Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK
* Author for correspondence (e-mail: sjb32{at}mole.bio.cam.ac.uk)
Accepted 27 August 2003
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SUMMARY |
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Key words: Limb development, Tarsus, Drosophila, Notch, odd-skipped, Zinc-finger protein
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Introduction |
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A final stage in translating the P/D patterning into the definitive
segmented structure of the insect adult leg is the formation of the
inter-segmental joints. The leg consists of six true segments or podites
(coxa, trochanter, femur, tibia, tarsus and pretarsus), which are
independently moveable by muscles. In Drosophila, the tarsus is
further subdivided into five tarsomeres (t1-t5), which have distinct
characteristics but lack independent musculature
(Snodgrass, 1935). Development
of both `true' joints and inter-tarsomere joints requires Notch activity,
shown by the loss of joints and fused segments in Notch mutant cells,
and by the ectopic joints that are formed when extra sites of Notch activity
are engineered (de Celis et al.,
1998
; Bishop et al.,
1999
; Rauskolb and Irvine,
1999
). Consistent with its pivotal role in specifying joint
development, Notch activity is detected at all segment/subsegment boundaries
at the end of larval development, using transcription of the Enhancer of
split target genes as a measure (de
Celis et al., 1998
). However, expression of Notch ligands is first
observed at a subset of locations at a much earlier stage shortly after the
initial `regional' domains of gene expression are established
(Rauskolb, 2001
). There are
two explanations for this. One is that the specification of joints occurs
sequentially, with some joints being determined early and others (e.g.
tarsomere joints) much later. Alternatively, Notch activity might have both
earlier roles in P/D regionalization and patterning and later roles that build
on these earlier events to establish the segmental boundaries and joints at
the correct locations.
To investigate further the mechanisms involved in P/D limb development, we
have looked for genes whose expression is dependent on Notch activity that
could allow us to establish whether it has roles in the initial P/D patterning
as well as in the subsequent establishment of joints. The zinc-finger protein
encoded by the gene bowl is detected at a subset of sites of Notch
activity and its expression is dependent on Notch. The bowl gene is
closely related to the segmentation gene odd-skipped, and is required
for development of the embryonic hindgut
(Wang and Coulter, 1996;
Iwaki et al., 2001
). Our
analysis of bowl and odd-skipped function in the developing
leg indicates that these genes are involved in the elaboration of pattern in
the tarsus, leading us to propose that Notch is important for patterning as
well as for joint formation. The effects of Bowl on tarsal development suggest
that P/D tarsal identities are determined progressively and might also explain
how different numbers of tarsomeres could have arisen from an ancestral limb
that is thought to have contained an unsegmented tarsus
(Snodgrass, 1935
).
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Materials and methods |
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The bowl or odd alleles were recombined onto w1118; P{mW+mw=pM}36F P{ry+t7.2=neoFRT}40A and were crossed to the following marked strains for inducing clones:
Clones were induced by a 1-hour heat shock at 38°C at 48-72 hours of development. Large mutant clones were also induced by X-irradiation at a dose rate of 1.28 Rads per second for 900 seconds using the Minute stock f36a; M(2)z P{f+}30B/CyO.
Notch mutant clones were generated using directed FLPase expression by crossing the stocks N81k P{ry+t7.2=neoFRT}101 / FM7 females with y w GFPXI P{ry+t7.2=neoFRT}101/Y; ptc-Gal4; UAS-FLPase/ SM6a-TM6B males (gift of T. Klein and A. Martinez-Arias).
UAS-bowl was generated using the full-length cDNA clone LD15614 obtained from the Berkeley Drosophila Genome Project. The bowl cDNA was excised using NotI and XhoI, and ligated into pUAST digested with the same enzymes. Transgenic flies were obtained by injection into y w, following standard P-element transformation procedures. Independent lines were analysed and graded according to the strength of phenotypes elicited with GAL4 drivers as follows: UAS-bowl1.1 (strong) > UAS-bowl6.1 (moderate) > UAS-bowl9.1 (weak)
Immunofluorescence
Leg discs were dissected from wandering third-instar larvae and from pupae
(up to 18 hours after pupation). Indirect immunofluorescence was carried out
as previously described (de Celis et al.,
1998). Anti-Bowl antibodies were generated in rabbits by Sigma
Genosys. Peptides used for immunization were [Cys]-PPIAPPPAPPRRTGFSIEDIMRR and
[Cys]-DLPRVHDLPREEDDD-FDPEDEEQ. Anti-Bowl serum was used at a final dilution
of 1/1000. Other primary antibodies were rabbit anti- GFP (Molecular Probes),
1:1000; rabbit anti-BarH1 (Higashijima et
al., 1992
), 1:1000; rabbit anti-ß-galactosidase (Cappel),
1:500; rabbit anti-Serrate (Thomas et al.,
1991
), 1:20; rat anti-Bab2
(Couderc et al., 2002
), 1:2000;
and mouse anti-Dac, 1:5, and anti-Dl, 1:5 [developed by Kooh et al.
