Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
* Author for correspondence (e-mail: saigo{at}biochem.s.u-tokyo.ac.jp)
Accepted 31 October 2002
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SUMMARY |
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Key words: Drosophila melanogaster, Visceral mesoderm, Visceral mesoderm parasegment, hedgehog, wingless, branchless, tinman, decapentaplegic, Trachea, Gastric caecum
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INTRODUCTION |
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During gastrulation and subsequent stages, future mesoderm invaginates,
spreading laterally and dorsally to form a rather uniform cell monolayer
(mesoderm), in close contact with overlaying ectoderm
(Leptin and Grunewald, 1990).
As with ectoderm, the mesoderm consists of parasegmental units (PSs), each
divided into even-skipped (eve) and sloppy-paired
(slp) domains along the anteroposterior (AP) axis
(Borkowski et al., 1995
;
Azpiazu et al., 1996
;
Riechmann et al., 1997
). The
concerted action of Decapentaplegic (DPP), which emanates from the dorsal
ectoderm, and Wingless (WG) and Hedgehog (HH), both of which are secreted from
alternating ectodermal sources along the AP axis, must be available for
further subdivision of mesodermal PS and subsequent mesodermal cell
differentiation (reviewed by Baylies, 1998).
VM development begins with VM competent cell specification within the
dorsoanterior quarter of each mesodermal parasegment. VM competent regions may
be defined using expression of bagpipe (bap), a homeobox
gene, which is positively regulated by hh, dpp and tinman
(tin), and negatively by wg
(Staehling-Hampton et al.,
1994; Frasch,
1995
, Azpiazu et al.,
1996
; Lee and Frasch,
2000
). During stage 10, the anterior terminus of each VM competent
region always appears to touch an anteroposterior compartment border (AP
border) or ectodermal WG source; the putative posterior terminus, which is
initially situated halfway between two flanking AP borders, approaches the
neighboring ectodermal WG source at late stage 10
(Borkowski et al., 1995
;
Azpiazu et al., 1996
;
Weiss et al., 2001
). Each VM
competent region splits into two regions that consist of peripheral columnar
cells, which may constitute a VM progenitor region destined for founder
myoblasts of circular muscles, and another comprising hexagonal cells, future
fusion-competent visceral myoblasts (San
Martin et al., 2001
; Klapper
et al., 2002
). A string-like VM arch structure, positive for
Fasciclin 3 (FAS3), is formed through head-to-tail connection of neighboring
VM progenitor regions. VM progenitor cells eventually fuse with
fusion-competent myoblasts to form circular visceral muscles
(San Martin et al., 2001
;
Klapper et al., 2002
).
As with mesoderm, VM possesses segmental modules (trunk visceral mesodermal
parasegments; VM-PSs). Connection (CON), a cell adhesion molecule, is
expressed in VM and its expression is well aligned with ectodermal parasegment
borders (Bilder and Scott,
1998). Bilder and Scott (Bilder
and Scott, 1998
) considered VM-PSs to consist of CON-positive and
-negative regions, and suggested that con expression in VM-PSs is
positively and negatively regulated by ectodermal hh and wg,
respectively.
VM confers positional cues to the endoderm
(Szuts et al., 1998).
dpp and wg are expressed in VM-PS7 and VM-PS8, respectively.
They are regulated by a positive feedback loop and, together, control
labial expression in underlying endoderm
(Hoppler and Bienz, 1995
;
Bienz, 1997
).
Attention in this study is directed to cell fate diversification within
each VM-PS. We first show that VM-PSs are subdivided into five or six regions
based on differences in the expression of five VM-PS genes, which include
hh and branchless (bnl)
(Sutherland et al., 1996).
Shift-up/down experiments indicate that VM-PS gene expression is regulated by
ectodermal HH and WG signals in different ways. Finally, we show that
hh expression in VM-PS3 is required for normal gastric caecum
development, while metameric expression of bnl in VM serves as a
guidance of the initial budding of visceral branches (VB) of the trachea, an
ectodermal organ.
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MATERIALS AND METHODS |
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Histochemistry
Immunohistochemistry was carried out as described previously
(Suzuki and Saigo, 2000).
Primary antibodies used are rabbit anti-HH (1:1000)
(Tabata and Kornberg, 1994
),
mouse anti-WG (mab4D4, DSHB), rabbit anti-TIN (1:900; generously provided by
Manfred Frasch), mouse anti-CON (1:5)
(Meadows et al., 1994
), mouse
anti-PTC (1:200) (Capdevila et al.,
1994
), mouse anti-FAS3 (mab7G10, DSHB), mouse anti-FAS2 (1:10)
(Grenningloh et al., 1991
) and
anti-lacZ protein [rabbit polyclonal (Cappel); mouse monoclonal
(Promega)] antibodies. TSA indirect amplification kit (Renaissance) was used
if necessary. Double fluorescence labeling with riboprobe and antibody was
carried out as described by Suzuki et al.
(Suzuki and Saigo, 2000
). cDNA
clones used as hybridization probes were bnl
(Sutherland et al., 1996
),
bap (Azpiazu and Frasch,
1993
), Wnt4 (Graba et
al., 1995
), hh
(Tashiro et al., 1993
),
dpp (Sato and Saigo,
2000
) and vein
(Yarnitzky et al., 1997
).
Temperature shift experiments
Temperature shift-up/down experiments were carried out essentially as
described (Matsuzaki and Saigo,
1996). hh9K/hh13C and
wgIL114 flies were used as hhts and
wgts mutants, respectively. Developmental times for
shift-up/down experiments are normalized to growth rate at 25°C and shown
by hours after egg laying (AEL).
