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Effects of Altered Gtf2i and Gtf2ird1 Expression on the Growth of Neural Progenitors and Organization of the Mouse Cortex

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Effects of Altered Gtf2i and Gtf2ird1 Expression on the Growth of Neural Progenitors and Organization of the Mouse Cortex Effects of altered Gtf2i and Gtf2ird1 expression on the growth of neural progenitors and organization of the mouse cortex by Hyemin Amy Oh A thesis submitted in conformity with the requirements For the degree of Master of Science Institute of Medical Science University of Toronto © Copyright by Hyemin Amy Oh (2013) Effects of altered Gtf2i and Gtf2ird1 expression on the growth of neural progenitors and organizations of the mouse cortex Hyemin Amy Oh Master of Science Institute of Medical Science University of Toronto 2013 Abstract Williams-Beuren syndrome (WBS) and 7q11.23 Duplication Syndrome (Dup7) are rare neurodevelopmental disorders associated with a range of cognitive and behavioural symptoms, caused by the deletion and duplication, respectively, of 26 genes on human chromosome 7q11.23. I have studied the effects of deletion or duplication of two candidate genes, GTF2I and GTF2IRD1, on neural stem cell growth and neurogenesis using cultured primary neuronal precursors from mouse models with gene copy number changes. I found that the number of neuronal precursors and committed neurons was directly related to the copy number of these genes in the mid-gestation embryonic cortex. I further found that in late-gestation embryos, cortical thickness was altered in a similar gene dose-dependent manner, in combination with layer-specific changes in neuronal density. I hypothesize that some of the neurological features of WS and Dup7 stem from these impairments in early cortical development ii Acknowledgement I would like to take this opportunity to thank Dr. Lucy Osborne for her continuous support and guidance. She is not just a supervisor but also a mentor as well as a motherly figure for me. The bright mood of the lab was the reason why I chose this lab for my masters and is the reason to continue to do my studies in the same lab for doctorial degree. I also like to extend my appreciation to supervisory committee members Dr. Freda Miller and Dr. Vincent Tropepe for their insightful suggestions, technical assistance and guidance. I give my deepest gratitude to Dr. Freda Miller for providing a research location, and to David for continuous guidance and feedbacks, without the support I would have not been able to finish my project. All the former and current members of the Osborne lab have made every corner of my master life pleasurable and exciting, allowing me to persevere despite all the curved bullets thrown by both negative and positive results I have encountered throughout the course of my masters. In particular, I owe a big gratitude to Emma for unconditional support, companionship (let us sail together in this boat and we will reach the new land), and irresistible art on the lab wall, to Elaine for maintaining animal colony and delightful lab- licious lunch, to Jennifer for delicious baked goods and exquisite suggestions for the thesis writing, to Emily for the kindly advices and directions especially when I faced with animosity, to Eli for brining colourful smiles through a stacks of intelligence and refinement, and lastly to Joana for allowing me to grow patience and extend goals to the level that I have never imagined before. I would also like to thank Victor, my previous supervisor, and mentor for inspiring me to become an elegant and insightful scientist. I also like to thank my friends especially BK, for listening to my abysmal concerns and doubts, and giving assurance and mental therapy sessions to solve problems, and for relieving the distress through countless numbers of sweets and food visits. None of this would have been even possible without support and unconditional love from my family, especially my parents. They encouraged me to be the best of myself and to iii persevere until I achieve my goals. Their love, sacrifice and wisdom are the reason for who I am and how I stand here. iv Table of Contents Abstract------------------------------------------------------------------ ii Acknowledgements--------------------------------------------------- v Table of Contents----------------------------------------------------- vi List of Tables ---------------------------------------------------------- ix List of Figures --------------------------------------------------------- x List of Abbreviations ------------------------------------------------ xii Chapter 1. Introduction --------------------------------------------- 1 1.1 Williams-Beuren Syndrome------------------------------------------------------ 1 1.1.1 History of Williams-Beuren Syndrome: 1 1.1.2 Williams-Beuren syndrome clinical phenotype 2 1.1.3 Williams-Beuren syndrome cognitive and behavioral phenotype. 