Identification and Characterization of a Novel Line ofDrosophila Schneider S2 Cells That Respond to Wingless Signaling*

Wingless (Wg) treatment of Drosophilawing disc clone 8 cells leads to Armadillo (Arm) protein elevation, and this effect has been used as the basis of in vitro assays for Wg protein. Previously analyzed stocks of DrosophilaSchneider S2 cells could not respond to added Wg, because they lack the Wg receptor, Dfrizzled-2. However, we found that a line of S2 cells obtained from another source express Dfrizzled-2 and Dfrizzled-1. Thus, we designated this cell line as S2R+ (S2 receptor plus). S2R+ cells respond to addition of extracellular Wg by elevating Arm and DE-cadherin protein levels and by hyperphosphorylating Dsh, just as clone 8 cells do. Moreover, overexpression of Wg in S2R+, but not in S2 cells, induced the same changes in Dsh, Arm, and DE-cadherin proteins as induced in clone 8 cells, indicating that these events are common effects of Wg signaling, which occurs in cells expressing functional Wg receptors. In addition, unphosphorylated Dsh protein in S2 cells was phosphorylated as a consequence of expression of Dfrizzled-2 or mouse Frizzled-6, suggesting that basal structures common to various frizzled family proteins trigger this phosphorylation of Dsh. S2R+ cells are as sensitive to Wg as are clone 8 cells but can grow in simpler medium. Therefore, the S2R+ cell line is likely to prove highly useful for in vitro analyses of Wg signaling.

The Drosophila segment polarity gene wingless (wg, see Refs. 1-4) is a member of the Wnt gene family, which encodes a secreted glycoprotein involved in cell-cell signaling in a number of basic developmental processes in a wide range of animal phyla (5)(6)(7)(8). Genetic screening of Drosophila for mutations in segmental patterning has revealed several genes acting sequentially in the Wg signal transduction pathway (9). These genes are dishevelled (dsh, see Refs. 10 and 11), zeste-white 3 (zw-3, see Refs. 12 and 13), and armadillo (arm, see Refs. 14 -16), and the order in which they act in the pathway has been defined by genetic and molecular studies (17,18). wg and dsh negatively regulate zw-3, which is a down-regulator of arm. Thus, the net effect of Wg signaling is the accumulation of a cytoplasmic pool of Arm protein.
The in vitro assay for Wg (19) using soluble Wg protein and the Drosophila imaginal disc cell line, clone 8 (20), has greatly contributed to biochemical analysis of the Wg signaling pathway (21)(22)(23)(24). In particular, this system led to the identification of Drosophila frizzled 2 (Dfz2) as a receptor for Wg (24). For identification of Dfz2, Bhanot et al. (24) took advantage of the fact that Schneider S2 cells (25) lack Dfz2 protein and thus do not respond to soluble Wg and that transfection of this new member of the frizzled family (26) makes S2 cells sensitive to Wg. However, we found another line of S2 cells that does respond to Wg by elevating the Arm protein levels.
In this study, we characterized this novel S2 cell line biochemically, and we found that our cell line expresses Dfz2 and Dfz1 proteins, which function as Wg receptors. Therefore, we designated this line as S2Rϩ (S2 receptor plus). To identify the biochemical changes commonly induced by Wg signaling, we compared the Wg-induced changes in S2Rϩ and clone 8 cells, both of which express Dfz2 and Dfz1, with those in S2 cells, which express neither. These analyses revealed that Wg signaling induces a common set of biochemical changes as follows: hyperphosphorylation of Dsh, accumulation of Arm, and elevation of DE-cadherin mRNA and protein levels, in S2Rϩ and clone 8 cells, which express functional Wg receptors, but not in S2 cells, which lack them. We propose S2Rϩ as a novel cell line for Wg assays in vitro.

EXPERIMENTAL PROCEDURES
Cell Lines and Cell Culture-The Drosophila wing imaginal disc cell line clone 8 (20) and Drosophila Schneider S2 cells (25) were obtained from Dr. R. Nusse at Stanford University and cultured as described (19,21). Another line of Drosophila Schneider S2 cells (later named as S2Rϩ, which referred to S2 cells with Wg receptors) were obtained from Dr. Tadashi Miake at Mitsubishi-kagaku Institute for Life Sciences and cultured in the same manner as the S2 cells in our laboratory. According to Dr. Miake, the S2Rϩ cells were obtained directly from Dr. Schneider and stored frozen in his laboratory. To use a homogeneous cell population, a clone of S2Rϩ cell was isolated from the original S2Rϩ cells by limited dilution and used throughout these experiments. Wg treatment of clone 8, S2, and S2Rϩ cells was performed by incubating cells with soluble Wg protein secreted in conditioned medium of S2-HS-wg cells (S2 cells expressing Wg under the control of heat-shock promoter, see Ref. 27), details of which were described previously (19).
