Transcriptional Regulation (part 2/2)

Spalt in imaginal discs

Through its receptors Thickveins and Punt, Decapentaplegic targets 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. The finding of an extended range of action for DPP is unexpected, yet DPP diffusion away from its site of expression may be limited by its tendency to be sequestered by components of the extracellular matrix. 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).

Localized expression of the transforming growth factor-beta (TGF-beta) homologue decapentaplegic (dpp) is crucial for Drosophila wing development. spalt and spalt-related (sal and salr), two closely related genes that encode transcription factors, are expressed in response to dpp in a central territory of the wing imaginal disc, where they are required for the patterning of the wing. They are among the first identified elements that act downstream of dpp in wing development. The phenotypic consequences of misexpression of sal and salr suggest that an important outcome of dpp activity is the subdivision of the wing disc into territories smaller than lineage compartments, through the regulation of transcription-factor-encoding genes such as sal and salr (de Celis, 1996).

The imaginal disk expression of the TGF-ß superfamily member DPP in a narrow stripe of cells along the anterior-posterior compartment boundary is essential for proper growth and patterning of the Drosophila appendages. DPP receptor function was examined to understand how this localized DPP expression produces its global effects on appendage development. Clones of saxophone (sax) or thick veins (tkv) mutant cells, defective in one of the two type I receptors for DPP, show shifts in cell fate along the anterior-posterior axis. In the adult wing, clones that are homozygous for a null allele of sax or a hypomorphic allele of tkv show shifts to more anterior fates when the clone is in the anterior compartment and to more posterior fates when the clone is in the posterior compartment. The effect of these clones on the expression pattern of the downstream gene spalt-major also correlates with these specific shifts in cell fate. The shift in cell fate is explained by assuming that the cells in mutant clones act as though they see a lower than normal DPP concentration. Thus cell fate along the A/P axis is directly related to the perceived DPP level. It is concluded that cell fate is directly related to the distance of cells from the source of DPP at the A/P axis and that DPP is responsible for patterning of the entire wing blade in direct response to the long-range DPP signal. The similar effects of sax null and tkv hypomorphic clones indicate that the primary difference in the function of these two receptors during wing patterning is that TKV transmits more of the DPP signal than does SAX. These results are consistent with a model in which a gradient of DPP reaches all cells in the developing wing blade to direct anterior-posterior pattern. While current evidence suggests that TKV is absolutely required for DPP signaling, there appears to be no such absolute requirement for SAX. Thus DPP receptor complexes that lack a TKV subunit cannot transmit a sufficient level of DPP signal to trigger a biological response in the receiving cell. In contrast, receptor complexes lacking SAX subunits are still capable of significant signal reception and downstream signaling (Singer, 1997).

The Vestigial and Scalloped proteins act together to directly regulate wing-specific gene expression in Drosophila: Targeted expression of Vg with the Gal4-UAS system induces ectopic expression of Sal and SRF in developing leg imaginal discs

A small number of major regulatory (selector) genes have been identified in animals that control the development of particular organs or complex structures. In Drosophila, the vestigial gene is required for wing formation and is able to induce wing-like outgrowths on other structures. Because ectopic expression of Vg in many imaginal discs induces the outgrowth of wing tissue, the expression of various wing patterning genes was examined to see if they are induced in ectopic growths. Vg is expressed in the entire developing wing pouch whereas Sal and SRF have specific expression patterns within this domain but are not expressed in wild-type leg discs. Targeted expression of Vg with the Gal4-UAS system induces ectopic expression of Sal and SRF in developing leg imaginal discs. Similarly, the nubbin (nub) gene (which is also expressed and required during wing development ) is ectopically induced in leg discs by Vg expression. In each case, only a subset of the cells expressing Vg activate the target gene, which suggests that additional factors control the expression pattern of each gene. In a first step toward elucidating the molecular mechanism by which Vg regulates gene expression, the response of wing-specific enhancers to ectopic Vg expression was examined. Attention was focused on both the boundary and quadrant enhancers of the vg gene and the enhancer from the SRF gene that drives expression specifically in the intervein region between veins three and four. All three enhancers are induced by ectopic Vg expression in leg and other imaginal discs. Importantly, ectopic expression of Vg in clones of cells induces the enhancers only within the clones. However, gene expression is not induced in all cells within clones nor in all clones. In addition, each individual enhancer is expressed in different regions of these discs that appear to correlate with the spatial distribution of the different signaling inputs known to be required for activation of these enhancers (Halder, 1998).

