The entire functional even-skipped locus of Drosophila is contained within a 16 kilobase region. As a transgene, this region is capable of rescuing even-skipped mutant flies to fertile adulthood. Detailed analysis of the 7.7 kb of regulatory DNA 3' of the transcription unit reveals ten novel, independently regulated patterns. Most of these patterns are driven by non-overlapping regulatory elements, including ones for syncytial blastoderm stage stripes 1 and 5, while a single element specifies both stripes 4 and 6. Expression analysis in gap gene mutants shows that stripe 5 is restricted anteriorly by Krüppel and posteriorly by giant, the same repressors that regulate stripe 2. Consistent with the coregulation of stripes 4 and 6 by a single cis-element, both the anterior border of stripe 4 and the posterior border of stripe 6 are set by zygotic hunchback, and the region between the two stripes is ëcarved outí by knirps. Thus the boundaries of stripes 4 and 6 are set through negative regulation by the same gap gene domains that regulate stripes 3 and 7, but at different concentrations (Fujioka, 1999).

Based on the regions of overlap of larger transgenes, these studies suggest that early stripes 1 and 5 might be driven by the region from +4.8 to +8.4 kb, and stripes 4 and 6 by the +4.8 to +6.6 kb region. Consistent with a composite element driving stripes 4 and 6, both the anterior border of stripe 4 and the posterior border of stripe 6 are determined by zygotic hb expression. In addition, in a kni mutant, the isolated stripe 4+6 composite element drives expression throughout the interstripe region. The spatial and temporal expression patterns of zygotic hb and kni are consistent with the products of these loci exerting direct repression on the element. Thus, as for stripes 2, 3 and 7, much of the spatial regulation of stripes 4, 5 and 6 appears to be due to repression by gap gene products. The sequences of these regulatory elements contain potential binding sites for the gap gene products that may directly regulate them. However, further analysis will be required to determine if these regulatory interactions are indeed direct (Fujioka, 1999).

The above observations concerning regulation of stripes 4 and 6 include a striking parallel with the regulation of stripes 3 and 7. The stripe 7 element is not separable from that of stripe 3, although full activation of stripe 7 requires sequences outside of the minimal stripe 3 element. Like the 4+6 element, a combined stripe 3+7 element directs expression throughout the interstripe region in a kni mutant, and both the anterior and posterior borders (of stripes 3 and 7, respectively) are set by hb-dependent repression. Thus, an intriguing situation exists in which the stripe 4+6 element is repressed by a higher concentration of Knirps protein than is the stripe 3+7 element and, at the same time, by a lower concentration of Hunchback protein. The differential sensitivity of these elements to repressor concentrations might be due to simple mechanisms, such as differential affinities of binding sites, or to more complex mechanisms, such as combinatorial interactions with different cofactors. Whatever the mechanism, this differential sensitivity is precise enough to allow three gap protein domains (those of Knirps and the anterior and posterior Hunchback domains), acting as repressor gradients, to regulate the positioning of eight distinct expression boundaries, thus helping to define four of the early stripes of eve expression. In a similar vein, stripe 5 is negatively regulated by the same gap genes that regulate stripe 2. The Kr domain represses both the posterior border of stripe 2 and the anterior border of stripe 5, while the anterior and posterior domains of giant expression are involved in setting the anterior and posterior borders of stripes 2 and 5, respectively (Fujioka, 1999).

Recently, several genes were reported to show stripe-specific effects on eve activation. lacZ expression driven in stripes 4, 5 and 6 by the eve 3' region were weakened in a fish-hook mutant (fish, also known as Dichaete). It was also shown that the product of this gene can bind within this large regulatory region, as determined by gel mobility shift assays. While expression from the minimal elements also shows some reduction in fish embryos, expression from these elements is clearly activated by other proteins as well. In a marelle mutant (encoding D-STAT), lacZ expression from a stripe 3 element was seen to be weakened. D-STAT is a primary activator of stripe 5, since expression from the stripe 5 element is absent in this mutant. Consistent with a direct effect on this element, several consensus sequences for D-STAT binding were found within the +7.4 to +8.2 kb region (unpublished observations). None of the gap and pair-rule mutants tested have a strong effect on stripe 1 element expression. hb mutants weaken reporter gene expression, but not severely. In a buttonhead mutant (btd), endogenous eve expression in the stripe 1 region was seen to be reduced. In beetles, as in Drosophila, eve forms stripes with anterior borders that coincide with parasegment boundaries but, rather than forming multiple stripes at once, stripe 1 is formed first, followed by sequential progression toward the posterior. Further analysis of the regulation of stripe 1 may reveal regulatory relationships that predate the divergence of Diptera. Recent analyses of eve stripe elements among Drosophila species suggests that many of the regulatory mechanisms are evolutionarily conserved. The growing body of information from various species may soon support detailed hypotheses for how the regulatory mechanisms of segmentation evolved (Fujioka, 1999 and references).

evenskipped is expressed in the nervous system, initially in GMCs 1- 1a, 4-2a and 7-1a, and later in the aCC/pCC, RP2, CQ and EL neurons. In an analysis of the eve promoter, elements for GMC 1-1a, its cellular progeny the aCC/pCC neurons, GMC 4-2a and its progeny neuron RP2 could not be separated. This is surprising, since these cells originate from different neuroblasts. A single element drives lacZ expression strongly in these neurons, at least through stage 11. However, by stage 15, transgene expression is reduced, particularly at the protein level, when both endogenous Eve expression and expression from rescue constructs (in an eve- background) remain strong. The eve 3' UTR, which the initial lacZ transgenes did not contain, appears to affect the efficiency of translation in these cells. Transgenic lines in which the standard 3' UTR (from the alpha-tubulin gene) is replaced by that of eve (while they show reduced mRNA levels at stage 11 and similar levels at later stages) give lacZ protein levels that remain high through stage 15 in the RP2 and aCC/pCC neurons. The eve 3' UTR confers a rapid turnover rate in early cycle 14 of the blastoderm stage. Thus it appears that the eve 3' UTR has functions in controlling protein levels in several tissues, at various stages, and probably through multiple mechanisms. Elements for EL cells and for GMC 7-1a and its progeny CQ neurons were also localized. However, the CQ and EL elements overlap those for posterior region expression and for even-numbered parasegment expression, respectively, suggesting that common activators may be utilized in these different tissues (Fujioka, 1999).

