4107
Development 126, 4107-4115 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
DEV8617
Mutual antagonism between signals secreted by adjacent Wingless and
Engrailed cells leads to specification of complementary regions of the
Drosophila parasegment
Uwe Gritzan*,‡, Victor Hatini* and Stephen DiNardo§
Cell & Developmental Biology, University of Pennsylvania Medical School, Philadelphia, PA 19104, USA
*These authors made an equal contribution
‡Present address: EMBL, Heidelberg, Germany
§Author for correspondence (e-mail: [email protected])
Accepted 7 July; published on WWW 23 August 1999
SUMMARY
Specialized groups of cells known as organizers govern the
establishment of cell type diversity across cellular fields.
Segmental patterning within the Drosophila embryonic
epidermis is one paradigm for organizer function. Here
cells differentiate into smooth cuticle or distinct denticle
types. At parasegment boundaries, cells expressing
Wingless confront cells co-expressing Engrailed and
Hedgehog. While Wingless is essential for smooth cell fates,
the signals that establish denticle diversity are unknown.
We show that wg mutants have residual mirror-symmetric
pattern that is due to an Engrailed-dependent signal
specifying anterior denticle fates. The Engrailed-dependent
signal acts unidirectionally and Wg activity imposes
this asymmetry. Reciprocally, the Engrailed/Hedgehog
interface imposes asymmetry on Wg signaling. Thus, a
bipartite organizer, with each signal acting essentially
unidirectionally, specifies segmental pattern.
INTRODUCTION
secreted by this organizer is the Wnt gene family member,
Wingless (Wg) (Cabrera et al., 1987; Rijsewijk et al., 1987;
Baker, 1988). It is expressed anterior to cells expressing the
homeoprotein Engrailed (En; Ingham et al., 1988). The Enexpressing cells co-express Hedgehog (Hh), member of another
signaling family (Lee et al., 1992; Mohler and Vani, 1992;
Tabata et al., 1992; Krauss et al., 1993; Riddle et al., 1993).
There are two phases to patterning the epidermis. First,
reciprocal signaling occurs between Wg- and En/Hh-expressing
cells, serving to consolidate parasegmental subdivision of the
body plan (DiNardo et al., 1988; Martinez-Arias et al., 1988;
Bejsovec and Martinez-Arias, 1991; Heemskerk et al., 1991;
Cumberledge and Krasnow, 1993; Ingham, 1993).
Subsequently, cells fates are established (Bejsovec and
Martinez-Arias, 1991; Dougan and DiNardo, 1992).
Ventrally, Wg signaling is required to establish fate of half of
the parasegment, specifically the region that differentiates into
smooth cuticle (Baker, 1988; Bejsovec and Martinez-Arias,
1991; Dougan and DiNardo, 1992; Noordermeer et al., 1992; Pai
et al., 1997). The signals specifying remaining fates within each
parasegment, those that differentiate into diverse denticle types
in abdominal segments 2-7, are less clear, although Wg signaling
has been implicated (Bejsovec and Martinez-Arias, 1991;
Bejsovec and Wieschaus, 1993; Yoffe et al., 1995; Lawrence et
al., 1996). One difficulty in establishing a direct role for Wg in
generating denticle diversity is to separate that contribution from
its early role in maintaining En/Hh expression; especially since
Organizers specify pattern across cellular fields (Spemann, 1938).
Work has focussed on elucidating the properties of organizers
because, aside from revealing how the specific tissue is patterned,
principles uncovered for one organizer will apply to others. In
Drosophila, cellular fields are established with the subdivision of
the body plan into parasegments (Martinez-Arias and Lawrence,
1985). Signals emanating from cells at the boundary between
adjacent parasegments guide patterning across each parasegment
in the epidermis (Baker, 1988; Martinez-Arias et al., 1988;
Bejsovec and Martinez-Arias, 1991; Heemskerk and DiNardo,
1994; Bokor and DiNardo, 1996). Similar mechanisms act in
patterning the imaginal discs, as signals emanate from the
compartment boundaries that are inherited from embryonic
parasegment boundaries (Garcia-Bellido, 1975; Basler and
Struhl, 1994; Capdevila and Guerrero, 1994; Tabata and
Kornberg, 1994; reviewed in Lawrence and Struhl, 1996). Since
these signals act to establish cell type diversity across the field,
the parasegment and compartment boundary are analogous to
classical pattern organizers. Thus, identifying signals from
parasegment or compartment boundaries and understanding their
control will provide general insight into organizer function.
Screens have identified mutations in the segment polarity
genes involved in parasegmental patterning (Nusslein-Volhard
and Wieschaus, 1980), and their molecular analyses is
providing a paradigm for organizer function. One of the signals
Key words: Engrailed, Wingless, Hedgehog, Drosophila, Segment
pattern
4108 U. Gritzan, V. Hatini and S. DiNardo
we previously found that Hh plays a significant, Wgindependent role in organizing dorsal epidermal pattern
(Heemskerk and DiNardo, 1994). That work coupled with two
studies on ventral pattern (Bejsovec and Wieschaus, 1993;
Sampedro et al., 1993) lead us to consider the contributions of
the En- and Hh-expressing cells in generating denticle diversity.
