Biochemical studies suggest that axon guidance activity requires cell-surface heparan sulfate to promote binding of mammalian Slit/Robo homologs (Liang, 1999; Hu, 2001). The Drosophila homolog of Syndecan (reviewed in Couchman, 2003), a heparan sulfate proteoglycan (HSPG), is required for proper Slit signaling. Slit, the ligand for the Roundabout (Robo) receptors, is secreted from midline cells of the Drosophila central nervous system (CNS). It acts as a short-range repellent that controls midline crossing of axons and allows growth cones to select specific pathways along each side of the midline. In addition, Slit directs the migration of muscle precursors and ventral branches of the tracheal system, showing that it provides long-range activity beyond the limit of the developing CNS. Syndecan (Sdc) mutations have been generated; they affect all aspects of Slit activity and cause robo-like phenotypes. sdc interacts genetically with robo and slit, and double mutations cause a synergistic strengthening of the single-mutant phenotypes. The results suggest that Syndecan is a necessary component of Slit/Robo signaling and is required in the Slit target cells (Steigemann, 2003).

Genetic assays provide a sensitive means of detecting an in vivo interaction between different components in a pathway, but they do not show that the association is direct. Thus, a biochemical assay was developed to determine whether Sdc binds to Slit and/or Robo in cellular extracts in which all three proteins are endogenously expressed. Immunoprecipitation of either Slit or Robo and subsequent detection with anti-Sdc antibodies reveals that Sdc associates with both Slit and its receptor, suggesting the possibility of a ternary complex. This association is specific because no Sdc is trapped by nonspecific IgG or N-Cadherin antibodies that successfully IP other signaling molecules. Thus, Sdc participates directly in a complex with Slit and Robo (Johnson, 2004).

Mammalian Slit proteins are capable of binding to the HSPG Glypican, and this in vitro interaction is sensitive to heparinase III treatment (Liang, 1999). Furthermore, heparinase III treatment significantly reduced the binding of human Slit2 to Robo1-transfected cells and reduced its biological activity (Hu, 2001). In contrast to mammals, the Drosophila genome contains only a single Slit-coding gene, which acts through three receptors: Robo, Robo2 (Leak), and Robo3. These receptors are expressed in an overlapping set of axons of the longitudinal tracts that run parallel to the midline of the developing CNS. In order to identify an endogenous HSPG that might participate in Slit signaling, the expression of HSPG-encoding genes was examined by whole-mount in situ hybridization, looking for HSPG encoding genes that are expressed in patterns that include the expression domains of Slit and/or Robo. syndecan (sdc), which encodes a conserved transmembrane protein containing a heparan-sulfate-modified extracellular domain (Spring, 1994), shows zygotic patterns overlapping all tissues affected in slit mutants and in domains directly adjacent to regions of slit expression (Steigemann, 2003).

If Sdc participates in the control of Slit/Robo signaling and is not expressed in Slit-expressing cells, it is likely to be coexpressed with the Robo receptors. Robo2 and Robo3 are expressed in a subset of the Robo-expressing axons. Thus, the anti-Robo antibody labels all axons in which the three Drosophila Slit receptors are active. In order to localize the Sdc-expressing cells, Sdc-specific antibodies were generated to immunostain embryos in combination with anti-Robo antibodies. Specificity of the antibody staining is indicated by the absence of staining in homozygous sdc mutant embryos and by panneural overexpression using the GAL4/UAS system . Panneural Sdc expression was achieved with a UAS-dependent transgene containing an sdc cDNA (composed of the translated exons; sdc-RA in response to the elav-GAL4-driver). Sdc and Robo are coexpressed in the longitudinal axons from stage 13 onward. Sdc protein is also expressed across the midline in positions corresponding to the commissural axons that lack Robo. The heparinase sensitivity of the mammalian Slit/Robo interaction, the molecular nature of Sdc, and its coexpression with Robo (and thus also with Robo2 and Robo3) are consistent with a role of Sdc in Slit signaling (Steigemann, 2003).

In order to test this proposal, sdc mutants were generated by imprecise excision of a P element inserted in the first intron of the sdc gene. Two sdc mutations were generated, the alleles sdc⁹⁷ and sdc²³, both lacking the first exon, the transcription start site, and parts of the first intron. In contrast to sdc⁹⁷, the translation start site as well as parts of the signal peptide encoded by the second exon are removed in the sdc²³ allele. Both deletion mutants fail to express the sdc transcript in all regions of the embryo where transcripts are detected in wild-type embryos. Furthermore, Sdc could not be detected by anti-Sdc antibodies in the mutant embryos. These findings indicate that the newly generated sdc mutants are either strong hypomorphic or amorphic alleles (Steigemann, 2003).

