InteractiveFly: GeneBrief

clawless: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

The distal region of the Drosophila leg, the tarsus, is divided into five segments (ta I-V) and terminates in the pretarsus, which is characterized by a pair of claws. Several homeobox genes are expressed in distinct regions of the tarsus, including aristaless (al) and lim1 in the pretarsus, Bar (B) in ta IV and V, and apterous (ap) in ta IV. This pattern is governed by regulatory interactions between these genes; for example, Al and Bar are mutually antagonistic, resulting in exclusion of Bar expression from the pretarsus. Although Al is necessary, it is not sufficient to repress Bar, indicating another factor is required. This factor has been identified as the product of the C15 gene, also termed clawless, a homeodomain protein that is a homolog of the human Hox11 oncogene. C15 is expressed in the same cells as al -- together, C15 and Al appear to directly repress Bar and possibly to activate Lim1. C15/Al also act indirectly to repress ap in ta V, i.e., in surrounding cells. To do this, C15/Al autonomously repress expression of the gene encoding the Notch ligand Delta (Dl) in the pretarsus, restricting Dl to ta V and creating a Dl+/Dl− border at the interface between ta V and the pretarsus. This results in upregulation of Notch signaling, which induces expression of the bowl gene, the product of which represses ap. Similar to aristaless, the maximal expression of C15 requires Lim1 and its co-factor, Chip. Bar attenuates aristaless and C15 expression through Lim1 repression. Aristaless and C15 proteins form a complex capable of binding to specific DNA targets, which cannot be well recognized solely by Aristaless or Clawless (Campbell, 2005; Kojima, 2005).

The role of morphogen gradients in regulating spatial patterns of differentiation in developing tissues is supported by an increasing body of experimental data. Gradients of secreted signaling polypeptides can be visualized in developing tissues and target genes have been identified whose expression is differentially sensitive to the intracellular activity of signaling pathways regulated by these polypeptides. However, the final pattern of expression of these targets usually requires further refinement, often by regulatory interactions between the targets themselves, in particular, direct cross-repressive interactions when the targets encode transcription factors (Campbell, 2005 and references therein).

Mutual repression results in sharp boundaries between expression domains; such boundaries are difficult to establish simply by differential threshold responses to graded information, which usually result in overlapping domains. Establishing sharp boundaries is often essential to the subsequent generation of precise patterns of cell differentiation. For example, in the vertebrate neural tube, a gradient of Sonic Hedgehog protein activates or represses the expression of several homeobox genes, such as Nkx2.2 and Pax6, but their final pattern of expression is dependent upon mutual repression resulting in sharp boundaries of expression between targets. This establishes non-overlapping domains of homeobox gene expression along the dorsoventral axis of the neural tube that is translated into the differentiation of specific neuronal subtypes at precise positions along this axis. Another example of this phenomenon can be found in the early Drosophila embryo, where gradients of the transcription factors Bicoid, Hunchback, and Caudal establish the initial expression domains of different gap genes at distinct positions along the anteroposterior axis of the embryo. However, their final expression pattern is dependent upon asymmetric cross-repression between adjacent gap gene products (Campbell, 2005 and references therein).

Another example of this phenomenon occurs in the developing tarsus of the Drosophila leg, the distal-most region of this appendage. Patterning along the proximodistal (P/D) axis of the tarsus is controlled by a distal-to-proximal gradient of EGF-receptor (EGFR) signaling activity, established by a source of ligands in the center of the leg imaginal disc, which corresponds to the presumptive tip of the adult appendage. The adult tarsus is divided into five segments (ta I to ta V, from proximal to distal) and terminates in the pretarsus that is characterized by a pair of claws. High levels of EGFR activity are required for development of the claws, while progressively lower levels are needed for development of more proximal segments (Campbell, 2002). Similarly, high levels are required to activate expression of the distal-most gene aristaless (al), which is required for development of the claws and is expressed in the very center of the leg disc, while lower levels are sufficient to activate more proximally expressed genes, such as Bar (Campbell, 2005 and references therein).

If Bar expression was regulated only through activation by EGFR signaling, it would be expressed throughout the central region of the disc, but it is, in fact, excluded from the cells in the center of the disc that express al, and consequently is expressed as a ring surrounding al, with no overlap. In late third instar discs, this ring corresponds to ta IV and V. Both al and Bar encode for homeodomain containing transcription factors and previous studies have demonstrated that al and Bar are mutually antagonistic so that Al is required to repress Bar, while Bar can repress al, thus accounting for the sharp boundary between their expression domains and the exclusion of Bar expression from the center of the disc. However, although loss of al results in expansion of the Bar domain into the center, ectopic expression of al does not repress Bar, indicating that, although Al is required for repression of Bar, it is not sufficient and at least one additional factor must be required. Another homeobox gene, lim1, is also expressed in the same cells as al, but since lim1 mutants are much weaker than those of al, it does not appear to encode for this missing factor (Campbell, 2005 and references therein).

This missing factor has been identified as the product of the C15 gene, a homolog of the Hox11 protooncogene of humans (Dear, 1994 and Reim, 2003). C15 is expressed in the same cells as al, and legs from C15 mutants have an identical phenotype as do those from al mutants. Data are presented to support the proposal that a combination of C15 and Al is required to repress Bar directly. As well as directly repressing Bar, C15/Al can also repress expression of genes such as apterous (ap) non-autonomously, in surrounding cells. This is achieved through upregulation of Notch signaling in surrounding cells, paradoxically through direct repression of the gene encoding the Notch ligand Delta (Dl) in the pretarsus by C15/Al (Campbell, 2005).

The center of the leg imaginal disc, the presumptive tip of the leg, is characterized by the co-expression of three homeobox genes, al, lim1, and C15. al and C15 are expressed here because EGFR signaling levels are highest in this location, while it is unclear if this is also true for lim1 or if it is just a target of C15 and Al. The center of the leg disc is also characterized by the absence of expression of several genes, including Bar and ap, which are expressed more proximally but which would be expected to extend into the center because they are also activated by EGFR signaling (Campbell, 2002). Both Bar and ap are repressed in the center by a combination of C15 and Al but Bar is repressed by a mechanism different from ap, and this accounts for the observation that ap is absent from a wider domain in the center than Bar (Campbell, 2005).

Neither C15 nor Al is sufficient to repress alone, as shown, for example, in al mutant discs where C15 is still expressed, and in C15 mutant discs where al is still expressed, but in both mutants Bar and Ap expression extends into the very center, i.e., they overlap with C15 or Al. Although Lim1 is co-expressed with C15 and Al, Bar and Ap are still repressed in lim1 mutants, which also have almost normal expression domains of Al and C15. However, there can be minor derepression of Bar in the center of lim1 mutant discs, suggesting it does have a minor role in augmenting C15 and Al activity, that may account for the defective development of the claws (Campbell, 2005).

Bar is repressed autonomously by C15/Al, consistent with one or both of these factors binding directly to cis-regulatory sequences at the Bar locus. There is indirect evidence that Al can bind to these sequences in the absence of C15, because ectopic expression of al can occasionally induce ectopic expression of Bar. This would imply that Al cannot act as a transcriptional repressor alone, at least for Bar, and that it may recruit C15 for this purpose (Campbell, 2005).

Other genes expressed in the developing tarsus, such as ap and bab, are also excluded from the very center of the disc, but in these cases, this exclusion zone is larger than that of Bar, so they are absent from the region fated to form ta V as well as the cells expressing C15/al in the very center. Consequently, there is a clear gap between their expression domains and that of C15/al. However, C15/Al are also required to repress expression of these genes in the center of the leg and do this non-autonomously, suggesting they regulate the expression or activity of a signaling molecule that leads to upregulation of a signaling pathway in the cells surrounding those expressing C15/Al. This appears to be the Notch pathway because upregulation of this in ta V results in loss of ap expression (Campbell, 2005).

The majority of these results are consistent with a model in which C15/Al upregulates Notch signaling in surrounding cells in ta V (and those at the edge of the pretarsus) through direct repression of the gene encoding the Notch ligand Dl in the pretarsus. This results in high levels of Dl expression only in ta V surrounding the pretarsus. Previous studies have shown that if a cell expresses Dl, it is often unresponsive to Dl in adjacent cells. The results presented in this study on the ability of Dl to induce expression of bowl indicate that, in the distal leg, expressing D1 cells in ta V can signal to adjacent Dl− cells in the pretarsus, but also appear to be able to signal to adjacent Dl+ cells in ta V, but only those at the distal edge of the Dl domain, i.e., cells that are also bordering Dl− cells in the pretarsus. Thus, Notch signaling is upregulated in a ring of cells straddling the ta V/pretarsus boundary. The key event that facilitates this is the repression of Dl expression in the center of the leg by C15/Al because this creates a Dl+/Dl− border that is essential for Dl to activate Notch. Notch signaling upregulates expression of bowl, which encodes for a transcription factor that appears to directly repress ap in ta V (Campbell, 2005).

