Wingless is a morphogen required for the patterning of many Drosophila tissues. Several lines of evidence implicate heparan sulfate-modified proteoglycans (HSPGs) such as Dally-like protein (Dlp) in the control of Wg distribution and signaling. dlp is required to limit Wg levels in the matrix, contrary to the expectation from overexpression studies. dlp mutants show ectopic activation of Wg signaling at the presumptive wing margin and a local increase in extracellular Wg levels. dlp somatic cell clones disrupt the gradient of extracellular Wg, producing ectopic activation of high threshold Wg targets but reducing the expression of lower threshold Wg targets where Wg is limiting. Notum encodes a secreted protein that also limits Wg distribution, and genetic interaction studies show that dlp and Notum cooperate to restrict Wg signaling. These findings suggest that modification of an HSPG by a secreted hydrolase can control morphogen levels in the matrix (Kirkpatrick, 2004).

By a number of cellular and molecular markers, compromising dlp function produced ectopic activation of high-threshold Wg target genes in the wing imaginal disc. dlp adult escapers have ectopic mechanosensory bristles and dlp third instar larvae show expanded expression of Ac, a marker for sensory organ precursor cells. Wg signaling was abnormally elevated along the DV boundary of dlp mutant discs by a number of other measures, including expansion of the zone of nonproliferating cells (ZNC) and ectopic expression of Cyclin A. These findings are consistent with the expansion of sensory organ precursor cells produced by RNAi inhibition of dlp function. These phenotypes show that Dlp serves to limit activation of high-threshold target genes near the DV boundary (Kirkpatrick, 2004).

The expansion of Wg signaling found in dlp mutants is accompanied by locally elevated levels of extracellular Wg. The increase in Wg in the matrix of dlp mutant wing discs is not associated with increases in Wg production or altered expression of D-fz2, a known regulator of Wg levels. These findings argue against the model that dlp controls Wg levels indirectly via control of Wg expression or other regulatory genes. It is well established that Wg binds heparan sulfate, and therefore it is likely that Dlp controls Wg levels through a direct interaction (Kirkpatrick, 2004).

The expansion of Wg-directed patterning in the wing disc of dlp mutants was a surprising finding on a number of counts: (1) mutations in genes encoding heparan sulfate biosynthetic enzymes compromise rather than increase the levels of Wg in the matrix; (2) ectopic expression of Dlp results in higher levels of Wg in the matrix. These findings suggest that loss of Dlp would destabilize Wg on cell surfaces and in the matrix, thereby reducing Wg signaling. However, exactly the opposite result was obtained in dlp mutants: loss of Dlp increases extracellular Wg and signaling. These findings emphasize that while mutations compromising synthesis of all HSPGs might destabilize Wg in the matrix, loss of any one proteoglycan can have very different effects (Kirkpatrick, 2004).

The phenotypes of dlp mutants suggest that Dlp serves to limit Wg distribution and/or alter Wg stability, thus affecting the Wg gradient in the wing disc. Genetic mosaic analysis of dlp clearly demonstrates that the ectopic activation of Wg signaling, as measured by Arm, Cyclin A, and Ac expression, occurs non-cell autonomously, providing support for this model. Recent mathematical modeling of morphogen gradients demonstrates that binding of morphogens to receptors or coreceptors on cell surfaces or in the matrix can have profound effects on diffusion and subsequently limit morphogen activity. Consistent with such modeling, it has been demonstrated experimentally that a nonheparin binding isoform of the secreted growth factor VEGF adopts a broader, shallower distribution than heparin binding forms and the differential localization of VEGF-A isoforms in the matrix controls the vascular branching pattern. It is also well established that heparan sulfate proteoglycans mediate endocytosis of extracellular ligands, and the findings suggest the possibility that Dlp might mediate endocytic control of Wg levels in the matrix. dlp mutants display locally elevated levels of extracellular Wg without an increase in production, and therefore Wg turnover must be affected in some way (Kirkpatrick, 2004).

In contrast to the higher levels of extracellular Wg found near the DV boundary in dlp mutant wing discs, there is no apparent change in the low level of Wg at the edges of the gradient. However, genetic mosaic analysis of dlp in wing development shows that loss of dlp can modestly reduce the activation of a low-threshold Wg target gene in those regions where Wg levels are low. Since this effect on Wg signaling is not a consequence of reduced levels of Wg in the matrix, it must result from an alteration of cell responsiveness or the bioactivity of the Wg that is present. The findings are consistent with studies showing that RNAi of dlp in the wing disc also inhibits transcriptional activation of Wg target genes in regions where Wg is low. It is interesting to note that dlp expression, like that of the Wg receptor D-fz2, is highest outside the presumptive wing margin. Perhaps the high level of dlp expression in this region serves, in part, to enhance cellular responses to Wg. The ability of Dlp to enhance Wg signaling as well as limit Wg distribution is not unprecedented: the other glypican in Drosophila, Dally, enhances Dpp signaling in regions of the wing disc where Dpp levels are modest in addition to regulating the distribution of Dpp protein (Kirkpatrick, 2004).

Mutations in Notum produce ectopic activation of Wg signaling and alter the Wg gradient, similar to what was observed in dlp mutants. Notum encodes a protein with homology to plant hydrolases and has been proposed to alter the structure of Dlp, thus affecting its affinity for Wg. Coexpression of Notum with Dlp decreases Dlp-mediated stabilization of Wg protein in the wing pouch. dlp and Notum mutants fail to complement: this is strong genetic evidence that Dlp and Notum cooperate to control Wg-mediated patterning. Biochemical studies show that Notum can cleave Dlp from the cell surface. The finding that heterozygosity for dlp ameliorates patterning abnormalities produced by ectopic Notum suggests that Dlp is a principal substrate for Notum activity. The results suggest that Notum-mediated release of Dlp from the cell surface is required for Dlp to limit Wg signaling (Kirkpatrick, 2004).

Notum and dlp are expressed in complementary patterns in the wing disc, with Notum levels highest near the DV boundary and dlp levels highest elsewhere. Thus, a gradient of Dlp cleavage may be established across the disc, with some Dlp remaining on cell surfaces in regions distant from the DV boundary, enhancing cellular responses to Wg. Cleavage of Dlp would remove this coreceptor activity, but shed Dlp-Wg complexes also could limit Wg signaling by promoting Wg clearance or could serve as dominant-negative inhibitors of signaling. Coexpression of Notum and Dlp in the wing disc produced more intracellular vesicles compared to expression of Dlp alone: this is consistent with a role for shed Dlp in promoting endocytosis and clearance of Wg from the matrix (Giraldez, 2002). Such a role is also consistent with the non-cell-autonomous effects of dlp somatic cell clones on extracellular Wg distribution. Loss of dlp in clones might locally reduce Wg turnover as well as reducing cell surface binding sites for Wg, permitting increased diffusion of Wg and accumulation on wild-type cells at clone boundaries. Spatial regulation of Dlp activity by Notum can explain how Dlp primarily limits Wg signaling near the DV boundary and enhances signaling away from the boundary, but other Wg-dependent factors may also influence the ability of Dlp and Notum to downregulate Wg levels. Notably, recent mathematical modeling suggests that elevated degradation of Wg close to its source is necessary to enhance the robustness of the morphogen gradient. Together, Dlp and Notum may provide this locally increased turnover and hence stabilize the Wg gradient. Enzymatic modification of a proteoglycan to influence its cell surface localization may enable it to play both positive and negative roles in signaling and provides another potential mechanism for regulating morphogen distribution in tissues (Kirkpatrick, 2004).

