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First published online September 1, 2004
doi: 10.1242/10.1242/dev.01315
Howard Hughes Medical Institute, Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers The State University of New Jersey, Piscataway, NJ 08854, USA
* Author for correspondence (e-mail: irvine{at}waksman.rutgers.edu)
Accepted 15 June 2004
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SUMMARY |
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 TOP SUMMARY IntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Key words: Fat, Cadherin, Limb, Growth, Drosophila
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Introduction |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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;
Irvine and Rauskolb, 2001;
Klein, 2001). Studies of
signaling along the AP and DV axes have established paradigms for tissue
patterning, and have also been instrumental in the identification of many key
components of the Hedgehog, Notch, Wingless (WG; Wnt) and Decapentaplegic
(DPP; TGF-ß) signaling pathways. More recently, it has become clear that
normal wing development is also dependent upon signaling along the
proximodistal (PD) axis (Liu et al.,
2000; del Álamo
Rodriguez et al., 2002; Kolzer
et al., 2003), but the identity of the genes that actually effect
signaling along this axis remains unknown.
There is a progressive elaboration of patterning along the PD axis over the
course of wing development (reviewed by
Klein, 2001). During the
second larval instar, interactions among the Epidermal Growth Factor Receptor,
DPP and WG signaling pathways divide the wing disc into a dorsal region, which
will give rise to notum, and a ventral region, from which the wing will arise
(Ng et al., 1996;
Baonza et al., 2000;
Wang et al., 2000;
Cavodeassi et al., 2002;
Zecca and Struhl, 2002). An
initial PD subdivision of the wing is then effected by signaling from the AP
and DV compartment boundaries, which promotes the expression of two genes,
scalloped and vestigial, that encode subunits of a
heterodimeric transcription factor (SD-VG) in the center of the wing
(Kim et al., 1995;
Kim et al., 1996;
Zecca et al., 1996;
Neumann and Cohen, 1997;
Halder et al., 1998;
Klein and Martinez Arias,
1998; Simmonds et al.,
1998). This subdivides the wing into distal cells, which give rise
to the wing blade, and surrounding cells, which give rise to proximal wing and
wing hinge structures (Fig. 1) (Kim et al., 1996;
Klein and Martinez Arias,
1998; Azpiazu and Morata,
2000; Casares and Mann,
2000; Liu et al.,
2000). The proximal wing is further subdivided into a series of
molecularly distinct domains (Fig.
1B). Studies of SD-VG function in the wing led to the realization
that the elaboration of this finer pattern depends in part upon signaling from
the distal, SD-VG-expressing cells, to more proximal cells
(Liu et al., 2000). Thus,
mutation of vg leads to elimination, not only of the wing blade,
where VG is expressed, but also of more proximal tissue
(Williams et al., 1991;
Williams et al., 1993;
Klein and Martinez Arias,
1998; Liu et al.,
2000). Conversely, ectopic expression of VG in the proximal wing
reorganizes the patterning of surrounding cells
(Liu et al., 2000;
del Álamo Rodriguez et al.,
2002; Kolzer et al.,
2003).
|
;
del Álamo Rodriguez et al.,
2002), and which later becomes expressed in a second, more
proximal ring (Fig. 1). WG
expression in the inner, distal ring within the proximal wing is regulated by
an enhancer called spade-flag (spd-fg), after an allele of
wg in which this enhancer is deleted
(Neumann and Cohen, 1996).
Studies of this allele, together with ectopic expression experiments, revealed
that WG is necessary and sufficient to promote growth of the proximal wing
(Neumann and Cohen, 1996;
Klein and Martinez Arias,
1998). WG also plays a role in proximal wing patterning, as it
acts in a positive-feedback loop to maintain expression of Homothorax (HTH)
(Azpiazu and Morata, 2000;
Casares and Mann, 2000;
del Álamo Rodriguez et al.,
2002). The rotund (rn) gene has been identified
as an additional target of distal signaling
(del Álamo Rodriguez et al.,
2002).
