scalloped
Regulation of early scalloped expression in the wing disc has not been characterized. Late expression of scalloped appears to be reciprocally activated by vestigial in the wing disc. Late expression of sd is absent in vestigial mutants (Williams, 1993).
Both vestigial and scalloped are overexpressed in shaggy/zeste white mutant clones. shaggy may be acting downstream of localized wingless expression to specify or maintain marginal identity in the wing (Blair, 1994).
The wingless product is required to restrict the expression of the apterous gene to
dorsal cells and to promote the expression of the vestigial and scalloped genes that demarcate the
wing primordia and act in concert to promote morphogenesis.
Two genes expressed along the normal wing margin, vestigial and scalloped, are
overexpressed at margin-like levels in shaggy-zeste white 3 clones. This phenotype does not depend
upon the formation of ectopic bristle precursors and occurs in clones lacking both shaggy/zeste
white 3 and the entire achaete-scute complex. As vestigial and scalloped are both involved in early
patterning events prior to the stages of bristle specification, these results strongly suggest that
shaggy/zeste white 3 is required for the normal specification or maintenance of regional identity in the
developing wing blade. However, the margin-like transformation is only partial, since the expression of
apterous (in pupal wings) and wingless and cut (at late third instar) is not reliably altered in
shaggy/zeste white 3 clones. shaggy/zeste white 3 may act
downstream of localized apterous and wingless expression to specify or maintain margin identity in
the wing (Williams, 1993 and Blair, 1994). Expression of scalloped appears to be reciprocally activated by vestigial in the wing disc (Williams, 1993).
In Drosophila, the Vestigial-Scalloped (VG-SD) dimeric transcription factor is required for wing cell identity and proliferation. Previous results have shown that VG-SD controls expression of the cell cycle positive regulator dE2F1 during wing development. Since wing disc growth is a homeostatic process, the possibility was investigated that genes involved in cell cycle progression regulate vg and sd expression in feedback loops. The experiments focused on two major regulators of cell cycle progression: dE2F1 and the antagonist Dacapo (Dap). The results reinforce the idea that VG/SD stoichiometry is critical for correct development and that an excess in SD over VG disrupts wing growth. Transcriptional activity of VG-SD and the VG/SD ratio are both modulated by down-expression of cell cycle genes. A dap-induced sd up-regulation was detected that disrupts wing growth. Moreover, a rescue was observed of a vg hypomorphic mutant phenotype by dE2F1 that is concomitant with vg and sd induction. This regulation of the VG-SD activity by dE2F1 is dependent on the vg genetic background. The results support the hypothesis that cell cycle genes fine-tune wing growth and cell proliferation, in part, through control of the VG/SD stoichiometry and activity. This points to a homeostatic feedback regulation between proliferation regulators and the VG-SD wing selector (Legent, 2006).
Cell proliferation relies on the tight control of cell cycle genes, and, in the wing pouch, VG–SD is also critically required. Accordingly, vg up-regulates dE2F1 expression and antagonizes the CKI dap. This study investigated the effects of these two antagonistic proliferation regulators in the wing pouch of the disc, and tested the hypothesis that cell cycle genes fine-tune proliferation, through regulation of the respective expressions of vg and sd and VG–SD dimer activity, thereby providing a feedback control (Legent, 2006).
Combined loss and gain of function experiments has ascertained the requirement of a precise VG/SD ratio for normal wing development and has shown that an excess in SD disrupts VG–SD function in wing growth, and probably acts as a dominant-negative through titration of functional VG–SD dimers. Therefore, sd induction may efficiently restrain VG–SD function in vivo, and a similar effect may also be physiologically achieved down-regulating vg. Moreover, since SD DNA target selectivity is modified upon binding of VG to SD in vitro, the hypothesis cannot be discarded that, in vivo too, VG–SD targets might be different from the targets of SD alone. This could explain to some extent the phenotypes observed when sd is induced (Legent, 2006).
The results show that the CKI member DAP, homogeneously expressed in the wing disc, regulates VG–SD function. dap heterozygotes display a wild type wing phenotype, reduced levels of both vg and sd transcripts, but an almost normal vg/sd ratio, thus VG–SD activity is normal. Consistently, no abnormal wing phenotype could be detected. Therefore, the relative vg/sd stoichiometry, rather than absolute vg and sd expression levels, determines wing growth. Interestingly, it had been observed that dap homozygous mutant adult escapers display duplication of the wing margin, indicating a role of DAP at the D/V boundary. This phenotype could be linked to an enhanced proliferation due to the absence of CKI function. Moreover, D/V-specific over-expression of dap alters wing margin structures. This dap over-expression triggers both ectopic expression of sd and subsequent impairment of VG–SD activity associated with a proliferation decrease.The associated wing phenotype is clearly enhanced in vg heterozygous flies, providing evidence that dap over-expression affects VG/SD stoichiometry and represses VG–SD activity in wing development. This reveals a model in which, in the wing pouch, cell proliferation down-regulation through cyclin/CDK inhibition by DAP, may be enhanced by an additive reduction of VG–SD proliferation function. Such a mechanism probably participates in vivo in the control of balanced wing growth (Legent, 2006).
The results also demonstrate that dE2F1-DP regulates VG–SD: the dE2F1 heterozygote displays a reduced vg/sd ratio due to a decrease in vg and an increase in sd transcripts, associated with reduced dimer activity, comparable to the vgnull/+ context. Thus, dE2F1 is required for vg normal expression. This supports the hypothesis that the slower proliferation observed in these contexts is linked to an imbalance in the dimer ratio (Legent, 2006).
Conversely, over-expressing dE2F1-DP-P35, in a vg83b27 hypomorphic mutant context, rescues expression of both vg and sd and normal VG–SD function, wing appendage specification and growth. This is not observed in vgnull flies implying the necessity for vg sequences, but the second intron, missing in the vg83b27 mutant. In addition, it was ascertained that not all the genes triggering cell cycle progression or cell proliferation can induce vg expression. Neither ectopic expression of CYC E, which promotes dE2F1-induced G1/S cell cycle transition, nor the growth regulator Insulin receptor (InR) is sufficient to elicit VG expression and wing growth in the vg83b27 mutant. These results demonstrate that vg induction is a prerequisite for vg83b27 wing pouch growth in response to dE2F1 activity (Legent, 2006).
In a vg+ genetic background, dE2F1 over-expression induces only sd, disrupting VG/SD stoichiometry. Consistently, at the D/V boundary, wing notching was observed. Therefore, although dE2F1 basically displays a positive role in proliferation, this sd induction in response to dE2F1 over-expression is clearly associated with wing growth impairment. This effect is significantly weaker in a vg heterozygote background, and a rescue of the wing phenotype was observed, supporting the hypothesis that VG/SD stoichiometry is restored. Therefore, sd induction by dE2F1 depends on the vg genetic context. This indicates that the effects of over-expressing dE2F1 differ depending on the growth-state of the wing pouch, which is tightly linked with the vg genotype (Legent, 2006).
Clearly, feedback regulations rule the growth of the wing disc. Regulation has been noted in three different vg genetic contexts that can be analyzed in the light of a homeostasis hypothesis. In the vg83b27under-proliferative wing pouch, ectopic dE2F1 expression coordinately increases vg and sd expression in a positive feedback loop. This triggers VG–SD activity, and induces both cell proliferation and wing specification in the mutant. Conversely, no such crosstalk occurs in a correctly grown vg+ disc, where over-growth should be prevented. In this latter case, sd induction (VG/SD decrease) probably restrains the proliferation function of dE2F1. Consistently, wings were not overgrown, but notches were observed. This phenotype was partially suppressed in a vg heterozygote background. As a whole, these results support the hypothesis that VG–SD/dE2F1 coordination tends to ensure normal wing growth and that the dimer does not trigger unrestricted cell proliferation in a vg+ context, since an excess in dE2F1 attenuates VG–SD function in a negative feedback loop. Thus, molecular interactions between dE2F1, vg and sd, display a clear plasticity depending on the vg genetic context (Legent, 2006).
