Download Homeotic genes regulate the spatial expression

1031
Development 110, 1031-1040 (1990)
Printed in Great Britain © The Company of Biologists Limited 1991
Homeotic genes regulate the spatial expression of putative growth factors
in the visceral mesoderm of Drosophila embryos
ROLF REUTER 1 ' 2 , GRACE E. F. PANGANIBAN 3 , F. MICHAEL HOFFMANN 3
and MATTHEW P. SCOTT 1 *
1
Howard Hughes Medical Institute and Department of Molecular, Cellular and Developmental Biology, University of Colorado,
Colorado 80309-0347, USA
2
Max-Planck-Institut fiir Entwicklungsbiologie, Abteilung III, Spemannstrasse 35, 7400 Tubingen, Germany
3
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706, USA
Boulder,
*To whom correspondence should be addressed. Present address: Department of Developmental Biology, Stanford University School of
Medicine, Stanford, California 94305-5427, USA
Summary
During Drosophila embryogenesis homeotic genes control the developmental diversification of body structures.
The genes probably coordinate the expression of as yet
unidentified target genes that carry out cell differentiation processes. At least four homeotic genes expressed
in the visceral mesoderm are required for midgut
morphogenesis. In addition, two growth factor homologs
are expressed in specific regions of the visceral
mesoderm surrounding the midgut epithelium. One of
these, decapentaplegic (dpp), is a member of the
transforming growth factor /? (TGF-fi) family; the other,
wingless (wg), is a relative of the mammalian protooncogene int-1. Here we show that the spatially
restricted expression of dpp in the visceral mesoderm is
regulated by the homeotic genes Ubx and abd-A. Vbx is
required for the expression of dpp while abd-A represses
dpp. One consequence of dpp expression is the induction
of labial (lab) in the underlying endoderm cells. In
addition, abd-A function is required for the expression of
wg in the visceral mesoderm posterior to the dppexpressing cells. The two growth factor genes therefore
are excellent candidates for target genes that are directly
regulated by the homeotic genes.
Introduction
reduced (Scr), and Antennapedia (Antp), and three in
the BX-C, Ultrabithorax (Ubx), abdominal-A (abd-A),
and Abdominal-B (Abd-B) (reviewed in Mahaffey and
Kaufman, 1988). The lab, pb, Dfd, and Scr genes act in
anterior parts of the fly, while Antp is required in the
thoracic segments and to some extent in the abdomen.
Ubx is required for the proper formation of the
posterior thorax and the anterior abdomen, abd-A for
the central abdomen, and Abd-B for the posterior
abdomen.
The mechanisms by which homeotic genes influence
morphogenesis remain largely unknown. All of the
ANT-C and BX-C homeotic genes encode proteins with
homeodomains,
sequence-specific
DNA-binding
domains (reviewed in Scott et al. 1989), and several of
the protein products of the homeotic genes have been
shown to function as transcription factors (Jaynes and
O'Farrell, 1988; Han et al. 1989; Driever and NiissleinVolhard, 1989; Hanes and Brent, 1989; Krasnow et al.
1989; Struhl et al. 1989; Winslow et al. 1989). These
observations strongly suggest that the genes control
Mutations in Drosophila homeotic genes transform one
part of the fly into another. The best studied
phenotypes involve changes in the formation of the
appendages and cuticle of the adult and of the larval
cuticle, but the genes affect internal structures as well.
We have used the development of the midgut to analyze
the roles of homeotic genes in forming a normal
embryo, to characterize interactions among the homeotic genes, and to identify genes that are regulated by
the homeotic genes.
Many of the Drosophila homeotic genes are grouped
in two clusters known as the Antennapedia complex
(ANT-C) and the bithorax complex (BX-C) (Lewis,
1978; Kaufman et al. 1980; reviewed in Akam, 1987;
Ingham, 1988). Apparently homologous gene clusters
exist in vertebrates (Gaunt et al. 1988; Duboule and
Dolle, 1989; Graham et al. 1989; reviewed in Akam,
1989). Five homeotic genes are in the ANT-C, labial
(lab), proboscipedia (pb), Deformed (Dfd), Sex combs
Key words: Ultrabithorax, decapentaplegic, labial,
abdominal-A, wingless, visceral mesoderm, homeotic,
growth factor, Drosophila.
1032 R. Renter and others
morphology by coordinating the spatial and temporal
transcription patterns of target or 'realizator' genes
(Garcia-Bellido, 1977). In the epidermis, the nervous
system, and the visceral mesoderm, cross-regulatory
interactions have been observed among the homeotic
genes; thus the first target genes to have been identified
are homeotic genes (Hafen et al. 1984; Struhl and
White, 1985; Carroll et al. 1986; Casanova and White,
1989). In addition some of the homeotic genes have
been found to autoregulate, including one, Ubx, that
activates its own expression in the visceral mesoderm
(Bienz and Tremml, 1988). There must, however, be
other genes that are controlled by homeotic genes but
are more directly linked to the processes of cell
differentiation and morphogenesis. In an attempt to
identify such target genes we have looked for genes that
are transcribed in restricted regions of the embryo
overlapping with domains of homeotic gene expression.
Two genes, wingless (wg) and decapentaplegic (dpp),
that encode putative growth factors are expressed in the
visceral mesoderm of the developing midgut in patterns
that suggest they may be regulated by homeotic genes.
Several of the homeotic genes, including lab, Scr,
Antp, Ubx, andabd-A, are expressed in discrete regions
in the developing midgut. The midgut is made up of two
cell layers: an inner layer of large polyploid endoderm
cells encased in a sheath of smaller, euploid visceral
mesoderm cells. The four homeotic genes Scr, Antp,
Ubx, and abd-A are expressed in non-overlapping
domains in the visceral mesoderm cells in an order
along the anterior-posterior axis similar to that in the
epidermis, i.e. Scr is active in the most anterior region
of the mesoderm, then Antp, then Ubx, and abd-A in
the most posterior domain (LeMotte et al. 1989;
Tremml and Bienz, 1989; Reuter and Scott, 1990).