Gut phenotype analysis
Larvae with appropriate genotypes were identified by the aid of the
GFP-balancer and dissected guts were stained with phalloidin-FITC (Molecular
Probes) as described previously (Hoppler
and Bienz, 1994).
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RESULTS |
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Metameric RNA expression of bnl, which encodes a ligand for
Breathless FGF receptor (Sutherland et
al., 1996), was first observed as 12 patches at mid stage 11
(Fig. 2A). bnl RNA
expression became homogeneous and then diminished during stage 12 (data not
shown). tin is a homeobox gene that is required for dorsal mesodermal
development (Azpiazu and Frasch,
1993
; Bodmer,
1993
). At early stage 10, tin is expressed throughout the
dorsal mesoderm from which VM is derived. Metameric TIN expression became
evident by early stage 11 (Azpiazu and
Frasch, 1993
) (Fig.
2B, Fig. 3L). TIN
expression decreased during stage 12. Expression of bap, another
homeobox gene required for VM development, can be monitored by bap
4.5#23 (bap-lacZ) (Fig.
2C). Staining for TIN and bap-lacZ or bnl RNA
(Fig. 2F,G) indicated that
tin, bap and bnl were co-expressed in VM-PS3-12 during stage
11; in VM-PS2, only bnl was expressed. Stage 11-12 VM was also
stained for TIN and VM-hh-lacZ
(Fig. 2H). TIN and
VM-hh-lacZ expression partially overlapped. VM-hh expression
in the anterior terminal region of VM-PSs (see
Fig. 1D) indicated that each
tin/bnl/bap trio expression domain straddles the VM-PS boundary (see
Fig. 2I).
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In summary, VM-PSs in thorax and abdomen, respectively, was found to be subdivided into five or six regions with respect to differential expression of VM-metameric genes at stages 11-12 (Fig. 2I).
Requirements of hh and wg signals for VM-PS gene
expression
VM-PS cell fates may be governed by ectodermal HH and WG signals
(Bilder and Scott, 1998). Thus,
we examined the effects of changing hh or wg activity on
VM-PS gene expression at late stage 11 to early stage 12. For misexpression of
hh or wg, 24B-GAL4 or twi-GAL4 were used as
mesodermal-GAL4 drivers (Brand and
Perrimon, 1993
; Greig and
Akam, 1993
).
As shown in Fig. 3A-C,
VM-hh expression was abolished in both hh13C and
wgCX4 embryos, while VM-hh expression was
expanded throughout VM in response to misexpression of wg
(Fig. 3D) and some expansion
was noted due to hh misexpression
(Fig. 3N,O). As hh and
wg expression is mutually regulated in early embryogenesis
(Ingham, 1993), the above
finding may indicate that either hh or wg, or both, is
required for VM-hh expression. Should WG be a primary effector but HH
not, VM-hh-lacZ misexpression may be expected to occur in hh
mutants that misexpress wg. To provide clarification of this point,
hh and wg were misexpressed on wg and hh
mutant backgrounds, respectively. No positive VM-hh signals could be
found in either case (Fig.
3E,F). Previous studies suggested that hh mutants with
ectopic wg fail to form VM due to earlier patterning functions of
these genes (Azpiazu et al.,
1996
). However, we found that VM, incompletely expressing TIN, is
formed under our experimental conditions
(Fig. 3G). It may thus follow
that the concerted action of WG and HH, both of which serve as primary
positive regulators, is required for VM-hh expression.
The effects of change in hh and wg activity on tin, bnl and bap expression in VM were very similar, if not identical, to each other. The expression of tin, bnl and bap at late stage 11 was significantly reduced or hardly present in the absence of wg activity (Fig. 3H,I). The expression of these VM metameric genes expanded into nearly all VM cells on misexpression of wg (Fig. 3J), indicating that WG serves as a positive regulator of tin/bnl/bap expression in VM. HH may have little or no role in tin/bnl/bap expression in stage 11 VM. Neither appreciable expansion nor reduction in tin/bnl/bap domains could be observed with mesodermal hh misexpression (compare Fig. 3M with 3L). In hh13C embryos, tin/bnl/bap expression appeared to have expanded in VM (Fig. 3K). But in hh13C embryos, the number of FAS3-positive VM-PS cells was reduced by about a half (see below) and the number of tin/bnl/bap-positive cells per VM-PS was virtually the same as that in wild type (data not shown). The absence of tin/bnl/bap-negative cells in hh13C embryos may thus be a reflection of partial failure of VM cell formation in hh mutants.
It is evident from the above that wg is required for tin/bnl/bap trio expression. Why was TIN was detected in hh13C embryos, which possibly lack wg expression owing to mutual regulation between wg and hh? In hh13C embryos, only dorsalmost WG, which is required for tin/bnl/bap expression, was still found to be expressed in hh13C embryos at stages 10-11, even with virtual elimination of other wg signals (data not shown).
The fact that hh misexpression does not change tin expression in VM allowed us to examine whether VM-hh expression occurred anteriorly or posteriorly subsequent to hh misexpression. VM cells misexpressing hh were stained for TIN and VM-hh-lacZ. As shown in Fig. 3N,O, hh misexpression brought about the anterior expansion of VM-hh.
Crucial periods of ectodermal HH and WG action for VM metameric gene
expression
To determine when ectodermal HH and WG required for VM-metameric gene
expression is produced, hh and wg activity was transiently
altered using temperature sensitive hh (hh9K/13C;
hhts) and wg (wgIL4;
wgts) mutants. hh activity was eliminated by
shifting-up hhts embryos from permissive (18°C) to
non-permissive (29°C) temperatures at stage 10 to early stage 11. As shown
previously for con expression
(Bilder and Scott, 1998),
metameric VM-hh (VM-hh-lacZ) expression, which normally
becomes discernable at mid- to late stage 11, disappeared
(Fig. 4A,B). By contrast,
con and VM-hh expression was normal after temperature
shift-up at mid stage 11 or later (Fig.
