Research Article

Identification of Hedgehog Pathway Components by RNAi in Drosophila Cultured Cells

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Science  28 Mar 2003:
Vol. 299, Issue 5615, pp. 2039-2045
DOI: 10.1126/science.1081403


Classical genetic screens can be limited by the selectivity of mutational targeting, the complexities of anatomically based phenotypic analysis, or difficulties in subsequent gene identification. Focusing on signaling response to the secreted morphogen Hedgehog (Hh), we used RNA interference (RNAi) and a quantitative cultured cell assay to systematically screen functional roles of all kinases and phosphatases, and subsequently 43% of predicted Drosophila genes. Two gene products reported to function in Wingless (Wg) signaling were identified as Hh pathway components: a cell surface protein (Dally-like protein) required for Hh signal reception, and casein kinase 1α, a candidate tumor suppressor that regulates basal activities of both Hh and Wg pathways. This type of cultured cell–based functional genomics approach may be useful in the systematic analysis of other biological processes.

The secreted protein signal Hedgehog (Hh) elicits cellular proliferation and differentiation responses during normal embryonic development, and inappropriate pathway activation can contribute to tumorigenesis. In Drosophila, this pathway is regulated by a series of repressive interactions between protein components that ultimately result in gene activation mediated by the transcription factor Cubitus interruptus (Ci) (Fig. 1A ) (1, 2). Ci is regulated by a cytoplasmic complex consisting of the kinesin-like protein Costal 2 (Cos2), the serine-threonine kinase Fused (Fu), and Suppressor of fused [Su(fu)], a protein that lacks known functional motifs. This complex prevents activation and nuclear localization of Ci and stimulates its proteolytic processing to a truncated form (Ci75) that represses gene targets. The activity of this complex is suppressed by Smoothened (Smo), a seven-transmembrane protein, and Smo activity in turn is suppressed by catalytic action of the transporter-like protein Patched (Ptc). Hh protein releases these sequential repressive interactions by binding and inactivating Ptc, thus permitting Smo-mediated suppression of the regulatory complex and releasing Ci for activation of target genes.

Figure 1

RNAi in Drosophila cultured cell assays for Hh and Wg signaling. (A) Schematic view of Hh and Wg signaling pathways. For clarity, only components tested by RNAi in (B) and (D) are shown. Green or red text denotes predominantly positive or negative effects of a particular component in the pathway response, respectively. (B) Effects of RNAi of known pathway components on Hh response in cl-8 cells (10). Su(fu) RNAi in combination with Fu RNAi reverses the loss of Hh response caused by Fu RNAi alone. yfp, yellow fluorescent protein. (Inset) Western blot analysis of Ptc protein levels afterptc dsRNA treatment. RNAi of ptc in S2 cells, which do not express Ci, results in a decrease in Ptc protein levels and stabilization of Smo protein (42). A Western blot of kinesin heavy chain (Khc) is shown as a control for protein loading. (C) Epistasis analysis of Ci relative to Cos2 in cl-8 cells. RNAi of Ci inhibited Hh induction of reporter activity, whereas RNAi of Cos2 increased basal reporter activity. The epistatic effect exerted by RNAi of Ci on RNAi of Cos2 prevailed, even at levels of cos2 dsRNA 10-fold higher than those ofci dsRNA. (D) Effects on Wg response of RNAi of pathway components in Kc cells. RNAi effects of known Wg pathway components on Wg response were monitored with an assay similar to that described for Hh response using the Wg-responsive reporter Super TopFlash (10). The inset shows the accumulation of Arm in Kc cells after 2 hours of incubation in the presence of Wg (in duplicate).

Classical genetic approaches to the study of embryonic processes such as Hh signaling have been subject to limitations imposed by the selectivity of mutagenesis methods, by the difficulty of identifying mutations whose zygotic phenotypes are cloaked by maternal contributions, and by difficulties in identifying mutated genes. The Hh pathway suffers from the additional complication that its embryonic loss-of-function phenotype is similar to that controlled by the signaling pathway regulated by the secreted protein Wingless (Wg), thus hindering correct assignment of gene function. Like Hh, Wg signaling employs a series of repressive interactions in which receptor activity antagonizes a cytoplasmic complex that in turn causes degradation of Armadillo (Arm), a key component in the activation of the Wg transcriptional response (Fig. 1A). Indeed, certain components of these pathways are shared, including the F-box protein Slimb (Sli) (3) and glycogen synthase kinase 3 (GSK3β or Sgg) (4, 5), each with dual roles in the proteolytic degradation of Arm and in the proteolytic formation of Ci75. In addition, a casein kinase 1 (CK1) activity triggers these proteolytic events (5), although the actual CK1 family member that functions in Ci75 formation remains to be identified.

