.. .EFFECTS OF HIGH GLUCOSE CONCENTRATIONS ON THE EXPRESSION OF GENES INVOLVED IN PROLIFERATION AND CELL- FATE SPECIFICATION OF MOUSE EMBRYONIC NEURAL STEM CELLS FU JIANG (MD, MMed) A THESIS... Illustration Schematic summary of the effects of high glucose on the expression of developmental control genes that are involved in proliferation, survival and differentiation of neural stem cells. .. promote the differentiation of autonomic neurons and induce the expression of Ascl1 indicating that BMP2 and BMP4 control the specification of autonomic neurons through the induction of Ascl1 expression
EFFECTS OF HIGH GLUCOSE CONCENTRATIONS ON THE EXPRESSION OF GENES INVOLVED IN PROLIFERATION AND CELL-FATE SPECIFICATION OF MOUSE EMBRYONIC NEURAL STEM CELLS FU JIANG NATIONAL UNIVERSITY OF SINGAPORE 2006 EFFECTS OF HIGH GLUCOSE CONCENTRATIONS ON THE EXPRESSION OF GENES INVOLVED IN PROLIFERATION AND CELL-FATE SPECIFICATION OF MOUSE EMBRYONIC NEURAL STEM CELLS FU JIANG (MD, MMed) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF ANATOMY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2006 ACKNOWLEDGEMENTS I would like to express my deepest appreciation to my supervisor, Dr S Thameem Dheen, Department of Anatomy, National University of Singapore, for his invaluable guidance, innovative ideas, friendly criticisms and constant encouragement throughout the course of the study I am greatly indebted to Professor Ling Eng Ang, Head, Department of Anatomy, National University of Singapore, for his full support in providing me with the excellent research facilities and a fascinating academic environment I am also grateful to Associate Professor Tay Sam Wah Samuel, Deputy Head, Department of Anatomy, National University of Singapore, for his constant encouragement, valuable advice and constructive criticism I must acknowledge my gratitude to Mrs Yong Eng Siang and Mrs Ng Geok Lan for their excellent technical assistance, Mr Yick Tuck Yong for his assistance in computer work, Mr Lim Beng Hock for looking after the experimental animals, and Ms Ang Lye Geck Carolyne, Ms Diljit Kaur d/o Bachan Singh, and Ms Teo Li Ching Violet for their secretarial assistance I would like to thank all other staff members, my fellow postgraduate students in the Department of Anatomy, National University of Singapore for their help and support I am also thankful to Sanwa Kagaku Kenkyusho Co., Ltd., Japan for providing fidarestat Certainly, without the financial support from the National University of Singapore, this work would not have been brought to a reality I would like to take this opportunity to express my heartfelt thanks to my parents, my sister and brother for their full and endless support through the years i Finally, I am greatly indebted to my wife, Ms Gao Qing for her understanding and encouragement during my study ii This thesis is dedicated to my beloved family iii PUBLICATIONS Various portions of the present study have been published or submitted for publication International Journals: J Fu, S S W Tay, E A Ling, S T Dheen High glucose alters the expression of genes involved in proliferation and cell-fate specification of embryonic neural stem cells Diabetologia (2006) 49: 1027-1038 J Fu, S S W Tay, E A Ling, S T Dheen Aldose reductase is implicated in high glucose-induced oxidative stress in mouse neural stem cells J Neurochem (In revision) Conference Abstracts: J Fu, S S W Tay, E A Ling, S T Dheen (2004) Analysis of proliferation and differentiation of neural stem cells in a high glucose environment American Society for Biochemistry and Molecular Biochemistry Annual Meeting and 8th International Union of Biochemistry and Molecular Biology Conference, Abstract Number 254, 1216 June, Boston, MA, USA J Fu, S S W Tay, E A Ling, S T Dheen (2004) Characterization of embryonic neural stem cells exposed to high glucose concentration 8th NUS-NUH Annual Scientific Meeting, Abstract Number P-22, Page 89, 7-8 October, Singapore J Fu, S S W Tay, E A Ling, S T Dheen (2004) Effects of high glucose concentration on the proliferation and differentiation of neural stem cells International Biomedical Science Conference, Abstract Number P08, Page 78, 3-7 December, Kunming, China J Fu, S S W Tay, E A Ling, S T Dheen (2005) High D-glucose induces oxidative stress and alters expression of genes involved in proliferation and cell fate specification of embryonic neural stem cells Society for Neuroscience 35th Annual Meeting, Abstract Number 24.10 (24 Neural Stem Cells in Brain Injury and Disease, Theme A, A10), 12-16 November, Washington, DC, USA J Fu, S S W Tay, E A Ling, S T Dheen (2005) Abnormal proliferation and differentiation of neural stem cells exposed to high glucose are associated with altered gene expression and oxidative stress International Neuroscience Conference, Abstract Number AP32, 26-29 November, Al Ain, United Arab Emirates iv TABLE OF CONTENTS ACKNOWLEDGEMENTS ·····················································································i DEDICATION·········································································································iii PUBLICATIONS ····································································································iv TABLE OF CONTENTS ························································································v ABBREVIATIONS ·······························································································xiii SUMMARY ···········································································································xvi LIST OF TABLES ·································································································xx LIST OF ILLUSTRATIONS ···············································································xxi LIST OF FIGURES ·····························································································xxii CHAPTER 1: INTRODUCTION··········································································· 1 General background of diabetes mellitus······························································2 1.1 Complications associated with diabetes mellitus ·········································3 1.2 Maternal diabetes and congenital malformation ··········································4 1.3 Maternal diabetes-induced neural tube defects ············································5 Molecular mechanisms of neural tube development·············································6 2.1 Development of the neural tube···································································6 2.2 Factors involved in neural tube development ··············································9 2.2.1 Morphogens······················································································10 2.2.1.1 Sonic hedgehog········································································11 2.2.1.2 Bone morphogenetic proteins ··················································12 2.2.2 Notch signaling pathway ··································································14 v 2.2.3 bHLH transcription factors ·······························································15 2.2.3.1 Hes1 and Hes5 ·········································································15 2.2.3.2 Neurog1/2 and Ascl1 ·······························································17 2.2.3.3 Olig1 and Olig2 ·······································································19 Hyperglycemia-associated molecular and