An element for muscle precursor cell expression is separable from those of other tissues. However, its expression at stage 15 becomes weaker than that of endogenous Eve, as observed for the RP2+aCC/pCC element. The eve 3' UTR may provide for a high level of protein expression in this tissue, at a similar time as that in the nervous system. Expression in the posterior region of the embryo is apparently a highly conserved feature of eve function, since it is shared by eve homologs in C. elegans, zebrafish and mice. While it was reported that posterior structures are not affected in certain eve mutants at the non-permissive temperature, the possibility remains that eve has some function in this region. eve homologs have been shown to have important functions in specifying posterior cell fates in C. elegans and zebrafish. The regulation of eve expression in the posterior region is complex. Initially, the late stripe element is responsible for expression in this region, which appears as an 8th stripe corresponding to parasegment 15. Later, expression is driven in a ring near the posterior end of the embryo by two separable elements, one active through germband retraction and the other after dorsal closure. The latter expression corresponds to the anal plate ring. Just downstream of the eve-coding region (+1.5 kb to +2.6 kb) lies an element that, when assayed by itself, drives lacZ expression strongly in the even-numbered parasegments, where only very weak eve expression is normally observed. As suggested previously, the upstream late element may be responsible for long-range repression of these ftz-like stripes in the endogenous eve gene. The biological function of this element, if any, is unclear, although eve expression does extend into this region, where it is required to clear odd-skipped expression from the anterior ftz domain, allowing activation of engrailed. This element may serve a function in this context (Fujioka, 1999 and references).

The regulatory DNA that was characterized downstream of the transcription unit, in combination with upstream regions described above, is sufficient to functionally rescue eve null mutants. In most cases, a single copy of the rescue transgene is not sufficient for full rescue. Many mutant embryos exhibit a weak eve hypomorphic phenotype when they carry only one copy of the transgene, suggesting that transgene expression is below that of the endogenous gene when inserted at most chromosomal locations. This might indicate that the transgene is missing a general enhancer of early eve expression. Alternatively, sequences within the P-element vector may repress eve expression at early stages. It is also possible that a chromosomal environment exists around the eve locus that is required for full activity which most insertion sites do not provide. The PSR element described below might participate in providing such an environment. The genomic region downstream of the RP2+aCC/pCC element causes strong pairing-sensitive repression (PSR) of the mini-white gene. Similar PSR is observed when Polycomb-group gene responsive elements are introduced into the genome with mini-white. Recently, it was reported that a region from the engrailed gene that exhibits PSR is bound directly by the Drosophila YY1 homolog, encoded by the pleiohomeotic gene. Consensus sites for YY1/Pho binding, as well as for GAGA factor, which are also seen in the engrailed element, exist within this region. Consistent with chromatin-based regulation of eve, a Polycomb-group protein, Polyhomeotic, was found to bind to polytene chromosomes in the region of the eve locus, and eve expression in the NB4-2 lineage is affected by Polycomb-group activity. Nonetheless, the function of the eve PSR element is unclear, since rescue transgenes that lack it do not show abnormal eve expression, and since including it does appear to enhance either expression or rescue. It is possible that the PSR element is only required in the context of the eve locus, perhaps to prevent inappropriate activation of eve by enhancers from a neighboring gene, or of a neighboring gene by eve enhancers. Other regions of the eve locus are also capable of repressing mini-white expression, since the -6.4 to +8.4 kb transgenes consistently gives transformants with very weak eye color. The utilization of Glass activator binding sites to enhance mini-white expression facilitates the identification of transformants, but these also show weak eye color relative to other Glass-mini-white transgenes. Although this repression is not consistently pairing sensitive, it may represent a function that is redundant with that of the PSR region in some aspect of eve regulation (Fujioka, 1999 and references).

Ras signaling elicits diverse outputs, yet how Ras specificity is generated remains incompletely understood. Wingless and Decapentaplegic confer competence for receptor tyrosine kinase-mediated induction of a subset of Drosophila muscle and cardiac progenitors by acting both upstream of and in parallel to Ras. In addition to regulating the expression of proximal Ras pathway components, Wg and Dpp coordinate the direct effects of three signal-activated transcription factors (dTCF, Mad, and Pointed that function in the Wg, Dpp, and Ras/MAPK pathways, respectively) and two tissue-restricted transcription factors (Twist and Tinman) on a progenitor identity gene enhancer. The integration of Pointed with the combinatorial effects of dTCF, Mad, Twist, and Tinman determines inductive Ras signaling specificity in muscle and heart development (Halfon, 2000).

Cell fate specification in the somatic mesoderm of the Drosophila embryo has been examined as a model for dissecting the molecular basis of combinatorial signaling involving receptor tyrosine kinases (RTKs). The somatic musculature and the cells that compose the heart develop from specialized cells called progenitors. Each progenitor divides asymmetrically to produce two founder cells that possess information that specifies individual muscle fate and that seed the formation of multinucleate myofibers. The focus of this study has been a small subset of somatic mesodermal cells that express the transcription factor Even skipped. Eve is expressed in the progenitors and founders of both the dorsal muscle fiber DA1 and a pair of heart accessory cells, the Eve pericardial cells or EPCs. Since eve is the earliest known marker for these cells and is required for their formation, eve is referred to here as a progenitor identity gene (Halfon, 2000).

