Development 111, 657-665 (1991)
Printed in Great Britain ©'The Company of Biologists Limited 1991
657
An extensive 3' c/s-regulatory region directs the imaginal disk expression
of decapentaplegic, a member of the TGF-/J family in Drosophila
RONALD K. BLACKMAN1*, MICHELE SANICOLA1, LAUREL A. RAFTERY1, TRUDY GILLEVET2
and WILLIAM M. GELBART1
1
Department of Cellular and Developmental Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138-2097, USA
Department of Cell and Structural Biology, University of Illinois, 506 Morrill Hall, 505 S. Goodwin Avenue, Urbana, IL 61801, USA
2
•Present address: Department of Cell and Structural Biology, University of Illinois, 506 Morrill Hall, 505 S. Goodwin Avenue, Urbana,
IL 61801, USA
Summary
The decapentaplegic (dpp) gene in Drosophila melanogaster encodes a TGF-/Mike signalling molecule that is
expressed in a complex and changing pattern during
development. One of dpp's contributions is to proximaldistal outgrowth of the adult appendages, structures
derived from the larval imaginal disks. Appendage
specific mutations of dpp fall in a 20 kb interval 3' to the
known dpp transcripts. Here, we directly test the
hypothesis that these mutations define an extended 3'
cis-regulatory region. By analysis of germ-line transformants expressing a reporter gene, we show that
sequences from this portion of the gene, termed the
dppdhk region, are capable of directing expression
comparable to that defined by RNA in situ hybridiz-
ation. We localize two intervals of the dppf°sk region that
appear to account for much of the dpp spatial pattern in
imaginal disks and discuss the positions of these
important elements in terms of the genetics of dpp.
Finally, we provide evidence to suggest that one of our
constructs expresses /J-galactosidase in the early imaginal disk primordia in the embryo, at approximately
the tune when they are set aside from surrounding larval
epidermal tissues. Thus, dpp may be involved directly in
the determination of the imaginal disks.
Key words: Drosophila, decapentaplegic, imaginal disks,
enhancer elements, ci?-regulation, TGF-/3 superfamily.
The dpp gene product contributes to numerous
developmental events in the fly. In the early embryo, it
The decapentaplegic gene in Drosophila melanogaster is is a major determinant of dorsal versus ventral
ectodermal fate (Irish and Gelbart, 1987; St. Johnston
one of the few well-characterized genes that contribute
and Gelbart, 1987; Ray, Arora, Nusslein-Volhard and
to pattern formation through their action as a comGelbart, in preparation). Later in embryogenesis, dpp
ponent of an intercellular communication system. Its
participates in an inductive interaction between the
protein product is a member of the transforming growth
visceral mesoderm and the endoderm in the morphofactor-/? (TGF-/3) superfamily and it is thought to act as
genesis of the larval gut (Immergllick et al. 1990;
a secreted signalling factor by binding to cell surface
Panganiban et al. 1990). It is also required in the
receptors on target cells (Padgett et al. 1987; Panganiimaginal disks for proper proximal-distal development
ban et al. 1990). Numerous factors in the TGF-/? family
of adult appendages (Spencer et al. 1982; Posakony et
have been implicated in profound developmental
al. 1990). In addition, the gene is expressed in the pupa
events, such as mesoderm induction in Xenopus (Smith,
and the adult (St. Johnston et al. 1990), suggesting other
1989), bone morphogenesis in mammals (Wozney et al.
as yet uncharacterized functions.
1988; Lyons et al. 1990) and regression of the Mtillerian
ducts in male mammalian fetuses (Cate et al. 1986). We
The developmental complexity of dpp is reflected in
are exploiting the genetic advantages of Drosophila
the structure of the gene. The dpp gene has been
melanogaster to attempt to understand how members of functionally divided into three domains: shv, Hin and
this protein family contribute to developmental events.
disk (St. Johnston et al. 1990) (Fig. 1). Transcripts are
One aspect of this work is to define in detail the spatial
entirely restricted to the shv and Hin domains. Multiple
and temporal expression of the gene, and to relate its
promoters in the shv and Hin regions are utilized, but
expression pattern to specific mutant phenotypes.
each of the RNAs contains the same protein coding
Introduction
658
R. K. Blackman and others
information (located in the Hin region). Thus, dpp
encodes a single polypeptide (St. Johnston et al. 1990;
Padgett et al. in preparation).
By a combination of classical mutant analysis and
germ-line transformation studies, it has been possible to
localize regions of the gene that direct dpp's several
developmental events. In addition to its protein coding
capacity, the Hin region is specifically required for
dorsal-ventral patterning of the embryo (Hoffmann
and Goodman, 1987; Irish and Gelbart, 1987; St.
Johnston et al. 1990). The shv region controls proper
gut morphogenesis in the larva, and wing vein and head
capsule formation in the adult (Segal and Gelbart, 1985;
Immergliick et al. 1990; Hursh and Gelbart, in
preparation). Uniquely, the disk region is required for
elaboration of proximal-distal adult appendage development (Spencer etal. 1982). This report focuses on the
contributions of the disk region to dpp function in the
appendages.
The disk region is at least 20 kb in size and is located
entirely 3' to the transcribed shv and Hin regions. It is
defined by an array of mutations that engender defects
confined to adult appendages, structures derived from
the imaginal disks of the larva. Most of these mutations
are associated with gross chromosomal rearrangements
- inversions and translocations - or with deletions
breaking within the disk region and extending out of the
gene into adjacent loci (St. Johnston et al. 1990). These
rearrangement dppd"k alleles can be sorted into three
phenotypic groups (Spencer et al. 1982).