This model is supported by the following observations: loss of bowl results in ap expression in cells immediately surrounding C15/Al, while ectopic expression of bowl can repress ap expression. bowl expression is dependent upon Dl, while being lost in Dl mutant clones, apart from mutant cells immediately adjacent to wild-type Dl-expressing cells. bowl expression can also be induced by clones of cells misexpressing Dl, both in cells adjacent to the clone and cells within the clone, but only those at the edge; cells in the center of large Dl+ clones do not express bowl. In wild-type discs, Dl expression is upregulated in ta V, while the Bowl expression domain is usually two cells in width with one cell in the pretarsus (overlapping with C15/Al) and one in ta V (overlapping with Dl). In C15 mutants, Dl expression extends into the center and in common with large clones ectopically expressing Dl, there is no bowl expression in the center. The lack of bowl expression at the proximal border of the central Dl domain appears to be due to repression of either Notch signaling or bowl itself, by an as yet unidentified factor (Campbell, 2005).

There are, however, some inconsistencies in this model. First, upregulation of Notch in ta V does not always repress all of the ap expression, in particular at the edge of a clone. Second, although clonal analysis shows that Bowl represses ap strictly autonomously, there is always a gap between cells expressing Bowl and those expressing Ap. It is possible that the antibody being used to monitor Bowl expression cannot detect lower levels of protein present in the gap. Alternatively, Bowl may only be transiently expressed in the gap. Consequently, further studies are required to investigate these problems (Campbell, 2005).

This study also addresses more general questions about how signaling gradients can generate expression of mutually antagonistic targets that are activated above different signaling thresholds, such as Bar and C15/al (Campbell, 2002), with Bar being activated above a lower threshold of EGFR signaling activity than C15/al. Consider what happens as a gradient of signaling activity is established across a group of cells following expression of a secreted signal. Initially, signaling levels will be low and the low-threshold target should be expressed close to the source, while the high-level target should not be expressed yet. This is supported by observations in the early leg disc where Bar expression can be detected in the center of the leg prior to expression of C15/al (Campbell, 2005).

However, Bar represses expression of the high-threshold targets al and C15, the expression of which expands slightly when Bar function is removed, so how are al and C15 ever expressed in cells already expressing Bar even when signaling levels rise? Expression of high-threshold targets such as C15/al is probably a balance between one negative and two positive influences: (1) repression by the low-threshold target, Bar; (2) activation from the signaling pathway, here, the EGFR pathway; and (3) the ability of C15/Al to repress Bar once they are expressed. Presumably, at high ligand levels, activation by EGFR signaling is sufficient to overcome any repression from Bar and C15/al will be expressed even in the presence of Bar. This is supported by observations in this study: in al mutants, for example, C15 is still expressed in the very center of the disc where EGFR signaling levels are highest, even though Bar is co-expressed there. However, the size of the C15 domain in al mutants is much smaller than in wild-type discs; this may be explained by the apparent inability of C15 to repress Bar on its own so now there is only a single positive influence, EGFR signaling, disturbing the normal balance in favor of repression by Bar. Alternatively, the smaller C15 domain in al mutants may reflect a reduction in cell proliferation or increase in cell death in the very center following loss of Al (Campbell, 2005).

Hox11 is required for development of the spleen in mice (Roberts, 1994), while misexpression is associated with specific T-cell leukemias in humans (Dube, 1991; Hatano, 1991; Kennedy, 1991). Consequently, uncovering the mechanisms it uses to regulate gene expression is crucial for understanding these processes, in particular transformation. Like C15, Hox11 appears to be capable of repressing gene expression (Owens, 2003). There is, as yet, no evidence that Hox11 interacts with any homologs of Al, but studies on C15 in Drosophila may provide further insight into the mechanisms it uses to regulate gene expression (Campbell, 2005).

Complex gene interactions in the establishment and maintainence of the pretarsus and distal tarsus

clawless/C15 (cll) is essential for pretarsus specification. The establishment and maintenance of pretarsus and distal tarsus regions require a concerted action of five homeobox genes, al, cll, Lim1 and Bar (BarH1 and BarH2), whose expression is regulated through a homeobox gene/homeodomain protein network involving Al/Cll complex formation (Kojima, 2005).

In early third instar, al and Bar (BarH1 and BarH2) expression is induced in a mutually independent manner according to a distal-to-proximal gradient of EGFR signaling activity. cll expression becomes discernible simultaneously with al and Bar expression in the future distal leg region, and al, a gene co-expressing with cll in the future pretarsus, cannot solely induce cll expression in early third instar. Thus, although it remains to be clarified, it is also considered that cll expression is also initiated by EGFR signaling (Kojima, 2005).

The results of previous (Kojima, 2000 and Tsuji, 2000) and present studies show that the expression domains of al, cll, Lim1 and Bar are considerably modulated and eventually established through homeobox gene/homeodomain protein interactions, which, in detail, may include the repression of Bar through a concerted action of al and cll, cll-dependent al activation, al/cll-dependent positive regulation of Lim1, the positive regulation of al and cll through Lim1 and Chi, the negative regulation of Lim1 by Bar and the auto-regulation of Bar (Kojima, 2005).

In the pretarsus, the absence of either al or cll activity is sufficient for Bar de-repression, indicating that al and cll activity is required for Bar expression. This notion is further supported by a misexpression experiment using blk-GAL4, in which the repression of the endogenous Bar expression on the ventral side of the distal tarsus region simultaneously requires al and cll activity. Biochemical analyses indicate that Al and Cll form a complex capable of binding to specific DNA targets, which may not be well recognized solely by Al or Cll. Two 11-bp long Al/Cll complex binding sites have been identified in the putative Bar enhancer. It is thus considered that al/cll-dependent Bar repression in the future pretarsus is most likely to be carried out through direct binding of the Al/Cll heterodimer to the putative Bar enhancer (Kojima, 2005).

Consistent with the notion that Bar repression requires a concerted action of al and cll, the sole misexpression of al failed to repress Bar expression. In contrast, endogenous Bar expression on the dorsal side of the future distal tarsus is completely repressed by the sole misexpression of cll driven by blk-GAL4. al misexpression cannot induce cll expression but cll is capable of inducing al expression in some cells in cll-misexpressing flip-out clones generated in the proximal region lacking endogenous Bar expression, indicating that cll may induce al expression independent of Bar activity. The sole cll misexpression brought about by a blk-GAL4 driver induces al expression on the dorsal side of the future distal tarsus. Thus, the repression of endogenous Bar in the dorsal tarsus cells by the sole misexpression of cll may be accounted for by a concerted action of misexpressed cll and induced al (Kojima, 2005).

Lim1 expression in the future pretarsus is initiated right after Al, Cll and Bar proteins are produced, and accordingly, may be regulated by these homeodomain proteins. A previous experiment showed that Bar can repress Lim1 (Tsuji, 2000). Since Bar is de-repressed in the pretarsus in al or cll mutant leg discs (Kojima, 2000; Tsuji, 2000), it is difficult to determine whether al and cll are involved in a positive regulation of Lim1, simply by examining the possible change in Lim1 expression in the future pretarsus. However, Lim1 expression is quite likely to be activated by a concerted action of al and cll, since Lim1-lacZ misexpression was found in all and only clones simultaneously expressing al and cll but not Bar (Kojima, 2005).

cll expression significantly reduces in Lim1 mutant clones, indicating that the maximal level of cll expression requires Lim1 activity as in the case of al (Tsuji, 2000). Lim1 has been shown to form a complex with Chip, and a considerable reduction of al and cll expression is observed in Chip mutant clones. Thus it is considered that the Lim1/Chi complex serves as a transactivator for al and cll expression. Interestingly, in contrast to al, cll is not ectopically induced upon Lim1 misexpression. An unknown transactivator (X) functioning in concert with the Lim1/Chi complex may be additionally required for cll expression. Alternatively, cll may be less sensitive to activation by Lim1 than al. In previous experiments, Lim1 misexpression was shown to be incapable of repressing Bar (Tsuji, 2000). This may be due to the absence of cll induction by Lim1 misexpression, since, as described above, a concerted action between al and cll appears essential for Bar repression (Kojima, 2005).

