bagpipe: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - bagpipe

Synonyms - NK3

Cytological map position - 93E1

Function - transcription factor

Keyword(s) - selector, visceral mesodermal

Symbol - bap

FlyBase ID:FBgn0004862

Genetic map position - 3- [72]

Classification - homeodomain - NK-2 class

Cellular location - nuclear

NCBI links: Entrez Gene

bap orthologs: Biolitmine
Recent literature
Seyres, D., Ghavi-Helm, Y., Junion, G., Taghli-Lamallem, O., Guichard, C., Roder, L., Girardot, C., Furlong, E. E. and Perrin, L. (2016). Identification and in silico modeling of enhancers reveals new features of the cardiac differentiation network. Development 143(23): 4533-4542. PubMed ID: 27899510
Developmental patterning and tissue formation are regulated through complex gene regulatory networks (GRNs) driven through the action of transcription factors (TFs) converging on enhancer elements. As a point of entry to dissect the poorly defined GRN underlying cardiomyocyte differentiation, an integrated approach was appled to identify active enhancers and TFs involved in Drosophila heart development. The Drosophila heart consists of 104 cardiomyocytes, representing less than 0.5% of all cells in the embryo. By modifying BiTS-ChIP for rare cells, H3K4me3 and H3K27ac chromatin landscapes were examined to identify active promoters and enhancers specifically in cardiomyocytes. These in vivo data were complemented by a machine learning approach and extensive in vivo validation in transgenic embryos, which identified many new heart enhancers and their associated TF motifs. These results implicate many new TFs in late stages of heart development, including Bagpipe, an Nkx3.2 ortholog, which is shown to be essential for differentiated heart function.

Two homeo box genes, tinman (tin) and bagpipe, spatially subdivide the mesoderm and determine cell fates in the dorsal mesoderm. These two genes are components of a cascade of genetic interactions that result in the spatial restriction of TIN mRNA to the dorsal mesoderm and the activation of bap in segmental clusters which segregate, under the control of bagpipe, to form visceral mesoderm that differentiates into gut musculature (Azpiazu, 1993).

In wild-type embryos of stage 12 [Image], the visceral mesoderm of the future midgut forms a continuous band of cells along the germ band where it comes into contact with the yolk sac. This splanchnopleura (gut musculature) has been separated from the somatopleura (somatic musculature) and expresses Fasciclin III. The visceral mesoderm of the midgut never forms in tin mutants. In bap mutants, the band of visceral mesoderm displays segmental interruptions, while the number of visceral mesoderm cells is reduced by about 70%. The cells do not form the normal columnar shape (Azpiaza, 1993).

The functional evolution of tinman and bagpipe is a classic example of regional specialization. tinman is clearly in charge, but delegates the resposibility for visceral musculature to bagpipe, while retaining control of heart muscles for itself. The downstream targets of bagpipe are not yet known, but are likely to include Fasciclin III, B3-tubulin, and connectin, all proteins characteristic of visceral muscle (Azpiazu, 1993).

Integration of positive Dpp signals, antagonistic Wg inputs and mesodermal competence factors and thir impact of Bagpipe expression during Drosophila visceral mesoderm induction

Tissue induction during embryonic development relies to a significant degree on the integration of combinatorial regulatory inputs at the enhancer level of target genes. During mesodermal tissue induction in Drosophila, various combinations of inductive signals and mesoderm-intrinsic transcription factors cooperate to induce the progenitors of different types of muscle and heart precursors at precisely defined positions within the mesoderm layer. Dpp signals are required in cooperation with the mesoderm-specific NK homeodomain transcription factor Tinman (Tin) to induce all dorsal mesodermal tissue derivatives, which include dorsal somatic muscles (the dorsal vessel and visceral muscles of the midgut). Wingless (Wg) signals modulate the responses to Dpp/Tin along anteroposterior positions by cooperating with Dpp/Tin during dorsal vessel and somatic muscle induction while antagonizing Dpp/Tin during visceral mesoderm induction. As a result, dorsal muscle and cardiac progenitors form in a pattern that is reciprocal to that of visceral muscle precursors along the anteroposterior axis. The present study addresses how positive Dpp signals and antagonistic Wg inputs are integrated at the enhancer level of bagpipe (bap), a NK homeobox gene that serves as an early regulator of visceral mesoderm development. An evolutionarily conserved bap enhancer element requires combinatorial binding sites for Tin and Dpp-activated Smad proteins for its activity. Adjacent binding sites for the FoxG transcription factors encoded by the Sloppy paired genes (slp1 and slp2), which are direct targets of the Wg signaling cascade, serve to block the synergistic activity of Tin and activated Smads during bap induction. In addition, binding sites for yet unknown repressors are essential to prevent the induction of the bap enhancer by Dpp in the dorsal ectoderm. These data illustrate how the same signal combinations can have opposite effects on different targets in the same cells during tissue induction (Lee, 2005).

