et al., 1996,

1999; Bernard et al., 1999). Instead, Area X is regulated by afferents from HVC, which provide IGF-II to this nucleus (Holzenberger et al., 1997). Since LMAN receives only indirect connections through Area X and RA (Nottebohm et al., 1976; Nottebohm and

Arnold 1976), whether its regulation is possible through HVC is unknown.

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In sum, we have shown that rpS6 is a sex-linked gene in avians with a potential role in masculinization of HVC and RA in males. This is one of very few genes to show a male- biased expression throughout life suggesting that it may be essential to initiate and maintain masculinization of the song system. However, its function in brain dimorphisms and its possible interaction with E2 has not been previously explored in other vertebrates, thus requiring future research.

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CHAPTER V

GLOBAL DISCUSSION

Traditionally, rodents have been used as the models for studying brain sex differences, and based on these species it was demonstrated that hormones play a primary role. That conclusion, however, has been challenged by research in the zebra finch which has identified that not just hormones, but rather a combined effect of sex-linked genes and hormones are most likely the major contributors of brain dimorphisms in vertebrates.

Currently, however, very little is known about what the exact factors are and how they act to create sex differences within the brain. My work suggests that the hormonal actions may occur through the membrane bound estrogen receptor GPR30, and the non- hormonal effects through the Z-linked gene rpS6. Additionally, my research has provided evidence suggesting a role for the interaction of the rpS6 gene and estrogens, most likely through nuclear estrogen receptors.

Based on conclusions derived from my data and existing studies, I propose a model on how GPR30 and rpS6 may contribute to dimorphic development of the songbird brain

(Figure 5.1). I hypothesize that rpS6 acts during the first and second week post-hatching to direct the formation of song control nuclei. In particular, it may induce masculinization of HVC and RA, possibly by facilitating male-biased neuron and glia addition, respectively. Even though morphological differences in the song system are not detectable until around P12 (Bottjer et al., 1985), sex differences in rpS6 that were

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Figure 5.1. Proposed mechanism of action for the membrane bound estrogen receptor

GPR30 and the rpS6 gene in dimorphic development of the neural song system in zebra finches.

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detected prior to this age may be important for initiating processes necessary for the impending dimorphisms. The overall dimorphic effect of rpS6 on incorporation of new cells begins to decrease around the third week after hatching, but still remains male- biased through adulthood. In contrast, the dimorphic actions of GPR30 are restricted to a more limited period of development. More specifically, its differential effect(s) begin in the third week after hatching and continue up until the fifth week, which corresponds to a period when neuronal loss is elevated in females. Based on this and direct evidences on neuroprotective effect of GPR30 in mammals, I hypothesize that this membrane-bound estrogen receptor induces neural dimorphisms by mediating greater neuronal protection in males. GPR30 is dimorphic in HVC, therefore its effects on directing sex differences are most likely specific to this nucleus. In RA, where it shows a moderate expression, the receptor is not dimorphic between the sexes, making it unlikely to be the direct mediator of sex differences within this nucleus. GPR30 can act to increase neuronal protection only after it is bound by estrogens. Whereas rpS6 may act by itself to facilitate neuron and glia addition and/or have its effects enhanced by E2. Given that GPR30 is not known to bind to ERE, and the nuclear receptor ERβ is absent in the song system (Bernard et al.,

1999), estrogenic influences on rpS6 gene are likely mediated through ERα, which can directly bind to the ERE. Thus, rpS6 and GPR30 may both contribute to dimorphic processes associated with sex differences in the neural song system.

In addition to a possible role for GPR30 in dimorphic development of the song control system, this receptor may also have critical functions outside of this circuit, where

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morphological sex differences have not been identified. In many of these brain regions, estrogens are utilized for various physiological functions, but these areas contain very limited or no nuclear receptors. However, GPR30 is abundantly expressed (although not dimorphically), and thus my work provides additional support for its role in regulating estrogenic responses that are not different between the sexes. For example, GPR30 may provide estrogenic input for the hippocampus whose function in memory formation in both sexes can be modulated by E2 in zebra finches and mammals (Luine et al., 1998;

Oberlander et al., 2004; Fernandez et al., 2008). Additional brain regions may also require GPR30 for E2 mediated functions. For example, rapid neuronal firing in response to E2 has been reported in the auditory region NCM, when a bird is exposed to species- specific as well as its own song (Stripling et al., 1997; Remage-Healey et al., 2010). This rapid neuronal activity in NCM is monomorphic and parallels the lack of dimorphism in

GPR30 immunoreactivity in this region (Tremere et al., 2009). Although ERα and ERβ have both been identified in NCM (Saldanha and Coomaralingam, 2005; Jeong et al.,

2011), rapid activation of neurons after a stimulus is characteristic of non-genomic signaling, which is primarily known to be mediated by membrane bound receptors. Thus, by producing specific neuronal responses to various song types in NCM, this receptor may facilitate song discrimination in both sexes.

Work in this dissertation has also demonstrated that E2 can up-regulate the rpS6 gene.