As with al, cll expression invades into Bar mutant clones in the distal tarsus and is attenuated by Bar misexpression in the pretarsus, indicating that Bar is capable of repressing both al and cll. Bar serves as a repressor for Lim1, and Lim1 is a transactivator for al and cll. Thus, it is quite feasible that Bar represses al and cll through repressing Lim1. Bar misexpression experiments carried out in a Lim1 mutant background indicate that Bar represses al mainly through Lim1 repression. cll expression appears, however, negatively regulated through Lim1-dependent and independent mechanisms. At early third instar, in which Lim1 is not expressed, the expression of al overlaps Bar expression but that of cll does not. This difference might be due to Bar-dependent repression of cll through the Lim1-independent mechanism (Kojima, 2005).

A previous experiment (Kojima, 2000) has shown that Bar expression at late third instar is positively regulated by an auto-regulation mechanism. Thus, the homeobox gene/homeodomain protein regulatory network in the future distal leg region appears to include two types of positive feedback loops: Bar auto-regulation and a mutual activation between al/cll and Lim1. The former and the latter, respectively, are considered to be the most fundamental for fate determination of the future distal tarsus and the future pretarsus. The homeobox gene/homeodomain protein regulatory network also includes two major negative interactions, Bar repression by al/cll and Lim1 repression by Bar. These negative interactions are considered to be essential for precise demarcation between the future pretarsus and the future distal tarsus regions (Kojima, 2005).

Bar is de-repressed in the pretarsus cells lacking the activity of al or cll at early third instar, indicating that Bar may possess a potential activity to be expressed in the pretarsus region, but may be normally repressed by al and cll so that a doughnut-like expression pattern is produced from the beginning of expression. Morphogen activity may accordingly directly specify only the proximal extent of Bar expression and the distal extent may be determined indirectly through a concerted action of al and cll expressed in a more distal region. At the beginning of its expression, Bar limits the distal extent of dachshund expression, which occurs at that time just outside the Bar domain. Morphogen signaling in the developing leg may thus directly determine only the proximal extent of the expression domain of each region-specific transcription factor gene, while distal extent is delimited by transcription factor(s) specific to the distally neighboring region. This simple mechanism may serve as one means by which concentric, doughnut-like patterns of gene expression are generated in the leg disc. If morphogen directly determines both distal and proximal boundaries of a gene expression domain, it would also control the activation and repression of the expression of the same gene by its signaling activity. But then, this would involve a much more complex molecular mechanism (Kojima, 2005).

al and cll appear to act cooperatively in the pretarsus development. Moreover, extensive similarity in expression pattern and mutant phenotype between al and cll in the antenna and notum implies that al and cll function cooperatively also in these tissues. In contrast, wing pouch development requires al but not cll, while cll but not al is essential for normal oceller development, indicating that Al or Cll, solely expressed, may be required for wing-pouch and oceller development. Thus, al and cll function solely or cooperatively in a developmental-context-dependent manner (Kojima, 2005).

In vertebrate, no genetic interactions between Hox11/tlx genes (vertebrate cll homologs) and vertebrate al homologs or physical bindings between these gene protein products have been reported to date. The results suggest that at least human Cart1 and human Hox11L1 are capable of forming complex not only with each other but also Drosophila putative partners. Thus, it is quite feasible that the complex or heterodimer formation is an evolutionally conserved feature of Al-type and Hox11/tlx/Cll-type proteins and that vertebrate al homologs and cll homologs function solely or in various combinations depending on developmental contexts (Kojima, 2005).

REGULATION

Transcriptional Regulation

To determine if C15 lies downstream of Al or vice versa, their expression was examined in discs from the reciprocal mutant. Each was still expressed, but its expression domain was significantly reduced. In contrast, Lim1 expression is lost completely in both C15 and al mutant discs. In addition, although there is some variation, the expression domains of C15 and Al are only mildly reduced in lim1 mutants (Campbell, 2005).

If C15 is not downstream of the other homeobox genes expressed in the center of the disc, it must be activated by another mechanism. al expression is induced by EGFR signaling, raising the possibility that C15 may also be under EGFR control. This was confirmed by loss and gain of function experiments, as follows: (1) C15 expression was lost in discs from an Egfr^ts mutant grown at the restrictive temperature (29.1°C) at which al expression is lost; (2) misexpression of a constitutively active form of the EGFR (UAS-Egfr.lambdatop) results in ectopic expression of C15; similar to other EGFR targets, this ectopic expression is restricted to the ventral region (Campbell, 2005).

Requirements of Lim1 and Chip for maximal al and cll/C15 expression: Lim1 expression becomes discernible slightly later than expression al, cll and Bar, and maximal al expression in late third instar depends on Lim1 function (Tsuji, 2000). Cll expression is also significantly reduced in Lim1^7B2 (a null allele) clones in late third instar discs, indicating that not only al but also cll is positively regulated by Lim1 in the late third instar. Chi encodes a LIM domain binding protein and has been suggested to act as a co-factor for Lim1. Cll and Al signals are significantly reduced in clones of Chi^e5.5, a null allele of Chi. The concerted action of Lim1 and Chi is thus shown to be required for the maximal expression of cll and al in the late-third-instar pretarsus region (Kojima, 2005).

When Lim1 is misexpressed using blk-GAL4, the expression of al but not cll is induced (Kojima, 2005; Tsuji, 2000). Thus, unlike al, cll may require an additional component for its maximal expression. Alternatively, cll may be less sensitive to activation by Lim1 than al (Kojima, 2005).

Bar attenuates al and cll expression through Lim1 repression: Previous experiments have shown that al expression invades into Bar mutant clones generated in the distal tarsus and that Bar misexpression attenuates al expression in the pretarsus (Kojima, 2000), indicating that al expression is negatively regulated by Bar. As with al, cll is also under a negative regulation of Bar, since cll expression not only intrudes into Bar mutant clones generated in the distal tarsus but is also attenuated in the pretarsus cells misexpressing Bar (Kojima, 2005).

That Bar represses Lim1 expression (Tsuji, 2000) and that al and cll expression is positively regulated by Lim1 may indicate that Bar represses both al and cll expression through Bar-dependent repression of Lim1 expression (Tsuji, 2000) (Kojima, 2005).

To test whether the attenuation of al and cll expression due to Bar misexpression is caused only through Lim1 repression, Al and Cll expression was examined in Lim1^7B2 leg discs misexpressing Bar with blk-GAL4. Should Lim1 repression be the sole cause for al and cll repression, Al and Cll signal reduction in Bar-misexpressing cells would be no more than in surrounding cells under a Lim1 mutant background. This was the case for Al but not for Cll, indicating that Bar negatively regulates al expression mainly through Lim1 repression, while cll expression is repressed by Bar both through Lim1 repression and that independent of Lim1 (Kojima, 2005).

Genetic interactions among scribbler, Atrophin and groucho in Drosophila uncover links in transcriptional repression; A protein complex provides C15 with maximal repressive activity

In eukaryotes, the ability of DNA-binding proteins to act as transcriptional repressors often requires that they recruit accessory proteins, known as corepressors, which provide the activity responsible for silencing transcription. Several of these factors have been identified, including the Groucho (Gro) and Atrophin (Atro) proteins in Drosophila. Strong genetic interactions are seen between gro and Atro and also with mutations in a third gene, scribbler (sbb), which encodes a nuclear protein of unknown function. Mutations in Atro and Sbb have similar phenotypes, including upregulation of the same genes in imaginal discs, which suggests that Sbb cooperates with Atro to provide repressive activity. Comparison of gro and Atro/sbb mutant phenotypes suggests that they do not function together, but instead that they may interact with the same transcription factors, including Engrailed and C15, to provide these proteins with maximal repressive activity (Wehn, 2006; full text of article).

Previous studies demonstrated that Atro acts as a corepressor in Drosophila, the most convincing of these being the demonstration that fusion of Atro to a heterologous DNA-binding domain confers repressive activity to the chimera. Atro has been shown to interact directly with two transcription factors, Even-Skipped (Eve) and Huckebein, and the repressive ability of Eve is compromised in Atro mutants, probably accounting for the loss of en expression in even-numbered parasegments in Atro mutant embryos (Wehn, 2006).

