Distal-less

Promoter Structure

There are independent enhancers, arrayed over 40 kb of DNA. Head and leg synthesis is independently regulated. One distal 5' enhancer sequence, an early enhancer regulating Dll expression in germ band extended embryos, is subject to repression by Ultrabithorax and abdominal-A (Vachon, 1992).

A new technique is described using fluorescent probes to simultaneously detect an mRNA and a protein in the Drosophila embryo. For in situ hybridization, 3-hydroxy-N-2'-biphenyl-2-naphthalenecarboxamide phosphate ester (HNPP)/Fast Red TR is used as a fluorescent substrate for alkaline phosphatase. It is possible to compare protein and mRNA expression on a cell by cell basis with a laser scanning confocal microscope. This technique was applied to analyse the dynamics of Distal-less (Dll) enhancer activity in the thoracic limb primordium in the early Drosophila embryo. Embryos were stained bearing the Dll early enhancer (Dll-304) fused to the Escherichia coli lacZ gene. LacZ mRNA is detectable in the ventral region of the limb primordium; beta-galactosidase protein is detectable in the dorsal region. In the middle, both mRNA and protein are detectable. These results suggest that the Dll enhancer is activated in the ventral region of the limb primordium and that Dll-positive cells migrate from a ventral position to a dorsal one within a single limb primordium (Goto, 1997b).

Activity regulation of Hox proteins, a mechanism for altering functional specificity in development and evolution

The closely related Hox transcription factors Ultrabithorax (Ubx) and Antennapedia (Antp) respectively direct first abdominal (A1) and second thoracic (T2) segment identities in Drosophila. It has been proposed that their functional differences derive from their differential occupancy of DNA target sites. A hybrid version of Ubx (Ubx-VP16), which possesses a potent activation domain from the VP16 viral protein, no longer directs A1 denticle pattern in embryonic epidermal cells. Instead, it mimics Antp in directing T2 denticle pattern, and it can rescue the cuticular loss-of-function phenotype of Antp mutants. In cells that do not produce denticles, Ubx-VP16 appears to have largely retained its normal repressive regulatory functions. These results suggest that the modulation of Hox activation and repression functions can account for segment-specific morphological differences that are controlled by different members of the Hox family. These results also are consistent with the idea that activity regulation underlies the phenotypic suppression phenomenon in which a more posterior Hox protein suppresses the function of a more anterior member of the Hox cluster. The acquisition of novel activation and repression potentials in Hox proteins may be an important mechanism underlying the generation of subtle morphological differences during evolution (Li, 1999).

Interestingly, although Ubx-VP16 acquires an Antp-like ability in denticle patterning, it preserves the Ubx ability to repress Keilin's organ development in thoracic segments. Therefore, Ubx-VP16 displays a mix of Antp-like and Ubx-like functions, dependent on tissue types and cell positions. Since development of Keilin's organs requires the appendage-promoting gene Distalless (Dll), the regulation of Dll by Ubx-VP16 was examined. The expression of Dll in thoracic appendage primordia cells is repressed by Ubx by means of the Dll304 element, presumably by eliciting the Ubx repression function on the element. In ectopic Ubx-VP16 embryos, both Dll expression and the activity of the Dll304 element are partially repressed. However, unlike ectopic Ubx, Ubx-VP16 is capable of activating Dll304 in other cells outside the appendage primordia. Thus, the Ubx-like function of Ubx-VP16 in repressing Keilin's organ development stems from retaining the Ubx repressive function upon Dll transcription. Since this repression appears specific for appendage primordia cells, the repression function of Ubx-VP16 is not constitutive but rather generated in a regulated manner. Taken together, the above results suggest that Ubx-VP16 functions are due to normal Ubx repressive effects on some targets (e.g., Dll), despite the attached VP16 activation domain, as well as a novel activation function on other targets (e.g., Antp) caused by the VP16 domain. The mix of functions that Ubx-VP16 exhibits is also often observed for natural Hox proteins (Li, 1999).

In these experiments the strength of activation function in Ubx is artificially varied. However, the partial change in segmental identity conferred by the Ubx-VP16 protein suggests that regulating the activity state of Ubx may modulate its functional specificity in denticle patterning. The fact that the Ubx-VP16 denticle patterning function is Antp-like suggests that the functional difference between the Ubx and Antp proteins in diversifying denticle patterns may reside in differences in activation and repression strengths on similar target genes rather than in differences in target occupancy. This suggestion is consistent with results indicating that Ubx and Antp recognize identical DNA sequences in vitro and regulate several common target genes in embryos. This evidence indicates that the segment identity functions of Ubx and Ubx-VP16 are distinct, but it does not eliminate the possibility that the VP16 domain increases activation function by altering the binding selectivity of the hybrid protein in developing embryos. This is thought to be unlikely because the specific Ubx targets such as dpp, Antp, and Dll are all regulated, and thus presumably occupied at similar Hox sites, by both Ubx and Ubx-VP16 (Li, 1999 and references).

How can it be examined whether this process has occurred in evolution? In the embryo of the crustacean Artemia, the Antp, Ubx, and abd-A homologs are coexpressed in a trunk region that is composed wholly of appendage-bearing segments. In contrast to Drosophila, the Artemia Ubx and Abd-A homologs do not repress Dll transcription and do not repress appendage development. There are a variety of reasons why the Artemia Ubx and Abd-A proteins might be incapable of repressing appendages, but one possibility is that sequence motifs within the proteins that would allow them to repress the appendage enhancer of Dll are missing. This possibility may be testable by placing the Artemia versions of Ubx and Abd-A proteins in the context of Drosophila early embryonic cells and assaying their effects on appendage development (Li, 1999 and references).

The design and analysis of a homeotic response element

The 26 bp bx1 element from the regulatory region of Distal-less is capable of imposing control by the homeotic genes Ultrabithorax and abdominal-A on a general epidermal activator in Drosophila. This provides an assay to analyze the sequence requirements for specific repression by these Hox genes. Both the core Hox binding site, 5'-TAAT, and the adjacent Exd 5'-TGAT core site are required for repression by Ultrabithorax and abdominal-A. The Distal-less bx1 site thus fits with the model of Hox protein binding specificity based on the consensus PBX/HOX-family site TGATNNAT[g/t][g/a], where the key elements of binding specificity are proposed to lie in the two base pairs following the TGAT. A single base pair deletion in the bx1 sequence generates a site, bx1:A^-mut, which on the consensus PBX/HOX model would be expected to be regulated by the Deformed Hox gene. It has been observed, however, that the bx1:A^-mut site is regulated predominantly by Sex combs reduced, Ultrabithorax and abdominal-A. The analysis of this site indicates that the specificity of action of Hox proteins may depend not only on selective DNA binding but also on specific post-binding interactions (White, 2000).

A homeotic response element that mediates repression by the Hox genes Ubx and abd-A has been built. The element is based on two short sequence modules; the 21 bp binding site Grainyhead binding site element (Gbe) for the transcription factor GRH and the 26 bp UBX/ ABD-A bx1 footprint site from the Dll regulatory sequences. On its own the Gbe mediates uniform epidermal activation. However, the combination of the Gbe and the bx1 element produces a homeotically modulated response. Specifically in the domain of expression of the Hox genes Ubx and abd-A the epidermal expression is repressed. This homeotic element thus successfully recapitulates the features of endogenous regulatory elements from homeotic target genes giving tissue-specific regulation by multiple homeotic genes (e.g. connectin). However, while endogenous target gene regulatory elements tend to be large (typically covering several kb) and are correspondingly difficult to analyze, this constructed element is appealingly simple and provides a manageable system for analyzing the specificity of the homeotic response (White, 2000).

The bx1 element contains the notable sequences 5' - TAAT (the core Hox binding site) and 5' -TGAT (the core Exd binding site). These two sequences are contained within the 5' -TGATTTAATT which is similar to the proposed PBX/HOX consensus site 5' -TGATNNAT[g/t][g/a]. Mutation of the 5' -TAAT site within bx1 abolishes the abdominal repression conferred by this element, presumably by reducing the affinity of the site for Ubx and Abd-A proteins. Thus, both Ubx and Abd-A appear to bind in vivo to the same site in the bx1 element. Mutation of the 5' -TGAT putative Exd binding site also abolishes the abdominal repression, suggesting that both Ubx and Abd-A require the binding of the cofactor Exd in order to function as repressors on this construct (White, 2000).

The basis of Hox target specificity has been proposed to depend on the nucleotides immediately following the Exd core site in the 5' -TGATNNAT[g/t][g/a] PBX/HOX consensus target site. In vivo studies have indicated that NN=GG produces a lab-specific response whereas NN=TA elicits a response to Dfd. Previous in vitro studies on the optimum target sequences for the whole range of PBX/HOX heterodimers have emphasized the importance of the nucleotide at position 7 in the PBX/HOX consensus sequence 5' - ATGATTNATGG. Preference for a particular nucleotide at N7 varies across the Hox complex. The most anteriorly-expressed proteins, HOXB1 and 2, prefer G at this position, the next more posteriorly-expressed Hox proteins allow N7=A, and from HOXB6-10 preference grows for N7=T. The optimum consensus for binding site for the PBX/HOXB7 heterodimer, equivalent to Ubx or Abd-A/Exd in Drosophila, is 5' -ATGATTTATGG. This is similar to the sequence ATGATTTAatt (differences in lower case) in the bx1 element. In particular, the nucleotide at N7 is T and thus fits with the predictions from the in vitro binding data. Taking the two elements previously studied in vivo into account, N7=G is in a lab (Hoxb1) element; N7=A in a Dfd (Hoxb4) element, and now N7=T is present in the bx1 element, which responds to Ubx and abd-A. Thus, the specificity of regulation of the bx1 element fits well with the expectations of the PBX/HOX model where the nucleotides immediately 3' of the TGAT PBX core provide the specificity for a particular subset of Hox proteins through interactions with the specific residues of the N-terminal arm of the homeodomain (White, 2000).

The bx1 element on its own does not drive reporter gene expression. It acts as a control module that together with the Grh activation module constitutes a homeotic response element. This may represent a general model for a target gene regulatory element, where proteins bound to a PBX/ HOX site interact with neighbouring regulatory complexes to modulate their activity. The PBX/HOX modules may act to overlay homeotic control on a whole variety of tissue-specific, signal transduction pathway-specific, temporal or other regulatory elements. It is worth noting that the regulatory regions that have been identified in homeotic target genes all drive highly tissue-restricted patterns, indicating the collaboration between tissue-specific and homeotic control. These observations give an insight into the modular construction of complex enhancers and also have important implications for the specificity of homeotic gene function. If the PBX/HOX complexes in target gene regulatory sequences act by interacting with other regulatory complexes, it seems very likely that different Hox proteins will vary in their ability to mediate specific interactions. Thus, there is clearly the potential for much of the specificity of responses to homeotic gene control to reside in post-binding interactions (White, 2000).

Specificity of Distalless repression and limb primordia development by Abdominal Hox proteins

In Drosophila, differences between segments, such as the presence or absence of appendages, are controlled by Hox transcription factors. The Hox protein Ultrabithorax (Ubx) suppresses limb formation in the abdomen by repressing the leg selector gene Distalless, whereas Antennapedia (Antp), a thoracic Hox protein, does not repress Distalless. The Hox cofactors Extradenticle and Homothorax selectively enhance Ubx, but not Antp, binding to a Distalless regulatory sequence. A C-terminal peptide in Ubx stimulates binding to this site. However, DNA binding is not sufficient for Distalless repression. Instead, an additional alternatively spliced domain in Ubx is required for Distalless repression but not DNA binding. Thus, the functional specificities of Hox proteins depend on both DNA binding-dependent and -independent mechanisms (Gebelein, 2002).

This work begins with a characterization of a Ubx binding site in the Dll gene that is critical for Dll repression; both Exd and Hth play a role in Ubx binding and repression. The Dll304 enhancer is sufficient to recapitulate the early expression pattern of Dll in the embryonic leg primordia. In addition to activation functions, Dll304 contains two Hox binding sites, Bx1 and Bx2, that repress enhancer activity in the abdomen and thereby restrict Dll expression to the thorax. Most of the repression activity is conferred by Bx1, a sequence bound by Ubx and Abd-A. In agreement with this result, a Distalless minimal element (DME) that lacks the Bx2 site accurately recapitulates the expression of Dll304 in the embryonic thorax. The DME enhancer also shows no derepression within the abdomen, suggesting that Bx1 is sufficient to fully repress Dll (Gebelein, 2002).

