DV patterning in the Drosophila abdomen - Development

13 Pages·1999·1.42 MB·English

Development 126, 3495-3507 (1999) 3495
Printed in Great Britain ©The Company of Biologists Limited 1999
DEV5330
Wingless, Decapentaplegic and EGF Receptor signaling pathways interact to
specify dorso-ventral pattern in the adult abdomen of Drosophila
Artyom Kopp1,*, Ronald K. Blackman2,‡ and Ian Duncan1,§
1Department of Biology, Washington University, St Louis, MO 63130, USA
2Department of Cell and Structural Biology, University of Illinois, Urbana, IL 61801, USA
*Present address: University of Wisconsin-Madison, R. M. Bock Laboratories, 1525 Linden Drive, Madison, WI 53706-1596, USA
‡Present address: Millennium Pharmaceuticals, Inc., 75 Sidney Street, Cambridge, MA 02139, USA
§Author for correspondence (e-mail: [email protected])
Accepted 28 May; published on WWW 19 July 1999
SUMMARY
Adult abdominal segments of Drosophila are subdivided Wg and Dpp interact antagonistically at two levels. First,
along the dorso-ventral axis into a dorsal tergite, a ventral their expression is confined to complementary domains by
sternite and ventro-lateral pleural cuticle. We report that mutual transcriptional repression. Second, Wg and Dpp
this pattern is largely specified during the pupal stage by compete directly with one another by exerting opposite
Wingless (Wg), Decapentaplegic (Dpp) and Drosophila effects on cell fate. DER signaling does not affect the
EGF Receptor (DER) signaling. Expression of wg and dpp expression of wgor dpp, indicating that it interacts with Wg
is activated at the posterior edge of the anterior and Dpp at the level of cell fate determination. Within the
compartment by Hedgehog signaling. Within this region, tergite, the requirements for Wg and DER function are
wg and dpp are expressed in domains that are mutually roughly complementary: Wg is required mainly in the
exclusive along the dorso-ventral axis: wg is expressed in medial region, whereas DER is most important laterally.
the sternite and medio-lateral tergite, whereas dpp Finally, we show that Dpp signaling at the dorsal midline
expression is confined to the pleura and the dorsal midline. controls dorso-ventral patterning within the tergite by
Neither gene is expressed in the lateral tergite. Shirras and promoting pigmentation in the medial region.
Couso (1996,Dev. Biol.175, 24-36) have shown that tergite
and sternite cell fates are specified by Wg signaling. We find
that DER acts synergistically with Wg to promote tergite Key words: wingless, decapentaplegic, EGFR, torpedo, Pattern
and sternite identities, and that Wg and DER activities are formation, Dorso-ventral, Abdominal histoblasts, Drosophila
opposed by Dpp signaling, which promotes pleural identity. melanogaster
INTRODUCTION from imaginal discs in two important respects. First, abdominal
histoblasts do not invaginate during embryogenesis, but remain
While much effort has been devoted to understanding the part of the larval epidermis and secrete larval cuticle. Second,
mechanisms of pattern formation in the embryo and imaginal they do not proliferate during the larval stages. After
discs of Drosophila, the adult abdomen has attracted little pupariation, histoblasts multiply rapidly and migrate to replace
attention. However, its unique mode of development raises the polyploid larval epidermal cells (LEC). As the individual
many intriguing questions about its patterning and offers a nests grow and merge, LEC are destroyed only upon contact
number of practical advantages. The abdominal epidermis has with histoblasts, so that the continuity of the pupal epidermis
a simple geometry (each segment can be regarded as a two- is maintained at all times. The replacement of LEC by
dimensional rectangular field), a wide range of identifiable cell histoblasts is completed by 40-42 hours after puparium
fates, and completely uniform polarity. The absence of cell formation (APF) (Madhavan and Madhavan, 1980). The
competition in the abdomen greatly simplifies clonal analysis. epidermis of each abdominal segment is produced by three
Most importantly, in contrast to the imaginal discs (Serrano and bilateral pairs of histoblast nests: the anterior dorsal nests
O’Farrell, 1997), patterning and growth in the abdomen appear produce the tergite, the posterior dorsal nests form the flexible
to be uncoupled. Even drastic pattern changes have little effect intertergal cuticle, and the ventral nests produce the sternite
on cell proliferation, and do not affect the final shape and size and pleura. In addition, a spiracular nest produces a small patch
of abdominal segments. of epidermis around each spiracle.
The adult abdominal epidermis develops during the pupal The antero-posterior (AP) patterning of abdominal segments
stage from groups of cells called histoblast nests, which differ is controlled partly by the Hedgehog (Hh) morphogen (Struhl
3496 A. Kopp, R. K. Blackman and I. Duncan
et al., 1997a,b; Kopp et al., 1997) and partly by an hh- and hs-FLP1 (Dang and Perrimon, 1992). Clones were induced by heat
independent mechanism of unknown identity (Kopp et al., shocking hs-FLP122; Tub>f+>dpp or hs-FLP1/Act>CD2>GAL4;
1997; A. Kopp and I. Duncan, unpublished). The patterning UAS-wg/UAS-lacZ or hs-FLP1/ Act>CD2>GAL4; UAS-dpp/UAS-
function of Hh is mediated primarily by optomotor-blind(omb) lacZ first or second instar larvae at 37(cid:176) C for 10-30 minutes in a
circulating water bath.
