The Role of Signaling Pathway Integration in Neurogenesis

iii List of Figures . xi List of Tables . xv List of Abbreviations . xvi Acknowledgements . xxvi Chapter 1: Introduction . 1 1.0 Introduction. 2 1.1 The neural retina a model for CNS development . 5 1.2 The invertebrate eye – a valuable model for retinal development . 11 1.3 Key Regulators of RPC cell fate and competence . 12 1.4 Critical regulators of RPC proliferation, maintenance and differentiation. 15 1.5 Extrinsic control of RPCs . 19 2.0 Hedgehog pathway – Overview . 20 v 2.1 Hh signaling and disease. 21 2.2 Hh ligand . 22 2.3 Hh signal transduction . 22 2.4 Gli family transcription factors . 27 2.5 Hh signaling in the retina . 31 3.0 Receptor Tyrosine Kinase pathway Overview . 33 3.1 RTK signal transduction . 33 3.2 The Epidermal Growth Factor pathway. 34 3.3 Fibroblast Growth Factor Signaling . 40 3.4 EGF and FGF signaling and progenitor growth in the retina . 40 4.0 Notch Signaling pathway – Overview . 43 4.1 Notch signaling cascade . 43 4.2 Two-way Notch signaling . 44 4.3 Notch signaling in the retina . 49 5.0 Integration of Hh signaling with the RTK and Notch pathways . 51 5.1 Synergistic Hh and RTK signaling . 51 5.2 Co-operative Notch and Hh signaling during neurogenesis . 52 vi 5.3 Mechanisms of Hh-Notch integration . 53 5.4 Phenotypic Hh-Notch overlap in the retina . 53 6.0 Objectives . 54 Chapter 2: Combinatorial Hedgehog and mitogen signaling promotes the in vitro expansion but not retinal differentiation potential of retinal progenitor cells . 56 2.1 Author Contribution Statement. 58 2.2 Abstract. 59 2.3 Introduction . 60 2.4 Materials and Methods . 62 2.4.1 Mice and retinal cell culture . 62 2.4.2 In situ hybridization and immunohistochemistry . 63 2.4.3 Western Blot . 64 2.4. 4 In vivo transplantation . 64 2.5 Results. 66 2.6 Discussion. 88 2.7 Acknowledgements . 91 vii 2.8 Supplemental Material . 92 2.8. 1 Supplemental Methods . 92 2.8. 1.1 Immunohistochemistry . 92 2.8. 1.2 RNA extraction and RT-PCR analysis . 92 2.8. 1.3 In vitro Hh-RPC differentiation . 93 2.8. 1.4 Microarray analysis . 93 2.8. 2 Supplemental Figures . 94 2.8. 3 Supplementary Table. 99 Chapter 3: A Notch-Gli2 axis sustains Hedgehog responsiveness of neural progenitors and Müller Glia . 100 3.1 Author contribution statement . 102 3.2 Abstract. 103 3.3 Introduction . 104 3.4 Materials and Methods . 107 3.4.1 Experimental animals . 107 3.4.2 Retinal explant culture, cell dissociation and EdU/BrdU labeling . 107 viii 3.4.3 RNA extraction and qRT-PCR . 108 3.4. 4 Tissue preparation . 109 3.4. 5 Immnocytochemistry, in situ hybridization, ß-galactosidase and EdU detection . 110 3.4. 6 Electroporation and expression vectors . 111 3.4. 7 Luciferase assay . 112 3.4. 8 Western blot . 112 3.4. 9 Florescent activated cell sorting . 113 3.4.10 Statistical analyses . 113 3.5 Results . 115 3.5.1 Gli transcription factor expression is associated with RPCs . 115 3.5.2 Gli2 is sufficient to promote RPC proliferation . 121 3.5.3 Notch regulation of Gli protein and Hh responsiveness . 124 3.5.4 Notch signaling biases cells to a Müller glial fate and maintains Gli2 protein levels, but does not fully activate the Hh pathway . 129 3.5. 5 Notch and Hh pathways can be active simultaneously in the same cell . 132 3.5. 6 Notch extends the competence of Müller glia to respond to an Hh ix signal via Gli2 . 136 3.6 Discussion. 143 3.7 Acknowledgements . 150 3.8 Supplemental Material . 151 3.8. 1 Supplemental Figures . 151 Chapter 4: Discussion . 166 4.0 Discussion.