The disk-V alleles are the most severe. These alleles,
in homozygotes, lead to such extensive loss of imaginal
tissue that death occurs early in pupariation. The diskIll alleles are intermediate in severity. In essence, these
alleles engender the loss of distal material from all of
the adult appendages. The disk-II alleles are the mildest
of the disk region breakpoint mutations. These alleles
cause milder defects in the wing, haltere and male
genitalia. The breakpoints within dpp of these disk
alleles fall in an order that is co-linear with the severities
of the mutant phenotypes they engender (Fig. 1). The
most severe disk-V alleles have breakpoints in a 8 kb
region nearest to the shv-Hin transcription unit. The
milder disk-Ill alleles are all broken within an adjacent
10 kb region. Finally, the mildest disk-II alleles interrupt a 2 kb region furthest from shv-Hin. These and
other data have led us to propose that the disk region
represents an array of 3' cis-regulatory elements
necessary to drive proper expression of the shv-Hin
transcription units in the larval imaginal disks (St.
Johnston et al. 1990).
In this report, we directly test this hypothesis by an
analysis of reporter gene constructs introduced into flies
by germ-line transformation. We demonstrate that
segments of the dppdisk region, when adjacent to a
heterologous promoter and a lacZ reporter gene, can
direct expression of /3-galactosidase in localized patterns in the imaginal disks, correlating well with the
distribution of dpp transcripts. Our findings show that
important regulatory information falls at the boundaries between the three classes of disk breakpoint
alleles. Finally, embryonic /3-galactosidase patterns
displayed by one of our constructs suggest that dpp may
be expressed in imaginal disks from the time they are
set aside from surrounding larval epidermal tissues.
This raises the possibility that dpp may contribute
directly to the determination of the imaginal disks.
Materials and methods
Plasmid constructions
The present studies have made use of the following
P-element-based /3-galactosidase vectors: pSXhLac-7 (Logan
and Wensink, 1990), KiSHZ194 (kindly provided by JeanPaul Vincent (UCSF, San Francisco)) and HZ50PL (Hiromi
and Gehring, 1987). In the first two plasmids, the hsp70
promoter begins at -194 and in the third, the promoter begins
at -50. KiSHZ194 differs from pSXhLac-7 in that the lone
Sail site of pSXhLac-7 has been replaced with a Kpnl site.
Each of the vectors contains the rosy+ gene to serve as a
selectable marker for germ-line transformation. In the
plasmids described below (see also Fig. 1), the dpp sequences
are inserted immediately 5' of the hsplO promoter.
BS1.0: An 8.2 kb Sail fragment (dpp coordinates 92-100.2)
from phage Aho-2 was inserted in the Sail site of pSxhLac-7 to
yield BS1.0.
BS1.1: A 1.8kb EcoRI fragment (98.4-100.2) was subcloned into pBluescript. The DNA was digested with Sail and
Xbal (sites in the polylinker) and inserted into the same sites
in pSXhLac-7.
BS2.0: A Sail fragment (100.2-104.5), obtained from
phage Aho-2, was cloned into the Sail site of pSXhLac-7.
BS3.0: A 12 kb EcoRI fragment (106.9-116.9) from phage
22-8 was subcloned into plasmid pUC1813 (Kay and
McPherson, 1987). This DNA was digested with Kpnl (106.9)
and Xbal (in the polylinker next to 116.9) and the dpp
fragment inserted at the Kpnl and Xbal sites of KiSHZ194.
BS3.1: A Hindm-PstI fragment (109.5-110.3) from clone
pUC109H was subcloned into plasmid pPolyII-sfi/not-14
(Lathe et al. 1987). This DNA was digested with Xbal and
Notl (sites in the polylinker) and the disk region sequence
inserted into the same sites of HZ50PL.
Germ-line transformation
Transgenic animals were constructed using standard procedures
(Spradling,
1986).
BS
plasmid
DNA
(400-500 ^g ml"1) and pjr25.7wc DNA (100 jig ml"1) (Karess
and Rubin, 1984) were co-injected into either en; ry42 or ry506
embryos. Homozygous or balanced stocks were prepared for
each line. In some cases, additional transgenic lines were
prepared by the mobilization of an extant insert with the A2-3
P-element (Robertson et al. 1988).
Histochemical fi-galactosidase staining
Transgenic larvae were bissected in PBS (340 mM NaCl,
6.7 mM KC1, 3.7 mM KH2HPO4, pH7) and each half was
turned inside-out. The tissues were fixed for 3min in 4%
formaldehyde in PBS, washed in PBS, and transferred to
X-Gal Solution (150 mM NaCl, lmM MgCl2, 3.3 mM
K4(Fe[CN]6), 3.3 mM K3(Fe[CN]6), 10mM sodium phosphate,
pH7, saturated with 5-bromo-4-chloro-3-indolyl-/£-D-galactopyranoside). Incubations continued for 2-24 h at room
temperature, after which the tissues were washed in PBS, and
mounted in 50% glycerol, lxPBS, and photographed under
Nomarski optics.
dpp expression in imaginal disks
Antibody staining of embryos
Antibody staining of embryos was done according to
MacDonald and Struhl (1986) using rabbit anti-/S-galactosidase from Cappel, biotinylated goat anti-rabbit IgG from
Vector, and a streptavidin-horseradish peroxidase conjugate
from Zymed. In some cases the chromogenic reaction was
carried out in the presence of 0.03 % NiCl2. Embryos were
mounted in 50% glycerol, lxPBS and photographed using
Nomarski optics.
To examine the staining patterns produced by BS3.0 in
dppdbkv mutant embryos, progeny were generated from the
following cross. Df(2L)dppdY" Bl TnBS3.0/+(Jdl were
crossed to In(2L)dpp" /+$?$. To examine the staining
patterns of BS3.0 in embryos deleted for the achaete-scute
complex (AS-CT), Df(1)260.1 was employed. Df(1)260.1
(Lindsley and Zimm, 1987) is deleted for the 1A1 to 2B4-6
region, including the AS-C. First, Df(l)260.1/In(l)FM4 $ ?
were crossed to line 6727 cfcf. (Line 6727 (provided by Y.