(Kopp and Duncan, 1997), which encodes a putative T-box
transcription factor (Pflugfelder et al., 1992). Surprisingly, the
Other mutations used
secreted morphogens Wingless (Wg) and Decapentaplegic
topCA/top2W74 heterozygotes show a severe loss of DER function at
(Dpp), which act downstream of Hh in the long-range AP 25(cid:176) C, but not at 18(cid:176) C (Clifford and Schupbach, 1992). Mad12 is a
patterning of imaginal discs, are not involved in AP patterning presumed null allele of Mothers against dpp, the transcription factor
in the abdomen (Shirras and Couso, 1996; Struhl et al., that activates dpptarget genes (Sekelsky et al., 1995). stubby chaete
1997a,b; Kopp et al., 1997). Rather, Hh functions directly to (stc), a mutation that reduces the size of bristles and trichomes (Struhl
specify cell fates along the AP axis (Struhl et al., 1997b). et al., 1997b), was used as a cell-autonomous marker for Mad12
The functions of Wg and Dpp in the adult abdomen are clones.
limited to dorso-ventral (DV) patterning. Wg has been shown
by Shirras and Couso (1996) to promote tergite and sternite, as
opposed to pleural, cell fates. Here, we report that signaling by RESULTS
the Drosophila EGF receptor (DER) acts in concert with Wg
to promote tergite and sternite identities, and that Wg and DER The wild-type cuticular pattern in the adult abdomen
activities are opposed by Dpp signaling, which promotes The cuticle of the adult abdomen is differentiated into three
pleural identity. We show that Wg and Dpp limit each other’s distinct regions along the DV axis (Fig. 1A). The ventralmost
expression domains by mutual transcriptional repression, and structure of a typical abdominal segment is the sclerotized,
that cell fates are determined by the balance between unpigmented sternite. The ventro-lateral position is occupied
competing Wg and Dpp activities. Finally, we demonstrate that by the flexible, unsegmented pleural cuticle, which is also
Dpp signaling controls tergite pigmentation near the dorsal present mid-ventrally between the sternites of consecutive
midline. segments. Dorsally, each segment is composed of a sclerotized,
pigmented tergite and flexible, unpigmented intertergal cuticle
that is normally folded underneath the tergite. All cells in the
MATERIALS AND METHODS sternites, pleura and tergites secrete 3-4 trichomes per cell. The
wide-based, curved trichomes secreted by pleural cells are
Staining procedures and cuticular preparations distinct from the thinner, straighter sternal and tergal trichomes
In all figures, anterior is up and dorsal is to the right. Preparation of (Fig. 1B). In addition, sternites and tergites, but not the pleura,
adult abdominal cuticles and pupal body walls, antibody and X-Gal contain arrays of bristles. Dorsoventral patterning is also
staining and in situ hybridization were performed as described (Kopp present within tergites, as the dark pigment band at the
et al., 1997). For X-Gal/antibody double staining, unfixed pupal posterior edge of each tergite is wider medially than laterally.
abdomens were stained in standard X-Gal staining solution for 10-20
Some segments deviate from the typical pattern. For example,
minutes at room temperature, fixed in 3% formaldehyde in PBS for
the first abdominal segment (A1) lacks a sternite. Also, in the
20 minutes, and processed according to standard antibody staining
male, A7 lacks both a sternite and a tergite, A6 lacks bristles
procedures.
on its sternite, and A5 and A6 have uniformly darkly
wg expression was detected using the enhancer trap lines
P{ry+t7.2=PZ}wgr0727 (G. Rubin, BDGP) and P{ry+t7.2=PZ}wg02657 pigmented tergites (Fig. 1A).
(A. Spradling, BDGP). Both lines (referred to as wg-lacZ) accurately The adult abdominal epidermis is formed during the first 40-
reproduce the pattern of wgtranscript expression in the pupal abdomen 42 hours of pupal development. At pupariation, the abdominal
(not shown), and were used interchangeably. dpp expression was epidermis is composed predominantly of the polyploid LEC,
detected using the P{ry+t7.2=PZ}dpp10638 enhancer trap (dpp-lacZ; which are easily distinguishable from the much smaller, diploid
Twombly et al., 1996) and by RNA in situ hybridization. The omb-
GAL42 enhancer trap was isolated by Y. H. Sun (unpublished). The
expression of GAL4 lines was monitored using the UAS-lacZreporter
of Brand and Perrimon (1993). We find that most enhancer traps,
including dpp-lacZ and wg-lacZ, become expressed ubiquitously in
LEC after 10-12 hours APF, and thus cease to be useful for monitoring
gene expression in LEC.
Ectopic expression
The UAS lines used were UAS-DN-DER (O’Keefe et al., 1997); UAS-
wg(Lawrence et al., 1995); UAS-dpp(dppUAS.cSa) (Staehling-Hampton
et al., 1994); UAS-DN-dTCF (dTCFD N) (van de Wetering et al., 1997);
UAS-l top (constitutively activated DER; Queenan et al., 1997); and
UAS-armS10 (constitutively activated Armadillo; Pai et al., 1997). Fig. 1. Adult abdominal cuticle of Drosophila melanogaster.
