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First published online 18 July 2007
doi: 10.1242/dev.006445
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Howard Hughes Medical Institute, Department of Genetics and Development, Columbia University College of Physicians and Surgeons, 701 W 168th Street, New York, NY 10032, USA.
* Author for correspondence (e-mail: gs20{at}columbia.edu)
Accepted 12 June 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Drosophila wing, Morphogen, Organ growth, Selector gene, Vestigial
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Williams et al., 1993;
Kim et al., 1996;
Halder et al., 1998;
Liu et al., 2000). Shortly
after the wing primordium is first apparent as a cluster of 
40
vg-expressing cells (Wu and
Cohen, 2002), it is subdivided into dorsal (D) and ventral (V)
compartments by the heritable activation of the selector gene
apterous (ap) in the D compartment
(Diaz-Benjumea and Cohen, 1993;
Williams et al., 1993;
Blair et al., 1994).
Short-range Delta/Serrate/LAG-2 (DSL)-Notch signaling across the D-V boundary
then initiates a dramatic 200-fold expansion of the wing primordium to a
population of 8000 vg-expressing cells under the control of the
long-range morphogens Wingless (Wg) and Decapentaplegic (Dpp)
(Diaz-Benjumea and Cohen, 1995;
Kim et al., 1995;
Zecca et al., 1995;
de Celis et al., 1996;
Doherty et al., 1996;
Lecuit et al., 1996;
Nellen et al., 1996;
Zecca et al., 1996;
Neumann and Cohen, 1997).
Here, we examine how morphogens and selector genes control organ growth, using
the Wg-driven expansion of the population of vg-expressing cells -
the wing primordium - as a paradigm.
In the accompanying study (Zecca and
Struhl, 2007), we focused on how Wg signaling controls vg
expression and wing growth, taking advantage of ap mutant discs.
Normally, short-range DSL-Notch signaling across the D-V boundary induces
`border' cells flanking the boundary to express both wg and
vg (Williams et al.,
1994; Couso et al.,
1995; Diaz-Benjumea and Cohen,
1995; Kim et al.,
1995; de Celis et al.,
1996; Kim et al.,
1996; Neumann and Cohen,
1996; Rulifson et al.,
1996). However, in ap mutant discs, border cells are not
specified, the early expression of vg that normally precedes the D-V
segregation dissipates, and the presumptive wing primordium fails to develop
(Williams et al., 1993). By
generating clones of cells that ectopically express Vg, Wg or both, we showed
that cells within ap mutant discs could be recruited to express
vg in response to Wg, but only if they were located near or next to
cells that already express Vg. These results defined a previously unknown
feed-forward mechanism of vg autoregulation, and led us to propose
that D-V border cells normally control the expansion of the wing primordium by
providing both a long-range morphogen, Wg, as well as the initial Vg-dependent
feed-forward signal that entrains neighboring cells to express vg in
response to Wg.
Here, we extend our results in ap mutant discs by testing whether this autoregulatory circuit is required for normal wing growth in wild-type discs. We first demonstrate that the previously identified quadrant enhancer (QE) of the vg gene mediates vg autoregulation in response to Wg, the feed-forward signal, and a newly defined third input: `priming' of the vg locus by pre-existing low levels of Vg. We then present evidence that QE-driven expression of vg is necessary and sufficient for the expansion of the wing primordium organized by D-V border cells. These findings support our hypothesis that wing growth normally depends on a non-autonomous autoregulatory circuit of vg gene expression triggered by short-range DSL-Notch signaling and fueled by long-range Wg signaling.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Transgenes and protocols unique to this study are as
follows.
5XQE>Tub
1-flu-GFP>vg and
5XQE>CD2,y2>vg transgenes were assembled using DNAs described
in Zecca and Struhl (Zecca and Struhl,
2007). 5XQE>vg derivatives of these transgenes
generated by germ-line excision of the intervening Flp-out cassette resulted
in dominant larval lethality. Similarly, induction of 5XQE>vg
clones in first instar larvae carrying such transgenes resulted in late larval
or pupal lethality. Hence, our analysis of 5XQE>vg transgene
activity was restricted to the behavior of Flp-out clones in the wing
disc.
The rp49>CD2,y2>vg transgene was assembled using the
rp49 promoter, as well as the destabilizing Hsp70 3'
UTR (Greenwood and Struhl,
1997; Casali and Struhl,
2004). rp49>vg derivatives (referred to subsequently
as rp49-vg) of each of several rp49>CD2,y2>vg
transgene insertions were generated by germ-line excision of the
>CD2,y2> cassette. All but one of these resulted in pupal
lethality when present in one copy. However, the exceptional rp49-vg
derivative was viable and fertile in one copy, albeit pupal lethal when
homozygous, indicating that this transgene expresses a lower level of
exogenous Vg than the others, and on this basis we selected it for use in all
subsequent experiments.
Generation of clones of cells that ectopically express, or lack, gene activity
Clones of vg or arrow (arr) mutant cells were
generated by Flp-mediated mitotic recombination
(Golic, 1991). The
Minute technique (Morata and
Ripoll, 1975) was used to generate vg83b27
(vgb) clones with a growth advantage
(Fig. 2E). Clones expressing
exogenous Vg were generated using the Flp-out technique
(Struhl and Basler, 1993). In
some cases, two types of clones were generated in the same disc to yield
either (1) adjacent clones of different type (e.g. 5XQE>vg clones
next to Tub1>vg clones; e.g.
