The expression of Lim1 is very similar to that of the PD gene aristaless. al is expressed in the most distal part of the leg discs, and in two rings in the peripheral and medial regions. Confocal immunofluorescence staining has shown that the expression of Lim1 and al are coincident in the most distal parts of the leg and antenna disc. Furthermore, there are similarities between the al and Lim1 mutant phenotypes, since in al mutants the claw organ, the sternopleural bristles in the leg and the arista are missing or reduced. A possible functional relationship between the two genes was explored. Since al is expressed earlier than Lim1, one possibility is that Lim1 expression is regulated by al. The expression of Lim1 was examined in al mutants, and Lim1 was found to be lost in the presumptive tip of the leg where both genes are co-expressed. In contrast, al expression is normal in Lim1 mutants. These results suggest that the Lim1 locus is regulated by the al gene, one possibility being that the Lim1 gene is a direct downstream target of the homeodomain Al protein (Pueyo, 2000).
In both al and Lim1 mutants, the strongest pupal lethal alleles eliminate the claw and reduced the rest of the pretarsal organs, but do not eliminate the whole pretarsus in all legs. This could be due to a functional cooperation between al and Lim1, such that the presence of any one product in the absence of the other would still provide enough function for some organs of the pretarsus to occasionally develop. Alternatively, the remnant pretarsal organs observed in al and Lim1 mutants could be due to hypomorphy of the mutants available and no functional relationship needs to be implied between the two genes. It was reasoned that if a functional relationship does exist, a double mutant should show an enhanced phenotype: a double mutant Lim1R12.4;alice was generated, and found to be embryonic lethal. Therefore, al and Lim1 double mutants show a synergistic effect that might betray a functional relationship. This synergistic relationship is also shown in ectopic expression experiments. Ectopic expression of either Lim1 or al driven by the 30AGal4 line produces only small defects in the joint between tarsus four and five. However, simultaneous expression of both al and Lim1 in 30AGal4;UASLim1;UASal flies produces stronger defects, including partial or complete fusion of tarsus four and five (Pueyo, 2000).
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 (cll), 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).
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 alice, 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).
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, is de-repressed in the pretarsus of al or cll mutants. 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. 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).
Lim1 expression becomes discernible slightly later than expression al, clawless (cll/C15) and Bar, and maximal al expression in late third instar depends on Lim1 function. Cll expression is also significantly reduced in Lim17B2 (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 Chie5.5, 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. 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).
At the sequence level, Lim1 is highly related to its vertebrate homologs. Given their conservation, it was of interest to see if this sequence homology translates into functional similarities at a molecular level. In Xenopus, Xlim-1 and the LIM-domain-binding protein (Xldb-1/NLI/CLIM- 2) interact in vitro, and cooperate in vivo to induce secondary axis structures (Agulnick, 1996). As the name implies, this association takes place through the LIM domains. More recently, the Drosophila homolog of Xldb1/NLI/CLIM-2, Chip has been cloned and shown to interact with the Apterous protein (Morcillo, 1997; Fernandez-Funez, 1998). To determine if Lim1 and Chip interact in vitro, co-immunoprecipitation experiments were carried out. Using the Lim1 antibody, the ability of Chip to be immunoprecipitated by full-length and truncated Lim1 proteins was carried out. The results show that Chip can be immunoprecipitated in the presence of full-length Lim1, and a truncated Lim1 protein that contains the LIM domains (LIM-Lim1). HD-Lim1, which lacks the LIM domains and includes the homeodomain fails to coimmunoprecipitate Chip. Additionally, Chip by itself is not immunopreciptated by the Lim1 antibody. These results show that Lim1 has the capacity to interact with the LIM-domain-binding protein, Chip. This interaction requires the LIM domains of Lim1 and is independent of the Lim1 homeodomain. Similar to its vertebrate counterparts, and Apterous in Drosophila, Lim1 and Chip may cooperate in vivo to modulate the transcriptional activity of its downstream target genes. Chip is ubiquitously expressed and therefore is present in all Lim1-expressing cells suggesting that an in vivo interaction is possible (Lilly, 1999).
Proximodistal patterning in Drosophila requires division of the developing leg into increasingly smaller, discrete domains of gene function. The LIM-HOM transcription factors apterous (ap) and Lim1 (also known as dlim1), and the homeobox genes Bar and aristaless (al) are part of the gene battery required for the development of specific leg segments. Genetic results show that there are posttranslational interactions between Ap, Bar and the LIM-domain binding protein Chip in tarsus four, and between Al, Lim1 and Chip in the pretarsus, and that these interactions depend on the presence of balanced amounts of such proteins. In vitro protein binding is observed between Bar and Chip, Bar and Ap, Lim1 and Chip, and Al and Chip. Together with evidence for interactions between Ap and Chip, these results suggest that these transcription factors form protein complexes during leg development. It is proposed that the different developmental outcomes of LIM-HOM function are due to the precise identity and dosage of the interacting partners present in a given cell (Pueyo, 2004).
Biochemical studies in vitro have shown that LIM-HOM transcription factors confer little transcriptional activation of target genes on their own. LIM-HOM proteins interact with a variety of proteins, including members of the bHLH family, the POU family and also other LIM family members, to control specific developmental processes. It has been suggested that these protein interactions confer specificity and modulate LIM-HOM activity. For example, Dlmo proteins reduce LIM-HOM activity, and Lbd proteins such as Chip modulate LIM-HOM activity by acting as a bridge between LIM-HOM proteins and Chip-binding cofactors, thus leading to the formation of heteromeric complexes. LIM-HOM protein activity functions in different contexts is the development of Drosophila (Pueyo, 2004).