Defects in gut morphogenesis are seen when any of
these four homeotic genes is defective. The morphological alterations seen in the gut in the homeotic
mutants generally correspond well with where the
products are found in wild-type embryos. The gastric
caeca, four tentacle-like protrusions of the midgut at its
anterior end, do not form if Scr function is lacking
(Reuter and Scott, 1990). Constrictions that divide the
gut into sections normally form at three positions along
the anterior-posterior axis of the gut tube, and each
constriction is dependent on homeotic gene function
(Tremml and Bienz, 1989; Reuter and Scott, 1990). The
anterior constriction that underlies the mesoderm cells
that express Antp does not form in mutant embryos that
lack Antp function. The middle constriction that
normally forms near the interface between Ubx and
abd-A expression fails to develop in either Ubx or abdA mutants, and the posterior constriction that underlies
abd-A expression does not form if abd-A function is
lacking. There is evidence that these morphological
features are formed by the imposition of the structure
upon the endoderm by the mesoderm (Reuter and
Scott, 1990); however, it is possible that both cell layers
are active participants in the formation of the constrictions. The lab gene is expressed only in a subset of the
endoderm cells, and defects in the formation of the
second midgut constriction have been reported in lab
mutants (Immergliick et al. 1990). Thus, a key element
of the control of differentiation by homeotic genes in
the midgut, as well as in the epidermis, is the
differential spatial expression pattern of each gene.
The sequencing of wg revealed that its product is
closely related to the product of the mammalian gene
int-1 (Rijsewijk et al. 1987). The wg gene is expressed in
early embryos in stripes corresponding to the posterior
part of each parasegment (PS; Baker, 1987) and is one
of several Drosophila genes involved in establishing
polarity of the embryo (Ingham, 1988). The protein is
made in about one-quarter of the cells in each segment
primordium, where the RNA is seen, and then some of
the protein appears to move into adjacent cells (van den
Heuvel et al. 1989). Studies of mosaic adult flies in
which some of the cells lack all wg function while other
surrounding cells have one functional copy of the gene
have shown that mutant cells can be rescued by the
neighboring wild-type cells (Morata and Lawrence,
1977). These observations, as well as the change in the
polarity of cell structures in parts of segments in wg
mutant embryos where its RNA is not transcribed,
suggest that the protein is involved in cell-cell
communication processes. The wg products are also
detected in the developing midgut, in a narrow band of
visceral mesoderm cells (van den Heuvel et al. 1989)
suggesting that it also has a function in the differentiation of the gut. The location of wg products in the
midgut led us to examine whether wg is regulated by
homeotic genes of the BX-C. We demonstrate that wg
transcription is activated by abd-A, but only in a
discrete subset of the visceral mesoderm cells where
abd-A is expressed, indicating that abd-A is necessary,
but not sufficient, for wg expression.
The decapentaplegic (dpp) gene was first recognized
for its effects on the development of adult structures
derived from imaginal discs (Spencer et al. 1982; St
Johnston etal. 1990), and was subsequently found to be
involved in other processes such as the development of
proper dorsal-ventral patterning (Irish and Gelbart,
1987). It encodes a product that is a member of the
TGF-/3 family of growth factors (Padgett et al. 1987).
The protein, synthesized in cultured cells, has been
found to be cleaved and secreted, suggesting that it is
involved in carrying information between cells (Panganiban et al. 1990a). The pattern of expression of the
gene is complex, in keeping with the gene's functions in
many different cells and tissues (St Johnston and
Gelbart, 1987; Masucci et al. 1990; Jackson and
Hoffmann, in preparation). One of the places that dpp
RNA and protein is found is in the developing midgut.
The RNA is detectable only in the visceral mesoderm
cells in particular positions, while the protein is found in
the same cells as the RNA and additionally in some of
the underlying endoderm cells (Panganiban et al.
1990b).
We have explored the relationship between Ubx and
dpp further by studying the induction of dpp by ectopic
expression of Ubx. We demonstrate that Ubx induces
the expression of dpp in visceral mesoderm cells of the
Homeotic genes regulate growth factor homologs
anterior half of the midgut. Induction of dpp by Ubx is
restricted to the mesoderm cells suggesting that Ubx
must act together with other factor(s) limited to the
visceral mesoderm to induce dpp, or that the action of
Ubx upon dpp is blocked in all tissues but the visceral
mesoderm. dpp expression in the posterior of the
midgut is inhibited by abd-A, even in the presence of
Ubx protein. The action of abd-A upon dpp is therefore
not due to repression of Ubx by abd-A. The ectopic
production of Ubx protein, and hence dpp protein, in
the visceral mesoderm leads to ectopic expression of lab
in many of the underlying endoderm cells.
Materials and methods
Fly strains
Stocks are described in Lindsley and Grell (1968) and
additional references as follows. The mutant stocks used were
st Ubx6-28 e ca sbd/TMl [(Kerridge and Morata, 1982), no
detectable Ubx protein due to the deletion of 32 bp in the 5'
exon which causes a frameshift at codon 27 and premature
stop of translation (Beachy et al. 1985; Weinzierl et al. 1987)],
abdAMXl/TM6B [(Sanchez-Herrero etal. 1985) no detectable
abd-A protein (Karch et al. 1990)], Df(3R)bxdl0O/TMl
[deficient for the Ubx transcription unit (Lewis, 1978; Bender
et al. 1983)], Df(3R)Ubx109 gl e s /Dp (3;3) P5, Sb [(Lewis,
1978), deficient for the Ubx and abd-A transcription units
(Karch etal. 1985)], Df(3R)P9/Dp(3;3)P5, Sb [(Lewis, 1978),
deficient for the complete bithorax complex (Karch et al.
1985)], Scrwl1 KI/TM3 Sb and Scrk6 cu fjTM3, Sb [(Wakimoto and Kaufman, 1981), no detectable Scr protein (Riley et
al. 1987)], AntpwW red e/TM3, Sb Ser [(Wakimoto and
Kaufman, 1981), no detectable Antp protein (Carroll et al.
1986)], Df(3R)AntpNs+RC3
th st cu eKx/TM3,
Sb Ser [De-
ficiency which takes out the Antp protein-coding region
(Struhl, 1981; Garber et al. 1983)].
The transformant line hsUbx which carries an Ubx cDNA
under the control of the hsp70 promoter on a rosy~
chromosome was provided by G. Struhl (Gonzalez-Reyes et
al. 1990); a similar construct was provided by R. Mann and D.
Hogness. The transformant line HSU-109 which carries a heat
shock promoter-controlled Ubx cDNA on a Df(3R)£/fct109
chromosome was provided by A. Gonzalez-Reyes (GonzalezReyes et al. 1990).