RNAi in Hh and Wg cultured cell assays.

To identify additional components in Hh signal response while also circumventing the difficulties of classical genetic screens, we used a cultured cell assay (6) and RNA interference (RNAi)–mediated disruption of gene function (7,8) to systematically screen Drosophila genes. This assay is based on transfection of wing imaginal disc–derived cl-8 cells with a control reporter and a Hh-responsive luciferase reporter. Unlike Drosophila S2 cultured cells, simply bathing cl-8 cells in double-stranded RNA (dsRNA) had no effect on Hh pathway response (9). However, transfection of dsRNA together with both reporter constructs affected pathway response in such a way that RNAi targeting of the positive regulatory components Smo, Fu, and Ci inhibited response to the Hh signal (Fig. 1B), and targeting of the negative regulatory components Cos2 and Ptc resulted either in basal activation or enhanced responsiveness to Hh (10,11). Consistent effects on the protein levels of several pathway components, including Smo and Ptc, were also observed in S2 cells after treatment with dsRNA (Fig. 1B, inset). RNAi inDrosophila cultured cells thus provides a functional test for gene products of known or predicted sequence. The Hh signaling assay in cl-8 cells is quantitative and is specific for cellular response, because the addition of exogenous Hh protein eliminates the requirement for functions involved in Hh protein synthesis or distribution.

We also found that multiple dsRNA species could be combined in these transfection experiments, facilitating large-scale screening and tests of gene interactions and epistasis. RNAi of Su(fu) in cl-8 cells, for example, produced little effect on Hh response but reversed the reduction in responsiveness elicited by RNAi of Fu (Fig. 1B), thus mimicking phenotypic suppression of fu mutations by Su(fu) mutations in flies (12). In addition, combined RNAi of Cos2 and Ci yielded a loss of responsiveness (Fig. 1C), indicating that Ci is epistatic to Cos2, in agreement with phenotypic analysis of mutant combinations (13). Ci remained epistatic to Cos2 even when the amount of Cos2 dsRNA was 10 times greater than that of Ci dsRNA (Fig. 1C). This indicates that the RNAi machinery was not overwhelmed by the transfection of excess dsRNA, even at levels 10-fold higher than those routinely used.

To facilitate independent testing of gene function in response to Wg and Hh, we also established a Drosophilacultured cell–based reporter assay for Wg response based on stabilization of Arm in the presence of Wg protein (10,14). In contrast to S2 cells, both embryo-derived Kc cells and cl-8 cells were responsive to Wg (Fig. 1D, inset) (9, 15), but the Kc cells consistently displayed a stronger transcriptional response (9). RNAi of positively acting components Frizzled 1 and 2 receptors (Fz1 and Fz2), Arm, or Pangolin [Pan, the Drosophila T cell–specific transcription factor (Tcf) homolog] caused loss of Wg responsiveness, and RNAi of GSK3β increased the response to Wg (Fig. 1D). Taken together, RNAi of 11 known components of the Hh and Wg pathways produced the effects predicted by classical genetic analyses, indicating that RNAi in these assays can provide a rapid and reliable indication of gene function.

Genomewide RNAi screen for kinases and phosphatases in Hh signaling.

To test this assay system and establish high-throughput methods for dsRNAi synthesis and screening, a library was prepared containing dsRNAs corresponding to all kinases and phosphatases predicted from the completed Drosophila genome sequence (fig. S1A and table S2) (10, 16). The initial screen identified several dsRNA pools that showed either a gain in basal reporter activity in the absence of Hh (Fig. 2, top panel), as expected for components with a predominantly negative regulatory role in the pathway, or a reduction in average fold induction (the ratio of reporter activity in the presence and absence of Hh; Fig. 2, bottom panel), as expected for components with either positive or negative pathway-regulatory roles (10). When pooled RNAs and individual dsRNAs were rescreened, three dsRNAs emerged that had reproducible effects on pathway activity. These dsRNAs targeted the putative serine-threonine kinase Fu and cAMP-dependent kinase 1 (PKA-C1), two known regulators of Hh signaling (1,2). CK1α, not previously implicated in Hh signaling, was also identified in the screen, because it was required to maintain the regulated state of the pathway. RNAi of CK1α elevated basal reporter activity in the absence of Hh (Fig. 2, left in inset) and expanded the segmental domain of Wg expression in embryos injected with CK1α dsRNA (Fig. 2, right in inset) (10).