Bar and ap genes are expressed in the fourth tarsal segment and are required for its proper development, whereas the al and Lim1 genes are expressed and required in the pretarsus. All of these genes encode putative transcription factors and display canonical regulatory relationships. Thus, al activates lim1 expression and then both genes cooperate to repress Bar expression in the pretarsus. Reciprocally, Bar represses al and lim1 expression while activating the expression of ap in tarsus four. After the refinement of their gene expression domains by these regulatory interactions, Bar directs tarsus five development, whereas cooperation between al and lim1 directs pretarsus development, and cooperation between Bar and ap directs tarsus four. The results of this study offer more evidence for the existence of this regulatory network, but also suggest an interesting role for direct protein interactions in its mechanism (Pueyo, 2004).
The cooperation between Bar and Ap on the one hand, and Al and Lim1 on the other, is likely to be carried out by transcriptional complexes involving Chip. The Chip protein is required for development of the tarsus four, five and pretarsus, and Gst (Glutathione-S-transferase-Chip fusion construct) experiments reveal Chip's ability to bind Ap, Bar, Lim1 and Al. However, the results also show that modulation of LIM-HOM protein activity by Chip alone does not explain distal leg development. For example, Ap function is not modulated primarily by Chip and Dlmo. The relative amount of Chip and Ap has to be grossly unbalanced before a phenotype is obtained in the leg, and dlmo is not expressed or required in leg development. Furthermore, the interaction between Ap and Chip does not confer the developmental specificity that allows LIM-HOM proteins to produce different outcomes in different parts of the leg. (1) Ap and Chip also interact in the wing and the CNS. (2) A chimaeric Lim3-Ap protein containing the LIM domains of Lim3 and the HOM domain of Ap does not behave as a dominant negative when expressed in tarsus four, and is even able to fulfil Ap function and rescue ap mutants. In the distal leg, developmental specificity seems to be achieved at the level of DNA binding and the transcriptional control of targets genes, mediated by partnerships between LIM-HOM and HOM proteins (Pueyo, 2004).
The evidence for this is presented first by dosage interactions between LIM-HOM and HOM proteins. Whereas there seems to be a relative abundance of endogenous Ap in tarsus four, an excess of Bar or Chip leads to a mutant phenotype, which is rescued by restoring the normal balance between Ap, Bar and Chip proteins in co-expression experiments. The effects observed could be explained simply by independent competition and the binding of Bar and Ap to Chip, leading, for example, to an excess of Bar-Chip complexes and a reduction of the pool of Chip available for Ap-Chip complexes. However, this hypothesis alone does not explain the additional dominant-negative effects of ectopic LIM-HOM and HOM proteins in tarsus four (Lim3, Islet and Al), which are also not mediated by transcriptional regulation but are nonetheless rescued by co-expression of appropriate endogenous proteins. For example, ectopic expression of UAS-islet or UAS-Lim3 in the ap domain produces loss of tarsus four without affecting Ap or Bar expression, and simultaneous co-expression of UAS-Bar partially suppresses this phenotype. If the sole effect of both UAS-Bar and UAS-Lim3 or UAS-islet were to quench Chip away from Ap, then simultaneous co-expression of Bar and Lim3 or Islet should worsen the phenotype, not correct it as observed. Moreover, ectopic expression of Islet or Lim3 proteins is not corrected by simultaneous co-expression of either UAS-Chip or UAS-ap. Altogether these results show instead that UAS-islet and UAS-Lim3 must interfere posttranslationally with Bar. The most direct explanation is that Islet and Lim3 have the ability to quench Bar protein into a non-functional state. Interestingly, the hybrid UAS-Lim3:ap does not behave as dominant negative but as an endogenous Ap protein in these experiments, since it does not produce a mutant phenotype on its own and it rescues UAS-Bar overexpression. This suggests that the LIM domains are not very specific when it comes to interaction with Bar, and points to the involvement of a common LIM-binding intermediary such as Chip. These results suggest that a protein complex involving Ap, Chip and Bar is the correct functional state of these proteins in tarsus four, and deviations from this situation into separate Bar-Chip, Ap-Chip, or Bar-Chip-Lim3 or Bar-Chip-Islet complexes leads to a mutant phenotype (Pueyo, 2004).
The notion of a protein complex involving Ap, Chip and Bar together is also supported by the Gst pull-down assays. The domain of Chip involved in Ap binding, the LIM interaction domain (LID), is not involved in Bar binding. However, the LID and the dimerisation domains of Chip are necessary to rescue the dominant-negative effect of UAS-Bar on tarsus four, suggesting a requirement for the formation of a complex with a LIM-HOM protein such as Ap. In agreement with this view, the Ap protein, and the LIM domains of Ap alone, are able to retain Bar protein in a Gst assay (Pueyo, 2004).
In the pretarsus, Al and Lim1 are possibly engaged in a partnership with Chip similar to that suggested for Ap, Chip and Bar. Synergistic cooperation between Al and Lim1 is required to direct pretarsus development and to repress Bar expression and function. Their cooperation entails a close functional relationship because a proper balance of Al, Lim1 and Chip is required, as is shown by the loss of pretarsal structures in UAS-Chip or UAS-Lim1 flies. Ectopic expression of LIM-HOM proteins in the pretarsus also disrupts pretarsal development without affecting Lim1 and Al expression. The possibility of direct protein interactions between Al, Lim1 and Chip is also suggested by the reciprocal ability of Al to interfere posttranscriptionally with Bar and Ap in tarsus four, and by the binding of Chip to Lim1 and to Al in in vitro experiments (Pueyo, 2004).
Comparison of tarsal development with other developmental processes illustrates how LIM-HOM proteins are versatile factors to regulate developmental processes. It had been observed that the outcome of LIM-HOM activity depends on their developmental context. This context can now be analysed as being composed of the presence, concentration and relative affinities of other LIM-HOM proteins, Ldb adaptors, and other cofactors such as LMO proteins and HOM proteins. It is proposed that the different developmental outcomes of LIM-HOM protein function could be due to the precise identity and dosage of cofactors available locally (Pueyo, 2004).
