Interactive Fly, Drosophila

Recent advances have shed new light on how the Q50 homeoproteins act in Drosophila. Q50 homeoproteins all contain a glutamine residue at position 50 of the homeodomain. These transcription factors, encoded by the segmentation genes even-skipped, fushi-tarazu and engrailed, have remarkably similar and promiscuous DNA-binding specificities in vitro, yet they each specify distinct developmental fates in vivo. One current model suggests that because the Q50 homeoproteins have distinct biological functions, they must each regulate different target genes. According to this 'co-selective binding' model, significant binding of Q50 homeoproteins to functional DNA elements in vivo would be dependent upon cooperative interactions with other transcription factors (cofactors). If the Q50 homeoproteins each interact differently with cofactors, they could be selectively targeted to unique, limited subsets of their in vitro recognition sites and thus control different genes. Thus cofactors would selectively target different Q50 homeoproteins to bind to different DNA sites. However, a variety of experiments question this model. Molecular and genetic experiments suggest that the Q50 homeoproteins do not regulate very distinct sets of genes. Instead, they mostly control the expression of a large number of shared targets. The distinct morphogenic properties of the various Q50 homeoproteins may result principally from the different manners in which they either activate or repress these common targets. Further, in vivo binding studies indicate that at least two Q50 homeoproteins, Eve and Ftz, have very broad and similar DNA-binding specificities in embryos, a result that is inconsistent with the 'co-selective binding' model. Based on these and other data, it is suggested that Q50 homeoproteins bind many of their recognition sites without the aid of cofactors. In this 'widespread binding' model, cofactors act mainly by helping to distinguish the way in which homeoproteins regulate targets to which they are already bound (Biggin, 1997).

Whereas the segmental nature of the insect head is well established, relatively little is known about the genetic and molecular mechanisms governing this process. The phenotypic analysis is reported of mutations in collier (col), which encodes the Drosophila member of the COE family of HLH transcription factors and is activated at the blastoderm stage in a region overlapping a parasegment (PS0: posterior intercalary and anterior mandibular segments) and a mitotic domain, MD2. col mutant embryos specifically lack intercalary ectodermal structures. col activity is required for intercalary-segment expression both of the segment polarity genes hedgehog, engrailed, and wingless, and of the segment identity gene cap and collar. The parasegmental register of col activation is controlled by the combined activities of the head-gap genes buttonhead and empty spiracles and the pair-rule gene even skipped; it therefore integrates inputs from both the head and trunk segmentation systems, which were previously considered as being essentially independent (Crozatier, 1999b).

Even-skipped as a transcriptional repressor

Even-skipped is a transcriptional repressor of a number of genes including fushi-tarazu (Carroll, 1986), Ultrabithorax (Martinez-Arias, 1988) and wingless (Ingham, 1988).

The regulatory functions of eve have been studied in vivo. Transcripts encoded by eight other segmentation genes were monitored for changes in distribution and abundance following short pulses of ectopic eve expression. Two tiers of response times appear to distinguish between genes that are direct targets of eve, (fushi tarazu , odd-skipped , runt, paired, and wingless) and indirect targets (hairy, engrailed and eve itself ). Genes that appear to be directly regulated by eve are differentially repressed in a concentration-dependent fashion (Manoukian, 1992).

The early bell-shaped gradient of even-skipped expression is sufficient for generating stable parasegment borders. The anterior portion of each early stripe has morphogenic activity, repressing different target genes at different concentrations. These distinct repression thresholds serve to both limit and subdivide a narrow zone of paired expression. Within this zone, single cell rows express either engrailed, where runt and sloppy-paired are repressed, or wingless, where they are not. While the early eve gradient is sufficient to establish parasegmental borders without refined, more sharply defined late expression, late eve expression has a role in augmenting this boundary to provide for strong, continuous stripes of engrailed expression. The early eve gradient is sufficient, at its posterior edge, for subdividing the ftz domain into engrailed expressing and non-expressing cells (Fujioka, 1995).

Thus even-skipped, through its ability to repress, is an indirect activator of engrailed. Eve carries out this function through direct action on runt and odd-skipped. Ectopic runt expression leads to rapid repression of eve stripes and a somewhat delayed expansion of ftz stripes. Ectopic runt is a rapid and potent repressor of odd-numbered en stripes. runt is required to limit the domains of en expression in the odd-numbered parasegments, while the odd-skipped gene is required to limit the domains of en expression in the even-numbered parasegments. Activation of en at the anterior margins of both sets of parasegments requires the repression of runt and odd by the product of the eve gene (Manoukian, 1993).

There are several distinct phases of runt expression in the early embryo. Each phase depends on a different set of regulators. In a second phase, three pair-rule genes, hairy, even-skipped, and runt itself, affect runt's seven stripe pattern. The effects of runt are stripe specific; the effects of hairy are more uniform; and the patterns obtained in even-skipped mutant embryos show a combination of both stripe specific and uniform regulatory effects (Klingler, 1993).

