BarH1 and BarH2


Transcriptional Regulation

Expression of BarH1 and BarH2 in the eye is regulated by rough and glass (Higashijima, 1992a). Considerably more BarH1 mRNA was detected in the Bar mutant than in wild type, suggesting that BarH1 might regulate its own synthesis (Kojima, 1991).

Lozenge is implicated in the regulation of Bar proteins, which are required specifically in R1/R6 cells to specify their fate. The expression of Bar in R1/R6 cells in dramatically reduced but not completely eliminated. The antibody used to detect Bar was raised against BarH1. Upon lz overexpression, Bar expression was no longer restricted to R1/R6, but ectopically staining cells were consistently detected in the developing cluster (Daga, 1996).

lozenge mutants do not express the two Bar genes, and the enhancer-trap O32 (associated with an unknown gene specific to cells R3/4 and R7) is expressed in too many cells. Thus the defective recruitment that occurs in lozenge mutants can be attributed to abnormalities in the expression of genes like Bar, the gene marked by O32, and seven-up, which are essential for establishing the correct cell fate for the final three photoreceptor cells, R1, R6 and R7. seven-up is derepressed in R7 cells in lozenge mutants. The derepression of seven-up is reminiscent of the derepression of svp in rough mutants. rough normally represses svp in R3/R4. Thus Lozenge both actively represses some genes and activates others (Crew, 1997).

A new Drosophila Pax gene, sparkling (spa), implicated in eye development, has been isolated and shown to encode the homolog of the vertebrate Pax2, Pax5, and Pax8 proteins. It is expressed in the embryonic nervous system, and in cone, primary pigment, and bristle cells of larval and pupal eye discs. Transcripts are expressed in the posterior portion of the eye disc, with the anterior boundary of expression lagging clearly behind the morphogenetic furrow. In spa(pol) mutants, a deletion of an enhancer abolishes Spa expression in cone and primary pigment cells and results in a severely disturbed development of non-neuronal ommatidial cells. Because Spa is not expressed in R7 cells, its expression in newly recruited cone cells distinguishes their fate from that of R7 cells. Lozenge may be the transcription factor whose synthesis would have to precede that of Spa, which is required for the specification of the R7 equivalence group, including R1/R6, R7 and the cone cells. Lozenge helps define the R7 equivalence group by repressing seven-up (Fu, 1997).

Spa expression is further required for activation of cut in cone cells and of the Bar locus in primary pigment cells. Cut expression is strongly reduced in cone cell of spa(pol) mutants compared to wild type. Interestingly, Cut expression recovers; by 45 hours after pupariation it has risen to levels even above those of wild-type. The lack of Spa protein in cone cells appears to delay the development of the cells, since the shape of their nuclei and the nuclear accumulation of Cut resemble those of earlier stages in wild-type pupal discs. This delay may be caused by a late larval and early pupal requirement of Spa for cut activation, which later becomes independent of Spa. Expression of cut in bristle cells, many of which are mispositioned, appears unaffected during these stages. Expression of both Bar proteins in primary pigment cells is abolished completely in spa(pol) mutants. However, it remains unaffected in the irregularly positioned bristle cells, which continue to express Spa protein. Thus Spa exerts at least part of its control of primary pigment cell development through its regulation of Bar expression. Bar is also expressed in R1 and R6 precuror cells, where Lozenge rather than Spa is one of its activators. It is suggested that close functional analogies exist between Spa and Pax2 in the development of the insect and vertebrate eye. In the absence in Pax2, the optic stalk epithelium develops into pigmented retina and fails to proliferate and differentiate into glial cells, which populate the optic nerve and are essential for guidance of the retinal axons. Thus the cone cell in Drosophila might be considered as a kind of neuronal support, or glial --a cell that may have evolved from a more primitive ancestral glial cell. In favor of such a hypothesis, it is observed that spa is expressed in glial cells in the developing PNS (Fu, 1997).