Previous genetic experiments have defined multiple intercellular signaling events that govern the progressive determination of the Eve progenitors. Signaling from both the Wnt family member Wingless (Wg) and the TGF family member Decapentaplegic (Dpp) prepatterns the mesoderm and renders cells competent to respond to Ras/MAPK activation. Localized Ras activation within the competence domain determined by the intersection of Wg and Dpp expression occurs through the action of two RTKs: the Drosophila epidermal growth factor receptor (Egfr) and the Heartless (Htl) fibroblast growth factor receptor. This RTK signaling induces two distinct equivalence groups, each of which expresses Eve. Lateral inhibition mediated by Notch then selects a single progenitor from each equivalence group (Halfon, 2000).

The present study explores how the prepattern genes wg and dpp establish competence for mesodermal cells both to activate and to respond to the Ras/MAPK cascade; how multiple intercellular signals are integrated to establish Eve progenitor fates, and how muscle- and cardiac-specific responses to Ras signaling are generated. Wg provides competence for the generation of the Ras/MAPK inductive signal by regulating the expression of key proximal components of the Egfr and Htl RTK pathways. Wg and Dpp then create competence for a specific response to the inductive signal both through their own respective downstream transcriptional effectors, dTCF and Mothers against dpp (Mad), and through their regulation of the mesoderm-specific transcription factors Tinman (Tin) and Twist (Twi). Specificity of the Ras/MAPK response is achieved though the integration of these signal-activated and tissue-restricted transcription factors, along with the Ras/MAPK-activated Ets domain transcription factor PointedP2 (Pnt), at a single transcriptional enhancer. These results provide a direct link between the initial axis patterning processes in the early embryo and the subsequent combinatorial signaling events that lead to the progressive determination of muscle and cardiac progenitors (Halfon, 2000).

The Eve progenitors in each mesodermal hemisegment arise during embryonic stage 11 in a dorsal region demarcated by the intersecting domains of Wg and Dpp expression. The cells exposed to both Wg and Dpp are competent to respond to localized Ras signaling, which induces the initial expression of Eve in two clusters of equipotent cells. In each of these equivalence groups, activity of the Notch pathway leads to the rapid refinement of Eve expression to a single muscle or cardiac progenitor. The two Eve equivalence groups arise sequentially. Cluster C2, from which progenitor P2 derives, is first to form. P2 divides asymmetrically, with one daughter maintaining Eve expression and becoming the founder of the two EPCs (F2_EPC), and the other losing Eve expression and becoming the founder of muscle DO2. The second Eve-expressing cluster, C15, forms slightly later and produces the progenitor P15, which in turn divides to yield the founder of the Eve-expressing muscle, DA1, and an Eve-negative cell of as-yet-undetermined identity. Activation of the Ras/MAPK pathway in C15 depends on both the DER and Htl RTKs, but only Htl signaling is required for C2 formation (Halfon, 2000).

The progressive determination of Eve mesodermal progenitors requires that Wg prepattern the mesoderm, rendering cells competent to respond to inductive RTK/Ras signaling. To further investigate the basis of this competence, whether or not the Ras pathway is active in the absence of Wg signaling was examined by monitoring the expression of the activated, diphosphorylated form of MAPK in wg mutant embryos. Diphospho-MAPK is expressed in progenitor P2 in early stage 11 wild-type embryos. Not only is this progenitor missing from wg mutant embryos, but activation of MAPK in the C2 equivalence group, which is dependent on Htl, fails to occur. Similarly, Wg is essential both for P15 formation and for the DER- and Htl-dependent activation of MAPK in the equivalence group from which this progenitor is derived (Halfon, 2000).

Next to be determined was at what level in the RTK/Ras pathway Wg is required for MAPK activation. In wg mutant embryos, there is loss of (1) the P2-specific expression of Htl; (2) its specific downstream signaling component, Heartbroken (Hbr, also known as Dof and Stumps), and (3) Rhomboid (Rho), a protein involved in the presentation of the Egfr ligand Spitz. Conversely, constitutive Wg signaling, achieved by ectopic expression of Wg or an activated form of the downstream Wg pathway component Armadillo (Arm), induces Htl, Hbr, and Rho expression in more dorsal mesodermal cells than the single P2 progenitor found at a comparable developmental stage. This effect is less prominent for Rho than for Htl and Hbr, which may reflect different threshold responses to Wg. Alternatively, the effect on Htl and Hbr may be more pronounced because ectopic Wg signaling prolongs their earlier expression in the entire C2 cluster; Rho, in contrast, is normally expressed in P2 but not in C2, possibly making it more refractory to a prepattern factor such as Wg. Expanded expression of these RTK pathway components is associated with increased MAPK activation and Eve expression. However, these effects of Wg hyperactivation are transient, with a normal number of Eve progenitors eventually segregating. Moreover, activated Arm is able to fully rescue Htl, Hbr, Rho, diphospho-MAPK, and Eve expression in wg mutant embryos. Htl, Hbr, and Rho expression, as well as MAPK activation, are also Dpp dependent. In summary, Wg and Dpp regulate the production of several key proximal components of the DER and Htl signal transduction pathways (Halfon, 2000).

Given the involvement of Wg in the expression of Htl, Hbr, and Rho, it was reasoned that a constitutively activated form of Ras1 might bypass the requirement of Wg for MAPK activation. Constitutively activated Ras1, when targeted to the mesoderm of wild-type embryos, leads to an overproduction of Eve progenitors, as well as to the expected hyperactivation of MAPK in these cells. In the absence of Wg signaling, diphospho-MAPK expression is restored by activated Ras1. However, despite this recovery of MAPK activation, constitutive Ras1 does not rescue Eve progenitor formation in a wg mutant background. This is in marked contrast to the ability of activated Arm to fully rescue RTK signaling and Eve progenitor specification in a wg mutant. These results suggest that, in addition to enabling activation of Ras/MAPK signaling as a result of the induction of Htl, Hbr, and Rho expression, Wg signaling must contribute other factors that are essential for the specification of mesodermal Eve progenitors (Halfon, 2000).