4A,C). With temperature shifted down from non-permissive to
permissive temperatures at the end of stage 9 to early stage 10, expression
patterns of VM-hh and all other VM genes examined here were very
similar or even identical to those in wild type
(Fig. 4A,D), although
progenitor cell number per VM-PS unit (VM-PS cell number) was reduced to
60-70% that of wild type (data not shown). HH produced during stage 10 to
early stage 11 should thus be considered essential for initiation of
VM-hh as well as con expression. HH secreted before the end
of stage 9 or after mid-stage 11 onwards should be dispensable at least for
initiation of VM-hh expression. Similar results have been reported in
the case of con expression by Bilder and Scott
(Bilder and Scott, 1998
). In
contrast to VM-hh and con expression, the expression of
tin and bnl and bap
(Fig. 4A,E and data not shown)
was normal with or without hh activity; this again demonstrated that
hh is not involved in bnl, tin or bap
expression.
As in the case of HH, the shift-up study indicated that WG produced during stage 10 was required for hh and tin expression in VM (Fig. 4F,G,N) but not expression of con (Fig. 4F,H). Nevertheless, WG produced at early stage 11 is essential for tin expression but dispensable for that of VM-hh (Fig. 4F,I). WG produced at mid stage 11 or later was apparently unnecessary for either (Fig. 4F). Shift-down experiments indicated that WG produced at and before the end of stage 9 to early stage 10 is dispensable for VM-hh and tin expression (Fig. 4F,J) and it follows that WG produced during stage 10 to early stage 11 regulates the expression of hh and tin expression.
hh and wg, expressed in ectoderm from stage 5 onwards
(Tabata et al., 1992;
Baylies et al., 1995
), is not
detectable in mesoderm by stages 9-10 (data not shown). Mesodermal hh
was again seen at mid-stage 11 (see Fig.
1A1,B), while that of wg is reported to start at
mid-stage 11 only in VM-PS8 (Immergluck et
al., 1990
). Our results would thus mean that HH and WG emanating
from the ectoderm during stage 10 to early stage 11 regulate the expression of
VM-metameric genes.
Each tin/bnl/bap domain consists of a posterior terminal region of
one VM-PS and anterior terminal region of its posterior neighbor (p
and a regions, respectively; Fig.
2I, Fig. 4K). The
latter is always close to the ectodermal AP border during early stage 10 to
mid-stage 11, while the former is so only at late stage 10 to mid-stage 11
(Borkowski et al., 1995) (see
also Fig. 8). Consistent with
this, examination of wgts embryos shifted-up at late stage
10 to early stage 11 indicated that an appreciable fraction of embryos possess
VM with TIN and bnl signals only in the a region of VM-PSs
(Fig. 4L,M). While no
tin expression occurred in embryos shifted-up from early stage 10
onwards (Fig. 4N), TIN signals
were detected only in a few p-region cells near the VM-PS border in a
fraction of embryos shifted-up at late stage 10 to early stage 11 and
shifted-down at mid-stage 11 (see Fig.
7F1). In addition, in contrast to an earlier report
(Azpiazu and Frasch, 1993
), we
found that, in wild-type VM, tin RNA signals change much more
dynamically than do tin protein signals at late stage 10 to early
stage 11 (compare Fig. 4O1 with
4O2,3). In slightly younger embryos, tin RNA signals were
detected only in the a region of each VM-PS
(Fig. 4O2), while, in older
ones, tin RNA signals were detected in both a and p
regions (Fig. 4O3). No
corresponding protein signal change could be detected possibly because TIN is
much more stable than tin RNA
(Fig. 4O1 and data not shown).
The tin/bnl/bap expression within each VM-PS is thus reasonably
concluded under the control of WG signals emanating from two distinct sources
at different times (see Fig.
8).
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Effects of hh and wg activity change on VM cell
formation
FAS3 is expressed throughout VM from mid stage 11 onwards
(Fig. 3P). Thus, using
anti-FAS3 antibody staining, VM-PS cell number in mid stage 11 embryos
differing in HH and WG signaling activity was examined. In
hh13C and wgCX4 embryos, this cell
number was reduced to about a half and two thirds that of wild type (17
cells), respectively (Fig.
3P-R), but was not affected by mesodermal overexpression of
hh or wg or transient loss of hh and wg
activity at stage 10 (Fig. 3S
and data not shown). Variation in hh or wg activity at stage
10 thus has no significant effect on VM-PS cell formation.
VM-hh is required for maintenance of its own expression
At mid-stage 11 and later, VM cells are situated far away from ectoderm
and, consequently, HH signals possibly required for VM development from
mid-stage 11 onwards may not come from hh ectodermal domains. The
activity of VM-hh, whose expression starts at mid stage 11, was
eliminated by shifting-up hhts embryos from permissive to
non-permissive temperatures. A 90-minute elimination of hh activity
during mid-stage 11 to mid-stage 12 resulted in loss of both VM-hh
RNA and CON signals at stage 13 (Fig.
5A-D) but not those at stage 12. VM-hh RNA expression was
also abolished by a 90-minute shifting-up from mid- to late stage 12 (data not
shown). The VM expression of ptc, a general target gene of HH
signaling, was also abolished upon elimination of hh activity from
mid stage 11 onwards (data not shown). It may thus follow that VM-hh,
the expression initiation of which is under the control of ectodermal HH, is
essential for the maintenance of its own expression and that of ptc,
and stage 13 con expression in VM.