(Kooh et al., 1993
) and Mardon
et al. (Mardon et al., 1994
),
respectively, and obtained from the Developmental Studies Hybridoma Bank,
University of Iowa, Department of Biological Sciences].
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Results |
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Given the profound effect on tarsal segmentation and similarities with
Notch phenotypes, we expected that bowl would be expressed
at the sites where Notch is active in the tarsus. We therefore compared the
expression of bowl with E(spl)mß, a known target of
Notch signalling in the leg, using an E(spl)mß-lacZ
transgene (Cooper et al., 2000)
and an antibody that recognizes Bowl. Although Bowl and ß-galactosidase
are clearly co-expressed at some positions, including the t5/pretarsus
boundary and the tibia/t1 boundary, Bowl was not detected at sites of Notch
activity within the tarsus (Fig.
1I-I''). Indeed, the distribution of Bowl and Odd appears to
be identical and neither is detected at tarsomere boundaries
(Fig. 1J-K'')
(Rauskolb and Irvine, 1999
).
Both are present at all the proximal joints (coxa/femur, femur/tibia,
tibia/t1) and at a distal site, the t5/pretarsal boundary
(Fig. 1J,K; the latter has not
previously been documented as a site of Notch activity, although it clearly
expresses E(spl)mß and gives rise to an articulated joint). In
summary, therefore, Bowl and Odd are present at a subset of the segmental
boundaries where Notch is active in the developing leg. These correspond to
the boundaries between `true' segments and not to those between
tarsomeres.
bowl is regulated by Notch in the developing leg
Expression of the Notch ligands is a key step in regulating Notch activity
in the developing leg (Mishra et al.,
2001; Rauskolb,
2001
). To investigate the relationship between Bowl and Notch
activity, we first compared the timing and distribution of Bowl expression
with that of Serrate and Delta, which both regulate Notch activity in the leg
disc (Mishra et al., 2001
;
Rauskolb, 2001
). By monitoring
expression from early third instar, we found that the evolution of
Bowl/odd-lacZ expression closely parallels that of the Notch ligands
(Fig. 3A-D). The only
significant discrepancy appears late in the third instar, when Serrate and
Delta are detected at intertarsomere boundaries but Bowl and odd-lacZ
are not (Fig. 3C,C' and
data not shown). Before that stage, Bowl/Odd expression occurs distal to each
domain of Delta that is established. For example, the central t5/pretarsal
ring of Bowl appears at
86 hours (Fig.
3A-C) and correlates with the appearance of Delta in the tarsus
and a transient expression of Serrate on the distal, pretarsal, side
(Fig. 3A-C and data not shown)
(Rauskolb, 2001
).
|
Mutations in bowl alter the expression of genes involved in
tarsal patterning
Neither Bowl nor Odd appear to be present within the tarsus
(Fig. 1J-K, Fig. 3C)
(Rauskolb and Irvine, 1999),
yet mutations in either gene produce defects in this part of the leg (fusion
of tarsomeres and growth defects; Fig.
1D-H). There are three models to explain this. (1) Bowl and Odd
influence tarsomere segmentation indirectly, by regulating production of a
long-range signal. (2) The two genes are expressed at intertarsomere joints
but at a level too low to be detected. (3) They are involved in an earlier
patterning event that influences subsequent tarsal segmentation. We can rule
out the first hypothesis, because fusions only occur within tarsal
bowl clones (Fig.
1D,F; Fig. 2) and
the effects on downstream genes are autonomous, as is clearly seen in
Fig. 4 (e.g.
Fig. 4D). Although it is
difficult to rule out the low levels of tarsal expression implied by the
second model, our data argue that this is an unlikely explanation for several
reasons. First, bowl clones that only spanned intertarsomere joints
(e.g. t2/t3/t4) appeared normal (Fig.