To better understand how Bx1 represses Dll, the presence of Exd and Hth binding sites were sought near the previously characterized Hox binding site. A consensus Exd site and a near consensus Hth site are in close proximity to the Hox site of Bx1. The Hox/Exd site (5'-AAATTAAATCA-3'), however, is unlike other previously characterized Hox/Exd binding sites because it contains an additional base pair in between the Hox and Exd half-sites. The Bx1 region containing this Hox/Exd/Hth site is referred to as the Distalless repression element (DllR). To determine whether DllR is required to repress DME expression in the abdomen, it was deleted from the DME enhancer (DME^act) and its ability to activate a reporter gene was tested in vivo. DME^act drives gene expression in all abdominal segments as well as in the thoracic region. Because the thoracic expression driven by DME^act is similar to that of DME, the DllR region is not required for DME activation but solely functions in the repression of Dll in the abdomen (Gebelein, 2002).

To determine whether Exd and Hth stimulate Hox binding to DllR, electrophoretic mobility shift assays (EMSAs) were performed with purified Ubx, Exd, and Hth proteins. Unless stated otherwise, all of these experiments were performed with UbxIa, the most widely expressed of several Ubx isoforms. By themselves, Ubx or an Exd/Hth heterodimer are capable of weakly interacting with DllR. The combination of all three proteins results in a slower migrating band indicating the formation of a Ubx/Exd/Hth/DNA complex. The formation of this protein/DNA complex is highly cooperative when compared to the amount of binding observed with Ubx or Exd/Hth alone. To test the contribution of each binding site, point mutations were introduced within the individual Hox, Exd, and Hth sites. Mutation of any one of these sites results in a decrease in the formation of the trimeric protein/DNA complex, suggesting that all three are required for optimal binding to DllR (Gebelein, 2002).

To test whether the Hox, Exd, and Hth binding sites are also required for Dll repression in vivo, reporter constructs were created containing the lacZ gene under the control of mutant versions of the DME enhancer. Mutation of the Hox site (DME^Hox) results in a similar level of derepression of reporter gene expression throughout the abdomen, as does the the complete deletion of DllR. Mutation of the Exd (DME^Exd) and Hth (DME^Hth) sites individually also results in derepression, albeit slightly weaker than mutation of the Hox site. However, if both the Exd and Hth sites are mutated together, full derepression is observed. Taken together, these results demonstrate that the efficient formation of a Hox/Exd/Hth trimeric complex on DllR is required for Dll repression within the abdomen (Gebelein, 2002).

These above data support a model in which a Ubx/Exd/Hth complex bound to DllR is necessary for Dll repression. Whether a single copy of DllR is sufficient to repress a heterologous enhancer element was tested. An artificial enhancer, called fkh(250^con), is activated by Scr, Antp, and Ubx (with Exd and Hth), and thus provides a useful heterologous activator to test for DllR function. A reporter construct under the control of both fkh(250^con) and DllR was created. Unlike fkh(250^con), which is expressed in parasegments (PS) 2-6, the composite enhancer (fkh250^con-DllR) is not expressed in PS 6, where Ubx is expressed. Ubx-mediated repression of fkh(250^con)-DllR is more obvious in embryos mutant for abd-A, which derepress Ubx and, consequently, fkh(250^con) throughout the abdomen. In this genetic background, fkh(250^con)-DllR is still active only in PS 2-5. Furthermore, misexpression of Ubx throughout the embryo activates fkh(250^con) but represses fkh(250^con)-DllR. Taken together, these results indicate that DllR is sufficient to confer Ubx-mediated repression of a heterologous enhancer. In addition, these results also illustrate that Ubx/Exd/Hth complexes can mediate repression through DllR in the same cells as it mediates activation through fkh(250^con) (Gebelein, 2002).

A general question for all transcription factors is how they achieve specificity in vivo. For the Hox proteins, a large number of studies have implicated sequences both within and outside the homeodomain as being important for their in vivo specificities. But how do these sequences function? Because DNA binding domains, including homeodomains, can also be protein interaction domains, studies that map the domains necessary for target gene regulation cannot answer this question by themselves. Instead, direct transcriptional targets must be identified and, once binding sites are characterized, DNA binding, in addition to target gene regulation, must be measured. The results allow two steps to be discriminated in the repression of Dll by Ubx. First, Exd and Hth stimulate Ubx, but not Antp, binding to DllR. In contrast, Ubx/Exd/Hth and Antp/Exd/Hth have similar affinities for a different "consensus" binding site (5'-CCATAAATCA-3'), suggesting that subtle differences in the DNA sequence, in addition to differences between Ubx and Antp, contribute to specificity. A C-terminal peptide in Ubx stimulates this cofactor-dependent binding to DllR. DNA binding, however, is not sufficient for Dll repression. Instead, an additional linker domain included in only a subset of Ubx isoforms is required for repression. Thus, a second step, the recruitment of additional factors to the Ubx/Exd/Hth complex bound to DllR, is implied by these data. In addition to the UbxIa linker, this step also requires the specific sequences and conformation imposed on the Ubx/Exd/Hth trimer by DllR (Gebelein, 2002).

Although the Ubx C terminus plays an important role in cofactor-dependent binding to DllR, additional domains contribute to optimal binding. In the presence of Exd and Hth, the AAUU chimera, but not heterologous AAUA or AAAU, binds DllR, suggesting that both the Ubx homeodomain and C terminus are important for optimal DNA binding to this site. The C terminus is not absolutely required for binding because a Ubx protein that lacks this domain (UUU*) is still able to bind well to DllR. Last, the finding that UUU*, but not AAU*, binds DllR suggests that a domain N terminal to the homeodomain also enhances DllR binding. Based on the crystal structures of Hox/Exd/DNA complexes, this difference could be due to the YPWM motif. Taken together, the data suggest that multiple regions of Ubx contribute to binding DllR and that no one domain is sufficient for full binding activity. This finding may be understood in light of the fact that the entire Ubx coding sequence has been constrained over millions of years of insect evolution to maintain leg (and Dll) repression in the abdomen (Gebelein, 2002).

How might the Ubx C terminus and YPWM motifs contribute to DNA binding? It is suggested that these regions could make additional protein-DNA contacts and/or protein-protein interactions that help stabilize the DllR-bound form of the trimeric complex. In support of this idea, the C termini of other homeodomain proteins also contribute to DNA binding. The Exd C terminus, for example, consists of an alpha helix that packs against its homeodomain and contributes to DNA binding. The C terminus of the MATalpha2 protein from yeast forms an alpha helix that contacts the MATa1 homeodomain to stabilize heterodimer formation on DNA. Interestingly, the two Hox proteins that repress Dll expression, Ubx and Abd-A, share sequence homology in their C termini, and are the only Drosophila Hox proteins predicted to form an alpha helix after their homeodomains (Gebelein, 2002).

The Ubx YPWM motif may also help stabilize complex formation on DllR. In the Hox/Exd/DNA crystal structures, this motif, together with flanking amino acids, directly contacts a hydrophobic pocket within the Exd homeodomain. These protein-protein contacts are thought to stabilize protein-DNA contacts made by the complex. The amino acids surrounding the YPWM motifs are different in Ubx and Antp and thus could contribute to DNA binding specificity by such an indirect mechanism (Gebelein, 2002).

The finding that UbxIa, but not UbxIVa, is able to repress Dll suggests that the linker region in UbxIa is required for repression. In addition, these results suggest that alternative splicing has the potential to modulate Ubx's control of gene expression. In support of this view, the expression of Ubx isoforms is temporally and spatially regulated. In addition, misexpression experiments using UbxIa and UbxIVa have shown that while both perform many of the same functions, only UbxIa efficiently transforms the peripheral nervous system. The finding that UbxIa and UbxIVa have different transcriptional regulatory properties provides a possible explanation for their distinct abilities to transform this tissue (Gebelein, 2002).

One argument against the idea that the different Ubx isoforms have distinct functions is that flies containing a genetic inversion that prevents the inclusion of the second microexon are, for the most part, normal. Although this mutation prevents the expression of UbxIa, it is unclear which other Ubx isoforms are expressed in this mutant because the inversion does not include both microexons. Furthermore, the effect that this mutation has on Dll expression has not been examined. A definitive test of the idea that Ubx isoforms have unique functions will require determining whether a Ubx allele in which both microexons are eliminated can provide all Ubx functions in vivo (Gebelein, 2002).

As in Drosophila, Dll expression is a marker for leg primordia in many animal phyla. Animals with appendages on their abdominal segments, such as crustaceans and onychophora, coexpress Ubx with Dll, demonstrating that Ubx is not a repressor of Dll in these species. The ability of Ubx to repress Dll probably arose in a subset of arthropods, the hexapods. Consistent with these findings, two recent studies suggest that one relevant difference between Ubx orthologs that repress Dll (for example, Drosophila Ubx) and Ubx orthologs that do not repress Dll (for example, onychophoran Ubx) maps to the C-terminal regions of these Hox proteins (Galant, 2002; Ronshaugen, 2002). These two groups, however, propose different mechanisms for how these sequences function. Galant suggests that the Drosophila Ubx C terminus actively represses transcription via a polyalanine motif that is present in the Ubx orthologs from all hexapods. Ronshaugen suggests that the Drosophila Ubx C terminus is only permissive for repression. Instead, they argue that crustaceans, which have abdominal legs, evolved a C-terminal sequence that inhibits Dll repression. However, neither study analyzed the binding of these proteins to the relevant binding sites in Dll, leaving open the possibility that the effects they observe could also be due to effects on DNA binding (Gebelein, 2002).

The data provide additional insights into how repression mechanisms may have evolved in these different species. It was found that the Drosophila Ubx C terminus contributes to DllR binding but is not sufficient for Dll repression in vivo. Thus, the positive role, observed by Galant, that the Drosophila sequence plays in Dll repression, could be due to an effect on DNA binding. These experiments also implicate the linker region of UbxIa as important for repression, but not DNA binding. Because some of the onychophora/ Drosophila and crustacean/ Drosophila chimeras lack this linker but are able to repress Dll, the crustacean and onychophoran Ubx orthologs must have repression domains that are different from the one identified in Drosophila Ubx (Gebelein, 2002).

Ronshaugen suggests that the phosphorylation of serine and threonine residues in the crustacean Ubx C terminus is necessary for it to prevent Dll repression (Ronshaugen, 2002). This is an intriguing possibility in light of the fact that phosphorylation of a Hox C terminus can inhibit cooperative DNA binding with Exd. Taken together with the current data that the C terminus of Ubx enhances DNA binding to DllR, it is suggested that the inhibition of Dll repression by the crustacean C terminus may be due to a reduced ability to bind DllR with Exd and Hth. This model accounts for why a Drosophila UbxIa protein containing the crustacean C terminus is unable to repress Dll (Ronshaugen, 2002) and for the inability of onychophora Ubx, which also contains a putative phosphorylation site in its C terminus, to repress Dll. Taken together, it is suggested that the evolution of limb suppression by Hox proteins, and probably many other Hox functions, depended upon the modification of both DNA binding-dependent and -independent mechanisms controlling Hox specificity (Gebelein, 2002).

Although these experiments focused on understanding why Antp is different from Ubx, the results provide some insights into the mechanism of transcriptional repression. The data strongly argue that a DNA-bound Ubx/Exd/Hth complex is necessary, but not sufficient, for repression. First, in addition to repressing Dll, Ubx/Exd/Hth activates fkh(250^con). When both fkh(250^con) and DllR simultaneously regulate the same reporter gene, DllR is able to repress gene expression in the same cells in which fkh(250^con) normally activates gene expression. This result suggests that the repressor proteins required for DllR activity are not cell type specific and are widely expressed in the embryo. Further, these results suggest that differences between the fkh(250^con) and DllR sequences determine whether transcription is activated or repressed. These sequences may recruit additional DNA binding factors that interact with the trimeric complex. These factors, which have not yet been identified, might provide or reveal a latent activation or repression domain within the Hox/Exd/Hth complex. Alternatively, another DNA binding factor may not be needed. Instead, the unique arrangement or spacing of the Hox, Exd, and Hth sites in these two elements may result in distinct conformations of the trimeric complex that recruit different coactivators or corepressors. Such a mechanism has been suggested for the nuclear receptor family of transcription factors and for the POU domain protein Pit-1, where a difference in spacing in a Pit-1 dimer binding site regulates the recruitment of a corepressor. Consistent with such a mechanism, it was found that the DllR^con binding site, which has one less base pair between the Hox and Exd half-sites than the DllR binding site, fails to repress transcription despite having a higher affinity for Ubx/Exd/Hth complexes. In addition, although repression activity for the UbxIa linker and C terminus in S2 cells can be measured, the experiments suggest that their activities are context dependent. The abdominal expression of DME^con-lacZ suggests that the mere presence of these domains is not sufficient for repression. Thus, the data suggest that transcription factor domains have distinct properties when assayed by themselves versus when they are part of a multiprotein complex. Further, it is concluded that the unique architecture of the complex assembled on DllR is necessary for efficient repression (Gebelein, 2002).