In this work, we identify Four-jointed (FJ), Dachsous (DS), Fat and Dachs
as proteins that influence signaling to proximal wing cells to regulate WG and
rn expression. FJ is a type II transmembrane protein, which is
largely restricted to the Golgi (Villano
and Katz, 1995; Brodsky and
Steller, 1996; Buckles et al.,
2001; Strutt et al.,
2004). Null mutations in fj do not cause any obvious
defects in the proximal wing (Villano and
Katz, 1995; Brodsky and
Steller, 1996). However, fj plays a role in the
regulation of tissue polarity, yet acts redundantly with some other factor(s)
in this process (Zeidler et al.,
1999; Zeidler et al.,
2000; Casal et al.,
2002). Mutations in fat or ds can also influence
tissue polarity (Adler et al.,
1998; Casal et al.,
2002; Rawls et al.,
2002; Strutt and Strutt,
2002; Yang et al.,
2002; Ma et al.,
2003), and both genes encode large protocadherins
(Mahoney et al., 1991;
Clark et al., 1995). Although
the molecular relationships among these proteins are not well understood,
genetic studies suggest that fj and ds act via effects on
fat, and both fj and ds can influence Fat
localization in genetic mosaics (Strutt
and Strutt, 2002; Yang et al.,
2002; Ma et al.,
2003).
Interestingly, alleles of fj, ds and fat, as well as
alleles of another gene, dachs, can result in similar defects in wing
blade and leg growth (Mohr,
1923; Waddington,
1943). The similar requirements for these genes during both
appendage growth and tissue polarity, together with the expression patterns of
fj and ds in the developing wing, led us to investigate
their requirements for proximal wing development. We find that all four genes
influence the expression of WG in the proximal wing, and genetic experiments
suggest a pathway in which FJ and DS act to modulate the activity of Fat,
which then regulates transcription via a pathway that includes Dachs. Our
observations lend strong support to the hypothesis that FJ, DS and Fat
function as components of an intercellular signal transduction pathway,
implicate Dachs as a key downstream component of this pathway, and identify a
normal role for these genes in proximodistal patterning during
Drosophila wing development.
|
Materials and methods |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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),
fat8 (fatfd) and
fatG-rv (Bryant et al.,
1988; Mahoney et al.,
1991), dsUA071 and ds38k
(Clark et al., 1995;
Adler et al., 1998),
d210 and d1
(Buckles et al., 2001),
wgspd-fg (Couso et
al., 1994), vg83b27r
(Williams et al., 1990) and
sd58 (Campbell et al.,
1991). fat8 d1, fat8
vg83b27r, fatG-rv wgspd-fg and
fat8 wgspd-fg double mutant chromosomes were
generated by meiotic recombination; a fjd1
dsUA071 chromosome was a gift of D. Strutt
(Strutt et al., 2004). UAS and
Gal4 transgenes used were: UAS-vg[49]
(Kim et al., 1996);
UAS-fj[6a.2] and UAS-fj[146.3]
(Zeidler et al., 1999);
UAS-GFP, AyGal4[17b] and AyGal4[5a] (actin>y+>Gal4)
(Ito et al., 1997); and
GS-ds. GS-ds contains an insertion of the UAS-containing Gene Search
transposon (Toba et al.,
1999), approximately 700 bp upstream of ds (flanking
sequence includes GTGTACAGTGAAGTGCGGAAAAGAGGTCGAGGGG), and was isolated in a
gain-of-function screen for genes that influence cell affinity in the wing (O.
Dunaevsky, C. Rauskolb and K.D.I., unpublished). Its influence on ds
expression was confirmed by in situ hybridization. Reporter genes employed
were spd-lacZ (Neumann and Cohen,
1996), fj-lacZ[P1]
(Brodsky and Steller, 1996),
rn-lacZ (St Pierre et al.,
2002) and ds-lacZ
(Clark et al., 1995). Mutant
clones were generated by mitotic recombination, and marked by the absence of a
MYC-tagged protein or GFP. They were induced at 24-48 and at 48-72 hours after
egg laying (AEL), and then allowed to grow for 48 or 72 hours, using the
following stocks:
-Myc] FRT40A M -Myc] FRT40A :MYC} FRT18A; hs-FLP Sb/TM6b
Clones of cells ectopically expressing genes of interest (Flip-out) were
generated by combining transgenes that provide expression under UAS control
with transgenes that allow the generation of clones of cells expressing the
Gal4 protein (AyGal4) (Ito et al.,
1997). Gal4-expressing clones were marked using a UAS-GFP
transgene. Flip-out clones were induced at 24-48 and 48-72 hours AEL.
