Download brachyenteron function in the mesoderm

3991
Development 126, 3991-4003 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
DEV7738
Functions for Drosophila brachyenteron and forkhead in mesoderm
specification and cell signalling
Thomas Kusch1 and Rolf Reuter1,2,*
1Institut für Genetik, Universität zu
2Abt. Genetik der Tiere, Institut für
Köln, Weyertal 121, D-50931 Köln, Germany
Zellbiologie, Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany
*Author for correspondence (e-mail: [email protected])
Accepted 1 July; published on WWW 23 August 1999
SUMMARY
The visceral musculature of the larval midgut of
Drosophila has a lattice-type structure and consists of an
inner stratum of circular fibers and an outer stratum of
longitudinal fibers. The longitudinal fibers originate from
the posterior tip of the mesoderm anlage, which has been
termed the caudal visceral mesoderm (CVM). In this
study, we investigate the specification of the CVM and
particularly the role of the Drosophila Brachyuryhomologue brachyenteron. Supported by fork head,
brachyenteron mediates the early specification of the
CVM along with zinc-finger homeodomain protein-1. This
is the first function described for brachyenteron or fork
head in the mesoderm of Drosophila. The mode of
cooperation resembles the interaction of the Xenopus
homologues Xbra and Pintallavis. Another function of
brachyenteron is to establish the surface properties of the
CVM cells, which are essential for their orderly migration
along the trunk-derived visceral mesoderm. During this
movement, the CVM cells, under the control of
brachyenteron,
induce
the
formation
of
one
muscle/pericardial precursor cell in each parasegment.
We propose that the functions of brachyenteron in
mesodermal development of Drosophila are comparable
to the roles of the vertebrate Brachyury genes during
gastrulation.
INTRODUCTION
during the evolution of different taxa and some of the original
functional aspects might have been lost. In addition, the
complexity of an organism might mask common functions,
making detailed functional analyses necessary.
Such a dynamic functional diversification has recently been
proposed for the Brachyury genes. The Brachyury proteins are
a subfamily of the T-domain transcription factors, which are
classified by a highly conserved DNA-binding domain (for
reviews, see Smith, 1997; Papaioannou and Silver, 1998).
Brachyury relatives have been found in the diploblastic
Cnidarian as well as triploblasts like chordates, protochordates,
echinoderms, hemichordates and insects, suggesting that
evolutionarily conserved mechanisms are regulated by these
factors (Peterson et al., 1999; Technau and Bode, 1999).
However, initial comparisons of the expression and function of
Brachyury genes between deuterostomes and protostomes
indicated diverse rather than common functions. In
deuterostomes, Brachyury seemed to play a critical role in
mesoderm development, whereas the protostome Brachyury
proteins have been shown to be involved in hindgut
development. For instance, in vertebrates, Brachyury is
transiently expressed in all nascent mesendodermal cells. As
development proceeds, the expression becomes restricted to the
notochord and tailbud. In cephalochordates and urochordates,
Many of the factors that play important roles in the ontogenesis
of different taxa share high structural similarites in functionally
relevant domains, such as DNA-binding or protein-protein
interaction domains. These similarities allow factors to be
grouped into families such as homeodomain proteins or Wnt
proteins (for reviews see e.g. McGinnis and Krumlauf, 1992;
McMahon et al., 1992). In some exceptional cases, members
of these families are associated with the regulation of a
particular developmental process. For instance, the Pax-6
subfamily of the Pax proteins are possibly involved in the
regulation of photoreceptor development in all bilaterians,
suggesting that this function was adopted early during
evolution (for a review see Gehring, 1996). Another case are
the gene products of the Hox complex, a subfamily of
homeodomain proteins, which are involved in patterning the
anteroposterior axis in most – if not all – higher metazoans (for
a review see Slack et al., 1993). Often, a common function in
development is far less obvious, in spite of high structural
similarities of the factors within one particular subfamily,
making it difficult to distinguish between original and
secondarily acquired functions. Such factors might have been
assigned to the regulation of multiple developmental processes
3992 T. Kusch and R. Reuter
the distinct aspects of mesodermal expression have been
divided between two paralogues, one expressed in the posterior
mesoderm and the other expressed in the notochord, whereas
no expression is detectable in the gut (Yasuo et al., 1996). In
echinoderms, Brachyury has only been reported to be
expressed in the migrating secondary mesenchyme cells
(Yasuo et al., 1995). However, it has recently been shown that
the hemichordate Brachyury homologue is not only expressed
in the mesoderm but also in the oral and anal region of the gut
(Tagawa et al., 1998; Peterson et al., 1999). Since Brachyury
is transiently expressed in vertebrates in the developing hindgut
epithelium of the tailbud region, it has been proposed that the
expression of Brachyury in the posterior gut reflects its original
function in development. Intriguingly, the Hydra Brachyury
gene is expressed in the cells of the gut opening (Technau and
Bode, 1999).
The Drosophila Brachyury homologue brachyenteron (byn)
is essential for the development of hindgut, anal pads and
Malpighian tubules (Kispert et al., 1994; Singer et al., 1996).
byn is activated by the terminal gap gene tailless (tll; Pignoni
et al., 1990) in a region of 0-20% egg length of the syncytium
(0% = posterior tip). With completion of cellularization, the
byn expression becomes downregulated in the posteriormost
cap of the embryo, which will later form the posterior midgut,
by the terminal gap gene huckebein (hkb, Weigel et al., 1990).
Thus, the expression of byn is confined to a ring of cells from
about 10-20% egg length. The dorsal and the lateral aspects of
that ring correspond to the proctodeum from which the
hindgut, the anal pads and the Malpighian tubules later develop
(for details see Skaer, 1993). Intriguingly, hkb also determines
the posterior extent of the ventral mesoderm primordium by
repressing the mesodermal determinant snail (sna; Brönner et
al., 1994; Reuter and Leptin, 1994). This suggests that the
ventralmost aspect of byn expression might comprise the
posterior tip of the mesoderm primordium. Recently, it has
been reported that a population of cells develops from the
posterior region of the mesoderm, which migrate in an orderly
movement anteriorly and eventually form an outer layer of
longitudinal muscle fibers surrounding the midgut (Georgias et
al., 1997; Campos-Ortega and Hartenstein, 1997). The cells
have been termed caudal visceral mesoderm (CVM; Nguyen
and Xu, 1998) in order to distinguish them from the progenitors
of the inner sheet of circular muscles of the midgut (for an
overview see Bate, 1993). The latter are recruited from 11
parasegmentally arranged clusters of dorsal mesoderm in the
trunk region and were therefore referred to as trunk visceral
mesoderm (TVM).
In this study, we wished to elucidate the role of byn and
other genes in the specification of the CVM during early
embryogenesis. In addition, we addressed the byn-mediated
functions of the CVM during later stages of development. The
CVM is specified through the interaction of byn and fork head
(fkh; Weigel et al., 1989a,b) and high levels of the gene
product of zinc-finger homeodomain protein 1 (zfh-1; Fortini
et al., 1991; Lai et al., 1991; Broihier et al., 1998).
