1 European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg,
Germany
2 Department of Molecular Genetics, Weizmann Institute of Science, Rehovot,
Israel
3 Department of Biology, New York University, New York, USA
Authors for correspondence (e-mail:
milan{at}embl.de
and
cohen{at}embl.de)
Accepted 28 October 2003
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SUMMARY |
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Key words: Homothorax, Distal-less, Nubbin, Limb development, Imaginal disc
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Introduction |
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Given that there is a common evolutionary origin of legs and wings
(Averof and Cohen, 1997), and
that legs and wings derive from a common imaginal disc primordium in the
Drosophila embryo (Cohen et al.,
1993
), we might expect that Wg and Dpp would act through the same
transcription factor(s) to specify the appendage-forming region in the leg and
wing discs. In this context we became interested in the zinc-finger proteins
encoded by the elbow (el) and no ocelli
(noc) loci. We present evidence that Elbow and Noc proteins are
expressed in the presumptive distal cells of both leg and wing imaginal discs,
where they are required to repress the expression of the body wall genes
homothorax (hth) and teashirt (tsh) and
promote appendage formation.
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Materials and methods |
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Antibodies
El and Noc protein expression was visualized using polyclonal antibodies
raised against full-length proteins in rat (El) or guinea pig (Noc). The
protein expression patterns were faithfully reflected by the
nocGal4 and elGal4 enhancer trap
lines, which were useful for double-labeling experiments.
Rat anti-Hth, Rabbit anti-Tsh, mouse anti-Nub and rat anti-Dll were
described previously (Wu and Cohen,
1999; Wu and Cohen,
2000
; Wu and Cohen,
2002
). Other antibodies are commercially available.
Genotypes of larvae used for genetic mosaic analysis
hs-FLP (I);noc64 FRT40/arm-lacZ
FRT40.
hs-FLP (I); el3.3.1
noc64 FRT40/arm-lacZ FRT40.
hs-FLP (I); el3.3.1
noc64 FRT40/M(+) arm-lacZ
FRT40.
f36ahs-FLP (I);
el3.3.1noc64
FRT40/P(f+) FRT40.
f36ahs-FLP (I);
el3.3.1noc64
FRT40/P(f+) M(+) FRT40.
Clones were generated by giving a 1 hour heat shock at 38°C at the indicated stages.
Genotypes of larvae used for ectopic expression of el and noc
ap4/+; uas-el
uas-noc.
dpp-gal4/uas-el uas-noc.
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Results |
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The early onset and position of El and Noc was consistent with their genes
being early targets of Wingless (Wg) and Decapentaplegic (Dpp), which specify
the appendage primordia. Expression of GSK3/Shaggy, a repressor of the Wg
signaling pathway (Diaz-Benjumea and
Cohen, 1994), or Brinker, a repressor of the Dpp signaling pathway
(Campbell and Tomlinson, 1999
;
Jazwinska et al., 1999
;
Minami et al., 1999
), under
control of dpp-Gal4, repressed El and Noc expression
(Fig. 2A,C,D and not shown).
Likewise ectopic expression of Wg caused ectopic expression of El and Noc
(Fig. 2B and not shown). These
observations indicate that Wg and Dpp signaling are required for induction of
el and noc expression in early leg and wing discs. The late
expression of el and noc in the wing disc
(Fig. 1F) is also under Dpp and
Wg control, with Wg signaling inducing their expression and Dpp repressing it
(data not shown).
|
To assess their role in early wing development, clones of
el3.3.1 noc64 double mutant
cells were generated in early second instar larvae and their distribution was
compared with their wild-type twins in the wing disc (e.g.
Fig. 3A). The homozygous mutant
cell and its wild-type "twin" are products of a single cell
division, so the two clones are normally recovered at equal frequency and in
close proximity to one another after a period of growth. This was the case for
el and noc double mutant clones in the presumptive body wall
portion of the wing disc (Fig.
3B, 26/26 pairs). When the clone pair was close to the edge of the
endogenous Nub expression domain, mutant clones were recovered outside the Nub
domain while their twin clones were found in the Nub expression domain (8/8
pairs). In some cases the mutant clone was separated from its twin
(Fig. 3A), suggesting that the
mutant clone had been displaced to the edge of the Nub domain. In other cases,
the mutant clones appear to have been lost. Only five double mutant clones
were recovered for 31 wild-type twins within the Nub domain. These
observations suggested that el and noc double mutant clones
sorted out from the Nub domain.