Typically both the coding and regulatory sequences of developmentally important genes are conserved through evolution among related species. For example, the genes and enhancer elements of tin and Mef2 are highly conserved between Drosophila melanogaster (D. melanogaster) and Drosophila virilis (D. virilis), two related species that separated evolutionarily about 60 million years ago. Expression pattern of bap is also fully conserved between these two Drosophila species, suggesting that the corresponding regulatory elements of bap are also conserved. DNA sequence comparisons of these regulatory elements between the two species would be expected to facilitate the identification of important regulatory sites, as these should display the highest degrees of sequence conservation. Based upon this premise, flanking genomic sequences were isolated from both D. melanogaster and D. virilis, and tested for their ability to drive lacZ reporter gene expression in embryos. Constructs with upstream sequences of the bap gene, baplac4.5 in D. melanogaster and bapUS3.5-R in D. virilis, direct metameric lacZ expression within the visceral mesoderm from stage 11, after the segmented bap-expressing cell clusters have merged into continuous visceral mesoderm bands. In addition, the flanking genomic DNA fragments downstream of bap, bapDS3.5 in D. melanogaster and bapDS4.6-R in D. virilis, are able to activate lacZ expression strongly in the primordia of foregut and hindgut visceral mesoderm (FVM and HVM) from stage 11 until late embryogenesis, and very weakly in the trunk visceral mesoderm. The focus of this study is on the regulation of bap expression in the early trunk visceral mesoderm (TVM) precursors, which is driven by regulatory sequences within a 1.2 kb genomic DNA fragment, bapH2-1.2, downstream of the bap-coding region in D. melanogaster. This enhancer, as well as a truncated version of it, bap3 (460 bp), is active in 11 metameric domains on either side of the dorsal mesoderm at stage 10-11. A similarly active element, bapDS2.7-R, was also found downstream of bap in D. virilis. Hence, the activity of these elements recapitulates the early endogenous bap expression pattern of segmented domains in the dorsal mesoderm, i.e., in the presumptive trunk visceral mesoderm (TVM). Overall, the similar spatial and temporal activities of the different bap regulatory elements as well as their genomic arrangements with respect to the coding sequences in D. melanogaster and D. virilis illustrate the high degree of evolutionary conservation of bap regulatory elements and suggest that the regulatory mechanisms between the two species are also conserved (Lee, 2005).

To further dissect this early bap regulatory element, bap3, from D. melanogaster, several overlapping shorter constructs were made to examine their enhancer activities in embryos. A minimal 267 bp DNA fragment, termed bap3.2, was able to drive reporter expression similar to the endogenous bap expression pattern in the TVM precursors. DNA sequence alignments of the 267 bp bap3.2 element of D. melanogaster with the 2.7 kb bapDS2.7-R element of D. virilis, as well as with corresponding genomic DNA sequences from D. yakuba, D. pseudoobscura and D. ananassae, revealed high levels of similarities within a stretch of ~150 to 200 bp of genomic sequences in all five species. In particular, the DNA sequences from D. melanogaster and D. pseudoobscura share ~90% identity within a DNA stretch from nucleotide 85 to 230 of the D. melanogaster bap3.2 element. Consistent with this strong sequence conservation, a 540 bp genomic DNA fragment within the bapDS2.7-R element, bapV1 of D. virilis, which contains the highly conserved 150 bp sequences in its center, was capable of driving lacZ expression in the dorsal mesoderm similar to bap3.2-lacZ from D. melanogaster (Lee, 2005).