Establishing and maintaining homeostasis is critical during development. This is achieved in part through a balance between cell proliferation and death. In mammals E2F1 and p21, the dacapo homolog, play a key role in this process. In the wing disc compensatory proliferation induced by cell death has been observed. However, the role of cell cycle genes in this process has not yet been established. How patterns of cell proliferation are generated during development is still unclear. It seems nevertheless likely that the gene responsible for regulating differentiation also regulates proliferation and growth. For instance, Hedgehog (HH) induces the expression of Cyclins D and E. This mediates the ability of HH to drive growth and proliferation. In the same way, other data support a direct regulation of dE2F1 by the Caudal homeodomain protein required for anterio-posterior axis formation and gut development. Wingless (WG) also displays both patterning and a cell cycle regulator function during Drosophila development (Legent, 2006).
Growth control in the wing pouch seems to be achieved through both positive and negative feedback regulations linking dE2F1 and VG–SD, but also via additive impairment of VG–SD by DAP. In fact, in a vg+ background, over-expression of both dap and dE2F1 induces sd, impairs VG–SD and alters wing development. Nevertheless, clear opposite behaviors are observed in vgnull/+ flies where dap-induced nicks are enhanced, while those of dE2F1 are partially rescued. This highlights the functional discrepancy between these two types of feedback regulation. It is suggested that dap expression inhibits cell proliferation through a process involving both Cyclin-CDK inhibition and VG–SD impairment in the wing pouch. In contrast, it is proposed that dE2F1 over-expression triggers a homeostatic response. It will either induce vg and sd to ensure proliferation (in a vg83b27 genotype), or decrease the VG/SD ratio in a vg+ context. In this latter genotype, down-regulation probably counteracts fundamental proliferative properties of dE2F1 and governs homeostatic wing disc growth (Legent, 2006).
At late third instar, wing discs display a Zone of Non-proliferating Cells (ZNC) along the wing pouch D/V boundary. It has been shown that, although dE2F1-DP is expressed in this area, its proliferative function is inactivated late, because of RBF1-induced G1 arrest. Accordingly, although expression of vg and sd presents a peak at the D/V boundary, in late third instar, VG–SD activity is decreased in D/V cells, and it was suggested to result from an excess of SD. Therefore, the ZNC setting may also reflect a VG–SD/dE2F1 coordinated dialogue that triggers a decrease in proliferation signals in this area (Legent, 2006).
Previous studies of homeostatic control of cell proliferation in the wing reported that, to some extent, over-expression of positive or negative cell cycle regulators only weakly affects the overall division rate. For instance, although dap over-expression alters dE2F1 function in G1-S cell cycle transition, it also promotes dE2F1 expression and function in G2-M transition, preventing a decrease in the overall rate of cell division. Strikingly, the cells seemed able to monitor each phase length and maintain cell cycle duration and normal proliferation in the wing pouch of the disc. Therefore, dE2F1 is a central component that enables cells to ensure normal proliferation in the wing disc and prevents imbalance in the process. The fact that dE2F1 triggers quite different or opposite responses in vg+ or vg hypomorphic contexts suggests that the VG–SD/dE2F1 crosstalk plays a role in the same sort of homeostatic process that ensures entire wing growth (Legent, 2006).
Such regulations are likely to reveal a precise physiological fine-tuning of vg and sd by cell cycle effectors, promoting an exquisite control of wing growth. Feedback loops between the developmental selector VG–SD and cell cycle effectors may stand for a control mechanism to guarantee that the tissue can sustain balanced growth and a reproducible size. Such a subtle mechanism, on a local scale, would correct the alterations in cell proliferation that may occur during development (Legent, 2006).
scalloped appears to be a specific activator of vestigial in the wing disc (Williams 1993).
A number of wing scalloping mutations have been examined to determine their effects on the mutant
phenotype of cut mutations and on the expression of the Cut protein. The mutations fall into two
broad classes, those which interact synergistically with weak cut wing mutations to produce a more
extreme wing phenotype than either mutation alone and those that have a simple additive effect with
weak cut wing mutations. The synergistically interacting mutations are alleles of the Notch, Serrate
and scalloped genes. These mutations affect development of the wing margin in a manner similar to
the cut wing mutations. The mutations inactivate the cut transcriptional enhancer for the wing margin mechanoreceptors and noninnervated bristles and prevent differentiation of the organs. Surprisingly, reduction of Notch activity in the wing margin does not have the effect of converting epidermal cells to a neural fate as it does in other tissues of ectodermal origin. Rather, it prevents the differentiation of the wing margin mechanoreceptors and noninnervated bristles (Jack, 1992).
A small number of major regulatory (selector) genes have been identified in animals that control the development of
particular organs or complex structures. In Drosophila, the vestigial gene is required for wing formation and is able to
induce wing-like outgrowths on other structures. Because ectopic expression of Vg in many imaginal discs induces the outgrowth of wing tissue, the expression of various wing patterning genes was examined to see if they are induced in ectopic growths. Vg is expressed in the entire developing wing pouch whereas Sal and
SRF have specific expression patterns within this domain but are not expressed in wild-type
leg discs. Targeted expression of Vg with the Gal4-UAS system induces ectopic expression
of Sal and SRF in developing leg imaginal discs. Similarly,
the nubbin (nub) gene (which is also expressed and required during wing development ) is ectopically induced in leg discs by Vg expression. In each case, only a subset of the cells expressing Vg activate the target gene,
which suggests that additional factors control the expression pattern of each gene. In a first step toward elucidating the molecular mechanism by which Vg regulates gene expression, the response of wing-specific enhancers to ectopic Vg expression was examined. Attention was focused on
both the boundary and quadrant enhancers of the vg gene and the enhancer from the SRF gene
that drives expression specifically in the intervein region between veins three and four. All three enhancers are
induced by ectopic Vg expression in leg and other imaginal discs. Importantly, ectopic expression of Vg
in clones of cells induces the enhancers only within the clones. However, gene
expression is not induced in all cells within clones nor in all clones. In addition, each individual
enhancer is expressed in different regions of these discs that appear to correlate with the spatial
distribution of the different signaling inputs known to be required for activation of these enhancers (Halder, 1998).
Scalloped is required for Vg function. In the notum primordia of the wing disc, the vg enhancers, as well as the sal, SRF, and nub genes
are not induced by ectopic Vg even though the known required extracellular signals are present. Target gene activation could depend then on the function of another
gene(s). One candidate for such a factor is the product of the sd gene, which is expressed in a pattern
similar to Vg in the wing disc and is required for wing formation and the proper expression of Vg and other genes. In other discs, such as the leg and eye discs,
sd is endogenously expressed and is upregulated wherever ectopic Vg is able to induce
wing-specific gene expression and trigger wing development. It is noted, however, that
a sd enhancer trap line and the SRF-intervein C enhancer transgene are also ectopically induced by
Vg in the presumptive notum, although at levels lower than those observed in the wing pouch. This is consistent with the inability of Vg to trigger wing development and induce other wing
patterning genes in the developing notum. Indeed, mis-expression studies show that Sd function is required in parallel with Vg in order for Vg to
exert its wing inducing activity. The three wing-specific enhancers from the SRF and vg genes are activated synergistically
when Sd and Vg are coexpressed in Drosophila S2 cells. Although each individual protein
has some effect on reporter gene expression, this is significantly less than that observed in
the presence of both Vg and Sd. Titration of the amounts of transfected Vg and Sd plasmids
with all enhancers shows that the relative concentration of the two factors is critical and, at any
given Vg concentration, high levels of Sd reduce activation (Halder, 1998).
To define the sequences of the enhancers that respond to Vg/Sd, the activation of smaller
fragments from the 704-bp SRF intervein C enhancer, the 806-bp vg quadrant enhancer, and the
754-bp vg boundary enhancer in tissue culture were analyzed. A 125-bp fragment (SRF-A)
derived from the 5' end of the SRF enhancer is activated, whereas an adjacent 131-bp fragment
(SRF-B) is not activated. A 65-bp fragment from the vg quadrant enhancer (MD2) has
been identified that, when multimerized, produces an expression pattern very similar to the full-length
enhancer in wing discs. When assayed in tissue culture, MD2 is
activated by Vg and Sd. Within the vg boundary enhancer, a 120-bp fragment sufficient to
drive reporter gene expression in the wing pouch (vg-A) as well as a nonoverlapping 90-bp fragment
(vg-B) are also activated synergistically by cotransfection of Vg and Sd. Sd was shown, using mobility shift and DNase I footprinting assays, to bind specifically to essential sites for target gene activation (Halder, 1998).