Direct integration of Hox and segmentation gene inputs during Drosophila development

During Drosophila embryogenesis, segments, each with an anterior and posterior compartment, are generated by the segmentation genes while the Hox genes provide each segment with a unique identity. These two processes have been thought to occur independently. This study shows tha abdominal Hox proteins work directly with two different segmentation proteins, Sloppy paired and Engrailed, to repress the Hox target gene Distalless in anterior and posterior compartments, respectively. These results suggest that segmentation proteins can function as Hox cofactors and reveal a previously unanticipated use of compartments for gene regulation by Hox proteins. The results suggest that these two classes of proteins may collaborate to directly control gene expression at many downstream target genes (Gebelein, 2004).

The segregation of groups of cells into compartments is fundamental to animal development. Originally defined in Drosophila, compartments are critical for providing cells with their unique positional address. The first compartments to form during Drosophila development are the anterior and posterior compartments and the key step to defining them is the activation of the gene engrailed (en). Expression of en, which encodes a homeodomain transcription factor, results in a posterior compartment fate, and the absence of en expression results in an anterior compartment fate. Once activated by gap and pair-rule genes, en expression and, consequently, the anterior–posterior compartment boundary later become dependent upon the protein Wingless (Wg), which is secreted from adjacent anterior compartment cells. Concurrently with anterior–posterior compartmentalization and segmentation, the expression of the eight Drosophila Hox genes is also initially established by the gap and pair-rule genes. The Hox genes, however, which also encode homeodomain transcription factors, do not contribute to the formation or number of segments but instead specify their unique identities along the anterior–posterior axis (Gebelein, 2004).

This flow of genetic information during Drosophila embryogenesis has led to the idea that anterior–posterior compartmentalization and segment identity specification are independent processes. In contrast to this view, this study shows that these two pathways are interconnected in previously unrecognized ways. Evidence is provided that Hox factors directly interact with segmentation proteins such as En to control gene expression. Moreover, Hox proteins collaborate with two different segmentation proteins in anterior and posterior cell types to regulate the same Hox target gene, revealing a previously unknown use of compartments to control gene expression by Hox proteins (Gebelein, 2004).

Distalless (Dll) is a Hox target gene that is required for leg development in Drosophila. In each thoracic hemisegment, wg, expressed by anterior cells adjacent to the anterior–posterior compartment boundary, activates Dll in a group of cells that straddle this boundary. A cis-regulatory element derived from Dll, called DMX, drives accurate Dll-like expression in the thorax. The abdominal Hox genes Ultrabithorax (Ubx) and abdominalA (abdA) directly repress Dll and DMX-lacZ in both compartments, thereby blocking leg development in the abdomen. DMX is composed of a large activator element (DMXact) and a 57-base-pair (bp) repressor element referred to here as DMX-R. Previous work demonstrated that Ubx and AbdA cooperatively bind to DMX-R with two homeodomain cofactors, Extradenticle (Exd) and Homothorax (Hth). In contrast, the thoracic Hox protein Antennapedia (Antp) does not repress Dll and does not bind DMX-R with high affinity in the presence or absence of Exd and Hth. Thus, repression of Dll in the abdomen depends in part on the ability of these cofactors to selectively enhance the binding of the abdominal Hox proteins to DMX-R (Gebelein, 2004).

Exd and Hth, as well as their vertebrate counterparts, are used as Hox cofactors at many target genes. Moreover, Hox/Exd/Hth complexes are used for both gene activation and repression, raising the question of how the decision to activate or repress is determined. One view posits that these complexes do not directly recruit co-activators or co-repressors, but instead are required for target gene selection. Accordingly, other DNA sequences present at Hox/Exd/Hth-targeted elements would determine whether a target gene is activated or repressed. Consistent with this notion, DMX-R sequences isolated from six Drosophila species show extensive conservation outside the previously identified Hox (referred to here as Hox1) Exd and Hth binding sites, suggesting that they also play a role in Dll regulation (Gebelein, 2004).

To test a role for these conserved sequences, a thorough mutagenesis of DMX-R was performed. Each mutant DMX-R was cloned into an otherwise wild-type, full-length DMX and tested for activity in a standard reporter gene assay in transgenic embryos. Thoracic expression was normal in all cases. However, surprisingly, many of the DMX-R mutations, such as X5, resulted in abdominal de-repression only in En-positive posterior compartment cells, whereas other mutations, such as X2, resulted in abdominal de-repression only in En-negative anterior compartment cells. Single mutations in the Hox1, Exd, or Hth sites also resulted in de-repression predominantly in posterior cells. In contrast, deletion of the entire DMX-R (DMXact-lacZ), or mutations in both the X2 and X5 sites (DMX[X2 + X5]-lacZ), resulted in de-repression in both compartments. These results suggest that distinct repression complexes bind to the DMX-R in the anterior and posterior compartments and that segmentation genes play a role in Dll repression (Gebelein, 2004).

One clue to the identity of the proteins in these repression complexes is that the sequence around the Hth site is nearly identical to a Hth/Hox binding site that had been identified previously by a systematic evolution of ligands by exponential enrichment (SELEX) approach using vertebrate Hox and Meis proteins. This similarity suggested the presence of a second, potentially redundant Hox binding site, Hox2. In agreement with this idea, mutations in both the Hox1 and Hox2 binding sites resulted in de-repression in both the anterior and posterior compartments of the abdominal segments. Similarly, although individual mutations in the Exd and Hth binding sites lead predominantly to de-repression in the posterior compartment, mutation of both sites resulted in de-repression in both compartments. These results suggest that a Hox/Exd/Hth/Hox complex may be used for repression in both compartments. Furthermore, they suggest that although single mutations in these binding sites are sufficient to disrupt the activity of this complex in the posterior compartment, double mutations are required to disrupt its activity in the anterior compartment (Gebelein, 2004).

To provide biochemical evidence for a Hox/Exd/Hth/Hox tetramer, DNA binding experiments were performed using DMX-R probes and proteins expressed and purified from E. coli. Previous experiments demonstrated that a Hox/Exd/Hth trimer cooperatively binds to the Hox1, Exd and Hth sites. The function of the Hox2 site was tested in two ways. First, binding was measured to a probe, DMX-R2, that includes the Exd, Hth and Hox2 sites, but not the Hox1 site. It was found that Exd/Hth/AbdA and Exd/Hth/Ubx trimers cooperatively bind to this probe and that mutations in the Hth, Exd or Hox2 binding sites reduced or eliminated complex formation (Gebelein, 2004).

Second, if both the Hox1 and Hox2 sites are functional, the full-length DMX-R may promote the assembly of Hox/Exd/Hth/Hox tetramers. Using a probe containing all four binding sites (DMX-R1 + 2), the formation of such complexes was observed. Mutation of any of the four binding sites reduced the amount of tetramer binding whereas mutation of both Hox sites or both the Exd and Hth sites eliminated tetramer binding. Furthermore, Antp, which does not repress Dll, formed tetramers with Exd and Hth that were approximately tenfold weaker than with Ubx or AbdA, but bound well to a consensus Hox/Exd/Hth trimer binding site. Because mutation of both Hox sites or both the Exd and Hth sites resulted in de-repression in both compartments, these experiments correlate the binding of a Hox/Exd/Hth/Hox complex on the DMX-R with the ability of this element to mediate repression in both compartments (Gebelein, 2004).

Although binding of a Hox/Exd/Hth/Hox tetramer is sufficient to account for the necessary abdominal Hox-input into Dll repression, it does not explain the compartment-specific de-repression exhibited by some DMX-R mutations. The X2 and X5 mutations, for example, result in abdominal de-repression but do not prevent the formation of the Hox/Exd/Hth/Hox tetramer. Sequence inspection of the DMX-R revealed that the X2 mutation, which resulted in de-repression specifically in the anterior compartment, disrupts two partially overlapping matches to a consensus binding site for Forkhead (Fkh) domain proteins. With this in mind, the expression pattern of Sloppy paired 1 (Slp1), a Fkh domain factor encoded by one of two partially redundant segmentation genes, slp1 and slp2, was examined. The two slp genes are expressed in anterior compartment cells adjacent and anterior to En-expressing posterior compartment cells. In the thorax, cells expressing Dll and DMX-lacZ co-express either Slp or En at the time Dll is initially expressed. In the abdomen, the homologous group of cells, which express DMXact-lacZ (a reporter lacking the DMX-R), co-express either Slp in the anterior compartment or En in the posterior compartment. The expression patterns of Slp and En were compared with Ubx and AbdA. Ubx levels are highest in anterior, Slp-expressing cells whereas AbdA levels are elevated in posterior, En-expressing cells. In contrast, both Exd and Hth are present at similar levels in both compartments throughout the abdomen (Gebelein, 2004).

On the basis of these data, a model is presented for Hox-mediated repression of Dll in both the anterior and posterior compartments of the abdominal segments. In the anterior compartment it is proposed that Slp binds to DMX-R directly with a Ubx/Exd/Hth/Ubx tetramer. In the posterior compartment it is suggested that En binds to DMX-R directly with an AbdA/Exd/Hth/AbdA tetramer. One important feature of this model is that Antp/Exd/Hth/Antp complexes fail to form on this DNA, thereby accounting for the lack of repression in the thorax. Furthermore, the model proposes that Slp and En should, on their own, have only weak affinity for DMX-R sequences because repression does not occur in the thorax, despite the presence of these factors. The Hox/Exd/Hth/Hox complex, perhaps in conjunction with additional factors, is required to recruit or stabilize Slp and En binding to the DMX-R. Both Slp and En are known repressor proteins that directly bind the co-repressor Groucho. Thus, the proposed complexes in both compartments provide a direct link to this co-repressor and, therefore, a mechanism for repression. DNA binding and genetic experiments are presented that test and support this model (Gebelein, 2004).

To test the idea that En is playing a direct role in Dll repression, the ability of En and Hox proteins to bind to DMX-R probes was examined. On its own, En binds to DMX-R very poorly. Surprisingly, it was found that En binds DMX-R with the abdominal Hox proteins Ubx or AbdA in a highly cooperative manner. The thoracic Hox protein Antp does not bind cooperatively with En to this probe. Mutations in the Hox1 or X5 binding sites block AbdA/En binding in vitro, consistent with these mutations showing posterior compartment de-repression in vivo. In contrast, the X6, X7 and Hth mutations do not affect AbdA/En complex formation (Gebelein, 2004).

On the basis of DMX-R's ability to assemble a Hox/Exd/Hth/Hox tetramer, whether En could bind together with an AbdA/Exd/Hth/AbdA complex was tested. Addition of En to reactions containing AbdA, Exd and Hth resulted in the formation of a putative En/AbdA/Exd/Hth/AbdA complex. This complex contains En because its formation is inhibited by an anti-En antibody. A weak antibody-induced supershift is also observed. Moreover, this complex fails to form on the X5 mutant, which causes posterior compartment-specific de-repression. It is noted that En/Exd/Hth complexes also bind to the DMX-R and that it cannot be excluded that an En/Exd/Hth/AbdA complex may be important for Dll repression. The model emphasizes a role for an En/AbdA/Exd/Hth/AbdA complex because it better accommodates the cooperative binding observed between En and AbdA on the DMX-R (Gebelein, 2004).

Repression in the anterior compartments of the abdominal segments requires the sequence defined by the X2 mutation, which is similar to a Fkh domain consensus binding site. The model predicts that this sequence is bound by Slp. Consistent with this view, Slp1 binds weakly to wild type, but not to X2 mutant DMX-R probes. However, in contrast to En, no cooperative binding was observed between Slp and Hox or Hox/Exd/Hth/Hox complexes, suggesting that additional factors may be required to mediate interactions between Slp and the abdominal Hox factors (Gebelein, 2004).

Together, these results suggest that En and Slp play a direct role in DMX-lacZ and Dll repression. However, these experiments do not unambiguously determine the stoichiometry of binding by these factors. Furthermore, in vivo, additional factors may enhance the interaction between these segmentation proteins and Hox complexes, thereby increasing the stability and/or activity of the repression complexes (Gebelein, 2004).

The model for Dll repression is supported by previous genetic experiments that examined the effect of Ubx and abdA mutants on Dll expression in the abdomen. Ubx abdA double mutants de-repress Dll in both compartments of all abdominal segments. In contrast, Ubx mutants de-repress Dll in the anterior compartment of only the first abdominal segment, which lacks AbdA. abdA mutant embryos de-repress Dll in the posterior compartments of all abdominal segments, where Ubx levels are low (Gebelein, 2004).