Immunostaining
Imaginal discs from third instar larvae were fixed and stained as
previously described (Liu et al.,
2000), using as primary antibodies: rabbit anti-VG (1:600, S.
Carroll, University of Wisconsin-Madison), mouse anti-WG 4D4 (1:1000,
Developmental Studies Hybridoma Bank), mouse anti-NUB (1:100, S. Cohen,
European Molecular Biology Laboratory), rabbit anti-ß-gal (1:2000, ICN),
Goat anti-ß-gal (1:1000, Biogenesis), mouse anti-ß-gal (1:1000,
Sigma), rabbit anti-MYC (1:100, Santa Cruz), rat anti-MYC (1:1000, Serotec),
rat anti-DS (1:200, M. Simon, Stanford University) and rat anti-Fat (1:100, H.
McNeill, Cancer Research UK). For precise timing, larvae were collected in
intervals after the second to third instar molt, but in some cases ages were
estimated based on the size and morphology of the disc.
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Results |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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).
However, the expression and phenotypes of fj and ds
(described below), together with prior studies suggesting a functional
relationship among these genes, suggested that Fat might have a role in
proximal wing development. Indeed, the two known targets of distal signaling,
WG and rn, are both strongly upregulated within fat mutant
clones in the proximal wing (Fig.
2). The influence of fat on rn and WG is
strictly cell autonomous. This autonomous action suggests that Fat is not
involved in sending a signal to proximal wing cells, but might be regulated by
signals from neighboring cells.
|
;
del Álamo Rodriguez et al.,
2002). These observations suggest that the VG-dependent signal
might activate WG expression by inhibiting Fat activity.
To further examine this possibility, we analyzed vg fat double
mutants. vg mutant wing discs contain a single ring of WG expression,
which, based on the NUB expression domain, appears to correspond to the outer
WG ring (Fig. 3A)
(Liu et al., 2000). Expression
of WG in the inner ring, which normally overlaps NUB, is either not detected
(8/14 discs), or is reduced to a small central spot (6/14 discs) in
vg mutants (Fig. 3A).
Importantly, in vg fat double mutants, WG expression is always
observed in the center of the disc (7/7 discs), and this expression is
substantially enlarged (Fig.
3B). The observation that mutation of fat can promote WG
expression even in the absence of VG is consistent with the hypothesis that
Fat is normally repressed downstream of a VG-dependent signal.
|
; Mahoney et al.,
1991; Garoia et al.,
2000). Although fat can influence the growth of most, and
perhaps all imaginal cells, examination of fat mutant wing discs
nonetheless indicates that there is a disproportionate overgrowth of the
proximal wing, which is particularly evident in older larvae
(Bryant et al., 1988;
Garoia et al., 2000)
(Fig. 4B,G). The observation
that mutation of fat results in ectopic WG expression in the proximal
wing (Fig. 2), together with
the knowledge that ectopic WG expression promotes overproliferation of
proximal wing tissue (Neumann and Cohen,
1996), suggested that the disproportionate overgrowth of the
proximal wing in fat mutants might be due to ectopic expression of
WG. To test this possibility, we recombined fat alleles with the
wgspd-fg allele. Indeed, disproportionate overgrowth of
the proximal wing disc is suppressed in fat mutant animals that also
carry wgspd-fg (Fig.
4D,H; compare with Fig.
4B,G). Thus, two distinct processes contribute to overgrowth in
fat mutant wing discs: a broad-based process that results in
enlargement of the entire disc, and a local upregulation of WG in the proximal
wing. This latter process emphasizes the importance of WG regulation by
fat to normal wing development.
|
;
Brodsky and Steller, 1996). In
order to investigate FJ as a potential contributor to distal-to-proximal
signaling, we first confirmed that its expression is similar to VG throughout
the third instar (Fig. 5A,C).
