Targets of Activity

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).

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 (IKfast) and amplitude of quantal cholinergic synaptic input. Charybdotoxin was used to pharmacologically separate the individual components of IKfast to 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).

Molecular dissection of cis-regulatory modules at the Drosophila bithorax complex reveals critical transcription factor signature motifs. Dev. Biol

At the Drosophila bithorax complex (BX-C) over 330kb of intergenic DNA is responsible for directing the transcription of just three homeotic (Hox) genes during embryonic development. A number of distinct enhancer cis-regulatory modules (CRMs) are responsible for controlling the specific expression patterns of the Hox genes in the BX-C. While it has proven possible to identify orthologs of known BX-C CRMs in different Drosophila species using overall sequence conservation, this approach has not proven sufficiently effective for identifying novel CRMs or defining the key functional sequences within enhancer CRMs. This study demonstrates that the specific spatial clustering of transcription factor (TF) binding sites is important for BX-C enhancer activity. A bioinformatic search for combinations of putative TF binding sites in the BX-C suggests that simple clustering of binding sites is frequently not indicative of enhancer activity. However, through molecular dissection and evolutionary comparison across the Drosophila genus it was discovered that specific TF binding site clustering patterns are an important feature of three known BX-C enhancers. Sub-regions of the defined IAB5 and IAB7b enhancers were both found to contain an evolutionarily conserved signature motif of clustered TF binding sites which is critical for the functional activity of the enhancers. Together, these results indicate that the spatial organization of specific activator and repressor binding sites within BX-C enhancers is of greater importance than overall sequence conservation and is indicative of enhancer functional activity (Starr, 2011).

The clustered organization of TF binding sites has been shown to be crucially important to the functional activity of enhancers. However, despite detailed studies of a small set of enhancers in Drosophila, including the eve stripe 2 (S2E) enhancer, the precise rules of cis-regulatory grammar have yet to be fully elucidated. In an effort to investigate the role of clustering of predicted TF binding sites for the identification of enhancers in the 330 kb Drosophila BX-C, a search for simple clusters of HB and KR binding sites was performed. The search algorithm returned 26 putative enhancers (PCRMs), of which 6 (23%) overlapped previously identified enhancers. The overlapping regions for four of these confirmed enhancers (BRE, IAB2, IAB5 and IAB8) were tested in transgenic reporter gene assay and recapitulated the known domains of regulatory activity in the embryo. Furthermore, the 1037 bp R10 region that was tested, that is able to recapitulate IAB2 enhancer functional activity, refines the boundaries of the previously characterized 1970 bp IAB2 sequence. The search also identified 20 additional PCRM sequences. Twelve of these previously uncharacterized genomic regions were analyzed for enhancer activity and only one (R8 from the bxd/pbx region) was found to be a novel embryonic enhancer capable of driving expression in a pattern indicative of Ubx gene expression. This result indicates that the approach of searching for novel enhancers in the BX-C using simple clustering may have significant limitations (Starr, 2011).

A key question is why 11 of the 16 PCRMs tested (69%) are false positives. Two possibilities include; a) that the PCRMs may in fact be actively regulating expression of the BX-C genes at later stages of development or in very specific patterns in post-embryonic tissues, and b) that in testing a specific ~ 1 kb genomic fragment from each PCRM critical regulatory sequences in neighboring regions may have been removed. However, the recent availability of in vivo TF binding data may also offer some potential answers. The binding of anterio-posterior restricted gap/terminal and pair-rule transcription factors in stages 4-5 embryos appears to correlate strongly with the functional activity of the PCRMs. When scored for ten specific TFs which are potential regulators of the BX-C enhancers, all the PCRMs tested in the transgenic assay that had chromatin immunoprecipitation (ChIP) binding peaks for 6 or more of the protein factors function as embryonic enhancers. For each of these confirmed enhancers, both KR and HB demonstrate in vivo binding at the endogenous genomic region corresponding to the enhancer. In contrast, all the false positive PCRMs do not have binding peaks for more than 5 of the TFs and most have less than 3, often reflecting an absence of binding for KR or HB (Starr, 2011).

One interpretation of this data is that the predicted TF binding sites in many of the false positive PCRMs do not represent actual in vivo embryonic binding sites and, as a result, the PCRM is not functional. In addition to KR and HB repressor binding sites, it is also important to consider the presence of potential binding sites for an appropriate activator (FTZ or EVE) necessary for the functional activity of the enhancer. Analysis of the 5 PCRMs that demonstrate in vivo activity reveals that each contains at least 3 strong predicted binding sites for the appropriate pair-rule activator. However, in many cases the false positive PCRMs tested also appear to contain putative activator binding sites. In these cases it is possible that additional architectural requirements (for example, close spacing between multiple activator and/or repressor binding sites) may be necessary for in vivo embryonic activity to occur. In support of this idea, analysis of the genomic fragments that were tested from the iab-2 to iab-8 genomic regions (R10, 11, 12, 13, 14, 15, 17, 20, and 21), predicts that R15 (overlapping IAB5) has a closely-spaced cluster of FTZ-KR sites and that R10 (overlapping IAB2) and R20 (overlapping IAB8) possess a closely spaced cluster of EVE-KR sites within 150 bp of one another, whereas the other regions do not appear to harbor pair-rule activator (FTZ or EVE) and repressor (KR) clusters in such close proximity. A third possibility is that additional protein factors may be involved which may affect the ability of TFs to access the binding sites within the predicted enhancer sequence. Such proteins, which control the recruitment of chromatin components and nucleosome positioning, are thought to be critical to the regulation of embryonic gene expression through the modulation of TF binding affinity at enhancers (Starr, 2011).