Figure 2

An RNAi screen of Drosophila kinases and phosphatases in Hh signaling. The effects of dsRNA corresponding to all known and predicted kinases and phosphatases in theDrosophila genome are plotted as basal luciferase activity (top) and fold induction by Hh (bottom). STDEV, standard deviation. Data points represent averages of three independent Hh signaling assays using pools of three dsRNAs, with each dsRNA targeting a separate transcript. Cutoffs for selection of dsRNA pools for identification of the gene of interest are indicated by the horizontal dotted lines (10). Data points in which a single gene could be identified are colored as indicated in the figure and labeled with the gene name. Controls included in the screen were dsRNA targeting Smo, Cos2, and a genomic noncoding sequence (700 base pairs in length). Fu and PKA-C1, kinases that are known to affect the pathway, were identified in this screen. In addition, a dsRNA pool targeting CK1α and CK1ɛ induced basal reporter activity (red dot in upper panel). (Inset) Examination of individual dsRNAs in this pool revealed that a decrease in the expression of CT6528, a transcript encoding CK1α, resulted in activation of the Hh pathway (29). Injection of this ck1α dsRNA into preblastodermDrosophila embryos resulted in an expansion of Wg expression domains at the extended germ band stage, consistent with a role in regulating basal Hh pathway activity.

Systematic screen of Drosophila Gene Collection Release 1.

The screen was extended with a dsRNA library based on theDrosophila Gene Collection Release 1 (DGCr1), which contains approximately 43% of the predicted genes in the Drosophilagenome (Fig. 3A and fig. S1B) (10). When pooled RNAs and individual dsRNAs were rescreened, 20 dsRNAs with reproducible effects on apparent pathway activity were identified, and these effects were confirmed with dsRNA from distinct sets of gene-specific primers (10). Nineteen of these genes [not includingthread (Fig. 3)] were classified into three groups (Fig. 3B). Class I genes encoding proteins with known roles in Hh response include Smo,Cos2, PKA, and combgap, which encodes a zinc finger protein that regulates Ci expression (17). Our screen identified all genes within DGCr1 that are known to regulate expression of Ptc, the Hh pathway target on which our reporter is based. Our screen thus is highly reliable and is not affected by pooling of dsRNAs.

Figure 3

RNAi screen of 43% of predictedDrosophila genes for components of the Hh pathway. (A) A screen of the DGCr1 dsRNA library. The upper panel shows relative basal luciferase activity and the lower panel shows relative fold induction, as described in the Fig. 2 legend. (Inset) Dlp was identified as a positive component in pathway activation in a dsRNA pool that also included dsRNA targeting Mbt and Mus210 transcripts. Injection of dlp dsRNA into Drosophila embryos resulted in decreased Wg expression, consistent with a reduction in response to Hh signaling. (B) Genes and gene products identified in the screen are grouped into three different classes: known pathway components and regulators (class I), components of large multifunctional protein complexes that are not likely to be involved exclusively in Hh signaling (class II), and genes with previously unrecognized roles in the Hh pathway (class III). The apparent effect of RNAi of thread, an antiapoptosis gene, is unreliable because of a dramatic reduction in control reporter activity, indicative of cell death.

The 11 genes in class II encode individual components of complexes with broad cellular roles that are not restricted to Hh signaling. This group includes nine genes encoding proteins that likely affect ribosome function, which, when targeted by RNAi, reduced reporter activity, probably because of loss of luciferase expression in response to Hh. Two additional proteins, DeltaCOP and BetaCOP, are involved in retrograde vesicle trafficking from the Golgi complex to the endoplasmic reticulum. RNAi targeting of these proteins increased basal reporter activity, but the effects were weaker and more variable than that produced by RNAi of Cos2. Other components of the COP1 complex also produced similar variable effects when targeted (9). How these proteins function in a pathway-regulatory trafficking event remains to be determined.

