Individual cardiac progenitors emerge at defined positions within each segment in the trunk mesoderm. Their specification depends on segmental information from the pre-patterned ectoderm, which provides positional information to the underlying cardiac mesoderm via inductive signals. This pattern is further reinforced by repressive interactions between transcription factors that are expressed in neighboring sets of cardiac progenitors. For example, even-skipped (eve) and ladybird early (lbe) gene products mark adjacent cardiac cell clusters within a segment, and their antagonistic interaction results in mutually exclusive expression domains. Lbe acts directly on the eve mesodermal enhancer (eme) to participate in restricting its expression anteriorly. It is hypothesized that additional repressive activities must regulate the precise pattern of eve expression in the cardiac mesoderm via this enhancer. In this study, two additional repressor motifs: 4 copies of an 'AT'-rich motif (M1a-d) and 2 copies of an 'GC'-rich motif (M2a,b), were identified which when mutated cause expansion of eme-dependent reporter gene expression. Potential negative regulators of eve and were examined and it was found that their overexpression is sufficient to repress eve as well as the eme enhancer via these sites. These data suggest that a combination of factors is likely to interact with multiple essential repressor sites to confer precise spatial specificity of eve expression in the cardiac mesoderm (Liu, 2008).

Although each of the identified repressor sites is necessary, none is individually sufficient for restricting the eme enhancer activity to the eve expression domain. Several additional homeodomain proteins, including Msh, C15 and Lim3, are capable of repressing mesodermal eve expression by interacting with specific sites within the enhancer element. While the repression of mesodermal eve expression by Msh, C15 and Lim3 is likely mediated by the AT-rich M1 sites and the Lb2 site, the repression of eve expression by Lbe requires both the AT-rich M1 and the Lb2 sites as well as the GC-rich M2a site. Therefore, each of the four repressor sites apparently is required on order to confer sensitivity to repression by Lbe. This raises the possibility that repression is the result of a complex in which the cooperation of all four repressor elements is required for successful repression (Liu, 2008).

A prominent feature of the Drosophila is its segmental polarity that includes distinct cardiac cell types that are precisely positioned within each segment. These cardiac progenitors are specified along the anterior-posterior axis during development and are marked by Lbe, Eve or Svp. As the embryo develops, a linear heart tube is formed and this metameric arrangement of cardiac cells types continues to be maintained. Within each hemi-segment, the anterior two pairs form the tinman-expressing 'working myocardium', while the posterior pair that expresses svp and the T-box transcriptional factor Doc form the ostia. Previous studies suggested that repressive interactions between cardiac factors expressed in non-overlapping subtypes of cardiac cells likely contribute to the diversification and maintenance of cellular identities. Svp and Tin have been shown to repress each other's expression during heart tube formation, and the current data suggest that antagonistic interactions between Lbe and Eve are also a part of this mutual repression network. In addition, the data show that eve expression within the cardiac mesoderm is negatively regulated by multiple repressor sites, thus further supporting the idea that transcriptional repression mechanisms play a prominent role in the generation of cellular diversity in the developing heart. Roles were demonstrated for two potential repressors, C15 and Lim3. Although they do not seem to be essential for patterning mesodermal eve expression, they are normally expressed in the cardiac mesoderm in the vicinity of the Eve cells and they do repress the eme enhancer via the identified repressor sites when ectopically expressed. Therefore, it is also possible that they function redundantly with other negative regulators yet to be identified (Liu, 2008).

Default repression is a common mechanism utilized by major signaling pathways, including Wnt, Shh and Notch pathways, to restrict target gene expression. In the absence of signaling, signal-regulated transcription factors function mainly as transcriptional repressors, thus preventing low levels of target gene expression that might be activated by weak, local activators ('default repression'). In response to signals, some transcription factors are then converted into transcriptional activators to promote target gene expression. Thus, transcriptional repression and activation can be mediated by the same binding sites. Default repression mechanisms may also contribute to the restricted mesodermal eve pattern. It has been reported that mesodermal eve expression is under the direct transcriptional control of Wg signaling. Mutating several putative binding sites for dTCF, the transcriptional mediator of Wg signaling, results in an expansion of low-level reporter gene expression within the cardiac mesoderm that is unaffected by reduced wg activity. Thus, dTCF may serve as a default signal to restrict mesodermal eve expression in the absence of wg signaling (Liu, 2008).

It has been shown that Hh signaling not only promotes eve and svp but also inhibits lbe expression in the dorsal mesoderm. One mechanism for Hh signaling may be via inhibition of Cubitus interruptus (Ci)-mediated repression. Interestingly, there is some similarity between the M2a sequence examined in this study (TGGGCCCT) and the consensus sequence for Ci (TGGGTGGTC). This raises the interesting possibility that M2a site may be a putative Ci binding site in eme. Thus, mutations of M2a site, which result in the anterior expansion of eme activity into Lbe expressing cells, may reflect a lack of repression by Ci. Alternatively, the M2a site may mediate transcriptional repression by Lbe or its potential cofactors. The latter hypothesis is more consistent with the observation that reporter gene expression is rendered insensitive to inhibition by Lbe overexpression when the M2a site is mutated in eme. As the M2a site does not resemble the Lbe consensus sequence, the idea is favored that another factor binds to the M2a site, which then cooperates with Lbe in repressing mesodermal eve expression. This interaction may be facilitated by the close proximity of the two sites (Liu, 2008).

In sum, the in vivo functional dissection of eme has revealed that each of two AT-rich sites, M1b or M1c and the previous studied Lb2 site, when mutated, causes reporter gene expansion that encompasses the entire cardiac mesoderm, overlapping with Tinman protein at late stage 12. In addition, the GC-rich site M2a is required for repression anterior to the Eve cluster. The absolute requirement of each repressor site for successful restriction of eve expression within the cardiac mesoderm is in striking contrast to the mechanism of incremental activation of this enhancer in the cardiac mesoderm by activators such as Tinman, dTCF, Mad, E-box and ETS sites. Repression through these repressor sites may require cooperation between the sites, perhaps via a repressor complex. Thus, eliminating the function of any of these sites will disrupt interactions with the complex causing de-repression within the 'activator'-dependent cardiac mesoderm (Liu, 2008).