Interactive Fly, Drosophila

d Interactive Fly, Drosophila

Chip was cloned and found to encode a homolog of the recently discovered mouse Nli/Ldb1/Clim-2 and Xenopus Xldb1 proteins, which bind nuclear LIM domain proteins. Chip protein physically interacts with the LIM domains in the Apterous homeodomain protein, and Chip interacts genetically with apterous, showing that these interactions are important for Apterous function in vivo. Importantly, Chip also appears to have broad functions beyond interactions with LIM domain proteins (Morcillo, 1997).

Dorso-ventral axis formation in the Drosophila wing requires the localized accumulation of the Apterous LIM/homeodomain protein in dorsal cells. dLdb/Chip encodes a LIM-binding cofactor that controls Ap activity. Both lack and excess of dLdb/Chip function cause the same phenotype as apterous (ap) lack of function; i.e. dorsal to ventral transformations, generation of new wing margins, and wing outgrowths. These results indicate that the normal function of Ap in dorso-ventral compartmentalization requires the correct amount of the Chip co-factor, and suggest that the Ap and Chip proteins form a multimeric functional complex. In support of this model, it has been shown that the dLdb/Chip excess-of-function phenotypes can be rescued by ap overexpression (Fernández-Fúnez, 1998).

Chip mutations behave as strong enhancers of wing phenotypes produced by hypomorphic ap mutations. This synergistic interaction suggests that Chip and ap have related functions. To investigate further the function of Chip during wing development, genetic mosaics were generated by induced mitotic recombination using the Minute technique. Clones of Chip mutant cells in the wing ventral compartment show a wild-type phenotype and appear with normal frequencies, indicating that Chip is not required in this compartment. In contrast, Chip clones in the dorsal compartment are associated with wing outgrowths and ectopic wing margins. Cells within these clones have a ventral identity revealed by the morphology of the wing margin bristles they differentiate. Normal cells abutting the mutant clones are induced to form the dorsal structures characteristic of the wing margin. The ectopic margins can be visualized in undifferentiated imaginal discs with the use of molecular markers that label the wing margin. The largest wing outgrowths correspond to clones far from the normal wing margins. These clones cause the outgrowth of wild-type tissue, with the mutant clones located at the tip of the outgrowth . Thus, although Chip is expressed in all wing cells, Chip mutations produce specific phenotypes that are indistinguishable from ap phenotypes in clones. One possibility is that normal Chip function is required for ap expression. To test this possibility, ap expression was monitored in Chip mutant clones induced in wing imaginal discs. Ap protein is shown to accumulate normally in Chip mutant cells. Thus Chip does not regulate ap expression but it does show genetic interactions with ap, and Chip also produces the same mutant phenotypes in genetic mosaics. Taken together, these results are consistent with the hypothesis that Chip encodes a co-factor required for ap function as a dorsal selector gene (Fernández-Fúnez, 1998).

If Ap and Chip physically interact forming a functional complex, their stoichiometry may be important for the formation of the complex and for dorso-ventral patterning. To test whether the levels of Chip expression are important for dorso-ventral patterning, Chip was overexpressed in various patterns. Overexpression of Chip using a decapentaplegic GAL4 driver results in wing outgrowths, the creation of an ectopic wing margin on the dorsal compartment and a cut in the wing. These phenotypes are also evident in imaginal discs using wingless-lacZ (wglacZ) expression as a marker of the wing margin. Because lack of ap function in clones also causes wing outgrowths and ectopic wing margins, the distribution of Ap protein was examined in these wing imaginal discs. Chip overexpression does not alter the distribution of Ap protein; these results indicate that the phenotypes produced by Chip overexpression are not caused by Chip repressing ap. Interestingly, overexpression of ap using the drivers dppGAL4 or apGAL4 described above does not result in wing abnormalities in the dorsal compartment. Thus overexpression of Chip results in the same phenotype as its lack of function, i.e. transformation of dorsal into ventral cells, and, as a consequence, wing margin formation and outgrowth. These observations suggest that the relative amounts of Ap and Chip are important for dorso-ventral patterning (Fernández-Fúnez, 1998).

If the relative amounts of Ap and Chip are critical for dorso-ventral patterning, then it should be possible to rescue the excess of Chip phenotype by overexpressing ap. The proposed domain structure of the LDB/NLI family of proteins provides a conceptual framework to understand the phenotypes produced by altering the dosage of Chip and ap. The presence of homodimerization and LIM-interacting domains in LDB/NLI proteins suggests that LDB and LIM domain proteins may form tetrameric complexes. These complexes would be formed by two LDB molecules interacting through the N-terminal homodimerization domain; in addition, each LDB molecule would interact with a LIM domain through the C-terminal LIM-interacting domain. The occurrence of these complexes has been demonstrated between murine LDB and the hamster LIM/homeodomain protein LMX1. In the case of Chip-Ap, this tetrameric complex may be the functional complex carrying out the dorso-ventral patterning functions. This model predicts that Chip overexpression would lead to the formation of non-functional complexes, and it also predicts that functional Chip-Ap complexes would be reconstituted by overexpressing ap in addition to Chip. To test this model, a GAL4 insertion in ap was used. Expression from this GAL4 driver faithfully reproduces ap expression in the wing imaginal disc. When Chip is expressed from the ap promoter, the wing is reduced or eliminated depending on the UAS:Chip transgenic line used. Most lines reduce the wings, whereas the strongest line completely eliminates them. The reduced wing phenotype can be completely rescued by ap overexpression. These results provide further evidence for the idea that the stoichiometry of Ap and Chip is critical for dorso-ventral patterning (Fernández-Fúnez, 1998).

Two distinct functional domains have been identifed within NLI, the vertebrate homolog of Chip; the amino-terminal 200 amino acids (aa) mediate homodimerization, and 38 aa near the carboxyl terminus are required for high-affinity binding to LIM domains of LIM-HD proteins. These two domains within Chip are predicted to be present by primary sequence similarities between NLI, Chip, and orthologs from other species. To perform biochemical assays in vitro and misexpression studies in vivo, recombinant mutant Chip proteins were prepared in which either the predicted dimerization domain (DD) or the predicted LIM interaction domain (LID) was deleted. In addition, a control full-length Chip with no mutations was used. Five copies of the c-myc epitope were fused to the carboxyl terminus of each protein to monitor each protein's expression (van Meyel, 1999).

To determine whether the predicted DD and LID of Chip are indeed required for self-dimerization and LIM-HD interaction, respectively, double-immunoprecipitation assays were performed to biochemically detect high-affinity protein interactions. A 32-amino acid LID has been shown to be required for Chip to interact with Apterous, Islet, and Lim3. Deletion of 156 amino acids of the putative DD severely impairs the ability of Chip to dimerize. The DD is dispensable for LIM-HD interactions, and the LID is not required for Chip self-dimerization, indicating that these two domains of Chip function independently. Because deletion of the DD (aa 221-376) does not completely abolish the ability of Chip to self-dimerize, it is suspected that in conservative efforts to avoid disturbing nearby motifs, not all components of the dimerization domain were removed. Chip contains nearly 200 unique N-terminal amino acids not present in NLI that could participate in Chip dimerization (van Meyel, 1999).