Fig. 4); (2) coincident clones
of different type [e.g. vg83b27R (vg0)
5XQE>vg clones; e.g. Fig.
3C]; or (3) `clones within clones' (e.g. arr0
clones inside 5XQE>vg clones;
Fig. 3D,E). Unless otherwise
stated, clones were induced by heat shocking first instar larvae [24-48 hours
after egg laying (AEL)] at 36°C for 30 minutes; for `clones within
clones', larvae were heat shocked, as above, during the first instar, and then
given a second heat shock of the same length and temperature at 60-84 hours
AEL (late second to early third instar), or 48-72 hours AEL (second instar).
In all of the experiments in this study, mature wing discs were dissected from
late third instar larvae, and fixed and analyzed as previously described
(Zecca and Struhl, 2002).
Twin spot analysis of vgb and vg0 clones
Larvae were heat shocked (35°C 10 minutes) at the times indicated in
Fig. 2. Mutant clones were
marked by loss of GFP expression, whereas their wild-type sibling (twin)
clones were marked by strong GFP expression (owing to homozygosity of the
Hsp70-flu-GFP transgene). All, and only those, wild-type clones that
contributed to the presumptive wing pouch area (marked by 1XQE-lacZ
expression) were scored for the presence and contribution of their associated
mutant twins. For further details, see Fig.
2.
Genotypes
Genotypes are listed by figure panel; except where stated otherwise, the X
chromosome was y w Hsp70-flp.
1B: 1XQE-lacZ vg83b27/vg83b27.
1C: 1XQE-lacZ vg83b27R/vg83b27R.
1D: 1XQE-lacZ vg83b27/vg83b27; rp49-vg/rp49-vg.
1E: 1XQE-lacZ vg83b27R/vg83b27R; rp49-vg/rp49-vg.
1F: 1XQE-lacZ vg83b27/vg83b27R;
Tub1>flu-GFP, y+>vg/+.
1G: 1XQE-lacZ vg83b27R/vg83b27R;
Tub1>flu-GFP, y+>vg/+.
2A,B,F: FRT42D vg83b27/1XQE-lacZ FRT42D Hsp70-flu-GFP.
2C: y w 5XQE-DsRed/y w Hsp70-flp; FRT42D vg83b27R/1XQE-lacZ FRT42D Hsp70-flu-GFP; rn-lacZ/+.
2D,F: FRT42D vg83b27/1XQE-lacZ FRT42D Hsp70-flu-GFP; rp49-vg/rp49-vg.
2E: FRT42D Minute(2)IK Hsp70-flu-GFP/FRT42D vg83b27; 1XQE-lacZ/+.
2F: 1XQE-lacZ FRT42D Hsp70-flu-GFP/FRT42D
Tub1-DsRed (for vg+ clones).
3B,C: y w 5XQE>CD2,y2>vg/y w Hsp70-flp; FRT42D vg83b27R/1XQE-lacZ FRT42D Hsp70-flu-GFP.
3D: y w 5XQE>CD2,y2>vg/y w Hsp70-flp; FRT42D
arr2/FRT42D Tub1-DsRed; 1XQE-lacZ/+.
3E: y w 5XQE-DsRed/y w Hsp70-flp; FRT42D arr2/FRT42D
Hsp70-CD2; Tub1>flu-GFP, y+>vg/+.
4A: y w 5XQE>CD2,y2>vg/y w Hsp70-flp; FRT42D
vg83b27R/1XQE-lacZ vg83b27R;
Tub1>flu-GFP, y+>vg/+.
4B,C: 1XQE-lacZ ap56f vg83b27R/1XQE-lacZ
vg83b27R; Tub1>DsRed,
y2>vg/5XQE>Tub1-flu-GFP>vg.
5A: FRT42D vg83b27R/1XQE-lacZ vg83b27R; BE-vgGFP/+.
5B,C: y w 5XQE>CD2,y2>vg/y w Hsp70-flp; FRT42D vg83b27R/1XQE-lacZ ap56f vg83b27R; BE-vgGFP/+.
5D: FRT42D vg83b27R/1XQE-lacZ vg83b27R;
BE-vgGFP/5XQE>Tub1-flu-GFP>vg.
5E: FRT42D vg83b27R/1XQE-lacZ vg83b27R; BE-vgGFP rp49-vg/+.
5F,G: y w 5XQE>CD2,y2>vg/y w Hsp70-flp; FRT42D vg83b27R/1XQE-lacZ vg83b27R; BE-vgGFP rp49-vg/+.
5H: FRT42D vg83b27R/1XQE-lacZ vg83b27R;
BE-vgGFP rp49>vg/5XQE>
Tub1-flu-GFP>vg.
6A,C: y w 5XQE>CD2,y2>vg/y w Hsp70-flp.
6B: y w 5XQE>CD2,y2>vg/y w Hsp70-flp; 1XQE-lacZ FRT42D Hsp70-flu-GFP/1XQE-lacZ ap56f vg83b27R.
6D: y w 5XQE>CD2,y2>vg/y w Hsp70-flp; Dll-lacZ/+; C765-Gal4/+.