A recent study has called into question the role of eve in the establishment of ftz pair-rule pattern, and has also questioned the classification of ftz as a secondary pair-rule gene. The so-called primary pair-rule genes are involved in the refinement rather than establishment of the fushi tarazu stripes. eve and ftz stripes alternate as odd and even expression domains in the developing embryo. The order of appearance of ftz stripes is not inversely correlated with the order of appearance of hairy stripes, as would be expected if ftz stripes were generated by h repression. Furthermore, the seven ftz stripes are correctly established in embryos carrying mutations in h, even-skipped or runt, with normal expression patterns decaying only after cellularization in the absence of primary pair-rule genes (Yu, 1995). For further discussion of this work, see the Biological Overview of the fushi tarazu site.

Sloppy paired (Slp) and Even-skipped are involved in cell fate determination and segmentation in the Drosophila mesoderm. bagpipe and serpent expressing mesodermal domains corresponding to the ectodermal even-skipped domains, alternate with the sloppy-paired expressing high-twist mesodermal domains. Ectodermal even-skipped is thought to act through engrailed and subsequently hedgehog to promote bagpipe expression in cardiac and dorsal muscle and serpent in the fat body (Azpiazu, 1996). Ectodermal Dpp is required for the maintenance of mesodermal tinman, which in turn activates bap expression in the eve domain. The visceral muscle and fat body primordia require even-skipped for their development and the mesoderm is thought to be unsegmented in even-skipped mutants. However, it has been found that even-skipped mutants retain the segmental modulation of the expression of twist. Both the domain of even-skipped function and the level of twist expression are regulated by sloppy-paired, and eve serves reciprocally to regulate the slp domain. sloppy-paired thus controls segmental allocation of mesodermal cells to different fates (Riechmann, 1997).

The Drosophila tinman homeobox gene has a major role in early mesoderm patterning: it determines the formation of visceral mesoderm, heart progenitors, specific somatic muscle precursors and glia-like mesodermal cells. These functions of tinman are reflected in its dynamic pattern of expression, which is characterized by initial widespread expression in the trunk mesoderm, then refinement to a broad dorsal mesodermal domain, and finally restricted expression in heart progenitors. Each of these phases of expression is driven by a discrete enhancer element, the first being active in the early mesoderm, the second in the dorsal mesoderm and the third in cardioblasts. Surprisingly, each of these elements are located at positions downstream of the transcription start site. The early-active enhancer element is a direct target of twist, a gene necessary for tinman activation that encodes a basic helix-loop-helix (bHLH) protein. This 180 bp enhancer includes three E-box sequences that bind Twist protein in vitro and are essential for enhancer activity in vivo. Binding of Even-skipped to these sequences appears to reduce twist-dependent activation in a periodic fashion, thus producing a striped tinman pattern in the early mesoderm. In addition, these sequences prevent activation of tinman by twist in a defined portion of the head mesoderm that gives rise to hemocytes. This repression requires the function of buttonhead, a head-patterning gene. The second expression domain, restricting tin mRNA expression in the dorsal mesoderm, is triggered by Dpp-mediated induction events (Yin, 1997).

Genetic and molecular analyses of patterning in the Drosophila embryo have shown that the process of head segmentation is fundamentally different from the process of trunk segmentation. The cephalic furrow (CF), one of the first morphological manifestations of the patterning process, forms at the juxtaposition of these two patterning systems. The initial step in CF formation is a change in shape and the apical positioning of a single row of cells. The anteroposterior position of these initiator cells may be defined by the overlapping expression of the head gap gene buttonhead (btd) and the primary pair-rule gene even-skipped. The position of the furrow coincides with the second row of Even-skipped-expressing cells in stripe 1. Re-examination of the btd and eve phenotypes in live embryos indicates that both genes are required for CF formation. Eve expression in initiator cells is found to be dependent on btd activity. The control of eve expression by btd in these cells is the first indication of a new level of integrated regulation that interfaces the head and trunk segmentation systems. In conjunction with previous data on the btd and eve embryonic phenotypes, these results suggest that interaction between these two genes both controls initiation of a specific morphogenetic movement, which separates the two morphogenetic fields, and contributes to patterning of the hinge region, which demarcates the procephalon from the segmented germ band. The position and size of Eve stripe 1 is determined by repressor elements acting downstream of Bicoid and overriding buttonhead dependent activation. These results strengthen the conclusion that btd might be a 'generic transcriptional activator' required for transcriptional activation of specific target genes, such as eve and collier , an ortholog of mammalian early B-cell factor/Olfactory-1 (Crozatier, 1996). collier's expression is restricted to a single stripe of cells corresponding to part of the intercalary and mandibular segment primordia, anterior to the CF domain, possibly parasegment 0. However, buttonhead's expression is not instructive for head development (Vincent, 1997).