The only significant difference observed was that along the DV boundary,
expression of VG remains at high levels outside of the distal wing, whereas
fj expression drops to lower levels
(Fig. 5C). At early third
instar, WG expression is directly adjacent to fj and VG, but as the
wing grows they separate (Fig.
5E-G) (Klein and Martinez
Arias, 1998; del Álamo
Rodriguez et al., 2002; Kolzer
et al., 2003). Because SD-VG is required for the growth and
viability of wing cells, clones of cells that are mutant for null alleles of
sd or vg fail to proliferate and die
(Kim et al., 1996;
Liu et al., 2000). However, in
clones of cells mutant for a hypomorphic allele of sd,
sd58, a reduction in fj expression is detectable
(Fig. 6A).
|
|
). However, in the wing, ectopic expression of FJ does not
result in a detectable induction of fj expression
(Fig. 7A), nor does ectopic
expression of fj exert any detectable influence on VG expression
(Fig. 7B). Although the
mechanism by which FJ becomes induced non-autonomously is not known, our
results nonetheless indicate that FJ is a normal downstream target of VG in
the distal wing, and that it is expressed in association with ectopic VG in
the proximal wing.
|
;
del Álamo Rodriguez et al.,
2002). Despite these differences, the spd-fg enhancer
also responds to FJ, both targets of distal signaling, rn and WG, are
similarly affected by FJ, and ectopic FJ is only able to modulate WG and
rn expression within the NUB-expressing cells in the distal half of
the proximal wing (Fig. 7). The
observations that FJ regulates the same genes, in the same place, and through
the same enhancer as VG suggest that FJ contributes to signaling from distal
cells. Similar reasoning (above) suggests that the distal signal acts through
Fat, and FJ has been suggested to influence Fat in regulating tissue polarity
(Strutt and Strutt, 2002;
Yang et al., 2002;
Ma et al., 2003). Together
then, these observations imply that FJ influences WG and rn
expression by modulating Fat activity, and, consistent with this, ectopic FJ
expression also modulates Fat protein staining in the developing wing
(Fig. 7G).
Mutation of fj impairs the initiation of WG expression in the proximal wing
The proximal wing appears normal in fj null mutant animals
(Villano and Katz, 1995;
Brodsky and Steller, 1996).
Thus, if FJ contributes to distal signaling, it must do so redundantly.
Nonetheless, we considered the possibility that some reduction in WG
expression might be detectable in fj mutants. In order to enhance our
ability to detect subtle changes, WG was examined in fj genetic
mosaics. In this situation, regions of the disc composed of wild-type cells
provide an internal control for normal levels of staining. Importantly, at
early to mid third instar, WG expression in the distal ring was reduced in
cells adjacent to fj mutant distal wing cells
(Fig. 8A,B; 10/13 early discs
with clones had detectable alterations in WG expression). WG expression was
never completely eliminated, consistent with notion that FJ contributes to,
but is not absolutely required for, WG expression. The influence of FJ on WG
expression depended on the genotype of distal wing cells rather than proximal
wing cells (Fig. 8A,B),
consistent with the fj expression pattern
(Fig. 5). Intriguingly, WG
expression sometimes (7/32 clone edges) also appeared elevated in mutant cells
immediately adjacent to wild-type cells
(Fig. 8A). The altered
expression of WG indicates that FJ contributes to normal distal signaling, but
is not solely responsible for it.
|
Expression of dachsous during wing development
Studies of tissue polarity suggest a close functional relationship among
fj, fat and ds. ds is expressed preferentially by proximal
wing cells (Clark et al.,
1995), but low levels have been reported in more distal cells
(Strutt and Strutt, 2002;
Ma et al., 2003). During third
instar, both DS protein expression and ds transcription, as detected
by a lacZ enhancer trap line, appear graded, with the highest levels
in proximal wing cells and the lowest levels in distal wing cells
(Fig. 5B,D). When the inner
ring of WG expression is first detected, at early third instar, it appears on
the slope of DS expression, with the highest levels of DS more proximal, and
the lowest levels of DS more distal.
