dorsal

Transcriptional Regulation

RTK signaling modulates the Dorsal gradient

The dorsoventral (DV) axis of the Drosophila embryo is patterned by a nuclear gradient of the Rel family transcription factor, Dorsal (Dl), that activates or represses numerous target genes in a region-specific manner. This study demonstrates that signaling by receptor tyrosine kinases (RTK) reduces nuclear levels and transcriptional activity of Dl, both at the poles and in the mid-body of the embryo. These effects depend on wntD, which encodes a Dl antagonist belonging to the Wingless/Wnt family of secreted factors. Specifically, it was shown that, via relief of Groucho- and Capicua-mediated repression, the Torso and EGFR RTK pathways induce expression of WntD, which in turn limits Dl nuclear localization at the poles and along the DV axis. Furthermore, this RTK-dependent control of Dl is important for restricting expression of its targets in both contexts. Thus, the results reveal a new mechanism of crosstalk, whereby RTK signals modulate the spatial distribution and activity of a developmental morphogen in vivo (Helman, 2012).

Specification of body axes in all metazoans is initiated by a small number of inductive signals that must be integrated in time and space to control complex and unique patterns of gene expression. It is therefore of utmost importance to unravel the mechanisms underlying crosstalk between different signaling cues that concur during early development. This study has elucidated a novel signal integration mechanism that coordinates RTK signaling pathways with the Dl nuclear gradient, and thus with terminal and DV patterning of the Drosophila embryo (Helman, 2012).

Previous work had identified an input by Torso signaling into specific transcriptional effects of Dl. The current results establish a general mechanism, which involves RTK-dependent control of the nuclear Dl gradient itself, and thus affects a large group of Dl targets. This regulatory input is based on RTK-dependent derepression of wntD, a Dl target that encodes a feedback inhibitor of the Dl gradient. Thus, Dl activates wntD effectively only when accompanied by RTK signaling, enabling region-specific negative-feedback control of the nuclear Dl gradient. In the absence of RTK signaling, wntD is not expressed and the levels of nuclear Dl are elevated. Consequently, Dl target genes are ectopically expressed, both at the poles and along the DV axis (Helman, 2012).

Torso RTK signaling depends on maternal cues and is independent of the Dl gradient. Thus, it can be viewed as a gating signal that operates only at the embryonic poles, where it controls Dl-dependent gene regulation. However, the activity of the EGFR RTK pathway later on in development crucially depends on Dl, which induces the neuroectodermal expression of rhomboid, a gene encoding a serine protease required for processing of the EGFR ligand Spitz. In this case, EGFR-dependent induction of WntD represents a negative feedback loop that reduces nuclear levels of Dl laterally and, consequently, limits the expression of multiple Dl targets along the DV axis (Helman, 2012).

It should be noted that the regulatory interactions that have been characterized do not preclude the existence of other mechanisms modulating nuclear Dl concentration or activity. For example, the progressive dilution or degradation of maternal components involved in Toll receptor activation upstream of Dl should cause reduced Dl nuclear accumulation and retraction of its targets as development proceeds. It is also possible that Torso- or EGFR-induced repressors block transcription of Dl target genes directly. Accordingly, the ectopic sna expression observed in embryos mutant for components of the Torso pathway such as DSor and trunk probably reflects both loss of WntD activity on Dl and loss of Hkb-mediated repression of sna. In this context, it is interesting to note that sna expression expands and colocalizes with Hkb at the poles of wntD mutants; perhaps repression of sna by Hkb is not sufficient to override increased Dl activation in this genetic background. Thus, the Torso pathway probably employs more than one mechanism to exclude Dl target expression from the termini. Furthermore, the existence of such additional regulatory mechanisms could explain why wntD mutants do not have a clear developmental phenotype, despite the broad effects on Dl-dependent gene expression patterns caused by the genetic removal of wntD. It is proposec that corrective mechanisms are present, which make the terminal and DV systems robust with respect to removal of the WntD-based feedback, such as RTK-induced repressors. Understanding the basis of this robustness will require additional studies (Helman, 2012).

This work shows that RTK-dependent relief of Gro- and Cic-mediated repression is essential for transcriptional activation of wntD by Dl. Correspondingly, in the absence of cic or gro, the early expression of wntD expands ventrally throughout the domain of nuclear Dl. The early onset of this derepression, and the presence of at least one conserved Cic-binding site in the proximal upstream region of wntD, indicate that repression of wntD may be direct. Interestingly, it is thought that Gro and Cic are also involved in assisting Dl-mediated repression of other targets such as dpp and zen, as gro and cic mutant embryos show derepression of those targets in ventral regions. However, as ectopic wntD expression in these mutants leads to reduced nuclear localization of Dl along the ventral region, it is conceivable that decreased Dl activity also contributes to the derepression of dpp and zen (Helman, 2012).

In conclusion, the data presented in this study demonstrate RTK-dependent control of nuclear Dl via wntD, based on multiple regulatory inputs, including negative gating, feed-forward loops and negative feedback control. Together, these mechanisms provide additional combinatorial tiers of spatiotemporal regulation to Dl target gene expression. Future studies will show whether other signal transduction cascades and/or additional developmental cues also impinge on the Dl morphogen gradient (Helman, 2012).

Nucleosome-mediated cooperativity between transcription factors

The Drosophila genome activator Vielfaltig (Vfl), also known as Zelda (Zld), is thought to prime enhancers for activation by patterning transcription factors (TFs). Such priming is accompanied by increased chromatin accessibility but the mechanisms by which this occurs are poorly understood. This study analyzed the effect of Zld on genome-wide nucleosome occupancy and binding of the patterning TF Dorsal (Dl). The results show that early enhancers are characterized by an intrinsically high nucleosome barrier. Zld tackles this nucleosome barrier through local depletion of nucleosomes with the effect being dependent on the number and position of Zld motifs. Without Zld, Dl binding decreases at enhancers and redistributes to open regions devoid of enhancer activity. It is proposed that Zld primes enhancers by lowering the high nucleosome barrier just enough to assist TFs in accessing their binding motifs and promoting spatially controlled enhancer activation if the right patterning TFs are present. It is envisioned that genome activators in general will utilize this mechanism to activate the zygotic genome in a robust and precise manner (Sun, 2015).

An important finding from this study is that early enhancers acquire high nucleosome occupancy about the length of typical enhancers in the absence of Zld. These regions have high predicted nucleosome occupancy based on underlying DNA sequences and acquire high nucleosome occupancy in wild-type embryonic tissues when Zld is no longer present during late embryogenesis. Taken together, these data show that early enhancers generally have a strong intrinsic nucleosome barrier (Sun, 2015).

Previous evidence on the intrinsic nucleosome occupancy at enhancers has been conflicting since it has been reported as either low or high. The current results unambiguously demonstrate high intrinsic nucleosome occupancy at early Drosophila enhancers since this study not only predicts intrinsic nucleosome occupancy but also demonstrates high nucleosome occupancy experimentally (as observed in zld minus embryos and in wild-type late muscle tissue). This has important implications for the well-studied function of early Drosophila enhancers (Sun, 2015).

The simplest model is that the high nucleosome occupancy in the absence of appropriate TFs protects enhancers from inappropriate binding and activation. However, a more intriguing model proposed by Mirny poses that high nucleosome occupancy promotes a specific type of TF cooperativity called cooperative nucleosome binding (Mirny, 2010). Experimental evidence showed that TFs can dramatically enhance each others' binding to nucleosomal DNA simply by competing against a common nucleosome. Thus, the higher the nucleosome barrier, the more TFs are required to break the histone-DNA contacts. This in turn makes the enhancer activity dependent on multiple TFs without requiring direct  physical interactions between them. This model fits well for the current system since early Drosophila enhancers are strongly controlled by the combinatorial input of multiple TFs, and no strict motif grammar has been found between their binding motifs (Sun, 2015).

Since ChIP results show that Dl binding depends on Zld, but not the other way around, there is a hierarchy by which TFs activate enhancers in a combinatorial manner. It is proposed that Zld's pioneering role is its ability to lower (or prevent) the very high nucleosome barrier in each enhancer and that it does so just enough to allow patterning TFs to bind and to help antagonize the remaining nucleosome barrier. Such partial nucleosome depletion by Zld is supported by findings that binding of Zld only leads to a relatively local depletion of about 1-2 nucleosomes within an enhancer, that multiple Zld binding motifs lead to stronger depletion and that the position of the Zld motifs within the enhancer matters. The degree of nucleosome depletion by Zld thereby sets a threshold required for patterning TFs such as Dl to achieve robust transcriptional activation (Sun, 2015).

It should be noted that the mechanism by which Zld induces nucleosome depletion remains unknown. In the simplest scenario, Zld might bind to its targets very  early during the rapid nuclear cycles, when the chromatin may not be as densely packed and thus more accessible, and then prevent nucleosomes from being assembled nearby. Alternatively, Zld may bind, destabilize and eject nucleosomes, thereby acting as a more classical pioneer factor. Regardless of whether Zld can bind its motifs embedded in nucleosomes, Zld's ability to reduce nucleosome occupancy and facilitate the binding of TFs certainly fulfills a pioneering role (Sun, 2015).

The pioneering role presented in this study for Zld during Drosophila ZGA may be a general feature of key zygotic genome activators. For example, Pou5f3, which controls  zygotic genome activation (ZGA) together with Nanog and SoxB1 family proteins in zebrafish also binds before ZGA. Interestingly, the mammalian homolog of Pou5f3, Oct4, is a pluripotency factor that, along with Sox2 and Klf4, gains initial access to closed chromatin at enhancers of genes promoting reprogramming from fibroblasts to induced pluripotent stem cells. This points to a mechanistic link between ZGA and cellular reprogramming, the center of which may be the pioneering activity to potentiate TF binding and gene expression as exemplified by Zld (Sun, 2015).

Taken together, the following temporal working model is proposed for how Zld primes  early embryonic enhancers during ZGA. As the Zld protein level rises in the first hour of development, Zld begins to locally reduce nucleosome occupancy at target enhancers that normally have a high intrinsic  nucleosome barrier. This is unlikely to be solely an effect of histone acetylation, which accompanies early Zld binding, since acetylated histones are more  broadly found over Zld-bound regions (Sun, 2015).

Starting 1-2h and peaking at 2-3h, patterning TFs such as Dl gain access to these enhancers. In certain embryonic regions, where the right combination of patterning  TFs is present, Zld and these TFs then strongly bind through collaborative nucleosome  binding and activate transcription in a distinct pattern in the embryo. In this process,  some TFs such as Dl might be more strongly dependent on prior chromatin accessibility.  A recent genome-wide analysis identified NF-κ B, the mammalian homolog of Dl, as a 'settler' TF whose binding is strongly governed by the accessible chromatin created by 'pioneer' TFs (Sun, 2015).

In the absence of Zld, binding of Dl is severely diminished. This is accompanied  by a redistribution of Dl to other regions in the genome that remain accessible. Such TF redistribution in the absence of a key activator has been observed previously in yeast, flies and mammalian systems. The simplest explanation for this phenomenon is the law of mass action, i.e. given that the nuclear Dl concentration remains the same, more unbound Dl is available to drive ectopic binding. A good candidate for facilitating ectopic Dl binding in the absence of Zld is GAF since this study found the ectopic Dl bound regions to be enriched for the GAGA motif (Sun, 2015).

When this early pattern formation phase ends and Zld levels begin to decrease, the nucleosome-favoring sequences promote high nucleosome occupancy at these regions, closing enhancers and reducing transcriptional output. Thus, Zld acts as a timer of ZGA in that it controls the engagement and decommission of TFs at target enhancers by transiently reducing the nucleosome barrier. Since the Zld-mediated nucleosome depletion strongly correlates with early enhancer activity, it is likely a central mechanism by which Zld specifies and primes enhancers across the genome. It will be interesting to analyze whether this is a general property of zygotic genome activators and whether other pioneer factors play similar roles at later stages of development (Sun, 2015).

Targets of Activity

The initial distribution of Decapentaplegic in the dorsal ectoderm of the developing fly is established by a gradient of the maternal Dorsal protein, which is asymmetrically distributed to the ventral portion of the fly. The Dorsal protein regulatory gradient initiates the differentiation of the mesoderm, neuroectoderm and dorsal ectoderm in the early Drosophila embryo. There are two primary Dorsal target genes: snail and dpp, which define the limits of the presumptive mesoderm and dorsal ectoderm, respectively. After gastrulation the Dorsal regulatory gradient defines the limits of inductive interactions between germ layers. Thus dorsal controls the subdivision of the mesoderm and dorsal ectoderm (Maggert, 1995).

The contributions made by maternal and zygotic genes to the establishment of the expression patterns of four zygotic patterning genes have been examined: decapentaplegic (dpp), zerknüllt (zen), twist (twi), and snail (sna). All of these genes are initially expressed at the poles and either dorsally or ventrally in the segmented region of the embryo. In the segmented region of the embryo, correct expression of these genes depends on cues from DL. The DL gradient appears to be interpreted on three levels: dorsal cells express dpp and zen, but not twi and sna; lateral cells lack expression of all four genes; ventral cells express twi and sna, but not dpp and zen. DL appears to activate the expression of twi and sna and repress the expression of dpp, zen and tolloid (Kirov, 1994).

The VRE sequence of zerknüllt is located between -1.6 and -1.0. kb upstream of the transcriptional start site. In order to examine the range of action of the VRE, a evenskipped minimal stripe 2 enhancer (MSE) was placed upstream of a reporter gene, and a VRE was placed downstream of a reporter gene. In these experiments, the closest Dorsal binding-sites in the VRE map nearly 5 kb from the MSE activators. Nonetheless, stripe 2 repression is repressed in ventral regions of early embryos. Repression takes place irrespective of orientation of the VRE. This repression appears to be distinct from that mediated by snail, Krüppel and knirps repressors that function in a local fashion to inhibit, or quench nearby activators within the enhancer to which it is bound (Cai, 1996).

Dorsal functions as both an activator and repressor of transcription to determine dorsoventral fate in the Drosophila embryo. Repression by Dorsal requires the corepressor Groucho (Gro) and is mediated by silencers termed ventral repression regions (VRRs). A VRR in zerknullt (zen) contains Dorsal binding sites as well as an essential element termed AT2. An AT2 DNA binding activity has been identified (called ZREB) and purified in embryos. It consists of cut (ct) and dead ringer (dri) gene products. dri was isolated as a novel gene encoding a sequence-specific DNA-binding protein. Dri is a founding member of a growing protein family whose members share a conserved DNA binding domain termed the A/T-rich interaction domain. dri is developmentally regulated, being expressed in a restricted set of cells including some neural cells and differentiating cells of the gut and salivary gland ducts. Dri is a member of the recently defined ARID family of DNA binding proteins, a family that includes the B-cell-specific factor Bright and the Drosophila factor Eyelid. Although Bright is thought to function as a transcriptional activator, genetic data suggest that Eyelid functions to repress transcription in response to activation of the wingless pathway (Valentine, 1998 and references).

Studies of loss-of-function mutations in ct and dri demonstrate that both genes are required for the activity of the AT2 site. Dorsal and Dri both bind Gro, acting cooperatively to recruit it to the DNA. Thus, ventral repression may require the formation of a multiprotein complex at the VRR. This complex includes Dorsal, Gro, and additional DNA binding proteins, all of which appear to convert Dorsal from an activator to a repressor by enabling it to recruit Gro to the template. By showing how binding site context can dramatically alter transcription factor function, these findings help clarify the mechanisms responsible for the regulatory specificity of transcription factors (Valentine, 1998).

To determine if cut and dir are required for the activity of the AT2 site in vivo, the effects of mutations in these genes were examined on the activity of the lacZ transgene under control of the minimal zen VRR. For both cut and dir, germ line clones were generated to test the effects of eliminating maternally contributed gene products, and, in addition, the effects of eliminating zygotically produced gene products were examined. A null mutation in ct (which is an X-linked gene) results in strong ventral derepression of the transgene. This ventral derepression is observed in about one-half the embryos derived from a cross between females containing ct germ line clones and hemizygous males. It was never observed in a cross between heterozygous females and hemizygous males, suggesting that derepression requires simultaneous elimination of both maternal and zygotic Ct. A strong hypomorphic mutation in dri (which is an autosomal gene) also results in strong derepression. In contrast to the results observed with ct, this effect is strictly zygotic. It is observed in a cross between heterozygous dri males and females but not in a cross between females carrying dri germ line clones and wild-type males. Most strikingly, in the absence of zygotic Dri, the zen VRR directs strong ventral expression in the blastoderm embryo, reminiscent of the results observed when the AT2 element is mutagenized. These results strongly suggest that, in the context of the minimal zen VRR, Dri plays an essential role in converting Dorsal from an activator into a repressor. The dri mutation results in a significant weakening of the transverse eve stripe (generated by the minimal even skipped (eve) stripe 2 enhancer (MSE) as well as a shift in the position of the stripe toward the anterior pole of the embryo, presumably due to a role for Dri in anteroposterior pattern formation. Despite the strong effects of the cut and dir mutations on the activity of the minimal zen VRR, both genes make only minor contributions to the ventral repression of the endogenous zen gene in the stage 4 embryo. In the absence of both zygotic and maternal Ct or in the absence of zygotic Dri, zen expression in the stage 4 embryo is still largely restricted to the dorsal 40 to 50% of the embryo, although weak ventral patches of zen expression are observed with high frequency. Such patches are never observed in wild-type embryos stained in parallel with these embryos. The contrast between the strong effect observed for the minimal VRR and the weak effect observed for the endogenous zen gene suggests redundancy in the zen locus. In other words, there may be additional unidentified ventral repression regions in the zen locus that function in a Ct- and Dri-independent manner. Although neither Ct nor Dri is essential for ventral repression of the endogenous zen gene in the stage 4 embryo, both factors appear to play essential roles in the refinement of the zen pattern that normally occurs in stage 5 embryos. Normally, zen expression refines during cellularization to a stripe approximately three to five cells in width. However, in the absence of both maternal and zygotic Ct or in the absence of zygotic Dri, a severe refinement defect is observed (Valentine, 1998).

Both Dorsal and Dri bind to the corepressor Gro in vitro, suggesting a possible mechanism for repression in which Dorsal and Dri recruit Gro to the template. This model is strengthened by results showing that Dorsal and Dri bound to DNA can cooperatively recruit Gro to the zen VRR in vitro. However, the magnitude of the cooperativity observed in vitro is small (twofold) and therefore does not completely account for the absolute requirement for the Dorsal and AT2 sites observed in germ line transformation assays. This suggests that factors in addition to Dorsal and Dri are required for the efficient recruitment of Gro in vivo. For example, it is possible that the addition of Ct would enhance cooperative recruitment, an idea that could not be tested due to difficulty obtaining sufficient amounts of recombinant Ct. It is also likely that elements in addition to Dorsal sites and AT2 are required for efficient Gro recruitment and therefore for efficient repression, since previous experiments indicate that, while these sites are required for repression, they are not sufficient for repression. Finally, it is possible that the cooperativity of Gro recruitment would be enhanced in the context of chromatin templates rather than naked DNA templates (Valentine, 1998).

Polar expression of dpp and zen requires the terminal system to override repression by DL, while that of twi and sna requires the terminal system to augment activation by DL (Ray, 1991).

capicua (cic) is involved in gene repression in Drosophila terminal and dorsoventral patterning. Torso signaling at the embryonic poles regulates repressor processes that operate during dorsoventral patterning. Such patterning depends on Dorsal: Dorsal activates ventral-specific genes [for example, twist (twi)] and represses dorsal-specific genes, such aszerknullt. Repression by Dorsal requires its association with Gro and other postulated corepressors that bind next to Dorsal in the zen promoter. This repressor complex is under negative regulation by Tor signaling at the embryonic termini, allowing zen expression at each pole of the embryo (Jimenez, 2000 and references therein).

The mechanism of repression by Dorsal is not fully understood. Dead-Ringer (Dri) and Cut (Valentine, 1998) function as corepressors that assist Dorsal (and Gro) in Dorsal's function as a repressor. However, the effects of removing either of these two factors appears weaker than those caused by the loss of Dorsal or Gro function, suggesting that other factors may also contribute to Dorsal repression. Because cic is involved in a Gro-mediated process that is inactivated by Tor signaling, it was of interest to see if cic could also be involved in Dorsal repression. Consistent with this idea, zerknullt expression is expanded ventrally in cic¹ mutant embryos. Although this expansion is not as strong as in dorsal or gro mutants, ectopic zen transcripts are clearly detected in lateral and ventral regions of the embryo, especially in its posterior half. In contrast, activation of twi by Dorsal is normal in cic¹ embryos, suggesting that cic only participates in repression, not activation, by Dorsal (Jimenez, 2000).

To test further the role of cic in ventral repression of zen, an examination was carried out of a lacZ transgene carrying an even-skipped (eve) stripe 2 enhancer coupled to a silencer from the zen promoter: the zen Ventral Repression Element (VRE), which includes binding sites for Dorsal and adjacent regulatory sites. In wild-type embryos, lacZ expression directed by the eve stripe 2 enhancer is repressed ventrally by the VRE. This repression is clearly attenuated in cic¹ mutant embryos, permitting stripe 2 activation in the ventral-most side of the embryo. In addition, significant ectopic lacZ expression is observed in ventral and lateral regions of the embryo, as expected if repression by Dorsal bound to the VRE is switched in favor of activation. These results suggest that cic encodes one of the cofactors required for VRE activity and the conversion of Dorsal from an activator to a repressor of transcription. Because Dri and Cut also function as Dorsal corepressors, it appears that this role is shared by several factors with overlapping activities (Jimenez, 2000).

Cis-acting elements for the expression of buttonhead head stripe expression are contained in a 1 kb DNA fragment, located about 3 kb upstream of the promoter, The four maternal coordinate systems are necessary for correct btd head stripe expression, most likely by acting through the 1 kb cis-acting control region. Expression of the btd head stripe depends on bicoid. bcd-dependent activation also involves the activity of the morphogens of the posterior and dorsoventral systems, hunchback and dorsal, respectively, which act together to control the spatial limits of the expression domain. Finally, tailless, a torso dependent repressor of btd, takes part in the regulation of btd head stripe expression by enhancing activation at low levels of activity and repression at high levels of activity (Wimmer, 1995).

The anterior, the dorsoventral and terminal systems, are required for the activation of crocodile expression and for the spatial control of the anterior cap domain, while the posterior system is not required for the regulation of the croc expression pattern. In the absence of bcd activity, croc fails to be expressed in the anterior cap domain. Although Bcd acts in a concentration-dependent manner, croc expression can only be expanded ventrally in the absence of dorsal activity. In fact, the lack of dorsal activity causes a strong reduction of the croc expression domain to a single spot, corresponding in position to the peak of bicoid activity at the anterior pole. Conversely, Dorsal activity along the entire dorsoventral axis, as in embryos laid by Toll mutant females, causes an expansion of the croc expression domain towards the dorsal-most position. In embryos lacking tor activity, croc expression is abolished in the dorsal region. However, if tor is activated ectopically due to a dominant tor mutation, the croc expression domains are expanded significantly on the ventral side. Thus Dorsal postively regulates croc and Bcd requires Dl to set the spatial limit of the croc anterior expression domain (Häcker, 1995).

In ventral regions of early embryos the first step is taken in the differentiation of Drosophila mesoderm: the activation of two regulatory genes, twi and sna . SNA is a transcriptional repressor, uniformly expressed throughout the presumptive mesoderm. Its sharp lateral limits help to establish the boundary between the mesoderm and neuroectoderm. How does it achieve such sharp limits? sna is the target of combined activation by Dorsal and Twist, and this interaction provides a model for determining how a morphogen gradient establishes a sharp, on/off threshold response. Site-directed mutagenesis of DL- and TWI-binding sites within defined regions of the sna promoter suggest that the two proteins function multiplicatively to ensure strong, uniform expression of sna, particularly in ventral-lateral regions where there are diminishing amounts of DL. These results are consistent with the possibility that the sharp sna borders are formed by multiplying the shallow DL gradient and the steeper TWI gradient (Ip, 1992b).

snail exhibits little flexibility in its expression domain. Narrowing the limits of the presumptive mesoderm (resulting from artificially reducing the size of snail's expression domain) leads to a loss of visceral and heart lineages. These effects are seen in the reduced expression of the heart specific markers tinman and even-skipped, as well as reduced Fasiculin III protein. Derivatives of the ventral mesoderm are not as severely disrupted (Maggert, 1995).

