bifid
The 3'-regulatory region is divided into three domains each having specific effects on optic lobe development (Brunner, 1992).
In the mutation In(1)ombH31, 40 kb of regulatory DNA,
located downstream from the transcription unit are removed from the omb gene. In(1)ombH31 is
characterized by the lack of a set of giant interneurons from the lobula plate of the adult optic lobes.
Already during embryogenesis, there is a drastic difference between wild type and
In(1)ombH31 in the level of the omb transcript in the optic lobe primordia. The adult mutant phenotype
may thus be caused by omb misexpression during embryonic development (Poeck, 1993b).
The gradient morphogen Decapentaplegic (Dpp) organizes pattern by inducing the transcription of different target genes at distinct
threshold concentrations during Drosophila development. An important, albeit indirect, mode by which Dpp controls the spatial extent of
its targets is via the graded downregulation of brinker, whose product in turn negatively regulates the expression of these targets. The molecular dissection is reported of the cis-regulatory sequences of optomotor-blind (omb), a Dpp target gene in the wing. A minimal 284 bp Dpp response element is described and it is demonstrated that this element is subject to Brinker (Brk) repression. Using this omb wing enhancer,
it has been shown that Brk is a sequence-specific DNA binding protein. Brk contains a DNA binding domain, which is located within its N-terminal 175 amino acids. Mutations in the high-affinity Brk binding site abolish responsiveness of
this omb enhancer to Brk and also compromise the input of an unknown transcriptional activator. These results therefore identify Brk as a novel transcription factor
antagonizing Dpp signaling by directly binding target genes and repressing their expression (Sivasankaran, 2000).
To identify a consensus sequence for Brk target sites, a Dpp-responsive enhancer, lab550, was analyzed using the same footprinting strategy used on the omb enhancer. A consensus sequence, GGCGC/TC/T, for high-affinity Brk binding was obtained from both the lab and omb enhancers. To assess the importance of each nucleotide, the WF12 Brk binding site of the omb enhancer was scanned
with single point mutations and the ability of these mutants to bind in vitro synthesized full-length Brk protein was tested. Mutations in the five central
nucleotides of the Brk site abolish Brk binding, indicating that these residues form the core recognition motif and
that Brk is a DNA binding protein with a high target site specificity (Sivasankaran, 2000).
Loss of Brk activity in vivo results in ectopic activation of the WF12 wing enhancer, so one would predict that mutations in WF12 that abolish Brk binding should also
lead to ectopic enhancer activity. To test this hypothesis, the core of the Brk binding site from GGCGCC to GATATC was mutated. As
expected, this change completely abolishes Brk binding in EMSA analysis. The same 4 bp mutation was introduced into the WF12-lacZ reporter
transgene. Unexpectedly, this mutation completely abolishes lacZ expression rather than expanding it. This result is interpreted as an indication that the Brk site
overlaps with that of an activating input. Single base pair mutations that interfere with Brk binding were introduced into WF12-lacZ, anticipating that
at least some of these mutations would still allow the unknown activator to bind, resulting in an uncoupling of the two inputs (Sivasankaran, 2000).
Two of the five point mutations that prevent Brk binding, mut271 and mut272, completely abolish WF12 enhancer activity as observed with the
4 bp mutation. However, mutations mut273, mut274 and mut275 still express lacZ, albeit only in a narrow stripe along the dorsoventral boundary. This
pattern unravels a hitherto masked, strong input into WF12 from a dorsoventral patterning system. Although these mutant enhancers show a reduced
extent of expression along the dorsoventral axis, all of them exhibit a clear expansion in expression along the anteroposterior axis. This latter property
corresponds to the behavior expected from the loss of a functional Brk binding site in the WF12 enhancer. These observations are interpreted to indicate that
mut273-mut275 represent mutations that completely abolish Brk binding but only partially prevent input by the activator. Mutations flanking the
Brk binding site (mut269, mut270, mut276 and mut277) abolish expression of WF12-lacZ, indicating that the binding site of the activator extends
beyond that of Brk (Sivasankaran, 2000).
Attempts were made to validate the assumption that mut273-mut275 exhibit an extended expression along the dorsoventral boundary due to loss of Brk-mediated repression.
Both the wild-type WF12 enhancer and the mutant derivatives were examined in cells that ectopically express Brk protein from a tub>CD2>brk flip-out transgene.
While Brk potently represses the transcriptional activity of WF12, it does not repress the mutant enhancers. Thus, the ability to bind Brk in
vitro, the lateral repression by endogenous Brk and the responsiveness to ectopic Brk in vivo all correlate with single nucleotide exchanges in the Brk core binding
site. Together, these results are taken as evidence that the wild-type omb WF12 enhancer is a direct target of Brk repression (Sivasankaran, 2000).
Decapentaplegic, through its receptors Thickveins and Punt target optimotor blind and spalt transcription in the wing imaginal disc. The range of DPP action is wide, affecting spalt and omb expression on both sides of the anterior-posterior compartment boundary. spalt and omb respond differently to the DPP concentration gradient, with omb showing a wider range of response due to its greater sensitivity to low DPP concentrations (Nellen, 1996).
The broad domain of omb expression in the wing imaginal disc corresponds to a wedge of expression that extends anteriorly from midway between veins 1 and 2, across the compartment boundary, to midway between veins 4 and 5 in the posterior compartment. Reduced omb activity leads to deletion of a central domain of the wing (Lecuit, 1996).
Overexpression of dpp causes an expansion of the wing along the AP axis and the width of the spalt domain is expanded relative to the DPP stripe. Cells with reduced Mothers against dpp (Mad), which is involved in transduction of the DPP signal, fail to express spalt, suggesting that cells must be able to transduce the dpp signal to express spalt (Lecuit, 1996).
It is suggested that the broader domain of omb expression relative to that of spalt could be generated by persistence of omb expression in the progeny of cells in which the gene was activated at an earlier time, when the cells were within the range of the DPP signal.
The reason for this belief comes from an experiment in which dpp was express ectopically. In this case spalt is expressed at a considerable distance away from the ectopic source of DPP while omb expression is more restricted to a region near the clone. These observations suggest that the effective range of DPP in activating omb is actually less than it is for spalt. Therefore, a domain of gene expression (and therefore of fate specification) may be defined by the history of a cell at least as much as by its direct responsiveness to secreted signals (Lecuit, 1996).
In wing development,
decapentaplegic is expressed along the anterior-posterior compartment boundary.
Early wingless expression is involved in setting up the dorsoventral boundary.
Interaction between dpp- and wg-expressing cells promotes appendage outgrowth.
optomotor-blind expression is required for distal wing
development and is controlled by both dpp and wg. Ectopic omb expression can lead
to the growth of additional wings. Thus, omb is essential for wing development and is
controlled by two signaling pathways (Grimm, 1996).
An investigation has been carried out of how Drosophila imaginal disc cells establish and maintain their
appendage-specific determined states. Ectopic
expression of wingless (wg) induces leg disc cells to activate expression of the wing
marker Vestigial (Vg) and to transdetermine to wing cells. Ectopic
wg expression non-cell-autonomously induces Vg expression in leg discs;
activated Armadillo, a cytosolic transducer of the Wg signal, cell-autonomously
induces Vg expression in leg discs, indicating that this Vg expression is directly
activated by Wg signaling. Ubiquitous expression of wg in leg discs can
induce only dorsal leg disc cells to express Vg and transdetermine to wing. Dorsal leg
disc cells normally express high levels of decapentaplegic (dpp) and its downstream
target, optomotor-blind (omb). Ectopic omb expression is sufficient to dorsalize leg cells but is not sufficient to induce transdetermination to wing. Dorsalization of ventral leg disc cells, through targeted expression of either dpp or omb, is sufficient to allow wg to induce Vg
expression and wing fate. Leg cells dorsalized by omb are competent to transdetermine to wing, as shown when wingless is expressed ubiquitously. Under these circumstances Vg is expressed in both dorsal and ventral regions. A non-autonomous effect of omb was observed on wg-induced Vg expression, suggesting that omb induces the expression of another signal that acts with wg to induce Vg expression (Maves, 1998).
High levels of dpp expression, which are both necessary and sufficient for dorsal leg development, are required for wg-induced transdetermination. Thus, dpp and omb promote both dorsal leg cell fate as well as transdetermination-competent leg disc cells. In leg discs, antagonist interactions between Wg and Dpp normally prevent wg expression from overlapping with high levels of dpp expresssion and with omb expression. It is thought that the interaction between Wg and Dpp in transdetermination mimics the interaction between Wg and Dpp normally used to establish the wing disc primordium. It is suggested that Wg signaling directly activates the vg boundary enhancer during wing disc development, presumably in conjunction with Notch signaling through Suppressor of Hairless. Taken together, these results show that the Wg and Dpp signaling pathways cooperate to induce Vg expression and leg-to-wing transdetermination. A specific vg regulatory element, the vg boundary enhancer, is required for transdetermination. It is proposed that an interaction between Wg and Dpp signaling can explain why leg disc cells transdetermine to wing and that these results have implications for normal leg and wing development (Maves, 1998).