We recently showed that the ligands Spitz and Wingless
emanate from distinct borders of the En domain, and proper
patterning across the En domain is guided by the balance between
these two antagonistic signals (O’Keefe et al., 1997). Further
results suggested that Spitz was also required for some denticle
cell fates just posterior to the En domain (Szüts et al., 1997).
We now bypass the need for Wg in the stabilization of En/Hh
expression to investigate the role of En and Hh in generating
denticle diversity. This establishes definitive evidence for Wgindependent specification of anterior denticle fates. In addition,
we uncover evidence that Wg and Hh activity each ensures that
signaling from the organizer is asymmetric, thereby generating
proper intrasegmental pattern.
MATERIALS AND METHODS
smo3, hh13C, wgcx4 and wgIL (wgts) were from Bloomington;
Df(3R)GR2 was from J. Mohler; Df(2L)NL Df (2R) enSF31 was from
A. Bejsovec. A smo3 FRT 40A and a wgcx4 smo3 FRT 40A
chromosome was used to generate germline clones (Chou et al.,
1993), and the resulting females crossed to wgcx4 smo3 FRT 40A /
CyO males. UAS-Arm-S10 is described in Pai et al. (1997); UASdTCF delta N dominant negative and En-GAL4 were from M. Peifer
(Cavallo et al., 1998) and A. Brand. Ptc-GAL4 is described in
Speicher et al. (1994). To bypass the requirement for Wg in
maintaining En expression, we constructed and crossed wgcx4 En-Gal4
/ CyO × wgcx4 UAS-ArmS-10/CyO.
Cuticles were prepared as usual (van der Meer, 1977). βgalactosidase activity stains on cuticle preparations (Heemskerk and
DiNardo, 1994) were performed using Wg-lacZ (wg1-en11; Kassis et
al., 1992) or En-lacZ (Xho-25; Hama et al., 1990) reporters.
RNA in situ antibody double labeling was as in Dougan and
Fig. 2. Cell fates specified by Wg and En. (A) WglacZ; registration of Wg-expressing cells (X-Gal
activity stain, white arrow) relative to first row
denticles (small black arrow). Large black arrow, row
5 denticles. (B) En-lacZ; registration of En-expressing
cells (white arrow) relative to first row denticles (small
black arrow). (C) wg en double mutants differentiate a
homogenous field of large, tapered denticles, generally
pointing inward to midline. There are a few smaller,
less differentiated denticles. (D) Ptc-GAL4 UASdTCFDN; inhibiting Wg signal transduction outside of
the En domain leaves a fairly normal denticle belt
(small right-pointing arrow, row 1; large right-pointing
arrow, row 5). Cells that normally differentiate smooth
cuticle now adopt character of a mirror-image belt,
with small denticles progressively leading toward
larger row 5-type denticles (left-pointing arrows).
(E) wg mutants differentiate row 5 denticles (large
arrows), alternating in mirror-image with small, row 2to 4-type denticles (small arrows). (F) wg En-GAL4
UAS-ArmS10 embryos have small hooked row 2-4
denticles (arrows) usually pointing away from smooth
cuticle regions. Bar, 10 µm.
Fig. 1. Control of pattern by Wg and En Cuticles, dark field, anterior
left. (A) Wild type. Arrow, smooth cuticle; arrowhead, denticle belt.
(B) wg en; no segmental pattern. (C) wg; alternating denticle pattern
(arrowheads). (D) wg En-GAL4 UAS-ArmS10; maintaining En
expression leads to smooth cuticle specification (arrow). Bar, 50 µm.
DiNardo (1992). Digoxigenin-labeled RNA probes were from cDNAs
for Wg, Rhomboid and Serrate (Baker, 1987; Bier et al., 1990;
Thomas et al., 1991). Anti-mAb4D9 (En/Inv) was used undiluted
(Patel et al., 1989); anti-Patched at 1:200 (Capdevila et al., 1994); antiβ-galactosidase (Cappel) at 1:1,000; biotinylated antibodies (Vector),
Cy2- and Cy3-labeled secondary antibodies (Jackson), and HRPconjugated streptavidin (Chemicon) at 1:400. Embryos were filletted
in 80% glycerol; some data were collected on a Zeiss LSM 510
confocal microscope; images were processed in Adobe Photoshop.
RESULTS
Ventrally across each segment of the second through seventh
abdominal segments six rows of epidermal cells choose to
Wingless and Hedgehog constitute a bipartite organizer 4109
(Molenaar et al., 1996; Brunner et al., 1997; van de Wetering
et al., 1997), thereby inhibiting Wg transduction in cells
outside the En domain, leads to loss of smooth cell fates (Fig.
2D). The pattern within the denticle belt is quite normal in such
embryos (Bejsovec and Martinez-Arias, 1991).
Furthermore, in place of smooth cuticle, there is an
approximate mirror-image duplication of denticle pattern (Fig.