GAL4 drivers used were T155-GAL4 (Harrison et al., 1995) and dpp- (A)Abdominal cuticle of a wild-type male. The abdomen has been
GAL4 (GAL4dpp.blk1) (Staehling-Hampton et al., 1994). UAS/GAL4 cut open along the dorsal midline and mounted flat. (B) High
combinations were kept at 17(cid:176) C until the late larval or early pupal magnification of the sternite-pleura boundary. Note the difference
stage, when they were shifted to the desired temperature (25-31(cid:176) C). between the thin, straight sternal and the wide-based, curved pleural
Flp-out clones were generated using Tub>f+>dpp and hs-FLP122 trichomes. Abbreviations in this and following figures: St, sternite;
(Zecca et al., 1995), Act>CD2>GAL4 (Pignoni and Zipursky, 1997) Pl, pleura; T, tergite.
DV patterning in the Drosophilaabdomen 3497
Fig. 2. wgand dppexpression in the early pupa. In this and following figures, anterior is up and dorsal is to the right. (A) Schematic
representation of wgand dppexpression at pupation (12 hours APF). enexpression is shown in pink, dppexpression in purple, wgexpression in
yellow, and overlapping dppand wgexpression in green. aDHN is located within the anterior compartment, the pDHN is within the posterior
compartment, and the VHN straddles the compartment boundary. The posterior limit of dppexpression coincides with the compartment
boundary (Fig. 5H). The same is likely to be true for wgexpression. In (B-E) histoblast nests are outlined by red broken lines. Two consecutive
segments are shown in each of these panels; thick vertical arrowheads point to specialized larval border cells (Kopp et al., 1997) that mark
segmental boundaries. (B) At approx. 18 hours APF, dpp-lacZis expressed in a dorsal sector in the VHN and in a few cells at the dorsal aDHN
margin (thin arrows); no expression is seen in the pDHN. All LEC are also labelled, since dpp-lacZbecomes ubiquitously expressed in LEC at
approx. 10-12 hours APF. (C) dpptranscript expression at approx. 20 hours APF detected by RNA in situ hybridization. Note the expression in
a dorsal sector in the VHN and in the LEC at the ventral and dorsal margins of the DHN (curved arrows). (D) wg-lacZexpression in the VHN
and ventral LEC at approx. 20 hours APF. (E) wg-lacZexpression in the aDHN and dorsal LEC at approx. 18 hours APF; no expression is seen
in the pDHN (arrow). Abbreviations in this and following figures: VHN, ventral histoblast nest; aDHN, anterior dorsal histoblast nest; pDHN,
posterior dorsal histoblast nest; DHN, combined dorsal nest; DM, dorsal midline; VM, ventral midline; LM, lateral midline.
histoblasts. At this stage, the anterior dorsal histoblast nest posterior region of the anterior compartment (Fig. 2). Within
(aDHN) contains 13-19 cells, the posterior dorsal nest (pDHN) this zone, the patterns of wg and dpp are largely
contains 5-8 cells and the ventral nest (VHN) contains 9-13 complementary along the DV axis. wgis expressed in a dorsal-
cells (Madhavan and Madhavan, 1980). Histoblasts begin to posterior sector of the aDHN and the adjacent dorso-lateral
proliferate and migrate to supplant the LEC soon after LEC, as well as in a ventral sector of the VHN and the adjacent
pupariation. At 18-20 hours APF, the aDHN and pDHN merge ventro-lateral LEC (Shirras and Couso, 1996; Fig. 2A,D,E). wg
to form a single dorsal histoblast nest (DHN) (Fig. 2). The is not expressed in the dorsal part of the VHN, in the ventral
DHN merges with the VHN and the spiracular anlage between part of the aDHN or in the lateral LEC between the two nests.
20 and 28 hours APF (Fig. 3). The spiracle, located at the wgexpression is also weak or absent in the LEC near the dorsal
lateral midline, marks the boundary between ventral and dorsal and ventral midlines. dppis expressed in a dorsal sector of the
histoblasts, and eventually the boundary between the pleura VHN and in a few cells at the dorsal margin of the aDHN. dpp
and the tergite. The fusion of histoblast nests of consecutive expression is also seen in the lateral LEC between the VHN
segments begins at 28 hours APF and proceeds until 40-42 and aDHN, and in the dorsal LEC between contralateral dorsal
hours APF, when the formation of a continuous adult epidermis nests (Fig. 2A-C).
is completed by the fusion of contralateral nests at the dorsal The pupal expression of wgevolves from the pattern present
and ventral midlines. Morphological differentiation of the in the larva, where wg is expressed in a circumferential stripe
epidermis into sternite, tergite and pleural territories becomes along the AP compartment boundary (not shown). The early
evident shortly thereafter. These regions can be distinguished pupal pattern develops by gradual elimination of expression at
at 45 hours APF by differences in the shape and arrangement the ventral, dorsal and lateral midlines (Shirras and Couso,
of cells and by the pattern of developing adult muscles (Fig. 1996, and our observations). dpp is not expressed in the
4). epidermis of third instar larvae, as judged by in situ
hybridization and lack of expression of the dpp10638 enhancer
Expression of wgand dppin the pupal abdomen trap. However, the expression of dppin the pupa is reminiscent
The origin of the adult wg and dpp patterns can be traced to of the embryo, where it is expressed in mid-dorsal and ventro-
the early pupal stage. At the time of fusion of the aDHN and lateral stripes (St Johnston and Gelbart, 1987). Thus, the pupal
pDHN (18 hours APF), wg and dpp expression domains expression of dppmay reflect some memory of this embryonic
encompass both adult and larval cells, and are limited to the pattern.
