Targets of Activity

Expression of hairy stripes can be generated in a two-step process involving regulatory interactions between the primary pair rule genes hairy and runt. Expression of h stripes 3 and 4 is directed by a common cis-acting element that results in an initial broad band of gene expression covering three stripe equivalents. Subsequently, this expression domain is split by repression in the forthcoming interstripe region, a process mediated by a separate cis-acting element that responds to Runt activity (Hartmann, 1994).

The so called primary pair-rule genes are involved in refinement rather than establishment of the fushi tarazu stripes. The order of appearance of ftz stripes has no relationship 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 in the absence of primary pair-rule genes only after cellularization (Yu, 1995).

Transient over-expression of runt under the control of a Drosophila heat-shock promoter caused stripe-specific defects in the expression patterns of the pair-rule genes hairy and even-skipped but had a more uniform effect on the secondary pair-rule gene fushi tarazu. The expression of the gap segmentation genes, upstream of runt in the segmentation hierarchy is also altered in heat shock/runt embryos. A subset of these effects are interpreted as due to an antagonistic effect of Runt on transcriptional activation by the maternal morphogen bicoid. Regulation of gap gene expression by runt is a normal component of the regulatory program that generates the segmented body pattern of the Drosophila embryo (Tsai, 1994).

Runt and Hairy act on ftz through fDE1, a common 32 base-pair element. The pair-rule expression of reporter gene constructs containing multimerized fDE1 elements depends on activation by Runt and repression by Hairy. Examination of reporter genes with mutated fDE1 elements provides further evidence that this element mediates both transcriptional activation and repression. Genetic experiments indicate that the opposing effects of runt and hairy are not due solely to cross-regulatory interactions between these two genes and that fDE1-dependent expression is regulated by factors in addition to runt and hairy (Tsai, 1995).

The runt gene is required to generate asymmetries within parasegmental domains. 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. Two remarkably different segmental phenotypes are generated as a consequence of these effects. The positioning of EN stripes is largely determined by the actions of negative regulators. 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).

Do Hairy and Runt repress target gene transcription independently of DNA binding, or as promoter bound regulators? Hairy-related transcriptional repressors show similar basic and HLH domains, and all terminate with an identical C-terminal tetrapeptide (WRPW), mutations of which largely or completely abolish repressor activity. It has proved difficult to define the precise molecular mechanism of Hairy action during segmentation. Although Hairy's embryonic patterning activity requires an intact basic (DNA binding) domain, none of the sequences in fushi tarazu promoter implicated in ftz repression by Hairy contain Hairy consensus binding sites. It is uncertain whether Runt acts primarily as a gene repressor or activator, as it behaves as a repressor of even-skipped and as an activator of fushi tarazu. In order to explore the ability of Hairy and Runt to act as promoter-bound transcriptional regulators, heterologous transcriptional activation domains (Act) were substituted for the WRPW repression domain (of Hairy) and the activation domain of Runt and the effects of such substitution were examined on presumed targets of Hairy and Runt. Expression of Hairy-Act during the blastoderm stage disrupts embryonic segmentation by driving ectopic expression of ftz, runt and odd-skipped. Activation depends on an intact basic domain, indicating that direct regulation occurs via sequence-specific binding to DNA. Expression of Runt-Act during the blastoderm stage likewise drives ectopic even-skipped, and shows that the normal apparent activation of fushi-tarazu by Runt is indirect, suggesting that Runt acts predominantly as a repressor. Hairy-Act has also been used to study sex determination. Ectopic Hairy mimics the activity of Deadpan in repressing early Sex-lethal transcription. Expression of Hairy-Act activates Sxl and causes male lethality, implying that Deadpan recognizes the Sxl promoter directly, and excludes models for Sxl regulation in which DPN functions as a passive repressor (Jiménez, 1996).

Ectopic expression of the pair-rule gene runt in the anterior end of the Drosophila embryo antagonizes transcriptional activation of the head gap gene orthodenticle (otd) by the anterior morphogen bicoid. The relevance of runt's activity as a repressor of otd in normal Drosophila embryogenesis has been investigated. otd expression is activated in the posterior region of embryos that are mutant for runt. This posterior expression domain of otd depends on the activity of the orphan nuclear receptor protein Tailless. Repression of otd by runt does not require the conserved VVVRPY motif, which mediates interaction between Runt and the co-repressor protein Groucho. It is speculated that the genetic interactions between runt and tll involve physical interactions between the two proteins. It is interesting to note that interactions between Runt and another orphan nuclear receptor protein, Ftz-F1 have been invoked to explain runt's regulation of the pair-rule gene fushi tarazu. However, in this case runt functions to activate, rather than repress Ftz-F1 dependent transcription. It will be interesting to determine if there are binding sites for Tll that are essential for the activation of otd in the posterior region and whether these sites respond to the repressive activity of runt. It is noted that the activity of tll is necessary, but not sufficient for otd expression in the posterior region of the embryo. The observed functional interactions between runt and tailless on otd expression may indicate there are other contexts where members of these two families of transcriptional regulators interact to regulate gene expression during development (Tsai, 1998).

The X-linked gene runt plays a role in the regulation of Sex lethal. Reduced function of runt results in female-specific lethality and sexual transformation of XX animals that are heterozygous for Sxl. The presence of a loss-of-function runt mutation masculinizes triploid intersexes. However, runt duplications cause a reduction in male viability by ectopic activation of Sex-lethal/runt is needed for the initial step of Sex-lethal activation, but does not have a major role as an X-counting element (Torres, 1992).

Three X-linked genes have been identified (scute, sisterless-a and runt) that determine the initial functional state of Sex lethal in the soma. These three X-linked genes do not seem to be required to activate Sex-lethal in the germ line (Granadino, 1993).