2). Almost all clones that resulted in observable phenotypes
spanned the tibia/t1 boundary and, even within these clones, there was
considerable variation in the number of tarsomere joints affected
(Fig. 2). This suggests that
the effect on tarsomere joints is a secondary consequence of the mutations.
Second, in the case of Odd, the pattern of expression detected in the larva
persists throughout pupal leg morphogenesis
(Mirth and Akam, 2002
), ruling
out later expression in the tarsomeres. Third, when we produce high levels of
bowl mRNA using GAL4/UAS system, very little protein is detected in
the tarsal domain of late-third-instar/early-pupal legs, suggesting either
that the protein is unstable or that the mRNA is poorly translated in this
region (e.g. see Fig. 6 and
data not shown).
|
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Second, we asked whether mutations in bowl alter the expression of
genes involved in the initial regionalization of the leg. Several genes have
been identified that confer distinct regional identities and are expressed in
broad domains within the leg disc. These include dachshund
(dac), which is expressed in more proximal regions including t1
(Mardon et al., 1994;
Lecuit and Cohen, 1997
), the
two genes of the bric-a-brac (bab) complex (bab1
and bab2), which are expressed in the presumptive tarsal region
(Godt et al., 1993
;
Couderc et al., 2002
;
Galindo et al., 2002
), and two
Bar genes that are expressed in distal tarsal segments t4 and t5
(BarH1 and BarH2)
(Kojima et al., 2000
). We find
that expression of all three types of regional genes is affected by mutations
in bowl. In late-third-instar/early-pupal leg discs, Bab2 expression
normally extends to the proximal edge of t1
(Fig. 4A). In bowl
clones, ectopic Bab2 is detected in proximal parts of t1 and the levels in t2
are also altered (Fig. 4B,D,E).
Conversely, when these clones also encompass the distal part of Dac domain,
there is a reduction in Dac (Fig.
4C,D). Mutant clones that lie at the distal side of the tarsal
domain again show derepression of Bab2 (in distal t5, where Bab2 expression is
low or absent), and this is coupled with a reduction in the levels of BarH1
(Fig. 4F). In all cases, the
effects are autonomous to the clone and precisely follow clone boundaries.
We therefore conclude that Bowl regulates the expression of patterning genes, promoting development of the proximal (t1/t2) (Fig. 4B,D,E) and distal (t5) extremities of the tarsus (Fig. 4F). Thus, bowl mutations lead to an expansion of a `central tarsal fate' that is characterized by uniform Bab2 and decreased BarH1 and Dac. This disruption in tarsal patterning would in turn affect the expression of Notch ligands in the tarsus and hence lead to defects in tarsomere joints, as seen in the disruption of E(spl)mß-CD2 (Fig. 4B). However, there is still a discrepancy between the apparent function of Bowl and its site of expression: Bowl is necessary to inhibit/lower Bab2 expression in t1/t2 and t5, but is not present in these regions in late-third-instar/early-pupal discs. Nevertheless, the effects of bowl mutations on Bab2 and Dac are strictly cell autonomous (Fig. 4B,D-F). These observations can be reconciled if Bowl (and likewise Odd) is expressed within the cells that give rise to t1/t2 and t5 at early stages when the domains of the regional genes (bab, dac and Bar) are first established. This expression must subsequently disappear from these regions and become restricted to the boundaries of the tarsus.
Early bowl/odd expression
If Bowl and Odd are regulating tarsomere segmentation via effects on
regional genes like bab2, they should be expressed at the boundaries
of the Bab2 domain during early stages. At 76-80 hours, both Bowl and Odd
are first detected in a 2-3-cell-wide ring that surrounds the Bab2 expressing
cells and corresponds to the proximal edge of the Dll domain
(Fig. 5A-A'') and the
distal edge of the Dac domain (data not shown). Most of these Bab2-expressing
cells also express BarH1; on the proximal side only a 1-2-cell-wide ring
contains Bab2 and not BarH1 (Fig.
6D). At this stage, therefore, the tarsus consists primarily of
one identity, which has Bab2 and BarH1 expression and appears to approximate
to t4. This early domain is surrounded by cells expressing Bowl and Odd.