dachsous influences WG expression in the proximal wing
Neither ds mutant discs (not shown), nor fj ds double
mutant discs (Fig. 3C), exhibit
obvious changes in WG expression, nor do they display the overgrowths of wing
tissue observed in fat mutants. Nonetheless, clones of cells mutant
for a strong ds allele, dsUA071, can exert a
subtle influence on WG expression. At early third instar, this influence is
most often detected as a slight decrease in WG within ds mutant
cells, and a slight increase in WG in wild-type cells that border the clone
(18/37 early to mid third instar clones revealed this effect)
(Fig. 9A), although in some
cases (8/37) WG expression appeared slightly elevated within mutant cells. At
late third instar a slight increase in WG expression is most often (19/35
clones) observed within ds mutant cells
(Fig. 9B), and a decrease in WG
expression is only rarely (3/35 clones) observed. Similarly, at early to mid
third instar, ectopic expression of DS was often (12/23 cases) associated with
upregulation of WG within DS-expressing cells at the edge of clones
(Fig. 9C), although
occasionally (4/23 cases) WG was upregulated in neighboring cells
(Fig. 9D). At late stages
elevation of WG expression in neighboring cells was observed (12/12 cases).
Although the influence of DS is complex (see Discussion), its ability to
modulate WG expression in the proximal wing is consistent with the suggestion
that it can influence Fat activity. It has been reported previously that Fat
localization is altered by ds mutant clones
(Strutt and Strutt, 2002;
Ma et al., 2003), and we find
that clones of cells ectopically expressing DS can also influence Fat
localization (Fig. 9F). To
investigate possible interactions between ds and fj, we also
examined clones of cells co-expressing both genes. These are associated with
non-autonomous upregulation of WG at all stages
(Fig. 9E).
|
). To investigate whether it functions in distal-to-proximal
signaling, we examined WG expression in gug35 mutant
clones in the wing. Although the clones exhibited other defects consistent
with previously described roles for gug
(Erkner et al., 2002;
Zhang et al., 2002), no
influence on WG expression in the proximal wing was detected
(Fig. 10A).
|
; Buckles et al.,
2001). To determine whether dachs also influences
distal-to-proximal signaling, we first attempted to generate clones mutant for
a strong allele (d210), but were unable to recover any
mutant clones, even when we gave them a growth advantage by using the
Minute technique. As an alternative, we examined clones mutant for a
hypomorphic allele of dachs, d1. When examined at early
stages of wing development, these clones are always (7/7 clones) associated
with a dramatic reduction of WG expression in the proximal wing
(Fig. 10B). The reduction in
WG expression is cell autonomous, suggesting that dachs is required
for receiving, rather than sending, the distal signal. Intriguingly however,
later in third instar, WG expression partially recovers within dachs
mutant clones (17 clones, the older the disc the more normal WG staining
appears) (Fig. 10C). This
recovery suggests again that WG expression in the hinge is regulated by
distinct initiation and maintenance mechanisms.
dachs is epistatic to fat
dachs has recently been found to encode an unconventional myosin
(F. Katz, personal communication), and thus is presumably a cytoplasmic
protein. The autonomous influence of dachs on WG expression, together
with its presumed cytoplasmic location, suggested that it might act downstream
of fat. Since mutation of fat and mutation of dachs
have opposing effects on WG, this possibility could be tested genetically. In
d1 fat8 double mutant clones, the influence of
dachs on WG expression is epistatic, as clones in early third instar
discs exhibit the same reduction in WG expression that is observed in
d1 mutant clones (12/12 clones)
(Fig. 10D). At later stages,
WG expression partially recovers (18 clones), but at no time do the clones
exhibit significant ectopic WG expression
(Fig. 10E). Interestingly, the
dachs phenotype is also epistatic for the growth effects of
fat, as the overgrowth phenotype of fat mutant discs is
partially suppressed in animals that are also heterozygous for
d1 (data not shown), and completely suppressed in animals
that are homozygous for d1
(Fig. 4F).
|
Discussion |
|---|
|
TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
|---|
;
Klein, 2001). However,
important aspects of wing patterning rely on signaling from distal cells to
proximal cells. In this work, we have identified a set of genes that influence
this process, and provide genetic evidence that in doing so they act as
components of an intercellular signal transduction pathway. Studies described
here and elsewhere suggest that Fat functions as a key component in this
pathway, which is regulated by FJ and DS, and which then modulates
transcription via intracellular pathways that include Grunge and/or Dachs
(Fig. 11A).