Antibodies
The origins of the antibodies used in this study are as follows.
The anti-dpp antibody was generated as described by
Panganiban et al. (1990a). The hybridoma line FP3.38 (antiUbx) was kindly provided by White and Wilcox (1984), the
hybridoma line 4C3 (anti-Antp) by D. Brower (J. Condie and
D. Brower, unpublished results) and the hybridoma line 6H4
(anti-5cr) by Glicksman and Brower (1988). The anti-lab
antibody was kindly provided by T. Kaufman (Diederich et al.
1989), the anti-abd-A antibody by W. Bender (Karch et al.
1990) and the anti-wingless antibody by R. Nusse (van den
Heuvel et al. 1989). The antibodies were used as hybridoma
supernatants diluted 1:10 {anti-Ubx) or 1:3 (anti-Scr and antiAntp) in PBT (see below) or as affinity-purified antibodies
diluted 1:200 (ant\-abd-A), 1:500 (anti-wingless) or to
1 ^g ml" 1 (anti-dpp) in PBT.
Heat-shock expression of the Ultrabithorax protein
Eggs were collected for 3h or overnight at 25 °C and the
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embryos were heat-shocked at 37 °C for two times 20 min with
a 20 min period at 18°C in between. Subsequently, the
embryos were kept for three hours or the time period given in
the text at 25°C for recovery.
Immunostaining of embryos
Embryos were fixed as described by Karr and Alberts (1986)
and were immunostained as described by Reuter and Scott
(1990). Staging was according to Campos-Ortega and Hartenstein, 1985. Antibodies raised in rabbits were immunolocalized using biotinylated secondary antibodies (Vector
Lab.) and the ELITE peroxidase detection kit from Vector
Lab. For the double label immunostaining the procedure was
modified as follows. Fixed and PBT-saturated embryos were
first simultaneously incubated with two primary antibodies
from different species, for example an\\-Ubx and ant\-dpp,
and then, after extensive washing, with a dilution of goat antimouse IgG peroxidase conjugate (Biorad) and of biotinylated
goat anti-rabbit IgG (Vector Lab.). The embryos were
washed again, and the bound murine antibodies were
detected by oxidation of diaminobenzidine (DAB) in the
presence of nickel and cobalt ions which gives a dark gray
stain (Lawrence et al. 1987). The DAB solution was
thoroughly removed before peroxidase activity was destroyed
by the addition of hydrogen peroxide to 3 %. Subsequently,
the rabbit antibody was detected after incubation with an
avidin-peroxidase complex (Vector Lab) by the oxidation of
DAB in the absence of heavy metal ions which gives a brown
stain.
Results
Ultrabithorax expression is required for the expression
of decapentaplegic in the visceral mesoderm
dpp is expressed in three regions of the visceral
mesoderm surrounding the midgut: anteriorly, in the
mesoderm covering the developing gastric caeca,
centrally, close to the position where the middle
constriction of the midgut forms, and posteriorly, at the
site of the third midgut constriction (St Johnston and
Gelbart, 1987; Panganiban et al. 19906; Fig. 1A). The
third constriction expression is relatively weak and
usually not detected by the dpp antibody. The homeotic
gene Ubx was also found to be expressed in the central
region of the visceral mesoderm, in PS 7 (White and
Wilcox, 1984; Bienz and Tremml, 1988). Indeed, when
dpp and Ubx protein are simultaneously detected in
embryos by a double label experiment, they are
expressed in overlapping domains (Fig. 1C). The
posterior borders of their expression coincide precisely;
anteriorly dpp protein is detected half a parasegment
anterior of the Ubx protein.
Because Ubx is known to regulate transcription
(Krasnow et al. 1989), the overlapping domains of
expression of dpp and Ubx led us to investigate whether
Ubx controls dpp. In embryos that lack Ubx protein,
dpp protein is virtually absent from the visceral
mesoderm in PS 6 and 7 (Fig. IB). Only a faint band of
dpp protein remains detectable around the midgut. This
is located in the posterior part of the normal dpp
expression domain (Fig. IB), directly adjacent to the
region where wg and abd-A are expressed in the visceral
mesoderm (Fig. ID).
1034 R. Reuter and others
The second domain of dpp expression in the visceral
mesoderm (Fig. 1A), in cells that later cover the
budding gastric caeca, is less than half a parasegment
wide and is located anterior to the domain of Scr
protein expression (data not shown), dpp expression
here is not influenced in embryos by the lack of Ubx
function (Fig. IB) and also persists in embryos mutant
for either of the homeotic genes Scr or Antp, both of
which are normally expressed in the visceral mesoderm
(data not shown).
Ultrabithorax expression is sufficient for the
expression of decapentaplegic in the visceral
mesoderm
To more conclusively test whether or not dpp is
activated by Ubx we examined the expression of dpp in
embryos that carry an Ubx cDNA under the control of
the heat shock hsp70 promoter (HSU) (Mann and
Hogness, 1990; Gonzalez-Reyes et al. 1990). One hour
after a heat shock Ubx protein can be detected in every
cell nucleus of the embryo at high levels (data not
shown). In these embryos dpp is ectopically expressed
in the visceral mesoderm from the anterior end of the
midgut to PS 7 (Fig. 2C). In other tissues, the
expression of dpp protein changes little if at all.
In embryos homozygous for mutations in the abd-A
gene, Ubx is expressed at high levels in posterior
regions where abd-A would normally be expressed
(Struhl and White, 1985; Bienz and Tremml, 1988). In
such embryos dpp is also ectopically expressed in the
posterior visceral mesoderm, but not in other tissues
(Fig. 2B; Immergliick et al. 1990; Panganiban et al.