Several genetic experiments were performed to provide in vivo support for the idea that Slp and En work directly with Ubx and AbdA to repress Dll. The design of these experiments had to take into consideration that the activation of Dll in the thorax depends on wg, and that wg expression depends on both slp and en. Consequently, Dll expression is mostly absent in en or slp mutants, making it impossible to characterize the role that these genes play in Dll repression from examining en or slp loss-of-function mutants. However, some of the mutant DMX-Rs described here provide the opportunity to test the model in alternative ways (Gebelein, 2004).

According to the model, DMX[X5]-lacZ is de-repressed in the posterior compartments of the abdominal segments because it fails to assemble the posterior, En-containing complex. Repression of DMX[X5]-lacZ in the anterior compartments still occurs because it is able to assemble the anterior, Slp-containing complex. According to this model, DMX[X5]-lacZ should be fully repressed if Slp is provided in posterior cells. A negative control for this experiment is that ectopic Slp should be unable to repress DMX[X2]-lacZ because this reporter gene does not have a functional Slp binding site. To mis-express Slp, paired-Gal4 (prd-Gal4), which overlaps both the Slp and En stripes in the odd-numbered abdominal segments, was used. As predicted, ectopic Slp repressed DMX[X5]-lacZ but not DMX[X2]-lacZ, providing strong in vivo support for Slp's direct role in Dll repression in the anterior compartments (Gebelein, 2004).

Conversely, the model posits that DMX[X2]-lacZ is de-repressed in the anterior compartment because it cannot bind Slp, but remains repressed in the posterior compartment because it is able to assemble the En-containing posterior complex. Thus, providing En in the anterior compartment should repress DMX[X2]-lacZ. A complication with this experiment is that En is a repressor of Ubx, which is the predominant abdominal Hox protein in the anterior compartment. It was confirmed that prd-Gal4-driven expression of En represses Ubx and that AbdA levels remain low at the time Dll is activated in the thorax. Consequently, ectopic En expression is not sufficient to repress DMX[X2]-lacZ, consistent with the observation that low levels of abdominal Hox proteins are present. Therefore, to promote the assembly of the posterior complex in anterior cells, En was co-expressed with AbdA using prd-Gal4. As predicted, this combination of factors repressed DMX[X2]-lacZ but not DMX[X5]-lacZ, providing strong in vivo evidence for En playing an essential role in Dll repression in the posterior compartments (Gebelein, 2004).

Several observations provide additional support for the model. First, ectopic expression of AbdA or Ubx in the second thoracic segment (T2) represses DMX[X5]-lacZ in the anterior compartment, but not in the posterior compartment. Conversely, expression of AbdA or Ubx in T2 represses DMX[X2]-lacZ only in posterior compartment cells. Second, co-expression of Slp with Ubx completely represses DMX[X5]-lacZ in T2 but does not repress DMX[X2]-lacZ in T2. Third, in those cases where repression is incomplete (for example, En + AbdA repression of DMX[X2]-lacZ in the abdomen), cells that escape repression have low levels of either an abdominal Hox protein or Slp/En. Together, these data provide additional evidence that the abdominal Hox proteins work together with Slp and En to repress Dll (Gebelein, 2004).

The segregation of cells into anterior and posterior compartments during Drosophila embryogenesis is essential for many aspects of fly development. The results presented in this study reveal an unanticipated intersection between anterior–posterior compartmentalization by segmentation genes and segment identity specification by Hox genes. Specifically, it is suggested that the abdominal Hox proteins collaborate with two different segmentation proteins, Slp and En, to mediate repression of a Hox target gene (Dll) in the anterior and posterior compartments of the abdomen, respectively. This mechanism of transcriptional repression suggests a previously unknown use of compartments in Drosophila development. The mechanism proposed here contrasts with the alternative and simpler hypothesis in which the abdominal Hox proteins would have used the same set of cofactors to repress Dll in all abdominal cells, regardless of their compartmental origin (Gebelein, 2004).

These results provide further support for the view that Hox/Exd/Hth complexes do not directly bind co-activators or co-repressors but instead indirectly recruit them to regulatory elements. Consistent with previous analyses, it is suggested that Hox/Exd/Hth complexes are important for the Hox specificity of target gene selection. Additional factors, such as Slp or En in the case of Dll repression, are required to determine whether the target gene will be repressed or activated. In the future, it will be important to dissect in similar detail other Hox-regulated elements, to assess the generality of this mechanism (Gebelein, 2004).

These results also broaden the spectrum of cofactors used by Hox proteins to regulate gene expression. Although the analysis of Exd/Hth in Drosophila and Pbx/Meis in vertebrates has provided some insights into how Hox specificity is achieved, there are examples of tissues in which these proteins are not available to be Hox cofactors and of Hox targets in which Exd and Hth seem not to play a direct role. This study shows that En, a homeodomain segmentation protein, is used as a Hox cofactor to repress Dll in the abdomen. Although the complex defined at the DMX-R includes Exd and Hth, the DNA binding studies demonstrate that Hox and En proteins can bind cooperatively to DNA in the absence of Exd and Hth. These findings suggest that En may function with Ubx and/or AbdA to regulate target genes other than Dll, and perhaps independently of Exd and Hth. Consistent with this idea are genetic experiments showing that, in the absence of Exd, En can repress slp and this repression requires abdominal Hox activity. Although these experiments were unable to distinguish whether the Hox input was direct or indirect, the results suggest that En may bind directly with Ubx and AbdA to repress slp, and perhaps other target genes (Gebelein, 2004).

Finally, these results raise the question of why a compartment-specific mechanism is used by Hox factors to repress Dll. The activation of Dll at the compartment boundary by wg may be important for accurately positioning the leg primordia within each thoracic hemisegment, but this mode of activation requires that Dll is repressed in both compartments in each abdominal segment. The utilization of segmentation proteins such as En and Slp may be the simplest solution to this problem. Compartment-specific mechanisms may also provide additional flexibility in the regulation of target genes by Hox proteins by allowing them to turn genes on or off specifically in anterior or posterior cell types. For these reasons, compartment-dependent mechanisms of gene regulation may turn out to be the general rule instead of the exception (Gebelein, 2004).

Transcriptional Regulation

Three signaling molecules are required for Distal-less induction in leg and antennal discs. hedgehog (hh) is locally expressed in posterior compartments, under the control of engrailed. wingless (wg) is expressed in ventral-anterior cells having been induced earlier by HH signals. decapentaplegic (dpp) is expressed in dorsal-anterior compartments, also having been induced by HH signals. All three, hh, wg and dpp are required for the induction of Distal-less and the establishment of the proximal-distal axis of the developing leg and antenna (Diaz-Benjumea, 1994).

Limb development requires the formation of a proximal-distal axis perpendicular to the main anterior-posterior and dorsal-ventral body axes. The secreted signaling proteins Decapentaplegic and Wingless act in a concentration-dependent manner to organize the proximal-distal axis. Discrete domains of proximal-distal gene expression are defined by different thresholds of Decapentaplegic and Wingless activities. distal-less is expressed in a central domain that corresponds to the presumptive tarsal segments and the distal tibia. The dachshund gene is required for development of the femur and tibia. Dac is expressed in a ring corresponding to the presumptive femur, tibia and first tarsal segment, but is absent from the more distal tarsal segments of the leg disc. Although there is little or no overlap between Dll and Dac domains at early stages, by mid third instar the combination of Dac and Dll expression defines three regions along the P-D axis. Dll and Dac are expressed in circular domains centered on the point at which the ventral Wg domain and the dorsal Dpp domain meet. Dll expression in the center of the disc depends on the combined activities of wg and dpp. Wg and Dpp act directly to induce Dll, as analysis of constitutively active Thick-veins clones has shown (Tkv is the receptor for Dpp); analysis of shaggy/zeste white 3 clones (Sgg is required for transduction of the Wingless signal) reveals that both Wg and Dpp transduction pathways are activated cell autonomously. Continuous signalling is not required to maintain Dll or Dac expression. The spatial domains of Dac and Dll expression are defined by different threshold levels of both Wg and Dpp activities. Both Dpp and Wg act to directly repress Dac in the center of the disc. Dac repression is actively maintained by Wg and Dpp signaling long after Dac and Dll have been induced and are stably expressed in the absence of further signaling. Subsequent modulation of the relative sizes of these domains by growth of the leg is required to form the mature pattern (Lecuit, 1997).

Two thoracic limbs of Drosophila, the leg and the wing, originate from a common cluster of cells that include the source of two secreted signaling molecules, Decapentaplegic and Wingless. Wingless, but not Decapentaplegic, is responsible for the initial distal identity specification of the limb primordia. Proximal limb precursors expressing escargot encircle the Distal-less expressing distal primordium. Dll expressing cells show a dynamic cell migration in the early stage of limb formation, migrating basally during stage 12. Cells that have just started to express Dll also express thickveins. This suggests a requirement for regulated Dpp signaling at the level of receptor expression. Limb formation is restricted to the lateral position of the embryo through exertion of negative control by Decapentaplegic and the EGF receptor, both of which determine the global dorsoventral pattern. dpp specifies proximal cell identities. In the absence of dpp Escargot and Snail are lost. A late function of Decapentaplegic locally determines additional cell identities in a dosage dependent manner. Loss of Decapentaplegic activity results in a deletion of the proximal structures of the limb, in contrast to the deletion of distal structures when decapentaplegic mutations affect the imaginal disc. The limb pattern elements appear to be added in a distal to proximal direction in the embryo, which is just the opposite of what is happening in the growing imaginal disc. It is proposed that Wingless and Decapentaplegic act sequentially to initiate the proximodistal axis. This model is contrary to that of Cohen (1993) who argues that Dpp and Wingless are both required to induce the limb. Since Dll expression persists and expands dorsally in the absence of Dpp, it is clear that Dpp plays no role in inducing initial Dll expression but that the dorsoventral limit of Dll expression is defined by repression as a result of Dpp expression. Similarly, EGF-R is required to repress Dll expression in the ventral ectoderm (Goto, 1997a).

Two lines of evidence suggest that Distal-less and vestigial expression at the wing margin, as well as bristle specification, are organized by WG. First, using a temperature-sensitive mutation of wg, it is possible to remove wg activity at chosen times during wing development. Dll expression is abolished within 48 hours following a shift to the nonpermissive temperature, and vg expression is eliminated except for a thin stripe of cells straddling the D/V boundary compartment boundary. Second, ectopic expression of wg as well as ectopic activation of the Wg-signal transduction pathway, caused by eliminating the Shaggy/Zeste-white 3 activity, up-regulates the expression of Dll and Vg within the wing-blade primordium. Expression of a teathered WG protein, genetically engineered to be attached to the cell surface and thus unable to diffuse like normal WG protein, drastically alters the up-regulation of vg and Dll in surrounding cells. Normal WG upregulates Dll in wild-type cells up to 10 or more cell diameters away from the WG source, while teathered WG up-regulates these genes only in their immediate wild-type neighbors. Thus normal WG can act as a long range morphogen, exerting a graded influence on vg and Dll in surrounding cells, while teatherd WG exerts only a short-range, all-or-non influence on surrounding cells (Zecca, 1996).

Armadillo is required autonomously and continuously to mediate the response of wing cells to WG-Secreting cells located at a distance. Clones of arm mutant cells were generated in wing discs. These cells stop dividing and either die or are actively eliminated from the disc epithelium. When stained for either VG or DLL expression 36 hours after mitotic recombination is induced, none of the cells within such clones express either protein (Zecca, 1996).

Short-range interaction between dorsal and ventral (D and V) cells establishes an organizing center at the DV compartment boundary that controls growth and specifies cell fate along the dorsal-ventral axis of the Drosophila wing. The secreted signaling molecule Wingless (WG) is expressed by cells at the DV compartment boundary and has been implicated in mediating its long-range patterning activities. Does WG acts directly at a long-range to specify cell fates in the wing? To investigate this question, mutant clones of two components of the WG transduction pathway, dishevelled and armadillo were examined. Cells mutant for dsh show reduced levels of Dll and vg expression. Cells mutant for a temperature sensitive hypomorphic allele of arm, likewise show loss of expression of Dll and vg when larvae are shifted to non-permissive temperatures. Reducing WG levels at the margin reduces both the maximum level of Dll expression near the DV boundary and the distance from the DV boundary at which Dll can be activated. An intermediate level of WG activity is not sufficient to support the specification of wing margin bristles, suggesting that WG has fallen below a critical threshold for activation of AS-C gene expression, while remaining above the respective thresholds of activation for both Dll and vg. Thus, WG acts directly, at long range, to define the expression domains of its target genes, Distal-less and vestigial. Expression of the Achaete-scute genes, Distal-less and vestigial at different distances from the DV boundary is controlled by WG in a concentration-dependent manner, with AS-C requiring the highest levels of WG. Dll, expressed in a wider range, requires the next highest level, and vg, which is expressed across the entire wing pouch, requires the lowest levels. It is proposed that WG acts as a morphogen in patterning the D/V axis of the wing (Neumann, 1997).