Expression Constructs and Transfection-Expression plasmids constructed with the pMK33 vector (28), which contains a hygromycinresistant gene, were transfected into clone 8, S2, and S2Rϩ cells using the calcium phosphate method as described (19). pMK-Dfz2 (24), a Dfz2 expression plasmid constructed with pMK33, was obtained from Dr. J. Nathans (Johns Hopkins University). A plasmid expressing Wg (named pMK-Wg) was constructed by isolating a 1.9-kb 1 BamHI-ClaI fragment containing the entire coding region of wg from Wg cDNA (1) and inserting it into the EcoRV site of pMK33. A plasmid-expressing mouse frizzled 6 (Mfz6) protein, named pMK-Mfz6, was made by synthesizing a 2.7-kb double-stranded cDNA fragment containing the entire coding region of Mfz6 (26) by reverse transcriptase-mediated polymerase chain reaction (RT-PCR) and inserting it into the EcoRV site of pMK33. The expression of Dfz2, Mfz6, and wg mRNA in each transfectant was confirmed by Northern blotting with probes specific for each gene (data not shown). The pMK-Wg and the pMK-Mfz6 transfectants used in this study were mixtures of stable clones selected with hygromycin (200 M). On the other hand, two independent stable clones of pMK-Dfz2 trans-fectants were used. Wg overexpression was induced in pMK-Wg transfectants by adding 0.5 mM CuSO 4 as described (19).
RNA Blots-Total RNAs were extracted by the guanidinium/acid phenol method from an overnight collection of Drosophila embryos, clone 8, S2, and S2Rϩ cells, and stable transfectants of those cell lines made with pMK-33 or pMK-Wg. Total RNA (20 g) was separated on 1% formaldehyde-agarose gels and Northern blotted as described (33). The probe for Dfz2 was the 2.6-kb BamHI fragment from pMK-Dfz2. The Dfz1 (34) probe was the 1.0-kb cDNA prepared by RT-PCR, the details of which are described below. The probes for DE-cadherin, Arm, and D-␣-catenin were described previously (22).
Immunohistochemistry-S2Rϩ cells transfected with pMK-Wg or pMK33 were seeded in 4-well chamber slides and treated with CuSO 4 for 24 h. They were fixed, permeabilized, and blocked (22). The samples were processed for indirect immunostaining with the mouse monoclonal anti-Arm antibody N2-7A1 followed by fluorescein isothiocyanate-conjugated rabbit anti-mouse immunoglobulin (Dako, Denmark), with the rat monoclonal anti-DE-cadherin antibody DCAD2 followed by fluorescein isothiocyanate-conjugated goat anti-rat immunoglobulin (Cappel, Durham, NC), or with the affinity purified rabbit anti-Wg IgG (30) followed by fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin (Cappel, Durham, NC). The samples were mounted in Vectashield medium (Vector Laboratories, Burlingame, CA) and observed using a Bio-Rad confocal laser system (MRC 600) attached to a Zeiss Axioscope microscope.

