Molecular and morphological characterization of caudal neural tube defects in embryos of diabetic swiss albino mice

MOLECULAR AND MORPHOLOGICAL CHARACTERIZATION OF CAUDAL NEURAL TUBE DEFECTS IN EMBRYOS OF DIABETIC SWISS ALBINO MICE LOH WAN TING A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF ANATOMY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2009 Acknowledgements ACKNOWLEDGEMENTS After several years of studying how maternal diabetes induces spina bifida while poking around embryos with a guilt-ridden feeling from all the killings, words cannot describe how elated I felt as I pen this section with whatever sanity that is left in me. First and foremost, I would like to express my deepest gratitude and heartfelt appreciation to Associate Professor Samuel Tay Sam Wah for his invaluable guidance and advice, the occasional pep talks and the sharing of his life’s philosophies with me. I also deeply appreciate that he has given me free reign in my project. I am thankful to him for believing in me and giving me the opportunity to continue my candidature as a graduate student under his wing. This work would not have been possible without him and I could not ask for a better supervisor. I am also greatly indebted to Associate Professor S Thameen Dheen for who he is: an open-minded and encouraging co-supervisor with a constant flow of ideas and constructive comments who provides support as a friend in the most difficult of times. An enormous amount of time was spent on weekly meetings and several drafts of my manuscript and thesis and I really appreciate all that he has done for me from the bottom of my heart. In addition to my supervisor, two people have played an instrumental role in saving me from leaving research after a year as a graduate student in the Department of Surgery. I am particularly grateful to Professor Bay Boon Huat for facilitating the transfer by introducing and recommending me to my supervisor and to Professor Ling Eng Ang for giving me the chance to join the Department of Anatomy as a graduate student. i Acknowledgements Special thanks go out to Mr. Jiang Boran for all the helpful discussions and taking time out to show me the technical know-hows when I first joined the department. I would also like to thank Dr. Dinesh Kumar for his help and support during my time in the department. I would like to express my heartfelt thanks to Mrs Ng Geok Lan for her excellent technical expertise in histology work, Mrs Yong Eng Siang for her troubleshooting help in the lab, Ms. Chan Yee Gek for her assistance in the learning of confocal microscopy, Mr. Lim Beng Hock for toiling in the hot and humid animal house looking after my experimental animals, Mdm Ang Lye Gek Carolyne, Mdm Teo Li Ching Violet and Mdm Mohan Singh for their secretarial assistance. My association with the Department of Anatomy has been an extremely pleasant one. Life is nothing without friends and I am grateful to Mr. Li Wenbo for the occasional frantic borrowing of reagents, Mr. Li Lv for providing a listening ear for all my complaints and his help in animal work, Mr. Lai Yiyang for giving the greedy me gastronomic bliss during those lunch outings and Mr. Guo Chunhua for his expertise in certain technical aspects of research. I am also happy and lucky to forge friendships with Ms. Yvonne Teng, Ms. Koo Chuay Yeng, Ms. Lim Daina, Ms. Yu Ying Nan and Mr. Li Zhaohui during my candidature in the department. I wish to extend my thanks to all other fellow graduate students and staff members in the Department of Anatomy for rendering help in one way or another. I also greatly acknowledge the National University of Singapore for giving me a chance to study my Ph.D. with a Research Scholarship. ii Acknowledgements Finally, I would like to thank my family for putting up with me as a ‘perpetual student’ for the last 20 odd years. I owe them for