6E: y w 5XQE>CD2,y2>vg/y w Hsp70-flp; Dll-lacZ/5XQE-DsRed vg83b27R; UAS-wg rp49-vg/C765-Gal4.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Williams et al.,
1994; Kim et al.,
1995; Kim et al.,
1996). Border cell and pouch expression are associated,
respectively, with the activity of distinct boundary enhancer (BE)
and quadrant enhancer (QE) elements
(Williams et al., 1994;
Kim et al., 1996). Here, we
have sought to determine whether QE elements are responsible for
mediating the feed-forward propagation of vg in response to Wg, and
if so, whether operation of this autoregulatory circuit is necessary and
sufficient for the normal expansion of the wing primordium that occurs
following the D-V segregation. Our main approach has been to generate a vg transgene that is expressed under QE control, validate that it is activated in response to the combined inputs of Wg and the vg-dependent feed-forward signal, and then test whether it is necessary and sufficient to mediate vg expression and wing growth away from the D-V compartment boundary. Crucial to the success of this approach has been our discovery of a third input necessary for the vg autoregulatory response: priming of the vg gene by pre-existing low levels of Vg. We begin by describing our evidence for priming, which arose unexpectedly from experiments designed to test the capacity of a BE-deficient allele of vg to mediate feed-forward autoregulation.
vg feed-forward autoregulation requires `priming' by cryptic, low levels of Vg
vg83b27 (henceforth vgb) is an
internal deletion of the intron 2 segment of vg that removes the
previously defined BE as well as adjoining sequences, but leaves
intact the rest of the gene, including the QE
(Williams et al., 1993;
Kim et al., 1996). Mature
vgb mutant discs, like vg-null (henceforth
vg0) discs, lack the wing primordium
(Fig. 1A-C)
(Williams et al., 1993), as
expected if D-V border cells require BE activity to express
vg and to initiate propagation of vg expression into
neighboring tissue. However, clones of vgb cells should
retain the capacity to propagate vg expression in response to
wild-type border cells and, hence, to contribute normally to the developing
wing. In testing this prediction, we obtained evidence that the
vgb mutation deletes a previously unknown `priming'
enhancer (PE), in addition to the BE, and that cryptic, low
levels of Vg, expressed under the control of this enhancer, are a prerequisite
for feed-forward autoregulation.
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)
failed to express 1XQE-lacZ and appeared to be excluded from the wing
pouch, forming non-wing tissue at its expense
(Fig. 2E). Thus, early-induced
vgb clones appear compromised for the ability to activate
the QE and develop as wing tissue, even when they abut wild-type
Vg-expressing cells and are in a position to receive Wg as well as Dpp.
One explanation for this unexpected result is that vgb
cells lack an additional component of the proposed vg autoregulatory
circuit. The QE contains binding sites for Scalloped (Sd), the
DNA-binding protein that combines with Vg to form a composite transcriptional
activator (Halder et al.,
1998; Paumard-Rigal et al.,
1998; Simmonds et al.,
1998; Guss et al.,
2001; Halder and Carroll,
2001). Moreover, Sd and the presence of its binding sites are
necessary for QE activity (Halder
et al., 1998; Guss et al.,
2001). Hence, the QE might need to be `primed' by
cryptic, low-level Vg to mediate feed-forward autoregulation, and the presence
of such pre-existing Vg might depend on a distinct `priming' enhancer
(PE) deleted in the vgb allele. According to this
hypothesis, sufficient Vg would perdure in vgb cells
induced after the D-V segregation to supply the requisite priming function,
but not in the descendents of vgb cells induced before D-V
segregation.
To test this, we generated an rp49-vg transgene in which the
vg coding sequence is expressed at exceptionally low level, under the
control of the uniformly active, but weak, ribosomal protein 49
(rp49; also known as RpL32 - Flybase) promoter
(Greenwood and Struhl, 1997;
Casali and Struhl, 2004), and
asked whether such low-level expression is sufficient to rescue normal wing
development and endogenous vg expression in early-induced
vgb clones.
Wing discs homozygous for the rp49-vg transgene express so little Vg protein that we were unable to detect it by antibody staining in wild-type or vg0 discs. In addition, homozygosity for the transgene failed to rescue wing development in vg0 discs. Instead, vg0; rp49-vg discs formed abnormally small wing pouches composed of cells that appeared to correspond to the periphery of the normal pouch, where neither Vg nor 1XQE-lacZ expression were readily detectable (Fig. 1E). Nevertheless, homozygosity for the rp49-vg transgene almost completely rescued the capacity of early-induced vgb clones to express endogenous Vg, as well as the 1XQE-lacZ reporter, and to contribute to the wing pouch (Fig. 2D,F). Indeed, it restored normal vg expression and wing development in the wing pouch of entirely mutant vgb discs, including in D-V border cells, despite the absence of the well-defined and evolutionarily conserved BE (Fig. 1D).
Thus, the capacity of vgb cells to express vg and to develop as normal wing tissue appears to depend on cryptic, low-level Vg activity, defining a third input, `priming', that is required together with Wg and the feed-forward signal, for upregulation of vg away from the D-V boundary. These findings also indicate that the vgb allele retains at least one additional BE, and that activity of this BE depends on priming. Hence, the primary cause of the vgb `no wing' phenotype appears to be the deletion of the PE, not the BE, in intron 2. In subsequent experiments, we used the rp49-vg transgene to satisfy the requirement for priming in the absence of the endogenous vg gene.