Identification of a Novel S2 Cell Line That Responds to the
Wg Signal-During our experiments to establish S2 cells that express various mouse frizzled proteins, we found that a Schneider S2 cell line (later designated S2Rϩ) obtained from Dr. T. Miake responds to soluble Wg protein produced by S2-HS-wg cells. Therefore, we obtained the S2 cell line that had been used for identification of Wg receptors (Dfz2) from Dr. R. Nusse and compared these two Schneider S2 cell lines biochemically. In addition, we established S2 cells expressing Dfz2 protein (named Dfz2/S2) and mouse Fz6 protein (named Mfz6/ S2) by transfecting S2 cells with the pMK-Dfz2 and pMK-Mfz6 plasmids, respectively. We then compared the effects of soluble Wg treatment on Arm accumulation in clone 8, S2Rϩ, S2, Dfz2/S2, and Mfz6/S2 cells (Fig. 1A). We thereby reproduced the published results (24) that clone 8 and Dfz2/S2, but not S2 cells, respond to Wg. In contrast to S2 cells, however, S2Rϩ cells did elevate Arm (especially the hypophosphorylated form) protein levels in response to soluble Wg protein. In addition, in agreement with the report that Mfz6 does not bind Wg (24), Mfz6/S2 cells did not respond to soluble Wg (Fig. 1A, right panel). Comparison of the Arm protein induction kinetics in clone 8 and S2Rϩ cells demonstrated that S2Rϩ cells showed slower onset of Arm accumulation (Fig. 1B). However, both cell lines exhibited marked elevation of Arm protein levels ( Fig. 1B) which plateaued between 150 and 180 min (data not shown) after Wg treatment. In addition, comparison of the Wg doseresponse curves of Arm accumulation in clone 8 and S2Rϩ cells (Fig. 1C) showed that S2Rϩ and clone 8 cells are equally sensitive to Wg. Fig. 2A shows photomicrographs of confluent and subconfluent cultures of these three cell lines. Each cell line has unique cell morphology. Moreover, S2Rϩ cells do not grow in the medium for clone 8 cells, M3 medium supplemented with 2.5% (v/v) fly extract, 5 g/ml insulin, and 2% (v/v) fetal calf serum. In contrast, clone 8 cells cannot be maintained in Schneider's medium supplemented with 10% fetal calf serum (data not shown). Protein electrophoretic profiles of the lysates from S2Rϩ, S2, and clone 8 cells (Fig. 2B) revealed different protein profiles (S2 and clone 8 cells are rich in 64-and 48-kDa proteins, respectively), further supporting the idea that the S2Rϩ cell line is morphologically and biochemically distinct from the S2 and clone 8 cell lines.
Expression of Dfz1 and Dfz2 mRNAs in S2Rϩ Cells-Studies using Dfz2/S2 cells demonstrated that expression of Dfz2 protein could confer Wg responsiveness on the S2 cell line (see Ref. 24 and Fig. 1A). Furthermore, Dfz1 protein functioned as a Wg receptor in S2 cells (S2 cells expressing Dfz1 protein respond to soluble Wg protein and elevated Arm, see Ref. 24). These results suggested that the Wg responsiveness of the S2Rϩ cell line could be explained by the expression of Dfz2 or Dfz1 genes. Therefore, expression of Dfz2 and Dfz1 mRNA was examined in S2Rϩ cells. Northern blot hybridization with probes for Dfz1 and Dfz2 (Fig. 3, upper panels) and RT-PCR experiments with specific sets of primers for Dfz1 and Dfz2 (Fig. 3, lower panels) showed that both Dfz1 and Dfz2 mRNAs were expressed in S2Rϩ and clone 8 cells but not in S2 cells. Densitometric analyses of Northern blots showed that expression levels of Dfz2 and Dfz1 mRNAs in S2Rϩ cells were about 1/3 and 1/4 of those in clone 8 cells, respectively.

Characterization of Dsh Protein in S2Rϩ, S2, and S2 Cells
Expressing Dfz2 or Mfz6 Proteins-We have reported that stimulation of clone 8 cells with Wg-conditioned medium led to the increased phosphorylation of Dsh on serine/threonine residues (these hyperphosphorylated Dsh species migrate more slowly than unphosphorylated Dsh) and that this hyperphosphorylation of Dsh correlated with Arm accumulation (21). These results led to the hypothesis that this hyperphosphorylated form of Dsh is the active form. By using the two Wg-responsive cell lines, S2Rϩ and Dfz2/S2, we examined whether or not the Wg signaling induces hyperphosphorylation of Dsh and concomitant Arm elevation. Clone 8, S2Rϩ, S2, Dfz2/S2, and Mfz6/S2 cells were treated with the conditioned media from either S2-HS-wg or S2 cells for 3 h, and each cell lysate was subjected to Western blotting with anti-Dsh antibody (Fig. 4). We confirmed that naive clone 8 cells display several bands of phosphorylated and unphosphorylated Dsh and that Wg treatment induces hyperphosphorylation of Dsh (increase of lower mobility bands with concomitant decrease of the highest mobility band) in clone 8 cells. The Dsh image from the longer exposure film (Fig.  4, middle panel) showed that a