Aus der Abteilung für Neurogenetik (Prof. Klaus-Armin Nave, Ph. D.) des Max-Planck-Instituts für Experimentelle Medizin in Göttingen Roles of bHLH Transcription Factors Neurod1, Neurod2 and Neurod6 in Cerebral Cortex Development and Commissure Formation INAUGURAL - DISSERTATION zur Erlangung des Doktorgrades der Medizinischen Fakultät der Georg-August-Universität zu Göttingen vorgelegt von Ingo Bormuth aus Offenbach am Main Göttingen 2015 D e k a n: Prof. Dr. rer. nat. Heyo Klaus Kroemer I. Berichterstatter: Prof. Dr. med. Mikael Simons II. Berichterstatter: Prof. Dr. rer. nat. Jörg Großhans III. Berichterstatter: Prof. Dr. mult. Thomas Meyer Tag der mündlichen Prüfung: 7. April 2016 Contents Summary 1 1 Introduction 3 1.1 Short History of Neurosciences....................4 1.2 Cerebral Cortex Development.....................6 1.2.1 Axis Specification......................6 1.2.2 Arealization.........................7 1.2.3 Radial Migration......................9 1.2.4 Neuronal Identity...................... 11 1.2.5 Axon Growth........................ 11 1.2.6 Connectivity......................... 13 1.3 bHLH Transcription Factors...................... 14 1.3.1 bHLH Domain........................ 14 1.3.2 Classification........................ 15 1.3.3 Functions.......................... 16 1.3.4 Neuronal bHLH Proteins.................. 17 1.4 The NeuroD-Family........................... 18 1.4.1 Neurod1........................... 18 1.4.2 Neurod2 and Neurod6................... 19 1.4.3 Neurod4........................... 20 1.5 Genetic Inactivation Studies...................... 20 1.5.1 Inactivation of Neurod1.................. 21 1.5.2 Inactivation of Neurod2.................. 22 1.5.3 Inactivation of Neurod6.................. 23 1.6 Functional Redundancy........................ 24 1.7 The Cre-LoxP System.......................... 25 2 Results 27 2.1 The Neurod6-Lineage of Cells..................... 28 2.2 Inactivation of Neurod6......................... 30 2.3 Inactivation of Neurod2......................... 31 2.3.1 Lethality........................... 31 2.3.2 Cortical Connectivity.................... 32 2.4 Simultaneous Inactivation of Neurod2/6.............. 33 2.4.1 Brain Anatomy....................... 34 2.4.2 Cortex Development.................... 35 2.4.2.1 Overview................... 36 2.4.2.2 Radial Migration............... 37 2.4.2.3 Subplate.................... 39 2.4.2.4 Upper Layers................. 40 2.4.2.5 Deeper Layers................ 41 i Contents 2.4.3 Adult Brain......................... 43 2.4.4 Cortical Connectivity.................... 44 2.4.4.1 Callosal Projections............. 44 2.4.4.2 Axon Growth................. 45 2.4.4.3 Fasciculation................. 46 2.4.4.4 Midline Glia.................. 47 2.4.4.5 Midline Crossing............... 49 2.4.4.6 Subcortical Projections........... 51 2.4.5 Synaptic Function..................... 52 2.4.6 Arealization of the Neocortex............... 54 2.4.7 Upregulation of Neurod1.................. 55 2.5 Simultaneous Inactivation of Neurod1/2/6............. 56 2.5.1 Breeding........................... 57 2.5.2 Brain Anatomy....................... 58 2.5.3 Hippocampus........................ 59 2.5.3.1 Granule Neuron Differentiation...... 59 2.5.3.2 Pyramidal Neuron Differentiation..... 60 2.5.4 Neocortex.......................... 61 2.5.4.1 Radial Migration and Laminarization... 63 2.5.4.2 Determination and Early Differentiation. 64 2.5.4.3 Terminal Differentiation and Identity... 65 2.5.5 Developmental Cell Death................. 66 2.5.6 Cortical Connectivity.................... 67 2.5.6.1 Intracortical Projections........... 67 2.5.6.2 Subcortical Projections........... 68 2.5.6.3 Thalamocortical Handshake........ 70 2.6 Other Observations........................... 72 3 Discussion 73 3.1 Determination.............................. 74 3.2 Differentiation.............................. 76 3.2.1 Ventricular Zone...................... 76 3.2.2 Subventricular Zone.................... 77 3.2.3 Intermediate Zone..................... 78 3.2.4 Cortical Plate........................ 78 3.3 Migration................................. 80 3.4 Arealization............................... 81 3.5 Axon Growth............................... 83 3.6 Apoptosis................................. 85 3.7 Genetic Background.......................... 87 3.8 Adult Functions............................. 87 3.9 Outlook.................................. 89 3.9.1 Inducible Neurod6-Cre Mice................ 89 3.9.2 Neurod6 Overexpression.................. 90 3.10 Closing Words.............................. 91 4 Material and Methods 93 4.1 Transgenic Mice............................. 93 4.2 Genotyping................................ 93 4.2.1 Tissue Lysis......................... 