Dorsal interacts with specific DNA sequences in the regulatory regions of its target genes. These DL binding sites, when taken from the context of either an activated or repressed promoter, mediate transcriptional activation (but not repression) of a heterologous promoter. T-rich sequences close to the DL binding sites in the silencer region of the zen promoter are conserved among three Drosophila species. A minimal element that can mediate repression of a heterologous promoter interacts with at least two factors present in embryonic extracts, one of which is DL protein. The other factor binds to the T-rich site (Kirov, 1993).

The distal portion of the zen promoter acts as a silencer that can mediate repression of zen . It contains four DL binding sites, sufficient for activation but not repression when tested out of context (Kirov, 1994).

Approximately 800 bp of 5'-flanking sequences upstream of the tolloid coding region drive an expression pattern indistinguishable from the wild-type pattern. A 423-bp fragment located within these sequences contains two DL binding sites and acts as a silencer to mediate ventral repression. (Kirov, 1994).

The three maternal systems (anterioposterior (bicoid); terminal (torso); dorsoventral (dorsal) control the early expression of Goosecoid. The GSC stripe never appears in bicoid mutants, the stripe is shifted anteriorly in torso mutants and the ventral repression of the stripe is abolished in dorsal mutants (Goriely, 1996).

The Torso receptor tyrosine kinase modulates DL activity. Torso pathway selectively masks the ability of DL to repress gene expression but has only a slight effect on activation. Intracellular kinases that are thought to function downstream of Torso, such as D-raf and the Rolled MAP kinase, mediate this selective block in repression. Normally, the Toll and Torso pathways are both active only at the embryonic poles, and consequently, target genes (zen and dpp) that are repressed in middle body regions are expressed at these sites. Constitutive activation of the Torso pathway causes severe embryonic defects, including disruptions in gastrulation and mesoderm differentiation, as a result of misregulation of DL target genes (Rusch, 1994).

Three different maternal morphogen gradients regulate expression of the gap gene tailless , which is required to establish the acron and telson of the Drosophila embryo. Identified thus far are regions mediating activation by the terminal system, regions mediating both activation and repression by Bicoid and regions mediated by Dorsal repression (Liaw, 1993).

Anterior repression of orthodenticle is carried out by Huckebein which in turn receives input for the torso system, from Dorsal and from Bicoid. Dorsal functions in the anterior repression of otd expression. The repression function of Dorsal is mediated, at least in part, through Huckebein, since anterior hkb expression is lost in dorsal mutants. Contrary to early models of embryonic pattern formation, high levels of Bicoid are not required for otd activation or for the establishment of anterior head structures (Gao, 1996).

All three maternal systems active in the cephalic region are required for proper sloppy-paired expression. The terminal and anterior patterning systems appear to be closely linked. This cooperation is likely to involve a direct interaction between the BCD morphogen and the terminal system. Low levels of terminal system activity seem to potentiate BCD as an activator of slp, whereas high levels down-regulate bcd, rendering it inactive. Dorsal, the morphogen of the dorsoventral system, and the head-specific gap gene empty spiracles act as repressor and corepressor in the regulation of slp (Grossnicklaus, 1994) .

rhomboid (rho) encodes a putative transmembrane receptor that is required for the differentiation of the ventral epidermis. Dorsal acts in concert with basic helix-loop-helix (b-HLH) proteins, possibly including Twist, to activate rhomboid in both lateral and ventral regions. Expression is blocked in ventral regions (the presumptive mesoderm) by snail, which is also a direct target of the DL morphogen (Ip, 1992a).

Drosophila dorsoventral patterning and mammalian hematopoiesis are regulated by related signaling pathways (Toll, interleukin-1) and transcription factors (Dorsal, nuclear factor-kappa B). These factors interact with related enhancers, such as the rhomboid neurectoderm element (NEE) and kappa light chain enhancer, that contain similar arrangements of activator and repressor binding sites. The NF-kappa B enhancer can generate lateral stripes of gene expression in transgenic Drosophila embryos in a pattern similar to that directed by the rhomboid NEE. Drosophila DV determinants direct these stripes through the corresponding mammalian cis regulatory elements in the NF-kappa B enhancer, including the kappa B site and kappa E boxes. These results suggest that enhancers can couple conserved signaling pathways to divergent gene functions (Gonzalez-Crespo, 1994).

The Drosophila DC2 gene was isolated on the basis of sequence similarity to DC0, the major Drosophila Protein kinase A (PKA) catalytic subunit gene. The 67-kD DC2 protein behaves as a PKA catalytic subunit in vitro. DC2 is transcribed in mesodermal anlagen of early embryos. This expression depends on dorsal but on neither twist nor snail activity. DC2 is also expressed in subsets of cells in the optic lamina, wing disc and leg discs of third instar larvae. Mutants are viable and fertile. The absence of DC2 does not affect the viability or phenotype of imaginal disc cells lacking DC0 activity or embryonic hatching of animals with reduced DC0 activity. These observations indicate that DC2 is not an essential gene and is unlikely to be functionally redundant with DC0 (Meléndez, 1995).

Zygotic expression of modifier of variegation modulo depends on the activity of genes which pattern the embryo along dorsoventral and anteroposterior axes and specify diversified morphogenesis. Dorsal and the mesoderm-specific genes twist and snail direct modulo expression in the presumptive mesoderm. The homeotic genes Sex combs reduced and Ultrabithorax positively regulate the gene in the ectoderm of parasegment 2 and abdominal mesoderm (Graba, 1995).

Dorsoventral (DV) patterning of the Drosophila embryo is initiated by a broad Dorsal (Dl) nuclear gradient, which is regulated by a conserved signaling pathway that includes the Toll receptor and Pelle kinase. What are the consequences of expressing a constitutively activated form of the Toll receptor, Toll(10b), in anterior regions of the early embryo? Using the bicoid 3' UTR, localized Toll(10b) products result in the formation of an ectopic, anteroposterior (AP) Dl nuclear gradient along the length of the embryo. The analysis of both authentic Dorsal target genes and defined synthetic promoters suggests that the ectopic gradient is sufficient to generate the full repertory of DV patterning responses along the AP axis of the embryo. For example, mesoderm determinants are activated in the anterior third of the embryo, whereas neurogenic genes are expressed in central regions. These results raise the possibility that Toll signaling components diffuse in the plasma membrane or syncytial cytoplasm of the early embryo (Huang, 1997).

The Huang (1997) paper also clearly summarizes what is known about the regulation of genes involved in dorsal/ventral patterning. There are five distinct thresholds of gene activity in response to the Dorsal nuclear gradient in early embryos. The type I target gene folded gastrulation is activated only in response to peak levels of the Dl gradient, so that expression is restricted to a subdomain of the presumptive mesoderm. The PE enhancer from the twist promoter region exhibits a similar pattern of expression. This enhancer contains a cluster of low-affinity Dl binding sites that restrict expression to the ventral-most regions of early embryos. The type II target gene snail contains a series of low-affinity Dl-binding sites, as well as binding sites for the bHLH activator, Twist. The Dl and Twist proteins appear to make synergistic contact with the basal transcription complex, so that snail is activated throughout the presumptive mesoderm in response to both peak and high levels of the Dl gradient. The ventral midline arises from the mesoderm, which is derived from the ventral-most regions of the neuroectoderm. Mesectoderm differentiation is controlled by the bHLH-PAS gene, sim. Some of the E(spl) complex also exhibit early expression in the presumptive mesectoderm. A synthetic enhancer containing high-affinity Dl-binding sites and Twist binding sites exhibits expression in this region. The type IV target gene rhomboid is expressed in lateral stripes that encompass the ventral half of the presumptive neuroectoderm. These stripes are regulated by a 300-bp enhancer (NEE) that contains high-affinity Dl-binding sites, Twist-binding sites, and "generic" E-box sequences that appear to bind ubiquitously distributed bHLH activators (Daughterless and Scute), which are present in the unfertilized egg. The fifth and final threshold response is defined by the lowest levels of the Dl gradient. The zerknullt target gene is repressed by high and low levels of the gradient, so that expression is restricted to the presumptive dorsal ectoderm. The zen promoter region contains high-affinity Dl-binding sites and closely linked "corepressor" sites. Efficient occupancy of the Dl sites appears to depend on strong, cooperative DNA-binding interactions between Dl and the corepressors. The same low levels of Dl that repress zen also repress sog. The sim, E(spl), rho and sog expression patterns are restricted to the neurogenic ectoderm and excluded from the ventral mesoderm by Snail, which encodes a zinc finger repressor (Huang, 1997).

This study also provides evidence that neurogenic repressors may be important for the establishment of the sharp mesoderm/neuroectoderm boundary in the early embryo. About half of the embryos carrying the Toll anteriorly expressed transgene exhibit a ventral gap in the endogenous ventral expression pattern of snail behind the ectopic anterior staining pattern. Although the identity of the repressor creating this gap is unknown, it is conceivable that members of the E(spl) complex encode putative snail repressors because previous studies have shown that the m7 and m8 genes are expressed in the lateral neuroectoderm of early embryos. Proteins coded for by these genes are known to repressors. These proteins might be regulated by the gene hierarchy responsible for D/V polarity (Huang, 1997).

The ventrolateral expression of brinker (brk) in early embryos suggests that brk, like sog and rho, is a target gene of the maternal Dorsal protein gradient. In support of this notion, brk expression is completely abolished in maternally dorsalized embryos. Conversely, in maternally ventralized embryos derived from Toll 9Q heterozygous mothers, brk expression is initiated along the entire embryonic circumference except in the presumptive mesoderm. In sna twi mutant embryos and in sna single mutants brk expression is uniform at the ventral side. Thus, as is known for rho, sna might be a ventral repressor of brk transcription. The complementarity between brk expression and regions of Dpp signaling in the embryo might arise if brk is itself negatively regulated by Dpp, as occurs in imaginal discs. To test this idea, brk expression was examined in dpp mutant embryos. Here, brk expression is normal before the onset of gastrulation, but subsequently expands toward the dorsal side of the embryo so that brk becomes uniformly expressed in the entire ectoderm. The opposite phenotype results if dpp expression expands into the ventrolateral region, as in a sog mutant embryo with extra wild-type copies of dpp. These embryos exhibit a strong repression of brk transcription in the ventrolateral region although a small domain of brk expression is maintained close to the border of the mesoderm. This residual expression might be responsible for the narrow stripe of neuroblasts that still forms in sog embryos with four copies of dpp+. Does the expansion of brk expression in dpp mutants require the previous Dl-dependent activation of brk transcription? In dl;dpp double mutant embryos, brk is initially not expressed; nevertheless, uniform brk expression is initiated during gastrulation. Thus, absence of dpp leads to derepression of brk irrespective of whether Dl is present, indicating that other mechanisms of transcriptional activation of brk exist that are normally counteracted by Dpp signaling (Jazwinska, 1999).

Transcriptional control of the Drosophila terminal gap gene huckebein (hkb) depends on Torso (Tor) receptor tyrosine kinase (RTK) signaling and the Rel/NFB homolog Dorsal (Dl). Dl acts as an intrinsic transcriptional activator in the ventral region of the embryo, but under certain conditions, such as when it is associated with the non-DNA-binding co-repressor Groucho (Gro), Dl is converted into a repressor. Gro is recruited to the enhancer element in the vicinity of Dl by sequence-specific transcription factors such as Dead Ringer (Dri). The interplay between Dl, Gro and Dri on the hkb enhancer was examined and it was shown that when acting over a distance, Gro abolishes rather than converts Dl activator function. However, reducing the distance between Dl- and Dri-binding sites switches Dl into a Gro-dependent repressor that overrides activation of transcription. Both of the distance-dependent regulatory options of Gro -- quenching and silencing of transcription -- are inhibited by RTK signaling. These data describe a newly identified mode of function for Gro when acting in concert with Dl. RTK signaling provides a way of modulating Dl function by interfering either with Gro activity or with Dri-dependent recruitment of Gro to the enhancer (Hader, 1999).

The cis-acting element has been identified that mediates expression of the Drosophila gene hkb, which is necessary for terminal pattern formation and to size the mesoderm anlage in the blastoderm embryo. Deletion analysis of this element reveals a 162 base pair (bp) sub-element that integrates the activities of the Tor-dependent RTK signaling cascade and the morphogen Dl. This element, termed hkb ventral element (VE), comprises a 112 bp ventral activator element (VAE) and a 50 bp ventral repressor element (VRE) (Hader, 1999).

The VAE contains a Dl-binding site, identified in vitro, and mediates gene activation along the ventral side of the embryo. VAE-mediated gene expression is absent in embryos lacking Dl activity and extends throughout Toll^10b mutants, in which Dl is present in all nuclei of the embryo. The expression pattern is not altered in embryos lacking snail and twist, the zygotic mediators of Dl. It is also not affected in embryos that lack Tor or express constitutively active Tor^Y9, which causes RTK signaling throughout the embryo. In contrast, the VE fails to activate in the absence of Tor and mediates broad ventral expression in tor^Y9 embryos not seen in the absence of Dl activity. This indicates that VAE mediates transcriptional activation by Dl, that the VRE, which by itself fails to activate transcription, is necessary to prevent Dl-dependent activation in the central region of the embryo, and that the activity of the unknown repressor, mediated by the VRE, is relieved by RTK signaling (Hader, 1999).

The evolutionarily conserved co-repressor Gro acts as a repressor of Dl activity, since both hkb expression and VE-driven gene expression expand along the ventral side of embryos lacking groucho (gro) activity. However, VAE-driven gene expression and the terminal expression domains of hkb are not significantly affected by lack of Gro. Thus, Gro functions as a repressor of VAE-directed, Dl-dependent transcriptional activation in the ventral region of the embryo and must act through the VRE (Hader, 1999).

Previous results have shown that Gro switches the transcriptional activator Dl into a potent silencer of transcription. This requires the formation of a multiprotein repressor complex of which Dl and Gro are obligatory components. Complex formation requires that Gro is recruited next to Dl by sequence-specific transcription factors such as Cut or Dri. Lack of either Gro or Dri activity results in VE-driven gene expression along the ventral axis of the embryo, indicating that both factors are necessary for repression of Dl-dependent activation. A single binding site has been identified for Dri in the VRE. Replacement of 5 bp in this site (VE-^DRI) results in loss of repression in the central region of the embryo, indicating that Dri is necessary for recruitment of Gro to the VE (Hader, 1999).

The VE differs from the cis-acting elements of the genes zerknullt (zen) and decapentaplegic (dpp), both of which mediate long-range Dl-dependent transcriptional silencing by Gro. In these elements, binding sites for Dri and Dl are directly adjacent, whereas in the VE they are some 90 bp apart. This distance suggested the possibility that Gro cannot associate with Dl on the VE, implying that Gro must prevent Dl-dependent activation by a means other than formation of a long-range silencing complex, for example, by short-range quenching. This proposal was tested by monitoring gene expression patterns directed by a cis-acting activator element of the gene knirps (kni-element) to which the VRE, the VAE, the VE or molecularly defined variants of the VE were fused (Hader, 1999).

The kni-element drives gene expression throughout the embryo except in the posterior pole region. It mediates activation in response to the transcriptional activators Bicoid (Bcd) and Caudal (Cad) and acts in a Dl-independent fashion. Addition of the VRE to the kni-element does not cause ventral repression, nor does addition of the VE or the VAE. This indicates that within the VE, Gro abolishes the activator function of Dl instead of converting Dl into a long-range repressor that interferes with transcriptional activation by Bcd and Cad (Hader, 1999).

To investigate whether this action of Gro on Dl is determined by the arrangement of Dri- and Dl-binding sites in the VE, the transcription patterns driven by a modified VE-kni-element were examined in which the normal distance of 91 bp between the binding sites was reduced to 45 bp. This reduction results in Dl-dependent repression along the ventral side of wild-type embryos. Repression is not observed in the absence of Gro or Dl or in embryos expressing the constitutively active Tor^Y9 protein. In contrast, the repression domain expands anteriorly in tor mutant embryos, which lack RTK signaling, and is found to be Dl-dependent. This suggests that the spatial arrangement of the Dl- and Dri-binding sites dictates the mechanism by which Gro and Dl act within the enhancer element. In one case, Dl is suppressed by Gro, in the other, Dl is converted into a potent silencer of transcription that can override activation by Bcd and Cad. Both modes of repression are controlled by Tor-dependent RTK signaling (Hader, 1999).

In the zen and dpp cis-acting elements, Gro causes Dl-mediated long-range silencing. Gro functions either by inhibiting the assembly and function of the core RNA polymerase II complex, by positioning nucleosomes over the core promoter and/or by recruiting the histone deacetylase Rpd3 to the template, where the enzyme can modulate local chromatin structure. However, in the VE, Gro only inhibits Dl-dependent activation without converting Dl into a repressor. The different modes of Gro function, that is, long-range silencing and short-range quenching, as shown here, are dependent on the distance between the Dl- and Dri-binding sites and/or their orientation on the enhancer, since shortening of the spacer distance converts the VE into a dpp- or zen-like element. This suggests that the way in which Gro regulates Dl activity depends on whether or not the two proteins can directly interact in vivo. Furthermore, both regulatory options of Gro on Dl are abolished by RTK signaling, a phenomenon that corresponds to the observation that Dl-dependent repression of dpp and zen is relieved by local Tor activity in the pole regions of the embryo. RTK-dependent phosphorylation may therefore interfere with the binding of Dri to the DNA template, the recruitment of Gro, or with both. Phosphorylation of the vertebrate Gro homolog TLE1 has been demonstrated, and many potential phosphorylation sites have been noted in Dri. Thus, local RTK-dependent phosphorylation may render one or both factors inactive, preventing Gro-dependent repression of Dl in the termini of the wild-type embryo (Hader, 1999).

These results establish that the cooperation between two maternal signaling systems, which determines the spatial limits of the Drosophila mesoderm anlage through hkb expression, is based on the management of the ubiquitously distributed factors Gro and Dri by local RTK signaling and that Gro can act through different modes on Dl. Lack of dead ringer (dri) activity does not result in an overt expansion of hkb expression on the ventral side of the embryo. However, as has been observed for VE-dependent gene expression, it causes only weak defects in mesoderm formation as compared with Gro-deficient embryos or embryos that express hkb under the control of the VAE. Thus, the interactions shown here represent only the Dri-dependent aspect of Gro's effect on hkb expression. The full picture of hkb control is likely to involve additional and redundantly acting factor(s) that recruit Gro to sites flanking the VE within the hkb control region (Hader, 1999).

An important question in neurobiology is how different cell fates are established along the dorsoventral (DV) axis of the central nervous system (CNS). The origins of DV patterning within the Drosophila CNS have been investigated. The earliest sign of neural DV patterning is the expression of three homeobox genes in the neuroectoderm -- ventral nervous system defective (vnd), intermediate neuroblasts defective (ind), and muscle segment homeobox (msh) -- which are expressed in ventral, intermediate, and dorsal columns of neuroectoderm, respectively. Previous studies have shown that the Dorsal, Decapentaplegic (Dpp), and EGF receptor (Egfr) signaling pathways regulate embryonic DV patterning, as well as aspects of CNS patterning. This study describes the earliest expression of each DV column gene (vnd, ind, and msh), the regulatory relationships between all three DV column genes, and the role of the Dorsal, Dpp, and Egfr signaling pathways in defining vnd, ind, and msh expression domains. The vnd domain is established by Dorsal and maintained by Egfr, but unlike a previous report vnd is found not to be regulated by Dpp signaling. ind expression requires both Dorsal and Egfr signaling for activation and positioning of its dorsal border, and abnormally high Dpp can repress ind expression. The msh domain is defined by repression: it occurs only where Dpp, Vnd, and Ind activity is low. It is concluded that the initial diversification of cell fates along the DV axis of the CNS is coordinately established by Dorsal, Dpp, and Egfr signaling pathways. Understanding the mechanisms involved in patterning vnd, ind, and msh expression is important, because DV columnar homeobox gene expression in the neuroectoderm is an early, essential, and evolutionarily conserved step in generating neuronal diversity along the DV axis of the CNS (Von Ohlen, 2000).

Early stage 5 embryos express vnd in a narrow domain similar to its final width; ind and msh are not detected. By the end of stage 5, both vnd and ind are expressed with a one to two cell wide gap; again, this expression is seen in domains similar to their final widths. The gap fills in during development resulting in the precise juxtaposition of the vnd and ind domains. Expression of msh in the trunk is not detected until stage 7. Thus, the timing of gene expression progresses from ventral to dorsal: vnd is detected first, ind appears soon after, and msh is observed last (Von Ohlen, 2000).

There is a gap between the initial vnd and ind domains, suggesting that each gene is independently activated at a precise DV position. Subsequently, ind can be expressed in the ventral domain, but this is normally prevented by vnd-mediated repression. Because ind is capable of repressing vnd expression, if ind were to be expressed first in both the ventral and the intermediate columns, it might fully inhibit the expression of vnd. Thus, the temporal pattern of vnd and ind expression is likely to be important for establishing their final spatial pattern of gene expression. The activation and borders of vnd expression appear to be wholly dependent on the Dorsal morphogen gradient. High levels of Dorsal in the mesoderm/mesectoderm anlagen can activate twist, snail, and vnd, but Snail activity represses vnd expression. Intermediate levels of Dorsal are sufficient to activate vnd, but not snail, thus establishing the ventral column of neuroectoderm. It is unclear how the dorsal border of vnd is positioned, but it may be dependent on the concentration of nuclear Dorsal, because if Dorsal levels are increased in dorsal cells, there is a corresponding expansion of the vnd domain. In contrast to a previous report, no evidence has been found that Dpp signaling establishes the dorsal border of the vnd domain. No change was observed in the width of the vnd domain in dpp embryos, and repression of vnd in ectopic Dpp embryos was not observed. In fact, elevated Dpp activity in the neuroectoderm (in sog 4xdpp embryos) gives a slight expansion of the vnd domain, and even higher levels of Dpp (in brk;sog embryos) still fail to repress vnd expression, despite eliminating much of the remaining CNS. The reason the vnd domain is expanded in sog 4xdpp embryos remains unclear; however, it is felt that the combined results clearly demonstrate that Dpp signaling does not repress vnd and therefore cannot position the dorsal border of vnd. All existing data are consistent with Dorsal acting as a direct, concentration-dependent activator of vnd expression. In contrast, the Egfr and Dpp signaling pathways have no role in establishing the correct vnd expression pattern, although Egfr is required to maintain vnd expression later in embryogenesis (Von Ohlen, 2000 and references therein).

Initiation and maintenance of ind expression require both Dorsal and Egfr signaling pathways, but not Dpp activity. The ventral border of ind expression is established by the dorsal limit of vnd expression. The dorsal border of ind expression has more complex regulation. Dpp repression does not establish the dorsal border of ind, since the ind domain is normal in dpp embryos. In contrast, both Dorsal and Egfr are required to activate ind and set its dorsal border. In wild-type embryos, the domains of ind and activated Egfr have identical dorsal borders. When Egfr activity is increased throughout the embryo, ind expression shows a partial dorsal expansion, showing that the dorsal border of Egfr activity sets the precise dorsal border of ind expression. Ectopic Dorsal activity can also expand the ind domain (without affecting the Egfr activation domain), showing that sufficiently high levels of nuclear Dorsal protein can independently activate ind expression. As expected, when Egfr activity and nuclear Dorsal levels are simultaneously increased there is a complete dorsal expansion of the ind domain. The data presented here suggest that ind expression is activated by both Dorsal and Egfr pathways, limited ventrally by vnd, and limited dorsally by lack of Dorsal and Egfr activity. The data do not distinguish between a linear pathway in which Egfr signaling activates or potentiates Dorsal to allow ind transcription and a parallel pathway in which Dorsal and Egfr signaling act independently to activate ind expression (Von Ohlen, 2000).