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To express Vg under the control of QE sequences, we generated
transformants of a 5XQE>CD2>vg Flp-out transgene in which five
copies of the QE drive the expression of either rat CD2 or Vg. In the
absence of Flp recombinase, the 5XQE>CD2>vg transgene
insertions behaved like the previously described 1XQE-lacZ and
5XQE-DsRed reporter transgenes, showing expression of CD2 that was
tightly restricted to the wing pouch but excluded from the D-V border cells
within the pouch (Fig. 3A)
(Kim et al., 1996;
Zecca and Struhl, 2007). Upon
heat shock-induced expression of Flp, the >CD2> cassette is
excised in single cells, generating clones of 5XQE>vg cells marked
in the prospective wing pouch by the absence of CD2 and the expression of
exogenous Vg. Different transgene insertions showed some variation in the
level and extent of Vg expression following excision of the
>CD2> cassette. For the most strongly active insertions, clones
of 5XQE>vg cells that were also vg0 developed
as wing tissue and expressed levels of exogenous Vg protein similar to those
of endogenous Vg expressed in surrounding wild-type cells
(Fig. 3B,C). By contrast,
clones of 5XQE>vg vg0 cells generated using the less
active insertions showed weaker, patchy expression of Vg and rescued wing
development less well (data not shown). We therefore focused our analysis on
one such strongly active 5XQE>CD2>vg insertion, and performed
the experiments described below to validate that its activity depends on Wg
and the Vg-dependent feed-forward signal.
Requirement for Wg input
To test whether expression of the chosen 5XQE>CD2>vg
transgene requires Wg input, we heat shocked first instar larvae to generate
large clones of 5XQE>vg cells in otherwise
5XQE>CD2>vg wing discs, and then heat shocked them again during
the late second to early third larval instar to generate smaller clones of
cells mutant for arr (arr0), which encodes a
co-receptor essential for transducing Wg
(Wehrli et al., 2000). We
observed that surviving arr0 clones showed greatly reduced
or no expression of both CD2 and Vg, irrespective of whether the clones were
located within the 5XQE>vg territories
(Fig. 3D) or in surrounding
5XQE>CD2>vg tissue (data not shown). These results indicate
that expression of both the excised and intact forms of the transgene require
Wg input.
Because arr0 clones are associated with the loss of
endogenous vg expression (data not shown), it is possible that
activity of the 5XQE element depends only on the presence of Vg
protein, and hence might `report' Wg input indirectly, via activation of
other, as yet unidentified, Wg-responsive enhancers in the endogenous
vg gene. To assess this, we replaced the 5XQE>CD2>vg
transgene with a Tub1>GFP>vg transgene
(Fig. 1G)
(Tub1 is also known as Tub84B -
Flybase) (Zecca and Struhl,
2007) to create clones of Tub1>vg
cells that continuously express moderate levels of exogenous Vg, irrespective
of Wg input. We then generated clones of arr0 cells within
such Tub1>vg clones and asked whether activity of
the 5XQE element still requires Wg input, using expression of a
5XQE-DsRed reporter to monitor 5XQE activity.
Tub1>vg clones make sufficient Vg protein to
rescue expression of both the 5XQE-DsRed and
5XQE>CD2>vg transgenes, as well as wing development, in
vg0 discs (Fig.
1G; data not shown). Nevertheless, arr0 clones
generated within Tub1>vg clones ceased to express
the 5XQE-DsRed transgene (Fig.
3E). We conclude that 5XQE transgene activity does not
merely reflect the presence of Vg protein, but instead depends on Wg input
even when cells are supplied continuously with exogenous Vg. Significantly,
such arr0 clones are subsequently lost from the wing
pouch, within 12 hours after they cease to express the
5XQE-DsRed reporter, despite being independently and continuously
supplied with exogenous Vg (data not shown). Hence, cells within the wing
primordium still require continuous Wg input to survive and grow, even when
they are provided with Vg protein by other means (see Discussion).
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1>vg clones concomitantly in these
same discs (by excision of a Tub1>GFP>vg
transgene) and asked if such exogenous Vg-expressing clones could act
non-autonomously to induce either 5XQE>vg or
5XQE>CD2>vg expression.
Activity of the intact 5XQE>CD2>vg transgene in this
experiment was monitored by CD2 expression and that of the excised
5XQE>vg transgene was monitored by either Vg (data not shown) or
1XQE-lacZ expression (which is robustly expressed in all cells that
express the 5XQE>vg transgene, e.g.
Fig. 3B,
Fig. 4A,
Fig. 6B). Accordingly, the
presence of 5XQE>vg clones can only be visualized if the
experiment gives a positive result: namely, that
Tub1>vg clones (marked by the absence of GFP) can
indeed induce 5XQE>vg expression in neighboring clones of
5XQE>vg cells (as monitored by Vg or 1XQE-lacZ
expression). Nevertheless, we identified many such positively responding
clones (lavender-colored clone in diagram in
Fig. 4A; data not shown).
Importantly, in all cases, these clones were adjacent to
Tub1>vg clones (green clone in diagram,
Fig. 4A). Thus, it appears that
clones of Tub1>vg cells can induce
5XQE>vg expression in neighboring 5XQE>vg clones.
Moreover, induction appears to depend on contact between the two clones.
Significantly, 5XQE>vg expression was not restricted to those
5XQE>vg cells that abut the neighboring
Tub1>vg clone. Instead, 5XQE>vg
expression appeared to spread many cell diameters into the 5XQE>vg
clone, away from the abutting Tub1>vg clone, and
was associated with an expansion of the rescued wing primordium. In addition,
such 5XQE>vg-expressing cells were also able to induce neighboring
5XQE>CD2>vg cells far from the abutting
Tub1>vg clone to express CD2 (yellow cells in
Fig. 4A). Thus, the
5XQE>vg transgene appears to have the capacity not only to respond
to the feed-forward signal, but also to propagate feed-forward signaling from
one cell to the next. As evident in Fig.