Additionally, byn confers specific adhesive and signalling
properties on the CVM which are prerequisite for the
inductive and migratory behaviour of the CVM. In the light
of these findings, the role of byn in the mesoderm of
Drosophila is reminiscent of proposed mesodermal functions
of vertebrate Brachyury genes.
MATERIALS AND METHODS
Fly stocks
byn4 and byn5: intermediate and strong hypomorphic brachyenteron
alleles, respectively (Singer et al., 1996). Df(2L)TE116(R)GW11: used
as snail deficiency (Ashburner et al., 1990). twiEY53: amorphic twist
allele (Simpson, 1983; Thisse et al., 1987). zfh-12: allele of zinc-finger
homeodomain gene 1 (Lai et al., 1993). hkbXM9: amorphic huckebein
allele (Brönner et al., 1994). tllL10 and Df(3R)tllg: strong hypomorphic
tailless allele and deficiency of the tll locus, respectively (Strecker et
al., 1988; Jürgens et al., 1984; Pignoni et al., 1990). fkhXT6: small
deficiency of the fork head locus (Weigel et al., 1989a). byn fkh:
double mutant, generated by meiotic recombination of byn5 and
fkhXT6. croc-lacZ: transgene that directs lacZ reporter gene expression
in the CVM under the control of a 7 kb BglII fragment from the 3′region of the croc transcription unit (Häcker et al., 1995). cpo-lacZ:
P[ry+; cpo10kb]2A, enhancer trap in the couch potato (cpo) locus that
confers β-galactosidase (β-gal) expression in the developing
longitudinal muscle fibers (Bellen et al., 1992; Campos-Ortega and
Hartenstein, 1997). htlAD326: amorphic heartless allele (Gisselbrecht
et al., 1996). dpp37: amorphic dpp allele (Hoffmann and Goodman,
1987). G447.2: GAL4 enhancer trap that directs expression in the
CVM from stage 10 onward (Georgias et al., 1997). twi-GAL4:
transgene on the second chromosome for mesodermal GAL4
expression (Giebel et al., 1997). UAS-lacZ: transgene for the detection
of GAL4 activity in the embryo (Brand and Perrimon, 1993).
For the generation of UAS-byn transgenes, the byn cDNA (Kispert
et al., 1994) was isolated from the pNB40 vector (Brown and
Kafatos, 1988) as a HindIII-EcoRI fragment, cloned into pBluescript
KS II (+), isolated again as a KpnI-XbaI fragment and finally cloned
into the pUAST vector (Brand and Perrimon, 1993). This construct
was introduced into the germline by P-element-mediated
transformation (Spradling, 1986) and several independent transgenic
lines were isolated. For misexpression experiments, the UAS-byn
lines Is(2)P[UAS-byn]18 or Is(3)P[UAS-byn]11 were used. For the
CVM-specific rescue, the line Is(2)P[UAS-byn]18 and the GAL4driver line G447.2 were crossed into byn5 mutant background,
respectively. The UAS-fkh line Is(X)P[UAS-fkh] E10.2 was kindly
provided by K.-P. Rehorn. This transgene comprises a 2.2 kb
HindIII-PstI genomic subclone of the fkh locus (for a genomic map,
see Weigel et al., 1989).
Generation of anti-Byn antibodies
For the generation of the anti-Byn antiserum, the coding region of the
byn cDNA (Kispert et al., 1994) was amplified by PCR (Saiki et al.,
1988) using the oligonucleotides 5′-GAAGATCTATGACCACATCGCACATCC-3′, and 5′-GAAGATCTCTGCAGCGTGCTCTGTGGCG-3′. The primers introduce BglII sites flanking the byn-coding
region. PCR products were subcloned via these BglII sites into the
bacterial expression vector pQE12 (Qiagen, Hamburg, Germany) and
were confirmed by sequencing. The resulting bacterially expressed
full-length Byn protein has a C-terminal His6-tag, which allowed the
purification of the fusion protein over Ni2+-NTA-agarose (Qiagen,
Hamburg, Germany) columns. The E. coli strain M15 carrying the
pREP4 repressor plasmid (Qiagen, Hamburg, Germany) was used for
the overexpression of the 711 aa fusion protein with a molecular mass
of 76 kDa. Expression was induced at an OD600=0.5 by adding 0.5
mM IPTG, and the bacteria were harvested after 3 hours of induction.
The fusion protein was purified under denaturing and reductive
conditions (10 mM β-mercaptoethanol) according to the
manufacturer’s protocol (Qiagen, Hamburg, Germany). It was
renatured by stepwise dialysis against 25 mM Tris, pH 7.2, 5 mM
DTT, 0.5 mM MgCl2, 60 mM KCl containing 8 M, 4 M, 2 M, 1 M
and no urea. Renatured Byn-His6 protein was affinity-purified over
Hitrap heparin-columns (Pharmacia, Uppsala, Sweden) by eluting the
binding protein fraction with 500 mM NaCl, 25 mM Tris, pH 7.2, 5
mM DTT, 0.5 mM MgCl2, 60 mM KCl. The desalted and lyophilized
brachyenteron function in the mesoderm 3993
fusion protein was sent to Eurogentec (Seraing, Belgium) for the
immunization of rabbits.
In situ detection of mRNA, proteins and of apoptotic cells
For detection of zfh-1 transcripts a 0.4 kb EcoRI fragment of the zfh-1
transcription unit was used (kindly provided by Dr Z. Lai). The 412
retrotransposon was used to detect gonadal mesoderm (Brookman et al.,
1992). The probes were labelled with digoxigenin-dUTP (Boehringer,
Mannheim, Germany) and used for in situ hybridization as described by
Tautz and Pfeifle (1989). Protein was detected in situ using standard
immunohistochemical techniques with avidin-biotinylated peroxidase
complexes. For the dissection of guts, devitellinized embryos were
filleted with a glass needle under PBS. The gut was then laid out on the
glass, fixed with 4% formaldehyde for 15 minutes at room temperature
and processed for immunostaining as usual. The following antibodies
were used in the study: anti-FasIII (Snow et al., 1989), anti-Mef2 (rabbit;
Lilly et al., 1995), anti-muscle MHC (rabbit 722; Kiehart and Feghali,
1986), anti-Eve (rabbit, Frasch et al., 1987), anti-α-Tubulin
(mouse monoclonal 3A5; Wolf et al., 1988), anti-Vasa (rabbit,
Broihier et al., 1998), anti-Srp (Riechmann et al., 1997), antiKr (Gaul et al., 1987) and anti-β-galactosidase (mouse
polyclonal; Sigma, St. Louis, USA).
For sectioning, immunostained embryos were embedded
in Araldite and cut on a Leica microtome RM 2065 in slices
of 10 mm thickness. The sections were then transferred to
a glass slide and covered with Araldite and a coverslip.
Apoptotic cells were detected by end-labelling the DNA
of fragmented chromatin by the TUNEL assay (see White
et al., 1994).
migration substratum (Georgias et al., 1997; Fig. 1C,D). During
the last phase of the migration, which takes place as the midgut
encloses the yolk, the progenitors of the longitudinal muscle fibers
spread regularly over the underlying circular muscle fibers (Fig.