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Both the expression patterns and the phenotypes suggest that cells within the t1/t2/t3 and t5 regions of the tarsal domain contain Bowl/Odd at 76-86 hours. We propose that Bowl/Odd expression is gradually lost from the tarsal cells as they proliferate, giving rise to a temporal gradient of Bowl/Odd (prolonged expression in t1, shorter period of expression in t3/t2). If this is the case, expression from the odd-lacZ line might be visible within t1/t2/t3 because of the endurance of ß-galactosidase. Indeed, in 96-hour-old odd-lacZ discs, we detect ß-galactosidase at lower levels within many cells of the tarsus (Fig. 5G). We cannot definitively show a temporal gradient by this method, but the expression of odd-lacZ is most persistent close to the final domain of Bowl and Odd, consistent with this model.
Ectopic bowl causes expansion of proximal and distal tarsal
fates
Because bowl mutations result in expansion of `central tarsal'
(t3/t4) fates, we anticipated that persistent expression of Bowl within the
tarsus would have the converse effect, expanding proximal (t1/t2) and distal
(t5) fates. To test this we used GAL4 driver lines to direct expression of
UAS-bowl within the tarsus, scoring phenotypes in the adult male
pro-thoracic legs, using the sex comb as a marker of t1
(Fig. 5A-D). Expression of
UAS-bowl throughout the tarsal region (Dll-Gal4) gave rise to legs
with expanded t1 fates manifest in the ectopic sex-combs on distal tarsal
segments. In the more strongly expressing lines, the tarsus became severely
distorted and carried sex-comb bristles throughout its length
(Fig. 6C,D). Even with more
restricted production of Bowl (e.g. klumpfuss-GAL4)
(Klein and Campos-Ortega,
1997), similar transformations occurred, with ectopic sex-comb
bristles present in t2 and t3 (Fig.
6B).
To determine whether expansion of t1 fates occurs at the expense of
`central tarsal' fates, we assayed the effects of ectopic Bowl on Bab, Dac and
BarH1. In leg discs from Dll-GAL4/UAS-bowl, levels of Bab2 were
strongly reduced and more patchy than in the wild type, consistent with
central tarsal identity being compromised
(Fig. 6E,F'). Conversely,
the domains of Dac and BarH1 were extended so that they were almost contiguous
in the middle of the tarsus (Fig.
6F,F'), demonstrating the expansion of t1 and t5 fates.
Ectopic expression of Bowl in a more restricted domain (e.g. with
Ptc-Gal4) also reduced Bab (Fig.
5G-G'') specifically within the domain of ectopic expression.
The inhibition of bab2 by Bowl fits with the phenotypes of
bab/bab2 loss-of-function alleles, which are similar to that of
ectopic Bowl (ectopic sex combs on distal segments)
(Godt et al., 1993;
Couderc et al., 2002
). In
analysing the levels of Bowl produced by the directed misexpression, we noted
that high levels of protein only accumulated close to the normal sites of
expression. Elsewhere, such as within the tarsus, protein levels remain low
and patchy (Fig. 6G'),
even though mRNA levels are fairly uniform throughout the domain of
misexpression (data not shown). Despite the low levels of protein, we still
see inhibition of Bab within the tarsus
(Fig. 6G'').
Further support for the role of bowl in patterning the proximal tarsus
comes from analysing spineless mutant legs. This gene is essential
for antenna development but is also expressed transiently in the tarsus of the
early third-instar leg (Duncan et al.,
1998). The phenotype observed in weak spineless mutants
(ssa/ss114;
Fig. 5H,H') resembles
that of ectopic Bowl (with ectopic sex combs in t2 and an ectopic joint within
t1), and we find that the domain of Bowl expression remains broader in
spineless larval discs and that ectopic patches of Bowl persist in t1
and t2 of early pupal discs (Fig.
6I,I'). Sometimes, the ectopic Bowl forms a discrete ring
within t1 that corresponds to the site of the ectopic joint. Persistent Bowl
therefore alters P/D patterning, promoting t1-like fates and, in some cases,
resulting in an ectopic tibia/t1-like joint. These data suggest that
spineless is involved in keeping Bowl absent from in the tarsus. In
agreement with this, ectopic Spineless results in loss of Bowl (data not
shown), although these conditions also result in transformation to antenna
fates, complicating the interpretations.
