Characterisation of the zebrafish cerebellar efferent system [Elektronische Ressource] / Andreas N. Babaryka

Adm.Briel

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 33
• Langue: Deutsch
• Poids de l'ouvrage: 9 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN
Lehrstuhl für Entwicklungsgenetik





Characterisation of the zebrafish cerebellar efferent system






Andreas N. Babaryka










Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur
Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.



Vorsitzender: Univ.-Prof. Dr. H. Luksch
Prüfer der Dissertation: 1. Univ.-Prof. Dr. W. Wurst
2. Univ.-Prof. A. Schnieke, Ph.D.



Die Dissertation wurde am 29.12.2008 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 15.05.2009 angenommen. Table of contents
Table of contents ..........................................................................................................2
Abbreviations ...............................................................................................................4
Abstract.........................................................................................................................6
Zusammenfassung........................................................................................................7
1. Introduction..............................................................................................................8
1.1. Morphology of the cerebellum .................................................................................................8
1.1.1. Topology and gross anatomy ................................................................................................8
1.1.2. Histology of the cerebellum..................................................................................................9
1.1.3. Comparison between mammals and fish.............................................................................11
1.2. Development of the cerebellum ............................................................................................13
1.2.1. Establishment of the cerebellar anlage................................................................................13
The isthmic organiser (IsO)......................................................................................................13
Rotation of the cerebellar neuroepithelium establishes the cerebellar anlage ..........................14
1.2.2. The two germinal zones of the cerebellar anlage................................................................15
The Rhombic Lip and its associated transcription factor atonal homologue 1.........................15
The Ventricular Zone and its associated transcription factor ptf1a..........................................16
1.2.3. Migration and cerebellum morphogenesis ..........................................................................18
Migration of RL- derived neurons19
Migration of VZ- derived neurons ...........................................................................................20
DCN neuron are both VZ- and URL-derived...........................................................................21
1.3. PCs control GPC proliferation and maturation of the cerebellum ....................................22
Modes of MYC action..............................................................................................................25
1.4. Aim of this study ......................................................................................................................25
2. Materials and methods ..........................................................................................27
2.1. Materials ...................................................................................................................................27
2.1.1. Equipment...........................................................................................................................27
2.1.2. Consumables.......................................................................................................................29
2.1.3. Chemicals............................................................................................................................30
2.1.4. Enzymes and Kits ...............................................................................................................32
2.1.5. Antibodies33
2.1.6. Zebrafish strains..................................................................................................................35
2.1.7. Bacteria35
2.1.8. Primers.35
2.1.9. Vectors.37
2.1.10. Antisense oligo nucleotides (morpholinos).......................................................................39
2.1.11. Buffers, media, solutions ..................................................................................................39
2.2. Methods.....41
2.2.1. Molecular biology...............................................................................................................41
2.2.1.1. Purification of nucleic acids........................................................................................41
2.2.1.2. Amplification of DNA by PCR (polymerase chain reaction)......................................42
2.2.1.3. Restriction digest DNA ...............................................................................................43
2.2.1.4. Site-Directed Mutagenesis ..........................................................................................43
2.2.1.5. Blunting 5 ´-ends of DNA...........................................................................................43
2.2.1.6. Dephosphorylation of DNA43
2.2.1.7. Ligation of DNA .........................................................................................................44
2.2.1.8. TA-Cloning, TOPO.....................................................................................................44
22.2.1.9. Transformation of bacteria ..........................................................................................44
2.2.1.10. Preparation of DNA ..................................................................................................46
2.2.1.11. Determination of nucleic acid concentration.............................................................48
2.2.1.12. Separation of nucleic acids using agarose gel electrophoresis ..................................48
2.2.1.13. cDNA synthesis by reverse transcription ..................................................................49
2.2.1.14. DNA sequencing .......................................................................................................50
2.2.1.15. Analysis of DNA sequences......................................................................................51
2.2.1.16. in vitro synthesis of RNA51
2.2.2. Manipulation of zebrafish embryos ....................................................................................53
2.2.2.1. Cytoplasmic injection of nucleic acids........................................................................53
2.2.2.2. Cytoplasmic injection of morpholinos54
2.2.2.3. Single cell transplantation ...........................................................................................54
2.2.2.4. In vivo retrograde labeling of neurons in the zebrafish larva ......................................55
2.2.2.5. Cyclopamine treatment ...............................................................................................55
2.2.3. Histological techniques55
2.2.3.1. Sectioning of embryos, larvae and adult brains...........................................................55
2.2.3.2. Whole-mount in-situ-hybridisation (ISH) ...................................................................57
2.2.3.3. Double in-situ-hybridisation........................................................................................59
2.2.3.4. Immuno-histo-chemistry .............................................................................................60
2.2.3.5. Morphological stainings..............................................................................................62
2.2.4. Microscopic Analysis..........................................................................................................62
3. Results .....................................................................................................................63
3.1. Characterisation of Eurydendroid Cells– the zebrafish´s equivalent of the deep
cerebellar nuclei neurons...............................................................................................................63
3.1.1. Expression of olig2 in the developing cerebellum ..............................................................63
3.1.2. olig2-expressing precursors differentiate into Eurydendroid Cells.....................................65
3.1.3. Development of Eurydendroid Cells...................................................................................77
3.2. Analysis of the protooncogene nmyc in the developing zebrafish cerebellum...............87
3.2.1. Cloning of the zebrafish homologue of nmyc and functional variants of zf nmyc87
3.2