Identification of Hedgehog pathway components by RNAi in ...

Identification of Hedgehog Pathway Components by 

RNAi in Drosophila Cultured Cells 

Lawrence Lum, et al. 

Science 299, 

2039 (2003); 

DOI: 10.1126/science.1081403 

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Identification of Hedgehog 

Pathway Components by RNAi in 

Drosophila Cultured Cells 

Lawrence Lum, 1 Shenqin Yao, 1 Brian Mozer, 2 

Alessandra Rovescalli, 2 Doris Von Kessler, 1 Marshall Nirenberg, 2 

Philip A. Beachy 1 * 

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 1, 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. 

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 diffi- 

1Department of Molecular Biology and Genetics, 

Howard Hughes Medical Institute, Johns Hopkins University 

School of Medicine, Baltimore, MD 21205, 

USA. 2Laboratory of Biochemical Genetics, NHLBI, 

NIH, Bethesda, MD 20892, USA. 

*To whom correspondence should be addressed. Email: 

pbeachy@jhmi.edu 

culty 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-offunction 

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 (GSK3 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 

R ESEARCH A RTICLES 

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 in Drosophila 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 Drosophila cultured 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 embryoderived 

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 

GSK3 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 identi- 

www.sciencemag.org SCIENCE VOL 299 28 MARCH 2003 2039 



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fied 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 serinethreonine 

kinase Fu and cAMP-dependent 

kinase 1 (PKA-C1), two known regulators of 

Hh signaling (1, 2). CK1, 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 CK1 elevated basal reporter activ- 

Fig. 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 after ptc 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 

ity in the absence of Hh (Fig. 2, left in inset) 

and expanded the segmental domain of Wg 

expression in embryos injected with CK1 

dsRNA (Fig. 2, right in inset) (10). 

Systematic screen of Drosophila Gene 

Collection Release 1. The screen was extended 

with a dsRNA library based on the 

Drosophila Gene Collection Release 1 

(DGCr1), which contains approximately 43% 

of the predicted genes in the Drosophila genome 

(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 including thread (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 pro- 

28 MARCH 2003 VOL 299 SCIENCE www.sciencemag.org 

tein 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. 

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 

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 

of ci 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). 



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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). CK1 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 predicted Drosophila HSPGs, including 

a second glypican, Dally, Syndecan (Sdc), 

and Perlecan (Pcan). Only RNAi of Dlp affected 

Hh signal response (Fig. 4A) (20), and the 

Fig. 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 the Drosophila 

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 

CK1 and CK1ε 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 CK1, resulted in activation of the Hh pathway (29). Injection of this ck1 dsRNA into 

preblastoderm Drosophila 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. 


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). 

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 cellautonomous 

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 




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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 CK1 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-phospha- 

tase and DGCr1 library screens suggested that 

CK1 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 

CK1ε was proposed on the basis of the effects 

of CK1ε overexpression in imaginal discs (5). 

To investigate the role of CK1 in Hh pathway 

regulation, RNAi of CK1 and CK1ε in cl-8 

cells were compared. Three dsRNAs containing 

distinct portions of the CK1 open reading 

frame (ORF) increased basal reporter activity 

(29). DsRNA corresponding to either the catalytic 

region or the extracatalytic region of CK1ε 

had no effect (Fig. 5A). In contrast, overexpression 

of either CK1 or CK1ε suppressed Hhinduced 

pathway activation (Fig. 5B). These 

results indicate that both CK1 and CK1ε affect 

basal pathway activity when overexpressed, 

but that physiological regulation depends 

primarily on CK1. 

Both CK1 and CK1ε 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 

Fig. 3. RNAi screen of 

43% of predicted Drosophila 

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. 




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CK1ε elevated basal pathway activity (Fig. 5D, 

right panel). In contrast, RNAi targeting revealed 

that only loss of CK1 function increased basal 

pathway activity (Fig. 5E, left panel), indicating 

that physiological regulation of basal Wg pathway 

activity depends on CK1. The increase in 

pathway activity produced by RNAi of CK1 

was blocked by RNAi of Arm, consistent with 

the proposed role of CK1 in Arm degradation 

(31, 32). Similarly, Ci function is required for 

basal activation of the Hh pathway by RNAi of 

CK1, whereas other positively acting components 

Smo and Fu were not (Fig. 5C, left 

panel). The increase in basal reporter activity 

produced by RNAi of CK1 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 CK1 

acts downstream of Smo and Fu, and at or 

upstream of Ci. The role of CK1 in maintaining 

the quiescent state of both Wg and Hh 

pathways suggests that these pathways might 

be simultaneously activated upon loss of CK1 

activity (Fig. 5D, table). 

