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 by 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 ii 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 iii 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 iv 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 v 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 vi 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 vii 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 viii Tn…….……………………………………..……..….… nucleus taeniae of the amygdala TP……………………………………………………….………... testosterone propionate Uva…….……………………………………………….…….………..uvaeformis nucleus V…………………………………………..……..…...……………………..….….ventricle ix 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 x 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