Daughters against dpp (Dad), whose
transcription is induced by Dpp shares, weak homology with Drosophila Mad (Mothers against
dpp), a protein required for transduction of Dpp signals. Dad is expressed in a wide stripe that straddles the A/P compartment boundary of the imaginal discs, in contrast to Dpp, whose expression is confined to the anterior side. This pattern of expression suggests that Dad expression is positively regulated by the secreted Dpp molecule, and in fact ectopic Dpp expression results in abnormally large discs and in ectopic expression of Dad. In contrast to Mad or the activated Dpp
receptor, whose overexpression hyperactivates the Dpp signaling pathway, overexpression of Dad
blocks Dpp activity. Dpp target gene optomotor blind is absent in Dad-overexpressing cells. Expression of Dad together with either Mad or the activated receptor rescues
phenotypic defects induced by either protein alone. Dad can also antagonize the activity of a vertebrate
homolog of Dpp, bone morphogenetic protein, as evidenced by induction of dorsal or neural fate
following overexpression in Xenopus embryos. It is concluded that the pattern-organizing mechanism
governed by Dpp involves a negative-feedback circuit in which Dpp induces expression of its own
antagonist, Dad. This feedback loop appears to be conserved in vertebrate development (Tsuneizumi, 1997).
The role of Medea in tranmission of the Dpp signal is exemplified by the finding of a position-specific requirement for Medea in wing development. dpp is expressed in a line along the anterior-posterior compartment boundary; In comparison, Thick veins and Mad are required throughout the wing primordium or pouch for cell proliferation and expression of the gene optomotor
blind. A second Dpp type I receptor gene, saxophone, is dispensable for proliferation, but is required throughout the wing pouch to boost the final level of the Dpp signal for pattern formation. Therefore, the requirement for Medea was examined in these two aspects of wing development (Wisotzkey, 1998).
Clones of cells homozygous for either Medea 8 or Medea 2 were generated. Medea mutant
clones in the wing pouch do not survive when induced during
the first larval instar (96-72 hours before pupariation), although mutant clones are recovered elsewhere in
the wing disk. A similar position-dependent requirement for
proliferation in the wing disk has been found for tkv. However, Medea mutant clones of variable size are recovered in the wing pouch when induced in the middle of the second larval instar (66 hours before pupariation). In contrast, clones of cells homozygous for null alleles of Mad cannot be recovered in the wing imaginal disk when they are induced at this stage of development.
Thus, there is a weaker requirement for Medea in cell proliferation than for Mad.
Analysis of the expression of omb reporter within Medea mutant clones indicates that loss of Medea function does not simply mimic the phenotype of clones
homozygous for a weak Mad allele. Most Medea mutant clones
show a strong reduction in omb expression; in all cases omb
expression is altered only in mutant cells. Thus,
Medea is required in cells responding to Dpp. Within the wing
pouch, the reduction in omb expression depends on where the clone is located. Clones that fall along the anterior-posterior midline, where dpp is expressed, show only a slight reduction. Clones distant from the midline displayed the greatest reductions in expression, many having undetectable
levels of omb expression. In contrast to the position-dependent effects on omb
expression in Medea clones, all clones homozygous for a leaky allele of Mad fail to express omb. Similarly, omb expression is lost in tkv clones induced 24 hours before
pupariation, while Medea clones induced at this stage have little effect (Wisotzkey, 1998).
Together, these observations indicate that Medea is not required for all Dpp-dependent signaling; instead, the requirement for Medea varies with its position in the wing pouch.
Since omb expression is only weakly affected in Medea mutant clones
located at the anterior-posterior midline of the wing pouch, and given that dpp is expressed in this vicinity, it is possible that the requirement for Medea in maintaining omb expression can be bypassed in the presence of stable, high level expression of Dpp. These observations suggest that while Mad is absolutely required for Dpp signaling, Medea enhances or modifies the signal (Wisotzkey, 1998).
The role of the zinc finger
transcription factor Schnurri (Shn) in mediating the nuclear response to Dpp during adult patterning has been investigated. Using clonal analysis, it has been shown that wing imaginal disc cells mutant for shn fail to transcribe the genes spalt, optomotor blind, vestigial, and Dad,
that are known to be induced by dpp signaling. shn clones also ectopically express brinker, a gene that is downregulated
in response to dpp, thus implicating Shn in both activation and repression of Dpp target genes. Loss
of shn activity affects anterior-posterior patterning and cell proliferation in the wing blade, in a manner that reflects the
graded requirement for Dpp in these processes. Furthermore, shn is expressed in the pupal wing and plays a distinct role in mediating dpp-dependent vein differentiation at this stage. The absence of shn activity results in defects that
are similar in nature and severity to those caused by elimination of Mad, suggesting that Shn has an essential role in dpp
signal transduction in the developing wing. These data are consistent with a model in which Shn acts as a cofactor for Mad (Torres-Vazquez, 2000).
A study by S. D. Weatherbee (1998) is arguably the best study yet published about how gene regulation differs in homologous structures, and points to future studies for how differential gene regulation will be shown to account for the structural differences between species. The differentiation of the Drosophila haltere from the wing through the action of the Ultrabithorax (Ubx) gene is a classic example of Hox regulation of serial homology. This study reveals several features of the control of haltere development by Ubx which, in principle, are likely to apply to the Hox-regulated differential development of other serially homologous structures in other animals. Specifically, it has been shown that Ubx acts: (1) at many levels of regulatory hierarchies, on long-range signaling proteins and their target genes, as well as genes further downstream; (2) selectively on a subset of downstream target genes of signals common to both wing and haltere, and (3) independently on these diverse targets. This information is presented in terms of the effects of Ubx on gene expression in the three axes of appendage formation, since these axes are to a large extent independently regulated and independent gene regulation in the axes serves to structure the entire wing (Weatherbee, 1998).
In the anterior posterior axis, Ubx represses selected Dpp target genes. The expression pattern of en is essentially the same in the haltere disc as in the wing disc, indicating that Ubx is not regulating haltere identity by altering the expression of this compartmental selector gene. Similarly, the expression of dpp in the developing haltere on the anterior side of the AP compartment boundary resembles that in the wing disc. Because these discs give rise to very different appendages, there may be genes downstream of the Dpp signal that are regulated by Ubx. To identify these, an examination was carried out of how a number of genes involved in the development of specific wing characters are expressed and regulated in the developing haltere (Weatherbee, 1998).
From its cellular site of secretion, Dpp acts as a morphogen to organize wing growth, AP pattern, and to activate target gene expression over a distance. The optomotor blind (omb), spalt (sal), and spalt related (salr) genes are expressed in nested patterns centered on the Dpp stripe and are necessary for proper development of the central wing region, including veins II-IV. The expression of these Dpp target genes was examined in the haltere disc: although omb is expressed in the developing haltere pouch (straddling the Dpp stripe as it does in the wing disc), salr and sal are not expressed in the haltere pouch. These results show that the Dpp signal transduction machinery operates in the haltere disc but that selected wing target genes are not activated by the Dpp signal. To determine whether Ubx represses salr expression in the haltere disc, homozygous Ubx clones were generated. Indeed, salr is derepressed in Ubx clones in the anterior compartment of the haltere disc. As in the wing disc, salr expression in these clones depends on their distance from the Dpp source. To determine whether Ubx is sufficient to repress salr, salr expression was examined in CbxM1/+ wing discs in which Ubx is ectopically expressed along part of the DV boundary. In these wing discs salr expression is repressed in a cell autonomous fashion. Because sal/salr are required for the induction of vein development, the selective repression of salr by Ubx suppresses part of the Dpp-mediated AP wing patterning program in the haltere. As with the spatial patterning of wing veins, the pattern of intervein tissue is also determined by specific regulatory genes and critical for morphogenesis. The Drosophila Serum Response Factor (DSRF, or blistered) gene is expressed in future intervein tissue and required for the adhesion of the dorsal and ventral surfaces of the flat wing. The haltere, however, is more balloon-like; interestingly, DSRF expression is absent from the haltere pouch except for two crescents at the extreme dorsal and ventral edges of the anterior compartment. This difference is caused by Ubx regulation, because in Ubx clones in the haltere disc, repression of DSRF is relieved and a pattern of DSRF expression homologous to that in the wing forms within the boundaries of the clone. Conversely, ectopic expression of Ubx in wing discs extinguishes DSRF expression in a cell-autonomous manner (Weatherbee, 1998).
This study reports the expression pattern of Dll in the genital disc, the requirement of Dll activity for the development of the terminalia and the activation of Dll by the combined action of the morphogenetic signals Wingless (Wg) and Decapentaplegic (Dpp). In Drosophila, the terminalia comprise the entire set of
internal and external genitalia (with the exception of the
gonads), and includes the hindgut and the anal structures. They arise from a single imaginal disc of ventral
origin which is of complex organization and shows bilateral symmetry. The genital disc
shows extreme sexual dimorphism. Early in development,
the anlage of the genital disc of both sexes consists of three
primordia: the female genital primordium (FGP); the male
genital primordium (MGP), and the anal primordium (AP).
In both sexes, only two of the three primordia develop: the
corresponding genital primordium and the anal primordium.
These in turn develop, according to the genetic sex, into
female or male analia. The undeveloped genital primordium
is the repressed primordium (either RFP or RMP,
for the respective female and male genital primordia) (Gorfinkiel, 1999).
During the development of the two components of the anal primordium -- the hindgut and the analia -- only the latter is dependent on Dll and hedgehog (hh) function. The hindgut is defined by the expression of the homeobox gene even-skipped. The lack of Dll function in the anal primordia transforms the anal tissue into hindgut by the extension of the eve domain. Meanwhile targeted ectopic Dll
represses eve expression and hindgut formation. The Dll requirement for the development of both anal plates in males and only for the dorsal anal plate in females, provides further evidence for the previously held idea that the analia arise from two primordia. In addition, evaluation was made of the requirement for the optomotor-blind (omb) gene which, as in the leg and antenna, is located downstream of Dpp. These results suggest that the terminalia show similar behavior as the leg disc or the antennal part of the eye-antennal disc, consistent with both the proposed ventral origin of the genital disc and the evolutive consideration of the terminalia as an ancestral appendage (Gorfinkiel, 1999).
