Modelling Kallmann Syndrome in the Zebrafish by Steven Mark Cadman A thesis submitted to University College London for the degree of Doctor of Philosophy December 2010 Centre for Neuroendocrinology University College London Medical School Royal Free Campus, London NW3 2PF Declaration I, Steven Mark Cadman, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. .................................................................... .............................. Steven Mark Cadman Date 2 Abstract Kallmann syndrome (KS) is a human genetic disorder characterised by delayed/absent pubertal development, associated with lack of olfaction. KS is proposed to result from disrupted migration and targeting of olfactory sensory axons and hypothalamic gonadotrophin releasing hormone (GnRH1) neurons during early embryogenesis. Mutations in anosmin-1 (KAL1), fibroblast growth factor receptor 1 (FGFR1) and fibroblast growth factor 8 (FGF8) are responsible for some cases of KS. Previously, in ex vivo human GnRH neuroblast culture, anosmin-1 was shown to enhance FGFR1 signalling in an FGF-dependent manner. Here, using a zebrafish in vivo system, the biological functions of anosmin-1- and FGF-mediated signalling during olfactory and GnRH system development have been investigated. Characterisation of the zebrafish GnRH system, and the role of olfactory axonogenesis in its development, was aided by the generation of a transgenic reporter line: pGnRH3:mCherry. Two notable mCherry populations were visualised by 36 hours post-fertilisation (hpf): the well-characterised terminal nerve cells, and an early, hitherto unreported, hypothalamic cluster of cells. Antisense morpholino approaches were used to demonstrate that knocking down both Kal1a and Kal1b genes, the two zebrafish KAL1 orthologues, caused noticeable deficiency in the number of olfactory sensory neurons accurately projecting to the olfactory bulbs, concomitant with disruption in the terminal nerve GnRH cells and presence of fewer presumptive hypothalamic GnRH cells by 36hpf. Moreover, there was a notable failure in formation of one or both of the two forebrain commissures in these morphants. In parallel experiments, knocking down one of the two FGF8 orthologues, Fgf8a, or specific temporal pharmacological inhibition of FGFR signalling at 14-22hpf, resulted in similar phenotypes by 36hpf. Interestingly, co-injection of Kal1a/Kal1b and Fgf8a morpholinos at concentrations which would give no phenotype individually was able to replicate the commissural mutant phenotype. Combined, these data strongly suggest that Kal1a/Kal1b may act via the Fgf8a pathway in vivo. 3 Acknowledgements I would firstly like to thank my two supervisors, Prof. Pierre Bouloux and Prof. Ivor Mason, for their continued help, support, and guidance throughout my PhD. I am so grateful to them both for giving me the opportunity to carry out my PhD studies in their labs. I‟m also greatly indebted to Soo-Hyun Kim, Laxmi Iyengar, Youli Hu, and Suba Poopalasundaram, for all their helpful discussions and advice; as well as their endless encouragement and moral support. I‟d also like to thank Panna, Rajit, Naila, James M, Annabelle, Sheona, and Suresh, as well as many others from the MRC Centre for Developmental Neurobiology (King‟s College London) and Royal Free Medical School who have helped to ensure that my experiences over the past few years have been so enjoyable and memorable. I am also very grateful to Gregory Philp for helping to initiate this project at King‟s College, and for teaching me some of the techniques, including zebrafish embryo micro-injection. I‟d also like to acknowledge Sharon, David, and Swapna for helping to ensure that my zebrafish were healthy and always happy to spare me some of their eggs! Finally, I‟d like to thank my parents for their patience and endless love and support; this PhD thesis is dedicated to them both. 4 “Nothing is more memorable than a smell. One scent can be unexpected, momentary and fleeting, yet conjure up a childhood summer beside a lake in the mountains.” Diane Ackerman 5 Table of Contents Abstract ......................................................................................... 3 Acknowledgements ....................................................................... 4 Table of Contents ......................................................................... 6 Figure list ..................................................................................... 13 Table list ...................................................................................... 17 Abbreviations .............................................................................. 18 Chapter 1: Introduction ............................................................ 20 1.1 Kallmann Syndrome ........................................................... 20 1.2 Human reproductive axis ................................................... 21 The onset of puberty ......................................................................................................... 21 1.3 Human olfactory & vomeronasal systems ........................ 25 Odorant detection .............................................................................................................. 25 Olfactory system development and regeneration .............................................................. 25 Pheromone detection ......................................................................................................... 28 1.4 An olfactory origin for GnRH neurons ............................. 29 GnRH neuronal migration in the mouse forebrain ............................................................ 31 Evidence from an aborted X-KS embryo .......................................................................... 31 1.5 The known KS genetic loci ................................................. 34 KAL1 (anosmin-1) ............................................................................................................. 34 KAL2 (FGFR1) ................................................................................................................. 35 FGF8 (KAL6) .................................................................................................................... 43 NELF ................................................................................................................................. 45 PKR2 (KAL3) and PK2 (KAL4) ........................................................................................ 46 CHD7 (KAL5) ................................................................................................................... 47 Non-KS loci ...................................................................................................................... 48 1.6 The role of anosmin-1 in KS .............................................. 49 In vitro and ex vivo analyses ............................................................................................. 49 6 In vivo studies ................................................................................................................... 50 Invertebrate X-KS model (fruitfly & nematode worm) ................................................. 50 Vertebrate X-KS model (rodents, chicken, fish) ............................................................ 54 Chicken anosmin-1 ........................................................................................................ 55 Zebrafish/medaka anosmin-1 ....................................................................................... 56 The advantages of a zebrafish model ........................................................................... 56 1.7 The role of FGF signalling in forebrain development ..... 58 1.8 Anosmin-1 modulates FGF signalling ............................... 61 1.9 Aims of thesis ....................................................................... 69 Chapter 2: Materials & Methods ............................................. 70 2.1 Buffers and solutions .......................................................... 70 2.2 Animals ................................................................................. 72 2.2.1 Adult zebrafish ........................................................................................................ 72 2.2.2 Harvesting zebrafish & medaka embryos ............................................................... 72 2.2.3 Chicken embryos .................................................................................................... 72 2.3 Immunohistochemistry of whole embryos ........................ 73 2.3.1 The standard protocol ............................................................................................. 73 2.3.2 Anti-GnRH (LRH13) .............................................................................................. 75 2.3.3 Anti-anosmin-1a/-1b and anti-pERK ...................................................................... 75 2.3.4 Anti-acetylated tubulin ............................................................................................ 76 2.3.5 Cryostat sections ..................................................................................................... 76 2.3.6 Vibratome sections .................................................................................................. 76 2.4 In situ hybridisation for whole embryos ..........................