Ectopic expression experiments distort these contexts and lead to non-functional or misplaced LIM-HOM activities. In the wing, a finely balanced amount of functional Ap protein is modulated by Dlmo and Chip. Over-abundance of Chip stops the formation of functional tetramers in the wing but not in the CNS, where the relative amount of Ap, which is not modulated by Dlmo, is limiting for the formation of Ap-Chip functional complexes. In tarsus four, the Ap-Chip-Bar partnership is affected by experimentally induced over-abundance of Chip, presumably also because ectopic Ap-Chip tetramers typical of the CNS and the wing, and Bar-Chip complexes typical of tarsus five, are produced. Similarly, an excess of Bar might be interpreted by the cells as being a wrong developmental outcome, since high levels of Bar in the absence of Ap direct tarsus five development. Overexpression of Ap rescues this Bar dominant-negative effect, by restoring the relative amounts of Bar and Ap, which are determinant and limiting for tarsus four development. Finally, the dominant-negative effects produced by overexpression of either Chip or Lim1 in the pretarsus could either prevent the formation of Al-Chip-Lim1 complexes, or could favor the existence of Lim1-Chip complexes typical of the CNS (Pueyo, 2004).
The wing and the CNS models have postulated that Ap function is carried out by an Ap-Chip tetramer; however, the molecular scenario might be more complex. A new component of Ap-Chip complexes, named Ssdp, has been identified and is required for the nuclear localisation of the complex. Thus it is possible that an Ap-Chip tetramer also contains two molecules of Ssdp. In addition, different types of Chip-mediated transcriptional complexes and different regulators have been identified in other developmental contexts, such as in sensory organ development and thorax closure, in which the GATA factor Pannier forms a complex with Chip and with the bHLH protein Daughterless. Heterodimers of this complex are negatively regulated by a protein interaction with Osa. Thus, although the current results indicate that in different segments of the leg there exist specific interactions between LIM-HOM, Chip and HOM proteins, the involvement of further elements in these multiprotein complexes is not excluded (Pueyo, 2004).
The results support a partnership between HOM and LIM-HOM proteins in the specification of distinct segments of the leg, and the results are compatible with Ap-Chip-Bar, Bar-Chip and Lim1-Chip-Al forming transcriptional complexes. Although the characterisation of the target sequences, followed by further biochemical and molecular assays, is necessary to study the transcriptional mechanism of these interactions, it has been shown that LIM-HOM proteins can interact specifically and directly with other transcription factors to regulate particular genes. For instance, mouse Lim1 (Lhx1) interacts directly with the HOM protein Otx2. In addition, the bHLH E47 transcription factor interacts with Lmx1, and both synergistically activate the insulin gene. This interaction is specific to Lmx1, since E47 is unable to interact with other LIM-HOM proteins such as Islet. Moreover, Chip is able to bind to other Prd-HOM proteins, such as Otd, Bcd and Fz, to activate downstream genes. Chip also complexes with Lhx3 and the HOM protein P-Otx, increasing their transcriptional activity. The current results reinforce the notion of Chip as a multifunctional transcriptional adaptor that has specific domains involved in each interaction (Pueyo, 2004).
Experiments in Drosophila have demonstrated a conservation of LIM-HOM activity at the functional and developmental level in the CNS between Drosophila and vertebrates. In addition, xenorescue experiments have shown that the mechanism of action of Ap and its vertebrate homolog Lhx2 is very conserved in Drosophila wings, whereas ectopic expression of dominant-negative forms of chick Lim1, Chip, Ap and Lhx2 mimic both Ap and Lhx2 loss-of-function phenotypes. The developmental role of Ap, Bar and Al in the fly leg, and their putative molecular interactions may also have been conserved because their vertebrate homologs Lhx2, Barx and Al4 are also co-expressed in the limb bud. It is expected that the interactions between the LIM-HOM and Prd-HOM proteins shown here represent a conserved mechanism to specify different cellular fates during animal development (Pueyo, 2004).
Lim1 protein, detected using a specific antiserum, is first detected at the cellular blastoderm stage in a circumferential stripe just anterior to the cephalic furrow. Prior to this, no expression is evident within the syncytium, indicating a lack of maternal contribution. Zygotic expression persists in the procephalic lobes as gastrulation proceeds and becomes compartmentalized to defined regions within the head segments. At maximum germ band extension, transcripts are detected in clusters of cells in the thoracic and abdominal segments. Expression is confined to the cells at the neuroectoderm border where the neuroblasts are segregating from the ectoderm. In later stages of embryogenesis, high levels of Lim1 are present in the head segments, particularly in the clypeolabrum, the mandible, and maxilla. High levels of expression are also observed in the cells of the ventral nerve cord, as well as the embryonic brain lobes. In addition, the Lim1 protein is present in small groups of cells in the peripheral nervous system and cells of the ventral epidermis. The expression of Lim1 RNA was analyzed by whole mount in situ hybridization. In comparing the expression of RNA and protein no obvious differences were observed between their individual patterns (Lilly, 1999).
To further define the Lim1 expressing cells within the nervous system, double labeling experiments were carried out with the Lim1 antibody and cell specific markers. Using an antibody against the Repo protein, which is expressed exclusively in glial cells, no overlap was observed between Lim1 and Repo expression. Double stainings for Lim1 and the Elav protein, which is expressed in all neural cells, shows that Lim1 is confined to a subset of neuronal cells within the ventral nerve cord. To help identify the neurons within the nerve cord that are positive for Lim1, double labelling using an antibody that recognizes the Evenskipped (Eve) protein has been carried out. Lim1 was localized to motoneurons, RP2, aCC, and the U neurons. All of these motorneurons innervate the dorsal-most muscle groups within each segment. Interestingly, the motorneurons in which Isl is expressed innervate only ventral muscle groups. Lim1 is localized to a subset of interneurons that express the Derailed protein. Thus, within the ventral nerve cord Lim1 is expressed in both motorneurons and interneurons, and is absent from the glial cell population (Lilly, 1999).