Homozygous sdc mutants are semilethal and show identical phenotypes in the CNS and in the muscle pattern. In order to unambiguously demonstrate that the lack of sdc activity is responsible for the mutant phenotype observed, Sdc-RA was panneurally expressed using the GAL4/UAS system in sdc mutant individuals. The neural phenotype of the mutants was rescued, indicating that the mutant phenotype was caused by the lack of Sdc and that the transgene provides functional Sdc activity (Steigemann, 2003).

In order to examine the possible defects in axonal guidance and muscle patterning, sdc mutant embryos were stained with both Fasciclin II (FasII; mAB 1D4) antibodies, which label three longitudinal axon tracts at each side of the midline, and anti-Mhc antibodies, which visualize the muscle pattern. The results show that the lack of sdc activity causes phenocopies of robo and robo2 mutants; i.e., it affects both midline guidance of axons and the establishment of the muscle pattern. The defects in CNS axon guidance were strikingly similar to robo2 mutants but less pronounced than in robo mutants. The muscle and CNS phenotypes were also weaker than in slit mutants, in which signaling through all Robo receptors is impaired (Steigemann, 2003).

It was next asked whether sdc activity participates in the control of robo and/or slit expression, or vice versa, by examining the strength and patterns of expression of each gene in sdc, robo, and slit mutant embryos. The results showed that there was no crossregulatory effect on gene expression and localization of the proteins. In addition, it was asked whether panneural overexpression of sdc interferes with axonal guidance and the establishment of the muscle patterns. Panneural sdc expression was strongly induced in transgene-bearing wild-type embryos, but no effect on the FasII-expressing axons, the commissures, and the mytotube pattern could be observed. Collectively, the results suggest that in the absence of sdc activity, both slit and robo expression as well as the production and localization of the proteins were not affected, but the effectiveness of the Slit signal is strongly reduced in sdc mutants. In addition, panneural sdc overexpression does not interfere with Slit signaling (Steigemann, 2003).

In order to link embryonic Sdc requirement genetically to Slit/Robo signaling, it was next asked whether sdc mutations can enhance loss-of-function slit and robo phenotypes. It was found that the number of ventral muscles, which cross the midline dorsal of the CNS in homozygous sdc and robo2 single mutants, is significantly increased in double mutant combinations of sdc and robo2, resulting in a muscle phenotype indistinguishable from slit and homozygous robo, robo2 double mutants. In the CNS, the FasII-expressing longitudinal fascicles of robo2, sdc double mutants converged into a single thick axon bundle at the ventral midline, resembling the effects seen with slit mutants. Similar observations were obtained with the mAb BP102 against all CNS axons, showing strongly condensed fascicles in robo2, sdc double mutant embryos. The synergistic strengthening of both the muscle and the CNS phenotypes in robo2, sdc double mutants, which are similar to a weak slit mutant phenotype, indicates that only some Slit-derived repellent activity is received along the midline. In contrast to robo2, the robo mutant phenotype was not significantly enhanced by the simultaneous lack of sdc. The data suggest that Robo can, in part, compensate for the lack of Robo2 and vice versa and that Robo is more sensitive to reduced Sdc-dependent Slit activity than Robo2 (Steigemann, 2003).

The results imply that sdc, slit, and robo are components of the same genetic circuitry. This proposal was tested by genetic means, asking whether the gene activities interact in vivo. Loss of only one copy of sdc led to the development of a normal muscle pattern, whereas the simultaneous absence of one copy of both slit and sdc in slit/+, sdc/+ double heterozygous embryos caused an increase in the number of longitudinal transverse muscles. Furthermore, the number of FasII-expressing inner fascicles that cross the midline is increased (3.3%) as compared to slit heterozygous embryos (0.6%;). More clearly, homozygous sdc mutant embryos, which also lack one copy of either robo, robo2, or slit, show an enhanced axonal guidance defect with multiple midline crossings of the fascicles (72%, 47%, and 95%, respectively), a phenotype very similar to the robo mutant. These results establish that Sdc acts in the same genetic circuitry as Slit and the Robo receptor family and represents a critical component of the Slit/Robo signaling pathway (Steigemann, 2003).

