bagpipe-expressing domains are defined by the intersecting dorsal activities of dpp/tin, which act positively, and segmentally modulated activities of wg/slp, which have repressing effects. bin also requires tin activity for normal expression in the trunk visceral mesoderm primordia. Whereas bap expression is virtually absent in these cells upon loss of tin activity, residual bin expression is observed in small clusters of cells. To test the possibility that residual expression of bin in tin mutant embryos is due to direct inputs from Dpp, bin expression was examined in embryos in which dpp expression was induced ectopically in the entire mesoderm. Ectopic dpp in a wild-type background, which causes tin expression to be expanded ventrally, results in an analogous expansion of the bin domains. Notably, ventral expansion of the bin domains is also observed upon ectopic dpp expression in the absence of tin activity, although the domains are narrow. Thus, Dpp is able to induce bin in the absence of tin, although tin activity is required for normal expression levels. The residual expression of bin in tin mutant embryos is unstable and not maintained in later stages of development (Zaffran, 2001).
Similar to tin, bap activity is also required for normal bin expression. This result is in agreement with the temporal sequence of bap and bin expression and with the observed expansion of bin throughout most of the dorsal mesoderm upon ectopic bap expression in the mesoderm. These data suggest that bin is furthest downstream within a mesoderm-intrinsic cascade of gene activation: twist -> tin -> bap -> bin. Moreover, bin itself is required for normal bin expression. Although bin expression initiates normally in stage 10 bin mutant embryos, it disappears at early stage 11 in the trunk visceral mesoderm primordia of bin mutants, except for those in PS1 and 2. bin expression in these two parasegments is also less sensitive to the loss of tin and bap activity. Furthermore, the expression of bin in foregut, hindgut, and caudal visceral mesoderm does not depend on any of the genes examined in the present study (Zaffran, 2001).
Whereas the above data show that maintenance of bin expression in most of the presumptive trunk visceral mesoderm requires positive autoregulation, they do not establish whether this autoregulatory loop is direct or indirect. Of note, maintenance of bap during stage 11 (but not its initiation during stage 10) also requires bin activity. Therefore, it is possible that, at least during stage 11, bin and bap maintain each other's expression through a cross-regulatory feedback loop (Zaffran, 2001).
Besides tissue-specific differentiation genes that are expressed throughout the trunk visceral mesoderm, several key regulators of midgut morphogenesis are known to be expressed in a spatially restricted manner within this tissue. This type of gene product includes the homeotic factor Ubx and the secreted factor Dpp, both of which are expressed in PS7 of the visceral mesoderm. Although it has been established that Ubx and Dpp maintain their expression in PS7 through a crossregulatory loop and the action of Wg from the adjacent PS8, there is evidence that their expression requires at least one additional, visceral mesoderm-specific cofactor, for which Bin may be a candidate. To test this possibility, Ubx and dpp expression were examined in bin mutant embryos, which carried bap3-lacZ, to allow the unambiguous identification of the disrupted visceral mesoderm layer. Visceral mesoderm expression of Ubx in bin mutant embryos is similar to that of wild-type embryos until at least stage 13, although there is a low level of ectopic expression. Likewise, Ubx expression is also observed in ß-gal-positive cells in bap mutant embryos, albeit with reduced levels and an expanded domain: These conditions are comparable to those in the somatic mesoderm. These data demonstrate that the establishment of Ubx expression in the visceral mesoderm requires neither bin nor bap activity. In contrast, dpp is not expressed at any stage in PS7 in the visceral mesoderm of bin mutant embryos, indicating that Bin may serve as a critical tissue-specific cofactor for the regulation of dpp expression. The expression of wg in PS8 is also absolutely dependent on bin activity. The absence of these morphogenetic factors is likely to contribute to the defective midgut morphology in bin mutant embryos (Zaffran, 2001).
The identification of visceral mesoderm-specific enhancer elements of dpp allowed a test of the possibility that bin might be a direct upstream regulator of dpp in the visceral mesoderm. Attention was focused on two minimal enhancer elements: the 130 bp element BM and the 231 bp element. PB is able to drive reporter gene expression in PS3 and PS7 of the visceral mesoderm in a pattern that is similar to that of endogenous dpp, although PB-lacZ expression in PS7 is less robust. In contrast to PB, BM is active in a broad region extending from PS7 to PS12 in the visceral mesoderm. In addition, the combination of BM and PB results in a significant enhancement of PS7 expression compared to PB alone. Because of the broad activity of BM in the visceral mesoderm and its enhancing effect on PB (or longer versions thereof), BM has been proposed to act as a general visceral mesoderm enhancer (GVME), whereas PB is predominantly targeted by spatially restricted activities that include Ubx and Exd (Zaffran, 2001).
