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Determining TrkB intracellular signalling pathways required for specific aspects of gustatory development Juraj Koudelka Thesis submitted to the School of Biomedical Sciences, College of Medicine and Veterinary Medicine at the University of Edinburgh for the degree of Doctor of Philosophy 2013 I have read and understood The University of Edinburgh guidelines on plagiarism and declare that this thesis is the result of my own work except where indicated by references. This thesis has been submitted to The University of Edinburgh for the degree of Doctor of Philosophy only. Juraj Koudelka ABSTRACT Neurotrophins BDNF and NT4 influence the development of the rodent gustatory system. Despite binding to the same receptor, TrkB, they have different roles. BDNF is chemo-attractive for gustatory neurons and regulates gustatory neuron targeting and number during development. NT4 regulates gustatory neuron number earlier in development than BDNF, but it is not chemo-attractive and does not regulate gustatory neuron targeting. To elucidate the mechanisms that regulate these processes we have examined which TrkB intracellular signalling pathways are required for specific aspects of gustatory development by studying the effect of specific point mutations in TrkB docking sites. We found that the TrkB/Shc docking site is involved in regulating the survival of geniculate ganglion neurons as a point mutation in this adaptor site (TrkbS/S) caused large losses of these neurons as early as E12.5. These losses were exacerbated throughout development until after birth. A point mutation in the TrkB/PLCγ (TrkbP/P) docking site did not cause loss of geniculate ganglion neurons at any point during development. Animals with a point mutation in both docking sites (TrkbD/D) caused a further decrease in neuron numbers compared to animals with a mutation in only one of the docking sites, similarly to what has previously been shown in Trkb null animals. We concluded that the TrkB/Shc docking site is crucial for determining the survival of geniculate ganglion neurons during mouse gustatory development, while the TrkB/PLCγ docking site does not affect the neuronal survival directly and likely plays a role in maintenance of these neurons. Examining the targeting of geniculate ganglion afferents into the tongue revealed large deficits in innervated neural bud and taste bud numbers in TrkbS/S animals both before and after birth. This was concluded to be reflecting the lack of neuronal survival in this ganglion, a result that was mirrored in TrkbD/D animals. TrkbP/P animals, on the other hand, exhibited a developmental delay in innervation. This was indicated by a low amount of innervated neural buds following the initial innervation period, which was compensated for by a large increase in the number of innervated taste buds by birth. By adulthood, the numbers of taste buds present on the tongues of TrkbP/P animals reached normal numbers compared to control animals. This suggested that the TrkB/PLCγ docking site is involved primarily in innervation. Finally, we examined the morphology of taste buds in newly born and adult animals. We found that the low amount of geniculate ganglion afferents innervating the tongue in TrkbS/S and TrkbD/D animals caused a decrease in size of taste buds. This effect was seen to be partially rescued by adulthood in TrkbS/S animals but not in TrkbD/D animals due to lack of viability. The morphology of taste buds was unaffected in TrkbP/P animals until adulthood, at which point the size of the taste buds was increased. These results are in agreement with previous findings showing dependency of taste bud morphology on the amount of innervation. Overall, our findings show a differential role of TrkB adaptor sites in gustatory development. Despite activated by the same ligands, the docking sites on this receptor are able to exert different influence on signalling pathways downstream of TrkB affecting neuronal survival, targeting and morphology of taste buds. ACKNOWLEDGEMENTS First and foremost, I would like to thank my PhD supervisor, Dr. Liliana Minichiello for the guidance and advice throughout my PhD. Her help was invaluable, particularly during the last months of this project. I would also like to thank all the former and present members of the Minichiello lab, especially Dr. Mirjam Geibel and Dr. Dario Besusso for their encouragement, support, and help through my PhD, as well as their patience while teaching me all the methods I would be using during my project. I am also grateful to Dr. Jacqui Horn for her assistance with the maintenance and genotyping of the mouse lines. All the members of the lab created a nice working environment and it was a pleasure to work with them for the past three years. Furthermore, I would like to thank Professor Peter Brophy and the Centre for Neuroregeneration for their financial support particularly during the last six months, which enabled me to finish my project. There are also many people who I am thankful to for their support outside of the university, and I will try to thank them in person. Particularly my family, who often offered emotional support and encouragement that made this project and the write up of the thesis possible, as well as my friends in Edinburgh who made sure I would not spend all my free time working in the labs. Finally, I would like to thank my partner, Sigrid, who has provided me with encouragement, love and support during the PhD but most importantly the write up as the project came to an end. TABLE OF CONTENTS 1. INTRODUCTION ..................................................................................................................... 1 1.1 Neurotrophins and their receptors ............................................................................ 3 1.1.1 Structure of neurotrophins ............................................................................................ 3 1.1.2. Neurotrophin receptors ................................................................................................ 4 1.1.3 The role of neurotrophins and their receptors in development: neuronal survival ...10 1.1.4 TrkB signalling pathways .............................................................................................12 1.2 Rodent gustatory system ...............................................................................................19 1.2.1 Structure of the taste system .......................................................................................19 1.2.2 Facilitating taste ...........................................................................................................22 1.2.3 Development of the taste system ................................................................................28 1.2.4. Influence of neurotrophins on geniculate ganglion neuronal survival .......................42 1.3 Aims of experiments ......................................................................................................48 2. MATERIALS AND METHODS ................................................................................................50 2.1 Chemicals and consumables ............................................................................................52 2.1.1 Chemicals .....................................................................................................................52 2.1.2 Consumables ................................................................................................................53 2.1.3 Buffers ..........................................................................................................................55 2.2 Animals .............................................................................................................................57 2.3 Genotyping .......................................................................................................................59 2.4 Geniculate ganglion neuron counts .................................................................................62 2.5 Analysis of tongue innervation ........................................................................................66 2.6 Statistical analysis ............................................................................................................73 3. RESULTS ...............................................................................................................................75 3.1 Geniculate ganglion neuron survival ............................................................................77 3.1.1 TrkB/Shc docking site point mutation causes loss of geniculate ganglion neurons at E14.5; point mutation in the