Runt functions as a transcriptional regulator in multiple developmental pathways in Drosophila melanogaster. Recent evidence indicates that Runt represses the transcription of several downstream target genes in the segmentation pathway. runt also functions to activate transcription. This paper documents the direct activation of Sex-lethal transcription by the Drosophila Runt protein. The initial expression of the female-specific sex-determining gene Sex-lethal in the blastoderm embryo requires runt activity. Male embryos mutant for deadpan show ectopic activation of Sxl expression, preferentially within the central, pre-segmented region of the embryo. Thus, it is possible that a major role for runt in the regulation of Sxl transcription is to counteract repression by dpn. Groucho is required to repress Sxl in male embryos. Thus it is possible that Runt bound to Sxl interacts with Groucho in a manner that blocks Groucho-mediated repression (Kramer, 1999 and references).

In situ hybridization was used to define the earliest effects of runt on transcription from the Sxl early embryonic promoter (SxlPe). Wild-type female embryos containing a SxlPe:lacZ reporter gene begin to express lacZ transcripts during the syncitial nuclear division cycles preceding formation of the cellular blastoderm. Expression at nuclear division cycle 12 is observed in punctate dots distributed throughout the embryo except in pole cells. Later, this expression is seen as uniform staining throughout the embryo except in pole cells. Females homozygous for the amorphic runtLB5 mutation fail to express the SxlPe:lacZ reporter gene within a broad central region of the embryo. This defect is observed concomitant with the earliest detectable expression of this reporter gene, demonstrating an early requirement for runt in SxlPe activation. The alterations in Sxl expression observed in runt mutants correspond well to the initial expression of runt in a broad central domain of syncitial blastoderm stage embryos. This expression precedes the formation of the seven-stripe pair-rule pattern during cellularization, suggesting that runt’s function in Sxl activation can be temporally separated from its role in segmentation. To test this idea, a temperature-sensitive runt mutation, runtYP17, was used. Female embryos homozygous for runtYP17 display normal SxlPe expression when reared and collected at the permissive temperature. At the restrictive temperature of 29°C, these embryos show non-uniform SxlPe expression identical to that observed in embryos deleted for runt. To examine runt’s effects on segmentation, the expression pattern of the segment polarity gene engrailed (en) was examined in these embryos. In runtYP17 embryos maintained at 18°C, En is expressed in a regular, well-spaced 14-stripe pattern, whereas at 29°C this pattern is disrupted. In collections of embryos aged at the non-permissive temperature for two hours and then shifted to the permissive temperature, female embryos with the abnormal SxlPe expression pattern typical of runt mutants show normal En expression. In reciprocal temperature-shift experiments, female embryos, aged at the permissive temperature to the cellular blastoderm stage and then shifted to the non-permissive temperature, show normal SxlPe expression and abnormal En expression. These results demonstrate that runt’s role in the activation of SxlPe is temporally distinct from and precedes the requirement for runt in segmentation, and provide strong evidence that runt’s role as an activator of Sxl transcription occurs prior to cellularization, during the earlier syncitial blastoderm stages of Drosophila embryogenesis (Kramer, 1999).

Consistent with a role as a direct activator, Runt shows sequence-specific binding to multiple sites in the Sex-lethal early promoter. The early regulation of Sxl transcription by runt is readily explained if Runt interacts directly with the Sxl early promoter to activate transcription. Previous work has identified a 1.1 kb fragment of the SxlPe promoter that contains sequences essential for sex-specific transcriptional activation. A test was performed for direct interactions between Runt and these DNA sequences. Probes that span this DNA fragment were generated and tested in electrophoretic mobility-shift assays (EMSAs). Runt binds only weakly to each of these DNA fragments. However, upon addition of the Brother partner protein (Bro, a homolog of mammalian PEBP2/CBF beta, a protein unrelated to Runt), multiple complexes are obtained with each of these probes. These complexes are Runt-dependent as they are not detected when only Bro protein is added. Competition with a bona fide CBF-binding site from the Polyoma enhancer prevents detection of these complexes. Competition is not observed when a mutant CBF-binding site is used, indicating that the binding is sequence specific. Recombinant mammalian CBF also recognizes multiple sites within these fragments from the SxlPe promoter. Inspection of the sequence for matches to the consensus CBF-binding sequence TG(T/C)GGT(T/C) has identified ten sites that match this consensus at positions two through five that also match at least one of the three other, less critical positions. Interestingly, no perfect matches to the consensus are found. The presence of multiple binding sites is consistent with the hypothesis that activation of Sxl transcription involves direct interactions between Runt and the Sxl promoter. One prediction of this hypothesis is that Runt’s DNA-binding activity should be required for Sxl activation: an in vitro assay shows this to be true (Kramer, 1999).

The 128 amino acid Runt domain confers sequence-specific DNA binding as well as heterodimerization with Brother, Runt's cofactor. As an initial test of the importance of Runt’s DNA-binding domain, a form of runt that is deleted for its Runt domain, runtdeltaRD was injected into the central region of female homozygous runtLB5 embryos. No evidence of rescue is seen in runtdeltaRD-injected embryos, indicating that the DNA-binding domain is important for runt’s function as an activator of SxlPe. However, since this is a large deletion, the effects could be attributed to improper folding and/or protein stability. Random- and site-directed mutagenesis experiments have identified several amino acids within the Runt domain that specifically affect DNA binding without disrupting association with the partner protein CBFbeta. Two conserved amino acids in Runt that are important for DNA binding correspond to a cysteine at position 127 and a lysine at position 199. In order to obtain a DNA-binding-defective form of Runt, a protein containing mutations at both of these sites (C127S, K199A) was generated. The DNA-binding activity of this mutant was compared with that of wild-type Runt in EMSAs with the high-affinity CBF-binding site from the Polyoma virus enhancer. The mutant protein, Runt[CK] shows only very low levels of complex formation on this DNA, and this is only in the presence of Brother. Similar experiments with a DNA probe from the Sxl promoter confirm the reduced DNA-binding activity of Runt[CK]. It is estimated that these mutations reduce DNA-binding affinity at least 50-fold. The observation that Brother stimulates DNA binding by Runt[CK] suggests that the two mutations do not disrupt interaction between the Runt and Brother proteins. Thus, these two mutations specifically impair DNA binding without affecting the overall structure of the Runt domain. The mRNA injection assay was used to examine the in vivo activity of this DNA-binding- defective form of Runt, and no evidence for rescue of SxlPe expression was found in runt mutant female embryos. These results are consistent with the hypothesis that Runt activates Sxl transcription by binding to sequences in the SxlPe promoter. Additional experiments further reveal that increasing the dosage of runt alone is sufficient for triggering the transcriptional activation of Sex-lethal in males. In addition, a Runt fusion protein, containing a heterologous transcriptional activation domain activates Sex-lethal expression, indicating that this regulation is direct and not via repression of other repressors. A small segment of the Sex-lethal early promoter that contains Runt-binding sites mediates Runt-dependent transcriptional activation in vivo (Kramer, 1999).