Vestigial contributes Distal-less gene expression in the wing blade. In the wild-type wing, Dll is expressed in the wing pouch in a circle of cells that lies within an area defined by the expression of vestigial. Ectopic expression of vg elevates the levels of Dll expression within the developing wing blade and triggers ectopic expression of Dll outside this area. This differs from the effects of ectopic expression of wg, which are always restricted to the developing wing blade and never induce ectopic expression of Dll. Coexpression of both, wg and vg, results in synergistic effects similar to those described before for other targets and an increase in the area of ectopic expression. The effects of vg are independent of wg, since they are achieved even in apterous mutants, where wg is never expressed along the DV boundary. These results indicate that Vestigial can indeed regulate gene expression in the wing blade. In addition, the correlation between levels suggests that different concentrations of Vestigial elicit different effects and that, in some instances, these effects seem to be independent of the levels of Wingless. Thus Vestigial might act in concert with Wingless, and not simply as an effector of wg, in the regulation of gene expression. Very high levels of Vestigial expression, or long-term exposure to vestigial expression, results in the cessation of proliferation, loss of gene expression and, eventually, in cell death. This might account for the small wings that are often visible when vg is overexpressed in the developing wing and would correlate with the zone of non-proliferation that appears at the wing margin, where the levels of vg expression are elevated (Klein, 1999).

Wnt/Wingless directs many cell fates during development. Wnt/Wingless signaling increases the amount of beta-catenin/Armadillo, which in turn activates gene transcription. The Drosophila protein D-Axin is shown to interact with Armadillo and Drosophila APC. D-Axin was identified in a yeast two-hybrid screen for proteins that bind the Armadillo repeat domain of Arm. d-axin codes for a protein of 743 amino acids. A region near its N-terminus shows similarity to the regulator of G protein signaling (RGS domain), whereas its C-terminus contains a region homologous to a conserved sequence near the N-terminus of Dishevelled. Thus D-Axin has a domain structure very similar to that of proteins of the mammalian Axin family. Unlike mammalian Axin family members, which bind to GSK-3beta, D-Axin does not bind to the homologous protein Shaggy/Zeste white3. d-axin is expressed maternally and is ubiquitously expressed during development. Embryos devoid of maternal and zygotic d-axin have completely naked ventral cuticle, lacking all denticles (Hamada, 1999).

During wing disc development, Wg signaling is induced along the dorsoventral compartment boundary in the wing imaginal disc. Arm accumulates in the cytoplasm, associates with its partner Pangolin, and activates expression of target genes such as Distal-less. Mutation of d-axin results in the accumulation of cytoplasmic Armadillo and results in elevation of Distal-less. Ectopic expression of d-axin inhibits Wingless signaling. Hence, D-Axin negatively regulates Wingless signaling by down-regulating the level of Armadillo. It is speculated that the Axin family of proteins functions to establish a threshold to prevent premature signaling events caused by Wg/Wnt and to restrict areas that are capable of responding to Wg/Wnt. These results establish the importance of the Axin family of proteins in Wnt/Wingless signaling in Drosophila (Hamada, 1999).

Dll expression in imaginal discs of leg and labial limb primordia, requires both wg and dpp, whereas expression in the antennal and labral limbs does not. Only the primordia of legs and labial limbs are bisected into compartments by the parasegment boundary under the control of wg and en. Dll expression occurs in cells surrounding and including the lateral tips of the ventral wg stripes in the thoracic segments of cells also expressing engrailed (B. Cohen, 1993).

Rows of cells expressing wg in an array of dorsal to ventral stripes, one per segment, intersect cells expressing dpp in a long stripe running anterior to posterior. Dll is expressed in surrounding cells as well. Dll is not required for allocation of imaginal cells. Thus Dll is required in only a subset of cells in the leg disc primordium, (to distinguish the presumptive leg cells from the surrounding body wall). Perhaps leg and wing are derived from a common imaginal primordium. ( B. Cohen, 1993).

The leg primordium enhancer is 12Kb upstream of the Dll gene. The head enhancer region is adjacent to and downstream of this location. The leg enhancer is a target of bithorax-complex mediated repression (B. Cohen, 1993).

Distal-less is a downstream gene of the HOM gene Deformed, and Distal-less function is required for the elaboration of a subset of the maxillary epidermal identities specified by Deformed. The regulatory effect of Deformed on Distal-less is mediated by a ventral maxillary-specific enhancer located 3' of the Distal-less transcription unit. Deformed and Distal-less, both of which encode homeodomain transcription factors that are persistently expressed in ventral maxillary cells, combinatorially specify a subsegmental code required for a group of cells to differentiate maxillary cirri. Ectopic dfd is sufficient to activate ectopic Dll in other segments. Whether the dfd activation is direct or indirect is unknown (O'Hara, 1993).

Distal-less is a target of Ultrabithorax in parasegment 6. Ubx is shown to be down-regulated by Engrailed in the posterior compartment of this parasegment. In the posterior compartment of parasegment 6, Dll is normally expressed in a small cluster of cells. If Ubx is expressed uniformly via a heat-shock promoter, Dll is inappropriately repressed in these posterior compartment cells. In the anterior compartment of parasegment 6, Dll is normally repressed by high levels of UBX. However, if en is expressed uniformly via a heat-shock promoter, Ubx is repressed and Dll is derepressed. Because Dll is required for the development of larval sensory structures, these results demonstrate that EN-mediated repression of Ubx in the posterior compartment is necessary for the morphology of parasegment 6 (Mann, 1994).

dwnt-5 is a putative downstream target of Distal-less. dwnt-5 may then act in the specification of the limb primordia (Eisenberg, 1992)

Regulation of Hox target genes by a DNA bound Homothorax/Hox/Extradenticle complex

To regulate their target genes, the Hox proteins of Drosophila often bind to DNA as heterodimers with the homeodomain protein Extradenticle (Exd). For Exd to bind DNA, it must be in the nucleus, and its nuclear localization requires a third homeodomain protein, Homothorax (Hth). A conserved N-terminal domain of Hth directly binds to Exd in vitro, and is sufficient to induce the nuclear localization of Exd in vivo. However, mutating a key DNA binding residue in the Hth homeodomain abolishes many of its in vivo functions. Hth binds to DNA as part of a Hth/Hox/Exd trimeric complex, and this complex is essential for the activation of a natural Hox target enhancer. Using a dominant negative form of Hth evidence is provided that similar complexes are important for several Hox- and exd-mediated functions in vivo. These data suggest that Hox proteins often function as part of a multiprotein complex, composed of Hth, Hox, and Exd proteins, bound to DNA (Ryoo, 1999).

During leg development, expression of the homeobox gene Distal-less, which is required for ventral limb development, is mutually antagonistic with Hth/Exd function: Dll is a repressor of hth and Hth can also repress Dll. Hth's ability to repress Dll requires Hth's homeodomain. From ectopic expression assays, it is concluded that although the Hth homeodomain is not required to induce Exd's nuclear localization, it is necessary for many Hth functions, including the regulation of specific target genes such as Dll. The one known exception is that all forms of Hth, including GFP-Hth 51A and GFP-HM, are able to interfere with distal leg development when expressed with the Dll:Gal4 driver. This phenotype, however, is also observed when wild-type Exd is expressed with this driver, and therefore does not require any Hth input. The different in vivo activities of Hth and Hth 51A indicate that Hth has functions in addition to localizing Exd to nuclei, and that these functions require Hth to bind DNA (Ryoo, 1999).

Proximodistal axis formation in the Drosophila leg: subdivision into proximal and distal domains by Homothorax and Distal-less

The proximal distal axis of the Drosophila leg is patterned by expression of a number of transcription factors in discrete domains along the axis. The homeodomain protein Homothorax and the zinc-finger protein Teashirt are broadly coexpressed in the presumptive body wall and proximal leg segments. Homothorax has been implicated in forming a boundary between proximal and distal segments of the leg. Evidence is presented that Teashirt is required for the formation of proximal leg segments, but Tsh has no role in boundary formation (Wu, 2000).

The leg disc consists of a single epithelial sheet in which the presumptive distal segments are specified in the center and the presumptive proximal segments are specified in the periphery. Cross-sections show that proximal segments, which express Hth and Tsh, fold back over the distal segments, which express Dll and Dac. Hth and Tsh expression is limited to the proximal region of the disc through repression by the combined activities of Wg and Dpp. Although the Hth and Tsh expression domains overlap through much of the proximal region, Hth expression extends more distally than Tsh. This is visible as a band of Hth expression that does not overlap Tsh in a basal optical section. This band coincides with the outer ring of Dll expression. The Tsh domain overlaps the proximal edge of the Dll ring by one or two cells. Tsh expressing cells are also found beneath the disc epithelium. Their location suggests that these may be adepithelial cells. Hth functions as a repressor to modulate Tsh expression. More distally located hth mutant clones lose Tsh expression. Loss of Tsh expression in hth correlates with ectopic expression of Dachshund. hth mutant clones cause ectopic expression of Dac close to the endogenous Dac domain, but do not do so in more proximal regions. The differential effect on Dac expression of hth clones located at different positions along the PD axis has been attributed to a role of Hth as a repressor of Wg and Dpp signaling. Thus the paradoxical loss of Tsh in more distal hth clones can be explained as an indirect effect of Hth on Dac expression. Dac can repress both Tsh and Hth when overexpressed. Thus the different distal limits of the Hth and Tsh expression domains presumably reflect a difference in their sensitivity to repression by Dac. The observation that Tsh levels increase in proximal hth clones suggests that Hth serves as a repressor of Tsh. Thus Hth modulates Tsh expression levels in the proximal leg in two ways. Hth may act directly to reduce Tsh expression levels in the proximal leg, and indirectly via repression of Dac to define the distal limit of Tsh expression (Wu, 2000).

Reducing Tsh activity produces a phenotype quite distinct from that of removing Hth activity. Tsh is required for the development of trochanter and coxa but does not appear to have a role in segment boundary formation. Hth and its partner Extradenticle are required to prevent fusion of coxa and trochanter with the femur. To better understand the basis for the defects in tsh mutant legs, Hth, Dll and Dac expression were studied in tsh^GAL4/tsh^HD1 and tsh^GAL4/tsh^GAL4 mutant discs. In wild-type discs Hth and Dac expression overlap in the proximal ring of Dll expression. Hth function in this ring is required for the affinity boundary between proximal and distal regions of the leg. This basic relationship holds in the tsh^GAL4/tsh^HD1leg disc. Hth, Dll and Dac expression overlap, and the affinity boundary between proximal and distal leg segments appears to be intact. The principal difference in these discs is expansion of the Dll domain into the proximal, Hth-expressing region. The spatial relationship between Hth and Dac is normal. Ectopic Dll expression is not sufficient to repress Hth but does appear to reduce the size of the coxa and trochanter and to cause problems that result in loss of pattern elements from the remaining portions of these segments. Even slight reductions in Tsh activity causes loss of sensory bristles from the coxa. In contrast, small clones of hth mutant cells are capable of differentiating bristles (Wu, 2000).

Taken together, these observations suggest that Tsh and Hth have distinct functions in the proximal leg. Hth limits the proximal extent of Dac expression, and is required for the affinity boundary between trochanter and femur. Tsh limits the proximal extent of Dll expression and is required for proper growth and differentiation of proximal segments, but does not appear to have a role in PD boundary formation (Wu, 2000).

The establishment of segmentation in the Drosophila leg

Segmentation is a developmental mechanism that subdivides a tissue into repeating functional units, which can then be further elaborated upon during development. In contrast to embryonic segmentation, Drosophila leg segmentation occurs in a tissue that is rapidly growing in size and thus segmentation must be coordinated with tissue growth. Segmentation of the Drosophila leg, as assayed by expression of the key regulators of segmentation, the Notch ligands and fringe, occurs progressively and this study defines the sequence in which the initial segmental subdivisions arise. The proximal-distal patterning genes homothorax and dachshund are positively required, while Distal-less is unexpectedly negatively required, to establish the segmental pattern of Notch ligand and fringe expression. Two Serrate enhancers that respond to regulation by dachshund are also identified. Together, these studies provide evidence that distinct combinations of the proximal-distal patterning genes independently regulate each segmental ring of Notch ligand and fringe expression and that this regulation occurs through distinct enhancers. These studies thus provide a molecular framework for understanding how segmentation during tissue growth is accomplished (Rauskolb, 2001).