19906). Embryos that lack both Ubx and abd-A
expression (Df(3R)Ubx109) or the entire bithorax
complex (Df(3R)P9) express dpp in a pattern that is
different from any dpp pattern seen in wild-type
embryos (Fig. 2D). The dpp expression that normally
overlaps with Ubx is gone completely, but dpp protein
is found in a segmentally modulated pattern, in five
faint stripes, in the region of the visceral mesoderm
where abd-A is normally expressed. This observation
indicates that abd-A negatively regulates dpp in the
absence of Ubx. dpp expression in the posterior part of
the midgut that is not dependent on the homeotic genes
Ubx and abd-A might result from influences of earlier
acting genes such as segmentation genes.
abdominal-A negatively regulates decapentaplegic in
the visceral mesoderm, but not via Ultrabithorax
In spite of the ubiquitous nuclear Ubx protein in the
HSU embryos after a heat shock treatment, dpp protein
is not found in the posterior part of the visceral
mesoderm where the abd-A protein is present
(Fig. 2C). The posterior border of dpp expression is not
changed by the heat shock-driven ectopic Ubx expression (compare embryos in Figs 2A and C). In order
to test whether abd-A is the negative regulator that
prevents the ectopic Ubx from activating dpp, dpp
expression in HSU embryos that lack abd-A function
was examined. For this purpose embryos were used that
carry HSU on a third chromosome that lacks both Ubx
and abd-A function (Df(3R)UbxlO9\ This chromosome
will hereafter be referred to as HSU-109 (GonzalezReyes et al. 1990). The absence of the endogenous Ubx
gene is required for the experiment because otherwise
dpp protein would be ectopically expressed in the
posterior part of the visceral mesoderm even without
the heat shock-driven expression of Ubx, just due to the
derepression of Ubx in abd-A mutant embryos
(Fig. 2B). The genotype of the embryos that have dpp
protein all through the midgut was established by using
an anti-abd-A antibody (Karch et al. 1990) to show that
they lack the abd-A protein (data not shown).
About one-quarter of the heat-shocked embryos
obtained from HSU-109 heterozygous parents, those
that lack Ubx and abd-A, have ectopic dpp protein
throughout the entire visceral mesoderm of the midgut
(Fig. 2E). Therefore the removal of a single gene
function, abd-A, allows HSU, or the ectopic Ubx in
abd-A mutants, to induce dpp expression in the
posterior visceral mesoderm. Consequently, dpp is a
likely candidate for a target gene directly negatively
regulated by abd-A.
Induction of labial requires direct contact with visceral
mesoderm cells during early midgut development
The study described in the accompanying paper
(Panganiban et al. 1990ft) shows that the endoderm
receives dpp as a signal from the mesoderm. dpp
protein can be detected at the apical side of the
endoderm; it moves from one germ layer to the other,
and it is required for labial {lab) expression in the
central part of the midgut endoderm (Panganiban et al.
1990£>). A close inspection of the lab expression in wildtype embryos revealed some interesting details of the
signal transduction from visceral mesoderm to endoderm. In stage 13 embryos the endoderm forms a multilayered cluster of cells beneath the expression domain
of Ubx and dpp. Of these cells only the outer layer,
which is in direct contact with the visceral mesoderm,
accumulates lab protein; the adjacent cells of the inner
layers do not (Fig. 3A,F). Later, from stage 15 on, the
inner endodermal cells become integrated into the
single cell layer of the endoderm (Fig. 3E; diagrammed
in Fig. 3F). In stage 17 endodermal cells within the
epithelial sheet that do not express lab can be clearly
distinguished from cells that do express it
(Fig. 3B,D,E). Thus even though at stages 15-17 dpp
protein is made and is moving between germ layers,
some cells still do not produce lab protein in response.
Ectopic decapentaplegic protein activates labial
expression in the endoderm
Here we provide further evidence that dpp protein
spatially controls lab expression in the endoderm. In
HSU embryos lab is detectable three hours after heat
shock in most of the anterior endoderm of the midgut,
underlying the cells where dpp is ectopically activated
in the visceral mesoderm by Ubx. (Fig. 4A). The
ectopic lab expression is transient; five hours after the
heat shock the amount of lab protein in the anterior
part of the midgut has decreased (Fig. 4B). Even in
Ubx
Fig. 1. Expression of decapentaplegic protein in the visceral
mesoderm of wild-type and Ubx null mutant embryos.
Optical sections of whole mount preparations viewed by
differential interference contrast microscopy. (A,B) dpp
protein in the visceral mesoderm of wild-type (A) or
homozygous Ubx6 M embryos (B) at stage 13, shown in a
dorsolateral view (between arrowheads: dpp expression
domains; arrows: endodermal cell cluster; ed: endoderm;
vm: visceral mesoderm; hg: hindgut). Ubx and dpp protein
(C) or Ubx, dpp, and wg proteins (D) have been
simultaneously detected in wild-type or homozygous Ubx 628
embryos (D) by double-label immunolocalization. Ubx
protein is visualized in black and indicated between
arrowheads in the visceral mesoderm. dpp (C,D) and wg (D)
protein are visualized in brown and pointed out between
arrows. No Ubx protein is visible in D due to the Ubx
mutation.
Fig. 2. Ectopic expression of decapentaplegic in various mutant and
transformant embryos, dpp protein in the visceral mesoderm of (A) a
wild-type embryo, (B) an abd-A1**1, and (D) a Df(3R)Ubx109 homozygous
mutant embryo or (C) a HSU and (E) a HSU-109 embryo three hours
after a heat shock. In HSU-109/TM1 progeny about three-quarters of the
embryos in the collection make Ubx protein ubiquitously after a heat
shock treatment; the others are TM1/TM1. Of those carrying HSU about
two-thirds have ectopic dpp expression similar to that of the HSU embryo
in C. About one-third of the embryos, presumably the HSU-109 embryos
that lack Ubx and abd-A, have ectopic dpp protein throughout the entire
visceral mesoderm of the midgut as in E. The homozygous mutant
embryos have been identified among their siblings due to their lack of
Ubx (D) or of abd-A (B,E) protein expression in double staining
experiments. The arrows point to the cluster of large endodermal cells.
The arrowheads (A,C) indicate the approximate anterior end of the
visceral mesoderm surrounding the developing midgut.
HSU109
3/V
yolk
stage 17
Fig. 3. Expression of /afew/ protein in the endodermal cells of embryos after germ band retraction, lab protein in the
endoderm of (A,C) a stage 14 and (B,D,E) a stage 17 embryo. (C) A close view of the middle region of the midgut of a
stage 14 embryo (cells in the same region delimited by arrowheads in panel A, but from a different embryo). Arrows in C
point to the nuclei of the large endodermal cells that are located beneath the lab expressing cells and do not express lab.