The presence of a simple cluster of KR and HB binding sites in many of the enhancers of the BX-C argues that certain precise patterns of TF binding site clusters may be responsible for functional activity among similarly-regulated enhancers. In the IAB8 enhancer, a distinct cluster of EVE-KR binding sites (one KR, two EVE sites) is highly conserved across different Drosophila species. The 3' third of IAB8 harboring the EVE-KR motif (minIAB8) is able to drive reporter gene expression in the characteristic IAB8-pattern in the presumptive A8 segment of transgenic Drosophila. Deletion of the pair of EVE binding sites (∆EVE) significantly weakens enhancer activity in A8, suggesting that while the these two EVE sites are important, cryptic weak EVE binding sites in the remaining sequence of the enhancer (which are sufficiently low scoring to escape computational prediction at the ln(p-value) cutoff of - 6.0) are capable of partially compensating for the loss of the two strong predicted EVE sites. In support of this idea is the recent discovery that even weak affinity binding sites contribute to TF occupancy at regulatory regions in Drososphila embryos. In that study it was found that the level of factor occupancy in vivo correlates more strongly with the degree of chromatin accessibility at a given site, rather than in vitro measurements of the affinity of a factor for a particular DNA sequence (Li, 2011). This observation may be especially relevant in the case of pair-rule factors (such as EVE), where a high localized concentration of the protein in each stripe may also facilitate the increased occupancy of low affinity binding sites (Starr, 2011).

A 141 bp fragment (EK) from within the minIAB8 region containing only the EVE-KR cluster drives strong reporter gene expression in A8, but also ectopic expression immediately posterior of A8 and weaker expression immediately anterior of A8. Ectopic reporter gene expression is also observed in the anterior head domain of the embryo. This result indicates that the EK fragment by itself lacks important binding sites responsible for repression in the anterior head domain of the embryo (such as HB) and for the region immediately anterior of A8 (such as KNI). Several predicted HB and KNI repressor sites capable of performing this role are present within the 602 bp minIAB8 enhancer. Importantly, in the C3-A4 domain of the embryo where the KR repressor protein is expressed, there is a lack of enhancer-driven reporter gene expression from the EK fragment, suggesting that the single KR site within the EVE-KR cluster is sufficient to allow KR-mediated repression in that domain of the embryo. The continued presence of the EVE-KR cluster within the IAB8 enhancer, despite extensive reorganization of TF binding sites across the Drosophila orthologs, is reminiscent of the architectural constraints in the Drosophila and Sepsid eve S2E orthologs, which possess a highly conserved cluster of overlapping BCD activator and KR repressor binding sites necessary for enhancer function (Starr, 2011).

To extend the analysis of the functional role of clustered TF binding sites the IAB5 and IAB7b enhancers from the Drosophila BX-C were also analyzed. Chimeric enhancers assembled from the D. melanogaster and D. pseudoobscura IAB5 orthologs appear to have their functional activity entirely preserved and drive reporter gene expression in presumptive abdominal segments A5, A7 and A9. This result contrasts with an earlier study in which chimeric enhancers assembled from reciprocal halves of D. melanogaster and D. pseudoobscura S2E orthologs did not accurately recapitulate enhancer activity. It is possible that the regulatory output for the chimeric IAB5 enhancers may be subject to very subtle modifications. Such modifications may result in changes to expression patterns that are beyond the detection of the reporter gene assay. However, one explanation for the difference in functional output between these two examples is that in the case of the S2E the organization of TF binding sites within the chimeric enhancer was sufficiently modified so as to destroy the functional activity of the enhancer, whereas for IAB5 this was not the case (Starr, 2011).

To further dissect the organization of TF binding sites in IAB5 the predicted TF binding sites in the sequence were examined. This approach reveals a highly evolutionarily conserved signature TF binding site motif consisting of two strong FTZ activator sites close to two strong KR repressor sites in the center of the defined 1 kb enhancer. The FTZ-KR signature motif is present and intact in both the functional IAB5 chimeric enhancers, cMP and cPM. In the case of the cMP enhancer, the signature motif is present in the IAB5.2 half from D. pseudoobscura, while in the case of the reciprocal cPM enhancer, the signature motif is present in the IAB5.2 half from D. melanogaster. Molecular dissection of IAB5 shows that the individual IAB5.2 halves from Drosophila and D. pseudoobscura each show functional enhancer activity, while the corresponding IAB5.1 halves that lack the FTZ-KR signature motif do not. Furthermore, the 424 bp region containing the center peak of sequence conservation of IAB5 (cIAB5) and the FTZ-KR signature motif drives reporter gene expression in the characteristic three-stripe IAB5-pattern in transgenic Drosophila. In support of the critical functional role of this region, previous functional studies showed that the strongest predicted KR binding site within this signature motif is in fact critical to regulate the spatially restricted expression directed by IAB5 to the posterior presumptive A5, A7, and A9 segments in the Drosophila embryo. In the context of the endogenous gene complex a single point mutation in this KR repressor binding site (Superabdominal mutation) causes an anterior expansion of the embryonic domain of Abd-B expression and results in a homeotic transformation of the A3 segment into the more posterior A5 segment. This result confirms that the strong KR binding site in the signature motif is essential for the in vivo functional activity of the IAB5 enhancer (Starr, 2011).

The IAB7b enhancer, which is expressed in the presumptive A7 segment of the Drosophila embryo, is thought to be regulated by many of the same activators and repressors as IAB5. Bioinformatic analysis reveals that a highly conserved FTZ-KR signature motif, very similar to the one identified in IAB5, is also present in the IAB7b enhancer. Molecular dissection of IAB7b to test the role of the signature motif in the activity of the enhancer demonstrates that a 154 bp region containing the motif (2F2K, with two FTZ and two KR sites) from within the Drosophila IAB7b enhancer is able to drive reporter gene expression in the presumptive A5, A7 and A9 segments of transgenic Drosophila, with notably stronger expression in A7. This expression pattern is very similar to that driven by the IAB5 enhancer. A 114 bp region (2F1K, containing two FTZ and one KR site) from within the Drosophila IAB7b enhancer also drives this same pattern of reporter gene expression, suggesting that the 3' KR site is dispensable for repression of enhancer activity in the central domains of the embryo. Despite the fact that the 3' KR site also overlaps predicted BCD and HB repressor binding sites, no ectopic anterior enhancer-driven expression is observed in the 2F1K construct when tested in transgenic embryos, suggesting that the single remaining 5' KR binding site is sufficient for repression. In fact, in more distantly related Drosophila species, the presence of two KR sites positioned near the pair of FTZ sites is lost, and only a single KR site remains (Starr, 2011).