The up-regulation was greater in males than in females suggesting that E2 may have a greater effect through this gene on the male brain. The interaction between E2 and rpS6

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most likely occurs through nuclear receptors since the ERE in its promoter serves as a binding site for these receptors only. Whereas estrogens acting through GPR30 may not influence rpS6 expression since G-protein coupled receptors neither act as transcription factors, nor contain a binding site for ERE. Since ERβ is absent in song control nuclei

(Jacobs et al., 1999; Metzdorf et al., 1999), the interaction in at least HVC between E2 and rpS6 is most likely mediated through ERα since this receptor’s expression is greatest in this song nucleus. In RA, ERα is very minimally expressed (Jacobs et al., 1999;

Metzdorf et al., 1999) and is questionable whether it is functionally significant. Instead, trophic signals such as BDNF from HVC initiated by E2 binding in HVC may facilitate masculinization of RA (Dittrich et al., 1999). In Area X where GPR30 is very minimal, and nuclear receptors are absent, direct estrogenic effects on masculinization is also unlikely. While rpS6 labeling is abundant in the region corresponding to Area X, its borders could not be outlined in males, and its analogous region in females also contained the labeling. Thus, it is currently not known whether rpS6 exerts any dimorphic effects in Area X. Instead, similar to what I hypothesize occurs in RA, dimorphisms in the nucleus may result because of connections from HVC which have been identified using retrograde tracers (Benton et al., 1998; Sakaguchi et al., 1999; Walton et al., 2012).

Neurons that project from HVC to Area X supply growth factors such as insulin growth factor (IGF) II, which can enhance neuronal survival (Holzenberger et al., 1997).

Although my dissertation work is the first to provide a mechanism for how estrogens can have dimorphic influences on the brain, and identifies a specific sex-linked gene that may

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participate in creating sex differences, some questions still remain unanswered. For example, how sex differences in LMAN are regulated largely remains unknown. This nucleus is masculinized by E2 but contains limited and monomorphic GPR30 immunoreactivity, and completely lacks ERα and ERβ. Interestingly, global blockage of nuclear estrogen receptors with the antagonist ICI,182,780 decreases neuron soma sizes in the area (Bender and Veney, 2008). Although it can be hypothesized that HVC may direct sex differences in this nucleus similar to how it may affect RA and Area X, it receives only indirect projections from HVC, through RA and Area X (Nottebohm and

Arnold, 1976). Whether such connections affect the development of LMAN is unknown and warrant future research. Another alternative is that non-hormonal components (such as rpS6) regulate dimorphic development of this nucleus. This too would need to be further investigated because at the age that we examined, rpS6 mRNA is abundant in the region of the brain that corresponded to where LMAN would develop; however, we were unable to measure whether expression differed between the sexes.

In sum, my research suggests that the regulation of brain dimorphisms in zebra finches

(similar to what is hypothesized for vertebrates) may involve hormone dependent as well as independent mechanisms. What we are left with is the conclusion that differential development of HVC and RA may likely depend on GPR30 as well as the sex-linked gene rpS6. Masculinization of RA may further be enhanced due to trophic support from

HVC. In contrast, development of Area X and LMAN are more indirect and may completely depend on factors secreted from HVC and/or yet to be identified genes or

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hormonal pathways. Why the song system is modeled in this fashion, with each region having seemingly different factors important for its masculinization, is not entirely clear.

FUTURE DIRECTIONS

Although existing studies in other mammals suggest a role for GPR30 in neuroprotection, its exact functions in zebra finches still needs to be verified. Examining the effects of the

GPR30 antagonist G15 on neuronal survival will provide more information on its function in this system. Similarly, the proposed role of rpS6 in neuron and glia addition needs to be investigated. This gene is greater in male HVC and RA where more cells are added. The rate of their incorporation within these nuclei can be examined by knocking down rpS6 in animals. In mammals, rpS6 has been characterized to be important for the proliferation of various cell types including neurons, fibroblasts and hepatic cells

(Volarevic et al., 2000; Yamashita et al., 2007; Palazeulos et al., 2012). Whether this gene has such roles in the zebra finch brain is unknown. Since neuronal proliferation is dimorphic and mostly restricted to the VZ and SVZ (DeWulf and Bottjer, 2002, 2005;

Scott and Lois, 2007), knowing whether newly born neuronal precursors in those regions express rpS6, and whether these cells are more abundant in males than females, will be very crucial in exploring its role in specific processes associated with neuron addition

(e.g. cell migration). Moreover, rpS6 is a critical component of the 40 S ribosomal subunit, which is a part of the ribosomal complex required for protein synthesis

(reviewed in Meyuhas O, 2008). However, it is not known whether it mediates the synthesis of proteins with specific functions, or is required for global protein synthesis.

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This question could be investigated by examining the change in global gene expression using microarray chips following knock down of the gene.

Another direction that future studies can take is to further identify how dimorphisms in

LMAN arise. To date only a few studies have provided mechanisms to explain this. One possibility that has been suggested is that it is supported by HVC. To examine if HVC is in fact mediating such effects, estrogen receptors in HVC (nuclear or membrane-bound) can be blocked and the effects on dimorphisms in LMAN examined. Alternatively, the possible presence of another known membrane estrogen receptor ER-X (which was initially identified in the mammalian brain; Toran-Allerand et al., 2002), can be explored in LMAN where nuclear receptors are absent and membrane receptor GPR30 shows a very limited expression. And lastly, although it has been hypothesized throughout my dissertation, another question that can be further explored is whether ERα directly binds to the ERE in the rpS6 promoter. This can be verified by direct binding assays such as chromatin immuno-precipitation (ChIP) which will allow the co-precipitation of ERα with the ERE in the gene promoter.

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