1E-G). The cells acquire a spindle shape, then stretch in an
anteroposterior direction and form about 16-20 regularly spaced
longitudinal muscle fibers. These fibers reach from the
proventriculus to the midgut-hindgut transition where the ureters
of the Malpighian tubules insert (Fig. 1G-I). The foregut and the
hindgut lack any longitudinal muscles and are solely covered by
the inner layer of circular muscles (Fig. 1H,I).
byn and fkh are essential for the specification of the
caudal visceral mesoderm
Previous studies have shown that byn is expressed at the
blastoderm stage in a ring of cells at the posterior terminus of
Microscopy
Embryos were mounted and oriented in polymerizing
Araldite, the dissected guts in glycerol. Pictures were either
taken on a Zeiss Axiophot on Kodak Ektachrome 64T film
and then digitized by transfer to Kodak PhotoCDs or
recorded by a Kontron ProgRes 3008 digital camera on a
Zeiss Axioplan. The figures were assembled in Adobe
Photoshop.
RESULTS
Development of the longitudinal muscle
fibers surrounding the midgut
The visceral musculature surrounding the midgut
consists of two layers: the inner layer of circular fibers
and the outer, longitudinally oriented fibers. During
germband retraction and midgut closure, the progenitors
of these latter fibers, the so-called caudal visceral
mesoderm (CVM), perform an ordered movement that
can be subdivided into three phases. The first migratory
phase starts at early germband retraction when the cells
begin to move anteriorly from their position at the
posterior tip of the mesodermal germ layer and split into
two tightly packed, bilaterally symmetrical clusters on
each side of the posterior midgut primordium (Fig.
1A,B). When these clusters have reached the anterior tip
of the posterior midgut primordium, the cells detach
from each other and disperse anteriorly as two rows
along the germband, the second phase of the migration.
During this movement, the cells are arranged along the
dorsal and ventral edge of the midgut primordia and are
in close contact with the band of progenitors of the
circular muscle fibers. The band seems to serve as a
Fig. 1. Development of the longitudinal muscle fibers surrounding the midgut.
(A-F) Embryos carrying the croc-lacZ reporter were immunostained for β-gal
protein. (A,B) With onset of germband retraction, the CVM cells move
anteriorly and split into two clusters ventrolaterally to the posterior midgut
rudiment. (C,D) Later, the CVM cells (blue; white arrowhead) disperse along the
strip of TVM cells (brown, black arrowhead), visualized with an anti-FasIII
antibody. (E) During midgut closure, the cells spread as longitudinally oriented
fibers around the circumference of the midgut (ventral view, stage 13).
(F) Magnification of a dissected midgut of a stage 16 embryo. β-gal highlights
the longitudinal fibers. (G, H) A midgut immunostained for α-Tubulin to
visualize the visceral musculature. The inner stratum of circular muscles is
covered by the longitudinal fibers (white arrowheads), which reach from the
foregut-midgut transition over the tips of the gastric caeca (gc) to the midguthindgut transition where the ureters (ur) of the Malpighian tubules (mt) insert.
(I) The nuclei of the splanchnopleura are visualized with an antibody against
Mef2. All embryos are shown laterally in sagittal optical sections if not stated
otherwise and are staged according to Campos-Ortega and Hartenstein (1997).
3994 T. Kusch and R. Reuter
Fig. 2. byn is expressed in the CVM. (A) At
blastoderm stage, Byn is expressed (bracket) in the
posterior tip of the mesoderm anlage comprising the
CVM anlage. (B) This expression is maintained
during gastrulation (arrowhead). (C,D) The fading
Byn protein signal marks the anteriorly migrating
CVM cells during early germband retraction (open
arrowheads). All embryos were stained with antiByn antiserum.
the embryo, which is posteriorly delimited by hkb (Kispert et
and do not distribute along the germband (Fig. 4F). During stage
al., 1994, see also Fig. 2A). The posterior border of the
11, most of the cells acquire a condensed appearance resembling
mesoderm anlage is also set by the repressor function of hkb,
apoptotic bodies (Fig. 4D,F). We detect a high level of apoptosis
suggesting that the ventral aspect of the byn expression domain
in the proctodeum of byn embryos as well as in the posteriormost
comprises the posteriormost region of the mesoderm anlage.
mesoderm (Fig. 4H). By stage 13, cells with the properties of
In fact, this aspect is internalized by the ventral furrow and
the CVM are not detectable any longer in the mutants (data not
forms the posterior end of the mesodermal germ layer shortly
shown) and, as expected from that, the dissected midguts of byn
after the onset of gastrulation (Fig. 2B). The byn RNA
embryos lack the outer, longitudinal muscle fibers (Fig. 4J).
expression ceases in these cells when the germband extends
byn embryos show morphological aberrations at a time before
(data not shown; see also Kispert et al., 1994), but an anti-Byn
the CVM begins to migrate anteriorly (Singer et al., 1996; Fig.
antiserum can detect the Byn protein far longer. We were able
4H). The severely shortened hindgut causes a significant shift in
to follow the Byn-expressing cells until they migrate anteriorly
the spatial relationship of the various primordia at the posterior
as progenitors of the longitudinal muscle fibers at late stage 11
region of the embryo and thereby might indirectly affect the
(Fig. 2C,D). This observation confirms the proposed caudal
migration of the CVM. In order to exclude such an indirect
origin of the progenitors of the longitudinal visceral muscle
influence, we generated byn embryos expressing byn in the
fibers (Georgias et al., 1997), and thus it seems suitable to
CVM precursors, but not in the hindgut (Fig. 4L). In such
adopt the suggested name caudal visceral mesoderm (CVM)
embryos, the CVM survives and disperses virtually the same as
for this mesodermal cell population (Nguyen and Xu, 1998).
in wild type along the TVM, whereas the proctodeum remains
In order to distinguish these cells from the progenitors of the
rudimentary as in ordinary byn mutants (compare Fig. 4L,N).
circular muscle layer the latter are referred to in the following
These results demonstrate that the defective migration and the
as the trunk visceral mesoderm (TVM).
death of the CVM cannot be attributed to the disordered
We investigated the specification of the CVM
and monitored its fate by the detection of Byn
protein or the expression of CVM-specific
markers like croc-lacZ and cpo-lacZ (Häcker et
al., 1995; Campos-Ortega and Hartenstein,
1997). The initial byn expression at the posterior
pole is regulated by tll and hkb (Kispert et al.,
1994). Thus it was likely that the CVM cells are
specified under the control of the same genes. In
fact, in hkb embryos, the size of the CVM
primordium is enlarged and comprises more cells
than normal (Fig. 3A,B). This corroborates the
notion that the CVM primordium constitutes the
most posteriorly located mesoderm primordium.
tll expression reaches more anteriorly than the
hkb domain and encompasses the primordia of
the proctodeum and of the CVM. One would
therefore expect that the formation of the CVM
is entirely dependent on tll. Indeed, this is the
case: the CVM is missing in tll mutant embryos
(Fig. 3C). Part of the function of tll seems to be
Fig. 3. byn and fkh are essential for the development of the CVM. Embryos carrying
mediated by byn. In byn mutants, a significantly
the croc-lacZ reporter were immunostained for β-gal. (A) Wild-type background.
reduced number of CVM cells is seen, and these
(B) In hkb mutants, supernumerary CVM cells are specified. (C) tllL10 embryos lack
few cells form clusters that are less compact and
the CVM. (D) In byn5 mutants, only a few CVM cells develop. (E) In fkh embryos, a
migrate significantly slower than in wild type
normal number of CVM cells are specified that fail to migrate along the TVM. (E) In
byn fkh double mutants, no CVM is formed.