DNaseI protection assays were performed with recombinant Bin protein to test for the presence of Bin binding sites within BM and PB. These experiments identified two protected regions within BM, termed Bin I and Bin II, which are about 50 bp apart from one another. PB contains a third strongly protected sequence, Bin III, and two minor binding sites which overlap with the Exd binding sites e1 and e2. All three of the strongly protected sequences and the weaker e1 contain sequence motifs that perfectly match forkhead domain binding sites, including the optimal binding site of a vertebrate ortholog, HFH-8. The presence of overlapping inverted and direct repeats of this sequence motif in Bin II and Bin III, respectively, may indicate that these two sites represent dimeric binding sites. Interestingly, the sequences of the three strong and two weak Bin binding sites within PB are highly conserved between D. melanogaster and D. virilis, suggesting that they are functionally important (Zaffran, 2001).
To test whether any of the strong Bin binding sites are required for enhancer activity in vivo, nucleotide exchanges that completely abolished in vitro binding of Bin were introduced. Mutation of Bin III results in an almost complete loss of PB enhancer activity in PS7, suggesting that Bin binding to Bin III plays an important role for the activation of dpp in this parasegment. The presence of two weak Bin binding sites in the mutated PB derivative may allow residual expression in a few visceral mesoderm cells within PS7. The fact that PS3 expression is not affected significantly upon Bin III mutation may be due to the activity of Exd binding sites, of which one was previously shown to regulate PS3 expression (Zaffran, 2001).
BM enhancer activity in the visceral mesoderm is completely lost when both Bin I and Bin II are mutated. When this mutated version of BM is combined with a wild-type version of PB, there is no enhancement of PS7 expression and the same pattern observed as that with PB alone. Finally, the combination of BM and PB with mutated Bin I, II, and III binding sites does not exhibit any significant enhancer activity in PS7. These data suggest that both BM and PB contain functionally important Bin binding sites. Bin binding to Bin I and Bin II may be key to providing BM with its general visceral mesoderm enhancer activity, whereas binding to Bin III is required in concert with spatially restricted activities to provide the PB enhancer with a basal level of activity in PS7 (Zaffran, 2001).
The NK homeobox gene bagpipe and the FoxF fork head domain gene biniou have been identified as essential regulators of visceral mesoderm development in Drosophila. Additional genetic and molecular information is presented on the functions of these two genes during visceral mesoderm morphogenesis and differentiation. Both genes are required for the activation of ß3Tub60D in the visceral mesoderm. A 254 bp derivative of a previously defined visceral mesoderm-specific enhancer element, vm1, from ß3Tub60D contains one specific in vitro binding site for Bagpipe and two such sites for Biniou. While the wild-type version of the 254 bp enhancer is able to drive significant levels of reporter gene expression within the entire trunk visceral mesoderm, mutation of either the Bagpipe or the Biniou binding sites within this element results in a severe decrease of enhancer activity. Moreover, mutation of all three binding sites for Bagpipe and Biniou, respectively, results in the complete loss of enhancer activity. Together, these observations suggest that Bagpipe and Biniou serve as direct, partially redundant, and tissue-specific activators of the terminal differentiation gene ß3Tub60D in the visceral mesoderm (Zaffran, 2002).
To test whether the expression of ßTub60D in the trunk visceral mesoderm depends on the activity of two known visceral mesoderm regulators, bap and bin, ßTub60D protein expression was examined in embryos that were mutant for the respective gene. In addition, the embryos carried a bap-lacZ transgene as an independent marker for the early visceral mesoderm which; in a wild-type background bap-lacZ is co-expressed with ß3 tubulin. In embryos lacking bap, ß3 tubulin expression is severely reduced in the visceral mesoderm and at early stage 12 only trace amounts remain detectable in this cell layer. Likewise, loss of bin activity also results in an almost complete loss of ß3 tubulin expression in the visceral mesoderm layer. These data show that the activities of both bap and bin are required for normal ß3-tubulin expression in the trunk visceral mesoderm (Zaffran, 2002).