Interestingly, Engrailed expression in PS1 requires btd but does not require eve, contrary to the setuation in parasegments 2 and 14. Despite having no effect on En expression in PS1, eve may still have a role in patterning this parasegement, since its expression and placement in relation to En is conserved between long germ-band and short germ-band insects. Recent data on the activation of collier (Crozatier, 1996) in PS0/mitotic domain 2, suggests a possible mechanism by which btd and eve cooperate to pattern PS1. Activation of col requires btd. Conversely, in the absence of eve, col expression is expanded posteriorly to overlap a region roughly corresponding to PS1, indicating that Eve acts as a repressor of col in this parasegment. Likewise, expression of string in mitotic domain 2, which also requires btd, is expanded posteriorly in eve mutant embryos. The current working model holds that the activation of eve by btd in anterior PS1 cells allows for differential gene expression between PS0 and PS1. In addition to the control of CF formation, the btd/eve interaction may thus assign separate gene expression and mitotic programs to cells on either side of the pro-cephalon/posterior head border (Vincent, 1997 and references).

The LIM homeobox gene islet is sufficient to direct motor axons via the ventral branch of the ISN (ISNb/d) into the ventral muscle field (Thor, 1997). The implications of the findings with respect to Eve are that together eve and islet might constitute a bimodal switch that directs motor axon growth either to ventral (islet) or dorsal (eve) regions of the muscle field. One prediction of such an interpretation would be that the expression patterns of these two genes in motorneurons are mutually exclusive. In the wild type, this is the case. Moreover, while the expression pattern of Eve remains unchanged when Islet is either absent or ectopically expressed, it is found that ectopic Eve expression throughout the CNS suppresses Islet expression in most motorneurons. In the wild type, Islet is expressed medially in the dorsal RP1, 3, and 4 neurons and one ventral VUM motorneuron, and laterally, in approximately four to five motorneurons. In stage 16 elav-GAL4; UAS-eve embryos, the Islet expression pattern is markedly reduced: medially, Islet expression is consistently lost from the VUM and from two of the three RP motorneurons; laterally, Islet expression is lost from a further four to six cells. However, the Islet expression pattern does not expand when Eve function is removed (Landgraf, 1999).

Our knowledge of the mechanism of gene activation and repression is still primitive. The simple view of transcription factors acting from a proximal promoter and directly interacting with the transcription apparatus is naive. There are at least three added levels of complexity: 1) enhancers act at a distance to influence the assembly of the transcription apparatus, 2) activation and silencing involves rearrangement of chromatin and 3) activation and silencing can involve Polycomb and trithorax group proteins. Eve contains domains which inhibit transcriptional activators present at the Ultrabithorax (Ubx) proximal promoter when bound up to 1.5 kb away from these activators. Three adjacent regions of EVE binding contribute to silencing. Repression in vitro correlates with binding of EVE protein to two low-affinity sites in the Ubx proximal promoter. Occupancy of these low-affinity sites is dependent upon cooperative binding of other EVE molecules to a separate high-affinity site. Some of these sites are separated by over 150 bp of DNA. The intervening DNA is bent to form a looped structure similar to those caused by prokaryotic repressors. One of the low-affinity sites overlaps an activator element bound by the Zeste transcription factor. Binding of EVE protein is shown to exclude binding by Zeste protein (TenHarmsel, 1993).

The early expression of the Drosophila segment polarity gene gooseberry is under the control of the pair-rule genes. A 514-bp enhancer, -5.3 to -4.8 kb interval (called fragment IV), has been identified that reproduces the early gsb expression pattern in transgenic flies. The transcription factor Paired (Prd) is the main activator of this enhancer in all parasegments of the embryo. It binds to paired-and homeodomain-binding sites, which are segregated on the enhancer. Using site-directed mutagenesis, sites critical for Prd activity have been identified. Negative regulation of this enhancer is mediated by the Even-skipped protein (Eve) in the odd-numbered parasegments and by the combination of Fushi-tarazu (Ftz) and Odd-skipped proteins in the even-numbered parasegments. The organization of the Prd-binding sites, as well as the necessity for intact DNA binding sites for both the paired- and homeodomain-binding sites, suggests a molecular model whereby the two DNA-binding domains of the Prd protein cooperate in transcriptional activation of gsb. This positive activity appears to be in competition with Eve and Ftz on Prd homeodomain-binding sites (Bouchard, 2000).

In principle, the consensus sequences identified by DNase I protection with Eve could represent binding sites of other homeodomain proteins regulating gsb expression. However, the fact that Eve and Ftz are the only known homeodomain proteins expressed in a double segment periodicity at the blastoderm stage strongly argues against this. Moreover, a direct action of Eve on gsb regulation is supported by short-pulse heat-shock experiments that favor direct regulatory effects. Using this assay, the ectopic overexpression of prd could override the repression by Eve in the odd-numbered parasegments, while a heat-shock eve could abrogate Prd activation of gsb in all parasegments. Altogether these results suggest that gsb responds to the Eve morphogenetic gradient in the odd-numbered parasegments (Bouchard, 2000).