In addition to Dlp and CK1, class III 

components with previously unrecognized 

roles in Hh signaling include caupolican, 

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 

in Drosophila can have implications for human 

disease. For example, the role of CK1 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 a Drosophila 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 identi- 

Fig. 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. 




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Fig. 5. CK1 regulates basal activity of both 

the Hh and Wg pathways. (A) Specificity of 

dsRNA targeting CK1. (Left) Schematic diagram 

of the different dsRNAs targeting CK1 

and its closest homolog CK1ε. 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 CK1 but 

neither of the two dsRNAs targeting CK1ε results in pathway 

activation. Three additional dsRNAs targeting CK1ε (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 CK1 or CK1ε suppresses the Hh pathway. 

CK1, CK1ε, or known pathway components were expressed 

in cl-8 cells and pathway response was measured. 

Expression of both CK1 and CK1ε blocked pathway response. 

(C) Mapping of CK1 activity within the Hh pathway. Pathway 

components were targeted by RNAi individually or in combination 

with RNAi of CK1. Only Ci was epistatic to CK1. RNAi 

of Sli, an F-box protein that regulates Ci proteolytic processing, 

in combination with RNAi of CK1 increased both basal and Hh-induced pathway activity more than did either individually. (D) CK1 also acts 

as a negative regulator of the Wg pathway. RNAi of CK1 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 CK1, consistent with CK1 action upstream of Arm. Expression of 

CK1 did not suppress Wg pathway response, and expression of CK1ε activated the pathway. (Right) Summary of the effects of RNAi or 

overexpression (OEX ) of CK1 or CK1ε in Hh and Wg pathways. 

fication of CK1 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. 

References and Notes 

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10. Materials and methods are available as supporting 

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11. In contrast to Ptc inactivation by mutation, dsRNA 

directed against Ptc does not cause constitutive pathway 

activation, probably because RNAi-induced loss 

of ptc mRNA and protein is balanced by a consequent 

increase in transcription of the still intact ptc gene, 

resulting in sufficient levels of Ptc protein to maintain 

pathway regulation. In S2 cells, which lack Ci and 

consequently this regulatory feedback, Ptc-directed 

RNAi treatment results in a decrease in Ptc protein 

and an increase in Smo protein similar to that produced 

by Hh stimulation (Fig. 1B, inset). 

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20. Injection of dally dsRNA used in cell-based assays 

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kinase-phosphatase library, only one of these targeting 

the ORF resulted in a gain in basal reporter 

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43. We thank A. Kaykas and R. Moon for providing the 

Super TopFlash reporter; F. Weiss-Garcia and the 

Sloan-Kettering Hybridoma Core Facility (NY) for 

development of monoclonal antibodies; R. Gong 

for technical assistance; S. Celniker for help with 

annotation of DGCr1; R. L. Johnson and S. M. Cohen 

for antibodies; S. Zusman at Genetic Services (MA) 

for help with dally dsRNA embryo injections; and J. 

Taipale for critical review of the manuscipt. Supported 

by grants and fellowships from NIH and a 

Life Sciences Research Foundation Fellowship (L.L). 

Polymer Replicas of Photonic 

Porous Silicon for Sensing and 

Drug Delivery Applications 

Yang Yang Li, 1 Frédérique Cunin, 1 Jamie R. Link, 1 Ting Gao, 1 

Ronald E. Betts, 1 Sarah H. Reiver, 1 Vicki Chin, 2 

Sangeeta N. Bhatia, 2 Michael J. Sailor1 Elaborate one-dimensional photonic crystals are constructed from a variety of 

organic and biopolymers, which can be dissolved or melted, by templating the 

solution-cast or injection-molded materials in porous silicon or porous silicon 

dioxide multilayer (rugate dielectric mirror) structures. After the removal of the 

template by chemical dissolution, the polymer castings replicate the photonic 

features and the nanostructure of the master. We demonstrate that these 

castings can be used as vapor sensors, as deformable and tunable optical filters, 

and as self-reporting, bioresorbable materials. 