The large cells have a remarkably pronounced nucleoli (yk: yolk; ed: endoderm; vnc: ventral nerve chord). (D) Qose
tangential view of a stage 17 embryo similar to the one in panel B. Cells of thetofc-expressingmidgut loop viewed with a
low depth of field. Arrows indicate non-/afe-expressing cells that probably had been part of the endodermal cluster and are
now integrated into the single-layered endoderm. (E) Optical section of a similar midgut loop. Arrow has same meaning as
in D. (F) Schematic summary of the proposed integration of the cluster of endodermal cells (({]))) into the midgut
epithelium within the lab expressing domain (•%£})•
Fig. 4. Expression of labial protein in HSU embryos after heat shock. Expression of lab protein in a HSU embryo of
approximately stage 13 three hours after heat shock (A) and a HSU embryo of approximately stage 16 five hours after heat
shock (B). The arrowheads indicate the relatively sharp anterior border of the ectopic lab domain within the midgut
endoderm. (C) High magnification view of a HSU embryo three hours after heat shock at approximately stage 13. The
arrows point to the cells of an endodermal cluster which, in the heat-shocked embryos as in normal embryos, do not
express lab protein. (D) Lateral view of the anterior midgut of a stage 14 HSU embryo after heat shock. (E,F) Close
lateral view in two focal planes of the midgut of a stage 14 HSU embryo after heat shock which reveals the large
endodermal cells that do not express lab (F, arrows) beneath the endodermal cells that contain large amounts of lab
protein.
fbxd
Fig. 5. wingless protein expression in the visceral
mesoderm depends on abdominal-A function, wg
protein in the visceral mesoderm of (A) a wild-type
embryo, (B) a Ubx628, (C) a abd-A™xi, and (D) a
Df(3R)P9 homozygous mutant embryo. The wgexpressing visceral mesoderm cells are indicated by
arrowheads. The arrows point at the endodermal
clusters. While the wg expression in the visceral
mesoderm of the Ubx628 embryo is indistinguishable
from the wild-type pattern, wg protein is absent from
this tissue in all mutant embryos that lack abd-A
function (C,D). (E) In embryos deficient for abx, bx,
Ubx, pbx, and bxd functions, i.e. Df (3R)bxdlC0
homozygotes, wg expression is absent from the visceral
mesoderm. The embryos are between stages 13 and 14
of development; anterior is oriented to the left.
DfP9
Fig. 6. wg protein migrates to the endoderm. (A) In stage 17
embryos wg protein (brown stain) is detectable at the apical side of
the endoderm (arrowheads), i.e. the side towards the lumen of the
midgut, indicating that it has moved from the visceral mesoderm
where wg protein is seen at an earlier stage (Fig. 3B in van den
Heuvel et al. 1989). The adjacent visceral mesoderm cells express
Ubx (black staining; arrows) The wg expressing midgut loop is
visible to the posterior of the Ubx domain, to the left in the
photograph, because the loop has inverted its position during the
convolution of the midgut. (B) High magnification view of the
central region of the midgut of a stage 15 embryo. Ubx protein in
the visceral mesoderm nuclei is stained in black (arrowheads); wg
protein is visualized in brown (arrows). The more posteriorly
located Ubx protein-containing cells (to the right) also display wg
protein on their surfaces. (C) Central region of the visceral
mesoderm of a stage 13 embryo which has been stained for both
dpp and wg protein (in brown) and Ubx protein (in black).
Homeotic genes regulate growth factor homologs 1035
HSU embryos after a heat shock treatment the lab
protein is restricted to the outer endodermal layer,
where the cells are in direct contact with cells that are
producing dpp protein'(Fig. 4E,F). The anterior constriction of the midgut and the constrictions separating
the gastric caeca are not formed in these embryos,
confirming that the appropriate spatial distributions of
homeotic and dpp gene products are required for
midgut morphogenesis.
In the part of the midgut endoderm posterior to the
developing gastric caeca, ectopic dpp protein is
sufficient to induce lab expression in the neighboring
germ layer (Fig. 4D). However, the ectopic domain of
lab protein does not extend as far to the anterior as the
ectopic dpp in the heat-shocked HSU embryos
(Fig. 4A,C). lab is not expressed in the endoderm of the
budding caeca or anterior to them.
abdominal-A expression is required for the expression
of wingless in the visceral mesoderm
In regions of the midgut where cells do not make dpp
protein, communication between germ layers is most
likely to be mediated by other growth factors. The only
other Drosophila growth factor known to be produced
in the midgut is the protein encoded by the wg gene.
The segment polarity gene wg, the Drosophila homolog
of the int-1 oncogene, was found to be expressed in PS 8
of the visceral mesoderm (van den Heuvel et al. 1989).
abd-A is required for wg expression in the visceral
mesoderm. Embryos homozygous for a null mutation of
abd-A (Fig. 5C), or lacking part of (not shown) or the
entire bithorax complex (Fig. 5D), do not accumulate
wg protein in the visceral mesoderm. Thus wg looks like
a good candidate for a target gene positively regulated
by abd-A. However, abd-A cannot be sufficient to
control the spatial distribution of wg because wg is only
expressed in the most anterior part of the broad visceral
mesoderm region where abd-A protein is made
(Fig. 5A). This function is probably not Ubx as Ubx628
mutants retain wild-type wg expression (Fig. 5B).
However, at least one other BX-C function is required
for wg expression because Df(3R) bxdlOO embryos lack
wg products in PS 8 (Fig. 5E). In contrast to another
report (Immergluck et al. 1990), we find that the wg
domain overlaps those of Ubx and dpp (Fig. 6A, B, C).
Some visceral mesoderm cells at the anterior rim of the
wg domain have Ubx protein in their nuclei and wg
protein at the cell periphery (Fig. 6B). This could be
due to movement of the wg protein rather than to
transcription of both genes in some of the same cells.
We have observed movement of wg protein from the
visceral mesoderm to the apical side of the endoderm in
older embryos (Fig. 6C).
Ubx may not be required for spatially restricted
expression of wg in the visceral mesoderm despite the
(limited) physical overlap of Ubx and wg products.
Embryos homozygous for the Ubx628 allele, which has
32 base pair deletion at codon 27 (Beachy et al. 1985;
Weinzierl et al. 1987) and therefore probably has little
or no Ubx function, have wg protein in an unchanged
pattern (Fig. 5B) as do HSU embryos in which ectopic
Ubx expression has been induced (not shown). However, a function within the bithorax complex other than
abd-A is required for normal wg expression. In embryos
homozygous for Df(3R)bxd100, which removes the Ubx
and the bxd transcription units from the third chromosome, or in embryos bearing Df(3R)bxd'°° in trans to
Df(3R)P9, which removes all of the BX-C, not only are
Ubx and dpp expression gone from the visceral
mesoderm, but wg expression is also severely reduced
(Fig. 5E).
