GENE EXPRESSION OF THYROID HORMONE RECEPTORS TRα AND TRβ DURING EARLY DEVELOPMENT IN TILAPIA, OREOCHROMIS MOSSAMBICUS A SHARMILA NATIONAL UNIVERSITY OF SINGAPORE 2003 GENE EXPRESSION OF THYROID HORMONE RECEPTORS TRα AND TRβ DURING EARLY DEVELOPMENT IN TILAPIA, OREOCHROMIS MOSSAMBICUS By A Sharmila M.Sc., M.Phil A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NATIONAL UNIVERSITY OF SINGAPORE 2003 ACKNOWLEDGEMENTS I would like to express my deep gratitude to Prof T J Lam, for his guidance, enthusiasm and support throughout the course of this project Most of all, his unfailing encouragement is very much appreciated I am thankful to A/Prof W K Chan and A/Prof A D Munro for their advice and help in carrying out the studies I am extremely grateful to Dr J Walford, Dr Konda P Reddy, Dr P Sukumar and Dr B Sivaloganathan for their helpful discussions, constant support and practical help and most importantly for their invaluable suggestions during the preparation of this thesis I would like to take this opportunity to express my gratitude to Dr Y W Liu, for her help in guiding me in whole mount in situ hybridization work I would also like to thank my friends, Sudha and Shanti for their timely help during the course of this study Finally, the award of a Research scholarship by the National University of Singapore is gratefully acknowledged This study was supported by a NUS Research Grant RP3972374 to Prof T J Lam i TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii LIST OF ILLUSTRATIONS v LIST OF TABLES viii SUMMARY ix Chapter GENERAL INTRODUCTION Chapter CLONING AND SEQUENCING OF THYROID HORMONE RECEPTORS TRα AND TRβ IN TILAPIA, OREOCHROMIS MOSSAMBICUS 2.1 INTRODUCTION 2.2 MATERIALS AND METHODS 10 2.2.1 Cloning of TRα1 10 2.2.2 Partial cloning of TRβ1 11 2.2.3 Cloning of TRβ2 11 2.2.3.1 5' and 3' -Rapid amplification of cDNA ends 11 2.2.3.2 Long distance PCR (LD PCR) 12 2.2.4 Analysis of TRα and TRβ PCR Products 12 2.2.4.1 Agarose gel electrophoresis 12 2.2.4.2 Extraction of PCR products from agarose gel 13 2.2.4.3 Quantification of DNA by spectrophotometry 13 2.2.4.4 DNA ligation 13 2.2.4.5 Preparation of competent cells 13 2.2.4.6 Transformation 14 2.2.4.7 Colony screening PCR 15 2.2.4.8 Isolation and purification of plasmid DNA 15 2.2.4.9 Automatic sequencing 16 2.2.4.10 Database search and sequence analysis of TRα and TRβ 17 2.3 RESULTS 17 2.3.1 Cloning and sequencing of TRα1 17 2.3.2 Cloning and sequencing of TRβ 18 2.3.2.1 Partial cloning and sequencing of TRβ1 18 2.3.2.2 Cloning and sequencing of TRβ2 18 2.4 DISCUSSION 29 ii Chapter EXPRESSION PROFILES OF THYROID HORMONE RECEPTORS TRα1 AND TRβ1 32 DURING EARLY DEVELOPMENT IN TILAPIA, OREOCHROMIS MOSSAMBICUS 3.1 INTRODUCTION 32 3.2 MATERIALS AND METHODS 33 3.2.1 3.2.2 Total RNA isolation 34 3.2.3 RNA gel electrophoresis 34 3.2.4 Synthesis of first strand cDNA 34 3.2.5 Semi-quantitative RT-PCR analysis 35 3.2.6 3.3 Source of tilapia eggs/larvae 33 Statistical analyses 35 RESULTS 36 3.3.1 Changes in the levels of TRα1 and TRβ1 expression during early development 36 3.4 DISCUSSION 40 Chapter LOCALIZATION OF THYROID HORMONE RECEPTORS TRα1 AND TRβ1 IN EMBRYOS AND THEIR TISSUE-SPECIFIC EXPRESSION IN TILAPIA, 44 OREOCHROMIS MOSSAMBICUS 4.1 INTRODUCTION 44 4.2 MATERIALS AND METHODS 46 4.2.1 Whole mount in situ hybridization 46 4.2.1.1 Synthesis of labeled RNA probe 46 4.2.1.1.1 Linearization of plasmid DNA 46 4.2.1.1.2 Probe incubation and precipitation 47 4.2.1.2 Preparation of tilapia embryos for in situ hybridization 48 4.2.1.3 Proteinase K treatment of embryos 48 4.2.1.4 Prehybridization 48 4.2.1.5 Hybridization 49 4.2.1.6 Post-hybridization washes 49 4.2.1.7 Antibody incubation 49 4.2.1.7.1 Preparation of preabsorbed DIG and fluorescein antibody 49 4.2.1.7.2 Incubation with preabsorbed antibodies 49 4.2.1.8 Color development 50 4.2.1.9 Mounting and photography 50 4.2.2 Extraction of total RNA from adult tissues 51 4.2.3 Statistical analyses 51 iii 4.3 RESULTS 51 4.3.1 4.3.2 4.4 Localization of TRα1 and TRβ1 expression using in situ hybridization 51 Expression of TRα1 and TRβ1 in various adult tissues 52 DISCUSSION 56 Chapter AUTOINDUCTION OF THYROID HORMONE RECEPTORS TRα1 AND TRβ1 59 DURING EARLY DEVELOPMENT IN TILAPIA, OREOCHROMIS MOSSAMBICUS 5.1 INTRODUCTION 59 5.2 MATERIALS AND METHODS 61 5.2.1 Source of tilapia larvae 61 5.2.2 Hormone treatment 62 5.2.2.1 Preparation of treatment media 62 5.2.2.2 Experiment 62 5.2.2.3 Experiment 62 5.2.2.4 Experiment 62 5.2.2.5 Preparation of samples 63 5.2.3 Quantification of mRNA using real-time RT-PCR 63 5.2.3.1 In vitro transcription 63 5.2.3.2 The Standard curve 64 5.2.3.3 Real-time RT-PCR analysis 64 5.2.4 5.3 Statistical analyses 65 RESULTS 65 5.3.1 5.3.2 Experiment 66 5.3.3 Experiment 66 5.3.4 5.4 Quantification of mRNA using real-time RT-PCR 65 Experiment 66 DISCUSSION 72 Chapter GENERAL DISCUSSION AND CONCLUSIONS 77 LITERATURE CITED…………………………………………………………………………… 81 iv LIST OF ILLUSTRATIONS FIGURE PAGE NO NO 2.1 Nucleotide and amino acid sequence of the O mossambicus TRα1 cDNA The open reading frame encodes a polypeptide of 409 amino acids Putative zinc fingers are underlined The presumptive translation start is indicated by ATG codon and TGA codon is marked by asterisk…………………………………………………… 21 2.2 Comparison of tilapia TRα1 with those of salmon, flounder, halibut and zebrafish A dot represents a gap insertion to maximize matches in the multiple alignment Amino acids identical to tilapia TRα1 are shown as dashes Numbers represent amino acid number starting with methionine……………………………………………………… 22 2.3 Partial nucleotide sequence (669 bp) of tilapia TRβ1 with the predicted amino acid sequence The nine amino acid insertion is represented as broken lines Degenerate primer sequences are in bold letters Gene specific primers and nested primers are also shown……………………………………………………………… 2.4 23 Nucleotide and deduced amino acid sequence of a cDNA encoding tilapia TRβ2 The sequence contains an open reading frame of 1185 nucleotides which encodes a polypeptide of 395 amino acids An insertion sequence of nine amino acids in the ligand binding domain is represented as broken lines Putative zinc fingers are underlined… 2.5 24 Comparison of tilapia TRβ2 with those of seabream, flounder, zebrafish and salmon Amino acids identical to tilapia TRβ1 are represented as dashes A dot represents a gap insertion to maximize the number of matches The amino acid insertion is indicated in bold letters Numbers represent amino acid number starting with methionine …………….