Although the truncated reporter gene (SxlPe0.4kb:lacZ), isolated from the proximal 400 basepair fragment of SxlPe, exhibits an abnormal pattern of expression in wild-type females, with higher levels found in the anterior and posterior, the expression is sex-specific. There are several putative Runt-binding sites found within this 400 bp fragment. Deletion of a small 70 bp segment within this fragment, which contains at least two putative binding sites for Runt, results in a loss of SxlPe expression. Conversely, a reporter gene that contains multiple copies of this segment, SxlPeGOF:lacZ, is expressed at high levels in WT female embryos. Interestingly, the SxlPeGOF:lacZ reporter gene is also expressed in males, however, at much lower levels and not in the anterior regions of the embryo. EMSA with Runt and Brother proteins demonstrates that Runt binds to sequences within this small segment. This interaction is sequence specific as it is competed by a DNA fragment from the Polyoma enhancer containing a wild-type CBF-binding site, but not by a similar DNA fragment with a mutant CBF-binding site. The differential expression in female and male embryos indicates that this reporter gene retains the ability to respond to numerator gene dosage. The observation that this transgene is expressed in males suggests that the activation mediated by multimerization of this small segment of DNA is sufficient to overcome the repression that is normally established in males for the parental SxlPe0.4kb:lacZ reporter gene. Furthermore, the preferential expression within the segmented region of the embryo strongly suggests that this reporter gene is responding to runt. To test this, SxlPeGOF:lacZ expression was examined in embryos mutant for runt. Expression is reduced in most, but not all, regions of runt mutant male embryos. Thus, the region that is multimerized in the SxlPeGOF:lacZ reporter gene mediates runt-dependent transcriptional activation (Kramer, 1999).

Low-level ectopic expression of the Runt transcription factor blocks activation of the Drosophila melanogaster segmentation gene engrailed (en) in odd-numbered parasegments and is associated with a lethal phenotype. By using a genetic screen for maternal factors that contribute in a dose-dependent fashion to Runt-mediated repression, it is shown that there are two distinct steps in the repression of en by Runt. The initial establishment of repression is sensitive to the dosage of the zinc-finger transcription factor Tramtrack. By contrast, the co-repressor proteins Groucho and dCtBP, and the histone deacetylase Rpd3, do not affect establishment but instead maintain repression after the blastoderm stage. The distinction between establishment and maintenance is confirmed by experiments with Runt derivatives that are impaired specifically for either co-repressor interaction or DNA binding. Other transcription factors can also establish repression in Rpd3-deficient embryos: this indicates that the distinction between establishment and maintenance may be a general feature of eukaryotic transcriptional repression (Wheeler, 2002).

Ftz modulates Runt-dependent activation and repression of segment-polarity gene transcription

A crucial step in generating the segmented body plan in Drosophila is establishing stripes of expression of several key segment-polarity genes, one stripe for each parasegment, in the blastoderm stage embryo. It is well established that these patterns are generated in response to regulation by the transcription factors encoded by the pair-rule segmentation genes. However, the full set of positional cues that drive expression in either the odd- or even-numbered parasegments has not been defined for any of the segment-polarity genes. Among the complications for dissecting the pair-rule to segment-polarity transition are the regulatory interactions between the different pair-rule genes. An ectopic expression system that allows for quantitative manipulation of expression levels was used to probe the role of the primary pair-rule transcription factor Runt in segment-polarity gene regulation. These experiments identify sloppy paired 1 (slp1), most appropriately classified as segment polarity genes, as a gene that is activated and repressed by Runt in a simple combinatorial parasegment-dependent manner. The combination of Runt and Odd-paired (Opa) is both necessary and sufficient for slp1 activation in all somatic blastoderm nuclei that do not express the Fushi tarazu (Ftz) transcription factor. By contrast, the specific combination of Runt + Ftz is sufficient for slp1 repression in all blastoderm nuclei. Furthermore Ftz is found to modulate the Runt-dependent regulation of the segment-polarity genes wingless (wg) and engrailed (en). However, in the case of en the combination of Runt + Ftz gives activation. The contrasting responses of different downstream targets to Runt in the presence or absence of Ftz is thus central to the combinatorial logic of the pair-rule to segment-polarity transition. The unique and simple rules for slp1 regulation make this an attractive target for dissecting the molecular mechanisms of Runt-dependent regulation (Swantek, 2004).

