Global gene expression analysis of cranial neural tubes in embryos of diabetic mice

GLOBAL GENE EXPRESSION ANALYSIS OF CRANIAL NEURAL TUBES IN EMBRYOS OF DIABETIC MICE JIANG BORAN NATIONAL UNIVERSITY OF SINGAPORE 2008 GLOBAL GENE EXPRESSION ANALYSIS OF CRANIAL NEURAL TUBES IN EMBRYOS OF DIABETIC MICE JIANG BORAN MBBS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF ANATOMY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2008 ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS I would like to express my deepest appreciation to my supervisor, Associate Professor S Thameem Dheen, Department of Anatomy, National University of Singapore, for his innovative ideas, invaluable guidance and constant encouragement throughout this study I am greatly indebted to Professor Ling Eng Ang, Head of Department of Anatomy, National University of Singapore, for his full support in providing me with the first class research facilities and an excellent academic environment, as well as for his valuable suggestions to my career I am also thankful to Associate Professor Samuel Tay Sam Wah and Dr S Dinesh Kumar, Department of Anatomy, National University of Singapore, for their kind suggestions and constructive criticisms I am happy to acknowledge my gratitude to Mrs Yong Eng Siang (Neuroscience Lab), Mrs Ng Geok Lan (Histology Lab), Miss Chan Yee Gek (Confocal Microscopy Lab), Mdm Wu Ya Jun (Electron Microscopy Unit), Mr Yick Tuck Yong (Multimedia Unit), Mr Poon Zhung Wei (Autoclave Room), and Mr Lim Beng Hock (Animal House) for their technical assistance and Mdm Ang Lye Gek, Carolyne, Ms Teo Li Ching Violet and Mdm Diljit Kour for their secretarial assistance i ACKNOWLEDGEMENTS I would like to express my thanks to Miss Evelyn Loh Wan Ting for her kind help and collaboration in the research I would like to extend my thanks to all staff members, my fellow graduate students in Department of Anatomy, National University of Singapore for their help and support one way or another I would like to express my special thanks to my friends, Dr Jayapal Manikandan and Dr Peter Natesan Pushparaj at the Department of Physiology, National University of Singapore for their friendly help and advice Certainly, without the financial support from the National University of Singapore in terms of Research Scholarship and Research grant (R-181-000-038-112 to Associate Professor S T Dheen), this work would not have been brought to a reality I would like to express my heartfelt thanks to my parents for their full and endless support for my study Finally, I would like to thank my wife, Mdm Yin Na, for her full support, encouragement and assistance during my study ii ACKNOWLEDGEMENTS This thesis is dedicated to my beloved family iii TABLE OF CONTENTS CONTENTS ACKNOWLEDGEMENTS………………………………………………… ………i TABLE OF CONTENTS………………………… iv LIST OF ABBREVIATIONS…………………… … xi SUMMARY…………………………………… ……………………………….…… xiii PUBLICATIONS…………………………………………………………………… xvii Chapter 1: Introduction…………………………………………………… … ……1 Diabetes Mellitus…………………………………………………………………… 1.1 Definition and classifications of diabetes mellitus………………………… 1.2 The complications of diabetes mellitus………………………………………… 1.3 Maternal diabetes………………………………………………………… …… 1.3.1 Congenital malformations in embryos of diabetic mother……………… Neural tube development…………………………………………………………… 2.1 Neurulation……………………………… 2.1.1 Neural tube defects……………… 2.2 Neurogenesis in the developing cranial neural tubes………………………… 10 11 2.2.1 Developmental control genes in the process of neurogenesis…………… 2.2.1.1 Doublecortin (Dcx)…………………………………………… 12 2.2.1.2 Brain lipid binding protein (Blbp)……… ………………………12 2.2.1.3 Paired box gene (Pax6)…………………………… 13 iv TABLE OF CONTENTS 2.2.1.4 Neurogenin (Ngn2)…………………………………………… 15 2.3 Glucose metabolism and hypoxia in the developing cranial neural tubes……… 16 2.3.1 Glycolysis pathway…………………………………………………….…17 2.3.2 Hypoxia in the developing cranial neural tubes…………………….…….18 2.3.2.1 Hypoxia-inducible factor (Hif-1)……………………………… 19 2.3.2.2 Vascular endothelial growth factor (Vegf)……………………… 21 2.4 Choroid plexus development………………………………………………… 21 2.4.1 Choroid plexus (CP)………………………………………………………21 2.4.2 Development of CP in mouse embryos…………………………… ……22 2.4.3 Factors involved in CP development…………………………… …… 23 2.4.3.1 Transthyretin (Ttr)……………………………………………… 23 2.4.3.2 Insulin-dependent growth factor (Igf-2)…………………… …24 Analysis of global gene expression profile using DNA microarray…………………25 3.1 Overview of DNA microarray technology……………………………………… 25 3.2 Basic principle of DNA microarray……………………………………… …… 26 3.3 Applications of DNA microarray………………………………………… …… 28 3.4 Data analysis strategies……………………………………………….