The respective contribution of Heparan Sulfate Proteoglycans (HSPGs) and Frizzled (Fz) proteins in the establishment of the Wingless (Wg) morphogen gradient has been examined. From the analysis of mutant clones of sulfateless/N-deacetylase-sulphotransferase in the wing imaginal disc, it was found that lack of Heparan Sulfate (HS) causes a dramatic reduction of both extracellular and intracellular Wg in receiving cells. These studies reveal that the Glypican molecule Dally-like Protein (Dlp) is associated with both negative and positive roles in Wg short- and long-range signaling, respectively. In addition, analyses of the two Fz proteins indicate that the Fz and DFz2 receptors, in addition to transducing the signal, modulate the slope of the Wg gradient by regulating the amount of extracellular Wg. Taken together, this analysis illustrates how the coordinated activities of HSPGs and Fz/DFz2 shape the Wg morphogen gradient (Baeg, 2004).

Sfl encodes a homolog of the Golgi enzyme HS N-deacetylase/N-sulfotransferase that is required for the modification of HS. It was of interest to determine whether the retention of Wg at the cell surface involves HSPGs in receiving cells, since it has been proposed that HSPGs are unlikely to be required in Wg-receiving cells. In the wing blade, Wg, originating from the D/V border, is detected in an irregular pattern of puncta in receiving cells; these puncta correspond to the internalized Wg protein. The intensity and number of puncta decreases from the source of Wg. Furthermore, using an extracellular labeling method, a gradient of Wg protein that appears broader, shallower, and with less puncta is observed. Both in sfl mutant wing discs and in large sfl mutant clones, a striking decrease in the number of Wg puncta was observed. This decrease is not due to a change in wg transcription because it is not affected in sfl mutant cells. Further, lack of Sfl activity does not appear to disrupt the overall amount of Wg produced by wg-expressing cells but is associated with a dramatic decrease in extracellular Wg. These results suggest that HSPGs are required for sequestering extracellular Wg in receiving cells (Baeg, 2004).

To gain further insights into the role of HSPGs in receiving cells to shape the Wg gradient, an examination has been made of the distribution of Wg in patches of WT cells located within a large sfl mutant territory. In such cases, bright spots of Wg were be detected within the patch of WT cells, indicating that sfl-expressing cells are able to sequester extracellular Wg, unlike neighboring cells that lack sfl. This result is consistent through an analysis of more than 10 clones. Further, clones of cells that overexpress Notum-GT (Golgi-tethered), which acts cell autonomously in receiving cells, were generated. Notum, which encodes a member of the α/β-hydrolase superfamily, antagonizes Wg signaling and it has been proposed that Notum acts by altering the ability of the cell surface glypican molecules Dally and Dlp to stabilize extracellular Wg (Giraldez, 2002). Consistent with the conclusion that HSPGs are required in receiving cells to capture extracellular Wg, a decrease in the formation of Wg puncta was observed in cells overexpressing Notum-GT. These results are consistent with at least two nonexclusive models: (1) HSPGs could be required for Wg stability and/or trapping of Wg at the cell surface such that it does not diffuse away; (2) HSPGs could be involved in promoting Wg movement throughout tissues. The role of HSPGs in sequestering and/or stabilizing the ligand is supported by previous observations that overexpression of either Dlp or Dally results in the accumulation of extracellular Wg (Baeg, 2004).

Because HSPGs have been implicated in endocytosis of ligands such as FGF, possibly HSPGs also play a role in Wg internalization. A role for HSPG in Wg endocytosis would be consistent with the absence of puncta in sfl clones, and also the observed accumulation of extracellular Wg following overexpression of Dlp-HA (Giraldez, 2002). However, much of Wg proteins appeared in intracellular vesicles, instead of outlining the cell surface, in discs overexpressing both Dlp-HA and Notum. If HSPGs were directly involved in Wg internalization, fewer intracellular vesicles would be detected in discs overexpressing Dlp-HA and Notum since Notum acts to decease the affinity of Dlp for Wg. Furthermore, if the primary function of HSPGs were to internalize Wg, then extracellular Wg accumulation in cells lacking HSPGs activity would be expected, which is not the case. However, the data does not rule out the possibility that HSPGs play a direct role in Wg endocytosis, and, thus, further analysis will be required to clarify this issue. Taken together, these results suggest that the primary role of HSPGs is to trap and/or stabilize extracellular Wg in receiving cells where it is then able to interact with its signaling receptor as well as other factors that are responsible for its internalization, and thus contributes to shaping the Wg gradient (Baeg, 2004).

Previous ectopic expression studies have shown that Dlp can trap extracellular Wg and prevent activation of the Wg signaling pathway. Because Dlp appears to be a major HSPG required to regulate Wg signaling, its endogenous distribution was examined in the wing imaginal disc using a polyclonal antibody against Dlp and a staining method that primarily detects extracellular proteins. The specificity of the Dlp antibody was confirmed by misexpressing dlp using the ap-Gal4 driver. In the third instar wing imaginal disc, Dlp was detected throughout the disc; however, a significant decrease in the level of Dlp was detectable at the D/V border. This domain of low Dlp expression correlates with the region where high level of Wg signaling is required to induce the expression of short-range target genes. It is noted that since dlp mRNA expression is uniform throughout the disc, the down-regulation of Dlp at the D/V border must occur post-translationally. Interestingly, an optical cross section of the disc has revealed that endogenous Dlp localizes mostly on the basolateral surface of the cell where extracellular Wg is detected. The subcellular localization of Dlp protein was also examined using a GFP-dlp expressed under the control of a Gal4 driver. Consistent with the Dlp antibody result, it was found that GFP-Dlp localizes predominantly to the basolateral membrane. Altogether, these observations suggest that Dlp can bind to extracellular Wg and that Dlp levels need to be reduced for high-level Wg activity in cells near the D/V boundary (Baeg, 2004).

