Cooperation between GDNF-Ret and EphrinA-EphA4 signals for motor axon pathway selection in the limb [Elektronische Ressource] / von Laura Franziska Knott

Laura Knott

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 31
• Langue: English
• Poids de l'ouvrage: 18 Mo

Extrait

Für Ingo und Evelyn

Acknowledgements

First and foremost I wish to thank my parents for supporting me in all my
endeavors, they deserve my deep gratitude.

I would like to thank my thesis supervisor Rüdiger Klein, who gave me the
opportunity to work in a great scientific environment. I am grateful for his
constant support and interest in my project and especially for his encouragement
when I almost gave up.

I thank my thesis committee members, Rüdiger Klein, Francoise Helmbacher and
Edgar Kramer for their interest, critical discussions and suggestions. Special
thanks are dedicated to Francoise Helmbacher, who sparked my interest in motor
neurons. I am grateful for her constant support in Munich and Marseille and for
teaching me so many techniques.

I wish to acknowledge my collaborators for their excellent experimental
contribution and scientific interest, especially Cathy Krull. I want to thank Artur
Kania, Klas Kullander, Silvia Arber and Carlos Ibañes for providing mouse lines
and constructs.

I thank all past and present lab members for a great working atmosphere, help
whenever needed, discussions, criticism and lots of fun on retreats, ski-trips and
Oktoberfest visits. My special thanks got to Francoise Helmbacher, Katrin
Deininger, Irina Dudanova, Joaquim Egea, Christian Erlacher and Sónia Paixao.

My thanks go to the people of all the service units at the MPI, who make our
work much easier. Special thanks to Anna Dengler, Bettina Hoisl und Patty
Ziehlke from the animal facility for their excellent work.

I want to thank Till Marquardt for introducing me into the secrets of explant
cultures and motor neuron tracings and for his interest, suggestions and scientific
discussions.

Special thanks are dedicated to Irina Dudanova and Louise Gaitanos for critically
reading this thesis and Tom Gaitanos, Katrin Deininger and Fabian Loschek for
helping me with formatting matters.





Cooperation between
GDNF/Ret and EphrinA/EphA4 signals
for motor axon pathway selection in the limb










Dissertation
Der Fakultät für Biologie der Ludwig-Maximilian-Universität München



















Eingereicht am 26.Oktober 2007 von Laura Franziska Knott aus München
Erstgutachter: Prof. Dr. Rüdiger Klein
Zweitgutachter: George Boyan

Tag der mündlichen Prüfung: 13.Dezember 2007







































The work presented in this thesis was performed in the laboratory of Prof. Dr.
Rüdiger Klein, Department of Molecular Neurobiology, Max-Planck Institute of
Neurobiology, Martinsried, Germany. Ehrenwörtliche Versicherung


Ich versichere hiermit ehrenwörtlich, dass die vorgelegte Dissertation von mir
selbständig und ohne unerlaubte Beihilfe angefertigt ist.
Hiermit erkläre ich, dass ich mich anderweitig einer Doktorprüfung ohne Erfolg
nicht unterzogen habe.


München, den ................................................. ................................................................
(Unterschift)
Table of contents
Table of Contents

Abstract.......................................................................................................... 1

Publication from the work presented in this dissertation..............................................3
Abbreviations ...............................................................................................................5
List of Figures...............................................................................................................9

1 Introduction ............................................................................................... 11
1.1 Motor neurons...................................................13
1.1.1 The “birth” of motor neurons.........................................................................13
1.1.2 Motor neuron organization – colonization of the ventral spinal cord.............14
1.1.3 The LIM code and motor axon trajectories...................................................16
1.1.4 Programmed cell death ....................................18
1.2 GDNF and Ret .....................................................................................................19
1.2.1 Receptor tyrosine kinase Ret and its signaling crew....................................19
1.2.2. Expression pattern of Ret, Gfrα1 and GDNF ..............................................23
1.2.3 Biological functions of GDNF and Ret..........................................................23
1.3 Eph receptors and ephrin ligands ........................................................................24
1.3.1 Eph receptor family ......................................................................................25
1.3.2 Ephrin ligands...............................................................................................26
1.3.3 Roles of Ephs and ephrins in axon guidance ...............................................28
1.4 The thesis project.................................................................................................31

2 Results...................................................................................................... 33
2.1 Differential expression of Ret and GDNF in motor neurons and hindlimb
mesenchyme.......................................................................................................33
2.1.1 Ret resembles EphA4 expression pattern on hindlimb innervating motor
axons ...........................................................................................................33
2.1.2 Ret mRNA expression in lumbar motor neurons before and during the period
of dorsoventral pathway selection................................................................35
2.1.3 GDNF expression at the pathway selection point.........................................37
2.2 Defective dorsal hindlimb innervation in Ret and GDNF mutant embryos ...........40
2.2.1. Ret null embryos display a reduction of the peroneal nerve........................40
2.2.2 Conditional inactivation of Ret reveals a cell-autonomous function of Ret in
motor neurons..............................................................................................42
Table of Contents
2.2.2.2 Recombination of Ret using Nestin-Cre ....................................................44
2.2.3 The hindlimb phenotype analysis .................................................................48
2.2.4 GDNF null embryos resemble Ret knockout and conditional mutants .........52
2.3 Teamwork between GDNF/Ret and ephrinA/EphA4 signals for the guidance
hindlimb innervating axons..................................................................................54
2.3.1 Protein expression of EphA4 and Ret receptors remains at high levels in
misguided axons ..........................................................................................54
2.3.2 EphA4-Ret double mutants display an enhanced hindlimb phenotype ........57
2.3.3 Activity-dependent interaction of Ret and EphA4.........................................58
2.3.4 Trans-phosphorylation between Ret and EphA4..........................................59
2.3.5 Motor neuron culture system set up .............................................................64
2.4 The requirement of axonal and mesenchymal EphA4 for pathway selection in the
hindlimb...............................................................................................................69
2.4.1. Specific and robust recombination activity of Tbx4-Cre ..............................69
2.4.2 Motor neuron backfill technique allows the detection of single misguided
axons ...........................................................................................................71

3 Discussion................................................................................................. 75
3.1. Differential expression pattern of Ret and GDNF suggests a function in
topographic mapping of hindlimb innervating motor axons.................................76
3.2. GDNF/Ret signaling is required for motor axon growth into the dorsal hindlimb 77
3.2. Cooperation between GDNF/Ret and ephrinA/EphA4 in motor axon guidance..79
3.4. Molecular mechanisms underlying the cooperation between EphA4 and Ret ....81
3.5. Deciphering EphA4/Ret interaction at the cellular level ......................................82
3.6. The role of axonal and mesenchymal EphA4 in the dorsoventral choice of
hindlimb innervating axons..................................................................................83

4 Materials and Methods..........................................