The absence of Drosophia Frizzled-3 produces no apparent phenotype. Binding studies reveal that Wg can interact with Dfz3 in cultured cells. In order to reveal a role for Dfz3 in development, the possiblity of a genetic interaction of Dfz3 with wingless has been investigated. Dfz3 may be involved in Wg signaling required for adult appendage formation. For example, Dfz3 may serve as an attenuator of Wg signaling, at least in a wg hypomorphic mutant background; the absence of Dfz3 may increase Wg signaling and stimulate wing formation. For analysis of this possiblility, a study was made to find possible interaction between Dfz3 and Wg signaling in various wg mutant backgrounds. Wing blades are frequently absent from flies mutant for wg 1. Thus, the first question to be examined was is the wg 1 phenotype affected by the absence of Dfz3? The absence of wing blades is partially rescued through the elimination of Dfz3 activity. On a wg 1/wg CX4 background, fractions of flies with two wings increased from 46% to 87%, while those flies with one wing and wing-less flies, respectively, reduced from 44% and 10% to 13% and 0.5%. The wing-less phenotype of wg 1 is enhanced in a heterozygous apterous (ap) mutant background: no wing blade is generated at approx. 90% of the presumptive wing-blade-forming sites in wg 1 homozygous flies heterozygous for ap. Wing blade formation increases 3-fold in the absence of Dfz3 activity. Since wg CX4 and wg 1 are null and regulatory mutant alleles, respectively, these effects are not due to possible change in Wg protein conformation. Thus, wild-type Dfz3 may serve as an attenuator of Wg signaling at least in a wg hypomorphic mutant background; the absence of Dfz3 may increase Wg signaling and stimulate wing formation (A. Sato, 1999).

To confirm that Dfz3 attenuates Wg signaling, an examination was made of the effects of Dfz3 absence in a different developmental context. Nearly all wg11en/wgCX4 flies lack antennal structures. This antenna-less phenotype is significantly rescued by removing Dfz3 activity; complete antennal structures, as well as incomplete ones, areregenerated at more than 70% of putative antennal sites. Distal antennal segment formation requires the circular expression of Bar homeobox genes. Dachshund (Dac) is required for the formation of proximal leg structures and expressed circularly in leg and antenna discs. Thus, wg 11en/wgCX4 fly discs with or without Dfz3 activity were stained for Wg, BarH1 and Dac. When there is Dfz3 activity, antennal discs are small and no or little expression of BarH1 and Dac is detected. In the absence of Dfz3 activity, about 10% of the discs, probably corresponding to the completely rescued type, exhibit circular BarH1 and Dac expression similar to that of wild-type discs. In about 50% of discs, presumably corresponding to the partially rescued type, Dac expression is partially restored without recovery of BarH1 expression. In contrast to BarH1 and Dac, no Wg expression is detected in the rescued mutant discs, indicating that wg expression is not enhanced by the absence of Dfz3. That wgCX4 and wg 11en are regulatory mutant alleles of wg suggests again that the genetic interactions found here would not be due to possible change in Wg protein structure, but simply to reduction in transcription products of wg. Thus it follows that in wg hypomorphic mutants, Dfz3 reduces Wg signaling activity required for antennal formation without changing wg expression; accordingly, Dfz3 would appear to function as a negative factor or attenuator of Wg signaling at least on a wg hypomorphic mutant background (A. Sato, 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 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).

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 specify distal leg cell fates in Drosophila (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).

Arthropods and higher vertebrates both possess appendages, but these are morphologically distinct and the molecular mechanisms regulating patterning along their proximodistal axis (base to tip) are thought to be quite different. In Drosophila, gene expression along this axis is thought to be controlled primarily by a combination of transforming growth factor-ß and Wnt signalling from sources of ligands, Decapentaplegic (Dpp) and Wingless (Wg), in dorsal and ventral stripes, respectively. In vertebrates, however, proximodistal patterning is regulated by receptor tyrosine kinase (RTK) activity from a source of ligands, fibroblast growth factors (FGFs), at the tip of the limb bud. This study revises understanding of limb development in flies and shows that the distal region is actually patterned by a distal-to-proximal gradient of RTK activity, established by a source of epidermal growth factor (EGF)-related ligands at the presumptive tip. This similarity between proximodistal patterning in vertebrates and flies supports previous suggestions of an evolutionary relationship between appendages/body-wall outgrowths in animals (Campbell, 2002).

Ectopic activation of EGFR signalling results in autonomous, ectopic expression of al and B in mid-third-instar discs. Curiously, not all regions of the disc respond identically, with ventral regions being the most responsive and lateral regions the least. The reason for this is unclear but it indicates that factors in addition to EGFR may be regulating expression of tarsal genes such as al, at least outside of their normal domains. Only the presumptive tarsus is responsive to ectopic EGFR activity; this is most evident in adult appendages where no defects in patterning can be observed outside of here. Other regions may be refractive to ectopic EGFR activity because expression of tarsal genes requires Dll, which is expressed only in distal regions under Wg and Dpp control (Campbell, 2002).