Discussion
The requirement for homeotic gene function in
establishing a wild-type body plan is well documented
(reviewed in Mahaffey and Kaufman, 1988); however,
the mechanism by which these genes act is just
beginning to be elucidated. Part of this mechanism
clearly involves inter-regulation among the homeotic
genes (Hafen et al. 1984; Struhl and White, 1985;
Carroll et al. 1986; Riley et al. 1987; Casanova and
White, 1987). Another part of this mechanism is likely
to involve the regulation of target genes which
implement the program set up by the homeotic genes.
These target genes could mediate intercellular interactions as well as intracellular differentiative events.
Recently, two putative targets that are not homeotic
genes, dpp and wg, have been postulated based on their
altered expression patterns in the visceral mesoderm of
embryos mutant for Ubx or abd-A (Immergluck et al.
1990; Panganiban et al. 19906). In this report, we
examine the effect of ectopic expression of Ubx during
gastrulation on the expression of dpp. We conclude that
Ubx and abd-A are sufficient to delimit the spatial
expression of dpp in the middle part of the visceral
mesoderm. Although we have not proven that dpp is
directly regulated by the Ubx protein, this possibility is
the simplest explanation for our observations. Further
evidence consistent with a direct interaction model
comes from experiments in which binding sites for the
Ubx protein were found in or near the dpp gene (P. A.
Beachy; J. Botas and D.S. Hogness, personal communications).
Ubx is required for dpp expression
In embryos bearing the putative Ubx protein null
mutation, Ubx628, the expression of dpp is reduced in
the visceral mesoderm of PS 6 and 7 to a faint band in
posterior PS 7. In embryos homozygous for deficiencies
of the bithorax complex that remove the Ubx transcript,
e.g. Df(3R)bxd100, dpp expression is completely eliminated in the visceral mesoderm of PS 6 and 7. These
data indicate that Ubx is required for dpp expression in
the central region of the midgut, and are essentially in
agreement with the report of Immergluck et al. (1990).
They reported that in embryos bearing a Ubx1 mutation
in trans to a triple mutation of the bithorax complex that
eliminates all its known protein-coding functions
(Casanova et al. 1987) there is no dpp expression in the
middle of the midgut visceral mesoderm. We are not yet
1036 R. Renter and others
sure why in embryos homozygous for Ubx628 there is
some spatially limited dpp expression, while in the
other cases mentioned dpp expression in the midgut has
vanished completely. Ubx6 has a deletion of 32 bp
which creates a stop codon after only 27 codons and
therefore is likely to be a null mutation in Ubx
(Weinzierl et al. 1987), but perhaps it has residual
function below detectable levels. Df(3R)bxd100 removes functions in addition to Ubx such as abx, bx,
pbx, or bxd; so perhaps one or more of these can affect
dpp expression and is also defective in the Ubx1 /triple
mutant. Another mystery is why dpp expression in the
posterior part of PS 6, where no Ubx protein is
detected, is dependent upon Ubx function. One
possibility is that dpp expression in posterior PS 6 is due
to limited paracrine autoactivation by the dpp protein
coming from PS 7. Such a localized effect would be
prevented in more posterior regions by the negative
influence of abd-A on dpp.
Ubx is sufficient for dpp expression
When the synthesis of Antp or Ubx products is induced
throughout the embryo using a heat shock promoter,
the effects of the proteins are limited to specific tissues
and to specific regions of the embryo (Gibson and
Gehring, 1988; Kuziora and McGinnis, 1988; Mann and
Hogness, 1990; Gonzalez-Reyes et al. 1990). For
example, only the cuticle of an embryo anterior to the
normal region where Ubx is expressed is affected by
expression of Ubx in all cells. Ectopic Ubx protein in
the posterior abdomen has no detectable effect on
cuticle patterning. Thus cells in certain positions are
permissive for the actions of the regulator while others
are not. The restriction of the effect is at least in part
due to competition between homeotic proteins. The
presence of abd-A products prevents Ubx protein from
having an effect in the posterior abdomen (GonzalezReyes et al. 1990; Mann and Hogness, 1990). Thus it is
not simply the negative regulation of Ubx by abd-A
(Struhl and White, 1985) that prevents Ubx from
deciding the fate of posterior abdominal cells. Rather,
groups of cells that contain both abd-A and Ubx protein
form structures characteristic of abd-A function, apparently ignoring the presence of the Ubx protein.
A transposon containing the Ubx cDNA under the
control of the heat shock promoter was used to
ectopically express Ubx throughout the embryo and the
pattern of dpp expression was assayed. Within 3h after
the heat shock, dpp expression in the HSU embryos
extends along the entire anterior end of the midgut in
the visceral rhesoderm. The rapid time course of dpp
induction is consistent with a direct interaction. We
observe ectopic dpp and ectopic labial expression in
stage 15 and stage 16 embryos. At the time of heat
shock, these embryos would have been older than 9 h, a
stage which is refractory to HSU induced segmental
transformations (Gonzalez-Reyes et al. 1990). Thus the
response of dpp to Ubx probably occurs in the absence
of general changes in segment identity.
The cellular context affects whether a homeodomain
protein is capable of activating a particular target gene.