A 110 bp region (1F2K, containing 1 FTZ and two KR sites) from IAB7b does not drive gene expression, demonstrating that the outer FTZ site is required for activation of the enhancer. One possible molecular explanation for the necessity of the outer FTZ binding site is that FTZ may be acting as a dimer in order to activate IAB5 and IAB7b. In both enhancers a pair of strong FTZ sites are present in the FTZ-KR signature motif. While the ability of FTZ to dimerize has not been reported in the literature, other homeodomain-leucine zipper proteins have been shown to function as dimers. In many such cases the protein factors are also able to bind DNA target sequences as monomers, albeit with comparatively lower affinity. There is also evidence that FTZ is capable of interaction with other proteins, namely the orphan nuclear receptor FTZ-F1 through its LXXLL leucine zipper motif. In this case the heterodimer is capable of co-activation of target genes. Given that the consensus binding sites for the two factors are very different; FTZ (NNYAATTR), FTZ-F1 (BSAAGGDKRDD, it is perhaps to be expected that none of the predicted FTZ and FTZ-F1 binding sites in the IAB5 or IAB7b enhancers directly overlap. However, in future studies it will be of interest to explore the role of FTZ homo- and hetero-dimerization in regulating IAB5 and IAB7b activity (Starr, 2011).

The ability of the 2F2K and 2F1K regions to drive reporter gene expression in an IAB5-like manner in the presumptive A5, A7 and A9 segments of transgenic Drosophila suggests that additional inputs into IAB7b are required to spatially restrict endogenous enhancer-driven gene expression to only the A7 segment. A likely candidate for repression of IAB7b activity in the A5 segment of the embryo is KNI, which is expressed in the presumptive A1-A6 segments. Bioinformatic analysis predicts several candidate KNI binding sites in the full length 728 bp IAB7b enhancer, whereas the 2F2K and 2F1K regions lack any such predicted KNI sites. Previous studies revealed that the repression of the IAB7b enhancer in A5 is mediated by sequences in the 728 bp fragment and does not require additional flanking 5' or 3' regions. In addition, while disruption of the two KR sites in the signature motif does result in reporter gene activation by IAB7b in anterior regions of the embryo, repression persists in the A5 segment. This result indicates that a factor other than KR is responsible for repression in A5. In the entire 728 bp enhancer only three strong KNI sites are predicted, all located in the ~ 300 bp region on the abd-A side of the signature motif. These sites all lie within an evolutionarily conserved genomic region and some of the sites are conserved in distantly related Drosophila species. The significance of these KNI sites in restricting the IAB7b mediated-expression pattern is currently under investigation (Starr, 2011).

A key question in understanding cis-regulatory grammar is why certain arrangements of TF binding sites confer functional enhancer activity while others fail to do so. The turnover of binding sites is common during the evolution of enhancers in different species, yet the functional activity of rapidly-evolving enhancer orthologs from different species is often robust, even across several hundred million years of evolutionary divergence. In the case of the BX-C, bioinformatic analysis demonstrates that there is extensive binding site turnover in the IAB5, IAB7b, and IAB8 enhancers across the Drosophila genus, particularly in more distantly related species. Despite this turnover of TF binding sites, the newly identified FTZ-KR signature motif present in both IAB5 and IAB7b and the functionally important EVE-KR cluster within IAB8 are composed of similar patterns of conserved binding site architecture. Specifically, the organization of sites is such that a pair of strong activator (FTZ or EVE) binding sites and at least one strong repressor (KR) site are in close proximity (< 116 bp) to each other. Notably, the spacing between the FTZ and KR sites in the signature motif is largely unchanged across IAB5 and IAB7b enhancer orthologs in distantly related Drosophila species, although in the case of IAB7b there is the loss of the secondary KR binding site in species more distantly related to Drosophila. Conservation of genomic architecture of these TFBSs in the BX-C enhancers does not directly indicate that the specific spacing between sites is essential. However, the functional activity of genomic regions containing these motifs supports previous findings that closely spaced activator and repressor binding sites are critical for enhancer function and suggests that the architecture of binding sites within an enhancer is subject to significant evolutionary constraint (Starr, 2011).

It has recently been suggested through computational synthetic evolution studies that the inherent bias for deletions over insertions in the genome of Drosophila (and many other species) may result in the gradual loss of nucleotide space between TF binding sites. In effect, this deletion bias helps to artificially cluster binding sites together. In this case, although clustering of TF binding sites may not itself be a feature originally selected for in evolution on the basis of its functional significance, once established in the genome it may still play a functional role in enhancer activity. Molecular dissection of IAB5, IAB7b, and IAB8 enhancer function argues that specific clusters of activator and repressor binding sites do play a key role in enhancer activity. As a result, such clusters, once present in enhancers, may well be under positive evolutionary selective pressure, as evidenced by the largely invariant organization of the binding sites in the IAB5 and IAB7b FTZ-KR signature motif. This selection does not preclude the possibility that if binding sites arise nearby in the genome de novo, these new binding sites may also contribute to enhancer functional activity. In this scenario, the original TF binding site cluster may no longer be necessary for enhancer function. Indeed, in the case of the IAB8 enhancer, the ∆EVE region tested in s transgenic assay may be an example of this phenomenon. This fragment is able to exhibit a weak IAB8-like enhancer function even with the deletion of the pair of strong predicted EVE binding sites, potentially through the activity of weaker EVE binding sites that are present in the remaining sequence (Starr, 2011).

Although the precise spatial arrangement of TF binding sites within an enhancer may not exactly mirror the ancestral arrangement, computational predictions suggest that functional clusters of TF binding sites are likely to result from the spatial re-organization of older pre-existing sites during evolution. Such clusters therefore also likely indicate genomic regions with robust enhancer activity. The fact that enhancer activity in the BX-C appears to be dependent on signature motifs that represent specific spatial arrangements of TF binding sites in minimal modular regions, indicates that the physical patterns of binding site clustering are functionally significant in terms of enhancer architecture (Starr, 2011).