One possible reason for the importance of the concentration of Sd on Vg function concerns the
localization of the Vg protein. It was observed that in S2 cells transfected with the vg expression plasmid
alone, the Vg protein appears to be localized to both the cytoplasm and the nucleus. In
contrast, in cells cotransfected with the Vg and Sd expression plasmids, Vg is clearly localized to nuclei. Vg localization is more diffuse in sd mutant clones than in sd+
cells; this is also true of ectopic Vg localization in regions of imaginal discs that lack endogenous Sd
expression. Furthermore, deletion of the Sd interaction domain of Vg results in cytoplasmic
accumulation of Vg in vivo. Thus, Sd may facilitate the transport or
retention of Vg protein in the nucleus and, coupled with the concentration-dependent, synergistic
effects of Vg and Sd on target gene expression, these results suggest that the proteins form a complex
in vivo (Halder, 1998).
These results demonstrate that the activation of several genes in the wing field requires Vg/Sd function.
It is also known that for each of the cis-regulatory elements analyzed here, direct input(s) of particular
signaling pathways are also required. Specifically, the activation of the SRF intervein C element
requires both Vg/Sd and Hh signaling; the activation of the vg boundary enhancer requires Vg/Sd and N signaling, and the activation of the vg quadrant enhancer requires Vg/Sd and Dpp signaling. Because these regulatory elements are not expressed in all tissues in which the signals are
active, nor in all wing cells in which Vg/Sd are active, it is deduced that neither the input of various
signals nor of Vg/Sd alone are sufficient for gene activation in vivo. Rather, the results suggest that the
various wing-specific cis-regulatory elements require a combination of direct inputs, comprising the
Vg/Sd selector function, which restricts expression to the wing field, and at least one signal transducer
that mediates signaling inputs and hence, the pattern of gene expression within the wing field. One
prediction of this model is that gene expression patterns within the wing field may be changed by
altering the signal-transducer binding sites within a cis-response element. To test this, the
Suppressor of Hairless [Su(H)] binding site that mediates the N input in the vg boundary enhancer
was changed to sites for the Cubitus interruptus (Ci) protein that transduces Hh signaling.
This switches the pattern of gene expression from a N-induced dorsoventral stripe to a Hh-induced
anteroposterior stripe while retaining the restriction of gene activation to the wing disc (Halder, 1998 and references).
These results demonstrate that the role of the Vg/Sd selector function is to directly regulate wing-specific
cis-regulatory elements that also require particular signaling inputs. The patterns of gene expression
induced in the wing disc are limited to cells in which both the selector genes and specific signaling
pathways are active. The response of the SRF-A, vg boundary, and vg quadrant enhancers to Hh, N, and Dpp signaling are limited to the wing pouch by Vg/Sd and occur in different patterns because of
their direct regulation by the Ci, Su(H), and Mad proteins, respectively. Furthermore, the
finding that the changing of the Su(H) binding site into a Ci binding site in the vg boundary enhancer
switches the pattern from a wing-specific dorsoventral N-regulated stripe to a wing-specific
anteroposterior Hh-regulated stripe suggests that spatial expression patterns are determined by the sites
for individual DNA-binding signal transducers. One corollary of this model is that for any given signaling protein, different selector proteins may be
involved in directing tissue-specific responses in different organs and tissues. For example, other studies have shown that tissue-specific enhancers in the embryo that are regulated by Dpp also require the action of
the Labial/Extradenticle or Tinman selector proteins to
limit expression to the endoderm or mesoderm, respectively. It is suggested that, in general, combinatorial
control by selector proteins and common signal transducers at a cis-regulatory level is required for the
tissue- and organ-specific responses of target genes to widely deployed signaling systems (Halder, 1998 and references).
The development of the Drosophila wing involves progressive patterning events. In the second larval instar, cells of the wing
disc are allotted wing or notum fates by a wingless-mediated process and dorsal or ventral fates by the action of apterous and
wingless. Notch-mediated signaling is required for the expression of the genes vestigial and scalloped in the presumptive wing
blade. Later, wingless, Notch and cut are involved in cell fate specification along the wing margin. The function of scalloped in
this process is not well understood and is the focus of this study. Patterning downstream of Notch and wingless
pathways is altered in scalloped mutants. Reduction in scalloped expression results in a loss of expression of wing blade- and
margin-specific markers. An enhancer element in the second intron of the vg gene is found influence the level of sd expression. Misexpression of scalloped in the presumptive wing causes misexpression of scalloped, vestigial and
wingless reporter genes. However, high levels of scalloped expression have a negative influence on wingless, vestigial and its own
expression. These results demonstrate that scalloped functions in a level-dependent manner in the presumptive wing blade in a
loop that involves vestigial and itself. It is suggested that wing development requires the regulated expression of scalloped, together
with vestigial: the 'wing formation' effects of Vestigial in other imaginal discs are probably due to its interaction with the
scalloped gene product normally expressed in these discs. sc and vg respond to wg and N signaling in a manner very similar to that characterized for vg in the developing wing. This activation is maintained by auto-regulatory events. It is also suggested that sd and vg serve to regulate and modulate wg expression and to change the pattern observed in the second larval instar to the very different pattern seen in the third instar wing disc (Varadarajan, 1999).
In Drosophila, the imaginal discs are the primordia for
adult appendages. Their proper formation is dependent
on the activation of the decapentaplegic gene in a
stripe of cells just anterior to the compartment boundary.
In imaginal discs, the dpp gene has been shown to be
activated by Hedgehog signal transduction. However, an
initial analysis of its enhancer region suggests that its
regulation is complex and depends on additional factors.
In order to understand how multiple factors regulate dpp
expression, focus was placed on a single dpp enhancer
element, the dpp heldout enhancer, from the 3' cis
regulatory disc region of the dpp locus. A molecular analysis of this 358 bp wing- and
haltere-specific dpp enhancer is presented that demonstrates a direct
transcriptional requirement for the Cubitus interruptus
(Ci) protein. The results suggest that, in addition to
regulation by Ci, expression of the dpp heldout enhancer is
spatially determined by Drosophila TCF (dTCF) and the
Vestigial/Scalloped selector system and that temporal
control is provided by dpp autoregulation. Consistent with
the unexpectedly complex regulation of the dpp heldout
enhancer, analysis of a Ci consensus site reporter construct
suggests that Ci, a mediator of Hedgehog transcriptional
activation, can only transactivate in concert with other
factors (Hepker, 1999).
The dppho enhancer (so named because mutations in the region
result in a 'held out' wing phenotype) was chosen for detailed analysis because this small
region contains a cluster of putative transcription factor binding
sites that is conserved in Drosophila virilis. The
dppho enhancer is located from map position 111.9 to 112.3, approximately 18 kb from the 3' terminus of the dpp structural gene.
The enhancer shares 52% sequence identity with the homologous
region from D. virilis. Within the conserved sequences are found
reasonable matches for the binding sites of several known
transcription factors, including Engrailed, Ci, dTCF, Mothers against Decapentaplegic (Mad) and Scalloped.
Of particular interest is the presence of potential Ci
consensus binding sites. Gel mobility shift assays were
performed with the DNA-binding domain of Ci and they
demonstrated sequence-specific binding to the dppho fragment (Hepker, 1999).
The expression pattern of dppho-lacZ is consistent with this
reporter being restricted by the extent of overlap between Hh
and Wg signals in the wing pouch. For example, the dppho enhancer directs
expression of lacZ reporter in a stripe coincident with high-level
full-length Ci and endogenous dpp expression in the wing
primordium of the wing imaginal disc. Furthermore,
its expression is most robust in early larval stages and fades in
a manner complementary to the dynamic pattern of wg
expression in the wing disc. This enhancer also
directs expression of a reporter ventrally in an analogous stripe
in the haltere disc.
Indeed ectopic expression data, together with clonal
analysis, demonstrates that Ci and dTCF regulate dppho-lacZ
expression, and this regulation is shown to be direct (Hepker, 1999).
Regulation of the dppho enhancer cannot be solely dependent
on Wg and Hh signals since this element directs expression
specifically in presumptive wing tissue. A candidate for a
wing-specific factor involved in dppho regulation is the Vestigial/Scalloped
transcriptional complex. The dppho sequence contains a weak match to the Sd/TEA DNA-binding site consensus, therefore a test was performed to see
whether the Vg/Sd selector system is involved in restricting
dppho-lacZ expression to the wing.
A 30AGAL4 or an apterousGAL4 driver was used to direct
expression of UAS-vg. In both cases, ectopic expression of vg
induces expression of dppho-lacZ, but only near the A/P
boundary. Similar experiments performed with
UAS-sd result in loss of dppho-lacZ expression (Hepker, 1999).
blistered is expressed in the precursors of the terminal tracheal cells and in the future intervein
territories of the third instar wing imaginal disc. Dissection of the blistered regulatory region reveals that a single enhancer element, which is
under the control of the fibroblast growth factor (FGF)-receptor signaling pathway, is sufficient to induce blistered expression in the terminal
tracheal cells. In contrast, two separate enhancers direct expression in distinct intervein sectors of the wing imaginal disc. One element is
active in the central intervein sector and is induced by the Hedgehog signaling pathway. The other element is under the control of
Decapentaplegic and is active in two separate territories, which roughly correspond to the intervein sectors flanking the central sector.
Hence, each of the three characterized enhancers constitutes a molecular link between a specific territory induced by a morphogen signal and
the localized expression of a gene required for the final differentiation of this territory (Nussbaumer, 2000).
An blistered enhancer (the boundary enhancer) has been identified
that is activated by Hh signaling in the cells anterior to the
A/P compartment boundary. In agreement with previous
reports demonstrating a direct morphogenic role of Hh in
the central region of the wing, this might indicate that the Hh signaling
is required to trigger intervein differentiation through blistered
expression in the intervein C domain. However, in contradiction to the activity of the boundary enhancer, blistered is
expressed in smo mutant clones analyzed during pupal
development. Noteworthy, the clones that were generated were analyzed during third instar, whereas blistered expression is detected later, 24-36 h
after puparium formation. At this time, gene interactions
between vein- and intervein-specific genes might be sufficient to maintain their respective, mutually exclusive expression domains. Thus, Hh would
be required only for the early setting of blistered expression
as a result of patterning the intervein sector C. Indeed, beta-galactosidase expression directed by the boundary enhancer
is not detected in the wing of newly emerged flies. This indicates that in the presumptive intervein sector C, the early setting of blistered is controlled
through the boundary enhancer, whereas the later expression might recruit another cis-regulatory element. The fact that the expression of blistered is observed in the posterior compartment of pupal wings, whereas the boundary enhancer is restricted to the anterior compartment in third instar discs, further supports this idea (Nussbaumer, 2000).
The boundary enhancer is directly regulated by Vestigial (Vg) and
Scalloped (Sd) which form a complex on a 120-bp DNA
sub-element. The wing-specific Vg-Sd
complex restricts the activation of the boundary enhancer to
the future wing, consistent with the finding that Ci can only
activate it in the pouch region. Hence, the boundary enhancer integrates positional cues from the Vg-Sd transcriptional complex and the Hh signal. The gene knot/collier (kn), which encodes a putative DNA-binding protein acting
downstream of the Hh signaling pathway, has been found to
be required for the expression of blistered in the intervein
sector C. Therefore, the Hh responsiveness of the boundary enhancer may be indirect and mediated by Kn. Alternatively, activation of the enhancer may require a molecular interaction between Ci and Kn. Therefore, it will be of prior interest to determine whether Kn and Ci directly regulate the boundary enhancer and
cooperate for its activation. Further analysis of how the
boundary enhancer integrates input from the Vg-Sd
complex and Hh signaling will contribute to a molecular
understanding of the synergistic activation of enhancers by
signaling input and selector genes, a strategy that may be
widely used to regulate gene expression during development (Nussbaumer, 2000).
In the Drosophila wing the cut gene is activated by Notch signaling along the dorso-ventral boundary but not in other cell types. Additional regulatory components, scalloped and strawberry notch, that are targets of the Notch pathway, are expressed specifically within the wing anlagen. As suggested by physical interactions, these proteins could be co-factors of the cut trans-regulator Vestigial. Additional
regulatory input comes from the Wingless pathway. These data support a model whereby context specific involvement of distinct co-regulators modulates Notch
target gene activation (Nagel, 2001).
These data show that the complex regulation of ct along the D/V boundary is based on a bifurcation of the Notch signaling pathway. Most signals from the Notch pathway are mediated by Su(H), which seems to act as a repressor on its own that is converted to an activator by Nact. Since Su(H) has the capacity to bind directly to the ct wing margin enhancer, the repression of ct by Su(H) and the activation by Nact/Su(H) might be direct. However, although sufficient for the activation of ct along the D/V boundary, a number of additional factors downstream of Nact are required. These include the products of wing fate selector genes vg and sd, that seem to be, together with Sno, part of a multi-factor trans-activation complex that binds to the ct wing margin enhancer. Thereby, Sd binds directly to the ct promoter, presumably recruiting the other factors by protein-protein interactions. In agreement with this hypothesis, respective physical interactions are observed between Vg and Sd or Sno. However, all three genes are targets of the Notch signaling pathway and are activated upon the overexpression of Su(H) specifically within presumptive wing tissue. Activation of Vg is also observed also within the wing pouch, although Su(H) acts as a repressor on the vg quadrant enhancer, indicating that the isolated enhancer elements reveal only a subset of the normal pattern and might contribute differently in a wild type context (Nagel, 2001).
A combination of Sd and Su(H) binding sites is sufficient to drive expression along the D/V boundary within the wing anlagen. This synthetic enhancer is too simplified to faithfully model ct regulation. Since the overexpression of Su(H) affects the accumulation of all the important trans-activator components, Vg, Sd, Sno and Su(H) itself, ct expression would be expected. Instead, repression of ct is observed: this might be due to a lack of Nact as co-activator of Su(H). However, repression can be overcome by concurrent expression of Wg resulting in strong ct activation. It is concluded that factors downstream of the Wg signaling cascade are able to convert Su(H) from a repressor to an activator, maybe by supplying a respective co-activator or by a cooperative combinatorial activity, e.g. together with the Wg signaling mediator dTCF, in accordance with a presumptive dTCF binding site within the ct wing margin enhancer
(Nagel, 2001).
These signaling events appear to be unique to the activation of ct along the D/V boundary of the wing disc. Another important role of ct is the specification of external sensory organ cells during embryogenesis and imaginal development alike. Although Notch signaling is essential for setting up the correct number of neuronal cells in the peripheral nervous system by lateral specification, it appears not to be involved in the transcriptional activation of ct within these cells. The complex mechanism of ct trans-activation from the wing margin enhancer is, therefore, not a general paradigm for ct gene regulation. Moreover, neither wg, sd nor sno are under the direct regulatory influence of the Notch pathway in various embryonic tissues suggesting that this remarkably complex control is strictly tissue specific (Nagel, 2001).
These data confirm and extend the model of context dependent activity of Notch signaling towards the regulation of ct expression along the presumptive wing margin. The regulation of ct requires the combined input of components downstream of Su(H) and Wg, including Vg, Sd and Sno. The latter three components have the potential to form a multi-protein complex which seems to be a pre-requisite for the trans-activation of the ct wing margin enhancer. Whether Su(H) is part of this specific complex or other, similar complexes has to be elucidated in the future. Although there are no indications for direct interactions between Su(H) and Sd, Vg or Sno, Su(H) has the capacity to bind to the ct wing margin enhancer and act in a combinatorial manner together with the Sd/Vg/Sno transactivation complex and components of the Wg pathway. Presumably, in many instances of Notch signaling, where Su(H) acts as a DNA-binding molecule and signal transducer, a number of additional positive or negative co-regulators confers tissue and cell specificity. Therefore, the identification of corresponding factors should help to further the understanding of the context dependent outcome of Notch signaling events (Nagel, 2001).