(Fig. 4B,D). Later, they fail to contact the TVM
brachyenteron function in the mesoderm 3995
Fig. 4. byn is essential for migration and
survival of the CVM. CVM development in
(A,C,E,G,I) wild type and (B,D,F,G,J) byn
mutants. (A,B) The CVM cells (arrowheads)
of a (B) hypomorphic byn4 embryo form lesscondensed clusters and migrate significantly
slower than in (A) wild type. Both embryos are
at the same developmental stage and are
immunostained for Byn protein (dorsal view).
(C,D) Embryos carrying croc-lacZ stained for
β-gal (dorsal view). The number of CVM cells
is strongly reduced in (D) byn5 mutants.
(E,F) Magnification of the tail region of stage
12 embryos double-labelled for FasIII (brown)
and β-gal (blue). (E) In the wild type, the
CVM cells (blue, white arrowheads) disperse
along the TVM (brown, black arrowheads)
whereas they fail to contact the TVM in (F) a
byn5 embryo. (G,H) Apoptotic cells detected
by the TUNEL assay. (H) Massive apoptosis is
seen in the proctodeum (arrow) and in the
CVM (open arrowhead) of byn5 embryos.
(I,J) Dissected guts at stage 16 immunostained
for MHC protein. (I) In wild-type midguts, the
cell bodies of the longitudinal muscle fibers
are regularly spaced (arrowheads). (J) No
longitudinal muscle fibers can be detected in
the byn5 mutant. The circular muscle layer
shows sporadic ruptures (grey arrowheads). No
midgut constrictions are formed. (K-N) Rescue
of CVM development of byn embryos by the
forced expression of byn in the CVM
primordium. (K) An embryo double
heterozygous for the GAL4 driver G447 and
UAS-byn. Endogenous Byn protein marks the
hindgut (arrow; also see insert), whereas the
G447-controlled expression of Byn from the
UAS-byn transgene is seen in the CVM (white
arrowheads). (L) A byn mutant carrying G447
and UAS-byn and stained for Byn protein. The
CVM cells have survived and migrate along
the TVM almost as in wild type, while the
hindgut is still rudimentary (arrow; see insert).
(M) Expression of the G447 driver detected by
β-gal. (N) Non-transgenic byn5 mutant stained
for Byn protein.
morphology of the posterior gut structures. We therefore
conclude that byn in the mesoderm is essential for the adhesive
and migratory properties of the CVM precursors.
byn cannot be the only gene that mediates the function of tll
in the specification and further development of the CVM since
the lack of tll causes a far stronger phenotype than the lack of
byn. In addition to byn, the gene fkh is known to act downstream
of tll in the posterior gut (Weigel et al., 1990; Casanova, 1990).
fkh is expressed in a large domain at the posterior pole (Weigel
et al., 1989) that encompasses the byn expression domain
including the ventral, mesodermal aspect. In fkh mutants, the
CVM specification seems less impaired than in byn mutants: the
number of CVM cells is initially quite normal. However, as in
byn mutants, the cells fail to migrate along the germband (Fig.
3E) although differentiation of the migration substratum, the
TVM, is not affected (data not shown). By stage 14, most of the
CVM cells have been eliminated by apoptosis. On this level of
analysis, fkh mutants resemble embryos homozygous for weak
byn alleles. However, the phenotype of byn fkh double mutants
shows that byn and fkh either have distinct functions in the
specification of the CVM or act synergistically. In double
mutants, no CVM cells are distinguishable (Fig. 3F), just as in
tll mutants (Fig. 3C). Therefore, the function of tll in the
specification of the CVM appears to be mediated by byn and fkh.
3996 T. Kusch and R. Reuter
byn and fkh act synergistically in the specification
The caudal visceral mesoderm is specified
of the caudal visceral mesoderm
independent of twist function
We asked whether byn and/or fkh would be sufficient in the
The overlapping expressions of the two zygotic genes twist (twi)
mesoderm to specify the development of the CVM. We
and snail (sna) are essential for gastrulation and specification of
therefore expressed either byn, fkh or a
combination of both genes throughout the
mesoderm shortly after gastrulation. In the
first case, after unrestricted mesodermal
byn expression, a few CVM-like cells
emerge from the mesoderm of the head
region (Fig. 5A). We did not observe any
gross changes in the specification of other
mesodermal derivatives such as somatic
mesoderm, fat body or TVM (data not
shown, but also see below). In contrast, the
ectopic expression of fkh within the
mesoderm does not lead to the formation
of additional CVM-like cells (Fig. 5E).
Rather, the differentiation of other
mesodermal derivatives like somatic
mesoderm and the TVM is severely
affected (K.-P. Rehorn and R. R.,
unpublished). This might explain why in
these embryos the CVM cells only
distribute over the posterior midgut
primordium where the cells later undergo
apoptosis (data not shown). Intriguingly,
the combined overexpression of byn and
fkh leads to a drastic increase in the
number of anteriorly derived CVM-like
cells (Fig. 5F). Hence, fkh strongly
enhances the effects of byn in the
specification of mesodermal cells as
CVM.
Only the anterior and the posterior
mesoderm are competent to be specified by
byn in conjunction with fkh as CVM (Fig.
5A,F). Therefore, at least one other gene
must exist that confines the competence to
form CVM to these two regions. A good
candidate for this gene is zinc finger
homeodomain protein-1 (zfh-1; Lai et al.,
1991). At the blastoderm stage, zfh-1 is
expressed in high levels in the terminal
regions of the mesoderm including the
Fig. 5. The CVM is specified by the combined activity of byn, fkh and high levels of zfh-1.
primordium of the CVM (Fig. 5I) and zfh(A-H) Embryos carrying croc-lacZ were stained for β-gal. (A,B,D) Embryos double
1 is essential for the migration of the CVM
heterozygous for twi-GAL4 and UAS-byn. (A) A few ectopic CVM-like cells are formed in
(Broihier et al., 1998): in zfh-1 mutant
the head mesoderm at stage 11 (white arrowhead). (B) At germband retraction stage, no
embryos, CVM-specific gene expression
CVM cells have reached the anterior trunk region. The fibers have formed, but are randomly
such as croc-lacZ is deleted (Fig. 5H).
distributed. (C,D) Dorsal views of stage 11 embryos. (C) In the wild type, the CVM cells
(blue) are exclusively attached to the TVM (labelled for FasIII, brown). (D) Upon
From the restricted effects of ectopic
ubiquitous expression of byn in the mesoderm, many CVM cells are attached to somatic
byn/fkh, we propose that the two genes are
mesoderm and fat body rather than to the TVM (arrowheads). (E) Overexpression of fkh in
capable of specifying CVM development
the mesoderm abolishes the dispersion of the CVM along the TVM, but does not lead to the
only in the region of high zfh-1 expression.