Selective spatial regulation of gene expression lies at the core of pattern formation in the embryo. In Drosophila, localized transcriptional regulation accounts for much of the embryonic pattern. Properties of a newly identified gene, partner of paired (ppa), suggest that localized receptors for protein degradation are integrated into regulatory networks of transcription factors to ensure robust spatial regulation of gene expression. The Ppa protein interacts with the Pax transcription factor Paired (Prd) and contains an F-box, a motif found in receptors for ubiquitin-mediated protein degradation. In normal development, Prd functions only in cells in which ppa mRNA expression has been repressed by another segmentation protein, Even-skipped (Eve). When ppa is expressed ectopically in these cells, Prd protein, but not mRNA, levels diminish. When ppa function is removed from cells that express PRD mRNA, Prd protein levels increase. It is concluded that Ppa coordinates Prd degradation and is important for the correct localization of expressed Prd. In the presence of Ppa, Prd protein is targeted for degradation at sites where its mis-expression would disrupt development. In the absence of Ppa, Prd is longer-lived and regulates downstream target genes (Raj, 2000).

To assess the possible functional relationships with Prd and Eve, embryo fillets were double-stained for ppa mRNA and Prd or Eve protein. During the early stages of cycle 14, when ppa expression is being restricted to stripes, there are significant levels of ppa expression overlapping the stripes of Prd protein. As cycle 14 proceeds, the posterior regions of the forming ppa stripes transiently overlap the anterior regions of the primary Prd stripes but, by early gastrulation, the Prd and ppa stripes are almost distinct. This transient but limited overlap in the expression of ppa and Prd is consistent with the model that Ppa negatively regulates Prd protein function (Raj, 2000).

Comparison of ppa mRNA with Eve protein shows almost reciprocal expression of the two genes (ppa interbands coincide with Eve stripes), raising the possibility that Eve might repress ppa expression, thereby giving rise to the ppa interbands. This interpretation is supported by examination of eve mutant embryos, which have uniform instead of striped ppa expression during cycle 14 and germ-band elongation. Moreover, adding back a transgene (P[eve.2,3,7] that expresses eve stripes 2, 3 and 7 in an otherwise eve mutant background, results in ppa interbands corresponding to these three Eve stripes (Raj, 2000).

Because the Drosophila embryo develops very quickly, the segmentation gene products are expected to be short lived. This is indeed the case for those products examined and is also likely to be true for the Prd protein, perhaps even in the absence of ppa function. Indeed, it is difficult to assess whether Prd protein levels are reduced in hs-ppa embryos because of the fairly broad range of immuno-staining signals observed between different embryos, a problem inherent to the detection technique. To overcome this problem, ppa was ectopically expressed over only part of the embryo, so that the effects of ectopic ppa could be assessed relative to regions of the same embryo where ppa expression is normal. eve mutant embryos with a transgene P[eve.2,3,7] that expresses only eve stripes 2, 3 and 7 have well-formed ppa interbands at these locations. Thus, it is possible to compare Prd protein expression at stripe 2, which overlaps the ppa interband at eve stripe 2, with Prd expression at stripe 4, where ppa is expressed ectopically. Examination of prd mRNA signals in eve-;P[eve.2,3,7] embryos reveals strong expression of stripe 4 when compared with stripe 2, consistent with previous observations that eve represses prd transcription, thereby contributing to refinement of Prd stripes. In contrast, Prd protein signal at stripe 4 is significantly lower than at stripe 2, correlating with the ectopic ppa expression at stripe 4, and suggesting that Ppa regulates Prd protein levels. Even though there is 50% more mRNA signal at stripe 4 than stripe 2 after ppa upregulation, there is 25% less protein. Note that it is formally possible that the reduced Prd protein levels result from changes in genes other than ppa that are regulated by eve. Nevertheless, these analyses of hs-ppa and ppa mutant embryos suggest that regulation by ppa is responsible (Raj, 2000).

The equivalent analysis of wild-type embryos shows similar mRNA signals at stripes 4 and 2, whereas the Prd protein signal at stripe 4 is somewhat reduced compared with stripe 2, correlating with the residual ppa expression normally still present at gastrulation at the ppa interband corresponding to Prd stripe 4. This decrease in Prd protein is less pronounced than in eve-;P[eve.2,3,7] embryos, presumably because the difference in ppa expression at stripes 2 and 4 is smaller. To confirm the interpretation that Ppa regulates Prd protein levels, the stripe 4 to stripe 2 ratios for mRNA and protein would be expected to be similar in heat-treated hs-ppa embryos, in which ppa is expressed ectopically at both stripes 2 and 4. This was indeed observed, supporting the conclusion that ppa regulates Prd protein levels. Because Ppa has an F-box, this regulation is most likely through targeted protein degradation rather than translational repression. Consistent with these data, Western analysis of embryo extracts indicates that Prd protein levels are reduced by approximately 50% in hs-ppa embryos, as compared with the wild type (Raj, 2000).