The formation of many complex structures is controlled by a special class of transcription factors encoded by selector genes. It has been shown that Scalloped, the DNA binding component of the selector protein complex for the Drosophila wing field, binds to and directly regulates the cis-regulatory elements of many individual target genes within the genetic regulatory network controlling wing development. Furthermore, combinations of binding sites for Scalloped and transcriptional effectors of signaling pathways are necessary and sufficient to specify wing-specific responses to
different signaling pathways. The obligate integration of selector and
signaling protein inputs on cis-regulatory DNA may be a general mechanism by which selector proteins control extensive genetic regulatory
networks during development (Guss, 2001).
The discovery of genes whose products control the formation and identity of various fields, dubbed 'selector genes', has enabled the recognition and redefinition of fields as discrete territories of selector gene activity. Although the term has been used somewhat liberally, two kinds of selector genes have been of central interest to understanding the development of embryonic fields. These include the Hox genes, whose products differentiate the identity of homologous fields, and field-specific selector genes such as eyeless, Distal-less, and vestigial-Scalloped (vg-sd) whose products have the unique property of directing the formation of entire complex structures. The mechanisms by which field-specific selector proteins direct the development of these structures are not well understood. In principle, selector proteins could directly regulate the expression of only a few genes, thus exerting much of their effect indirectly, or they may regulate the transcription of many genes distributed throughout genetic regulatory networks (Guss, 2001).
In the Drosophila wing imaginal disc, the Vg-Sd selector protein complex regulates wing formation and identity. Sd is a TEA-domain protein that binds to DNA in a sequence-specific manner, whereas Vg, a novel nuclear protein, functions as a trans-activator. To determine whether direct regulation by Sd is widely required for gene expression in the wing field, the regulation of several genes that represent different nodes in the wing genetic regulatory network and that control the development of different wing pattern elements were analyzed. Focus was placed in particular on genes for which cis-regulatory elements that control expression in the wing imaginal disc have been isolated, including cut, spalt (sal), and vg (Guss, 2001).
First it was tested whether sd gene function is required for the expression of various genes in the wing field. Mitotic clones of cells homozygous for a strong hypomorphic allele of sd were generated and the expression of gene products or reporter genes was assessed within these clones. Reduction of sd function reduces or eliminates the expression of the Cut and Wingless (Wg) proteins and of reporter genes under the control of the sal 10.2-kb and the vg quadrant enhancers, demonstrating a cell-autonomous requirement for selector gene function for the expression of these genes in the wing field (Guss, 2001).
These results, however, do not distinguish between the direct and indirect regulation of target gene expression by Vg-Sd. To differentiate between these possibilities, whether the DNA binding domain of Sd could bind to specific sequences in cut, sal, and vg wing-specific cis-regulatory elements were tested. Using DNase I footprinting, Sd-binding sites were identified in all of the elements assayed. Thus, Sd may control the expression of these genes by binding to their cis-regulatory elements (Guss, 2001).
To determine whether Sd binding to these sites is necessary for the function of these cis-regulatory elements in vivo, specific Sd-binding sites within each of the elements were mutated such that they reduced or abolished Sd binding in gel mobility-shift assays. The mutation of tandem Sd-binding sites in the cut and sal elements results in complete loss of reporter gene expression in vivo. Similarly, mutation of the four single Sd-binding sites identified in the vg quadrant enhancer eliminated or dramatically reduced reporter gene expression. These results show that Sd binds to and directly regulates the expression of four genes (cut, sal, vg, and DSRF) in the wing genetic regulatory network. This molecular analysis and the genetic requirement for Sd function for the expression of other genes suggest a widespread requirement for direct Vg-Sd regulation of genes expressed in the wing field (Guss, 2001).
Each of the Sd targets analyzed is activated in only a portion of the wing field, in patterns controlled by specific signaling pathways. For instance, cut is a target of Notch signaling along the dorsoventral boundary, and the sal and vg quadrant enhancers are targets of Dpp signaling along the anteroposterior axis. Binding sites for the transcriptional effectors of the Notch- and Dpp-signaling pathways, Suppressor of Hairless [Su(H)], and Mothers Against Dpp (Mad), and Medea (Med), respectively, have been shown to be necessary for the activity of a number of wing-specific cis-regulatory elements, and occur in these elements. This observation, coupled with the data demonstrating a direct requirement for Sd binding, suggests that gene expression in the wing field requires two discrete inputs on the cis-regulatory DNA: one from the selector proteins that define the field, and one from the signaling pathway that patterns the field (Guss, 2001).
These findings also raised the possibility that the combination of selector and signal inputs may be sufficient to drive field-specific, patterned gene expression. To test this, there were built a number of synthetic regulatory elements comprised of combinations of Sd binding sites with binding sites for Su(H) or Mad/Med. The activity of these elements was compared with those composed of tandem arrays of just selector- or signal effector-binding sites, or combinations of different signal effector sites. Each of the binding sites used in these constructs was selected from sequences found in native Drosophila cis-regulatory elements that have been demonstrated to function in vivo (Guss, 2001).
Elements containing only single classes of binding sites for the selector or signal effectors were unable to drive reporter gene expression in the wing. There are several potential mechanisms whereby selector proteins and signaling effectors might operate in a combinatorial manner to regulate transcription. One mechanism is through cooperative interactions that increase the occupancy of transcription factor-binding sites on the DNA. Such a scenario appears unlikely in this case, because it would require that each selector protein be able to interact directly with many different signaling pathway transcriptional effectors. Furthermore, cooperative filling of binding sites alone is insufficient to explain selector-signal synergy, because Sd alone binds cooperatively to DNA, and yet the presence of multiple Sd-binding sites alone is insufficient to generate transcriptional activation (Guss, 2001).
A second, more likely mechanism underlying selector-signal synergy is the formation of complexes between the two classes of transcription factors and required transcriptional coactivators. Coactivators facilitate transcription by relieving repression by chromatin and/or by mediating interactions with the basal transcriptional machinery. It is suggested that gene activation by selector proteins and signaling pathways may require both of these activities, and these proteins may form complexes with coactivators on the cis-regulatory DNA. These complexes could include coactivators such as the multifunctional protein CBP, which has been shown to interact directly with three signaling pathway transcriptional effectors, Mad, Ci, and Pangolin, and also appears to interact with Sd. Alternatively, synergy between Sd and signaling pathway transcriptional effectors could be mediated by different coactivators, with independent functions. The obligate requirement for combined inputs from selector genes and signaling pathways, seen here in the wing, may be a general mechanism whereby a universally deployed set of signals can elicit field, tissue, and cell type-specific genetic responses (Guss, 2001).
The two genes vestigial and scalloped are required for wing development in Drosophila. They present similar patterns of expression in second and third instar wing discs and
similar wing mutant phenotypes. vg encodes a nuclear protein without any recognized nucleic
acid-binding motif. Sd is a transcription factor homologous to the human TEF-1 factor, whose promoter
activity depends on cell-specific cofactors. It is postulated that Vg could be a cofactor of Sd in the wing
morphogenetic process and that, together, they could constitute a functional transcription complex. Genetic interactions between the two genes have been investigated. vg and sd are shown to co-operate in
vivo in a manner dependent on the structure of the Vg protein. The vg79d5 mutation is recessive. Surprisingly, in an sdETX4 background, the flies heterozygous for the vg79d5 allele exhibit a mutant phenotype that is almost as strong as that of the sdETX4 flies homozygous for this allele. The same phenotype is observed in a sd1 background. Morover, the sdETX4 flies heterozygous for the vgnull homozygous mutants have a very extreme phenotype. Therefore, in a hypomorphic sd background, the vg79d5 mutation has a dominant effect that leads to an enhancement of the sd mutant phenotype. The vg79d5 allele is known to encode a protein with an internal deletion corresponding to the 5' end of exon 3, which includes a poly-alinine-rich region, the correct reading frame being preserved. When the expression of sd is reduced, the presence of the vg79d5 allele encoding such a deleted protein is more drastic than the complete loss of one vg allele. Therefore, the genetic interactions observed between vg and sd are dependent on the structure of the Vg protein (Paumard-Rigal, 1998).
vg was ectopically expressed in
patched (ptc) domains. Wing-like outgrowths induced by ectopic expression of vg
are severely reduced in vg or sd mutant backgrounds. Accordingly, it was demonstrated that
ptc-GAL4-driven expression of vg induces both expressions of the endogenous vg and sd genes and
that the two Vg and Sd proteins have to be produced together to promote wing proliferation. It is unknown whether vg alone is able to initiate sd expression in the ptc domain or if vg requires Sd protein. Indeed, both proteins are ubiquitously expressed at a low level throughout the wing disc in early second instar larvae. It is concluded that vg activates its own transcription
Futhermore, an interaction between the two proteins is demonstrated by double hybrid experiments in yeast. The C-terminal region of the Vg protein contains the domain involved in the formation of the Vg-Sd complex.
