Identification of Hedgehog pathway components by RNAi in ...

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.
R ESEARCH A RTICLES
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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