Zebrafish Trap230-Med12 is required for Sox9 activity and limb induction [Elektronische Ressource] / vorgelegt von Marlene Juliane Rau

Marlene Rau

Sujets

• Biologie

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2006
• Nombre de lectures: 41
• Langue: English
• Poids de l'ouvrage: 29 Mo

Extrait

Zebrafish Trap230/Med12 is required for
Sox9 activity and limb induction

DISSERTATION
zur Erlangung des Doktorgrades der Naturwissenschaften

Doctor rerum naturalium
(Dr. rer. nat.)

dem Fachbereich Biologie
der Philipps-Universität Marburg
vorgelegt von
Dipl.-Biol. Marlene Juliane Rau
aus Marburg

Marburg/ Lahn 2005
Vom Fachbereich Biologie
der Philipps-Universität Marburg als Dissertation am
angenommen.

Erstgutachterin: Prof. Dr. Renate Renkawitz-Pohl
Zweitgutachter : Prof. Dr. Michael Bölker

Tag der mündlichen Prüfung: 07.12.2005





Zebrafish Trap230/Med12 is required for
Sox9 activity and limb induction










To all the people who continuously encouraged me to keep going



Acknowledgements
I want to thank everyone who supported me during the last three years while this work was
ongoing. In particular, I'd like to thank…

Carl Neumann, for giving me the opportunity to pursue this work in his lab and for unbroken
optimism throughout the whole time.

The members of the Neumann lab, past and present, who contributed significantly to this
work in many little ways, from encouragement and helpful discussions over providing a
great humourous atmosphere up to sharedly proofreading this "masterpiece". Special thanks
go to Sabine Fischer and Gillian Brunt for continous technical support and to Sarika Sharma
for participating in the Neumann lab age of mapping.

The members of my thesis advisory committee at EMBL, Dr. Steve Cohen, Dr. Damian
Brunner and also Dr. Jürg Müller, for strong support and interest, and helpful advice.

My external supervisors at the Philipps-Universität Marburg, Prof. Dr. Renate Renkawitz-
Pohl, Prof. Dr. Michael Bölker, Prof. Dr. Monika Hassel und PD Dr. Detlev Buttgereit, for
agreeing to referee this thesis. Very special thanks to Prof. Dr. Renate Renkawitz-Pohl for
continous support over the last seven years, kick-starting my interest in developmental
biology in the first place, and being a great example of integrating science, life and family to
me.

Florian Raible, Thomas Sandmann, Bernadett Papp, Nadine Keller, Corinna Berger, Manuel
Klang and Paulo Cunha for special scientific and moral support over the last years – I
promise not to talk to you about mapping any more!

My friends, who make life (in Heidelberg) worth living; Florian Wintgens and Frank
Wigger, who unexpectedly shared the manifold burden of encouraging me to start, pursue
and finish this thesis – und schliesslich meinen Eltern, für alles.
Table of contents
1 INTRODUCTION .................................................................................................... 1
1.1 Zebrafish as a model system ....................................................................................... 2
1.2 Screening .................................................................................................................... 2
1.3 Mapping and positional cloning ................................................................................. 3
1.4 The Mediator complex .............................................................................................. 10
1.4.1 Trap230 and the kinase subcomplex ............................................................................ 15
1.5 Sox9........................................................................................................................... 25
1.6 Pectoral fin development .......................................................................................... 29
1.7 Aim of this thesis ....................................................................................................... 31
2 RESULTS ................................................................................................................ 33
2.1 Mapping and cloning the zebrafish trapped mutant ................................................. 34
2.1.1 Rough mapping ............................................................................................................ 34
2.1.2 Fine mapping 35
2.1.3 Cloning.........................................................................................................................39
2.1.4 Expression of Trap230 ................................................................................................. 41
2.1.5 Phenotype of the embryo.............................................................................................. 42
2.1.6 Phenocopy by morpholino injections ........................................................................... 43
2.2 Trap230 and Sox9..................................................................................................... 44
2.2.1 The Trap230 loss-of-function phenotype resembles loss of Sox9 activity in many
different tissues ............................................................................................................ 44
2.2.2 Trap230 is required for Sox9-dependent neural crest fates and craniofacial cartilage
formation...................................................................................................................... 47
2.2.3 The role of Trap230 in Sox9-dependent ear development ........................................... 50
2.3 Trap230 is required for Sox9 activity ....................................................................... 52
2.3.1 Sox9 expression in tpd is mostly normal...................................................................... 52
2.3.2 Sox9 overexpression cannot rescue tpd mutants .......................................................... 53
2.4 A Sox9-independent role of Trap230 in forelimb development ................................ 54
2.5 Other Phenotypes...................................................................................................... 56
2.5.1 Brain phenotype ........................................................................................................... 57
2.5.2 Eye and axon guidance phenotypes.............................................................................. 58
2.5.3 Heart and circulation phenotype................................................................................... 63
2.5.4 Somite phenotype......................................................................................................... 64 TABLE OF CONTENTS iii

2.5.5 Overall downregulation of Fgf signalling and gut phenotype ...................................... 64
3 DISCUSSION .......................................................................................................... 66
3.1 tpd encodes a mutation in the Mediator complex component Trap230 .................... 67
3.2 Interaction of Sox9 + Trap230 ................................................................................. 70
3.2.1 The trapped mutation shares many phenotypes with Sox9 mutants............................. 70
3.2.2 The role of Trap230 in Sox9 activity ........................................................................... 72
3.2.3 Trap230 as a transcriptional co-regulator during vertebrate development ................... 76
3.3 The role of Trap230 in forelimb initiation................................................................ 77
3.4 Other phenotypes ...................................................................................................... 79
3.4.1 Brain and muscle phenotypes....................................................................................... 79
3.4.2 Eye and axon guidance phenotypes.............................................................................. 80
3.4.3 Heart and circulation phenotype................................................................................... 82
3.4.4 Somite phenotype......................................................................................................... 83
3.4.5 Overall downregulation of Fgf signalling and gut phenotype ...................................... 83
3.4.6 Outlook.........................................................................................................................85
4 MATERIALS AND METHODS ........................................................................... 88
4.1 Fish stocks................................................................................................................. 89
4.2 Chemicals and solutions ........................................................................................... 89
4.3 Linkage analysis and genetic mapping ..................................................................... 90
4.3.1 Quick lysis of embryos................................................................................................. 90
4.3.2 Finclipping ................................................................................................................... 90
4.3.3 SSLP markers used....................................................................................................... 90
4.3.4 Bulked segregant analysis ............................................................................................ 91
4.3.5 Fine mapping............................................................................