In order to find other genes involved in the development
of the terminal structures, the expression
pattern and the functional requirement for optomotor-blind
(omb) were examined. This gene encodes a protein with a DNA-binding
domain (T domain) and behaves
as a downstream gene of the Hh pathway in other imaginal
discs. In the genital disc,
Omb is detected in the dpp expression domains, abutting
the wg expressing cells. This behaviour
of omb expression is similar to that found in the leg and
antennal discs. In the genital
disc, omb is also regulated by the Hh signaling pathway
since Pka2
clones also ectopically express omb.
The phenotypes produced due to omb lack of
function using the allele omb282 were examined; homozygous females for this allele could not be obtained
but some male pharates were analyzed. In males, the dorsal
bristles of the claspers and the hypandrium bristles are
absent. Also, the hypandrium is
devoid of hairs and the hypandrium fragma is reduced. Surprisingly, the anal plates are mostly
somewhat enlarged in the ventral region and reduced in the
dorsal areas. The structures affected in omb2
are
duplicated when omb is overexpressed in the dpp domain
using the dpp-GAL4/UAS-omb combination. In males, the
dorsal bristles of the clasper and the hypandrium
bristles are duplicated. These phenotypes are
similar to the ones obtained as a result of ectopic Dpp (Gorfinkiel, 1999).
In Drosophila wing discs, a morphogen gradient of Dpp has been proposed to be a determinant of the transcriptional response thresholds of the downstream genes sal and omb. Evidence is presented that the concentration of the type I receptor Tkv must be low to allow long-range Dpp diffusion. However, low Tkv receptor concentrations result in low signaling activity. To enhance signaling at low Dpp concentrations, a second ligand, Tgf-beta-60A, has been found to augment Dpp/Tkv activity. Tgf-beta-60A signals primarily through the type I receptor Sax, which synergistically enhances Tkv signaling and is required for proper Omb expression. Omb expression in wing discs is found to require synergistic signaling by multiple ligands and receptors to overcome the limitations imposed on Dpp morphogen function by receptor concentration levels (Haerry, 1998).
The phenotypic consequences of overexpressing constitutively active forms of Tkv and Sax receptors in the developing wing was investigated using the GAL4-UAS system. The A9-Gal4 line was used: this drives high-level expression of Gal4 in the entire wing disc before it is restricted to the dorsal pouch at late third instar stage. In wild-type discs, the Sal and Omb products are symmetrically expressed along the anterior/posterior (A/P) boundary in response to Dpp. Normally, the Sal domain is restricted to cells in the wing pouch that are in close proximity to the Dpp-expressing cells, while Omb responds to lower levels of Dpp and is expressed in cells further away from the A/P boundary. The anterior boundary of Sal has been shown to specify the location where the longitudinal vein 2 (L2) is formed, while the formation of L5 coincides approximately with the posterior boundary of the Omb domain, but a causal relationship has not yet been established. When Dpp is ubiquitously expressed in wing discs, they become overgrown and the expression of both Sal and Omb is expanded. Like Dpp, overexpression of constitutively active Tkv (TkvA) also leads to disc overgrowth and ectopic induction of Sal and Omb. All cells in wings derived from animals expressing either Dpp or activated Tkv appear to differentiate into vein tissue, as exemplified by production of vein-specific morphological markers such as dark pigment and longer bristles. In contrast to TkvA, expression of either one or two copies of SaxA or development at 30°C, which results in an approximately twofold increase of Gal4 activity, is not sufficient to expand either Sal or Omb and produces only weak adult phenotypes consisting primarily of ectopic and thickened veins with a small amount of wing blistering in the region of the posterior cross vein. This phenotype is similar to that seen in animals raised at 18°C, which express low levels of TkvA. Although these findings suggest that Sax function may be qualitatively similar to that of Tkv but simply weaker, higher levels of activated Sax (four copies) still cannot mimic the effects of activated Tkv, such as the expansion of Sal and Omb (Haerry, 1998).
When high levels of activated Sax activity are combined with low levels of activated Tkv, the result is more than additive. The combination of one copy of saxA and low levels of Tkv leads to overgrowth, with the expansion of Omb (but not Sal), and results in a strong wing phenotype. The interaction of Sax and Tkv is synergistic. Taken together, these data suggest that Sax and Tkv synergistically interact and control the expression of a common target gene, omb. Activation of omb expression requires a level of signaling that can be activated by either high levels of Tkv activity alone or by a synergistic interaction between low levels of Tkv and high levels of Sax activity. In contrast, Sal activation requires a higher level of signaling, which can only be achieved by high levels of Tkv activity (Haerry, 1998).
Since both Tkv and Sax, as well as the type II receptor Put, have been implicated in mediating Dpp signaling, whether the loss in signal activity of these receptors would cause similar patterning defects in the wing was investigated. If these three receptors all bind the same ligand and signal to the same sets of downstream genes, it would be expected that a reduction in the activity of any individual receptor should result in qualitatively similar phenotypes that differ in severity only. Increasing levels of dominant negative receptors were expressed in different regions of the developing wing disc. Similar to using an allelic series of hypomorphic mutations, it was expected that expression of increasing copy numbers of dominant negative receptors should result in progressively more severe phenotypes. Ubiquitous expression of 3-4 copies of either form of two dominant negative Tkv1 constructs results in small wings with partial loss of L4 and both cross veins. In addition, L2 and L3 are closer together and the triple-row margin bristles are shifted more distally/posteriorly, as expected if the level of Dpp signal is reduced by titration of Dpp into nonproductive complexes. At higher levels (6-8 copies) of dominant negative Tkv1, very small adult wings are produced that show fusion of L2 and L3 as well as L4 and L5. Similar phenotypes are produced by expressing dominant negative versions of the alternative Tkv isoform that have an N-terminal extended extracellular domain, and also by expression of dominant negative Put. Both the Sal and the Omb domains are strongly reduced: the adult wings show fusion of L2 with L3 and L4 with L5. The wing phenotypes obtained with increasing levels of dominant negative Tkv and Put resemble those of certain combinations of dpp loss-of-function alleles, which is consistent with the notion that Dpp is primarily signaling through the combination of the Tkv and Put receptors. In contrast to these observations, dominant negative Sax constructs produce different results. When increasing copy numbers (1-8 copies) of dominant negative Sax are expressed, the discs become smaller and the Omb domain is reduced to the size of the normal Sal domain. But unlike expressing dominant negative Tkv, the Sal domain is not affected. In the adult wing, L5 and the posterior cross vein are lost compared to losing L3 and L4 after expression of dominant negative Tkv or Put. In addition, L2 is shifted more proximally and the proximal triple-row bristles that expand more distally/posteriorly in dominant negative Tkv wings are replaced by more proximal costa bristles. While the distance between L3 and L4 is normal, the overall shape of the wing becomes more strap-like, suggesting loss of peripheral tissue rather than the central tissue that is deleted in animals expressing Tkv or Put dominant negative receptors. These results suggest that dominant negative Sax acts in a qualitatively different manner from dominant negative Tkv (Haerry, 1998).
These results indicate that while the reduction of Tkv and Put activity affects the whole disc (Sal, Omb and growth), the expression of dominant negative Sax only affects the peripheral region of the disc (Omb and peripheral growth). If the dominant negative receptors function primarily by titrating Dpp, then it is curious why the overexpression phenotypes of dominant negative Sax are different. One possibility is that these receptors do not simply signal in response to Dpp but also in response to the binding of other ligands as well. Of the other two BMP-type ligands that have been described in Drosophila, scw shows no detectable expression at this stage. However, Tgf-beta-60A is expressed broadly in wing discs, and mutant analyses indicate that Tgf-beta-60A is required for normal wing development. Given its role in wing patterning, the effects of heteroallelic Tgf-beta-60A mutations were examined on Sal and Omb expression. Similar to discs expressing dominant negative Sax, Sal expression in Tgf-beta-60A mutant discs is normal while the Omb domain is reduced, particularly in the dorsal compartment. These observations are consistent with the notion that a second BMP-type ligand, Tgf-beta-60A, is required in addition to Dpp for proper Omb expression. Furthermore, the similarity of the Tgf-beta-60A loss-of-function and the dominant negative Sax phenotypes is consistent with recently described genetic interactions between Tgf-beta-60A and sax mutations and suggests that Tgf-beta-60A could signal in part through Sax (Haerry, 1998).
Ubiquitous overexpression of moderate levels of Tgf-beta-60A does not result in excessive disc overgrowth and does not alter the distribution of Sal and Omb. The resulting wings are slightly larger and exhibit minor venation defects along L2 and L5. However, similar to Dpp or TkvA, higher levels of Tgf-beta-60A overexpression expands both Sal and Omb and results in blistered and pigmented adult wings. Since only activated Tkv but not Sax is able to expand Sal and Omb expression, these findings are consistent with the notion that expression of moderate levels of Tgf-beta-60A leads to signaling preferentially through Sax, producing relative mild phenotypes, while higher concentrations of Tgf-beta-60A may also result in signaling through Tkv, producing phenotypes similar to activated Tkv (Haerry, 1998).