2D). This suggests that, in the absence of late Wg function,
whatever signal(s) organizes denticle pattern now acts bidirectionally, generating a mirror-image pattern. It has been
suggested that early Wg function specifies denticles diversity.
However, even in wg null mutants, there is remaining mirrorpattern (Fig. 1C), as large row 5-type denticles (Fig. 2E, large
arrowheads) alternate with small row 2-4 type denticles (Fig.
2E, small arrowheads). Thus, in the absence of Wg, any
remaining signal(s) now acts symmetrically, generating a
roughly mirror-image pattern.
Fig. 3. Serrate expression confirms cuticle interpretations Stage
12/13 embryos; En protein (brown), Serrate RNA (blue). (A) Serrate
(white arrow) is expressed in a 3-cell-wide stripe located about 1 cell
row posterior and several rows anterior to En domain (black arrow).
(B) wg; Serrate expression expands. En-expressing neurons
underlying the epidermis (black arrow) show that Ser expands
posteriorly(Chu-LaGraff and Doe, 1993). (C) wg en; Serrate is
expressed almost globally. (D) wg En-GAL4 UAS-ArmS10; with En
maintained and no Wg signaling, Serrate expression is lost (asterisk)
or reduced (white arrowhead), correlating with a loss of row 5
denticles. The En stripe is broad, as expected, since more cells have
the capacity to maintain En than usually do. Normally, only Enexpressing cells near the Wg source maintain En (Vincent and
O’Farrell, 1992). In wgcx4 En-Gal4 UAS-ArmS10 embryos, intrinsic
activation of the Wg pathway in En cells maintains the broader
stripe. Bar, 10 µm.
differentiate smooth cuticle, while the next six rows secrete
cuticle that has protrusions called denticles (Fig. 1A; LohsSchardin et al., 1979). These denticle-secreting cells form a
belt, wherein denticle rows can usually be distinguished from
each other based on size and polarity. Row five denticles are
largest, with tapered, posteriorly pointing tips. Row six has tiny
denticles of indeterminate polarity. Denticles of the first four
rows are all smaller than row five, and their tips are hooked
more than tapered. Rows one and four point anteriorly, while
two and three point posteriorly.
Wingless and Engrailed are essential in organizing this
pattern. Wg-expressing cells lie anteriorly adjacent to the En
cells. While the Wg-expressing cells differentiate into smooth
cuticle (Fig. 2A; O’Keefe et al., 1997), the posterior-most Enexpressing cells differentiate into row 1 denticles (Fig. 2B;
Dougan and DiNardo, 1992). Embryos null for both wg and en
have no smooth cuticle, and the remaining denticles are row 5like, mostly large with tapered tips (Figs 1B, 2C; Bejsovec and
Martinez-Arias, 1991). While this demonstrates that Wg and
En together are essential for normal pattern, the individual
contribution made by the En-expressing cells is not clear.
In contrast, Wg activity is necessary and sufficient for
smooth fates (Wieschaus and Riggleman, 1987; Baker, 1988;
Lawrence et al., 1996; Hays et al., 1997; Pai et al., 1997).
Smooth fates are specified late, after the Wg-dependent
stabilization of en gene expression. Removing Wg function
after about 5 hours after egg laying (AEL) by wgts upshifts, or
using Ptc-GAL4 to express a dominant negative form of dTCF
An En-dependent signal
A signal made by the En cells could be responsible for
production of the now symmetrically acting signal. In wg
mutants, this signal would persist only transiently, as En
expression decays early. If true, then maintaining En expression
should generate more organized pattern. We therefore examined
the pattern generated when En expression is maintained in the
absence of Wg function. Previously, we accomplished this in
dorsal epidermis by precociously activating En autoregulation
using Hs-En in wg mutants (Heemskerk and DiNardo, 1994).
Here we use En-GAL4 to express an activated form of the Wg
signal transducer Armadillo in the En-expressing cells in wg
nulls. This bypasses the transient requirement for Wg in
maintaining En expression, leading to significant stabilization
of En-expressing cells (see Fig. 3D). By cell intrinsically
activating the Wg pathway in En cells, this maintains Hedgehog
and other Wg-responsive genes in the En cells, while removing
any contribution of Wg to patterning any other cells.
Maintaining En-expressing cells leads to obvious differences
comparing the pattern of wgcx4 En-Gal4 UAS-ArmS10
embryos with wg mutants. First, strips of segmentally repeated
smooth cuticle are often found (Fig. 1D). These regions of
smooth cuticle mark the cell-autonomous differentiation of En
cells expressing activated Armadillo (O’Keefe et al., 1997; Pai
et al., 1997; Sanson et al., 1999). Second, there are fewer large,
tapered row 5-type denticles; nor are there any tiny row 6-type
denticles. This suggests a loss of posterior belt identities (Fig.
2F). Instead, the denticles formed are small and hooked, row
2- or 3-type denticles (Fig. 2F, arrows).