Additionally, the expression of Lim1 was analyzed in dissected larvae by immunostaining. Lim1 protein is present in discrete cells of the larval nerve cord, and in the lobes of the brain. There is particularly strong expression in the cells of brain from which the optic stalk emerges to attach to the eye disc. Lim1 immunoreactivity is detected in the antennal disc and the leg disc. No protein was observed in the eye disc, or the wing and haltere discs. Lim1 in the leg and antennal disc are expressed in concentric rings, corresponding to the major segmental folds in the disc epithelium. The center spot in each disc represents the distal-most segment of what will become the adult leg and antennae. The outer rings correspond to the progenitors of more proximal segments. In addition, Lim1 expression is detected in the labial disc. All discs that express Lim1 are derived from imaginal progenitors that originate from the ventral region within the embryo. Thus, as in the embryo, Lim1 shows a very defined pattern of expression within subsets of imaginal discs and adult progenitors (Lilly, 1999).
The unique expression of Lim1 in a subset of motoneurons and interneurons led to an examination of whether this expression overlaps with other LIM homeodomain members. Characterization of the expression of a group of vertebrate LIM homeodomain genes (Isl-1, Isl-2, Lim-1 and Lim-3) along the chick spinal column has demonstrated that these genes overlap with one another in very distinct patterns (Tsuchida, 1994). Their spatial overlap demarcates regions of motorneuron subclasses, suggesting that the LIM homeodomain genes confer an identity to pools of motorneurons by Lim protein combinatorial expression. More recently, a combinatorial code for motorneuron pathway selection has been demonstrated for isl and lim3 in Drosophila (Thor, 1999). In order to evaluate the expression of Lim1 with respect to its LIM homeodomain relatives, a series of double labeling experiments were carried out using late stage embryos. These embryos carried enhancers from either islet (Thor, 1997), lim3 (Thor, 1999) or apterous (O'Keefe, 1998) that recapitulated their expression using a tau-LacZ or tau-c-myc fusion construct as a reporter. By double staining for enhancer expression and the Lim1 protein, no overlap of expression was observed between Lim1 and Isl, Ap or Lim3. All neurons that stain positively for Lim1 in the nuclei lack enhancer expression within their cell bodies. Similar to what is observed in vertebrates, the LIM homeodomain genes that were analyzed in Drosophila are expressed in distinct subclasses of neurons within the ventral nerve cord. The expression of Lim1 is confined to a subset of motorneurons and interneurons that are lacking the other LIM homeodomain genes tested. To assess the possibility that the absence of Lim1 in cells expressing other LIM homeodomain proteins was due to repression by these family members, the expression of Lim1 was analyzed in ap and lim3 mutant embryos. In embryos with a null mutation in ap, the expression of Lim1 remains unchanged. Likewise, analysis of the Lim3-expressing RP neurons in lim3 mutants shows no upregulation of Lim1. These results indicate that the exclusion of Lim1 from cells expressing other LIM homeodomain proteins is not a result of repression by these LIM homeodomain family members (Lilly, 1999).
In addition to the exclusive expression of Lim1, Ap and Isl do not overlap (Thor, 1997), while Lim3 fails to overlap with Ap, but is found in a subset of Isl positive cells (Thor, 1999). Thus, as in vertebrates, the expression of these genes in the Drosophila nerve cord may provide instructional cues for proper pathfinding and target identity in the embryo (Lilly, 1999).
Proper information processing in neural circuits requires establishment of specific connections between pre- and postsynaptic neurons. Targeting specificity of neurons is instructed by cell-surface receptors on the growth cones of axons and dendrites, which confer responses to external guidance cues. Expression of cell-surface receptors is in turn regulated by neuron-intrinsic transcriptional programs. In the Drosophila olfactory system, each projection neuron (PN) achieves precise dendritic targeting to one of 50 glomeruli in the antennal lobe. PN dendritic targeting is specified by lineage and birth order, and their initial targeting occurs prior to contact with axons of their presynaptic partners, olfactory receptor neurons. A search was performed for transcription factors (TFs) that control PN-intrinsic mechanisms of dendritic targeting. Two POU-domain TFs, acj6 and drifter have been identified as essential players. After testing 13 additional candidates, four TFs were identified, (LIM-homeodomain TFs islet and lim1, the homeodomain TF cut, and the zinc-finger TF squeeze) and the LIM cofactor Chip, that are required for PN dendritic targeting. These results begin to provide insights into the global strategy of how an ensemble of TFs regulates wiring specificity of a large number of neurons constituting a neural circuit (Komiyama, 2007).
For technical simplicity, larval born GH146-Gal4-positive PNs, originating from three neuroblast lineages, anterodorsal (adPNs), lateral (lPNs), and ventral (vPNs), were studied. Out of ~25 classes defined by their glomerular targets, focus was placed on 17 classes whose target glomeruli are reliably recognized across different animals. The MARCM technique allows visualization and genetic manipulation of PNs in neuroblast and single-cell clones in otherwise heterozygous animals, so PN-intrinsic programs can be studied for dendritic targeting. GH146 is expressed only in postmitotic PNs (Komiyama, 2007).
acj6 and drifter have been identified as lineage-specific regulators of PN dendritic targeting. To identify additional transcription factors (TFs) that regulate dendritic targeting of different PN classes, candidates were tested that have been shown to regulate neuronal subtype specification and targeting specificity and have available loss-of-function mutants. The following was tested; (1) the expression of candidate genes in PNs at 18 hr after puparium formation (APF) when PN dendrites are in the process of completing their initial targeting, and/or (2) their requirement in PNs by examining dendritic targeting in homozygous mutant MARCM clones (Komiyama, 2007).
In addition to the eight genes described below, five other TFs were examined that were not pursued because of the lack of expression in GH146-PNs at 18 hr APF (aristaless and pdm-1) or the lack of targeting defects in homozygous mutant PNs (abrupt [abk02807], kruppel [Kr1], and Dichaete [Dichaete87]) (Komiyama, 2007).