Therefore, these results support the hypothesis that Sd and Vg directly interact in vivo to form a complex regulating the proliferation of wing tissue (Paumard-Rigal, 1998).
The formation and identity of organs and appendages are regulated by specific selector genes that encode
transcription factors that regulate potentially large sets of target genes. The DNA-binding domains of selector
proteins often exhibit relatively low DNA-binding specificity in vitro. It is not understood how the target selectivity of most selector proteins is determined in vivo. The Scalloped selector protein controls wing development in Drosophila
by regulating the expression of numerous target genes and forming a complex with the Vestigial protein. The binding of Vestigial to Scalloped switches the DNA-binding selectivity of Scalloped. Two conserved domains of the Vestigial protein that are not required for Scalloped binding in solution are required for the formation of the heterotetrameric Vestigial-Scalloped complex on DNA. It is suggested
that Vestigial affects the conformation of Scalloped to create a wing cell-specific DNA-binding selectivity. The modification of selector protein
DNA-binding specificity by co-factors appears to be a general mechanism for regulating their target selectivity in vivo (Halder, 2001).
Essential native Sd-binding sites have been identified in several cis-regulatory elements that control the wing field-specific expression of Sd-regulated target genes. These sites were identified by DNaseI footprinting using the TEA domain of Sd. In these analyses, the finding that essential sites occurred most often as tandem double sites, for example, in the cut, spalt and DSRF (bs) genes, was particularly striking. Despite substantial differences in sequence, the TEA domain of Sd binds cooperatively to all of these doublet sites with high affinity, and with similar affinity to single, nonessential sites and to native single vertebrate TEF-1-binding sites in muscle-specific cis-regulatory elements and the SV40 enhancer. From these studies, a consensus binding site sequence of T/A A/G A/G T/A AT G/T T for the TEA domain of Sd, which is very similar to that of the TEA domain of TEF-1, has been inferred (Halder, 2001).
In contrast to the isolated TEA domain, however, the full-length Sd protein (produced by in vitro translation) does not bind equivalently to all of these sites but rather shows a restricted DNA-binding specificity. Full-length Sd binds specifically to the doublet site in the DSRF enhancer and to most of the single binding sites, but binding to the cut, sal, kni and other native templates with doublet sites is weak or nearly undetectable. The difference in DNA-binding activity between the TEA domain and Sd protein indicates that there are motifs within the native Sd protein that affect the activity of the TEA domain and restrict its binding to certain sites. Sites that are bound by Sd are referred to here as A-sites (Halder, 2001).
The finding that most of the doublet-binding sites are not bound by the full-length Sd protein is surprising, considering that these templates are bound with high affinity by the TEA domain and that these sites are essential for enhancer activity in vivo. The observations that the activity of these cis-regulatory elements in vivo and in cell culture depends on co-expression of Vg with Sd, and the finding that Vg and Sd interact physically, raises the possibility that interaction of Vg with Sd changes Sd's DNA-binding properties and enables binding to these sites. However, previous Vg-Sd protein interaction studies have been performed in the absence of DNA and the possible effect of the interaction between Vg and Sd on DNA-binding has thus not been addressed. Whether Sd and Vg form a complex on DNA in vitro and whether this complex has different DNA-binding properties from the Sd protein alone has now been tested (Halder, 2001).
Co-translation of Sd with Vg produces a Vg-Sd complex that binds to these other sites (referred to as B-sites). In contrast to Sd alone, complexes containing Sd and Vg bind strongly to the cut, sal and kni elements. Quantification of the bound complexes shows that Vg increases Sd binding to these doublet sites by about 10-fold. In addition to enabling binding to B-sites, interaction with Vg reduces Sd binding to the single site templates by at least fivefold. Importantly, binding of Vg alone to any of the binding sites described in this report or to any other DNA templates tested has not been detected. Therefore, Vg binding to Sd switches the DNA target preference of Sd from the single A-sites to the doublet B-sites (Halder, 2001).
Four key findings are reported in this study: (1) it was found that the Sd protein has a more restricted DNA-binding specificity than its isolated TEA
domain; (2) the Vg-Sd complex binds well to sites in native cis-regulatory elements to which Sd alone does
not bind well; (3) two domains of the Vg protein are required for Vg-Sd complex formation on DNA that are not required for Vg binding to Sd in solution, and (4) that this complex is a heterotetramer on DNA while apparently a heterodimer in solution. A mechanistic model is presented for the control of Vg-Sd DNA target selectivity that considers these findings (Halder, 2001).
This model proposes that Vg binding to Sd switches the DNA target selectivity of Sd. The Sd protein alone binds to sites with a particular
composition, termed A-sites, which exist singly or as doublets. In the latter case, Sd may bind cooperatively if the two sites are arranged in tandem. When Vg is also present, Vg and Sd interact and form a dimer in solution. This complex has two distinct properties. First, the Vg-Sd dimer has a greatly reduced affinity for A-sites. Vg may either induce a conformational change in Sd that inhibits the TEA domain from interacting with DNA, or Vg could directly mask the TEA domain. Second, the dimer forms a higher order complex on a different set of binding sites, termed B-sites. These two activities of Vg are distinguished by
their structural requirements. While the Sd-interaction (SID) domain of Vg is sufficient to inhibit Sd DNA-binding to A-sites, additional domains N- and C-terminal to the SID are required for complex formation on B-sites. Importantly, B-sites are poorly bound by Sd in the absence of Vg. Thus, Vg binding to Sd inhibits binding to A-sites while enabling binding to B-sites, that is, Vg switches the DNA-binding preference from A-sites to B-sites (Halder, 2001).
How does Vg binding affect the target selection of Sd? Two, not necessarily mutually exclusive models, may be postulated. First, Vg may influence Sd through global effects on Sd DNA binding. That is, Vg may act to reduce the DNA binding affinity of Sd to any target DNA, while also enhancing cooperativity of neighboring Vg-Sd complexes on DNA. It was found that Vg and Sd form dimers in solution and that these dimers do not bind single A-sites. In spite of the negative effect of Vg on DNA binding, two Vg-Sd dimers bind strongly to doublet B-sites. Apparently, strong cooperative interactions between two Vg-Sd dimers allow binding to B-sites. The N- and C-terminal protein domains of Vg that are required in addition to the SID for complex formation on DNA may be required for these interactions, which could involve Vg-Sd and/or Vg-Vg interactions between the two dimers on DNA (Halder, 2001).
Alternatively, Vg interaction may specifically enhance binding to doublet B-sites. This model is favored because Vg-Sd has a similar affinity for several
B-sites such as those in cut and 2xGT, even though 2xGT is a much better Sd binding site. The affinities of Sd for these sites therefore do not translate directly into the relative affinities observed for Sd-Vg binding, as would be expected if Vg only enhances cooperativity. In addition, it was found that the TEA domain binds several A- and B-sites with high affinity, but that full-length Sd has a strong preference for A-sites over B-sites. Thus, in the absence of any co-factor, Sd is in a conformation
in which a domain of Sd separate from the TEA domain inhibits the TEA domain from binding to B-sites specifically. In vitro, Vg interaction appears to be
able to alleviate this inhibition because Vg-Sd complexes bind strongly to B-sites. This alleviation only occurs when complexes form on doublet sites, since Vg-Sd complexes do not bind to DNA as a dimers. It is suggested that some sort of conformational change is associated with binding to doublet B-sites. The model is
supported by the finding that the region of Sd that binds to the SID of Vg is homologous to a region of the vertebrate TEF-1 that negatively affects DNA binding. This model is analogous in part to the role of Exd overcoming the inhibitory effect of the YKWM motif in the Labial Hox protein (Halder, 2001).