It is striking that Ubx does not govern dpp expression in
embryonic tissues outside of the anterior half of the
midgut visceral mesoderm. The restriction of the
activating effect of Ubx could be due to a transcriptional
cofactor present only in the visceral mesoderm cells that
is required for the Ubx protein to turn on dpp. This
cofactor could be another DNA-binding protein, a
protein that affects the alternative RNA splicing events
that produce different Ubx proteins, a protein that
modifies the Ubx protein, or an adaptor protein that
interfaces between Ubx and the basic transcription
machinery. Alternatively, there could be a block to Ubx
activation of dpp in all tissues except the visceral
mesoderm. The block could take the form of a
repressor protein that competes with Ubx for binding to
the dpp gene, a repressor that competes with Ubx
protein for contacting the basic transcription apparatus,
an enzyme that modifies the dpp gene or the Ubx
protein, or a chromatin component that keeps the dpp
gene in an inactive or inaccessible state.
abd-A represses dpp via a \Jbx-independent
mechanism
We have previously shown that in the absence of abd-A
function dpp is derepressed in visceral mesoderm of the
posterior midgut (Panganiban et al. 19906). In this
report, we demonstrate that the repression of dpp by
abd-A is independent of Ubx. In the heat-shocked HSU
embryos, dpp is ectopically expressed only in the
anterior midgut and not in the visceral mesoderm of the
posterior midgut, where abd-A is expressed. This
indicates that the repression of dpp by abd-A can take
place even in cells that are also expressing Ubx. That
abd-A can repress dpp even in the presence of large
amounts of Ubx protein indicates that the mechanism
by which abd-A negatively regulates dpp is not by
negatively regulating Ubx. In heat-shocked HSU-109
embryos, which are deficient for Ubx and abd-A in
addition to harboring the HSU transposon, ectopic dpp
expression extends over the posterior as well as the
anterior midgut, confirming that abd-A represses Ubx
induction of dpp in the posterior half of the midgut. The
domination of abd-A over Ubx in the control of dpp
provides a specific example of target gene regulation
that parallels the observations on the effects of ectopic
Ubx on cuticle pattern discussed above. It will be
interesting to determine whether Ubx protein does in
fact bind to dpp DNA in vivo and whether the presence
of abd-A interferes with this interaction.
A further indication of the negative regulation of dpp
by abd-A is the appearance of a striped pattern of dpp in
Df(3R)Ubx109 embryos which are deficient for Ubx and
abd-A. This pattern is reminiscent of segment polarity
gene expression patterns and suggests that, in the
absence of normal negative regulation of dpp, the gene
exhibits an aberrant response to one or more segment
polarity genes. In the lateral ectoderm, dpp exhibits a
segmentally repeated pattern; thus the gene does
possess mechanisms for responding to this sort of
pattern information. Presumably, this response is
normally overridden in the visceral mesoderm by the
Homeotic genes regulate growth factor homologs 1037
PS 2
7
8
9
10
11
12
visceral
mesoderr
wt
Ubx
abd-A
Ubx 109
HSU
HSU-109
labn.d.
dpp
Ubx
lab
M abd-A
wg
Fig. 7. Summary of the expression patterns of dpp, wg, Ubx, abd-A, and lab proteins in the visceral mesoderm of various
mutant or transformant embryos. The graph summarizes the expression patterns of Ubx, abd-A, dpp, wg, and lab (see
Panganiban et al. 1990ft) in the visceral mesoderm or the endoderm of the midgut in wild-type (wt), in Ubx null mutant
embryos (Ubx628' Ubx~), in abd-A null mutant embryos (abd-AMXl,abd-A~) or in embryos (Df(3R)Ubxm lacking both
Ubx and abd-A function (Ubx-109), and in heat-shocked HSU or HSU-109 embryos. The approximate parasegmental
positions of expression were determined relative to the segmental grooves of the ectoderm during germ band retraction.
The expression patterns of lab in HSU-109 embryos, and those of wg in Ubx109 and HSU-109 embryos were not
determined.
action of the homeotic regulatory proteins. In addition,
weak dpp expression is normally present in the visceral
mesoderm of PS 9 (Jackson and Hoffmann, in
preparation). The PS 9 expression of dpp in the visceral
mesoderm is regulated by a completely different set of
regulatory elements than those required for dpp
expression in PS 7 and the gastric caeca (Jackson and
Hoffmann, in preparation). It is not known what
regulates dpp expression in PS 9, but in the absence of
regulatory input from the homeotics, these unknown
factors may be able to generate the striped pattern of
dpp expression observed in the Df(3R)Ubx mutants.
That abd-A can have positive as well as negative
effects on target genes is indicated by the observation
that embryos null for abd-A, or for the entire BX-C, do
not express wg in PS 8 of the visceral mesoderm.
However, at least one other function within the BX-C is
also required for wg expression: Embryos that are
homozygous for Df(3R)bxd100 or trans-heterozygous
for Df(3R)bxd1O0/Df(3R)Ubx109' and therefore still
have abd-A function, also lack wg expression. This
function may not be Ubx because, in Ubx628 mutant
embryos, wg expression in PS 8 of the visceral
mesoderm is normal. However, as is discussed above,
Ubx62s may not be null for all Ubx functions. The
absence of ectopic wg products in regions of HSU
embryos where abd-A, Ubx, and dpp are all expressed
is consistent with the hypothesis that Ubx is not the BXC function required for wg expression. Immergluck et
al. (1990) have reported that dpp expression in PS 7 is
required for wg expression in PS 8. Perhaps there is
sufficient movement of dpp protein, at levels undetected in the present studies, from PS 7 into the visceral
mesoderm of PS 8, to induce wg expression in cells
expressing abd-A and not expressing Ubx. The overlap
we have observed in the Ubx and wg protein domains
might be due to an anterior movement of the wg protein
from its site of synthesis into the most posterior part of
PS 7. Experiments using heat shock promoter driven
ectopic expression of dpp could be used to test this
hypothesis.
Reciprocal regulation of dpp and Ubx
Although Ubx is responsible for localized expression of
dpp in posterior PS 6 and PS 7 of the visceral
mesoderm, we have also observed an effect of dpp on
Ubx in the visceral mesoderm (Panganiban et al.
19906). We have not established whether Ubx protein
or dpp protein is present first in the visceral mesoderm
of PS 7. dpp RNA is clearly detected in the visceral
mesoderm by stage 11 as germ band shortening begins
(Jackson and Hoffmann, in preparation). We think it
most likely that Ubx initiates dpp expression, and that
dpp is required to maintain Ubx expression in PS 7.
Secreted factors, like dpp, with a limited capacity for
moving from their site of synthesis, may provide an
ideal means of mediating intercellular interactions in a
specific region of the embryo. The intercellular signals
1038 R. Reuter and others
dpp—\Scr
Gastric
caeca
n
Scr
I
Antp \—Ubx Ubx
Mesoderm
?