In a genome-wide RNAi screen in S2R+ cells to identify genes that either up- or down-regulate Wg signaling, Dlp was identified as both a negative and positive regulator of Wg signaling under stimulated and nonstimulated conditions, respectively. The cell-based assay devised consists of the activation of a Tcf/Arm-dependent Wg-reporter gene upon induction of S2R+ cells by expressing a wg cDNA by transient transfection. The activity of the Wg pathway and the effect of the addition of various dsRNAs on the pathway were assayed by monitoring Luciferase-reporter activity using a luminescence-based plate reader. Using this assay, the addition of dsRNAs of positive transducers of Wg signaling, such as arm, decrease the Top12X-HS-Luciferase reporter activity, while dsRNAs to negative Wg regulators, such as Daxin, increase its activity. Interestingly, under condition of Wg induction, dlp was found to act as a negative regulator of Wg signaling, since dlp dsRNA led to a twofold increase in luciferase activity. This increase is significant since it is comparable to that of daxin dsRNA. In the absence of Wg induction, dlp was found to positively regulate Wg signaling, since dlp dsRNA leads to a fivefold decrease in luciferase activity, a decrease that is similar to that observed by the addition of arm dsRNA. These results suggest Dlp acts as a positive regulator of the Wg pathway when Wg level is low and negatively influences signaling when Wg is abundant. These results are consistent with in vivo results that demonstrate that Dlp has both a positive and negative role in Wg signaling (Kirkpatrick, 2004). The observations in S2R+ are consistent with the hypothesis that low Dlp levels at the D/V boundary supports high-level Wg signaling, while further away from the D/V boundary where the Wg concentration is lower, Dlp positively influences Wg signaling. Overall, the result from the S2R+ RNAi experiments indicates that (1) Dlp is not an essential component of the Wg signal transduction pathway; and (2) Dlp can either have a negative or positive impact on Wg signaling depending on the level of Wg available. The negative effect of Dlp is consistent with previous in vivo studies that have shown ectopic Dlp expression can trap extracellular Wg and prevent activation of the Wg signaling pathway. Therefore, a reduction of Dlp levels at the D/V border would be expected to contribute to high-level Wg signaling. The positive effect of Dlp in Wg signaling needs to be understood in the context of the previous findings that loss of HSPG activity results in wg loss-of-function phenotypes, as shown by a decrease in dll expression in clones mutant for enzymes involved in GAG biosynthesis. One attractive model is that Dlp would act as a co-receptor that traps/stabilizes extracellular Wg and facilitates its association with the signal transducing Fz receptors in cells located at a distance from the D/V boundary where low level of Wg is available. Finally, these observations are consistent with a recent study (Kirkpatrick, 2004) that showed that (1) ectopic activation of Wg signaling at the wing margin occurs in dlp mutant tissues, and (2) a cell autonomous reduction in Wg signaling in dlp clones located distal to the Wg-producing cells (Baeg, 2004).

The distribution of Dlp protein is reminiscent of the down-regulation of Dfz2 transcription near the D/V border. Wg-mediated repression of DFz2 expression has been shown to affect the shape of the Wg gradient, resulting in a gradual decrease in Wg concentration. Because these results indicate that HSPGs affect Wg distribution, the functions of the two seven transmembrane Wg receptors, Fz and DFz2, were examined to evaluate how the signal transducing receptors cooperate with HSPGs in shaping the Wg gradient. To determine the role of Fz and DFz2 in Wg movement, the distribution of Wg in fz DFz2 double-mutant clones was examined. In these clones, an expansion of wg expression was observed, which is consistent with the previously described Wg “self-refinement” process, by which Wg signaling represses wg expression in cells adjacent to wg-expressing cells. Unexpectedly, within these clones, Wg puncta are still present, indicating that Fz/DFz2 receptor activities are not required for Wg spreading (Baeg, 2004).

To determine whether Wg is present in endosomes in the absence of Fz/DFz2 activities, wing discs were labeled with the endosomal marker Texas-red dextran. More than 50% of Wg puncta co-localize with red dextran, indicating that Wg is internalized in the absence of Fz/DFz2 activities. These observations are consistent with results in the embryo, and altogether suggest that internalization of Wg can be accomplished by proteins other than Fz/DFz2. Interestingly, this observation contrasts with the role of HSPGs in Wg distribution, since wing discs lacking GAGs show alteration in Wg puncta in receiving cells. The extracellular distribution of Wg was examined in fz DFz2 mutant clones. Interestingly, accumulation of extracellular Wg was detected throughout these clones, thus revealing that Wg can bind to the cell surface and that Fz/DFz2 receptors are required somehow for Wg degradation. To exclude the possibility that accumulation of extracellular Wg results from increased wg expression or secretion in fz DFz2 clones that cross D/V boundary, small clones that do not include the D/V boundary were generated. Accumulation of extracellular Wg was detected in these clones, which is reminiscent of the finding that overexpression of a dominant-negative form of DFz2 (ΔDFz2-GPI) driven by en-Gal4 in embryonic tissue prevents Wg decay within the en domain. It has been proposed that endocytosis of a Wg/receptor complex is responsible for down-regulating Wg levels. Further, because Wg is still organized in a graded manner in these clones, as shown by the distribution of the Wg puncta, it indicates that Wg movement can occur in the absence of Fz/DFz2 (Baeg, 2004).

There is a third member of the Frizzled family encoded by DFz3 that could influence the distribution of Wg in tissues. DFz3 expression is similar to that of wg, and a constitutively activated form of Arm up-regulates its expression in the wing disc, suggesting that DFz3 is transcriptionally regulated by Wg signaling. Based on these observations, little or no DFz3 protein would be expected to be present in cells that lack Fz/DFz2 activity, suggesting that internalization of Wg in Fz/DFz2 mutant cells is unlikely to be mediated by DFz3. Another candidate that could affect Wg distribution is Arrow, which is a Drosophila homolog of a low-density lipoprotein (LDL)-receptor-related protein (LRP) and has been shown to be essential in cells receiving the Wg signal. However, because a soluble form of the Arrow fails to bind Wg and Fz receptors in vitro, and because Arrow functions after DFz2 engages Wg, it is unlikely that Wg internalization in Fz/DFz2 mutant cells is mediated by Arrow. Finally, as is case for FGF endocytosis, HSPGs themselves possibly play a role in Wg internalization (Baeg, 2004).

In summary, there is a Fz/DFz2 receptor-independent mechanism that organizes Wg distribution, and Fz/DFz2 proteins play a role in Wg gradient formation by decreasing the level of extracellular Wg. Regulation of extracellular Wg levels by Fz/DFz2 may occur through receptor-mediated endocytosis, or by some other mechanisms. If Wg degradation occurs by receptor-mediated endocytosis, it indicates that there may exist more than one way to generate Wg puncta since these are still present in the absence of Fz/DFz2 receptor activity. These findings also emphasize that the amount of Fz/DFz2 receptors at the cell surface must be precisely regulated to achieve the proper spreading of Wg, an observation that is underscored by the transcriptional down-regulation of DFz2 expression near the source of Wg (Baeg, 2004).

To further examine the role of Fz/DFz2 receptors in Wg gradient formation, DFz2 was overexpressed at the D/V boundary using the C96-Gal4 driver, and the effect on Wg distribution and wing patterning was examined. Analysis was focused on DFz2 since DFz2 has been shown to bind Wg with high affinity and to stabilize it. Further, DFz2 expression is down-regulated by Wg signaling, and this regulation has been shown to play a critical role in the overall shape of the Wg gradient. Interestingly, ectopic expression of DFz2 results in wing notching and ectopic bristles at the wing margin of adult wing. Previous studies have shown that wing nick phenotypes result from an inhibition in Wg signaling activity while the presence of ectopic bristles on the wing blade corresponds to an increase in Wg signaling. Thus, based on the wing phenotypes, it appears that overexpression of DFz2 paradoxically both increases and decreases Wg signaling (Baeg, 2004).