Hiromi) contains the transposon P[ry+,ftz/lacF] in the
polytene subdivision IB. This transposon produces a /8galactosidase pattern in embryos which is easily distinguishable from that produced by the BS3.0 transposon.)
Df(1)260.1/6727 $ $ were then mated with cTcf homozygous
for an insertion of the BS3.0 transposon on the second
chromosome. The lack of the 6727 pattern in an embryo
stained for the ^-galactosidase protein indicated the deletion
of the AS-C in that embryo.
In situ hybridizations to whole-mount larval tissues
In situ hybridization to intact imaginal disks and larval brains
were performed by the protocol of Masucci et al. (1990),
except that the hybridization time was reduced to 24 h.
Digoxigenin labelling of the dpp cDNA E55 was performed as
detailed by Masucci et al. except that the entire E55 cDNA
clone was used for the hybridizations and that Alul and
Haelll were used to digest the DNA before labelling.
V / III
boundary
72
78
—I-
86
—r-
94
—h
T102
659
Results
As defined by the molecular localization of mutant
lesions, the disk region of dpp minimally extends from
kilobase coordinates 92 to 112 on the molecular map of
the gene (Fig. 1). We have scanned nearly all of this
region for evidence of ds-regulatory information
capable of activating a reporter gene construct. The
disk region was divided into three segments according
to convenient restriction sites (BS1.0, BS2.0 and BS3.0)
(Fig. 1), and each was inserted just 5' to an hsp70
promoter-/acZ reporter gene fusion in a P elementcontaining plasmid. Multiple germ-line transformants
of these constructs were obtained (see Materials and
methods for details) and balanced or homozygous
stocks of each transposon line were derived. At least 3
lines were examined for each construct. For each of the
lines, histochemical staining was used to analyze the
patterns of expression of /J-galactosidase in late third
instar larvae. These patterns were compared with dpp
transcript accumulation as assayed by whole-mount
RNA in situ hybridization.
The locations of major enhancer elements controlling
spatial patterning in the imaginal disks
For constructs BS1.0 and BS3.0, the spatial distribution
of /S-galactosidase expression is consistent within
several independent lines and is restricted to regions
which show localization of authentic dpp transcripts.
Expression is observed in every imaginal disk, typically
in a stripe running roughly along the anterior-posterior
(A-P) compartment boundary, consistent with the
results of earlier studies (Posakony et al. 1990; Spencer,
III / II
boundary
* 10
118
—t—
coordinate* (kb)
disk
Fig. 1. Schematic representation of the dpp gene with diagrams of the reporter gene constructs. The coordinates of the
dpp gene (St. Johnston et al. 1990) are listed across the top. The extent of the genetically defined shv, Hin and disk regions
are noted by boxes. The disk region box is subdivided into the class V, class III and class II regions. The limits of these
regions are shown below the disk region box. The locations of the boundaries between the class V-class III and the class
Ill-class II regions are highlighted by two large arrows above the coordinate line. Transcripts from the two major dpp
promoters are shown beneath the shv and Hin regions. At least three other minor promoters, located further 5', have been
identified. The reporter gene constructs described in this paper are shown below and to the right. The black box indicates
the extent of the DNA from the disk region that was used in each construct, flanked by its map coordinates. In each case
the disk region fragment is located 5' to the hsp70 promoter (gridded) and the lacZ gene (hatched). The name of each
construct is listed to the right.
660
R. K. Blackman and others
Fig. 2. Expression of dpp is detected in the brain of third
instar larvae. Two lateral and two medial spots of dpp
expression are observed in the larval brain. (A) dpp
transcription pattern detected in the brain by RNA in situ
hybridization. Stronger hybridization is seen to the lateral
spots which are on the cortex and nearer to the surface
than to the subcortical medial spots. (B) dpp-lacZ
construct BS3.0 directs the expression of /S-galactosidase in
a spatial pattern that reflects the distribution of dpp
transcripts seen in the brain.
Raftery and Gelbart, in preparation). Some sites of
expression are common to both BS1.0 and BS3.0, while
others are distinctive. Neither construct alone expresses
the full pattern of dpp transcript accumulation. BS3.0 is
also expressed in 4 spots in each lobe of the larval brain;
dpp transcripts accumulate in at least two and possibly
all four of these spots (Fig. 2). With the exception of
some artifactual expression due to sequences in the
basic reporter gene module (data not shown), no other
expression of BS1.0 or BS3.0 is seen in the larva.
In contrast to these results, BS2.0 has no /Jgalactosidase expression in the imaginal disk epithelium. Weak expression is seen only in limited regions
of the peripodial membrane of the wing, haltere and
second leg disks (data not shown). These sites of
expression may reside at or near the locations of the
A-P compartment boundary on the peripodial membrane side of each disk (Hama et al. 1990). In addition,
each of 6 independent lines of BS2.0 shows expression
in the larval epidermis. Such expression is specific to
this construct, but we have not detected any corresponding accumulation of dpp transcripts in this tissue.
The lack of spatial regulatory elements within BS2.0
which can promote reporter gene expression is not
surprising given that the phenotypes of mutants which
have rearrangement breakpoints in the 100 to 105
region are not substantially different from those broken
in the 106 to 108 region (St. Johnston et al. 1990). All of
these lesions have similar disk-Ill phenotypes, indicating that major m-regulatory elements do not reside
in the interval between 100 and 108.
