Scheper, Christoph Tobias: The role of Drosophila Apaf-1-related-killer (dark) in establishing synaptic specificity in the olfactory system. 2009
Inhalt
- 1 Introduction
- 1.1 Development of neuronal networks
- 1.2 The olfactory system
- 1.3 Development of the Drosophila olfactory system
- 1.3.1 Early development and specification
- 1.3.2 Targeting and synaptic specificity
- 1.3.3 Zonal organization in-growing ORN axons
- 1.4 Identification of genes involved in olfactory targeting in Drosophila
- 1.5 Aim of this work
- 2 Experimental procedures
- 2.1 Materials
- 2.2 Methods
- 2.2.1 Cultivation of flies
- 2.2.2 Identifications of novel genes involved in ORN targeting
- 2.2.3 Generation of recombinant fly lines / chromosome “cleaning”
- 2.2.4 Clonal analysis
- 2.2.5 Selection of homozygous mutant embryos
- 2.2.6 Preparation technique of adult brains
- 2.2.7 Preparation of pupal brains and antennae
- 2.2.8 Cryo sections of adult antenna
- 2.2.9 Antibody staining
- 2.2.10 Acridin Orange staining
- 2.2.11 Imaging of adult antenna
- 2.2.12 DNA isolation from adult flies
- 2.2.13 DNA isolation from single flies or embryos
- 2.2.14 PCR
- 2.2.15 Inverse PCR
- 2.2.16 DNA gel elektrophoreses
- 2.2.17 Sequencing
- 3 Results
- 3.1 Mutations in verpeilt cause ectopic mis-targeting of Or47a neurons
- 3.2 Identification of the verpeilt locus
- 3.2.1 Complementation analysis
- 3.2.2 P-element mutagenesis
- 3.2.3 Generation of verpeilt deletions
- 3.2.4 Sequencing of candidate genes
- 3.3 Apoptosis and its effect on Or47a axon targeting
- 3.3.1 The effect of the darkV8 allele on apoptosis
- 3.3.2 Mutations in darkverpeilt do not affect ORN numbers
- 3.3.3 Suppression of apoptosis does not cause Or47a mis-targeting
- 3.3.4 Dark induced ORN mis-targeting is allele specific
- 3.3.5 Over-expression of dark does not suppress the OR47a mis-targeting
- 3.3.6 Loss of function of apoptosome components does not cause ORN mis-targeting
- 3.4 Projection domains
- 3.4.1 Domain organization in the olfactory system of Drosophila
- 3.4.2 Projection domain specificity of Or 47a axons
- 3.5 Characterization of the ORN targeting phenotype in darkverpeilt
- 3.5.1 Not all ORN classes are affected in darkverpeilt
- 3.5.2 The mis-targeting phenotype in darkverpeilt is Cell autonomous
- 3.5.3 The ectopic glomerulus needs a certain amount of axons to be stabilized
- 3.5.4 Mis-projecting Or47a axons sort out from other classes
- 3.5.5 Misprojecting Or47a axons interact with projection neurons
- 3.6 LRR Cell adhesion molecules are predominantly restricted to one projection domain
- 3.6.1 Connectin is mainly expressed in the trichoid sensilla
- 3.6.2 Con expression during development
- 3.6.3 The ectopic Or47a glomerulus in darkV8 mutants is Con positive
- 3.6.4 Dark mutant Or47a neurons express Con
- 3.6.5 Con mutants do not affect OR47a targeting
- 3.6.6 Knock down in dark mutant ORNs rescues the mis-targeting phenotype
- 3.6.7 Con over-expression in 47aORNs does not affect OR47a targeting
- 3.6.8 Over expression of Con in PNs leads to changes in ectopic Or47a target specificity
- 4 Discussion
- 4.1 Mapping of the verpeilt mutations to the dark gene
- 4.2 Phenotypic implications
- 4.3 Projection domains
- 4.3.1 In-growing ORN axons sort out in projection domains in Drosophila
- 4.3.2 Domain organization is not sufficient for precise ORN targeting
- 4.4 ORN targeting requires dark function only in some ORN classes
- 4.4.1 The darkverpeilt induced targeting phenotype is cell autonomous
- 4.4.2 Darkverpeilt affects ORN projection identity
- 4.5 Impacts of apoptosis on ORN targeting
- 4.5.1 Lack of apoptosis does not cause ORN mis-targeting
- 4.5.2 The ORN-mis-targeting phenotype is allele specific
- 4.5.3 The darkverpeilt mutations show dominant negative properties
- 4.6 Connectin is required but not sufficient for lateral OR47a targeting in darkverpeilt mutants
- 4.6.1 Con is not expressed in all darkverpeilt mutant Or47a neurons
- 4.6.2 Ectopic Con expression in darkverpeilt is due to transcriptional up-regulation
- 4.7 Precise regulation of dark is required for olfactory targeting
- 5 Abstract
- 6 Literature
- 7 Appendix