Ap is the only known LIM-HD family member expressed in the developing wing disc. Chip interacts genetically with ap to cause disruptions of the wing margin, suggesting that these two genes act in the same pathway (Morcillo, 1997). In vitro analysis of Chip interactions with LIM-HD factors further suggests that the functionally relevant complexes required for transcriptional regulation in the wing disc are comprised of two Ap molecules bridged by two dimerized molecules of Chip. Alternatively, it is possible that Ap function is independent of Chip and that Chip, although expressed coincidentally with Ap, participates in an independent pathway and modulates Ap function indirectly by sequestering it from alternative complexes (van Meyel, 1999).

To distinguish whether Ap and Chip form functional complexes in vivo, or whether Ap works independently of Chip, the GAL4-UAS system was used to express Chip (ChipFL) and mutant Chip proteins (ChipdeltaDD and ChipdeltaLID). ap^GAL4, a P[GAL4] insertion in the ap locus, was used to direct reproducibly high levels of UAS transgene expression in the ap cells of the wing disc. As assayed by staining for the c-myc epitope, all Chip variants were expressed and were localized to the nucleus. Control ap^GAL4/+ heterozygotes display no wing defects, nor do they in the presence of a UAS-ap transgene. The latter observation is consistent with the hypothesis that a Chip/Ap tetrameric complex is functionally relevant, since it would not be compromised by Ap overexpression. In contrast, overexpression of ChipFL in the wing discs of ap^GAL4/+ heterozygotes results in defects resembling hypomorphic ap mutants. Wings were severely compromised in size and structure, and the wing margin was poorly demarcated. These wing defects are suppressed by simultaneous overexpression of Ap using a UAS-ap transgene. This indicates that the stoichiometry between Ap and Chip is an important factor in wing development and further suggests that overexpression of ChipFL titrates endogenous Ap to form incomplete complexes in which LID domains of Chip molecules remain vacant. The suppression of wing defects caused by co-overexpression of Ap is consistent with the idea that these incomplete complexes can be rendered fully functional by providing additional Ap to occupy vacant LID sites (van Meyel, 1999).

Overexpression of ChipdeltaDD results in severe wing defects that mimic those of extreme ap mutants. Like ChipFL, ChipdeltaDD is predicted to bind and sequester endogenous Ap but is unable to dimerize. The relative severity of ChipdeltaDD suggests that it renders Ap completely nonfunctional and blocks it from forming further Chip-bridged multimolecular interactions. Simultaneous overexpression of Ap suppresses the wing defects induced by ChipdeltaDD, suggesting that a pool of endogenous, dimerized Chip molecules exists to which ectopic Ap molecules bind and ''fill in'' unoccupied LID sites to form functional complexes. The suppression was not to the same extent as that achieved by Ap coexpressed with ChipFL and suggests that the poisoned ChipdeltaDD/Ap complexes can compete with fully functional complexes for binding to control elements in target genes (van Meyel, 1999).

Overexpression of ChipdeltaLID causes wing defects resembling those induced by overexpression of ChipFL. This experiment demonstrates a key role for Chip in wing development, since ChipdeltaLID is unable to interact with Ap and thus cannot simply sequester it from biological function. Importantly, these defects are not suppressed by simultaneous overexpression of Ap, as are those induced by ChipFL or ChipdeltaDD. Therefore, Ap cannot reconstitute function of complexes containing ChipdeltaLID, strongly supporting a role for direct physical interaction between Ap and Chip for function in vivo (van Meyel, 1999).

These results suggest that a tetrameric Chip/Ap complex mediates Chip and Ap function in the wing. In a simple model, the Ap LIM domains and the Chip LID domain bind one another and permit the homeodomains of two Ap molecules to be bridged by Chip dimerization. To test this model directly, the domains responsible for the interaction were removed and the remainder of each protein was tethered by fusing ChipdeltaLID directly to a LIM domain deleted version of Ap. This chimeric molecule should allow reconstitution of the Chip/Ap complex independently of the LID and LIM domains and rescue ap mutant wing defects if Chip and Ap are indeed required to bind one another. ap^GAL4 has been shown to act as a strong hypomorphic allele of ap. Using ap^GAL4 as a driver in an ap mutant background, it was found that ApdeltaLIM and ChipdeltaLID are incapable of rescuing any ap mutant wing defects. In contrast, the ChipdeltaLID:ApdeltaLIM chimera does rescue ap mutants. While the extent of rescue is not as complete as for Ap, flies expressing ChipdeltaLID:ApdeltaLIM exhibit significant wing outgrowth and a clearly demarcated margin with a triple row of sensory bristles. It is concluded from this result that physical interactions between Ap and Chip are required for appropriate margin formation and wing outgrowth (van Meyel, 1999).

In experiments to be reported elsewhere, it has been found that loss-of-function Chip mutations reveal ap-like defects in axon pathfinding and neurotransmitter production by Ap interneurons. Having implicated a role for Chip in three distinct functions of Ap, and given the widespread expression of Chip, it is speculated that Chip is an obligate cofactor for other LIM-HD activities. In fact, it would appear that the only way to exclude Chip from participating in complexes with LIM-HD factors is to selectively render it ineffective, perhaps by sequestration or by the formation of more favorable interactions between LIM-HDs and other factors. Indeed, both of these processes may act to limit the range of activity of Chip. (1) Chip may be sequestered in cells that coexpress dLMO (Beadex), a member of the LIM-only subclass of nuclear LIM-containing proteins that have no homeodomain. dLMO can selectively inhibit Chip interactions with Ap in vitro and can modulate Ap function in the wing. (2) LIM-HD proteins may directly interact with proteins other than Chip. For example, in the absence of NLI, the LIM domains of Lhx-3 mediate direct heterodimerization with Isl-1 and Isl-2 but not with other LIM-HD family members. Drosophila Lim3 and Isl are likewise capable of forming heterodimers in the absence of Chip, suggesting that they can participate in both Chip-dependent and Chip-independent heterodimeric complexes in cells in which they are coexpressed. In fact, the data show that direct Islet/Lim3 interactions may be of higher affinity than Islet/Chip interactions, raising the possibility that under certain conditions Islet/Lim3 interactions may be favored over interactions with Chip. Recent studies have shown that Islet and Lim3 function in a combinatorial manner to specify motor axon pathway selection in flies (Thor, 1999), and analyses of Lhx-3/4 mutant mice indicate that a similar code operates in vertebrates. The implementation of any LIM-HD combinatorial code relies not only on the availability, concentration, and relative affinities of LIM-HDs; in addition, Chip/NLI and possibly LMO cofactors are also involved (van Meyel, 1999).

To test whether the active form of Ap is a complex involving two molecules of Ap and two molecules of Chip, the effects of expressing dominant-negative forms of Chip that differ in their ability to bind Ap were compared. Overexpression of wild-type Chip has dominant-negative activity in vivo. It has been suggested that this could be due to formation of trimeric complexes (Ap:Chip:Chip) that lack a second Ap molecule because the dominant-negative activity of Chip can be suppressed by overexpression of Ap. It was reasoned that a truncated form of Chip lacking the LIM interaction domain (ChipdeltaLID) would also serve as a dominant negative but that its activity should not be suppressed by overexpression of Ap. Before testing the activity of the ChipdeltaLID construct in vivo, it was verified that the molecular interactions between Ap and Chip in vitro are consistent with the expectations based on analysis of the human LDB protein, NLI (Milan, 1999).