Overexpression of the DFz2 could interfere with Wg signaling and its distribution in a number of ways. For example, an increase in DFz2 could increase the efficiency of Wg signaling, if the amount of receptor is limiting. Further, since wg expression in the wing disc is restricted to the D/V margin, and Wg diffuses from it, trapping of Wg near these cells most likely will have an effect on Wg short- and long-range activity since the shape of the Wg gradient will be disrupted. To distinguish between these possibilities, Wg distribution was examined in discs with clones of cells that overexpress DFz2 at the D/V boundary. These clones were associated with two effects on Wg distribution. (1) The level of Wg was increased in the clones of cells where DFz2 was overexpressed, indicating that an increase in the level of DFz2 in receiving cells leads to an increase in trapping extracellular Wg. This observation is consistent with the occurrence of extra bristles on the wing blade since they reflect high levels of Wg signaling activity. (2) A dramatic reduction in Wg puncta was detected in WT cells located adjacent to the cells overexpressing DFz2, suggesting that Wg movement from the D/V margin into the wing blade is impaired as a result of the excess trapping of Wg by cells that overexpress DFz2. To demonstrate that Wg accumulation correlates with an increase in Wg signaling and that the absence of Wg puncta correlate with an absence of Wg signaling, the effect of DFz2 overexpression on the expression of senseless was examined. sen expression is expanded in cells overexpressing DFz2, yet sen is not expressed in WT cells near a clone of cells overexpressing DFz2. This is consistent with the observation that more Wg can be detected in cells overexpressing DFz2 and that less Wg puncta are present in WT cells near a clone of cells overexpressing DFz2 (Baeg, 2004).

It has been proposed that Fz proteins contribute to Wg turnover. Thus, it is intriguing to note that overexpression of DFz2 leads to an accumulation of extracellular Wg. This may reflect saturation of the endocytotic pathway when DFz2 is overexpressed or an inability of the regulatory pathways that normally control Dfz2 endocytosis in the wing disc to appropriately respond under this overexpressed condition. Another possibility is HSPGs themselves might play an important role. Wg endocytosis and the stoichiometry of Fz to HSPGs is essential to promote proper Wg internalization. Detailed biochemical and cell biological studies are now required to clarify the role(s) of these receptors in Wg movement (Baeg, 2004).

Finally, whether overexpression of DFz2 at the D/V boundary could affect long-range activity of Wg was examined, using wing disc overexpressing DFz2 driven by C96-Gal4 driver. Interestingly, Dll expression is dramatically shortened in wing disc overexpressing DFz2 at the D/V boundary when compared to that of WT disc. It is concluded that DFz2 has multiple roles in Wg signaling: First, it transduces Wg signaling and its level is limiting in amount; and second, it affects Wg short- and long-range activity by modulating the availability of extracellular ligand (Baeg, 2004).

In this study, the respective roles of HSPGs and Fz/DFz2 receptors in Wg distribution and gradient formation were examined. Interestingly, it was found that loss of Dlp activity significantly increases the level of Wg activity in S2R+ cells upon Wg induction, indicating that Dlp acts as a negative regulator in Wg signaling and that it is not required for transducing the Wg signal. Interestingly, the in vivo results show that Dlp protein levels are low near the D/V boundary. Thus, low levels of Dlp near the source of Wg production may allow for activation of high threshold Wg target gene. It is of interest to note that Notum is highly expressed along the D/V boundary (Giralez, 2002), which would be predicted to further diminish HSPGs activity. In addition, it was found that Dlp positively influences Wg signaling in S2R+ cells when Wg is not induced, suggesting that it is required for Wg signaling in cells where Wg level is low. A possible explanation for this result is that Dlp may act as a co-receptor that traps/stabilizes extracellular Wg and facilitates its association with signal transducing Fz receptors. In the wing imaginal disc, given that Dlp is required for Wg signaling in cells where Wg levels are low, HSPG activity is possibly required for Wg signaling by somehow facilitating Wg movement. The binding of extracellular Wg to the low-affinity HSPG receptors in receiving cells may result in the association of Wg to cell membranes. Ligand movement could then occur by a mechanism that directly involves HPSGs where subsequent cycles of Wg dissociation/reassociation with HSPGs might promote the movement or require other yet to be identified extracellular molecules. To distinguish these possibilities, the role of HSPGs in Wg movement will require further detailed analysis. Regardless, these results clearly indicate that the primary role of HSPGs is to sequester and/or stabilize extracellular Wg in receiving cells. The imposition of the HSPG-mediated Wg accumulation and the Fz-dependent degradation mechanism would thus contribute to the Wg morphogen gradient. It is important to note that the expression levels of some of the critical components of each systems (i.e., dally, DFz2) are also regulated by the Wg pathway itself, indicating that the slope of the Wg gradient is established by the delicate balance between these two systems (Baeg, 2004).

Heparan sulfate proteoglycans (HSPGs), a class of glycosaminoglycan-modified proteins, control diverse patterning events via their regulation of growth-factor signaling and morphogen distribution. In C. elegans, zebrafish, and the mouse, heparan sulfate (HS) biosynthesis is required for normal axon guidance, and mutations affecting Syndecan (Sdc), a transmembrane HSPG, disrupt axon guidance in Drosophila embryos. Glypicans, a family of glycosylphosphatidylinositol (GPI)-linked HSPGs, are expressed on axons and growth cones in vertebrates, but their role in axon guidance has not been determined. This study demonstrates that the Drosophila glypican Dally-like protein (Dlp) is required for proper axon guidance and visual-system function. Mosaic studies reveal that Dlp is necessary in both the retina and the brain for different aspects of visual-system assembly. Sdc mutants also show axon guidance and visual-system defects, some that overlap with dlp and others that are unique. dlp⁺ transgenes are able to rescue some sdc visual-system phenotypes, but sdc⁺ transgenes are ineffective in rescuing dlp abnormalities. Together, these findings suggest that in some contexts HS chains provide the biologically critical component, whereas in others the structure of the protein core is also essential (Rawson, 2005).

The distribution of Dlp was examined in the developing visual system by using a monoclonal antibody that specifically recognizes Dlp in tissues. In Drosophila, the adult eye is comprised of approximately 800 sensory units, or ommatidia, each with eight distinct photoreceptors, R1-R8. In the eye imaginal disc, Dlp was found on photoreceptor cell bodies and on cells within the morphogenetic furrow. Axons from R1-R6 terminate in the lamina, the first optic ganglion, whereas those from R7 and R8 project to the medulla. In the optic lobe, Dlp is present on photoreceptor axons at the boundary between the lamina and adjacent tissues and along the lamina plexus. Dlp is also observed in the medulla neuropil, medulla glia, medulla neuropil glia, neuroblasts of the proliferative centers, and in the mushroom body neuropil (Rawson, 2005).