With the recent cloning of F-box proteins and the realization that they provide specific links between substrates and the protein degradation machinery, it has been predicted that F-box proteins would play important roles in development. Because F-box-regulated degradation normally depends on phosphorylation of substrates, localized action of signal transduction systems can, in principle, lead to localized protein degradation. This is likely to be the case for the signal-dependent localized degradation of Drosophila Cactus, a homolog of vertebrate IkappaB, whose degradation is a prerequisite for nuclear import of the Dorsal transcription factor (a homolog of NFkappaB) in the ventral portion of the embryo. Degradation of Cactus is mediated by the F-box protein Slimb (a homolog of ß-TrCP), which is also implicated in Wingless and Hedgehog pathways. In contrast to these signal transduction systems, the localized protein degradation in the Ppa system depends on spatially regulated expression of the Ppa F-box protein itself. By having its transcription regulated by a segmentation protein (Eve), and by targeting other segmentation proteins for degradation (Prd), the Ppa F-box protein forms an integrated link in the segmentation protein regulatory cascade that serves to strengthen the spatial refinement required for pattern formation. It is predicted that integration into transcriptional cascades may be a property of an important subfamily of F-box proteins, which, as suggested above, may also have recruited transcriptional repression functions to optimize their negative regulation of targeted transcription factors (Raj, 2000).

Ppa is the first example of a localized F-box receptor for protein degradation that works alongside transcription factors to ensure localized gene expression in the Drosophila segmentation cascade. These analyses suggest that Ppa targets the Prd transcription factor for degradation in cell rows in which Prd function is inappropriate, and that it is crucial that ppa expression is removed, through repression by eve, from cell rows in which Prd function is required for normal embryonic development (Raj, 2000).

Characterization of Even-skipped repressive domains

During segmentation of the Drosophila embryo, even skipped is required to activate engrailed stripes and to organize odd-numbered parasegments. A 16 kb transgene containing the even skipped coding region can rescue normal engrailed expression, as well as all other aspects of segmentation, in even skipped null mutants. To better understand its mechanism of action, the Even-skipped protein was functionally dissected in the context of this transgene. Even-skipped utilizes two repressor domains to carry out its function. Each of these domains can function autonomously in embryos when fused with the Gal4 DNA-binding domain. A chimeric protein consisting only of the Engrailed repressor domain and the Even-skipped homeodomain, but not the homeodomain alone, is able to restore function, indicating that the repression of target genes is sufficient for even skipped function at the blastoderm stage, while the homeodomain is sufficient to recognize those target genes. When Drosophila Even skipped is replaced by its homologs from other species, including a mouse homolog, these homologs are able to provide substantial function, indicating that these proteins can recognize similar target sites and also provide repressor activity. Using this rescue system, it has been shown that broad, early even skipped stripes are sufficient for activation of both odd- and even-numbered engrailed stripes. Furthermore, these 'unrefined' stripes organize odd-numbered parasegments in a dose-dependent manner, while the refined, late stripes, which coincide cell-for-cell with parasegment boundaries, are required to ensure the stability of the boundaries (Fujioka, 2002).

Eve has two distinct repressor domains, one dependent on the corepressor Gro and the other Gro-independent. For this study Eve was divided into 4 domains: an N-terminal region; the HD, which includes a conserved flanking region; a repressor domain identified in transient assays in cultured Drosophila cells, and the remaining C terminus, which includes a Gro interaction domain. Paradoxically, a primary function of Eve in segmentation is to allow activation of en stripes in both even- and odd-numbered parasegments. The ability to functionally replace the endogenous eve gene with a transgenic copy was used to evaluate the relative contribution of these and other domains to the function of Eve in this process. Neither repressor domain is sufficient to properly organize the odd-numbered parasegments, although all (or most) en stripes can be restored by either one alone. However, the relative width of the odd-numbered parasegments is reduced, so that they are unstable, and are deleted at later developmental stages. This gives rise to the pair-rule phenotype that earned even skipped its name (the even-numbered abdominal denticle bands are in odd-numbered parasegments) (Fujioka, 2002).

The Gro-independent repressor domain has been defined in repression assays in cultured cells, based on transient transfections with artificial reporter genes. This region has been shown to interact physically with the TATA-box binding protein TBP, and to repress transcription in vitro. A similar region interacts physically with the human Atrophin homolog, which acts as a corepressor. A phosphorylation-dependent function of down-regulating the repressor activity of this region in vitro has been ascribed to the N-terminal domain of Eve. Consistent with this result, deletion of the N terminus causes an increase in Eve activity in vivo. One possible explanation for this effect is that the deleted protein is more stable than wild-type Eve, since PEST sequences are deleted, although antibody staining against the Flag-tagged proteins indicates only a minor, if any, increase in protein levels. When both repressor domains are removed, neither the Eve HD alone (with conserved flanking regions) nor the HD with the N terminus are able to provide any significant functional activity in segmentation (Fujioka, 2002).