The Dpp gradient regulates spalt

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).

Synergistic signaling by two BMP ligands through the SAX and TKV receptors controls wing growth and patterning in Drosophila: Regulation of Spalt by the Dpp gradient

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).

Brinker regulates spalt

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).

Distal-less and homothorax regulate spalt to pattern the Drosophila antenna

The Distal-less gene is known for its role in proximodistal patterning of Drosophila limbs. However, Distal-less has a second critical function during Drosophila limb development, that of distinguishing the antenna from the leg. The antenna-specifying activity of Distal-less is genetically separable from the proximodistal (PD) patterning function because certain Distal-less allelic combinations exhibit antenna-to-leg transformations without proximodistal truncations. Distal-less has been shown to act in parallel with homothorax (a previously identified antennal selector gene) to induce antennal differentiation. While mutations in either Distal-less or homothorax cause antenna-to-leg transformations, neither gene is required for the others expression, and both genes are required for antennal expression of spalt. Coexpression of Distal-less and homothorax activates ectopic spalt expression and can induce the formation of ectopic antennae at novel locations in the body, including the head, the legs, the wings and the genital disc derivatives. Ectopic expression of homothorax alone is insufficient to induce antennal differentiation from most limb fields, including those of the wing. Distal-less therefore is required for more than induction of a proximodistal axis upon which homothorax superimposes antennal identity. hth encodes a TALE-class homeodomain protein required for the nuclear localization of a PBC-class homeodomain protein encoded by extradenticle. Based on their genetic and biochemical properties, it is proposed that Homothorax and Extradenticle may serve as antenna-specific cofactors for Distal-less (Dong, 2000).

The Drosophila antenna is a highly derived appendage required for a variety of sensory functions including olfaction and audition. To investigate how this complex structure is patterned, the specific functions of genes required for antenna development were examined. The nuclear factors, Homothorax, Distal-less and Spineless, are each required for particular aspects of antennal fate. Coexpression of Homothorax, necessary for nuclear localization of its ubiquitously expressed partner Extradenticle with Distal-less is required to establish antenna fate. This study tests which antenna patterning genes are targets of Homothorax, Distal-less and/or Spineless. Antennal expression of dachshund, atonal, spalt, and cut requires Homothorax and/or Distal-less, but not Spineless. It is concluded that Distal-less and Homothorax specify antenna fates via regulation of multiple genes. Phenotypic consequences of losing either dachshund or spalt and spalt-related from the antenna are reported. dachshund and spalt/spalt-related are essential for proper joint formation between particular antennal segments. Furthermore, the spalt/spalt-related null antennae are defective in hearing. Hearing defects are also associated with the human diseases Split Hand/Split Foot Malformation and Townes-Brocks Syndrome, which are linked to human homologs of Distal-less and spalt, respectively. It is therefore proposed that there are significant genetic similarities between the auditory organs of humans and flies (Dong, 2002).

There are only a few genes expressed in either the antenna or the leg but not in both. Among these are sal and salr, which are identically expressed in a ring pattern in presumptive a2, but are detected at low levels only in leg imaginal disc cells that contribute to the body wall and not to the leg itself (Dong, 2002).

sal and salr have similar sequences and are identically expressed in the antennal imaginal disc in presumptive a2. However, functions for sal and salr in the antenna have not yet been described. To investigate whether sal and/or salr are required for normal antenna development, clones null either for sal alone or for both sal and salr in the adult head were examined. Clones null for only sal in the antenna have no obvious cuticular phenotypes. However Df(2L)32FP-5 clones, which are null for both sal and salr, exhibit cuticular defects in the antenna. This supports the view that sal and salr have some redundant functions. The areas affected in the mutants are correlated with their expression domains in the antennal disc (Dong, 2002).

a2 normally forms a cup, in which a3 sits and must rotate along the PD axis, to transmit sound vibrations from the arista. An overall reduction in a2 is observed in salFCK–25/Df(2L)32FP-5 transheterozygous null antennae. In addition, a2 appears to be fused to a3 and a portion of the stalk that connects a3 to a2 is exposed. The circular outline of the a2/a3 joint, to which the chordotonal organs of the JO attach, is defective in Df(2L)32FP-5 clones and lost in salFCK–25/Df(2L)32FP-5 mutant antennae. Furthermore, a3 is unable to rotate in a2. The same antenna phenotypes are observed in salFCK–25 homozygous flies. However, these phenotypes are not observed in sal null clones generated using a sal16 FRT40A chromosome or in salFCK–25/sal16 transheterozygous antennae, that do not express sal but do express salr in the antenna. Together, the loss of the a2/a3 joint and the loss of the freedom of rotation of a3 in a2 indicate that sal/salr null antennae are defective in hearing and implicate both sal and salr in normal development of the auditory organ (Dong, 2002).