much of what I have become and I dedicate this work to them for their love, patience and unwavering support during these years. iii Table of Contents TABLE OF CONTENTS ACKNOWLEDGMENTS . i TABLE OF CONTENTS iv SUMMARY ix LIST OF ABBREVIATIONS . xi LIST OF PUBLICATIONS xiv CHAPTER 1.1 INTRODUCTION Diabetes mellitus 1.1.1 General background of diabetes mellitus 1.1.2 Complications associated with diabetes mellitus 1.1.3 Maternal diabetes and congenital malformations .4 1.1.4 1.2 Maternal diabetes-induced neural tube defects Development of the neural tube 1.2.1 Gastrulation: From cells to embryo 1.2.2 Mechanisms of neurulation .7 1.2.3 Closure of the neural tube .8 1.2.4 Molecular factors involved in neural tube development .10 1.2.4.1 Dorso-ventral patterning of the neural tube .10 1.2.4.1.1 Sonic hedgehog .10 1.2.4.1.2 Bone morphogenetic proteins .12 1.2.4.1.3 Wingless-related MMTV integration site .13 1.2.4.2 Specification of neuronal fates in the neural tube 14 1.2.4.2.1 Basic helix-loop-helix proteins .17 1.2.4.2.1.1 Mammalian atonal homolog 18 1.2.4.2.1.2 Neurogenin and Neurogenin .19 1.2.4.2.1.3 Achaete-scute complex-like .20 1.2.4.2.1.4 Oligodendrocyte lineage transcription factor 21 iv Table of Contents 1.2.4.2.2 Homeobox genes .22 1.2.4.2.2.1 Pax2 .22 1.2.4.2.2.2 Islet .23 1.2.4.2.3 Other molecular factors .25 1.2.4.2.3.1 Brain lipid-binding protein .25 1.2.4.2.3.2 Doublecortin .26 1.3 Development of dorsal root ganglia neurons .26 1.3.1 Sensory neuron specification 28 1.4 Diabetic mouse models 29 1.5 Hypotheses and objectives .31 1.5.1 Specific aims .34 CHAPTER MATERIALS AND METHODS 36 2.1 Experimental animals 37 2.2 Induction of diabetes mellitus in mice 37 2.2.1 Materials .37 2.2.2 Procedure 38 2.3 Blood glucose test .38 2.4 Collection of embryos 39 2.4.1 Materials .39 2.4.2 Procedure 40 2.5 Storage of embryos 40 2.5.1 Materials .40 2.5.2 Procedure 41 2.6 Histology .41 2.6.1 Materials .41 2.6.2 Procedure 42 2.7 Fluorescent immunohistochemistry .44 2.7.1 Principle 44 v Table of Contents 2.7.2 Materials .45 2.7.3 Procedure 46 2.8 Tdt-mediated dUTP nick end labeling (TUNEL) assay .47 2.8.1 Principle 47 2.8.2 Materials .48 2.8.3 Procedure 49 2.9 Analysis of cell proliferation by BrdU labeling .49 2.9.1 Principle 49 2.9.2 Materials .50 2.9.3 Procedure 51 2.10 Whole mount in situ hybridization .52 2.10.1 Principle 52 2.10.2 Preparation of cRNA probes .53 2.10.3 Preparation of competent cells 53 2.10.3.1 Materials 53 2.10.3.2 Procedure .54 2.10.4 Transformation of competent cells with plasmid 55 2.10.4.1 Materials 55 2.10.4.2 Procedure .55 2.10.5 Linearization of plasmid .56 2.10.5.1 Materials 56 2.10.5.2 Procedure .57 2.10.6 In vitro transcription .57 2.10.6.1 Materials 57 2.10.6.2 Procedure .58 2.10.7 Whole mount in situ hybridization .59 2.10.7.1 Materials 59 2.10.7.2 Procedure .62 2.11 Isolation of total RNA and real time RT-qPCR 64 vi Table of Contents 2.11.1 Principle 64 2.11.2 Extraction of total RNA 66 2.11.2.1 Materials 66 2.11.2.2 Procedure .66 2.11.3 Synthesis of first-strand cDNA .67 2.11.3.1 Materials 67 2.11.3.2 Procedure .67 2.11.4 Real time RT-qPCR 68 2.11.4.1 Materials 68 2.11.4.2 Procedure .68 2.12 Statistical analysis 69 CHAPTER RESULTS .70 Part I 3.1 Maternal diabetes induces neural tube closure defects in embryos .71 3.2 Differentiation of neuronal cell types (moto- and inter-neurons) in the caudal neural tube of embryos from diabetic mice 72 3.3 Cell cycle progression is altered in the caudal neural tube of embryos from diabetic mice 74 3.4 Oligodendrocyte progenitors are increased in the caudal neural tube of embryos of diabetic mice 75 3.5 Development of radial glial cell lineages and migration of neurons are disrupted in the caudal neural tube of embryos from diabetic mice 75 3.6 Altered expression of developmental control genes in the caudal neural tube of embryos from diabetic mice .76 