In addition to activating genes, EGFR signalling is required to repress genes in distal regions, and again different genes appear to be differentially sensitive, with some, such as B and rn, possibly being both activated and repressed above different thresholds. B, rn and dac are repressed in the center of wild-type discs, with dac being repressed over a wider region than B and rn. Lowering EGFR activity in Egfrts discs to a level sufficient only for loss of al, results in expression of B and rn in the center, but not dac. Raising the temperature still further results in extension of the dac domain to fill the center. Clonal analysis shows that Egfr acts autonomously to repress dac. Ectopic EGFR activity can also repress B, dac and rn but again predominantly in ventral regions (B is repressed mainly at later stages). Previous studies have shown that repression of dac in distal regions requires high levels of Wg and Dpp signalling, so all three pathways appear to be required to achieve this (Campbell, 2002).

Al and C15 repress Bar in the leg disc

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).

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).

As with strong al mutants, Bar is de-repressed in the putative cll/C15 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, these findings may indicate that a cooperative action of al and cll is required for Bar repression in the future pretarsus (Kojima, 2005).

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 alex or clldl9-2 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).

Induction and autoregulation of the anti-proneural gene Bar during retinal neurogenesis

Neurogenesis in the Drosophila eye imaginal disc is controlled by interactions of positive and negative regulatory genes. The basic helix-loop-helix (bHLH) transcription factor Atonal (Ato) plays an essential proneural function in the morphogenetic furrow to induce the formation of R8 founder neurons. Bar homeodomain proteins are required for transcriptional repression of ato in the basal undifferentiated retinal precursor cells to prevent ectopic neurogenesis posterior to the furrow of the eye disc. Thus, precise regulation of Bar expression in the basal undifferentiated cells is crucial for neural patterning in the eye. Evidence is shown that Bar expression in the basal undifferentiated cells is regulated by at least three different pathways, depending on the developmental time and the position in the eye disc. (1) At the time of furrow initiation, Bar expression is induced independent of Ato by Hedgehog (Hh) signaling from the posterior margin of the disc. (2) During furrow progression, Bar expression is also induced by Ato-dependent EGFR (epidermal growth factor receptor) signaling from the migrating furrow. (3) Once initiated, Bar expression can be maintained by positive autoregulation. Therefore, it is proposed that the domain of Bar expression for Ato repression is established and maintained by a combination of non autonomous Hh/EGFR signaling pathways and autoregulation of Bar (Lim, 2004).

To identify activators of Bar expression in the basal undifferentiated cells, focus was placed on two different transcription factors, Lozenge (Lz) and Glass (Gl), as candidates. Both proteins are known to be required for normal Bar expression in R1/6 photoreceptor cells, but it has not been demonstrated whether they are also required for Bar expression in the basal undifferentiated cells. Lz is expressed in R1, 6 and 7 photoreceptor cells and is required for normal level of Bar expression in R1/6 cells. In the basal undifferentiated cells, Lz is co-expressed with Bar in a majority of Bar-expressing cells, except in a group of cells just posterior to the furrow. To test whether Lz is also required for Bar expression in the basal undifferentiated cells, Bar expression was examined in homozygous lzr15 mutants and loss-of-function (LOF) clones of lzr15, a null allele of lz. It was found that the expression level of Bar is strongly decreased but not completely eliminated in R1/6 photoreceptor cells within lzr15 mutant clones. However, Bar expression in the basal undifferentiated cells is little changed compared with its expression level in adjacent wild-type cells. These results suggest that Lz is necessary to activate Bar expression in R1/6 cells, but not in the basal undifferentiated cells behind the furrow (Lim, 2004).

Next, the requirement for Gl was examined in undifferentiated cells. Gl is a zinc-finger protein expressed in all cells posterior to the furrow. Gl is not necessary for Bar expression in the basal undifferentiated cells although it is essential for Bar expression in R1/6 photoreceptor cells. Taken together, these results suggest that Bar expression requires other activators in the basal undifferentiated cells (Lim, 2004).

Based on the evidence presented in this study, a model is proposed for the regulation of Bar expression in the basal undifferentiated cells. Prior to photoreceptor differentiation at the time of furrow initiation, Bar expression in the basal undifferentiated cells near the posterior region of the disc is induced by secreted signaling factors from the posterior margin. Hh signaling from the posterior margin is required for the initial induction of Bar expression. During furrow progression, a narrow region of Bar expression immediately posterior to the furrow depends on Ato from the furrow. EGFR signaling may partially mediate Ato effects on Bar expression. Hh produced by photoreceptor cells generated behind the furrow may also be required in part for Bar expression near the furrow during furrow progression. Finally, Bar is autoregulated to maintain its expression. The properly expressed Bar proteins repress ato transcription in the basal undifferentiated cells, thereby preventing ectopic photoreceptor differentiation posterior to the furrow (Lim, 2004).