Since ato is expressed within a subset of the sal/salr domain and is activated later than sal and salr in the antenna, tests were performed to see whether Dll and hth activate ato via sal/salr. No detectable reduction of ato expression is found in a2 either in Df(2L)32FP-5 clones or in salFCK–25/Df(2L)32FP-5 transheterozygous animals. This allelic combination lacks detectable sal and salr expression in the antenna, but retains sal and salr expression in the eye. The normal expression of ato in the antennae of these mutants suggests that the activation of ato expression by Dll and hth is independent of sal/salr. Antennal sal/salr expression is also unaffected in ato null imaginal discs. Therefore, sal/salr and ato are required in parallel for development of antennae that are functional in audition (Dong, 2002).

Dll and hth are required for the expression of sal in the antenna. sal expression does not appear to be affected in ss null antenna. The fact that Ss is not required for the expression of either ato or sal in a2 is consistent with the observation that the a2/a3 joint is still present in the ss null antenna (Dong, 2002).

sal and salr, like ato, are required for normal auditory functions. Since both Dll and hth are required for the antennal expression of ato and sal, Dll and hth mutant antennae are also hearing defective. In contrast, ss null antennae exhibit normal expression of both ato and sal and normal morphology of the a2/a3 joint, leading to the idea that ss mutants are likely to be functional in audition (Dong, 2002).

Homeotic genes, Dll and hth, regulate multiple targets during antennal development. These targets function in specifying antenna structures and/or in repressing leg development. For example, the ss mutant phenotype suggests that it represses leg tarsal differentiation. But ss is also required for the formation of olfactory sensory sensilla normally found in a3. Although Dll and hth repress distal leg development via activation of ss, their repression of medial leg development appears to be, at least in part, independent of ss. Instead, this is achieved via their regulation of the medial leg gene, dac, to a narrower domain of expression with lower levels in the antenna as compared to the leg. sal/salr and ato are required for proper differentiation of a2. However, no transformation phenotypes are associated with the sal/salr and ato null antenna. This indicates that while sal/salr and ato are required to make particular antenna-specific structures, they do not appear to repress leg fates. Therefore homeotic genes such as Dll and hth repress the elaboration of other tissue fates in addition to activating genes required for the differentiation of particular tissues (Dong, 2002).

In third instar imaginal discs, coexpression of Dll and Hth activates sal/salr and ato in a2 where they, in turn, are needed for JO development. The expression of ato is required for the formation of the JO and the a2/a3 joint to which it is attached. Although sal and salr are not required for the expression of ato, the a2/a3 joint is lost in the sal/salr null antenna. It is expected that this leads to improper formation of the JO, although it is also possible that defects in a2/a3 joint formation preclude JO differentiation. In addition, because sal is not lost in ato null antennae, it is concluded that sal/salr and ato are required in Drosophila parallel for proper formation of the JO. Furthermore, in the sal/salr null antenna, a3 cannot freely rotate within a2. This rotation is necessary for transmission of sound vibrations from the arista to the JO. Taken together, these findings implicate sal/salr in Drosophila audition. Interestingly, mutations associated with the human homolog of sal, SALL1 cause the human autosomal dominant developmental disorder, Townes-Brocks Syndrome (TBS). Auditory defects are also associated with the human genetic disorder, Split Hand/Split Foot Malformation (SHFM), and the SHFM1 locus is linked to the Dll homologs, DLX5 and DLX6. The sensorineural hearing defects associated with the Distal-less and spalt genes in both Drosophila and Homo sapiens, in conjunction with a recent finding that atonal functions in mouse as well as fly audition, leads to the proposal that insect and vertebrate hearing share a common evolutionary origin. Further developmental genetic dissection of the Drosophila auditory system should therefore provide additional insights into human ear development and suggest that Drosophila could provide a useful model system for studying both TBS and SHFM (Dong, 2002).