To evaluate the function of Dlp in axon guidance, a photoreceptor-specific monoclonal antibody (24B10) was used to visualize photoreceptor projections in dlp mutants. In 50% of dlp mutant hemispheres, the lamina plexus was irregular and thickened. Additionally, 80% of dlp mutant larvae had fibers that aberrantly crossed between ommatidial bundles and/or photoreceptor process expansions outside the normal termination zone of the lamina plexus. Examination of dlp mutant pupae revealed that 80% of optic lobes contain irregularities in the R7 and R8 medulla termini. Crossover of R7 axons to neighboring medulla cartridges was observed (~50%) and misrouting of R7/R8 axons (~20%) (Rawson, 2005).

Visual-system function was assessed in dlp mutants by recording electroretinogram (ERG) profiles in adult flies. A wild-type ERG is composed of the photoreceptor response generated by a light-induced depolarization of the photoreceptor neurons and of the two transient voltage changes, the 'on-transient' and the 'off-transient', resulting from currents related to synaptic transmission during the initiation and termination of the light stimulus. dlp mutants show statistically significant defects in the photoreceptor response and in both on- and off-transients, suggesting that Dlp is required for proper photoreceptor currents and synaptic transmission (Rawson, 2005).

Axon guidance in the visual system depends on photoreceptor specification, as well as on glial-cell migration and lamina-neuron differentiation. Although dlp mutants have reduced and roughened eyes, thin sections of dlp eyes demonstrate that all photoreceptors are present and retain proper polarity in each ommatidium. Staining with several photoreceptor-specific markers confirm that dlp mutant photoreceptors differentiate properly. Likewise, glial cells and lamina neurons were found in the correct number and location in dlp mutants, indicating that patterning defects of these critical cells cannot account for the observed axon-guidance defects (Rawson, 2005).

Because Dlp is expressed in several visual-system elements and cell types, somatic mosaic studies were conducted to determine which cells require dlp for visual-system assembly. Using a method that generates clones encompassing a majority of cells in the retina, crossover of axons between ommatidial bundles and photoreceptor process expansions outside the lamina plexus was observed in 67% of animals with dlp mutant photoreceptors projecting to a heterozygous brain. Conversely, R7/R8 termination defects were absent from the medulla of 40 hr pupae with dlp mutant retinas and dlp/+ optic lobes. These results indicate that Dlp is required in the eye to specify proper axon guidance to the lamina, but not to the medulla (Rawson, 2005).

The photoreceptor-specific requirement for Dlp was further evaluated via the mosaic analysis with a repressible cell marker (MARCM) technique to visualize axons from small dlp mutant clones. dlp mutant photoreceptors display ectopic axon outgrowths directed away from their proper targets in the lamina (37% of dlp/dlp axons). Ectopic axon processes were four times more prevalent on axon bundles near the boundaries of the lamina than on those in more-central regions. This suggests that expression of Dlp on photoreceptors may be important for the detection of repellant cues that prevent aberrant axon outgrowth (Rawson, 2005).

Finally, the tissue-specific requirement was evaluated for Dlp in physiological function of the visual system. Using a mosaic strategy that generated eyes composed solely of dlp mutant photoreceptors, no see statistically significant defects were seen in ERG recordings compared to controls. These results demonstrate that the ERG defects of dlp mutants are produced by loss-of-function in the optic lobe, not in the eye (Rawson, 2005).

Drosophila Syndecan (Sdc) represents another class of HSPG, and it is required for normal axon guidance in the embryo. Antibody specific for Sdc revealed that this proteoglycan is present on photoreceptors in the retina and on photoreceptor projections to the optic lobe. Like Dlp, Sdc is enriched throughout the lamina plexus and at the boundary between the lamina and adjacent tissues. Sdc immunoreactivity was also detectable at a low level on cells just medial to the lamina plexus, the medulla glia, but was absent from some Dlp-expressing cells such as the mushroom body and the medulla neuropil glia (Rawson, 2005).

Analysis of sdc null mutant larvae revealed that 50% of optic lobes had photoreceptor-projection abnormalities and/or lamina-plexus defects including gaps (29%) and gross disorganization (21%, n = 28). Compared to dlp mutants, sdc mutants showed a low penetrance of the lamina-thickening (4%) and lamina-axon-crossover phenotypes. Additionally, some sdc mutants had R7/R8-axon misrouting in the larval stage. sdc mutant pupae showed crossover of R7 axons between medullary cartridges (100%) and defective axon pathfinding to the medulla (86%) but a low penetrance of R7/R8-termini disruption (~10%), a phenotype common in dlp mutants (80%-100%). As is the case for dlp, sdc mutants do not show defects in the specification of photoreceptors, glia, or lamina neurons, confirming that the above phenotypes are not secondary to other overt patterning deficiencies. Overall, dlp and sdc mutants share similar axon-guidance phenotypes, but each has a largely distinct level of penetrance for a given defect (Rawson, 2005).

Electrophysiological analysis revealed that sdc mutants have grossly abnormal ERGs, with defective photoreceptor depolarization and complete absence of on- and off-transients. These ERG abnormalities -- particularly the virtual loss of photoreceptor depolarization -- are distinct from those found in dlp animals. In addition, whereas dlp null mutants have a reduced and roughened eye, sdc mutants do not. These findings are consistent with distinct molecular functions for Dlp and Sdc during visual-system assembly (Rawson, 2005).

Sdc and Dlp are both heparan sulfate-modified proteoglycans, and Dlp expression is capable of limited rescue of sdc axon-guidance abnormalities in the embryo. To determine whether these two cell-surface molecules have overlapping functions in the visual system, a series of rescue experiments was performed. With or without a GAL4 driver, dlp mutant animals bearing a UAS-dlp⁺ construct show complete rescue of pupal axon-pathfinding defects and ERG abnormalities, suggesting that low levels of Dlp expression are sufficient to provide the function necessary for normal axon guidance and visual-system assembly. Despite this, ubiquitous expression of Sdc was unable to rescue the axon-guidance abnormalities of dlp mutants, demonstrating that Dlp and Sdc are not functionally interchangeable during photoreceptor axon guidance (Rawson, 2005).

The ability of UAS-dlp⁺ transgenes to rescue axon-guidance phenotypes of sdc mutant larvae and pupae was tested. Pupal sdc mutant phenotypes, including misrouting of R7/R8 photoreceptors to the medulla and crossover of R7 to neighboring medulla cartridges, are largely rescued with neuronal-directed expression of Sdc. Conversely, neuron-specific expression of Sdc in sdc mutants is not sufficient to rescue photoreceptor projection defects to the larval lamina or the ERG abnormalities in adults, suggesting that expression of Sdc in additional cell types is critical for complete restoration of axonal patterning and visual-system function. In contrast to the inability of Sdc expression to rescue dlp mutant phenotypes, neuron-specific expression of Dlp restores proper R7/R8 photoreceptor projection to the medulla of sdc mutant pupae. This rescue demonstrates that a structurally distinct HSPG can provide some of the functions normally served by Sdc (Rawson, 2005).