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Few larvae in which we generated large el and noc double
mutant clones survived to adulthood and we did not recover adult flies in
which all derivatives of the leg disc were mutant. The most severe defects
recovered corresponded to nearly complete loss of distal leg tissue, including
reduction of femur and tibia (Fig.
4A,C). Although El and Noc activity are not strictly required in
femur- or tibia-producing cells, the defects observed in these segments in
discs with large areas of mutant tissue presumably reflect a requirement at
earlier stages. As for Dll the domain in which El and Noc are required may
retract distally as the disc develops
(Campbell and Tomlinson, 1998;
Cohen et al., 1989
;
Cohen and Jürgens, 1989a
;
Gorfinkiel et al., 1997
). Dll
is required early for formation of tibia and femur as well as tarsus, even
though Dll mutant clones induced later can be recovered in tibia and
femur.
These observations suggest that the combined activity of El and Noc is
required to prevent Tsh and Hth expression in the presumptive
appendage-forming region of the early leg and wing imaginal discs. As neither
gene produces a defect when mutated alone, it appears that either protein is
sufficient to mediate repression of Hth and Tsh. We have attempted to test
this by Gal4-dependent ectopic expression of UAS-noc or UAS-el constructs. To
our surprise, ectopic expression of El or Noc or both together, resulted in a
reduced expression of Nub and up-regulation of Tsh (data not shown). As these
effects are the same as those produced by the loss-of-function mutant clones,
we infer that overexpression of either protein causes a paradoxical dominant
negative effect, reducing net activity. While unexpected, this is not
unprecedented. For example, when overexpressed, dLDB/CHIP, a cofactor of
Apterous, behaves as a dominant negative, apparently by disrupting the
stoichiometry of a multi-protein complex that is required for transcription
factor activity (Fernandez-Funez et al.,
1998; Milan and Cohen,
1999
).
Taken together our observations suggest that the early activities of
el and noc are required for specification of both, wings and
legs in Drosophila. In the wing disc, repression of Hth and Tsh is
required to allow Wg and Dpp to induce Nub expression in the early wing
primordium. In the leg Wg and Dpp repress Hth and Tsh and independently induce
Dll and Dac (Abu-Shaar and Mann,
1998; González-Crespo
et al., 1998
; Wu and Cohen,
1999
; Wu and Cohen,
2000
). We suggest that Wg and Dpp act through el and
noc to define the appendage-forming region of the early wing disc by
repression of body-wall specific genes. Cells lacking El/Noc activity appear
to adopt proximal identity by default, by virtue of not being able to repress
Hth and Tsh.
Late functions of el and no ocelli
Transformation of el and noc mutant cells toward body
wall identity occurred when clones were induced in first or early second
instar. Clones induced later did not alter their proximal-distal identity,
indicating that the role of el and noc in repressing body
wall-specific genes is transient. This is consistent with the alteration in
their patterns of expression as development proceeds
(Fig. 1E,F). In the wing disc,
El and Noc expression shifts to a ring near the base of the wing pouch and to
a wedge shaped stripe along the dorsal-ventral boundary. Consistent with these
changes, clones induced during early third instar lost proximal (hinge)
structures (Fig. 6B,C). These
phenotypes resemble those seen in the wings of the homozygous viable
el1 mutant (compare
Fig. 6C and D)
(Davis et al., 1997).
el1 is a deletion that removes approximately 25 kb of DNA
from the region between the el and noc genes
(Ashburner et al., 1999
;
Davis et al., 1997
), but does
not affect the coding region of either gene. By antibody labeling, we found
that the el1 deletion causes loss of El and Noc protein
expression in the wing pouch, while their expression in the hinge region seems
to be broader than in wild-type discs (Fig.
7). On this basis we conclude that el1 deletes
a common regulatory element that controls expression of el and
noc in the wing pouch. As such el1 should be
considered a regulatory allele of both loci and not just as an allele of
el (Davis et al.,
1997
). The broader ring of El and Noc expression in the hinge
might explain the adult hinge phenotype
(Fig. 6).
|
|
In the leg, large el and noc double mutant clones
extended from coxa, through femur and tibia and caused failure of leg
segmentation (Fig. 6E,F). These
defects resembled those produced by hth or exd mutant
clones, which fail to maintain an affinity border between body wall and leg
(González-Crespo and Morata,
1996; Wu and Cohen,
1999
). Thus the el and noc genes appear to serve
distinct functions in proximal wing and proximal leg later in development that
differ from their primary early role in appendage specification.