It has been argued that Sd and the Vg-Sd complex differentiate between A- and B-sites. What then are the distinguishing features of these sites? The sequences of the A- and B-sites are quite diverse and their alignment does not reveal different consensus sequence motifs. However, Sd clearly prefers binding to A-sites, and the inability of Sd to bind strongly to B-sites, such as that in the cut element, must therefore be due to the sequence of the template site. Vg-Sd complexes bind with high affinity to only two sites when arranged in tandem, and do not form on single A- or B-sites. Thus, Sd discriminates between A- and B-sites based on sequence, while the binding of Vg-Sd complex depends both on sequence and the arrangement of the sites. Two sites, DSRF and 2xGT, have been identified that have A- as well as B-site properties, so these properties are not mutually exclusive. However, many sites exist that are bound well by Sd or Vg-Sd, but not by both. Most of the essential sites for Vg-Sd regulation in vivo have mainly B-site character and are bound poorly by Sd. The identification of the exact sequence requirements that distinguish native essential Sd sites from the known Vg-Sd target sites will require some knowledge of Sd-regulated target genes in other tissues (Halder, 2001).
Vg binding and its effect on the DNA target selectivity of Sd plays a major role in distinguishing the biological specificity of Sd action in the developing wing from Sd function in other tissues. Sd is required for the development of tissues other than the wing -- for example, the eye and the PNS -- where it is not co-expressed with Vg. Based on the results of this study, it is postulated that Sd selects a different set of target genes in organs other than the wing, at least in part because its
DNA-binding specificity is different in the absence of Vg (Halder, 2001).
No direct target genes for Sd in these other tissues have been identified. However, many target genes for the vertebrate Sd homolog TEF-1 are known. Sd and TEF-1 may function very similarly, since their TEA domains are 99% identical and have indistinguishable DNA-binding properties in vitro, and TEF-1 can substitute for Sd in Drosophila. In mammals, TEF-1 directly regulates many genes expressed during muscle differentiation by binding to A-sites containing the so-called 'm-CAT' motif (CATTCCT). Importantly, this motif is bound by a single TEF-1 molecule. Two of these m-CAT sites were tested for Sd binding and it was found that, as for other single A-sites, Sd alone binds well, but the presence of Vg inhibits Sd binding and does not result in complex formation on DNA. Because these sites are in vivo targets of TEF-1, this suggests that TEF-1 and Sd may directly regulate gene expression by binding to single A-sites alone or in complexes with other factors, but not in complexes containing the Vg/Fdu proteins. Interestingly, it has been
found that vertebrate TEF-1 forms a complex with the bHLH protein Max in vivo, and that Max, or another bHLH protein, may be an obligatory co-factor for
TEF-1 function during muscle differentiation. Because Max contacts DNA sequence specifically, it increases the target selectivity of TEF-1 in
muscle cells. The association of TEF-1 with Max may present another example of a tissue-specific co-factor that differentiates the DNA-target selectivity of a TEF
transcription factor family member between different tissues (Halder, 2001).
One of the major aims of genome sequence analysis is to decipher genetic regulatory sequences involved in development and differentiation. One critical challenge in achieving this goal is the ability to correctly predict the in vivo target genes of transcription factors. Several types of data may be considered for such predictions, including the presence or absence of transcription factor binding sites in potential regulatory regions, gene expression profiles and detailed protein function studies.
Searching genomic sequences for binding sites is obviously important; however, binding site consensus sequences are often short and degenerate, so that potential binding sites are predicted to occur in regulatory regions of virtually any gene. This also holds true for Sd. The consensus binding site of the TEA domain (T/A A/G A/G T/A AT G/T T) is found once about every 2 kb, on average. However, many, if not all, Vg-Sd-regulated target genes possess a doublet of
Sd-binding sites. Requiring a second binding site in tandem decreases the frequency of potential biologically relevant Vg-Sd binding sites by a factor of
~2000. The fact that most of the Vg-Sd sites would not have been found using full-length Sd protein in footprint assays and that the Sd DNA-binding domain
alone binds promiscuously therefore sounds a note of caution. Understanding the role of tissue-specific co-factors may be imperative to deciphering transcription
factor-regulated networks on a genome-wide scale. Efforts are under way, using these new insights into the selectivity of the Vg-Sd complex, towards defining the network of Vg-Sd-regulated genes in the developing wing (Halder, 2001).
The development of the Drosophila wing requires both scalloped and vestigial functions. Using a fusion between full-length Vestigial and the Scalloped TEA domain, the fusion protein can rescue scalloped wing mutations because within wing development, Scalloped and Vestigial cooperatively act as a transcription complex. Scalloped provides the necessary DNA binding function via the TEA domain and Vestigial promotes the activation of target genes. The putative nuclear localization signal contained in the TEA domain of Scalloped is likely responsible for the nuclear localization of Vestigial. The fusion protein is also capable of activating a known target gene of the native complex and thus represents a tool that will be helpful in rapidly identifying target genes of the Sd/Vg complex that are involved in wing differentiation. The functionality of the fusion suggests that only the TEA domain of Scalloped is critical for wing development and the rest of the protein (about 70%) is dispensable. This result is novel and should stimulate further studies of sd in other tissues in view of the fact that scalloped is a vital gene in Drosophila (Srivastava, 2002).
Lunde, K., et al. (2003). Activation of the knirps locus links patterning to morphogenesis of the second wing vein in Drosophila. Development 130: 235-248. 12466192
The adjacent knirps and knirps-related
(knrl) genes encode functionally related zinc finger transcription
factors that collaborate to initiate development of the second longitudinal
wing vein (L2). kni and knrl are expressed in the third
instar larval wing disc in a narrow stripe of cells just anterior to the broad
central zone of cells expressing high levels of the related spalt
genes. A 1.4 kb cis-acting enhancer element from
the kni locus has been identified that faithfully directs gene expression in the L2 primordium. Three independent ri alleles have
alterations mapping within the L2-enhancer element; two of these
observed lesions eliminate the ability of the enhancer element to direct gene
expression in the L2 primordium. The L2 enhancer can be subdivided into
distinct activation and repression domains. The activation domain mediates the
combined action of the general wing activator Scalloped and a putative locally
provided factor, the activity of which is abrogated by a single nucleotide
alteration in the ri53j mutant. Misexpression of genes in L2 that are normally expressed in veins other than
L2 results in abnormal L2 development. These experiments provide a mechanistic
basis for understanding how kni and knrl link AP patterning
to morphogenesis of the L2 vein by orchestrating the expression of a selective
subset of vein-promoting genes in the L2 primordium (Lunde, 2003).
An enhancer element upstream
of the kni coding region selectively directs gene expression in
the L2 primordium in third instar larval wing discs. Three
separate ri alleles have defects mapping within a minimal 1.4 kb L2
enhancer element. Two of these mutations eliminate
activity of the L2 enhancer, kniri[1], which contains a
252 bp deletion, and kniri[53j], which harbors a single
base-pair substitution. Truncation of the minimal L2 enhancer to
a 0.69 kb fragment leads to ectopic reporter gene expression in the extreme
anterior and posterior regions of the wing, indicating that repression
contributes to restricting activation of the L2 enhancer. In addition, the general wing promoting transcription factor Scalloped (Sd) binds with
high affinity to several sites in the L2 enhancer and sd is
required for kni expression in the wing disc. The L2 enhancer element has been employed as a tool to drive expression of various UAS
transgenes in the L2 primordium. The loss of the L2 vein in
ri mutants can be rescued by L2-specific expression of either the
kni or knrl genes, or the downstream target gene
rho. In addition, misexpression of genes in the L2
primordium that are normally expressed in veins other than L2 results in
abnormal L2 development. These results provide a framework for understanding
how positional information is converted into morphogenesis of the L2 wing vein
by 'vein organizing genes' such as kni and knrl (Lunde, 2003).
A hypothetical transcription factor that binds the kni promoter
and mediates an inductive signal presumably collaborates with the more
generally required wing selector Sd, since
mutation of four of the Sd binding sites (the doublet and two single sites) in
the L2 activation domain completely eliminates enhancer activity in the wing
disc. Clonal analysis with a hypomorphic sd allele also indicates
that sd is required for high-level expression of the full 4.8 kb L2
enhancer element in the wing disc. It is notable that the reduction in
lacZ expression in these clones is not as dramatic as the complete
loss of L2 activity observed when Sd binding sites in the activation domain
are mutated. There are several possible explanations for this discrepancy.