These studies here are consistent with Atro acting as a corepressor since it was shown that several genes, including run, tkv, al, and B, are completely or partially derepressed in Atro mutant clones in imaginal discs, suggesting that transcriptional repressors required to silence these genes recruit Atro. Atro-dependent repression of Bar (B) in the center of the leg disc is very likely due to interaction with the transcription factor C15, which is expressed in the center of the leg and is required for repression of B. Similarly, Atro-dependent repression of al in the posterior of the wing is very likely due to interaction with En, which is expressed in the posterior and required to exclude al from this compartment. At present it is unclear which transcription factors recruit Atro to repress run in the eye or tkv in the wing, although a strong candidate for run would be the Rough homeodomain protein, which is expressed in the same cells, R2 and R5, that exhibit ectopic run expression in Atro mutant clones. Whether Atro can, in fact, bind directly to C15, En, and possibly Rough, needs to be tested biochemically, since previous studies with Eve and Hkb did not identify a possible interaction motif for Atro nor do sequence comparisons among C15, En, Eve, and Hkb suggest a common motif (Wehn, 2006).

The sbb gene encodes a nuclear protein with unknown function. sbb mutations have many different phenotypes affecting multiple tissues. sbb and Atro interact very strongly genetically and that many of the phenotypes of sbb mutants are very similar to those of Atro mutants, including derepession of run, tkv, al, and B in imaginal discs. Thus, Atro is unable to silence these genes in the absence of Sbb, suggesting that it is required for Atro activity either to recruit Atro to transcription factors or possibly to assist binding of these factors to DNA. Since these transcription factors appear to function normally in some respects in the absence of Sbb (or Atro), it appears more likely that Sbb and Atro function together in a corepressor complex (Wehn, 2006).

One problem with the proposal that Atro activity is dependent upon Sbb is that the phenotypes of Atro and sbb mutants are not identical. For example, embryos lacking both maternal and zygotic Atro have a very severe, almost uncharacterizable phenotype, while embryos lacking both maternal and zygotic Sbb have a much less severe phenotype, characterized by a reduced number of abdominal segments, that is similar to that of embryos lacking only maternal Atro. This could be explained if Atro is partially active in the absence of Sbb, or if it is dependent upon Sbb for repression of some genes but not others. Alternatively, the difference between Atro and sbb mutant phenotypes could be related to Atro having functions other than that of a corepressor. It is has been implicated in positive regulation of Hox gene expression, and it also functions in the cytoplasm to control planar cell polarity. This analysis of sbb mutants does not reveal any potential involvement of Hox gene expression or planar cell polarity and, consequently, if Sbb is required only for Atro to act as a corepressor, then it is not surprising that Atro and sbb mutant phenotypes are not identical. Further experiments are required to determine the nature of the Atro dependence on Sbb for transcriptional repression and how direct any interactions might be (Wehn, 2006).

Mutations in sbb and Atro were originally uncovered in a genetic screen for enhancers of al. It is likely that they act as enhancers because they are utilized by the C15 transcription factor to repress genes such as Bar; C15 is expressed in the same cells as Al and it is thought that they bind together to regulate gene expression. Strong genetic interactions were uncovered among sbb, Atro, and en mutations, that could be explained if En also recruits Atro/Sbb (Wehn, 2006).

Curiously, genetic studies also revealed strong interactions among gro, sbb, and Atro. This could be explained if Gro was also required for Atro activity; i.e., all three proteins may form a corepressor complex. However, this appears to be unlikely because, in contrast to the similar phenotypes of sbb and Atro mutants, there are several distinct differences among the phenotypes of gro mutants and those of sbb and Atro mutants. For example, repression of tkv in the anterior of the wing is dependent on both Sbb and Atro but not on Gro, while repression of run in the antennal disc is dependent upon Gro but not upon Atro or Sbb. This suggests that a specific transcription factor recruits Atro/Sbb to repress tkv in the wing and another transcription factor recruits Gro to repress run in the antenna. The identity of these transcription factors remains to be uncovered (Wehn, 2006).

In some cases gro mutants do have a similar phenotype to those of Atro and sbb; this includes partial derepression of al expression in the posterior of the wing and Bar in the center of the leg. This can be explained if C15 (expressed in the center of the leg) and En (expressed in the posterior of the wing) recruit both Gro and Atro/Sbb and if each imparts some but not all the repressive activity to these transcription factors. Consistent with this, both C15 and En possess eh1-type Gro-interaction motifs and previous studies have revealed that En can repress in the absence of Gro. Further biochemical studies are required to determine if C15 and En can indeed recruit Atro (Wehn, 2006).

At present it is unclear whether Atro and Gro provide all the repressive activity to C15 and En; this will await the generation of Atro gro double-mutant clones. sbb gro double-mutant clones have been analyzed and these reveal that some targets of C15 and En are still at least partially repressed, although En activity appears to be somewhat compromised following the simultaneous loss of Sbb and Gro, in comparison to loss of one of these alone. Either Atro has some activity in the absence of Sbb or C15 and En can use mechanisms other than recruitment of Gro and Atro to repress transcription. Many transcription factors have been shown to have the ability to repress by several mechanisms; for example, although Brk recruits both CtBP and Gro, it can repress some genes in the absence of both, using additional repression domains (Wehn, 2006).

Why do C15 and En need to recruit both Gro and Atro? En can repress some genes completely in the absence of either Gro or Atro, for example, ci and dpp in the wing. However, for repression of al, the activity of En is clearly reduced in the absence of either, indicating that it needs to recruit both to completely repress this gene. This would suggest a quantitative explanation; i.e., En recruits both Gro and Atro to increase its activity, rather than to allow it to repress specific genes repressed more efficiently by one or the other. This is consistent with the suggestion that both corepressors function via a similar mechanism: both Gro and a vertebrate homolog of Atro have been shown to recruit a histone deacetylase. The recruitment of both may decrease histone acetylation to a level that cannot be achieved with either alone (Wehn, 2006).

Targets of Activity

Lim1 may be a direct target of C15: To investigate regulatory interactions between C15, Al, and Lim1, each was misexpressed in the leg and expression of the other two was examined. This was achieved initially with a UAS-C15 line and by generating Gal4 expressing clones using the FLPout technique and Tub-Gal4; the clones were monitored with UAS-GFP. This revealed that ectopic C15 could, in fact, induce ectopic expression of both Al and Lim1, although this was somewhat random with Al and Lim1 being expressed only in some cells ectopically expressing C15. Ectopic C15 can also repress Bar and loss of Bar results in expansion of the Al expression domains, but only in the cells immediately surrounding their normal domains. Repression of Bar does not appear to account for the ectopic Al and Lim1 expression induced by C15, because, Al and Lim1 can be induced some distance from their endogenous domains. In contrast, misexpression of al or lim1 in Tub-Gal4 clones has no effect on expression of the other genes. It has been shown that driving higher levels of lim1 can induce ectopic expression of al and this was confirmed using dpp-Gal4. However, there was no ectopic C15 in the UAS-lim1; dpp-Gal4 discs. Similarly, driving higher levels of al with dpp-Gal4 does not induce ectopic expression of C15 (Campbell, 2005).

Therefore, although Al is still expressed in C15 mutants, and vice versa, indicating that both are probably activated independently by EGFR signaling, C15 can induce expression of al and lim1. This may act as a feedback mechanism to ensure all three are expressed in the same cells. Since expression of Lim1 is completely lost in the center of discs from C15 and al mutants, it may simply be a direct target of either or both and may not be directly activated by EGFR signaling (Campbell, 2005).

Since al is still expressed, albeit in a much smaller domain, in C15 mutants and C15 is still expressed in al mutants, it appears possible that each may play an additional, redundant role, in patterning the leg. This was ruled out by examining al^ice, C15² double mutants (both alleles are either null or very close to being null), which have legs and antennae that are indistinguishable from either single mutant, indicating that, in the absence of the other, neither Al nor C15 provides any function during leg development (Campbell, 2005).

C15 acts directly to repress Bar in the center of the leg: In late third instar discs, Bar is expressed in the cells immediately surrounding C15, as has already been described for Al. In partially everted discs, this corresponded to C15 at the very tip and Bar in ta V and IV. The nubbin (nub) gene is expressed in ta V overlapping with Bar in ta V but not in IV. With antibody staining, Bar and C15 can first be detected in very early third instar and both appear to be expressed at the same time. Bar is already excluded from the center at this stage. However, using a lac-Z enhancer trap in Bar, which is more sensitive than antibody staining, β-gal expression was detected even earlier in late second instars. At this stage, when no C15 can be detected, β-gal expression is found throughout the center of the disc. Slightly later when C15 becomes detectable, β-gal is excluded from the center. Al is first detected at approximately the same time as C15 (Campbell, 2005).