3498 A. Kopp, R. K. Blackman and I. Duncan
Fig. 3. wgand dppexpression at the time of fusion of the ventral and dorsal histoblast nests. In (B-E) histoblast nests are outlined by red broken
lines, and segmental boundaries are indicated by thick arrowheads. (A) A summary of en, wgand dppexpression patterns, colored as in Fig.
2A. wgand dppexpression in the LEC could no longer be monitored at this stage due to the ubiquitous derepression of the enhancer traps.
(B)At approx. 28 hours APF, dpp-lacZexpression is seen in the dorsal part of the VHN and in a few cells at the ventral DHN margin (arrow);
the latter expression is transient (compare with Fig. 4B). (C) dpp-lacZexpression at the dorsal DHN margin at approx. 30 hours APF. lm,
persisting larval muscle. (D) wg-lacZexpression in the VHN at approx. 30 hours APF; note the low level of expression near the ventral midline.
(E) wg-lacZexpression in the DHN at approx. 30 hours APF; note the low level of expression near the dorsal midline.
The patterns of wg and dpp expression established by 18 3A,D,E). dppis expressed in a stripe in the dorsal two-thirds of
hours APF are maintained during the subsequent growth of the the VHN and in a group of 30-40 cells at the dorsal DHN
histoblast nests. At the time of fusion of the VHN and DHN margin (Fig. 3A-C). dppexpression also extends transiently into
(24-28 hours APF), wg expression is seen in sectors in the the ventral DHN margin; this expression lasts for only a few
ventral third of the VHN and in the dorsal half of the DHN (Fig. hours, and encompasses about 15 cells at its peak (Fig. 3B).
Fig. 4. wgand dppexpression in the pharate adult. (A) A summary of en, wgand dppexpression patterns, colored as in Fig. 2A. (B) dpp-lacZ
expression at approx. 45 hours APF. The spiracle marks the boundary between the pleura, produced by the VHN, and the tergite, produced by
the DHN (staining in the spiracle in this and following figures is artifactual). Note the differences in the arrangement of cells between the pleura
and the tergite, the creasing of the pleural epidermis, and the emergence of adult muscles (arrows) and intersegmental folds in the tergites. In
this and following panels, thick arrowheads mark segmental boundaries. (C) dpp-lacZis expressed in the pleura, but not in the sternite at
approx. 40 hours APF. (D) dpp-lacZexpression at the dorsal midline at approx. 40 hours APF. (E) At approx. 40 hours APF,wg-lacZis
expressed in most of the medial tergite, but not in the lateral tergite or at the dorsal midline. (F) wg-lacZexpression in the sternite at approx. 40
hours APF. Abbreviations in this and following figures: Sp, spiracle; lm, persisting larval muscle; am, developing adult muscle.
DV patterning in the Drosophilaabdomen 3499
The complementary expression patterns of wg and dpp are Competing activities of dppand wgpromote
retained in the newly formed adult epidermis at 40-42 hours opposite cell fates in the adult abdomen
APF. dppis expressed in a transverse stripe in the presumptive Shirras and Couso (1996) presented evidence that wgfunctions
pleura (Fig. 4A-C) and in a wedge-shaped stripe along the in the pupal abdomen to promote tergite and sternite identities.
dorsal midline of the tergite (Fig. 4A,D). The limits of pleural They found that ectopic wgexpression in hs-wganimals causes
expression of dpp coincide precisely with the sternite-pleura the expansion of tergite and sternite territories at the expense
and tergite-pleura boundaries. wg is expressed in the sternite of the pleura, whereas loss of wg function in wgts animals
and in the medial tergite, but is excluded from the dorsal causes the reciprocal phenotype. We have obtained similar
midline (Fig. 4A,E,F). Neither gene is expressed in a large results. Almost the entire pleura is transformed to tergite and
lateral region of the tergite (Fig. 4A,B,E). The expression of sternite identities following ectopic expression of wg in the
wg and dpp remains restricted to the posterior region of the pleural territory in dpp-GAL4/UAS-wg pupae (Fig. 6C).
anterior compartment, with sharply defined posterior and Expression of a constitutively active form of the Wg
graded anterior boundaries.
Double labelling with Engrailed
(En) shows that the posterior
limit of dppexpression coincides
with the compartment boundary
(Fig. 5H). Based on
morphological landmarks and on
the pattern of lacZexpression in
wg-lacZ/dpp-lacZ pupae (not
shown), the same appears to be
true for wg.
wgand dppare activated
by hhand repressed by en
The expression of wg and dpp
in the pupal abdomen is
regulated along the AP axis by
hh, which activates both genes,
and en, which represses them.
The gain-of-function allele
hhMircauses ectopic expression
of hh in a stripe at the anterior
edge of the DHN, and low level
ectopic expression in the
anterior compartment of the
VHN (Kopp et al., 1997; Fig.
5A). In hhMirheterozygotes, wg
expression in the presumptive
tergite is duplicated, and dpp
expression in the pleura is
expanded to the anterior (Fig.