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Cubitus interruptus (Ci) is a downstream transcription factor of HH
signaling and ectopic expression of the Ci repressor form in the posterior
compartment of imaginal discs results in elimination of hh expression
(Methot and Basler, 1999).
Similarly, VM-hh was abolished by twi-GAL4-driven
UAS-ciNZn encoding the Ci repressor
(Hepker et al., 1997
) (data
not shown). 48Y-GAL4 is a GAL4 driver originally thought to be specific for
endodermal expression (Martin-Bermudo et
al., 1997
) but capable of driving target genes in VM after mid
stage 11 (data not shown). When UAS-ciNZn was driven by 48Y-GAL4,
maintenance but not initiation of VM-hh expression was impaired
(Fig. 5E,F). Thus, both
initiation and maintenance of VM-hh appear to be negatively regulated
by Ci repressor misexpression.
Requirements of VM-hh for normal gastric caecum
formation
As with dpp and wg expressed in VM-PS7 and 8,
respectively (Szuts et al.,
1998), VM-hh may be required for endodermal cell
differentiation. To clarify this point, VM-hh activity was abolished
and possible morphological changes of VM derivatives were observed at 25 hours
AEL. hhts embryos were shifted up from permissive to
non-permissive temperatures for 3 hours during mid-stage 11 to late stage 12.
The majority of embryos were associated with the short gastric caecum
phenotype (Fig. 6A,D), while,
in about 10% of the total (n=57), one or a few gastric caeca were
lost and the size of those remaining was considerably reduced
(Fig. 6F). A similar short
gastric caecum phenotype was also observed in virtually all embryos with
UAS-ciNZn driven by 48Y-GAL4 (Fig.
6G). As described above, in these embryos, VM-hh RNA
expression is normally initiated but not maintained, while ectodermal
hh expression appears normal, indicating that they possess normal
ectodermal HH activity but lack VM-HH activity during most of late
developmental stages. VM-hh thus appears essential for normal gastric
caecum formation.
Gastric caeca are derivatives of precursors consisting of VM-PS3 cells and
immediately adjacent endodermal cells
(Reuter and Scott, 1990;
Bate, 1993
;
Szuts et al., 1998
) and all
four gastric caecum invaginations become apparent by stage 17 (16 hours AEL)
(Reuter and Scott, 1990
).
While a considerable fraction of gastric caecum precursors express
DWnt-4 (Graba et al.,
1995
), anterior VM-PS3 cells are positive for VM-hh
(Fig. 6C). Because, in VM, PTC
signal, which serves as a marker for the area with high levels of HH
reception, was restricted to cells expressing VM-hh and their
vicinity (see Fig. 1C), we
believe that VM-PS3 must provide HH required for normal gastric caecum
formation.
It should, however, be noted that the above conclusion does not necessarily
exclude the possibility that ectodermal hh may have some essential
roles in gastric caecum formation at earlier stages. Indeed, gastric caecum
phenotypes more severe than those described above were found in
hhts embryos shifted-up during stage 10-11, and in those
with UAS-ciNZn driven by 24B-GAL4 or bap-GAL4
(Zaffran et al., 2001)
(Fig. 6I and data not shown).
Gastric caecum was virtually completely abolished in hh13C
and twi-GAL4-driven UAS-ciNZn embryos
(Fig. 6J,L). We interpret these
findings as suggesting that there are multiple steps requiring hh
activity for normal gastric caecum formation.
Short gastric caecum phenotypes have also been reported in dpp
hypomorphic mutants and vein (vn) mutants
(Bate, 1993;
Szuts et al., 1998
).
dpp is expressed in VM-PS3 from stage 11 onwards and its expression
area virtually overlaps that of VM-hh-lacZ expression
(Bate, 1993
)
(Fig. 6B), while
VM-vn, whose expression is maintained by VM-PS3 dpp
(Szuts et al., 1998
), is
restricted to VM-PS2-4 from stage 13 onwards
(Szuts et al., 1998
). The
complete absence of dpp activity from VM-PS3 has been reported to
induce the gastric caecum-less phenotype
(Hursh et al., 1993
;
Masucci and Hoffmann, 1993
).
Thus, examination was made of whether dpp expression is reduced or
abolished in hhts embryos shifted up during midstage 11 to
late stage 12 and 48Y-Gal4-driven UAS-ciNZn embryos, along with
hh13C and twi-GAL4-driven UAS-ciNZn
embryos. dpp expression was observed at stage 13. As shown in
Fig. 6B,E,H,K, the dpp
RNA expression levels in these HH signaling mutants were roughly proportional
to the gastric caecum length. That is, in the hhts embryos
shifted up during mid-stage 11 to late stage 12 and 48Y-Gal4-driven
UAS-ciNZn embryos, both exhibiting the short gastric caecum phenotype
(see Fig. 6D,G), VM-PS3
dpp expression was significantly reduced
(Fig. 6E,H), whereas, in
hh13C embryos and twi-GAL4-driven
UAS-ciNZn embryos, both of which completely lack the gastric caecum
(see Fig. 6J,L), no
dpp signals were detected in VM-PS3
(Fig. 6K and data not shown).
Moreover, vn expression in VM-PS2-4 was significantly reduced or
virtually completely abolished in these HH signaling mutants (data not shown).
It may thus follow that the concerted action of HH secreted at stages prior to
stage 11 and HH from VM-PS3 at later stages establishes the normal expression
level of dpp in VM-PS3, and most, if not all, gastric caecum defects
of these HH signaling mutants are attributed to reduction or loss of
dpp expression in VM-PS3.
