Physiological roles of Robo receptor during dendrite development of the multidendritic arborization neurons of the Drosophila peripheral nervous system [Elektronische Ressource] / vorgelegt von Svetla Dimitrova

Svetla Dimitrova

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 41
• Langue: Deutsch
• Poids de l'ouvrage: 28 Mo

Extrait

Physiological Roles of Robo

Receptor during dendrite development of the

multidendritic arborization neurons of

the Drosophila peripheral nervous system








Dissertation


Zur Erlangung des Doktorgrades
der Naturwissenschaften (Dr. rer. nat)
der Fakultät für Biologie
der Ludwig- Maximilians- Universität München


Angefertigt am Max Planck Institut für Neurobiologie,
Abteilung Molekulare Neurobiologie,
Abteilungsgruppe Dendritische Differenzierung
Vorgelegt von
Svetla Dimitrova
München 2007



1


Hiermit, erkläre ich, dass ich die vorliegende Dissertation selbständig und ohne
unerlaubte Hilfe angefertigt habe. Sämtliche Experimente wurden von mir selbst
durchgeführt, au βer wenn explizit auf Dritte verwiesen wird. Ich habe weder
anderweitig versucht, eine Dissertation oder Teile einer Dissertation einzureichen
bzw. einer Prüfungskommission vorzulegen, noch eine Doktorprüfung
durchzuführen.


München, den 18.10.2007 …………………………………
(Unterschrift)






























1st Gutachter: Prof. Tobias Bonhoeffer
2nd Gutachter: Prof. John Parsch
Tag der mündlichen Prüfung: 12.12.2007


2




Die vorliegende Arbeit wurde zwischen November 2003 und August 2007 unter
der Leitung von Dr.Gaia Tavosanis am Max- Planck Institut für Neurobiologie in
Martinstried durchgeführt.


































3

Table of Contents…………………………………………………………………...…4

Abbreviations…..……………………………………………………………………….7

Figure and Tables….………………………………………………………………….11

Preface…………………………………………………………………………………..13

Summary……………………………………………………………………………….15

1. Introduction……………………………………………………………………….17
1.1 Molecular and cellular mechanisms underlying axongenesis……………….17
1.2 s controlling dendrite formation………...19
1.3 Roundabout receptors and their ligand Slit……………………………….....25
1.4 Drosophila melanogaster and its PNS as a model system to study
dendritogenesis…………………………………………………………………..30
1.5 The Role of Robo during dendrite morphogenesis…………………………..34

The aim of my PhD work……………………….…………………………………...35

2. Results ……………………………………………………………………………...37

2.1 Identification and characterization of girandola mutant…………………….37
2.1.1 Mapping the girandola mutation ………………………………….37 2.1.2 Characterization of the girandola mutant………………………….46
2.2 Developmental analyses of dendrite arborization neurons of control animals
during late embryonic/larval stages……………………………………………...48
2.3 Dendrite field developmental analyses of robo, robo2 and slit single mutants
and robo,robo2 double mutants………………………………………………….51
2.4 Robo and Robo2 are differentially expressed in the PNS of Drosophila during
late embryogenesis……………………………………………………………….56
2.5 Overexpression of Robo affects dendrite branch distribution……………….60
2.6 Robo acts in a cell-class specific manner to mediate the dendrite field
formation of Class IV but not Class I neurons…………………………………...62
4

2.7 Robo overexpression results in less branched dendritic shafts of Class IV
neurons …………………………………………………………………………..64
2.8 Time lapse imaging of Class IV neurons overexpressing Robo …………….68
2.9 Robo regulation of the dendrite field of Class IV neurons is
cell-autonomous………………………………………………………………….73
2.10 Differentiation of Class IV neuron…………………………………………77
2.11 How is the specific activation of Robo via Slit mediated during dendrite field
development of Class IV neurons?........................................................................80
2.12 The role of Ena and Dock during dendrite field
development…………….......................................................................................83
2.13 Robo2 functions during axon guidance of md-da neurons to specifically
downregulate activation…………………………………………………………86

3. Discussion………………………………………………………………………….91
4. Materials and Methods………………………………………………………..111

4.1 Fly stocks…………………………………………………………………...111
4.2 Immunohistochemistry……………………………………………………..112
4.2.1 General antibody staining for Drosophila mounts………………..112
4.3 Instruments………………………………………………………………….114
4.4 Consumables………………………………………………………………..115
4.5 Solutions and media………………………………………………………...115
4.6 Fly maintenance…………………………………………………………….116
4.7 Molecular biology…………………………………………………………..116
4.8 MARCM Analyses…………………………………………………………118
4.9 The Gal4-UAS-System……………………………………………………..120
4.10 Imaging and image processing……………………………………………120
4.11 Time-lapse analyses……………………………………………………….120
4.12 Quantitative analyses of md-da neuron dendrites ………………………...121

5. Bibliography……………………………………………………………………...123
5


6. Acknowledgements …………………………………………………………….133

7. Curriculum Vitae……………………………………………………………….134
































6


Abbreviations


2L second left chromosome
Abl Ableson Tyrosine Kinase
AEL after egg laying
bd bipolar neuron
BDNF brain derived neurotrophic factor
cAMP cyclic adenosine monophosphate
CC conserved cytoplasmic sequence motifs
CLASP CLIP associated protein
CLIP cytoplasmic linker protein
CNS central nervous system
Comm Drosophila Commissureless
CREB cAMP response element binding protein
CREST calcium response transactivator
da dendritic arborization
DCC deleted in colorectal carcinoma
Df Deficiency
DNA deoxyribonucleic acid
Dock Drosophila Dreadlocks
DRG dorsal root ganglion cells
DSCAM Down Syndrome Cell Adhesion Molecule
EcR Drosophila Ecdyson Receptor
7

EGF Epidermal Growth Factor
EGFP enhanced Green Fluorescence Protein
elav embryonic lethal abnormal vision
EMS ethyl methane sulfonate
Ena Drosophila Enabled
Eph Ephrin receptor tyrosine kinase
ER endoplasmatic reticulum
FAS Drosophila Fasciclin
Fmi Drosophila Flamingo
FN3 Fibronecting type 3
Fry Drosophila Furry Kinase
GAL4 Galactosidase protein 4
GFP Green Fluorescent Protein
GTPase Guanosine Triphosphatase
h hours
HSPG Heparane Sulfate Proteoglycan
Ig immunoglobulin domains
kb kilo bases
LN cholinergic local neurons
LRRs leucine-reach repeats
MAP Microtubule Associated Protein
MARCM mosaic analyses with a repressible cell marker
MASs muscle attachment sites
8

Mb mega bases
md-da multiple dendritic dorsal arborisation neurons
mRNA messenger RNA
Nck non-catalytic region of Tyrosine kinase
Neuro D neuronal determination factor
Nos Drosophila Nanos
Orbit/MAST Drosophila Microtubule-associated protein
ORN olfactory receptor neurons
Pak p21-activated serine/threonine kinase
PB primary branch
PN projection neurons
PNS peripheral nervous system
ppk Drosophila Pickpocket
PRC polycomb repressor complex
RGC retinal ganglion cells
RNA ribonucleic acid
Robo Roundabout receptor protein
SB secondary branch
Sema3A Semaphorin 3A
SH Src homology domain
Shh Sonic Hedgehog
Sos Drosophila Son of Sevenless
Src Sarcoma Virus Oncogene
9

TB tertiary branch
Trc Drosophila Tricornered
UAS upstream activating sequence
UPS ubiquitin-proteasome system
UCSF University of California San Francisco
VASP vasodilator-stimulated phosphoprotein



















10