Constructs spanning the dppdisk boundary regions
The two constructs that direct strong patterns of figalactosidase expression in appropriate locations of the
imaginal disks are those that include the DNA segments
defining the boundaries between different classes of
dppdM mutant alleles (St. Johnston et al. 1990). BS1.0
includes the disk-V-disk-IH boundary, which is within
the 98.0 to 100.0 region, and BS3.0 includes the diskIH-disk-II boundary region, which falls between 109.5
and 110.0. To determine if these boundary regions
make major contributions to the reporter gene expression patterns, smaller segments spanning the
boundary regions were also tested.
BS1.1 extends from 98.4 to 100.2, spanning the diskV-disk-III boundary, and BS3.1 extends from 109.5 to
110.3, spanning the disk-III-disk-H boundary. The
BS1.1 larval jS-galactosidase pattern is indistinguishable
from that of the larger BS1.0 construct, consistent with
the idea that enhancer-like elements within the disk-Vdisk-III boundary region are determining the spatial
patterns displayed by both constructs. Larvae containing the BS3.1 reporter gene likewise display expression
patterns similar to those produced by the larger BS3.0
construct, but some important distinctions can be seen.
First, the BS3.1 pattern is subject to position effects.
The wing disk expression, in particular, displays
variability among the transformed lines; some transformants show little or no expression, whereas others show
expression that is as intense as in the BS3.0 constructs.
Second, even in the BS3.1 lines showing strong
expression, the pattern is only a subset of the BS3.0
pattern. These observations suggest that important
regulatory elements for dpp's spatial expression lie in
the disk-IH-disk-H boundary region, but that additional elements must reside elsewhere in the 107 to
117 interval.
Patterns of dpp expression in the imaginal disks
The wing disk
In the wing disk, BS3.0 directs expression in a noncontinuous band of cells that extends the length of the
disk, marking out a path reminiscent of the A-P
boundary. A narrow stripe of staining cells is also
observed in the peripodial membrane (Fig. 3C). In situ
hybridization to whole-mount wing disks shows that the
pattern of /J-galactosidase expression driven by BS3.0 is
an accurate subset of the sites at which dpp RNA
accumulates (Fig. 3A).
Reporter gene constructs BS1.0 and BS1.1 also direct
expression of a subset of the dpp transcription pattern
in the wing disk. Analysis of these constructs reveals
that cells in a portion of the presumptive notum region
of the wing disk are expressing /3-galactosidase
(Fig. 3B). The staining of these cells appears at least
partially to overlap the staining observed in the
presumptive notum region of BS3.0 lines.
The wing disk staining pattern in the different BS3.1
lines is variable, although it is uniform among
individuals of a given line. No staining is observed in
four of the lines, while in another five, expression is
dpp expression in imaginal disks
661
Fig. 3D shows a wing disk from the transformed line
displaying the most extensive expression.
Several aspects of the jS-galactosidase expression
patterns of these lines correlate with phenotypes
elicited by the three classes of disk mutations. The
strong /J-galactosidase expression in the wing pouch
region of the wing disk of BS3.0 and some lines of
BS3.1 is consistent with the severe wing defects
displayed by mutations broken between the dpp
transcription unit and the 109.5-110 region. All of the
transposons except BS2.0 produce staining in the
presumptive notal region of the disk, suggesting that
dpp expression in some cells may be controlled by
multiple regulatory elements. However, genetic evidence has shown that the presence of both elements is
not required for the formation of a normal notum.
Mutations that remove the BS3.0 elements but retain
those of BS1.1 produce normal nota while alleles that
delete both sets of elements engender notal defects
(Spencer et al. 1982; St. Johnston et al. 1990;
unpublished). Because we lack mutations that delete
only the BS1.1 region, we are unable to determine if
these two regions possess redundant information for
notum development or if the BS1.1 contains only
information used in vivo.
D
Fig. 3. Expression of dpp in wing imaginal disks of late
third instar larvae. (A) Whole-mount wing disk in situ
hybridization with a dpp cDNA probe (see Materials and
methods for details). (B-D) Spatial distribution of pgalactosidase-expressing cells in wing disks of dpp-lacZ
transformant lines. Each construct directs a subset of the
wild-type dpp RNA in situ pattern. (B) BS1.1. Staining is
observed in the presumptive notum region of the disk (for
review of imaginal disk fate maps see Bryant, 1978).
(C) BS3.0. Expression in this line extends the length of the
disk in a pattern remininscent of the A-P compartment
boundary. An arrow indicates the peripodial membrane
(pm) staining observed. (D) BS3.1. A subset of the BS3.0
/j-galactosidase expressing cells are seen. Staining is still
noted in the wing pouch and presumptive notal region
while expression in the hinge and pleural regions of the
disk is altered. For Figs 2-7 staining patterns were
visualized using the X-gal histochemical assay described in
Materials and methods. Disks are oriented with anterior to
the left. Adult structures derived from regions of the disk:
wp=wing pouch; n=notum.
seen in the wing pouch and notum areas. The notal
staining is identical in each of the five lines, and is a
subset of that seen for BS3.0. Staining in the wing
pouch region is different for each of the five lines.
The eye-antennal disk
The staining pattern in the eye disk is more dynamic,
reflecting the unusual growth properties of the disk
(Tomlinson, 1988). Ahead of the furrow, the cells
remain undifferentiated. As the furrow passes, the cells
differentiate, in a multi-step process, and organize into
ommatidial precursors. Starting early in the third instar,
a dorsoventral indentation, the morphogenetic furrow,
moves anteriorly across the eye disk (i.e., from the
bottom to the top of the disk pictured in Fig. 4D). In
BS3.0 lines, /J-galactosidase activity is localized in a
narrow stripe just behind the morphogenetic furrow,
i.e., in the cells just beginning to differentiate (Fig. 4C).