Part II 3.7 Maternal diabetes induced defects in the developing DRG .78 3.8 Cell proliferation and specification of mature neurons were impaired in the DRG of embryos from diabetic mice .78 3.9 Development of the sympathetic chains in embryos of diabetic mice are affected .79 vii Table of Contents CHAPTER DISCUSSION .80 CHAPTER CONCLUSIONS 89 REFERENCES .95 FIGURES AND FIGURE LEGENDS .121 viii Summary SUMMARY Embryos from diabetic mice exhibit several forms of neural tube defects including non-closure of the caudal neural tube. In the present study, embryos collected at embryonic day 11.5 from diabetic pregnancies displayed open neural tube with architectural disruption of the surrounding tissues and impaired development of dorsal root ganglia (DRG). The percentage of proliferating cells was found to be increased in the dorsal and ventral domains of the spinal neural tube of embryos from diabetic mice, indicating a defect in the precise timing of cell-cycle exit. The development of various cell types including motoneurons, interneurons, oligodendrocytes and migrating neurons as well as radial glial cells in the open neural tube using specific molecular markers have also been analyzed. Immunofluorescence results revealed a significantly reduced number of Pax2+ interneurons and increased number of Isl-1+ motoneurons as well as Olig2+ oligodendrocytes in the caudal neural tube of embryos from diabetic mice as compared to controls. In addition, these embryos exhibited a decreased number of doublecortin-positive migrating neurons and Glast/Blbp positive radial glial cells with shortened processes in the neural tube. Expression levels of several developmental control genes involved in generation of different neuronal cell types (such as Shh, Ngn, Ngn2, Ascl1) were also found to be altered in the caudal neural tube of embryos from diabetic mice. Sensory neurons in the DRGs of embryos from diabetic mice were found to have a lower proliferation index as compared to controls. The development of the sympathetic chains in the PNS was also affected by maternal diabetes as evident with the decrease in expression of the molecular marker tyrosine hydroxylase. ix Fig.12. Whole mount in situ hybridization for Ascl1 mRNA expression in embryos of normal (A-C) and diabetic (D-F) mice. Transverse sections (plane shown in A,D) through caudal neural tube of whole mount in situ hybridization embryos from normal (G) and diabetic (H) mice. Ascl1 expression domain has been found to have expanded in the caudal neural tube of embryos from diabetic mice. Scale bar: 200µm. 145 146 Fig.13(A-E). Wnt1 (221bp), Atoh1 (170bp), Ngn1 (164bp), Ngn2 (238bp) and Ascl1 (225bp) mRNA products were amplified by PCR and specificity of the products were confirmed with the respective melting curve analysis (A-E). 147 148 Fig.13(F). Real time RT-qPCR analysis of Wnt1, Atoh1, Ngn1, Ngn2 and Ascl1. Expression levels of Ngn1, Ngn2 and Ascl1 show an increase in the neural tubes of embryos from diabetic mice in comparison to the controls. Data represent mean ± S.E.M. *p[...]... List of Abbreviations PUBLICATIONS Articles in Journals 1 WT Loh, ST Dheen, B Jiang, SD Kumar and SSW Tay Molecular and morphological characterization of caudal neural tube defects in embryos of diabetic Swiss Albino mice (BMC Developmental Biology, in revision, 2008) 2 B Jiang, SD Kumar, WT Loh, J Manikandan, EA Ling, SSW Tay and ST Dheen Global gene expression analysis of cranial neural tubes in embryos. .. SSW Tay Molecular analysis of malformations in the neural tube of embryos derived from diabetic mother Symposium on Trends in Molecular and Applied Approaches to Reproduction and 15th Annual Meeting of ISSRF, 34 (Invited paper) (2005) 4 – 6 Feb 2005, Kolkata, Kolkota, India 12 WT Loh, ST Dheen, SSW Tay Analysis of malformations in the spinal neural tube and pancreas in embryos of diabetic mice