5B,D). In addition, dpp
expression at the dorsal midline
becomes wider, while wg
expression recedes laterally
(Fig. 5C,D). In the gain-of-
function mutant enApa, en Fig. 5. Regulation of wgand dppexpression along the AP axis. T3 and A1-4 indicate the third thoracic
is expressed ectopically in segment and the abdominal segments 1 through 4. (A) hhtranscript expression in the DHN in hhMir.
anterior compartment histoblasts Note the expression at the anterior edge of the anterior compartment, in addition to the normal
in all abdominal segments posterior compartment expression. Arrowheads mark segmental boundaries. (B) dpp-lacZexpression in
except A1, where its expression the pleura in hhMiris strongly expanded to the anterior (compare with Fig. 4B,C). (C) dpp-lacZ
is nearly normal (Kopp et al., expression at the dorsal midline in hhMiris expanded laterally (compare with Fig. 4D). (D) wg-lacZ
1997; Fig. 5E). In enApa expression in the tergite in hhMiris duplicated along the AP axis. The double peaks of expression are
indicated by curved arrows. Also note that expression is absent near the dorsal midline. (E) Patchy En
heterozygotes, dpp and wg
expression in the anterior compartment in enApa. Arrow points to the AP compartment boundary in A3
are almost completely
(see Kopp et al., 1997). (F) dpp-lacZexpression at the dorsal midline in enApais repressed in A2 and
repressed in A2 and more the more posterior segments, but is normal in A1. (G) wg-lacZexpression in the tergite in enApais
posterior segments, but are repressed in A2 and the more posterior segments, but is normal in A1. (H) Expression of En (brown)
expressed normally in A1 (Fig. and UAS-lacZdriven by dpp-GAL4 (blue). The posterior limit of dppexpression coincides with the AP
5F,G). compartment boundary.
3500 A. Kopp, R. K. Blackman and I. Duncan
Fig. 6.(A) The wild-type cuticular pattern of a typical abdominal segment. The spiracle (Sp) marks the tergite-pleura boundary. (B) T155-
GAL4/ UAS-dpp. The lateral tergite is transformed to pleura, while the sternite is reduced to about one-quarter of normal size. Note that some
bristles develop in the midst of pleural tissue (arrows). A similar phenotype is seen in dpp-GAL4/UAS-dpp (not shown). (C) dpp-GAL4/ UAS-
wg. The pleura is almost entirely transformed to sternite and tergite identities. (D) T155-GAL4/ UAS-DN-dTCF. The sternites are completely
transformed to pleura, although occasional bristles remain (arrowhead). The tergite is also transformed to pleura, with the exception of a small
patch of lateral tergite tissue (arrow). (E) T155-GAL4/ UAS-wgUAS-dppflies are restored almost to wild type. The sternite is slightly
widened, while the lateral tergite margin recedes from the spiracle. (F) A Mad12stc- mitotic clone in the pleura (circled) is transformed to
sternal or tergal identity, as indicated by the formation of an stc- bristle and narrow-based trichomes. Such transformed clones are a small
minority; most display more subtle phenotypes, or retain pleural identity.
transcriptional effector Armadillo (ArmS10; Pai et al., 1997) in Interestingly, the number of bristles remains unchanged,
dpp-GAL4/UAS-armS10 pupae produces an identical resulting in a higher than normal bristle density in the tergite
phenotype (not shown). Interference with Wg signaling has the and in the development of bristles in the pleural tissue near the
opposite effect. dTCF, the product of the pangolin gene, is reduced sternite (Fig. 6B). A similar, but weaker phenotype is
required to activate transcription of wg target genes (Bienz, seen following overexpression of dpp in dpp-GAL4/UAS-dpp
1997; Cavallo et al., 1997; Nusse, 1997; Riese et al., 1997; van pupae (not shown).
de Wetering et al., 1997). We used the GAL4 enhancer trap To examine the effects of loss of dppfunction, we generated
T155 (Harrison et al., 1995), which is expressed ubiquitously mitotic clones homozygous for Mad12, a null allele of Mothers
in the pupal abdomen (not shown), to drive the expression of against dpp. The Mad gene product is required for the
a dominant-negative form of dTCF (DN-dTCF; van de transcription of dpptarget genes (Sekelsky et al., 1995; Heldin
Wetering et al., 1997). When T155/UAS-DN-dTCF pupae are et al., 1997). Surprisingly, only six out of several hundred
raised at 29(cid:176) C, the entire sternite and most of the tergite are clones located in the pleura were transformed to sternite or
transformed to pleura (Fig. 6D). Strikingly, a small patch of tergite identity, as gauged by trichome morphology and the
lateral tergite retains tergal identity, while more medial regions presence of bristles (Fig. 6F). Other clones retained pleural
are completely transformed. identity, or displayed a weak phenotype such as mild
We find that dppfunctions reciprocally and antagonistically sclerotization (not shown). Furthermore, the Dpp receptor
to wgin the pupal abdomen. Ectopic expression of dppexpands components encoded by punt (put) and thickveins (tkv)
the pleura at the expense of sternites and tergites. In (Ruberte et al., 1995) also appear dispensable for pleural