This accurately reflects endogenous dpp transcription in
the eye disk. BS3.1 also produces /S-galactosidase
expression in the eye disk, but its staining pattern does
not resemble normal dpp RNA accumulation
(Fig. 4D,A). Staining is present in cells posterior to and
away from the furrow, in the cells of the maturing
ommatidial array. BS3.1 probably contains only a
subset of the eye disk regulatory elements, and
additional elements found in BS3.0 are needed to
reproduce the normal dpp pattern. These additional,,
regulators are likely to lie within the 2.5 kb region
between 107 (the left end of BS3.0) and 109.5 (the diskH-disk-III boundary), since disk-II adults have normal
eyes but disk-Ill flies do not. Consistent with this,
previous genetic analysis of the dppd'blk mutation,
deleted in the region 106.0 to 111.4, has shown that
most of the eye disk regulatory information hes within
this 5.4kb interval (St. Johnston et al. 1990). Furthermore, BS1.0, BS1.1 and BS2.0 show no expression in
the eye disk, indicating the absence of eye-specific
regulatory elements in the 92-104 region.
In the antennal disk, BS3.0 staining is observed in the
662
R. K. Blackman and others
ant
eye
same antennal /3-galactosidase activity described for
BS3.0 and BS3.1, but additional staining extends across
the width of the antennal disk, through the second and
first antennal regions towards the medial side of the disk
(Fig. 4B).'
The leg disks
Staining patterns are shown for mesothoracic leg disks
(Fig. 5). Pro- and metathoracic legs have identical
staining patterns. In BS1.0 and BS1.1, /3-galactosidase
is expressed along the length of the disk in a series of
patches that define a line near the A-P boundary.
BS1.0 and BS1.1 each direct /3-galactosidase expression
in a pattern that is a subset of the in situ pattern of dpp
RNA in the leg disk (Fig. 5B.A). BS3.0 and BS3.1 also
direct a subset of the RNA pattern and some of the
observed expression may overlap BS1.0 and BS1.1
(Fig. 5C,D). Interestingly, the most intensely staining
cells in BS3.0 are the epithelial cells of the leg disk fated
to become the prospective tarsi, precisely the leg tissues
that are affected by mutations lacking the 107-117
sequences.
Genital disks
Consistent with the accumulation of dpp RNA in the
genital disks of the two sexes (Fig. 6A,D), there is
much more extensive /S-galactosidase staining in the
male than in the female disk in BS3.0 and BS1.0
transformants (Fig. 6E,F versus 6B,C respectively).
The BS3.0 pattern appears accurately to reflect dpp
transcript accumulation. Again, the /3-galactosidase
patterns appear to lie near the A-P boundaries of the
genital disks, as defined by engrailed//3-galactosidase
expression (Hama et al. 1990). The greater expression
in the male genital disks is consistent with the
observation that in homozygotes for disk-III mutations,
the genitalia and analia of the male are absent or
severely reduced, whereas the females suffer only anal
plate defects. In disk-II homozygotes, males have
partially defective genitalia, while female terminalia are
normal. Our evidence suggests that the elaboration of
the male genital disk may require a greater quantitative
need for the dpp product than does its female
counterpart.
Fig. 4. Expression of dpp in eye-antennal disks of third
instar larvae. (A) Spatial distribution of dpp transcripts in
eye-antennal disk detected by whole-mount RNA in situ
hybridization experiments. (B-D) X-gal staining patterns
observed in eye-antennal disks of dpp/lacZ staining lines:
(B) BS1.1 directs the expression of /5-galactosidase across
the width of the eye-antennal disk from the region fated
to become the arista through the antennal segment folds.
Staining is also observed in the stalk region. Note: no /Sgalactosidase activity is seen in the eye part of the
eye-antennal disk. (C) BS3.0. Staining is observed in the
antennal disk in the region fated to become the arista and
third antennal segment. Expression of /3-galactosidase is
seen in the eye disk along the morphogenetic furrow.
(D) BS3.1. /5-galactosidase-expressing cells in the antennal
disk reflect a subset of the dpp RNA pattern. In contrast,
BS3.1 does not direct a wild-type dpp eye-disk expression
pattern. Adult structures derived from regions of the disk:
ant=antennal region; eye=eye; mf= morphogenetic furrow.
Other disks
While the small size of these disks precludes detailed
analysis, we have seen spots of BS 1.0, BS1.1 andBS3.0
expression in the remaining disks of the larva: the
labial, clypeo-labral, dorsal prothoracic and haltere
disks. Expression patterns in the haltere disks were
similar to the wing disks. Thus, the regulatory elements
included in these constructs are responsible for localized dpp expression in all of the imaginal disks.
region of the disk fated to become the arista and third
antennal segment, and some staining is also seen in the
stalk region (Fig. 4C). BS3.1 shows a similar expression
pattern (Fig. 4D). Again, this is a subset of the dpp
RNA pattern (Fig. 4A). BS1.0 and BS1.1 display the
Reporter gene expression at earlier stages of
development
We have noted that BS3.0 displays extensive expression
during embryogenesis. Embryonic /3-galactosidase activity is first detected at the beginning of germband
retraction. Expression is seen in a series of dorsal and
dpp expression in imaginal disks
Fig. 5. Expression of dpp in mesothoracic leg disks of third
instar larvae. (B-D) X-gal staining patterns observed in
mesothoracic leg disks of dpp/lacZ transformant lines
represent a subset of the dpp RNA in situ expression
profile seen in A. (B) BS1.1. Intense areas of staining are
seen in the presumptive tarsal regions. However, faint
staining is observed that mimics the entire leg disk in situ
transcript pattern. (C) BS3.0. Expression extends from the
end knob (presumptive tarsal region) of the disk towards
the stalk. (D) BS3.1 exhibits the spatial pattern of /3galactosidase expression seen in BS3.0 with additional
expressing cells in the medial part of the disk. Disks are
oriented with anterior to the left. Abbreviation: ek=end
knob.
lateral spots in each segment of the embryo (Fig. 7A).