Hh expression is dynamic, depending on the time and the position in the developing eye disc. In the early third instar eye disc, Hh is expressed in the posterior margin and is required for the furrow initiation. During furrow progression, Hh is also produced in the differentiating photoreceptor cells generated posterior to the furrow and secreted anteriorly to promote furrow progression. During this process, Bar is specifically expressed in the basal undifferentiated cells posterior to the furrow and inhibits ectopic retinal neurogenesis by repressing proneural gene ato expression (Lim, 2004).

Hh signaling is required for Bar expression in the basal undifferentiated cells during initial eye development because Bar expression is strongly reduced or absent within smo LOF clones generated near the furrow or close to the posterior margin of the disc. Prior to the photoreceptor differentiation, Hh expressed in the posterior margin of the disc is responsible for Bar expression at specific distances from the posterior region of the eye disc proper. A graded expression of Bar near the posterior region in ato1 mutant eye disc might be the effects of Hh secreted by the posterior margin (Lim, 2004).

During furrow progression, Hh signaling is required for Ato expression in the furrow, and Ato-mediated EGFR signaling is required for Bar activation. Therefore, it is possible that the loss of Bar expression near the furrow in smo LOF clones might be caused by indirect effects of reduced Ato expression rather than by direct effects of Hh signaling on Bar expression. Hh may partially contribute to Bar expression by activating normal levels of Ato expression in the furrow. Thus, the Hh-Ato-EGFR cascade activates Bar expression just posterior to the furrow. Alternatively, since Hh signaling may also affect furrow progression, it is possible that the loss of Bar expression near the furrow in smo LOF clones might be caused by indirect effects of slow furrow migration rather than by direct effects of Hh signaling on Bar expression (Lim, 2004).

The results suggest that Ato is required nonautonomously for the induction of Bar expression just posterior to the migrating furrow. Although Ato acts as an activator for Bar expression, expression of these proteins always show a juxtaposed complementary pattern along the furrow. This suggests that some mediator(s) is required for transducing Ato effects on Bar expression. EGFR activated by Ato in the furrow is required for Bar expression, suggesting that nonautonomous effects of Ato on Bar expression may be partially mediated by EGFR, as revealed by analysis of Egfr negative clones and temperature senstive mutants. Furthermore, EGFR is required for Bar expression not only in the eye disc but also in the antenna and leg discs in Drosophila, suggesting that EGFR signaling may be a common activator for Bar expression in different tissues or even in higher organisms (Lim, 2004).

Notch (N) signaling is also known to contribute to neuronal differentiation together with Hh and Dpp pathways. Thus, N signaling may play a role for Bar expression in the basal undifferentiated cells during furrow progression. Bar expression is strongly downregulated when N function is removed with a temperature-sensitive mutation (Nts) or using the Enhancer-of-split [E(spl)] mutant clones in the eye disc. This suggests that N signaling may be required for Bar expression in the basal undifferentiated cells. However, it is equally possible that loss of Bar expression in the E(spl) LOF clones or in the Nts eye disc may be an indirect secondary effect of the lack of the basal undifferentiated cells because nearly all cells in the basal region of the eye disc differentiate into photoreceptor cells without N function. Further analysis of Bar regulation at the molecular level will be helpful to identify direct regulators of Bar expression in the undifferentiated cells of the eye disc (Lim, 2004).

odd-skipped genes and lines organize the notum anterior-posterior axis using autonomous and non-autonomous mechanisms>

The growth and patterning of Drosophila wing and notum primordia depend on their subdivision into progressively smaller domains by secreted signals that emanate from localized sources termed organizers. While the mechanisms that organize the wing primordium have been studied extensively, those that organize the notum are incompletely understood. The genes odd-skipped (odd), drumstick (drm), sob, and bowl comprise the odd-skipped family of C2H2 zinc finger genes, which has been implicated in notum growth and patterning. This study shows that drm, Bowl, and eyegone (eyg), a gene required for notum patterning, accumulate in nested domains in the anterior notum. Ectopic drm organized the nested expression of these anterior notum genes and downregulated the expression of posterior notum genes. The cell-autonomous induction of Bowl and Eyg required bowl, while the non-autonomous effects were independent of bowl. The homeodomain protein Bar is expressed along the anterior border of the notum adjacent to cells expressing the Notch (N) ligand Delta (Dl). bowl was required to promote Bar and repress Dl expression to pattern the anterior notum in a cell-autonomous manner, while lines acted antagonistically to bowl posterior to the Bowl domain. These data suggest that the odd-skipped genes act at the anterior notum border to organize the notum anterior–posterior (AP) axis using both autonomous and non-autonomous mechanisms (Del Signore, 2012).