Class III comprises four genes with previously unrecognized roles in the Hh pathway, two of which have been characterized in other contexts. Dally-like protein (Dlp) is a member of the glypican family of heparan sulfate proteoglycans (HSPGs) and is required for Hh responsiveness in cl-8 cell–based assay and maintenance of normal Wg expression in embryos (Fig. 3A, inset). CK1α was also identified in the kinase and phosphatase library screen as a negative regulator of Hh pathway activity. A potential role for HSPGs in Hh signaling is suggested by binding of the Hh signaling domain to heparin (18) and by the requirement in Hh signaling for tout velu (ttv), which encodes a heparan sulfate polymerase that is important in synthesis of the glycosaminoglycan (GAG) chains of HSPGs (19). To characterize the requirement for Dlp in Hh signal response, we examined the roles all four known or predictedDrosophila HSPGs, including a second glypican, Dally, Syndecan (Sdc), and Perlecan (Pcan). Only RNAi of Dlp affected Hh signal response (Fig. 4A) (20), and the effect was comparable to RNAi of Smo. Expression of Dlp also increased the response to Hh, comparable to the increase caused by expression of Ci (9).

Figure 4

A role for the cell surface HSPG Dlp in Hh response. (A) A specific role of Dlp in Hh signaling. Of four characterized Drosophila HSPGs (Sdc, Pcan, Dally, and Dlp), only RNAi of Dlp resulted in loss of pathway responsiveness (left panel). Similar effects were observed with dsRNA corresponding to the Dlp 5′ UTR. YFP and Smo dsRNAs were negative and positive controls. Conversely, expression of Dlp but not of Dally or YFP resulted in increased responsiveness to Hh (10) (right panel). (B) Dlp is a cell surface–localized, heparan sulfate–modified protein. (Top) A diagram of the Dlp precursor indicating the 14 conserved cysteines in the globular domain, several putative GAG attachment sites, a hydrophobic GPI attachment sequence, and the region recognized by the monoclonal antibody to Dlp (10). (Left) Immunofluorescence analysis of nonpermeabilized or detergent-permeabilized cl-8 cells expressing Dlp and green fluorescent protein (GFP) (10). Epitopes for both Dlp and GFP were accessible in permeabilized cells, whereas only Dlp protein was detected in nonpermeabilized cells. (Right) Analysis of the GAG modifications of Dlp in S2 cells. Control Arm protein (left lane) or Dlp (right lanes) was immunoprecipitated and treated before Western blot analysis with heparinase III (HepIII), chondroitinase ABC (ChABC), and the reducing agent dithiothreitol (DTT) as indicated (10). The vertical line indicates migration of the heparinase III–treated juxtamembrane subunit of Dlp in DTT-treated samples. Heparinase III–treated Dlp migrates substantially more slowly in the absence of DTT (arrow), suggesting that Dlp undergoes proteolytic processing to yield a disulfide-linked product. Background immunoglobulin G bands are indicated by asterisks. (C) Mapping of Dlp activity within the Hh pathway. Pathway activation resulting from RNAi of Ptc or Cos2 was not affected by RNAi of Dlp, placing Dlp function upstream of both components. (D) Dlp is not required when Hh is expressed within responding cells. Hh responsiveness in cl-8 cells was measured when Hh was supplied either in conditioned medium or by transfection of a construct for the expression of full-length Hh. Hh response was not affected by loss of Dlp in Hh-transfected cells, demonstrating that delivery of membrane-tethered Hh directly into responding cells circumvents the requirement for Dlp.

Dlp, a cell surface HSPG, is required for reception of the Hh signal.

The glypican Dlp has 14 conserved cysteines that are predicted to form an N-terminal globular domain and several putative GAG modification sites next to a consensus glycosylphosphatidylinositol (GPI) attachment sequence. A monoclonal antibody directed against the juxtamembrane portion of the protein (10) revealed Dlp at the surface of cl-8 cells (Fig. 4B). The sensitivity of Dlp to treatment with heparinase III, but not chondroitinase ABC, indicates modification by heparan sulfate (Fig. 4B). Treatment with heparinase III reduced the broad electrophoretic mobility of Dlp to a more compact band that migrates faster than the predicted molecular weight of the mature protein (∼78 kD), suggesting that, like other glypicans, maturation of Dlp may involve proteolytic processing (21,22).

