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On the Role of CCDC66 Gene Products in Retina and Extraretinal Tissues Of ON THE ROLE OF CCDC66 GENE PRODUCTS IN RETINA AND EXTRA- RETINAL TISSUES OF THE CCDC66 -DEFICIENT MOUSE MODEL FOR RETINAL DEGENERATION by Sabrina Schreiber A thesis submitted in partial fulfilment of the requirements for the degree of Philosophiae Doctoris (PhD) in Neuroscience from the International Graduate School of Neuroscience Ruhr University Bochum March 31 st 2015 This research was conducted at the Department of Human Genetics within the Faculty of Medicine at the Ruhr University under the supervision of Prof. Dr. J. T. Epplen Printed with the permission of the International Graduate School of Neuroscience, Ruhr University Bochum Statement I certify herewith that the dissertation included here was completed and written independently by me and without outside assistance. References to the work and theories of others have been cited and acknowledged completely and correctly. The “Guidelines for Good Scientific Practice” according to § 9, Sec. 3 of the PhD regulations of the International Graduate School of Neuroscience were adhered to. This work has never been submitted in this, or a similar form, at this or any other domestic or foreign institution of higher learning as a dissertation. The abovementioned statement was made as a solemn declaration. I conscientiously believe and state it to be true and declare that it is of the same legal significance and value as if it were made under oath. Name / Signature Bochum, 31.03.2015 PhD Commission Chair: 1st Internal Examiner: Prof. Dr. J. T. Epplen 2nd Internal Examiner: Dr. habil. M. Schmidt External Examiner: Non-Specialist: Date of Final Examination: PhD Grade Assigned: Table of Contents Table of Contents I. List of Figures V II. List of Tables VII III. List of Abbreviations VIII IV. Abstract XI Chapter 1 - General introduction 1 1.1 The mammalian retina 2 1.1.1 The outer nuclear layer and photoreceptor structure 3 1.1.2 Subsequent layers and signal transmission 3 1.2 Inherited retinal degeneration 4 1.2.1 Non-syndromic inherited retinal degeneration 5 1.2.1 Syndromic inherited retinal degeneration 6 1.3 Animal models for retinal degeneration 7 1.4 Ccdc66 in retinal degeneration 8 1.4.1 Ccdc66 gene and products in the mouse 9 1.4.1.1 Ccdc66 RNA 9 1.4.1.2 CCDC66 protein 9 1.4.1.3 Localization 10 1.4.2 The Ccdc66 -deficient mouse – a retinal degeneration model 11 1.5 CCDC66 mutations in humans 12 1.6 Objectives 13 Chapter 2 - Functional investigation of Ccdc66 gene products in the mouse retina 16 2.1 Ccdc66 RNA and reporter gene expression 16 2.1.1 Introduction 16 2.1.2 Materials and methods 17 2.1.2.1 Animals 17 2.1.2.2 Tissue preparation 17 2.1.2.3 Preparation of cryosections 17 2.1.2.4 In situ hybridization 18 2.1.2.5 X-gal staining (enzyme histochemistry) 20 2.1.3 Results 21 2.1.3.1 Ccdc66 RNA expression in the postnatal Ccdc66 +/+ and Ccdc66 -/- mouse retina 21 I Table of Contents 2.1.3.2 Ccdc66 reporter gene expression in the postnatal Ccdc66 -/- mouse retina and comparison to Ccdc66 RNA expression (2.1.3.1) 22 2.1.3.3 Ccdc66 reporter gene expression during postnatal retinal development of the Ccdc66 -/- mouse retina 24 2.1.4 Discussion 28 2.1.4.1 Methodological aspects of Ccdc66 RNA, reporter gene expression and protein CCDC66 in the mouse retina 28 2.1.4.2 Retinal processes in parallel with temporal and spatial Ccdc66 reporter gene expression changes – functional association of Ccdc66 expression 32 2.2 Interaction partners of protein CCDC66 in the mouse retina 34 2.2.1 Introduction 34 2.2.2 Materials and methods 34 2.2.2.1 Yeast strains 35 2.2.2.2 Generation of CCDC66 bait constructs 35 2.2.2.3 Chemical transformation of yeast cells 37 2.2.2.4 Auto activity test of baits and prey 38 2.2.2.5 Yeast two-hybrid screen 39 2.2.2.6 Confirmation of positive interaction 40 2.2.3 Results 41 2.2.3.1 Auto activity tests of the baits 41 2.2.3.2 Interaction partner candidates 42 2.2.3.3 Verification of the candidates open reading frame (ORF) 44 2.2.3.4 Auto activity test of the prey and validation of their positive interaction with CCDC66 constructs 46 2.2.4 Discussion 48 2.2.4.1 Mpdz and Eps8 – prime candidates to interact with CCDC66 49 2.2.4.2 ZnHit3, C21orf2, Ppdpf and Med4 – false positive interaction partners? 