An investigation was carried out to determine if Tgf-beta-60A contributes to wing development primarily in the form of homodimers or Tgf-beta-60A/Dpp heterodimers. Results: (1) the level of Tgf-beta-60A mRNA appears to be significantly less than that of DPP, based on RNA in situ hybridization, indicating that heterodimers are not likely to be very abundant assuming similar translational efficiencies. (2) Localized overexpression of Tgf-beta-60A in the dpp-expressing cells does not result in any mutant phenotypes. (3) Expression of Tgf-beta-60A in the posterior compartment results in overgrowth, an expansion of the Sal and Omb domains, and restriction all adult wing defects exclusively to the posterior compartment. Since Tgf-beta-60A expression in this experiment does not overlap with Dpp-secreting cells, no Dpp/Tgf-beta-60A heterodimers should form, since heterodimer formation requires expression of both proteins in the same cell. Therefore, Tgf-beta-60A functions most likely as a homodimer. This finding is consistent with recent genetic analysis showing that clones of Tgf-beta-60A mutant cells that do not include dpp-expressing cells nevertheless produce patterning defects. It has been shown that dominant negative Tkv is more potent than Sax for inhibiting Dpp signaling, while dominant negative Sax is a stronger suppressor than Tkv of Tgf-beta-60A signaling. High levels of Tkv receptor limit Dpp diffusion and restrict Omb expression (Haerry, 1998).
Dpp, a TGFbeta, organizes pattern in the Drosophila wing by acting as a graded morphogen, activating different targets
above distinct threshold concentrations. Like other TGFbetas, Dpp appears to induce transcription directly via activation of
Mad. However, Dpp can also control gene expression indirectly by downregulating the
expression of the brinker gene, which encodes a putative transcription factor that functions to repress Dpp targets. The
medial-to-lateral Dpp gradient along the anterior-posterior axis is complemented by a lateral-to-medial gradient of Brinker,
and the presence of these two opposing gradients may function to allow cells to detect small differences in Dpp
concentration and respond by activating different target genes (Campbell, 1999).
Dpp controls patterning along the A-P axis in the wing of Drosophila by activating a number of
downstream targets, including sal, omb, and vg. These targets are activated cell autonomously by Dpp signaling, and there is
evidence, at least for vg, that Dpp induces gene transcription directly through activation of the SMAD
Mad, which may act as a transcription factor. Expression of
these three targets is also regulated negatively by brk: loss-of-function mutations in brk lead to ectopic and
inappropriate levels of expression of sal, omb, and vg. Wing discs from the pupal lethal hypomorph brkXA
are greatly enlarged along the A-P axis, phenocopying the ubiquitous expression of Dpp. These discs
show expanded domains of sal, omb, and vg expression in the expanded wing pouch. Null
mutants, including brkXA, are embryonic lethal, but mutant clones can result in outgrowths in adult wings
when the clone is located in the anterior or posterior extremes of the wing. These outgrowths
are comprised entirely of mutant tissue but are similar to outgrowths produced by misexpression of Dpp. Examination of such clones in wing discs reveals autonomous activation of
sal, omb, and vg outside of their normal expression domains. Thus, Brk functions in the
developing wing to repress the expression of Dpp targets such as sal, omb, and vg (Campbell, 1999).
Why is the indirect method involving Brk used to activate expression of Dpp targets? In other words, if
Dpp can directly activate these genes via Mad, then why is this not sufficient? It is speculated that it is
directly related to Dpp acting as a morphogen. Activation of sal, omb, and vg is not simply all or none,
but each is induced above a distinct threshold concentration of Dpp, with sal requiring the highest level
and vg the lowest. The gradient of Dpp will be transduced into a gradient of activated Mad, but it is
possible that cells cannot perceive small differences in activated Mad reliably enough to faithfully define
the expression domains of Dpp targets and that the introduction of the Brk intermediary provides the
necessary information. This type of dual control of gene expression may turn out to be a common feature of many morphogen
systems. The possibility is raised that other TGFßs may also use indirect mechanisms to control expression
of target genes, possibly even Brk-related proteins, especially if they also induce multiple targets in a
concentration-dependent manner. One relevant observation in this regard is that brk is also expressed in
the early embryo where its expression also appears to be regulated by Dpp; null mutant embryos are
partially dorsalized, suggesting it has a similar function here as in the wing. Unlike the wing,
control of D-V patterning by TGFßs is probably a conserved feature of almost all animal embryos and strengthens the possibility that brk homologs will be typical regulators of
TGFß target genes (Campbell, 1999).
decapentaplegic functions as a long-range
morphogen in patterning of the embryo and the adult appendages. Dpp signals via the SMAD proteins Mad and Medea.
In the absence of brinker (brk), Mad is not required for the activation of Dpp target genes that depend
on low levels of Dpp. brk encodes a novel protein with features of a transcriptional repressor. brk itself is negatively
regulated by Dpp. Dpp signaling might relieve brk's repression of low-level target genes either by transcriptional
repression of brk or by antagonizing a repressor function of brk at the target gene promoters (Jazwinska, 1999).
brk could be a transcription factor based both on its epistatic position in the pathway and on some
features of the protein sequence. If brk specifically represses only the promoters of low- and
intermediate-level target genes of Dpp, then loss of brk would lead to the activation of these genes at
ectopic positions. At these positions, structures would form that correspond to low or intermediate levels
of Dpp signaling, not because signaling has occurred, but instead because a specific subset of target
genes had been activated in a signaling-independent way. If it is assumed that brk is a target gene-specific transcriptional regulator, then two models can be
envisaged describing how Dpp regulates the target genes controlled by brk. In both models, the transcriptional
control of brk by Dpp plays an important role. Dpp signaling is a potent repressor of brk
transcription and seems to be required throughout wing development. As soon as Dpp signaling is abolished, strong brk expression can be seen at
any position in the wing pouch. If brk is ectopically expressed in the center of the wing, then induction of
omb and sal is suppressed even in regions of high Dpp signaling. All these observations suggest that Dpp
signaling, at least in part, counteracts brk repression by reducing the amount of repressor. The promoter
regions responsible for omb and low-level sal expression might even have only Brk-binding sites, so that
their activation would be completely dependent on downregulation of brk expression. Alternatively, these
promoters might integrate both the activation by SMAD proteins and repression by Brk (Jazwinska, 1999).
The morphogenesis of specialized structures within the CNS relies on the nonautonomous activity of cell populations that play the role of organizers. In the
Drosophila visual system, cells on the dorsal and ventral margins of the developing visual cortex express the Wnt family member Wingless (Wg) and the TGF-beta Decapentaplegic (Dpp). The activity of these morphogens in
establishing cortical cell fates sets the stage for the guidance of photoreceptor axons to their retinotopic destinations in the Drosophila brain. One role for Wg in cortical development is to induce and maintain the expression of Dpp, a key step in the assignment of dorsoventral cell identities. Dpp is induced early in cortical development, shortly after the onset of Wg expression in a few dorsal and ventral margin cells, and is maintained by
Wg activity until at least the time of retinal axon pathfinding. Wg is a developmental signal in many different tissues, and acts by regulating different target gene sets to
elicit a constellation of different cell fates. Wingless-controlled targets include distal-less and vestigial in the wing, engrailed in the embryonic ectoderm, labial in the gut, and sloppy-paired in the embryonic CNS. Conversely, Dpp
belongs to a Hedgehog-controlled circuit in the wing (Song, 2000 and references therein).
A regulatory mechanism is described that relays Wg signal reception to the tissue-specific expression of target genes in the visual cortex. In a screen for mutants
in which photoreceptor axons project aberrantly to their destinations in the brain, a mutation in combgap was discovered. Retinal axon navigation defects in combgap animals are due to the role of cg in the establishment of
cortical cell identity. cg represses the expression of Wg target genes in a positionally restricted manner in the
visual cortex. wg+ induction of its cortical cell targets occurs via the downregulation of cg. Combgap is thus a tissue-specific relay between Wingless and its target genes for the determination
of cell fate in the visual cortex (Song, 2000).
A combgap mutation was recovered in a screen for mutants with aberrations in retinal axon projections. On the basis of its effects on target region gene expression and the outcome of mosaic analysis, it is evident that a role for combgap in the specification of cortical cell identity underlies its requirement for the establishment of retinotopic connectivity in the visual system. In cg loss of function animals, three markers under wg+ control are expressed in expanded dorsal and ventral portions of the retinal axon target field. The requirement for cg to repress the markers within these domains is autonomous. The lamina midline region, however, appears phenotypically normal in homozygous or mosaic cg animals. This positionally restricted requirement for cg+ activity is correlated with the pattern of cg expression, since cg is not expressed in the midline region where it is not required. Since wg+ misexpression is sufficient to induce wg+-dependent markers in the midline region, another regulatory system must control these markers there. Hence, the consequences of wg signal reception at different dorsoventral positions within the cortical precursor field would appear to involve a set of regulatory molecules that divide the cortex into specific domains for pattern formation (Song, 2000).
At hatching, approximately 40 cortical cell precursors form a disc-shaped epithelium on the ventrolateral surface of each brain hemisphere. The epithelium is divided into lamina and medulla precursor zones, which can be distinguished by the expression of Cubitus interruptus (Ci) in the prospective lamina cortex. Cells in two domains at the prospective dorsal and ventral margins of the adult optic lobe begin to express wingless in the mid-first instar stage. dpp expression begins after the onset of wg expression and continues in domains immediately adjacent to the Wg-positive cells. Two additional dorsoventral-specific markers are optomotor blind (omb) and aristaless. Omb is expressed in dorsal and ventral domains that include both the Wg- and Dpp-positive cell populations. Omb-positive glia migrate from these domains toward the lamina midline. aristaless, as assayed by the al04352 enhancer trap insertion (al-lacZ), is expressed in a graded pattern with respect to distance from the Wg-positive cells. The expression of omb, dpp, and al-lacZ is induced by ectopic wg+ expression and absent under conditions of wg loss of function. These observations indicate that Wg is responsible for the expression of these three markers (Song, 2000).
