Analyse axonaler Lenkungsmechanismen in Drosophila
Final Report Abstract
Innervation of contractile muscle fibers is a prerequisite for locomotion and controlled movements in any higher animal. Reduced or lack of innervation leads to uncoordinated, spastic movements or even paralysis in certain muscle groups. Connections between innervating motoneurons and their target muscles are established during embryogenesis in a process called axon guidance and synapse formation. The transmembrane proteins of the immunoglobulin family Beaten path (Beat) and Sidestep (Side) play fundamental roles in this process since a high percentage of muscle fibers (30-40%) permanently lack innervations or are innervated at wrong positions in beat and side mutants. In previous work, we have shown that Side is expressed ahead of motor axonal growth cones and serves as an intermediate target, implying that Beat-expressing motor axons selectively recognize and grow along Side-labeled cell surfaces. In fact, in the absence of Beat, motor axons are not attracted to Side-expressing cells, and ectopic expression of Side in tissues not involved in the guidance process strongly attracts motor axons to these tissues. Biochemically, transfecting S2 suspension cells with Beat and Side cDNAs, leads to the formation of large cell aggregates, indicating that such adhesives complexes may steer motor axons towards their target fields in vivo. To better understand the formation of these complexes and to uncover functionally important protein domains, we proposed to perform a structure function analysis of Beat and Side. In addition, we were curious about potential regulatory mechanisms, because of the precisely controlled expression pattern of side and the inability of Side-specific antibodies to detect Side after contact with motor growth cones. Furthermore, since little is known about the consequences of misinnervated muscles on adult locomotion, we proposed to study the impact of side mutations on locomotor performance and the adult innervation pattern. Using various full-length and truncated constructs we found that Beat and Side interact with their first IG-domains in vivo, consistent with a point mutation identified in an EMS mutagenesis screen (sideI306). In addition, the first IG domain of Side is both necessary and sufficient to attract motor axons. Expression of tagged Beat constructs also showed that full-length Beat is not released into the hemolymph of living embryos. However, upon deletion of a C-terminal domain, the truncated proteins accumulated in the hemolymph. Screening regulatory elements identified a single short promotor fragment in the beat locus that was sufficient to rescue the beat mutant phenotype completely. In adult flies, we stained motor nerves in wild-type and side mutant legs and flight muscles, finding that side also guides adult motor axons. Overexpression of Side in adult muscles strongly attracted motor axons and caused exuberant branching. These innervation defects caused severe alterations in leg postures, locomotion and flight behavior. In summary, Beat and Side have profound effects on the wiring of the neuromuscular system in Drosophila. Further deciphering their molecular functions these molecules will hopefully shed some light on the basic axon guidance principals in Drosophila and possibly also in other organisms.