formation of ectopic CVM cells in the head region. (F) Embryos expressing both byn and
zfh-1, byn and fkh act in parallel
fkh throughout the mesoderm form almost the same number of CVM-like cells in the
downstream of tll. High levels of caudal
anterior region as they do in the posterior. (G) croc-lacZ in wild type. (H) In a zfh-1 mutant,
zfh-1, as for byn and fkh, are dependent on
croc-lacZ expression is not detectable. (I-L) zfh-1 expression in (I,K) wild type, (J) tll and
tll (Fig. 5J), and there is no crossregulation
(L) byn fkh mutants. (I) High levels of zfh-1 mRNA expression can be detected in the
between zfh-1, byn and fkh (Fig. 5L; data
termini of the mesoderm of the wild type. (J) zfh-1 expression in the posterior tip of the
not shown; Singer et al., 1996; Kispert et
mesoderm is weakened in tll mutants. (L) It is unchanged in byn fkh embryos, which at late
stage 9 can be distinguished by their short proctodeum.
al., 1994).
brachyenteron function in the mesoderm 3997
the mesoderm (for a review see Leptin, 1995). twi is an activator
of mesodermal gene expression, whereas sna mainly functions as
a repressor of neuroectodermal gene expression in the mesoderm.
Deviating from this rule, mesodermal zfh-1 expression is missing
in sna mutants (Hemavathy et al., 1997), whereas high zfh-1
expression is unaffected in the termini of the mesoderm in twi
mutants (Lai et al., 1991; see also Fig. 6G). The expression of
byn and posterior fkh does not depend on sna or twi function (data
not shown), raising the question whether the CVM might be at
least partially specified in twi or sna mutants.
In sna embryos, no byn-expressing cells can be detected in a
mesoderm-specific position, i.e. between the epidermis and the
midgut epithelium. Such embryos lack CVM-specific gene
expression (data not shown), which is in accordance with the
findings that zfh-1 depends on sna (Hemavathy et al., 1997) and
CVM development on zfh-1 (Broihier et al., 1998). Strikingly,
in twi mutants, byn-expressing cells can be found at an internal,
mesoderm-typical position in the tail region (Fig. 6B,D). It is
not clear how the cells find their way into the twi embryos,
which are characterized by the failure to form a ventral furrow.
The cells probably immigrate after they have been internalized
together with the adjoining posterior gut anlagen. Later the cells
begin to express CVM-specific marker genes and undergo the
first phase of the normal migration movement: they arrange as
two clusters ventrolaterally to the posterior midgut (Fig. 6F;
data not shown). The cells initiate the second phase of migration
as well; they become migratory, disperse
and acquire the typical spindle shape of
normal CVM cells. However, most likely
because of the absence of their putative
migration substratum, the TVM, they
merely spread over the posterior midgut
in twi embryos. Thus, the CVM is not
only internalized during gastrulation
independent of twi function, but also
acquires at least some of its adhesive and
migratory properties. This view is
consistent with the finding that the
expressions of byn, fkh and zfh-1, which
are required for the specification of the
CVM, are not affected in twi mutants.
byn specifies surface properties
of the caudal visceral
mesodermal cells
The defects in the CVM of a byn mutant
suggest that byn is not only involved in
the early specification of the CVM, but
also plays an essential role in establishing
the adhesive and migratory properties of
the CVM cells. These do not properly
form the two bilaterally symmetrical
clusters and later fail to disperse along the
TVM in byn mutants (Fig. 4B,D,F).
Moreover, the ubiquituous mesodermal
byn expression strongly affects the
normal migratory behaviour of the CVM
(Fig. 5A-D). The cells do not exclusively
migrate along the TVM towards the
anterior (Fig. 5D), but attach to any other
cell in the mesoderm. As a consequence,
migration is not restricted dorsoventrally and the cells do not
reach the anterior half of the midgut (Fig. 5B). In these
experimental embryos, it is impossible to physically separate
splanchopleura and somatopleura, since they are firmly attached
to each other (data not shown). Normally this is not the case.
We conclude from these observations that ectopic byn
expression changes the adhesive properties of the mesoderm.
The specific effects of ectopic byn on the surface properties
of other mesoderm cells also include the rescue of the germ cell
migration defect of byn mutants. Normally, the germ cells pass
through the posterior midgut epithelium, as it becomes
mesenchymal prior to germband retraction. From the basal side
of the endoderm, the germ cells migrate to the adjoining
mesoderm of the body wall. Later, they migrate anteriorly and
intermingle with the somatic gonadal mesoderm (Fig. 7A). It has
been suggested that the CVM plays an important role in directing
or facilitating this transition of the germ cells to the mesoderm
(Broihier et al., 1998). During the first phase of migration, the
CVM cell clusters are located at a position where the germ cells
pass through the epithelium of the posterior midgut before they
migrate to the gonadal mesoderm. In byn mutants, the germ cells
pass through the midgut epithelium as in wild type, but virtually
all cells distribute over the ventral surface of the posterior midgut
rather than contacting the mesoderm (Fig. 7B). byn is neither
expressed in the germ cells nor in the gonadal mesoderm and
the latter develops normally in byn mutants (data not shown).
Fig. 6. twist is dispensable for the internalization and specification of the CVM. CVM
development is shown for (A,C,E) wild-type and (B,D,F,G) twi embryos by the detection of
(A-D) Byn protein, (E,F) croc-lacZ expression or (G) zfh-1 RNA. (A) In the wild-type
embryo, the CVM migrates along the germband while Byn protein expression diminishes
(arrowhead). (B) In a twi embryo, Byn-expressing cells are distinguishable in the position
from which the CVM originates. (C,D) Transversal sections through the posterior region of
the germband. In (C) wild type and (D) twi mutants, the CVM (arrowheads) is located
between hindgut epithelium and epidermis. (E,F) Embryos carrying the croc-lacZ reporter
were double-labelled for β-gal (black) and for FasIII (brown) protein in order to follow the
specification of the CVM (arrowhead) and the TVM, respectively. (E) In wild type, the
migrating CVM is closely attached to the TVM. (F) In twi mutants, the CVM expresses croclacZ and spreads over the midgut. (G) zfh-1 expression is detectable in the CVM of a twi
embryo (arrowhead). All embryos are at stage 11, except for C (stage 10).
3998 T. Kusch and R. Reuter
Therefore, the observed phenotype is most likely due to defects
in a byn-dependent signalling or adhesive properties of the
CVM. This idea is supported by our finding that ubiquitous
expression of byn in the mesoderm rescues the defective germ
cell migration in byn mutants almost completely (Fig. 7C): only
a few germ cells do not coalesce with the somatic gonadal
precursors. Thus, either the few rescued CVM cells of byn
embryos at the normal position close to the migrating germ cells,
or the adhesive or signalling properties that ectopic byn confers
to other mesodermal cells, are sufficient for the transition of the
germ cells from the midgut to the gonadal mesoderm.
the fibers are oriented perpendicularly to the constriction planes.