4A, the range over which 5XQE>vg cells can induce
5XQE>CD2>vg expression across the clone border is tightly
restricted to only a few cell diameters, consistent with the feed-forward
signal being dependent on cell contact.
In principle, activation of the 5XQE>vg transgene by
feed-forward signaling should require priming by low levels of Vg protein in
the responding cells, and hence is unexpected in vg0
discs. However, the 5XQE>vg cells in this experiment carry the
5XQE>vg as well as the Tub1>GFP>vg
transgene, either one of which could provide cryptic Vg expression and hence
the requisite priming activity. The same explanation also applies to
activation of the 5XQE>CD2>vg transgene by adjacent
5XQE>vg-expressing cells, as even the intact
5XQE>CD2>vg transgene was able to provide a cryptic, priming
activity in other experiments (Fig.
5A,B,E).
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1>vg cells
(Fig. 4), whereas that of the
5XQE>CD2>vg transgene was not (data not shown). We infer that
both the 5XQE>vg and 5XQE>CD2>vg transgenes
initially respond only weakly to feed-forward input from abutting
Tub1>vg cells, but that the initial weak response
of the 5XQE>vg transgene raises the level of Vg protein in these
cells (and hence the strength of the priming input), thereby initiating an
autoregulatory amplification of 5XQE>vg expression induced by the
feed-forward signal. By contrast, 5XQE>CD2>vg cells would lack
the capacity to autoregulate in this way, preventing them from mounting a
robust response to Tub1>vg cells. It is also
notable that the response of 5XQE>CD2>vg cells depended on the
level of Vg expressed in the inducing cells.
Tub1>vg cells express only moderate levels of Vg,
well below peak endogenous levels (Fig.
1F',G'), and were ineffective. However,
5XQE>vg-expressing cells make much higher levels
(Fig. 3C) and were able to
strongly activate 5XQE>CD2>vg expression in abutting cells
(yellow cells in Fig. 4A).
Hence, we infer that 5XQE>vg-expressing cells are more potent
inducers of 5XQE>CD2>vg expression because they provide a
correspondingly stronger feed-forward signal.
Although the experimental design of using the 5XQE>CD2>vg
transgene to generate 5XQE>vg clones has the virtue that it allows
the 5XQE>CD2 response to be assayed in cells outside of the clone,
it suffers from the fact that cells within such clones can only be identified
if they respond positively, by expressing the 5XQE>vg transgene.
We therefore repeated the experiment using an equivalent transgene,
5XQE>Tub1-GFP>vg, which allows all of the
5XQE>vg cells to be scored independently by the loss of a
Tub1-GFP transgene within the Flp-out cassette (see
Materials and methods). We again found that the resulting 5XQE>vg
transgene was only activated in 5XQE>vg clones that abut
Tub1>vg clones (the latter being marked
independently in this experiment by excision of a >DsRed>
cassette; Fig. 4B,C),
confirming the requirement for the Vg-dependent feed-forward signal.
Control of wing growth by the quadrant enhancer
The experiments described above establish that activation of the
5XQE>vg transgene requires both Wg and the Vg-dependent
feed-forward signal. In the following experiments, we use this transgene to
test our hypothesis that feed-forward autoregulation mediated by the
QE is necessary and sufficient for the dramatic expansion of the wing
primordium organized by D-V border cells. To do so, we asked whether the
presence of the 5XQE>vg transgene can rescue wing growth in discs
in which Vg expression is otherwise driven only by the BE.
To generate such `BE-vg-only' wing discs, we used a
BE-vgGFP transgene that expresses a functional Vg-GFP
chimeric protein under the control of a minimal form of the intron 2
BE (Zecca and Struhl,
2007). In otherwise wild-type discs, this transgene behaves like
the standard BE-lacZ reporter gene
(Williams et al., 1994;
Kim et al., 1996), being
expressed in a thin stripe of border cells flanking the D-V compartment
boundary within the wing pouch, and in a broader stripe in the surrounding
hinge and notum primordia (Fig.
3A). In vg0 discs, the
BE-vgGFP transgene was expressed only weakly and
sporadically in D-V border cells within the pouch, affording detectable, but
very limited, rescue of wing development
(Fig. 5A). This minimal
response appeared to reflect a requirement for priming for efficient
activation of the BE-vgGFP transgene, as adding the
rp49-vg transgene significantly enhanced border cell expression of
VgGFP, as well as local rescue of wing development along the D-V
boundary (Fig. 5E; data not
shown).
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These results indicate that the 5XQE>vg transgene is both necessary and sufficient to restore wing growth in discs in which vg expression is otherwise dependent only on BE and cryptic priming activity. However, it is apparent that growth is not fully rescued, as the expansion of wing tissue associated with 5XQE>vg clones was significantly less than the expansion that normally occurs following D-V segregation in wild-type discs. One explanation is that both BE- and QE-driven expression of Vg are compromised by inadequate Vg priming that derives from the BE-vgGFP and 5XQE>vg transgenes (the only possible sources of pre-existing Vg activity in the 5XQE>vg clones). We therefore repeated the experiment in the presence of the rp49-vg transgene, to ensure adequate priming in all cells.