Given the importance of Ras/MAPK signaling in Eve progenitor determination, a determination was made of whether Pnt, an Ets domain transcriptional activator that functions downstream of MAPK, is also involved in this process. In pnt mutant embryos, there is a severe reduction in the number of both Eve progenitors, although this loss is more pronounced for the P15 lineage. Since mesoderm migration is normal in pnt embryos, Pnt must only be required for the progenitor specification function of Htl. Consistent with this conclusion, an activated form of Pnt induces extra Eve progenitors (Halfon, 2000).

In embryos mutant for yan, which encodes a MAPK-regulated Ets-domain transcriptional repressor, there is an increase in the number of Eve progenitors and their differentiated derivatives. Conversely, a constitutively activated form of Yan inhibits Eve progenitor formation. Thus, two MAPK-regulated transcription factors are involved in the development of Eve progenitors (Halfon, 2000).

One mechanism that would ensure the convergence of the multiple regulatory inputs required for the formation of P2 and P15 is integration by a transcriptional enhancer. Since Eve expression is the feature that uniquely identifies these progenitors, an investigation was made of whether eve itself is a direct target for regulation by both signal-activated and tissue-specific transcription factors. Regulatory sequences responsible for mesodermal eve expression are located approximately 6 kb downstream of the transcription start site. Deletions of this region were generated and a 312 bp minimal enhancer was defined that has been termed the eve Muscle and Heart Enhancer (MHE). When fused to a nuclear-lacZ reporter gene, the MHE drives expression in a mesodermal pattern identical to that of the endogenous eve gene. Reporter expression initiates at early stage 11, coincident with the onset of Eve expression in the equivalence group C2. Following formation of P2, MHE activity is observed in P15 and in the P2 daughters, F2_EPC and F2_DO2, then in the EPCs and the F15 daughters of P15, and finally in muscle fiber DA1. Colocalization of MHE-driven ß-galactosidase expression with Runt, which marks the F2_DO2 founder and muscle DO2, establishes that the reporter gene expression present in Eve-negative sibling cells is a result of ß-galactosidase perdurance. Of note, the MHE mimics endogenous Eve expression despite its lack of a consensus binding site for the transcription factor Zfh-1 that had previously been proposed to play a role in mesodermal eve regulation (Halfon, 2000).

Strikingly, the MHE is only active in a single nucleus of the mature DA1 and DO2 muscles. It is inferred that these are the original nuclei of the F15_DA1 and F2_DO2 founders based on prior reporter expression in those cells. Similar results were obtained when DNA flanking the MHE by several hundred base pairs on either side (+4.96 to +7.36 kb), including the previously described Zfh-1 site, was included in the reporter construct, or when the MHE was placed 3' to a reporter gene fused to the endogenous eve promoter. Thus, additional sequences are required for eve expression in non-founder myofiber nuclei. Of critical importance to the present study, the MHE fully recapitulates mesodermal Eve expression during the signal-dependent induction of progenitor and founder cells (Halfon, 2000).

Genetic manipulation of the Wg, Dpp, and RTK/Ras signaling pathways causes predictable alterations of endogenous mesodermal Eve expression. A determination was made of whether the isolated MHE responds appropriately to these signals. In all genetic backgrounds, reporter gene expression corresponds precisely to that of endogenous eve. For example, constitutively activated Arm transiently increases the expression of both genes. However, Wg hyperactivation does not have a stable effect on MHE function. In contrast, both endogenous eve and the MHE-driven reporter are induced throughout the initial competence domain by constitutively activated Pnt, and expression of both markers extends laterally in the presence of activated Arm plus Pnt. Ectopic Dpp leads to both endogenous Eve and MHE-driven reporter expression in the ventral mesoderm, while coexpression of Dpp and activated Ras1 induces expression of both genes in a dorsal-ventral stripe. These results demonstrate that the isolated MHE is responsive to all of the known signals that are essential for the specification of Eve progenitors (Halfon, 2000).

Given that the MHE recapitulates early mesodermal Eve expression, a determination was made of whether this enhancer contains binding sites for candidate signal-dependent and mesoderm-specific transcription factors. Focus was placed on two mesoderm-specific factors, Tin and Twi, as well as the nuclear factors that act downstream of Wg (dTCF), Dpp (Mad) and Ras (Pnt, Yan). A computer-based search of the MHE sequence has suggested the presence of potential binding sites for each of these transcription factors. Gel-shift assays confirm that these putative sites actually bind the relevant factors. This analysis establishes the existence of one binding site for dTCF, six for Mad, two for Twist, and four each for Tin and Pnt. Since Yan binds to each of the Pnt sites, these are referred to as Ets sites (Halfon, 2000).

To ascertain whether these in vitro binding sites have in vivo functional significance, the sites were mutated, both singly and in combination, within the context of the entire MHE. All mutagenesis was by base substitution so as not to affect the spacing between other potential cis-regulatory elements. The ability of the mutated MHEs to drive reporter gene expression was tested in transgenic embryos and this expression was compared to that of endogenous Eve. Of the six Mad sites, only Mad4, 5, and 6 are critical for MHE function when inactivated singly or in combination. Mutation of the single dTCF site or of individual binding sites for Twi, Tin, or the Ets factors also lead to loss of reporter gene expression in some, but not all, Eve-expressing cells, with some mutant sites associated with a more severe loss than others. Of note, both the EPC and DA1 lineages are affected equally by all of the mutations. In addition, the activity level in those Eve-expressing cells that do maintain reporter gene expression is on average lower than that seen with the wild-type MHE. In contrast to the single site mutants, mutation of the two Twi, all four Tin, or all four Ets sites completely eliminate MHE activity. It is concluded that binding sites for two tissue-specific and three signal-responsive transcription factors are required for full activity of the MHE in both the muscle and the heart lineages (Halfon, 2000).