LIM-HD factors and PN targeting: LIM-homeodomain (LIM-HD) TFs are involved in multiple events during neuronal development. Most functions of LIM-HD factors require the LIM domain-binding cofactor, which is represented in Drosophila by ubiquitously expressed Chip. Chip antibody revealed ubiquitous expression of Chip in cells around the antennal lobe (AL) including all GH146-PNs at 18 hr APF (Komiyama, 2007).
The requirement of Chip in PN dendritic targeting was tested. Wild-type adPNs, lPNs, and vPNs target stereotyped sets of glomeruli. PNs homozygous for a Chip null allele (Chipe5.5) failed to target most of the correct glomeruli and occupied inappropriate glomeruli. Most adPN and lPN clones (12/13) also mistargeted a fraction of dendrites to the structure ventral to the AL, the suboesophaegeal ganglion (SOG). Thus, Chip is required for targeting specificity of most, if not all, PN classes studied here, and Chip-interacting proteins including LIM-HD factors likely play important roles in PN dendritic targeting (Komiyama, 2007).
Five LIM-HD factors have been characterized in Drosophila: apterous, arrowhead, islet, lim1, and lim3. apterous, arrowhead, or lim3 were not pursued because they are not expressed in GH146-PNs at 18 hr APF (apterous) or they do not have targeting defects in PNs homozygous for null alleles (lim337Bd6 and awh16) (Komiyama, 2007).
Islet antibody detected Islet expression in ~50% adPNs and most lPNs but not in vPNs at 18 hr APF and adult. isl−/− adPNs failed to target many (but not all) of the normal target glomeruli, including VA1lm, VA3, and VM7. In addition, DA1, a lPN target, was often specifically mistargeted. Defects of isl−/− lPNs were very similar to Chip−/− lPN defects. A fraction of dendrites often mistargeted to the SOG. Within the AL, dendrites were diffusely spread, although DA1 and DL3 were always correctly innervated. Targeting of isl−/− vPNs was normal, consistent with their lack of Islet expression (Komiyama, 2007).
Lim1 antibody revealed Lim1 expression in most or all vPNs, but not in adPNs or lPNs in adults. The expression pattern appears similar at 18 hr APF, although vPNs are difficult to identify unambiguously at early stages. lim1−/− adPNs showed no defects, consistent with the lack of Lim1 expression. lim1−/− lPNs rarely showed a cell number decrease, but in clones in which the cell number was normal, lim1−/− lPNs targeted correct glomeruli. In contrast, lim1−/− vPNs showed a specific targeting defect. Wild-type vPNs innervate DA1 and VA1lm densely because of the single vPNs that specifically innervate these glomeruli, in addition to the diffuse innervation all over the AL contributed by the pan-AL vPN. In lim1−/− vPNs, DA1 innervation was greatly reduced and sometimes undetectable. Therefore, lim1 is required for dendritic targeting by a single vPN class, vDA1, despite its general expression in vPNs. lim1 might be redundant with other factors in non-DA1 vPNs. It was note that phenotypes of islet and lim1 combined are only a subset of the Chip phenotype. Additional Chip phenotype may be explained by non-Lim-HD molecules interacting with Chip (Komiyama, 2007).
cut is required for targeting of several lPN and all vPN classes: cut encodes a homeodomain TF that regulates sensory organ identity and dendritic morphogenesis in Drosophila peripheral nervous system. A monoclonal antibody detected Cut in subsets of adPNs and lPNs (~8 for each) and in all vPNs. The expression pattern appeared similar at 18 hr APF. Costaining with Mz19-Gal4 and various single-cell clones with GH146-Gal4 further narrowed down Cut-expressing PNs; Cut-positive adPNs are likely embryonically born and thus not included in the functional analysis, while DM1 and DM2 lPNs express Cut, but DA1, DL3, and DM5 lPNs do not (Komiyama, 2007).
cut−/− adPNs targeted all their normal glomeruli correctly, consistent with their lack of expression. cut−/− lPNs failed to target DM1, DM2, and VA5. cut−/− vPNs were severely affected, with their cell numbers reduced from 4–6 in wild-type to 2–3 in cut−/− clones. cut−/− vPNs failed to elaborate their dendrites correctly in the AL and mistargeted the SOG. In summary, cut is required by a specific subset of lPNs and all vPNs that express Cut (Komiyama, 2007).
cut appears to control global targeting of PNs along mediolateral axis, as indicated by the fact that loss and gain of cut in lPNs causes a lateral and medial shift of dendrites, respectively. adPNs do not show a cut loss-of-function defect, consistent with the lack of expression. Nevertheless, cut misexpression in adPNs shifted their dendrites medially. Interestingly, adPNs misexpressing cut usually avoid DM1 and DM2, suggesting that cut controls global targeting, rather than simply promoting innervation of these glomeruli (Komiyama, 2007).
Postmitotic expression of a cut transgene only in labeled cut−/− lPNs completely rescued targeting of DM1, DM2, and VA5. There were also gain-of-function phenotypes, and DA1 and DL3 innervation was often lacking in these clones. Thus, cut postmitotically rescues dendritic targeting defects of lPNs that normally express cut, whereas postmitotic misexpression in other lPNs disrupts their targeting fidelity (Komiyama, 2007).
The vPN rescue phenotype was more complex. The cell number decrease was not rescued by postmitotic cut expression. However, the targeting defect was partially rescued. 71% of vPN rescue clones examined sent some dendrites to the AL (the rest completely failed to innervate the AL), and 68% innervated VA1lm. This is markedly better than cut−/−, in which only 51% entered the AL and 23% innervated VA1lm. DA1 targeting was not rescued, raising the possibility that the DA1 vPN was never born or correctly specified in these animals (Komiyama, 2007).