The heparan sulfate modification of Dlp could involve activity of the Ttv heparan sulfate polymerase, whose protein targets are unknown. Loss of Ttv function, however, primarily affects movement of the Hh protein signal through target tissues (18, 23), whereas our assays show that Dlp plays a cell-autonomous role in response to the Hh signal. Although it remains possible that Dlp could also have a role in extracellular Hh transport, perhaps alongside of other HSPGs, the cell-autonomous function of Dlp in signal response is distinct from that of Ttv targets in Hh transport. Consistent with an extracellular transport role for the GAG chains elaborated by Ttv, we failed to observe an effect on Hh response when RNAi of Ttv or its relatives Sotv (Dext2) and Botv (Dext3) were applied, either singly or in combinations (9). Similarly, although RNAi of Dally or Dlp produced segment polarity defects in embryos, there was no effect in the Kc cell Wg assay (9), suggesting that the previously reported roles of these proteins in Wg signaling (24–26) may be largely restricted to non–cell-autonomous extracellular effects on Wg transport.

To further investigate the mechanism of Dlp action on Hh signal response, we examined RNAi of Dlp in combination with RNAi of other pathway components. Dlp function was not required for the increased basal pathway activity produced by loss of Cos2 (Fig. 4C). Moreover, the requirement for Dlp in Hh response was suppressed by RNAi of Ptc, suggesting that Dlp may act upstream or at the level of the Ptc receptor (Fig. 4C). Given its localization, Dlp could function to concentrate Hh on the surface of responding cells, perhaps aiding in the delivery of Hh to Ptc. Preliminary biochemical analysis shows that a soluble fusion protein containing the extracellular domain of Dlp can associate with a tagged form of Hh (9). Also consistent with such a role, RNAi of Dlp did not block signal response when Hh was expressed in responding cells (Fig. 4D). In this case, mature Hh is anchored to the plasma membrane via its lipid modifications (27, 28). Circumvention of the requirement for Dlp by expression of a tethered Hh signal in responding cells suggests that Dlp may function normally to concentrate Hh released from producing cells.

A dual role for CK1α in regulation of Hh and Wg pathway activity.

In contrast to the positive role of Dlp in Hh response, our initial observations from the kinase-phosphatase and DGCr1 library screens suggested that CK1α may control basal pathway activity (Figs. 2 and 3). A role for kinases other than PKA in regulation of Ci processing was proposed (6), and CK1 sites essential for processing Ci to Ci75 were reported (5). However, no CK1 gene required for Hh pathway regulation has been identified, although the gene encoding CK1ɛ was proposed on the basis of the effects of CK1ɛ overexpression in imaginal discs (5). To investigate the role of CK1α in Hh pathway regulation, RNAi of CK1α and CK1ɛ in cl-8 cells were compared. Three dsRNAs containing distinct portions of the CK1α open reading frame (ORF) increased basal reporter activity (29). DsRNA corresponding to either the catalytic region or the extracatalytic region of CK1ɛ had no effect (Fig. 5A). In contrast, overexpression of either CK1α or CK1ɛ suppressed Hh-induced pathway activation (Fig. 5B). These results indicate that both CK1α and CK1ɛ affect basal pathway activity when overexpressed, but that physiological regulation depends primarily on CK1α.

Figure 5

CK1α regulates basal activity of both the Hh and Wg pathways. (A) Specificity of dsRNA targeting CK1α. (Left) Schematic diagram of the different dsRNAs targeting CK1α and its closest homolog CK1ɛ. Highly homologous regions and nucleotides corresponding to the catalytic domains are indicated. CT numbers corresponding to transcripts and lengths of exon sequences targeted by RNAi are noted for dsRNAs included in the kinase/phosphatase library (thin line); only the length of the targeted sequence is noted for dsRNAs derived from a separate synthesis (thick line) (10). (Right) Transfection of three out of four dsRNAs targeting CK1α but neither of the two dsRNAs targeting CK1ɛ results in pathway activation. Three additional dsRNAs targeting CK1ɛ (left panel, thin lines that are not numbered) were included in the screen described in Fig. 2 and had no effect on pathway responsiveness. (B) Expression of CK1α or CK1ɛ suppresses the Hh pathway. CK1α, CK1ɛ, or known pathway components were expressed in cl-8 cells and pathway response was measured. Expression of both CK1α and CK1ɛ blocked pathway response. (C) Mapping of CK1α activity within the Hh pathway. Pathway components were targeted by RNAi individually or in combination with RNAi of CK1α. Only Ci was epistatic to CK1α. RNAi of Sli, an F-box protein that regulates Ci proteolytic processing, in combination with RNAi of CK1α increased both basal and Hh-induced pathway activity more than did either individually. (D) CK1α also acts as a negative regulator of the Wg pathway. RNAi of CK1α resulted in increased basal activity of the Wg pathway, whereas RNAi of Arm resulted in loss of pathway responsiveness. RNAi of Arm was epistatic to RNAi of CK1α, consistent with CK1α action upstream of Arm. Expression of CK1α did not suppress Wg pathway response, and expression of CK1ɛactivated the pathway. (Right) Summary of the effects of RNAi or overexpression (OEX) of CK1α or CK1ɛ in Hh and Wg pathways.