Complex formation between Ap, Chip, and ChipdeltaLID was assayed using in vitro translated proteins. Ap was expressed with a T7-epitope tag, incubated with 35S-labeled Chip or ChipdeltaLID, and immunoprecipitated with anti-T7. Full-length Chip coprecipitates with T7-Ap. ChipdeltaLID is not recovered above background levels when incubated with T7-Ap, confirming that Chip needs the LID to bind effectively to Ap. ChipdeltaLID coprecipitates when incubated with T7-Ap and full-length Chip, demonstrating formation of a three part Ap:Chip:ChipdeltaLID complex. To verify that a Chip dimer can bridge two Ap molecules, a tagged version of Ap (Ap-TAP) was used. The biological activity of Ap-TAP is comparable to that of wild-type Ap when ectopically expressed in vivo under GAL4 control. Ap-TAP was in vitro translated and bound to IgG beads. The beads were washed and incubated with labeled Ap with or without unlabeled Chip. Without Chip, only background levels of 35S-Ap are recovered. When Chip is present, Ap-TAP beads bind 35S-Ap, indicating formation of the tetrameric complex in vitro (Milan, 1999).

Overexpression of Chip represses the Ap targets fringe-lacZ and dLMO. Overexpression of ChipdeltaLID using patched GAL4 also represses fringe-lacZ and dLMO, indicating that both forms of Chip suppress Apterous activity when overexpressed. Overexpression of Chip under control of ap-GAL4 interferes with wing formation, producing a phenotype resembling the lack of ap function. This can be suppressed by coexpression of UAS-Chip and UAS-Ap. Overexpression of ChipdeltaLID using ap-GAL4 causes a mild apterous phenotype: distal notching of the wing margin and dorsal-to-ventral transformation of the alula. Although the phenotype suggests only partial reduction of Ap activity, the defects caused by overexpression of ChipdeltaLID cannot be suppressed by coexpression of Ap. These results suggest that ChipdeltaLID acts as a dominant negative for Ap activity in vivo by promoting the formation of a trimeric Ap:Chip:ChipdeltaLID complex that cannot bind another Ap molecule (Milan, 1999).

The LIM domain protein dLMO can compete with Ap for binding to its cofactor Chip. If the active form of Ap in vivo is a LIM-HD dimer bridged by a dimer of cofactor, dLMO might compete for Ap activity by displacing an Ap molecule from the Ap:Chip complex to form an Ap:dLMO heterodimer bridged by the cofactor. This model was tested by preparing a form of Ap that could dimerize but that could not be displaced by dLMO. To do so, a fusion protein consisting of the N-terminal dimerization domain of Chip linked to a C-terminal fragment of Ap containing the homeodomain (aa 270-469) was expressed. The new protein, called ChAp, lacks the LIM interaction domain of Chip and the LIM domains of Ap. Its structure should allow it to form an Ap dimer (Milan, 1999).

A test was performed to see whether ChAp has activity comparable to Ap in vivo. When expressed along the anteroposterior compartment boundary under control of dpp-GAL4, UAS-ap and UAS-ChAp produce essentially identical phenotypes. In both cases, ectopic wing margins are induced on both sides of the dpp-GAL4 stripe in the ventral compartment. The ectopic wing margin is due to the ectopic expression of Wingless in the ventral compartment. This correlates with ectopic induction of the dorsally expressed Ap targets fringe-lacZ and dLMO in ventral cells. These observations show that ChAp can mimic the effects of Ap in ectopic expression assays. A rescue assay was used to ask whether ChAp can functionally substitute for Ap in vivo. The wing defect in ap^GAL4/ap^rk568 flies is completely suppressed when wild-type Ap is expressed in dorsal cells using ap-GAL4. Dorsal expression of ChAp produces a comparable rescue. These results show that ChAp behaves like wild-type Ap when ectopically expressed and that ChAp can substitute for Ap in vivo (Milan, 1999).

According to the dimer model, ChAp should be sensitive to the dominant-negative activity of ChipdeltaLID but should not be subject to regulation by dLMO in vivo. dLMO is induced by Ap in the wing disc, and loss-of-function dLMO mutants produce defects that are thought to result from overactivation of Ap. ChAp overexpression in the dorsal compartment of an otherwise wild-type wing disc gives a phenotype that closely resembles the dLMO loss-of-function phenotype. The wings are smaller than wild type and are held in an abnormal position. The dorsal compartment is typically smaller than the ventral compartment, giving the wing a slightly curled appearance. The pattern of veins is also abnormal. Coexpression of the dominant-negative form of the cofactor, ChipdeltaLID, suppresses the ChAp overexpression phenotype. This indicates that dimerization is required for ChAp activity in vivo (Milan, 1999).

To ask whether ChAp is subject to regulation by dLMO, the ability of Ap and ChAp to suppress the effects of dLMO overexpression in vivo were compared. apGAL4/+; UAS-dLMO/+ wings show loss of the normal wing margin and sporadic patches of ectopic wing margin, thus promoting local overgrowth. Overexpression of wild-type Ap does not suppress the dLMO overexpression phenotype. Antibody staining shows that Wg is not expressed at the DV boundary in these discs. The wing pouch is very small, and the normally straight boundary between cells expressing Ap and adjacent nonexpressing cells is uneven. These observations suggest that Ap is nonfunctional in these discs in spite of being overexpressed. In contrast, coexpression of ChAp and dLMO restores Wg expression at the DV boundary even though dLMO is expressed at high levels. The resulting wings have a normal wing margin and resemble the dLMO mutant wing. These results suggest that ChAp overexpression phenocopies the dLMO loss-of-function mutant because ChAp is not sensitive to downregulation by dLMO. Consequently, ChAp remains active in the presence of excess dLMO (Milan, 1999).

The bridged dimer model suggests that removing dLMO activity would result in excess Ap activity. To test this, the properties of Chip and Ap interaction were exploited to regulate Ap activity in a dLMO mutant background. dLMO loss-of-function mutants were generated by excision of a GAL4-P element insertion in the second intron of the dLMO gene. Fortuitously, excision line hdp^R590 strongly reduces dLMO expression but leaves GAL4 and the cis-regulatory region unaffected, so that the mutant expresses GAL4 in the normal pattern of dLMO. hdp^R590 causes aberrant Serrate expression and a reduced dorsal wing pouch. The dLMO loss-of-function phenotype in this mutant can be suppressed by expression of ChipdeltaLID. The small wing size of hdp^R590 is fully rescued, and the abnormal venation is partially suppressed. Likewise, expression of a mutant form of Ap lacking only the homeodomain completely suppresses the hdp^R590 phenotype. Both of these constructs have mild dominant-negative effects that reduce Ap activity in vivo. These results confirm that the dLMO loss-of-function phenotype results principally from excess Ap activity at later stages of wing development. Further, they support the proposal that the normal function of dLMO is to regulate Ap activity levels by interfering with formation of an active complex consisting of two Ap molecules bridged by a dimer of Chip molecules (Milan, 1999).

The finding that ChAp can completely replace Apterous function in vivo suggests that the relevant feature of this tetrameric complex is the formation of a dimer of Ap. This molecule is not subject to negative regulation by dLMO and so remains constitutively active. The consequence is a failure to downregulate Ap activity as development proceeds. The phenotypic consequences of excess Apterous activity are comparable to those of the dLMO lack-of-function mutant. These findings support the view that the tetrameric complex between Ap and its cofactor Chip provides a means to generate the requisite bridged dimer of Ap, while allowing the activity of the complex to be regulated by the competitive inhibitor dLMO. It is suggested that this may provide a general model for regulation of LIM-HD activity. LMO family proteins may be generic antagonists of LIM homeodomain proteins through binding to their common LDB cofactors. The active complexes may be cofactor-bridged homodimers (as is the case for Ap in wing development) or heterodimers with other LIM-HD transcription factors or other types of LDB-binding transcription factors. Combinations of different transcription factors bridged by a cofactor dimer might broaden the range of possible target genes (Milan, 1999).