A visceral mesoderm-specific enhancer element from the ßTub60D gene, vm1, has been described that is contained in the reporter construct pWHß3-14 and consists of 515 bp of enhancer sequences from the first intron of this gene (+3154 to +3669). While two Ubx binding sites within this enhancer are involved in increasing enhancer activity within parasegments (PS) 6 and 7, bap and/or bin may act as direct regulator(s) of the broad basal activity of this enhancer in the entire trunk visceral mesoderm. To test this possibility a derivative of pWHß3-14, in which the Ubx sites were deleted (pWHß3-14/DeltaUbx1+2), was crossed into bap and bin mutant backgrounds. While in the wild-type background this enhancer derivative is driving significant (though anteroposteriorly graded) levels of ßgal expression in a continuous row of visceral mesoderm cells, in a bap null mutant background enhancer activity is completely lost in this tissue. Likewise, a strong reduction of enhancer activity driven by pWHß3-14/DeltaUbx1+2 is also observed in a bin null mutant background, although in this case some residual visceral mesoderm cells are still expressing low levels of the reporter gene (Zaffran, 2002).
In order to clarify whether these genetic interactions reflect any direct interactions of the bap or bin products with vm1 enhancer sequences in vitro DNA-binding experiments were performed with the two proteins. DNaseI protection assays with bacterially expressed Bin fusion proteins revealed two strongly protected sequences, termed BIN-I and BIN-II, within vm1. Closer inspection of these sequences showed that BIN-I contains overlapping tandem copies and BIN-II a single copy of a canonical binding motif for fork head domain proteins. The specificities of these in vitro binding activities are further corroborated by the results from gel mobility shift experiments. In particular, these data show that both BIN-I and BIN-II oligonucleotides can compete for binding of Bin to vm1, whereas analogous oligonucleotides in which the canonical fork head domain binding sequence was mutated fail to compete (Zaffran, 2002).
Bap fusion proteins also produce a strongly protected region in DNaseI footprinting experiments. The protected sequence contains an overlapping tandem repeat of a canonical NK-homeodomain binding motif, which has been shown to bind Tinman. Indeed, the footprints produced with Bap and Tin on this sequence are almost indistinguishable (Zaffran, 2002).
In preparation for functional tests of the Bin and Bap binding sites in vivo, a shorter version of vm1, termed ß3-17, was generated that lacks 5' and 3' sequences that have been shown to be dispensable for driving basal levels of visceral mesoderm expression (+3252 to +3506). As predicted, ß3-17-driven ßgal expression occurs in a uniform pattern and at intermediate levels within the visceral mesoderm. Next the effects of mutant Bin and Bap binding sites on the in vivo activity of the ß3-17 enhancer element were tested. Mutation of either BIN-I (ß3-17 bin-Imt) or BIN-II ß3-17 (bin-IImt) results in a strong decrease of ß3-17 enhancer activity. Although it was expected that the activities of BIN-I and BIN-II may be partially redundant, simultaneous mutation of both binding sites did not result in a significant reduction of enhancer activity beyond the levels seen with mutations in either binding site alone, particularly BIN-II, (ß3-17 bin-I+IImt) (Zaffran, 2002).
Mutation of the Bap binding site also results in a strong reduction but not a complete loss of enhancer activity within the visceral mesoderm. To determine whether the residual enhancer activity of the mutated elements is due to functional redundancy between the Bin or Bap binding sites the effects of mutations in all three binding sites were tested. Simultaneous disruption of all Bin and Bap binding sites within the ß3-17 enhancer element results in the complete loss of enhancer activity, thus confirming that Bin and Bap have partially redundant roles in activating the vm1 enhancer of ßTub60D (Zaffran, 2002).
The residual enhancer activity upon mutation of Bin or Bap binding sites is largely observed in the middle portion of the visceral mesoderm, suggesting the influence of spatially restricted regulator(s). Indeed, the close spatial correlation between residual enhancer activity and Dpp-signaling activity as well as the presence of putative Smad binding sites within vm1(+3265: GGGCCG; +3289: CAGAC; +3431: CAGACGGCAGAC) suggests a role for direct inputs from Dpp in the regulation of vm1 enhancer activity. Thus, Smad complexes and Bap bound to vm1 sequences may act in a synergistic fashion, a situation that may be analogous to the synergistic activity of Smad and Tin during the induction of the Dpp-responsive enhancer of the tin gene. However, the fact that this effect is only observed with a weakened version of the enhancer indicates that the Dpp-input plays a minor role during the normal activation of the ßTub60D gene in the visceral mesoderm. Additional inputs, which may also be insignificant for ßTub60D regulation in the normal situation, could come from Wg and/or Hh and result in low levels of metameric expression with weakened enhancer constructs (Zaffran, 2002).