In the wild-type brain, retinal axons project in a crescent-shaped array into the lamina target field. Domains of dpp-expressing cortical cell precursors lie at the ends of the crescent-shaped retinal axon array. In cgk11504 animals, ingrowing retinal axons form an irregular pattern of projections, with axons often straying outside the normal target field. When assayed by either introduction of the dpp-lacZ reporter construct BS3.0 or by staining with anti-Dpp antibody, dpp expression was found to extend, in the cg mutant, toward the midline beyond the normal positions of its dorsal and ventral domains. The domains of omb expression also extend beyond their usual boundaries toward the midline. Expression of the al-lacZ reporter does not diminish in a graded fashion with distance from the Wg domains in cg brains. With respect to all three markers, and on the basis of morphology, a region centered about the dorsoventral midline is relatively unaffected by cg loss of function. Similar results have been obtained with the stronger cgDelta10 deletion allele and with cg1/cgk11504 heterozygotes. Thus cg loss of function results in an extension of dorsal and ventral cell identities toward the midline, while a region centered about the midline remains relatively unaffected (Song, 2000).
The cell autonomy of combgap function was determined by generating somatic cgk11504 clones using the FLP, FRT method. Within cg clones outside of the midline region, Omb, Dpp, and al-lacZ are all expressed ectopically. Clones or portions of clones that fall within the midline region (30% of those examined) appeared phenotypically normal, consistent with the lack of a cg requirement for the midline region in homozygous animals. There are also position-specific effects observed within cg clones. For example, not all cells within a cg clone expressed the marker Dpp. The position-specific ectopic gene activation in cg clones might reflect the activity of other signals involved in cortical cell fate determination. cg thus behaves as an autonomous repressor of omb, dpp, and al-lacZ expression, except in the midline region where it is not required (Song, 2000).
To place cg+ activity in the context of the Wg signal transduction cascade, cg homozygotes were examined in which Wg signaling was suppressed by the misexpression of the Drosophila axin gene homolog, Axn. It was supposed that expressing a UAS-Axn construct under the control of the omb-GAL4 (P{GawB}bimd65) driver would create a negative feedback loop in which omb expression would be suppressed. That is, wg+ activation of omb-GAL4 transcription would be countered by GAL4 driven Axn expression. This was indeed the case. Omb expression in omb-GAL4, UAS-Axn brains is greatly reduced in both dorsal and ventral domains and in the glia that migrate into the lamina field . The reduction of Omb expression varies among specimens; the complete absence of Omb expression in at least one domain is observed in ~30% of specimens. Dpp expression is also greatly reduced or undetectable in omb-GAL4, UAS-Axn brains. These effects on Omb and Dpp expression are associated with a defect in the projection pattern of the photoreceptor axons. Similar axon projection phenotypes have been observed with wg loss of function (Song, 2000).
The loss of cg function is epistatic to Axn misexpression. The developing visual ganglia of animals homozygous for cgk11504, harboring the omb-GAL4 and UAS-Axn transgenes, displays ectopic expression of the wg targets Dpp and Omb like that found in cgk11504 animals. Ectopic Axn does suppress the high level of Omb expression in the normal Omb domains in the cgk11504 background, consistent with the notion that high-level Omb expression remains wg+ dependent in cgk11504. A second set of experiments were performed utilizing the heat shock-inducible P{hsGAL4} driver to express the P{UAS-Axn} transgene. Multiple heat shocks during the larval period were applied to animals harboring the transgenes in either a wild-type or cg background. Similar results were obtained to those with the omb-GAL4 driver. These observations argue that cg functions downstream of Axn (Song, 2000).
The constellation of genes under Wingless control displays considerable tissue specificity. Wingless-controlled targets include Distal-less and vestigial in the wing, engrailed in the embryonic ectoderm, and sloppy-paired in the embryonic CNS. Though Dpp and Omb belong to a Hedgehog-controlled circuit in the wing, they are under Wg control in the visual cortices of the brain. With respect to the control of cell fate, Wg signal transduction apparently follows a canonical pathway from a pair of redundant receptors at the cell surface to the cytoplasmic control of Armadillo stability and nuclear translocation. This raises the question of how the tissue specificity of wg target gene expression is achieved (Song, 2000).
The observations that cg regulates dpp, optimotor blind and aristaless in the visual cortex place cg in a second tier of regulation, as a component of a tissue-specific relay mechanism between the Wg signal transduction pathway and the target genes that are wg dependent in visual system cortical cells. The evidence in support of this hypothesis is as follows: (1) epistasis analysis with the wg pathway negative regulator Axn places the requirement for cg downstream of the cytoplasmic complex that includes APC, GSK-beta, and Armadillo; (2) the induction of at least three downstream effectors of wg+ activity is mediated by negative regulation of cg expression -- cg expression is reduced in the dorsal and ventral domains of the cortical lamina where these wg target genes are expressed and ectopic cg expression blocks wg target gene expression within these domains; (3) ectopic wg+ clones repress cg expression, yielding Cg-negative domains in which wg target genes are ectopically expressed. The presence of consensus Pangolin binding sites in the first intron of cg suggests cg may be a direct target of Wg signal transduction. How the Armadillo/Pangolin complex might participate in the negative regulation of cg is unclear. Cg might act by binding directly to wg target gene regulatory elements as a transcriptional repressor (Song, 2000).
Responses to graded Dpp activity requires an input from a complementary and opposing gradient of Brinker (Brk), a transcriptional repressor protein encoded
by a Dpp target gene. Brk harbours a functional and transferable repression domain, through which it recruits the corepressors Groucho and CtBP. By analysing transcriptional outcomes arising from the genetic removal of these corepressors, and by ectopically expressing Brk variants in the embryo, it has been demonstrated that these corepressors are alternatively used by Brk for repressing some Dpp-responsive genes, whereas for repressing other distinct target genes they are not required. These results show that Brk utilizes multiple means to repress its endogenous target genes, allowing repression of a multitude of complex Dpp target promoters (Hasson, 2001).
In the wing imaginal disc, cells in the posterior compartment are programmed by the engrailed selector gene product to secrete Hedgehog (Hh), which induces dpp in a stripe of anterior cells along the A/P boundary. Dpp then acts as a long-range morphogen that governs patterning across the entire imaginal disc field. To determine whether Gro participates in the implementation of Hh signaling, clones overexpressing gro, or clones that are homozygous for the strong groE48 allele, were stained for dpp-lacZ expression. In all clones, even those overlapping with the Hh activity domain, there are no noticeable alterations in the dpp expression pattern, indicating that Gro is not required downstream of Hh for dpp transcriptional regulation. In striking contrast, however, three distinct targets of the Dpp pathway, expressed either in the wing pouch [optomotor-blind (omb) and vestigial (vg) or in the periphery of the wing disc (brk)], are repressed in clones overexpressing gro. Expression of omb-lacZ, as well as that of a lacZ reporter driven by vg's Dpp-responsive enhancer (vgQ-lacZ), is completely abrogated in these clones, whereas expression of brk-lacZ is only reduced. All three Dpp targets are repressed in a cell autonomous manner, i.e. only in the clones but never in adjacent cells. These results, together with an extensive gro loss-of-function clonal analysis detailed below, implicate Gro specifically as a downstream effector of Dpp signaling (Hasson, 2001).
Recent genetic and molecular studies have shown that brk encodes a repressor acting downstream of the Dpp pathway, which helps define the low end of the Dpp gradient. In particular, the Dpp targets omb and vgQ are both derepressed in brk- mutant clones and in brk- wing imaginal discs, suggesting that they are normally subjected to Brk repression. More directly, Brk binds to specific sequences within defined omb and vgQ enhancer elements, bringing about their silencing by outcompeting the Mad-Medea complex, or some other activator, from binding to overlapping DNA sites (Hasson, 2001).
Brk competes with an activator for binding to an omb wing enhancer, suggesting that, for this promoter, Brk should act independently of corepressors. Consistent with this, omb-lacZ is not ectopically expressed in cells homozygous for groE48 (hereafter referred to as gro- clones), nor is it affected by CtBP loss-of-function clones, generated using the l(3)87De-10 allele (CtBP-), or by CtBP-, gro- double mutant clones). Thus, single and double mutant clones for gro and CtBP do not phenocopy the omb derepression seen in brk- clones, implying that Brk can repress omb even in the absence of these corepressors. Repression of the Dpp target gene spalt (sal) is also independent of Gro and CtBP. Nonetheless, in gro overexpression clones, omb is repressed, suggesting that, even for the omb promoter, Gro reinforces Brk repressor function (Hasson, 2001).
omb and vgQ expression is completely shut off in clones of cells overexpressing gro, whereas that of brk is only reduced, suggesting that Brk might be repressing its own transcription via a negative autoregulatory loop. To establish whether, in negating its own expression, Brk is assisted by Gro and/or CtBP, gro- and CtBP- single, or CtBP-, gro- double mutant clones were stained for brk-lacZ expression. brk is never ectopically expressed in any of the single mutant clones, whereas ectopic brk expression is clearly observable in double mutant clones. Thus, in the absence of one corepressor, repression is adequately mediated by the other, suggesting that negative autoregulation by Brk is robust, relying on either Gro or CtBP (Hasson, 2001).