The LIM homeodomain (LIM-HD) protein Apterous (Ap) and its cofactor DLDB/CHIP control dorso-ventral (D/V) patterning and growth of the Drosophila wing. To investigate the molecular mechanisms of Ap/CHIP function, their relative levels of expression were altered and mutants were generated in the LIM1, LIM2 and HD domains of Ap, as well as in the LIM-interacting and self-association domains of CHIP. Using in vitro and in vivo assays it was found that: (1) the levels of CHIP relative to Ap control D/V patterning; (2) the LIM1 and LIM2 domains differ in their contributions to Ap function; (3) Ap HD mutations cause weak dominant negative effects; (4) overexpression of ChipDeltaSAD mutants mimics Ap lack-of-function, and this dominant negative phenotype is caused by titration of Ap because it can be rescued by adding extra Ap; and (5) overexpression of ChipDeltaLID mutants also causes an Ap lack-of-function phenotype, but it cannot be rescued by extra Ap. These results support the model that the Ap-CHIP active complex in vivo is a tetramer (Rincon-Limas, 2000).

Regulation of Apterous activity in Drosophila wing development

Apterous is a LIM-homeodomain protein that confers dorsal compartment identity in Drosophila wing development. Apterous activity requires formation of a complex with a co-factor, Chip/dLDB. Apterous activity is regulated during wing development by dLMO, which competes with Apterous for complex formation. Complex formation between Apterous, Chip and DNA stabilizes Apterous protein in vivo. A difference in the ability of Chip to bind the LIM domains of Apterous and dLMO contributes to regulation of activity levels in vivo (Weihe, 2001).

Apterous activity levels are spatially and temporally regulated in the wing disc by expression of dLMO. Comparing expression of Ap protein and mRNA in the wing imaginal disc suggested that Ap might be subject to post-transcriptional regulation. ap mRNA is expressed at similar levels in the presumptive wing hinge and wing pouch. By contrast, Ap protein levels are considerably lower in the wing pouch than in the hinge region. The region where Ap levels are low coincides with the region in which dLMO is expressed. This suggests that the difference in Ap protein levels reflects a post-transcriptional consequence of dLMO expression. To ask whether dLMO is responsible for reducing Ap levels where the two proteins are co-expressed, genetic mosaics were produced in which dLMO activity was removed from clones of cells. Ap protein was more abundant in cells homozygous mutant for dLMO^Delta39. The increased level of Ap protein in the clone was similar to the level detected in the hinge (Weihe, 2001).

These observations suggest that dLMO protein is responsible for the reduced level of Ap protein in the dorsal wing pouch. To further test this possibility, clones of cells overexpressing dLMO were created and Ap protein levels were assessed. As expected from the loss-of-function data, dLMO expression reduced Ap levels in the hinge region, where Ap levels are usually high. The lower level of Ap in the dorsal pouch was further reduced by elevated dLMO expression. It is therefore concluded that dLMO reduces Ap levels in third instar imaginal wing discs. To determine whether Ap protein might be degraded in dLMO-expressing cells by a proteasome-dependent mechanism, wing discs were incubated with the proteasome inhibitor MG-132. Ap protein levels were increased in the wing pouch relative to the levels in the hinge in drug treated. This suggests that Ap protein is more susceptible to proteasome-mediated degradation in cells expressing dLMO (Weihe, 2001).

Since dLMO competes with Ap for binding to Chip, the possibility that Ap protein may be protected when it is in a complex with Chip was examined. To test this, Chip^e5.5 mutant clones, which lack Chip protein and therefore lack Ap activity, were created. Ap protein levels were reduced in Chip mutant clones, and increased in the wild-type twin spots which contain a higher level of Chip protein. To verify that reduced Chip activity does not affect ap mRNA levels ap-lacZ reporter gene expression was examined in discs expressing the dominant negative form of Chip, ChipDeltaLID. Ap protein levels were reduced in cells expressing ChipDeltaLID but ap-lacZ levels were unaffected. Thus, loss of Chip leads to reduced levels of Ap protein. It was noted that Chip mutant clones also lack dLMO expression. Thus, loss of Ap protein in Chip mutant clones does not correlate with expression of dLMO, as in wild-type cells. Rather, reduction of Ap levels correlates with the availability of Chip as a binding partner. This suggested that binding to Chip contributes to stabilization of Ap (Weihe, 2001).

ChipDeltaLID is capable of binding to full-length Chip through its dimerization domain, but cannot bind to Ap. Consequently, ChipDeltaLID leads to formation of trimeric complexes and thereby blocks Ap activity in vivo. The observation that ChipDeltaLID leads to reduced Ap stability without affecting ap-lacZ expression suggests that stabilization might require formation of tetrameric complexes between Chip and Ap. The tetrameric form of Chip and Ap is thought to be the active DNA-binding complex. Overexpression of ChipDeltaLID does not decrease the availability of LIM-binding sites in wild-type Chip, but does compete for tetramer formation. This raises the possibility that Ap stability might depend on whether it is able to form a DNA-binding complex with Chip (Weihe, 2001).

If Ap stability decreases when it is unable to bind DNA, it was reasoned that providing additional binding sites might stabilize the protein. To test this possibility a cell culture system was used in which the number of Ap-binding sites could be varied by transfection. It was first verified that co-expression of dLMO would decrease Ap stability in transfected cells. A constant amount of a plasmid directing expression of a Myc-tagged Ap protein was co-transfected with varying amounts of a plasmid directing expression of myc-tagged dLMO. As observed in the wing disc, overexpressed dLMO reduces Ap protein levels in S2 cells. It was noted that high levels of dLMO are required to reduce Ap levels. The relative levels of the two proteins can be directly compared in this assay by virtue of the myc-epitope tag. Comparison of relative levels of the endogenous proteins is not possible (Weihe, 2001).

To test the effect of Ap-binding sites on Ap protein stability, a constant amount of a plasmid directing expression of a myc-tagged Ap protein was co-transfected with varying amounts of a plasmid carrying nine tandem repeats of a binding-site for the mammalian Ap-homolog Lhx2. This DNA sequence binds Drosophila Ap in S2 cells. The total amount of DNA in the transfection assay was held constant by varying the ratio of plasmid containing the binding sites and empty vector. Increasing the ratio of the plasmid containing the binding sites results in dose-dependent stabilization of Ap-myc protein. This observation supports the idea that availability of binding sites limits the amount of Ap protein that is stable in the cell when mRNA levels are held constant (Weihe, 2001).

Another means to test this possibility is by competition between Ap and a related protein for a fixed number of binding sites. For these experiments use was made of a fusion protein between Chip and Ap (called ChAp). In this protein the dimerization domain of Chip mediates dimerization of the DNA-binding domains of Ap. Thus, ChAp dimers should compete with endogenous Chip:Ap tetramers for DNA-binding sites. Use of the Myc tag versions of both proteins allowed direct comparison of their relative levels in co-transfected cells. Using this assay it was verified that increasing the level of ChAp-myc decreases the level of co-transfected Ap-myc in a dose-dependent manner. Expression of Chip-myc as a control has little effect on Ap-myc levels. Note that the level of Ap-myc construct was held constant in all samples. ChAp-myc and Chip-myc expression levels were controlled by varying the ratio of the expression constructs to the empty expression vector in the transfections (Weihe, 2001).