The role of Runt as a primary pair-rule gene complicates interpreting the alterations in segment-polarity gene expression that are observed in run mutants. Recent experiments utilizing a GAL4-based NGT-expression system [the transgene construct used to express GAL4 maternally contains the nanos promoter and the 3' untranslated region of an alpha-tubulin mRNA and is thus referred to as an NGT transgene (nanos-GAL4-tubulin)] to manipulate expression in the blastoderm embryo have demonstrated that low levels of Runt repress en in odd-numbered parasegments without altering expression of the pair-rule genes eve and ftz. This observation suggested that this approach might provide a useful tool for defining the role of Runt in regulating other segment-polarity genes. A systematic survey was undertaken of the response of other segmentation genes to increasing levels of NGT-driven Runt expression. These experiments revealed significant differences in sensitivity as well as interesting differences in the nature of the response of different genes to ectopic Runt. The odd-numbered en stripes are repressed at both intermediate and high levels of ectopic runt. After en, the second most sensitive target is slp1. This gene shows a partially penetrant and subtle defect in the spacing of the segmentally repeated stripes in embryos with low levels of NGT-driven Runt. A more pronounced alteration is obtained in embryos with intermediate levels of Runt. In these embryos the slp1 pattern is converted from a segment-polarity-like, 14-stripe pattern to a pair-rule-like, seven-stripe pattern. At this level, expression of other segmentation genes is normal although there are subtle changes in the spacing of the wg stripes and a partial loss of the odd-numbered hh stripes. All three of these genes show clearer alterations at higher levels of NGT-driven Runt, with wg responding in a manner similar to slp1 and hh responding in a manner similar to en. High Runt levels also produce spacing defects in the expression of odd and gsb, as well as a more subtle effect on prd. Several of the changes observed at high levels of ectopic Runt are likely to be indirect and due to alterations in the expression of eve, ftz and hairy. The response of slp1 to ectopic Runt is notable both because of its sensitivity and apparent simplicity, thus suggesting that Runt plays a pivotal role in regulating slp1 transcription (Swantek, 2004).

The differential combinatorial effects of Runt and Ftz on segment-polarity gene regulation emerged as a result of analyzing the sensitive and relatively simple response of slp1 to ectopic Runt. The slp1 transcription unit is one of two redundant genes that comprise the slp locus. This locus was initially characterized as having a pair-rule function in the segmentation gene hierarchy based on a weak pair-rule phenotype associated with loss of slp1 function. The slp1 and slp2 genes are expressed in similar patterns during early embryogenesis. Embryos deficient for both slp1 and slp2 have an unsegmented lawn cuticle phenotype similar to that produced by wg mutations. This raises the question of whether it is most appropriate to consider slp as a pair-rule or segment-polarity locus. In the most straightforward interpretation of the segmentation hierarchy, the role of the pair-rule genes is to establish the initial metameric expression patterns of the segment-polarity genes. The initial expression of the key segment polarity genes en and wg is normal in gastrula stage embryos that are deleted for both slp1 and slp2. The expression of wg begins to become abnormal and is lost during early germband extension. These observations are consistent with the proposal that slp expression identifies cells that are competent to maintain wg expression subsequent to the blastoderm stage. Based on these observations, it is concluded that slp1 and slp2 are most appropriately classified as segment polarity genes, not pair-rule genes (Swantek, 2004).

The expression of slp1 (and slp2) differs from several other segment-polarity genes in that the metameric pattern is comprised of two-cell wide, rather than single-cell wide stripes. These two cell-wide stripes comprise the posterior half of each parasegment. slp1 activation in odd-numbered parasegments requires the cooperative action of Runt and Opa, whereas in even-numbered parasegments Runt works together with Ftz to repress slp1 expression. The simple rules involving these three factors fully account for slp1 regulation in all of the Runt-expressing cells in the blastoderm embryo but also raise a question regarding the positional cues that regulate slp1 expression in cells that do not express Runt (Swantek, 2004).

There are four other pair-rule transcription factors that could be involved in slp1 regulation: Eve, Hairy, Odd and Prd. Expression of both Odd and Prd overlaps the slp1 stripes in a manner that suggests that neither of these factors provides positional information crucial for slp1 regulation. Consistent with this, there are no substantial changes in the early 14-striped slp1 pattern in embryos mutant for either odd or prd. By contrast, elimination of either Eve or Hairy leads to changes in both the number and spacing of the slp1 stripes. However, as these are both primary pair-rule genes some of these changes are certainly indirect and due to alterations in Runt and Ftz expression (Swantek, 2004).

Several lines of evidence indicate that Eve has a direct role in slp1 repression. Experiments with the temperature-sensitive eve[ID19] mutation indicate that transient elimination of Eve at the cellular blastoderm stage leads to expanded six cell-wide slp1 stripes because of de-repression in the anterior two cells of each odd-numbered parasegment. These two are the cells with the highest level of Eve, indicating that the primary role of Eve at this stage is to repress slp1 expression. Complementary experiments with an inducible hs-Eve transgene reveal that ectopic Eve blocks slp1 activation in both odd- and even-numbered parasegments. This result not only confirms Eve's role as a repressor, but also reveals a crucial difference between Eve and Ftz-dependent repression. Ftz-dependent repression is restricted to odd-numbered parasegments unless Runt is also ectopically expressed. This same restriction is observed in experiments with hs-Ftz transgenes, indicating that the difference between Eve and Ftz is not due to the mode of ectopic expression. Taken altogether these results indicate that Eve and Ftz normally have comparable roles in repressing slp1 transcription in the anterior half of the odd- and even-numbered parasegments, respectively, in late blastoderm stage embryos. The key distinction in the regulation of slp1 by these two homeodomain transcription factors is the critical role that Runt plays in Ftz-dependent repression (Swantek, 2004).