Brk utilizes a self-reliant mechanism, which need not depend on tethered corepressors, by competing with activators over coinciding DNA-binding sites. In the absence of both Gro and CtBP, Brk represses not only omb and zen, but also sal, suggesting that the Brk-binding site(s) in the sal promoter overlap with those employed by activators. Transcription of both sal and vgQ requires activation by Mad, yet, although both promoters are exposed to identical levels of pMad, the sal expression domain is spatially more restricted than that of vgQ, presumably because activation of sal requires higher levels of pMad than that of vgQ. Hence, 'passive' competition-based repression should efficiently block activation of sal but may not be sufficient for promoters like vgQ, which are activated even by low amounts of Mad. For silencing such promoters, alternative mechanisms such as recruitment of corepressors have evolved and are employed (Hasson, 2001).
Significantly, the overexpression of gro results in ectopic omb repression, suggesting that, even for promoters that are switched off in a 'passive', competitive manner, excess Gro can over-potentiate Brk-mediated negative transcriptional regulation. Thus, Gro and/or CtBP might reinforce Brk repression of those promoters on which it initially acts by competing with activators for binding to DNA, via recruitment of histone deacetylases and alterations to chromatin structure, or by some other mechanism (Hasson, 2001).
In summary, these data suggest that Brk uses multiple means to negate target gene expression, such as competition and the varied recruitment of long- and short-range corepressors. It is proposed that this versatility is, biologically, most significant given Brk's role in Dpp signaling, since it facilitates the negative regulation of diverse, complex Dpp target promoters (Hasson, 2001).
The abdomen of adult Drosophila consists of a chain of alternating anterior (A) and posterior (P) compartments which are themselves subdivided into stripes of different types of cuticle. Most of the cuticle is decorated with hairs and bristles that point posteriorly, indicating the planar polarity of the cells. This study has focused on a link between pattern and polarity. Previous studies have shown that the pattern of the A compartment depends on the local concentration (the scalar) of a Hedgehog morphogen produced by cells in the P compartment. Evidence is presented in this study that the P compartment is patterned by another morphogen, Wingless, which is induced by Hedgehog in A compartment cells and then spreads back into the P compartment. Both Hedgehog and Wingless appear to specify pattern by activating the optomotor blind gene, which encodes a transcription factor. A working model that planar polarity is determined by the cells reading the gradient in concentration (the vector) of a morphogen 'X' which is produced on receipt of Hedgehog, is re-examined. Evidence is presented that Hedgehog induces X production by driving optomotor blind expression. X has not yet been identified and data is presented that X is not likely to operate through the conventional Notch, Decapentaplegic, EGF or FGF transduction pathways, or to encode a Wnt. However, it is argued that Wingless may act to enhance the production or organize the distribution of X. A simple model that accommodates these results is that X forms a monotonic gradient extending from the back of the A compartment to the front of the P compartment in the next segment, a unit constituting a parasegment (Lawrence, 2002).
It has been concluded that Hh acts indirectly via another system (a gradient of 'X') to effect polarity. The evidence was based on clones that lacked such downstream genes as patched (ptc) or cAMP-dependent protein kinase 1 (Pka). In the A compartments, Ptc and Pka proteins act within cells to prevent the Hh pathway from being activated inappropriately; if either protein is removed the Hh pathway becomes constitutively activated within the mutant cells themselves. With respect to the type of cuticle (the scalar output of Hh) the results fit the model; the mutant cells make the cuticle normally made by cells responding strongly to Hedgehog and all the cells outside the clone make the normal type of cuticle (a cell-autonomous effect). However, with respect to polarity (the vectorial output of Hh), the results are different; polarity is altered in the wild-type cells up to several cell diameters away from the clone (a cell non-autonomous effect). Although it has been argued that these effects were not due to Hh itself, the possibility was not eliminated that low levels of ectopic Hh might be produced by the clone and diffuse out, being sufficient to repolarize the cells without changing the scalar. This study now disproves this possibility by making clones that lack both effective Ptc protein and the hh gene. These clones still cause repolarization in the back half of the clone and behind it arguing strongly that the Hh protein is a component of 'X' and raising again the question, what is X? X should be engendered downstream of Hh receipt, which is where the search is started (Lawrence, 2002).
omb encodes a transcription factor that is activated on receipt of high amounts of Decapentaplegic (Dpp) in both the A and the P compartments of the wing and elsewhere. It is expressed in each segment, both dorsally and ventrally, as a single stripe spanning the AP border and including the rear of the A compartment and the front region of the P. Accordingly, omb- clones in other parts of the segment are normal (Lawrence, 2002).
Within the posterior half of the A compartment, Omb is required for the normal scalar response to Hh. At the extreme back, in the a6 region, where the Hh concentration is highest, the omb- cells develop only a little abnormally: the unpigmented cuticle of that region (a6) is expanded a little anteriorly in the clone, but sometimes contains small 'a3' bristles. Note that specification of a6 cuticle normally requires engrailed activity, which is induced in A cells by peak levels of Hh. However, in omb- clones that are situated more anteriorly, in the pigmented region at the back of the A compartment (a4, a5), there is a big effect: it appears that Hh acts through omb, because omb- cells never make a4 cuticle or a5 bristles (pattern elements that signal a response to Hh), and in their stead make a3 cuticle (the type of cuticle made where there is little or no response to Hh). Also, Hh directly upregulates expression of ptc, which encodes a component of the Hh receptor and this also occurs in omb- clones. This finding indicates that Omb is not required for Hh signal transduction per se, but for the appropriate response of cells (Lawrence, 2002).
With regard to polarity, the clones confined to the anterior and middle part of the A compartment are normal. However, clones just behind the middle of the A compartment usually show reversal at the front, with normal polarization at the back. More strikingly, clones confined to the very back of the A compartment, in the a6, a5 and a4 domains can be largely or entirely reversed and this reversal usually extends anterior to the clone (Lawrence, 2002).
To explain these polarity changes, it is suggested that Hh induces X production through the agency of Omb. It follows that little or no X can be produced within omb- clones and therefore that the polarities of cells in or near such clones depend on X produced outside. Clones in the middle of the A compartment behave normally because most X is produced behind them and the gradients of X concentration are little changed. Clones located a little further back will have peaks of X both behind and in front, and this can cause localized reversal at the front of the clone. For a clone extending back to the AP boundary, the only source of X will be anterior to the clone, presumably because omb+ cells there will 'see' Hh protein that has passed through the clone. These cells should make X that spreads backwards into the clone, setting up a gradient of reversed polarity. There is corroborating evidence: in some clones there is dark pigmentation and large bristles develop anterior to the clone, confirming that Hh has indeed been received there. However, many omb- clones are associated with anterior repolarizations that occur even where there is no dark pigmentation anterior to the clone, suggesting that the level of Hh required to stimulate some X production anterior to the clone is less than that needed to make a4 pigment. It follows that, in normal flies, some X is produced by cells anterior to the a4 pigmented zone. Finally, it is found that some clones, which extend nearly to the back of A, show reversed territory behind the clone, perhaps due to the domination of the X source that is anterior to the clone over any production of X behind it (Lawrence, 2002).
It is noted that the reversed polarity associated with omb- clones located at the back of the A compartment usually extends only to the AP boundary, with polarity in the P compartment being normal. This result suggests that the AP boundary coincides with a barrier to the movement or action of X. The existence of such a barrier would provide an explanation for why X normally produced in cells at the back of the A compartment does not spread posteriorly into the P compartment, reversing the polarity in P. However, in rare cases, some reversed hairs were seen in what appeared to be adjacent P compartment cells, as marked independently by ptc.lacZ staining. It is not known whether these rare cases are artifactual, due to a slight posterior shift -- during mounting -- of the cuticle relative to the underlying epidermis, or are frank reversals of cells within the P compartment. If the reversed cells are indeed P cells, this raises a problem for the notion that the AP boundary constitutes a barrier to X movement (Lawrence, 2002).
If the production of X depends at least in part on omb, then ptc- clones, in which the Hh pathway has been constitutively activated, should produce little or no X if they also lack omb. Clones were made that were both ptc- and omb-; these clones form a6 cuticle as do ptc- clones. However, in the middle of the A compartment and unlike ptc- clones in that position, they fail to repolarize behind, but reverse their polarity in front -- as do omb- cells. Similarly, omb- ptc- clones situated at the back of the A compartment behave like omb- clones, the whole being reversed in polarity (and not like ptc- clones in the same location, that have normal polarity). Thus in terms of the type of the cuticle (the scalar), omb- ptc- behave as ptc- clones, but in terms of the vector they behave as omb- clones. These results confirm that Hh induces X production through the action of omb (Lawrence, 2002).
The model for X suggests that, if omb were ectopically activated in cells at the front of the A compartment, those cells could become a source of X. Indeed omb-expressing clones can repolarize the cells behind them -- as if there were a local peak in the X distribution (Lawrence, 2002).
smoothened (smo), is an essential component of Hh transduction; without it the cells cannot see Hh protein. As regards polarity, one would expect neither omb- nor smo- clones to produce X and for their phenotype to be the same. Although this is generally the case, the effects of smo- and omb- differ for clones located at the back of the A compartment. Polarity within these omb- clones is completely reversed, consistent with the model, whereas the corresponding smo- clones are reversed only within the anterior portion of the clone, polarity returning to normal at the very back of the A compartment. The preferred explanation for this discrepancy is that Smo protein perdures in smo- clones, allowing partial rescue of the smo mutant phenotype, particularly at the back of the A compartment, where Hh is most abundant. This rescue could allow production of X, enough to restore normal polarity at the back of the clone, but not enough to specify a4 cuticle or to upregulate ptc.lacZ. For both smo- and omb- clones, some Hh would be expected to move forward across the clone and induce an ectopic peak of X production in more anterior, wild-type cells, accounting for the polarity reversals that are observed in both cases (Lawrence, 2002).
