The receptor guanylyl cyclase Npr2 regulates the bifurcation of cranial sensory axons Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) Submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by Gohar Ter-Avetisyan from Gyumri, Armenia Berlin, 2013 This thesis was prepared between July 2009 and November 2012 at the Max Delbrück Center for Molecular Medicine, Department of Developmental Neurobiology, under the supervision of Prof. Dr. Fritz G. Rathjen. 1st Reviewer: Prof. Dr. Fritz G. Rathjen 2nd Reviewer: Prof. Dr. Carmen Birchmeier-Kohler Disputation on __ 06.02.2013 _____ In honor of my grandparents Hakob Ter-Avetisyan Zina Ter-Avetisyan Hovhannes Manukyan Kalipse Manukyan Acknowledgments I would like to express my sincere appreciation to my supervisor, Prof. Dr. Fritz G. Rathjen, for giving me the great opportunity to pursue a PhD in his group and for all the encouragement and advice he gave me during the course of my PhD. My deepest thank to PD Dr. habil. Hannes Schmidt for all the invaluable advice and discussions, thought provoking questions and giving me countless guidance. I want to thank present and past members of the Rathjen Lab for their feedbacks on my research and for making me feel at home in the lab. I would like to thank Karola Bach for her help in organisation of mice breedings. My gratitude to Mechthild Henning, Madlen Driesner and Anne Köhn for their excellent technical assistance. I am also grateful to Birchmeier-Kohler Lab for their help by Southern blot experiments. For discussions and encouragement I gratefully mention Dr. Hagen Wende. I extend my gratitude to Kira Balueva for all the fruitful discussions and beyond. Special thanks to Andrea Leschke for her support with ES cell culture. I thank SFB665 for funding my conference travels. I’m also indebted to my former supervisors Prof. Dr. Cristina M. Cardoso and Dr. Gilla Lättig-Tünnemann. I feel they given me the tools to be successful in my future scientific career. I’m very grateful to my dear friend, Dr. Annekathrin Möller, for accurate proofreading and valuable comments. Thank you, Anne, your sympathetic ear was a great moral encouragement. I would also like to thank the people who have been instrumental in my future and I think that each of them have helped lay the foundation that was necessary for me to make it to this point: My beloved grandaunts, Gohar, Anahit, Aspram Ter-Avetisyan: I will always cherish those memories how you have nurtured my love for education and science. My parents, Larisa and Sargis Ter-Avetisyan, I could not thank you enough for all that you have done for me and continue to do. Your unconditional love livens my spirit! My heartfelt thanks to my husband, Dr. Constantin Rüder, for his support and endless patience. A very special place in my heart is always reserved for my grandparents, who shaped me to be the person I am today. Իմ կարոտած սրտի համար ո՛չ մի ուրիշ հեքիաթ չկա․ Նարեկացու, Քուչակի պես լուսապսակ ճակատ չկա․ Աշխա՛րհ անցի՛ր, Արարատի նման ճերմակ գագաթ չկա․ Ինչպես անհաս փառքի ճամփա՝ ես իմ Մասիս սա՛րն եմ սիրում. Եղիշե Չարենց Table of Contents Table of Contents LIST OF ABBREVIATIONS ............................................................................................. IV ABSTRACT ................................................................................................................... VIII ZUSAMMENFASSUNG ................................................................................................... IX 1. INTRODUCTION ........................................................................................................... 1 1.1 Axonal pathfinding .................................................................................................... 1 1.2 Axonal branching ...................................................................................................... 2 1.3 Axonal projections of dorsal root ganglion (DRG) neurons ....................................... 3 1.4 The cGMP-dependent signaling pathway in axonal branching .................................. 4 1.5 Characterization of CNP, Npr2 and cGKI.................................................................. 7 1.6 Cre and Flp recombination in gene targeting ............................................................ 8 1.6.1 The inducible Cre recombination system ............................................................. 10 1.6.2 Inducible Cre-mediated reporter gene expression ............................................... 11 1.6.3 Single neuron labeling using inducible Cre reporter lines..................................... 12 2. PREFACE AND OBJECTIVE ...................................................................................... 14 3. MATERIALS ................................................................................................................ 16 3.1 Buffers, solutions and media .................................................................................. 16 3.2 Restriction enzymes ............................................................................................... 18 3.3 Polymerases .......................................................................................................... 19 3.4 Embryonic stem (ES) cells ..................................................................................... 19 3.5 Kits ......................................................................................................................... 19 3.6 Antibiotics ............................................................................................................... 19 3.7 Bacterial strains ...................................................................................................... 20 3.8 Antibodies for immunohistochemistry ..................................................................... 20 3.9 Chemicals .............................................................................................................. 20 3.10 Special equipment ................................................................................................ 22 4. METHODS ................................................................................................................... 23 4.1 Molecular biology ................................................................................................... 23 4.1.1 Bacterial culture ................................................................................................... 23 4.1.2 Transformation of E. coli ...................................................................................... 24 4.1.3 Chemical transformation of E. coli ....................................................................... 24 4.1.4 Transformation of E. coli by electroporation ......................................................... 24 4.1.5 Homologous recombination in E. coli ................................................................... 25 4.1.6 Purification of mini-λ DNA .................................................................................... 25 4.1.7 Induction of mini-λ recombination function ........................................................... 26 4.1.8 E. coli strains EL250 and EL350: confirmation of functional Flp and Cre target sites ............................................................................................................................. 26 4.1.9 Isolation of Plasmid DNA ..................................................................................... 27 4.1.10 Isolation of Bacterial Artificial Chromosome (BAC) DNA .................................... 27 4.1.11 Determination of DNA concentration.................................................................. 27 4.1.12 Restriction enzyme digestion of plasmid DNA ................................................... 28 4.1.13 Agarose gel electrophoresis .............................................................................. 28 i Table of Contents 4.1.14 Isolation of DNA from agarose gels (gel extraction) ........................................... 28 4.1.15 Polymerase-chain-reaction (PCR) ..................................................................... 29 4.1.16 PCR for molecular cloning ................................................................................. 29 4.1.17 Blunting of DNA fragments ................................................................................ 30 4.1.18 Dephosphorylation and Phosphorylation ........................................................... 31 4.1.19 Ligation .............................................................................................................. 31 4.1.20 Control of the directional cloning ........................................................................ 32 4.2 Mouse embryonic stem cell culture and establishing the mouse models Npr2LacZ and Npr2CreERT2........................................................................................................ 32 4.2.1 Culturing of feeder cells ....................................................................................... 33 4.2.2 Culturing of ES cells ............................................................................................ 34 4.2.3 Electroporation of ES cells ................................................................................... 34 4.2.4 Selection, picking and culturing of geneticin resistant clones ............................... 35 4.2.5 Extraction of genomic ES cell DNA .....................................................................