The histone deacetylase Rpd3 affects eve function. In Rpd3 mutant embryos, although the expression pattern of eve is not changed, even-numbered en stripes are very weak or missing owing to a lack of repression of odd. However, odd-numbered en stripes are expressed with only minor alterations. This is in contrast to the relative effects on odd- versus even-numbered en stripes when the eve dose is reduced, or in hypomorphic mutants, suggesting that Rpd3 may affect the repression of odd more than that of slp and prd. The Rpd3 effect similarly contrasts with the effects of removing either of the Eve repressor domains, suggesting that Rpd3 specificity cannot be explained by a selective effect on one of the Eve corepressors. This is true despite the fact that Rpd3 has been shown to mediate Gro repressor activity. Therefore, the apparent specificity of action of Rpd3 during segmentation is not easily explained solely through an effect on Eve activity. Conceivably, Rpd3 might affect the target specificity of the Eve HD, perhaps through selective effects on chromatin structure at different target sites. Another possibility is that it might affect the activities of other pair-rule gene products in addition to Eve. For example, it has been shown that Slp interacts with Gro in vitro. If Rpd3 reduces slp activity, then the effect of Rpd3 on Eve repressor function might be partially antagonized at the odd-numbered parasegment boundaries by its effect on slp (Fujioka, 2002).

Both of the repressor domains of Eve have autonomous activity, since they can repress an artificial target gene in vivo when fused with the Gal4 DNA-binding domain. Transgenes expressing such fusion proteins with either domain alone are capable of repressing transgenes containing a UAS target site for binding by Gal4. However, maximal repression activity requires both repressor domains, consistent with the fact that Eve requires both domains for full function in segmentation (Fujioka, 2002).

Repression of the Gal4 binding site-containing transgene by the Eve-Gal4 fusion proteins shows a consistently stronger effect on stripe 1 than on stripe 5. Although the stripe 5 element in the reporter is further away from the Gal4 binding sites and is also less well repressed than the stripe 1 element, the apparent specificity of repression is probably not due to a distance effect. This is inferred from the fact that a similar stripe preference is seen when Gal4 binding sites are inserted upstream of the same stripe elements, this time closer to the stripe 5 region. The stronger repression activity on stripe 1 expression may be due to the earlier activity of the stripe 1 enhancer, relative to that of stripe 5. Since these elements are also used to drive the expression of the repressors, the earlier activity of the stripe 1 element causes earlier accumulation of the repressors in the stripe 1 region, which may result in more effective repression. Alternatively, the Eve repressor domains may have some functional specificity that allows them to work more effectively on the stripe 1 enhancer (Fujioka, 2002).

A chimeric protein consisting of the Eve HD (including the conserved flanking regions) and a heterologous repressor domain from the En protein is able to fully rescue segmentation, while the HD region alone shows no activity. This suggests that repression of its direct target genes is sufficient for the function of Eve as a segmentation gene, and that the HD region is sufficient to recognize those target genes (Fujioka, 2002).

Eve homologs have been studied in several species. Is there a functional conservation in the recognition of specific target sites as well as in transcriptional activity, and if so, do these aspects of conservation extend to mammals? The strongest conservation is found in the HD and the immediate flanking sequences, with recognizable homology also in the C-terminal region. The ability of several homologs to function in early Drosophila development was analyzed. Expression of each protein was driven by the complete Drosophila regulatory region, and each protein's ability to rescue the phenotype of eve null mutants was assessed. This provides a sensitive assay for function, since proteins with reduced activities give a range of distinctive hypomorphic phenotypes. The Caenorhabditis ortholog, Vab-7, was expressed at very low levels at the blastoderm stage, presumably due to protein instability, so that its activity could not be determined. Homologs from the flour beetle (Tc-Eve), grasshopper (Sa-Eve), and mouse (Evx1) do, however, provide varying degrees of rescuing activity, paralleling their evolutionary relatedness to Drosophila. Tc-Eve rescues all of the en stripes, and parasegments are well organized, reflecting the relatively close evolutionary distance. Tc-Eve is expressed in stripes in the beetle, and has been shown to have a role in segmentation in that organism. In contrast, Sa-Eve is not normally expressed in stripes, and, correspondingly, provides a less complete rescue than does Tc-Eve. Nonetheless, Sa-Eve is capable of rescuing all of the en stripes, with the parasegments being better organized than in eve hypomorphic mutants (Fujioka, 2002).

Evx1, the mouse homolog that is expressed in early development, is able to provide a very significant rescuing activity in Drosophila. This suggests that it not only recognizes endogenous Eve target sites, but that it also has transcriptional repressor function, since this function is required for any such rescue. Although Evx1 apparently acts as a repressor in Drosophila embryos, it may also exhibit other activities in other contexts (Fujioka, 2002).

Interestingly, the Gro interaction motif of Eve (LFKPY), located at the C terminus, is conserved in the flour beetle (Tribolium) and the grasshopper (Schistocerca), and appears to be recognized by the monoclonal antibody 2B8. Without this motif, Eve is no longer recognized by the antibody, which recognizes the Eve homologs in other arthropods, including crustaceans. This suggests that the motif is functionally conserved and that interaction with Gro homologs is thus likely to be a conserved feature of Eve function. The repressor activity of Evx1 may also reflect, at least in part, a conserved interaction, since the C terminus also shows sequence similarity to the Gro interaction domain of Eve (Fujioka, 2002).