Regulation of tracheal visceral branch migration through VM metameric
BNL
Tracheal cells are formed from 10 clusters (Tr1-10) of ectodermal cells on
each side of the embryo. Future tracheal cell clusters invaginate, and at late
stage 11, migrate to produce six primary branches, one being VB
(Manning and Krasnow, 1993;
Samakovlis et al., 1996
). The
ordered development of trachea is a consequence of communication between
tracheal cells and surrounding tissues (reviewed by
Zelzer and Shilo, 2000
).
bnl acts at least as a motogen for all primary branches
(Sutherland et al., 1996
). In
parallel to and/or prior to bnl signaling, DPP, EGF, WG and HH are
required for proper invagination of tracheal cells and guidance of specific
branches (Glazer and Shilo,
2001
). Integrin proteins have been recently shown involved in VB
guidance after VB reaches VM (Boube et
al., 2001
). Mesodermal cells may also be required for assisting or
constraining tracheal branching
(Franch-Marro and Casanova,
2000
; Wolf and Schuh,
2000
). No branch formation occurs in bnl mutants and
ectopic bnl expression redirects some branches
(Sutherland et al., 1996
), but
whether restricted bnl expression in VM is essential for VB guidance
remains unclear.
VB cells, though not those in Tr3 or Tr9
(Manning and Krasnow, 1993;
Sutherland et al., 1996
),
begin budding towards corresponding bnl expression domains in a 1:1
fashion at late stage 11 (Fig.
7A). The tip of VB of Tr-(i-3) (i; integer)
first appeared to touch the vicinity of the posterior end of the a
region or bnl/TIN-expressing anterior region of VM-PS-i
(Fig. 7B) and then, VB/VM
contact expanded to the entire VM-PS-(i-1) p-region and
VM-PS-(i) a-region (Fig.
7C). During stage 12, with bnl expression becoming rather
homogeneous throughout VM (data not shown), VB extends further anteriorly.
This extension depends on integrin genes
(Boube et al., 2001
).
The expression of bnl was changed to examine how this would affect
initial VB budding at stage 11 to early stage 12, which is independent of
integrin genes (Boube et al.,
2001). First, bnl was misexpressed in the entire VM with
bap-GAL4. Should VM-BNL serve as a chemoattractant for VB guidance,
considerable VB misrouting would be expected to occur upon bnl
misexpression. We found 48 VB bifurcations (23%) and five simple VB
misroutings (2.4%) in 210 VM-PSs normally capable of coming into contact with
VB (Fig. 7D,E). These defects
appear to be persisted at least until stage 15.
Fig. 7D,E also shows that no
appreciable change in tin expression was induced by bnl
misexpression in VM. Thus, VB misrouting and bifurcation appear solely due to
bnl misexpressed in VM.
The bnl/tin expression domain in VM-PSs was reduced in area in two
ways, both dependent on WG being a positive regulator of metameric expression
of bnl and tin in VM. As TIN and bnl are
co-expressed in both wild-type and wg hypomorphic mutant conditions,
TIN was used as an area marker for bnl expression.
dTCF-N is a dominant negative form of d-TCF and
serves as an antagonist of WG signaling
(Cavallo et al., 1998
).
twi-GAL4-driven UAS-dTCF-
N, which blocks WG signaling
in a mesoderm-specific manner, restricted TIN/bnl expression only to
cells in the vicinity of the VM-PS border
(Fig. 7H,I). In these mutant
flies, the remnant TIN/bnl-expressing p/a-border-cells
served as targets for the initial VB contact
(Fig. 7H); unlike the situation
in wild type, no initial VB/VM contact was detected at the vicinity of the
putative posterior end of the VM-PS a region
(Fig. 7I).
A similar redirection of VB migration was also observed in wgts mutants shifted-up for 60 minutes from late stage 10 to stage 11. In this case, the remnant TIN/bnl-expressing p-region cells near the VM-PS border served as targets for the initial VB contact (Fig. 7F). Subsequently, VM/VB contact expanded, as in wild type (Fig. 7G). Based on these results, we conclude that metameric BNL within VM serves as a chemoattractant for initial VB migration.
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DISCUSSION |
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Reiterative use of hh and wg signals in VM
development
VM is presently considered to develop in two steps under the control of
ectodermal HH and WG signals. First, by stage 10 (when four mesodermal
primordia have become specified), VM competent or bap expression
regions are promoted by hh but repressed by wg, via a direct
targetor, slp (Lee and Frasch,
2000). The second surge of hh and wg activity at
stages 10-11 is responsible for subdividing VM-PSs into two regions:
con positive and negative (Bilder
and Scott, 1998
). Our results indicate that the expression of
other four VM-metameric genes, hh, tin, bnl and bap, is also
regulated by the second surge of hh and wg activity at
stages 10-11.
To examine the regulation of VM-metameric genes with changing the activity of hh and/or wg, it may be necessary to evaluate the effects of possible change in cell number on VM-PS subdivision or VM-PS cell specification. In temperature-sensitive mutants of hh and wg shifted-up from stage 10, the number of VM cells positive to FAS3 at mid stage 11 on was essentially identical to that of wild type, indicating that VM-cell number change is negligible under the conditions used, while the expression of some VM metameric genes appeared compromised. In hhts mutants, VM-hh and con were not expressed, though tin, bnl and bap were (see Fig. 4A). In wgts mutants, VM-hh and tin were not expressed, but con was (see Fig. 4F). All these observations are totally in agreement with those found in simple loss-of-function and overexpression experiments, as described in the Results; under these conditions, the formation of a VM competent region should be hindered. Thus, our results may indicate that ectodermal HH and WG regulate directly, but in different ways, the expression of metameric genes in VM; VM-hh expression requires both HH and WG. tin, bnl and bap are positively regulated by WG alone, and con is activated by HH and repressed by WG.