Synthesis of materials using nanostructured 

templates has emerged as a useful and versatile 

technique to generate ordered nanostructures 

(1). Templates consisting of microporous membranes 

(2, 3), zeolites (4), and crystalline colloidal 

arrays (5–7) have been used to construct 

elaborate electronic, mechanical, or optical 

structures. Porous Si is an attractive candidate 

for use as a template (8) because the porosity 

and average pore size can be tuned by adjusting 

the electrochemical preparation conditions that 

allow the construction of photonic crystals, 

dielectric mirrors, microcavities, and other 

optical structures (9). For many applications, 

porous Si is limited by its chemical and mechanical 

stability. The use of porous Si as a 

template eliminates these issues while providing 

the means for construction of complex 

optical structures from flexible materials that 

are compatible with biological systems or 

harsh environments. 

Multilayered porous Si templates containing 

nanometer-scale pores are prepared (10) byan 

1 Department of Chemistry and Biochemistry, University 

of California, San Diego, 9500 Gilman Drive, 

Department 0358, La Jolla, CA 92093–0358, USA. 

2 Department of Bioengineering, University of California, 

San Diego, 9500 Gilman Drive, Department 0412, 

La Jolla, CA 92093–0412, USA. 

anodic electrochemical etch of crystalline silicon 

wafers with the use of a pseudosinusoidal current-time 

waveform, according to published procedures 

(9, 11–16). The thickness, pore size, and 

porosity of a given layer is controlled by the 

current density, duration of the etch cycle, and 

etchant solution composition (17). The multilayer 

templates possess a sinusoidally varying 

porosity gradient, providing sharp features in the 

optical reflectivity spectrum (Fig. 1) that approximate 

a rugate filter (18). The porous Si is converted 

to porous SiO 2 by thermal oxidation, and 

the oxidized nanostructure (fig. S1) (10) is used 

as a template for solution-cast or injection-molded 

thermoplastic polymers. 

Removal of the porous SiO 2 template 

from the polymer or biopolymer imprint by 

chemical dissolution provides a freestanding 

porous polymer film with the optical characteristics 

of the photonic crystal master (figs. 

S2 to S4). Reflection spectroscopy (Fig. 1) 

and scanning electron microscopy (SEM) 

(Fig. 2) confirm that the photonic structure of 

the porous Si master is retained in the polymer 

casting. The sharp optical reflectivity 

feature expected of a rugate filter is observed 

in both the template and the polymer casting 

(Fig. 1), confirming that the process replicates 

the microstructure. Cross-sectional 


P.A.B. is an investigator of the Howard Hughes 

Medical Institute. 

Supporting Online Material 

www.sciencemag.org/cgi/content/full/299/5615/2039/ 

DC1 

Materials and Methods 

Fig. S1 

Tables S1 and S2 

References 

11 December 2002; accepted 6 March 2003 

REPORTS 

SEM measurements (Fig. 2 and fig. S5) corroborate 

the optical data. 

Vapor dosing experiments confirm that the 

microporous nanostructure is retained in the 

castings. The position of the spectral feature for 

a rugate filter depends on the periodicity and 

refractive index gradient of the structure. When 

porous Si multilayers are exposed to condensable 

vapors such as ethanol or hexane, microcapillary 

condensation in the nanometer-scale 

pores produces an increase in the average refractive 

index of the matrix and a spectral red shift of 

the photonic feature (11, 19, 20). The shift of the 

spectral peak correlates with partial pressure of 

the analyte in the gas stream, following the 

Kelvin equation for condensible vapors (15, 19– 

21). Dose-response curves for ethanol vapor for 

the porous Si template and for the polystyrene 

Fig. 1. Reflectivity spectra of an oxidized porous 

Si rugate film (top) and a polystyrene film 

cast from the porous Si template (bottom). The 

spectral peaks correspond to the second-order 

diffraction peak of the template and the second- 

and third-order diffraction peaks of the 

imprint. The porous Si template was etched 

using a sinusoidal current varying between 38.5 

and 192.3 mA/cm 2 , with 70 repeats and a 

periodicity of 8 s. The total thickness of the 

porous Si film is 40 m. The reflected light 

spectra were obtained using an Ocean Optics 

SD2000 charge-coupled device spectrometer 

using tungsten light illumination. Spectra are 

offset along the y axis for clarity. 




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