small amount of modified Dsh was also present in naive S2Rϩ cells. In contrast, naive S2 cells and pMK-33-transfected S2 cells (data not shown) display a single band of unphosphorylated Dsh protein. Consistent with published results (23), expression of Dfz2 in S2 cells (even the basal level of Dfz2 expression in the absence of CuSO 4 ) led to phosphorylation of Dsh, and further induction of Dfz2 expression by CuSO 4 led to predominant modification of Dsh with an additional mobility shift, but this Dfz2 overexpression could not induce Arm accumulation by itself (data not shown). In addition, we found the following changes in Dsh for the first time in this study. First, expression of Mfz6 in S2 cells, like Dfz2, led to phosphorylation of Dsh, suggesting that basal structures common to all frizzled family proteins (such as the seven transmembrane segments) trigger basal phosphorylation of Dsh in some way. Second, Wg treatment had no effect on phosphorylation of Dsh in S2 cells. This is in agreement with the fact that S2 cells can neither receive the Wg signal nor induce Arm accumulation due to the lack of Wg receptors. In contrast, Wg treatment induced hyperphosphorylation of Dsh (Fig. 4, upper and middle panels) and concomitant Arm elevation (Fig. 1A) in S2Rϩ cells, indicating that the presence of functional Wg receptors and the Wg-induced hyperphosphorylation of Dsh are strongly correlated. Third, similar to clone 8 cells, Wg treatment resulted in an increase in the levels of hyperphosphorylated forms of Dsh (the Wg-induced hyperphosphorylation of Dsh) in Dfz2/S2 cells. Therefore, Dfz2/S2 and clone 8 cells showed very similar Dsh phosphorylation profiles before and after Wg treatment (Fig. 4, upper panel). However, Wg treatment did not result in the Wg-induced hyperphosphorylation of Dsh (Fig. 4, upper panel) or Arm elevation (Fig. 1A) in Mfz6/S2 cells. This may be related to the reported inability of Mfz6 protein to bind to Wg (24). Altogether, expression of Dfz2 protein in S2 cells appeared to first promote basal phosphorylation of Dsh, and next, binding of Wg to Dfz2 further induced hyperphosphorylation of Dsh. The results from clone 8, S2Rϩ, and Dfz2/S2 cells indicate that Wg-induced Dsh hyperphosphorylation is a common biochemical readout in cells expressing functional Wg receptors.
Wg Overexpression Induces Arm and DE-cadherin Accumulation in S2Rϩ and Clone 8 Cells, but Not in S2 Cells-Overexpression of the Wg protein in cells is another way to transmit the Wg signal into cells (19). In this case, the Wg proteins synthesized in a cell bind to the Dfz2 or Dfz1 proteins of that cell itself or neighboring cells and thereby stimulate the Wg pathway in an autocrine or paracrine way, respectively. Therefore, the effects of Wg overexpression on protein levels and modification status of Dsh, Arm, DE-cadherin, and D-␣-catenin were examined in clone 8, S2Rϩ, and S2 cells (Fig. 5A). Three cell lines were transfected with the Wg expression plasmid, pMK-Wg, or the negative control vector, pMK-33, and pools of stable transfectants were used. In addition, similar analyses were performed in S2Rϩ cells treated with soluble Wg for 24 h (Fig. 5A, right panels). In pMK-Wg-transfected clone 8 cells, addition of CuSO 4 induced high levels of Wg expression, whereas very little Wg protein expression was detected in the absence of CuSO 4 . In pMK-Wg-transfected S2 and S2Rϩ cells, however, cells not induced with CuSO 4 already expressed significant levels of Wg protein, and CuSO 4 further elevated Wg expression. In clone 8 and S2Rϩ cells, which express Wg receptors, pMK-Wg transfectants showed elevated levels of Arm and DE-cadherin proteins compared with their pMK-33-transfected counterparts. In contrast, Wg overexpression had no effect on Arm and DE-cadherin protein levels in S2 cells, which lack Wg receptors (Fig. 5A). Despite their poor Wg expression, the pMK-Wg-transfected clone 8 cells not induced with CuSO 4 also produced increased levels of Arm and DE-cadherin proteins. This suggests that a very small amount of Wg protein is sufficient to stimulate the Wg pathway in this system. Similar to the results presented in Fig. 4, Wg overexpression led to modification of Dsh protein in S2Rϩ and clone 8 cells but not in S2 cells. D-␣-Catenin protein levels were not affected in any cell type except for the pMK-Wg-transfected S2Rϩ cells. Probably, in pMK-Wg-transfected S2Rϩ cells, very high levels of DEcadherin protein stimulated cadherin adhesion complex formation, and D-␣-catenin proteins were stabilized and thus accumulated in these cadherin adhesion complexes. Treatment of S2Rϩ cells with soluble Wg also elevated both Arm and DEcadherin protein levels (Fig. 5A, right panels).
