MITF in the mouse central nervous system Mitf expression and target genes in the olfactory bulb Anna Þóra Pétursdóttir Thesis for the degree of Master of Science University of Iceland Faculty of Medicine Department of Biomedical Sciences School of Health Sciences MITF in the mouse central nervous system Mitf expression and target genes in the olfactory bulb Anna Þóra Pétursdóttir Thesis for the degree of Master of Science Supervisor: Eiríkur Steingrímsson Instructor: Pétur Henry Petersen Masters committee: Jón Hallsteinn Hallsson Faculty of Medicine Department of Biomedical Sciences School of Health Sciences University of Iceland December 2010 MITF í miðtaugakerfi músarinnar Tjáning Mitf og markgen í lyktarklumbu Anna Þóra Pétursdóttir Ritgerð til meistaragráðu Umsjónarkennari: Eiríkur Steingrímsson Leiðbeinandi: Pétur Henry Petersen Meistaranámsnefnd: Jón Hallsteinn Hallsson Læknadeild Námsbraut í Líf- og læknavísindum Heilbrigðisvísindasvið Háskóla Íslands Desember 2010 Ritgerð þessi er til meistaragráðu í líf- og læknavísindum og er óheimilt að afrita ritgerðina á nokkurn hátt nema með leyfi rétthafa. © Anna Þóra Pétursdóttir 2010 Prentun: Háskólaprent Reykjavík, Ísland 2010 Mitf in the mouse central nervous system 3 ÁGRIP Mitf (microphthalmia-associated transcription factor) genið er vel varðveitt og er tjáning þess nauðsynleg fyrir þroskun litfrumna og einnig er það mikilvægt fyrir myndun sortuæxla. Mitf stökkbreyttar mýs skortir litfrumur í húð, hári og augum; þær eru blindar, með pínulítil augu (microphthalmia) og heyrnarlausar. Samkvæmt Allen Brain Atlas gagnagrunninum er Mitf tjáð í lyktarklumbu músa, en lyktarklumban er sá hluti heilans sem tekur á móti lyktarskilaboðum frá nefinu. Ekkert er vitað um hlutverk MITF í miðtaugakerfinu. Fyrra markmið þessa verkefnis var að staðfesta Mitf tjáningu í lyktarklumbu músa. Það var gert með RT-PCR og magnbundnum PCR mælingum. Seinna markmiðið var að ákvarða hvaða gen MITF hefur áhrif á í lyktarklumbunni og komast þannig nær því að ákvarða hlutverk þess í lyktarskyni. Annars vegar voru þekkt MITF markgen úr öðrum frumugerðum skoðuð og hins vegar framkvæmd genatjáningargreining á örflögu. Ekki reyndust nein af þeim þekktu markgenum sem valin voru vera markgen fyrir MITF í lyktarklumbu músa, nema hugsanlega Cma1. Úr örflögugreiningunni kom genið Sgcg helst til greina sem MITF markgen. Niðurstöður úr luciferasa prófi á stýrilsvæði Sgcg í HEK 293T frumum benda til þess að MITF virkji ekki tjáningu frá stýrilsvæði mSgcg. Hlutverk MITF í lyktarskyni var athugað með einföldum lyktarprófum. Niðurstöður þeirra sýndu að Mitfmi-vga9 arfhreinu mýsnar hafa ekki misst lyktarskynið, en mýs með minna MITF magn (Mitfmi-vga9 arfblendnar og arfhreinar mýs) virðast þó mögulega hafa minna þefskyn heldur en villigerðarmýs. Anna Þóra Pétursdóttir 4 Mitf in the mouse central nervous system Anna Þóra Pétursdóttir Mitf in the mouse central nervous system 5 ABSTRACT The Mitf (microphthalmia-associated transcription factor) gene is very well conserved and its expression is crucial for the development of both types of pigment cells and it is important for the development of melanoma skin cancer. Loss of function mutations in this gene cause loss of pigmentation in skin, hair and eyes, microphthalmia, blindness, and deafness. According to the Allen Brain Atlas database, Mitf is expressed in the mouse olfactory bulb (OB), the part of the brain that recieves olfactory information from sensory neurons in the nose. Nothing is yet known about the function of MITF in the CNS. The first aim of this project was to confirm Mitf expression in the mouse OB. This was done using RT-PCR and real time PCR. The second aim was to determine which genes MITF affects in the OB and thereby getting a better understanding of its function in olfaction. This was assessed by using both candidate gene approach and a microarray gene expression analysis. None of the genes selected through the candidate gene approach however appear to be MITF target genes in the mouse OB, except possibly Cma1. From the microarray analysis data one gene, Sgcg, is the most promising MITF target gene candidate. Co-transfection assay results for Sgcg promoter in HEK 293T cells suggest that MITF does not activate expression from the mSgcg promoter. MITF function in olfaction was tested with simple olfactory tests. The results showed that Mitfmi-vga9 homozygous mice have not lost their ability to smell, although their olfactory capabilities are perhaps not as good as that of Mitfmi-vga9 heterozygous and wt mice. Anna Þóra Pétursdóttir 6 Mitf in the mouse central nervous system Anna Þóra Pétursdóttir Mitf in the mouse central nervous system 7 ACKNOWLEDGEMENTS All the work in this project was performed in Læknagarður and the project was partly funded by Rannís. First of all