Significantly, the denticle pattern exhibits some mirror
symmetry. An axis of symmetry lies within each strip of Enexpressing smooth cells as denticles point away from the
nearest region of smooth cuticle (Fig. 2F). Thus, maintaining
En in the absence of Wg leads to loss of posterior fates and
duplication of anterior denticle identities. Given that En
maintenance by activated Armadillo is cell autonomous, the
changes in denticle patterning outside the En domain suggest
strongly that En-expressing cells have a non-autonomous effect
in generating this symmetrical pattern.
Expression of Rhomboid and Serrate reveal
asymmetry in signaling
Molecular markers confirm our interpretation of the cuticle
4110 U. Gritzan, V. Hatini and S. DiNardo
phenotype. Since there is a decrease in row 5 cell types in wgcx4
En-Gal4 UAS-ArmS10 embryos, we first examined the
expression of Serrate. At 8 hours AEL Serrate is activated in
about 3 rows of cells (Fig. 3A; Thomas et al., 1991), the
anterior of which will correspond to the row 4/5 border. Serrate
normally acts as a Notch ligand and, although the role of Notch
in specifying denticle diversity has not been investigated,
Serrate is required for posterior belt identities, such as row 4
(Wiellette and McGinnis, 1999). In wg mutants, where there is
an expansion in posterior row cell types, Serrate expression is
also expanded (Fig. 3B; Wiellette and McGinnis, 1999).
Expression does not fill each segment, reflecting the remaining
mirror-image pattern in wg mutants (Fig. 1C). Indeed, in wg
en double mutants, where only row 5 denticles are specified,
Serrate expression virtually fills the parasegment (Fig. 3C).
This further confirms that the remaining pattern in wg mutants
is due to En (and Hh) function. In wgcx4 En-Gal4 UAS-ArmS10
embryos, Serrate expression is greatly reduced (Fig. 3D),
consistent with a loss in posterior denticle rows.
We next examined Rhomboid, which at 7 hours AEL is
expressed in a row of cells posterior to the En domain (Fig.
4A; Bier et al., 1990). Rhomboid induces EGF-Receptor
signaling, which is required for row 1, as well as row 2-3
denticle cell types (O’Keefe et al., 1997; Szüts et al., 1997),
the rows that we suspect to be duplicated in the wgcx4 En-Gal4
UAS-ArmS10 embryos. In the wgcx4 En-Gal4 UAS-ArmS10
embryos, En-expressing cells are now flanked by Rho stripes
anteriorly (Fig. 4B), as well as posteriorly. Thus, the mirrorimage cuticle pattern is paralleled molecularly by symmetrical
expression of Rho. Occasionally Rho-expressing cells
surround En-expressing cells (Fig. 4C), a topology suggesting
that induction of Rho is En-dependent and short range. These
data suggest that Wg acts to ensure that signaling from the En
domain is asymmetric, leading to the induction of Rhomboid
only posteriorly.
Asymmetry in Hh signaling is governed by Wg
Since Hh is the known ligand secreted by En cells, we
examined the effects of removing Wg function on symmetry
of Hh signaling. The transmembrane protein Patched binds Hh
(Hooper and Scott, 1989; Nakano et al., 1989; Stone et al.,
1996). The incorporation of Patched into responding cells in
multivesicular bodies is a reflection of the strength of Hh
signaling (Capdevila et al., 1994). In wild type, Patched protein
is most strongly expressed in cells adjacent to En-expressing
cells (Fig. 4D). From 6 hours AEL, there is normally
asymmetry in Hh signaling as revealed by the increased
number of dots in cells just posterior compared to anterior to
En cells (Fig. 4D). However, in wgcx4 En-Gal4 UAS-ArmS10
embryos, dots are symmetrically distributed (Fig. 4E),
suggesting that, in the absence of Wg function, Hh now signals
equally strongly anterior to En cells. We conclude that Wg
plays an important role in maintaining (or establishing)
asymmetry in signaling from En cells.
Hedgehog affects Rhomboid expression
Our results suggest that a locally acting signal expressed from
En cells controls part of the cuticle pattern by inducing
Rhomboid only in cells posterior to the En domain. To test
whether this signal was Hh, we first asked whether increasing
Hh expression in En cells would affect Rho expression. In
embryos carrying En-GAL4 and UAS-Hh, Rho expression
expands posteriorly to 2- to 3-cell rows (Fig. 5B). Fate
specification was also affected as we observed an excess of
small denticles, apparently of row 2-4 type at the expense of
row 5 and 6 (Fig. 5C; Lee et al., 1994; Fietz et al., 1995). So
excess Hh can broaden Rho expression, and this has a
consequence on fate selection. Thus, the level of Hh signaling
from En cells can limit the Rho expression domain.
Reciprocally, in hh mutants, Rho expression is reduced (Fig.
5D). Thus, both loss- and gain-of-function experiments
implicate hh as a positive regulator of Rho. The hh mutant
cuticle pattern is consistent with this since, in hh or smo
mutants, mostly type 5 denticles (Fig. 5E) are observed and
Serrate expression is broadened encompassing all but the
residual En-expressing cells (Fig. 5F; Wiellette and McGinnis,
1999).