The loss of Bar and Nub expression from the center of the disc can be explained by repression by Al and C15. Loss of al has been shown to result in expansion of Bar expression into the center of the disc, indicating Al is required to repress Bar in this position. Not surprisingly, C15 null mutant discs have the same phenotype. The diameter of the domain of Bar is slightly smaller than the diameter of the Bar ring in wild-type discs. Nub expression is also found in the center of C15 mutant discs, but in a smaller domain than Bar, indicating that there are still distinct differences between ta IV and V in C15 mutants (Campbell, 2005).

Repression of Bar by C15 is strictly autonomous, as shown in discs containing C15 mutant clones, where Bar expression expands into all the cells in the center that has lost C15. In addition, ectopic expression of C15 resulted in autonomous repression of Bar. Curiously, although studies that showed ectopic al cannot repress Bar, it was also found that it can actually induce ectopic expression of Bar in more proximal regions of the disc (Campbell, 2005).

C15 acts indirectly to repress ap in the center of the leg: Bar expression is absent from the center of the leg, specifically from the cells expressing Al and C15. However, other genes, including ap and bab, are absent from a more extensive region in the center, and there is a gap between the C15 expression domain and Ap and Bab. Consequently, Ap expression is restricted to presumptive tarsal segment IV, where it overlaps with Bar. It has been suggested that, as well as activating genes such as al and Bar, EGFR signaling may directly repress genes in the center of the disc, possibly accounting for the absence of ap and bab in this location. Surprisingly, ap and bab expression, as well as Bar, is regulated by C15/Al. In both C15 and al mutant discs, Ap and Bab expression expands into the center of the disc. Consequently, in regard to Ap expression, the distal region of the leg adopts a tarsal segment IV-like fate. However, Nub, which is normally only expressed in ta V, is now co-expressed with Ap in the very center, indicating that the distal-most segment in C15 legs has characteristics of both ta IV and V (Campbell, 2005).

In wild-type discs, Ap expression is first detected slightly later than Bar, Al, or C15, but even at this time there is a clear gap between Ap expression and C15, indicating that C15/Al acts non-autonomously to repress ap. This is supported by two further studies: (1) unless there is a complete loss of C15 in homozygous mutant discs, Ap expression is not derepressed in C15 mutant clones in the center if the clones are not too large, indicating surrounding wild-type C15-expressing cells can rescue the mutant tissue; (2) ectopic expression of C15 results in non-autonomous repression of Ap (Campbell, 2005).

These results suggest that EGFR signaling represses gene expression in the center of the disc only indirectly through activation of C15/Al. This is also supported by two other observations. (1) Al is still expressed in C15 mutant discs, indicating that EGFR signaling levels are still very high in the center of these discs, but ap is not repressed (if ap is repressed directly by EGFR, its threshold for this would be lower than the threshold for activation of al because ap is repressed further from the source in the center than al is activated). (2) Ectopic expression of C15 results in non-autonomous repression of ap, but, if this is due to increased EGFR signaling in surrounding cells, then it should result in activation of EGFR targets such as Bar immediately adjacent to the cells expressing C15 (outside of the normal Bar domain), but does not. Consequently, it seems very likely that C15 uses an alternative mechanism to repress ap, most likely by upregulation of a signaling pathway in surrounding cells (i.e., ta V) (Campbell, 2005).

Notch signaling can repress ap expression:

The ability of different signaling pathways to repress ap expression was tested and it was discovered that upregulation of the Notch pathway in ta IV (by misexpression of the Notch intracellular domain) results in loss of Ap expression. Curiously, however, Ap expression is not upregulated in Notch mutant cells, and is, in fact, lost or downregulated; the phenotype is somewhat variable), indicating low-level Notch signaling is required for Ap expression, possibly indirectly, because loss of Notch can also lead to downregulation or loss of Bar expression in ta IV and Bar is required for expression of ap (Campbell, 2005).

Bowl can repress ap and is activated non-autonomously by C15: Notch signaling usually represses gene expression indirectly by inducing expression of repressors, so known Notch targets in the distal leg were tested to determine if they are required for repression of ap. The best candidate appeared to be the bowl gene which encodes a zinc finger transcription factor that is expressed in a ring in the distal leg under the control of Notch signaling and can both activate and repress gene expression. To investigate if bowl is involved in repressing ap expression, mutant clones were generated in leg discs. In these clones, cells expressing high levels of Ap now directly abut those expressing C15, i.e., there is no gap between them. Low-level Ap expression is also detected in clones that extend into the C15 domain, indicating Bowl is also required here but that an additional factor, possibly C15/Al, can partially repress ap in this location (if so, C15/Al would be acting autonomously in a similar fashion to repression of Bar). Ectopic bowl expression can also repress Ap expression. The response to ectopic bowl is fairly weak, but it appears that ectopic expression of this gene does not result in high levels of protein expression (Campbell, 2005).

bowl represses ap and C15 regulates bowl expression: Examination of Bowl and Ap expression in leg discs reveals that there is a gap between their expression domains, even at a time when Ap expression is first detected in mid-third instars. This could indicate that Bowl acts non-autonomously to repress ap. However, the clonal analysis clearly shows that Bowl acts autonomously: any wild-type cells expressing Bowl has no influence on Ap expression in surrounding mutant tissue. It is possible that there is low-level Bowl expression in the 'gap' that cannot be detected with antibody staining. Another possible explanation is one of timing, and that Bowl is expressed in the cells in the 'gap' slightly earlier and that this is sufficient to silence the ap gene even before its expression can be detected more proximally. The possibility that bowl is expressed transiently in cells has been proposed to explain the observation that bowl mutant clones have effects in central regions of tarsus, i.e., in regions where its expression cannot be detected later (Campbell, 2005).

Thus, Bowl is required to repress ap expression in tarsal segment V and this predicts that C15 regulates bowl expression. This was confirmed by analysis of C15 mutant discs, in which Bowl expression in the center is lost, although other, more proximal, domains of expression are normal. The ring of Bowl in the distal tarsus is usually just two cells in width with the inner cell overlapping with C15, but the outer cell being outside the C15 domain, suggesting C15 can induce bowl non-autonomously. This is supported by the ability of cells ectopically expressing C15 to activate Bowl expression in surrounding cells. This ability is fairly limited, but would be expected because the endogenous C15-expressing cells only appear able to induce bowl in their immediate neighbor (resulting in a ring of bowl expression in a single row of cells surrounding the C15 domain (Campbell, 2005).

Delta activates bowl, but Delta expression is repressed by C15: If Notch signaling induces bowl expression and C15 is also required for bowl expression, it was predicted that C15 upregulates Notch signaling by regulating the expression of the Notch ligand responsible for activation of bowl. Although, both Notch ligands, Delta (Dl) and Serrate are expressed in leg discs, it was discovered that only Dl is required to induce expression of bowl. bowl expression is lost in homozygous Dl mutant clones, although, if positioned appropriately, wild-type cells can rescue bowl expression in adjacent cells laterally and distally. Curiously, nub, which was also thought to be a target of Notch signaling, is still expressed in Dl mutant cells (even far from wild-type cells), albeit in an irregular pattern (it is expressed at normal levels in some cells, but at lower levels or not at all in others. Misexpression of Dl can also induce ectopic bowl expression both in adjacent cells and in the cells misexpressing Dl. However, in large clones, cells in the center of the clone do not express bowl, which is only expressed in the cells at the edge of the clone and in the cells immediately adjacent to the clone. Nub is not ectopically expressed following misexpression of Dl (Campbell, 2005).

In wild-type mid-third instar discs, Dl expression is upregulated in ta V, overlapping with Nub, but not with C15. Distally, it overlaps partially with Bowl, although Bowl is also expressed even more distally. Proximally, however, Dl does not appear to induce expression of Bowl, suggesting there is a repressor of Bowl expressed in this location. This is supported by the inability of cells misexpressing Dl in this position (proximal ta V, ta IV) to activate Bowl. Although it might be predicted that C15 would induce expression of Dl, in fact the opposite was found, and C15 actually represses Dl in the center of the disc. In C15 mutants, Dl expression expands into the center of the disc and misexpression of C15 can repress expression of Dl. How C15-repression of Dl can result in upregulation of Notch signaling in cells in ta V surrounding the pretarsus is discussed below (Campbell, 2005).

clawless/C15 represses Bar expression cooperatively with al in the pretarsus: As with strong al mutants (Tsuji, 2000), Bar is de-repressed in the putative cll mutant pretarsus at late third instar. Cell-autonomous Bar de-repression is also observed in al and cll mutant clones generated in the pretarsus region. Since the sole misexpression of al could not induce Bar repression (Kojima, 2000), these findings may indicate that a cooperative action of al and cll is required for Bar repression in the future pretarsus.

blk-GAL4 (a dpp enhancer driven GAL4) is a GAL4 driver capable of inducing UAS-gene expression strongly on the dorsal side and weakly on the ventral side along the anterior/posterior boundary. cll was misexpressed using a moderate UAS-cll line and blk-GAL4. In contrast to al, cll misexpression causes endogenous Bar repression on the dorsal side of the future distal tarsus region. Furthermore, unexpectedly, ectopic al is found in the dorsal-side tarsal cells that have lost Bar expression, possibly suggesting that cll misexpression or Bar elimination induces the ectopic expression of al (Kojima, 2005).

