Functional genomics of the Drosophila melanogaster X chromosome and the role of DWnt5 during development

The collection of X chromosome insertions (PX) lethal lines, which was isolated from a screen for essential genes on the X chromosome, was characterized by means of cloning the insertion sites, mapping the sites within genomic DNA and determination of the associated reporter gene expresssion patterns. The established STS flanking the P element insertion sites were submitted to EMBL nucleotide databases and their in situ data together with the enhancer trap expression patterns have been deposited in the FlyView database. The characterized lines are now available to be used by the scientific community for a detailed analysis of the newly established lethal gene functions. One of the isolated genes on the X chromosome was the Drosophila gene Wnt5 (DWnt5). From two independent screens, one lethal and three homozygous viable alleles were recovered, allowing the identification of two distinct functions for DWnt5 in the fly. Observations on the developing nervous system of mutant embryos suggest that DWnt5 activity affects axon projection pattern. Elevated levels of DWNT5 activity in the midline cells of the central nervous system causes improper establishment and maintenance of the axonal pathways. Our analysis of the expression and mutant phenotype indicates that DWnt5 function in a process needed for proper organization of the nervous system. A second and novel function of DWnt5 is the control of the body size by regulation of the cell number rather than affecting the size of cells. Moreover, experimentally increased DWnt5 levels in a post-mitotic region of the eye imaginal disc causes abnormal cell cycle progression, resulting in additional ommatidia in the adult eye when compared to wild type. The increased cell number and the effects on the cell cycle after exposure to high DWNT5 levels is the result of a failure to downregulate cyclin B and therefore the unsuccessful establishment of a G1 arrest.

Role of Rab5 in synaptic vesicle recycling

During synaptic transmission, NT-filled synaptic vesicles are released by Ca2+-triggered exocytosis at the active zone. Following exocytosis, SV membrane is immediately re-internalized and synaptic vesicles (SVs) are regenerated by a local recycling mechanism within the presynaptic terminal. It is debated whether an endosomal compartment is involved in this recycling process. In contrast, it is well known from cultured mammalian cells, that endocytic vesicles fuse to the early sorting endosome. The early endosome is a major sorting station of the cell where cargo is send into the degradative pathway to late endosome and lysosome or towards recycling. Each trafficking step is mediated by a certain protein of the Rab family. Rab proteins are small GTPases belonging to the Ras superfamily. They accumulate at their target compartments and have thereby been used as markers for the different endocytic organelles in cultured mammalian cells. Rab5 controls trafficking from the PM to the early endosome and has thereby been used as marker for this compartment. A second marker is based on the specific binding of the FYVE zinc finger protein domain to the lipid PI(3)P that is specifically generated at the early endosomal membrane. This study used the Drosophila NMJ as a model system to investigate the SV recycling process. In particular, three questions were addressed: First, is an endosomal compartment present at the synapse? Second, do SVs recycle through an endosome? Third, is Rab5 involved in SV recycling? We used GFP fusions of Rab5 and 2xFYVE to visualize endosomal compartments at the presynaptic terminal of Drosophila third instar larval NMJs. Furthermore, the endosomes are located within the pool of recycling SVs, labeled with the styryl-dye FM5-95. Using the temperature-sensitive mutation in Dynamin, shibirets, we showed that SV recycling involves trafficking through an intermediate endosomal compartment. In cultured mammalian cells, interfering with Rab5 function by expressing the dominant negative version, Rab5SN causes the fragmentation of the endosome and the accumulation of endocytic vesicles. In contrast, when Rab5 is overexpressed enlarged endosomal compartments were observed. In Drosophila, the endosomal compartment was disrupted when loss of function and dominant negative mutants of Rab5 were expressed. In addition, at the ultrastructural we observed an accumulation of endocytic vesicles in Rab5S43N expressing terminals and enlarged endosomes when Rab5 was overexpressed. Furthermore, interfering with Rab5 function using the dominant negative Rab5S43N caused a decrease in the SV recycling kinetics as shown by FM1-43 experiments. In contrast, overexpression of Rab5 or GFP-Rab5 caused an increase in the FM1-43 internalization rate. Finally, standard electrophysiological techniques were used to measure synaptic function. We found that the Rab5-mediated endosomal SV recycling pathway generates vesicles with a higher fusion efficacy during Ca2+-triggered release, compared to SVs recycled when Rab5 function was impaired. We therefore suggest a model in which the endosome serves as organelle to control the SV fusion efficacy and thereby the synaptic strength. Since changes in the synaptic strength are occuring during learning and memory processes, controlling endosomal SV recycling might be a new molecular mechanism involved in learning and memory.