Specification of muscle identity in Drosophila is a multistep process: early positional information defines competence groups termed promuscular clusters, from which muscle progenitors are selected, followed by asymmetric division of progenitors into muscle founder cells (FCs). Each FC seeds the formation of an individual muscle with morphological and functional properties that have been proposed to reflect the combination of transcription factors expressed by its founder. However, it is still unclear how early patterning and muscle-specific differentiation are linked. This question was addressed using Collier (Col; also known as Knot) expression as both a determinant and read-out of DA3 muscle identity. Characterization of the col upstream region driving DA3 muscle specific expression revealed the existence of three separate phases of cis-regulation, correlating with conserved binding sites for different mesodermal transcription factors. Examination of col transcription in col and nautilus (nau) loss-of-function and gain-of-function conditions showed that both factors are required for col activation in the 'naive' myoblasts that fuse with the DA3 FC, thereby ensuring that all DA3 myofibre nuclei express the same identity programme. Together, these results indicate that separate sets of cis-regulatory elements control the expression of identity factors in muscle progenitors and myofibre nuclei and directly support the concept of combinatorial control of muscle identity (Dubois, 2007).

col belongs to the class of Drosophila regulatory genes with numerous introns, large amounts of flanking sequence and multiple expression sites. During embryogenesis, col is expressed in the MD2/PS0 head region, the somatic DA3 muscle, precursor cells of the lymph gland, a small set of multidendritic (md) neurons of the peripheral nervous system and specific neurons of the central nervous system (CNS). A lacZ reporter transgene (P{5col::lacZ}, abbreviated P5cl, contains 5 kb of col upstream DNA, which faithfully reproduced col transcription both in the MD2/PS0 and the DA3 muscle, starting at the progenitor stage and not in promuscular cluster(s). To identify the missing cis-regulatory information, a longer construct was tested containing the entire 9 kb region separating col from CG10200, the next predicted upstream gene. In addition to the head and DA3 muscle, P9cl expression reproduced col expression in md neurons and a subset of neurons in the CNS. A DNA fragment located further upstream, between CG10200 and the next predicted gene CG10202, was independently shown to drive col expression in the anteroposterior organiser of the wing imaginal disc (Hersh, 2005). However, neither this construct nor P9cl reproduced Col expression in promuscular clusters. The col transcription unit is immediately flanked at its 3' end by another gene, BEAF32, making rather unlikely the presence of cis-regulatory elements within this region. However, it contains ten different introns, of total length around 30 kb, the cis-regulatory content of which remains to be assessed (Dubois, 2007).

To delineate more precisely the CRM driving col expression in the DA3 muscle, a series of constructs was tested containing 2.6, 2.3, 1.6 and 0.9 kb of DNA upstream of the col transcription start site, respectively. P2.6cl retained the information necessary for col expression in MD2/PS0 and the DA3 progenitor and muscle, although it was noted that P2.6cl expression in muscle progenitors was less robust than P9cl. P2.3cl was also activated in MD2/PS0 at stage 6 and the DA3 muscle. However, unlike P9cl or P2.6cl, P2.3cl was not activated in the DA3/DO5 progenitor but only at the FC stage; ectopic lacZ expression was observed in clusters of neuroectodermal cells at embryonic stage 11). This difference indicated that cis-regulatory elements required for col expression in the DA3/DO5 progenitor reside between positions -2.6 and -2.3 and act separately from those required for expression in the DA3 FC and muscle. P1.6cl was active only in MD2/PS0, whereas no expression at all could be detected with P0.9cl. Together, expression data from this series of reporter constructs allowed the mapping of the CRM required for col-specific expression in the DA3/DO5 muscle progenitor and DA3 FC/myofibre to a DNA fragment between positions -2.6 and -1.6 upstream of the col transcription start (Dubois, 2007).