To further clarify these points, whether cll is capable of inducing al expression was examined. Since pretarsus al expression intrudes into Bar mutant clones generated in the tarsus region in the late third instar (Kojima, 2000), cll-misexpressing clones were generated outside of the Bar domain using a flip-out technique, and the presence or absence of Al misexpression in the cll-misexpressing clones was examined. Al was induced in a considerable fraction of cll-misexpressing clones outside of the Bar domain, indicating that cll is capable of inducing al misexpression independent of Bar repression (Kojima, 2005).

Examined next was whether endogenous Bar expression in the future tarsus is repressed on the ventral side when al and cll are simultaneously misexpressed using blk-GAL4. Note that neither Bar reduction (Kojima, 2000) nor cll misexpression occurs upon blk-GAL4-driven al misexpression. A simultaneous misexpression of al and cll using blk-GAL4 causes Bar repression not only on the dorsal side but on the ventral side as well, strongly supporting the notion that endogenous Bar expression in the future tarsus is repressed by a concerted action of al and cll. It is concluded that cll is capable of inducing al and that Bar is repressed by a concerted function of al and cll (Kojima, 2005).

At early third instar, al, cll and Bar expression in the wild-type leg disc became discernible simultaneously. From the very beginning of the expression onward, Al and Cll signals localize in the disc center, while Bar signals are in a circular region immediately adjacent to the Al/Cll domain (Kojima, 2000), possibly suggesting that Bar is negatively regulated by Al and Cll from an early stage of expression. To test this possibility, Bar expression was examined in al^ex or cll^dl9-2 clones in early third instar discs. Bar is de-repressed cell-autonomously in both mutant clones, indicating that Bar has a potentiality to be expressed not only in the future distal tarsus but in the future pretarsus as well. It is concluded that, in the wild-type leg discs, pretarsus Bar expression is repressed by a concerted action of Al and Cll from a very early stage of expression (Kojima, 2005).

Positive regulation of Lim1 expression via concerted action of al and cll: That Lim1 expression becomes discernible in the future pretarsus shortly after the appearance of Al and Cll signals may imply that Lim1 is positively regulated by al and/or cll. However, it is difficult to directly determine whether al and cll are required for Lim1 expression, since Bar, serving as a repressor for Lim1 (Tsuji, 2000), is de-repressed in the pretarsus of al or cll mutants (Kojima, 2000; Tsuji, 2000). Thus, the effect was examined of cll or al misexpression on Lim1-lacZ, whose expression is essentially identical to that of Lim1. cll-misexpressing flip-out clones were generated in first-second instar, and Lim1-lacZ signals were detected in late third instar. Lim1-lacZ misexpression occurred in some cll-misexpressing cells, indicating that Cll can serve as a positive regulator of Lim1 (Kojima, 2005).

A previous experiment showed the sole misexpression of al is incapable of inducing Lim1 misexpression (Tsuji, 2000). However, this does not necessarily mean that al is not involved in Lim1 regulation. Rather the notion is preferred that Lim1 is positively regulated by a concerted action of al and cll, since Lim1-LacZ signals are detected in all cll-misexpressing cells, only in those cells in which al expression is simultaneously observable (Kojima, 2005).

Spatial specificity of mesodermal even-skipped expression relies on multiple repressor sites

Individual cardiac progenitors emerge at defined positions within each segment in the trunk mesoderm. Their specification depends on segmental information from the pre-patterned ectoderm, which provides positional information to the underlying cardiac mesoderm via inductive signals. This pattern is further reinforced by repressive interactions between transcription factors that are expressed in neighboring sets of cardiac progenitors. For example, even-skipped (eve) and ladybird early (lbe) gene products mark adjacent cardiac cell clusters within a segment, and their antagonistic interaction results in mutually exclusive expression domains. Lbe acts directly on the eve mesodermal enhancer (eme) to participate in restricting its expression anteriorly. It is hypothesized that additional repressive activities must regulate the precise pattern of eve expression in the cardiac mesoderm via this enhancer. In this study, two additional repressor motifs: 4 copies of an 'AT'-rich motif (M1a-d) and 2 copies of an 'GC'-rich motif (M2a,b), were identified which when mutated cause expansion of eme-dependent reporter gene expression. Potential negative regulators of eve and were examined and it was found that their overexpression is sufficient to repress eve as well as the eme enhancer via these sites. These data suggest that a combination of factors is likely to interact with multiple essential repressor sites to confer precise spatial specificity of eve expression in the cardiac mesoderm (Liu, 2008).

Although each of the identified repressor sites is necessary, none is individually sufficient for restricting the eme enhancer activity to the eve expression domain. Several additional homeodomain proteins, including Msh, C15 and Lim3, are capable of repressing mesodermal eve expression by interacting with specific sites within the enhancer element. While the repression of mesodermal eve expression by Msh, C15 and Lim3 is likely mediated by the AT-rich M1 sites and the Lb2 site, the repression of eve expression by Lbe requires both the AT-rich M1 and the Lb2 sites as well as the GC-rich M2a site. Therefore, each of the four repressor sites apparently is required on order to confer sensitivity to repression by Lbe. This raises the possibility that repression is the result of a complex in which the cooperation of all four repressor elements is required for successful repression (Liu, 2008).

A prominent feature of the Drosophila is its segmental polarity that includes distinct cardiac cell types that are precisely positioned within each segment. These cardiac progenitors are specified along the anterior-posterior axis during development and are marked by Lbe, Eve or Svp. As the embryo develops, a linear heart tube is formed and this metameric arrangement of cardiac cells types continues to be maintained. Within each hemi-segment, the anterior two pairs form the tinman-expressing 'working myocardium', while the posterior pair that expresses svp and the T-box transcriptional factor Doc form the ostia. Previous studies suggested that repressive interactions between cardiac factors expressed in non-overlapping subtypes of cardiac cells likely contribute to the diversification and maintenance of cellular identities. Svp and Tin have been shown to repress each other's expression during heart tube formation, and the current data suggest that antagonistic interactions between Lbe and Eve are also a part of this mutual repression network. In addition, the data show that eve expression within the cardiac mesoderm is negatively regulated by multiple repressor sites, thus further supporting the idea that transcriptional repression mechanisms play a prominent role in the generation of cellular diversity in the developing heart. Roles were demonstrated for two potential repressors, C15 and Lim3. Although they do not seem to be essential for patterning mesodermal eve expression, they are normally expressed in the cardiac mesoderm in the vicinity of the Eve cells and they do repress the eme enhancer via the identified repressor sites when ectopically expressed. Therefore, it is also possible that they function redundantly with other negative regulators yet to be identified (Liu, 2008).

Default repression is a common mechanism utilized by major signaling pathways, including Wnt, Shh and Notch pathways, to restrict target gene expression. In the absence of signaling, signal-regulated transcription factors function mainly as transcriptional repressors, thus preventing low levels of target gene expression that might be activated by weak, local activators ('default repression'). In response to signals, some transcription factors are then converted into transcriptional activators to promote target gene expression. Thus, transcriptional repression and activation can be mediated by the same binding sites. Default repression mechanisms may also contribute to the restricted mesodermal eve pattern. It has been reported that mesodermal eve expression is under the direct transcriptional control of Wg signaling. Mutating several putative binding sites for dTCF, the transcriptional mediator of Wg signaling, results in an expansion of low-level reporter gene expression within the cardiac mesoderm that is unaffected by reduced wg activity. Thus, dTCF may serve as a default signal to restrict mesodermal eve expression in the absence of wg signaling (Liu, 2008).

It has been shown that Hh signaling not only promotes eve and svp but also inhibits lbe expression in the dorsal mesoderm. One mechanism for Hh signaling may be via inhibition of Cubitus interruptus (Ci)-mediated repression. Interestingly, there is some similarity between the M2a sequence examined in this study (TGGGCCCT) and the consensus sequence for Ci (TGGGTGGTC). This raises the interesting possibility that M2a site may be a putative Ci binding site in eme. Thus, mutations of M2a site, which result in the anterior expansion of eme activity into Lbe expressing cells, may reflect a lack of repression by Ci. Alternatively, the M2a site may mediate transcriptional repression by Lbe or its potential cofactors. The latter hypothesis is more consistent with the observation that reporter gene expression is rendered insensitive to inhibition by Lbe overexpression when the M2a site is mutated in eme. As the M2a site does not resemble the Lbe consensus sequence, the idea is favored that another factor binds to the M2a site, which then cooperates with Lbe in repressing mesodermal eve expression. This interaction may be facilitated by the close proximity of the two sites (Liu, 2008).