The finding that the three Wg-dependent factors, dTCF, Twi, and Tin, that directly regulate eve could explain why activated Ras is incapable of bypassing Wg in the induction of Eve progenitors. Therefore attempts were made to rescue Eve expression in wg mutant embryos by ectopically expressing Twi and Tin together with activated Ras. However, Eve progenitors were not recovered by this manipulation, perhaps due to the direct requirement of dTCF for eve MHE activity. While activated Arm can supply the missing downstream Wg transcription factor in this rescue experiment, Arm alone is capable of fully rescuing not only the Eve progenitors but also all of the Wg-dependent factors that regulate the MHE, including Twi, Tin, and the RTK/Ras pathway components. Thus, the combined effects of the MHE transcription factors could not be further evaluated in the absence of Wg signaling. Nevertheless, the rescue and enhancer mutagenesis data strongly support the involvement of Wg as a mesodermal competence determinant both upstream of the Ras pathway and directly (via dTCF) as well as indirectly (via Twi and Tin) in the transcriptional response to inductive RTK signaling (Halfon, 2000).

Since mutation of any single transcription factor binding site in the MHE causes only a partial loss of enhancer activity, it was considered whether different sites might function together synergistically. To test this possibility, binding site mutations for two different activators were combined. Simultaneous mutation of the dTCF and Twi1 sites led to reporter gene expression in approximately 5-fold fewer cells than would be expected from the additive independent effects of each mutation. A similar, though slightly less robust, synergy was observed when the dTCF and Ets3 mutations were combined (Halfon, 2000).

An assessment was made of whether ectopic coexpression of individual transcription factors or upstream signals would lead to cooperative effects on endogenous Eve expression. As previously reported, ectopic Wg has no effect on Eve expression at late stage 11, activated Ras1 induces extra Eve progenitors, and ectopic Wg plus activated Ras1 cause a lateral expansion of the progenitor clusters. When Twi is expressed using a twi-Gal4 driver, a few Eve-positive cells develop at ectopic positions. The magnitude of this effect is increased by coexpression of Wg and Twi, and even more so by coexpression of Twi with activated Ras1. The latter effect strikingly resembles that of Wg plus activated Ras1. With the simultaneous ectopic expression of Wg, Twi, and activated Ras1, Eve progenitors form an almost continuous anteroposterior stripe confined to the dorsal mesoderm. These results demonstrate a synergistic induction of Eve progenitors by various combinations of Wg, Twi, and activated Ras1 that parallels the synergistic loss of MHE activity seen by mutating the dTCF, Twi, and Ets binding sites. Taken together, these loss- and gain-of-function findings suggest that dTCF, Twi, and Pnt cooperate at the MHE to synergistically regulate Eve transcription and, by extension, to induce the specification of Eve progenitor fates (Halfon, 2000).

It is concluded that Wg and Dpp coordinate a series of signal-activated (dTCF and Mad) and mesoderm-specific (Twi and Tin) transcription factors in a temporal and spatial pattern that facilitates cooperation with the Ras transcriptional effector Pnt. The synergistic integration of these five transcription factors by a single enhancer generates a specific developmental response to Ras/MAPK signaling. Moreover, Wg and Dpp exert proximal effects in this signaling network by enabling Ras/MAPK activation through the regulated localized expression of upstream components of the RTK signal transduction machinery. A model governing the acquisition of developmental competence, signal integration and response specificity in this system is presented. Wg and Dpp provide competence through the regulation of tissue-specific transcription factors (Tin and Twi), signal-responsive transcription factors (Mad and dTCF), and proximal components of the RTK/Ras pathways (Htl, Hbr, and Rho). The Ras signaling cascade leads to activation of the inductive transcription factor, Pnt, and inactivation of the Yan repressor. While a direct role for Mad in regulating Tin expression has been demonstrated, Wg regulation of Tin, Twi, Htl, Hbr, and Rho may be either direct or indirect. Dpp has additional effects on the proximal RTK factors. The five transcriptional activators assemble at and are integrated by the MHE, where they function synergistically to promote eve expression. Specificity of the response to inductive RTK/Ras signaling derives from the combinatorial effects of the tissue-restricted and signal-activated transcription factors that converge at the MHE. In the absence of inductive signaling, Yan would repress eve by binding to the Ets sites. Since eve is a muscle and heart identity gene, the regulatory mechanisms are inferred to have a more general function in determining progenitor fates. Additional complexity attendant upon the control of RTK activity in this system derives from positive feedback regulation of the Ras/MAPK cascade and from reciprocal regulatory interactions between the Ras and Notch pathways (Halfon, 2000).

Individual somatic muscles and heart progenitors are specified at defined positions within the mesodermal layer of Drosophila. The expression of the homeobox gene even-skipped (eve) identifies one specific subset of cells in the dorsal mesoderm, which give rise to particular pericardial cells and dorsal body wall muscles. Genetic analysis has shown that the induction of eve in these cells involves the combined activities of genes encoding mesoderm-intrinsic factors, such as Tinman (Tin), and spatially restricted signaling activities that are largely derived from the ectoderm, particularly those encoded by wingless and decapentaplegic. A Dpp-activated Smad protein, phosphorylated Mad, is colocalized in eve-expressing cells during an extended developmental period. A mesodermally active enhancer of eve contains several Smad and Tin binding sites that are essential for enhancer activity in vivo. This enhancer also contains a number of binding sites for the Wg-effector Pangolin (Pan/Lef-1), that are required for full levels of enhancer activity. However, the main function of these sites is to prevent ectopic enhancer activity in the dorsal mesoderm. This suggests that, in the absence of Wg signaling, Pan binding serves to abrogate the synergistic activities of Smads and Tin in eve activation, while in cells that receive Wg signals, Pan is converted into a coactivator that promotes eve induction. Together, these data show that the eve enhancer integrates several regulatory pathways via the combinatorial binding of the mesoderm-intrinsic regulator Tin and the effectors of the Dpp and Wg signals (Knirr, 2001).