Although Dpp is not required for any aspect of ind expression in wild type embryos, ectopic Dpp signaling in the neuroectoderm can repress ind expression. This shows that Dpp signaling must be kept low in the intermediate column to allow ind transcription and raises the possibility that the loss of ind expression seen in dorsal embryos is an indirect effect, due to the de-repression of Dpp activity within the neuroectoderm. dorsal;dpp double mutants fail to express ind, however, proving that loss of ind expression in dorsal mutants is not due to de-repression of Dpp within the neuroectoderm. It is proposed that Dorsal must both activate ind expression and repress Dpp signaling to allow ind expression (Von Ohlen, 2000).

Determining the position of ventro-lateral neuroectoderm versus dorsal non neural ectoderm is controlled by maternal (dorsal) and zygotic genes (dpp, sog, brk, sna, twi). SoxNeuro (SoxN) expression is specifically affected in these mutants. dl mutants lack early SoxN expression. Embryos mutants for dpp show a dorsal expansion of SoxN expression, as also observed when misexpressing sog by means of the Gal4 system. Inversely, misexpressing dpp early in embryogenesis leads to severe reduction of SoxN expressing-cells, as observed in sog mutants and sog, brk double mutants. Finally, twi mutants are characterized by a ventral expansion of SoxN expression into the presumptive mesoderm. These experiments are consistent with a role for the D/V patterning genes in the control of SoxN expression, with SoxN being negatively regulated dorsally and ventrally by dpp and mesoderm genes, and positively by sog and brk in the neuroectodermal region. A similar situation has been reported in Xenopus, with SoxD, an essential mediator of neural induction, being negatively regulated by BMP4 and positively by chordin (the vertebrate homologs of dpp and sog, respectively) (Cremazy, 2000).

IkappaB kinase (IKK; see Drosophila Ird5) and Jun N-terminal kinase (Jnk) signaling modules are important in the synthesis of immune effector molecules during innate immune responses against lipopolysaccharide and peptidoglycan. However, the regulatory mechanisms required for specificity and termination of these immune responses are unclear. Crosstalk occurs between the Drosophila Jnk and IKK pathways; this leads to downregulation of each other's activity. The inhibitory action of Jnk is mediated by binding of Drosophila activator protein 1 (AP1) to promoters activated by the transcription factor NF-kappaB. This binding leads to recruitment of the histone deacetylase dHDAC1 to the promoter of the gene encoding the antibacterial protein Attacin-A and to local modification of histone acetylation content. Thus, AP1 acts as a repressor by recruiting the deacetylase complex to terminate activation of a group of NF-kappaB target genes (Kim, 2005).

Genome-wide survey for potential Dorsal targets

Genome-wide analysis of clustered Dorsal binding sites was used to examine the distribution of Dorsal recognition sequences in the Drosophila genome. The homeobox gene zerknullt (zen) is repressed directly by Dorsal, and this repression is mediated by a 600-bp silencer, the ventral repression element (VRE), which contains four optimal Dorsal binding sites. The arrangement and sequence of the Dorsal recognition sequences in the VRE were used to develop a computational algorithm to search the Drosophila genome for clusters of optimal Dorsal binding sites. There are 15 regions in the genome that contain three or more optimal sites within a span of 400 bp or less. Three of these regions are associated with known Dorsal target genes: sog, zen, and Brinker. The Dorsal binding cluster in sog is shown to mediate lateral stripes of gene expression in response to low levels of the Dorsal gradient. Two of the remaining 12 clusters associated with genes that exhibit asymmetric patterns of expression across the dorsoventral axis. These results suggest that bioinformatics can be used to identify novel target genes and associated regulatory DNAs in a gene network (Markstein, 2002).

zen is an immediate target of the maternal Dl gradient. The gene is activated initially at nuclear cleavage cycle 11-12 within 1 h after the Dl gradient is formed. zen initially exhibits a broad pattern of expression in the presumptive dorsal ectoderm and at the termini. High and low levels of the Dl gradient keep zen off in ventral and lateral regions. sog exhibits a complementary pattern of expression because it is activated by Dl, whereas zen is repressed. As seen for zen, sog expression is detected shortly after the formation of the Dl gradient (Markstein, 2002).

The zen VRE contains four optimal Dl recognition sequences within a span of 400 bp. Three of the four Dl binding sites contained within the zen VRE conform to the following consensus sequence for high-affinity Dl binding sites: GGG(W)_nCCM (where W = A or T, M = C or A, and n corresponds to either four or five W residues). The fourth recognition sequence (binding site 3 within the VRE) contains a G residue in the AT-rich central region and is represented by the optimal consensus sequence GGGWDWWWCCM (where D = A, T, or G). To determine whether a similar density of optimal Dl sites might account for the regulation of sog, the entire Drosophila genome was scanned for clusters of any of the 208 unique Dl sequences that conform (either directly or by reverse complement) to two degenerate sequences: GGG(W)₄CCM and GGGWDWWWCCM (Markstein, 2002).

The genome was scanned for clusters of Dl binding sites in windows of 400 bp, the interval within which the sites are clustered in the zen VRE, and also for clustering in windows of 1,000 bp because the operational size of enhancers can generally be thought of as about 1,000 bp. Although the genome-wide occurrence of 676 clusters of two or more optimal Dl sites in 1,000 bp is not statistically significant, the occurrence of 55 clusters with at least three sites and of eight clusters containing four sites is enriched beyond what one would expect from random chance. However, none of the clusters within 1,000 bp identified known Dl targets that were missed by the more stringent screen for clustering within 400 bp. Therefore, this study focussed on the results from the more stringent screen (Markstein, 2002).

As expected, the occurrence of 400-bp windows containing at least two sites (327 clusters) is much greater than the occurrence of 400-bp windows containing at least three sites (15 clusters) or four sites (3 clusters). However, the statistical significance of the clusters increases with their rarity. For example, the occurrence of 15 clusters with three or more Dl sites is 6 standard deviations from expected, making the probability of finding 15 clusters by random chance less than one in a million. The probability of finding three 400-bp clusters with at least four Dl sites is less than 10^-49. Remarkably, two of the clusters in this rarest class are associated with the sog and zen genes, which exhibit the most sensitive response to the Dl gradient. Of the remaining 13 clusters containing three or more Dl sites, one is associated with the Brinker gene, which is expressed in lateral stripes and probably is a direct target of the Dl gradient. The Brinker site is located ~10 kb 5' of the transcription start site. Brinker probably is a direct target of the Dl gradient in that it exhibits lateral stripes of expression that are similar to those observed for rhomboid. The other remaining 12 clusters were found to neighbor genes that were not known previously to be involved in dorsoventral patterning (Markstein, 2002).

sog is expressed initially in broad lateral stripes that encompass the entire presumptive neurogenic ectoderm. Staining persists in these lateral stripes during cellularization and the onset of gastrulation but quickly refines within the mesectoderm at the ventral midline of elongating embryos. There are four optimal Dl binding sites located within a 263-bp region of sog intron 1. Three different DNA fragments that encompass this region of the sog gene were placed 5' of a lacZ reporter gene and expressed in transgenic embryos. The largest fragment is 6 kb in length and includes two-thirds of intron 1, whereas the smallest is just 393 bp and centered around the cluster of Dl binding sites. A 1.5-kb fragment that extends into the 5'-flanking region was also tested. All three fragments direct lateral stripes of lacZ expression that are similar to those seen for the endogenous gene. These broad stripes persist until gastrulation and then refine within the mesectoderm. Midline staining becomes weak and erratic in older embryos. The 393-bp sog fragment directs essentially the same staining pattern as those obtained with the 6-kb DNA fragment as well as the 1.5-kb fragment. These results suggest that the 393-bp fragment (hereafter called the sog lateral stripe enhancer) contains most of the cis elements responsible for regulating the early sog pattern (Markstein, 2002).

The sog lateral stripe enhancer shares a number of similarities with the previously characterized rhomboid NEE, which also mediates gene expression in the neurogenic ectoderm. However, the NEE stripes are narrower than those generated by the sog enhancer, suggesting that the sog enhancer responds to lower levels of the Dl gradient than does the NEE. This difference might be due, at least in part, to the quality or organization of the Dl binding sites in the two enhancers. For example, only two of the four Dl binding sites contained in the NEE are optimal sites, whereas all four sites are optimal in the sog enhancer. The NEE contains four binding sites for the zinc finger snail repressor, which is expressed selectively in the ventral mesoderm and thereby restricts rhomboid expression to lateral regions. The sog lateral stripe enhancer contains two potential snail repressor sites (CACCT) that might be responsible for attenuated staining in ventral regions (Markstein, 2002).

The preceding results indicate that computational methods can identify cis-regulatory DNAs. To determine whether these methods also identify target genes, the expression profiles of two of the genes that contain optimal Dl clusters: Phm and Ady were examined. Digoxigenin-labeled antisense RNA probes were prepared for each gene and hybridized to fixed embryos. Both genes exhibit expression in ventral regions of early embryos, although Ady appears to be expressed earlier than Phm. Staining persists in the developing mesoderm during cellularization and gastrulation. The Ady pattern may be somewhat broader than the Phm pattern and may extend into ventral regions of the presumptive neurogenic ectoderm. These results suggest that one or both genes represent direct targets of the Dl gradient (Markstein, 2002).

It seems that the computational genome-wide search for optimal Dl clusters successfully identified the full spectrum of Dl gradient thresholds including genes that are activated by high (Phm), intermediate (Ady), and low (sog) levels of the Dl gradient. Perhaps the exact sequence, arrangement, and density of the binding sites help to determine the response to these different thresholds. For example, the Dl binding sites in zen and sog appear to be helically phased, occurring every 60-80 bp. This phasing may lead to cooperative protein-protein interactions. The Phm gene might respond to only peak levels of the Dl gradient, because the binding sites exhibit a dispersed organization. Of course, it is likely that these thresholds also depend on the binding of additional regulatory factors within the different target enhancers (Markstein, 2002).

It is unclear whether any of the remaining 10 clusters are associated also with Dl target genes. One of the very best clusters, containing four optimal sites within a span of 400 bp, is located near two genes that do not exhibit asymmetric patterns of expression across the dorsoventral axis. Moreover, another cluster is associated with the Runt gene, which is involved in segmentation and almost certainly is not a target of the Dl gradient. However, Runt is a member of the AML family of transcription factors, which have been implicated in mammalian hematopoiesis. It is conceivable that the cluster of Dl binding sites located 3' of the Runt transcription unit are recognized by another Rel-containing transcription factors in Drosophila, Dif and/or Relish, which mediate immune responses (Markstein, 2002).

A regulatory code for neurogenic gene expression in the Drosophila embryo involves Dorsal, Twist and Su(H)

Bioinformatics methods have identified enhancers that mediate restricted expression in the Drosophila embryo. However, only a small fraction of the predicted enhancers actually work when tested in vivo. In the present study, co-regulated neurogenic enhancers that are activated by intermediate levels of the Dorsal regulatory gradient are shown to contain several shared sequence motifs. These motifs permit the identification of new neurogenic enhancers with high precision: five out of seven predicted enhancers direct restricted expression within ventral regions of the neurogenic ectoderm. Mutations in some of the shared motifs disrupt enhancer function, and evidence is presented that the Twist and Su(H) regulatory proteins are essential for the specification of the ventral neurogenic ectoderm prior to gastrulation. The regulatory model of neurogenic gene expression defined in this study permitted the identification of a neurogenic enhancer in the distant Anopheles genome. The prospects for deciphering regulatory codes that link primary DNA sequence information with predicted patterns of gene expression are discussed (Markstein, 2004).

Previous studies identified two enhancers, from the rho and vnd genes, that are activated by intermediate levels of the Dorsal gradient in ventral regions of the neurogenic ectoderm. The present study identified a third such enhancer from the brk gene. This newly identified brk enhancer corresponds to one of the 15 optimal Dorsal-binding clusters described in a previous survey of the Drosophila genome. Although one of these 15 clusters has been shown to define an intronic enhancer in the short gastrulation (sog) gene, the activities of the remaining 14 clusters were not tested. Genomic DNA fragments corresponding to these 14 clusters were placed 5' of a minimal eve-lacZ reporter gene, and separately expressed in transgenic embryos using P-element germline transformation. Four of the 14 genomic DNA fragments were found to direct restricted patterns of lacZ expression across the dorsoventral axis that are similar to the expression patterns seen for the associated endogenous genes (Markstein, 2004).

The four enhancers respond to different levels of the Dorsal nuclear gradient. Two direct expression within the presumptive mesoderm where there are high levels of the gradient. These are associated with the Phm and Ady43A genes. The third enhancer maps ~10 kb 5' of brk, and is activated by intermediate levels of the Dorsal gradient, similar to the vnd and rho enhancers. Finally, the fourth enhancer maps over 15 kb 5' of the predicted start site of the CG12443 gene, and directs broad lateral stripes throughout the neurogenic ectoderm in response to low levels of the Dorsal gradient. In terms of the dorsoventral limits, this staining pattern is similar to that produced by the sog intronic enhancer (Markstein, 2004).

The remaining ten clusters failed to direct robust patterns of expression and are thus referred to as 'false-positives'. Since analysis of spacing and orientation of the Dorsal sites alone did not reveal features that could discriminate between the false positives and the enhancers, whether additional sequence motifs could aid in this distinction was examined. A program called MERmaid was developed that identifies motifs over-represented in specified sets of sequences. MERmaid analysis identified a group of motifs, which was largely specific to the brk, vnd and rho enhancers, suggesting that the regulation of these coordinately expressed genes is distinct from the regulation of genes that respond to different levels of nuclear Dorsal (Markstein, 2004).

The rho, vnd and brk enhancers direct similar patterns of gene expression. The rho and vnd enhancers were previously shown to contain multiple copies of two different sequence motifs: CTGNCCY and CACATGT. A three-way comparison of minimal rho, vnd and brk enhancers permitted a more refined definition of the CTGNCCY motif (CTGWCCY), and also allowed for the identification of a third motif, YGTGDGAA. The CACATGT and YGTGDGAA motifs bind the known transcription factors, Twist and Suppressor of Hairless [Su(H)], respectively. All three motifs are over-represented in authentic Dorsal target enhancers directing expression in the ventral neurogenic ectoderm, as compared with the 10 false-positive Dorsal-binding clusters. Some of the false-positive clusters contain motifs matching either Twist or CTGWCCY; however, none of the false-positive clusters contain representatives of both of these motifs. The rho enhancer is repressed in the ventral mesoderm by the zinc-finger Snail protein. The four Snail-binding sites contained in the rho enhancer share the consensus sequence, MMMCWTGY; the vnd and brk enhancers contain multiple copies of this motif and are probably repressed by Snail as well (Markstein, 2004).

The functional significance of the shared sequence motifs was assessed by mutagenizing the sites in the context of otherwise normal lacZ transgenes. Previous studies have suggested that bHLH activators are important for the activation of rho expression, since rho-lacZ fusion genes containing point mutations in several different E-box motifs (CANNTG) exhibited severely impaired expression in transgenic embryos. However, it was not obvious that the CACATGT motif was particularly significant since it represents only one of five E-boxes contained in the rho enhancer. Yet, only this particular E-box motif is significantly over-represented in the rho, vnd and brk enhancers. vnd-lacZ and brk-lacZ fusion genes were mutagenized to eliminate each CACATGT motif, and analyzed in transgenic embryos. The loss of these sites causes a narrowing in the expression pattern of an otherwise normal vnd-lacZ fusion gene. By contrast, the brk pattern is narrower in central and posterior regions, but relatively unaffected in anterior regions. The brk enhancer contains two copies of an optimal Bicoid-binding site, and it is possible that the Bicoid activator can compensate for the loss of the CACATGT motifs in anterior regions (Markstein, 2004).

Similar experiments were performed to assess the activities of the Su(H)-binding sites (YGTGDGAA) and the CTGWCCY motif. Mutations in the latter sequence cause only a slight reduction and irregularity in the activity of the vnd enhancer, whereas similar mutations nearly abolish expression from the brk enhancer. Thus, CTGWCCY appears to be an essential regulatory element in the brk enhancer, but not in the vnd enhancer. Mutations in both Su(H) sites in the brk enhancer caused reduced staining of the lacZ reporter gene, suggesting that Su(H) normally activates expression. Further evidence that Su(H) mediates transcriptional activation was obtained by analyzing the endogenous rho expression pattern in transgenic embryos carrying an eve stripe 2 transgene with a constitutively activated form of the Notch receptor (Notch^IC). rho expression is augmented and slightly expanded in the vicinity of the stripe2-Notch^IC transgene. A similar expansion is observed for the sim expression pattern (Markstein, 2004).

To determine whether the shared motifs would help identify additional ventral neurogenic enhancers, the genome was surveyed for 250 bp regions containing an average density of one site per 50 bp and at least one occurrence of each of the four motifs for Dorsal, Twist, Su(H) and CTGWCCY. In total, only seven clusters were identified. Three of the seven clusters correspond to the rho, vnd and brk enhancers. Two of the remaining clusters are associated with genes that are known to be expressed in ventral regions of the neurogenic ectoderm: vein and sim. Both clusters were tested for enhancer activity by attaching appropriate genomic DNA fragments to a lacZ reporter gene and then analyzing lacZ expression in transgenic embryos. The cluster associated with vein is located in the first intron, about 7 kb downstream of the transcription start site. The vein cluster (497 bp) directs robust expression in the neurogenic ectoderm, similar to the pattern of the endogenous gene. The cluster located in the 5' flanking region of the sim gene (631 bp) directs expression in single lines of cells in the mesectoderm (the ventral-most region of the neurogenic ectoderm), just like the endogenous expression pattern. These results indicate that the computational methods define an accurate regulatory model for gene expression in ventral regions of the neurogenic ectoderm of D. melanogaster (Markstein, 2004).

To assay the generality of these findings, genomic regions encompassing putative sim orthologs from the distantly related dipteran Anopheles gambiae were scanned for clustering of Dorsal, Twist, Su(H), CTGWCCY and Snail motifs. One cluster located 865 bp 5' of a putative sim ortholog contains one putative Dorsal binding site, two Su(H) sites, three CTGWCCY motifs (or close matches to this motif), a CACATG E-box and several copies of the Snail repressor sequence MMMCWTGY. A genomic DNA fragment encompassing these sites (976 bp) was attached to a minimal eve-lacZ reporter gene and expressed in transgenic Drosophila embryos. The Anopheles enhancer directs weak lateral lines of lacZ expression that are similar to those obtained with the Drosophila sim enhancer. These results suggest that the clustering of Dorsal, Twist, Su(H) and CTGWCCY motifs constitutes an ancient and conserved code for neurogenic gene expression (Markstein, 2004).

This study defines a specific and predictive model for the activation of gene expression by intermediate levels of the Dorsal gradient in ventral regions of the neurogenic ectoderm. The model identified new enhancers for sim and vein in the Drosophila genome, as well as a sim enhancer in the distant Anopheles genome. Five of the seven composite Dorsal-Twist-Su(H)-CTGWCCY clusters in the Drosophila genome correspond to authentic enhancers that direct similar patterns of gene expression. This hit rate represents the highest precision so far obtained for the computational identification of Drosophila enhancers based on the clustering of regulatory elements. Nevertheless, it is still not a perfect code (Markstein, 2004).

Two of the seven composite clusters are likely to be false-positives: they are associated with genes that are not known to exhibit localized expression across the dorsoventral axis. It is possible that the order, spacing and/or orientation of the identified binding sites accounts for the distinction between authentic enhancers and false-positive clusters. For example, there is tight linkage of Dorsal and Twist sites in each of the five neurogenic enhancers. This linkage might reflect Dorsal-Twist protein-protein interactions that promote their cooperative binding and synergistic activities. Previous studies identified particularly strong interactions between Dorsal and Twist-Daughterless (Da) heterodimers. Da is ubiquitously expressed in the early embryo and is related to the E12/E47 bHLH proteins in mammals. Dorsal-Twist linkage is not seen in one of the two false-positive binding clusters (Markstein, 2004).

The regulatory model defined by this study probably fails to identify all enhancers responsive to intermediate levels of the Dorsal gradient. There are at least 30 Dorsal target enhancers in the Drosophila genome, and it is possible that 10 respond to intermediate levels of the Dorsal gradient. Thus, half of all such target enhancers might have been missed. Perhaps the present study defined just one of several 'codes' for neurogenic gene expression (Markstein, 2004).

The possibility of multiple codes is suggested by the different contributions of the same regulatory elements to the activities of the vnd and brk enhancers. Mutations in the CTGWCCY motifs nearly abolish the activity of the brk enhancer, but have virtually no effect on the vnd enhancer. Future studies will determine whether there are distinct codes for Dorsal target enhancers that respond to either high or low levels of the Dorsal gradient. Indeed, it is somewhat surprising that the sog and CG12443 enhancers essentially lack Twist, Su(H) and CTGWCCY motifs, even though they direct lateral stripes of gene expression that are quite similar (albeit broader) to those seen for the rho, vnd and brk enhancers (Markstein, 2004).

This study provides direct evidence that Twist and Su(H) are essential for the specification of the neurogenic ectoderm in early embryos. The Twist protein is transiently expressed at low levels in ventral regions of the neurogenic ectoderm. SELEX assays indicate that Twist binds the CACATGT motif quite well. The presence of this motif in the vnd, brk and sim enhancers, and the fact that it functions as an essential element in the vnd and brk enhancers, strongly suggests that Twist is not a dedicated mesoderm determinant, but that it is also required for the differentiation of the neurogenic ectoderm. However, it is currently unclear whether the CACATGT motif binds Twist-Twist homodimers, Twist-Da heterodimers or additional bHLH complexes in vivo. Su(H) is the sequence-specific transcriptional effector of Notch signaling. The restricted activation of sim expression within the mesectoderm depends on Notch signaling; however, the rho, vnd and brk enhancers direct expression in more lateral regions where Notch signaling has not been demonstrated. Nonetheless, mutations in the two Su(H) sites contained in the brk enhancer cause a severe impairment in its activity. This observation raises the possibility that Su(H) can function as an activator, at least in certain contexts, in the absence of an obvious Notch signal (Markstein, 2004).

The Dorsal gradient produces three distinct patterns of gene expression within the presumptive neurogenic ectoderm. It is proposed that these patterns arise from the differential usage of the Su(H) and Dorsal activators. Enhancers that direct progressively broader patterns of expression become increasingly more dependent on Dorsal and less dependent on Su(H). The sog and CG12443 enhancers mediate expression in both ventral and dorsal regions of the neurogenic ectoderm, and contain several optimal Dorsal sites but no Su(H) sites. By contrast, the sim enhancer is active only in the ventral-most regions of the neurogenic ectoderm, and contains just one high-affinity Dorsal site but five optimal Su(H) sites. The reliance of sim on Dorsal might be atypical for genes expressed in the mesectoderm. For example, the m8 gene within the Enhancer of split complex may be regulated solely by Su(H). The Anopheles sim enhancer might represent an intermediate between the Drosophila sim and m8 enhancers, since it contains optimal Su(H) sites but only one weak Dorsal site. This trend may reflect an evolutionary conversion of Su(H) sites to Dorsal sites, and the concomitant use of the Dorsal gradient to specify different neurogenic cell types. A testable prediction of this model is that basal arthropods use Dorsal solely for the specification of the mesoderm and Su(H) for the patterning of the ventral neurogenic ectoderm (Markstein, 2004).

Organization of developmental enhancers in the Drosophila embryo

Most cell-specific enhancers are thought to lack an inherent organization, with critical binding sites distributed in a more or less random fashion. However, there are examples of fixed arrangements of binding sites, such as helical phasing, that promote the formation of higher-order protein complexes on the enhancer DNA template. This study investigated the regulatory 'grammar' of nearly 100 characterized enhancers for developmental control genes active in the early Drosophila embryo. The conservation of grammar is examined in seven divergent Drosophila genomes. Linked binding sites are observed for particular combinations of binding motifs, including Bicoid-Bicoid, Hunchback-Hunchback, Bicoid-Dorsal, Bicoid-Caudal and Dorsal-Twist. Direct evidence is presented for the importance of Bicoid-Dorsal linkage in the integration of the anterior-posterior and dorsal-ventral patterning systems. Hunchback-Hunchback interactions help explain unresolved aspects of segmentation, including the differential regulation of the eve stripe 3 + 7 and stripe 4 + 6 enhancers. Evidence is presented that there is an under-representation of nucleosome positioning sequences in many enhancers, raising the possibility for a subtle higher-order structure extending across certain enhancers. It is concluded that grammar of gene control regions is pervasively used in the patterning of the Drosophila embryo (Papatsenko, 2009).