Axonal expression of Sdc suggests that its activity is required in the Robo-expressing Slit target cells. In order to test this inference, cell-specific rescue experiments were performed by expressing Sdc from either an elav-GAL4 or a sim-GAL4 driven UAS transgene in Robo-expressing neurons and Slit-expressing midline cells, respectively. The axon guidance defects of the sdc mutant were entirely rescued by Sdc expression in neurons, whereas no rescuing activity was seen in response to Sdc expression in midline cells. These findings indicate that sdc activity is not required for the production and/or secretion of Slit, but in the reception and/or the transmission of the signal in the target cells (Steigemann, 2003).

The results present evidence that axonal and myotube guidance in Drosophila require the transmembrane HSPG Sdc and that Sdc is an essential component of the Slit/Robo signaling pathway. These findings are consistent with earlier results showing that heparinase III treatment both weakens the Slit/Robo interaction and the biological effect of Slit signaling in mammalian cell culture, but is surprising in the light of the demonstration that mammalian Slit associates with the HSPG Glypican (Liang, 1999). Therefore dally and perlecan mutations were examined for potential interaction with Slit/Robo signaling and no effect was found. In addition, no defects were detected in sdc mutants reminiscent of impaired Hedgehog, Wingless, or FGF signaling, pathways previously shown to be sensitive to the levels of other HSPGs. Furthermore, Slit signaling in sdc mutants is also impaired in other tissues including the tracheal system. Thus, Slit/Robo signaling seems to primarily or even specifically require Sdc among the known Drosophila HSPGs. The difference in the nature of the HSPG that interacts with Slit in Drosophila and mammals may either reflect a species-dependent difference or a difference between the in vivo situation shown in this study and the previously published in vitro analysis. Other remarkable results are the spatially restricted requirement for Sdc as suggested by its expression domains adjacent to Slit-expressing cells and the finding that no defects, other than those related to Slit/Robo signaling, could be observed. Furthermore, overexpression of Sdc in the Slit target tissue did not interfere with proper Slit signaling. This result and the finding that overexpression of Sdc in the Slit-secreting midline glia cells also has no effect on axonal pathfinding and myotube guidance make it unlikely that Sdc functions as a simple ligand gatherer of soluble Slit. In addition, the rescue and protein localization studies indicate that Sdc functions in the target tissue and not in the Slit-secreting cells and acts in a Robo receptor-dependent manner. Sdc could, therefore, function to stabilize and/or transduce the Slit signal. Sdc may act as a coreceptor that presents Slit to Robo receptors or stabilizes the ligand receptor complex. Alternatively, signaling across the cell membrane might involve the Sdc-dependent organization of microdomains within the target cell membrane as has been proposed for vertebrate Sdc proteins (Steigemann, 2003).

GENE STRUCTURE

cDNA clone length - 1516 bp (one of three different isoforms with alternative splicing)

Bases in 5' UTR - 328

Exons - 6

Bases in 3' UTR - 183

PROTEIN STRUCTURE

Amino Acids - 395

Structural Domains

In mammals, cell-surface heparan sulfate is required for the action of basic fibroblast growth factor, fibronectin, antithrombin III, as well as other effectors. The syndecans, a gene family of four transmembrane proteoglycans that participates in these interactions, are the major source of this heparan sulfate. Based on the conserved transmembrane and cytoplasmic domains of the mammalian syndecans, a single syndecan-like gene was detected and localized in the Drosophila genome. As in mammals, Drosophila Syndecan is a heparan sulfate proteoglycan expressed at the cell surface that can be shed from cultured cells. Drosophila Dyndecan is expressed in embryonic tissues that correspond with those tissues in mammals that express distinct members of the syndecan family predominantly. Conservation of this class of molecules suggests that Drosophila, like mammals, uses cell-surface heparan sulfate as a receptor or coreceptor for extracellular effector molecules (Spring, 1994).

The extracellular domain of Drosophila Syndecan is rich in aspartic acid, threonine, serine and proline, but the transmembrane and cytoplasmic sequences are >50% identical to each vertebrate syndecan protein. As with the extracellular domains of the vertebrate syndecans, similarities with that of the Drosophila syndecan are hardly recognizable, except for a predominance of prolines and hydrophopic residues in regions that separate the conserved Ser-Gly glycosaminoglycan attachment sites from the transmembrane domain. The Drosophila gene is derived from a common ancestor of all four vertebrate syndecan genes, and syndecan 1 and 3 and syndecan 2 and 4, respectively, form two subfamilies. An intron near the junction between the extracellular and transmembrane domains is precisely conserved in the Drosophila gene, murine syndecan 1, chicken syndecan 3, consistent with common ancestory (Spring, 1994).

date revised: 30 November 2004

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