These spots fall within or near domains of dorsal and
lateral dpp transcript accumulation during the germband retraction stage of development (Fig. 7D,E>
Robert Ray, Harvard University, personal communication). More extensive expression is seen in the
cephalic lobes and the thoracic segments, both by in situ
hybridization and by /3-galactosidase assays. /S-galactosidase expression in similar locations continues until
dorsal closure is complete. At this point, all expression
663
in the epidermis is extinguished with the exception of
(artifactual) expression at the anterior end of the
embryo. Later in embryogenesis, staining is observed in
the brain and epiphysis of the developing larva (data
not shown). Expression in the disk primordia is clearly
observed as early as mid first larval instar.
To try to understand the nature of the embryonic
expression directed by BS3.0, its pattern was examined
in two mutant genotypes. First, dppdlsk'v embryos
carrying BS3.0 were examined (Fig. 7B). While several
of the segmentally repeated spots remain and the
abdominal pattern is basically unaltered, the segmental
pattern is now much more uniform, and the intense
spots of /5-galactosidase activity in the cephalic and
thoracic segments are not present. We conclude that
directly or indirectly, the expression of the reporter
gene construct is dependent upon normal activity
conferred by dppdtsk region function. At the very least,
this is the first demonstration of an embryonic
phenotype of mutations in the dppdlsk region.
Second, embryos containing BS3.0 but homozygous
for Df(1)260.1 were examined (Fig. 7C). Df(l)260.1 is
deleted for the achaete-scute complex (AS-C), causing
the loss of all external sense organs and most of their
peripheral neurons (Dambly-Chaudie're and Ghysen,
1987). The strong spots of activity in the cephalic and
thoracic segments remain intense. This suggests that
these sites of expression are not likely to represent
peripheral nervous system (PNS) elements. In contrast,
the pattern of the abdominal spots as well as the weak
spots normally found in the cephalic and thoracic
segments are dramatically altered in the animals lacking
the PNS, suggesting that these are PNS cells.
These observations suggest that the intense spots of
expression in the cephalic and thoracic segments are
within the primordia of the imaginal disks. Consistent
with this idea, residual staining in the eighth, ninth and
tenth abdominal segments of AS-C deletion homozygotes, in favorable preparations, is more intense than in
the more anterior abdominal segments. The genital disk
is thought to arise by a fusion of primordia derived from
each of these three segments (Schiipbach et al. 1978),
and we may be visualizing these primordia with our
BS3.0 construct. If our suggestion proves correct, the
BS3.0 construct would provide us with the earliest
marker yet available for all disk primordia in the
embryo. Furthermore, it raises the possibility that dpp
may contribute to the actual partitioning of the disk
primordia from adjacent larval epidermal cells.
BS1.0 and BS1.1 also display segmentally repeated
embryonic /S-galactosidase-staining patterns at similar
embryonic stages. These patterns do not include sites of
intense expression in the cephalic and thoracic segments. Further characterization of these patterns is
underway.
Discussion
Our results clearly demonstrate that the 3' disk region
contains enhancer-like cw-regulatory elements that are
664
R. K. Blackman and others
Fig. 6. Expression of dpp in male and female genital disks of third instar larvae. (Top row) female genital disks. (Bottom
row) male genital disks. (A,D) dpp transcription pattern detected by RNA in situ hybridization. (B,C,E,F) Spatial
distribution of /J-galactosidase-expressing cells from dpp-lacZ constructs: (B) BS1.1, female disk. (C) BS3.0, female disk.
(E) BS1.1 male disk. (F) BS3.0 male disk.
B
Fig. 7. Expression of dpp in embryos. (A-C) dpp-lacZ BS3.0 /3-galactosidase staining in embryos at approximately stage
15, just before dorsal closure: (A) wild-type embryo, (B) dppd" mutant embyro and (C) embryo lacking the AS-C.
Darker spots of staining are seen in abdominal segments A8, A9 and A10 (Arrows, C). (D-E) dpp transcript pattern in
wild-type embyros as detected by in situ hybridization: (D) lateral view of late stage 11 embyro, germ band fully extended.
(E) ventolateral view of stage 13 embyro. Circles indicate cephalic segments. Triangles demarcate the thoracic segments.
able to re-create the spatial patterns of expression
characteristic of dpp transcripts in imaginal disks.
Major patterning elements are located at or near the
regions defined as the boundaries of the classes of disk
breakpoint mutations. These boundary regions are
located 17 and 27 kb downstream of the nearest dpp
promoter, pointing to their ability to act at great
distance. From additional constructs, we also know that
these regulatory elements are able to function in an
orientation-independent fashion (data not shown).
Thus, these elements have all the properties expected
of enhancers.
The expression pattern of dpp in any single disk is
likely to result from the action of several independent
regulatory regions. For example, in the wing disk, the
specific patterns directed by the disk-IH-disk-II and the
disk-V-disk-IH boundary regions are partially overlapping but clearly different from one another. Additional
wing disk elements must also reside in the 106.9 to 116.9
region, as BS3.0 has a more extensive staining pattern
dpp expression in imaginal disks
than does BS3.1. Finally, BS2.0 has elements directing
expression in the peripodial membrane of the disk. The
separate regulation of the domains comprising the line
of dpp expression is reminiscent of the separate
regulation of each of the stripes of the pair-rule gene
even-skipped (Goto et al. 1989; Harding et al. 1989).
Indeed, even though the imaginal disk RNA expression
pattern of dpp is typically a continuous band, it appears
that the band is regulated as a series of independent
blocks.