In addition, we can exclude that byn acts by the regulation of
the homeotic genes Antennapedia or Ultrabithorax, for
instance, which are essential for the constrictions to form (Bienz
and Tremml, 1988; Tremml and Bienz, 1989; Reuter and Scott,
1990). They are not expressed in the CVM and are still
expressed in the TVM of byn embryos (data not shown).
Strikingly, other mesodermal tissues that are affected in
mutants lacking the CVM are not in obvious contact with the the
CVM during development. For instance, in byn mutants, the two
rows of cardiac cells do not unite to form the heart vessel (Fig.
7H). In addition, pericardial cells are missing (Fig. 8I) and the
most dorsal internal muscle (dorsal acute 1, DA1) is absent or
might be fused with DA2 in many segments (Fig. 7H, and data
not shown). The progenitors of DA1 and of a subset of pericardial
cells develop from a common cluster of dorsal mesodermal cells
byn-dependent signalling from the caudal visceral
mesoderm
There are a number of mesodermal tissues that do not properly
develop in embryos lacking the CVM, as in byn, fkh or tll
embryos. For instance, the TVM
develops
aberrantly
in
byn
mutants during late stages of
embryogenesis.
Although
the
inner layer of circular muscles
differentiates in the absence of the
CVM as in wild type, the
morphogenesis of this layer does
not proceed properly. The nuclei of
the TVM are normally arranged as
one broad band on each side of the
midgut during germband retraction
and subsequently split into two
bands when the midgut primordia
meet at stage 13. During this
movement, the nuclei pass the rows
of CVM cells, which are located at
the dorsal and the ventral edge
of the midgut primordium,
respectively (Fig. 4K, and data not
shown). In a byn mutant, however,
the movement of the TVM nuclei is
irregular, so that their organization
into bands is lost and they become
distributed over the entire gut
circumference (Fig. 7E,F). Since
byn is never expressed in the TVM,
we conclude that the proper
arrangement and integrity of the
Fig. 7. The CVM is required for germ cell migration, midgut morphogenesis, and dorsal mesoderm
circular muscle fibers requires the
patterning. (A-C) Gonad development in (A) wild type, (B) byn mutants or (C) byn mutants after
presence of the CVM. The irregular
enforced mesodermal byn expression. Magnifications of the tail region are shown after
dorsoventral extension of the fibers
immunostaining for Vasa protein to visualize the primordial germ cells (PGC, embryos in dorsal
results in an incomplete closure of
view at stage 14). (A) In the wild type, the PGC (arrowheads) have reached the gonadal mesoderm.
the layer and the circular muscle
(B) Most of the PGC fail to do so in the byn embryo and are distributed over the basal side of the
posterior midgut epithelium. (C) In byn mutants expressing byn under the control of twi-GAL4,
layer of the midgut in byn embryos
most of the PGC have reached the gonads. Dissected guts of (D) a wild-type embryo and (E,F) byn
shows sporadic ruptures (Fig. 4J).
mutants at stage 15 (D,E) and 16 (F) immunostained for Mef-2 protein. (D) In the wild-type
These defects might be the reason
midgut, nuclei of the longitudinal muscle fibers are regularly spaced and organized in rows
why the three constrictions that
(arrowheads). The nuclei of the TVM form four bands, one of which is visible (bracket). (E,F) The
normally subdivide the midgut tube
nuclei of the longitudinal muscle fibers are absent in the byn mutant, while the nuclei of the TVM
into four gastric chambers are not
become irregularly distributed. (G-I) Somatic muscles and heart of (G) wild type, (H) byn mutants
formed in byn mutants (Singer et al.,
or (I) byn mutants after enforced expression of byn in the CVM primordium (embryos at stage 16
1996). It seems rather unlikely that
stained for MHC, dorsal view). (G) The split arrowhead indicates the heart and the outlines of the
the longitudinal muscle fibers
dorsal muscle DA1 are marked with white dots. (H) In the mutant, the two rows of cardiac cells do
physically participate in the
not meet and DA1 is missing in numerous hemisegments. (I) These defects can be considerably
restored by the rescue of the CVM by byn expression under the control of G447.
formation of the constrictions, since
brachyenteron function in the mesoderm 3999
that can be followed from stage 10 on
by their even-skipped (eve) expression
(Frasch et al., 1987). Three cells per
hemisegment begin to express eve in each
of 11 dorsal clusters in the mesoderm
(Fig. 8A). By stage 12, the number of
mesodermal eve cells increases by one in
each cluster. We found that this additional
eve cell appears in succession from
posterior to anterior clusters (Fig. 8A,B).
Furthermore, we noted that the cells of the
CVM pass the mesodermal eve clusters at
a distance of about one cell diameter as
they migrate anteriorly along the TVM
(Fig. 8C). Shortly after the time when the
leading edge of the CVM had passed, the
fourth eve cell is added to the cluster. This
addition occurs towards the CVM and
by recruitment from neighbouring cells
rather than by cell division (data not
shown). Most importantly, the temporal
and spatial correlation between the
appearance of the fourth eve cell and the
migration of the CVM is not a mere
coincidence. In byn, tll or zfh-1 mutants in
which the CVM fails to migrate anteriorly
or is absent, the number of eve cells does
not increase during germband retraction
(Fig. 8E,G; data not shown). We propose
that this is the primary defect in the dorsal
mesoderm that causes the defects in heart
and dorsal muscle development of byn or
tll mutants, and that normally an inductive
signal emerging from the migrating CVM
triggers the addition of the fourth eve
cells. This view is supported by the
observation that the specific rescue of
CVM development in byn mutant
embryos (see above, Fig. 4L) restores
the dorsal mesodermal structures to a
considerable extent. byn is neither
expressed in the mesodermal eve cells nor
in other dorsal mesodermal derivatives
of the experimental embryos, but
nevertheless the number and position of
pericardial cells is essentially normal (Fig.
8J), the two rows of cardiac cells join and
DA1 muscles are detectable in many
segments (Fig. 7I).
We wondered whether byn is required
solely for the early specification and
migration of the CVM, or whether it is
more directly involved in the signalling to
the dorsal mesoderm. We therefore
expressed byn also outside the CVM,
throughout the mesoderm, and monitored
the number of mesodermal eve cells. In
such experimental embryos, a drastic
increase of eve cells is seen at the dorsal
edge of the mesoderm in the proximity to
the original eve clusters during stage 11
Fig. 8. byn is responsible for a signal coming from the CVM that is essential for dorsal mesoderm
development. (A-C) Embryos carrying croc-lacZ were double-labelled for β-gal (brown) and Eve
protein (blue). (A) The CVM has passed eve cluster No. IX in which a 4th cell becomes visible
(grey arrow). All eve clusters anterior to No. IX consist of 3 cells. (Dorsolateral view, early stage
11; the panel is generated from two different focal planes about 1 cell apart.) (B) All eve clusters
anterior to No. VI (black arrow) comprise 3 cells. The leading edge of the CVM has almost
reached cluster No. III (stage 12 embryo). (C) A ventral view reveals that the CVM cells pass the
eve clusters in a distance of about 1 cell (bracket). (D-J) Magnification of the mesodermal eve
cells in (D,F,H) wild-type, (E,G,I) byn or (J) byn embryos with specific rescue of the CVM.