52 Chapter 3 - Functional investigation of Ccdc66 gene products in the mouse brain 55 3.1 Ccdc66 RNA and reporter gene expression in the mouse brain 55 3.1.1 Introduction 55 3.1.2 Materials and methods 55 3.1.2.1 Animals, tissues and preparation of cryosections 55 3.1.2.2 In situ hybridization 56 3.1.2.3 X-gal staining 56 3.1.3 Results 56 3.1.3.1 Ccdc66 RNA expression in the postnatal Ccdc66 +/+ and Ccdc66 -/- mouse brain 56 II Table of Contents 3.1.3.2 Ccdc66 reporter gene expression in the postnatal Ccdc66 -/- mouse brain and comparison to Ccdc66 RNA expression 62 3.1.3.3 Ccdc66 reporter gene expression in the postnatal developing mouse brain 68 3.1.4 Discussion 78 3.1.4.1 Ccdc66 RNA, Ccdc66 reporter gene expression and protein CCDC66 in the mouse brain; on the role of potential short Ccdc66 transcripts and CCDC66 isoforms 78 3.1.4.2 Ccdc66 reporter gene expression in the mouse brain and parallelizing developmental processes – functional association of Ccdc66 expression 80 3.2 Degeneration in the Ccdc66 -/- mouse brain 85 3.2.1 Introduction 85 3.2.2 Material and methods 85 3.2.3 Results 86 3.2.3.1 Olfactory bulb 86 3.2.3.2 Brain ventricular ependyma 87 3.2.3.3 Hippocampus 87 3.2.4 Discussion 91 Chapter 4 - Compartment localization of protein CCDC66 in mouse retina and brain 93 4.1 Introduction 93 4.2 Materials and methods 93 4.2.1 Animals and tissues 93 4.2.2 Cell compartment fractionation and Western blot 94 4.2.3 Double immunofluorescence on neurospheres derived mixed neural cell culture 95 4.3 Results 97 4.3.1 Cell compartment fractionation 97 4.3.2 Co-localization of CCDC66 and neural marker proteins by double immune detection in a mixed neural cell culture 99 4.4 Discussion 101 Chapter 5 – Extra-retinal and extra-cerebral Ccdc66 reporter gene expression sites 104 5.1 Introduction 104 5.2 Materials and methods 104 5.2.1 Animals, tissues and preparation of cryosections 104 5.2.2 X-gal staining 104 5.3 Results 105 III Table of Contents 5.4 Discussion 110 Chapter 6 - General discussion 111 6.1 Summary of presented results 111 6.2 Concluding discussion and open questions 112 6.3 Conclusions 115 7. References 116 8. Appendices 137 8.1 Buffers and solutions 137 8.1.1 Chapter 2.1 137 8.1.2 Chapter 2.2 139 8.1.3 Chapter 3.2 140 8.1.4 Chapter 4 141 8.2 Curriculum vitae 143 8.3 List of publications 144 8.4 Acknowledgements 145 IV List of Figures I. List of Figures Figure 1: Structure of the mammalian retina .........................................................................................2 Figure 2: Prevalence of mutations in genes causing genetically heterogeneous retinal dystrophies in man. ................................................................................................................5 Figure 3: CCDC66 isoforms in the mouse. ......................................................................................... 10 Figure 4: 5`gene-trap-mediated interruption of the mouse Ccdc66 gene. .......................................... 11 Figure 5: Progressive degeneration of the Ccdc66 -deficient mouse retina ........................................ 12 Figure 6: Expression of Ccdc66 RNA in the Ccdc66 +/+ and Ccdc66 -/- mouse retina detected by in situ hybridization (postnatal day 17). ............................................................................... 22 Figure 7: Ccdc66 reporter gene expression in the retina of the Ccdc66 -/- mouse (postnatal day 17) ................................................................................................................ 23 Figure 8: Ccdc66 expression in the Ccdc66 +/+ mouse retina by in situ hybridization and by reporter gene expression in the Ccdc66 -/- mouse retina (postnatal day 17) ........................ 24 Figure 9: Ccdc66 reporter gene expression in the Ccdc66-deficient mouse during postnatal development ........................................................................................................................ 27 Figure 10: Ccdc66 constructs for the yeast two-hybrid screen. ........................................................... 35 Figure 11: Auto activity test of CCDC66-3 ............................................................................................ 42 Figure 12: Amino acid sequences of the prey proteins that showed positive interaction in the yeast two-hybrid screen with CCDC66 constructs .............................................................. 45 Figure 13: Auto activity test of prey C21orf2 and ZnHit3 and retransformation with CCDC66-1 and -5 ................................................................................................................................... 47 Figure 14: Auto activity test of prey Mpdz and Eps8 and proof of positive interaction with CCDC66-3 ........................................................................................................................... 48 Figure 15: Expression of Ccdc66 in the olfactory bulb of Ccdc66 +/+ and Ccdc66 -/- mice by in situ hybridization (P17) ............................................................................................................... 58 Figure 16: Ccdc66 in situ hybridization on hippocampus sections of the Ccdc66 +/+ and Ccdc66 -/- mouse (P17) .......................................................................................................................
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