In vertebrates, glypicans are expressed on axons and growth cones in the developing nervous system. Whereas previous findings have established a function for glypicans in growth-factor signaling, their role in axon guidance has not been reported. Consistent with the expression pattern of Dlp on axons and glial cells of the developing visual system, dlp mutants show defects in photoreceptor projections to the lamina and medulla. Mosaic analysis demonstrates that Dlp is required on photoreceptors for some, but not all, aspects of axon guidance, serving functions in both peripheral and central components of the visual system. dlp is not required in the retina for normal electrophysiological responses to light, indicating that the synaptic-transmission defects in dlp mutant adults are due to loss of Dlp activity in the optic lobes (Rawson, 2005).

Comparison of sdc and dlp mutants as well as transgene rescue experiments demonstrates that Sdc and Dlp have some overlapping functions in visual-system assembly but others that are unique. For example, photoreceptor misrouting to the medulla in sdc mutants can be rescued by neuron-specific expression of dlp⁺, consistent with the capacity of dlp⁺ to rescue midline-crossover defects in sdc mutant embryos. In contrast to the ability of dlp⁺ transgenes to rescue sdc mutants, UAS-sdc⁺ constructs are unable to rescue any of the dlp mutant phenotypes, even under conditions where very modest levels of Dlp rescues completely. These findings show that Dlp functions cannot be readily provided by another unrelated HSPG, and they argue that the conserved sequence elements of the Dlp core protein are critical for these unique functions (Rawson, 2005).

Effects of HSPGs on axon pathfinding have been considered principally in the context of classical axon-guidance pathways, Slit-Robo signaling in particular. Although there is genetic evidence from mouse, Drosophila, and C. elegans that HSPGs affect Slit-Robo signaling, there are many other possibilities. HSPGs have been extensively characterized as molecules that affect both morphogen signaling and distributions. Recent studies demonstrating that the classical morphogens Wnt, Hh, and BMP also play a bona fide role in axon guidance suggest the possibility that HSPGs govern axon guidance by affecting morphogen function or distributions during this process (Rawson, 2005).

Heparan sulfate is ubiquitous at the cell surface, where it is expressed predominantly as covalent modifications of proteoglycans of either the transmembrane syndecan family or the glycosylphosphatidylinositol (GPI)-anchored glypican family. Heparan sulfate has been proposed to function as a "coreceptor" for a number of "heparin-binding" growth factors. Although little is known about functional differences between individual members of the glypican gene family, mutations in both the Drosophila gene dally and the human gene for glypican-3 strongly suggest that at least some glypicans do function in cellular growth control and morphogenesis. In particular, deletion of the human glypican-3 gene is responsible for Simpson-Golabi-Behmel syndrome, and its associated pre- and postnatal tissue overgrowth, increased risk of embryonal tumors during early childhood, and numerous visceral and skeletal anomalies. An mRNA has been identified and characterized that encodes a 572-amino-acid member of the glypican gene family (glypican-5) that is most related (50% amino acid similarity, 39% identity) to glypican-3. Glypican-5 mRNA is detected as a 3.9- and 4.4-kb transcript in adult and neonatal mouse brain total RNA, and in situ hybridization results localize transcript primarily to restricted regions of the developing central nervous system, limb, and kidney in patterns consistent with a role in the control of cell growth or differentiation. Interestingly, glypican-5 localizes to 13q31-32 of the human genome, deletions of which are associated with human 13q- syndrome, a developmental disorder with a pattern of defects that shows significant overlap with the pattern of glypican-5 expression (Saunders, 1997).

The glypicans are a family of glycosylphosphatidylinositol (GPI)-anchored proteoglycans that, by virtue of their cell-surface localization and possession of heparan sulfate chains, may regulate the responses of cells to numerous heparin-binding growth factors, cell adhesion molecules, and extracellular matrix components. Mutations in one glypican cause a syndrome of human birth defects, suggesting important roles for these proteoglycans in development. Glypican-1, the first-discovered member of this family, was originally found in cultured fibroblasts, and later shown to be a major proteoglycan of the mature and developing brain. The pattern of glypican-1 mRNA and protein expression was examined in the developing rodent, concentrating on late embryonic and early postnatal stages. High levels of glypican-1 expression are found throughout the brain and skeletal system. In the brain, glypican-1 mRNA is widely, and sometimes only transiently, expressed by zones of neurons and neuroepithelia. Glypican-1 protein localizes strongly to axons and, in the adult, to synaptic terminal fields as well. In the developing skeletal system, glypican-1 is found in the periosteum and bony trabeculae in a pattern consistent with expression by osteoblasts, as well as in the bone marrow. Glypican-1 is also observed in skeletal and smooth muscle, epidermis, and in the developing tubules and glomeruli of the kidney. Little or no expression is observed in the developing heart, lung, liver, dermis, or vascular endothelium at the stages examined. The tissue-, cell type-, and in some cases stage-specific expression of glypican-1 revealed in this study are likely to provide insight into the functions of this proteoglycan in development (Litwack, 1998).

A study was carried out of the expression patterns and subcellular localization in nervous tissue of glypican, a major glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan that is predominantly synthesized by neurons, and of biglycan, a small, leucine-rich chondroitin sulfate proteoglycan. By laser scanning confocal microscopy of rat central nervous tissue and C6 glioma cells, it was found that a significant portion of the glypican and biglycan immunoreactivity colocalizes with nuclear staining by propidium iodide and is also seen in isolated nuclei. In certain regions, staining is selective, insofar as glypican and biglycan immunoreactivity in the nucleus is seen predominantly in a subpopulation of large spinal cord neurons. The amino acid sequences of both proteoglycans contain potential nuclear localization signals; these were demonstrated to be functional based on their ability to target beta-galactosidase fusion proteins to the nuclei of transfected 293 cells. Nuclear localization of glypican beta-galactosidase or Fc fusion proteins in transfected 293 cells and C6 glioma cells is greatly reduced or abolished after mutation of the basic amino acids or deletion of the sequence containing the nuclear localization signal, and no nuclear staining is seen in the case of heparan sulfate and chondroitin sulfate proteoglycans that do not possess a nuclear localization signal, such as syndecan-3 or decorin (which is closely related in structure to biglycan). Transfection of COS-1 cells with an epitope-tagged glypican cDNA demonstrates transport of the full-length proteoglycan to the nucleus, and there are also dynamic changes in the pattern of glypican immunoreactivity in the nucleus of C6 cells both during cell division and correlated with different phases of the cell cycle. Therefore these data suggest that in certain cells and central nervous system regions, glypican and biglycan may be involved in the regulation of cell division and survival by directly participating in nuclear processes (Liang, 1997).