Schnurri targets spalt

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).

Control of a genetic regulatory network by a selector gene: Vg regulates spalt

The formation of many complex structures is controlled by a special class of transcription factors encoded by selector genes. It has been shown that Scalloped, the DNA binding component of the selector protein complex for the Drosophila wing field, binds to and directly regulates the cis-regulatory elements of many individual target genes within the genetic regulatory network controlling wing development. Furthermore, combinations of binding sites for Scalloped and transcriptional effectors of signaling pathways are necessary and sufficient to specify wing-specific responses to different signaling pathways. The obligate integration of selector and signaling protein inputs on cis-regulatory DNA may be a general mechanism by which selector proteins control extensive genetic regulatory networks during development (Guss, 2001).

The discovery of genes whose products control the formation and identity of various fields, dubbed 'selector genes', has enabled the recognition and redefinition of fields as discrete territories of selector gene activity. Although the term has been used somewhat liberally, two kinds of selector genes have been of central interest to understanding the development of embryonic fields. These include the Hox genes, whose products differentiate the identity of homologous fields, and field-specific selector genes such as eyeless, Distal-less, and vestigial-Scalloped (vg-sd) whose products have the unique property of directing the formation of entire complex structures. The mechanisms by which field-specific selector proteins direct the development of these structures are not well understood. In principle, selector proteins could directly regulate the expression of only a few genes, thus exerting much of their effect indirectly, or they may regulate the transcription of many genes distributed throughout genetic regulatory networks (Guss, 2001).

In the Drosophila wing imaginal disc, the Vg-Sd selector protein complex regulates wing formation and identity. Sd is a TEA-domain protein that binds to DNA in a sequence-specific manner, whereas Vg, a novel nuclear protein, functions as a trans-activator. To determine whether direct regulation by Sd is widely required for gene expression in the wing field, the regulation of several genes that represent different nodes in the wing genetic regulatory network and that control the development of different wing pattern elements were analyzed. Focus was placed in particular on genes for which cis-regulatory elements that control expression in the wing imaginal disc have been isolated, including cut, spalt (sal), and vg (Guss, 2001).

First it was tested whether sd gene function is required for the expression of various genes in the wing field. Mitotic clones of cells homozygous for a strong hypomorphic allele of sd were generated and the expression of gene products or reporter genes was assessed within these clones. Reduction of sd function reduces or eliminates the expression of the Cut and Wingless (Wg) proteins and of reporter genes under the control of the sal 10.2-kb and the vg quadrant enhancers, demonstrating a cell-autonomous requirement for selector gene function for the expression of these genes in the wing field (Guss, 2001).

These results, however, do not distinguish between the direct and indirect regulation of target gene expression by Vg-Sd. To differentiate between these possibilities, whether the DNA binding domain of Sd could bind to specific sequences in cut, sal, and vg wing-specific cis-regulatory elements were tested. Using DNase I footprinting, Sd-binding sites were identified in all of the elements assayed. Thus, Sd may control the expression of these genes by binding to their cis-regulatory elements (Guss, 2001).

To determine whether Sd binding to these sites is necessary for the function of these cis-regulatory elements in vivo, specific Sd-binding sites within each of the elements were mutated such that they reduced or abolished Sd binding in gel mobility-shift assays. The mutation of tandem Sd-binding sites in the cut and sal elements results in complete loss of reporter gene expression in vivo. Similarly, mutation of the four single Sd-binding sites identified in the vg quadrant enhancer eliminated or dramatically reduced reporter gene expression. These results show that Sd binds to and directly regulates the expression of four genes (cut, sal, vg, and DSRF) in the wing genetic regulatory network. This molecular analysis and the genetic requirement for Sd function for the expression of other genes suggest a widespread requirement for direct Vg-Sd regulation of genes expressed in the wing field (Guss, 2001).

Each of the Sd targets analyzed is activated in only a portion of the wing field, in patterns controlled by specific signaling pathways. For instance, cut is a target of Notch signaling along the dorsoventral boundary, and the sal and vg quadrant enhancers are targets of Dpp signaling along the anteroposterior axis. Binding sites for the transcriptional effectors of the Notch- and Dpp-signaling pathways, Suppressor of Hairless [Su(H)], and Mothers Against Dpp (Mad), and Medea (Med), respectively, have been shown to be necessary for the activity of a number of wing-specific cis-regulatory elements, and occur in these elements. This observation, coupled with the data demonstrating a direct requirement for Sd binding, suggests that gene expression in the wing field requires two discrete inputs on the cis-regulatory DNA: one from the selector proteins that define the field, and one from the signaling pathway that patterns the field (Guss, 2001).