Relationship of cut and lim1 in vPNs: The lim1 phenotype in vPNs is a subset of the cut phenotype. Lim1 immunoreactivity in cut−/− vPNs was either absent or greatly reduced compared to wild-type. Therefore, Cut directly or indirectly controls Lim1 expression (Komiyama, 2007).
If a major function of Cut in vPNs is to upregulate Lim1, then transgenic lim1 expression in cut−/− vPNs might suppress part of the cut−/− phenotype. In cut−/− vPNs expressing a lim1 transgene, the reduction of cell number was not suppressed. However, 67% clones innervated the AL (compared to 51% in cut−/−). VA1lm innervation was also mildly improved (36% in UAS-lim1 versus 23% in cut−/−). Thus, UAS-lim1 expression partially suppresses cut−/− targeting defects, although not quite as well as UAS-cut. In contrast, UAS-lim1 expression in cut−/− lPNs, which normally do not express Lim1, did not suppress the cut−/− targeting defects. Therefore, Cut and Lim1 are not simply interchangeable, and the partial suppression of cut−/− defects by lim1 is specific to vPNs (Komiyama, 2007).
Although postmitotic expression of cut partially rescued the cut−/− vPN phenotypes, it failed to rescue Lim1 expression. In addition, postmitotic misexpression of cut in adPNs or lPNs did not lead to an ectopic expression of Lim1. Therefore, cut is not sufficient to upregulate Lim1 expression in postmitotic neurons. It is proposed that cut functions at two distinct stages of vPN development. First, cut controls the proliferation and/or fate specification of the vPN neuroblast, including Lim1 expression. Second, cut controls dendritic targeting by postmitotic VA1lm vPNs, partially redundantly with lim1. This partial redundancy may explain the observation that lim1−/− vPNs target VA1lm normally. These pre- and postmitotic functions of cut in the same neuronal lineage are reminiscent of its function in peripheral nervous system development (Komiyama, 2007).
Lim1 mutation is lethal. Lim1 mutant embryos hatch from their egg case and can develop to the 3rd instar larval stage. In good culture conditions most progress to the 3rd instar stage, and some will pupate. Two alleles appear to be hypomorphs, because 90% of the mutants pupate, compared to only 10%-20% for null allele E9 and allele E4. Heteorallelic combinations of the Lim1 mutations with each other and with Df(1)lz-90b24 give similar results. The mutant larvae exhibit no developmental delay: they molt and reach the wandering stage with no obvious defects. Movement of larvae appears typical until the 3rd instar wandering stage. At this stage the mutants appear sluggish and fail to wander normally. This causes them to either arrest or pupate on or near their food source. From analysis of dissected pupal cases, a few mutants are seen to develop into pharate adults, but are incapable of eclosion and survival beyond this point. These behavioral defects suggest that the Lim1 mutants have abnormal motor coordination, leaving them unable to wander properly and eclose into adult flies. Molecular analysis of these mutants has not revealed any striking defects. By using an array of molecular markers to analyze both embryos and larvae, the overall structure of the nervous system has been found to be normal. The mutants were analyzed with several nervous system markers including 22C10 (see Futsch), FasII BP102, and BP104; no structural defects or misguided axon projections were detected. In particular, the projections of the RP2 and aCC neurons were analyzed in dissected embryos and larvae and they were found to be morphologically normal. For each marker, approximately 5 individual hemisegments from 50 Lim1 mutant embryos were analyzed and compared to wild-type embryos using whole mount immunocytochemistry. For the dissected preparations, more than 20 mutant embryos and 20 mutant larvae were dissected, from which 5 to 6 hemisegments where analyzed in detail and compared in parallel to wild-type preparations (Lilly, 1999).
Although it is quite possible that Lim1 mutants have subtle pathfinding defects, as is the case with ap and isl, they could not be identified. Lim1 is not found in serotonin- and dopamine-producing neurons, and is absent from the ring gland in larva, suggesting that these defects are not due to disruptions in these neurosecretory pathways. Thus a clear explanation for the cause of lethality for the Lim1 mutants remains to be elucidated. Additionally the neuronal phenotype of Lim1 null mutants in embryos heterozygous for chip-/+ was examined. However, as with the Lim1 mutation alone, no phenotype was observed in the Lim1-/- null, chip-/+ heterozygous background (Lilly, 1999).
During Drosophila leg development, the distal-most compartment (pretarsus) and its immediate neighbor (tarsal segment 5) are specified by a pretarsus-specific homeobox gene, aristaless, and tarsal-segment-specific Bar homeobox genes, respectively; the pretarsus/tarsal-segment boundary is formed by antagonistic interactions between Bar and pretarsus-specific genes that include aristaless. Drosophila Lim1 is involved in pretarsus specification and boundary formation through its activation of aristaless. Ectopic expression of Lim1 causes aristaless misexpression, while aristaless expression is significantly reduced in Lim1-null mutant clones. Pretarsus Lim1 expression is negatively regulated by Bar and is abolished in leg discs lacking aristaless activity, which is associated with strong Bar misexpression in the presumptive pretarsus. No Lim1 misexpression occurred upon aristaless misexpression. The concerted function of both Lim1 and aristaless is required to maintain Fasciclin 2 expression in border cells and form a smooth pretarsus/tarsal-segment boundary. Lim1 is also required for femur, coxa and antennal development (Tsuji, 2000).
To isolate genes that possibly act with al in pretarsus specification, a search was made for genes expressed in the pretarsus but not the segment immediately adjacent to it at late third instar stages. P0092 is an enhancer trap line, in which lacZ expression in leg and antennal discs was found to be similar to al expression in these tissues. lacZ is coexpressed in virtually all Al-positive cells in the pretarsus, tibia, femur and possibly coxa in leg discs, and the arista and first antennal segment in antennal discs. Although Al expression is restricted to ventral cells in the tibia and dorsal cells in the femur, coxa and first antennal segment, lacZ expression has been noted in both ventral and dorsal cells uniformly, which gives rise to complete circular expression. In wing and haltere discs, in which al is also expressed, no appreciable expression of P0092-lacZ is observed. Nucleotide sequence analysis indicates that the putative P0092 gene encodes a LIM-homeodomain protein identical in amino acid sequence to Lim1 (Tsuji, 2000).