(D,E) At stage 12, there are 4 eve cells (arrow) in wild type (D) and 3 eve cells in the (E) byn
mutant. (F) At stage 14, the DA1 precursors (grey arrowhead) separate ventrally from the
pericardioblasts. (G) In a byn mutant, eve expression appears weaker and the number of the eve
cells is reduced. (H) The heart of a stage 17 embryo in a dorsal view. By then, Eve protein is only
visible in pericardial cells. (I) The heart of a byn mutant has not formed properly and less
pericardial cells are detectable. (J) The rescue of the CVM of a byn mutant restores
morphogenesis of the dorsal vessel and the number of pericardial cells has increased. (K,L) eve
cells (black) and CVM (brown) in embryos ectopically expressing byn throughout the mesoderm.
(K) Early stage 11. In proximity of the CVM (white arrowhead) a dorsal band of the eve cells
(arrow) has formed. (L) Late stage 11. The strip of eve cells has further thickened in proximity of
the CVM and extended to the entire dorsal trunk mesoderm. (M,N) eve expression in (M) htl
embryos and (N) htl embryos with ubiquitous byn in the mesoderm. (M) No eve cells are seen in
the htl mutant. (N) htl is epistatic to the formation of additional eve cells by ectopic mesodermal
expression of byn (grey arrowheads). Dorsal views of early stage 11 embryos.
4000 T. Kusch and R. Reuter
Fig. 9. Model for the early specification of the CVM. The CVM is
specified by byn, fkh and high levels of zfh-1. The expression of zfh-1
generally requires sna, whereas high levels of zfh-1 in the CVM are
depending on tll function. tll is also required to activate the
expressions of byn and fkh in the CVM. byn and fkh act
synergistically in the specification of the CVM, i.e. fkh enhances the
action of byn. hkb sets the posterior border of the CVM anlage by
repressing sna and byn.
(Fig. 8K,L). Initially, these additional cells only appear close to
the CVM, i.e. in the posterior half of the experimental embryos
(Fig. 8K). Later, they also fill the gaps between the anterior eve
clusters, to which the CVM fails to migrate upon ubiquitous
mesodermal byn expression (compare Fig. 5B), and form a band
of cells along the entire dorsal mesoderm (Fig. 8L). Only the
dorsal mesoderm appears to be competent to (directly or
indirectly) respond to byn. This notion is supported by our
finding that, in htl embryos that specifically lack derivatives of
the dorsal mesoderm (Beiman et al., 1996; Gisselbrecht et al.,
1996; Shishido et al., 1997), ubiquitous mesodermal expression
of byn does not lead to ectopic eve expression (Fig. 8M,N).
DISCUSSION
The specification of the caudal visceral mesoderm:
a cooperation between byn and fkh
The ventral aspect of the byn expression domain comprises the
primordium of the caudal visceral mesoderm (CVM), which
forms the longitudinal muscle fibers surrounding the midgut
and influences dorsal mesoderm development during its
migration along the germband. The specification of the CVM
occurs under the control of the genes byn, fkh and zfh-1 (Fig.
9). The CVM primordium is not specified in either zfh-1
mutants or byn fkh double mutants. Moreover, the unrestricted
expression of byn and fkh in the mesoderm leads to the
formation of ectopic CVM. This ectopic CVM is only found
where high levels of zfh-1 are present and we therefore assume
that the dose of zfh-1 matters.
The genes tll, hkb and sna act upstream of byn, fkh and zfh1. sna formally functions as an activator of zfh-1 and probably
sets the dorsoventral boundary of the CVM primordium.
Astonishingly, the CVM initially develops independently of
the twi gene product, which otherwise is essential for early
mesoderm development. This twi independence is consistent
with the finding that posterior zfh-1 expression is present in twi
mutants (compare Figs 5K and 6G). The CVM cells are
internalized in twi mutants and even acquire a number of CVM
characteristics, such as expression of markers like cpo-lacZ or
croc-lacZ, spindle shape and migratory behaviour (Fig. 6F, and
data not shown); however, the terminal differentiation to
muscles does not occur. The CVM also does not form fibers
since proper morphogenesis certainly requires the migration
along the TVM, which is missing in twi mutants.
tll has a central role in setting up the CVM primordium. tll
is not only required for the strong zfh-1 expression in the
posterior mesoderm region (Fig. 5J), but also for the expression
of byn. In addition, the expression of fkh is severely reduced
in tll mutants and is solely activated by hkb. As a consequence,
fkh is not expressed in the posterior part of the mesoderm
primordium. hkb in turn limits the CVM primordium towards
the posterior in more than one way. It represses sna at the
posterior terminus and thereby indirectly zfh-1, and it also
represses byn. We suggest that the CVM is derived from the
very posterior end of the ventral furrow since these cells
express byn and since both the ventral furrow and the CVM
primordium are expanded in hkb mutants.
byn and fkh act in parallel in the specification of the CVM. In
single mutants, the CVM still partially develops while, in the
double mutant, it is entirely absent. The consequences of
overexpression of the two genes in the mesoderm suggest that
fkh primarily enhances the effect of byn in the specification. The
unrestricted mesodermal expression of fkh shows no discernable
effect on the formation of CVM (Fig. 5E). This suggests that fkh
does not have the capacity to respecify the anterior mesoderm
since, in this experiment, the situation in the anterior mesoderm
(i.e. fkh and high zfh-1) should be created as is present in the
posterior mesoderm of byn embryos. Ectopic byn triggers the
formation of a few CVM cells in the anterior tip of the mesoderm
(Fig. 5A). Only the combined overexpression of the two genes
leads to a large number of CVM-like cells in the anterior region
of the embryo (Fig. 5F). Unfortunately, it is impossible to show
that these cells have the full potential to become longitudinal
muscle fibers since other requirements for their development,
such as the formation of the TVM, are severely disturbed by
ectopic fkh. Nevertheless, the results indicate that fkh enhances
the effects of byn in directing mesodermal development. This
mode of cooperativity is reminiscent of the functional logic
found between the fkh homologue Pintallavis (Ruiz i Altaba and
Jessell, 1992) and the byn homologue XBra (Smith et al., 1991)
in mesoderm specification of Xenopus. The two genes are coexpressed in the mesoderm primordium and in the dorsal
mesoderm. The misexpression of Pintallavis in animal pole
explants does not lead to the formation of ectopic mesodermal
derivatives (Ruiz i Altaba and Jessell, 1992; Ruiz i Altaba et al.,
1993), while ectopic Xbra only causes the formation of ventral
mesoderm (Cunliffe and Smith, 1992). However, when both
genes are simultaneously misexpressed in animal caps more
ventral and also dorsal mesoderm is formed (O’Reilly et al.,
1995). Thus, a similar functional relationship might exist
between the two genes in mesoderm development of Drosophila
and Xenopus. Both code for members of highly conserved DNAbinding protein families and thus might even act on homologous
target genes in insects and vertebrates.