To test this explanation Hh receipt was blocked by a different method that is not so subject to perdurance: a marked clone was made that contained no wild-type Ptc, but provided instead a mutant form of Ptc that is ineffective at transducing the Hh signal. Such clones behave like smo- clones in most respects, including making a3 cuticle instead of a4, a5 or a6 cuticle in the back half of the A compartment, and causing polarity reversals both within and anterior to the clone. However, unlike smo- clones, the polarity at the back of these clones does not return to normal. Instead, in the majority of cases, polarity remains reversed all the way to the back edge of the clone, and sometimes beyond, as observed for omb- clones in the same position. These results support the perdurance explanation for the smo- clones and are consistent with the working model, which is based mainly on the results with omb (Lawrence, 2002).
In clones mutant for arm or arrow, the expectation was that the Wg pathway in these two types of clones would be blocked. Two effects were noted. (1)The clones in the dorsal epidermis differentiated cuticle characteristic of the ventral epidermis: they made pleural hairs, and patches of sternite. Clones in all portions of the tergite, in both the A and P compartments, were transformed in this manner, indicating a general requirement for Wnt signaling to specify dorsal as opposed to ventral structures. Thus, in the wild type, all dorsal cells are probably exposed to at least low levels of Wg or some other Wnt protein.
(2) Such clones affect polarity: in the tergites, the mutant clones were normal at the rear of the clone but reversed in the front, with reversal extending outside the clone. One explanation for these polarity changes could be that, in the tergites, Wg normally acts to enhance the production of X. Thus cells deficient in the Wnt pathway would produce less X than normal, giving a dip in the concentration landscape for X, causing reversed polarity at the front of the clone. In the eye, both arm- and arrow- clones cause equivalent polarity reversals and a similar resolution has been offered: it is suggested that Wg might regulate the production of a secondary polarizing factor also dubbed X (Lawrence, 2002).
Thus, it is proposed that Wg helps to produce X, but that Wg itself is not X. If Wg were X, both arm- and arrow- clones should not be able to transduce it, and hence, should have random polarity within the clone. Moreover, the effects on polarity should be cell autonomous. Yet, as has been seen, these clones behave as if they have caused an altered distribution of X, rather than any failure to transduce X. Similar arguments apply to sgg- clones. In this case, the Wg pathway should be constitutively activated in all cells within the clone, preventing them from detecting a gradient of Wg protein. However such clones are not randomly polarized, indicating that they can still respond to graded X activity (Lawrence, 2002).
It is useful to compare the roles of Omb and Wg on X production. Omb is apparently essential for X production: omb- clones at the back of A show reversed polarity that extends all the way to the posterior edge of the compartment. By contrast, in arm- and arrow- clones, reversal occurs only in the anterior portions of such clones. Thus, it is inferred that arm- and arrow- cells located at the back of A can produce some X, even though they cannot activate the canonical Wnt pathway. Thus, it could be that Hh drives X production mainly through Omb, but also adds to the level of X produced through the induction and action of Wg. The combination of both Omb and Wg activity might extend the reach of the X gradient to encompass the whole A compartment, and possibly also further forward into the neighboring P compartment (Lawrence, 2002).
None of the previous studies has helped gain an understanding of how the P compartment is patterned or how its cells are polarized. smo- clones have no phenotype in the P compartment, confirming that Hh has no function there. In the embryo and imaginal discs, Hh crossing over from the P compartment induces the expression of Wg and Dpp in line sources along the back of A. Both proteins then spread back into the P compartment where they act as gradient morphogens to control P growth and pattern. Wg and Dpp are also produced at the back of the A compartment in each abdominal segment (albeit in distinct dorsal and ventral domains). Hence, by analogy with the embryo and imaginal discs, these morphogens seem to be the most likely candidates to pattern the P compartment here as well. If so, it would be supposed that in the tergites, Hh induces Wg and this Wg moves posteriorly across the AP compartment boundary into the P compartment where it activates expression of omb, thus specifying the zone of hairy cuticle (p3) and distinguishing it from p2 cuticle, which is bald. This hypothesis was tested in the following experiments (Lawrence, 2002).
Loss-of-function omb mutants tend to lose the hairy, unpigmented cuticle characteristic of both posterior A (a6) and anterior P (p3) regions, whereas gain-of-function mutations tend to acquire it. Since it was observed that omb- clones in the A compartment are able to make a6 cuticle, it seems likely that Omb is required specifically for the hairy, unpigmented cuticle (p3) that normally forms at the front of the P compartment. If so, one might expect omb- clones at the front of the P compartment to transform the anterior type of cuticle (p3) into that found more posteriorly (p2). Although most omb- clones were normal in this region, about one third of p3 clones lost some, but not all, of the hairs within the clone. Thus it appears that omb may be required in the p3 territory, as it is in the a5 and a4 territories, to specify the type of cuticle secreted (Lawrence, 2002).
If Wg activates omb in anterior regions of the P compartment, blocking the Wnt pathway in cells in the P compartment should block expression of omb. Expression of omb was therefore monitored in arrow- clones. omb is sometimes, but not always, turned off autonomously in the clone. Conversely, ectopic activation of the Wnt pathway should transform bald cuticle (p2) at the back of P into hairy cuticle (p3) normally found at the front of P. Indeed, some clones lacking the sgg gene become hairy if situated in the bald areas of P, apparently causing a transformation from p2 to p3 cuticle. But, clones expressing either tethered Wg or activated Arm, which should behave similarly, have no clear effects. Even so the positive results with arrow and sgg give support to the hypothesis that Wg stratifies the P compartment by working through Omb (Lawrence, 2002).
The heart of the model requires that a cell's polarity be determined by reading the local slope, the vector of a morphogen, X. Within the A compartment, it is proposed that X is produced in a gradient with its peak at the back of the A compartment and its minimum at the front. Hh is the primary morphogen that patterns the A compartment, and, at the rear of this compartment, it acts through omb to produce X. X spreads further to the anterior, forming a monotonic gradient that extends from the back of the A compartment and could go as far as the front of the next P compartment, thus encompassing a parasegment. In this model there might need to be a barrier to the movement of X across the AP (parasegment) border in order to isolate the X gradients in neighboring parasegments from one another. This model is speculative; for example there is no evidence for X spreading forward into the P compartment. In an alternative scenario, X might be made near the AP border, spreading forward into A and backward into P to form a reflected gradient. In that case, cells in the A and P compartments would have to make hairs that point in opposite directions relative to the vector of X, since all hairs point toward the posterior (Lawrence, 2002).
Although it is proposed that X is a long range morphogen, the results do not exclude models in which polarity depends on short range interactions between cells. Recent models for planar polarity concentrate mostly on this aspect of how cells become polarized, particularly on how proteins within cells become asymmetrically localized, and how such molecular polarity might propagate from cell to cell by localized recruitment of other proteins at the abutting cell membranes. These models can provide explanations for the local, non-autonomous perturbations of polarity that occur along the borders of mutant clones, but they do not readily explain the longer range effects of such clones nor how polarity is determined globally in the wild-type fly (Lawrence, 2002).
The model for X can be further elaborated, for example, polarity could depend on two cooperating morphogens, each operating in different directions. While X could emanate forward from the back of the A compartment, another polarizing gradient, 'Y' could be sourced from the front, or from the P compartment, and move backwards. Hairs would be subject to two separate and mutually supportive influences, pointing up the gradient of X and down the gradient of Y. More complex hypotheses of this sort have two main appeals: they might help explain how the polarity is determined across the AP border and they also might help in an understanding of why removal of genes needed for polarity, such as fz or four-jointed still gives near-normal flies with much of their polarity unscathed (Lawrence, 2002).
Patterning along developing body axes is regulated by gradients of
transcription factors, which activate or repress different genes above
distinct thresholds. Understanding differential threshold responses requires
knowledge of how these factors regulate transcription. In the
Drosophila wing, expression of genes such as omb and
sal along the anteroposterior axis is restricted by lateral-to-medial
gradients of the transcriptional repressor Brinker (Brk). omb is less
sensitive to repression by Brk than sal and is consequently expressed
more laterally. Contrary to previous suggestions, it has been shown that Brk cannot
repress simply by competing with activators, but requires specific repression
domains along with its DNA-binding domain. Brk possesses at least three
repression domains, but these are not equivalent; one, 3R, is sufficient to
repress omb but not sal. Thus, although sal and
omb show quantitative differences in their response to Brk, there are
qualitative differences in the mechanisms that Brk uses to repress them (Winter, 2004).
The simplest method of transcriptional repression involves competition with
an activator, and can operate at the level of DNA if the activator and the
repressor have the same, or overlapping, binding sites in an enhancer. In
theory, assuming a transcription factor is nuclear, it should only require a
DNA-binding domain to act in this fashion. Brk has been shown to possess an
N-terminal sequence-specific DNA-binding domain (DBD), and this study has identified several mutations in this domain that either completely
inactivate or reduce the activity of the protein, indicating that this
region is essential for Brk activity (Winter, 2004).
Previous studies suggested that Brk could function by competition, more
specifically, by competing with Mad for overlapping binding sites in vitro.
However, a nuclear localized Brk protein consisting primarily of the DBD,
BrkNLS, cannot repress any Brk target in vivo, including the
embryonic UbxB reporter, which has been shown to possess overlapping Brk and
Mad binding sites that Brk and Mad can compete for in vitro. It is
possible that BrkNLS cannot bind to DNA in vivo. However, a
modified protein, BrkNLSW, which is identical to BrkNLS
apart from the addition of the four amino acids WRPW that recruit the
co-repressor Gro, can repress targets, indicating that
BrkNLS should also be capable of binding to these targets in
vivo (Winter, 2004).