…………………………………………… 25 v 2.6 Phylogenetic tree of vertebrate TRα and TRβ receptors Phylogram of Bootstrap analysis (1000) replicates are identical to this tree Bootstrap percentages are given above the branches Species and their Genbank accession numbers for sequences included for analysis: Atlantic salmon TRα1 (AF146775), flounder TRαA (D16461), flounder TRαB (D16462), halibut TRα1 (AF143296), zebrafish TRα1 (U54796), Xenopus TRαA (M35343), chicken TRα1 (Y00987), human TRα1 (X55005), Atlantic salmon TRβ1 (AF302251), flounder TRβ1 (D45245); sea bream TRβ1 (Nowell et al., 2001); zebrafish TRβ1 (AF109732), conger eel TRβ1 and TRβ2 (Kawakami et al., 2003); Xenopus TRβ1 (M45245), chicken TRβ1 (X17504) and human TRβ1 (X04707)……………………………… 3.1 27 Developmental changes in TRα1 and TRβ1 mRNA levels during early life stages of tilapia, Oreochromis mossambicus Data presented as mean ± standard error from three batches of 10 pooled individuals…………………………………………………………… 3.2 38 Gel photographs showing changes in the expression of (A) TRα1 (B) TRβ1 genes during early development of tilapia and (C) β actin is used as an internal control………………………………………… 4.1 39 Localization of TRα1 and TRβ1 in dpf embryos by whole mount in-situ hybridization in O mossambicus (A) No expression with sense TRα1 probe (B) antisense-TRα1 expressed in optic tectum, cerebellum and medulla of brain and tail regions (C) No expression with sense TRβ1 probe (D) antisense-TRβ1 mRNA expressed in brain and tail regions Abbreviations: b- brain; ot- optic tectum; cecerebellum; md- medulla; t- tail…………………………………… 53 vi 4.2 Semi-quantitative RT-PCR analysis of tilapia TRα1 and TRβ1 expression in various adult tissues Data presented as mean ± standard error from triplicate experiments………………………… 4.3 54 Gel photographs showing the expression of (A) TRα1 (B) TRβ1 in various adult tissues of tilapia and (C) β actin used as an internal control……………………………………………………………… 55 5.1 Real time PCR of TRα1 standard curve using in-vitro transcribed RNA as template Ten fold serial dilutions of the templates, ranging from 2.38-7.38 log molecules (log molecules= log N, where N is the copy number of that molecule)……………………………………… 67 5.2 Real time PCR of TRβ1 standard curve using in-vitro transcribed RNA as template Ten fold serial dilutions of the templates, ranging from 2.4 -7.4 log molecules (log molecules= log N, where N is the copy number of that molecule)……………………………………… 68 5.3 Quantification of TRα1 and TRβ1 expression on day post treatment of newly hatched larvae with different concentrations of T3 Data presented as mean ± standard deviation from triplicate experiments Means with the same alphabet are not significantly 69 different (p>0.05, One Way ANOVA followed by SNKM) 5.4 Quantification of TRα1 and TRβ1 expression on day post treatment of newly hatched larvae with different concentrations of T4 Data presented as mean ± standard deviation from triplicate experiments Means with the same alphabet are not significantly 70 different (p>0.05, One Way ANOVA followed by SNKM).………………………………………………………… 5.5 Real time PCR analysis of expression of TRα1 and TRβ1 in different adult tissues 16 hrs after injection of tilapia with a dose of 0.05 µg T3/ g body weight (A) TRα1 (B) TRβ1 Data presented as mean ± standard deviation from triplicate experiments…………… 71 vii LIST OF TABLES TABLE PAGE NO NO 2.1 List of primers used in obtaining full 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CHAPTER CLONING AND SEQUENCING OF THYROID HORMONE RECEPTORS TR? ? AND TR? ? IN TILAPIA, OREOCHROMIS MOSSAMBICUS 2.1 INTRODUCTION... TR isoforms, including TR? ?1, TR? ?2 and TR? ?3 from the TR? ? gene and TR? ?1 and TR? ?2 from the TR? ? gene (Lazar, 1993) The TR? ? isoforms differ in their carboxyl-termini The TR? ?1 isoform binds to T3 and. .. distribution, their expression during early development and the autoinduction of these receptors in the tilapia, O mossambicus In order to understand gene expression of TRs during development in O mossambicus,