Interestingly, in hh mutants, we still observed residual Rhoexpressing cells, even in embryos homozygous for a deficiency
deleting hh, or embryos deficient for smoothened function, a
receptor that transduces the Hh signal (Alcedo et al., 1996;
Chen and Struhl, 1996; van den Heuvel and Ingham, 1996).
Thus, residual Rho expression cannot be due to any residual
Hh signaling. Therefore, Hh is important but is not absolutely
required for the induction of Rho expression, implicating an
unidentified signal partially redundant with Hh. This second
signal may also emanate from the En cells. In hh mutants, En
expression does not completely decay and the residual patches
of Rho expression are often found in close proximity to Enexpressing cells (Fig. 5D, arrows). Thus, Hh as well as a
second, likely En-dependent signal positively regulate Rho
expression, thereby contributing to the correct specification of
particular denticle identities.
Wg governs asymmetric Rhomboid expression
Further evidence for a second En-dependent signal derives
from analyzing Wg control of Rho expression. In wg null
mutants, we observed an ectopic stripe of Rho in each segment
(Fig. 6B). Thus, Wg negatively regulates Rho expression. The
induction of an ectopic Rhomboid stripe is consistent with the
wg mutant mirror-symmetric cuticle pattern.
Since Hh can positively regulate Rho, the ectopic Rho stripe
in wg mutants could be the result of the now symmetrical
signaling by Hh in the absence of Wg. To test this we removed
both Hh and Wg function. We and others have difficulty
constructing wg;hh doubly mutant stocks. To circumvent this,
we removed all Hh-dependent signaling by examining
smoothened (smo) mutants. We first made germline clones,
removing both maternal and zygotic contribution. Resulting
embryos show reduced Rho expression (Fig. 6C), identical to
hh mutants (Fig. 5D). We next examined embryos lacking wg
function as well as both maternal and zygotic smo activity
(functionally wg; hh double mutants). Rho is expressed in
duplicate stripes in these embryos (Fig. 6D), as in wg single
mutants. Thus, although hh plays a role in Rho regulation (Fig.
5), the robust Rho expression in wg mutants cannot be due to
symmetrical Hh signaling.
Note also that Rhomboid expression is still spatially
restricted in embryos lacking both Wg and Hh signaling, just
as in wg single mutants (Fig. 6B,D). Thus, some other localized
factor must be required for the spatially restricted activation of
Rhomboid. We could not use En as a registration marker to
Wingless and Hedgehog constitute a bipartite organizer 4111
determine the position of ectopic Rho expression, since En
expression is lost in wg null mutants. To circumvent this, we
either inactivated wg in later stage embryos, after early Wg
signaling has stabilized En expression, using wgts upshifts, or
examined Ptc-GAL4 UAS-dominant negative dTCF embryos
wherein Wg signal transduction is inhibited outside of the En
domain. This also resulted in an ectopic stripe of Rho (Fig. 6E),
positioned just anterior to now stabilized En-expressing cells.
Thus, in each case where Rho has been mapped, it is expressed
adjacent to En cells (Figs 4B,C, 6E). This, plus the observation
that Rhomboid is still spatially restricted in embryos lacking
both Wg and Hh signaling, strongly suggests that there exists
an En-dependent inducer besides Hh. This factor must be made
early by En cells. In the absence of Wg function, this inductive
signal now acts symmetrically, on both sides of the En domain,
just as we have shown that Hh can act symmetrically in the
absence of Wg function (Fig. 4E).
fates, proper ordering of their expression is essential to ensure
correct polarity of pattern across the segment.
Signaling from parasegment boundaries represents one
paradigm for organizer function. This segmental organizer is
bipartite, composed of adjacent rows of cells, one expressing
Wingless and the next co-expressing Engrailed and Hedgehog.
We now assign a specific, Wg-independent role to Hh, as well
as to an unidentified second signal, also likely expressed from
En cells. Coupled with prior analysis for the role of Wingless,
we conclude that these two rows of cells emit signals that each
specify a particular portion of the pattern. We also find that
each signal prevents the other one from acting bi-directionally
in order to create zones that are predominantly patterned by
either Wg or by signals from the En cells.
Organizer function: maintenance and patterning
Reciprocal signaling between Wg- and En/Hh-expressing
cells stabilizes one another’s expression, consolidating the
parasegmental body plan (DiNardo et al., 1988; MartinezArias et al., 1988; Bejsovec and Martinez-Arias, 1991;
Heemskerk et al., 1991; Cumberledge and Krasnow, 1993). At
this time, signaling is effective only locally, thereby restricting
the expression of the other signal to a narrow strip of cells
(Martinez Arias et al., 1988; Vincent and O’Farrell, 1992;
Ingham, 1993). This ensures that the bipartite organizer
remains a line source rather than broadening during patterning.