A general theme in patterning during development is the subdivision of tissues initially by genes expressed in broad, partially overlapping domains, which through combinatorial control, subsequently regulate the expression of downstream genes to generate a repeating pattern. The studies presented here demonstrate that leg segmentation follows this same theme. The 'leg gap genes' Hth, Dac, and Distal-less are expressed in broad domains in the leg disc that encompass more than a single segment. Initially expression of these genes is largely nonoverlapping, but as the leg disc grows, the expression patterns of the leg gap genes change such that five different domains of gene expression are established. The analysis of the regulation of Notch ligand and fringe expression during leg development reveals two fundamental aspects of leg development. (1) These leg gap genes are key components in regulating the expression of the molecules controlling segmentation. Indeed, the effect of these leg gap genes on leg segmentation and growth can be accounted for by their regulation of Serrate, Delta and fringe expression. (2) The expression of each ring of Serrate, Delta and fringe is controlled by its own unique combination of regulators, apparently acting through independent enhancers (Rauskolb, 2001).

How do these three transcription factors regulate the formation of nine segments? Since the requirements for and the expression of the leg gap genes encompasses all leg segments, it is unlikely that there are additional leg gap genes yet to be identified. Rather, a collection of distinct combinatorial approaches is used to establish a segmental pattern of Serrate, Delta and fringe expression (Rauskolb, 2001).

In early third instar leg discs, there are two domains of gene expression: proximal cells express Hth and distal cells express Distal-less. Hth autonomously promotes the expression of Serrate, while Distal-less may prevent expression more distally, giving rise to a ring of expression in the coxa. Additionally, Distal-less-expressing cells may signal to the Hth-expressing cells to restrict Serrate expression to the distal edge of the Hth domain. As the leg disc grows, cells in an intermediate position, lying between the Hth and Distal-less domains, begin to express Dac. Dac, as shown in this study, is both necessary and sufficient to induce the expression of Serrate, Delta and fringe within the femur. Since they are not expressed in all Dac-expressing cells, other factors appear to be required to promote their expression in the proximal femur. The nonautonomous induction of Serrate expression by Hth suggests that this may be accomplished by a signal (X) emanating from the neighboring Hth-expressing cells. By mid third instar stages, expression of Serrate, Delta and fringe is also observed in tarsal segments 2 and 5, within cells expressing Distal-less but not Dac. Given that Distal-less is necessary and sufficient to repress their expression, Serrate, Delta and fringe expression within the tarsus appears to be induced by a mechanism that overrides the repressive effects of Distal-less. Subsequently, expression of Serrate, Delta and fringe is observed within the tibia, in cells expressing both Dac and Distal-less. Dac is necessary for expression of Serrate within the tibia, and its role here may be to overcome the repressive effects of Distal-less. It is also worth noting that the tibia ring of expression is not established at the time when cells first express both Dac and Distal-less. This may be because Dac levels may not be sufficiently high enough to overcome the repression by Distal-less. Clearly levels of Dac expression are critical because simply increasing Dac levels is sufficient to promote Serrate expression in cells already expressing endogenous levels of Dac. This observation can be explained if high levels of Dac expression in cells already expressing Dac override the function of inhibitory regulators of Serrate expression, such as Distal-less, where the expression of these genes overlap. Although late stages of leg segmentation were not investigated in this study, it has been noted that Hth, Dac and Distal-less are co-expressed in the presumptive trochanter late in leg development. It is thus hypothesized that Serrate, Delta and fringe expression is established by the combined activities of the three leg gap genes in the trochanter (Rauskolb, 2001).

Although these here have focused on the regulation of Serrate expression, it is thought that not only Serrate, but also Delta and fringe, receive primary regulatory input from the leg gap genes. Delta and fringe expression, like Serrate, is positively regulated by Dac. Moreover, Dl and fringe mutants have stronger leg segmentation phenotypes than Ser mutants, and thus Delta and fringe expression cannot simply be regulated downstream of Ser. The identification of two separate Ser enhancers, directing expression in the proximal versus distal leg, argues against Serrate being regulated downstream of Dl and fringe. Thus, the simplest model is that expression of all three genes is regulated directly by the leg gap genes. The regulation of Serrate, Delta and fringe expression in each segment appears to occur through independent and separable enhancer elements, supported by the analysis of the Ser reporter genes. This is reminiscent of what occurs during Drosophila embryonic segmentation, where separable enhancer elements direct different stripes of pair-rule gene expression (Rauskolb, 2001).

Most of the tarsus of the Drosophila leg derives from cells expressing Distal-less, but not Dac or Hth. Surprisingly, the studies presented here have shown that Distal-less actually represses Notch ligand expression. This negative regulatory role for Distal-less contrasts with the positive promoting role of Dac and Hth, and further indicates that a distinct molecular mechanism must promote segmentation within the tarsus. One key gene is spineless-aristapedia (ss), since simple, unsegmented tarsi develop in ss mutant flies. Moreover, ss regulates the expression of bric-à-brac (bab), which is also required for the subdivision of the tarsus into individual segments. Together, ss and bab must, in some way, ultimately overcome the repression of Notch ligand and fringe expression by Distal-less. If the sole function of ss and bab is to overcome the inhibitory effects of Distal-less, then in the absence of ss and/or bab, Serrate expression is expected to remain repressed (Rauskolb, 2001).

Intriguingly, the only notable variation between insect species is in the number of tarsal segments, with an unsegmented tarsus believed to be the ancestral state. Thus, the combinatorial regulation of segmentation by the leg gap genes may represent an ancient mechanism common to all insect species, a hypothesis supported by the conserved expression of Hth, Dac and Distal-less in the developing legs of many insect species (Rauskolb, 2001 and references therein).

The Iroquois homeobox genes function as dorsal selectors in the Drosophila head

The Iroquois complex (Iro-C) genes are expressed in the dorsal compartment of the Drosophila eye/antenna imaginal disc. Previous work has shown that the Iro-C homeoproteins are essential for establishing a dorsoventral pattern organizing center necessary for eye development. In addition, the Iro-C products are required for the specification of dorsal head structures. In mosaic animals, the removal of the Iro-C transforms the dorsal head capsule into ventral structures, namely, ptilinum, prefrons and suborbital bristles. Moreover, the Iro-C minus cells can give rise to an ectopic antenna and maxillary palpus, the main derivatives of the antenna part of the imaginal disc. These transformations are cell-autonomous, which indicates that the descendants of a dorsal Iro-C minus cell can give rise to essentially all the ventral derivatives of the eye/antenna disc. These results support a role of the Iro-C as a dorsal selector in the eye and head capsule. Moreover, they reinforce the idea that developmental cues inherited from the distinct embryonic segments from which the eye/antenna disc originates play a minimal role in the patterning of this disc (Cavodeassi, 2000).

The expression of genes required to make antenna was examined in the Iro-C minus clones, such as the homeobox gene Distal-less (Dll). Using Dll-lacZ, Dll expression was found to start at the early third instar and is confined to a small group of cells in the centre of the antennal primordium. Later expression of Dll encompasses all the antennal segments with the exception of the most proximal one. In the non-overproliferating Iro-C minus clones, Dll is activated, although this only occurs in clones located at the dorsal/posterior quadrant of the eye disc. In the antennal primordium, activation of Dll depends on simultaneous signaling by Dpp and Wg. wg is expressed by most of the cells of dorsal Iro-C minus clones. Many of these clones also express Dpp, which could be provided by either the morphogenetic furrow or, in clones that are near the disc margin, by a source of Dpp induced by the ectopic eye organizer associated with the clone. Thus, the clone cells were exposed to both Dpp and Wg, which in combination should activate Dll. Iro-C minus cells at the dorsal/anterior region, which are also exposed to Wg and Dpp, probably fail to activate Dll because they are located within the domain of expression of homothorax (hth). The removal of the Iro-C does not appreciably modify the expression of this gene. The presence of Hth might impair the activation of Dll since, in the leg disc, this protein, by driving the nuclear localization of Extradenticle (a cofactor of many homeoproteins), appears to reduce the sensitivity of cells to Wg and Dpp signaling. Thus, at the dorsal eye disc, Iro-C and hth probably cooperate to repress Dll. Large outgrowths resulting from Iro-C minus clones encompass hth-expressing cells in their anterior part and display ectopic Dll expression in the posterior part. In summary, the results suggest that, upon removal of Iro-C in dorsal cells, the cells become exposed to Wg and Dpp, and this and the absence of Iro-C products promote Dll activation. The interaction of Dll with hth, a gene with an antenna-selector function, should allow the growth of an ectopic antenna (Cavodeassi, 2000).

Notch signaling and the determination of appendage identity

The Notch signaling pathway defines an evolutionarily conserved cell-cell interaction mechanism that throughout development controls the ability of precursor cells to respond to developmental signals. Notch signaling regulates the expression of the master control genes eyeless, vestigial, and Distal-less, which in combination with homeotic genes induce the formation of eyes, wings, antennae, and legs. Therefore, Notch is involved in a common regulatory pathway for the determination of the various Drosophila appendages (Kurata, 2000).

Because Distal-less in combination with extradenticle (exd) and homothorax (hth) specifies the antennae, Dll expression was monitored in the eye discs that are capable of forming ectopic antennae. In wild-type larvae, Dll protein is expressed in the antennal but not in the eye disc. In all of the tested discs in ey-GAL4 UAS-N^actey² animals that form ectopic antennae from the eye disc, significant Dll expression was detected ectopically. This indicates that Notch signaling directly or indirectly induces ectopic expression of Dll in the eye-antennal disc, leading to the ectopic induction of antennae (Kurata, 2000).

The observation that N^act can induce both ectopic eyes and, in a specific genetic background, antennae, led to a consideration of the possibility that Notch signaling also might induce the formation of other appendages in a different genetic context. To test this hypothesis, the activation of Notch signaling was combined with ectopic expression of Antennapedia (Antp). The latter is known to determine the identity of the second thoracic segment (T2), which on the dorsal side gives rise to a pair of wings and on the ventral side to a pair of second legs. For this purpose, transgenic flies of the constitution ey-GAL4 UAS-N^act UAS-Antp were generated. About 26 of the flies escaping pupal lethality were found to have ectopic wings on the head. Almost all ectopic wing structures consisted of dorsal and ventral wing blades bordered by bristles of the wing margin, but lacking wing veins. In contrast, in wing structures induced by the ectopic expression of vg, the wing margin is not formed, suggesting that Notch signaling and Antp are acting upstream of vg. Furthermore, about 17% of these flies show ectopic leg structures induced by secondary transformation of the ectopic antennal tissue into leg structures (e.g., arista into tarsus). Therefore, activation of Notch signaling when combined with the ectopic expression of Antp driven by ey-GAL4 is capable of inducing wing and leg structures on the head (Kurata, 2000).

In wild-type larvae, the vg gene is expressed in the wing but not in the eye disc. By contrast, in ey-GAL4 UAS-N^act UAS-Antp animals in which ectopic wing structures are induced in the eye disc all of the tested eye discs show significant ectopic expression of Vg protein. It therefore appears that activation of Notch signaling in the context of Antp expression induces vg expression in the eye discs and that there are synergistic effects between Notch signaling and Antp expression. Notch signaling pathway has been shown to be used to specifically activate the boundary enhancer of the vg gene necessary for dorso-ventral wing formation. The same enhancer also may be used for ectopic formation of the wing, a point that has to be investigated further. A dorso-ventral boundary also is established by Notch in the eye disc that controls growth and polarity in the Drosophila eye. In ey-GAL4 UAS-N^act UAS-Antp ectopic legs also are induced on the head; this is accompanied by Dll expression (Kurata, 2000).

In view of the above observations it is proposed that the effects of Notch signaling on the various appendages depend on the context provided by control genes such as ey and Antp. In the eye primordia, Notch signaling induces ey expression, which induces a cascade of downstream genes leading to eye morphogenesis. In conjunction with Antp, Notch signaling induces vg, leading to wing formation. At low levels of ey expression, Notch signaling induces Dll, leading to antenna morphogenesis. In the case of the leg, Notch also induces Dll expression that, in conjunction with Antp, leads to leg formation (Kurata, 2000).

Segmental identity is specified by the homeotic genes that are active in a particular combination in each segment. Within a given segment, the appendages are specified by a different set of subsidiary control genes; the eyes are specified by ey, the wings and haltere by vg; the legs by Dll and the antennae by Dll in combination with extradenticle (exd) and homothorax (hth). The results presented here indicate that they all are regulated by Notch signaling and that they share the same cell signaling pathway, which raises the possibility that the appendage specificity is provided by a combinatorial interaction between Notch and the homeotic and the subsidiary control genes (Kurata, 2000).