6 1.1.4 Genetic Basis of WBS 8 1.2 7q11.23 Duplication Syndrome ------------------------------------------------- 11 1.3 Genotype –Phenotype correlations--------------------------------------------- 14 1.3.1 Implications of GTF2I and GTF2IRD1 in behavioural and cognitive profiles of WBS 15 v 1.4 GTF2I gene family ------------------------------------------------------------------ 18 1.4.1 GTF2I 19 1.4.2 GTF2IRD1 22 1.5 GTF2I gene family animal models ---------------------------------------------- 23 1.5.1 Gtf2ird1 mouse model 24 1.5.2 Gtf2i mouse model 26 1.6 Development of the cerebral cortex ------------------------------------------ 30 1.6.1 Genesis of the cerebral cortex 30 1.6.2 Signaling pathways in cortical development 34 1.7 . Neural mechanisms in WBS ---------------------------------------------------- 36 1.7.1 Neural mechanisms of impaired social processing in WBS 36 1.7.2 Role of Serotonin in emotional behaviors 38 1.7.3 Altered brain connectivity in Gtf2ird1 targeted mice 39 1.8 Research Aims and hypothesis-------------------------------------------------- 41 Chapter II. Disturbance in early neuronal development 42 in mouse models of WBS 2.1 Introduction ------------------------------------------------------------------------- 42 2.1.1 Mouse models 42 2.1.2. Neural Precursor Cells 44 2.2 Material and Methods ------------------------------------------------------------ 45 2.4.1. Animals 45 2.4.2 Genotyping 46 2.4.3. Cortical Precursor cell culture 47 2.4.4. Immunohistochemistry 48 2.4.5. Microscopy and Quantification 50 vi 2.4.6. Statistical Analysis 51 2.3 Results --------------------------------------------------------------------------------- 53 2.3.1. Gtf2i and Gtf2ird1 copy number variation regulates cortical precursor physiology 53 2.3.2. Hemizygous deletion of Gtf2i and Gtf2ird1 disrupts cortical precursor physiology and neurogenesis 57 2.3.3 Gtf2i duplication promotes proliferation and differentiation of cortical precursors but does not affect survival 63 2.3.4. Effects of a single gene Gtf2i+/del on precursor maintenance and neurogenesis. 69 2.3.5. Altered cortical layer organization in mouse models of WBS 71 Chapter III. Discussion and conclusions ---------------------- 74 3.1 Effects of Gtf2i and Gtf2ird1 on precursor cell biology ------------------- 74 3.2 Possible interactions between Gt2i and Trk signaling in cortical development -------------------------------------------------------------------------- 77 3.3 Gtf2i and Gtfi2rd1 plays a role in laminar organization and cell density of cortices ------------------------------------------------------------------- 83 Chapter IV. Conclusions and Future Directions 88 4.1 Summary ------------------------------------------------------------------------------ 88 4.1.1. Overview 88 4.1.2. Gtf2i and Gtf2ird1 play a crucial role in radial glial cells maintenance and differentiation without affecting proliferation and survival 90 4.1.3. Gtf2i and Gtf2ird1 copy number variation regulates cortical architecture and cell density in developing cortex 92 vii 4.2 Future Directions ------------------------------------------------------------------- 95 4.2.1. Balance between different cellular components: 95 4.2.1.1 defining Precursor differentiation 95 4.2.1.2. Defining precursor population 96 4.2.2. Molecular mechanism: relationship between TFII-I and ERK1/2 in neural precursor physiology 97 4.2.3 Cortical cytoarchitecture 98 4.2.4. Circuits and networks - synaptic function 100 4.2.5. Contribution of individual genes to neural development during prenatal and postnatal periods 103 4.2.6. Human models of WBS - induced pluripotent stem cells 105 4.3 Conclusion ---------------------------------------------------------------------------- 106 viii List of Tables Table 1.1 Clinical presentations of Williams-Beuren syndrome Table 1.2 The percentage of diagnosed WBS with different deletion size due to nonallelic homologous recombination between respective LCRs. Table 1.3 Comparison between WBS Duplication and WBS deletion clinical phenotypes Table 1.4 The summary of mouse models with varying copy number of Gtf2i and Gtf2ird1. Table 2.1 List of primers used and its sequence. Table 2.2 List of observed genotypes in each cross. Table 3.1 The summary of research findings. Table 3.2 Altered cortical architecture in different developmental disorders with cognitive impairments. ix List of Figures Chapter I Figure 1.1 The distinctive facial features of WBS individuals. Figure 1.2 The drawings of Down syndrome (DS) and WBS individuals displaying dissociation in visuospatial construction cognition. Figure 1.3 Schematic diagrams of WBS region on chromosomal region 7q11.23. Figure 1.4 The comparison between individuals with WBS deletion and WBS duplication. Figure 1.5 The genetic mapping of the reported cases of atypical deletion and typical WBS deletion. Figure 1.6 The protein structure diagram of the GTF2I gene family
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