Non-additive interactions involving two distinct elements mediate sloppy-paired regulation by pair-rule transcription factors

The relatively simple combinatorial rules responsible for establishing the initial metameric expression of sloppy-paired-1 (slp1) in the Drosophila blastoderm embryo make this system an attractive model for investigating the mechanism of regulation by pair rule transcription factors. This investigation of slp1 cis-regulatory architecture identifies two distinct elements, a proximal early stripe element (PESE) and a distal early stripe element (DESE) located from −3.1 kb to −2.5 kb and from −8.1 kb to −7.1 kb upstream of the slp1 promoter, respectively, that mediate this early regulation. The proximal element expresses only even-numbered stripes and mediates repression by Even-skipped (Eve) as well as by the combination of Runt and Fushi-tarazu (Ftz). A 272 basepair sub-element of PESE retains Eve-dependent repression, but is expressed throughout the even-numbered parasegments due to the loss of repression by Runt and Ftz. In contrast, the distal element expresses both odd and even-numbered stripes and also drives inappropriate expression in the anterior half of the odd-numbered parasegments due to an inability to respond to repression by Eve. Importantly, a composite reporter gene containing both early stripe elements recapitulates pair-rule gene-dependent regulation in a manner beyond what is expected from combining their individual patterns. These results indicate interactions involving distinct cis-elements contribute to the proper integration of pair-rule regulatory information. A model fully accounting for these results proposes that metameric slp1 expression is achieved through the Runt-dependent regulation of interactions between these two pair-rule response elements and the slp1 promoter (Prazak, 2010).

This work identifies two distinct CRMs from the slp1 gene that drive early expression in response to pair-rule gene regulation. The observation that a composite reporter gene containing both elements faithfully emulates the initial metameric expression of slp1 in wild-type embryos as well as the response to manipulations in pair-rule activity strongly suggests these two CRMs together account for most of the early regulation of slp1 in response to pair-rule transcription factors. This view is supported by recent ChIP/chip results from the Berkeley Drosophila Transcription Network Project indicating there are two major regions of association for both Runt and Ftz in the 20 kb of DNA flanking the slp1 transcription unit, a distal region spanning from ~8.4 to ~6.7 kb and a proximal region spanning from ~3.7 to ~2.2 kb upstream of the transcription start site. These two intervals correspond extremely well to the minimal intervals defined by the functional analysis of ~8.1 to ~7.1 and ~3.1 to ~2.5 kb, respectively. Although this genome-wide analysis did not include results for either Eve or Opa it is interesting to note that Paired (Prd) and Hairy, the two other pair-rule transcription factors included in this study also show association with these same two regions. This observation further suggests Prd and Hairy may also participate in slp1 regulation, although genetic experiments do not provide any evidence indicating that either of these factors play important direct roles in regulating the early slp1 stripes (Prazak, 2010).

A most conspicuous finding from the work presented in this study is that pair-rule dependent regulation of slp1 transcription involves non-additive interactions between two distinct upstream CRMs. The ability of composite reporters containing both the DESE and PESE enhancers to mimic expression of the endogenous gene cannot be explained solely by the independent regulatory capabilities of the two elements as a simple addition of the two patterns will include inappropriate DESE-driven expression in anterior even-numbered parasegments. This non-additive interaction potentially conflicts with the generally accepted paradigm for the modular and independent action of distinct CRMs, a point that will be discussed further below (Prazak, 2010).

Although the early stripe elements need to be combined in order to fully recapitulate pair-rule regulation, studies on the independent elements provide new insights on the pair-rule to segment polarity gene transition. The homeodomain proteins Eve and Ftz both participate in slp1 repression. Several lines of evidence indicate differences in the cis-regulatory requirements for repression by these two structurally related transcription factors. DESE is insensitive to repression by Eve, but is capable of mediating repression by Ftz. The exact opposite specificity is demonstrated by the PESE:C1+ element, which is repressed by Eve but not by Ftz. The DNA-binding specificities of Eve and Ftz are similar both in vitro and in vivo and their specificity of action is thought to involve co-factor interactions that dictate the manner in which they regulate different targets. An established cofactor for Ftz is the orphan nuclear receptor protein Ftz-F1. Indeed, elimination of maternally provided Ftz-F1 results in alterations in slp1 expression that are identical to those seen in ftz mutants. The Ftz-dependent repression of slp1 also requires Runt, making this a second prospective co-factor for this activity of Ftz. Further studies on slp1 regulation should provide valuable information on the mechanisms that underlie repression by the Eve and Ftz proteins (Prazak, 2010).

These studies on the independent DESE and PESE reporters also provide information on the properties of the unidentified factor(s) that are responsible for slp1 activation in posterior even-numbered parasegments. In the case of PESE, expression of the minimal slp1[PESE:C1+]lacZatt reporter throughout the entire pre-segmental region of eve mutant embryos indicates that a factor(s) capable of activating this element is present in all cells within this region of the embryo. DESE also drives expression of even-numbered stripes, but interestingly fails to generate stripe 0. This difference between DESE and PESE suggests there are differences in the factors responsible for activating these two elements in even-numbered parasegments. DESE also generates the odd-numbered stripes, an aspect of slp1 expression that is normally driven by the combination of Runt and Opa. Runt is normally expressed in the posterior half of only the odd parasegments and not in the posterior half of even-numbered parasegments. However, the observation that transient elimination of Runt does not abrogate DESE-driven expression in odd parasegments suggests Opa may be capable of activation in the absence of Runt. This same proposal could account for the ectopic DESE-driven expression in the anterior half of the odd parasegments as Opa is uniformly expressed in all cells within the pre-segmental region that are posterior to the cephalic furrow. The observation that Opa expression is lost anterior to the head-fold in late blastoderm stage embryos may further account for the failure of DESE to generate stripe 0. Although the only Opa-expressing cells that do not activate the DESE-lacZ reporters are those that express the combination of Runt and Ftz, there are differences in the level of expression in different cells. The increased expression in posterior versus anterior odd-numbered parasegments may reflect a contribution from Runt in potentiating DESE-driven expression (Prazak, 2010).

A central issue raised by the results is to understand how interactions involving two distinct CRMs can account for their ability to faithfully recapitulate the regulation of slp1 in response to the pair-rule transcription factors. A major discrepancy between the expression of the composite [DESE+PESE] reporter and the pattern expected from the independent action of the separate CRMs is repression of DESE-driven expression in anterior odd parasegments. One potentially trivial explanation for inappropriate expression of the DESE-lacZ reporters in these cells is close juxtaposition of binding sites for DESE-interacting activators with the basal promoter region. However, the observations that this inappropriate expression is seen for DESE-lacZ reporters that have slp1 basal promoter segments that extend anywhere from ~71 bp to ~1.8 kb upstream of the transcription start site indicates it is not due to short range interactions between DESE-bound activators and the promoter region (Prazak, 2010).