Competition has been proposed as a mechanism for many transcriptional
repressors. However, direct in vivo support for or against such proposals is
rare, at least of the sort presented here, i.e., testing, in vivo, the ability
of a protein consisting largely of a functional DBD, which has access to the
nucleus, to repress a target for which there is in vitro evidence for
overlapping binding sites with an activator. There is some in vivo evidence
that the Drosophila embryonic repressor Kruppel can repress a
synthetic enhancer containing overlapping binding sites with the activators
Dorsal and Bicoid. However, although this repression is CtBP-independent, and
further studies are required to rule out additional domains outside of the DBD
being required in a similar fashion to the 3R domain in Brk. The paucity of
good examples of binding-site competition in vivo in eukaryotes is in stark
contrast to that in prokaryotes, and raises the question of how common this phenomenon
really is in eukaryotes (Winter, 2004).
If Brk cannot repress by competition it must possess repression
domains/motifs, and previous studies identified interaction motifs for the
co-repressors CtBP and Gro (CiM and GiM). However, repression of at least one Brk target, omb, has been shown shown not to require CtBP or Gro. This is consistent with the demonstration that the protein produced by the endogenous mutant brkF138, which is truncated before the CiM and GiM, can still repress omb. Truncated proteins
that lack the CiM and GiM, BrkStop1, BrkEC and
BrkA, can also repress omb, but only if they contain a specific region between the DBD and CiM that has been classified as a third repression domain, 3R. Further studies are required to determine if 3R is a true autonomous repression domain, i.e., if it can function outside of Brk, or if it is more specific (for example, antagonizing activators such as Mad), and to determine what its specific
properties are (for example, how close do Brk sites have to be to activator
sites for 3R to be effective?) (Winter, 2004).
The three repression domains/motifs of Brk are not equivalent. Wild-type Brk and
proteins containing only a GiM, BrkNLSW, or only a CiM,
BrkStop1NAC, can repress both sal and omb, and
they are more effective at repressing sal than omb. Analysis of
gro and CtBP single and double mutant clones reveals that
Gro is required for normal repression of sal in wing discs, and that
CtBP can provide some, but not always complete, activity for the repression of
sal in the absence of Gro. By contrast, Gro and CtBP are not required for repression of omb (Winter, 2004).
The 3R domain (the region between the DBD and the CiM) is sufficient for Brk to repress omb and the UbxB enhancer
in embryos, but is deficient for the repression of sal. Furthermore,
misexpression of proteins possessing only the 3R domain (plus the DBD) are
much more effective at repressing omb than sal, i.e., the
converse of wild-type Brk or Brk possessing only a GiM or a CiM. Although some
results suggested that 3R may confer a limited ability to repress sal, this is probably indirect, because a previous study
demonstrated that sal requires Omb to be expressed, and if
omb is repressed directly, sal will be lost also. However, the possibility cannot be ruled out that high levels of proteins possessing only the 3R domain can repress sal directly (Winter, 2004).
Contradictory results were obtained regarding the ability of 3R to repress
the vg-QE. Expression of the vg-QE did show expansion in some
brkF138 clones, indicating that the truncated protein produced in this mutant (which only has the 3R repression domain) cannot efficiently repress this
enhancer. However, similar in vitro truncated proteins, such as
BrkStop1, could efficiently repress vg-QE expression when
misexpressed using the UAS/Gal4 system. Such a difference
could simply be a reflection of the high levels of expression achieved with
the Gal4/UAS system, and that, at physiological levels, the 3R domain is not
sufficient for complete repression of the vg-QE (Winter, 2004).
Whether a single repression domain is sufficient for Brk to repress a
particular target may depend upon the positioning of Brk sites in relation to
activator sites (or possibly the promoter) at that target. The UbxB reporter
has overlapping Brk and activator (Mad) sites. Analysis of an omb enhancer revealed that an important Brk site may also overlap with an activator.
Conversely, analysis of the cis-regulatory elements of the sal gene
indicate that activator and Brk sites are separated.
Proteins possessing only 3R can repress UbxB and omb, but not
sal, suggesting that 3R may only be sufficient for the repression of
genes in which the Brk sites are situated very close to activator sites (Winter, 2004).
Why does Brk possess at least three, probably four, independent repression
domains/motifs? There are two obvious answers: qualitative, different
repression domains/motifs are required for the repression of different
targets; quantitative, more domains/motifs provide greater repressor
activity. Other transcription factors have multiple repression domains and
there is evidence that they have these for either qualitative or quantitative
reasons, and, in some cases, both. For example, in the Drosophila
embryo, the pair-rule protein Runt requires Gro for the repression of one
stripe of the pair-rule genes, even skipped (eve) and
hairy, but not for the repression of engrailed. The gap protein Knirps represses different stripes of eve; for stripes 4 and 6 it requires CtBP, but for stripes 3 and 7, it does not. However, this
appears to be a quantitative difference, because increasing the levels of
Knirps allows it to repress stripes 4 and 6 even in the absence of CtBP. Similarly, Gro appears to increase the repressor activity of the Eve protein (Winter, 2004).
There is some difference in the ability of the three
domains/motifs in Brk to repress different targets. For example, the 3R domain
is sufficient for the normal repression of omb but not sal.
However, either the CiM or GiM appear to be sufficient for the repression of
both sal and omb, so
why does Brk need the 3R domain? In the absence of Gro and CtBP, the Brk
protein appears fully active in its ability to repress omb, and
recruiting Gro and CtBP does not seem to increase its activity towards
omb; otherwise, the width of the omb domain would be
expected to shift in brkF138 mutant cells, which have no
CiM or GiM, or in CtBP gro double mutant cells, but Brk protein does not. It is
possible that, in regard to omb, the 3R domain is more efficient than
either of the other two and provides Brk with sufficient activity to establish
the omb domain in the correct position (Winter, 2004).
Brk needs to recruit either CtBP or Gro for the repression of some targets,
including sal and brk itself, or just
Gro for some others, including the vg-QE. Consequently, why does Brk need to recruit CtBP? Mutation of the CiM alone, in
common with mutation or deletion of just the GiM and 3R, does reduce activity
of Brk, as judged by its effect when misexpressed. However, there is no
evidence that CtBP is required specifically for the repression of any Brk
target in the wing, because CtBP mutant clones have no effect on the
expression of any known Brk target in the wing. The
CtBP and Gro motifs in Brk have been conserved over millions of years, and thus, recruiting CtBP is presumably important for Brk activity. It is possible that CtBP is
required outside of the wing -- for example in the embryo --
or for some other, as yet, uncharacterized targets in the wing (Winter, 2004).
Recruiting both CtBP and Gro does appear to be a little illogical from what
is known about their basic properties, CtBP acting only over a short range,
while Gro acts over much longer ranges. It might be assumed that different
transcription factors would use either Gro or CtBP,
because the primary advantage of recruiting CtBP is that it would allow a
transcription factor to repress one enhancer without disrupting the activity
of one nearby, which would be repressed if Gro was recruited, although this
simple model does not always hold. Consequently, most transcription factors do recruit only one of these co-repressors. However, there are two other exceptions,
Hairy and Hairless. In Hairy it appears that CtBP may actually be functioning
to antagonize Gro activity and not in its standard role as a co-repressor. There
is no evidence that it does this in Brk, where it can provide repressor
activity. For Hairless, there is genetic evidence that both CtBP and Gro
provide repressor activity to the protein,
although it is not clear if CtBP is required to increase the general activity
of Hairless, or for repression of specific targets that cannot be repressed
adequately by Gro (Winter, 2004).
With the exception of the brkF138 mutant, this analysis
has been limited to analyzing the effects of misexpressing modified Brk
proteins in positions where the endogenous protein is not found. Consequently,
further insights into the precise roles of individual repression domains will
require replacing the endogenous gene with one in which only one or two
repressions domain have been mutated or deleted (Winter, 2004).
To conclude, it is often assumed that the sensitivity of one enhancer to a
transcription factor compared with that of another enhancer is based largely
upon the number or the affinity of the binding sites for that transcription
factor in each enhancer. However, other factors are also important; for
example, the ability of the Giant transcription factor to repress a promoter
is related to how closely it binds. This study has shown that the two best characterized outputs of the Dpp morphogen gradient, sal and omb, appear to be regulated differently by Brk. Consequently, simply counting binding
sites and measuring their affinity will not reveal why one is more sensitive
to Brk than the other, and it is necessary to factor in what specific repressive
mechanisms are being used, and the relative efficiencies of each (Winter, 2004).
The development of the Drosophila wing is governed by the action of morphogens encoded by decapentaplegic and wingless that promote cell proliferation and pattern the wing. Along the anterior/posterior (A/P) axis, the precise expression of dpp and its receptors is required for the transcriptional regulation of specific target genes. The function of the T-box gene optomotor-blind (omb), a dpp target gene, was analyzed. The wings of omb mutants have two apparently opposite phenotypes: the central wing is severely reduced and shows massive cell death, mainly in the distal-most wing, and the lateral wing shows extra cell proliferation. Genetic evidence is presented that omb is required to establish the graded expression of the Dpp type I receptor encoded by the gene thick veins (tkv) to repress the expression of the gene master of thick veins and also to activate the expression of spalt (sal) and vestigial (vg), two Dpp target genes. optomotor-blind plays a role in wing development downstream of dpp by controlling the expression of its receptor thick veins and by mediating the activation of target genes required for the correct development of the wing. The lack of omb produces massive cell death in its expression domain, which leads to the mis-activation of the Notch pathway and the overproliferation of lateral wing cells (del Alamo Rodriguez, 2004).
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