It was next asked whether competition for DNA-binding sites would affect Ap stability in the wing disc. Fortuitously, the antibody raised against Ap does not recognize ChAp. This allows the level of the endogenous Ap protein to be measured in cells expressing ChAp. ChAp expression under dpp^Gal4 control leads to a decrease in the level of endogenous Ap protein. Together, these observations suggest that Ap protein is unstable in vivo unless bound to DNA as part of an active complex with its co-factor Chip (Weihe, 2001).

dLMO has been proposed to act as a competitive inhibitor of Ap in vivo. This model suggests that overexpression of Ap should be able to produce phenotypes similar to those caused by reduced levels of dLMO; however, this has not been observed. Overexpression of Ap in its endogenous domain does not produce alterations in the wing comparable with those caused by loss of dLMO activity. By contrast, expression of fusion proteins between Chip and Ap, which are insensitive to repression by dLMO, produce the expected phenotypes. This suggests that Ap does not compete effectively with dLMO for interaction with Chip, even when overexpressed. These observations could be explained by an intrinsic difference in the affinities of the two LIM domain proteins for Chip. To test this possibility, a fusion protein was constructed that contains the LIM domains of dLMO (100 amino acids) but otherwise consists entirely of Ap sequences. This protein was called dLAp to indicate LIM-domains of dLMO in Ap. To distinguish dLAp from endogenous Ap and dLMO proteins, a C-terminal flag tag was included (Weihe, 2001).

Two assays were performed to test the functionality of dLAp. (1) Use was made of the fact that ap^Gal4 is a mutant allele of ap. ap^Gal4/ap^UGO35 larvae have reduced ap activity and fail to develop normal wings. Expression of Ap protein under ap^Gal4 control rescues wing development in this mutant combination. Expression of dLAp was able to replace endogenous ap and rescue the ap^Gal4/ap^UGO35 mutant. (2) Ectopic expression of dLAp induces the formation of an ectopic DV boundary, as revealed by ectopic wing margin formation and ectopic Wingless expression. Ectopic Wingless is induced wherever Ap-expressing cells and non-expressing cells are juxtaposed. These results indicate that dLAp has Ap activity (Weihe, 2001).

To ask whether overexpression of dLAp in dorsal cells would compete effectively with dLMO to produce a net increase in Ap activity levels, wing development was compared in flies expressing dLAp or Ap under ap^Gal4 control. Ap overexpressing wings are normal. In ap^Gal4/+; uas-dLAp/+ flies defects were observed in wing veins, especially in the posterior crossvein and vein 5, and a held up wing phenotype. These defects resemble the dLMO mutant phenotype, which has been shown to be due to excess Ap activity. Another feature of dLMO mutant wings is overexpression of Serrate in the D compartment. Overexpression of wild-type Ap under ap^Gal4 control does not cause abnormal Serrate expression; however, expression of dLAp in ap^Gal4/+;uas-dLAp/+ wing discs induces ectopic Serrate in the dorsal compartment and causes mild reduction of the D compartment. Similar, though somewhat stronger effects were obtained by overexpression of the Chip/Ap fusion protein ChAp, which is insensitive to competition by dLMO. Thus, dLAp expression can increase Ap activity to levels above normal in the presence of dLMO (Weihe, 2001).

Ap activity can be abolished by overexpression of dLMO under ap^Gal4 control in the wing disc. Providing additional Ap protein by co-expression of Ap does not overcome the inhibitory effects of dLMO. Wingless is not expressed at the interface between D and V cells and the wing pouch is very small. By contrast, co-expression of dLAp restores Wingless expression along the DV boundary and wing pouch growth. This indicates that dLAp is able to restore Ap activity in the presence of dLMO. Taken together, these observations indicate that dLAp competes efficiently with dLMO for binding to Chip, whereas Ap does not. Since the only difference between Ap and dLAp is in the LIM domains, their different behavior is attributed to an intrinsic property of the LIM domains (Weihe, 2001).

This report addresses the problem of asymmetry in the competition between dLMO and Ap. The simplest model for competitive inhibition by dLMO would suggest that Ap should compete effectively with dLMO for binding to Chip when overexpressed. However, overexpression of Ap does not produce an excess of Ap activity. dLMO competes effectively for Ap activity, but the reverse is not true. Swapping the LIM domains of Ap for those of dLMO produces a functional Ap protein that is able to compete effectively with dLMO. This finding may provide an explanation for the non-reciprocal properties of Ap and dLMO. The effectiveness of dLMO as an inhibitor of Ap activity is attributed to an intrinsic difference in the ability of the LIM domains of these two proteins to bind to Chip. It is considered likely that the LIM domains of dLMO bind the LID of Chip with higher affinity than the LIM domains of Ap. However, these proteins have not been produced in soluble form at adequate concentrations and so the affinities of these binding interactions have not been determined directly (Weihe, 2001).

Other proteins might also contribute to stabilization of Chip-dLMO complexes or to destabilization of Chip-Ap complexes in vivo. Interactions involving Ap, Chip and other proteins have been reported. For example, Pannier interacts with Chip and competes with Apterous for patterning of the thorax. In this model, Chip is found in a complex with Pannier and dLMO, which promotes dorsal thorax formation. Chip is also found in a complex with Ap. The level of Chip is not in great excess, so competition occurs between Ap and Pannier for formation of Chip complexes, despite the fact that Pannier and Ap do not bind to Chip in the same way. It was noted that overexpression of dLAp-flag appears to interfere with Pannier complex formation, because it causes the formation of a cleft in the thorax, resembling a pannier loss-of-function mutant phenotype. Comparable overexpression of Ap does not do so. This suggests that dLAp competes more effectively than Ap for binding to Chip and so is more effective at sequestering Chip from Pannier-containing complexes. The relative affinity of these proteins appears to play an important role in maintaining the proper balance of complex formation in vivo. Numerous LIM-HD proteins have been found to play important roles in the development of a number of species. It seems likely that other LIM-homeodomain transcription factors will be regulated in similarly complex ways (Weihe, 2001).

Ssdp binds to Chip and regulates the activity of Apterous complexes in vivo

LIM-homeodomain transcription factors control a variety of developmental processes, and are assembled into functional complexes with the LIM-binding co-factor Ldb1 (in mouse) or Chip (in Drosophila). The identification and characterization is described of members of the Ssdp family of proteins, which are shown to interact with Ldb1 and Chip. The N terminus of Ssdp is highly conserved among species and binds a highly conserved domain within Ldb1/Chip that is distinct from the domains required for LIM binding and self-dimerization. In Drosophila, Ssdp is expressed in the developing nervous system and imaginal tissues, and it is capable of modifying the in vivo activity of complexes comprised of Chip and the LIM-homeodomain protein Apterous. Null mutations of the ssdp gene are cell-lethal in clones of cells within the developing wing disc. However, clones mutant for a hypomorphic allele give rise to ectopic margins, wing outgrowth and cell identity defects similar to those produced by mutant clones of Chip or apterous. Ssdp and Ldb/Chip each show structural similarity to two Arabidopsis proteins that cooperate with one another to regulate gene expression during flower development, suggesting that the molecular interactions between Ssdp and Ldb/Chip proteins are evolutionarily ancient and supply a fundamental function in the regulated control of transcription (van Meyel, 2003).