cellular changes ································20 3.1 Changes in cellular glucose uptake····························································21 3.2 Hyperglycemia-induced oxidative stress ···················································22 3.3 Alterations in signaling pathways······························································24 3.4 Cellular changes ························································································26 Experimental models to study the pathogenesis of maternal diabetes-induced neural tube defects ······································································································26 4.1 Diabetic animal models ·············································································27 4.2 Neural stem cell (NSC) culture··································································29 Specific Aims ·····································································································31 CHAPTER 2: MATERIALS AND METHODS ················································· 34 Animals ··············································································································35 Induction of diabetes mellitus in mice ································································36 2.1 Materials····································································································36 2.2 Procedure···································································································36 Blood glucose test ······························································································36 Collection of embryos ························································································37 4.1 Materials····································································································37 vi 4.2 Procedure···································································································38 Histology············································································································39 5.1 Materials····································································································39 5.2 Procedure···································································································39 Primary culture of neural stem cell (NSC) ·························································40 6.1 Materials····································································································40 6.2 Procedure···································································································41 Differentiation of NSCs······················································································41 7.1 Materials····································································································41 7.2 Procedure···································································································42 Treatment of NSCs·····························································································43 8.1 Materials····································································································43 8.2 Procedure···································································································43 Assay of cell viability·························································································44 9.1 Principle ····································································································44 9.2 Materials····································································································44 9.3 Procedure···································································································45 10 TUNEL analysis to detect apoptosis·································································46 10.1 Principle ··································································································46 10.2 Materials··································································································47 10.3 Procedure·································································································47 11 Analysis of proliferation index ·········································································48 11.1 Principle ··································································································48 vii 11.2 Materials··································································································49 11.3 Procedure·································································································50 12 Fluorescent immunohistochemistry··································································51 12.1 Principle ··································································································51 12.2 Materials··································································································53 12.3 Procedure·································································································54 13 Isolation of RNA and Real time reverse transcription- polymerase chain reaction (RT-PCR) ·········································································································55 13.1 Principle ··································································································55 13.1.1 Polymerase chain reaction (PCR) ···················································55 13.1.2 Isolation of total RNA·····································································57 13.2 Materials··································································································58 13.3 Procedure·································································································58 13.3.1 Extraction of total RNA ··································································58 13.3.2 cDNA synthesis ··············································································59 13.3.3 The real time RT-PCR ····································································59 13.3.4 Image of PCR products···································································59 14 In situ hybridization··························································································61 14.1 Principle ··································································································61 14.2 Preparation of cRNA probes····································································61 14.3 Preparation of competent cells·································································62 14.3.1 Materials·························································································62 14.3.2 Procedure························································································63 viii Figures Figure 30 Cell proliferation index by BrdU labeling in NSCs exposed to PG and HG (30mmol/l) with or without fidarestat (a-d, arrows) for 72h The percentage of proliferating cells appeared to be decreased in NSCs exposed to HG medium for 72h in comparison to that of NSCs exposed to PG medium for 72h (a, b, e) Addition of fidarestat increased the proliferation index of NSCs exposed to the HG medium in comparison to that of NSCs exposed to the HG medium without fidarestat (c-e) The data are presented as Mean ± SD (n=5) *p