Nearly 100 characterized enhancers and ~30 associated binding motifs control the patterning of the early Drosophila embryo, probably the best understood developmental process. These enhancers and sequence-specific TFs regulate the expression of ~50 genes controlling AP and DV patterning, including segmentation and gastrulation. The known TFs controlling embryogenesis represent less than ~10% of all TFs in the Drosophila genome. Thus, this analysis of regulatory grammar was restricted to the ~100 AP and DV enhancers and their ~30 TF inputs (31) (Papatsenko, 2009).

The recent completion of whole-genome sequence assemblies for 12 divergent Drosophila species has created an unprecedented opportunity for analyzing enhancer evolution. In this study 96 selected enhancer sequences from D. melanogaster were mapped to all 12 Drosophila genomes, using the UCSC Browser. The resulting collection combined 1420 kb of genomic sequence data in 1127 sequences, representing 60 enhancers in 23 AP genes and 36 enhancers in 31 DV genes. The entire collection of sequences and binding motifs is available at the Berkeley on-line resource (Papatsenko, 2009).

Inspection of aligned enhancer sequences among all 12 Drosophila species revealed strong conservation within the D. melanogaster subgroup (D. melanogaster, D. simulans, D. seichellia, D. yakuba and D. erecta) and also within the D. obscura group (D. pseudoobscura and D. persimilis). In order to focus on evolutionary changes in these enhancers the seven most divergent Drosophilids were analyzed: D. melanogaster, D. ananassae, D. pseudoobscura, D. willistoni, D. mojavensis, D. virilis and D. grimshawi. The remaining five species contain conservation patterns that are similar to those present in D. melanogaster or D. pseudoobscura (Papatsenko, 2009).

Short-range TF-binding linkages (0-80 bp) were examined in the collection of 96 enhancers from seven species for homo- and heterotypic pairs of binding motifs. Binding sites for the 30 most reliable TF motifs (see the Berkeley online resource) were mapped in enhancers using position weight matrices with match probability cutoff values set to ~2E-04. Distance histograms were generated for distances smaller than 80 bp, measured between the putative centers of each pair of neighboring site matches. Periodic signals were identified in the distance histograms using Fourier analysis, and statistical significance was estimated by bootstrapping positions of site matches in each enhancer sequence (Papatsenko, 2009).

Fourier analysis has identified helical phasing (~11 bp spacing) for several different homotypic activator-activator motif pairs. Such periodic signals were found in the distributions of Bcd-binding sites. Weaker helical-phasing signals were also identified for Caudal (Cad) and Dl-binding sites. Periodic signals close to two DNA turns (~20-22 bp) were found for Twi, Hb and Kruppel. Such helical phasing raises the possibility of direct protein-protein interactions (Papatsenko, 2009).

A weaker, ~11.4-bp periodic signal was detected in the distribution of heterotypic activator-activator site pairs, including Dl-Twi and Bcd-Cad. In contrast, there is a significant reduction in helical phasing signatures for activator-repressor motif pairs, and in fact, an over-representation of site pairs with 'anti-helical' spacing (15.2 bp). A similar 15.2 bp anti-helical signal was detected in distributions of all possible pair-wise combinations of the 30 binding motifs examined in this study. Thus, it would appear that any two randomly chosen binding sites are more likely to occupy the opposite sides of the DNA duplex as compared with helical phasing. This observation raises the possibility that most TFs function either additively or antagonistically to one another and just a special subset of TFs function in a synergistic fashion as reflected by helical phasing of the associated binding sites (Papatsenko, 2009).

The preceding analysis considered 'short-range' organizational constraints, involving linked binding sites separated by <25-30 bp. The possibility of 'long-range' constraints were also considered. The 96 enhancers under study possess characteristic 'unit lengths' of ~500 bp to 1.5 kb (300 bp minimum). The minimal/maximal sizes of the functional enhancers and the 'optimal' site densities can be determined by the amount of encoded information (pattern complexity), mechanisms of TF-DNA recognition such as lateral diffusion, or structural chromatin features like nucleosome positioning (Papatsenko, 2009).

Differential distance histograms reveal an over-representation of short-range linkages (<50 bp), but a depletion in mid-range distances (100-500 bp). These observations raise the possibility that TFs are distributed in a non-uniform manner across the length of the enhancer. That is, there may be sub-clusters, or 'hotspots', of binding sites within a typical enhancer. Such hotspots are observed in the prototypic eve stripe 2 enhancer, whereby 8 of the 12 critical binding sites are observed within two ~50-bp fragments located at either end of the minimal 480 bp enhancer. Homotypic motifs display the greatest propensity for such sub-clustering. Homotypic clusters (38) usually contain 3-5-binding sites distributed over 50-100 bp. Heterotypic activator-activator motif pairs also demonstrate sub-clustering, but these clusters are smaller (<25-30 bp) and usually contain just a pair of heterotypic sites. Heterotypic activator-repressor pairs show moderate enrichment over a distance of 50-70 bp, which is in agreement with the well-documented phenomenon of 'short-range repression'. Depletion of mid-range spacing constraints (around ~200 bp) is especially striking in the case of heterotypic motif pairs. Thus, activator synergy is like short-range repression: it appears to depend on closely linked binding sites (Papatsenko, 2009).

A possible explanation for this depletion of mid-range spacing is the occurrence of positioned nucleosomes, which might separate functionally distinct regions within an enhancer, and also separate neighboring enhancers. To test this hypothesis, nucleosome formation potential was compared with the distributions of TF-binding motifs in enhancers using the 'Recon' program. Three of the four eve enhancers that were examined (eve 1+5, eve 2 and eve 4+6) display a clear negative correlation between potential nucleosome formation and the distribution of TF-binding sites. This observation is consistent with the depletion of nucleosomes near TF-binding sites in vertebrates. This anti-correlation is especially striking in the case of the bipartite eve stripe 1+5 enhancer, where two enhancer regions (stripe 1 and stripe 5) are separated by a 400 bp 'spacer' DNA (in positions 600-1000), which might promote positioning of two nucleosomes and associated linker sequences (Papatsenko, 2009).

To investigate nucleosome positioning further, nucleosome-forming potential was measured in two sets of sequences, previously identified based on clustering of Dl sites and tested in vivo for enhancer activity. One set of sequences functioned as bona fide enhancers and produced localized patterns of gene expression across the DV axis of early embryos. The other set produced no expression in transgenic embryos, despite the presence of the same quality Dl-binding site clusters. The nucleosome-forming potential of the enhancers (true positives) was lower than that of the non-functional sequences (false-positives). These observations raise the possibility that the false Dl-binding clusters fail to function due to the formation of inactive nucleosomal structures (Papatsenko, 2009).

All 465 possible pairwise motif combinations for the 30 relevant binding motifs were tested for conservation in divergent drosophilids. Only linked binding sites, separated by a distance with small variations (max. distance bin = five bases) were considered. In the case of motif pairs, statistical significance was evaluated by bootstrapping columns in the binding motif alignments, thus preserving patterns of conservation. Pairs of homotypic motifs strongly prevailed in this type of analysis (28% of total pairs versus 6.5% expected), suggesting that homotypic interactions are important and pervasive in embryonic patterning. The strongest linkages were found for Bcd, Cad and Hb homotypic pairs. Each of these pairs was shared by five to six different enhancers and conserved in four to seven species. Among the identified heterotypic motif pairs, the most interesting were Bcd-Dl, Bcd-Cad and Dl-Twi (Papatsenko, 2009).

To identify cases of binding site pairs organized in a more flexible fashion, significant motif combinations were extracted using large distance bins or large distance variations. Along with the previously identified motif pairs, this analysis revealed several additional combinations, mainly involving the 'TAG-team' sequence motif, which is recognized by Zelda, a ubiquitous zinc finger TF. Zelda participates in the activation of the early zygotic genome and regulates a wide range of critical patterning genes. Indeed, significant combinations were identified for the TAG motif and Bcd, Dl and Hb. However, all of these TAG-X combinations exhibit spacing variability in different Drosophilids (Papatsenko, 2009).

It is conceivable that these results represent an underestimate of significantly linked motif combinations since very conservative cutoff values were used for statistical evaluation. A database of shared and/or conserved motif pairs, including those below the selected significance cutoff P = 0.03 is available from the Berkeley online resource (Papatsenko, 2009).

Conserved Bcd-Dl-binding site pairs were identified in the enhancers of several AP- and DV-patterning genes, including sal (AP), brk and sog (DV). The sites were found at similar distances, in the same orientation and were conserved in all seven species. It was suggested that the Bcd sites in the brk enhancer might augment gene expression in anterior regions, but this possibility was not directly tested. In wild-type embryos, both brk and sog exhibit significantly broader patterns of gene expression in anterior regions. This expanded pattern is lost in bcd mutants (Papatsenko, 2009).

Highly conserved Hb tandem repeats were detected in the regulatory regions of pair-rule genes, in the gap gene Kruppel, and in the Notch-signaling gene nubbin. Most of the homotypic Hb-Hb site pairs fall into two major groups, separated by either 6-8 or 13-15 bases. Some of the pair-rule enhancers selectively conserve either the 'short' or 'long' arrangement. For example, the eve stripe 4 + 6 enhancer contains two short Hb elements, while the stripe 3 + 7 enhancer contains a single long element. The odd 3 + 6 enhancer contains both short and long elements with various degrees of conservation. The hairy stripe 2,6,7 enhancer contains a single short element. Among the known gap genes, the long and short Hb elements were widely present in the enhancers of Kruppel, and in the blastoderm enhancer of nubbin, but not in any of the known knirps enhancers. It is conceivable that the distinct Hb site arrangements are important for the differential regulation of pair-rule genes by the Hb gradient (Papatsenko, 2009).

In conclusion, the systematic analysis of TF-binding sites in AP and DV patterning enhancers suggests a much higher degree of grammar, or fixed arrangements of binding sites, than is commonly believed. Developmental enhancers are thought to be highly flexible, with randomly distributed binding sites sufficing for the integration of multiple TFs. The results suggest that a large number of enhancers contain conserved short-range arrangements of pairs of binding sites. For instance, virtually all of the enhancers that respond to intermediate and low levels of the Dl gradient contain conserved arrangements of Dl-binding sites along with recognition sequences for other critical DV determinants, such as Twist and Zelda. Cooperating pairs of Bcd sites are found in enhancers responding to low Bcd concentrations, such as Knirps. Finally, distinctive arrangements of Hb-binding sites might influence whether the associated target genes are activated or repressed by high or low levels of the Hb gradient (Papatsenko, 2009).

Deciphering a transcriptional regulatory code: modeling short-range repression in the Drosophila embryo

Systems biology seeks a genomic-level interpretation of transcriptional regulatory information represented by patterns of protein-binding sites. Obtaining this information without direct experimentation is challenging; minor alterations in binding sites can have profound effects on gene expression, and underlie important aspects of disease and evolution. Quantitative modeling offers an alternative path to develop a global understanding of the transcriptional regulatory code. Recent studies have focused on endogenous regulatory sequences; however, distinct enhancers differ in many features, making it difficult to generalize to other cis-regulatory elements. This study applied a systematic approach to simpler elements and presents the first quantitative analysis of short-range transcriptional repressors, which have central functions in metazoan development. Fractional occupancy-based modeling uncovered unexpected features of these proteins' activity that allow accurate predictions of regulation by the Giant, Knirps, Krüppel, and Snail repressors, including modeling of an endogenous enhancer. This study provides essential elements of a transcriptional regulatory code that will allow extensive analysis of genomic information in Drosophila melanogaster and related organisms (Fakhouri, 2010).

In this study, by using a reductionist analysis of short-range repression, a relatively untouched, yet central aspect of gene regulation was explored in Drosophila. Earlier qualitative studies highlighted the extreme distance dependence of short-range repressors, and comparative analysis has shown many instances of evolutionary plasticity of regulatory regions controlled by these proteins. Knowing that transcription factors influence each other in a local manner permitted the identification of novel enhancers, based on the clustering of binding sites. Yet, clustering studies alone do not provide the basis for predicting evolutionary changes that reshape transcriptional output, or predicting activity of coregulated enhancers. For example, the original hypothesis that the affinity and or number of Bicoid-binding sites dictates the output of regulated genes has been replaced by an understanding that other, as-yet unknown features, seem to have more decisive functions (Fakhouri, 2010).

Earlier modeling studies focused on endogenous enhancers, which have complex arrangements of transcription factor-binding sites. The curret studies focused on detecting quantitative differences resulting from subtle differences in binding sites, allowing modeling with a tractable number of parameters. A common block of Dorsal and Twist activator sites was used, allowing a focus on changes made in the number and arrangement of repressor sites; clearly, differences in affinity, number, and arrangement of activator sites also have decisive functions in dictating transcriptional output; thus, future modeling efforts will need to integrate these elements as well. The tight focus on short-range repressors with the analysis of a relatively small number of reporter genes provided sufficient data for robust estimation of important parameters. From the comparison of repression by other short-range repressors, it is likely that the analysis of Giant can guide studies of other similarly acting repressors, including Krüppel, Knirps, and Snail (Fakhouri, 2010).

Relating to transcriptional regulatory code, this study uncovered specific quantitative features that seem to apply to short-range repressors in a general context. A complex non-linear quenching relationship was found that suggests that within the range of activity, Giant, and probably other short-range repressors, have an optimum distance of action that may reflect steric constraints. Multiple formulations of the model generated very similar predictions, suggesting that this non-linear distance function is a real feature of the system. Consistent with this notion, an earlier study of transcription factor-binding sites in Drosophila enhancers discovered an overall preference of Krüppel sites to be found 17 bp from Bicoid activator sites, which may be an indication that other short-range repressors also have preferred distances for optimal activity (Fakhouri, 2010).

The similar quenching efficiencies for repressors acting adjacent to Dorsal or Twist activator sites were an additional significant finding. The similar effect on disparate activator proteins indicates that the effects of short-range repression are general, and are likely to be translatable to distinct contexts. Earlier empirical tests had already pointed in this direction; for example, insertion of ectopic-binding sites for Knirps and Krüppel into rho NEE sequences is sufficient to induce repression, although these proteins do not usually cross-regulate. In addition, short-range repressors can counteract a variety of transcriptional activation domains with similar efficiency, suggesting that specific protein-protein contacts are not essential. In one area quantitative differences were found between parameters derived from the synthetic gene modules and the endogenous regulatory regions. The importance of homotypic cooperativity predicted for Snail sites in the context of the rho NEE was overall much higher than that found for Giant, Krüppel, and Knirps sites acting on the synthetic gene constructs; this might be an example in which the individual proteins do exhibit different context dependencies perhaps because the proteins differ in level of stickiness. Alternatively, the distance between the Snail sites in question, 23 bp, might facilitate cooperative interactions much more than the closely apposed spacing used in the genes genes used in this study, in which steric interference may have an opposing function (Fakhouri, 2010).

In modeling mutant forms of the endogenous rho NEE, several important features of the architecture of this regulatory region were uncovered. This enhancer seems to use redundancy in use of Snail to mediate repression; based on earlier experiments, it seems that even a single Snail site is sufficient to mediate repression. Such redundancy may provide the correct dynamical response, with a swift repression of rho at an early enough time in which Snail levels are still low, or it may ensure that gene output is robust to environmental and genetic noise (Fakhouri, 2010).

The rho NEE modeling also highlighted features of transcriptional activators. Activator-scaling factors for Dorsal were reproducibly lower than those of Twist, and this was apparent for several different assumptions of expression level. The relative differences in contribution to activation can be explained by examination of the structure of the enhancer; contribution by the low intrinsic values of Dorsal is amplified by strong cooperativity with Twist, setting up a chain of interacting weak sites that together are highly active. Experimental evidence bears out these conclusions: isolated Dorsal sites tested on reporter genes mediate relatively weak activation, and a rho NEE lacking Twist sites, but containing four Dorsal sites, is similarly compromised (Fakhouri, 2010).

Earlier studies suggested that many developmental enhancers, including those regulated by short-range repressors, may possess a flexible 'billboard' design, in which individual factors or small groups of proteins would independently communicate with the promoter region, so that the net output of an enhancer would reflect the cumulative set of contacts over a short time period. Such a view of enhancers would account for the evolutionary plasticity observed in regulatory sequences. No DNA-scaffolded superstructure, reflecting the formation of a unique three-dimensional complex, would be necessary in this scenario. Yet, the modeling suggests that the rho NEE might involve communication between relatively distant-binding sites, through sets of cooperative interactions. In this case, it is possible that such distant interactions might be compatible with a flexible structure, if many distinct configurations of binding sites provide such a cooperative network. Current studies have indeed highlighted potential frameworks involving Dorsal and interacting factors on same classes of enhancer. Application of a transcriptional regulatory code integrating activities of activators and repressors is a critical next step to illuminate enhancer design and evolution (Fakhouri, 2010).

Sequential patterns of vnd, ind, and msh expression respond to distinct thresholds of the Dorsal gradient

A nuclear concentration gradient of the maternal transcription factor Dorsal establishes three tissues across the dorsal-ventral axis of precellular Drosophila embryos: mesoderm, neuroectoderm, and dorsal ectoderm. Subsequent interactions among Dorsal target genes subdivide the mesoderm and dorsal ectoderm. The subdivision of the neuroectoderm by three conserved homeobox genes, ventral nervous system defective (vnd), intermediate neuroblasts defective (ind), and muscle segment homeobox (msh) has been investigated. These genes divide the ventral nerve cord into three columns along the dorsal-ventral axis. Sequential patterns of vnd, ind, and msh expression are established prior to gastrulation and evidence is presented that these genes respond to distinct thresholds of the Dorsal gradient. Maintenance of these patterns depends on cross-regulatory interactions, whereby genes expressed in ventral regions repress those expressed in more dorsal regions. This 'ventral dominance' includes regulatory genes that are expressed in the mesectoderm and mesoderm. At least some of these regulatory interactions are direct. For example, the misexpression of vnd in transgenic embryos represses ind and msh, and the addition of Vnd binding sites to a heterologous enhancer is sufficient to mediate repression. The N-terminal domain of Vnd contains a putative eh1 repression domain that binds Groucho in vitro. Mutations in this domain diminish Groucho binding and also attenuate repression in vivo. The significance of ventral dominance is discussed with respect to the patterning of the vertebrate neural tube, and ventral dominance is compared with the previously observed phenomenon of posterior prevalence, which governs sequential patterns of Hox gene expression across the anterior-posterior axis of metazoan embryos (Cowden, 2003).

The ability of Vnd to repress msh in addition to ind raises the possibility that transcriptional repressors expressed in ventral regions of the embryo can inhibit repressors active in more dorsal regions. Support for this hypothesis came from using the Krüppel enhancer to misexpress both ind and msh along the anterior-posterior axis. Ectopic Ind failed to repress vnd expression, while ectopic Msh did not repress either vnd or ind expression. To determine if 'ventral dominance' is restriced to the neuroectoderm, the mesodermal repressor snail was misexpressed in transgenic embryos using the even-skipped (eve) stripe 2 enhancer. The stripe2-snail transgene creates an ectopic domain of snail along the anterior-posterior axis. This ectopic expression leads to a gap in the sim expression pattern. The transgene also causes a gap in the vnd pattern, confirming the model that Snail excludes vnd expression in the ventral mesoderm and restricts expression to the neuroectoderm. The stripe2-snail transgene also creates a gap in the ind pattern. These results support the ventral dominance model, whereby repressors located in ventral regions inhibit repressors expressed in more dorsal regions. Consistent with this 'directionality' of repression, ectopic expression of Vnd, Ind, or Msh does not repress snail (Cowden, 2003).

Further support for ventral dominance of the Snail repressor was obtained by analyzing mutant embryos derived from CtBP germline clones. CtBP is a maternally deposited corepressor protein essential for snail-mediated repression. Removal of this corepressor results in ventral derepression of sim and vnd into the presumptive mesoderm due to loss of Snail mediated repression. However, this ventral expansion of vnd does not result in a transformation of mesoderm into medial neuroblasts. Instead, the expanded vnd pattern is lost at slightly later stages, and expression becomes restricted to lateral regions, similar to the endogenous expression pattern. This lateral restriction is consistent with the observation that neuroblasts are formed in lateral regions of CtBP^- mutants, and not in ventral regions that normally form the mesoderm. Neuroblast segregation can be visualized using a snail antisense RNA probe, which stains all neuroblasts following gastrulation. Sim may be responsible for the late repression of vnd, because vnd expands into the ventral midline of sim mutant embryos. Repression of vnd by Sim is probably indirect because a Krüppel-sim transgene does not alter vnd expression in the lateral neuroectoderm. Perhaps Sim activates an unknown repressor that ultimately inhibits vnd expression in the midline (Cowden, 2003).

It is conceivable that the cross-regulatory interactions among the Snail, Vnd, Ind, and Msh repressors are indirect. For example, perhaps Vnd activates an unknown repressor, which in turn inhibits the expression of ind and msh in medial neuroblasts. Several experiments were done to determine whether Vnd functions as a transcriptional repressor. The first examined whether Vnd binding sites mediate activation or repression in transgenic embryos (Cowden, 2003).

The IAB5 enhancer drives the expression of a lacZ reporter gene in a series of three adjacent bands in the presumptive abdomen of cellularizing embryos. This staining pattern is maintained through gastrulation and germ band elongation. Vnd binding sites were introduced into this IAB5-lacZ transgene by inserting a 220 bp genomic DNA fragment between the IAB5 enhancer and lacZ reporter. This genomic fragment is located 3' of the ind gene and contains three Vnd binding sites. Insertion of this fragment caused a ventrolateral gap in the IAB5-lacZ staining pattern. This gap coincides with the endogenous vnd expression pattern and is maintained during germ band elongation. At this stage, there is a clear loss of lacZ expression in medial regions of the developing ventral nerve cord. The importance of the Vnd binding sites in mediating this repression was examined by mutagenizing all three sites within the 220 bp DNA fragment. Each site was converted from the 5'-CAAGTG-3' consensus to 5'-CCCGGG-3'. The mutagenized IAB5-lacZ transgene exhibits expanded expression in medial regions of the presumptive nerve cord. This observation suggests that Vnd functions as a sequence-specific transcriptional repressor (Cowden, 2003).

Further evidence that Vnd is a repressor was obtained using an in vivo repression assay in transgenic embryos. The N-terminal region of Vnd contains a putative eh1 Groucho-interaction motif, FxIxxIL. This eh1 motif is present in two known transcriptional repressors, Engrailed and Goosecoid. It is also found in the Ind and Msh proteins. GST pull-down assays suggest that this motif mediates interaction between Vnd and Groucho. A GST-VEH1 fusion protein containing amino acid residues 183 to 226 from Vnd binds S³⁵-labeled Groucho protein produced via in vitro translation. This binding is lost when the GST-Vnd fusion protein is mutagenized to replace the phenylalanine in the FxIxxIL motif with an alanine. Various positive and negative controls were included in these experiments. For example, Groucho does not bind a GST-Ind fusion protein containing the Ind homeodomain. Weak binding is observed with a GST-Eve fusion protein containing the FKPY Groucho-interaction motif (Cowden, 2003 and references therein).

A Gal4-Vnd fusion gene containing the Gal4 DNA binding domain and the N-terminal 543 codons of Vnd was placed under the control of the Krüppel 5' regulatory region. The resulting fusion gene is expressed in central regions of cellularizing embryos. Similar levels of expression were obtained with a mutagenized version of the fusion gene that contains multiple alanine substitutions in the FxIxxIL motif. The regulatory activities of the two Gal4-Vnd fusion proteins were monitored with a lacZ reporter gene that contains a modified version of the rhomboid NEE lateral stripe enhancer. The modified NEE enhancer contains three Gal4 binding sites (UAS) and lacks Snail repressor sites. The reporter gene is expressed in ventral regions, including the mesoderm and portions of the lateral neuroectoderm (Cowden, 2003).