Anterior
Constriction
Middle
Constriction
Posterior
Constriction
Endoderm
lab
Anterior
Parasegment 7
Posterior
Fig. 8. Summary of gene functions in the visceral mesoderm. The diagram is a schematic representation of the visceral
mesoderm and endoderm of an embryo, not to scale, with anterior to the left. Results are incorporated from the present
paper, Tremml and Bienz, 1988; Bienz and Tremml, 1988; Panganiban et al. 1990b, Reuter and Scott, 1990; and
Immergluck et al. 1990. In the visceral mesoderm Scr and dpp are required for formation of the gastric caeca. Scr is
expressed in cells posterior to the cells that form the caeca and therefore appears to act at a distance to affect caeca
formation. The target genes acted upon by Scr are unknown, dpp is active in visceral mesoderm cells that overlie the
developing caeca but dpp protein has not been observed to move into the endoderm in this part of the embryo, dpp
prevents Scr expression from spreading to the anterior. Antp is expressed in the visceral mesoderm, acts upon unknown
targets, and is required for the formation of the anterior midgut constriction. Both Ubx and abd-A functions are required
for the formation of the posterior midgut constriction. In PS 7 Ubx prevents Antp transcription and activates dpp, setting
the anterior border of dpp expression, dpp protein moves across to the endoderm and activates lab. abd-A represses the
expression of both Ubx and dpp and sets the posterior border of dpp and Ubx expression. In PS 8 abd-A is essential for
the expression of wg, and the wgprotein moves to the endoderm. The factors controlling the posterior limit of wg
expression are not known, wg may activate residual dpp expression in the visceral mesoderm in the absence of Ubx
function (not shown in diagram). The regulation of the weak dpp expression in the vicinity of the third constriction has not
been studied and is not shown.
may be necessary to maintain particular differentiated
cell states among the population of cells, possibly by
causing maintenance of homeotic gene expression.
Requirements for lab expression in the midgut
endoderm
Expression of dpp in the midgut visceral mesoderm is
required for the expression of lab in the underlying
endoderm (Immergluck et al. 1990; Panganiban et al.
19906). We have extended this analysis and find that in
order for endoderm cells to express lab, they must have
been in contact with the visceral mesoderm cells which
express dpp relatively early (i.e. stage 13) during
midgut development. The apparent temporal requirement for early exposure to dpp for lab expression may
reflect a subsequent commitment by the endoderm cells
to specific patterns of differentiation, for which lab
expression is a marker.
Early exposure of the endoderm cells to dpp is
required, but clearly not sufficient to induce lab. In
heat-shocked HSU embryos, ectopic expression of dpp
extends over the entire anterior midgut, while lab
expression is restricted to the endoderm of the midgut
between the gastric caeca and the second constriction.
The distribution of other factors (e.g. dpp transport
protein or receptor, signal transduction machinery,
and/or specific transcription factors) may determine
whether particular endoderm cells can respond to dpp.
Differences in the responses of parts of the midgut
endoderm to dpp were noted previously (Panganiban et
al. 1990b). dpp is expressed in wild-type embryos in the
visceral mesoderm of the gastric caeca as well as the
visceral mesoderm of PS 6 and 7, yet lab expression is
only detected in the endoderm cells of PS 6 and 7. The
dpp protein has been shown to migrate from the visceral
mesoderm across the endodermal cell layer in PS 6 and
7, however, no migration of dpp protein has been
observed at the caeca (Panganiban et al. 1990;
Panganiban and Hoffmann, unpublished observations).
It may be that the dpp protein simply does not migrate
to the endoderm at the anterior end of the midgut, and
that if it did, lab would be induced in the underlying
endoderm.
Gene interactions in the midgut
The interactions among the genes discussed above and
by Panganiban et al. (1990£>) are summarized in Fig. 8.
Immergluck et al. (1990) have independently arrived at
many of the same conclusions. At the anterior end of
the midgut, both dpp and Scr expression are required
for the gastric caeca to form, dpp expression prevents
Scr expression in the cells that form the gastric caeca.
As the cells expressing Scr act at a distance on the cells
that form the gastric caeca, we expect that an as yet
unidentified secreted factor, perhaps another growth
factor homolog, may be made in response to Scr. Antp
expression positively regulates Scr expression, also at a
distance, and Antp expression is required for the
Homeotic genes regulate growth factor homologs 1039
anterior midgut constriction to form. Mutations in Scr
or Antp do not affect the expression of the other
homeotic genes, or of dpp or wg. In the adjacent region,
we propose that Ubx causes dpp expression, which
positively maintains Ubx expression in the visceral
mesoderm cells and induces labial expression in the
adjacent endodermal cells. Both dpp and Ubx expression are required for these cells to form the middle
constriction. In the posterior midgut, abd-A expression
negatively regulates Ubx and dpp, and positively
regulates wg. The domain of wg expression is restricted
to PS 8 - only a portion of the visceral mesoderm cells
expressing abdA - by as yet unidentified factors. It has
been reported that movement of the wg protein to the
apposing endoderm is involved in maintaining lab
expression in the endoderm (Immergliick et al. 1990).
The molecular basis of these interactions remains to
be determined. These studies present an initial picture
of the regulatory circuitry in the midgut of the embryo
and establish growth factor homologs in Drosophila as
target genes for the pattern information inherent in
localized homeotic gene expression. It is likely that the
homeotic gene products themselves also regulate the
production of other molecules including cytoskeletal
components, specialized enzymes, and cell surface
components. Our understanding of how specific morphological events occur in response to the homeotic
genes will require continued progress in identifying the
target genes of the homeotics themselves and in
identifying the genes regulated indirectly by molecules
like dpp and wg.
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Thorn Kaufman, Marcel van den Heuvel, Roel Nusse, and
Rob White for gifts of antibodies (W.B., anti-afod-^4; S.C.,
&ni\-Abd-B; T.K., anti-lab; R.N.; anti-wg; R.W., anti-Ubx)
and to Gary Struhl, Gines Morata, Acaimo Gonzalez-Reyes,
and Richard Mann for sending usflystocks (HSU transformant flies, G.S. and R.M.; HSU-109 transformantfliesG.M.
and A.G.-R.). R.R. would like to thank Maria Leptin for
space and support in her laboratory. We thank Mariann Bienz
for communication of results prior to publication and for
stimulating discussions. The research was supported by NIH
grant no. 18163 to M.P.S. and ACS grant no. NP-612 to
F.M.H. M.P.S. was an Investigator, and R.R. a Research
Associate, of the Howard Hughes Medical Institute.
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(Accepted 9 October 1990)