Cell fate diversification within VM-PSs through concerted hh
and wg signaling
In view of morphological changes in a VM competent region and consideration
of our findings on VM gene regulation, the following model for VM-PS cell
specification may be proposed (Fig.
8).
At stage 10 to early stage 11, anterior terminal cells of VM-PSs are presumed to be always situated near an ectodermal AP border, where they are capable of continuously receiving WG and HH signals, and WG confers competence on these cells to express tin/bnl/bap. WG and HH are responsible for inducing VM-hh, and HH, for con expression. In the anterior-most cells, con expression is reduced, which would be expected in view of repression by high WG signal. The different thresholds of hh for con and VM-hh expression may explain why the con area expands more posteriorly compared with that of VM-hh. Posterior terminal VM cells, when formed, are situated far from WG expressed on the ectodermal PS border (see `early to late stage 10' in Fig. 8). But as they migrate posteriorly and close to the posteriorly neighboring AP border by early stage 11, they become capable of receiving WG and acquire competence to express tin/bnl/bap (see `late stage 10 to midstage 11' in Fig. 8). Thus, the tin/bnl/bap domain would appear regulated by spatially and temporally distinct WG signals. The two-step induction of tin/bnl/bap expression is supported by experiments using the wgts mutant, where, either posterior or anterior expression within one patch could be differentially turned off (Fig. 4L-N). Indeed, we observed a stepwise activation of tin/bnl expression in VM-PSs around stage 11 (Fig. 4O). As schematically shown in Fig. 4A, tin and bnl metameric expression became apparent almost simultaneously at mid-stage 11, and our preliminary experiments showed that neither tin nor bnl misexpression could induce the ectopic expression of any other metameric genes examined here. Thus, tin and bnl expression might be initiated in a mutually independent manner.
This VM-PS subdivision model should be modified when applied to thoracic
segments, where hh may not be the sole determinant of con
expression (Zaffran et al.,
2001) (see Figs 2,
5; data not shown).
Requirements of VM-hh for segmental subdivision of VM-PSs
and gastric caecum formation
Our study strongly suggested that metameric VM-hh is required for
the maintenance of its own as well as metameric con expression (see
Fig. 5C,D), although the latter
becomes independent of VM-hh at late stages (C. H., unpublished).
That PTC, a direct target of hh, is upregulated in each
VM-hh expression domain at stage 12, at that time VM is far away from
the epidermis or ectodermal HH sources (see
Fig. 1C), is another evidence
supporting the notion that hh signaling caused by metameric
VM-hh is operative in VM.
Our results (Fig. 6) also
showed that HH is required for gastric caecum development. HH may operate in
multiple steps in mesoderm and its source for the last step is VM-HH emanating
from VM-PS3, a part of the future gastric caecum region.
Fig. 6 also indicated that
most, if not all, gastric caecum defects found in HH signaling mutants may be
due to the reduction or loss of VM-PS3 dpp, whose production is under
the control of VM-PS3 HH and HH at stages prior to stage 11 (this work)
(Hursh et al., 1993;
Masucci and Hoffmann, 1993
).
vn expression, which is positively controlled by VM-PS3 dpp
(Szuts et al., 1998
), was also
significantly reduced in HH signaling mutants (data not shown), while
Dwnt4 expression was not seriously affected even in hh null
mutants (C. H., unpublished). Thus, the effect of hh activity loss on
gastric caecum formation may be due to partial changes in fate/transcription
programs of VM-PS3 cell precursors.
VM BNL may serve as a chemoattractant for VB migration
Reiterative bnl expression in VM is likely to be a determinant of
the particular mode of VB migration. The tip of VB first came in touch with
the vicinity of the posterior end of the tin/bnl/bap expressing
a region, where all the five metameric genes examined are expressed
(Fig. 7A,B). BNL misexpression
with VM-specific-GAL4 drivers induced VB misrouting and bifurcation
(Fig. 7D,E) but neither
hh misexpression nor transient loss of HH activity during stage 11
had any effect on VB budding (C. H., unpublished). BNL misexpression brought
about no significant change in expression of tin
(Fig. 7D,E), while restriction
of the tin/bnl/bap expression domain using either
dTCF-N or wgts caused a shift in the first
VB/VM contact point (Fig. 7I).
Furthermore, under a wg mutant condition, no change could be detected
in VM-hh or in con expression (see
Fig. 4F). Thus, only the
bnl expression appears to be closely correlated with VB budding,
strongly suggesting that BNL serves as a chemoattractant for initial VB
migration.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Alcedo, J. and Noll, M. (1997). Hedgehog and its Patched-Smoothened receptor complex: a novel signaling mechanism at the cell surface. Biol. Chem. 378,583 -590.[Medline]
Azpiazu, N. and Frasch, M. (1993). tinman and bagpipe: Two homeobox genes that determine cell fates in the dorsal mesoderm of Drosophila. Genes Dev. 7,1325 -1340.[Abstract]
Azpiazu, N., Lawrence, P. A., Vincent, J. P. and Frasch, M. (1996). Segmentation and specification of the Drosophila mesoderm. Genes Dev. 10,3183 -3194.[Abstract]
Baylies, M. K., Arias, M. and Bate, M. (1995).
wingless is required for the formation of a subset of muscle founder
cells during Drosophila embryogenesis.
Development 121,3829
-3837.
Baylies, M. K., Bate, M. and Gomez, M. R. (1998). Myogenesis: a view from Drosophila.Cell 93,921 -927.[Medline]
Bate, M. (1993). The mesoderm and its derivative. In The Development of Drosophila melanogaster (ed. M. Bate and A. Martinez-Arias), pp1013 -1090. Plainview, NY: Cold Spring Harbor Laboratory Press.