The unmutagenized Gal4-Vnd fusion protein containing an intact FxIxxIL motif attenuates expression of the NEE-lacZ reporter gene. This result suggests that the fusion protein binds UAS sites in the modified NEE enhancer and mediates transcriptional repression, either by direct repression of the core promoter, or quenching Dorsal and other activators within the NEE. In contrast, the mutagenized Gal4-Vnd fusion protein (DeltaVEH1) fails to repress expression from the lacZ reporter gene. This result suggests that the FxIxxIL motif is essential for the repression activity of the normal Gal4-Vnd fusion protein. Altogether, these experiments, along with the analysis of Vnd binding sites, suggest that Vnd functions as a sequence-specific transcriptional repressor that might recruit the Groucho corepressor protein (Cowden, 2003).

Thus the Dorsal gradient directly subdivides the neuroectoderm into separate dorsal-ventral compartments through the differential regulation of three conserved homeobox genes, vnd, ind, and msh. Maintenance of sequential patterns of gene expression depends on cross-regulatory interactions, whereby repressors expressed in ventral regions inhibit repressors active in more dorsal regions. This ventral dominance is evocative of the posterior prevalence phenomenon that governs sequential patterns of Hox gene expression across the anterior-posterior axis of metazoan embryos. At least one of the cross-regulatory interactions is direct and evidence was presented that Vnd functions as a sequence-specific transcriptional repressor (Cowden, 2003).

The Dorsal gradient establishes at least three thresholds of gene expression across the dorsal-ventral axis of early embryos. High concentrations activate target genes such as twist and snail in ventral regions that form the mesoderm. Intermediate concentrations activate the rhomboid gene in ventral regions of the neuroectoderm. Finally, low levels of the gradient activate the sog gene in both ventral and dorsal regions of the neuroectoderm. The same low levels of Dorsal repress target genes important for the differentiation of the dorsal ectoderm, including dpp, zen, and tolloid (Cowden, 2003).

Mutant embryos lacking Dorsal fail to activate early expression of either vnd or ind. Conversely, ectopic Dorsal activity leads to a corresponding dorsal shift in the vnd and ind expression patterns. The lateral stripes of vnd expression encompass ventral regions of the neuroectoderm, similar to the rhomboid (rho) pattern. rho is a direct Dorsal target gene that is expressed in the neuroectoderm and encodes a membrane-associated protease that processes the EGFR ligand spitz. Like rho, vnd appears to be a direct target of the Dorsal gradient: an intronic enhancer containing clustered Dorsal and Twist binding sites directs lateral stripes of expression in transgenic embryos. The ind lateral stripes appear to straddle the region between the vnd/rhomboid ventrolateral stripes and the broad sog lateral stripes, and previous studies suggest that ind may be regulated in a different manner from vnd. The regulation of ind relies on both the Dorsal gradient and the EGF signaling pathway. Removal of either Dorsal or the EGF receptor results in the loss of ind expression from the neuroectoderm. It is unclear whether Dorsal directly activates ind or simply establishes a domain of EGF signaling through the regulation of rhomboid (rho). However, given the early onset of ind expression and the misexpression of ind by ectopic Dorsal, it is likely that Dorsal is essential for its regulation. Consistent with the possibility that early ind expression pattern might reflect a threshold readout of the Dorsal gradient is the finding that the low levels of Dorsal present in Toll^rm9/Toll^rm10 embryos are sufficient to activate ind, but not msh. Moreover, the ind lateral stripes do not extend beyond the sog expression pattern, which is known to be directly activated by vanishingly low levels of the Dorsal gradient. Finally, a 3' ind enhancer that encompasses the three Vnd binding sites used in this study contains optimal Dorsal and Twist binding sites, suggesting that it is directly regulated by the Dorsal and Twist gradients (Cowden, 2003).

The initial compartmentalization of the neuroectoderm appears to depend on threshold readouts of the Dorsal gradient. This strategy is different from the subdivision of the other two primary embryonic tissues, the mesoderm and dorsal ectoderm. Patterning the mesoderm depends on interactions between twist and dpp. The Snail repressor establishes the limits of mesoderm invagination, while the localized expression of Dpp restricts induction of the lateral mesoderm to dorsal-lateral regions. Similarly, subdivision of the dorsal ectoderm depends on the differential regulation of the Dorsal target genes sog and dpp. Both genes respond to the same low levels of the Dorsal gradient, but sog is activated by Dorsal, while dpp is repressed. Subsequent protein-protein interactions between Sog and Dpp establish a broad Dpp signaling gradient in the dorsal ectoderm (Cowden, 2003).

Transcriptional repression of ind by Vnd was predicted from previous genetic studies but lateral repression of msh was somewhat unexpected. Previous studies have shown that ectopic Vnd represses msh expression in the procephalic neuroectoderm, where the vnd and msh expression patterns overlap. This result was extended in the present study using a Krüppel-vnd transgene. It would appear that Vnd represses both ind and msh to specify medial neuroblasts. A similar result was seen using the eve stripe 2 enhancer to misexpress snail. Previous studies have shown that Snail acts as a transcriptional repressor to create the boundary between mesoderm and neuroectoderm. As expected, ectopic snail repressed vnd expression but surprisingly, ind was also repressed. These results suggest that the Dorsal gradient separates domains along the dorsal-ventral axis by activating a series of localized transcriptional repressors. According to this model, repressors located in ventral regions selectively repress those located more dorsally, while dorsal repressors do not inhibit ventral repressors. For example, ectopic Vnd represses ind but not snail, while ectopic Ind fails to repress vnd or snail. According to this model, ectopic Ind should repress msh expression. However, because none of the transgenic Krüppel-ind lines persisted until germband elongation when msh expression is uniform, it was not possible to determine if ectopic Ind repressed msh. Similarly, while ectopic Msh failed to repress snail, vnd, or ind expression, the lack of early target genes that are regulated by Msh prevents any definitive conclusions regarding its role as a transcriptional repressor. Both Ind and Msh contain putative eh1 domains, suggesting that they may function as Groucho dependent repressors and previous work supports such a role for Ind and Msh in the ventral nerve cord (Cowden, 2003).

'Ventral dominance' might govern the patterning of the ventral nerve cord in older embryos, in addition to the prepatterning of the neuroectoderm in pregastrulating embryos. Sim might exclude vnd, ind, and msh expression in the ventral midline. In embryos lacking maternal CtBP products, Snail fails to act as a repressor, allowing the ventral expansion of sim and vnd into the presumptive mesoderm. However, vnd expression is ultimately lost from ventral regions, while sim expression persists. As a result, ventral regions form an expanded mesectoderm, while neuroblasts arise from lateral regions. These observations suggest that Sim excludes vnd expression from ventral regions in CtBP mutants, either directly by acting through a CNS specific enhancer or indirectly by activating an unknown repressor. This putative repressor probably does not rely on the CtBP corepressor, as it is still capable of repressing vnd in CtBP germ line clones. According to a ventral dominance scenario, the misexpression of this unknown repressor should inhibit the expression of vnd, ind, and msh in the ventral midline. One potential target for the indirect repressor could be the EGF pathway. The ventral midline is a well-characterized source of EGF signaling and both vnd and ind rely upon EGF signaling for maintenance of expression. By eliminating EGF activation, this midline repressor could prevent vnd and ind expression (Cowden, 2003).

It is conceivable that the ventral dominance model governing cross-regulatory interactions among Vnd, Ind, Msh, Snail, and possibly sim, also applies to the patterning of the vertebrate neural tube. The vertebrate homolog of vnd, Nkx2.2, is expressed in ventral regions of the neural tube, while the homologs of ind (Gsh) and msh (Msx) are expressed in intermediate and dorsal regions, respectively. These neural tube expression patterns match the dorsal-to-ventral positions of vnd, ind, and msh in the ventral nerve cord of Drosophila. Furthermore, the vertebrate homolog of Vnd, Nkx2.2, also functions as a Groucho-dependent transcriptional repressor. A clear prediction of this study is that the misexpression of Nkx2.2 throughout the vertebrate neural tube should lead to the repression of both Gsh and Msx. In contrast, the misexpression of Gsh should repress Msx, but not Nkx2.2. Thus, a cascade of homologous localized transcriptional repressors could subdivide both the vertebrate and invertebrate CNS (Cowden, 2003).

pyramus and thisbe are targets of the Dorsal gradient

The Heartless (Htl) FGF receptor is required for the differentiation of a variety of mesodermal tissues in the Drosophila embryo, yet its ligand is not known. Two FGF genes, thisbe (ths; FGF8-like1) and pyramus (pyr; FGF8-like2), have been identified which probably encode the elusive ligands for this receptor. The two genes were named for the 'heartbroken' lovers described in Ovid's Metamorphoses because the genes are linked and the mutant phenotype exhibits a lack of heart. The genes exhibit dynamic patterns of expression in epithelial tissues adjacent to Htl-expressing mesoderm derivatives, including the neurogenic ectoderm, stomadeum, and hindgut. Embryos that lack ths⁺ and pyr⁺ exhibit defects related to those seen in htl mutants, including delayed mesodermal migration during gastrulation and a loss of cardiac tissues and hindgut musculature. The misexpression of Ths in wild-type and mutant embryos suggests that FGF signaling is required for both cell migration and the transcriptional induction of cardiac gene expression. The characterization of htl and ths regulatory DNAs indicates that high levels of the maternal Dorsal gradient directly activates htl expression, whereas low levels activate ths. It is therefore possible to describe FGF signaling and other aspects of gastrulation as a direct manifestation of discrete threshold readouts of the Dorsal gradient (Stathopoulos, 2004).

ths is directly activated by low levels of the Dorsal gradient in the neuroectoderm (Stathopoulos, 2002). ths is probably kept off in the ventral mesoderm by the localized Snail repressor because the neuroectoderm enhancer contains an optimal Snail-binding site. It is conceivable that the Dorsal gradient controls mesoderm spreading by differentially regulating the ths/pyr FGF ligands in the neuroectoderm and the FGF receptor and intracellular signaling components in the mesoderm (Stathopoulos, 2004).

To investigate this possibility, computational methods were used to identify putative Dorsal target enhancers for the htl and dof/hbr/sms genes. Both genes are activated in the presumptive mesoderm prior to the formation of the ventral furrow, and both are required for the spreading of the mesoderm after invagination. A survey of the htl locus identified a cluster of two putative Dorsal-binding sites and two copies of a distinct sequence motif, CACATGT, which probably binds the Twist activator and is found in several Dorsal target enhancers (Stathopoulos, 2002). The Dorsal-Twist binding cluster is located within the first intron of the htl gene. When expressed in transgenic embryos, this 800-bp fragment directs lacZ expression in the ventral furrow and invaginated mesoderm. A putative dof/hbr/sms enhancer was identified within the first intron of this gene as a cluster of two Dorsal-binding sites and a copy of a conserved sequence motif, RGGNCAG, which is seen in a variety of Dorsal target enhancers (Stathopoulos, 2002). When attached to the lacZ reporter gene, this cluster directs weak expression in the mesoderm of early embryos and tracheal pits of older embryos (Stathopoulos, 2004).

These results provide evidence that htl and dof/hbr/sms are direct target genes of the Dorsal gradient that are induced in response to peak levels of nuclear Dorsal present in ventral regions of early embryos. The previously identified ths enhancer (previously called the Neu4 enhancer) contains three high-affinity Dorsal-binding sites and a Snail repressor site (Stathopoulos, 2002). The ths enhancer directs expression throughout the neurogenic ectoderm during early stages of gastrulation in response to lower levels of nuclear Dorsal. It is, therefore, possible to describe gastrulation as a series of discrete threshold readouts of the Dorsal gradient (Stathopoulos, 2004).

WntD is a target and an inhibitor of the Dorsal/Twist/Snail network in the gastrulating embryo and acts as a repressor of Toll/Dorsal-mediated immunity

The maternal Toll signaling pathway sets up a nuclear gradient of the transcription factor Dorsal in the early Drosophila embryo. Dorsal activates twist and snail, and the Dorsal/Twist/Snail network activates and represses other zygotic genes to form the correct expression patterns along the dorsoventral axis. An essential function of this patterning is to promote ventral cell invagination during mesoderm formation, but how the downstream genes regulate ventral invagination is not yet known. wntD (FlyBase name: Wnt8) is shown to be a member of the Wnt family. The expression of wntD is activated by Dorsal and Twist, but the expression is much reduced in the ventral cells through repression by Snail. Overexpression of WntD in the early embryo inhibits ventral invagination, suggesting that the de-repressed WntD in snail mutant embryos may contribute to inhibiting ventral invagination. The overexpressed WntD inhibits invagination by antagonizing Dorsal nuclear localization, as well as twist and snail expression. Consistent with the early expression of WntD at the poles in wild-type embryos, loss of WntD leads to posterior expansion of nuclear Dorsal and snail expression, demonstrating that physiological levels of WntD can also attenuate Dorsal nuclear localization. The de-repressed WntD in snail mutant embryos contributes to the premature loss of snail expression, probably by inhibiting Dorsal. Thus, these results together demonstrate that WntD is regulated by the Dorsal/Twist/Snail network, and is an inhibitor of Dorsal nuclear localization and function. The closest homologs of Drosophila WntD, vertebrate Wnt8 proteins, regulate mesoderm patterning, neural crest cell induction, neuroectoderm patterning, and axis formation (Hoppler, 1998; Lekven, 2001; Lewis, 2004; Popperl, 1997). These vertebrate Wnt8 proteins may transmit the signal through the canonical pathway, but the exact mechanism remains unclear. So far, the downstream mediators of Drosophila WntD signaling are not known (Ganguly, 2005).

A second study (Gordon, 2005) confirms and extends Ganguly (2005) by inducing a mutation in wntD by homologous replacement. The Gordon study shows that WntD acts as a feedback inhibitor of the NF-kappaB homologue Dorsal, during both embryonic patterning and the innate immune response to infection. wntD expression is under the control of Toll/Dorsal signalling, and increased levels of WntD block Dorsal nuclear accumulation, even in the absence of the IkappaB homologue Cactus. The WntD signal is independent of the common Wnt signalling component Armadillo. By engineering a gene knockout, this study shows that wntD loss-of-function mutants have immune defects and exhibit increased levels of Toll/Dorsal signalling. Furthermore, the wntD mutant phenotype is suppressed by loss of zygotic dorsal (Gordon, 2005).

To identify novel components in the dorsoventral pathway, a microarray assay was carried out using embryos derived from gain-of-function and loss-of-function mutants of the Toll pathway. Among the novel genes identified, the expression and function of wntD was analyzed because the Wnt family of secreted proteins regulates patterning, cell polarity and cell movements. The results show that wntD is activated by Dorsal and Twist but repressed by Snail. Increased expression of WntD in wild-type early embryos inhibits ventral invagination. Thus, wntD is the first Snail target gene shown to have an interfering function in mesoderm invagination. The overexpressed WntD blocks invagination by inhibiting Dorsal nuclear localization. Loss-of-function analyses also show that physiological levels of WntD can attenuate Dorsal nuclear localization and function. Therefore, wntD is a novel downstream gene of the Dorsal/Twist/Snail network and can feed back to inhibit Dorsal (Ganguly, 2005).

The dynamic pattern of wntD expression in the early embryo is a combined result of activation by Dorsal/Twist and repression by Snail. Overexpressed WntD negatively regulates Dorsal nuclear localization, leading to an inhibition of ventral cell invagination. Physiological levels of WntD can also negatively regulate Dorsal, since loss of WntD leads to detectable expansion of both Dorsal nuclear localization and snail expression in the posterior regions. Furthermore, de-repressed WntD expression in the ventral region of snail mutant embryos can also attenuate Dorsal function. However, the loss of WntD could not rescue the invagination defect of the snail mutant embryo, suggesting that in the snail mutant embryo there are other de-repressed genes that can interfere with ventral invagination (Ganguly, 2005).

The wntD loss-of-function phenotype correlates with the expression of wntD at the poles of pre-cellular blastoderms. wntD is also expressed a bit later in the mesectoderm, and weakly in the mesoderm. Because WntD can inhibit Dorsal, one speculation is that WntD in the early mesectoderm may help to establish the sharp snail expression at the mesectoderm-neuroectoderm boundary. However, no changes were detected in the Dorsal protein gradient or snail pattern in the trunk regions of the Df(3R)l26c embryos. It is speculated that the timing of early expression of wntD, which may have additional input from the Torso pathway at the poles, is important for the feedback inhibition of Dorsal. By the time of cellularization, the Dorsal protein gradient is well established. This well-established Dorsal gradient activates the wntD gene in the trunk regions, but the subsequently translated WntD protein may not be capable of exerting a strong negative-feedback effect on the already formed Dorsal gradient. This timing argument is supported by the results of WntD-overexpression experiments. The use of maternal nanos-Gal4 caused a strong inhibition of Dorsal nuclear localization and of ventral invagination, whereas the use of zygotic promoters did not result in a significant phenotype (Ganguly, 2005).

Snail acts as a transcriptional repressor for at least 10 genes in the ventral region where mesoderm arises. In snail mutant embryos, all of these target genes are de-repressed in the ventral cells, concomitant with severe ventral invagination defects. However, no direct evidence has been reported on whether these de-repressed genes interfere with invagination. This study showed for the first time that a target gene of Snail, namely wntD, can block ventral invagination when overexpressed. If de-repressed WntD is solely responsible for inhibiting ventral invagination, it would be expected that, in the snail;Df(3R)l26c double-mutant embryos, ventral invagination would appear again. No rescue of ventral invagination was detected in the double-mutant embryos, suggesting that wntD is not the only de-repressed target gene that inhibits invagination. Nonetheless, the de-repressed WntD can attenuate Dorsal function, and may contribute to the ventral invagination defect (Ganguly, 2005).

In light of the interaction between WntD and Dorsal in the embryo, it was asked if WntD could have a role later in the fly's life as a repressor of Toll/Dorsal-mediated immunity. Polymerase chain reaction with reverse transcription (RT-PCR) was used to confirm expression of endogenous wntD RNA in adults. wntD mutant adults appear normal, with the exception that at low frequency (1%-2%) sites of ectopic melanization have been observed, most notably on the wing hinge. This is consistent with a role for WntD in maintaining low basal levels of Toll/Dorsal signalling; other mutations that hyper-activate Toll show increased levels of phenoloxidase-driven melanization. Furthermore, Dorsal has been shown to be an essential component of the melanization response in larvae (Gordon, 2005).

To investigate the role of WntD after septic injury, wntD and control flies were injected with a dilute culture of the gram-positive bacterium Micrococcus luteus, and the induction of antimicrobial peptide (AMP) transcripts were monitored over time using quantitative RT-PCR. It was observed that some, but not all, AMPs showed aberrant expression in wntD mutants. The AMP diptericin is most severely affected, with wntD flies displaying dramatically elevated basal levels of expression (approximately 15-fold), and significantly higher mRNA levels following infection. In contrast, Drosomycin mRNA levels were not significantly different from controls in either uninfected or infected wntD mutants. A third AMP, defensin, showed an intermediate pattern of expression, with elevated mRNA levels in wntD mutants at some time points (Gordon, 2005).

These results pose an apparent paradox, since previous experiments have characterized diptericin as a target of IMD/Relish, and drosomycin as a target of Toll signalling. Drosomycin expression is reported to be primarily regulated by Dif in adult flies, and appears to be unaffected by increased Dorsal activity. Thus, the results for Drosomycin are consistent with past work. The diptericin result initially appears puzzling, but existing data demonstrate that the signal transduction pathways regulating immunity are not as specific as initially described. For example, Relish is required for diptericin induction in response to infections in vivo, but constitutive activation of Toll signalling results in elevated levels of diptericin in adult flies. Furthermore, Dorsal is sufficient to activate the diptericin promoter in vitro. The simplest explanation for these observations is that diptericin transcription can be induced by Toll/Dorsal signalling. Taken together, these data support a model in which WntD signalling specifically represses Toll/Dorsal, and not Toll/Dif signalling (Gordon, 2005).

Given a role for WntD in the regulation of antimicrobial gene transcription, attempts were made to determine whether wntD mutants were immunocompromised. To test this, wntD and control adults were infected with the gram-positive, lethal pathogen Listeria monocytogenes. In response to infection, wntD mutants exhibit significantly higher levels of mortality when compared with parental lines. Importantly, this phenotype is suppressed by the introduction of dorsal mutations, with close to full suppression in the absence of both copies of dorsal and partial suppression in flies heterozygous for a dorsal mutation. These genetic interactions are consistent with the assertion that WntD specifically regulates Dorsal, and not other mediators of immunity. Recent reports have demonstrated that a fly's response to bacterial challenge includes factors that are damaging to the host, and that increased Toll signalling can render flies more susceptible to viral infection. It is therefore proposed that it is the deleterious hyper-activation of specific Dorsal target genes that is responsible for the increased mortality seen in wntD mutants. Furthermore, the susceptibility of wntD mutants to a lethal infection suggests a reason for the positive selection of wntD during evolution; immune responses have a cost, and their appropriate downregulation would be expected to provide flies with a selective advantage. Although wntD flies appear healthy in a lab environment, it is easy to imagine that under the more stressful, and septic, conditions in the wild, flies lacking wntD would suffer the perils of a hyperactive immune system (Gordon, 2005).

This study has presented evidence that WntD, a Wnt family member, produces a signal that blocks the nuclear translocation of Dorsal. Furthermore, WntD is a target of Toll/Dorsal signalling, and creates a negative feedback loop to repress Dorsal activation. wntD is not required for viability under lab conditions, but wntD mutants show defects in embryonic Dorsal regulation, and in the adult innate immune system. Since the WntD signal in the embryo is not mediated by Armadillo, it is supposed that the immune function of WntD is also Armadillo-independent, although immune defects have been observed in flies expressing a dominant-negative form of the Aramdillo partner DTCF. Further characterization of signalling events bridging WntD and Dorsal could yield valuable insight into the regulation of the therapeutically important NF-kappaB family of proteins (Gordon, 2005).

Computational models for neurogenic gene expression in the Drosophila embryo

The early Drosophila embryo is emerging as a premiere model system for the computational analysis of gene regulation in development because most of the genes, and many of the associated regulatory DNAs, that control segmentation and gastrulation are known. The comprehensive elucidation of Drosophila gene networks provides an unprecedented opportunity to apply quantitative models to metazoan enhancers that govern complex patterns of gene expression during development. Models based on the fractional occupancy of defined DNA binding sites have been used to describe the regulation of the lac operon in E. coli and the lysis/lysogeny switch of phage lambda. This study applies similar models to enhancers regulated by the Dorsal gradient in the ventral neurogenic ectoderm (vNE) of the early Drosophila embryo. Quantitative models based on the fractional occupancy of Dorsal, Twist, and Snail binding sites raise the possibility that cooperative interactions among these regulatory proteins mediate subtle differences in the vNE expression patterns. Variations in cooperativity may be attributed to differences in the detailed linkage of Dorsal, Twist, and Snail binding sites in vNE enhancers. It is proposed that binding site occupancy is the key rate-limiting step for establishing localized patterns of gene expression in the early Drosophila embryo (Zinzen, 2006).

Comprehensive identification of Drosophila dorsal-ventral patterning genes using a whole-genome tiling array

Dorsal-ventral (DV) patterning of the Drosophila embryo is initiated by Dorsal, a sequence-specific transcription factor distributed in a broad nuclear gradient in the precellular embryo. Previous studies have identified as many as 70 protein-coding genes and one microRNA (miRNA) gene that are directly or indirectly regulated by this gradient. A gene regulation network, or circuit diagram, including the functional interconnections among 40 Dorsal (Dl) target genes and 20 associated tissue-specific enhancers, has been determined for the initial stages of gastrulation. This study attempts to extend this analysis by identifying additional DV patterning genes using a recently developed whole-genome tiling array. This analysis led to the identification of another 30 protein-coding genes, including the Drosophila homolog of Idax, an inhibitor of Wnt signaling. In addition, remote 5' exons were identified for at least 10 of the ~100 protein-coding genes that were missed in earlier annotations. As many as nine intergenic uncharacterized transcription units (TUs) were identified, including two that contain known microRNAs, miR-1 and -9a. The potential functions of these recently identified genes are discussed and it is suggested that intronic enhancers are a common feature of the DV gene network (Biemar, 2006).

