Smad2 overexpression rescues the tgf-β3 null mutant mice cleft palate by increased apoptosis

II Preface . IV Table of contents . V List of tables. IX List of figures . X List of abbreviations . XI Acknowledgements . XIV Dedication . XV Chapter 1: Introduction: . 1 1.1 Overview of the research plan . 1 1.2 Prevalence of orofacial clefts . 6 1.3 The origin of cells in the palate. 8 1.4 The process of palatogenesis . 9 1.4.1 Human palatal development . 9 1.4.2 Mouse palatal development . 13 1.4.3 Disparities between mouse and human anatomical development. 16 1.5 Syndromic cleft lip, with or without cleft palate . 17 1.6 Non-syndromic cleft lip, with or without cleft palate . 19 1.6. 1 Epidemiology of non-syndromic cleft lip, with or without cleft palate . 19 1.6. 2 Genetic etiology . 20 1.6. 3 Environmental risk factors . 20 VI 1.6. 4 NSCL/P and genetic studies. 22 1.7 TGF-β signaling . 23 1.7. 1 SMAD-dependent TGF-β signaling pathway . 24 1.7. 2 SMAD-independent TGF-β signaling pathway . 24 1.8 Mouse craniofacial phenotypes and deletion of TGF-β signaling genes . 25 1.8. 1 TGF-β1 null (TGF-β1) mice . 25 1.8. 2 TGF-β2 null (TGF-β2) mice . 25 1.8. 3 TGF-β3 null (TGF-β3) mice . 26 1.8. 4 TβRI, TβRII and TβRII null mice. 26 1.8. 5 SMAD2 null mice . 27 1.8. 6 SMAD3 and SMAD4 null mice . 27 1.9 Experimental mammalian models: mice in the study of cleft lip and palate . 28 1.9. 1 Experimental mammalian models: K14-SMAD2 mouse phenotype. 29 1.9. 2 TGF-β3 and K14-SMAD2/TGF-β3 mice phenotype . 29 Chapter 2: Medial edge epithelium . 31 2.1 Fate of medial edge epithelium . 31 2.1.1 Apoptosis (programed cell death) . 32 2.1.2 Epithelial-mesenchymal transdifferentiation . 33 2.1.3 Cell migration . 36 2.2 The role of twist1 and collagen IV during palatal development . 37 2.3 How cells die: apoptosis, autophagy and necrosis . 41 2.3.1 Cell death . 41 2.3.2 Differences between mechanism of cell death. 42 VII 2.4 TGF-β and apoptosis. 43 2.5 Mechanism of apoptosis . 45 2.6 Apoptosis detection methods . 47 2.6. 1 TUNEL technique . 47 2.6. 2 Immunohistochemistry detection of apoptosis related caspase-3 . 48 2.7 Hypothesis. 49 2.8 Aims and objectives . 49 Chapter 3: Materials and methods . 50 3.1 Animal breeding. 50 3.2 Animal genotyping. 50 3.3 Histology and immunohistochemistry . 52 3.3.1 Fixation and paraffin embedding of the samples . 52 3.3.2 Hematoxylin and eosin . 53 3.3.3 Deparaffinization of tissue sections and rehydration . 54 3.3.4 Antigen retrieval and background stain blocking . 54 3.3.5 Analysis of MEE apoptosis rate TUNEL technique . 55 3.3. 6 Analysis of MEE apoptosis rate ccaspase3 technique . 56 3.3. 7 Analysis of MEE phosphorylated-SMAD2 rate . 57 3.3. 8 Analysis of MEE twist1 and collagen IV expression . 58 3.4 Confocal laser microscopy . 59 3.5 Statistical analysis . 60 Chapter 4: Results and conclusion . 61 4.1 Increased MEE cell apoptotic rate. 61 VIII 4.2 Increased levels of activated “phosphorylated” SMAD2 in the MEE . 67 4.3 Loss of basement membrane in the MEE cell and twist1 positive cells. 69 4.4 Discussion . 72 4.4.1 SMAD2 overexpression rescues palatal fusion by inducing apoptosis . 73 4.4.2 Evidence of EMT mechanism and basement membrane degradation. 74 4.5 Conclusion . 75 Bibliography . 76