Ventrally, the organizer specifies half the fates as smooth cell
types and compelling evidence demonstrates that Wg specifies
these (Wieschaus and Riggleman, 1987; Baker, 1988; Bejsovec
and Martinez-Arias, 1991; Dougan and DiNardo, 1992;
Noordermeer et al., 1992; Lawrence et al., 1996; Pai et al.,
1997). This step occurs after Wg stabilizes En/Hh expression.
In contrast, the identity of the signals responsible for
specifying the diverse denticle cell types has been less clear.
By bypassing the need for Wg input to En cells, we find row
2- to 4-type denticles specified in the absence of Wg. This
conclusion was confirmed by analyzing the control of
Rhomboid expression in cells flanking the En domain, as
Rhomboid is essential for the proper differentiation of row 14 fates (O’Keefe et al., 1997; Szüts et al., 1997).
Previous studies indicated that denticle diversity arises early,
around the time Wg stabilizes En/Hh expression (Bejsovec and
Martinez-Arias, 1991; Sampedro et al., 1993). Our data suggest
that the stabilization of En/Hh expression establishes the
conditions to generate denticle diversity, but that diversity is
not specified until later as reflected in the induction of Serrate
and Rhomboid (~7-8 hours AEL). Indeed, excess Hh delivered
at late times can broaden Rho stripes and this still affects
denticle diversity.
Due to a lack of molecular markers, the exact posterior
boundary of the En/Hh-dependent domain is unclear. However,
since row 5 cell types are specified in embryos lacking all
Hh/Smo signaling, En/Hh influence can only extend up to row
4. Consistent with this, Hh signaling sets the anterior Serrate
expression boundary. Since Wg signaling sets Serrate’s
posterior boundary, the Serrate domain defines a region of
positional values within the segment where Hh and Wg
cooperate in patterning. In fact, Serrate expression is perhaps
a molecular marker for a default state, as its expression is
almost global in cases where only row 5 cell types are
specified, such as in wg en doubly mutant embryos. We
presume it would indeed be globally expressed in a wg en; hh
triple mutant, which we have not been able to construct and
test.
Organizer establishment
The segmentation gene hierarchy establishes the asymmetric
composition of the organizer, with Wg-expressing cells
anteriorly adjacent to En/Hh-expressing cells. Sampedro and
Lawrence (1993) suggested that the strict spatial order of these
two signals did not matter for intrasegmental patterning, but
simply defined the position of the parasegment boundary.
However, since we find that Wg and En/Hh specify distinct
Wg and denticle diversity
Although previous work suggested that Wg signaling generates
denticle diversity, several observations made us question
whether this role is direct. First, Sampedro et al. (1993) found
that removing Hh function blocks the ability of Wg to generate
a “mirror phenotype”, a reflection of denticle diversity. This
Hh requirement supports the role that we find for En/Hh cells
in defining row 2-4 denticles. Second, although cells can
Hedgehog also ensures signaling asymmetry
The repression of Rhomboid by Wg also suggested why most
Rhomboid expression is lost in smo or hh mutants. When wg
function is removed from smo mutants, Rho expression is
restored (Fig. 6D). Thus, in the absence of Hh signaling, Wg
activity now represses Rho expression in the domain posterior
to En cells. This suggests that the asymmetric induction of Rho
normally comes about because Hh blocks Wg from signaling
to cells posterior to the En domain. To test this, we asked
whether activation of the Wg pathway in cells posterior to the
En domain would be sufficient to block Rho induction. In PtcGAL4 UAS-ArmS10 embryos, Rho is repressed (Fig. 6F).
Thus, during normal development, it is important to block
activation of the Wg pathway in cells posterior to the En
domain. We infer from this that one role for Hh is to establish
or maintain asymmetry of signaling during patterning. Thus,
wg and hh confer directionality upon each other’s signaling
from the segment organizer.
DISCUSSION
4112 U. Gritzan, V. Hatini and S. DiNardo
Fig. 4. Rhomboid expression
A
confirms cuticle interpretations
stage 12/13 embryos; En protein
(brown), Rhomboid RNA (blue).
(A) Rhomboid is expressed in a row
of cells, posterior to En domain.
(B) wg En-GAL4 UAS-ArmS10;
with En maintained and no Wg
signaling, Rhomboid is expressed
on both sides (arrows) of the En
domain. This correlates with the
specification of small, row 2- to 4type denticles on both sides of
smooth cuticle. Sometimes Rho
expression fills the region between
adjacent En domains, correlating
with those instances where Serrate
expression is completely lost.
(C) Occasionally, Rhomboid is
expressed in a ring surrounding En-expressing cells. (D) Wild type, Anti-Patched; ventrolateral confocal sections. Accumulation of dots
(arrow), reflecting Patched antigen in responding cells, shows more signaling to the posterior of the En domain (asterisk), marked by absence
of Patched expression. (E) wg En-GAL4 UAS-ArmS10; Patched accumulates more symmetrically relative to En-expressing cells (asterisk).
Bar, 10 µm in A-C; 20 µm in D, E.