In sum, the in vivo functional dissection of eme has revealed that each of two AT-rich sites, M1b or M1c and the previous studied Lb2 site, when mutated, causes reporter gene expansion that encompasses the entire cardiac mesoderm, overlapping with Tinman protein at late stage 12. In addition, the GC-rich site M2a is required for repression anterior to the Eve cluster. The absolute requirement of each repressor site for successful restriction of eve expression within the cardiac mesoderm is in striking contrast to the mechanism of incremental activation of this enhancer in the cardiac mesoderm by activators such as Tinman, dTCF, Mad, E-box and ETS sites. Repression through these repressor sites may require cooperation between the sites, perhaps via a repressor complex. Thus, eliminating the function of any of these sites will disrupt interactions with the complex causing de-repression within the 'activator'-dependent cardiac mesoderm (Liu, 2008).

Protein Interactions

The al/cll cooperation found in Bar repression in the pretarsus may possibly stem from the interactions between Al and Cll. GST pull-down assay was first conducted in vitro to confirm this possibility. Cll was tagged with GST, and a possible binding of Cll to Al was monitored by Western blotting of the eluents from a GST column with anti-Al antibody. GST-Cll was prepared using E. coli cells, and Al was synthesized using reticulocyte lysates. Al signals were detected only when a mixture of GST-Cll and Al was applied to and then eluted from the GST column, indicating that Al and Cll are capable of binding to each other in the absence of DNA (Kojima, 2005).

A polymerase chain reaction-based approach, the systematic evolution of ligands by exponential enrichment (SELEX), was undertaken to determine a possible consensus DNA sequence for the binding of the Al/Cll complex. The nucleotide sequence alignment of 48 fragments obtained after five rounds of enrichment revealed a consensus sequence of 5′-(T/C)TAATTAA(T/A)(T/A)G-3′, which differs from the consensus sequences for the vertebrate homologs of Al (TAATNNNATTA; Alx and Cart proteins; Qu, 1999) and those for Cll homologs (CGGTAA(T/G)(T/C)(G/C)G; Hox11/tlx proteins (Dear, 1993; Shimizu, 2000; Tang, 1995; Kojima, 2005).

Protein-DNA interactions were examined using the electrophoretic mobility shift assay (EMSA). A double-stranded oligonucleotide containing the SELEX consensus sequence was used as a probe. No or weak retardation bands were detected for Cll or All alone. In contrast, a very strong retardation signal was observed for a combination of Al and Cll. A few base substitutions in the consensus sequences results in a significant reduction in or the abolishment of retardation signals. Thus, the Al/Cll complex is significantly different in target-sequence specificity from Al and Cll, and only the Al/Cll complex can strongly bind to the SELEX-determined consensus sequence (Kojima, 2005).

It may thus be concluded that, in the pretarsus, Al and Cll form a complex capable of binding to specific sequences, which cannot be well recognized solely by Al or Cll, and that the resultant complex plays a central role in al/cll-dependent gene regulation in the future pretarsus. However, it should be noted that the possibility cannot be formally excluded that Al and Cll separately bind to their own consensus sequences and function cooperatively in the pretarsus (Kojima, 2005).

Thus al and cll seem to act cooperatively through the formation of the complex between their protein products. To determine whether vertebrate Al and Cll homologs possess similar properties, possible interactions between the Al/Cll consensus sequence and either one of vertebrate Al homolog, Cart1, or a Cll homolog, Hox11L1 (also called as Tlx2), were assessed. Cart1 is capable of binding to the Al/Cll consensus binding site to some extent, but Hox11L1 can not at all. A considerably strong signal is detected when a mixture of Cart1 and Hox11L1 is subjected to gel retardation. Moreover, strong retardation signals are detected for a mixture of Al and Hox11L1 and that of Cart1 and Cll. Thus, the formation of an Al/Cll-type complex may be an evolutionally conserved feature of Al-type and Hox11/tlx-type homeodomain protein family members (Kojima, 2005).

DEVELOPMENTAL BIOLOGY

For clarification of a possible role of C15/clawless in the pretarsus/distal-tarsus establishment, the temporal and spatial expression patterns of Cll were compared with those of Al in leg discs collected at various stages. Pretarsus Al expression is first detected in early third instar in a domain slightly overlapping the Bar domain (Kojima, 2000). Similar to Al, pretarsus Cll expression first becomes discernible in early third instar, at which time, Cll expression largely overlaps that of Al. A close examination indicates that the Cll domain is slightly narrower than that of Al and that, in contrast to the Al domain, the Cll domain possesses no apparent peripheral gradation. Since both anti-Bar and anti-Cll antibodies were prepared from immunized rabbits, Bar expression could not be directly compared with Cll expression. However, it is believed that the Cll domain possesses almost no overlap with the Bar domain, because no appreciable overlap was recognized between cll mRNA and Bar expression domains (Kojima, 2005).

By mid third instar, the peripheral Al gradation disappears and the proximal extents of the Al and Cll domains became identical to each other. From this stage onward, the future distal leg region is virtually completely separated into two regions: Bar-positive future distal tarsus and Al/Cll-positive future pretarsus (Kojima, 2005).

An antibody raised against C15 revealed that it was expressed in exactly the same cells as Al and Lim1 in the center of leg discs, so that its expression domain abuts that of Bar, which is expressed in ta IV and V (there are actually two Bar genes, H1 and H2, which are co-expressed) (Campbell, 2005).

Function of Dorsocross transcription factors in the amnioserosa as revealed by the expression of C15

Signals from the BMP family member Decapentaplegic (Dpp) play a role in establishing a variety of positional cell identities in dorsal and lateral areas of the early Drosophila embryo, including the extra-embryonic amnioserosa as well as different ectodermal and mesodermal cell types. Although a reasonably clear picture is available of how Dpp signaling activity is modulated spatially and temporally during these processes, a better understanding of how these signals are executed requires the identification and characterization of a collection of downstream genes that uniquely respond to these signals. Three novel genes, Dorsocross1, Dorsocross2 and Dorsocross3, are described that are expressed downstream of Dpp in the presumptive and definitive amnioserosa, dorsal ectoderm and dorsal mesoderm. These genes are good candidates for being direct targets of the Dpp signaling cascade. Dorsocross expression in the dorsal ectoderm and mesoderm is metameric and requires a combination of Dpp and Wingless signals. In addition, a transverse stripe of expression in dorsoanterior areas of early embryos is independent of Dpp. The Dorsocross genes encode closely related proteins of the T-box domain family of transcription factors. All three genes are arranged in a gene cluster, are expressed in identical patterns in embryos, and appear to be genetically redundant. By generating mutants with a loss of all three Dorsocross genes, it has been demonstrated that Dorsocross gene activity is crucial for the completion of differentiation, cell proliferation arrest, and survival of amnioserosa cells. In addition, the Dorsocross genes are required for normal patterning of the dorsolateral ectoderm and, in particular, the repression of wingless and the ladybird homeobox genes within this area of the germ band. These findings extend knowledge of the regulatory pathways during amnioserosa development and the patterning of the dorsolateral embryonic germ band in response to Dpp signals (Reim, 2003).

The expression of a novel amnioserosa marker, which is encoded by the homeobox gene C15, was examined. In the normal situation, C15 is expressed in the amnioserosa from stage 7 until stage 17, when the amnioserosa undergoes apoptosis. In addition, from early stage 10 onwards there is a narrow domain of expression at the leading edge of the dorsal germ band, which later becomes segmental. In DocA mutant embryos, the level of C15 expression in early amnioserosa cells is unaltered; this allows use of C15 protein as a marker for the development of this tissue in the absence of Doc activity (Reim, 2003).