Advantage was taken of the recently available genome sequences of several Drosophila species to search for conserved motifs in the col upstream DNA, as it has often proven to be effective to identify functionally important cis-regulatory elements. Among these species, D. virilis (D. vir) is the most distant from D. melanogaster (D. mel). It was first verified that Col expression in D. vir was similar to that in D. mel embryos and could infer from this that the regulatory information controlling col transcription in the DA3 muscle lineage has been conserved. Sequence comparison of 9 kb of the col upstream region between D. mel, D. vir and four other Drosophila species, D. yakuba, D. ananassae, D. pseudoobscura and D. mojavensis revealed numerous stretches of high sequence conservation, of sizes up to 100 bp. Ten conserved motifs of size >20 bp, numbered 1 to 10 from 5' to 3', were found in the same order and at the same relative position between position -2.6 and the start of transcription in all six Drosophila species. To test the relevance of this conservation, lacZ reporter constructs were created containing either D. vir or D. mel DNA (Dubois, 2007).

P.3.4cl^vir corresponds to D. mel P2.6cl, whereas P3.4-1.3cl^vir and P2.6-0.9cl are truncated versions covering motifs 1 to 10. All four reporter genes showed expression in the DA3 muscle, starting at the progenitor stage, confirming the evolutionary conservation of a DA3-muscle-specific CRM. A Gal4 driver line containing only the -2.6 to -1.6 region (P2.6-1.6cG), harbouring only motifs 1 to 7, was also specifically expressed in the DA3 muscle. This confirmed that the DA3 muscle CRM is located between positions -2.6 and - 1.6. It was noticed, however, that expression of P2.6-1.6cG was weaker and more sporadic than P2.6-0.9cl, suggesting the existence of cis-regulatory element(s) between positions -1.6 and -0.9 contributing to robust DA3 muscle expression. The conserved motifs 1 to 10 were searched for consensus binding sites of known TFs that could account for col activation in the DA3 muscle. This identified a binding site for the mesodermal basic helix-loop-helix (bHLH) protein Twi (within motif 2), correlating well with the position of the muscle progenitor cis-element and a potential EBF/Col-binding site within motif 7. Further visual inspection of the sequence alignments identified other conserved TF-binding sites, including one Mef2-binding site within the -1.6 to -0.9 fragment and one consensus binding site for Nau (Huang, 1996; Kophengnavong, 2000). In contrast, the position of the Mef2 site correlated well with the requirement of the -1.6 to -0.9 fragment for robust DA3 muscle expression. The presence of a Nau-binding site was particularly intriguing since Nau is required for DA3 muscle formation. Potential binding sites for other TFs could be found in the DA3 CRM, but the annotation to the conserved sites. The relative paucity of known TF-binding sites in the conserved sequence motifs found in the DA3 muscle CRM leaves largely open the question of the roles of these motifs in col regulation (Dubois, 2007).