A second potential explanation is that repression in these cells involves interactions that allow the Eve-sensitivity of PESE to be transmitted to DESE. In this version of the model, Eve-interacting PESE is acting as an insulator element that prevents DESE from communicating with the slp1 promoter by blocking propagation of signals that track along the chromosome. It is also possible to imagine insulator models involving looping, such as sequestration of DESE by Eve-interacting PESE, thus preventing DESE-dependent activation at the promoter. However, in both of these insulator models ectopic Eve expression would be expected to block expression of both the odd- and even-numbered stripes, an effect that is not observed for slp1 or for the composite [DESE+PESE] reporter (Prazak, 2010).

A second discrepancy between the single elements and the composite [DESE+PESE] reporter is the Runt-independent activation by DESE in odd parasegments. This observation strongly suggests that Runt’s role in activating the composite reporter involves enabling DESE-dependent activation. This could be due to Runt-dependent antagonism of the insulator-like activities of PESE depicted in. However, there is a perhaps more straightforward explanation that does not invoke Runt-dependent regulation of PESE insulator activity, but that instead involves regulating competition between the two upstream enhancers and the slp1 promoter. In this model it is proposed that Runt plays a role in switching the promoter from interacting with PESE to interacting with the further upstream DESE. This proposed promoter-targeting role of Runt is bypassed in the DESE-lacZ reporter due to the lack of competition from PESE, thus accounting for the expression of this reporter in all cells within the segmented region of the embryo except for those that express both Runt and Ftz. Importantly, this model fully accounts for expression of both slp1 and the composite [DESE+PESE] reporters in both wild-type and mutant embryos. All expression in anterior odd-parasegments of wild-type embryos is restricted to PESE-dependent regulation as these cells do not express Runt and thus will not reveal the activating potential of DESE. The role that Eve normally plays in repressing both slp1 and the [DESE+PESE] reporter in anterior odd parasegments is also clearly accounted for by Eve’s activity as a repressor of PESE. Further work is needed to determine whether an insulator or enhancer competition model more readily accounts for the non-additive interaction between these two cis-elements, but in either event the regulatory output would appear to be due to functional attributes of the Runt transcription factor (Prazak, 2010).

There are two aspects of the current results that have widespread implications for studies on cis-regulatory DNA elements and their role in developmentally regulated gene expression. The first point is that cis-elements (such as DESE) that have broad activities when tested as autonomous single elements can have more restricted roles in regulating gene expression in the context of their normal chromosomal environment. A second, and perhaps even more crucial point is that transcription factors (such as Runt) that are not essential for activation by a single autonomous enhancer, even when tested in a physiologically relevant context, may have critical roles in enabling the activity of this enhancer in a developmental setting. Further studies on the functional interplay between the slp1 early stripe elements during Drosophila segmentation should provide insights on a phenomenon of potentially far-reaching importance in understanding the developmental regulation of gene expression (Prazak, 2010).

Downstream of identity genes: muscle-type-specific regulation of the fusion process

In all metazoan organisms, the diversification of cell types involves determination of cell fates and subsequent execution of specific differentiation programs. During Drosophila myogenesis, identity genes specify the fates of founder myoblasts, from which derive all individual larval muscles. To understand how cell fate information residing within founders is translated during differentiation, this study focused on three identity genes, eve, lb, and slou, and how they control the size of individual muscles by regulating the number of fusion events. They achieve this by setting expression levels of Mp20, Pax, and mspo, three genes that regulate actin dynamics and cell adhesion and modulate the fusion process in a muscle-specific manner. Thus, these data show how the identity information implemented by transcription factors is translated via target genes into cell-type-specific programs of differentiation (Bataille, 2010).

Myoblast fusion is asymmetric and takes place between fusion cells (FCs) and fusion competent myoblasts (FCMs). Previous reports originated a hypothesis that FCMs are not 'naive' myoblasts and contribute to the modulation of fusion process. In contrast, the current results support a view that FCs rather than FCMs carry the instructive information and lead to the conclusion that FCMs do not play an active role in setting the number of fusion events. However, because the spatial distribution of FCMs seems to be nonuniform, it is conceivable that the local distribution of FCMs was coordinated with the requirements of FCs to facilitate fusion process (Bataille, 2010).

The identity genes lb, slou, and eve are required to specify FCs at the origin of five muscles the DA1, DT1, SBM, VA2, and VT1. This study providea evidence that these identity genes are also required for setting the muscle-specific number of fusions and demonstrate how this identity information is executed. After specification step, FCs fuse, between the embryonic stage 12 and 15, with a determined number of FCMs to generate muscles with a specific number of nuclei. During this time period eve, lb, and slou continue to be expressed in subsets of developing muscles, and the data show that they are sufficient to establish the muscle-specific fusion programs in DA1, SBM, and VT1 (11, 7, and 4 nuclei, respectively). Furthermore, slou in combination with other factors contributes to two other programs that end up with seven to eight fusion events in muscles DT1 and VA2. To regulate number of fusion events eve, lb, and slou act by modulating expression of genes involved in dynamics of actin cytoskeleton or cell adhesion. Starting from stage 13, they establish a muscle-specific combinatorial code of expression levels of three targets: Mp20, Pax, and mspo. The combination of expression of the targets leads to the muscle-specific control of the number of fusion events. This notion is supported by the fact that each of identity genes is able to impose at ectopic locations the combinatorial realisator code of Mp20, Pax, and mspo expression, and thus, ectopically execute its fusion program. Given that the code of Mp20, Pax, and mspo is not sufficient to explain fusion programs in all muscles, it is hypothesized that other identity gene targets exist that modulate fusion counting (Bataille, 2010).

Moreover, the data support a two-step model of myoblast fusion according to which a muscle precursor is formed between stage 12 and 13 by an initial fusion, and then, between stage 13 and 15, fuses with additional myoblasts until the muscle reaches its final size. The fact that Mp20, Pax, and mspo are expressed from stage 13 suggests that the transition point between the two steps depends not only on the timing of FCM migration but also on the activation of limiting factors such as the identity gene targets which modulate the number of additional fusions. Since no nuclear divisions were observed in FCs nor in growing myotubes in any of the genetic contexts analyzed, it is clear that the number of nuclei present in each muscle is determined only by the number of fusion events (Bataille, 2010).