Ap is expressed in the dorsal compartment of the wing disc and is required to establish the DV affinity boundary, the wing margin, wing outgrowth and dorsal-specific wing structures such as sensory bristles. In the absence of Ap, the wing fails to develop. Ap functions through a tetrameric complex in which two molecules of Ap are bridged by a homodimer of Chip. Chip mutants interact genetically with ap to cause disruptions of the wing margin, and clones of Chip mutant cells in the wing disc behave like ap mutant clones, causing ectopic wing margins and outgrowths (van Meyel, 2003).

In contrast to a previous study, no phenotypes were detected in simple trans-heterozygous combinations of a null allele of Chip with any ssdp alleles used here, including ssdp^KG03600 and the two null alleles ssdp^L5 and ssdp^L7. Nor were any phenotypes detected in transheterozygous combinations of ap and ssdp. Thus, to address the role of Ssdp in the function of Chip/LIM-HD complexes in vivo, the GAL4-UAS system was used to reduce Ap/Chip complex activity to levels that would be sensitive to the effects of reducing ssdp gene dosage. ap^GAL4, a GAL4 P-element insertion in the ap gene, which faithfully expresses GAL4 in Ap-expressing cells, was used to drive expression of UAS transgenes in the dorsal compartment of the wing disc (van Meyel, 2003).

Over-expression of UAS-Chip disrupts wing patterning by titrating endogenous Ap into incomplete complexes in which LID domains of Chip molecules remain vacant. Relative to controls, such wings are small and lack regular structure, and the wing margin is poorly demarcated. These phenotypes resemble hypomorphic ap mutants, and can be completely suppressed by simultaneous overexpression of UAS-ap. This indicates that the stoichiometry between Ap and Chip is an important factor in the formation of functional complexes. The effect of removing one copy of the ssdp gene was examined; the resulting flies have little or no residual wing tissue, consistent with a further reduction of the activity of the complex (van Meyel, 2003).

Fusion of Chip and Ap into one chimeric molecule, called ChipDeltaLID:ApDeltaLIM, results in a hyperactive complex, since it is not susceptible to downregulation of activity imposed by LMO, a LIM-only factor that competes efficiently with Ap for binding with Chip. Flies that overexpress ChipDeltaLID:ApDeltaLIM have blistered wings in which the dorsal and ventral surfaces fail to fuse, and which are held upward and away from the thorax in a fashion resembling LMO loss-of-function mutants. Removal of one copy of ssdp suppresses the blistered wing phenotype and the surfaces fuse properly, although the wings remain held up. Thus, Ssdp can modify the activity of Chip/Ap tetrameric complexes of both reduced and hyperactive function (van Meyel, 2003).

Finally, the effects of Chip overexpression were compared with those produced by expression of a Chip variant lacking the LCCD (ChipDeltaLCCD). ChipDeltaLCCD is capable of self-dimerization and binding to Ap, but it cannot bind Ssdp. If Ssdp were required for function of the complex, ChipDeltaLCCD would be predicted to have a more potent dominant-negative effect on the function of the complex than would Chip itself, since the latter can still recruit Ssdp. Expression of ChipDeltaLCCD with ap^GAL4 consistently produced more extreme wing defects than Chip. ChipDeltaLCCD sequesters Ap into nonfunctional complexes, but it cannot bind Ssdp. Therefore, removal of one copy of ssdp would not be expected to suppress the phenotype caused by ChipDeltaLCCD, and indeed it does not. Collectively, these results argue that in addition to forming the dimeric bridge for two molecules of Ap, Chip also recruits Ssdp to the complex (van Meyel, 2003).

Clones of ap mutant cells in the dorsal compartment of the wing disc induce an ectopic wing margin and therefore ectopic wing outgrowth. These ap mutant cells differentiate ventral wing margin structures, despite the fact that they remain in the dorsal compartment. Chip mutant clones induced in the dorsal compartment give rise to strikingly similar phenotypes. The effects of Chip clones are influenced both by the timing of their induction as well as their position within the disc. For example, clones induced later (third instar) result in ectopic margin tissue, but do not lead to outgrowth (van Meyel, 2003).

If Ssdp were an additional member of the Ap/Chip complex, then mutations of ssdp would be predicted to give rise to mutant phenotypes similar to those of ap and Chip. To test this, the FRT/FLP recombinase system was used to induce clones of cells mutant for ssdp in an otherwise heterozygous animal. Clones were generated in larvae at second and third instar by heat-shock induction at 36 hours, 48 hours, 72 hours or 96 hours after egg laying (AEL). The effects of clone induction were observed in newly eclosed adults. Clones of mutant cells were identified by the presence of the cell-autonomous marker pawn (pwn). Each of the mutant alleles ssdp^L7, ssdp^L5 and ssdp^l(3)neo48 were tested, as was a control chromosome with no mutation, and the experiment was repeated on four separate occasions, each time observing many individuals of each genotype (van Meyel, 2003).

In contrast to the cell lethality associated with ssdp null alleles, there were striking phenotypes observed in clones of cells mutant for the hypomorphic ssdp^l(3)neo48 allele. Many pwn mutant clones located both ventrally and dorsally were observed. However, as for ap and Chip clones, associated phenotypes were found only when the clone arose on the dorsal surface of the wing. ssdp^l(3)neo48 clones induced earlier (at 36 hours and 48 hours AEL) give rise to ectopic margins and occasional wing outgrowth. The outgrowths were associated with ssdp mutant cells but were not entirely made up of them, indicating that, as for ap and Chip, outgrowth results from the induction of wild-type tissues in proximity to the clone. Clones induced at 72 hours and 96 hours AEL give rise to margin defects but not outgrowth, indicating that there is a temporal restriction on the extent to which ssdp mutation is capable of inducing outgrowth, similar to what has been shown for Chip (van Meyel, 2003).

Induction of ectopic margin bristles was the most commonly observed effect of dorsal ssdp mutant clones. They were primarily observed in proximity to a clone near the native anterior wing margin and comprised at least one row of extra sensory bristles. Most ectopic bristles were not marked by pwn, indicating they were induced by the neighboring mutant (pwn) cells. ssdp^l(3)neo48 mutant clones that occurred within the margin, rather than near it, resulted in the loss of dorsal-specific sensory bristles. Occasionally a large clone was observed to straddle the dorsoventral boundary, and in these instances, the entire margin, including some nearby non-margin tissue, was lost (van Meyel, 2003).

In general, there is a striking resemblance between the phenotypes resulting from ssdp^l(3)neo48 mutant clones and those reported for clones of Chip or ap. This provides strong evidence that Ssdp is an important additional component of Chip/Ap transcriptional complexes in vivo (van Meyel, 2003).

Chip-mediated partnerships of the homeodomain proteins Bar and Aristaless with the LIM-HOM proteins Apterous and Lim1 regulate distal leg development

Proximodistal patterning in Drosophila requires division of the developing leg into increasingly smaller, discrete domains of gene function. The LIM-HOM transcription factors apterous (ap) and Lim1 (also known as dlim1), and the homeobox genes Bar and aristaless (al) are part of the gene battery required for the development of specific leg segments. Genetic results show that there are posttranslational interactions between Ap, Bar and the LIM-domain binding protein Chip in tarsus four, and between Al, Lim1 and Chip in the pretarsus, and that these interactions depend on the presence of balanced amounts of such proteins. In vitro protein binding is observed between Bar and Chip, Bar and Ap, Lim1 and Chip, and Al and Chip. Together with evidence for interactions between Ap and Chip, these results suggest that these transcription factors form protein complexes during leg development. It is proposed that the different developmental outcomes of LIM-HOM function are due to the precise identity and dosage of the interacting partners present in a given cell (Pueyo, 2004).