Functional dissection of eve flanking regions has identified a 900-bp fragment, located about 5.7-6.6 kb downstream of the eve transcription start site and named Eve Mesodermal Enhancer (EME) 0.9SS: this fragment is able to reproduce the full mesodermal pattern of endogenous eve expression between embryonic early stage 11 and stage 15. Between stages 11 and 12, reporter gene expression occurs exclusively in the 11 (plus one posterior) bilateral clusters of mesodermal eve progenitors. Perduring ßGalactosidase (ßGal) protein at stage 16 shows that these clusters include the progenitors of the eve pericardial cells and muscle 1 (DA1). Because the long half life of ßGal protein precludes accurate determination of the temporal dynamics of reporter gene expression, transgenic embryos were stained for both lacZ mRNA, which is less stable, and Eve. This analysis confirms that the EME 0.9SS enhancer is active in all pericardial cells and muscle 1 precursors until stage 15 and continues to be active in the eve pericardial cells, predominantly those surrounding the posterior portion of the dorsal vessel (the heart proper), until the end of embryogenesis. This pattern is similar to that of eve mRNA, except that eve continues to be expressed evenly in pericardial cells along the entire length of the dorsal vessel. A second difference is ectopic reporter gene expression, which is observed following stage 15 in muscle 2, several ventral muscles, and in ectodermal muscle attachment sites. From this it is inferred that EME 0.9SS lacks site(s) for negative regulators, which normally prevent eve activation by late-acting regulators outside of eve's normal domain of expression (Knirr, 2001).

The spatial and temporal activity of a subfragment comprising 709 bp of the 59 portion of EME 0.9SS, EME A, is identical to the full-length fragment. Both the 394-bp fragment EME B, from the center of EME 0.9SS, and the longer EME C have activities similar to that of EME 0.9SS, although their lacZ expression already terminates at stage 14 in muscle 1 precursors and there is little ectopic expression (Knirr, 2001).

To define minimally active enhancer fragments, EME B was further subdivided into two overlapping portions, EME B59 (232 bp) and EME B39 (361 bp). Both subfragments drive expression in mesodermal eve progenitors, although the levels of EME B59 activity are lower than those of the parental EME B fragment. Similar to EME B, but unlike EME 0.9SS, EME B59 and EME B39 exhibit low levels of ectopic activity in dorsal mesodermal cells outside of the endogenous early Eve clusters, while other embryonic regions show very little ectopic activity. Enhancer activity of EME B59 persists in eve pericardial cells until stage 16 (more strongly posteriorly), while dorsal muscle founders have already become negative at mid stage 12 (Knirr, 2001).

EME B39 enhancer activity ceases prematurely at early stage 13 in both pericardial and dorsal muscle progenitors. The identical spatial activity of EME B59 and EME B39 at stages 11 and 12 indicates that both fragments include a similar set of regulatory sequences that are necessary for normal early activation of mesodermal eve expression. Either all of these sequences are contained within the ~90-bp overlap between the two fragments or there is redundancy of individual regulatory sites within EME B. By contrast, only EME B59 contains regulatory sequences that are sufficient for late pericardial expression, and both EME B59 and EME B39 have to cooperate to drive expression in syncytial muscle 1 precursors at later stages (Knirr, 2001).

The expression of eve identifies a small subset of pericardial and somatic muscle progenitors and its onset coincides with the processes that determine their developmental fates. Although eve itself cannot be sufficient to specify the distinct identities of these cells, it may fulfill such a function in specific combinations with additional regulators, such as Krüppel (in muscle 1 progenitors) and Runt (in muscle 10 progenitors). Therefore, eve activation can serve as a paradigm for studying the genetic and molecular processes that determine the identities of individual muscle and heart progenitors (Knirr, 2001).

Previous work has provided insight into the regulatory cascades and some of their components that are critical for eve expression. These studies show that the combined activities of tin and slp, which themselves are induced in the mesoderm by Dpp and Wg, respectively, are required but not sufficient for eve activation. Further, ectopic expression of slp and wg in combination, but not of either component alone, results in uniform eve expression along the dorsal margin of the mesoderm. These and other data indicate that wg and possibly also dpp are required during multiple steps in the regulatory cascade of eve induction. The results of this study confirm that there is a renewed requirement for Wg and Dpp signaling at the level of eve activation. More generally, this means that these two signaling molecules first induce the spatially restricted and overlapping expression of prepatterning genes in the mesoderm and subsequently act again, this time in conjunction with the products of prepatterning genes, to induce genes that determine the identities of heart and muscle progenitors. During this downstream step, the restricted areas of overlap between prepatterning gene expression patterns determine the domains in which cells are competent to respond to signals (Knirr, 2001).

The integration of the regulators of eve involves the direct interaction of Dpp and Wg effectors as well as Tin (but not Slp) with a mesodermal eve enhancer. Tin and Dpp-activated Smads appear to synergize to allow eve induction, analogous to the situation that has been described for the broad induction of tin in the dorsal mesoderm. However, this raises the question of why, unlike the case of tin, the combined activities of Smads and Tin are unable to induce eve in the whole dorsal mesoderm. A likely explanation is that negative regulators bind to the eve enhancer and abrogate the synergistic activities of Smads and Tin. Based on this analysis, these negative regulators include Pangolin (Lef1), and the ETS protein Yan has also been identified as a negative factor in this process. In the current view, the role of Wg and RTK signaling would be to neutralize these negative regulators and convert them into positive ones, which would then enable Smads and Tin to activate eve exclusively in the cells that receive these additional signals (Knirr, 2001).