In many developmental processes, signals that emanate from field borders play a crucial instructive role in patterning morphogenetic fields. The early Drosophila embryo is patterned by opposing gradients of Bicoid and Nanos that are generated from localized translation of corresponding mRNAs at the anterior and posterior poles of the embryo. In the embryonic epidermis, the pattern of cell differentiation across each segment is regulated by the secreted Wg and Hh signals that emanate from localized sources at the anterior and posterior borders of each segment. Similarly, the dorsoventral axis of the vertebrate spinal cord is organized by Shh ventrally, and BMP and Wnt signals that emanate from localized dorsal sources. By contrast, current models of notum AP patterning focus mainly on the organizing influence of Dpp, which is secreted from the posterior border of the notum. Previous work has found that odd-skipped genes are expressed along the anterior border of the notum, and that broadly inhibiting their function in early wing discs caused a severe reduction or complete loss of the notum. As this reduction occurred despite the maintenance of dpp expression (Nusinow, 2008), whether the odd-skipped genes might define a second organizing center within the developing notum was investigated. The current findings indeed suggest that signals that emanate from the anterior border of the notum act reciprocally to Dpp to promote expression of anterior notum genes and repress expression of posterior genes. Through loss- and gain-of-function clonal analyses, it was demonstrated that the odd-skipped genes pattern the notum AP axis both locally through regulation of Eyg, Bar, and Dl, and broadly through the regulation of Eyg and Tup. Finally, it was shown that lines acts antagonistically to bowl in this process (see Model of the role odd-skipped genes in notum AP patterning) (Del Signore, 2012).

drm overexpression was sufficient to promote Eyg accumulation non-autonomously within the notum. This activity suggests that drm controls expression of an unidentified signal that spreads from the drm domain to induce Eyg accumulation non-autonomously. Alternatively, drm could initiate the propagation of a cascade of local inductive interactions to induce Eyg at a distance. Recent studies have shown that recruitment of cells to the wing field is accomplished by the propagation of a feed forward signal from the DV compartment boundary. In this process signaling at the border between Vestigial (Vg) and non-Vg expressing cells is used to recruit non-Vg expressing cells to the expanding wing field, a process dependent on signaling through the Fat-Dachsous pathway. Though a functional relationship between odd-skipped genes and Ft-Ds signaling has yet to be characterized, it is interesting to note that Ds accumulates in a complex graded AP pattern across the notum, consistent with such a role (Del Signore, 2012).

In addition to the broad induction of Eyg accumulation, it was surprising to find that drm overexpression also induced Bowl in cells just adjacent to clones. Though the effect was subtle, it is noted that this pattern of activation recapitulated the endogenous nested pattern of drm and Bowl expression in the presumptive prescutum. It is unclear whether the nested expression of odd-skipped genes plays a functional role in notum AP patterning. Despite this, the concordance of endogenous and ectopic expression patterns supports the hypothesis that ectopic drm induces a physiologically relevant program of anterior gene expression in the notum. One possible clue as to the relevance of this nested pattern may come from the observation that only drm was able to promote Bowl non-autonomously. In contrast, lines−/−, odd+, and sob+ clones each induced only cell-autonomous accumulation of Bowl. Notably, these clones rounded up and segregated from the epithelium, while drm expressing clones remained integrated with the surrounding epithelium. One interpretation of these data is that abrupt discontinuities in the level of Odd-skipped proteins may alter epithelial morphology. This interpretation is further supported by the observation that bowl mutant clones within the Bowl domain adopt a compact, round morphology relative to clones outside the Bowl domain. It is hypothesized that drm promotes lower levels of Bowl in nearby cells to dampen otherwise sharp discontinuities in Bowl activity to regulate local buckling of the epithelium (Del Signore, 2012).