T155/UAS-dpp pupae raised at 29°C, a large lateral region of specification. We tested the effects of loss of Put function in
the tergite is transformed to pleural identity, while the sternite clones homozygous for the strong hypomorphic allele put135
is reduced to about one-quarter of its normal size (Fig. 6B). (Ruberte et al., 1995) and in temperature-sensitive
DV patterning in the Drosophilaabdomen 3501
put135/put10460 heterozygotes (Theisen et al., 1996). Loss of and Struhl, 1996; Johnston and Schubiger, 1996; Penton and
Tkv function was examined in clones homozygous for tkv7, a Hoffmann, 1996; Theisen et al., 1996). wg-lacZ expression is
null (or possibly antimorphic) allele (de Celis, 1997; Twombly reduced dramatically in the abdominal histoblasts of
et al., 1996). None of these mutants had any effect in the pleura T155/UAS-dpppupae raised at 29(cid:176) C from the late third larval
(not shown). We conclude that, although Dpp is able to instar (Fig. 8A). Conversely, the expression of dpp-lacZ is
promote pleural identity, its function in pleural specification is virtually eliminated in the histoblasts of dpp-GAL4/UAS-wg
not essential. pupae raised under the same regime (Fig. 8B,C). The
To study the effects of localized dpp expression, we expression of wgand dppin the LEC was not visibly affected
generated ‘flp-out’ dpp-expressing clones in the abdomen of in these experiments. We see no change in the expression of
f36a hs-FLP122; Tuba 1>f+>dpp flies (Zecca et al., 1995). dpp-lacZin T155/UAS-dpppupae, or in the expression of wg-
When such clones, which are marked by f - bristles, are located lacZin dpp-GAL4/UAS-wgpupae (not shown). Thus, we find
in the sternite or lateral tergite, they cause transformation of no evidence that wg and dpp are subject to autoactivation, as
nearby sternal or tergal tissue to pleural identity (Fig. 7A,B). they are in the leg disc (Brook and Cohen, 1996).
This effect is opposite to that of wg-expressing clones, which Wg and Dpp also appear to compete directly with one
cause transformation of the pleura to tergite or sternite (Shirras another in specifying opposite cell fates. As described above,
and Couso, 1996, and Fig. 7D). To test the autonomy of ectopic Wg expression expands tergite and sternite territories
dpp and wg, we induced flp-out clones in hs- at the expense of the pleura (Fig. 6C), whereas ectopic Dpp
FLP1/Tuba 1>CD2>GAL4; UAS-dpp/UAS-lacZ and hs- expands the pleura at the expense of tergites and sternites (Fig.
FLP1/Tuba 1>CD2>GAL4; UAS-wg/UAS-lacZ flies. In these 6B). However, when wg and dpp are coexpressed in
genotypes, cells that express dpp or wg are marked by T155/UAS-wg UAS-dpp pupae, abdominal pattern is restored
coincident expression of lacZ. We observe that cells well away almost to wild type (Fig. 6E). Since the UAS-wg and UAS-dpp
from the source of Dpp or Wg are transformed, indicating that transgenes are not subject to mutual repression, we conclude
both genes function at a distance (Fig. 7D-F). that pleural and tergite/sternite cell fates are specified by the
We find that wg and dpp repress one another at the balance between competing Wg and Dpp activities.
transcriptional level in the pupal abdomen, as they do in the Direct competition between Wg and Dpp presumably
leg and antennal imaginal discs (Brook and Cohen, 1996; Jiang accounts for the incomplete transformations observed in flp-
Fig. 7. Phenotypes of dpp- and wg-expressing clones; all clones were induced during the first or second larval instar. (A) Tuba 1>dppflp-out
clone in the lateral tergite, marked by f- bristles (arrowheads), transforms nearby tergite tissue to pleura. Note, however, that part of the clone
secretes tergal cuticle. (B) A similar clone induces sternite to pleura transformation. (C) Large Tuba 1>dppclone in the medial tergite, marked
by f-bristles, has no effect on cell fates. (D) An Act5C>GAL4; UAS-wg/UAS-lacZflp-out clone, marked by lacZexpression, causes the
nonautonomous transformation of pleura to tergite. Note the tergal trichomes ventral to the spiracle (long arrows) and an ectopic bristle growing
out of the pleura (arrowhead); both are located in areas that do not express lacZ(blue stain). (E) An Act5C>GAL4; UAS-dpp/UAS-lacZflp-out
clone, marked by lacZexpression, causes the nonautonomous transformation of sternite to pleura. Several remaining sternal bristles are
embedded in the ectopic pleural tissue. (F) Higher magnification of the same clone under phase contrast. Note that the clone, marked by blue
lacZstaining, is surrounded by pleural trichomes. (G) Small wg-expressing clone in the pleura, marked by lacZstaining, has no effect on cell
fates.
3502 A. Kopp, R. K. Blackman and I. Duncan
Fig. 8.Mutual repression of wg
and dpp. Histoblast nests are
outlined by red broken lines;
segmental boundaries are
indicated by black arrowheads.
(A) wg-lacZexpression in
T155/UAS-dppat approx. 18
hours APF. No expression is
seen in the VHN, while
expression in the aDHN is
severely reduced (compare with
Fig. 2D,E). (B) dpp-lacZ
expression in dpp-Gal4/UAS-wg
at approx. 20 hours APF. No
expression is seen in the VHN.