International... called the neural tube in which the caudal region of the neural tube gives rise to the spinal cord and the rostral region becomes the brain Bending of the neural plate is based on the 7 Chapter 1: Introduction formation of three hinge points in the neural tissue: a median hinge point (MHP) where the neural plate is anchored to adjacent tissues, and paired lateral hinge points (LHP) where the neural plate... (represented by single asterisk) occurs in most mouse strains with more caudal and rostral locations of closure in some other strains (dashed lines with arrows) Modified from (Copp et al., 2003) 9 Chapter 1: Introduction 1.2.4 Molecular factors involved in neural tube development 1.2.4.1 Dorso-ventral patterning of the neural tube After neural induction and the formation of the neural tube, patterning along... that are involved in the control and coordination of motor output reside predominantly in the ventral half of the neural tube Past and recent experimental studies have culminated in a model in which graded Shh signaling in the ventral neural tube controls the expression of a group of homeodomain (HD) proteins, which in turn, establishes five domains of progenitor cells (Illustration 3) (Wilson and Maden,... growth in the developing neural tube is regulated by a balance between Wnt and BMP signaling in which Wnt signaling is ascribed a role in promoting cell cycle progression while BMP favors cell differentiation 1.2.4.2 Specification of neuronal fates in the neural tube Our understanding of the origin of neuronal cell types in the neural tube initially came from experiments conducted in the rat and chick... embryos of diabetic mice Journal of Neuroscience Research, July 2008, epub ahead of print 3 WT Loh, ST Dheen, B Jiang and SSW Tay Characterisation of developing dorsal root ganglia in embryos of diabetic mice (Manuscript in preparation, 2008) 4 ST Dheen, SSW Tay, B Jiang, WT Loh, SD Kumar and EA Ling Recent studies on neural tube defects in embryos of diabetic pregnancy: An overview Current Medicinal... of the neural folds Each hinge facilitates bending of the neural plate that results in convergence of the neural plate at the dorsal midline (Schoenwolf and Franks, 1984; Smith et al., 1994; Colas and Schoenwolf, 2001) Illustration 1 Mechanism of neural plate bending with the hinge point model Blue represents dorsolateral hinge points and red represents median hinge point Asterisks indicate furrowing... over-simplified and described as a continuous event that initiates at the level of the future cervical region and proceeds both rostrally and caudally (O'Rahilly and Muller, 1994) However, with multiple initiation sites of neural tube closure being described in mice and other species, it soon became clear that the closure of the human neural tube involves more than just a single site of initiation Indeed,... open neural tubes in embryos of diabetic mice are associated with defects in the timing of cell-cycle exit, cell migration and specification of different cell types including motoneurons and interneurons as well as glial cells along the dorsoventral axis of the developing spinal cord As these neuronal cell types in the spinal cord provide a network that modulates the sensory and motor output in both . altered in the caudal neural tube of embryos from diabetic mice 74 3.4 Oligodendrocyte progenitors are increased in the caudal neural tube of embryos of diabetic mice 75 3.5 Development of radial. percentage of proliferating cells was found to be increased in the dorsal and ventral domains of the spinal neural tube of embryos from diabetic mice, indicating a defect in the precise timing of cell-cycle. Jiang, SD Kumar and SSW Tay. Molecular and morphological characterization of caudal neural tube defects in embryos of diabetic Swiss Albino mice. (BMC Developmental Biology, in revision, 2008)