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Discussion |
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Previous studies have shown that bab1/bab2 expression is promoted
by Dll and that its proximal and distal limits are dependent on Dac proximally
and on epidermal-growth-factor-receptor signalling distally
(Campbell and Tomlinson, 1998;
Campbell, 2002
;
Couderc et al., 2002
;
Galindo et al., 2002
). We
propose that these activities not only define the initial domain of
bab1/bab2 expression but also indirectly regulate the
production of Bowl and Odd through their effects on Notch-ligand expression
(Rauskolb, 2001
). Bowl is then
necessary to fine tune bab2 expression so that its levels are low or
absent in the extremities of the tarsus, allowing these to adopt t1 and t5
characteristics (Fig. 7). If
one of the factors responsible for positively regulating bab1/bab2
expression was present transiently, its decay would also contribute to the
gradation in Bab2 expression and could explain why Bab2 is not turned on in
the t1 cells that have lost Bowl at late stages.
|
Notch activation appears to be one key factor in promoting the accumulation
of Bowl and Odd at the tarsal boundaries
(Fig. 3) (see
Rauskolb, 2001), but some data
indicate that other factors are required and that the regulation might be
indirect. First, Bowl and Odd can only be induced at a subset of the locations
where Notch is active, so Notch alone is not sufficient. Second, although all
Notch clones at the t5/tibia boundary result in a loss of Bowl protein, not
all clones at the more proximal boundaries have a phenotype. Because the
smaller clones tend to have the least effect on Bowl, Notch appears to
initiate but not to maintain Bowl expression at these locations. Third,
although regulation of odd can be fully explained by its effects on
transcription, Bowl might be subject to post-transcriptional regulation. When
we drive expression of bowl mRNA through the leg (using GAL4
drivers), we detect at best low levels of Bowl protein within the tarsus,
suggesting that the translation or the stability of the protein are regulated.
Candidates to participate in Odd and Bowl regulation include Spineless
(Fig. 6) and Lines, a protein
that acts antagonistically to Bowl and Drm in hindgut morphogenesis
(Iwaki et al., 2001
;
Green et al., 2002
). Although
the combined actions of Notch and these factors might explain the initial
expression of Bowl and Odd, the mechanism that maintains their expression
specifically at the boundaries of the tarsus is unclear. This aspect of
regulation is crucial for the diversification of the tarsomeres and, if our
model is correct, would be linked to proliferation. Our predictions are that
tarsal cells should show a bias in their patterns of proliferation, as is the
case in more proximal regions of the leg
(Weigmann and Cohen, 1999
),
and that the progeny of Bowl-expressing cells should occupy the t1/t2 and t5
tarsal segments. We have not yet been able specifically to monitor the
proliferation pattern and fate of Bowl-expressing cells to test these
predictions.
One extrapolation from our proposed model for tarsal development in
Drosophila is that the basal or ancestral state consisted of a single
tarsal segment, specified by uniform levels of Bab and directly flanked by
sites of Bowl expression prefiguring the tarsal/tibial and tarsal/pretarsal
joints. This is in agreement with the phylogenetic evidence, which points
towards the ancestral arthropod limb having an unsegmented tarsus (as remains
the case for many modern arthropods, including some insects)
(Snodgrass, 1935).
Furthermore, there is considerable variation in the extent of tarsal
subdivision, with most insects having between two and five tarsomeres (some
arachnids have further subdivisions;
Snodgrass, 1935
). These
differences in pattern could be explained by differences in either the
duration or the rate of proliferation during the crucial phase when
bowl/odd influence bab2 patterning. Although mutations in
Notch or bowl/odd affect the extent of tarsal proliferation,
as do mutations in spineless and bab2, none of these
activities alone is sufficient to cause an increased length of the tarsus
(although ectopic Notch activity can give ectopic outgrowth)
(Rauskolb and Irvine, 1999
).
Further investigation of how these factors combine to coordinate tarsal
patterning and proliferation should help us to unravel the mechanism
underlying the diversification of arthropod limb structure. Furthermore, as
modifications of bab2 expression are correlated with diversification
of pigmentation and trichome patterns in Drosophila species
(Gompel and Carroll, 2003
),
the possibility that bab2 expression is intrinsic to diversification
of tarsal patterning suggests that changes in the regulation of a single gene
could contribute to the diversification of many different morphological
traits.
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ACKNOWLEDGMENTS |
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Footnotes |
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A related paper by Hao et al. discussing the expression and function of
bowl-related genes in the Drosophila leg is currently in press
(Hao et al., 2003).
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