One aspect of slp1 expression not accounted for by the above rules is the factor (or combination of factors), referred to here as factor X, that is responsible for slp1 activation in the posterior half of the even-numbered parasegments. Activation in these cells is blocked either by the combination of Runt+Ftz or by ectopic Eve. Runt and Ftz are co-expressed anterior to these even-numbered stripes and presumably both play a role in defining the anterior margin of these stripes. Conversely, Eve is expressed posterior to these cells and probably has a role in defining the posterior margins of these stripes. The sole pair-rule transcription factor that remains as a candidate for Factor X is Hairy, which is expressed in the posterior half of even-numbered parasegments. However, it is not thought that factor X is Hairy for several reasons. All of the evidence to date indicates that Hairy functions as a repressor. Furthermore, NGT-driven expression of Hairy does not lead to slp1 activation in anterior blastoderm cells similar to that produced by the co-expression of Runt and Opa. Identification of factor X is clearly important for a complete understanding of slp1 regulation (Swantek, 2004).

Previous studies have indicated that Runt has roles in both activating and repressing transcription of different target genes in the Drosophila. The current results provide additional compelling evidence for this dual activity and also provide insight on factors that contribute to this context-dependent regulation. The dramatic effects of Ftz on Runt-dependent slp1 regulation clearly demonstrate that one important component of context is the specific combination of other transcription factors that are present in a cell. Indeed, the unique and relatively simple rules for slp1 regulation make this an especially attractive target for dissecting the molecular mechanisms whereby Ftz converts Runt from an activator to a repressor of transcription. It seems likely that the rules governing the Runt-dependent regulation of slp1 will provide a foundation for understanding the regulation of wg and gsb, two segment-polarity genes that are expressed in a subset of slp-expressing cells and that respond to Runt in a manner similar, but not identical to slp1 (Swantek, 2004).

The results also point to a second important component of context-dependent regulation by Runt. The specific combination of Runt + Ftz, which represses slp1, does not always give repression, since these same two factors work together to activate en in some of these same cells at the same stage of development. Thus, cellular context alone cannot fully account for the regulatory differences and there must be a target-gene specific component of context-dependent regulation. A similar gene-specific example of context-dependent regulation has recently been described for the Runx protein Lozenge. In this case, the presence of binding sites for the Cut homeodomain protein helps to stabilize a complex that leads to repression of deadpan transcription in the same cells in which Lozenge is responsible for activation of Drosophila Pax2. In a strict parallel of this model, it would be speculated that the slp1 regulatory region contains binding sites for some factor that helps to stabilize a repressor complex that includes the Runt and Ftz proteins. In a reciprocal, and not mutually exclusive model, perhaps there are binding sites for a factor in the en regulatory region that helps to stabilize a Runt- and Ftz-dependent transcriptional activation complex. Further studies on the en and slp1 cis-regulatory regions are needed in order to address these questions at the molecular level. This future work is crucial for understanding the context-dependent activity of Runt and thus the molecular logic of the control system that underlies the pair-rule to segment-polarity transition in Drosophila segmentation (Swantek, 2004).

The HMG-box protein Lilliputian is required for Runt-dependent activation of the pair-rule gene fushi-tarazu

lilliputian, the sole Drosophila member of the FMR2/AF4 (Fragile X Mental Retardation/Acute Lymphoblastic Leukemia) family of transcription factors, is widely expressed with roles in segmentation, cellularization, and gastrulation during early embryogenesis with additional distinct roles at later stages of embryonic and postembryonic development. This study identified lilli in a genetic screen based on the suppression of a lethal phenotype that is associated with ectopic expression of the transcription factor encoded by the segmentation gene runt in the blastoderm embryo. In contrast to other factors identified by this screen, lilli appears to have no role in mediating either the establishment or maintenance of engrailed (en) repression by Runt. Instead, it was found that Lilli plays a critical role in the Runt-dependent activation of the pair-rule segmentation gene fushi–tarazu (ftz). The requirement for lilli is distinct from and temporally precedes the Runt-dependent activation of ftz that is mediated by the orphan nuclear receptor protein Ftz-F1. A role is described for lilli in the activation of Sex-lethal (Sxl), an early target of Runt in the sex determination pathway. However, lilli is not required for all targets that are activated by Runt and appears to have no role in activation of sloppy paired (slp1). Based on these results it is suggested that Lilli plays an architectural role in facilitating transcriptional activation that depends both on the target gene and the developmental context (Vanderzwan-Butler, 2006).

This study uncovered Lilli's role in Runt-dependent transcriptional regulation based on the identification of lilli mutations as dose-dependent suppressors of the lethality produced by threshold levels of NGT-driven Runt expression. In contrast to all of the other Runt-interacting genes and deficiency intervals identified as suppressors in this genetic screen, a reduction in maternal lilli dosage has no effect on either the establishment or maintenance of Runt-dependent en repression. The target of Runt that provides the most dramatic and clearest evidence for a functional interaction between runt and lilli is the pair-rule gene ftz. It is notable that the ftz expression is not discernibly altered by the relatively low levels of NGT-driven Runt used in the genetic screen. This raises a question regarding the basis for lilli acting as a dose-dependent suppressor of the lethality associated with ectopic Runt expression. One explanation is that there are subtle changes in ftz expression at the threshold levels of NGT-driven Runt used in the viability assays that contribute to lethality. A second possibility is that there are other targets of Runt and Lilli that contribute to the lethality associated with ectopic Runt expression. Although Sxl would seem to be one obvious candidate for such a target, the developmental window for Sxl activation occurs prior to the stage during which the NGT-drivers are useful for manipulating gene expression. Indeed, it has not been possible to detect activation of Sxl by NGT-driven Runt, even at levels that are tenfold higher than the levels used in the genetic screen. Finally, it is possible that the effect on viability is in part due to a non-specific effect of Lilli on GAL4-dependent activation of Runt. There is some evidence for non-specificity, especially with Df(2L)C144; however, there is also a clear suppression of lethality with other lilli alleles that do not show a comparable reduction in NGT-driven UAS-lacZ expression. Thus it seems likely that the identification of lilli is due to a combination of specific and non-specific effects on the lethality produced by NGT-driven Runt expression. If this interpretation is correct, then it also seems likely that other deficiency intervals that were eliminated from further consideration due to apparent non-specific effects may in fact have specific and interesting effects that would be revealed by more directly assaying the effects of these mutations on the responses of different targets to NGT-driven Runt expression (Vanderzwan-Butler, 2006).