Biochemical studies in vitro have shown that LIM-HOM transcription factors confer little transcriptional activation of target genes on their own. LIM-HOM proteins interact with a variety of proteins, including members of the bHLH family, the POU family and also other LIM family members, to control specific developmental processes. It has been suggested that these protein interactions confer specificity and modulate LIM-HOM activity. For example, Dlmo proteins reduce LIM-HOM activity, and Lbd proteins such as Chip modulate LIM-HOM activity by acting as a bridge between LIM-HOM proteins and Chip-binding cofactors, thus leading to the formation of heteromeric complexes. LIM-HOM protein activity functions in different contexts is the development of Drosophila (Pueyo, 2004).

Bar and ap genes are expressed in the fourth tarsal segment and are required for its proper development, whereas the al and Lim1 genes are expressed and required in the pretarsus. All of these genes encode putative transcription factors and display canonical regulatory relationships. Thus, al activates lim1 expression and then both genes cooperate to repress Bar expression in the pretarsus. Reciprocally, Bar represses al and lim1 expression while activating the expression of ap in tarsus four. After the refinement of their gene expression domains by these regulatory interactions, Bar directs tarsus five development, whereas cooperation between al and lim1 directs pretarsus development, and cooperation between Bar and ap directs tarsus four. The results of this study offer more evidence for the existence of this regulatory network, but also suggest an interesting role for direct protein interactions in its mechanism (Pueyo, 2004).

The cooperation between Bar and Ap on the one hand, and Al and Lim1 on the other, is likely to be carried out by transcriptional complexes involving Chip. The Chip protein is required for development of the tarsus four, five and pretarsus, and Gst (Glutathione-S-transferase-Chip fusion construct) experiments reveal Chip's ability to bind Ap, Bar, Lim1 and Al. However, the results also show that modulation of LIM-HOM protein activity by Chip alone does not explain distal leg development. For example, Ap function is not modulated primarily by Chip and Dlmo. The relative amount of Chip and Ap has to be grossly unbalanced before a phenotype is obtained in the leg, and dlmo is not expressed or required in leg development. Furthermore, the interaction between Ap and Chip does not confer the developmental specificity that allows LIM-HOM proteins to produce different outcomes in different parts of the leg. (1) Ap and Chip also interact in the wing and the CNS. (2) A chimaeric Lim3-Ap protein containing the LIM domains of Lim3 and the HOM domain of Ap does not behave as a dominant negative when expressed in tarsus four, and is even able to fulfil Ap function and rescue ap mutants. In the distal leg, developmental specificity seems to be achieved at the level of DNA binding and the transcriptional control of targets genes, mediated by partnerships between LIM-HOM and HOM proteins (Pueyo, 2004).

The evidence for this is presented first by dosage interactions between LIM-HOM and HOM proteins. Whereas there seems to be a relative abundance of endogenous Ap in tarsus four, an excess of Bar or Chip leads to a mutant phenotype, which is rescued by restoring the normal balance between Ap, Bar and Chip proteins in co-expression experiments. The effects observed could be explained simply by independent competition and the binding of Bar and Ap to Chip, leading, for example, to an excess of Bar-Chip complexes and a reduction of the pool of Chip available for Ap-Chip complexes. However, this hypothesis alone does not explain the additional dominant-negative effects of ectopic LIM-HOM and HOM proteins in tarsus four (Lim3, Islet and Al), which are also not mediated by transcriptional regulation but are nonetheless rescued by co-expression of appropriate endogenous proteins. For example, ectopic expression of UAS-islet or UAS-Lim3 in the ap domain produces loss of tarsus four without affecting Ap or Bar expression, and simultaneous co-expression of UAS-Bar partially suppresses this phenotype. If the sole effect of both UAS-Bar and UAS-Lim3 or UAS-islet were to quench Chip away from Ap, then simultaneous co-expression of Bar and Lim3 or Islet should worsen the phenotype, not correct it as observed. Moreover, ectopic expression of Islet or Lim3 proteins is not corrected by simultaneous co-expression of either UAS-Chip or UAS-ap. Altogether these results show instead that UAS-islet and UAS-Lim3 must interfere posttranslationally with Bar. The most direct explanation is that Islet and Lim3 have the ability to quench Bar protein into a non-functional state. Interestingly, the hybrid UAS-Lim3:ap does not behave as dominant negative but as an endogenous Ap protein in these experiments, since it does not produce a mutant phenotype on its own and it rescues UAS-Bar overexpression. This suggests that the LIM domains are not very specific when it comes to interaction with Bar, and points to the involvement of a common LIM-binding intermediary such as Chip. These results suggest that a protein complex involving Ap, Chip and Bar is the correct functional state of these proteins in tarsus four, and deviations from this situation into separate Bar-Chip, Ap-Chip, or Bar-Chip-Lim3 or Bar-Chip-Islet complexes leads to a mutant phenotype (Pueyo, 2004).

The notion of a protein complex involving Ap, Chip and Bar together is also supported by the Gst pull-down assays. The domain of Chip involved in Ap binding, the LIM interaction domain (LID), is not involved in Bar binding. However, the LID and the dimerisation domains of Chip are necessary to rescue the dominant-negative effect of UAS-Bar on tarsus four, suggesting a requirement for the formation of a complex with a LIM-HOM protein such as Ap. In agreement with this view, the Ap protein, and the LIM domains of Ap alone, are able to retain Bar protein in a Gst assay (Pueyo, 2004).

In the pretarsus, Al and Lim1 are possibly engaged in a partnership with Chip similar to that suggested for Ap, Chip and Bar. Synergistic cooperation between Al and Lim1 is required to direct pretarsus development and to repress Bar expression and function. Their cooperation entails a close functional relationship because a proper balance of Al, Lim1 and Chip is required, as is shown by the loss of pretarsal structures in UAS-Chip or UAS-Lim1 flies. Ectopic expression of LIM-HOM proteins in the pretarsus also disrupts pretarsal development without affecting Lim1 and Al expression. The possibility of direct protein interactions between Al, Lim1 and Chip is also suggested by the reciprocal ability of Al to interfere posttranscriptionally with Bar and Ap in tarsus four, and by the binding of Chip to Lim1 and to Al in in vitro experiments (Pueyo, 2004).

Comparison of tarsal development with other developmental processes illustrates how LIM-HOM proteins are versatile factors to regulate developmental processes. It had been observed that the outcome of LIM-HOM activity depends on their developmental context. This context can now be analysed as being composed of the presence, concentration and relative affinities of other LIM-HOM proteins, Ldb adaptors, and other cofactors such as LMO proteins and HOM proteins. It is proposed that the different developmental outcomes of LIM-HOM protein function could be due to the precise identity and dosage of cofactors available locally (Pueyo, 2004).