Bienz, M. (1994). Homeotic genes and positional signaling in the Drosophila viscera. Trends Genet. 10,22 -26.[CrossRef][Medline]
Bienz, M. (1997). Endoderm induction in Drosophila: The nuclear targets of the inducing signals. Curr. Opin. Genet. Dev. 7, 683-688.[CrossRef][Medline]
Bilder, D. and Scott, M. P. (1998). Hedgehog and wingless induce metameric pattern in the Drosophila visceral mesoderm. Dev. Biol. 201, 43-56.[CrossRef][Medline]
Bodmer, R. (1993). The gene tinman is
required for specification of the heart and visceral muscles in
Drosophila. Development
118,719
-729.
Borkowski, M., Brown, N. H. and Bate, M.
(1995). Anterior-posterior subdivision and the diversification of
the mesoderm in Drosophila. Development
121,4183
-4193.
Boube, M., Martin-Bermudo, M. D., Brown, N. H. and Casanova,
J. (2001). Specific tracheal migration is mediated by
complementary expression of cell surface proteins. Genes
Dev. 14,2140
-2145.
Brand, A. H. and Perrimon, N. (1993). Targeted
gene expression as a means of altering cell fates and generating dominant
phenotypes. Development
118,401
-415.
Capdevila, J., Pariente, F., Sampedro, J., Alonso, J. and
Guerrero, I. (1994). Subcellular localization of the segment
polarity protein patched suggests an interaction with the wingless reception
complex in Drosophila embryos. Development
120,987
-998.
Campos-Ortega, J. A. and Hartenstein, V. (1985). The Embryonic Development of Drosophila melanogaster. Berlin: Springer-Verlag.
Campos-Ortega, J. A. and Hartenstein, V. (1997). The Embryonic Development of Drosophila melanogaster. Berlin: Springer-Verlag.
Cavallo, R. A., Cox, R. T., Moline, M. M., Roose, J., Polevoy, G. A., Clevers, H., Peifer, M and Bejsovec, A. (1998). Drosophila Tcf and Groucho interact to repress Wingless signaling activity. Nature 395,604 -608.[CrossRef][Medline]
Dettman, R. W., Turner, F. and Raff, E. (1996). Genetic analysis of the Drosophila ß3-Tubulin gene demonstrates that the microtubule cytoskeleton in the cells of the visceral mesoderm is required for morphogenesis of the midgut endoderm. Dev. Biol. 177,117 -135.[CrossRef][Medline]
Frasch, M. (1995). Induction of visceral and cardiac mesoderm by ectodermal Dpp in the early Drosophila embryo. Nature 374,464 -467.[CrossRef][Medline]
Franch-Marro, X. and Casanova, J. (2000). The alternative migratory pathways of the Drosophila Tracheal cells are associated with distinct subsets of mesodermal cells. Dev. Biol. 227,80 -90.[CrossRef][Medline]
Glazer, L and Shilo, B. Z. (2001). Hedgehog
signaling patterns the tracheal branches. Development
128,1599
-1606.
Graba, Y., Gieseler, K., Aragnol, D., Laurenti, P., Mariol, M.,
Berenger, H., Sagnier, T. and Pradel, J. (1995).
Dwnt4, a novel Drosophila Wnt gene acts downstream of
homeotic complex genes in the visceral mesoderm.
Development 121,209
-218.
Greig, S. and Akam, M. (1993). Homeotic genes autonomously specify one aspect of pattern in the Drosophila mesoderm. Nature 362,630 -632.[CrossRef][Medline]
Grenningloh, G., Rehm, E. J. and Goodman, C. S. (1991). Genetic analysis of growth cone guidance in Drosophila: Fasciclin II functions as a neuronal recognition molecule. Cell 67,45 -57.[Medline]
Hepker, J., Wang, Q. T., Motzny, C. K., Holmgren, R. and Orenic,
T. V. (1997). Drosophila cubitus interruptus forms a
negative feedback loop with patched and regulates expression of
Hedgehog target genes. Development
124,549
-558.
Hiromi, Y., Kuroiwa, A. and Gehring, W. J. (1985). Control elements of the Drosophila segmentation gene fushi tarazu. Cell 43,603 -613.[Medline]
Hoppler, S. and Bienz, M. (1994). Specification of a single cell type by a Drosophila homeotic gene. Cell 76,689 -702.[Medline]
Hoppler, S. and Bienz, M. (1995). Two different thresholds of wingless signaling with distinct developmental consequences in the Drosophila midgut. EMBO J. 14,5016 -5026.[Abstract]
Hursh, D., A., Padgett, R., W. and Gelbart, W. M.
(1993). Cross regulation of decapentaplegic and
Ultrabithorax transcription in the embryonic visceral mesoderm of
Drosophila. Development
117,1211
-1222.
Immergluck, K., Lawrence, P. A. and Bienz, M. (1990). Induction across germ layers in Drosophila mediated by a genetic cascade. Cell 62,261 -268.[Medline]
Ingham, P. W. (1993). Localized hedgehog activity controls spatial limits of wingless transcription in the Drosophila embryo. Nature 366,560 -562.[CrossRef][Medline]
Klapper, R., Stute, C., Schomaker, O., Strasser, T., Janning, W., Renkawitz-Pohl, R. and Holz, A. (2002). The formation of syncytia within the visceral musculature of the Drosophila midgut is dependent on duf, sns and mnc. Mech. Dev. 110, 85-96.[CrossRef][Medline]
Lee, H. and Frasch M. (2000). Wingless effects
mesoderm patterning and ectoderm segmentation events via induction of its
downstream target sloppy paired. Development
127,5497
-5508.