In the absence of 5XQE>vg clones, vg0 BE-vgGFP discs carrying the rp49-vg transgene, as well as the 5XQE>CD2>vg and 1XQE-lacZ transgenes, showed robust expression of the BE-vgGFP transgene in a narrow stripe of D-V border cells, and this was accompanied by weak expression of both the 5XQE>CD2>vg and 1XQE-lacZ transgenes in flanking cells (Fig. 5F). We note that in these discs, as well as in otherwise wild-type discs, both the BE-vgGFP and standard BE-lacZ transgenes were expressed at a low level in cells up to several cell diameters away from the D-V boundary (data not shown). Hence, the weak 5XQE>CD2>vg and 1XQE-lacZ expression detected in cells flanking the D-V boundary might reflect a response to this low-level VgGFP activity.
Strikingly, when clones of 5XQE>vg cells were generated in this
background, near or next to the D-V compartment boundary, they were associated
with an autonomous upregulation of the 1XQE-lacZ transgene and a
dramatic expansion of prospective wing tissue
(Fig. 5G). Furthermore, they
appeared to induce an equally dramatic, albeit short-range, induction of CD2
expression in neighboring 5XQE>CD2>vg cells (arrows in
Fig. 5G). Corresponding
experiments using the 5XQE>Tub1-GFP>vg
transgene instead of 5XQE>CD2>vg confirmed the rescue of wing
growth and also showed that it is an autonomous property of the
5XQE>vg clones (marked independently by the absence of GFP;
Fig. 5D,H).
|
Regulation of wing growth by the quadrant enhancer
If wing growth is governed by the capacity of QE sequences to
mediate vg feed-forward autoregulation, the size of the wing
primordium should depend on the strength of the QE response. To test
this, we assayed the effects of 5XQE>vg clones on wing growth in
otherwise wild-type discs, where they appear to generate a more sensitive and
potent upregulation of Vg expression driven by the combined
QE-dependent activities of the transgene and endogenous
vg.
Early-induced 5XQE>vg clones were associated with an abnormal,
cell-autonomous expansion of prospective wing tissue, extending beyond the
normal limit of detectable Vg expression into the surrounding `rotund
(rn)-only' territory of the wing pouch delimited by the inner ring of
Wg expression (Fig. 6A-C) (see
Zecca and Struhl, 2007). These
clones also induced adjacent 5XQE>CD2>vg cells that abutted the
abnormally expanded wing primordium to ectopically express CD2
(Fig. 6A,B). Note that these
CD2-expressing cells did not appear to express either Vg or
1XQE-lacZ, suggesting that the 5XQE transgene has a greater
capacity to respond to one or more of its normal inputs than either endogenous
vg or the 1XQE-lacZ transgene. Taken together, these results
suggest that strengthening and/or sensitizing the QE response by
introducing the 5XQE>vg transgene causes an enhanced expansion of
wing tissue.
We note that even though 5XQE>vg clones formed abnormally enlarged domains of wing tissue, other elements of wing pattern were not similarly expanded within the clones. In particular, the domain of Distal-less (Dll) expression, which normally depends on Wg but is less broad than that of vg, remained unaltered in such clones (Fig. 6D). However, the Dll domain could be expanded in response to ectopic Wg (Fig. 6E). It follows that the effects of Wg on wing size (via control of vg expression) can be dissociated from its effect on wing pattern (via control of other target genes such as Dll).
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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200-fold expansion
of the population of cells expressing the wing selector gene vg,
under the control of the long-range morphogens Wg and Dpp. This system thus
poses the fundamental question of how morphogens organize the increase in the
mass and number of cells that express a given selector gene, to yield an adult
appendage of appropriate size and shape. In the accompanying paper, we defined a novel autoregulatory property of vg that appears crucial for this process. We presented evidence that vg-expressing cells send a short-range feed-forward signal that neighboring cells must receive in order to express vg in response to Wg. This led us to hypothesize that Wg controls wing development by fueling this non-autonomous autoregulatory mechanism. Here, we establish that the vg quadrant enhancer (QE) can mediate vg autoregulation in response to Wg and then use a transgene that expresses Vg under QE control to provide a proof-in-principle that wing growth normally depends on the operation of the autoregulatory circuit.
vg autoregulation and expansion of the wing primordium in response to Wg
As illustrated in Fig. 7, we
envisage wing growth following D-V segregation as an outcome of vg
autoregulation, primed by cryptic, low-level Vg in all cells that is seeded by
DSL-Notch-mediated induction of specialized D-V border cells that express high
levels of vg and wg, and then propagated by the capacity of
vg-expressing cells to induce and sustain vg expression in
neighboring cells in response to Wg. In support, we have been able to restore
wing growth in vg0 discs in a step-wise manner by the
sequential addition of transgenes that provide, first priming
(rp49-vg), then initiation (BE-vgGFP), and
finally feed-forward propagation (5XQE>vg). As we observe
(Fig. 1E,
Fig. 5), priming is necessary
but not sufficient for wing development, initiation provides local rescue of
wing tissue, and propagation is responsible for the dramatic expansion in the
size of the prospective wing.
|
The self-reinforcing nature of this autoregulatory circuit, both between and within cells, helps explain how Wg spreading from D-V border cells normally fuels the expansion of the population of vg-expressing cells. It also helps account for the unexpected responses we observed in experiments using the rp49-vg, BE-vgGFP and 5XQE>vg transgenes to mimic the normal priming, initiation and feed-forward inputs (Figs 4,5). All of these transgenes depend on heterologous promoters and potentially complex enhancer elements operating outside of their normal genomic contexts. Consequently, weak, inappropriate activities of any of these transgenes (e.g. cryptic priming by BE-vgGFP and 5XQE>vg transgenes, or faint QE activity of the BE-vgGFP transgene) could be amplified by the autoregulatory circuitry, yielding spatially inappropriate responses. Nevertheless, despite these experimental limitations, our results indicate that the major factor governing the expansion of the wing primordium is feed-forward autoregulation mediated by the QE.