A shorter regulatory DNA fragment, bap3.2.1, was derived from the bap3.2 regulatory element by removing the first 57 bp from the 5'-end and the last 30 bp from the 3'-end of bap3.2. bap3.2.1 drives expression in the same segmented pattern as bap3.2; interestingly, however, this expression occurs not only in the dorsal mesoderm but also in the dorsal ectoderm. The lacZ expression patterns of bap3.2.1-lacZ in dorsal ectoderm and mesoderm can largely be superimposed onto one another. The only major difference between the two germ layers is observed in parasegments 13 and 14, where bap3.2.1-lacZ produces two additional expression clusters in the ectoderm that is neither seen with any of the reporter constructs nor with endogenous bap. Likewise, a 165 bp genomic DNA fragment, bapV2 from D. virilis, which corresponds to the bap3.2.1 element of D. melanogaster, also displays enhancer activity in both dorsal mesoderm and dorsal ectoderm with this pattern. The presence of ectopic lacZ expression in the ectoderm with bap3.2 derivatives was further confirmed in embryo cross-sections. Whereas bap3-lacZ embryos show no detectable lacZ expression in the ectoderm, there are traces of ectodermal lacZ expression in bap3.2-lacZ embryos and strong dorsal ectodermal lacZ expression in bap3.2.1-lacZ embryos (Lee, 2005).

Altogether, these observations imply that the first 57 bp and the last 30 bp of the bap3.2 regulatory element have a key role in the repression of bap enhancer induction in the dorsal ectoderm and show that the mechanism of repression of ectodermal bap induction is evolutionarily conserved. The similar patterns of enhancer activity in both germ layers upon deletion of these repressor sequences support the notion, based on genetic data, that the major spatial inputs regulating bap expression in the mesoderm are also active in the ectoderm. Indeed, one of these candidate inputs from the ectoderm, namely Dpp, leads to the activation of Mad in a dorsal domain in the mesoderm (and ectoderm): the ventral mesodermal border of Mad coincides with the ventral border of bap induction (Lee, 2005).

To investigate whether the bap regulators identified genetically, including tin, dpp, slp (downstream of wg) and bin, can act directly on the early TVM regulatory element of bap, DNaseI protection experiments with recombinant Tin, Bap, Smad (Mad and Medea), Slp and Bin proteins were performed on the 180 bp bap3.2.1 DNA sequence from D. melanogaster. The DNA footprinting results demonstrate that both Tin and Bap proteins can bind to the predicted Tin-binding site, which includes a perfect match to the canonical Tin-binding motif TCAAGTG. In addition to the Tin-binding site, a site with a TAAG core motif can strongly bind Bap but not Tin (CTTA in opposite strand; note that the same core motif is found in binding sites of a Bap ortholog, Nkx3.2). With regard to Dpp signaling mediators, there are five Mad-protected regions, three of which are also protected by recombinant Medea (Mad/Medea-1 to -3). Site 1 includes an AGAC motif that was initially identified as a Smad binding motif in vertebrates whereas sites 3-5 contain GC-rich sequences with CGGC motifs that were first shown to bind Smad proteins in Drosophila. Site 2 may be a combination of the two types (TGAC motif and CG-rich sequences). No clear correlation of either type of site was observed with the binding of Mad versus Medea. Finally, recombinant Slp proteins protect a wide stretch that includes an inverted repeat of core binding motifs for forkhead transcription factors (TAAACA), but extends further downstream. Slp can bind to tandem repeats of CAAA sequences, which are present in three copies in the 3' region of the protected region. Gel mobility shift and competition assays with Slp using wild-type oligonucleotides and a version in which the TAAACA motifs were mutated indicate that Slp can bind to both the TAAACA and the CAAA motifs with roughly equal affinity. In addition, the FoxF family protein Bin binds to the TAAACA inverted repeat region, but less well to the CAAA repeat region when compared with Slp. Taken altogether, these binding data are consistent with the hypothesis that the known mesodermal regulators of bap, namely Tin and Bin (and possibly autoregulatory Bap), as well as the signaling inputs from Dpp and Wg (through Smads and Slp, respectively) are integrated via direct binding to the early TVM enhancer of bap (Lee, 2005).