Fig. 5. Hedgehog affects Rhomboid expression. (A,B,D,F) Stage
12/13 embryos doubly labeled for En protein (brown) and
Rhomboid or Serrate RNA (blue). (C,E) Cuticle preparation.
(A) Rhomboid is expressed in a single row, just posterior to En
cells. (B) En-GAL4; UAS-Hh; extra Rho-expressing cells form a
2-3 cell-wide stripe, which is sometimes split. (C) En-GAL4;
UAS-Hh; excess rows of small denticles replace posterior belt
identities. (D) hh; Rho-expression decreases; the few remaining
Rho+ cells are posteriorly adjacent to residual En+ cells (arrows;
the asterisk notes an exception). (E) smoMat- Zyg-; most cells make
tapered, row 5 type denticles. Some small denticles are also
produced (not shown), presumably from residual En-dependent
signal. (F) hh; Ser expression almost fills parasegment.
Bar, 10 µm.
respond to different levels of Wg, the values specified are
limited to midregions of the segment, just anterior to and
within the Wg-expressing cells (Lawrence et al., 1996). This
is equivalent to the smooth region of the segment in A2-A7 and
does not bear on denticle diversity. Third, and most
compelling, is the isolation and characterization of wg alleles
that distinguish the specification of smooth cell fates from
denticle diversity (Bejsovec and Wieschaus, 1993; Hays et al.,
1997). Since the authors showed that generating denticle
diversity correlates with the ability to sustain En expression,
we interpret their data to suggest that Wg acts as a relay: the
stabilization of En/Hh allows, as we show, for the production
of signals necessary to specify some denticle diversity.
Bejsovec and Wieschaus (1993), in their comprehensive
analysis of segment polarity mutant phenotypes, implicated
several genes in specifying denticle diversity. Their evidence
came from modifications of denticle type in various double
mutants with wg. Three such mutants directly implicate En or
Hh: (1) ptc, which would lead to excess Hh pathway activity,
(2) en, which would lead to loss of the En-derived signals that
we identify, and (3) hh. Thus, our observations confirm their
conclusions, as we demonstrate that a Wg-independent En/Hh
signal is involved in generating denticle diversity. They also
implicate nkd, but we find two limitations in their
interpretation. First, they define the wg phenotype to be of
single, row 5 denticle type, while we present evidence for
alternating intervals of row 5 with row 2-4 type. Second, since
En/Hh expression is maintained for a longer period in wg; nkd
doubly mutants (Bejsovec and Wieschaus, 1993), we would
predict excess denticle diversity due to persistence of the
En/Hh-dependent signals defined here. This is indeed the case.
Thus, the contribution of nkd to denticle diversity is likely
indirect, relayed through the effect on En/Hh stabilization.
Relay and direct cell type specification by Hh
Together, Hh and a second En-dependent signal lead to Rho
induction, which, in turn activates EGF-R signaling. Since
EGF-R signaling is essential for row 1-4 denticles, this
Wingless and Hedgehog constitute a bipartite organizer 4113
A
Ant
*
B
Ant
*
Fig. 6. Wg signaling governs asymmetric Rhomboid expression. (AD) Stage 12/13 embryos labeled for Rhomboid RNA (blue); (E,F)
embryos doubly labeled for Rhomboid and En protein (brown). (A)
Wild type; (B) wg; duplicate Rho stripes (arrows). (C) smoMat−Zyg−;
fewer Rho-expressing cells. Occasionally, stripes are almost intact
(right-hand stripe), or the few remaining expressing cells round up
(arrow). (D) smoMat−Zyg− embryo also null for wg; duplicate Rho
stripes. (E) Ptc-GAL4 UAS-dTCFDN; Rho is also expressed anterior
to En domain. (F) Ptc-GAL4 UAS-Arm-S10; Rho expression is lost
posterior to En domain. The En domain is sometimes expanded
posteriorly in these embryos, and wg gene expression is induced
through autoactivation (not shown). Bar, 10 µm.
supports a relay mechanism for patterning. However, our data
suggest that Hh also plays a direct role. Specifically, wg
mutants have duplicate stripes of Rho, and duplicate domains
of row 2- to 4-type denticles. However, when smoothened is
also removed from the wg mutant embryos, thereby removing
Hh signaling, there are still duplicate stripes of Rho, but no
small denticles. Thus, Rho is not sufficient to specify row 2-4
fates without Hh having signaled these cells also. Perhaps Hh
acts early to induce Rho expression and separately acts to
confer competence to a domain of cells that will respond to
EGF-R pathway activation later. These data distinguish three
distinct functions of Hh in epidermal patterning: Wg
maintenance, Rho induction and a Rho-independent role in the
specification of rows 2-4. This is reminiscent of wing
patterning where Hh has been shown to have several functions
as well: Dpp induction (Zecca et al., 1995) and a Dppindependent role in the specification of cell types in the vicinity
of the compartment boundary (Mullor et al., 1997; Strigini and
Cohen, 1997).