Alternatively, differences in the total levels or ratios of Odd family proteins along the anterior border of the notum could elicit different transcriptional outcomes. Since Odd and Bowl have been shown to interact with the transcriptional co-repressor Groucho, variation in the levels of the Odd-skipped proteins could titrate Groucho and affect Groucho-dependent transcriptional outputs. Alternatively, given their distinct structure outside the zinc finger domain, the Odd-skipped proteins could interact with distinct sets of target genes to pattern the anterior border of the notum. Though additional experiments will be required to ascertain whether such mechanisms are active in the prescutum, this study provides evidence that bowl is strictly required for the early autonomous induction of Eyg, the later expression of Bar genes, and the repression of Dl. These results provide evidence that odd-skipped genes act both independently and redundantly to organize the notum AP axis (Del Signore, 2012).

bowl is essential for patterning the prescutum, but not for broadly patterning the notum AP axis. Previous studies have revealed a variety of essential and redundant functions for odd-skipped family genes in patterning embryonic and larval tissues. In the embryo, drm and bowl antagonize lines function to pattern the dorsal embryonic epidermis, foregut, and hindgut, while odd functions as a pair rule gene to promote embryonic segmentation. In the leg imaginal disc, bowl is essential for patterning the tarsal proximodistal axis at early stages, but acts redundantly with other odd-skipped genes to control leg segmentation later in developmen. In the eye, bowl is essential for the initiation of retinogenesis from the eye margin, while odd and drm have been proposed to activate Bowl redundantly (Del Signore, 2012).

Loss-of-function analysis revealed that neither drm nor odd is necessary to stabilize Bowl. At present the possibility cannot be excluded that sob is necessary to promote Bowl accumulation because a null sob mutant is not yet available. Biochemical and genetic analysis demonstrates that not only Drm, but also Odd and Sob can each outcompete the interaction of Lines with Bowl and stabilize the Bowl proteins in S2 cells and in vivo. These results suggest that different combinations of Odd-skipped proteins could be used to activate bowl depending on context (Del Signore, 2012).

Previous work suggested reciprocal roles for lines and odd-skipped genes in subdividing the early wing disc into disc proper and peripodial epithelium. The loss-of-function analysis described in this study suggests that the odd-skipped genes act redundantly to control the early specification of the PE and the subsequent expansion of the notum, while revealing an essential role for bowl in specification of the anterior prescutum. Redundancy can increase the robustness of essential developmental processes and provide a buffer against fluctuations in activity of single genes. The redundant role of the odd-skipped genes in PE specification and notum expansion could therefore serve to ensure the optimal growth of the wing disc at early stages and that of the notum at later stages and protect these critical processes from perturbations (Del Signore, 2012).

It is concluded that the growth and patterning of the wing field are coordinated with the elaboration of the wing PD axis. The developing notum lacks an obvious PD axis, and instead is subdivided into a series of AP and mediolateral domains. The establishment of organizers that act antagonistically from opposing field borders is a robust strategy to subdivide the notum AP axis. This work demonstrates that the odd-skipped genes act autonomously at the anterior border of the notum to specify the prescutum, and non-autonomously at short and long range to control the expression of transcription factors that prefigure the differentiation of the notum AP axis. Though further experiments will be required to characterize the mechanism by which this putative organizer acts, these studies provide evidence that the anterior border of the notum exhibits the functional attributes of an organizer (Del Signore, 2012).

Targets of Activity

BarH1 and BarH2 play essential roles in the formation and specification of the distal leg segments of Drosophila. In early third instar, juxtaposition of Bar-positive and Bar-negative tissues causes central folding that may separate future tarsal segments 2 from 3, while juxtaposition of tissues differentially expressing Bar homeobox genes at later stages gives rise to segmental boundaries of distal tarsi including the tarsus/pretarsus boundary. Tarsus/pretarsus boundary formation requires at least two different Bar functions: early antagonistic interactions with a pretarsus-specific homeobox gene, aristaless, and the subsequent induction of Fas II expression in pretarsus cells abutting tarsal segment 5. Bar homeobox genes are also required for specification of distal tarsi. Bar expression requires Distal-less but not dachshund, while early circular dachshund expression is delimited interiorly by BarH1 and BarH2 (Kojima, 2000).