The present study describes an example of an enhancer whose response to Dpp is suppressed by Wg signals. A comparison of the functional organization of these enhancers provides new insight into molecular strategies of nuclear signal integration to produce differential developmental responses. The data show that bap is a direct target of Dpp signals. Thus, an indirect pathway of bap being activated solely by tin, whose mRNA expression is known to depend on Dpp inputs during the time of bap activation, can be ruled out. Rather, tin acts simultaneously and synergistically with Dpp. In fact, recent data with tin alleles lacking the Dpp-responsive enhancer show that bap can be induced in the absence of Dpp-induced tin products, as long as the twist-activated tin products are present. The molecular basis for this observed synergism of tin and dpp relies on the combinatorial binding of Tin and Dpp-activated Smad proteins to the bap enhancer. Several possible molecular mechanisms could underlie the strict requirement for combinatorial binding of Tin and Smads. For example, the relatively low binding affinity and specificity of Smads might be enhanced by bound Tin, which can engage in protein interactions with Mad and Medea. The combined presence of Tin and Smads in close vicinity or in complexes may also be a prerequisite for the assembly of higher order complexes with transcriptional co-activators such as CBP/p300. In addition, Tin may counteract the function of yet unknown repressors of nuclear Dpp signaling activity so that they can only repress in the ectoderm (Lee, 2005).

Unlike Dpp, Wg signals act indirectly upon the early bap enhancer. Previous genetic and molecular data have shown that Wg induces the expression of the forkhead domain-encoding gene slp via crucial dTCF/Lef-1 binding sites in both mesoderm and ectoderm. slp, in turn, functions as a repressor of bap. The present data show that slp products exert this function by direct binding to the Dpp-responsive bap enhancer, which obviously results in a suppression of the synergistic activity of bound Tin and Smad complexes. Slp proteins contain eh1 motifs that can potentially bind the Groucho co-repressor and Slp has known repressor activities in other contexts. In addition, the vertebrate counterpart of Slp, FoxG (BF-1), is known to interact with Groucho and histone deacetylases (Yao, 2001). Thus, it is proposed that Slp overrides nuclear Dpp signaling activities by dominantly establishing an inactive state of the chromatin at the bap locus (Lee, 2005).

It is likely that additional components are involved in the antagonistic interaction of Slp with Tin/Smad complexes. The Slp-binding site includes sequences that are also required positively for the mesodermal response to Dpp, although not for ectopic responses in the ectoderm. In a genome-wide expression analysis, any forkhead domain genes other than bin were found that are mesoderm specific. However, the function of an essential co-activator in the mesoderm interacting with this site could be fulfilled by a ubiquitously expressed forkhead domain protein, and in part by Bin, which is required for the prolongation of the Dpp response. In the yeast one-hybrid screens with this site that yielded Slp clones, a clone of fd68A, a uniformly expressed ortholog of vertebrate FoxK1 (Myocyte Nuclear Factor) was isolated, but genetic confirmation of its involvement in bap induction is currently lacking. Regardless of the identity of this factor, Slp could either compete with this protein and with Bin for DNA binding, or it could disrupt their productive functional interactions with the Tin/Smad complexes. Interestingly, the latter type of mechanism has been proposed to operate during the interference of the slp ortholog BF-1 with TGFß signaling in the vertebrate cerebral cortex (Lee, 2005).

Inductive responses that are germ layer- or cell type-specific and exclude the signal-producing cells are a recurring theme in developmental systems. Although this type of target specificity can involve different levels of the signaling cascade, including the tissue-specific expression of receptors or signaling effectors, this study has shown that germ layer-specific induction of bap is controlled by nuclear events. This is crucial because activated Smads are present in dorsal nuclei of both germ layers. Two mechanisms that are probably functionally intertwined have been described that ensure mesoderm-specificity of the response to Dpp. The first is the requirement for Tin to synergize with activated Smads. Tin is present exclusively in the mesoderm and is therefore not available to fulfill such a function in the ectoderm. Hence, in developmental terms, Tin provides the mesoderm with the unique competence to respond to Dpp and induce bap. Perhaps surprisingly then, there is an additional component involved, which actively prevents induction of the bap enhancer by Dpp in the ectoderm. The Dpp-responsive core enhancer of bap is flanked by sequences that appear to function as binding sites for as yet unidentified repressor(s), which keep the enhancer silent in the ectoderm. A very similar situation has been described for the Dpp-responsive enhancer of tin and, as shown in this study by sequence comparisons as well as functional swapping of the putative ectodermal repressing sequences from the tin and bap enhancers, they appear to bind the same repressor(s). Brinker, a known nuclear repressor of Dpp signaling, can be excluded as a candidate because of its different sequence preference and absent expression in the dorsal ectoderm. The situation is reminiscent of an endodermal labial enhancer, in which a homeotic response element and a repressor element interact to control the spatially restricted activity of a minimal Dpp response element (Lee, 2005).