The Dl nuclear gradient differentially regulates a variety of target genes in a concentration-dependent manner. The gradient generates as many as five different thresholds of gene activity, which define distinct cell types within the presumptive mesoderm, neuroectoderm, and dorsal ectoderm. Total RNA was extracted from embryos produced by three different maternal mutants: pipe^–/pipe^–, Toll^rm9/Toll^rm10, and Toll^10B. pipe^–/pipe^– mutants completely lack Dl nuclear protein and, as a result, overexpress genes that are normally repressed by Dl and restricted to the dorsal ectoderm. For example, the decapentaplegic (dpp) TU is strongly "lit up" by total RNA extracted from pipe^–/pipe^– mutant embryos. The intron-exon structure of the transcribed region is clearly delineated by the hybridization signal, most likely because the processed mRNA sequences are more stable than the intronic sequences present in the primary transcript. There is little or no signal detected with RNAs extracted from Toll^rm9/Toll^rm10 (neuroectoderm) and Toll^10B (mesoderm) mutants. Instead, these other mutants overexpress different subsets of the Dl target genes. For example, Toll^rm9/Toll^rm10 mutants contain low levels of Dl protein in all nuclei in ventral, lateral, and dorsal regions. These low levels are sufficient to activate target genes such as intermediate neuroblasts defective (ind), ventral neuroblasts defective (vnd), rhomboid (rho), and short gastrulation (sog) but insufficient to activate snail (sna). In contrast, Toll^10B mutants overexpress genes (e.g., sna) normally activated by peak levels of the Dl gradient in ventral regions constituting the presumptive mesoderm (Biemar, 2006).

To identify potential Dl targets, ranking scores were assigned for the six possible comparisons of the various mutant backgrounds, pipe vs. Toll^rm9/Toll^rm10, pipe vs. Toll^10B, Toll^rm9/Toll^rm10 vs. Toll^10B, Toll^rm9/Toll^rm10 vs. pipe, Toll^10B vs. Toll^rm9/Toll^rm10, and Toll^10B vs. pipe, using the TiMAT software package. As a first approximation, only hits with a median fold difference of 1.5 and above were considered. For further analysis, the top 100 TUs were selected for each of the comparisons, with the exception of Toll^rm9/Toll^rm10 vs. pipe for which the TiMAT analysis returned only 43 hits that meet the cutoff. To refine the search for TUs specifically expressed in the mesoderm, where levels of nuclear Dl are highest, only those present in the Toll^10B vs. Toll^rm9/Toll^rm10 and Toll^10B vs. pipe, but not pipe vs. Toll^rm9/Toll^rm10 comparisons were selected. For TUs induced by intermediate and low levels of nuclear Dl in the neuroectoderm, those present in both the Toll^rm9/Toll^rm10 vs. Toll^10B and Toll^rm9/Toll^rm10 vs. pipe, but not pipe vs. Toll^10B comparisons were selected. For TUs restricted to the dorsal ectoderm, only those present in the pipe vs. Toll^rm9/Toll^rm10 and pipe vs. Toll^10B, but not Toll^rm9/Toll^rm10 vs. Toll^10B, were selected. Finally, the TUs corresponding to annotated genes already identified in the previous screen were eliminated to focus on annotated genes not previously considered as potential Dorsal targets, as well as transcribed fragments (transfrags) not previously characterized. Using these criteria, 45 previously annotated protein-coding genes were identified, along with 23 uncharacterized transfrags. Of the 45 protein-coding genes, 29 exhibited localized patterns of gene expression across the DV axis, whereas the remaining 16 were not tested (Biemar, 2006).

The previous microarray screen relied on high cutoff values for the identification of authentic DV genes. For example, only genes exhibiting 6-fold up-regulation in pipe^–/pipe^– mutant embryos were tested by in situ hybridization for localized expression in the dorsal ectoderm. Many other genes displayed >2-fold up-regulation but were not explicitly tested for localized expression. The whole-genome tiling array permitted the use of much lower cutoff values. For example, CG13800, which was identified by conventional microarray screens, falls just below the original cutoff value but displays 5-fold up-regulation in pipe^–/pipe^– mutants in the analysis. In situ hybridization assays reveal localized expression in the dorsal ectoderm. This pattern is greatly expanded in embryos derived from pipe^–/pipe^– mutant females, as expected for a gene that is either directly or indirectly repressed by the Dl gradient. Genes exhibiting even lower cutoff values were also found to display localized expression. Among these genes is a Wnt homologue, Wnt2, which is augmented only 2.25-fold in mutant embryos lacking the Dl nuclear gradient (Biemar, 2006).

The 4-fold cutoff value used in the previous screen for candidate protein-coding genes expressed in the neuroectoderm also excluded genes expressed in this tissue. The Trim9 gene exhibits just a 2-fold increase in mutant embryos derived from Toll^rm9/Toll^rm10 females. Nonetheless, in situ hybridization assays reveal localized expression in the neuroectoderm of WT embryos. As expected, expression is expanded in Toll^rm9/Toll^rm10 mutant embryos. Another gene, CG9973, displays just 1.8-fold up-regulation but is selectively expressed in the neuroectoderm. CG9973 encodes a putative protein related to Idax, an inhibitor of the Wnt signaling pathway. Idax inhibits signaling by interacting with the PDZ domain of Dishevelled (Dsh), a critical mediator of the pathway. A Wnt2 homologue is selectively expressed in the dorsal ectoderm. Recent studies identified a second Wnt gene, WntD, which is expressed in the mesoderm. Thus, the CG9973/Idax inhibitor might be important for excluding Wnt signaling from the neuroectoderm. Such a function is suggested by the analysis of Idax activity in vertebrate embryos (Biemar, 2006).

Additional genes were also identified that are specifically expressed in the mesoderm. Among these is CG9005, which encodes an unknown protein that is highly conserved in different animals, including frogs, chicks, mice, rats, and humans. It displays <2-fold up-regulation in Toll^10B embryos but is selectively expressed in the ventral mesoderm of WT embryos. Expression is expanded in embryos derived from Toll^10B mutant females (Biemar, 2006).

Other protein-coding genes were missed in the previous screen because they were not represented on the Drosophila Genome Array used at the time. These include, for instance, CG8147 in the dorsal ectoderm and CG32372 in the mesoderm (Biemar, 2006).

An interesting example of the use of tiling arrays to identify tissue-specific isoforms is seen for the bunched (bun) TU. bun encodes a putative sequence-specific transcription factor related to mammalian TSC-22, which is activated by TGFβ signaling. It was shown to inhibit Notch signaling in the follicular epithelium of the Drosophila egg chamber. Three transcripts are expressed from alternative promoters in bun, but it appears that only the short isoform (bun-RC) is specifically expressed in the dorsal ectoderm. A number of bun exons are ubiquitously transcribed at low levels in the mesoderm, neuroectoderm, and dorsal ectoderm. However, the 3'-most exons are selectively up-regulated in pipe^–/pipe^– mutants. It is conceivable that Dpp signaling augments the expression of this isoform, which in turn, participates in the patterning of the dorsal ectoderm (Biemar, 2006).

In addition to protein-coding genes, the tiling array also identified uncharacterized TUs not previously annotated. Some of them are associated with ESTs, providing independent evidence for transcriptional activity in these regions. For 14 of these transfrags (61%), visual inspection of neighboring loci using the Integrated Genome Browser suggested coordinate expression of a neighboring protein-coding region (i.e., overexpressed in the same mutant background). The N-Cadherin gene (CadN) has a complex intron-exon structure consisting of ~20 different exons. The strongest hybridization signals are detected within the limits of exons, but an unexpected signal was detected ~10 kb upstream of the 5'-most exon. It is specifically expressed in the mesoderm, suggesting that it represents a previously unidentified 5' exon of the CadN gene. Support for this contention stems from two lines of evidence: (1) in situ hybridization using a probe against the 5' exon detects transcription in the presumptive mesoderm, the initial site of CadN expression; (2) using primers anchored in the 5' transfrag as well as the first exon of CadN, confirmation was obtained by RT-PCR that the recently identified TU is part of the CadN transcript. This recently identified 5' exon appears to contribute to the 5' leader of the CadN mRNA. It is possible that this extended leader sequence influences translational efficiency as seen in yeast. Because there seems to be a considerable lag between the time when CadN is first transcribed and the first appearance of the protein, it is suggested that this extended leader sequence might inhibit translation. An interesting possibility is that it does so through short upstream ORFs, as has been shown for several oncogenes in vertebrates (Biemar, 2006).

A 5' exon was also identified for crossveinless-2 (cv-2), a component of the Dpp bone morphogenetic protein (BMP) signaling pathway. cv-2 binds BMPs and functions as both an activator and inhibitor of BMP signaling. It is specifically required in the developing wing disk to generate peak Dpp signaling in the presumptive crossveins. cv-2 is also expressed in the dorsal ectoderm of early embryos, but its role during embryonic development has not been investigated. The whole-genome tiling array identified a 5' exon located ~10 kb 5' of the transcription start site of the cv-2 TU. Using RT-PCR and in situ hybridization assays, it was confirmed that the exon is part of the cv-2 transcript. It is possible that the exon resides near an embryonic promoter that is inactive in the developing wing discs. Future studies will determine whether this 5' exon influences the timing or levels of Cv-2 protein synthesis (Biemar, 2006).

In addition to the identification of 10 5' exons associated with previously annotated genes such as CadN and cv-2, three other transfrags appear to correspond to 3' exons, and nine of the RNAs seem to arise from autonomous TUs. Three of these represent annotated computational RNA (CR) genes: CR32777, CR31972, and CR32957. CR32777 corresponds to roX1, which is ubiquitously expressed at the blastoderm stage, hence it represents a false positive. The other two potential noncoding RNAs were recently identified independently in two other studies, and although the expression of CR32957 could not be detected by in situ hybridization, CR31972 transcripts are detected in the mesoderm. There is no evidence that these transcripts are processed into miRNAs, but noncoding genes corresponding to known miRNA loci were also identified in the screen. Transfrag 22 corresponds to the miR-9a primary transcript (pri-mir9a) and is detected in both the dorsal- and neuroectoderm. Expression of pri-mir9a is ubiquitous in embryos derived from pipe^–/pipe^– or Toll^rm9/Toll^rm10 females. Transfrag 8 corresponds to pri-mir1, which is present in the mesoderm (Biemar, 2006).

A third noncoding transcript (Transfrag 12) maps next to a known miRNA, miR-184. It is selectively expressed in the mesoderm and overexpressed in Toll^10B mutants. The mesodermal expression of miR-184 has been reported. It is possible that Transfrag 12 corresponds to pri-mir-184, and that secondary structures in the miRNA region preclude detection on the array. This is seen for several other miRNA precursors expressed at various stages during embryogenesis. Alternatively, Transfrag 12 might represent the fragment resulting from Drosha cleavage of the pri-mir-184 to produce the miR-184 precursor hairpin (pre-miR-184). A similar situation has been observed for the iab4 locus. Like miR-1, miR-184 is selectively expressed in the ventral mesoderm. It will be interesting to determine whether the two miRNAs jointly regulate some of the same target mRNAs (Biemar, 2006).

The identity of the last three transfrags is less clear. Visual inspection using the Integrated Genome Browser suggests expression of Transfrag 10 in the mesoderm, Transfrag 21 in the neuroectoderm, and Transfrag 11 in both the dorsal ectoderm and neuroectoderm. However, in situ hybridization assays confirm the predicted expression pattern only for Transfrag 11. Computational analyses designed to estimate the likelihood of translation suggest a protein-coding potential for Transfrag 10 [Likelihood Ratio Test (LRT) P < 0.001] and possibly Transfrag 11 (LRT P < 0.01), whereas Transfrag 21 could not be analyzed because of lack of conservation in other Drosophila species (Biemar, 2006).

This work has attempted to identify nonprotein coding genes involved in patterning the DV axis of the Drosophila embryo using an unbiased approach to survey the entire genome. This study, along with earlier analyses, identified as many as 100 protein-coding genes and five to seven noncoding genes that are differentially expressed across the DV axis of the early Drosophila embryo. Roughly half of the noncoding RNAs correspond to miRNAs, although <1% of the annotated genes in the Drosophila genome encode miRNAs. Future studies will determine how these RNAs impinge on the DV regulatory network (Biemar, 2006).

Recent studies have identified large numbers of noncoding transcripts in the mouse and human genomes. If the present study is predictive, less than one-fourth of the transcripts correspond to novel noncoding RNAs of unknown function, akin to CR31972 and Transfrag 11 expressed in the mesoderm and ectoderm, respectively. Most of the noncoding transcripts are likely to derive from intronic sequences because of the occurrence of cryptic remote 5' exons as seen for the CadN and cv-2 genes. At least 10% of the DV protein-coding genes were found to contain such exons. As a result, these genes contain large tracts of intronic sequences that might encompass regulatory DNAs such as tissue-specific enhancers. The FGF8-related gene, thisbe (ths), represents such a case. A neurogenic-specific enhancer that was initially thought to reside 5' of the TU actually maps within a large intron because of the occurrence of a remote 5' exon. It is suggested that such exons are responsible for the evolutionary "bundling" of genes and their associated regulatory DNAs. Gene duplication events are more likely to retain this linkage when regulatory DNAs map within the TU. In contrast, enhancers mapping in flanking regions can be uncoupled from their normal target gene by chromosomal rearrangements (Biemar, 2006).

A core transcriptional network for early mesoderm development in Drosophila consists of Twist, Mef2, Tinman and Dorsal

Embryogenesis is controlled by large gene-regulatory networks, which generate spatially and temporally refined patterns of gene expression. This study reports the characteristics of the regulatory network orchestrating early mesodermal development in the fruitfly, where the transcription factor Twist is both necessary and sufficient to drive development. Through the integration of chromatin immunoprecipitation followed by microarray analysis (ChIP-on-chip) experiments during discrete time periods with computational approaches, >2000 Twist-bound cis-regulatory modules (CRMs) were identified and almost 500 direct target genes. Unexpectedly, Twist regulates an almost complete cassette of genes required for cell proliferation in addition to genes essential for morophogenesis and cell migration. Twist targets almost 25% of all annotated Drosophila transcription factors, which may represent the entire set of regulators necessary for the early development of this system. By combining in vivo binding data from Twist, Mef2, Tinman, and Dorsal an initial transcriptional network was constructed of early mesoderm development. The network topology reveals extensive combinatorial binding, feed-forward regulation, and complex logical outputs as prevalent features. In addition to binary activation and repression, it is suggested that Twist binds to almost all mesodermal CRMs to provide the competence to integrate inputs from more specialized transcription factors (Sandmann, 2007).

Twist and Dorsal collaborate much more extensively than previously predicted

One of the earliest functions of Twist within the pregastrula embryo is the coregulation of D-V patterning with the NFkappaB ortholog Dorsal. Dorsal acts as a morphogen by regulating its target genes at (at least) three threshold concentrations along the D-V axis. Type I-regulated Dorsal enhancers receive high levels of Dorsal, contain low-affinity Dorsal sites and drive expression in ventral mesodermal domains (e.g., sna, htl, twi). Type II enhancers receive intermediate levels of Dorsal and drive expression in mediolateral domains of different sizes (e.g., sim, brk, vn), while Type III enhancers receive low levels of Dorsal, contain high-affinity Dorsal sites, and can be either activated (sog, ths) or repressed (dpp, tld, zen) by Dorsal. This system has been studied so intensively that the level of knowledge is sufficient for quantitative modeling of cis-regulatory interactions. It was therefore of interest to determine whether global analysis could reveal new insights into this process. The data identified in vivo binding of Twist to both Type I and II Dorsal enhancers, as expected. The boundaries of Twist binding are in remarkable agreement with the limits of characterized minimal enhancers (e.g., htl, rho, and ths). More importantly, new CRMs were identified for several of these well-characterized genes (Sandmann, 2007).

Seven novel enhancers for D-V patterning genes reveal the regulatory complexity of Twist-bound CRMs: The cactus, stumps, and wntD enhancers drive expression in a domain overlapping Twist within the ventral blastoderm and likely represent Type I enhancers. Cactus, an IkappaB ortholog, is expressed both maternally and zygotically and sequesters Dorsal within the cytoplasm. While the regulation of zygotic cactus expression was previously not understood, these data reveal a Twist-bound CRM that is sufficient to drive expression in the presumptive mesoderm. Twist also binds to a CRM of Toll. Although the function of cactus'and Toll's zygotic regulation remains unclear in Drosophila, positive feedback regulation of zygotic Toll-receptor expression is required to refine the Dorsal nuclear gradient in the flour beetle Tribolium castaneum (Sandmann, 2007).

The stumps CRM is expressed in a subset of Twist-expressing cells, yielding a salt and pepper pattern that may reflect the requirement for a second, partially redundant enhancer (e.g., the 'stumps_early' enhancer) to give robust expression. The wntD CRM is highly expressed at the anterior and posterior poles of the ventral blastoderm, but is very weakly expressed within the central region. This mirrors the transient expression of the endogenous gene at this stage of development. This single enhancer reflects the regulatory logic deduced from genetic studies: The inputs from Twist and Dorsal activate WntD, while Snail represses its transcription within the presumptive mesoderm. The CRM for crumbs reproduces the endogenous genes expression. This 480-bp region can function as an enhancer in the ectoderm while acting as a silencer within the ventral blastoderm. This ventral repression is most likely due to direct input from Snail on this CRM. Therefore, even at the same stage of development, these four Twist-bound CRMs drive expression in different spatial patterns within a small population of cells. This complexity is clearly mediated by context-dependent integration of additional inputs. Three additional CRMs for mir-1 (Type I), vn, and sim (Type II Dorsal targets) drive expression later in development, reproducing part of the endogenous gene’s expression (Sandmann, 2007).

Unexpectedly, Twist also binds to characterized Dorsal Type III enhancers known to regulate dpp, ind, and ths. Dorsal and its associated corepressors Cut, Retained, and Capicua recruit Groucho to repress dpp, confining its expression to the dorsal blastoderm. The cobinding of Twist and Dorsal to Type III CRMs suggests that these factors may also collaborate in transcriptional repression. Interestingly, Twist binds to regulatory regions of all three Dorsal corepressors, providing another level at which Twist may modulate Dorsal-mediated repression. Overall, this exhaustive map of new CRMs for D-V patterning genes greatly extends previous knowledge and will likely improve predictive models for this system (Sandmann, 2007).

Twist targets functional modules required for diverse aspects of mesoderm development

Twist is not only required for D-V patterning. The 494 direct target genes are significantly enriched in functional groups of genes involved in cell communication, signal transduction, cell motility, and cell adhesion. Genes in these categories are essential for multiple aspects of development, including gastrulation and directed migration of mesodermal cells. Genetic studies have demonstrated a requirement for twist in these processes; however, the molecular mechanism remained ill-defined. These data reveals Twist binding to CRMs for entire functional modules necessary for both gastrulation and migration (the FGF pathway) (Sandmann, 2007).

The present study highlights a new direct connection between Twist and many key components involved in cell cycle progression and cell growth. Members of both the Cdk2/CyclinA/B and Cdk2/CyclinE complexes are targeted, as well as modifiers of their activity and genes involved in cytokinesis and replication. In many cases, Twist binds to several CRMs of these genes (e.g., cyclinE and E2f), revealing the complexity of their regulation. This surprising link between Twist and the cell cycle is highly likely to be of regulatory significance; twist mutant embryos have proliferative defects that can be genetically separated from the block in mesoderm gastrulation (Sandmann, 2007).

These three functional groups of target genes (involved in morphogenesis, migration, and cell proliferation) have been defined as essential developmental network 'plug-ins.' Twist orchestrates early mesoderm development by binding to CRMs of virtually all genes within functional groups essential for gastrulation, mesoderm proliferation, migration, and specification. In contrast, few CRMs for genes involved in terminal differentiation (e.g., sarcomere structure) are targeted by Twist (Sandmann, 2007).

Twist is a highly connected hub targeting a large repertoire of TFs

This global map of Twist-bound CRMs provides a first glimpse of Twist’s connectivity to the rest of the regulatory genome. Remarkably, TFs represent the largest group of Twist targets: Twist binds to CRMs of a striking 25% (113/454) of all sequence-specific Drosophila TFs. Among these are TFs essential for mesoderm development, including gap (hb, hkb, kr, kni), pair rule (eve, slp, opa, odd, prd, run), and segmentation genes (en, hh, ptc, wg), as well as homeotic genes (pb, Scr, Antp, Abd-A, Abd-B, Ubx). These classes of target genes implicate a new role for Twist in the establishment or maintenance of anterior-posterior patterning within the mesoderm in addition to its known role in D-V axis formation. Although the function of many of the remaining TFs is unknown, this data links these regulators to mesoderm development. The sheer number of TFs regulated by Twist does not support a simple hierarchical network, where Twist regulates a small set of TFs, which in turn control another layer of regulators, and so forth. Rather, the data suggests a model for Twist contributing to the regulation of the majority of TFs involved in every aspect of early mesoderm development (Sandmann, 2007).

Temporal enhancer occupancy by Twist reveals stage-specific coregulators

Although Twist is expressed during both developmental time periods assayed, it binds to CRMs in a temporally regulated manner. Approximately half of the enhancer regions are detected at both time periods, indicating continuous binding of Twist throughout these developmental stages. In contrast, 23% of Twist-CRMs are only bound in early development (2-4 h), while 26% are specific to later time periods (4-6 h). This dynamic occupancy reveals that the ability of Twist to bind to CRMs is tightly controlled beyond the mere presence of a suitable binding site, and is likely regulated by other TFs that aid or inhibit binding. To identify additional regulators that could differentiate between temporally bound CRMs, a search was performed for overrepresented sequence motifs, using two complementary computational approaches: statistical enrichment of position weight matrices (PWMs) for characterized TFs, and the de novo detection of overrepresented motifs (Sandmann, 2007).

Twist and Snail consensus motifs are significantly overrepresented in all three groups of CRMs, indicating a potential for extensive cobinding between these two TFs. In contrast, Dorsal motifs are exclusively enriched in the early-bound CRMs, and not in the late group. While Tinman motifs are specifically overrepresented in the continuous and late-bound CRMs. A number of other motifs were also uncovered, including sites for potential Twist/Daughterless heterodimers, suggesting additional mechanisms to generate diverse outputs from Twist-CRMs (Sandmann, 2007).

These data reveals Twist binding to almost all previously characterized Dorsal enhancers. Twist and Dorsal are known to interact physically and to coregulate enhancers in the early, but not the late, time window of this experiment. It is therefore hypothesized that Dorsal may be coregulating many of the newly discovered Twist CRMs, in keeping with the specific enrichment of Dorsal consensus motifs within these enhancers. To experimentally test Dorsal's presence on predicted sites in vivo, ChIP experiments were performed at 2-4 h of development. Significant binding of Dorsal was detected by quantitative real-time PCR to all seven predicted sites tested. Similarly, since Tinman consensus sites were significantly enriched in 4-6-h CRMs, the in vivo occupancy of predicted sites by Tinman was tested at this stage of development. ChIP experiments detected significant binding of Tinman to 10 of 11 sites tested. Given the large number of early and late CRMs, the enrichment of these motifs highlights extensive combinatorial binding of Dorsal and Twist at 2-4 h, and Tinman and Twist at 4-6 h. A substantial part of Twist's temporal specificity likely stems from its association with these upstream and downstream coregulators (Sandmann, 2007).

A core transcriptional network for early mesoderm development

To delineate the combinatorial relationships between Twist and other TFs, an initial transcriptional network was generated for early mesoderm development. The temporal binding map for Twist was integrated with in vivo binding data for Mef2, Dorsal, and Tinman. A previous study of Mef2-bound enhancers offers the largest collection of regulatory regions bound at this stage of development to date. As it is difficult to visualize all 494 Twist target genes, focus was placed on TFs whose CRMs are cobound by two or more regulators during these stages of development. Therefore, all links in this network represent direct connections to the same CRM at the same stages of development (Sandmann, 2007).