(C) dpp-lacZexpression in dpp-
Gal4/UAS-wgat approx. 40
hours APF. Expression is seen
in only a few scattered cells in
the pleura (curved arrow).
out clones. Although dpp-expressing clones in the lateral with Wg to promote tergite and sternite fates, and is
tergite are associated with tergite to pleura transformations, particularly important in the lateral tergite region.
parts of these clones can secrete tergite cuticle (Fig. 7A). We find that expression of wg-lacZ and dpp-lacZ is not
Moreover, even very large dpp-expressing clones located in the visibly affected in T155; UAS-DN-DER pupae at 29(cid:176) C (not
medial tergite have no effect on cell fates (Fig. 7C). In both shown). This suggests that DER signaling does not regulate wg
cases, it is likely that the effects of ectopic Dpp are or dppexpression, but rather interacts synergistically with Wg
overwhelmed by endogenous Wg. Conversely, small wg- and/or antagonizes Dpp directly at the level of cell fate
expressing clones surrounded by pleural tissue often secrete determination.
pleural cuticle (Fig. 7G), presumably because ectopic Wg is The involvement of DER in tergite specification may explain
out-competed by endogenous Dpp. the surprising resistance of the tergite to the effects of ectopic
dpp. While ectopic expression of wgcan cause transformation
DER and Wg signaling interact synergistically to of the entire pleura to tergite and sternite (Shirras and Couso,
promote tergite and sternite fate 1996; Fig. 6C), ectopic dppis able to transform only the lateral
The behavior of the lateral tergite was initially puzzling to us. edge of the tergite to pleura (Fig. 6B). No further
This region lies between wg- and dpp-expressing territories transformation is achieved by increasing the dosage of UAS-
(the medial tergite and pleura, respectively), but expresses dpp (not shown). However, a strong synergistic effect is
neither gene (Fig. 4). Because of this, and because the ventral observed when dpp and DN-DER are coexpressed in
DHN margin is closer to the source of Dpp than it is to the T155/UAS-dpp; UAS-DN-DER pupae. In the resulting adults,
source of Wg, we expected the lateral tergite to be very the tergite is reduced to a very small patch near the dorsal
sensitive to changes in wg expression. However, this region midline; importantly, the lateral tergite is completely
turns out to be the least sensitive to loss of Wg function. transformed to pleura (Fig. 9E).
Reduction in Wg activity in either wgts (Shirras and Couso, Madhavan and Madhavan (1995) report that histoblast
1996) or T155/UAS-DN-dTCF pupae (Fig. 6D) transforms the proliferation is inhibited in topCA/top2W74heterozygotes raised
entire medial tergite to pleura, yet fails to transform part of at the restrictive temperature. However, direct examination of
the lateral tergite. This observation cannot be explained by the the pupal abdominal epidermis shows that the patterning
effects of Wg and Dpp alone. Indeed, our results indicate that defects we observe in T155; UAS-DN-DER pupae are not due
signaling by the DrosophilaEGF Receptor (DER) pathway is to reduced histoblast proliferation (not shown). The
also involved in specifying tergite and sternite identities. proliferation defects observed by Madhavan and Madhavan
Ubiquitous expression of a dominant negative form of DER likely result from a stronger reduction in DER function than
(DN-DER; O’Keefe et al., 1997) in T155; UAS-DN-DER was achieved in our experiments.
pupae raised at 25-29(cid:176) C causes expansion of the pleura at the
expense of tergites and sternites (Fig. 9A). A similar phenotype dppcontrols tergite pigmentation near the dorsal
is caused by a temperature-sensitive combination of DER midline
alleles, topCA/top2W74, at 25(cid:176) C (Fig. 9B). When DN-DER is In addition to promoting pleural development, Dpp signaling
coexpressed with DN-dTCF, the entire tergite is transformed contributes to DV patterning within the tergite. The pigment
to pleura (Fig. 9D). Conversely, ubiquitous expression of a band that covers the posterior edge of the tergite widens toward
constitutively active form of DER (l top; Queenan et al., 1997) the anterior at the dorsal midline (Fig. 10A). This widening
in dpp-GAL4; UAS-l top or T155; UAS-l top pupae transforms coincides with the mid-dorsal stripe of dpp expression (Fig.
the pleura to tergite and sternite identities (Fig. 9C). These 4D). Two lines of evidence indicate that dppis responsible for
observations suggest that DER signaling functions in concert pigmentation near the dorsal midline. First, ectopic expression
DV patterning in the Drosophilaabdomen 3503
Fig. 9.The role of DER in the DV patterning of the abdomen. (A) T155-
GAL4; UAS-DN-DER transforms lateral tergite and most of the sternite
to pleura. (B) A similar phenotype is produced in topCA/top2W74
heterozygotes at 25(cid:176) C. (C) UAS-l top; dpp-GAL4 transforms the pleura to
tergite and sternite identities. Arrowhead points to a bristle in the ectopic
tergite tissue. (D) UAS-DN-DER; T155-GAL4/ UAS-DN-dTCF. Sternite
and tergite tissues are completely eliminated, including the lateral tergite
(compare with Fig. 6D). (E) UAS-DN-DER; T155-GAL4/ UAS-dpp. The
tergite is reduced to a small patch near the dorsal midline, pointing to a
strong synergy between ectopic dppand DN-DER (compare with A and
with Fig. 6B). The sternite is also greatly reduced.