The Drosophila pair-rule gene even skipped is required for embryonic segmentation and later in specific cell lineages in both the nervous system and the mesoderm. eve mesoderm-specific mutants have been generated by combining an eve null mutant with a rescuing transgene that includes the entire locus, but with the mesodermal enhancer removed. This allowed analysis in detail of the defects that result from a precisely targeted elimination of mesodermal eve expression in the context of an otherwise normal embryo. Absence of mesodermal eve causes a highly selective loss of the entire eve-expressing lineage in this germ layer, including those progeny that do not continue to express eve, suggesting that mesodermal eve precursor specification is not implemented. Despite the resulting absence of a subset of muscles and pericardial cells, mesoderm-specific eve mutants survive to fertile adulthood, providing an opportunity to examine the effects of these developmental abnormalities on adult fitness and heart function. In these mutants, flying ability, myocardial performance under normal and stressed conditions, and lifespan are all severely reduced. These data imply a nonautonomous role of the affected pericardial cells and body wall muscles in developing and/or maintaining cardiac performance and possibly other functions contributing to normal lifespan. Given the similarities of molecular-genetic control between Drosophila and vertebrates, these findings suggest that peri/epicardial influences may well be important for proper myocardial function (Fujioka, 2005).

Removing the mesodermal enhancer eme in the context of a rescue transgene removes all detectable mesodermal eve expression without affecting any other aspect of expression. When combined with a null mutation at the endogenous locus, this results in an eve 'mesoderm negative' (eve meso^–) mutant. A separate transgene containing eme upstream of a reporter gene (lacZ) can be used to faithfully mark the cells that would normally express eve. In the absence of mesodermal eve, the body wall muscles no longer show reporter gene expression. In cells associated with the heart, however, the stable reporter protein persists until the formation of a seemingly normal heart tube. Except for the lack of eve expression, other heart cell types, particularly the myocardial cells, appear to be present in their normal numbers and positions. In the absence of eve, the cells in which the reporter persists appear to be naïve and able to adopt other (cardiac) fates, consistent with eve acting as an essential factor for specification of their lineage. Interestingly, the vertebrate eve homolog, Evx2, is also expressed in the heart, in an epicardial cell line, and in cells from primary epicardial explants. Thus, eve/Evx may play a role in heart development in both insects and mammals (Fujioka, 2005).

Mesodermal eve expression normally occurs in founder cells that give rise to a subset of pericardial cells and to 2 muscles per hemisegment, DO2 and DA1. A cell within 1 eve-expressing cluster (cluster 2) initiates expression of Krüppel (Kr). This Kr- and eve-expressing cell (progenitor 2) divides to yield two founder cells that express runt, one of which is the founder of DO2 and continues to express runt, and the other of which is the EPC founder and turns off runt. The DO2 founder turns off eve shortly after runt is activated, whereas the EPC founder and the resulting EPCs continue to express eve. A cell within a second eve-expressing cluster (cluster 15) activates Kr, then divides to yield the DA1 founder and a second cell that is fated to die. The DA1 muscle maintains eve expression (Fujioka, 2005).

Previous studies have suggested that eve function is required for normal EPC differentiation and for the normal pattern of expression of ladybird. As a way to define the role of eve, both runt and Kr expression were examined in eve meso^– embryos. In the absence of eve, both runt and Kr expression are either completely absent from or dramatically reduced in these muscle founder lineages. This strongly suggests that eve is required for these cells to adopt their normal fates. Thus, eve has either a direct or an indirect role (repression of a repressor) in activating Kr and runt (Fujioka, 2005).

To determine the extent to which eve function is required for normal muscle formation, the musculature was examined in eve meso^– third instar larvae. The normal arrangement of dorsal muscles within each segment is clearly altered. Both DA1 and DO2 are severely defective or aberrant, and other muscles in the vicinity exhibit alterations in their placement and size. A simple interpretation of the effects seen in a majority of segments is that DA1 is missing, and a single, large muscle occupies the normal positions of DO1 and DO2. The muscle occupying the normal position of DA2 is also enlarged. These enlarged muscles suggest that myoblasts that normally fuse with the DA1 and DO2 founders may instead fuse with other muscles nearby, or, alternatively, that in the absence of DA1 and DO2, attachment sites for adjacent muscles expand (Fujioka, 2005).

The DA1 and DO1 muscles are innervated in wild-type embryos by motorneurons that express eve. Transgenes that express green fluorescent protein (GFP) in these neurons were used to label their axons in the muscle field. In eve meso^– embryos, one or both muscles in the normal positions of DA1/2 and DO1/2 are innervated by these eve-expressing neurons. It was also found that in eve meso^– third instar larvae, both DA1/2 and DO1/2 are innervated (Fujioka, 2005).

The changes in gene expression observed in eve meso^– embryos suggest that eve acts, directly or indirectly, as an activator of Kr and runt. Previous analyses of eve function suggested that it acts as a repressor of transcription. If this is true in the mesoderm, then at least one intermediary gene that is repressed by eve normally represses runt and Kr. To study the domain requirements of eve, transgenes were expressed in the eve meso^– background that contained the eve mesodermal enhancer driving expression of modified Eve proteins. With the wild-type Eve coding region, the mesodermal defect is completely rescued. In contrast, when the Eve homeodomain (HD)-containing region alone is so expressed, a very limited degree of rescue is observed. Importantly, when the heterologous Engrailed repressor domain is added to the HD construct, full rescuing ability is restored, implying that eve acts exclusively as a repressor in the mesoderm (Fujioka, 2005).

The ability of Eve to act as a direct repressor in the mesoderm was examined by targeting it to a reporter transgene using the Gal4 DNA binding domain. When a Gal4-Eve fusion protein containing both of the repressor domains of Eve is combined with an eve lineage-specific Gal4-UAS-containing reporter, the reporter is strongly repressed (Fujioka, 2005).

eve meso^– embryos develop into viable adults, providing an opportunity to examine the role of PC cells in larval and adult heart function. The absence of mesodermal eve does not noticeably affect the assembly of the myocardial cells at the dorsal midline, which will give rise to the contractile part of the heart tube. To examine the contribution of EPCs to larval heart development, eve meso^– larvae were dissected and PC cells were counted. Wild-type and wild-type eve-rescued larvae show 7 to 8 PC cells per segement, and eve meso^– heterozygotes show 6. In contrast, two independent eve meso^– lines displayed a marked reduction, with an average of only 3 to 4 PC cells per segment. Thus, eve is required to produce the normal complement of larval PC cells (Fujioka, 2005).