Flies neither homozygous nor hemizygous for the P0092 P insertion show any obvious morphological defects. Thus, Lim1 loss-of-function mutants generated by imprecise P-element excision and six independent larval or pupal lethal mutant lines were obtained. These frequently produce pharate adults with apparent defects in mouth parts, leg and antennal morphology, making it possible to examine the roles of Lim1 in leg and antennal development (Tsuji, 2000).
In legs and antenna completely lacking al activity, all pretarsus structures and arista are lost, respectively. In moderate hypomorphic al mutants, claws are frequently lost without loss of other pretarsus structures such as pulvilli and empodia, while in weak hypomorphic mutants, claws and aristae are not lost but only reduced in size. Lim1 minus mutants are very similar in leg phenotype to moderate al hypomorphic mutants. In about half of all cases, the antenna are absent from the Lim1 minus half head. When antennae is present, arista is deformed and reduced in size. That is, Lim1 minus arista are morphologically similar to those of weak hypomorphic al mutants. These findings indicate that Lim1 is essential for proper development of pretarsus and arista as well as al, although Lim1 mutant phenotypes are much less severe than a1 minus mutant phenotypes. In Lim1 minus legs that are simultaneously homozygous for al, not only claws but also empodia and pulvilli are frequently lost. The concerted function of Lim1 and al would thus appear to be required for normal pretarsus/aristal development (Tsuji, 2000).
Apart from the future pretarsus, Lim1 is expressed circularly in proximal segments such as the coxa, femur and tibia. In Lim1 minus flies, the femur is extensively reduced in size and the coxa is missing for the most part or present only as a small bulb-like structure, suggesting the requirement of Lim1 for proper development of the femur and coxa. Although the tibia is bent and fused with the femur, morphological analysis indicates the presence of essentially normal characteristic structures of the tibia, such as transverse rows of bristles, preapical bristles, tibial sense organs and tibial sensilla trichodea; tibial sense organs and tibial sensilla trichodea are structures situated near the proximal tibial end. The tibial phenotype may thus possibly derive from secondary effects of the femoral deformation. In late third instar, Dll expression is evident in the central region spanning from the most distal tip to distal half of the tibia and in the future trochanter. Consistent with shortening of the femur, appreciable reduction in mass has already taken place in the region flanked by the central Dll domain and the proximal Dll ring at late third instar. In Lim1 minus leg and antennal discs, Al expression in the proximal region, such as in the femur, coxa and first antennal segment, is virtually absent. In Lim1 mosaic clones in the femur or coxa, Al expression is abolished cell autonomously. Tibial Al expression remains in Lim1 discs but mosaic analysis clearly indicates substantial reduction in Al expression in Lim1 clones. al is expressed considerably prior to Lim1 and thus Lim1 may thus be involved in maintenance of pretarsus al expression. But loss of Al expression would not completely explain the femoral and coxal defects, since al is dispensable for normal development of the femur and coxa (Tsuji, 2000).
Mutually antagonistic interactions between al and Bar are essential for the strict separation of Al and Bar domains, leading to localized Fas2 induction by Bar in border cells. Although the absence of Lim1 shows little Bar misexpression in the pretarsus, increased Bar misexpression in Lim1;al leg discs could indicate the involvement of Lim1 in the repression of Bar expression in the pretarsus. Remarkable decrease in Fas2 expression in putative Lim1;al mutant border cells indicates that Fas2 expression requires al and Lim1 functions, in addition to cell non-autonomous functions of Bar. Lim1 may be involved in pretarsus specification and boundary formation only through its activation of al. Low al expression in Lim1 single mutants may still be sufficient for maintaining the normal expression of Bar and Fas2, but with further reduction in al expression in Lim1;al double mutants, Bar misexpression and loss of Fas2 expression may result. Alternatively, Lim1 may act independently of al, and simultaneous reduction in al and Lim1 expression may cause Bar misexpression and reduction of Fas2 expression in the double mutants. These considerations are not mutually exclusive (Tsuji, 2000).
Proximal-distal leg development in Drosophila involves a battery of genes expressed and required in specific proximal-distal (PD) domains of the appendage. apterous is required for PD leg development, and the functional interactions between ap, Lim1 and other PD genes during leg development have been explored. A regulatory network formed by ap and Lim1 plus the homeobox genes aristaless and Bar specifies distal leg cell fates in Drosophila (Pueyo, 2000).
Lim homeobox (Lhx) genes have been shown to interact functionally in the nervous systems of Drosophila and vertebrates. It has been suggested that different combinations of Lhx proteins shunt cells into different cell fates, and this model predicts that Lhx proteins can act combinatorially, possibly forming complexes to activate target genes. In appropriate experiments, ectopic generation of a given combination of Lhx proteins shunts cells into an ectopic, but coherent and predictable, cell fate. This study has explored the possibility of similar interactions between Lim1 and other Lhx genes in the appendages of Drosophila. A computer search of the Drosophila genome has identified four other Lhx genes. The Lhx genes Lim3 and Islet (tailup) have been characterized previously in Drosophila, but their mutant phenotypes and patterns of expression do not involve the appendages. A search has identified a new putative Lhx gene, homologous to vertebrate Lmx1, which is not expressed in legs either. The only other Lhx gene identified is the apterous (ap) gene, which is homologous to vertebrate Lhx2. ap is expressed in the leg in the presumptive tarsal segment four, near the tip of the leg close to where Lim1 is expressed. A mutant phenotype for ap in legs has not been described, but using allelic mutant combinations that produce extreme loss of function of ap the following phenotypes were observed: either fusion of tarsus four and five, reduction and deformities in tarsus four, or complete loss of tarsus four, the latter producing legs with only four tarsi but looking otherwise normal. Lim1 and ap expression was combined using UAS constructs to express ap and Lim1 ectopically in legs (Pueyo, 2000).