|
;
Yang et al., 2002;
Ma et al., 2003) (Figs
7,
9). The possibility that they
act as components of an intercellular signaling pathway has been suggested
based on studies of tissue polarity, but, at the same time, the nature of
tissue polarity has complicated attempts to assign distinct roles for these
genes in signaling versus receiving cells. Particularly important then, is the
identification of transcriptional outputs of Fat signaling. We have identified
here two genes, wg and rn, that are influenced
non-autonomously by FJ and cell autonomously by Fat in the proximal wing.
Similarly, expression of fj itself is influenced non-autonomously by
FJ (Zeidler et al., 1999) and
cell autonomously by Fat (Yang et al.,
2002) in the eye, and Serrate expression is influenced
non-autonomously by FJ (Buckles et al.,
2001) and cell autonomously by Fat (E.C. and K.D.I., unpublished)
in the leg. The observation that four different genes in three different
tissues are each influenced non-autonomously by FJ and cell autonomously by
Fat suggests strongly that FJ and Fat have common roles on the sending and
receiving sides, respectively, of a broadly deployed intercellular signaling
pathway. The identification of a normal developmental event in which one
population of cells (distal wing) signals to adjacent cells (proximal wing)
via these genes adds further support to this argument, and, at the same time,
provides a developmental context for further identifying and characterizing
roles of pathway components.
Regulation of Fat activity
The common feature of all of our manipulations of FJ and DS expression is
that WG expression, and by inference, Fat activity, can be altered when cells
with different levels of FJ or DS are juxtaposed. In the case of FJ, its
normal expression pattern, mutant clones and ectopic expression clones are all
consistent with the interpretation that juxtaposition of cells with different
levels of FJ is associated with inhibition of Fat in the cells with less FJ
and activation of Fat in the cells with more FJ
(Fig. 11A). The influence of
DS, however, is more variable. Studies of tissue polarity in the eye suggested
that DS inhibits Fat activity in DS-expressing cells, and/or promotes Fat
activity in neighboring cells (Yang et
al., 2002). The predominant effect of DS during early wing
development is consistent with this, but its effects in late discs are not.
Studies of tissue polarity in the abdomen suggest that the DS gradient might
be interpreted differently by anterior versus posterior cells
(Casal et al., 2002), and it is
possible that a similar phenomena causes the effects of DS to vary during wing
development.
The influence of ds mutation on gene expression and growth in the
wing is much weaker than that of fat. It has been suggested that FJ
might influence Fat via effects on DS
(Yang et al., 2002), and
fj mutant clones have been observed to influence DS protein staining
(Strutt and Strutt, 2002;
Ma et al., 2003). Our
observations are consistent with the inference that both DS and FJ can
regulate Fat activity, but they do not directly address the question of
whether FJ acts through DS. They do, however, indicate that even the combined
effects of FJ and DS cannot account for FAT regulation, and, assuming that the
strongest available alleles are null, other regulators of Fat activity must
exist. It is presumably because of the counteracting influence of these other
regulators that alterations in FJ and DS expression have relatively weak
effects. In addition, according to the hypothesis that Fat activity is
influenced by relative rather than absolute levels of its regulators, the
effects of FJ or DS could be expected to vary depending upon their temporal
and spatial profiles of expression, as well as on the precise shape and
location of clones.
Downstream signaling
Our observations, together with those of Fanto et al.
(Fanto et al., 2003), imply
the existence of at least two intracellular branches of the Fat signaling
pathway (Fig. 11A). One branch
involves the transcriptional repressor Grunge, influences tissue polarity,
certain aspects of cell affinity, and fj expression, but does not
influence growth or WG expression. An alternative branch does not require
Grunge, but does require Dachs. Dachs is implicated as a downstream component
of the Fat pathway, based on its cell autonomous influence on Fat-dependent
processes, and by genetic epistasis. The determination that it encodes an
unconventional myosin (F. Katz, personal communication), and hence presumably
a cytoplasmic protein, is consistent with this possibility. It also suggests
that it does not itself function as a transcription factor, and hence implies
the existence of other components of this branch of the Fat pathway. This
Grunge-independent branch influences WG expression in the proximal wing and
imaginal disc growth. However, further studies will be required to determine
whether Dachs functions solely in Grunge-independent Fat signaling, or whether
instead Dachs is required for all Fat signaling.