These observations confirm and extend findings regarding a role for Lilli in the transcriptional activation of the pair-rule gene ftz (Tang, 2001). Lilli does not appear to have any role in regulating other pair-rule genes, and the effects of eliminating maternal Lilli on segment-polarity gene expression have been interpreted as an indirect effect due to the loss of Ftz (Tang, 2001). Thus ftz appears to stand out as the sole gene in the segmentation pathway that shares a requirement for both Lilli and Runt. The previous work from Tang did identify two other targets for Lilli in the early Drosophila embryo, serendipity-α (sry-α) and huckebein (hkb). There is no evidence that either of these genes is regulated by Runt. Thus, just as there are targets of Runt in the segmentation pathway whose regulation is Lilli-independent, there are also targets of Lilli that do not involve interactions with Runt (Vanderzwan-Butler, 2006).

This work adds Sxl as an additional candidate target for Lilli. The elimination of maternal Lilli interferes with the activation of the SxlPE:lacZ reporter gene in all somatic cells of the female embryo. This global effect stands in contrast to the more localized role of Runt which is only required for Sxl activation in cells within the pre-segmental region of female embryos. The failure in Sxl activation observed in the absence of maternal Lilli is similar to the phenotype of embryos that are mutant for either sisA or sisB. Indeed the possibility is considered that the primary defect in lilli germline clone embryos was the failure to activate either of these two X-chromosome linked numerators. Expression of sisA was found to be normal in lilli germline clone embryos (VanderZwan, 2003). The low level of sisB expression in wild-type embryos made it difficult to unambiguously determine whether lilli was important for sisB activation. To further investigate the role of Lilli in Sxl activation the expression of the SxlGOF:lacZ reporter gene was examined. Both Runt and SisB contribute to the ectopic expression of this reporter in male embryos. The elimination of maternal Lilli has a more severe effect on the expression of the SxlGOF:lacZ reporter than is observed in embryos that are mutant for either runt or sisB (VanderZwan, 2003). The most straightforward interpretation of these results is that Lilli is directly involved in the transcriptional activation of the early embryonic Sxl promoter, in this case in cooperation with the four different X-linked factors that are responsible for the sex-specific expression of Sxl in female embryos (Vanderzwan-Butler, 2006).

The inclusion of Sxl gives four putative direct targets of Lilli in the Drosophila embryo. These four genes, Sxl, ftz, sry-α and hkb are normally activated at very early stages, and in all four cases this activation is reduced, if not abrogated, in the absence of maternally provided lilli. The notion that Lilli functions primarily in activation is consistent with observations on the properties of the mammalian homologs FMR2 and LAF4 (Hillman, 2001). However, early activation is clearly not the sole identifying characteristic of Lilli's targets. Indeed, for three of these targets, there are other genes in the same developmental pathway that are activated at the same time that do not require Lilli. In the cellularization pathway, Lilli is required for expression of sry-α but has no role in the activation of bottleneck (bnk) or nullo (Tang, 2001). In the segmentation pathway, the gap gene hkb is expressed in the anterior and posterior poles in response to signaling by the terminal pathway. Elimination of maternal Lilli greatly reduces hkb expression, but has no obvious effect on tailless (tll), another gap gene that is activated at the same stage in response to the terminal signaling pathway (Tang, 2001). Finally, the requirement for maternally provided Lilli that is observed for ftz is not shared with the pair-rule segmentation genes eve, hairy and runt (Tang, 2001). This last observation is perhaps most important as the wealth of information that exists on pair-rule gene regulation provides a useful framework for further considering the potential attributes of Lilli-dependent targets (Vanderzwan-Butler, 2006).

Elimination of maternal Lilli reduces, but does not eliminate ftz expression. The reduced expression that remains is similar to what is obtained in embryos that lack Runt, and is presumed to be in response to other activating factors. The complications presented by these other factors are bypassed in experiments in which Runt is over-expressed, either by heat-shock or by NGT-driven expression. Indeed, the inability of ftz to respond to ectopic Runt in the absence of maternal Lilli provides very compelling evidence for the importance of Lilli in ftz activation (Vanderzwan-Butler, 2006).

Lilli is acutely required for mediating Runt-dependent activation of ftz during the blastoderm stage, and this requirement precedes the temporal requirements for Ftz-F1. Ftz-F1 was initially identified as a factor that interacts with sequences within the ftz 'zebra element', a 669-bp, promoter proximal element that drives early expression in response to gap and pair-rule gene transcription factors. However, subsequent studies revealed that Ftz-F1 plays a more significant role in mediating Ftz-dependent auto-regulation by the so-called 'upstream element' during the early stages of germ-band extension. The earlier requirement for Lilli strongly suggests it contributes to the early 'zebra element'-dependent activation of ftz in response to activating inputs from Runt (Vanderzwan-Butler, 2006).

What is the role of Lilli in mediating Runt-dependent activation? Directed yeast two-hybrid assays fail to detect direct interactions between the full length Runt and Lilli proteins. This observation suggests that other factors contribute to the functional interactions described above. A notable conserved feature that Lilli shares with its mammalian homologs is an HMG-box. This structural DNA-binding motif interacts with the minor groove of DNA and modulates DNA structure by bending. These properties have been interpreted to reflect an architectural role for HMG-box proteins in facilitating the formation of higher order chromatin structures that contribute to the regulation of gene expression. It seems likely that chromatin architecture is important for ftz zebra element function. Although the 'zebra element' was one of the very first cis-regulatory elements in the segmentation pathway to be described, there is not yet a clear understanding of the rules that govern its activity. This is in contrast to the relatively simple combinatorial rules that have been elucidated for several of the stripe-specific elements of the pair-rule genes eve, hairy, and runt. It is proposed that the difficulty in identifying discrete regulatory modules within the zebra element reflects the importance of chromatin architecture in conferring high-fidelity regulation of the zebra element in response to inputs from Runt and other gap and pair-rule transcription factors (Vanderzwan-Butler, 2006).