Until stage 9, the large majority of amnioserosa nuclei in DocA mutant embryos appear large and flattened as in wild-type embryos. Together with data from alpha-tubulin staining, this observation indicates that the amnioserosa cells begin to acquire the normal features of a squamous epithelium. However, the amnioserosa does not display a properly folded morphology during stages 8-10, and the posterior germ band is forced to bend towards the inside in DocA mutant embryos. In addition, some small nuclei become detectable within the amnioserosa during this stage. Altogether, these observations indicate that the amnioserosa initiates its differentiation process in the absence of Doc gene activity but fails to complete it, thus leading to morphological and functional abnormalities of this tissue towards the end of germ band elongation. Much stronger alterations can be observed during subsequent stages, when there are an increasing number of C15-stained amnioserosa nuclei with much smaller diameters than regular amnioserosa nuclei. At late stage 12, almost all amnioserosa cells feature small nuclei that are difficult to distinguish from dorsal epidermal cells. Co-staining for race indicates that it is predominantly the cells with the small nuclei that lose race expression, while most normally-sized nuclei are still surrounded by race signals. From this stage onwards, non-stained 'holes' appear in the amnioserosa and the number of C15-stained amnioserosa nuclei decreases prematurely. Hence, unlike wild-type embryos, stage 14 DocA mutant embryos are not covered dorsally by C15-stained amnioserosa cells. In addition to the observed alterations in the amnioserosa, the C15 expression domain at the leading edge of the epidermis appears significantly broadened (Reim, 2003).

Is the increasing number of smaller nuclei in the amnioserosa of DocA mutant embryos connected with abnormal cell divisions? The M-phase marker phospho-Histone H3 can be detected in numerous amnioserosa nuclei of DocA mutant embryos after stage 10; this increase is not seen in wild-type embryos. In addition, there is significant incorporation of BrdU in amnioserosa nuclei of DocA mutant embryos (particularly in the small nuclei, whereas no incorporation is observed in wild-type embryos. Mitotic spindles are also present in the amnioserosa of DocA mutants. These observations indicate that the normal G2 arrest of amnioserosa cells has been released and the cells re-enter the cell cycle. Whether the subsequent disappearance of small C15-stained amnioserosa nuclei in DocA mutant embryos is a result of premature apoptosis of cells in this tissue was also tested. This possibility was confirmed by the results of TUNEL labeling experiments, which produced signals in many amnioserosa nuclei from 12 onwards. Most of the TUNEL-labeled nuclei have reduced or are lacking C15 expression, which shows that wild-type amnioserosa nuclei at late stage 12 are not apoptotic). Altogether, these observations suggest that loss of Doc activity prevents the normal differentiation of the amnioserosa to a fully functional tissue, suspends the cell cycle block of amnioserosa cells, and causes premature apoptotic cell death in this tissue (Reim, 2003).

REFERENCES

Search PubMed for articles about Drosophila clawless

Andermann, P. and Weinberg, E. S. (2001). Expression of zTlxA, a Hox11-like gene, in early differentiating embryonic neurons and cranial sensory ganglia of the zebrafish embryo. Dev Dyn. 2001 Dec;222(4):595-610. 11748829

Brake, R. L., Kees, U. R. and Watt, P. M. (2002). A complex containing PBX2 contributes to activation of the proto-oncogene HOX11. Biochem. Biophys. Res. Commun. 2002 May 31;294(1):23-34. 12054735

Brake, R. L., Chatterjee, P. K., Kees, U. R. and Watt, P. M. (2004). The functional mapping of long-range transcription control elements of the HOX11 proto-oncogene. Biochem. Biophys. Res. Commun. 313(2): 327-35. 14684164

Campbell G. (2002). Distalization of the Drosophila leg by graded EGF-receptor activity. Nature 418(6899): 781-5. 12181568

Campbell, G. (2005). Regulation of gene expression in the distal region of the Drosophila leg by the Hox11 homolog, C15. Dev. Biol. 278(2): 607-18. 15680373

Cande, J. D., Chopra, V. S. and Levine, M. (2009). Evolving enhancer-promoter interactions within the tinman complex of the flour beetle, Tribolium castaneum. Development. 136(18): 3153-60. PubMed Citation: 19700619

Dear, T. N., Sanchez-Garcia, I. and Rabbitts, T. H. (1993). The HOX11 gene encodes a DNA-binding nuclear transcription factor belonging to a distinct family of homeobox genes. Proc. Natl. Acad. Sci. 90(10): 4431-5. 8099440

Dear, T. N. and Rabbitts, T. H. (1994). A Drosophila melanogaster homologue of the T-cell oncogene HOX11 localises to a cluster of homeobox genes. Gene 141(2): 225-9. 7909304

Dear, T. N., et al. (1995). The Hox11 gene is essential for cell survival during spleen development. Development 121(9): 2909-15. 7555717

Dube, I. D., et al. (1991). A novel human homeobox gene lies at the chromosome 10 breakpoint in lymphoid neoplasias with chromosomal translocation t(10;14). Blood 78(11): 2996-3003. 1683261

Hatano, M., et al. (1991) . Deregulation of a homeobox gene, HOX11, by the t(10;14) in T cell leukemia. Science 253(5015): 79-82. 1676542

Kanzler, B. and Dear, T. N. (2001). Hox11 acts cell autonomously in spleen development and its absence results in altered cell fate of mesenchymal spleen precursors. Dev. Biol. 234(1): 231-43. 11356032

Kawabe, T., Muslin, A. J. and Korsmeyer, S. J. (1997). HOX 11 interacts with protein phosphatases PP2A and PP1 and disrupts a G2/M cell-cycle checkpoint. Nature 385: 454-458. 9009195

Kennedy, M. A., et al. (1991). HOX11, a homeobox-containing T-cell oncogene on human chromosome 10q24. Proc. Natl. Acad. Sci. 88(20): 8900-4. 1681546

Kojima, T., Sato, M. and Saigo, K. (2000). Formation and specification of distal leg segments in Drosophila by dual Bar homeobox genes, BarH1 and BarH2, Development 127: 769-778. 10648235

Kojima, T., Tsuji, T. and Saigo, K. (2005). A concerted action of a paired-type homeobox gene, aristaless, and a homolog of Hox11/tlx homeobox gene, clawless, is essential for the distal tip development of the Drosophila leg. Dev. Biol. 279(2): 434-45. 15733670

Langenau, D. M., (2002). Molecular cloning and developmental expression of Tlx (Hox11) genes in zebrafish (Danio rerio). Mech. Dev. 117(1-2): 243-8. 12204264

Liu, J., Qian, L., Han, Z., Wu, X. and Bodmer, R. (2008). Spatial specificity of mesodermal even-skipped expression relies on multiple repressor sites. Dev. Biol. 313(2): 876-86. PubMed Citation: 18067885

Logan, C., Wingate, R. J., McKay, I. J. and Lumsden, A. (1998). Tlx-1 and Tlx-3 homeobox gene expression in cranial sensory ganglia and hindbrain of the chick embryo: markers of patterned connectivity. J Neurosci. 18(14): 5389-402. 9651221

Owens, B. M., (2003). Specific homeodomain-DNA interactions are required for HOX11-mediated transformation. Blood 101(12): 4966-74. 12586625

Qu, S., Tucker, S. C., Zhao, Q., deCrombrugghe, B. and Wisdom, R. (1999). Physical and genetic interactions between Alx4 and Cart1. Development 126(2): 359-69. 9847249

Reim, I., Lee, H.-H. and Frasch, M. (2003). The T-box-encoding Dorsocross genes function in amnioserosa development and the patterning of the dorsolateral germ band downstream of Dpp. Development 130: 3187-3204. 12783790

Roberts, C. W., Shutter, J. R. and Korsmeyer, S. J. (1994). Hox11 controls the genesis of the spleen. Nature 368(6473): 747-9. 7908720

Shimizu, H., Kang, M., Iitsuka, Y., Ichinose, M., Tokuhisa, T. and Hatano, M. (2000). Identification of an optimal Ncx binding sequence required for transcriptional activation. FEBS Lett. 475(3): 170-4. 10869550

Tang, S. and Breitman, M. L. (1995). The optimal binding sequence of the Hox11 protein contains a predicted recognition core motif. Nucleic Acids Res. 23(11): 1928-35. 7596820

Tsuji, T., Sato, A., Hiratani, I., Taira, M., Saigo, K. and Kojima, T. (2000). Requirements of Lim1, a Drosophila LIM-homeobox gene, for normal leg and antennal development. Development 127(20): 4315-23. 11003832

Watt, P. M., (2003). Specific alternative HOX11 transcripts are expressed in paediatric neural tumours and T-cell acute lymphoblastic leukaemia. Gene 323: 89-99. 14659882

Wehn, A. and Campbell, G. (2006). Genetic interactions among scribbler, Atrophin and groucho in Drosophila uncover links in transcriptional repression. Genetics 173(2): 849-61. 16624911

date revised: 20 September 2008

Home page: The Interactive Fly © 2017 Thomas Brody, Ph.D.