Specification of FCs requires combinatorial code of activities of identity genes. This study shows that the same identity genes play instructive roles in subsequent muscle-type-specific differentiation process. Importantly, the data highlight the fact that the identity genes are not equivalent and have distinct, context-dependent mode of action. eve, lb, and slou are sufficient to set the fusion programs in DA1, SBM, and VT1 muscles; however, in VA2 and DT1 slou functions in a different way and seems not to have a decisive role in this process. Because the specification of the VA2 and DT1 FCs also involves functions of Poxm, Kr, and ap, it is hypothesized that they act together with slou in setting fusion programs of VA2 and DT1. This raises an important question about hierarchy of identity genes during execution of muscle identity programs and their roles in acquisition of specific properties of muscles such as number of nuclei, attachment points, and innervation (Bataille, 2010).

The data presented here demonstrate that the number of fusion events in developing muscles is regulated by a muscle-specific combinatorial realisator code of identity gene targets. In contrast to the previously identified fusion genes (e.g., Ruiz-Gomez et al., 2000; Chen and Olson, 2001; Rau et al., 2001) acting in all muscles, the identified identity targets, Mp20, Pax, and mspo, display muscle-type specific expression and modulate fusion in a muscle-type-specific manner proportionally to the level of their expression. The loss and gain of function of each of them lead to subtle fusion phenotypes indicating that the range of fusion events controlled by these three candidates is limited. Indeed, the loss of function of Mp20 results in loss of two nuclei in a subset of muscles, whereas its overexpression induces the recruitment of maximum two FCMs. A similar range of defects in number of fusion events is observed in Pax and mspo mutant embryos indicating that they influence fusion process at the same level (Bataille, 2010).

Mp20 encodes a cytoskeletal protein displaying restricted expression in adult muscles and sharing sequence homology with the lineage-restricted mouse proteins SM22alpha, SM22beta, and NP25. These proteins contain calponin-like repeats, and, in mammals, interact with F-actin and participate in the organization of the actin cytoskeleton. In Drosophila S2R cells, the RNAi knockdown of Mp20 induces a phenotype of round and nonadherent cells supporting its role in regulation of fusion process (Bataille, 2010).

The second candidate, Pax (DPxn37), is a scaffold protein that recruits structural and signaling molecules to the sites of focal adhesion. Pax has been shown to be involved in the actin cytoskeleton organization, cell adhesion, cell migration, and cell survival. In the developing Drosophila muscles, Pax protein localizes at muscle-tendon junctions, suggesting that it may play a role in muscle attachment. Analyses of Pax mutant embryos do not reveal muscle-tendon adhesion defects but show discrete myoblast fusion phenotypes, which correlate with differential musclespecific expression of Pax. The role of Pax in modulating fusion is consistent with previously described implications of Pax interacting proteins, including ARF6 in myoblast fusion in both Drosophila and vertebrates, and FAK in vertebrates. Finally, mspo belongs to the F-Spondins, a conserved family of ECM proteins, which maintain cell-matrix adhesion in multiple tissues. In vertebrates, F-Spondins have context-dependent effects on axon outgrowth and cell migration. As Mp20, Pax, and Mspo are expressed in FC cells and growing myotubes, one possibility is that they modify the spreading and/or motility of FC protrusions required to attract FCMs. Alternatively, by modulating actin cytoskeleton, Mp20, Pax, and Mspo may also influence the stability of adhesion between the growing muscle and the FCM creating permissive conditions or blocking the progression of fusion process (Bataille, 2010).

The muscle-type-specific regulation of fusion programs by the identity genes and their targets raises an intriguing question of how this regulation is executed from the mechanistic point of view. Because different levels of expression of Mp20, Pax, and mspo correlate with different fusion programs in both wild-type and genetically manipulated embryos, it was thought that by following kinetics of fusion in small and big muscles, insights may be gained into how the fusion programs are modulated. It turns out that the rate of fusion is proportional to the size of muscle, meaning the number of fusion events, thus revealing that the identity genes acting via their targets set up the frequency of fusion events. Accordingly, loss and gain of function of identity genes and their targets identified here results in modulations of fusion programs by accelerating or slowing down the fusion rate. This finding provides insights into mechanistic understanding of muscle-type-specific regulation of fusion process and raises an important question about whether this mechanism is broadly conserved (Bataille, 2010).

A GATA/homeodomain transcriptional code regulates axon guidance through the Unc-5 receptor

Transcription factor codes play an essential role in neuronal specification and axonal guidance in both vertebrate and invertebrate organisms. However, how transcription codes regulate axon pathfinding remains poorly understood. One such code defined by the homeodomain transcription factor Even-skipped (Eve) and by the GATA 2/3 homologue Grain (Grn) is specifically required for motor axon projection towards dorsal muscles in Drosophila. Using different mutant combinations, genetic evidence is presented that both Grn and Eve are in the same pathway as Unc-5 in dorsal motoneurons (dMNs). In grn mutants, in which dMNs fail to reach their muscle targets, dMNs show significantly reduced levels of unc-5 mRNA expression and this phenotype can be partially rescued by the reintroduction of unc-5. It was also shown that both eve and grn are required independently to induce expression of unc-5 in dMNs. Reconstitution of the eve-grn transcriptional code of a dMN in dMP2 neurons, which do not project to lateral muscles in Drosophila, is able to reprogramme those cells accordingly; they robustly express unc-5 and project towards the muscle field as dMNs. Each transcription factor can independently induce unc-5 expression but unc-5 expression is more robust when both factors are expressed together. Furthermore, dMP2 exit is dependent on the level of unc-5 induced by eve and grn. Taken together, these data strongly suggests that the eve-grn transcriptional code controls axon guidance, in part, by regulating the level of unc-5 expression (Zarin, 2012).