In Drosophila, the concentration of Eve within each early stripe forms a gradient, and this graded distribution has morphogenic activity, crucial to the repression of different target genes in different cell rows. A graded pattern of mouse Evx1 expression is also seen in the primitive streak, and has been suggested to play a role in specifying cell fates. Thus, the action of Eve as a morphogen to subdivide embryonic domains may be a conserved aspect of function (Fujioka, 2002).

Eve homologs share common features in their expression patterns, which include the posterior region of embryos and specific cells during neurogenesis. In Drosophila, posterior expression is seen in the proctodeum, and later in the anal plate ring. However, the function of this posterior expression has not been established. In the nervous system, Sa-Eve is expressed in identified neurons that are homologous to those expressing Eve in Drosophila, and this conserved expression pattern is also seen in crustaceans and Caenorhabditis, Eve has been shown to be important for correct neuronal fate specification, particularly in terms of axonal path finding. The functional importance of mouse Evx1 in the developing central nervous system has recently been established genetically by showing that in Evx1 mutant embryos, a majority of V0 interneurons fail to extend commissural axons. It will be interesting to determine whether the mechanisms connecting Eve function to axonal guidance are analogous between vertebrates and invertebrates (Fujioka, 2002).

In the complete absence of eve function, en is not expressed in the trunk region, and there is little evidence of segmentation at the end of embryogenesis. In hypomorphic eve mutants, the odd-numbered en stripes are expressed at posteriorly shifted positions, so that the odd-numbered parasegments are too narrow, and are deleted at later embryonic stages. Note: a contribution to the narrowing of odd-numbered parasegments may also come from an anterior shift of even-numbered en stripes. The positions of the odd-numbered parasegment boundaries, which are the anterior edges of odd-numbered en stripes, are foreshadowed by the anterior borders of refined, late eve stripe expression, prompting the suggestion that the late stripes are the more important functional aspects of expression, with the early, broad stripes serving only to help activate the late stripes. However, a previous model of eve function has suggested that the early stripes, acting as morphogenic gradients, independently set the anterior margins of both late eve and odd-numbered en stripes, which coincide because of their similar regulation by repressors (including slp) and the activator prd. These models were tested by removing late eve stripe expression while retaining normal early stripes. In eve null embryos rescued by a transgene deleted for the late expression element, although there is variable partial refinement under the influence of runt, which represses each early stripe from the posterior, the well-refined, late stripes never appear. In these embryos, odd-numbered en stripes form normally. However, they are variably lost during germband extension, coincident with an expansion of slp expression. Nonetheless, without refined, late eve stripes, many embryos are able to survive to fertility. Thus, it appears that the initial expression pattern of en and the overall organization of parasegments are determined primarily by the broad, early stripes. The late, refined stripes are required to maintain the pattern of slp, and to prevent partial repression of en shortly after it is activated. The expansion of slp is probably sufficient to explain the loss of en, since ectopic slp expression causes repression of these en stripes. The hypothesis that early stripes position odd-numbered en stripes in a concentration-dependent manner is also supported by the phenotype of embryos rescued by a transgene missing the stripe 4+6 element: these embryos have severely reduced levels of early stripes 4 and 6, and activate odd-numbered en stripes in those regions at posteriorly shifted positions. A model of these functions of early and late eve expression is presented (Fujioka, 2002).

The prevalence of repression as a mechanism of early developmental regulation among pair-rule and gap genes is striking. In the case of Eve, this activity provides not only for the activation of en with the appropriate spacing between cell rows, but also for the maintenance of en expression in the face of opposing repressive activities. One of these opposing activities is that of slp, which apparently helps to set the anterior boundary of both late Eve and en expression. Thus, spatially localized repressors may have advantages over activators in making and maintaining cell fate decisions, where mutually exclusive patterns of transcription factor expression help to establish and reinforce those decisions. Such mutually exclusive patterns can be directly established and reinforced by repressors acting to repress each others expression in adjacent domains, while activators can do this only indirectly (Fujioka, 2002).

Even-skipped as a transcriptional activator

There are persistant reports of EVE behaving as a gene activator. These are included here in order to have as complete a record as possible. The expression evidence conflicts with the biochemical evidence, but until shown unequivocably to be false, the reports of gene activation by EVE have to be taken seriously. Pair-rule genes even-skipped, runt and hairy, activate paired expression in stripes. With the exception of stripe 1, which is activated by even-skipped, and stripe 8, which depends upon runt, the primary pair-rule proteins are required for subsequent modulation rather than activation of the paired stripes (Gutjahr, 1993).

The activation of Deformed is achieved through a complex pattern of input. The simplest activation code sufficient to establish Deformed expression consists of a moderate level of expression from the coordinate gene bicoid, in combination with expression from both the gap gene hunchback, and the pair-rule gene even-skipped. Other pair-rule genes in addition to even-skipped can apparently act in combination with bicoid and hunchback for Deformed activation (Jack, 1990).