……… 28 Animal models…………………………………………………………….……… 29 4.1 Animal models of diabetes mellitus…………………………………….……… 29 4.1.1 Mechanism of action of streptozotocin (STZ)…………………………… 30 4.2 Animal model of maternal diabetes…………………………………….……… 30 Hypotheses and Objectives……………………………… 32 v TABLE OF CONTENTS 5.1 Specific aims…………………………………………………………………… 35 Chapter 2: Materials and Methods………………………………………………… 37 Animals………………………………………………………………………………38 Induction of diabetes mellitus……………………………………………………… 40 2.1 Blood glucose test……………………………………………………………… 41 2.2 Collection of embryos……………………………………………………… …41 Histology…………………………………………………………………………… 42 3.1 Fixation of embryos…………………………………………………………… 42 3.2 Preparation of silane coated slides………………………………………………43 3.3 Cryosection of embryos…………………………………………………………44 3.4 Haematoxylin and Eosin staining………………………………….……………44 RNA extraction and quantification…………………………………………… ……45 49 Microarray analysis………………………………………………………………… 49 5.1 Synthesis of double stranded cDNA from total RNA…………………………… 5.2 cDNA cleanup and precipitation…………………………………………… … 51 5.3 Synthesis of Biotin-labeled cRNA………………………………………………52 5.4 cRNA clean up and quantification…………………………………………… 52 54 5.5 Fragmentation the cRNA for target preparation………………………………… 5.6 Target hybridization…………………………………………………………… 54 5.7 Post-hybridization washing, staining and scanning of arrays……………… … 55 5.8 Data analysis……………………………………………………………… … 57 vi TABLE OF CONTENTS 5.8.1 Absolute data analysis……………………………………………………57 5.8.2 Comparison data analysis………………………………… ……………58 5.8.3 Gene filtration………………………………………………………….…58 5.8.4 Gene clustering and functional analysis based on Gene Ontology………58 Real time reverse transcriptase polymerase chain reaction (Real time RT-PCR)… 59 Immunohistochemistry (IHC)…………………………………………………….… 65 Immunofluorescence staining…………………………………………………….… 69 BrdU labeling analysis……………………………………………………………… 70 10 Terminal Deoxynucleotidyl Transferase -mediated dUTP Nick-End Labeling analysis (TUNEL)………………………………………………………………… 73 11 In situ Hybridization………………………………………………… ……………75 11.1 Plasmids for preparation of cRNA probes…………………………….………76 11.2 Preparation of competent cells……………………………………………… 76 11.3 Transformation of competent cells with plasmids…………………………… 78 11.4 Linearization of the plasmid………………………………………………… 79 11.5 Synthesis of digoxigenin-labeled RNA probe from the linear DNA…………81 11.6 Whole mount of in situ hybridization……………………………… ………82 12 Western blot analysis…………………………………………………………….…87 Chapter 3: Results…………………………………………………………………… 92 Neural tube defects in embryos of diabetic mice…………………………………… 93 Gene expression profile of cranial neural tubes in embryos of control vii TABLE OF CONTENTS and diabetic mice…………………………………………………………………… 94 Effect of maternal diabetes on expression of genes involved in glucose metabolism and that respond to physiological hypoxia in cranial neural tubes of mouse embryos…………………………………………………………… 95 3.1 Maternal diabetes altered the expression of genes involved in glycolysis in cranial neural tubes of mouse embryos………………………………………… 95 3.2 Maternal diabetes altered the expression of genes that respond to hypoxia in cranial neural tubes of mouse embryos………………………………………… 96 3.2.1 Expression of hypoxia-inducible factor 1α (Hif-1α) in cranial neural tubes of embryos from control and diabetic mice……………………… 96 3.2.2 Expression of Vegf in cranial neural tubes of embryos from control and diabetic mice…………………………………………………………97 Maternal diabetes induces apoptosis in the neuroepithelium of cranial neural tubes in mouse embryos… 98 Maternal diabetes inhibits proliferation index in the neuroepithelium of cranial neural tubes in mouse embryos………………………………………………… … 98 Maternal diabetes alters the expression of genes involved in neuronal migration and neurogenesis in cranial neural tubes of mouse embryos…………… 99 6.1 Expression pattern of developmental control genes is altered in embryos of diabetic mice…………………………………………………… 100 6.1.1 Paired box gene (Pax6)………………………………………….…… 100 6.1.2 Neurogenin (Ngn2)……………………………………………………100 viii FIGURES AND FIGURE LEGENDS Figure 21 Control E11.5 Diabetic E11.5 LV Ttr TC TC A LV B E13.5 IV E13.5 CP IV C D 191 CP FIGURES AND FIGURE LEGENDS Fig.22 Igf-2 gene product (138bp) is amplified in cranial neural tubes of mouse embryos by the real time RT-PCR (A) Melting curve indicates the specificity of the product (B) The quantitative real time RT-PCR (C) shows that expression level of Igf-2 is significantly decreased in cranial neural tubes of E11.5 and E13.5 embryos derived from diabetic mice in comparison to that of embryos from control mice The data are presented as mean ± S.E (n=4), *p