The regulatory information located at the two
boundary junctions is sufficient in each case to direct
dpp expression in every imaginal disk. Formally, there
are two possibilities for how these region could be
molecularly organized. In the first, each boundary
could comprise clusters of functionally related
enhancers, each specific for a particular disk. Alternatively, the boundary could contain a single enhancer
directing expression in a specific position globally in all
imaginal disks. As finer analysis of these regulatory
regions is accomplished, we will be able to resolve this
issue.
Together, the 98.4-100.2 and 106.9-116.9 constructs
recapitulate much or all of the normal dpp expression
pattern. This raises the question of the contributions of
other portions of the disk region to the ds-regulation of
dpp. The physical distance between the Drosophila
pseudoobscura equivalents of the Hin and disk-V-diskIII boundary regions is very similar to the distance in D.
melanogaster (unpublished observations). Indeed, by
cross-hybridization criteria, there are several highly
conserved colinear blocks in the disk regions of these
two species (Ira Clark, our laboratory, personal
communication). We presume that these regions
contain numerous cis-regulatory elements that contribute to dpp'?, expression in imaginal disks. In the present
study, our reporter gene assay reveals those elements
which are sufficient in isolation to activate transcription. Other types of regulatory information, e.g.,
negative regulators, elements affecting amounts of gene
product, would not have been identified.
The complexity of the dpp m-regulatory apparatus is
striking. This gene organization may reflect dpp'?,
evolutionary history, or it may reflect important
structural features for optimum expression of the gene.
Clearly, the regulatory regions contributing to the
major developmental events controlled by dpp are
compartmentalized. The middle region of the gene
(Hin) contains the embryonic dorsal-ventral control
elements. The 5' region contains the regulatory
elements for visceral mesoderm expression, as well as
adult wing vein and anterior-ventral head capsule
development. The 3' region is principally concerned
with imaginal disk-specific expression. It may be that
the regulatory signals for each of these rounds of
expression, activating any of several promoters (St
Johnston et al. 1990) need to be in their own domain, so
that specific expression patterns can be generated.
An unanticipated aspect of the reporter gene
expression of BS3.0 is that it may drive expression of
dpp in the disk primordia in 7-12 h embryos, during
665
germband retraction and dorsal closure. This time of
expression is within the interval that the disks may first
sort out from adjacent larval epidermal cells (Wieschaus and Gehring, 1976). If indeed this expression is
within the early disk primordia, it will be important to
determine if dpp is actually contributing to the process
of the acquisition of imaginal disk identity and if
expression is ubiquitous within the primordia. Attempts
are underway to align this embryonic expression to the
expression of the distalless gene, a marker for some
disks in these mid-embryonic stages (Cohen, 1990), and
also to align the expression to markers of the embryonic
peripheral nervous system.
Finally, we should note that the >20kb disk region
represents one of the largest 3' cw-regulatory domains
known. This region was identified solely through the
extensive genetic and molecular genetic techniques
available to analyze dpp in Drosophila. Such a region
would likely have gone undetected in experimental
systems lacking such tools. It will be interesting to see if
the large 3' regulatory domain of dpp is novel, or if
other genes deployed in multiple developmental patterns (such as many growth factor genes) have such
sequences.
We thank Robert Ray for discussions and for his embryonic
dpp in situ hybridization photomicrographs, David Bergstrom
for technical assistance and David Smouse for his help with
the characterization of the embryonic staining patterns of
BS3.0. We thank James Masucci and F. Michael Hoffmann
for sharing unpublished information. This work has been
supported by a grant from the National Institutes of Health
(NIH) to W.M.G. and from University of Illinois start-up
funds to R.K.B. During the course of this work, R.K.B.,
M.S. and L. A.R. were NIH postdoctoral fellows, and L. A.R.
was also a fellow of the Charles A. King Trust.
References
BRYANT, P. J. (1978). Pattern formation in imaginal disks. In The
Genetics and Biology of Drosophila, vol. 2c, M. Ashburner and
T. R. F. Wright (eds), Academic Press, New York. pp. 230-236.
CATE, R. L., MATTALLIANO, R. J., HESSION, C , TIZARD, R.,
FARBER, N. M., CHEUNG, A., NINFA, E. G., FREY, A. Z., GASH,
D. J., CHOW, E. P., FISHER, R. A., BERTONIS, J. M., TORRES,
G., WALLNER, B. P., RAMACHANDRAN, K. L., RAGIN, R. C ,
MANGANARO, T. F., MACLAUGHLIN, D. T. AND DONAHOE, P. T.
(1986). Isolation of the bovine and human genes for Mullerian
inhibiting substance and expression of the human gene in animal
cells. Cell 45, 685-698.
COHEN, S. M. (1990). Specification of limb development in the
Drosophila embryo by positional cues from segmentation genes.
Nature 343, 173-177.
DAMBLY-CHAUDIERE, C. AND GHYSEN, A. (1987). Independent
subparterns of sense organs require independent genes of the
achaete-scute complex in Drosophila larvae. Genes and Dev. 1,
297-306.
GOTO, T., MACDONALD, P. AND MANIATIS, T. (1989). Early and
late periodic patterns of even skipped expression are controlled
by distinct regulatory elements that respond to different spatial
cues. Cell 57, 413-422.
HAMA, C , A L I , Z. AND KORNBERG, T. B. (1990). Region specific
recombination and expression are directed by portions of the
Drosophila engrailed promoter. Genes and Dev. 4, 1079-1093.
HARDING, K., HOEY, T., WARRIOR, R. AND LEVINE, M. (1989).
Autoregulatory and gap gene response elements of the evenskipped promoter of Drosophila. EMBO J. 8, 1205-1212.
666
R. K. Blackman and others
HIROMI, Y. AND GEHRING, W. J. (1987). Regulation and function
of the Drosophila segmentation gene fushi tarazu. Cell 50,
963-974.