Ectopic expression experiments distort these contexts and lead to non-functional or misplaced LIM-HOM activities. In the wing, a finely balanced amount of functional Ap protein is modulated by Dlmo and Chip. Over-abundance of Chip stops the formation of functional tetramers in the wing but not in the CNS, where the relative amount of Ap, which is not modulated by Dlmo, is limiting for the formation of Ap-Chip functional complexes. In tarsus four, the Ap-Chip-Bar partnership is affected by experimentally induced over-abundance of Chip, presumably also because ectopic Ap-Chip tetramers typical of the CNS and the wing, and Bar-Chip complexes typical of tarsus five, are produced. Similarly, an excess of Bar might be interpreted by the cells as being a wrong developmental outcome, since high levels of Bar in the absence of Ap direct tarsus five development. Overexpression of Ap rescues this Bar dominant-negative effect, by restoring the relative amounts of Bar and Ap, which are determinant and limiting for tarsus four development. Finally, the dominant-negative effects produced by overexpression of either Chip or Lim1 in the pretarsus could either prevent the formation of Al-Chip-Lim1 complexes, or could favor the existence of Lim1-Chip complexes typical of the CNS (Pueyo, 2004).

The wing and the CNS models have postulated that Ap function is carried out by an Ap-Chip tetramer; however, the molecular scenario might be more complex. A new component of Ap-Chip complexes, named Ssdp, has been identified and is required for the nuclear localisation of the complex. Thus it is possible that an Ap-Chip tetramer also contains two molecules of Ssdp. In addition, different types of Chip-mediated transcriptional complexes and different regulators have been identified in other developmental contexts, such as in sensory organ development and thorax closure, in which the GATA factor Pannier forms a complex with Chip and with the bHLH protein Daughterless. Heterodimers of this complex are negatively regulated by a protein interaction with Osa. Thus, although the current results indicate that in different segments of the leg there exist specific interactions between LIM-HOM, Chip and HOM proteins, the involvement of further elements in these multiprotein complexes is not excluded (Pueyo, 2004).

The results support a partnership between HOM and LIM-HOM proteins in the specification of distinct segments of the leg, and the results are compatible with Ap-Chip-Bar, Bar-Chip and Lim1-Chip-Al forming transcriptional complexes. Although the characterisation of the target sequences, followed by further biochemical and molecular assays, is necessary to study the transcriptional mechanism of these interactions, it has been shown that LIM-HOM proteins can interact specifically and directly with other transcription factors to regulate particular genes. For instance, mouse Lim1 (Lhx1) interacts directly with the HOM protein Otx2. In addition, the bHLH E47 transcription factor interacts with Lmx1, and both synergistically activate the insulin gene. This interaction is specific to Lmx1, since E47 is unable to interact with other LIM-HOM proteins such as Islet. Moreover, Chip is able to bind to other Prd-HOM proteins, such as Otd, Bcd and Fz, to activate downstream genes. Chip also complexes with Lhx3 and the HOM protein P-Otx, increasing their transcriptional activity. The current results reinforce the notion of Chip as a multifunctional transcriptional adaptor that has specific domains involved in each interaction (Pueyo, 2004).

Experiments in Drosophila have demonstrated a conservation of LIM-HOM activity at the functional and developmental level in the CNS between Drosophila and vertebrates. In addition, xenorescue experiments have shown that the mechanism of action of Ap and its vertebrate homolog Lhx2 is very conserved in Drosophila wings, whereas ectopic expression of dominant-negative forms of chick Lim1, Chip, Ap and Lhx2 mimic both Ap and Lhx2 loss-of-function phenotypes. The developmental role of Ap, Bar and Al in the fly leg, and their putative molecular interactions may also have been conserved because their vertebrate homologs Lhx2, Barx and Al4 are also co-expressed in the limb bud. It is expected that the interactions between the LIM-HOM and Prd-HOM proteins shown here represent a conserved mechanism to specify different cellular fates during animal development (Pueyo, 2004).

Osa modulates the expression of Apterous target genes in the Drosophila wing

The establishment of the dorsal-ventral axis of the Drosophila wing depends on the activity of the LIM-homeodomain protein Apterous. Apterous activity depends on the formation of a higher order complex with its cofactor Chip to induce the expression of its target genes. Apterous activity levels are modulated during development by dLMO (Beadex). Expression of dLMO in the Drosophila wing is regulated by two distinct Chip dependent mechanisms. Early in development, Chip bridges two molecules of Apterous to induce expression of dLMO in the dorsal compartment. Later in development, Chip, independently of Apterous, is required for expression of dLMO in the wing pouch. A modular P-element based EP (enhancer/promoter) misexpression screen was conducted to look for genes involved in Apterous activity. Osa, a member of the Brahma chromatin-remodeling complex, was found to be a positive modulator of Apterous activity in the Drosophila wing. Osa mediates activation of some Apterous target genes and repression of others, including dLMO. Osa has been shown to bind Chip. It is proposed that Chip recruits Osa to the Apterous target genes, thus mediating activation or repression of their expression (Milan, 2004).

This study presents evidence that Osa, a member of a subset of Brahma chromatin remodeling complexes, behaves overall as a general activator of Apterous activity in the Drosophila wing. Overexpression of Osa rescues and loss of Osa enhances the Beadex¹ phenotype. It does so by modulating the expression levels of Apterous target genes, some of them being activated (e.g. Serrate and probably other unknown target genes) and some repressed (e.g. Delta, fringe). Chip has been shown to bind Osa. The fact that Osa has different effects on the transcription of Apterous target genes suggests that Chip recruits Osa to the promoters and in combination with other unknown factors mediates either transcriptional repression or activation. Osa mediates repression of both Apterous dependent and independent expression of fringe, suggesting a direct and probably Chip independent effect of Osa on fringe transcription (Milan, 2004).

Apterous activity is regulated during development by dLMO. Osa is required to mediate repression of dLMO expression. Since both early and late expression of dLMO depend on Chip, it is postulated that Chip forms a transcriptional complex with Apterous in the D compartment and an unknown transcription factor expressed in the wing pouch. Osa may interact with Chip thus recruiting the Brahma complex to the dLMO locus and remodeling chromatin in a way that limits dLMO transcriptional activation. High levels of dLMO protein reduce Apterous activity and the Notch dependent organizer is not properly induced along the DV boundary. Osa mediated repression of dLMO expression may ensure moderate levels of expression of dLMO in the wing, thus allowing proper wing development. Gain of function mutations that cause misexpression of vertebrate LMO proteins have been implicated in cancers of the lymphoid system. Truncating mutations in the human SWI-SNF complex, the human homologues of the Brahma complex, cause various types of human cancers. The SWI-SNF complex may be required to mediate repression of LMO expression in lymphoid tissues. Thus, it would be very interesting to analyze if truncating mutations in members of the human SWI-SNF complex cause higher levels of LMO expression and are associated with lymphoid malignancies (Milan, 2004).

It has been shown that the Brahma complex plays a general role in transcription by RNA Polymerase II. Then, is Osa having a general effect on the expression levels of every gene involved in wing patterning? Several observations indicate this is not the case. (1) Osa is a component of a subset of Brahma (Brm) chromatin complexes. (2) Brahma and Polycomb were shown to have non-overlapping binding patterns in polytenic chromosomes. Those genes involved in wing patterning and regulated by Polycomb (i.e. Hedgehog) may not be affected by overexpression of Osa. (3) Overexpression of Osa has different effects on the expression levels of Serrate, Delta and fringe. (4) Osa has been shown to specifically regulate the expression of Wingless target genes and the Achaete-scute complex genes, interestingly by restricting their expression levels (Milan, 2004).

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