The resulting core network of 51 TFs is already relatively complex, with nine genes [nau, E(spl), eve, bap, Ubx, lbe, odd, hth, and Ptx1] being targeted by three out of the four examined regulators. The topology of the network provides several insights into how Twist functions to regulate multiple aspects of early mesoderm development. Extensive combinatorial binding and feed-forward regulation are abundant features. Dorsal activates twist, which in turn coregulates the majority of known direct Dorsal targets. This network motif is even more prominent within the mesoderm: Twist regulates the expression of Mef2 and tinman, and cobinds with these TFs to many of their targets' enhancers. In fact, Twist co-occupies 42% of all Mef2-bound enhancers during early mesoderm development. Depending on the logical inputs from the two upstream regulators (transcriptional repression or activation), feed-forward loops can aid in cellular decision making by filtering out noisy regulatory inputs or control the timing of a transcriptional response. For example, early gene expression in the mesoderm (e.g., activation of tin) depends on Twist alone, while transcription of other genes initiated at a later stage may require the input from both Twist and Tinman proteins (Sandmann, 2007).

Twist-bound CRMs correspond to silencers as well as enhancers of transcription

Through the integration of ChIP-on-chip analysis with expression profiling data during early stages of Drosophila development, this study has identified >2000 Twist-bound regulatory regions and almost 500 direct target genes. This data, in combination with in vivo binding data for other TFs, lays the foundation of a transcriptional network describing early mesoderm development. The resulting network view reveals regulatory features that form the basis of Twist's functional versatility (Sandmann, 2007).

The data revealed extensive Twist binding to characterized Dorsal enhancers and also, surprisingly, to Dorsal-regulated silencers (e.g., dpp). Moreover, many of the new regulatory regions identified for D-V patterning genes can function either as enhancers or integrated enhancer-silencer modules (e.g., WntD and crumbs). This ability of Twist to act within the context of silencers, as well as enhancers, may partially explain the widespread recruitment of Twist to many regulatory regions and its ability to regulate diverse developmental processes (Sandmann, 2007).

An attractive molecular explanation for this bifunctionality is the potential of Twist to form both homodimers and heterodimers. Twist homodimers drive gene activation in Drosophila, while Twist-Daughterless heterodimers are associated with transcriptional repression. This model is supported by the significant overrepresentation of the Twist/Daughterless heterodimer consensus motif in both 2-4-h and 4-6-h CRMs. Direct protein-protein interactions between Twist and Dorsal is an alternative mechanism for Twist's incorporation into repressive complexes (Sandmann, 2007).

A network with unexpected topology governs early mesoderm development

Although the network generated in this study is far from complete, it represents the largest set of combinatorial-bound CRMs during these stages of development described to date, and therefore provides a comprehensive resource to decipher general regulatory principles. The resulting network topology was surprising. Instead of Twist regulating a restricted group of TFs, which in turn regulate a successive wave of transcription in a relay model, Twist directly impinges on CRMs for the vast majority of genes expressed in the early mesoderm (Sandmann, 2007).

The extent of combinatorial binding was also unanticipated. There is extensive cobinding of Twist and Dorsal to early 2-4-h CRMs. In fact, the presence of Dorsal binding may be a general prerequisite for Twist binding to enhancers specific for early development. The cooperative binding of Dorsal and Twist to the rho and sim CRMs supports this model. At 4-6 h of development, the composition of TFs impinging on Twist-bound CRMs changes. Although genome wide ChIP-on-chip data is currently not available for Tinman, the significant overrepresentation of Tinman motifs in Twist-bound CRMs and the ability of Tinman to bind to the majority of sites tested indicates prevalent combinatorial binding between these two TFs during 4-6 h of development. Comparing Twist-bound CRMs with a previously generated data set for Mef2 revealed extensive cobinding to enhancers early in development. Converging regulatory connections through combinatorial binding can produce diverse logical outputs, depending on the nature of the TFs. The cobinding of several pan-mesodermal TFs (Twist, Tinman, and Mef2) may ensure robust gene expression. While in other contexts (for example, the WntD-enhancer) the combined inputs of Twist and Snail allow for spatial fine-tuning of gene expression (Sandmann, 2007).

The core network also revealed an abundance of feed-forward loops, providing directionality during early mesoderm development. This is prevalent with both upstream regulators of Twist (Dorsal and Twist) and downstream regulators (Tinman and Twist and Mef2 and Twist). This network motif will likely become even more widespread as additional ChIP-on-chip data becomes available. Twist targets an astounding number of TFs, which may represent an almost complete repertoire of TFs required for early mesoderm development. It is tempting to speculate that Twist participates in feed-forward regulation, with many of these factors through combinatorial binding to different subsets of the ~2000 Twist-bound CRMs (Sandmann, 2007).

Temporal network dynamics reflect developmental progression

Both the composition and connectivity of regulatory networks describing developmental progression will naturally change over time. To capture dynamic changes within the early mesodermal network, these experiments were performed at consecutive time periods. The data reveals temporally regulated binding of Twist to three classes of CRMs: early, continuous, and late. Similar temporally restricted enhancer occupancy has also been observed for other regulators with broad temporal expression, suggesting that this may be a general feature of developmental networks—e.g., MyoD, PHA-4, and Mef2 (Sandmann, 2007).

The temporal occupancy of specific CRMs by Twist reflects the development of this tissue. At 2-4 h of development, Twist and Dorsal coregulate genes essential for D-V patterning. Twist also targets an almost complete set of genes essential for gastrulation and is required to progress to the next phase of development, mesoderm maturation. During this developmental window, the predominant target genes are part of functional modules essential for the cell migration, proliferation, patterning, and specification events occurring within the mesoderm at these stages. As expected for a TF essential for early aspects of mesoderm development, Twist does not bind to significant numbers of CRMs for genes involved in terminal differentiation. This is in sharp contrast to Mef2, which first co-occupies CRMs involved in early mesoderm development with Twist, and later selectively regulates an alternative group of CRMs driving genes involved in later aspects of differentiation; e.g., sarcomere structure or muscle attachment (Sandmann, 2007).

Conserved regulation of functional classes of genes by Twist

Integrating these data with genetic evidence from other species suggests that the regulation of several functional gene cassettes by Twist is conserved throughout evolution, from flies to man. These include (1) the FGF signaling pathway: Mutations in human FGF receptors phenocopy mutants in human twist (Htwist). (2) Genes implicated in epithelial-mesenchymal transitions (EMTs): In mice and humans, Twist facilitates tumor metastasis through the promotion of EMTs. (3) Cell proliferation and apoptosis: Htwist has been classified as a potential oncogene, since it maintains tissue culture cells in a proliferative state. Interestingly, ectopic expression of Htwist in Drosophila also induces proliferation and inhibits p53-dependent apoptosis, indicating that the ability to regulate these processes is conserved. However, for each process, only a few Twist-regulated genes have been known. Extrapolating from the current findings in flies points toward a role for Twist in the direct regulation of entire gene modules required for each process in vertebrates (Sandmann, 2007).

An emerging model for Twist as a global competence factor for mesoderm development

The results provide an initial global view of the transcriptional network describing early mesoderm development within the metazoan Drosophila. Twist resides at the top of this network and binds to CRMs for the vast majority of genes that need to be expressed during these stages. In many cases, Twist is essential and sufficient to drive expression of the target gene. In other cases, however, the contribution of Twist remains unclear (e.g., crumbs and NetA) . Rather than acting as a binary switch, Twist may act redundantly with other TFs. Alternatively, Twist may provide the competence for more specific TFs to bind to these CRMs; for example, by acting as a pioneer TF to facilitate chromatin remodeling (Sandmann, 2007).

In species as diverse as flies, jellyfish, and mice, Twist is only expressed in mesodermal cells when they are in an immature state, and loss of twist expression correlates with the initiation of differentiation. Moreover, overexpression of Twist-1 in mice is sufficient to block osteoblast differentiation. It is suggested that Twist provides the mesoderm with the competence to be pluripotent: first, by providing these cells with the components necessary to respond to inductive cues directing further specification; and second, by providing an almost universal repertoire of mesodermal CRMs with the competence to respond to other TFs. Once bound by Twist, these regulatory regions may be primed for activation by more specialized TFs, and thereby allow rapid developmental progression at the appropriate time (Sandmann, 2007).

NF-kappaB/Rel-mediated regulation of the neural fate in Drosophila

Two distinct roles are described for Dorsal, Dif and Relish, the three NF-kappaB/Rel proteins of Drosophila, in the development of the peripheral nervous system. First, these factors regulate transcription of scute during the singling out of sensory organ precursors from clusters of cells expressing the proneural genes achaete and scute. This effect is possibly mediated through binding sites for NF-kappaB/Rel proteins in a regulatory module of the scute gene required for maintenance of scute expression in precursors as well as repression in cells surrounding precursors. Second, genetic evidence suggests that the receptor Toll-8, Relish, Dif and Dorsal, and the caspase Dredd pathway are active over the entire imaginal disc epithelium, but Toll-8 expression is excluded from sensory organ precursors. Relish promotes rapid turnover of transcripts of the target genes scute and asense through an indirect, post-transcriptional mechanism. It is proposed that this buffering of gene expression levels serves to keep the neuro-epithelium constantly poised for neurogenesis (Ayyar, 2007).

The results suggest a dual role for the NF-kappaB/Rel proteins of Drosophila in the formation of SOPs. First, they could be recruited directly to the sc promoter and regulate transcription. The SOP enhancer of sc, required for auto-regulation of sc in the SOPs, contains α boxes (ACTAGA), consensus sequences for NF-kappaB/Rel. Evidence has been obtained for a role of these sequences in both activation and repression of sc. Expression of Rel-VP16, a potent transcriptional activator form of Relish, is able to ectopically activate a reporter gene containing the intact sc SOP enhancer but not one in which the α3 box is mutated. So activation in this experimental situation requires the presence of an intact α3 site. The experiment does not rule out indirect effects, so further work is required to verify whether activation is direct. It is suggested the NF-kappaB/Rel proteins participate in activation and repression of transcription of sc, a hypothesis consistent with dl, Dif and Rel mutant phenotypes of additional as well as missing bristles. Second, unexpected role is described of Rel in mRNA turnover of sc, ase and sens, neuronal genes required to specify and/or maintain the neuronal fate of SOP cells. In Rel mutants, transcripts of sc, ase and sens accumulate due to increased transcript stability. Therefore in the wild type, Relish promotes rapid mRNA turnover, presumably indirectly through an unidentified transcriptional target. A similar phenotype is observed in Toll-8 mutants, which furthermore, interact genetically with Rel mutants. Transcripts for Rel are reduced in the Toll-8 mutant suggesting a role for Toll-8 in maintaining the levels of Rel transcript. This might be the reason for the genetic interaction (Ayyar, 2007).

A number of differences are apparent between mutants of the three NF-kappaB/Rel-encoding genes of Drosophila. Mutants triply homo- or hetero-zygous have a normal complement of bristles, while single homo- or hetero-zygous animals have either additional or missing bristles. This suggests possible opposing functions for these genes. Furthermore bristle phenotypes due to loss or gain of function differ in detail between the three mutants. Together these results point to the importance of the stoichiometric relationships between the three NF-kappaB/Rel proteins and raise the possibility that different Dorsal/Dif/Relish homo- or hetero-dimers may have distinct binding sites and therefore different targets. This merits further investigation (Ayyar, 2007).

If NF-kappaB/Rel proteins both activate and repress sc, then they are expected to activate in SOP cells and repress in cells of the proneural clusters not chosen to be SOPs. Two possible ways that this could occur are discussed. First, activation in the SOP may rely on high levels of proneural protein and low levels of NF-kappaB/Rel protein; conversely repression may require low levels of proneural and high levels of NF-kappaB/Rel protein. Notch-mediated lateral inhibition results in high levels of Sc in the SOP and lower levels in surrounding cells. Toll-8 expression is excluded from SOP cells suggesting, that, if Toll-8 affects NF-kappaB/Rel activity, there would be lower levels of NF-kappaB/Rel in SOPs. NF-kappaB has been shown to activate transcription even without stimulus if IkappaB levels are low enough to allow NF-kappaB-dependent gene expression in the basal state. Interestingly, it has been shown that low levels of Dorsal can act synergistically with bHLH proteins to activate target genes in the embryo. This depends on direct association of Dorsal and bHLH proteins and cooperative binding to closely linked binding sites for the two respective proteins. Furthermore cooperative binding for Sc and Dorsal has been demonstrated. In the sc SOP enhancer one of the alpha boxes is indeed close to an E box, so perhaps high levels of Sc and low levels of NF-kappaB/Rel combine to activate transcription in the SOP. Two observations are consistent with this hypothesis: Rel-VP16 was able to ectopically activate sc-SOPE-lacZ only at sites where ac and sc are expressed and, after over-expression of NF-kappaB/Rel proteins, bristles are generally missing on the lateral notum (where Toll-8 levels are high), whereas ectopic bristles are found on the medial notum (where Toll-8 levels are low) (Ayyar, 2007).

A second means by which NF-kappaB/Rel proteins could act differently in SOP and in non-SOP cells, may be the presence/absence of co-factors. It has been shown that Dorsal can be converted from an activator to a repressor by association with the co-repressor Groucho. This bi-functionality is attributable to the fact that Dl only weakly interacts with Gro. During embryogenesis both Cut and Dead ringer bind an AT-rich silencer sequence, AT2, present in target genes of Dorsal and both Dorsal and Dead ringer bind the co-repressor Groucho and recruit it to DNA. A similar AT-rich sequence (the β box) is present in the sc SOP enhancer. Furthermore repression of sc by the E(spl) proteins, targets of Notch signalling in non-SOP cells, is already known to require the activity of Groucho (Ayyar, 2007).

Transcripts for sc, ase and sens (and GFP) accumulate in Rel and Toll-8 mutants as a result of increased transcript stability. Transcript stability correlates with the presence of a six or seven nucleotide motif in the transcribed sequence of these genes. The motif is present in sc, ase and sens, but not ac the transcription of which is unaffected in Rel mutants. The motif is almost identical to the heptamer in MyoD and Sox9 that is associated with transcript stability after inhibition of NF-kappaB/Rel signalling in C2C12 cells. A sc mutant with a truncated sc transcript lacking one of the two motifs present in the coding sequence of this gene, has a phenotype similar to Rel and Toll-8 mutants and an increase in sc mRNA. It has been suggested that increased stability of the transcripts rather than increased transcription underlies this phenotype. The presence of the heptamer is noted in a number of genes involved in sensory organ patterning suggesting possible regulation by NF-kappaB/Rel of a battery of genes in the imaginal epithelium. A similar motif is present in other vertebrate targets of NF-kappaB/Rel. Post-transcriptional regulation of target genes by NF-kappaB/Rel could therefore be an ancient feature common to Drosophila and mammals and possibly even jellyfish. It has been suggest that an unknown factor, presumably a transcriptional target of NF-kappaB/Rel, regulates messenger turnover through association with this sequence. In Rel and Toll-8 mutants the accumulated transcripts are not translated. This must be an effect of the mutants because ectopic expression in wild-type flies allows translation and ectopic bristle formation (Ayyar, 2007).

Promotion of a rapid turnover of transcripts of neuronal genes presumably does not take place in the SOPs where high levels of the protein products of these genes are required. Accordingly Toll-8 expression is extinguished in the SOPs after their formation. Factors specific to the SOP presumably allow translation of the transcripts. It is therefore suggested that high levels of Relish provided by Toll-8 in non-SOP cells might be required for post-transcriptional regulation of neuronal genes (Ayyar, 2007).

In wild-type animals expression of neuronal precursor genes such as sens and ase is restricted to SOPs where they are activated by high levels of Ac and Sc. The results suggest that they are in fact expressed over the entire neuro-epithelium but that mRNA turnover is rapid due to NF-kappaB/Rel activity. Activation of ac-sc in proneural clusters would counteract the effects of NF-kappaB/Rel to allow selection of SOPs. After selection of SOPs for the large sensory bristles is finished, Toll-8 expression is maintained in the epithelium, suggesting that high levels of NF-kappaB/Rel are still required for continued transcript turnover. Continuous buffering of neuronal gene expression presumably continues until the next round of neurogenesis that takes place after pupariation when precursors for the small bristles form. Therefore it is hypothesized that NF-kappaB/Rel plays a subtle role in maintaining steady state levels of expression of many genes required for neural development. The maintenance of low levels of expression of neuronal genes would keep the tissue poised for neurogenesis that takes place in repeated rounds. Perhaps low levels of expression of neuronal genes are characteristic of neuro-epithelia in general (Ayyar, 2007).

The hypothesis concerning the dual role of NF-kappaB/Rel in neurogenesis in Drosophila is as follows. The neuro-epithelium of the imaginal discs expresses neuronal genes. Prior to development of SOPs, high levels of Toll-8 maintain high levels of Rel and result in nuclear accumulation of NF-kappaB/Rel. Through an unknown transcriptional target(s), Relish promotes rapid turnover of neuronal transcripts by a post-transcriptional mechanism. This might be mediated by a specific sequence in the coding regions of target genes. Activation of ac and sc in proneural clusters by regulatory proteins of the notal prepattern counteracts the effects of Relish. After singling out of SOPs by Notch-mediated lateral inhibition, Toll-8 expression ceases in the SOPs. Reduced levels of signal uncover a trans-activator function for NF-kappaB/Rel that, synergistically with Sc, helps to maintain high levels of sc expression in the SOP, possibly through direct binding to consensus sequences in the sc SOP enhancer. The NF-kappaB/Rel proteins may also directly repress sc in non-SOP cells of the proneural clusters. It remains to be seen to what extent each of the three proteins participates in these two processes (Ayyar, 2007).

Design flexibility in cis-regulatory control of gene expression: synthetic and comparative evidence

In early Drosophila embryos, the transcription factor Dorsal regulates patterns of gene expression and cell fate specification along the dorsal-ventral axis. How gene expression is produced within the broad lateral domain of the presumptive neurogenic ectoderm is not understood. To investigate transcriptional control during neurogenic ectoderm specification, divergence and function was examined of an embryonic cis-regulatory element controlling the gene short gastrulation (sog). While transcription factor binding sites are not completely conserved, it has been demonstrated that these sequences are bona fide regulatory elements, despite variable regulatory architecture. Mutation of conserved sequences revealed that putative transcription factor binding sites for Dorsal and Vielfaltig, also known as Zelda, a ubiquitous maternal transcription factor, are required for proper sog expression. When Zelda and Dorsal sites are paired in a synthetic regulatory element, broad lateral expression results. However, synthetic regulatory elements that contain Dorsal and an additional activator also drive expression throughout the neurogenic ectoderm. These results suggest that interaction between Dorsal and Zelda drives expression within the presumptive neurogenic ectoderm, but they also demonstrate that regulatory architecture directing expression in this domain is flexible. A model for neurogenic ectoderm specification is proposed in which gene regulation occurs at the intersection of temporal and spatial transcription factor inputs (Liberman, 2009).

Through a comparative analysis of orthologous sog cis-regulatory modules from twelve Drosophilid species, core regulatory elements conserved in these sequences were identified. Considerable binding site turnover has occurred during the approximately 40 million years of evolution, yet some sequences are conserved. This observation supported the hypotheses that were investigated in this work which are, 1) that conserved sequences are functionally required and, 2) that variable architectures might generate the same or similar patterns of expression. Surprisingly, despite the opportunity for binding site turnover during the course of evolution, the sog regulatory regions from D. virilis can still be interpreted faithfully when used to drive reporter expression in D. melanogaster. It is concluded from these experiments, despite flexibility in the cis-regulatory element structure, regulatory logic has been conserved during evolution of the cis-regulatory module sequences to support sog expression (Liberman, 2009).

Though this comparative analysis identified limited sequence homology, what sequence conservation that was present facilitated efforts to examine the core regulatory elements required for patterning the neurogenic ectoderm. Using site-directed mutagenesis to eliminate sites within the sog cis-regulatory sequence, results were obtained that suggest that Dorsal functions together with the ubiquitous activator Zelda to control sog expression within the neurogenic ectoderm. Furthermore, synthetic cis-regulatory elements were constructed, consisting of Dorsal and Zelda or Dorsal and D-STAT sites, which are both able to support expression in the broad lateral domain of Drosophila early embryo. From these results it is concluded that broad lateral expression is achieved by a combination of Dorsal sites and sites for the ubiquitous activator Zelda, which suggests that a more general mechanism to create broad expression may involve interactions between Dorsal and other broadly expressed transcription factors (Liberman, 2009).

Mutagenesis and mutant analysis results demonstrate that Dorsal and Zelda support expression of sog along the dorsal-ventral axis. In the absence of Dorsal protein, expression of sog is gone; however when Dorsal binding sites were mutagenized, weak ventral-lateral reporter expression remains that could be due to unknown Dorsal binding sites that were not detected by PWM searches or due to input from another transcription factor. In the absence of Zelda binding sites or in Zelda mutants, expression is slightly broader than when Dorsal sites are eliminated. This residual expression could be due to Dorsal and/or another transcription factor (e.g. bHLH) functioning to direct expression, in a Zelda-independent manner, within the ventral-neurogenic ectoderm; however, the data suggests that Twist is not likely involved, as the domain of sog expression along the dorsal-ventral axis is not severely affected in twist mutants (Liberman, 2009).

Previous genetic studies have demonstrated that Dorsal is required for specification of the presumptive neurogenic ectoderm, but binding sites for Dorsal alone are not sufficient to generate expression within the broad lateral domain of embryos. Dorsal has been shown to function synergistically with Twist to pattern the presumptive mesoderm and ventral neurogenic ectoderm. This study presents evidence that Dorsal and Zelda function synergistically to regulate expression that is able to encompass the entire presumptive neurogenic ectoderm domain. Some method of cooperativity likely exists between Dorsal and Zelda, at the level of DNA binding or downstream, and is responsible for extending the expression domain into dorsal-lateral regions of the embryos, where the levels of nuclear Dorsal are low (Liberman, 2009).

It is proposed that Dorsal functions as a spatial regulator in the neurogenic ectoderm and that additional transcription factors like Zelda, act as co-activators to regulate the precise onset of expression. Furthermore, it is suggested that multiple ubiquitous or broadly expressed activators may function with Dorsal to support expression in a broad lateral domain (e.g. Zelda, STAT, and bHLH transcription factors such as Daughterless (Da). This study has demonstrated that STAT binding sites can also function together with Dorsal to drive expression in a broad lateral domain. Further support for this idea includes the observation that sog as well as ths exhibit broad expression early. Sites for Zelda are also present in the ths cis-regulatory module, and these sites likely direct the almost-ubiquitous early expression of ths observed. Interaction of Dorsal with distinct co-activators may not only regulate the spatial domain of expression supported, but also the temporal output. Zelda along with Dorsal or a Dorsal target initiates the earliest zygotic expression detected; perhaps interactions between Dorsal and other activators facilitate expression within a broad lateral domain (or other defined pattern) at later time-points. It is asserted that gene expression is achieved at the intersection of the Dorsal nuclear gradient and the additional activator which could either be ubiquitous in the case of Zelda or localized in the case of Twist (Liberman, 2009).

Even equipped with this new knowledge, other cis-regulatory modules that support co-expression of genes SoxN, pyramus and Neu3 have proven difficult to identify. To date, SoxN and pyramus regulatory elements remain unidentified. Flexible regulatory structures could account for some of the obscurity that has been encountered in the identification of cis-regulatory modules that support expression of genes within Drosophila early embryos. Flexibility in binding site composition, orientation and number of sites has also been demonstrated in the regulation of co-expressed genes in Ciona by extensive co-expression analyses. Possibly the observed flux in binding site composition and arrangement provides a mechanism that facilitates the introduction of mutations, which may be selected when a fitness advantage is provided to the developing embryo (Liberman, 2009).