As discussed in the accompanying paper
(Zecca and Struhl, 2007), wing
growth does not depend solely on the capacity of Wg to recruit and maintain
cells in the wing primordium by fueling vg autoregulation. Instead,
we show here that even when wing pouch cells are supplied constitutively with
exogenous Vg (thus bypassing the requirement for vg autoregulation),
they still depend on continuous Wg input to survive and grow within the
context of the wing primordium (Fig.
3) (see also Johnston and
Sanders, 2003). This is in contrast to cells in the more proximal
hinge and notum primordia, which survive and grow without Wg input
(Chen and Struhl, 1999;
Giraldez and Cohen, 2003).
Thus, Wg appears to promote wing growth via two distinct mechanisms: by
continuously `selecting' which cells enter and remain within the wing
primordium, and by allowing the survival and growth of cells so selected. We
cannot, at present, distinguish the relative contributions of these two
mechanisms. However, as we show here, both appear essential, as cells fail to
enter, or stay, within the wing primordium when either one is eliminated.
Dpp and feed-forward autoregulation of Vg
Wing growth depends not only on Wg emanating from D-V border cells, but
also on Dpp secreted by A compartment cells along the A-P compartment boundary
(Zecca et al., 1995;
Burke and Basler, 1996;
Lecuit et al., 1996;
Nellen et al., 1996;
Zecca et al., 1996;
Neumann and Cohen, 1997),
suggesting that the QE might mediate feed-forward autoregulation in
response to Dpp, as well as Wg. In support, the QE contains binding
sites for the Dpp transducer Mad, and there is evidence that these sites, as
well as Mad itself, contribute to QE activity
(Kim et al., 1997;
Halder et al., 1998;
Guss et al., 2001). Moreover,
clones of cells that cannot transduce Dpp behave like those that cannot
transduce Wg: they cease to express Vg and are lost specifically from the wing
primordium, in contrast to clones located in the more proximal hinge and notum
primordia (Burke and Basler,
1996; Martin-Castellanos and
Edgar, 2002; Moreno et al.,
2002; Gibson and Perrimon,
2005; Shen and Dahmann,
2005). Hence, we think it likely that Dpp and Wg act together to
fuel the feed-forward autoregulatory circuit, and by so doing, regulate the
size and shape of the developing wing.
Morphogen gradients and organ growth
The ability of Wg, and potentially Dpp, to promote wing growth by fueling a
non-autonomous autoregulatory circuit of vg expression is, to our
knowledge, novel, and has implications for the control of organ growth by
morphogens. As epitomized by the developing wing, a long-standing enigma is
that gradient morphogens drive relatively uniform growth and proliferation
across a tissue at the same time that they function in a
concentration-dependent manner to organize complex patterns of gene expression
and cell differentiation (Garcia-Bellido
and Merriam, 1971; Milan et
al., 1996; Resino et al.,
2002). We suggest that a minimum threshold level of morphogen
might be sufficient to fuel both feed-forward autoregulation of organ selector
genes and the growth and proliferation of cells so selected. Accordingly, as
illustrated in Fig. 7, organ
growth would be governed primarily by the progressive expansion in the range
of morphogen (a process that might itself depend on the ability of morphogen
to regulate expression of its receptors and other binding proteins) and by any
boundary conditions that limit the availability and capacity of surrounding
cells to respond.
|
ACKNOWLEDGMENTS |
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|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Blair, S. S., Brower, D. L., Thomas, J. B. and Zavortink, M. (1994). The role of apterous in the control of dorsoventral compartmentalization and PS integrin gene expression in the developing wing of Drosophila. Development 120,1805 -1815.[Abstract]
Burke, R. and Basler, K. (1996). Dpp receptors are autonomously required for cell proliferation in the entire developing Drosophila wing. Development 122,2261 -2269.[Abstract]
Casali, A. and Struhl, G. (2004). Reading the Hedgehog morphogen gradient by measuring the ratio of bound to unbound Patched protein. Nature 431,76 -80.[CrossRef][Medline]
Chen, C. M. and Struhl, G. (1999). Wingless transduction by the Frizzled and Frizzled2 proteins of Drosophila. Development 126,5441 -5452.[Abstract]
Couso, J. P., Knust, E. and Martinez Arias, A. (1995). Serrate and wingless cooperate to induce vestigial gene expression and wing formation in Drosophila. Curr. Biol. 5,1437 -1448.[CrossRef][Medline]
de Celis, J. F., Garcia-Bellido, A. and Bray, S. J. (1996). Activation and function of Notch at the dorsal-ventral boundary of the wing imaginal disc. Development 122,359 -369.[Abstract]
Diaz-Benjumea, F. J. and Cohen, S. M. (1993). Interaction between dorsal and ventral cells in the imaginal disc directs wing development in Drosophila. Cell 75,741 -752.[CrossRef][Medline]
Diaz-Benjumea, F. J. and Cohen, S. M. (1995). Serrate signals through Notch to establish a Wingless-dependent organizer at the dorsal/ventral compartment boundary of the Drosophila wing. Development 121,4215 -4225.[Abstract]
Doherty, D., Feger, G., Younger-Shepherd, S., Jan, L. Y. and
Jan, Y. N. (1996). Delta is a ventral to dorsal signal
complementary to Serrate, another Notch ligand, in Drosophila wing formation.