Distal-to-proximal signaling in the wing
The observations that fj expression is regulated by SD-VG, and
that fj is both necessary and sufficient to modulate the distal ring
of WG expression in the proximal wing, suggest that FJ influences the activity
of a distal signal, which then acts to influence Fat activity
(Fig. 11B). However, the
relatively weak effects of fj indicate that other factors must also
contribute to distal signaling (X in Fig.
11B), just as fj functions redundantly with other factors
to influence tissue polarity. As DS expression is downregulated in a domain
that is broader than the VG expression domain, a direct influence of VG on the
DS gradient is unlikely, and the essentially normal appearance of WG
expression in the proximal wing in fj ds double mutants implies that
DS is not a good candidate for Signal X. Rather, we suggest that DS acts in
parallel to signaling from VG-expressing cells to modulate Fat activity. This
VG-independent effect would account for the remnant of the distal ring that
sometimes appears in vg null mutants
(Fig. 3A)
(Liu et al., 2000).
Importantly though, the observation that the phenotypes of hypomorphic
dachs mutant clones on WG expression are more severe than fj
and ds suggests that the hypothesized additional factors also act via
the Fat pathway. We also note that the limitation of WG expression to the
proximal wing even in fat mutant clones implies that wg
expression both requires NUB, and is actively repressed by distally-expressed
genes (Fig. 11B).
The recovery of normal WG expression by later stages in both fj
and dachs mutant clones implies that the maintenance of WG occurs by
a distinct mechanism. Prior studies have identified a positive-feedback loop
between WG and HTH that is required to maintain their expression
(Azpiazu and Morata, 2000;
Casares and Mann, 2000;
del Álamo Rodriguez et al.,
2002). We suggest that once this feedback loop is initiated, Fat
signaling is no longer required for WG expression. Moreover, the recovery of
normal levels of WG at late stages suggests that this positive-feedback loop
can amplify reduced levels of WG to near normal levels.
The distinct consequences of VG expression and FJ expression in clones in
the proximal wing suggest that another signal or signals, which are
qualitatively distinct from the FJ-dependent signal, is also released from
VG-expressing cells. When VG is ectopically expressed, WG is often induced in
a ring of expression that completely encircles it
(Liu et al., 2000). However,
this is not the case for FJ-expressing clones. Both VG- and FJ-expressing
clones can activate rn and wg only within NUB-expressing
cells, but VG expression can result in non-autonomous expansion of the NUB
domain, and this expansion presumably facilitates the expression of WG by
surrounding cells (Liu et al.,
2000; del Álamo
Rodriguez et al., 2002;
Baena-Lopez and Garcia-Bellido,
2003). Another striking difference between VG- and FJ-expressing
clones is that in the case of ectopic FJ, enhanced WG expression is only in
adjacent cells. By contrast, in the case of VG, WG expression initiates in
neighboring cells, but often moves several cells away as the disc grows,
resulting in a gap between VG and WG expression. This gap suggests that a
repressor of WG expression becomes expressed there, and recent studies have
identified Defective proventriculus (DVE) as such a repressor
(Kolzer et al., 2003).
Growth regulation by the Fat signaling pathway
In strong fat mutants, the wing discs become enlarged and have
extra folds and outgrowths in the proximal wing
(Bryant et al., 1988;
Garoia et al., 2000). The
disproportionate overgrowth of the proximal wing is due to upregulation of WG
in this region, as demonstrated by its suppression by
wgspd-fg (Fig.
4). At the same time, clones of cells mutant for fat
overgrow in other imaginal cells, and fat wgspd-fg discs
are still enlarged compared with wild-type discs. Thus, Fat appears to act
both by regulating the expression of other signaling pathways (e.g. WG), and
via its own, novel growth pathway. The identification of additional components
of this pathway will offer new approaches for investigating its profound
influence on disc growth.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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