brachyenteron establishes adhesive and migratory
properties of the caudal visceral mesoderm
byn is not only relevant for the specification of the CVM in
conjunction with fkh. byn also establishes the surface properties
of the cells. The primary defect in the mesoderm of byn mutants
is a change in cell adhesion of the CVM rudiment as compared
to wild type. The CVM cells fail to form properly the two
bilateral clusters and later to contact the TVM. Thus, byn is
brachyenteron function in the mesoderm 4001
essential for some form of cell association and we propose that
it acts as a transcriptional regulator of the responsible adhesion
molecules. All the other defects observed, the failure to
distribute along the germband and the apoptosis, might be of
secondary nature. Moreover, the unrestricted mesodermal byn
expression severely affects the proper migration of the CVM
along the TVM (Fig. 5B,D). In such experimental embryos, the
cells are able to migrate, but fail to reach the anterior half of
the embryo. Instead of distributing along the TVM, the cells
attach to splanchnopleura as well as somatopleura without
dorsoventral restriction. We propose that ectopic byn expression
changes the adhesive properties of other mesodermal cells to
the probably homotypic form of adhesion, which might be the
basis for the byn-dependent formation of the two clusters during
the first phase of the CVM migration.
Regulation of cell adhesion and migration might be a general
feature of the Brachyury-type T domain transcription factors.
Studies on the function of the mouse Brachyury gene have
provided evidence that Brachyury affects the morphogenetic
movements of nascent mesodermal cells by influencing their
surface properties. Isolated mesodermal cells of Brachyury mice
show a decreased aggregation and migration rate (Yanagisawa
and Fujimoto, 1977; Hashimoto et al., 1987), and the extracellular
matrix of these cells is defective (Jacobs-Cohen et al., 1983).
These defects are cell autonomous: Brachyury mutant ES cells,
which are introduced into wild-type embryos, accumulate in the
primitive streak and later in the tail bud (Beddington et al., 1992;
Wilson et al., 1995). Furthermore, Brachyury orthologues are
expressed in migrating mesodermal cells in other taxa such as
echinoderms and enteropneusts (Yasuo et al., 1995; Peterson et
al., 1999). Our observations in Drosophila suggest that one
original and evolutionary conserved function of Brachyury
proteins is to confer particular adhesive and migratory properties
to mesodermal cells. Given this evolutionary conservation, the
identification of byn target genes coding for cell surface
molecules might give insights in the function of Brachyury
proteins in early mesodermal development.
brachyenteron is required for the inductive
properties of the caudal visceral mesoderm
Our data revealed an hitherto unknown inductive process during
Drosophila development: the specification of a single cell per
hemisegment within the dorsal mesoderm by the passing CVM.
This cell is the fourth of a group of eve-expressing cells, of
which two cells later form the dorsal muscle DA1 and two
become pericardial cells. In mutants lacking the CVM, this
fourth eve cell is not added to the original triplet and, as a
consequence, the number of pericardial cells and DA1 cells is
reduced. We propose that these defects are caused by an
impaired induction based on the following observations. (i) The
CVM passes the dorsal eve cells prior to the addition of the
fourth cell. During this passage, the distance appears close
enough for a signal from the CVM to trigger the recruitment of
the fourth eve cell (Fig. 8A-C). (ii) A common feature of byn,
tll or fkh mutants is that they lack the CVM (Fig. 3) and none
of these genes are expressed in the dorsal mesoderm itself. (iii)
No matter which marker we used to trace the cells of the CVM,
for instance, lacZ under the control of the GAL4-driver G447
(Georgias et al., 1997; compare Fig. 4M), they did not become
located to the dorsal mesoderm. (iv) The expression of byn
directed by G447 not only restores the defective migration of
the CVM, but also rescues the dorsal mesoderm patterning and
the heart defects in a byn mutant (Figs 4L, 7I, 8J).
We had one tool to manipulate the induction besides the
mutants lacking the CVM, i.e., forced byn expression. Ubiquitous
mesodermal byn expression led to a significant increase of eveexpressing cells at the dorsal rim of the mesoderm (Fig. 8K,L).
This is even the case in tll mutants, which lack the CVM entirely
(data not shown). We conclude that byn in the mesoderm is
sufficient to trigger the production of the signal that normally
emerges from the CVM. This would constitute the third
distinguishable function of byn in the development of the CVM
besides participation in the specification and the regulation of the
CVMs adhesive properties. We rule out the possibility that byn
is directly involved in transcriptionally activating eve in the dorsal
mesoderm, since byn is normally never expressed in the eve
clusters. Instead, we propose that byn regulates the expression of
the ligand in the signalling process. byn can only exert this
function on mesodermal cells, since a strictly ectodermal
misexpression of byn (for instance, under the control of the
ZKrGAL8-driver; Frasch, 1995) has no effect on mesodermal eve
expression (data not shown). In fact, only cells in the
neighbourhood of the eve cells begin to express eve upon
ubiquitous mesodermal byn expression, indicating that the
competence to perceive the byn-mediated signal is dictated by
contact to other eve cells (Fig. 8K,L).
The nature of the emerging signal and of its receptor are
unknown. The mesoderm-specific Drosophila FGF-receptor Htl
is essential for the formation of the eve clusters (Beiman et al.,
1996; Gisselbrecht et al., 1996), and also for the byn-mediated
formation of supernumerary mesodermal eve cells (Fig. 8N).
However, Htl is already required for the early step of the
migration of the mesoderm to dorsal positions where the cells
become specified as dorsal mesoderm by a Dpp signal (Frasch,
1995). Later, Htl is more directly involved in the specification
of heart and dorsal muscle progenitors within the dorsal
mesoderm (Michelson et al., 1998). We have not yet separated
these two functions of Htl with regard to the signalling from the
CVM. Nevertheless, it is tempting to speculate that Htl and a
corresponding growth factor are involved in the signalling from
the CVM. In Xenopus, the byn homologue Xbra acts as direct
activator of the eFGF gene and, in the mouse, FGF-4 seems to
be activated by Brachyury (Casey et al., 1998). FGF-4 in turn
has been shown to induce cardiogenesis in the so-called
precardiac mesoderm, a primordium that is specified by the Dpp
homologue BMP-2 (Lough et al., 1996). If indeed byn activated
a yet unidentified FGF gene in the CVM that signals via Htl to
the dorsal, pericardiac mesoderm, this would represent a
considerable degree of conservation of inductive properties
mediated by Brachyury homologues in mesoderm development.
We thank H. Thelen for expert technical assistance and Drs. H.
Bellen, M. Frasch, M. Fuller, B. Giebel, U. Häcker, U. Hinz, D.
Kiehart, R. Lehmann, A. Paululat, K.-P. Rehorn, V. Riechmann and T.
Seher for sending fly stocks or reagents. We are indebted to E. Foley,
T. Klein, F. Sprenger and R. Wilson for critical reading the manuscript.
The work was supported by the Deutsche Forschungsgemeinschaft as
part of the SFB 243 and by the Fond der Chemischen Industrie.
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