94 4.2.2 Polymerase Chain Reaction................ 94 ii Contents 4.2.3 Gel Electrophoresis..................... 95 4.2.4 Neurod1-Flox PCR..................... 95 4.2.5 Neurod2-Null PCR..................... 96 4.2.6 Neurod6-Cre PCR...................... 97 4.3 Cell Culture............................... 97 4.4 In Utero Electroporation........................ 99 4.5 Electrophysiology............................ 99 4.6 qRT-PCR................................. 100 4.7 Histology................................. 101 4.7.1 Tissue Preparation and Fixation............. 101 4.7.2 Tissue Sectioning...................... 101 4.7.2.1 Vibratome................... 101 4.7.2.2 Cryostat.................... 101 4.7.2.3 Paraffin.................... 102 4.7.3 X-gal Histochemistry.................... 102 4.7.4 Myelin Staining....................... 102 4.7.5 Immunohistochemistry.................. 103 4.7.6 In Situ Hybridization.................... 104 4.8 Software................................. 105 4.8.1 Statistical Analysis..................... 106 4.8.2 Sequence Alignment.................... 107 4.8.3 Genetic Modeling...................... 107 4.8.4 Image Processing...................... 107 4.8.5 Manuscript Preparation.................. 108 Bibliography 109 Appendix 128 Acknowledgments............................... 129 Abbreviations................................. 131 List of Figures................................. 134 List of Tables.................................. 135 Index...................................... 136 Zusammenfassung (German Summary).................. 140 CC-BY iii Summary The brain is deeper than the sea, For, hold them, blue to blue, The one the other will absorb, As sponges, buckets do. E. Dickison1 ASIC HELIX-LOOP-HELIX (bHLH) proteins constitute a diverse group of evolutionary well-conserved transcription factors. Many transactivating B bHLH proteins follow cell type- or tissue-specific expression patterns and act as key regulators of cellular determination and differentiation processes. The closely related neuronal bHLH genes Neurod1, Neurod2 and Neurod6 are expressed by differentiating pyramidal neurons in the developing cerebral cortex and have long been suspected to regulate the maturation of these cells. Each of the three genes was genetically inactivated in mice, but studies of single-deficient animals failed to identify important functions in embryonic pyramidal neurons. Considering high sequence similarity and overlapping expression patterns, most authors suggested functional redundancy amongst the NeuroD-family. To test this, I bred transgenic mice lacking the two most similarly expressed NeuroD genes, Neurod2/6; and analyzed cerebral cortex development with an emphasis on pyramidal neuron identity and neocortical connectivity. Neurod2 and Neurod6 indeed share several hitherto unknown functions and compensate for each other’s loss. At least one of the two genes is necessary for: (1) the control of radial migration in a subset of pyramidal neurons; (2) area determination in the neocortex; and (3) the formation of fiber tracts connecting the neocortex to the striatum, to the thalamus, and to the contralateral hemi- sphere. In Neurod2/6 double-deficient mice, callosal axons form fasciculated fiber bundles that grow tangentially into the medial neocortex, but stall and defasciculate before reaching the ipsilateral cingulum or any midline associated structure. This new variant of callosal agenesis implies the presence of a not yet identified axon guidance mechanism in the medial neocortex. Neocortical Neurod1 expression, which is normally restricted to the subventric- ular zone, persists in the intermediate zone and cortical plate of Neurod2/6 double-deficient embryos. Ectopically upregulated Neurod1 can provide redun- dant functionality to compensate for the loss of Neurod2/6. I went further and bred conditional Neurod1/2/6 triple-deficient mice, in which the Neurod1 gene is specifically inactivated in cells with Neurod6-promoter activity. 1Dickinson 2013, CXXVI, verse 2 1 Summary As hypothesized, Neurod1 shares additional functions with Neurod2 and Neurod6. At least one of the three genes is necessary for hippocampal pyramidal neuron differentiation and the prevention of developmental cell death in the medial cortex. While the simultaneous inactivation of Neurod1/2/6 results in the complete loss of archicortical pyramidal neurons, many neocortical pyramidal cells survive, migrate radially and settle in the cortical plate. However, terminal pyramidal neuron differentiation is incomplete and neocortical connectivity is dramatically reduced in the triple-deficient mice. Taken together, this work shows that NeuroD-family transcription factors coopera- tively regulate pyramidal neuron differentiation, survival,