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).

GENE STRUCTURE

C15 maps to a cluster of NK-homeobox genes (including lady-bird-late and lady-bird-early, as well as tinman/NK4, bagpipe and S59/NK1 (Dear, 1994).

Exons - 4

PROTEIN STRUCTURE

Amino Acids - 307

Structural Domains

The human homeobox gene HOX11 has been identified at the site of a chromosomal translocation in a subset of T-cell acute leukaemias. In the mouse genome, the hox11 family consists of at least three related genes, each of which possesses a highly conserved homeobox. To assist in elucidating the roles of this gene, a homologue was studied from Drosophila melanogaster. Drosophila C15 shares similar identity to all three murine family members and contains the threonine residue in helix 3 of the homeodomain characteristic of the Hox11 family (Dear, 1994).

date revised: 30 March 2005

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.