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Discussion |
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Dll expression is required for the formation of all leg and antenna
elements in the ventral (leg) discs, and until this work Dll was the earliest
known marker for the distal region leg disc
(Cohen et al., 1989;
Cohen and Jürgens, 1989a
;
Diaz-Benjumea et al., 1994
;
Gorfinkiel et al., 1997
;
Panganiban et al., 1994
).
Previous work has shown that repression of Hth and Tsh by Dpp and Wg was not
required for expression of Dll in the leg, nor could Dll repress Hth and Tsh
(Wu and Cohen, 1999
). Thus an
essential mediator of the effects of Wg and Dpp was missing. Our results
present evidence that El and Noc serve this function, as their removal leads
to ectopic expression of Hth and Tsh. Removal of El and Noc does not cause
loss of Dll expression, so we conclude that Wg and Dpp act independently to
induce El and Noc expression and Dll to define the distal region of the leg
disc.
The situation differs slightly in the wing. Repression of Tsh is the
earliest marker for specification of the distal wing region
(Wu and Cohen, 2002),
preceding the onset of Hth repression or of Nub induction
(Azpiazu and Morata, 2000
;
Casares and Mann, 2000
;
Ng et al., 1996
). Loss of Tsh
and Hth are required to allow Nub expression. We observed ectopic expression
of Hth and Tsh and loss of Nub in clones lacking El and Noc activity. Thus in
the wing, expression of the distal marker Nub cannot be demonstrated to be
independent of El and Noc (because ectopic Hth can repress Nub, but not Dll).
The vestigial gene is also important for wing development and has
been proposed to be a wing specifying gene
(Kim et al., 1996
;
Simmonds et al., 1998
;
Williams et al., 1991
).
However, Vestigial is expressed all along the DV boundary of the wing, both in
the wing primordium and in the body wall. This leads us to suggest that while
Vestigial is essential for wing development, its expression cannot be taken as
a molecular marker for wing identity per se, particularly at early stages [as
discussed previously (Wu and Cohen,
2002
)]. For this reason we have not focussed on analysis of the
relationship between El, Noc and Vestigial in this report.
Is the repression of trunk genes needed to specify appendage as opposed to
the body wall in wing and leg discs? In the wing disc the answer appears to be
yes; repression of `trunk genes' like hth is necessary to make the
remaining part of the disc competent to form the appendage. However, in the
leg the situation is more complex. Coexpression of Dll and Hth does not
disrupt proximal-distal axis formation, but leads to homeotic transformation
of leg tissue into antennal tissue (Casares
and Mann, 1998; Dong et al.,
2001
). Hth is not repressed and limited to proximal areas in the
antenna. However, loss of el and noc activities in the leg
disc leads to loss of distal leg tissue without any evident transformation
into antennal tissue. Thus, El and Noc may regulate the expression of other
`trunk genes', whose restricted expression are required to make the remaining
leg and antenna disc competent to form the appendage.
The regional requirements for El and Noc highlight another interesting
difference between leg and wing disc development. el noc double
mutant cells were excluded from contributing to the tarsal region of the leg
but not from contributing to the femur and tibia. As summarized above, lineage
tracing has shown a considerable net flux of cells from the proximal
(Tsh-expressing domain) into femur and tibia
(Weigmann and Cohen, 1999).
While there is no boundary of lineage restriction separating these domains,
cells must be able to change from expressing the proximal marker Hth to
expressing the distal marker Dll in order to move from one territory to the
other (Wu and Cohen, 1999
).
The wing in contrast does not appear to normally exhibit this large net flux
of cells from proximal to distal (K. Weigmann and S.M.C., unpublished data)
and the el noc double mutant cells were excluded from contributing to
the entire wing region. Our clonal analysis has suggested that el noc
double mutant cells attempt to sort out toward proximal territory, or if that
fails they can be lost from the disc, apparently by sorting out perpendicular
to the epithelium. These observations suggest that El and Noc activity may
contribute to the production of proximal-distal differences in cell affinities
and thereby may help to maintain segregation of these cell populations during
development.
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ACKNOWLEDGMENTS |
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Footnotes |
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