Tenascin major, the extracellular protein related to tenascin and a segment polarity gene, is under the positive control of Fushi tarazu and Even-skipped (Baumgartner, 1994).

DPTP61F is a non-receptor protein tyrosine phosphatase that is expressed during Drosophila oogenesis and embryogenesis. DPTP61F transcripts are alternatively spliced to produce two isoforms of the protein which are targeted to different subcellular locations. The two transcripts differ in the C-termini. There is an alternate splice site in exon 7, which is spliced to exon 8 to generate the transcript encoding DPTP61Fn. DPTP61Fn accumulates in the nucleus, and DPTP61Fm associates with the membranes of the reticular network and the mitochondria. The spatial and temporal expression of the two alternative transcripts of dptp61F has been examined during Drosophila embryogenesis. The two isoforms are expressed in distinct patterns. The DPTP61Fn transcript is expressed in the mesoderm and neuroblast layer during germband extension and later in the gut epithelia. In comparison, the transcript encoding DPTP61Fm accumulates in 16 segmentally repeated stripes in the ectoderm during germband extension. These stripes are flanked by, and adjacent to, the domains of engrailed and wingless gene expression along the anterior/posterior axis. In stage 10 embryos, the domains of DPTP61Fm transcript accumulation are wedge shaped and roughly coincide with the area lateral to the denticle belts that will give rise to naked cuticle. The DPTP61Fm transcript is also expressed later in embryogenesis in the central nervous system. The segmental modulation of DPTP61Fm transcript accumulation along the A/P axis of the germband is regulated by the pair-rule genes, and the intrasegmental pattern of transcript accumulation is regulated by the segment polarity genes. In hairy mutants, the complement of DPTP62Fm stripes is reduced by half, to approximately eight wide stripes. It is presumed that odd numbered stripes have been deleted. Within embryos homozygous for a strong eve allele, odd stripes are absent except for stripe 1. In odd paired mutants every even stripe is decreased. In paired mutants odd numbered domains of expression are shifted anteriorly towards the even numbered domains. wingless, hedgehog, naked and patched are involved in refining the pattern of mRNA accumulation within each parasegment (Ursuliak, 1997).

The 18 wheeler stripes require pair rule gene function for their establishment and later become dependent upon segment-polarity gene function for their maintenance. The establishment of the first even-numbered stripes of 18w depends on the function of the pair-rule gene fushi tarazu; the appearance of odd-numbered stripes depends on the function of even skipped. In wingless and hedgehog mutants, 18w expression rapidly declines as the germband reaches full extension, with the exception of small regions including a subset of neuroblasts along the midline. In engrailed mutant embryos, although general loss of expression is consistently observed, the loss of expression is not as striking as in wg and hh mutants. In naked and patched mutant embryos, the 18w stripes expand to about twice their normal width with occasional broadening in naked mutants such that the space between stripes is obliterated (Chiang, 1995).

The homeobox transcription factor Even-skipped regulates acquisition of electrical properties in Drosophila neurons

While developmental processes such as axon pathfinding and synapse formation have been characterized in detail, comparatively less is known of the intrinsic developmental mechanisms that regulate transcription of ion channel genes in embryonic neurons. Early decisions, including motoneuron axon targeting, are orchestrated by a cohort of transcription factors that act together in a combinatorial manner. These transcription factors include Even-skipped (Eve), islet and Lim3. The perdurance of these factors in late embryonic neurons is, however, indicative that they might also regulate additional aspects of neuron development, including the acquisition of electrical properties. To test the hypothesis that a combinatorial code transcription factor is also able to influence the acquisition of electrical properties in embryonic neurons the molecular genetics of Drosophila was used to manipulate the expression of Eve in identified motoneurons. Increasing expression of this transcription factor, in two Eve-positive motoneurons (aCC and RP2), is indeed sufficient to affect the electrical properties of these neurons in early first instar larvae. Specifically, a decrease was observed in both the fast K⁺conductance (I_Kfast) and amplitude of quantal cholinergic synaptic input. Charybdotoxin was used to pharmacologically separate the individual components of I_Kfastto show that increased Eve specifically down regulates the Slowpoke (a BK Ca²⁺-gated potassium channel), but not Shal, component of this current. Identification of target genes for Eve, using DNA adenine methyltransferase identification, revealed strong binding sites in slowpoke and nAcRα-96Aa (a nicotinic acetylcholine receptor subunit). Verification using real-time PCR shows that pan-neuronal expression of eve is sufficient to repress transcripts for both slo and nAcRα-96Aa. Taken together, these findings demonstrate that Eve is sufficient to regulate both voltage- and ligand-gated currents in motoneurons, extending its known repertoire of action beyond its already characterized role in axon guidance. These data are also consistent with a common developmental program that utilizes a defined set of transcription factors to determine both morphological and functional neuronal properties (Pym, 2006).

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