Identification of Hedgehog pathway components by RNAi in ...

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-
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