of dpp in T155/UAS-dpp pupae leads to expansion of the and Duncan, 1997), which encodes a putative T-box
pigment band (Fig. 10B). Second, Mad12 clones at or near transcription factor (Pflugfelder et al., 1992). ombexpression is
the dorsal midline show loss of pigmentation (Fig. 10C,D). activated along the posterior edge of the tergite by Hh protein
Surprisingly, a partial loss of pigmentation is often observed secreted by posterior compartment cells (Kopp and Duncan,
in Mad12clones at positions lateral to the anterior inflection of 1997). ombmay also be responsible for pigmentation along the
the pigment band, suggesting that Dpp signaling contributes to dorsal midline. We find that the omb-GAL42enhancer trap (Y.
pigmentation over a wide medial region of the tergite. H. Sun, unpublished), which appears to reflect accurately the
Away from the dorsal midline, the width of the pigment band pattern of omb transcript expression in the pupal abdomen, is
is controlled by the expression of optomotor-blind(omb) (Kopp expressed more extensively near the dorsal midline than
Fig. 10.dppcontrols tergite
pigmentation near the dorsal
midline. (A) Wild-type pigment
pattern at the dorsal midline
(DM). (B) Expansion of the
pigment band in the medial
tergite in T155-GAL4/ UAS-
dppflies. Increased bristle
density is due to a reduction in
tergite area, rather than to an
increase in bristle number.
(C)A Mad12stc- clone in the
medial tergite (outlined in red)
causes partial loss of
pigmentation. Note that the loss
of pigment is more severe
closer to the dorsal midline.
(D) An identical clone located further laterally (outlined in red) has no effect on pigmentation. (E) omb-GAL42/UAS-lacZexpression widens
near the dorsal midline. (F) Tergite pigmentation in D. mulleri. Note the patch of pigment at the lateral tergite margin, the anterior inflection of
the pigment band in the medial tergite and the gap at the dorsal midline.
3504 A. Kopp, R. K. Blackman and I. Duncan
elsewhere in the tergite (Fig. 10E). Also, omb- hemizygotes rather than transient, expression in the lateral tergite (Fig.
lack midline as well as posterior pigmentation (Kopp and 11B,E). The tergite expression is controlled in part by a distinct
Duncan, 1997). However, ectopic dppexpression in T155/UAS- element, located between 112.3 kb and 113.5 kb (Fig. 11C).
dpppupae does not consistently upregulate omb(not shown). A second enhancer region active in histoblasts (the
‘circumferential enhancer’) is located between 117.2 kb and
cis-regulatory sequences that control dpp 118.9 kb. This fragment drives expression in a stripe that
expression in the pupal abdomen extends around almost the entire segment, interrupted only at
To understand better the regulation of dpp in the abdomen, we the ventral midline and near the spiracle (Table 1; Fig. 11D,F).
tested genomic fragments from the 3¢ region of dpp(Blackman Presumably the activity of this enhancer is normally repressed
et al., 1991; R. K. B., unpublished) for the ability to drive lacZ in the tergite and sternite territories by other regulatory regions.
expression in the pupal epidermis. We find that dppexpression We have not identified sequences responsible for dpp
in the histoblasts and in the LEC is controlled by separate expression along the dorsal midline.
enhancer elements (Table 1; Fig. 11). Both the pleural and circumferential histoblast enhancers are
Histoblast expression is regulated by two distinct regions responsive to hh. Expression of the BS 3.21 reporter construct,
(Table 1; Fig. 11B-F). Fragments from between 109.5 kb and which is representative of the pleural enhancer (Table 1), is
113.5 kb on the standard dpp genomic map (Blackman et al., strongly expanded to the anterior in the hhMir gain-of-function
1991; Merli et al., 1996) drive lacZ expression in the mutant, whereas expression of the BS 4 construct, which contains
developing pleura, but not in the sternite or most of the tergite the circumferential enhancer, is duplicated (not shown). Both
(Fig. 11B,F). Accordingly, we refer to this region as the pleural enhancers are repressed by wg, although to differing extents. BS
enhancer. The dpp-GAL4 reporter of Staehling-Hampton et al. 4 expression in the tergite (but not in the pleura) is completely
(1994) is driven by a fragment that extends from 106 to eliminated in hs-wgpupae grown at high temperature overnight,
approximately 110.8 kb, and is expressed in a similar pattern whereas BS 3.21 expression is only weakly affected (not shown).
(Fig. 11E). Unlike the endogenous dpp pattern, some of the dpp expression in the LEC is controlled by an entirely
fragments from the 109.5-113.5 kb region drive persistent, separate region (Table 1; Fig. 11A,F). Fragments located
Fig. 11.Genomic fragments from the dpplocus driving lacZexpression. See Table 1 for construct information. (A) The BS 2 construct is
expressed in the dorsal and lateral LEC, but not in any of the histoblast nests (outlined in red). (B) BS 3.21 is expressed in the pleura and lateral
tergite margin, but not in the sternite or the remainder of the tergite. (C) BS 3.3 is expressed exclusively at the lateral tergite margin. (D) BS 4 is
expressed around the entire circumference of the segment, with only small gaps at the ventral midline and near the spiracle. (E) UAS-lacZ
expression driven by dpp-GAL4. Note that dpp-GAL4, unlikedpp-lacZ(Fig. 4B) or dpptranscript (not shown), shows stable expression at the
lateral tergite margin. (F) A summary of genomic regions that drive dpp expression in the pupal abdomen (modified from Blackman et al.,
1991).