Both CK1α and CK1ɛ have been implicated in Wnt pathway signaling in vertebrates, largely on the basis of overexpression studies (30). In the Wg signaling assay, only overexpression of CK1ɛ elevated basal pathway activity (Fig. 5D, right panel). In contrast, RNAi targeting revealed that only loss of CK1α function increased basal pathway activity (Fig. 5E, left panel), indicating that physiological regulation of basal Wg pathway activity depends on CK1α. The increase in pathway activity produced by RNAi of CK1α was blocked by RNAi of Arm, consistent with the proposed role of CK1α in Arm degradation (31, 32). Similarly, Ci function is required for basal activation of the Hh pathway by RNAi of CK1α, whereas other positively acting components Smo and Fu were not (Fig. 5C, left panel). The increase in basal reporter activity produced by RNAi of CK1α was enhanced by RNAi of Sli, which is thought to be involved in targeting Ci for processing to Ci75 (Fig. 5C, right panel). These results suggest that CK1α acts downstream of Smo and Fu, and at or upstream of Ci. The role of CK1α in maintaining the quiescent state of both Wg and Hh pathways suggests that these pathways might be simultaneously activated upon loss of CK1α activity (Fig. 5D, table).

In addition to Dlp and CK1α, class III components with previously unrecognized roles in Hh signaling includecaupolican, one of a cluster of homeodomain genes with roles in specifying wing vein and sensory organ pattern (33). A closely related gene within the cluster, araucan, also reduced Hh-induced reporter activity when targeted by RNAi (9). Curiously, both of these genes have been characterized as targets rather than mediators of Hh signaling. The remaining gene in class III, CG9211 (CT26314), encodes a putative cell surface protein that shares features with a subfamily of the immunoglobulin (Ig) Superfamily, including fibronectin type III and Ig domain repeats (34).

Implications for human disease.

The identification of signaling pathway components inDrosophila can have implications for human disease. For example, the role of CK1α in regulating basal activity of both Wg and Hh signaling pathways suggests that it could act as a tumor suppressor in colon cancer, basal cell carcinoma, rhabdomyosarcoma, or medulloblastoma. These tumors are associated with inappropriate activity of one or the other pathway, except medulloblastoma, which is associated with the activation of either (2). In the case of Dlp, GPC4 and GPC6 are the most closely related of the six mammalian glypican family members (35). GPC6 maps to 13q32 (36), a human chromosomal locus whose deletion (13q32 syndrome) is associated with defects, including holoprosencephaly (HPE), anogenital malformations, and an absent thumb (37); all of these malformations are consistent with loss of varying degrees of Sonic hedgehog signaling (38,39). If GPC6 levels are limiting in mammalian Hh responsiveness, then loss of GPC6 function may play a role in 13q32 syndrome malformations, possibly alongside other HPE genes in or near this region (40). Finally, mutation of CDO, the mammalian homolog of CG9211, results in a form of HPE (41), consistent with a role for CDO in signaling.

An RNAi-based approach to functional genomics in aDrosophila cultured cell assay has provided a rapid screen that is sufficiently sensitive to detect known maternal and zygotic functions in Hh signaling, as well as the functions of previously unknown pathway components. This screen, together with RNAi-based functional characterization, has allowed identification of CK1α and Dlp in Hh response. In the case of Dlp, the influence of possible effects on extracellular signal transport was avoided, because the assay monitors only responses within target cells. Furthermore, although both of these genes were previously implicated in Wg signaling, the use of a quantitative Hh signaling assay avoided the complexities of phenotypic analysis and elucidated roles in the Hh response. Activity assays for Hh, Wg, and other signaling pathways in a setting amenable to RNAi-based functional genomics approaches should make possible the systematic identification of components within each pathway, thereby providing the basis for a more complete view of the mechanisms of signaling. In addition, a fuller knowledge of common, as well as unique, components in Hh, Wg, and other pathways should lead to a better understanding of the relationships and regulatory cross-talk between pathways.

Supporting Online Material

Materials and Methods

Fig. S1

Tables S1 and S2


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