Assuring unidirectional signaling
Our data suggest that both Wg- and the En/Hh-expressing cells
Post
*
Ant
*
Fig. 7. Distinct organizer properties due to compartmentalization of
cellular field. (A) From Nellen et al. (1996); in imaginal discs such
as the wing, where anterior (non-En-expressing) and posterior (Enexpressing) compartments each make up a significant part of field, a
line source of Dpp morphogen is sufficient to establish pattern. A
cell (asterisk) equidistant from the morphogen source, adopts the
same positional value, reflected by its Omb+ Sal− state in the
example. However, the cell will adopt a distinct fate in anterior
compared to posterior compartment since one cell is uniquely En+.
(B) In embryonic parasegments, the posterior (En-expressing)
compartment makes up a trivial portion of the cellular field. Thus, if
one signal acted bidirectionally, a compartmentally restricted
transcription factor would not be sufficient to specify distinct cell
types on either side of the line source. Thus, a bipartite signaling
center is utilized (Wg and En/Hh), with each signal acting essentially
unidirectionally to specify a distinct part of the pattern. We do not
know if the En-dependent signal acts directly in a concentrationdependent manner.
establish a block so that each signal operates largely
unidirectionally. Rho is repressed by Wg signaling and it is
important to block activation of the Wg pathway from cells
posterior to the En/Hh domain. This block seems to be Hhdependent as Rho expression is greatly reduced in hh mutants
but maintained in wg smo double mutants. Elegant evidence
for Hh restricting the range of Wg has been presented by
Sanson et al. (1999). Our data also shows that Wg imparts
asymmetry to signals from En cells. Without Wg function, Hh
can signal more strongly to the anterior compared to wild type.
Importantly, the cuticle pattern generated is also now
symmetric relative to the En/Hh cells, strongly suggesting that
a normally biased signal is now sent or received bidirectionally.
Whereas we can show this directly for Hh signaling by
examining Patched protein distribution, proof for asymmetric
functioning of the unknown En-dependent signal awaits its
identification.
The signals expressed from an organizer are
developmentally potent, as they confer pattern over a large
cellular field. Thus, once the appropriate expression of these
signals is established, an important facet to organizer function
is the temporal and spatial restriction of signaling. In some
cases, activation of a signaling pathway induces an inhibitor of
4114 U. Gritzan, V. Hatini and S. DiNardo
that same pathway. For example, we previously showed that
inhibition of signaling was crucial for proper fate specification
by this parasegment organizer (O’Keefe et al., 1997). In
examining fate across the En domain, Rho-dependent
activation of the EGF-R pathway posterior to the En cells leads
to the induction of the diffusible inhibitor, Argos. Argos
attenuates EGF-R activation in anterior En cells, allowing Wg
signaling to win out, leading to proper fate specification of
anterior En cells. In this paper, our data do not address the
possible mechanism(s) that account for the bias in Wg or
En/Hh signaling. The investigation of other pattern organizers
has revealed several mechanisms that constrain signaling
function, some of which apply to Wg or to Hh, such as inhibitor
induction (Leyns et al., 1997), receptor sequestration (Chen
and Struhl, 1996; Cadigan et al., 1998) or directed transcytosis
(Bejsovec and Wieschaus, 1993; Dierick and Bejsovec, 1998).
Most of these rely on the cognate signal affecting its own
signaling properties or responses. The parasegment organizer
is different in one regard, however, as one signal restricts the
function of another.
Organizing pattern across parasegments and
compartments
In discs, signals emanate from compartment boundaries, which
are inherited from the embryonic parasegment boundaries
(Basler and Struhl, 1994; Capdevila and Guerrero, 1994;
Tabata and Kornberg, 1994; Zecca et al., 1995; reviewed in
Lawrence and Struhl, 1996). For the compartment organizer,
Hh locally induces a line source for a long-range morphogen,
either Wg or Dpp, which each act symmetrically. Cells exposed
to the same ligand concentration on opposite sides of the
source, adopt the same positional value (Fig. 7A, asterisks).
However, the anterior compartment cell will select a different
fate from the posterior compartment cell at the same positional
value. This is because the posterior compartment expresses a
unique transcription factor, En, and therefore is programmed
intrinsically with a different response repertoire to the
morphogen (Fig. 7A).
In parasegments, 10 of 12 rows of cells are intrinsically
equivalent anterior compartment cells, while the posterior Enexpressing compartment only accounts for 2 cells. Thus,
compartmental organization, with each compartment sporting
unique transcription factors, can only make a small contribution
toward distinguishing cell fate selection. Perhaps for this reason
patterning cannot rely on induction of one longer-range
morphogen. Instead, a bipartite organizer is used with each
signal acting essentially unidirectionally. Equivalent cells to
each side of the parasegment boundary develop differently
because they are exposed to different signals (Fig. 7b).
We thank Mark Peifer and Bloomington stock center for flies. J. P.
Vincent and E. Wiellette for communicating unpublished results.
Supported by NIH GM45747. U. G. is a German National Scholar
and V. H. is supported by the Cancer Research Fund of the Damon
Runyon-Walter Winchell Foundation Fellowship, DRG-1442.
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