During development of Drosophila legs, the disc epithelium folds concentrically; genes expressed in concentric circles in the leg disc just before the onset of folding may thus be essential for leg morphogenesis. BarH1 and BarH2, may belong to such a class of genes. Staining for BarH1, BarH2 and Bar-lacZ indicates that BarH1 and BarH2 are coexpressed circularly in all three types of third-instar leg discs. Since BarH1 and BarH2 are functionally redundant to each other, they are referred to as Bar collectively. Indications of circular Bar expression (Bar ring) are first evident at 76 hours AEL (after egg laying) at 25°C and become more evident at 80 hours AEL. The formation of the central fold, from which distalmost leg segments are generated, begins at 84 hours AEL as a circular indentation along the periphery of the Bar ring, starting dorsally and finishing ventrally by 88 hours AEL. As the indentation becomes deeper in mid third instar, graded Bar expression gradually becomes apparent along the proximodistal axis. In the central fold of a 112 hours AEL leg disc, future tarsal segment 3, lacking Bar expression, becomes clearly identifiable. At 5 hours APF (after puparium formation), Bar expression is closely related to lines of demarcation of tarsal segments. Bar expression is strongest in tarsal segment 5; clearly evident in tarsal segment 4, and not detected in tarsal segment 3 and the pretarsus except for future claw regions. From late third instar stages onwards, Bar is also expressed in putative claw cells. Staining for BarH1 and en-lacZ shows the anterior edge of the posterior claw region coincides with that of the posterior compartment, suggesting that the center of the Bar ring is situated in the anterior compartment, between the paired claw regions. During late third instar, Bar-negative patches in future tarsal segment 5 become detectable. Staining for BarH1 shows that these correspond to sensory organ precursors and/or their derivatives (Kojima, 2000).

Bar is also required for the formation of the distal antennal segment. In Drosophila, antennae possess segmental structures homologous to those in legs and similarly differentiate through circular folding. Arista and basal cylinder, probably corresponding to pretarsus and distal tarsus, are derivatives of the central knob of antennal discs. After the onset of third instar, Bar expression occurs initially in a central region of the antennal disc and gradually become a ring similar to that observed in the leg disc. As with legs, central folding occurs just outside the Bar ring. In antennal discs lacking Bar activity, no central fold is formed; Bar minus antennae frequently loose arista and basal cylinder. Thus, Bar is concluded to be essential not only for leg but also for antennal development (Kojima, 2000).

aristaless (al) is a homeobox gene expressed at the center of leg and antennal discs from early third instar onwards. Initially, the Al expression domain and early Bar ring overlap slightly in leg discs. Al/Bar overlapping can be more clearly seen in early antennal discs. Up to 90 hours AEL, the central part of the leg disc is strictly divided into two regions, Bar-positive/Al-negative and Bar-negative/Al-positive circular domains. In antennal discs, such discrimination in Bar/Al expression may be incomplete. Regionally exclusive expression of Bar and Al may be due to mutually antagonistic interactions between Bar and Al. When Al expression is examined in mid to late third instar larval leg discs having Bar minus clones, Al expression is seen to invade the Bar minus presumptive tarsus region, while Al expression is considerably attenuated by Bar misexpression along the anteroposterior compartment border in mid third instar discs. Ectopic patches of Bar expression are frequently observed in the presumptive pretarsus of hypomorphic al leg discs in late third instar. Bar derepression due to reduction in Al activity is more clearly observed in antennal discs; on a hypomorphic al mutant background, Bar is expressed in the centralmost region even at late third instar larval stages. That no appreciable repression of Bar is detected when al is misexpressed may indicate the involvement of factors other than Al in Bar repression in presumptive pretarsus. In addition, the lack of Al invasion into Bar minus clones in leg discs at early third instar larval stages may indicate that Bar is dispensable for repressing al when their expression is initiated (Kojima, 2000).

At late third instar, cells in the distalmost region of leg discs are densely packed at the apical surface and distinguishable from surrounding loosely packed cells. Double-staining with rhodamine-phalloidin and anti-BarH1 antibody reveal that the former corresponds to Bar-negative pretarsus cells, and the latter, Bar-positive tarsus cells. Proximalmost pretarsus cells (border cells) are frequently rectangular in apical shape. Staining for Fasciclin II (Fas II) shows that border cells prominently express Fas II at late third instar. Fas II expression is interrupted by Bar minus clones. Fas II misexpression is induced along Bar-misexpressing presumptive pretarsus, while endogenous Fas II expression is repressed. Thus, Bar upregulates and downregulates Fas II expression in Bar-negative border cells and Bar-positive non-border cells, respectively. It is concluded that Bar is essential for the establishment of the boundary between tarsal segment 5 and pretarsus. Thus, Bar may establish the boundary between the pretarsus (Bar-negative) and tarsal segment 5 (Bar-positive) by regulating the expression of cell adhesion molecules such as Fas II (Kojima, 2000). Interestingly, BarX2, a mouse gene encoding a Bar-related homeodomain protein, has been reported to regulate the expression of Fas II-like cell adhesion molecules (Kojima, 2000 and references therein).