The repression of one control gene by the expression of another seems to be a widespread mechanism to ensure that the developmental pathways are mutually exclusive so that the formation of intermediary cell types is prevented. Similar to the repression of ey by Antp, ey directly or indirectly represses Dll. In hypomorphic ey mutants, the activation of Notch signaling leads to ectopic expression of Dll in the eye disc, suggesting that ey might repress Dll in the wild-type eye disc. In dpp-GAL4 UAS-ey transheterozygous flies Ey is expressed on the ventral side of the posterior half of the antennal discs under the control of the dpp-enhancer, whereas Dll is not detectable in this area. A similar mutually exclusive expression is found in the leg discs of these flies, suggesting that ey represses Dll expression (Kurata, 2000).

Based on these findings, a model is proposed to explain the difference between the eye and antennal pathway starting from a common signaling mechanism. Notch signaling induces the expression of both ey and Dll. However, in the eye primordia ey represses Dll and induces eye morphogenesis. By contrast, in the antennal disc ey is repressed by a repressor, resulting in Dll expression that confers antennal (ventral appendage) specificity. Two of the possible candidates for the repressor are the homeobox genes exd and hth, because both exd^- and hth^- mutant clones in the rostral membrane region of the antennal disc can result in ectopic eye development, which presumably is caused by derepression of ey. Both exd and hth also may function in conjunction with Dll, serving as corepressors. The present study extends the fundamental role of Notch by indicating that the implementation of entire developmental programs leading to appendage formation and organogenesis may be controlled by Notch activity (Kurata, 2000).

Osa-containing Brahma chromatin remodeling complexes are required for the repression of Wingless target genes

The Wingless signaling pathway directs many developmental processes in Drosophila by regulating the expression of specific downstream target genes. The product of the trithorax group gene osa is required to repress such genes in the absence of the Wingless signal. The Wingless-regulated genes nubbin, Distal-less, and decapentaplegic and a minimal enhancer from the Ultrabithorax gene are misexpressed in osa mutants and repressed by ectopic Osa. Osa-mediated repression occurs downstream of the up-regulation of Armadillo but is sensitive both to the relative levels of activating Armadillo/Pangolin and repressing Groucho/Pangolin complexes that are present, and to the responsiveness of the promoter to Wingless. Osa functions as a component of the Brahma chromatin-remodeling complex; other components of this complex are likewise required to repress Wingless target genes. These results suggest that altering the conformation of chromatin is an important mechanism by which Wingless signaling activates gene expression (Collins, 2000).

To test whether Osa is acting directly on Wg target genes or regulating the expression of some other gene that interacts with the wg pathway, attempts have been made to determine at what level in the wg pathway Osa acts. In third-instar wing discs, wg is expressed in a narrow stripe of cells that straddles the dorsal/ventral (DV) boundary of the wing pouch and directs growth and patterning of the wing blade with respect to the DV axis. Cells adjacent to the DV boundary respond to the wg signal by posttranscriptionally up-regulating cytosolic Arm. Arm then translocates to the nucleus and binds to Pan to activate the expression of downstream target genes such as Distal-less (Dll). When an activated form of the protein kinase Sgg, which constitutively targets Arm for degradation (UAS-Sgg*) is expressed in the dorsal compartment using the ap-Gal4 driver, Arm is not up-regulated. Expression of UAS-Osa in the dorsal compartment similarly prevents the expression of Dll on the dorsal side of the DV boundary. However, these cells still respond to the Wg signal by up-regulating cytosolic Arm. Therefore, Osa represses Wg target genes without affecting the up-regulation of Arm. This places the activity of Osa in the nucleus and argues that Osa may directly repress the expression of Wg target genes (Collins, 2000).

The Hox gene Abdominal-B antagonizes appendage development in the genital disc of Drosophila

In Drosophila, the Hox gene Abdominal-B is required to specify the posterior abdomen and the genitalia. Homologs of Abdominal-B in other species are also needed to determine the posterior part of the body. The function of Abdominal-B in the formation of Drosophila genitalia has been studied, and the absence of Abdominal-B in the genital disc of Drosophila is shown to transform male and female genitalia into leg or, less frequently, into antenna. These transformations are accompanied by the ectopic expression of genes such as Distal-less or dachshund, which are normally required in these appendages. The extent of wild-type and ectopic Distal-less expression depends on the antagonistic activities of the Abdominal-B gene (as a repressor), and of the decapentaplegic and wingless genes (as activators). Absence of Abdominal-B also changes the expression of Homothorax, a Hox gene co-factor. These results suggest that Abdominal-B forms genitalia by modifying an underlying positional information and repressing appendage development. It is proposed that the genital primordia should be subdivided into two regions, one of them competent to be transformed into an appendage in the absence of Abdominal-B (Estrada, 2001).

Abd-B clones were induced, and they transform posterior abdominal segments into more anterior ones but are normal in the analia. Rare clones transform to distal antennae (second and/or third antennal segment and arista). Transformations to legs or antennae are cell autonomous. The formation of legs requires the activity of genes such as homothorax (hth), dac and Dll, which specify the proximal, medial and distal parts of the leg, respectively. Dll expression in wild-type discs is regulated by the combined activities of wingless and dpp in the genital primordia, and is confined to two groups of cells in male and female discs, the female domains being smaller and expressing lower levels of Dll protein. Since Abd-B is transcribed in the entire genital primordia of the two sexes, some cells co-express Abd-B and Dll. In the male disc, hth is not expressed in the Dll-expressing cells and is also excluded from a large group of cells surrounding them. Levels of antibody signal vary within the disc, and are higher in the female repressed primordium. In females, the hth domain of expression occupies the whole primordium. Lower levels of Hth are detected in a region encompassing the Dll-expressing cells, whereas higher levels are observed in the male repressed primordium. In both sexes, hth expression is absent from the anal primordium. dac is expressed differently in male and female genital primordia: in male discs, Dac protein is detected in two broad lateral bands, while in female discs it is found in the central region, almost coincident with the wg-expressing region. Therefore, the expression patterns of hth, dac and Dll differ substantially from those observed in legs (Estrada, 2001).

It is known that expression of Dll is not required to make male genitalia and that it has only a minor role in the formation of the female one. To ascertain the role of hth in the genitalia, hth minus clones were induced during the third larval period and they were examined in the adult structures. In the female genitalia, hth minus clones cause extra growths with additional vaginal teeth. In males, these clones show occasionally some abnormalities in the clasper teeth. hth clones in the analia are wild type. Possible interactions between Dll and hth in the genital disc were sought. In these experiments, unless stated, the results apply both to male and female genital primordia. Dll minus clones in the Dll domain of the male disc have no hth expression. Similarly, in hth minus clones Dll is not ectopically expressed. Dll was also expressed ectopically and the effect on hth expression was examined. Dll-expressing cells close to the wild-type Dll domain repress hth expression, although not all the cells do so. By contrast, clones far from the Dll domain do not affect hth expression (Estrada, 2001).

To characterize the transformation of genitalia into leg or antennal tissues, Abd-B minus clones were examined. Abd-B minus clones in the genital primordia tend to segregate from the rest of the tissue, round up and have smooth borders, suggesting they have acquired different affinities. By contrast, clones in the analia have indented borders and do not segregate. Abd-B minus clones in the genital primordium close to the normal Dll domain show ectopic, cell-autonomous Dll expression, whereas those far apart do not show such expression. dac is also activated cell autonomously in many Abd-B minus clones. As expected, Dll target genes, such as Bar, also become activated in these clones (Estrada, 2001).

Abd-B minus clones exhibit differential effects on hth, depending on their position: those close to the Dll domain show no hth expression, whereas those located away from the Dll domain show a slight increase in hth signal. Clones in intermediate positions do not significantly change hth levels. This distribution, however, is clearer in females, since in males there is a wide region with no hth expression. The repression of hth observed in some Abd-B minus clones may be mediated by the ectopic Dll (Estrada, 2001).

In the genital disc, the transcription of Dll depends, as in the leg disc, on dpp and wg signals. Abd-B represses Dll expression. Moreover, increasing Abd-B levels in the Dll domain suppresses Dll transcription. The antagonistic activities of dpp/wg and Abd-B in determining the Dll distribution was analyzed. Mutations in PKA ectopically activate wg and dpp expression. PKA minus clones in the genital primordia activate Dll, although only in some places. This activation is not mediated by changes in Abd-B levels. Similarly, although Dll is derepressed in many late Abd-B minus clones, derepression of either dpp or wg was not observed. It is concluded that there is an antagonism between the activation of Dll by dpp/wg signaling and its repression by Abd-B. This is not mediated by changes in the expression of either dpp, wg or Abd-B (Estrada, 2001).

To characterize this antagonism further, Abd-B minus clones that were made were also unable to transduce the dpp signal. This signal requires the presence of the type II receptor encoded by the gene punt. In put;Abd-B double mutant clones, Dll is not activated, indicating that, in the absence of Abd-B, Dpp (and possibly Wg) are still required to activate Dll. Abd-B minus clones far from the wild-type Dll domain fail to activate Dll ectopically, suggesting that activation of Dll in the absence of Abd-B depends on the range of diffusion of Dpp and Wg, as in the leg disc and in the anal primordium (Estrada, 2001).

Dll is required for the development of legs and antennae, and induces these structures when expressed ectopically in the wing or eye-antennal discs. However, although Dll is also expressed in the genital primordia this expression does not lead to the formation of any of these appendages. To test if Abd-B prevents Dll function Abd-B was eliminated in Dll-expressing cells; these cells formed leg tissue. However, it is possible that the high levels of Dll observed in these mutant cells account for the leg transformation. To test this, use was made of the GAL4/UAS system to increase Dll expression in the genital disc (dpp-GAL4/ UAS-Dll flies). Male and female genitalia of this genotype are abnormal, but not transformed into leg. To extend these observations, the ability of Dll to promote Bar transcription, a gene expressed in the leg disc and activated by Dll, was examined. Bar is not expressed in the female genital primordium and only in a few cells within the Dll domain in the male genital primordium; however, Abd-B minus clones show Bar expression in both sexes. When Dll is ectopically expressed in the genital disc, Bar expression is activated in some of the cells that express Dll. These results suggest that, in females, Dll levels are insufficient to activate Bar when Abd-B is present, but that increasing Dll expression or removing Abd-B activates Bar transcription. Abd-B, therefore, prevents some Dll activity in females. In males, although there is Bar transcription, leg tissue is not formed, probably because Abd-B modifies or prevents the activation of other Dll target genes. A similar case has been reported in the wing disc: ectopic Dll activates bric a brac, a gene downstream of Dll, both in the wing pouch and the body wall region of the wing disc; however, legs appear in the wing, but not in the notum (Estrada, 2001).

Pygopus, a nuclear PHD-finger protein required for Wingless signaling in Drosophila

The secreted glycoprotein Wingless (Wg) acts through a conserved signaling pathway to regulate expression of Wingless pathway nuclear targets. Wg signaling causes nuclear translocation of Armadillo, the fly ß-catenin, which then complexes with the DNA-binding protein TCF (Pangolin), enabling it to activate transcription. Though many nuclear factors have been implicated in modulating TCF/Armadillo activity, their importance remains poorly understood. A ubiquitously expressed protein, Pygopus, is required for Wg signaling throughout Drosophila development. Pygopus contains a PHD finger at its C terminus, a motif often found in chromatin remodeling factors. Overexpression of pygopus also blocks the pathway, consistent with the protein acting in a complex. The pygopus mutant phenotype is highly, though not exclusively, specific for Wg signaling. Epistasis experiments indicate that Pygopus acts downstream of Armadillo nuclear import, consistent with the nuclear location of heterologously expressed protein. These data argue strongly that Pygopus is a new core component of the Wg signaling pathway that acts downstream or at the level of TCF (Parker, 2002).

To examine targets that are positively regulated by Wg in the wing, the zinc-finger protein Senseless (Sens) and the homeodomain protein Distal-less (Dll) were chosen. Sens is expressed in the proneural clusters on either side of the dorsoventral border, immediately adjacent to the Wg expression domain. Inhibition of Wg signaling with a dominant-negative TCF blocks Sens expression, demonstrating that it is a short-range target of Wg action. pygo-expressing cells outside the Wg expression domain completely lack Sens expression. The long-range target Dll is also always lost in clones overexpressing pygo. For reasons that are not clear, occasionally some expression persists just inside the clonal border (Parker, 2002).

Coordinating proliferation and tissue specification to promote regional identity in the Drosophila head

The Decapentaplegic and Notch signaling pathways are thought to direct regional specification in the Drosophila eye-antennal epithelium by controlling the expression of selector genes for the eye (Eyeless/Pax6, Eyes absent) and/or antenna (Distal-less). The function of these signaling pathways in this process has been investigated. Organ primordia formation is indeed controlled at the level of Decapentaplegic expression but critical steps in regional specification occur earlier than previously proposed. Contrary to previous findings, Notch does not specify eye field identity by promoting Eyeless expression but it influences eye primordium formation through its control of proliferation. Analysis of Notch function reveals an important connection between proliferation, field size, and regional specification. It is proposed that field size modulates the interaction between the Decapentaplegic and Wingless pathways, thereby linking proliferation and patterning in eye primordium development (Kenyon, 2003).