The role of the T-box gene optomotor-blind in patterning the Drosophila wing: Omb activates spalt

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).

Repression of spalt in the Drosophila wing by Brinker

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).

Inverse regulation of target genes at the brink of the BMP morphogen activity gradient

BMP-dependent patterning in the Drosophila melanogaster wing imaginal disc serves as a paradigm to understand how morphogens specify cell fates. Profile of the transcriptional response to the graded signal of BMP, relies upon two counter active gradients of pMad and Brinker (Brk). This patterning model is inadequate to explain the expression of target genes, like vestigial and spalt, in lateral regions of the wing disc where BMP signal decline and Brk levels peak. This study shows that in contrast to the reciprocal repressor gradient mechanism, where Brk represses BMP targets in medial regions, in lateral regions target expression is downregulated by BMP signaling and activated by Brk. Brk induces lateral expression indirectly apparently through repression of a negative regulator. These findings provide a model explaining how the expression of an established BMP target is differentially and inversely regulated along the anterior-posterior axis of the wing disc (Ziv, 2012).

During Drosophila wing disc development the BMP morphogenetic gradient established by the collective actions of the two BMP ligands, Dpp and Gbb, patterns the cellular field by modulating gene expression in a concentration-dependent manner. How is the BMP concentration gradient translated into coordinated target gene expression? Current model relies upon two opposing activity gradients of the transcriptional regulators pMad and Brk, established in response to the BMP gradient. This model has been particularly successful in elucidating the regulatory influence exerted by BMP signaling although most of the attention has been focused on wing pouch, a region proximal to the peak of the gradient. Moreover, it is assumed that the target gene expression, in lateral regions where the BMP activity gradient decline, is independent of the signaling influence. Data presented in this study demonstrate that this supposition is incorrect. Alterations in pMAD signaling even in the lateral regions of the wing disc indeed lead to changes in positive target gene expression such as vg and sal albeit in an unexpected manner. In a classic 'role reversal' mode, the expression of the same targets is positively regulated by Brk and negatively by BMP signaling. Thus, while the classical morphogen model assumes that morphogens pattern a homogeneous field of responding cells, this study shows that in the developing wing disc interpretation and response to the BMP morphogenetic signal qualitatively differ along the anterior-posterior axis. The fact that sal is already expressed in both medial and lateral regions of the wing disc during early second larval stage, implies that this subdivision occurs early in development disc (Ziv, 2012).

By comparing sal expression in the different regions of the wing disc, this study has uncovered a novel circuitry underlying inverse regulation of an archetypal BMP target gene in distant regions where the morphogen levels decline. How is this counter regulation of sal achieved? Using the enhancer fragment that drives sal expression just in the lateral regions of the wing disc, evidence is provided that Brk induces expression of sal at the periphery of the wing disc indirectly through repression of a negative regulator (NRS). In one case, NRS represses sal expression by binding to a cis-regulatory element that contains neither Brk nor pMad-Med-Shn complex binding sites. In another, NRS is itself negatively regulated by Brk. The experiments described in this paper provide an initial working model to explain how the expression pattern of an established BMP target like sal is in fact differentially and inversely regulated in different regions along the anterior-posterior axis of the wing disc disc (Ziv, 2012).

Phenotypic consequences of the loss of brk on sal expression are qualitatively distinct and the effects vary in a position dependent manner. Compromising brk function results in upregulation of sal in the central region shows no effect towards the edge of the wing pouch and leads to a loss of sal expression in the periphery of the disc disc (Ziv, 2012).

Brk is known to repress lateral expression of classic BMP responsive genes such as omb and dad through direct binding to specific sequences within their enhancers, indicating that it is highly active in lateral regions. Since the medial enhancer of sal also contains two Brk consensus-binding sequences, this raises the question as to why the medial enhancer does not function to repress sal in the lateral zone? While the precise mechanism underlying this position dependent-repression is not known, the fact that a P-lacZ reporter of the sal medial enhancer is induced in lateral regions upon removal of Brk, indicates that in isolation the medial enhancer responds to Brk repressive activity also in the periphery of the wing disc. Similarly, other studies have found that mutating the relevant Brk binding sites in the isolated medial enhancer of sal expanded the expression to lateral regions. Combined together, these observations suggest that in the context of the full-length endogenous promoter of sal, (yet unknown) trans-factors are differentially distributed along the A-P axis of the developing wing disc to prevent repression by Brk (via the medial enhancer) in lateral regions and thus to confer position-dependent expression (Ziv, 2012).