Functional dissection of the DA3 muscle CRM present in the col upstream region showed that col expression in the DA3 FC can be separated from its expression in the DA3/D05 progenitor and the promuscular cluster. It thus revealed the existence of three steps in the transcriptional control of muscle identity. That col expression in the DA3/D05 progenitor could be uncoupled from that in promuscular clusters was in apparent contradiction with the previous conclusion from pioneering studies on Eve expression in dorsal muscle progenitors that this expression issued from Eve activation in promuscular clusters. Restriction of Eve expression to progenitors was considered a secondary step, mediated by N-signalling during progenitor selection by lateral inhibition. To reconcile these data and this model, it is proposed that the muscle DA3 CRM is active only in the DA3/D05 progenitor because it lacks some positively acting cis-elements necessary to counteract N-mediated repression of col transcription. It has been shown that col transcription is repressed by N during the progenitor selection process. It is also noted that a Twi-binding site is present in the 'progenitor' subdomain of the DA3 CRM. The functional importance of this site is supported by its in vivo occupancy in 4- to 6-hour-old embryos when selection of the DA3/DO5 progenitor takes place (Sandmann, 2007). Together, Twi in vivo binding and the col/P2.6cl/P2.3cl expression data suggest that Twi activity contributes to col expression in the DA3/DO5 progenitor but may not be sufficient to override N repression of col transcription before progenitor selection. Additional binding sites for Twi present in the col upstream region, between positions -8.7 and -8.3, are also bound by Twi in vivo (Sandmann, 2007) and probably contribute to the robustness of P9cl expression in progenitor cells, but the question of which cis-regulatory elements mediate col activation in promuscular clusters remains open. From Eve expression studies, a computational framework has been developed to identify other FC-specific genes (Estrada, 2006; Philippakis, 2006). This framework, named Codefinder, integrates transcriptome data and clustering of combinations of binding sites for five different TFs (Pnt, dTCF, Mad, Twi and Tin). col/kn was selected by Codefinder owing to the presence of five clusters of binding sites, four of which are located within introns (Philippakis, 2006). It remains to be determined which of these could be responsible for col activation in promuscular clusters, but it is interesting to note that another in vivo Twi-binding site in 4-6-hour-old embryos correlates with the 3'-most cluster (Sandmann, 2007). In addition to Twi, conserved binding sites for Nau and Mef2 are found within the DA3 CRM. The Mef2 binding site is located in a region required for robust DA3-muscle expression of a reporter gene. A direct control of col transcription by Mef2 during the muscle fusion process is further supported by the recent finding (Sandmann, 2006) that Mef2 binds in vivo to the col upstream region between 6 and 8 hours of embryonic development (Dubois, 2007).

Detailed analysis of col auto-activation revealed a reiterative, two-step process: import of pre-existing Col protein in the fusion competent myoblast nuclei that incorporate into the growing DA3 myofibre precedes activation of col transcription. This process ensures that all incorporated FCM nuclei acquire the same identity. Nau is required for maintaining col transcription in the DA3 muscle precursor and this control is probably direct. The presence of a putative EBF-binding site in the DA3 muscle CRM also correlates with the Col requirement for maintaining its own transcription beyond the FC stage. Thus, despite the failure to detect strong Col binding to this site in vitro, it appears to be essential for col auto-regulation in vivo. This suggests that in vivo binding is potentiated by one or more specific co-factor(s) present in the DA3 muscle. One co-factor is probably Nau, as the ability of Col to activate its own transcription in newly recruited fusion competent myoblasts is dependent upon Nau activity. Nau is not sufficient, however, as many muscles containing both Nau and Col proteins do not activate col transcription. Interestingly, mouse EBF (also known as Ebf1 and Olf1 - Mouse Genome Informatics) and E2A (Tcfe2a - Mouse Genome Informatics), a bHLH protein of the same subgroup as MyoD, have been shown to act on the same target promoter and synergistically upregulate transcription of B-lymphocyte-specific genes, although no direct physical interaction between EBF and E2A could be found in vitro. This suggested that functional interaction of EBF and E2A, similar to Col and Nau, requires yet another factor. Taking into account the restricted pattern of ectopic col activation in hs-col conditions, it is hypothesised that Vg could be another component of the DA3 combinatorial identity. However, we found that Vg is not required for DA3 muscle specification, leaving open the question of which factor may bridge Col and Nau functions (Dubois, 2007).

Unlike col or P2.6cl, P2.3cl is expressed in the DA3 FC and muscle precursor but not the DA3/DO5 progenitor, showing that col transcription in the progenitor and muscle precursor is under separate control. These two phases of col regulation are intimately linked, however, as Col is required for activating its own transcription in the nuclei of FCM recruited by the DA3 FC. This regulatory cascade may explain how pre-patterning of the somatic mesoderm and muscle identity are transcriptionally linked in the Drosophila embryo. As discussed above, the ability of Col to auto-regulate depends upon the presence of Nau, another muscle identity TF. Col and Nau act as obligatory co-factors fo maintenance/activation of Col expression in all nuclei of the DA3 muscle, thus bringing to light a clear case of combinatorial coding of muscle identity (Dubois, 2007).