(1) The sd mutation used in these experiments is a hypomorphic
allele and therefore has residual activity. Unfortunately, stronger
sd alleles produce even smaller viable clones in the wing disc and
thus were not used. (2) Since only small clones can be generated, they must
typically have been produced with only two or three intervening cycles of cell
division. Consequently, the sd- cells may still
contain functional levels of wild type Sd (protein perdurance). (3) Another
possibility is that other activators can partially substitute for Sd, at least
in certain regions of the wing. Based on the absence of L2 activity when Sd
binding sites are mutated and the reduction in L2 activity in
sd- hypomorphic clones, it is concluded that Sd plays an
important role as an activator of the L2 enhancer. These results support the
view that Sd functions as a general transcriptional activator of genes
expressed in the wing field (Lunde, 2003).
Considerable evidence indicates an obligate partnership of Vestigial (Vg) and Scalloped (Sd) proteins within the context of wing development.
It is evident that Sd and Vg act together as a transcriptional complex during wing formation, wherein Sd provides the DNA-binding activity and nuclear localization signal, while Vg provides the activation function.
A 56-amino-acid motif within Vg is necessary and sufficient for binding of Vg with Sd. While the importance of this Sd-binding domain has been clearly demonstrated both in vitro and in vivo, the remaining portions of Vg have not been examined for their in vivo function(s). Herein, additional regions within Vg were tested for possible in vivo functions. The results identify two additional domains that must be present for optimal Vg function as measured 1) by the loss of ability to rescue vg mutants, 2) by the ability to induce ectopic sd expression, and 3) by the ability to perform other normal Vg functions when these domains are deleted. An in vivo study such as this one is fundamentally important because it identifies domains of Vg that are necessary in the cellular context in which wing development actually occurs. The results also indicate that an additional large portion of Vg, outside of these two domains and the Sd-binding domain, is dispensable in the execution of these normal Vg functions (MacKay, 2003).
From results using ectopic sd-lacZ induction (which measures the ability of ectopic vg to induce ectopic sd expression), the ability to rescue vg mutations, and the ability to carry out other functions associated with normal vg, it can be discerned that certain portions of the vg ORF, in addition to the Sd-binding domain, are necessary to accomplish normal Vg function. These appear to be the critical regions, since other portions can be deleted without effect. More specifically, the N-terminal amino acids (approximately the first 65) and C-terminal residues from 335 to 453 seem to play an important role in the induction of sd-lacZ. When the N-terminal deletion Delta5'-5 (deleting amino acids 2-65) is assayed, the ectopic expression ability is reduced markedly compared to that seen with the full-length vg construct, although it is not eliminated completely. Moreover, the larger N-terminal deletions (amino acids 2-170 and 2-278, respectively) do not further lower the ability to express sd. Thus, it seems that the fundamentally important region is already removed with the Delta5'-5 construct. For C-terminal deletions Delta1-4 and Delta1-2 (amino acids 356-453 and 335-426, respectively), the ability to ectopically express sd is much less than that produced by full-length vg but somewhat stronger than that produced when the N-terminal deletion constructs are assayed. Deletions Delta5'-5, Delta5'-6, and Delta5'-7 retain the encoded amino acids missing from Delta1-4 and Delta1-2 and vice versa. Taken together, these data suggest the presence of two important functional domains for Vg: one within amino acids 1-65 (domain 1) and the other within amino acids 336-453 (domain 2). Although the precise boundaries of these domains have not yet been determined, domain 1 is very likely within the first 65 amino acids (deleted in vgDelta5'-5) since this is the region most highly conserved between D. melanogaster and the mosquito Aedes egyptii. There is 82% identity over the first 66 amino acids, but over the next 20 amino acids the identity drops to 35% and drops even further beyond that. In agreement with this notion, the extent of 'functional' loss in UASDeltavg 5'-6 and 5'-7 is no stronger than that exhibited by UASDeltavg 5'-5, which deletes the first 65 amino acids only. The activity of domain 2 appears to be weaker, since domain 1 deletions produce a slightly more drastic impairment of Vg function than do domain 2 deletions (amino acids 356-453 or 335-426). However, homology between Drosophila and mosquito Vg is also high within the Sd-binding domain of Vg and, in fact, remains strong to the carboxyl terminus of Vg (82% identity from residue 335 to 453. The data define the presence of two necessary functional domains for the Vg protein in vivo. These domains correlate well with data that predict two activation regions using in vitro experiments, including yeast one-hybrid assays. The regions identified in this study also complement more recent in vitro data, implicating these regions of Vg as necessary for binding of the Vg/Sd complex to target genes (MacKay, 2003).
Drosophila thoracic muscles are comprised of both direct flight muscles (DFMs) and indirect flight muscles (IFMs). The IFMs can be further subdivided into dorsolongitudinal muscles (DLMs) and dorsoventral muscles (DVMs). The correct patterning of each category of muscles requires the coordination of specific executive regulatory programs. DFM development requires key regulatory genes such as cut (ct) and apterous (ap), whereas IFM development requires vestigial (vg). Using a new vgnull mutant, a total absence of vg is shown to lead to DLM degeneration through an apoptotic process and to a total absence of DVMs in the adult. vg and scalloped (sd), the only known Vg transcriptional coactivator, are coexpressed during IFM development. Moreover, an ectopic expression of ct and ap, two markers of DFM development, is observed in developing IFMs of vgnull pupae. In addition, in vgnull adult flies, degenerating DLMs express twist (twi) ectopically. Evidence is provided that ap ectopic expression can induce per se ectopic twi expression and muscle degeneration. All these data seem to indicate that, in the absence of vg, the IFM developmental program switches into the DFM developmental program. Moreover, the muscle phenotype of vgnull flies can be rescued by using the activity of ap promoter to drive Vg expression. Thus, vg appears to be a key regulatory gene of IFM development (Bernard, 2003).
Vg interacts with Sd to form a transcription factor that binds DNA through the Sd TEA/ATTS domain and activates transcription through the Vg activation domain. Since vgnull mutants show drastic muscle degeneration phenotypes, Vg and sd expressions were examined. Vg is expressed in adepithelial cells. Vg is expressed in myoblasts around the forming DLMs and in some of the DLM nuclei. Moreover, sd expression is expressed in adepithelial cells and developing IFMs. Vg is present in all DLM nuclei and sd is coexpressed with Vg. It is therefore likely that in muscle, as in the wing disc, Sd and Vg are obligate partners. This result is supported by indirect arguments: (1) Vg dimerization with Sd is necessary for Vg activity. Protein interaction has been shown between Vg and Strawberry Notch (SNO), but the function of this new partner remains unknown; (2) Vg is localized to the nucleus in muscles, and nuclear relocalization of Vg in S2 cells requires the presence of Sd. However, no muscle phenotypes were found in sd strong hypomorphic viable mutants (sd58 and sd3L). It is concluded that if Sd is required for muscle development, a very low level of sd product is sufficient to fulfill its function. There is some precedent for this type of situation: for example, whereas Ct is necessary for DFMs development, viable ct mutant alleles do not exhibit any muscle phenotypes (Bernard, 2003).
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In contrast, the synthetic elements in which binding sites for both selector and signal effector were combined drove field-specific expression restricted to the wing and haltere discs in patterns predicted by the specific signaling inputs to each element. That is, the [Sd]2 [Su(H)]2 element drove wing-specific expression along the dorsoventral margin, consistent with Notch activation along this boundary, and the [Sd]2 [Mad/Med] element drove expression in a broad domain oriented with respect to the anteroposterior axis of the disc, consistent with Dpp-signaling activity along this boundary. These patterns of expression are similar to those of the native cut and vg quadrant cis-regulatory elements that also respond to Notch- and Dpp-signaling inputs, respectively. However, regulatory elements containing a combination of Su(H) and Mad/Med sites were not active in vivo, demonstrating that combinatorial input in the absence of selector input is not sufficient to drive gene expression. These results suggest that the Vg-Sd complex provides a qualitatively distinct function required to generate a wing-specific response to signaling pathways (Guss, 2001).
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