Leptin, M. and Grunewald, B. (1990). Cell shape changes during gastrulation in Drosophila. Development 10, 73-84.
Manning, G. and Krasnow, M. (1993). Development of the Drosophila tracheal system. In The Development of Drosophila melanogaster (ed. M. Bate and A. Martinez-Arias), pp.609 -686. Plainview, NY: Cold Spring Harbor Laboratory Press.
Martin-Bermudo, M. D., Dunin-Borkowski, O. M. and Brown, N.
H. (1997). Specificity of PS integrin function during
embryogenesis resides in the subunit extracellular domain.
EMBO J. 16,4184
-4193.
Masucci, J., D. and Hoffmann, F., M. (1993). Identification of two regions from the Drosophila decapentaplegic gene required for embryonic midgut development and larval viability. Dev. Biol. 159,276 -287.[CrossRef][Medline]
Matsuzaki, M. and Saigo, K. (1996).
hedgehog signaling independent of engrailed and
wingless required for post-S1 neuroblast formation in
Drosophila CNS. Development
122,3567
-3575.
Meadows, L. A., Gell, D., Broadie, K., Gould, A. P. and White,
R. (1994). The cell adhesion molecule, connectin, and the
development of the Drosophila nueromuscular system. J.
Cell Sci. 107,321
-328.
Methot, N. and Basler, K. (1999). Hedgehog controls limb development by regulating the activities of distinct transcriptional activator and repressor forms of Cubitus interruptus. Cell 96,819 -831.[Medline]
Reuter, R. and Scott, M. P. (1990). Expression and function of the homeotic genes Antennapedia and Sex combs reduced in the embryonic midgut of Drosophila.Development 109,289 -303.[Abstract]
Riechmann, V., Irion, U., Wilson, R., Grosskortenhaus, R. and
Leptin, M. (1997). Control of cell fates and segmentation in
the Drosophila mesoderm. Development
124,2915
-2922.
Samakovlis, C., Hacohen, N., Manning, G., Sutherland, D.,
Guillemin, K. and Krasnow, M. (1996). Development of the
Drosophila tracheal system occurs by a series of morphologically
distinct but genetically coupled branching events.
Development 122,1395
-1407.
San Martin, B., Ruiz-Gomez, M., Landgraf, M. and Bate, M.
(2001). A distinct set of founders and fusion-competent myoblasts
make visceral muscles in the Drosophila embryo.
Development 128,3331
-3338.
Sato, M. and Saigo, K. (2000). Involvement of pannier and u-shaped in regulation of Decapentaplegic-dependent wingless expression in developing Drosophila notum. Mech. Dev. 93,127 -138.[CrossRef][Medline]
Sutherland, D., Samakovlis, C. and Krasnow, M. (1996). branchless encodes a Drosophila FGF homolog that controls tracheal cell migration and the pattern of branching. Cell 87,1091 -1101.[Medline]
Staehling-Hampton, K., Hoffmann, F. M., Baylies M. K., Rushton, E. and Bate, M. (1994). dpp induces mesodermal gene expression in Drosophila. Nature 372,783 -786.[Medline]
Suzuki, T. and Saigo, K. (2000).
Transcriptional regulation of atonal required for Drosophila
larval eye development by concerted action of Eyes absent, Sine oculis and
Hedgehog signaling independent of Fused kinase and Cubitus interruptus.
Development 127,1531
-1540.
Szuts, D., Eresh, S. and Bienz, M. (1998).
Functional intertwining of dpp and EGFR signaling during Drosophila
endoderm induction. Genes Dev.
12,2022
-2035.
Tabata, T., Eaton, S. and Kornberg, T. B. (1992). The Drosophila hedgehog gene is expressed specifically in posterior compartment cells and is a target of engrailed regulation. Genes Dev. 6,2635 -2645.[Abstract]
Tabata, T. and Kornberg, T. B. (1994). Hedgehog is a signaling protein with a key role in pattering Drosophila imaginal discs. Cell 76,89 -102.[Medline]
Tashiro, S., Michiue, T., Higashijima, S., Zenno, S., Ishimaru, S., Takahashi, F., Orihara, M., Kojima, T. and Saigo, K. (1993). Structure and expression of hedgehog, a Drosophila segment-polarity gene required for cell-cell communication. Gene 124,183 -189.[CrossRef][Medline]
Tremml, G. and Bienz, M. (1989). Homeotic gene expression in the visceral mesoderm of Drosophila embryos. EMBO J. 8,2677 -2685.[Abstract]
Weiss, J., Suyama, K. L., Lee, H. and Scott, M. P. (2001). Jelly belly: a Drosophila LDL receptor repeat-containing signal required for mesoderm migration and differentiation. Cell 107,387 -398.[Medline]
Wolf, C. and Schuh, R. (2000). Single
mesodermal cells guide outgrowth of ectodermal tubular structures in
Drosophila. Genes Dev.
14,2140
-2145.
Yarnitzky, T., Min, L. and Volk, T. (1997). The
Drosophila neuregulin homolog Vein mediates inductive interactions
between myotubes and their epidermal attachment cells. Genes
Dev. 11,2691
-2700.
Zaffran, S., Kuchler, A., Lee, H. and Frasch, M.
(2001). biniou (FoxF), a central component in a
regulatory network controlling visceral mesoderm development and midgut
morphogenesis in Drosophila. Genes Dev.
15,2900
-2915.
Zelzer, E. and Shilo, B. Z. (2000). Cell fate choices in Drosophila tracheal morphogenesis. BioEssays 22,219 -226.[CrossRef][Medline]