Cell-surface proteoglycans have been implicated in cell responses to growth factors, extracellular matrix, and cell adhesion molecules. M12, one of the most abundant membrane-associated proteoglycans in the adult rat brain, is an approximately 65 kDa glycosylphosphatidylinositol-linked protein that bears heparan sulfate chains. To assess its identity, M12 was purified and internal peptide sequences obtained. Comparison of the results with the protein sequence predicted by a cDNA cloned from PC12 cells indicates that M12 is rat glypican, a proteoglycan first cloned from human fibroblasts. In addition, antibodies raised against a rat glypican fusion protein specifically detect the 65 kDa brain proteoglycan core protein, both by immunoprecipitation and by Western blotting. Northern blot analysis using a rat glypican probe also detects glypican message in the adult, as well as the developing rat brain. In situ hybridization with glypican RNA probes shows that glypican is expressed in a subset of structures in the adult rat nervous system. These include the hippocampus, dorsal thalamus, amygdala, cerebral cortex, piriform cortex, olfactory tubercle, several cranial nerve nuclei, the ventral horn of the spinal cord, and the dorsal root ganglia. Several other brain regions exhibit little or no hybridization over background. In most cases where glypican hybridization is observed, the signal can be localized specifically to the cell bodies of identifiable neurons, for example, spinal motoneurons and hippocampal pyramidal cells. In the cerebral cortex, glypican hybridization is found in layers 2/3, 5, and 6, but is missing from 1 and 4. The data suggest that glypican is expressed primarily by subpopulations of projection neurons in the adult rat nervous system (Litwack, 1994).

Heparan sulfate proteoglycans (HSPGs) are found on the surface of all adherent cells and participate in the binding of growth factors, extracellular matrix glycoproteins, cell adhesion molecules, and proteases and antiproteases. Reported here are the cloning and pattern of expression of cerebroglycan, a glycosylphosphatidylinositol (GPI)-anchored HSPG that is found in the developing rat brain. The cerebroglycan core protein has a predicted molecular mass of 58.6 kD and five potential heparan sulfate attachment sites. Together with glypican, it defines a family of integral membrane HSPGs characterized by GPI linkage and conserved structural motifs, including a pattern of 14 cysteine residues that is absolutely conserved. Unlike other known integral membrane HSPGs, including glypican and members of the syndecan family of transmembrane proteoglycans, cerebroglycan is expressed in only one tissue: the nervous system. In situ hybridization experiments at several developmental stages strongly suggest that cerebroglycan message is widely and transiently expressed by immature neurons, appearing around the time of final mitosis and disappearing after cell migration and axon outgrowth have been completed. These results suggest that cerebroglycan may fulfill a function related to the motile behaviors of developing neurons (Stipp, 1994).

The expression of cell surface heparan sulfate proteoglycans in rat heart was investigated by Northern blot analysis with specific cDNA probes. In adult heart mRNAs for both syndecan-3 and glypican are abundantly expressed. Lower levels of syndecan-2 mRNA and very low levels of syndecan-1 mRNA are also detected. Analysis of RNA isolated from hearts of rats of various ages reveals that the levels of both syndecan-3 and glypican mRNA increase dramatically at birth, and continued to be expressed at high levels in adult animals. To determine which of these proteoglycans is expressed in cardiomyocytes, primary cultures of cardiomyocytes and nonmyocytes isolated from neonatal rat hearts were analyzed for proteoglycan expression. Glypican mRNA is localized almost exclusively to cardiomyocytes. Syndecan-3 mRNA is not detected in myocytes, but is detected in the nonmyocyte cells. Biochemical characterization of cardiomyocyte glypican reveals that it is a phosphatidylinositol-anchored heparan sulfate proteoglycan. Results of immunofluorescent staining of rat hearts with anti-glypican antibodies are consistent with the Northern blot data, and localize glypican to the lateral regions of myocyte plasma membrane that contact the basement membrane, as well as sites of myocyte adhesion junctions. At the latter site glypican colocalizes with vinculin. Visualization of basic fibroblast growth factor binding sites by means of a tissue slice overlay assay also reveals colocalization with glypican. These results demonstrate developmental and cell-type-specific expression of membrane heparan sulfate proteoglycans in the heart. They also show that glypican is a major heparan sulfate proteoglycan expressed on the cardiomyocyte plasma membrane (Asundi, 1997).

An avian cDNA homolog of human and rat glypicans has been cloned from a stage 17 chicken heart cDNA library and used to analyze the distribution of this proteoglycan during development by Northern analysis and whole mount in situ hybridization. At stages 7-12, strong signals are detected in the cephalic region of the neural folds, rostral portion of paraxial mesoderm, and newly formed epithelial somites. At stages 20-25, strong expression is observed in the mantle zone of the telencephalon, the apical epidermal ridge and proximal region of developing limb. Transcripts also are found in the truncus arteriosus and arteriovenous-canal region of the heart, but not in the myocardium. This distribution pattern suggests that the avian glypican may be involved in the morphogenesis of limb, somite, heart, and brain. The expression of glypican also overlaps FGFs in limb bud, FGF receptors in heart and somite, and NGF receptors in forebrain. The affinity of heparan sulfate proteoglycans for growth factors and the distribution of the avian glypican are consistent with a role for this molecule in growth factor-mediated signals (Niu, 1996).

Transcripts for avian glypican are found in endocardial cushions, limb buds, somites and forebrain of early chick embryos. Since avian glypican is not well characterized, the cellular localization, regulation of expression, and possible function during cardiac development have been studied. A polyclonal antibody was raised against a 20-amino acid peptide corresponding to an antigenic sequence within avian glypican core protein. The antibody recognizes the expressed core protein in bacterial lysates and the endogenous HSPG in the proteoglycan fraction from chick forebrain. Immunolocalization studies indicate that the core protein is associated with cell membranes. The level of mRNA for avian glypican in MEQC (myc embryonic quail cardiomyocytes) grown in medium containing 10% fetal calf serum was compared to the message levels in cells grown without serum for 3 days. By Northern analysis, glypican transcripts increase markedly after serum starvation. Up-regulation of glypican transcripts by serum withdrawal is partially prevented by addition of TGFbeta-1 and bFGF, suggesting that these growth factors may regulate its expression. MEQC cells deprived of serum migrate into clumps that can be blocked by an antisense OND (oligodeoxynucleotide) to the mRNA encoding the avian glypican. The same antisense OND inhibits the migration of endothelial cells from chick tubular heart explants over the surface of collagen gels. These results indicate that avian glypican may play a role in cell migration during development of endocardial cushions (Niu, 1998).

Several processes that occur in the luminal compartments of the tissues are modulated by heparin-like polysaccharides. To identify proteins responsible for the expression of heparan sulfate at the apex of polarized cells, the polarity of the expression of the cell surface heparan sulfate proteoglycans was examined in CaCo-2 cells. Domain-specific biotinylation of the apical and basolateral membranes of these cells identifies glypican, a GPI-linked heparan sulfate proteoglycan, as the major source of apical heparan sulfate. Yet, most of this is expressed at the basolateral surface, an unexpected finding for a glypiated protein. Metabolic labeling and chase experiments indicate that sorting mechanisms, rather than differential turnover, account for this bipolar expression of glypican. Chlorate treatment does not affect the polarity of the expression of glypican in CaCo-2 cells, and transfectant MDCK cells express wild-type glypican and a syndecan-4/glypican chimera also in an essentially unpolarized fashion. Yet, complete removal of the heparan sulfate glycanation sites from the glypican core protein result in the nearly exclusive apical targeting of glypican in the transfectants, whereas two- and one-chain mutant forms have intermediate distributions. These results indicate that glypican accounts for the expression of apical heparan sulfate, but that glycanation of the core protein antagonizes the activity of the apical sorting signal conveyed by the GPI anchor of this proteoglycan. A possible implication of these findings is that heparan sulfate glycanation may be a determinant of the subcellular expression of glypican. Inverse glycanation-apical sorting relationships in glypican may ensure near constant deliveries of HS to the apical compartment, or, alternatively, "active" GPI-mediated entry of heparan sulfate into apical membrane compartments may require the overriding of this antagonizing effect of the heparan sulfate chains (Mertens, 1996).