It is interesting to note that the cis-regulatory element responsible for the initial activation of Sxl shares several properties with the ftz zebra element. The minimal DNA element necessary to faithfully recapitulate the strong, early sex-specific activation of the Sxl promoter is 1.7 kb in size. As found for the ftz zebra element, smaller reporter gene constructs do not function properly, although sub-elements that confer sex-specific regulation, and augment this activation have been identified. The on/off regulation of Sxl is in response to a twofold difference in the activity of four different DNA-binding transcription factors. It is easy to imagine that chromatin architecture may be critical in sensing this twofold difference in a robust and reproducible manner. There is one further similarity shared by Sxl and ftz that is intriguing. The initial Lilli-dependent activation of both genes is followed by a second phase of gene expression that involves distinct cis-regulatory components. In the case of ftz, the switch is from regulation by the zebra element to regulation by the upstream element, whereas for Sxl the switch is from expression at the SxlPe promoter to expression at SxlM a different promoter that is activated in all somatic cells of both male and female embryos . Perhaps the unique requirements for Lilli reflect architecture-dependent regulatory elements that retain the ability to be rapidly re-organized in a developmentally dynamic manner. Further studies on the mechanisms by which Lilli participates in the activation of ftz and Sxl in the early Drosophila embryo should provide new insights on the role of chromatin architecture in developmentally regulated gene expression (Vanderzwan-Butler, 2006).

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).

A system of repressor gradients spatially organizes the boundaries of Bicoid-dependent target genes

The homeodomain (HD) protein Bicoid (Bcd) is thought to function as a gradient morphogen that positions boundaries of target genes via threshold-dependent activation mechanisms. This study analyzed 66 Bcd-dependent regulatory elements, and their boundaries were shown to be positioned primarily by repressive gradients that antagonize Bcd-mediated activation. A major repressor is the pair-rule protein Runt (Run), which is expressed in an opposing gradient and is necessary and sufficient for limiting Bcd-dependent activation. Evidence is presented that Run functions with the maternal repressor Capicua and the gap protein Kruppel as the principal components of a repression system that correctly orders boundaries throughout the anterior half of the embryo. These results put conceptual limits on the Bcd morphogen hypothesis and demonstrate how the Bcd gradient functions within the gene network that patterns the embryo (Chen, 2012).

This study identified 32 enhancers that respond to Bcd-dependent activation and form expression boundaries at different positions along the AP axis of fly embryos. Adding these elements to the 34 previously known enhancers constitutes the largest data set of in vivo-tested and -confirmed enhancers regulated by a specific transcription factor in all of biology (Chen, 2012).

The 32 confirmed enhancers were identified among 77 tested genomic fragments, which were selected because they showed in vivo-binding activity, or they conformed to a stringent homotypic-clustering model for predicted Bcd-binding sites, or both. All seven previously unknown fragments showing in vivo binding and a predicted site cluster directed Bcd-dependent transcription in the early embryo. Other fragments from the top 50 ChIP-Chip signals (which do not conform to the clustering model) were also very likely (21 of 26) to test positive in the in vivo test, but this likelihood drops significantly (9 of 25) in a set of fragments from lower on the list of ChIP-Chip fragments. Interestingly, of 19 tested fragments that contain clusters of predicted sites, but no in vivo binding activity, not a single one tested positive in vivo. These results suggest that in ;vivo binding assays are much better predictors of regulatory function than simple site-clustering algorithms alone (Chen, 2012).

One explanation for the failure of these predicted site clusters to bind Bcd in vivo is that they lie in heterochromatic regions of the genome that prevent site access. However, because they fail to function when taken out of their normal context (in reporter genes), whatever is preventing activation must be a property of the fragment itself and not its location in the genome. Interestingly, a number of Bcd site cluster-containing fragments drive expression later in development. It is proposed that these fragments fail to bind Bcd because they lack sites for cofactors that facilitate Bcd binding. In preliminary experiments it was observed that Bcd-activated fragments contain on average more binding sites for the ubiquitous activator protein Zelda (Zld) than those that fail to activate. Zld has been shown to be critical for timing the zygotic expression of hundreds of genes in the maternal to zygotic transition (Chen, 2012).

These results suggest strongly that a gradient of Run protein plays a major role in limiting Bcd-dependent activation. Run seems to work as part of a repression system that also includes Cic and possibly Kr. Expression boundaries in the region anterior to the presumptive cephalic furrow shift toward the posterior in run and cic mutants, and the double mutant causes boundaries that are normally well separated to collapse into a single position (Chen, 2012).

The use of multiple repressors permits flexibility in binding site architecture within enhancers that establish boundaries at similar positions. For example type I enhancers show overrepresentations of both Run and Cic sites, but 27% lack strong matches to the Cic PWM, and 12% lack strong matches to the Run PWM. Importantly, however, all type I enhancers lacking Cic sites contain Run sites, and those lacking Run sites contain Cic sites. Multiple Kr sites were observed in a large number of Bcd-dependent enhancers, which suggests that Kr is also a major component of the repression system that orders Bcd-dependent expression boundaries. Taken together, these data suggest that antagonistic repression of Bcd-mediated activation is a key design principle of the system that organizes the AP body plan. The repressors identified so far (Run, Cic, and Kr) are expressed in overlapping domains with gradients at different positions, consistent with the formation and ordering of a relatively large number of boundaries throughout the anterior half of the embryo (Chen, 2012).