This analysis implies that Brk drives endogenous sal expression in region IV by repressing a negative regulator of sal (NRS), which targets the lateral enhancer. Brk levels, which decline medially, enable NRS to be active which in turn represses sal expression in lateral regions of the wing pouch (region III). By manipulating Brk levels and monitoring the activity of the AK-lacZ reporter, evidence is provided that NRS is normally expressed and active all along the wing pouch (regions I, II and III). This raises the question as to how endogenous sal in the center of wing pouch escapes repression mediated by NRS. In principle, high pMad activity in medial regions (I and II) could overcome the repressive function of NRS. While plausible, this is an unlikely scenario as in the absence of both brk and mad, sal is ectopically expressed in medial regions. It is therefore proposed that in the context of the endogenous promoter, the activity of NRS is antagonized specifically in center of the wing pouch (regions I and II) with the assistance from the localized, non-uniform distribution of trans-acting factors along the A-P axis of the wing disc to confer a position-dependent transcriptional response (Ziv, 2012).

In these experiments sal is ectopically induced near the edge of the wing pouch (region III) in tkv mutant clones. Even more perplexingly tkvQD overexpressing clones also behave in a similar manner. Both of these outcomes are difficult to reconcile with the current model describing how the BMP morphogen gradient is linearly interpreted as both extreme situations; either complete loss (tkv) or substantial gain (tkvQD) of endogenous pMad activity, results in ectopic expression of sal . However, this conundrum can be partially resolved by taking into account the activity of the newly invoked, additional component NRS into the BMP-dependent patterning system. In wild type wing disc, pMad activity in region III, although low, is still sufficient to downregulate Brk levels just enough to allow for concomitant rise in NRS levels ultimately resulting in repression of sal expression. Importantly, low levels of pMad in region III [acting through the medial enhancer (ME)] are inadequate to antagonize the repressor activity of NRS [mediated through the lateral enhancer; (LE)]. The absence of pMad activity in tkv mutant clones in region III increases the levels of Brk, which in turn represses NRS and thus de-represses sal expression. In the case of tkvQD overexpressing clones, the substantially elevated pMad activity represses brk expression leading to elevated NRS activity. In region III clones (periphery of the wing pouch) the high activity of pMad (acting from the ME) overcomes the repressing activity of NRS (acting from the LE), ultimately resulting in activation of sal. By contrast, in the vast majority of tkvQD overexpressing clones (57 out of 63 clones) located in region IV (periphery of the wing disc) endogenous expression of sal is either lost or diminished. Why in region IV (periphery of the wing disc) pMad inducing activity does not have an edge over NRS repressing activity, as is the case in region III? It is proposed that in the context of the endogenous promoter, the activity of pMad is antagonized specifically in region IV due to the activity of unevenly distributed trans-acting factors. The rare occasions, where in the tkvQD overexpressing clones located in region IV sal expression was upregulated could be due to a rare event leading to acquisition of wing pouch like identity by the cells at the wing disc periphery, presumably due to early exposure to high pMad activity. Nevertheless, differential transcriptional response behavior exhibited by the cells from regions III v/s IV supports the subdivision of the developing wing disc on the basis of distinct regional competence (Ziv, 2012).

How an exponentially decaying morphogen gradient of BMP gives rise to computable changes in gene expression ultimately leading to discreet morphological structures is a fascinating question. A steep slope of the BMP activity gradient near the peak allows sharp expression domains of target genes to be defined within the wing pouch area. However in the lateral regions of wing disc, the activity gradient of BMP dips considerably raising the question as to how small differences in signal strength provide discrete threshold responses. Indeed, it is believed that BMP/Brk patterning system does not regulate the lateral expression of sal. These data argue that not only the signaling is active in the lateral regions but the inverse regulatory mode adopted by the signaling circuitry is in fact responsible for generating distinct threshold responses (Ziv, 2012).

back to part 1/3

spalt: Biological Overview | Evolutionary Homologs | Targets of Activity | Developmental Biology | Effects of Mutation | References

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