OCI-5, the rat homolog of human glypican 3 (GPC3), is believed to be involved in morphogenesis and growth control during development. The finding that GPC3 is mutated in patients with the Simpson-Golabi-Behmel overgrowth syndrome is consistent with this idea. In this report, using RNA in situ hybridization, expression of OCI-5 in the developing intestine is shown to be present in both endoderm- and mesenchyme-derived cells in a phased manner related to age and proximal/distal position. To investigate the mechanism of its regulation during intestinal development, OCI-5 expression was studied in the primitive rat intestinal epithelial cell line IEC-18. The expression of the OCI-5 transcript is increased in IEC-18 cells at confluence, in low calcium media, and during spheroid culture, all conditions which result in the cells acquiring a more rounded cell shape. In contrast, cytoskeletal disruption with colchicine causes cells to flatten and spread and abolishes both the confluence- and the low calcium-dependent induction of OCI-5. Treatment with vanadate, a phosphatase inhibitor, causes cells to acquire a spindle-shaped morphology and prevents OCI-5 induction in all situations. Nuclear run-on analysis demonstrates that the rate of OCI-5 transcription is increased at confluence, in low calcium media, and during spheroid culture of IEC-18, and decreased by treatment of cells with colchicine. Together, these data suggest that OCI-5 expression is regulated in IEC-18 by cell shape. The pattern of expression of OCI-5 in the developing intestine is consistent with it playing a role in epithelial-mesenchymal interactions during intestinal morphogenesis, when cell shape changes are likely to occur (Li, 1997).

Glypican is a glycosylphosphatidylinositol (GPI)-anchored heparan sulfate proteoglycan (HSPG) releasable by phosphatidylinositol-specific phospholipase C (PI-PLC) from the surface of differentiated skeletal muscle cells. The processing, location and amount of this HSPG in skeletal muscle cells has been evaluated during differentiation. Immunoprecipitation of incubation medium obtained from differentiated cells incubated with [35S]sulfate by specific antibodies against glypican isolated from Schwann cells demonstrates that the antisera precipitates an intact HSPG. Immunoblot analysis of the proteins released by PI-PLC after heparitinase treatment reveals the presence of a main band of 64 and a faint band of 62 kDa, whereas the sizes of the core proteins for glypican present in the incubation media are 62 and 59 kDa. Pulse-chase experiments indicates that glypican present in the membrane is spontaneously released into the culture medium with a t1/2 of 12 h. The level of expression of glypican was analyzed during in vitro differentiation. The specific amount of the PI-PLC releasable HSPG increases about fourfold during cell differentiation. No changes are detected in the level of the mRNA for glypican. Indirect analysis reveals that in myotubes glypican is present on the cell surface as well as associated with the extracellular matrix (ECM). These results indicate that glypican is present, at least, in two different compartments on the surface of skeletal muscle cells (Brandan, 1996).

Skeletal muscle has the remarkable capacity to regenerate new muscle fibers in the event of injury or disease. This capacity lies in the satellite cells, which are myogenic stem cells residing in adult muscle. While the signals that activate satellite cells to divide in vivo are not fully understood, satellite cells grown in culture respond to the mitogenic action of fibroblast growth factor (FGF). Satellite cells from the dystrophic mdx mouse are more sensitive to FGF in culture than satellite cells from normal mice. The basis for this heightened sensitivity of mdx satellite cells to FGF was investigated by measuring the number and affinity of protein and heparan sulphate proteoglycan (HSPG) receptors for FGF. HSPG receptors are elevated over four-fold in the mdx cells, as compared with cells from normal animals. This observation was supported by measuring the synthesis of heparan sulphate (HS) and chondroitin sulphate (CS) by satellite cells in culture. Mdx satellite cells synthesized approximately ten times more of these sulphated glycosaminoglycans (GAGs) than did normal cells. For muscle fibroblasts, however, no significant differences were found between normal and mdx cells in the number or affinity of protein or HSPG receptors, or in the amount of sulphated GAGs synthesized. It is proposed that the increase in FGF HSPG receptors is the basis for the heightened response of mdx satellite cells to FGF in culture and may reflect exposure of the cells to growth factors in the degenerating mdx muscle (Crisona, 1998).

The glypicans compose a family of glycosylphosphatidylinositol-anchored heparan sulfate proteoglycans. Mutations in dally, a gene encoding a Drosophila glypican, and in GPC3, the gene for human glypican-3, implicate glypicans in the control of cell growth and division. So far, five members of the glypican family have been identified in vertebrates. By sequencing expressed sequence tag clones and products of rapid amplifications of cDNA ends, a sixth member of the glypican family has been identified. The glypican-6 mRNA encodes a protein of 555 amino acids that is most homologous to glypican-4 (63% identity). Expression of this protein in Namalwa cells shows a core protein of approximately 60 kDa that is substituted with heparan sulfate only. GPC6, the gene encoding human glypican-6, contains nine exons. Like GPC5, the gene encoding glypican-5, GPC6 maps to chromosome 13q32. Clustering of the GPC5/GPC6 genes on chromosome 13q32 is strongly reminiscent of the clustering of the GPC3/GPC4 genes on chromosome Xq26 and suggests GPCs arose from a series of gene and genome duplications. Based on similarities in sequence and gene organization, glypican-1, glypican-2, glypican-4, and glypican-6 appear to define a subfamily of glypicans, differing from the subfamily that thus far comprises glypican-3 and glypican-5. Northern blottings indicate that glypican-6 mRNA is widespread, with prominent expressions in human fetal kidney and adult ovary. In situ hybridization studies localize glypican-6 to mesenchymal tissues in the developing mouse embryo. High expressions occur in smooth muscle cells lining the aorta and other major blood vessels and in mesenchymal cells of the intestine, kidney, lung, tooth, and gonad. Growth factor signaling in these tissues might be regulated, in part, by the presence of glypican-6 on the cell surface (Veugelers, 1999).

Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked condition characterized by pre- and postnatal overgrowth with visceral and skeletal anomalies. To identify the causative gene, breakpoints in two female patients with X;autosome translocations were identified. The breakpoints occur near the 5' and 3' ends of a gene, GPC3, that spans more than 500 kilobases in Xq26; in three families, different microdeletions encompassing exons cosegregate with SGBS. GPC3 encodes a putative extracellular proteoglycan, glypican 3, that is inferred to play an important role in growth control in embryonic mesodermal tissues where it is selectively expressed. Initial western- and ligand-blotting experiments suggest that glypican 3 forms a complex with insulin-like growth factor 2 (IGF2), and might thereby modulate IGF2 action (Pilia, 1996).