Expression of UASap over the presumptive claw region using several different Gal4 lines produces no discernible phenotype. In contrast, expression of UASLim1 driven by apGal4, which faithfully reproduces ap expression, produces complete absence of tarsus four, thus mimicking extreme loss of function of ap. This loss of tarsus four fates is specific, since it is also accomplished by ectopic expression of Lim3, a close sequence paralogue of Lim1, but not by other, unrelated proteins, and it is accompanied by the loss of ap expression. However, this apparent dominant negative effect of Lim1 on ap was not rescued by simultaneous co-expression of extra ap in apGal4;UASLim1;UASap flies, as would be expected if the phenotype of UASLim1 were due to either loss of ap expression or competition with the Ap protein. Furthermore, mild tarsal fusions produced by expressing UASLim1 under the control of the weak line 30AGal4 were not made worse by simultaneous reduction of endogenous ap function in ap minus 30AGal4;UASLim1 flies. Altogether these results suggest that, although there exists an effect of ectopic Lim1 on ap expression, the Lim1 and Ap proteins are not interfering directly with each other. Rather, Lim1 must interact with another element involved in tarsus four development and related to ap function (Pueyo, 2000).
The Drosophila Chip gene has been shown to encode a ubiquitous transcriptional cofactor. Chip proteins bind to the Lim domains of Ap and the ap and Chip genes have to be present in similar doses to ensure normal wing development. Interestingly, Chip has been shown to bind the Lim domains of other Lhx proteins, among them Lim1. However, no such dose relationships were found, between chip and Lim1 or between chip and ap in the leg, or in flies expressing UASChip and UASLim1. Furthermore, intermediate ap or Lim1 mutants are not rescued by UASChip, and co-expression of UASChip together with UASLim1 in the ap domain does not rescue the dominant-negative effect of UASLim1 on ap. Therefore, it is concluded that Chip is either not required for Lhx protein function in leg development, or not present in either limited amounts or stoichiometric doses. Thus Chip is unlikely to be the putative ap partner affected by Lim1 (Pueyo, 2000).
Ectopic expression of Lim1 leads to loss of ap expression and of tarsus four, but a direct regulatory relationship between ap and Lim1 in the wild type does not need to exist, since they are never expressed in the same cells. Furthermore, expression of ap in Lim1 mutants, and of Lim1 in ap mutants is normal, which indicates the absence of long-range regulatory cell signals between these genes. However, expression of Bar in the presumptive tarsus abuts that of Lim1 and al. Bar encodes two redundant Hox proteins expressed in tarsus four and five, which are required for the development of these structures and for the expression of ap in tarsus four. When Lim1 is ectopically expressed in the Bar territory, a reduction of Bar expression occurs. This loss of Bar expression could explain the loss of ap expression seen in apGal4;UASLim1 flies and suggests that in the wild type, an important regulatory role of Lim1 is to restrict Bar expression to the presumptive tarsus five. The apparent paradox that apGal4;UASLim1;UASap flies still show a mutant phenotype can be understood if Bar also has a direct requirement for tarsus four development, one beyond simply activating ap expression (Pueyo, 2000).
PD patterning in Drosophila legs seems to proceed stepwise after it is initiated by the Wg- and Dpp-mediated activation of Dll, dac and al. Later on, these genes interact among themselves and with Hth to activate, in a Wg- and Dpp-independent phase, the expression of further PD genes in new domains of expression. Similar interactions of this kind must lead to the eventual allocation of all different PD fates. The genes downstream of the initial PD genes are still to be identified but Lim1 and ap may serve downstream functions. In early third instar, shortly after 72 hours AEL, al expression at the presumptive leg tip is possibly initiated by a combination of Wg and Dpp signaling, with a requirement for Dll. Around mid-third instar, al-expressing cells in the presumptive tip of the leg activate the expression of Lim1. At this time, the expression of Bar is present in a ring in the presumptive distal tarsal region, partially overlapping that of al and Lim1. This overlap then resolves into an abutment by late third instar. This refinement is important for proper development of the claw organ and tarsus five, and could be based on direct repressory action between the Hox transcription factors Bar and al. However, whereas ectopic Bar expression represses al expression, in the reciprocal experiment ectopic al does not repress Bar. Interestingly, ectopic expression of lim1 results in a reduction of Bar expression. It is concluded that al and Bar do have a mutual repressory relationship that involves Lim1 (Pueyo, 2000).
Whereas Bar might repress al expression directly, the repressory effect of al on Bar is mediated by Lim1. This regulatory circuit between Bar, al and Lim1 establishes the abutting fields of tarsus five cells expressing Bar, and claw organ cells expressing al and Lim1. This circuit also explains why although al mutants lead to an expansion of Bar expression, ectopic al does not reduce Bar expression. Whereas loss of al produces loss of Lim1 and hence leads to ectopic Bar expression, ectopic al on its own is not able to repress Bar. The final element in the determination of distal leg fates is the expression of ap, which is activated in the presumptive tarsus four around mid-third instar. Although ap expression is reduced by ectopic Lim1, this is probably an indirect consequence of the loss of Bar, because appropriate levels of Bar are responsible for the activation of ap. Whereas low levels of Bar are needed for ap expression in tarsus four, high levels of Bar in tarsus five prevent it. Thus, the tip of the leg gets divided into its three final domains during the second half of the third instar: the presumptive claw organ or pretarsus, defined by the expression of al and Lim1; the presumptive tarsus five, defined by the expression of high levels of Bar; and the presumptive tarsus four, defined by the expression of ap and low levels of Bar. During the subsequent pupal metamorphosis into an adult fly, these transcription factors must control the expression of appropriate downstream genes, leading to the differentiation of appropriate structures in each of these presumptive leg segments (Pueyo, 2000).
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Date revised: 25 October 2009
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