Circular Dac expression appears in second-instar leg discs before Bar ring appearance. This early Dac-ring is associated interiorly with Bar-positive Keilin's organ cells, which are situated along the interior circumference of or within the early Bar ring. Although they are separated from each other by a Bar-negative, Dac-negative region just before the onset of central fold formation Dac and Bar rings are immediate neighbors at earlier stages. Dac expression is derepressed in Bar minus clones observed in early third instar, while repressed by Bar misexpression, indicating that Bar is essential for distal restriction of Dac expression. Since early Bar expression normally occurs in dac minus clones, dac appears dispensable for proximal restriction of the early Bar ring. Interestingly, in dac minus mutants, Bar misexpression occurs in regions fated to become trochanter, indicating that Dac represses Bar in future trochanter (Kojima, 2000).

Protein Interactions

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).

Groucho interaction with Engrailed homology 1 (eh1) proteins

Drosophila Groucho, like its vertebrate Transducin-like Enhancer-of-split homologues, is a corepressor that silences gene expression in numerous developmental settings. Groucho itself does not bind DNA but is recruited to target promoters by associating with a large number of DNA-binding negative transcriptional regulators. These repressors tether Groucho via short conserved polypeptide sequences, of which two have been defined: (1) WRPW and related tetrapeptide motifs have been well characterized in several repressors; (2) a motif termed Engrailed homology 1 (eh1) has been found predominantly in homeodomain-containing transcription factors. A yeast two-hybrid screen is described that uncovered physical interactions between Groucho and transcription factors, containing eh1 motifs, with different types of DNA-binding domains. One of these, the zinc finger protein Odd-skipped, requires its eh1-like sequence for repressing specific target genes in segmentation (Goldstein, 2005).

The eh1 Gro recruitment domain was originally defined as a heptapeptide motif that is conserved in members of the En family of homeodomain proteins and their vertebrate homologues. More recently, eh1-dependent binding to Gro has also been demonstrated in vitro for various other Drosophila and mammalian proteins, nearly all of which contain homeodomains. Given that Bowl and Odd, two non-homeodomain ZnF transcription factors, contain this motif and interact with Gro, the possibility was explored that eh1 motifs are prevalent among additional non-homeodomain transcription factor families. Indeed, an unbiased yeast screen for Gro-interacting proteins selected two additional transcriptional regulators that contain eh1-like motifs, namely, Sloppy-paired (Slp; Forkhead related) and Dorsocross (Doc; T box). Alignment of the eh1-like sequences of Bowl, Odd, Slp, and Doc with those of En and Gsc revealed three conserved amino acids: phenylalanine-x-isoleucine-x-x-isoleucine (Phe-x-Ile-x-x-Ile, where x is any amino acid). Subsequent database searches for presumptive Drosophila transcription factors containing this minimal peptide sequence identified a wide range of potential negative regulators belonging to different superfamilies as classified by their distinct DNA-binding domain types. Remarkably, eh1-related motifs have been preserved in many human homologues of these fly proteins, indicating that the ability to bind Gro/TLE has been evolutionarily conserved in human transcriptional regulators and that this sequence may have been widely adopted throughout the proteome as a Gro recruitment domain (Goldstein, 2005).

Several representatives, corresponding to different transcription factor families, were tested for the ability to bind Gro in biochemical assays. Where possible, full-length expressed sequence tags encoding these proteins were obtained; otherwise, single exons containing the eh1-like sequence were PCR amplified from genomic DNA. Each polypeptide was assessed for the ability to pull down radiolabeled Gro in vitro. GST-tagged Slp and Doc (amino acids 254 to 391) readily retain Gro, as do Eyes absent (Eya) and the homeodomain proteins Ventral nervous system defective (Vnd, 1 to 465), Bagpipe (Bap, 1 to 129), BarH1, and Empty spiracles (Ems, 1 to 360), as well as the orphan nuclear hormone receptor DHR96. To confirm that these interactions rely on intact eh1-related sequences, the eh1 motif of one of these, BarH1, was mutated by substituting glutamic acid for Phe at position 1, finding that its binding to Gro is reduced by >60% (Goldstein, 2005).

BarH1 and BarH2 : Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

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