dorsal

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) 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).

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