During Drosophila embryogenesis, the attachment of somatic muscles to epidermal tendon cells requires heterodimeric PS-integrin proteins (alpha- and beta-subunits). The alpha-subunits are expressed complementarily, either tendon cell- or muscle-specific, whereas the beta-integrin subunit is expressed in both tissues. Mutations of beta-integrin cause a severe muscle detachment phenotype, whereas alpha-subunit mutations have weaker or only larval muscle detachment phenotypes. Furthermore, mutations of extracellular matrix (ECM) proteins known to act as integrin binding partners have comparatively weak effects only, suggesting the presence of additional integrin binding ECM proteins required for proper muscle attachment. Here, we report that mutations in the Drosophila gene thrombospondin (tsp) cause embryonic muscle detachment. tsp is specifically expressed in both developing and mature epidermal tendon cells. Its initial expression in segment border cells, the tendon precursors, is under the control of hedgehog-dependent signaling, whereas tsp expression in differentiated tendon cells depends on the transcription factor encoded by stripe. In the absence of tsp activity, no aspect of muscle pattern formation as well as the initial contact between muscle and tendon cells nor muscle-to-muscle attachments are affected. However, when muscle contractions occur during late embryogenesis, muscles detach from the tendon cells. The Tsp protein is localized to the tendon cell ECM where muscles attach. Genetic interaction studies indicate that Tsp specifically interacts with the alphaPS2 integrin and that this interaction is needed to withstand the forces of muscle contractions at the tendon cells.

Syndecan (Sdc) is a conserved transmembrane heparan sulfate proteoglycan (HSPG) bearing additional chondroitin sulfate (CS) modifications on its extracellular domain. In vertebrates, this extracellular domain of Sdc is shed and acts as a soluble effector of cellular communication events, and its cytoplasmic domain participates in intracellular signaling needed to maintain epithelial integrity. In Drosophila, Sdc has been shown to be necessary for Slit signaling-dependent axon and myotube guidance during CNS development and muscle pattern formation. We report that Sdc acts in a cell-autonomous manner in Slit-receiving cells and that its membrane-anchored extracellular domain is sufficient to mediate Slit signaling. Sdc activity can be replaced by the human homolog hsdc2. However, the HSPG Dally-like protein (Dlp), which lacks CS modifications at its extracellular domain, can only partially substitute for Sdc function, and its activity is not restricted to the Slit target cells. Our results suggest that Sdc and Dlp act in a cooperative but nonredundant fashion in axon and myotube guidance. We propose that Dlp, which lacks CS modifications, participates in the transfer of Slit from its site of expression to the target cells, where CS-modified Sdc concentrates and presents the ligand.Drosophila ͉ heparan sulfate ͉ Slit signal transduction ͉ axon guidance

Introduction__________________________________________________________ 1.1 Heparan Sulphate Proteoglycans (HSPGs) ___________________________________ 1.2 Roles of HSPGs in Signalling ______________________________________________ 1.3 The Drosophila ventral midline____________________________________________ 1.4 Slit/Robo signalling mediates axon guidance_________________________________ 1.5 Slit/Robo signalling mediates muscle patterning______________________________ 1.6 Syndecan regulates Slit/Robo signalling ____________________________________ 1.7 Domain structure of Syndecan ____________________________________________1.7.1 Cytoplasmic domain of Syndecan_______________________________________________ 1.7.2 Transmembrane domain of Syndecan ____________________________________________ 1.7.3 Extracellular domain of Syndecan ______________________________________________ 1.8 Mechanistic analysis of Syndecan function in Slit/Robo signalling _______________ 2 Materials and Methods ________________________________________________ 2.1 Molecular Biology ______________________________________________________ 2.1.1 Polymerase Chain Reaction ___________________________________________________ 2.1.2 DNA Restriction ____________________________________________________________ 2.1.3 DNA extraction from agarose gel _______________________________________________ 2.1.4 Dephosphorylation of vector DNA ______________________________________________ 2.1.5 DNA Ligation ______________________________________________________________ 2.1.6 Preparation of electro-competent Escherichia coli (E.coli) cells _______________________ 2.1.7 Preparation of chemical-competent E.coli cells ____________________________________ 2.1.8 Transformation of electro-competent E.coli cells ___________________________________ 2.1.9 Transformation of chemical-competent E.coli cells _________________________________ 2.1.10 Plasmid DNA purification____________________________________________________ 2.1.11 DNA Sequencing __________________________________________________________ 2.1.12 Site-directed mutagenesis ____________________________________________________ 2.1.13 DNA preparation for Embryo Injections_________________________________________ 2.2 Fly techniques __________________________________________________________ 2.2.1 Maintenance of flies _________________________________________________________ 2.2.2 Generation of stable transgenic fly lines __________________________________________ 2.2.3 Ectopic gene expression via the UAS/GAL4 system ________________________________ 2.2.4 Fixation of embryos _________________________________________________________ 2.2.5 Antibody (Ab) staining of embryos _____________________________________________ 2.2.6 Fluorescence microscopy _____________________________________________________ 2.3 Cell culture based techniques _____________________________________________ 2.3.1 Maintenance of cells _________________________________________________________ 2.3.2 Transfection of cells _____________________________________...