Different transcriptional codes regulate axon guidance but the guidance systems they regulate are still unknown. The GATA factor, Grn, is a major determinant of guidance within dorsal MNs. A strong guidance phenotype occurs in grn mutants, in which ISN axons fail to reach their targets in >85% of the segments. However, none of the downstream molecules required for guidance downstream of grn has been identified to date. Several lines of evidence indicate that the Unc-5 receptor mediates guidance downstream of the GATA transcription factor Grn. First, both genes interact genetically in trans and this type of genetic interaction is often seen between two genes the gene products of which directly interact, such as Slit and Robo. Second, there is also a partial requirement of grn for unc-5 expression in dMNs as unc-5 mRNA levels are reduced in aCC and RP2 in grn mutants. Further support for the role of unc-5 downstream of grn comes from the partial rescue of the grn phenotype obtained by exogenously providing unc-5 specifically in aCC and RP2 (Zarin, 2012).

Among transcriptional codes that regulate motor-axon pathfinding, specific Lim-HD codes are required for the proper guidance of vertebrate motoneurons, in part, through the regulation of the EphA. In Drosophila, Nkx6 (HGTX -- FlyBase) is important for vMN specification and has been proposed to promote guidance through the expression of fasciclinIII (Fasciclin 3 -- FlyBase). Similarly, eve regulates guidance of dMNs through unc-5. In at least some of these situations the regulation is unlikely to be direct because the regulators are thought to mediate their function through a repressive activity. Indeed, eve repressive activity has been shown to be responsible for its guidance function in aCC and RP2, suggesting that its function might be to repress the expression of other transcription factors, such as HB9 (Exex -- FlyBase), which would confer those motoneurons with a ventral fate. Analysis of the unc-5 neuronal enhancer regulated by eve failed to identify any conserved Eve consensus homeodomain binding site, suggesting that eve regulation of unc-5 is not mediated through a direct binding to this element (Zarin, 2012).

Combinatorial codes of transcription factors play an instructive role in the generation of subclass diversity within the vertebrate spinal cord. In invertebrates, in which it is possible to analyse individual motoneurons within a subclass, a further level of complexity is revealed. Within the subclass of motoneurons that project to dorsal muscles, aCC and RP2, subclass determinants (eve, grn and zfh1) can work in a sequential order in aCC specification or independently, in parallel pathways, within RP2. Whereas Zfh1 is a general factor required in all motoneurons, eve and grn are specific to dMNs. It is plausible that each one of them might be important for specific aspects of specification within the same subclass of neurons but together might be responsible for the regulation of common targets such as unc-5. Although grn is required in both aCC and RP2 for unc-5 expression, it is not the only factor required because unc-5 mRNA is not completely absent from those cells and it also requires the presence of eve. In fact, grn or eve can independently induce unc-5 expression in dMP2 neurons but only both factors expressed in combination are able to induce axon exit towards the muscle field. This combinatorial expression of eve and grn might bring unc-5 above the threshold required for exit. unc-5 levels are definitely important for dMP2 exit because removing 50% of the gene dosage significantly suppresses the exit phenotype and this suppression is almost complete in an unc-5 null background. Transheterozygous interactions identified between unc-5 and eve, or unc-5 and grn also suggest that their levels are tightly controlled. As both eve and grn can regulate unc-5 it is likely that their combined activity in aCC and RP2 is essential to express the required levels of unc-5 in both neurons. A model is presented of unc-5 regulation by grn and eve in each individual neuron (Zarin, 2012).

The combinatorial nature of substrate recognition during motoneuron guidance and targeting is well established. Mutants for guidance receptors or ligands often show partially penetrant phenotypes and this includes the unc-5 phenotype in dMNs. By contrast, phenotypes observed for transcription factor mutants that affect dMN guidance are far more severe. For example, Lim1 regulates EphA4 in vertebrate motoneurons but the EphA4 mutant phenotype is less severe than Lim1 mutant phenotype. Similarly, 100% of ISNs in eve mutants or 85% in grn mutants are affected, suggesting that they regulate several guidance systems. It will be interesting to investigate the full array of guidance systems regulated by the eve-grn code. One potential candidate is FasII (Fas2 -- FlyBase) as it is an essential molecule for the pioneer function of aCC and RP2 (Zarin, 2012).

In summary, this study has identified the guidance receptor Unc-5 as a novel target of the GATA transcription factor Grn. unc-5 is key common target that mediates guidance downstream of both eve and grn. Furthermore, both transcription factors can promote transcription of unc-5 independently, suggesting that their combined action is essential to attain the proper expression levels of the Unc-5 receptor in dMNs. Future investigations in cell type-specific expression profiling and chromatin immunoprecipitation sequencing (ChIP-seq) analysis will hopefully unravel other guidance systems regulated by the eve-grn code in dMNs. This knowledge will bring us closer to understanding the cell-specific transcriptional regulation of guidance (Zarin, 2012).

Cytoneme-mediated delivery of hedgehog regulates the expression of bone morphogenetic proteins to maintain germline stem cells in Drosophila

Stem cells reside in specialised microenvironments, or niches, which often contain support cells that control stem cell maintenance and proliferation. Hedgehog (Hh) proteins mediate homeostasis in several adult niches, but a detailed understanding of Hh signalling in stem cell regulation is lacking. Studying the Drosophila female germline stem cell (GSC) niche, this study shows that Hh acts as a critical juxtacrine signal to maintain the normal GSC population of the ovary. Hh production in cap cells, a type of niche support cells, is regulated by the Engrailed transcription factor. Hh is then secreted to a second, adjacent population of niche cells, the escort cells, where it activates transcription of the GSC essential factors Decapentaplegic (Dpp) and Glass bottom boat (Gbb). In wild-type niches, Hh protein decorates short filopodia that originate in the support cap cells and that are functionally relevant, as they are required to transduce the Hh pathway in the escort cells and to maintain a normal population of GSCs. These filopodia, reminiscent of wing disc cytonemes, grow several fold in length if Hh signalling is impaired within the niche. Because these long cytonemes project directionally towards the signalling-deficient region, cap cells sense and react to the strength of Hh pathway transduction in the niche. Thus, the GSC niche responds to insufficient Hh signalling by increasing the range of Hh spreading. Although the signal(s) perceived by the cap cells and the receptor(s) involved are still unknown, these results emphasize the integration of signals necessary to maintain a functional niche and the plasticity of cellular niches to respond to challenging physiological conditions (Rojas-Ríos, 2012).