Why would induction of tin and bap in the mesoderm require Tin as a co-factor of Smads, whereas in the ectoderm, which lacks Tin, the induction of tin and bap needs to be actively repressed? In the case of the tin enhancer, the ectodermal repressor elements are overlapping with the Tin-binding sites. Based upon this situation, a model is proposed in which the repressor would be present in both germ layers, but in cells of the mesoderm it is competed away from binding to the enhancer by Tin. This model is compatible with data showing that ectopic expression of Tin in the ectoderm is able to activate the Dpp-responsive enhancer of tin, even in the presence of the putative repressor binding elements. However unlike full-length Tin, an N-terminally truncated version with an intact homeodomain is not able to allow induction of the tin enhancer in the ectoderm. Furthermore, the putative repressor binding sites in the bap enhancer are separate from the Tin site. Hence, Tin does not compete for binding but may rather block or override the repressor factor(s) functionally. Thus, the positive activity of Tin would dominate over the negative action of this repressor in the mesoderm. By contrast, the repressing activity of Slp dominates over the positive action of Tin. Through this intricate balance of positive and negative switches, Tin could ensure that bap is induced by Dpp only in the mesoderm, while bound Slp prevents Tin from promoting Dpp inputs towards bap in striped domains within this germ layer. However, it can still not be fully explained why the absence of both the functional Tin and ectodermal repressor sites allows enhancer induction in the ectoderm, while preventing it in the mesoderm. The additional positive and negative binding factors involved will need to be identified to gain a full understanding of the germ layer-specific induction of these Dpp-responsive enhancers (Lee, 2005).

The bap enhancer described in this study represents the third example of well-characterized Dpp-responsive enhancers from mesodermal control genes. The other two are from tin, which is induced in the entire dorsal mesoderm, and eve, which is active in a small number of somatic muscle founder cells and pericardial progenitors in the dorsal mesoderm. The activities of the bap and eve enhancers along the anteroposterior axis are reciprocal, which is due to the fact that the eve enhancer requires inputs from Wg, whereas bap enhancer activity is suppressed by Wg. A comparison of the molecular architecture of these three enhancers reveals that they all share a number of important features. Most notably, all three enhancers feature several Tin- and Smad-binding sites in close vicinity that are essential for the activation of the enhancer in the mesoderm. Each enhancer includes both types of known Smad-binding motifs, which have 'AGAC' and 'CG'-rich cores, respectively. Hence, the basic activation mechanisms of each of the three enhancers downstream of Dpp are likely to be closely related. In the enhancers of both tin and bap, binding sites for a nuclear repressor of Dpp signals are key for the germ layer specificity of the inductive response. Although it is not known whether the same repressive mechanism operates at the eve enhancer, it is noted that motifs related to the presumed repressor binding motifs are present and their function can now be tested in vivo. As in the case of bap, the tin enhancer includes also additional sites that are required for Dpp-inducible enhancer activity, which may bind essential Smad co-factors. However, based upon the divergent sequences of these sites (C1 site in the bap and 'CAATGT' motifs in the tin enhancer), they appear to bind different types of factors in each case (Lee, 2005).