This paper analyzes the role of Dpp and Notch in the regional specification of the eye-antennal disc. This study makes four observations: (1) domains of regional identity emerge in a complex pattern starting early in L2; (2) formation of eye and antenna primordia depend upon specific domains of dpp expression that emerge in early-L2 (eye) and mid-L2 (antenna); (3) neither Notch nor Dpp control the establishment of separate eye and antennal fields; (4) Notch can influence the establishment of an eye primordium through its control of proliferation in the eye field. Current models of regional specification have been evaluated based on these results and a new perspective on the emergence of regional identity in this tissue is presented (Kenyon, 2003).

It has been proposed that allocation of eye field and antennal field identity occurs in the latter half of L2 through the restriction of eye selectors, such as Ey, and antennal selectors, such as Dll, to distinct regions of the disc. However, two observations reported in this paper are not consistent with this interpretation: (1) Dll is not expressed ubiquitously at any time during disc development; (2) eye and antennal fields are clearly established by mid-L2 as evidenced by the restricted expression of Ey (eye field) and Cut (antennal field), and by distinct Dpp/Wg patterning centers within each field. These observations place the emergence of separate eye and antennal fields in the first half of L2 and not in the second half as previously proposed. Moreover, onset of Eya occurs in early-L2 and so is expressed by mid-L2. The beginning of eye primordium formation in early-L2, prior to the appearance of distinct fields, indicates that regional specification within this disc does not follow a two-step mechanism (i.e., establishment of separate fields followed by induction of organ primordia) but occurs in a more complex pattern. Further analysis of the transcription factors and signaling molecules active in the late-L1 and early-L2 disc is necessary to better understand how the establishment of eye field identity relates to eye primordium formation and the emergence of an antennal field (Kenyon, 2003).

Analyses of hypomorphic dpp alleles and tissue mutant for Mad implicate Dpp in the control of eya and Dll expression during late larval development. The onset of Eya and Dll expression correlate with specific changes in dpp expression during normal development. Using temperature shift experiments, it has also been established that Dpp signaling in L2 is required for the proper induction of both Eya and Dll in their respective fields. Gain-of-function analyses show that Dpp is also sufficient to induce Eya expression within the eye field and Dll expression within the antennal field. Clearly, though, Dpp must function in the context of selector factors such as Ey in order to produce two independent primordia within the eye-antennal epithelium. In the presence of Ey, Dpp signaling induces Eya expression as opposed to Dll. The absence of Ey in the antennal field at the time of Dpp signaling is of crucial importance to ensure the proper induction of Dll and subsequent formation of an antenna primordium. Indeed, as described in this study, the restriction of Ey to the eye field precedes the emergence of dpp and Dll in the antennal field during normal development (Kenyon, 2003).

In conclusion, Dpp, unlike Notch, functions as an inducer of tissue identity during specification of the eye-antennal disc, and the spatial and temporal aspect of organ primordia formation is controlled at the level of dpp transcription (Kenyon, 2003).

buttonhead and Sp1 are activators of the headcase and Distal-less in ventral imaginal discs

The related genes buttonhead (btd) and Drosophila Sp1 (the Drosophila homolog of the human SP1 gene) encode zinc-finger transcription factors known to play a developmental role in the formation of the Drosophila head segments and the mechanosensory larval organs. A novel function of btd and Sp1 is reported: they induce the formation and are required for the growth of the ventral imaginal discs. They act as activators of the headcase (hdc) and Distal-less (Dll) genes, which allocate the cells of the disc primordia. The requirement for btd and Sp1 persists during the development of ventral discs: inactivation by RNA interference results in a strong reduction of the size of legs and antennae. Ectopic expression of btd in the dorsal imaginal discs (eyes, wings and halteres) results in the formation of the corresponding ventral structures (antennae and legs). However, these structures are not patterned by the morphogenetic signals present in the dorsal discs; the cells expressing btd generate their own signalling system, including the establishment of a sharp boundary of engrailed expression, and the local activation of the wingless and decapentaplegic genes. Thus, the Btd product has the capacity to induce the activity of the entire genetic network necessary for ventral imaginal discs development. It is proposed that this property is a reflection of the initial function of the btd/Sp1 genes that consists of establishing the fate of the ventral disc primordia and determining their pattern and growth (Estella, 2003).

In a search for genes with restricted expression in the adult cuticle, the MD808 Gal4 line was found to direct expression in the ventral derivatives of the adult body; proboscis, antennae, legs and genitalia. In the abdomen and analia no clear expression was discerned. It was also noticed that the insertion was located in the first chromosome and associated with a lethal mutation. The mutant larvae showed a head phenotype resembling that described for mutants at the btd gene: loss of antennal organ and the ventral arms of the cephalopharyngeal skeleton, and complementation analysis indicated that the chromosome carrying the insert contained a mutation at btd. The expression pattern found in MD808/UAS-lacZ embryos was also similar to that reported for btd, suggesting that the Gal4 insertion was located at this gene. In addition, the imaginal expression of MD808 and of btd was largely coincident (Estella, 2003).

Further to the genetic analysis and the expression data, DNA fragments at the insertion site were cloned to map the position of the P-element on the genome. It is located 753 bp 5' of the btd gene. The related gene Sp1 is immediately adjacent. It is likely that btd and Sp1 have originated by a tandem duplication of a primordial btd-like gene (Estella, 2003).

In early embryos btd is expressed in the head region, but by the extended germ band stage the expression domain has expanded to the ventral region of cephalic, thoracic and abdominal segments. During germ band retraction most of the abdominal and thoracic expression is lost, except in derivatives of the peripheral nervous system and the primordia of the imaginal discs. Sp1 is not expressed in early embryos, but from stage 11 onwards it shows the same pattern as btd (Estella, 2003).

Special attention was paid to the btd/Sp1 expression domain in the thoracic imaginal discs primordia, as it may suggest a novel function related to imaginal development. Double labelling with Dll and btd probes indicates that btd precedes Dll expression, but by stage 12 the two genes are co-expressed in a group of thoracic cells. However, the Dll domain is smaller and is included within the btd/Sp1 domain: there are cells expressing btd that do not show Dll activity, although all the cells expressing Dll express btd (Estella, 2003).

The ventral disc primordia include not only cells expressing Dll but also other cells containing expression of escargot (esg) and hdc, markers of the diploid cells that form the imaginal primordia. In late embryonic stages, esg is expressed in a ring domain surrounding the Dll-expressing cells and hdc is expressed in a similar pattern. Double label experiments were carried out with btd, hdc and esg probes; the expression of the two latter genes overlaps with that of btd (and with Sp1) in the thoracic disc primordia (Estella, 2003).

The overlap of the btd and of esg domains indicates that btd is also expressed in the hth domain, which is coincident with that of esg. As the hth/esg domain marks the precursor cells of the proximal region of the adult leg the embryonic expression data indicate that btd and Sp1 are active in the entire primordia of the ventral adult structures, including the distal and the proximal parts (Estella, 2003).

In the mature antennal disc, btd expression is restricted mostly to the region corresponding to the second antennal segment, where it co-localizes with both Dll and hth. In the leg disc btd also overlaps in part with Dll and with hth. The latter result is significant, for the expression of Dll and hth define two major genetic domains, which are kept apart by antagonistic interactions. The fact that btd is expressed in the two domains suggests that its regulation and function is independent from the interactions between the two domains. This observation is consistent with the results obtained in embryos and suggests that the btd domain includes the precursors of the whole ventral thoracic region from the beginning of development (Estella, 2003).

This work demonstrates a novel and also redundant function of btd and Sp1: they are responsible for the formation of the ventral imaginal discs by activating the genetic network necessary for their development. Furthermore, Btd protein retains the capacity of inducing the entire ventral genetic network during the larval period. It is proposed that the activation of btd/Sp1 is the crucial event in the determination of the ventral structures of the adult fly (Estella, 2003).

This argument is based on the finding that btd and Sp1 appear to mediate all events connected with the formation of the ventral discs. The discussion deals with the leg disc, but there is evidence that antennal primordium also requires btd. Moreover, the genital primordium is lacking in Df(1)C52 embryos, suggesting that this disc is also under the same control. Most of the experiments concern the function of btd but given the expression and functional similarities between the two genes, it is assumed that Sp1 fulfils the same or a very similar role. Therefore, btd/Sp1 will be considered to carry out a single function (Estella, 2003).

One crucial feature is that btd is an upstream activator of Dll and hdc, which are considered developmental markers of disc primordia: (1) btd expression precedes that of Dll and of hdc; (2) the btd expression domain includes those of Dll and hdc; (3) in btd mutants, Dll and hdc activity is much reduced, and completely absent in Df(1)C52 embryos; (4) ectopic btd function induces ectopic activation of Dll and hdc (Estella, 2003).

The role of btd in embryogenesis can be illustrated in the light of the models of Dll regulation. Dll is activated by wg and its expression modulated by the EGF spitz and by dpp, whereas it is repressed in the abdominal segments by the BX-C genes. The current experiments suggest that Dll regulation is mediated by btd: in wg mutants there is no btd expression and hence neither Dll nor hdc activity. In dpp mutant embryos, btd expands to the dorsal region resembling the effect on Dll. In Ubx^- embryos there is an additional group of cells in the first abdominal segment expressing btd; the same cells that also express Dll in those embryos. The interpretation of the role of btd mediating Dll regulation by Ubx is complicated by previous observations showing direct repression of Dll by the Ubx protein. It is possible that Ubx regulates Dll both directly and by controlling btd activity (Estella, 2003).

It is proposed that the localization of btd/Sp1 activity to a group of ventral cells is a major event in the specification of adult structures. btd and Sp1 are activated in response to spatial cues from Wg, Dpp, EGF and BX-C, and in turn their function induces the activity of the genes necessary for ventral imaginal development (Estella, 2003).

This hypothesis is strongly supported by the results obtained inducing ectopic btd activity in the dorsal discs; just the presence of the Btd product alone is sufficient to bring about ventral disc development. In the wing and the haltere discs, Btd induces a transformation into leg, whereas in the eye it induces antennal development. This indicates that it specifies ventral disc identity jointly with other factors, e.g., the Hox genes, possibly through the activation of subsidiary genes such as Dll, known to contribute to ventral appendage identity in combination with Hox genes (Estella, 2003).

The requirement for btd and Sp1 activity appears to be restricted only to the ventral discs, even during the early phases of the thoracic disc primordia. In this context it is worth considering the observation that in Df(1)C52 embryos there is esg expression in the wing and haltere disc primordia, even though it is absent in the leg discs. Thus, the wing and haltere discs are formed in the absence of btd and Sp1. Because in these embryos there is an almost complete lack of Dll expression, this observation raises the question of the origin of the dorsal thoracic discs, which are currently considered to derive from the original ventral primordium, formed by cells expressing Dll. Although some of the original group of ventral cells may contribute to the dorsal disc primordia, the data suggest that there may be cells recruited to form the dorsal discs that do not originate in the initial ventral primordium. Accordingly, it is worth considering that in the absence of Dll activity the leg and wing discs are formed, although the leg only differentiates proximal disc derivatives. Thus, the activity of Dll cannot be considered a reliable marker for imaginal discs (Estella, 2003).

RNA interference experiments also indicate that both btd and Sp1 are required for the growth of the antennal and leg discs. When the two gene functions are reduced simultaneously, leg segments fuse and there is an overall reduction in the size of antennae and legs. The reduction of growth affects the proximal and distal regions of the appendage, and assigns a role to the expression observed in the imaginal discs. The two genes are able to perform this function on their own, for the inactivation of only one is not sufficient to impair growth. This conclusion is also supported by the observation that mutant btd clones do not have any effect; they still possess Sp1 activity, which is sufficient for normal development. At this point the mechanism by which btd/Sp1 may affect growth is not known (Estella, 2003).

One particularly significant result about the mode of action of btd comes from the analysis of the ectopic leg patterns observed with ectopic btb expression in the wing and halteres. The clones of cells ectopically expressing btd tend to recapitulate the complete development of leg and antennal discs. For example, the whole genetic network necessary to make a leg appears to be activated. btd induces the activity of hth, dac and Dll, the domains of which account for the entire disc. Furthermore, hth, dac and Dll are activated in a spatially discriminated manner. The formation of the dac and Dll domains is dependent on signalling from Wg and Dpp, although they require different signal thresholds. In one clone, for example hth is expressed only in the peripheral region, resembling the normal expression in the leg disc; in another