The study of the mechanisms behind Hh signalling in the Drosophila ovary has allowed the identification of Hh-coated cytonemes in a cellular stem cell niche, emphasizing the idea that cytonemes mediate spreading of the activating signal from the producing cells. Recently, it has been reported that the Hh protein localises to long, basal cellular extensions in the wing disk (Callejo, 2011). In addition, filopodial extensions in the wing, eye, and tracheal system of Drosophila have been shown to segregate signalling receptors on their surface, thus restricting the activation of signalling pathways in receiving cells (Roy, 2011). Hence, cytonemes, as conduits for signalling proteins, may be extended by receiving cells (and so are involved in uptake) or may be extended by producing cells (and so are involved in delivery and release) (Rojas-Ríos, 2012).

Interfering with actin polymerisation in adult niches leads to a significant reduction in the number of cap cells (CpCs) growing Hh cytonemes, concomitant with precocious stem cell differentiation, demonstrating that these actin-rich structures are required to prevent stem cell loss and thus are functionally relevant. Importantly, because this study disturbed actin dynamics in post-mitotic CpCs that still produce wild-type levels of Hh protein and express CpC markers (but fail to activate the Hh pathway in ECs), the observed effects on stem cell maintenance are most likely specific to Hh delivery from CpCs to their target ECs via short cytonemes. This interpretation is further reinforced by the observation that CpCs can sense decreased Hh levels and/or a dysfunction in the transduction of the Hh pathway in the niche and respond to it by growing Hh-rich membrane bridges up to 6-fold longer than in controls. In this regard, it is interesting to note that the two lipid modifications found in mature Hh protein act as membrane anchors and give secreted Hh a high affinity for membranes and signalling capacities. In fact, it has been recently described that a lipid-unmodified form of Hh unable to signal does not decorate filopodia-like structures in the wing imaginal disc epithelium, confirming the link between Hh transport along cytonemes and Hh signalling (Callejo, 2011). Thus, cytonemes may ensure specific targeting of the Hh ligand to the receiving germline cells in a context of intense signalling between niche cells and the GSCs. Interestingly, in both en- and smo- mosaic niches, the long processes projected towards the signalling-deficient area of the niche, which showed that competent CpCs sense the strength of Hh signalling activity in the microenvironment. While the nature of the signal perceived by the CpCs or the receptor(s) involved in the process are unknown, it is postulated that Hh-decorated filopodial extensions represent the cellular synapsis required for signal transmission that is established between the Hh-producing cells (the CpCs) and the Hh-receiving cells (the ECs). In this scenario (and because Ptc, the Hh receptor, is a target of the pathway) the membranes of mutant ECs, in which the transduction of the pathway is compromised, contain lower Ptc levels. Thus, longer and perhaps more stable projections ought to be produced to allow proper signalling. In addition, the larger the number of en mutant cells (and hence the stronger the deficit in Hh ligand concentration or target gene regulation), the longer the cellular projections decorated with Hh, which indicates that the niche response is graded depending on the degree of signalling shortage (Rojas-Ríos, 2012).

Do the longer cytonemes found in mosaic germaria represent structures created de novo, or do they simply reflect a pre-existing meshwork of thin intercellular bridges that can regulate the amount of Hh protein in transit across them? Because an anti-Hh antibody was utilised to detect the cytonemes and all attempts to identify other markers for these structures have failed, it is not possible to discriminate between these two possibilities. In any case, since no increased was detected in Hh levels in wild-type CpCs that contained cytonemes relative to those that did not, it is clear that long filopodia do not arise solely by augmenting Hh production in the CpCs. Rather, if long cytonemes are not synthesised in response to a Hh signalling shortage and if they already existed in the niche, they ought to restrict Hh spreading independently of significant Hh production. Furthermore, because the strength of Hh signalling in the niche determines the distance of Hh spreading, either cytoneme growth or Hh transport (or both) are regulated by the ability of the CpCs to sense the Hh signalling output (Rojas-Ríos, 2012).

The demonstration that a challenged GSC niche can respond to insufficient signalling by the cytoneme-mediated delivery of the stem cell survival factor Hh over long distances has wider implications. Niche cells have been shown to send cellular processes to their supporting stem cells in several other scenarios: the Drosophila ECs of the ovary and the lymph gland, the ovarian niche of earwigs, and the germline mitotic region in the hermaphrodite Caenorhabditis elegans. Similarly, wing and eye disc cells project cytonemes to the signalling centre of the disc. However, definitive proof that the thin filopodia described in the lymph gland, the earwig ovary, or imaginal discs deliver signals from the producing to the effector cells is lacking. The current findings strongly suggest that cytonemes have a role in transmitting niche signals over distance, a feature that may underlie the characteristic response of more complex stem cell niches to challenging physiological conditions. Careful analysis of the architecture of sophisticated niches, such as the bone marrow trabecular zone for mouse haematopoietic stem cells, will be needed to further test this hypothesis and to determine whether it represents a conserved mechanism for stem cell niche signalling (Rojas-Ríos, 2012).

Multiple regulatory safeguards confine the expression of the GATA factor serpent to the hemocyte primordium within the Drosophila mesoderm

Serpent (srp) encodes a GATA-factor that controls various aspects of embryogenesis in Drosophila, such as fatbody development, gut differentiation and hematopoiesis. During hematopoiesis, srp expression is required in the embryonic head mesoderm and the larval lymph gland, the two known hematopoietic tissues of Drosophila, to obtain mature hemocytes. srp expression in the hemocyte primordium is known to depend on snail and buttonhead, but the regulatory complexity that defines the primordium has not been addressed yet. This study found that srp is sufficient to transform trunk mesoderm into hemocytes. Two disjoint cis-regulatory modules were identified that direct the early expression in the hemocyte primordium and the late expression in mature hemocytes and lymph gland, respectively. During embryonic hematopoiesis, a combination of snail, buttonhead, empty spiracles and even-skipped confines the mesodermal srp expression to the head region. This restriction to the head mesoderm is crucial as ectopic srp in mesodermal precursors interferes with the development of mesodermal derivates and promotes hemocytes and fatbody development. Thus, several genes work in a combined fashion to restrain early srp expression to the head mesoderm in order to prevent expansion of the hemocyte primordium (Spahn, 2013).

Interactive Fly, Drosophila even-skipped: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Post-transcriptional regulation | Protein interactions | Developmental Biology | Effects of Mutation | References

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