The close linkage between repressor sites and Bcd sites within discrete enhancers suggests that repression occurs via short-range interactions that interfere directly with Bcd binding or activation. Interestingly, Cic also shows repressive effects that seem to be binding site independent. For example some type I enhancers do not contain recognizable Cic sites, but their expression boundaries expand posteriorly in cic mutants. This could be caused by the reduced expression of run and Kr in cic mutants. However, genetically removing both Kr and run causes a less dramatic expansion than that seen in the absence of cic. This suggests that Cic binds these enhancers via suboptimal sites or that it is required for the correct patterning of another unknown repressor. Another possibility is that these expansions are caused indirectly by changing the balance of MAPK phosphorylation events that control terminal patterning (Chen, 2012).

These results do not strictly falsify the Bcd morphogen hypothesis, but they support the idea that the Bcd gradient can establish only a 'rough framework that is elaborated by the interaction of the zygotic segmentation genes'. What is the nature of this framework, and what role does it play in the network that precisely positions target gene boundaries (Chen, 2012)?

One component of the system, the Cic repression gradient, is maternally produced and formed by downregulation at the poles via the terminal patterning system. This gradient is formed independently of Bcd but is critical for establishing boundaries of Bcd-dependent target genes. In contrast, Bcd is involved in activating the expression patterns of run and Kr and in repressing them in anterior regions. Both run and Kr expand anteriorly in bcd mutants. There is no evidence that Bcd functions directly as a transcriptional repressor, so these repressive activities are probably indirect. Previous work showed that the Bcd target gene gt is involved in setting the anterior Kr boundary, and it is hypothesized that another Bcd target gene, slp1, encodes a forkhead domain (FKH) protein that sets the anterior boundary of the early run pattern. slp1 is expressed in a pattern reciprocal to the run pattern and was previously shown to position the anterior boundaries of several pair-rule gene stripes including run stripe 1 (Chen, 2012).

These results suggest that a major function of the Bcd gradient is the differential positioning of two repressors, Slp1 and Gt, which set the positions of the Run and Kr repression gradients, which then feedback to repress Bcd-dependent target genes. How are slp1 and gt differentially positioned? One possibility is that slp1 and gt enhancers respond to specific concentrations within the Bcd gradient, consistent with the original model for morphogen activity. However, the fact that the slp1 and gt expression domains form boundaries at the same positions in embryos lacking the Cic and Run repressors argues against this model for these genes (Chen, 2012).

It was also shown that Bcd target genes normally expressed in cephalic regions form and correctly position posterior boundaries in embryos containing flattened Bcd gradients. Run is still expressed in these embryos, specifically in a domain that consistently abuts the boundaries of the anterior Bcd target genes, regardless of copy number. This suggests that a mutually repressive interaction between Slp1 and Run is maintained in these embryos but does not explain how these boundaries are consistently oriented perpendicularly to the AP axis. The answer might lie in the fact that the flattened Bcd gradients in these embryos are not completely flat but are present as shallow gradients with slightly higher levels in anterior regions. In these embryos the slight changes in concentration along the AP axis might cause a bias that enables the orientation of the mutual repression interaction. In wild-type embryos, Bcd is much more steeply graded, which makes this bias stronger and the boundary between these mutual repressors more robust (Chen, 2012).

These results suggest that antagonistic repression precisely orders Bcd-dependent expression boundaries. However, repression may not be required for the activity of all morphogens. For example the extracellular signal activin has been shown to activate target genes in a threshold-dependent manner in isolated animal caps from frog embryos. Also, a gradient of the transcription factor Dorsal (Dl) is critical for setting boundaries between different tissue types along the dorsal-ventral (DV) axis of the fly embryo. It is thought that the major mechanism in Dl-specific patterning is threshold-dependent activation, which is quite different from the system described in this paper. One major difference between Bcd and Dl is the number of boundaries specified: three for Dl and more than ten for Bcd. It is proposed that the robust ordering of more boundaries simply requires a more complex system (Chen, 2012).

In general, though, it seems that antagonistic mechanisms are involved in controlling the establishment or interpretation of most morphogen activities. For example in the Drosophila wing disc, the TGF-N2 signal Dpp forms an activity gradient that is refined by interactions with multiple extracellular factors. Also, in vertebrates the signaling activity of the extracellular morphogen Sonic hedgehog (Shh) is affected by positive and negative interactions with specific molecules on the surfaces of receiving cells (Chen, 2012).

There is some evidence that transcriptional repression is also used for refining the patterning activities of extracellular molecules. Dpp acts as a long-range morphogen that activates two major target genes (optomotor blind [omb] and spalt [sal]) in nested patterns with boundaries at different positions with respect to the source of Dpp. Although these boundaries could in theory be formed by differential responses to the morphogen, it is clear that the transcriptional repressor Brinker (Brk), which is expressed in an oppositely oriented gradient, also plays an important role. The Brk gradient is itself positioned by Dpp activity in a manner analogous to positioning of the Run and Kr repressor gradients by Bcd. Also, a similar transcriptional network functions in Shh-mediated patterning of the vertebrate neural tube, where a series of spatially oriented repressors feeds back to limit the expression boundaries of Shh-mediated cell fate decisions (Chen, 2012).

Conceptually, these more complex systems are reminiscent of the reaction-diffusion model proposed by Turing, in which a localized activator would activate a repressor, which would diffuse more rapidly than the activator, and feed back on its activity. These systems strongly suggest that the patterning activity of a single monotonic gradient is insufficiently robust for establishing precise orders of closely positioned expression boundaries. By integrating gradients with repressive mechanisms that refine gradient shape or influence outputs, systems are generated that ensure consistency in body plan establishment while still maintaining the flexibility required for complex systems to evolve (Chen, 2012).

runt : Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | mRNA Transport | Developmental Biology | Effects of Mutation | References

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