I want to offer my thanks to my supervisors on this project, Eiríkur Steingrímsson and Pétur Henry Petersen, for allowing me the opportunity to work on this project and for their instruction and support. I would also like to thank Eiríkur for providing the mice used in this project. I want to really thank all the people at Læknagarður who supported this project. These include: Kristín Bergsteinsdóttir for teaching me all about working with RNA and for providing me with cDNA from mouse hearts and skin, Finnbogi R. Þormóðsson for his support, wisdom and amusing anecdotes, Indíana Elín Ingólfsdóttir for her help with the PC12 cell line and her undying support, Helga Eyja Hrafnkelsdóttir for her endless support, Marteinn Þór Snæbjörnsson for all his help, advice, and interesting conversations, the people at the Stem Cell Research Unit (SCRU) for all their help and advice, Alexander Schepsky for helping me with the luciferase assay and providing some of the plasmids used, Guðrún Valdimarsdóttir for her help on analyzing the microarray data, Christian Praetorius for his help and for providing lists of known MITF target genes, Christine Grill for helping me with the luciferase assay data analyzis, Benedikta S. Hafliðadóttir for her help and advice, Lilja Guðrún Steinsdóttir for providing the rats used in the project, and all the other people at Læknagarður who supported me in any way. I would also like to thank the third member of my master’s committee, Jón Hallsteinn Hallsson at the Agricultural University of Iceland for reading this thesis and providing me with good advice, Fulvio Celsi at Sector of Neurobiology, International School for Advanced Studies (SISSA), Anna Þóra Pétursdóttir 8 Mitf in the mouse central nervous system Trieste, Italy, for providing the PC12 cells, and Satoru Noguchi (Department of Cell Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Ogawahigashi, Kodaira, Tokyo, Japan and Inheritance and Variation Group, PRESTO, Japan Science and Technology Corporation, Kawaguchi, Saitama, Japan) for providing plasmids for the luciferase assay. Last but not least I want to thank my family: my boyfriend Óskar Jónsson, my parents Sigurveig Knútsdóttir and Pétur Örn Björnsson, my sister Eva Margrét Pétursdóttir, and also my friends – for always listening and supporting me, for their patience and encouragement. Thank you all! Anna Þóra Pétursdóttir Mitf in the mouse central nervous system 9 TABLE OF CONTENTS ÁGRIP ....................................................................................................................... 3 ABSTRACT .............................................................................................................. 5 ACKNOWLEDGEMENTS .................................................................................... 7 TABLE OF CONTENTS ......................................................................................... 9 FIGURES AND TABLES ..................................................................................... 11 ABBREVIATIONS ................................................................................................ 13 I. INTRODUCTION ............................................................................................. 15 1.1 MICROPHTHALMIA-ASSOCIATED TRANSCRIPTION FACTOR (MITF) ............... 15 1.1.1 MITF ..................................................................................................... 15 1.1.2 The discovery of Mitf ............................................................................ 16 1.1.3 Mitf conservation .................................................................................. 17 1.1.4 Isoforms ................................................................................................ 18 1.1.5 MITF – a key regulator in melanocytes ................................................ 19 1.1.6 Mitf mutations and pathology ............................................................... 20 1.1.7 MITF function – target genes ............................................................... 22 1.1.8 Background on transgenic mice used in this study ............................... 23 1.2 THE OLFACTORY BULB ................................................................................. 27 1.2.1 Olfaction ............................................................................................... 27 1.2.2 Olfactory bulb function and structure .................................................. 27 1.3 AIM ............................................................................................................... 33 II. MATERIALS AND METHODS .................................................................... 34 2.1 ANIMALS ......................................................................................................