Recently, a second regulatory element for sog located upstream of the gene was identified which also drives expression in a broad lateral stripe in the presumptive neurogenic ectoderm of cellularized embryos. This novel regulatory element as well as the known regulatory element, the intronic enhancer examined in this study, probably function together to control the full expression pattern of sog in the developing embryo. While both cis-regulatory sequences contain Dorsal and Zelda binding sites, the novel enhancer contains many more bHLH sites (L. Liberman, unpub. obs.), which is in stark contrast to the intronic sog regulatory element, which contains only one bHLH site and exhibits very little change of expression in twist mutant embryos. This new regulatory element presents further evidence that there exist multiple solutions for the developmental problem of producing spatially and temporally regulated expression. Future experiments will address whether these early embryonic enhancers controlling the expression of the sog gene within similar domains use the same mechanism (i.e. Dorsal + Zelda cooperativity) to support expression in a broad lateral stripe or whether different mechanisms are used (Liberman, 2009).

Evolutionary comparisons of sequences from diverged species can be very useful for the dissection of underlying cis-regulatory logic, as has been shown in this study; yet the important variable is that the proper comparisons of sequences must be made (i.e. species of appropriate evolutionary distance) and this is not always easy to define. In vertebrate systems, analyses of cis-regulatory modules usually focus on modules identified by methods that select for high degrees of conservation, which inherently have a low amount of flexibility. Arguments have been made that deciphering the underlying regulatory logic from evolutionary comparisons of sequences, when conservation is too high, is hard to interpret. However, it is contended that the relevant comparisons that provide insights into cis-regulatory logic are context-dependent. In analysis of the sog and Neu3 cis-regulatory modules, only limited sequence conservation was identified in comparisons of homologous sequences isolated from D. melanogaster and other Drosophilids. In the sog early embryonic regulatory element that was analyzed in this study, 71 (of 395) base-pairs of non-contiguous sequence exhibits conservation. The degree of conservation that was retained however was useful for dissecting the underlying regulatory logic (Liberman, 2009).

Identifying regulatory regions with flexible structure is more challenging than scanning for a stringent set of binding sites, but it may also reveal alternative mechanisms for specification that were not previously considered. It is predicted that studies that dissect the flexibility of cis-regulatory modules may one day provide insights to facilitate dissection of vertebrate regulatory elements in general, including ones that exhibit flexibility of sequence. It seems plausible that stringently conserved regulatory elements control gene expression of certain classes of genes, like those required for certain essential processes. Flexible regulatory architectures may provide a mechanism for generating variability throughout evolution. Ultimately it will prove useful to make evolutionary comparisons with both highly conserved sites and flexible architectures to determine how each contributes to establishment or maintenance of gene regulation (Liberman, 2009).

Conservation of enhancer location in divergent insects

Dorsoventral (DV) patterning of the Drosophila embryo is controlled by a concentration gradient of Dorsal, a sequence-specific transcription factor related to mammalian NF-kappaB. The Dorsal gradient generates at least 3 distinct thresholds of gene activity and tissue specification by the differential regulation of target enhancers containing distinctive combinations of binding sites for Dorsal, Twist, Snail, and other DV determinants. To understand the evolution of DV patterning mechanisms, Dorsal target enhancers from the mosquito Anopheles gambiae and the flour beetle Tribolium castaneum were identified and characterized. Putative orthologous enhancers are located in similar positions relative to the target genes they control, even though they lack sequence conservation and sometimes produce divergent patterns of gene expression. The most dramatic example of this conservation is seen for the 'shadow' enhancer regulating brinker: It is conserved within the intron of the neighboring Atg5 locus of both flies and mosquitoes. These results suggest that, like exons, an enhancer position might be subject to constraint. Thus, novel patterns of gene expression might arise from the modification of conserved enhancers rather than the invention of new ones. It is proposed that this enhancer constancy might be a general property of regulatory evolution, and should facilitate enhancer discovery in nonmodel organisms (Cande, 2009).

This study identified 5 different DV enhancers in A. gambiae and T. castaneum, representing a broad spectrum of patterning responses to the Dorsal gradient. These enhancers embody the largest collection of functionally defined regulatory DNAs engaged in a common process in divergent insects. Despite extensive differences in the DV regulatory networks of flies, mosquitoes, and beetles, the enhancers are located in similar positions relative to the promoters of the target genes they control. A constrained position is also observed for the previously identified vnd enhancer in Anopheles. Altogether, these 6 enhancers are located in all possible orientations, including the immediate 5' flanking region (twist, sim), remote 5' flanking region (sog), intron 1 (vnd), 3' intron (cactus), and within a neighboring gene (brinker) (Cande, 2009).

Given the rapid divergence of noncoding sequences due to the constant turnover of individual transcription-factor binding sites within enhancer, there are no arrangements of sites or even individual sites that can be thought of as orthologous in any of the pairs of enhancers described in this study. The most closely related pair of species examined in this study, Anopheles and Drosophila, last shared a common ancestor >200 million years ago. The 2 genomes have been rearranged to such an extent that only an estimated 34% of orthologous genes can be sorted into microsyntenic blocks. In light of this, it is somewhat surprising to see such conservation in enhancer positions. There are at least 2 possible explanations. First, the different enhancers identified in Anopheles and Tribolium might be orthologous, that is, they might derive from a common enhancer in the last shared ancestor. The position of the enhancer within the locus is apparently unchanged simply because no genomic events that would perturb its position have occurred. According to this view, selection might depend on the modification of preexisting enhancers rather than the creation of new ones, similar to the evolution of protein coding sequences. A nonexclusive alternative explanation is that the enhancer position is functionally constrained within a genomic locus. For example, enhancers might be able to communicate with the target promoter only when located in particular positions within the higher-order structure of a complex genetic locus. Thus, enhancers might be in a constant flux of death and birth, but de novo enhancers work only when located in particular positions. The Hox gene complexes represent extreme examples of constrained enhancer organization. Perhaps simpler loci are subject to similar, but somewhat less stringent, organizational constraints (Cande, 2009).

The conservation of enhancer location observed in this study applies to developmental control genes. It is certainly conceivable that enhancer turnover alters the locations of regulatory DNAs, particularly in the case of housekeeping genes. An interesting example is seen for the spec genes in the sea urchin Strongylocentrotus purpuratus, which have coopted repetitive elements to function as enhancers. In contrast, regulatory genes engaged in interlocking network interactions, as seen for the genes considered in this study, might tend to retain old enhancers rather than invent new ones. An implication of this study is that the evolution of novel patterns of gene expression depends on the modification of existing enhancers rather than the invention of new ones. This has already been documented for the evolution of the sim expression pattern and ventral midline of divergent insects. It is proposed that the modification of constrained or orthologous enhancers will prove to be a general mechanism for the evolution of gene expression patterns (Cande, 2009).

Shadow enhancers foster robustness of Drosophila gastrulation

Critical developmental control genes sometimes contain 'shadow' enhancers that can be located in remote positions, including the introns of neighboring genes. They nonetheless produce patterns of gene expression that are the same as or similar to those produced by more proximal primary enhancers. It was suggested that shadow enhancers help foster robustness in gene expression in response to environmental or genetic perturbations. This hypothesis was critically tested by employing a combination of bacterial artificial chromosome (BAC) recombineering and quantitative confocal imaging methods. Evidence is presented that the snail gene is regulated by a distal shadow enhancer located within a neighboring locus. Removal of the proximal primary enhancer does not significantly perturb snail function, including the repression of neurogenic genes and formation of the ventral furrow during gastrulation at normal temperatures. However, at elevated temperatures, there is sporadic loss of snail expression and coincident disruptions in gastrulation. Similar defects are observed at normal temperatures upon reductions in the levels of Dorsal, a key activator of snail expression. These results suggest that shadow enhancers represent a novel mechanism of canalization whereby complex developmental processes 'bring about one definite end-result regardless of minor variations in conditions (Perry, 2010).

Despite both intrinsic and environmental sources of noise, which introduce variability in complex developmental processes, the patterning of the Drosophila embryo unfolds with high fidelity. It has been postulated that genetic interactions in developmental regulatory networks can channel these variable inputs into faithful outcomes, as a ball bouncing inside of a funnel is channeled to the center, a process termed canalization. This paper presents evidence that shadow enhancers are important mediators of canalization, ensuring reliable and robust expression of critical patterning genes (Perry, 2010).

snail is a key determinant of dorsal-ventral patterning. It encodes a zinc finger repressor that establishes a sharp boundary between the presumptive mesoderm and neurogenic ectoderm and is essential for the formation of the ventral furrow and the invagination of the mesoderm. Whole-genome ChIP-chip assays identified a cluster of Dorsal and Twist (key activators of snail expression) binding sites in the immediate 5' flanking region of the snail transcription unit that coincide with the known enhancer. Unexpectedly, these studies also identified a second cluster of binding sites within the neighboring Tim17b2 locus, located ~7 kb upstream of snail. A small genomic DNA fragment (~1 kb) encompassing this second cluster of binding sites was attached to a lacZ reporter gene and was expressed in transgenic embryos. The fusion gene exhibits localized expression in the presumptive mesoderm, similar to that seen for the endogenous gene or obtained with the proximal enhancer (the first 2.8 kb of the 5' flanking region. The newly identified distal enhancer is arbitrarily referred to as the shadow enhancer and the original, proximal enhancer is referred to as the primary enhancer (Perry, 2010).

A snail fusion gene containing only the primary enhancer rescues the gastrulation of at least some snail mutants in a population of mutant embryos. Because snail is essential for the coordinated invagination of the mesoderm during early gastrulation, variability in expression could lead to occasional disruptions in morphogenesis. Perhaps the additional enhancer provides a mechanism for suppressing such variability, thereby ensuring robust expression in large populations of embryos. This hypothesis was motivated in part by previous preliminary evidence that neurogenic genes with shadow enhancers show less sensitivity to changes in activator concentration than similar genes lacking shadows (Perry, 2010).

An alternative view is that the proximal and shadow enhancers are primarily responsible for controlling distinct dynamic aspects of the snail expression pattern rather than functioning in an overlapping manner during mesoderm invagination. An expectation of the former robustness hypothesis is that transgenes containing either enhancer alone should be sufficient to induce gastrulation in snail mutant embryos. This possibility was tested by creating a series of recombineered bacterial artificial chromosomes (BACs) containing an ~25 kb genomic interval encompassing the snail and Tim17b2 loci. Comparable BACs were prepared that either contain or lack the proximal enhancer. This enhancer was not simply deleted, but an ~1 kb segment containing critical Dorsal activator elements was replaced with a spacer DNA sequence in order to retain normal spacing of the regulatory region (Perry, 2010).

To measure the effect that different enhancers have on transcriptional activity, a reporter system was developed for detecting nascent transcripts. The endogenous yellow gene is not transcribed until late in development and contains a large intron, making it an ideal reporter for the detection of de novo transcripts by in situ hybridization. In contrast, the snail transcription unit lacks introns and is therefore not amenable to quantitative in situ hybridization methods that rely on intronic probes. Consequently, a series of BACs was created that contains yellow in place of snail. These BACs contain both enhancers or have either the primary or shadow enhancer replaced with random DNA. All of the aforementioned BACs were inserted in the same chromosomal location on 2L using phiC31 targeted integration (Perry, 2010).

BACs containing the snail gene were crossed into a mutant background with a deletion spanning the entire snail transcription unit (Df (2L)osp²⁹), along with a marked balancer to identify homozygous snail null mutants. As noted earlier, the reciprocal situation, proximal enhancer without shadow, can sometimes rescue gastrulation. Mutant embryos homozygous for the snail deficiency chromosome (osp²⁹) are easily recognized by the absence of snail expression and ectopic single-minded (sim) expression, a key regulator of midline formation within the central nervous system that is normally excluded from the mesoderm by the Snail repressor (Perry, 2010).

There is neither a ventral furrow nor subsequent ingression of the mesoderm in these mutants or just the shadow enhancer alone rescue gastrulation of mutant embryos. In both cases, a complete ventral furrow is formed, followed by invagination of the mesoderm indistinguishable from that seen in wild-type embryos. Both BACs restore snail expression in the presumptive mesoderm, and sim transcripts are restricted to lateral regions that form the ventral midline of the central nervous system after gastrulation. These observations, along with previous studies, indicate that neither the primary nor shadow enhancer is necessary for the gastrulation of embryos raised at optimal, permissive conditions (Perry, 2010).

Although the shadow enhancer is sufficient for generating a qualitatively normal pattern of snail expression, additional assays were done to determine whether there might be subtle changes in expression. Quantitative confocal imaging methods were used to investigate this possibility. As mentioned earlier, BAC transgenes were prepared that contain the yellow reporter gene in place of the snail transcription unit. In situ hybridization assays with intronic probes permit direct detection of yellow de novo transcripts, and, hence, precise measurements of snail transcription with single cell (nucleus) resolution. At normal culturing temperatures (22°C), there is no discernible difference in the initial de novo transcription patterns of BAC transgenes containing both enhancers or containing just a single enhancer, either the primary enhancer or shadow enhancer. In the majority of cases, more than 90% of the nuclei in the presumptive mesoderm express yellow nascent transcripts (Perry, 2010).

Less-reliable expression is observed for BAC transgenes containing a single enhancer at elevated temperatures (30°C). More than 20% of the nuclei in the presumptive mesoderm lack yellow nascent transcripts in over half of the embryos expressing the BAC transgene without the shadow enhancer. This effect is even more pronounced upon removal of the primary enhancer. The same cutoff value, absence of yellow nascent transcripts in at least 20% of all mesodermal nuclei, occurs in over three-fourths of these embryos. In contrast, the BAC transgene containing both the primary and shadow enhancers continues to display nearly complete patterns of de novo transcription at the elevated temperature (Perry, 2010).

Similar results were obtained in response to genetic perturbations. For example, the yellow transgene BAC containing both enhancers exhibits a normal pattern of expression in embryos derived from dl/+ mothers containing half the normal dose of the Dorsal gradient. The distribution of nuclei failing to maintain active expression is similar to that seen for wild-type embryos. However, the comparable BAC transgene containing only the shadow enhancer exhibits erratic patterns of activation in these embryos, particularly in lateral regions. These results, along with the preceding analysis of embryos grown at elevated temperatures, suggest that the snail shadow enhancer helps ensure accurate and reproducible patterns of gene expression in large populations of embryos subject to genetic and environmental perturbations (Perry, 2010).

The preceding results document quantitative changes in the variability and reliability of snail expression upon removal of the primary or shadow enhancer. Next it was asked whether such variation causes changes in cellular morphogenesis, particularly the formation of the ventral furrow and subsequent invagination of the mesoderm. snail mutant embryos carrying BACs with both enhancers or just the shadow enhancer. Embryos carrying the transgene with both enhancers exhibit normal patterns of gastrulation. In contrast, comparable embryos lacking the primary enhancer display erratic patterns of gastrulation, including the formation of incomplete ventral furrows that do not extend along the entire germband and disruptions in the symmetry of the involuted mesodermal tube. As shown earlier, such defects are not observed at normal temperatures (22°C) (Perry, 2010).

This paper has presented evidence that the snail shadow enhancer located within the Tim17b2 locus helps ensure reliable and reproducible patterns of snail expression in the presumptive mesoderm during gastrulation. BAC transgenes lacking either the primary enhancer or the shadow enhancer display erratic patterns of de novo transcription at elevated temperatures. It is proposed that shadow enhancers come to be fixed in populations by ensuring robustness in the activities of key patterning genes such as snail. Increases in temperature should cause less-stable occupancy of critical binding sites, but an additional enhancer could suppress this noise by increasing the probability of gene activation. This increased time of active transcription per cell might augment the overall levels of expression, which could be an important function of shadow enhancers (Perry, 2010).

Other critical dorsal-ventral determinants also contain shadow enhancers, including brinker, vnd, and sog. The recent analysis of shavenbaby suggests that shadow enhancers are essential for the reliable morphogenesis of embryonic bristles in older embryos. There is also evidence that shadow enhancers might be a common feature of vertebrate systems such as zebrafish (Perry, 2010).

Shadow enhancers appear to represent a novel mechanism of canalization, whereby complex developmental processes lead to a fixed outcome despite genetic and environmental perturbations. Other mechanisms of canalization have been suggested, including recursive wiring of gene regulatory networks and 'capacitors' such as hsp90 that suppress both altered folding of mutant proteins and transpositioning of mobile elements (Perry, 2010).

It is conceivable that primary and shadow enhancers mediate overlapping patterns of activity only during early embryogenesis. They might come to possess distinctive regulatory activities at later stages of development. Nonetheless, during the time when their activities coincide during gastrulation, they maintain reliable patterns of snail expression in response to environmental and genetic variability. Although either enhancer might be sufficient, both enhancers are required for accurate and reliable patterns of expression in response to variability. This precise patterning enables rapid development, without delays arising from corrective feedback mechanisms. It is easy to imagine that delays in embryogenesis would result in selective disadvantages to the resulting larvae, which must compete for limiting sources of food. Regardless of the specific mechanisms that select for shadow enhancers, the occurrence of such enhancers provides an opportunity for the evolution of novel patterns of gene expression. As long as the two enhancers maintain overlapping activities during developmental hot spots such as gastrulation, they can drift or be selected to produce novel patterns of gene expression (Perry, 2010).

The full-length transcripts and promoter analysis of intergenic microRNAs in Drosophila melanogaster

MicroRNA (miRNA) transcription is poorly understood until now. To increase miRNA abundance, miRNA transcription was stimulated with CuSO(4) and Drosha enzyme was knocked down using dsRNA in Drosophila S2 cells. The full length transcripts of bantam, miR-276a and miR-277, the 5'-end of miR-8, the 3'-end of miR-2b and miR-10 were obtained. A series of miRNA promoter analyses was conducted to prove the reliability of RACE results. Luciferase-reporter assays proved that both bantam and miR-276a promoters successfully drove the expressions of downstream luciferase genes. The promoter activities were impaired by introducing one or multiple mutations at predicted transcription factor binding sites. Chromatin immunoprecipitation analysis confirmed that hypophosphorylated RNA polymerase II and transcription factor c-Myc physically bind at miRNA promoters. RNA interference of transcription factors Mad and Prd led to down-expression of bantam, miR-277 and miR-2b but not miR-276a, whereas RNAi of Dorsal had the opposite effect (Qian, 2011).

The core promoter composition establishes a new dimension in developmental gene networks

Developmental processes are highly dependent on transcriptional regulation by RNA polymerase II, which initiates transcription at the core promoter. The dorsal-ventral gene regulatory network (GRN) includes multiple genes that are activated by different nuclear concentrations of the Dorsal transcription factor along the dorsal-ventral axis. Downstream core promoter element (DPE)-containing genes are conserved and highly prevalent among Dorsal target genes. Moreover, the DPE motif is functional in multiple Dorsal target genes, as mutation of the DPE results in the loss of transcriptional activity. Furthermore, analysis of hybrid enhancer-promoter constructs reveals that the core promoter composition plays a pivotal role in the transcriptional output. Importantly, in vivo evidence is provided that expression driven by the homeotic Antennapedia P2 promoter during Drosophila embryogenesis is dependent on the DPE. Taken together, it is proposed that transcriptional regulation results from the interplay between enhancers and core promoter composition, thus establishing a novel dimension in developmental GRNs (Zehavi, 2014b).

Capicua is involved in Dorsal-mediated repression of zerknullt expression in Drosophila embryo

The maternal transcription factor Dorsal (Dl) functions as both an activator and a repressor in a context-dependent manner to control dorsal-ventral patterning in the Drosophila embryo. Previous studies have suggested that Dl is an intrinsic activator and its repressive activity requires additional corepressors that bind corepressor-binding sites near Dl-binding sites. However, the molecular identities of the corepressors have yet to be identified. This study presents evidence that Capicua (Cic) is involved in Dl-mediated repression in the zerknullt (zen) ventral repression element (VRE). Computational and genetic analyses indicate that a DNA-binding consensus sequence of Cic is highly analogous with previously identified corepressor-binding sequences and that Dl failed to repress zen expression in lateral regions of cic mutant embryos. Furthermore, electrophoretic mobility shift assay (EMSA) shows that Cic directly interacts with several corepressor-binding sites in the zen VRE. These results suggest that Cic may function as a corepressor by binding the VRE (Shin, 2014).

Quantitative single-embryo profile of Drosophila genome activation and the dorsal-ventral patterning network

During embryonic development of Drosophila melanogaster, the Maternal to Zygotic Transition (MZT) marks a significant and rapid turning point when zygotic transcription begins and control of development is transferred from maternally deposited transcripts. Characterizing the sequential activation of the genome during the MZT requires precise timing and a sensitive assay to measure changes in expression. This study used the NanoString nCounter instrument, which directly counts mRNA transcripts without reverse transcription or amplification, to study over 70 genes expressed along the dorsal-ventral (DV) axis of early Drosophila embryos, dividing the MZT into 10 time points. Transcripts were quantified for every gene studied at all time points, providing the first data set of absolute numbers of transcripts during Drosophila development. Gene expression was found to change quickly during the MZT, with early Nuclear Cycle (NC) 14 the most dynamic time for the embryo. twist is one of the most abundant genes in the entire embryo and mutants were used to quantitatively demonstrate how it cooperates with Dorsal to activate transcription and is responsible for some of the rapid changes in transcription observed during early NC14. Elements within the gene regulatory network were uncovered that maintain precise transcript levels for sets of genes that are spatiotemporally co-transcribed within the presumptive mesoderm or dorsal ectoderm. Using this new data, it was shown that a fine-scale, quantitative analysis of temporal gene expression can provide new insights into developmental biology by uncovering trends in gene networks including coregulation of target genes and specific temporal input by transcription factors (Sandler, 2016).

Quantitatively predictable control of Drosophila transcriptional enhancers in vivo with engineered transcription factors

Genes are regulated by transcription factors that bind to regions of genomic DNA called enhancers. Considerable effort is focused on identifying transcription factor binding sites, with the goal of predicting gene expression from DNA sequence. Despite this effort, general, predictive models of enhancer function are currently lacking. This study combine quantitative models of enhancer function with manipulations using engineered transcription factors to examine the extent to which enhancer function can be controlled in a quantitatively predictable manner. These models, which incorporate few free parameters, can accurately predict the contributions of ectopic transcription factor inputs. The effect of individual transcription factors can be considered as independent submodules of activity that are combined in a linear manner to produce a sigmoidal output. Individual submodules can also encode more complex inputs. For example, a submodule is represented by the saturating cooperativity displayed by Dorsal and Twist, whose combined output has an upper limit. The models allow the predictable 'tuning' of enhancers, providing a framework for the quantitative control of enhancers with engineered transcription factors (Crocker, 2016).

Epigenetic patterns of histone modifications contribute to the maintenance of tissue-specific gene expression. This study shows that such modifications also accompany the specification of cell identities by the NF-kappaB transcription factor Dorsal in the precellular Drosophila embryo. Evidence is provided that the maternal pioneer factor, Zelda, is responsible for establishing poised RNA polymerase at Dorsal target genes before Dorsal-mediated zygotic activation. At the onset of cell specification, Dorsal recruits the CBP/p300 coactivator to the regulatory regions of defined target genes in the presumptive neuroectoderm, resulting in their histone acetylation and transcriptional activation. These genes are inactive in the mesoderm due to transcriptional quenching by the Snail repressor, which precludes recruitment of CBP and prevents histone acetylation. By contrast, inactivation of the same enhancers in the dorsal ectoderm is associated with Polycomb-repressed H3K27me3 chromatin. Thus, the Dorsal morphogen gradient produces three distinct histone signatures including two modes of transcriptional repression, active repression (hypoacetylation), and inactivity (H3K27me3). Whereas histone hypoacetylation is associated with a poised polymerase, H3K27me3 displaces polymerase from chromatin. These results link different modes of RNA polymerase regulation to separate epigenetic patterns and demonstrate that developmental determinants orchestrate differential chromatin states, providing new insights into the link between epigenetics and developmental patterning (Boija, 2016).

Dorsal involvement in the immune response

Continued: see Dorsal: Regulation part 2/2

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dorsal continued: Biological Overview | Evolutionary Homologs | Protein Interactions | Developmental Biology | Effects of Mutation | References

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