Because eve meso^– animals are missing half or more of their PC cells, the effect on heart function was examined in pupae and adults. Neither wild-type eve-rescued pupae nor those heterozygous for eve and carrying one copy of a meso^– rescue transgene exhibited heart rates that differed significantly from controls. However, all 3 meso^– lines, which carry independent transgene insertions, exhibited a significant reduction (30% to 50%) in heart rate (Fujioka, 2005).

Adults of the same genotypes were examined to assess whether defective functions persist through partial remodeling of the heart during metamorphosis. The wild-type heart rate is 2.9 beats/second (Hz) in 1-week-old adults and 2.6 Hz in 3-week-old adults. Compared with wild type, both the eve null background rescued by a wild-type rescue transgene and the eve meso^– heterozygotes (J49/CyO) exhibit a reduced heart rate at both ages, probably attributable to a genetic background effect inherent to these eve rescue lines that serve as controls. eve meso^– flies, however, develop a dramatically lower heart rate with age, and one of the meso^– lines also shows a severely reduced heart rate at an early age (Fujioka, 2005).

Heart function can also be assayed by quantifying stress tolerance, using an external current to briefly pace the heart to about twice the normal rate, then charting the percentage that undergo either fibrillation or cardiac arrest (termed heart failure). In wild-type flies, the ability of the heart to withstand such stress is highly age-dependent, with stress-induced failure rates increasing dramatically (2- to 3-fold) between 1 and 5 weeks of age.Because eve meso^– flies seldom reach 5 weeks of age, flies at one and three weeks of age were examined. Neither heterozygous eve meso^– nor eve rescued flies differed from wild type. In contrast, one eve meso^– line showed a significantly increased failure rate at 1 week of age, whereas the other showed a disastrously high failure rate at 3 weeks of age. Although dorsal somatic muscle defects might also conceivably affect heart function, these results suggest that the reduction in PC cell number causes a slowed heartbeat and reduces cardiac stress resistance (Fujioka, 2005).

To further assess the importance of fully functional heart and muscle activity and potentially of other results of mesodermal eve expression, the life spans of eve meso^– flies was examined. Such flies display a significantly reduced mean and maximal lifespan, suggesting that the presence of mesodermal Eve is required not only for normal activity levels but also for a normal lifespan (Fujioka, 2005).

Thus eve is required not only in the eve-expressing lineages in which it is maintained during terminal differentiation (the eve-expressing pericardial cells and the DA1 muscle), but also in the lineage in which it is expressed in the progenitor but turned off in the muscle founder cell and the resulting DO2 muscle. Thus, both DA1 and DO2 require mesodermal eve to be specified, and without this specification, the pattern and size control of some remaining muscles are compromised. Importantly for heart function, in the absence of mesodermal eve expression, the majority of the large larval PC cells are missing (Fujioka, 2005).

It is intriguing that even though ectopic Eve expression can interfere with the DO2 fate, and eve is normally turned off as runt is activated in the lineage, eve function is nonetheless required for DO2 formation, apparently because of a requirement in the progenitor before the lineage divisions (Fujioka, 2005).

When normal eve expression in the mesoderm is replaced by expression of the eve HD (with repressor domains deleted), a similar but less severe muscle deficiency is observed compared with the complete absence of mesodermal eve. In particular, a muscle in the DO2 position is usually formed, whereas DA1 is still absent. Additionally, there is occasionally an extra muscle ventral to DO2, as if the DO2 founder was duplicated. Importantly, however, when the heterologous Engrailed repressor domain is added to the HD construct, full rescuing ability is restored. This suggests that Eve functions in the mesoderm primarily or exclusively as a repressor, and in turn that eve acts indirectly to activate Kr and runt in the mesoderm. Good candidates for intermediary repressors are ladybird and the muscle identity gene msh (Fujioka, 2005).

A reduced number of larval PC cells (and dorsal somatic muscles) caused by a lack of mesodermal eve expression results in severely compromised heart function and is likely to contribute to a shortened lifespan. A less drastic effect on cardiac performance and lifespan is observed when manipulating insulin signaling exclusively in the heart. The functional role of pericardial cells in insect hearts is not well understood, but they may contribute to heart function by secreting hormones or by gathering such peptides from circulating hemolymph and 'presenting' them to the myocardium. It has been suggested that pericardial cells may function as nephrocytes, and at this point it cannot be ruled out that a potential accumulation of toxic agents, as a consequence of fewer pericardial cells, contributes to the observed phenotypes (Fujioka, 2005).

As in insects, the developmental and functional interactions between the vertebrate epicardium and the myocardium are not well understood. Recent studies have suggested that the loss of epicardial function results in impaired growth of the myocardium at mid-gestation. The epicardium is thought to be a source of signals and secreted factors that affect myocardial proliferation and differentiation, as well as influencing formation of the conduction system. Even though it cannot yet be decided whether the mammalian epicardium has a developmental program in common with a fly’s pericardial cells, they both depend on GATA factors for formation. In addition, the Evx2 homolog of eve is indeed expressed in the mammalian heart, including in epicardial tissue. These findings are consistent with pericardial cells in Drosophila functioning as a source of signals that affect the myocardium. Possibly because the myocardium, which is maintained by proliferation in vertebrates, does not proliferate in flies after it is developmentally specified, pericardial deficiency does not appear to result in morphological heart defects. Rather, defects manifest themselves as functional deficits. This provides an opportunity to study the influence of these heart-associated cell types on cardiac physiology in the absence of myocardial defects. Epicardial lineages in vertebrates may contribute analogously to normal cardiac physiology and performance (Fujioka, 2005).