Genes Dev. 10,421
-434.
Garcia-Bellido, A. and Merriam, J. R. (1971). Parameters of the wing imaginal disc development of Drosophila melanogaster. Dev. Biol. 24,61 -87.[CrossRef][Medline]
Gibson, M. C. and Perrimon, N. (2005).
Extrusion and death of DPP/BMP-compromised epithelial cells in the developing
Drosophila wing. Science
307,1785
-1789.
Giraldez, A. J. and Cohen, S. M. (2003).
Wingless and Notch signaling provide cell survival cues and control cell
proliferation during wing development. Development
130,6533
-6543.
Golic, K. G. (1991). Site-specific
recombination between homologous chromosomes in Drosophila.
Science 252,958
-961.
Greenwood, S. and Struhl, G. (1997). Different levels of Ras activity can specify distinct transcriptional and morphological consequences in early Drosophila embryos. Development 124,4879 -4886.[Abstract]
Guss, K. A., Nelson, C. E., Hudson, A., Kraus, M. E. and
Carroll, S. B. (2001). Control of a genetic regulatory
network by a selector gene. Science
292,1164
-1167.
Halder, G. and Carroll, S. B. (2001). Binding
of the Vestigial co-factor switches the DNA-target selectivity of the
Scalloped selector protein. Development
128,3295
-3305.
Halder, G., Polaczyk, P., Kraus, M. E., Hudson, A., Kim, J.,
Laughon, A. and Carroll, S. (1998). The Vestigial and
Scalloped proteins act together to directly regulate wing-specific gene
expression in Drosophila. Genes Dev.
12,3900
-3909.
Johnston, L. A. and Sanders, A. L. (2003). Wingless promotes cell survival but constrains growth during Drosophila wing development. Nat. Cell Biol. 5, 827-833.[CrossRef][Medline]
Kim, J., Irvine, K. D. and Carroll, S. B. (1995). Cell recognition, signal induction, and symmetrical gene activation at the dorsal-ventral boundary of the developing Drosophila wing. Cell 82,795 -802.[CrossRef][Medline]
Kim, J., Sebring, A., Esch, J. J., Kraus, M. E., Vorwerk, K., Magee, J. and Carroll, S. B. (1996). Integration of positional signals and regulation of wing formation and identity by Drosophila vestigial gene. Nature 382,133 -138.[CrossRef][Medline]
Kim, J., Johnson, K., Chen, H. J., Carroll, S. and Laughon, A. (1997). Drosophila Mad binds to DNA and directly mediates activation of vestigial by Decapentaplegic. Nature 388,304 -308.[CrossRef][Medline]
Lecuit, T., Brook, W. J., Ng, M., Calleja, M., Sun, H. and Cohen, S. M. (1996). Two distinct mechanisms for long-range patterning by Decapentaplegic in the Drosophila wing. Nature 381,387 -393.[CrossRef][Medline]
Liu, X., Grammont, M. and Irvine, K. D. (2000). Roles for scalloped and vestigial in regulating cell affinity and interactions between the wing blade and the wing hinge. Dev. Biol. 228,287 -303.[CrossRef][Medline]
Martin-Castellanos, C. and Edgar, B. A. (2002). A characterization of the effects of Dpp signaling on cell growth and proliferation in the Drosophila wing. Development 129,1003 -1013.[Medline]
Milan, M., Campuzano, S. and Garcia-Bellido, A.
(1996). Cell cycling and patterned cell proliferation in the
Drosophila wing during metamorphosis. Proc. Natl. Acad. Sci.
USA 93,11687
-11692.
Morata, G. and Ripoll, P. (1975). Minutes: mutants of Drosophila autonomously affecting cell division rate. Dev. Biol. 42,211 -221.[CrossRef][Medline]
Moreno, E., Basler, K. and Morata, G. (2002). Cells compete for decapentaplegic survival factor to prevent apoptosis in Drosophila wing development. Nature 416,755 -759.[CrossRef][Medline]
Nellen, D., Burke, R., Struhl, G. and Basler, K. (1996). Direct and long-range action of a DPP morphogen gradient. Cell 85,357 -368.[CrossRef][Medline]
Neumann, C. J. and Cohen, S. M. (1996). A hierarchy of cross-regulation involving Notch, wingless, vestigial and cut organizes the dorsal/ventral axis of the Drosophila wing. Development 122,3477 -3485.[Abstract]
Neumann, C. J. and Cohen, S. M. (1997). Long-range action of Wingless organizes the dorsal-ventral axis of the Drosophila wing. Development 124,871 -880.[Abstract]
Paumard-Rigal, S., Zider, A., Vaudin, P. and Silber, J. (1998). Specific interactions between vestigial and scalloped are required to promote wing tissue proliferation in Drosophila melanogaster. Dev. Genes Evol. 208,440 -446.[CrossRef][Medline]
Resino, J., Salama-Cohen, P. and Garcia-Bellido, A.
(2002). Determining the role of patterned cell proliferation in
the shape and size of the Drosophila wing. Proc. Natl. Acad. Sci.
USA 99,7502
-7507.
Rulifson, E. J., Micchelli, C. A., Axelrod, J. D., Perrimon, N. and Blair, S. S. (1996). wingless refines its own expression domain on the Drosophila wing margin. Nature 384, 72-74.[CrossRef][Medline]
Shen, J. and Dahmann, C. (2005). Extrusion of cells with inappropriate Dpp signaling from Drosophila wing disc epithelia. Science 307,1789 -1790.