HOFFMANN, F. M. AND GOODMAN, W. (1987). Identification in
transgenic animals of the Drosophila decapentaplegic sequences
required for normal embryonic pattern formation. Genes and
Dev. 1, 615-625.
IMMERGLOCK, K., LAWRENCE, P. A. AND BIENZ, M. (1990).
Induction across germ layers in Drosophila mediated by a
genetic cascade. Cell 62, 261-268.
IRISH, V. F. AND GELBART, W. M. (1987). The decapentaplegic
gene is required for dorsal-ventral patterning of the Drosophila
embryo. Genes and Dev. 1, 868-879.
KARESS, R. E. AND RUBIN, G. M. (1984). Analysis of P
transposable element functions in Drosophila. Cell 38, 135-146.
KAY, R. AND MCPHERSON, J. (1987). Hybrid pUC vectors for
addition of row restriction enzyme sites to the ends of DNA
fragments. Nucleic Acids Res. 15, 2778.
LATHE, R., VILOTTE, J. L. AND CLARK, A. J. (1987). Plasmid and
bacteriophage vectors for excision of intact inserts. Gene 57,
193-201.
LINDSLEY, D. L. AND ZIMM, G. (1987). The Genome of
Drosophila melanogaster. Part 3: rearrangements. Dros. Inform.
Serv. 65.
LOGAN, S. K. AND WENSINK, P. C. (1990). Ovarian follicle cell
enhancers from the Drosophila yolk protein genes: different
segments of one enhancer have different cell-type specificities
that interact to give normal expression. Genes and Dev. 4,
613-623.
LYONS, K. M., PELTON, R. W. AND HOGAN, B. L. M. (1990).
Organogenesis and pattern formation in the mouse: RNA
distribution patterns suggest a role for Bone Morphogenetic
Protein-2A (BMP-2A). Development 109, 833-844.
MACDONALD, P. M. AND STRUHL, G. (1986). A molecular gradient
in early Drosophila embryos and its role in specifying the body
pattern. Nature 324, 537-545.
MASUCCI, J. D . , MlLTENBERGER, R . J. AND HOFFMANN, F . M .
(1990). Pattern specific expression of the Drosophila
decapentaplegic gene in imaginal disks is regulated by 3' cisregulatory elements. Genes and Dev. (in press).
PADGETT, R. W., ST. JOHNSTON, R. D. AND GELBART, W. M.
(1987). A transcript from a Drosophila pattern gene predicts a
protein homologous to the transforming growth factor-/3. Nature
325, 81-84.
PANGANIBAN, G. E. F., RASHKA, K. E., NETTZEL, M. D. AND
HOFFMANN, F. M. (1990). Biochemical characterization of the
Drosophila dpp protein, a member of the transforming growth
factor P family of growth factors. Molec. cell. Biol. 10,
2669-2677.
PANGANIBAN, G. E. F., REUTER, R., SCOTT, M. P. AND HOFFMANN,
F. M. (1990). A Drosophila growth factor homolog,
decapentaplegic, regulates homeotic gene expression within and
across germ layers during midgut morphogenesis. Development
110, 000-000.
POSAKONY, L. M., RAFTERY, L. A. AND GELBART, W. M. (1990).
Wing formation in Drosophila melanogaster requires
decapentaplegic function along the anterior-posterior
compartment boundary. Mech. of Devel. (in press).
ROBERTSON, H. M., PRESTON, C. R., PHILLIS, R. W., JOHNSONSCHLITZ, D. M., BENZ, W. K. AND ENGELS, W. R. (1988). A
stable genomic source of P element transposase in Drosophila
melanogaster. Genetics 118, 461—470.
SCHOPBACH, T., WIESCHAUS, E. AND NOTHIGER, R. (1978). The
embryonic organization of the genital disc studied in genetic
mosaics of Drosophila melanogaster. Wilhelm Roux' Arch, devl
Biol. 185, 249-270.
SEGAL, D. AND GELBART, W. M. (1985). shortvein, a new
component of the decapentaplegic gene complex in Drosophila
melanogaster. Genetics 109, 119-143.
SMITH, J. C. (1989). Mesoderm induction and mesoderm-inducing
factors in early amphibian development. Development 105,
665-677.
SPENCER, F. A., HOFFMANN, F. M. AND GELBART, W. M. (1982).
decapentaplegic: a gene complex affecting morphogenesis in
Drosophila melanogaster. Cell 28, 451-461.
SPRADLING, A. C. (1986). 'P-element mediated transformation.' In
Drosophila: A Practical Approach, (ed. D. B. Roberts). IRL
Press, Oxford, pp. 175-197.
ST JOHNSTON, R. D. AND GELBART, W. M. (1987). decapentaplegic
transcripts are localized along the dorsal-ventral axis of the
Drosophila embryo. EMBO J. 6, 2785-2791.
ST JOHNSTON, R. D., HOFFMAN, F. M., BLACKMAN, R. K., SEGAL,
D., GRIMAILA, R., PADGETT, R. W., IRICK, H. AND GELBAKT, W.
M. (1990). The molecular organization of the decapentaplegic
gene in Drosophila melanogaster. Genes and Dev. 4, 1114-1127.
TOMLJNSON, A. (1988). Cellular interactions in the developing
Drosophila eye. Development 104, 183-193.
WIESCHAUS, E. AND GEHRING, W. (1976). Gynandromorph analysis
of the thoracic disc primordia in Drosophila melanogaster.
WUhelm Roux's Arch, devl Biol. 180, 31-46.
WOZNEY, J. M., ROSEN, V., CELESTE, A. J., MITSOCK, L. M.,
WHITTERS, M. J., KRIZ, R. K., HEWICK, R. M. AND WANG, E.
A. (1988). Molecular cloning of novel regulators of bone
formation. Science 2442, 1528-1534.
(Accepted 16 November 1990)