Next we examined the amount of Wg protein secreted in the culture medium of the S2 and S2Rϩ cells transfected with pMK-Wg (Fig. 5B). Induction with CuSO 4 markedly increased the levels of secreted Wg protein in the culture medium of S2 and S2Rϩ transfectants. However, comparison of the Wg protein levels in cell lysate and in the conditioned medium indicated that less than 10% of the total Wg protein produced was secreted in the induced S2 transfectants. These results suggested that the paracrine mode of activation of the Wg pathway also occurred in S2Rϩ cells expressing Wg and probably in the clone 8 cell counterparts as well. On the other hand, Wg protein present either inside or outside of the S2 cells cannot transmit Wg signals into the S2 transfectants, because the Wg receptor is not present in these cells.
We have reported that Wg signaling caused elevated DEcadherin mRNA levels in clone 8 cells (22). Therefore, the effects of Wg overexpression on DE-cadherin, Arm, and D-␣catenin mRNA levels were examined in S2Rϩ cells. Fig. 5C shows that, as in clone 8 cells, Wg overexpression in S2Rϩ cells resulted in elevated levels of DE-cadherin mRNA but not Arm and D-␣-catenin mRNAs, further supporting the idea that DEcadherin is a target for gene expression regulation by the Wg signaling in a variety of cells (22), whereas Arm and D-␣catenin protein elevations are caused by post-transcriptional regulation.
Immunofluorescence Analysis of Wg, DE-cadherin, and Arm Protein Distribution in S2Rϩ Cells Expressing Wg-To analyze the subcellular localizations of Wg, DE-cadherin, and Arm proteins, S2Rϩ cells transfected with pMK-Wg or pMK33 were stained with antibody against each protein. The speckled staining observed over the entire cell except for the nucleus (Fig. 6A) indicated that the Wg protein is located in structures related to the secretory pathway, such as the endoplasmic reticulum, Golgi, and secretory vesicles. In agreement with the Western blotting results presented in Fig. 5A, Wg expression resulted in markedly elevated DE-cadherin staining at the cell-cell junctions (Fig. 6C) and cytoplasmic Arm staining (Fig. 6E) in S2Rϩ cells. This cytoplasmic localization of Arm is consistent with the notion that Wg/Wnt signaling up-regulates the cytoplasmic Arm/␤-catenin pools (16,19,21). These results showed that S2Rϩ cells exhibit the same biochemical changes, elevation of the cytoplasmic Arm pools, and DE-cadherin accumulation upon Wg overexpression as do clone 8 cells upon Dsh overexpression (22). DISCUSSION In this study, we identified a novel S2 cell line that expresses Dfz2 and Dfz1 proteins and responds to added Wg protein by elevating the levels of Arm protein. Judging from cell morphology, pattern of growth, and nutritional requirements, this cell line is of S2 type, and was thus designated S2Rϩ. Because the origin of S2Rϩ cells is poorly documented, we do not know why these two different S2 cell lines appeared. However, the results of this study give some indication of the variation in the behavior of different S2 cell lines. Therefore, if experimental results using S2 cells differ among laboratories, these differences should be evaluated with regard to whether the S2 cells used in different laboratories have different characteristics. By using two novel Wg-responsive cell lines, S2Rϩ and Dfz2/S2, we tried to evaluate whether or not the effects of Wg signaling that we previously discovered with clone 8 cells, i.e. hyperphosphorylation of Dsh (21) and DE-cadherin induction (22), are generally associated with Wg signaling. As described above, we have shown that they are. In addition, we confirmed that none of these biochemical changes were induced by Wg overexpression or extracellular soluble Wg in S2 cells that lack Wg receptors. Fig. 3 shows that Dfz2 and Dfz1 mRNA were expressed in S2Rϩ and clone 8 cells but not in S2 cells. Following the procedures described by Bhanot et al. (24), we tried to show that Wg protein binds to the surface of S2Rϩ and clone 8 cells but not to that of S2 cells. Whereas the Dfz2/S2 cells showed strong Wg binding, none of these cells showed any significant Wg binding (data not shown), suggesting that relatively little Dfz2 and Dfz1 protein is sufficient to transmit the extracellular Wg signal into cells.