On top of this basic arrangement that allows the enhancer to be active in the dorsal mesoderm, the enhancers from bap and eve, but not tin, include binding sites that make them respond to Wg inputs in an opposite fashion. In the case of bap, Wg-induced Slp binds and dominantly suppresses the activity of bound Smad effectors. For the eve enhancer it has been proposed that there is an analogous repressive activity; however, in this case, it is exerted by bound Wg signal effectors, i.e., dTCF/Lef-1, in the absence of Wg signals. In the domains with active Wg signaling, the repressive activity of dTCF/Lef-1 is neutralized by the Wg signaling cascade, which allows the Dpp effectors to be active at the eve enhancer (since it lacks Slp binding sites). Through these switches, the bap and eve enhancers become induced in reciprocal AP patterns. In addition, the eve enhancer includes binding sites for activators and repressors downstream of receptor tyrosine kinases and Notch, respectively, which serve to restrict eve activity to specific subsets of cells within the domains of overlapping Dpp and Wg activities. Clearly, many of the molecular details still need to be clarified. Nevertheless, the basic principles of how differential inputs from inductive signals and tissue-specific activities can be integrated at the enhancer level to achieve distinct patterns of target gene expression during early tissue induction in the Drosophila mesoderm are now beginning to be understood (Lee, 2005).

A transcription factor collective defines cardiac cell fate and reflects lineage history

Cell fate decisions are driven through the integration of inductive signals and tissue-specific transcription factors (TFs), although the details on how this information converges in cis remain unclear. This study demonstrates that the five genetic components essential for cardiac specification in Drosophila, including the effectors of Wg and Dpp signaling, act as a collective unit to cooperatively regulate heart enhancer activity, both in vivo and in vitro. Their combinatorial binding does not require any specific motif orientation or spacing, suggesting an alternative mode of enhancer function whereby cooperative activity occurs with extensive motif flexibility. A fraction of enhancers co-occupied by cardiogenic TFs had unexpected activity in the neighboring visceral mesoderm but could be rendered active in heart through single-site mutations. Given that cardiac and visceral cells are both derived from the dorsal mesoderm, this 'dormant' TF binding signature may represent a molecular footprint of these cells' developmental lineage (Junion, 2012).

Dissecting transcriptional networks in the context of embryonic development is inherently difficult due to the multicellularity of the system and the fact that most essential developmental regulators have pleiotropic effects, acting in separate and sometimes interconnected networks. This study presents a comprehensive systematic dissection of the cis-regulatory properties leading to cardiac specification within the context of a developing embryo. The resulting compendium of TF binding signatures, in addition to extensive in vivo and in vitro analysis of enhancer activity, revealed a number of insights into the regulatory complexity of developmental programs (Junion, 2012).

Nkx (Tinman in Drosophila), GATA (Pannier in Drosophila), and T box factors (Doc in Drosophila) regulate each other's expression in both flies and mice, where they form a recursively wired transcriptional circuit that acts cooperatively at a genetic level to regulate heart development across a broad range of organisms. The data demonstrate that this cooperative regulation extends beyond the ability of these TFs to regulate each other's expression. All five cardiogenic TFs (including dTCF and pMad) converge as a collective unit on a very extensive set of mesodermal enhancer elements in vivo (Tin-bound regions) and also in vitro (in DmD8 cells). Importantly, this TF co-occupancy occurs in cis, rather than being mediated via crosslinking of DNA-looping interactions bringing together distant sites. Examining enhancer activity out of context, for example, in transgenic experiments and luciferase assays, revealed that the TF collective activity is preserved in situations in which these regions are removed from their native genomic 'looping' context (Junion, 2012).

In keeping with the conserved essential role of these factors for heart development, the integration of their activity at shared enhancer elements may also be conserved. Recent analyses of the mouse homologs of these TFs (with the exception of the inductive signals from Wg and Dpp signaling) in a cardiomyocyte cell line support this, revealing a signifcant overlap in their binding signatures (He, 2011; Schlesinger, 2011), although interestingly not in the collective 'all-or-none' fashion observed in Drosophila embryos. This difference may result from the partial overlap of the TFs examined, interspecies differences, or the inherent differences between the in vivo versus in vitro models. Examining enhancer output for a large number of regions indicates that this collective TF occupancy signature is generally predictive of enhancer activity in cardiac mesoderm or its neighboring cell population, the visceral mesoderm expression patterns that cannot be obtained from any one of these TFs alone (Junion, 2012).

