ENGRAILED IS REQUIRED IN MATURING SEROTONIN NEURONS TO REGULATE THE CYTOARCHITECTURE AND SURVIVAL OF THE DORSAL RAPHE NUCLEUS by STEPHANIE R. FOX Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. Evan S. Deneris Department of Neurosciences CASE WESTERN RESERVE UNIVERSITY August 2012 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Stephanie R. Fox . candidate for the Doctor of Philosophy degree*. (signed) Jerry Silver . (chair of the committee) Evan Deneris . Heather Broihier . Stephen Maricich . (date) 5/21/2012 . *We also certify that written approval has been obtained for any proprietary material contained within. Table of contents List of tables ................................................................................................... 4 List of figures .................................................................................................. 5 Abstract ........................................................................................................... 7 Chapter 1 - Introduction to the central serotonin system and engrailed transcription factors ....................................................................................... 9 Central serotonin system .............................................................................. 9 5-HT neuron identity .......................................................................... 11 Molecular control of 5-HT neuron development ............................. 16 Setting up the A-P axis – Mid-hindbrain organizer .................... 17 D-V patterning ........................................................................... 22 Neurogenesis ............................................................................ 23 Terminal differentiation .............................................................. 27 Maturation and maintenance ..................................................... 31 Developmental neuroanatomy .......................................................... 33 Cell body movements and formation of the 5-HT nuclei ............ 34 Formation of ascending and descending projections ................ 35 5-HT transcriptional network and components - relevance to human health .................................................................................................. 38 Maternal care ............................................................................ 40 Mood and emotional behaviors ................................................. 41 Neurodevelopment–implications for autism spectrum disorder . 44 1 Engrailed .................................................................................................. 47 Roles in the CNS ................................................................................ 49 Tissue patterning ....................................................................... 50 Fate selection/ terminal cell type generation ............................. 53 Cell survival ............................................................................... 57 Axon guidance .......................................................................... 58 Engrailed and mental health ............................................................. 61 Aims of thesis ............................................................................................... 64 Chapter 2-Engrailed is required in maturing serotonin neurons to regulate the cytoarchitecture and survival of the dorsal raphe nucleus ................ 75 Summary ............................................................................................ 76 Introduction ........................................................................................ 77 Materials and methods ..................................................................... 79 Results ................................................................................................ 87 En1 and En2 are expressed in postmitotic 5-HT neurons. ........ 87 Conditional targeting of En in maturing 5-HT neurons .............. 89 Engrailed controls maturation of DRN cytoarchitecture ............. 90 Engrailed is required for perinatal maintenance of serotonergic neuron identity........................................................................... 94 En1/2 deficiency causes 5-HT neuron cell death postnatally .... 98 En1/25HTCKO dams display mild deficits in pup rearing ............... 99 Discussion ........................................................................................ 102 2 Chapter 3- Effect of genetic background on serotonergic system development and the Pet-1-/- phenotype ................................................... 131 Summary .......................................................................................... 132 Introduction ...................................................................................... 133 Materials and methods ................................................................... 135 Results .............................................................................................. 139 Comparison of C57BL/6, SJL and wild-type 129/Sv*C57BL/6 139 Interaction between Pet-1 expression and background .......... 140 Reduced pup survival from C57BL/6-Pet-1-/- dams ................. 142 Discussion ........................................................................................ 144 Chapter 4- Discussion ................................................................................ 156 5-HT neurogenesis........................................................................... 158 Cell migration and DRN formation ................................................. 159 Maintenance of 5-HT neuron identity ............................................. 164 Cell survival ...................................................................................... 167 Axon projections ............................................................................. 173 Behavior consequences of conditional En1/2 ablation ................ 175 Final thoughts .................................................................................. 176 References ................................................................................................ 179 3 List of tables Chapter 2 Table. Summary of pup survival .................................................................. 155 4 List of figures Chapter 1 Figure 1. Major components of 5-HT neuron identity .......................... 67 Figure 2. Determinants important for the development of 5-HT neurons ............................................................................................... 69 Figure 3. Neuroanatomical origins of 5-HT neurons ........................... 71 Figure 4. Engrailed is highly conserved amongst species and paralogs .............................................................................................. 73 Chapter 2 Figure 1. Mating strategies for generation of each of the genotypes used in analyses ........................................................................................ 108 Figure 2. Spatiotemporal expression patterns of En1 and En2 in the rostral hindbrain ........................................................................................... 110 Figure 3. En1 expression in 5-HT neurons is maintained postnatally ......................................................................................... 112 Figure 4. Conditional targeting of En1/2 in 5-HT neurons after neurogenesis .................................................................................... 114 Figure 5. Engrailed is required in rostral postmitotic 5-HT neurons to regulate formation of the DRN .......................................................... 116 Figure 6. Engrailed is not required for early maintenance of 5-HT neuron identity .............................................................................................. 119 5 Figure 7. Engrailed is required for maintenance of serotonergic identity in the perinatal period ........................................................................... 121 Figure 8. Engrailed is required in postmitotic 5-HT neurons for maintenance of brain 5-HT levels ..................................................... 123 Figure 9. En1 is the predominant functional engrailed paralog for maintenance of 5-HT neuron identity ................................................ 125 Figure 10. Engrailed is required for survival of postmitotic 5-HT neurons ............................................................................................. 127 Figure 11. Conditional targeting of En1/2 in 5-HT neurons negatively impacts pup outcome measures ....................................................... 129 Chapter 3 Figure 1. Comparison of wild-type C57BL/6, SJL and mixed 129/Sv*C57BL/6 backgrounds .......................................................... 147 Figure 2. Immunohistological comparison of congenic C57BL/6 and SJL and mixed 129/Sv*C57BL/6 Pet-1+/+ and Pet-1-/- mice ...................... 149 Figure 3. HPLC analysis of congenic and mixed background in wild-type and Pet-1 mutant mice ...................................................................... 151 Figure 4. Pup survival and nest building behavior in C57BL/6-Pet-1+/- and Pet-1-/- dams .................................................................................... 153 . 6 Engrailed is Required in Maturing Serotonin Neurons to Regulate the Cytoarchitecture