We have shown that expression of Dfz2 or Mfz6 induces phosphorylation of Dsh in S2 cells and that a small proportion of Dsh protein is phosphorylated in S2Rϩ and clone 8 cells.
These results suggest that expression of frizzled family proteins induces the basal phosphorylation of Dsh. In this regard, Willert et al. (23) have reported that casein kinase 2 (CK2), which binds to the PDZ domain of Dsh, is the major kinase responsible for phosphorylation of Dsh upon Dfz2 overexpression in S2 cells. Therefore, CK2 may take part in the basal phosphorylation of Dsh in Dfz2/S2, Mfz6/S2, clone 8 and S2Rϩ cells not stimulated with soluble Wg (Fig. 4). In addition, Axelrod et al. (35) have shown that Dfz1 overexpression led to translocation of Dsh from cytoplasm to plasma membrane. Moreover, Yang-Snyder et al. (36) have reported that overexpression of rat frizzled-1 results in recruitment of Xwnt-8 and XDsh to the plasma membrane in Xenopus embryos. Thus, it is possible that Dfz2 or Mfz6 expression induces translocation of at least a part of Dsh to the plasma membrane in S2 cells and that this Dsh translocation in some way stimulates Dsh phosphorylation by CK2. However, it is not clear whether CK2 also participates in Wg-induced hyperphosphorylation of Dsh or whether other kinase(s) were activated by the binding of Wg to Dfz2 in clone 8, S2Rϩ, and Dfz2/S2 cells and induced the hyperphosphorylation. In view of the reports indicating association (probably indirect) between frizzled family proteins and In addition, the right-most column shows similar analyses in S2Rϩ cells treated with soluble Wg. The S2Rϩ cells were treated for 24 h with conditioned medium from either S2 (Ϫ) or S2-HS-wg (Wg) cells. B, Wg protein secretion in the culture supernatant of S2 and S2Rϩ cells transfected with pMK-Wg. S2 or S2Rϩ cells transfected with pMK-Wg or pMK33 were induced (ϩ) or not (Ϫ) with CuSO 4 for 24 h, and the cell lysates and cell culture supernatants were prepared. One percent of either the cell lysate or the culture supernatant obtained from a confluent cell culture in a T24 flask was loaded on each lane of a 7.2% gel, and the gels were subjected to Western blot analysis with anti-Wg antibody. In S2 cells expressing Wg, Wg protein levels in cells were compared with those in culture supernatants. C, DE-cadherin protein accumulation was accompanied by mRNA elevation in S2Rϩ cells expressing Wg. Total RNA was isolated from uninduced (Ϫ) or induced (ϩ) S2Rϩ cells transfected with pMK-Wg, or from induced (ϩ) S2Rϩ cells transfected with pMK33, and subjected to Northern analysis. Steady-state levels of DE-cadherin, Arm, and D-␣-catenin mRNAs were analyzed with specific DNA probes. The bottom panel is an ethidium bromide staining of the gel showing that the same amount (20 g) of total RNA was loaded in each lane.
Dsh and the binding of Dsh to CK2, it is attractive to speculate that Wg binding induces aggregation of Dfz2 receptors, which, in turn, brings the Dsh-CK2 or other kinase complexes close together, and this aggregation stimulates the Dsh phosphorylation by CK2 or other kinases in these Dsh-kinase complexes. This could explain how Wg induces Dsh hyperphosphorylation in clone 8, S2Rϩ, and Dfz2/S2 cells. However, it is noteworthy that Dfz2 overexpression led to marked phosphorylation of Dsh, but not to elevation of Arm, in S2 cells (23), indicating that phosphorylation of Dsh, at least by Dfz2 overexpression, cannot activate the Wg signaling pathway by itself. Clearly, further detailed experiments are necessary to evaluate the function of Dsh phosphorylation in Wg signaling.
In vitro analyses of Wg signaling with clone 8 cells (19) have made great contributions to the biochemical analysis of the Wg pathway. However, clone 8 cells absolutely require fly extract and insulin for growth (20). Unfortunately, preparation of fly extract is not easy for biochemists who are not working with flies. In contrast, the S2Rϩ cells grow well in Schneider's medium supplemented only with 10% fetal calf serum and are as sensitive as clone 8 cells to the added Wg (Fig. 1). Therefore, we recommend S2Rϩ as a new cell line for analyses of Wg signaling in vitro.