There are currently two prevailing models of how enhancers function. The enhanceosome model suggests that TFs bind to enhancers in a cooperative manner directed by a specific arrangement of motifs, often having a very rigid motif grammar. An alternative, the billboard model, suggests that each TF (or submodule) is recruited independently via its own sequence motif, and therefore the motif spacing and relative orientation have little importance. The results of this study indicate that cardiogenic TFs are corecruited and activate enhancers in a cooperative manner, but this cooperativity occurs with little or no apparent motif grammar to such an extent that the motifs for some factors do not always need to be present. This is at odds with either the enhanceosome (cooperative binding; rigid grammar) or billboard (independent binding; little grammar) models and represents an alternative mode of enhancer activity, which was termed a 'TF collective' (cooperative binding; no grammar), and likely constitutes a common principle in other systems (Junion, 2012).

The data suggest that the TF collective operates via the cooperative recruitment of a large number of TFs (in this case, at least five), which is mediated by the presence of high-affinity TF motifs for a subset of factors initiating the recruitment of all TFs. The occupancy of any remaining factor(s) is most likely facilitated via protein-protein interactions or cooperativity at a higher level such as, for example, via the chromatin activators CBP/ p300, which interact with mammalian GATA and Mad homologs. This model allows for extensive motif turnover without any obvious effect on enhancer activity, consistent with what has been observed in vivo for the Drosophila spa enhancer and mouse heart enhancers (Junion, 2012).

Integrating the TF occupancy data for all seven major TFs involved in dorsal mesoderm specification (the five cardiogenic factors together with Biniou and Slp) revealed a very striking observation: the developmental history of cardiac cells is reflected in their TF occupancy patterns. Visceral mesoderm (VM) and cardiac mesoderm (CM) are both derived from precursor cells within the dorsal mesoderm. Once specified, these cell types express divergent sets of TFs: Slp, activated dTCF, Doc, and Pnr function in cardiac cells, whereas Biniou and Bagpipe are active in the VM. Despite these mutually exclusive expression patterns, the cardiogenic TFs are recruited to the same enhancers as VM TFs in the juxtaposed cardiac mesoderm. Moreover, dependent on the removal of a transcriptional repressor, these combined binding signatures have the capacity to drive expression in either cell type. This finding provides the exciting possibility that dormant TF occupancy could be used to trace the developmental origins of a cell lineage. It also explains why active repression in cis is required for correct lineage specification, which is a frequent observation from genetic studies. At the molecular level, it remains an open question why the VM-specific enhancers are occupied by the cardiac TF collective. It is hypothesized that this may occur through chromatin remodeling in the precursor cell population. An 'open' (accessible) chromatin state at these loci in dorsal mesoderm cells, which is most likely mediated or maintained by Tin binding prior to specification, could facilitate the occupancy of cell type-specific TFs in both CM and VM cells. Such early 'chromatin priming' of regulatory regions active at later stages has been observed during ES cell differentiation. The current data provide evidence that this also holds true for TF occupancy and not just chromatin marks. On a more speculative level, this developmental footprint of TF occupancy may reflect the evolutionary ancestry of these two organs. Visceral and cardiogenic tissues are derived from the splanchnic mesoderm in both flies and vertebrates. These complex VM-heart enhancers may represent evolutionary relics containing functional binding sites that reflect enhancer activity in an ancestral cell type (Junion, 2012).

Taken together, the collective TF occupancy on enhancers during dorsal mesoderm specification illustrates how the regulatory input of cooperative TFs is integrated in cis, in the absence of any strict motif grammar. This more flexible mode of cooperative cis regulation is expected to be present in many other complex developmental systems (Junion, 2012).


Both the Drosophila lady-bird-late gene, previously named nk4, and lady-bird-early reside directly upstream of labial. Together with tinman/NK4, bagpipe/NK3, S59/NK1, and 93Bal they compose the 93D/E homeobox gene cluster (Jagla, 1994). tinman and bagpipe are separated by 6500 bp (Azpiazu, 1993).

cDNA clone length - 1446

Bases in 5' UTR - 132

Bases in 3' UTR - 168


Amino Acids - 382

Structural Domains

The homeodomain is in the middle portion of the BAP polypeptide and is 59% identical to the TIN homeodomain. The protein contains serine/threonine rich regions and several regions rich in acidic and basic amino acids (Azpiaza, 1993).

bagpipe: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 10 May 2005

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