Activation of the vm1 enhancer during stage 11 is restricted to the ventral row of visceral mesodermal cells, but is missing in the remaining cells of this tissue that also express Bap and Bin. Hence, the combination of Bap and Bin is required, but not sufficient for activating ßTub60D expression through vm1. Previous observations have shown that the region defined by deletion 3 (e3, +3439 to +3471), which neither contains Bap nor Bin binding sites, is also required for normal enhancer activity. Therefore, this sequence may be a target of an as yet unknown activity within the ventral row of visceral mesodermal cells that is required in combination with Bap and Bin to trigger vm1 activation. Recent reports have shown that these ventral cells are the equivalent of founder cells in the visceral mesoderm; these cells subsequently fuse with adjacent dorsal cells into binucleate syncytia. Similar to the expression of dpp in PS 7 of the visceral mesoderm, vm1-lacZ expression spreads throughout the visceral mesoderm only upon fusion of founders with fusion-competent cells (Zaffran, 2002).
Combined with previous data, the current results define a continuous regulatory cascade of gene activation that initiates with the regulation of genes which pattern the early mesoderm; this process concludes with the activation of a terminal differentiation gene in the visceral mesoderm. Specifically, this pathway involves the activation of tin by twist, followed by the induction of dorsal mesodermal tin by dpp, then activation of bap by tin and dpp, activation of bin by bap and dpp, and finally activation of ßTub60D by the combined action of bap and bin. A second gene that is activated at the end of this cascade in the visceral mesoderm with a similar temporal, albeit more restricted spatial pattern as compared to ßTub60D, is dpp. In the case of dpp, a visceral mesoderm-specific enhancer requires only Bin, but not Bap, as a direct activator. Hence, genes controlling morphogenesis or differentiation of the visceral mesoderm differ in their requirement for either one or both of the ubiquitously distributed visceral mesoderm activators, Bap and Bin, as direct regulators. These differences may depend on the particular involvement of additional regulators, which in the case of dpp includes spatially-restricted activities such as Ubx, that may obviate a requirement for Bap in addition to Bin as a direct activator (Zaffran, 2002).
The visceral trunk mesoderm in Drosophila develops under inductive signals from the ectoderm. This leads to the activation of the key regulators Tinman, Bagpipe and Biniou that are crucial for specification of the circular visceral muscles. How further differentiation is regulated is widely unknown, therefore it seems to be essential to identify downstream target genes of the early key regulators. This study focuses on the analysis of the transcriptional control of the highly conserved transcription factor Hand in circular visceral muscle cells, providing evidence that the hand gene is a direct target of Biniou. A regulatory region has been identified in the hand gene that is essential and sufficient for the expression in the visceral mesoderm during embryogenesis. hand expression in the circular visceral mesoderm is abolished in embryos mutant for the FoxF domain containing transcription factor Biniou. Furthermore it is demonstrated that Biniou regulates hand expression by direct binding to a 300 bp sequence element, located within the 3rd intron of the hand gene, and marked by the presence of four putative motifs with homology to the HFH-8 consensus binding site A/G C/T A A A C/T A, recognized by Biniou. This regulatory element is highly conserved in different Drosophila species. In addition, evidence is provided that Hand is dispensable for the initial differentiation of the embryonic visceral mesoderm. This study shows that cross species sequence comparison of non-coding sequences between orthologous genes is a powerful tool to identify conserved regulatory elements. Combining functional dissection experiments in vivo and protein/DNA binding studies hand was identified as a direct target of Biniou in the circular visceral muscles (Popichenko, 2007; full text of article).
Smooth muscle plays a prominent role in many fundamental processes and diseases, yet understanding of the transcriptional network regulating its development is very limited. The FoxF transcription factors are essential for visceral smooth muscle development in diverse species, although their direct regulatory role remains elusive. A transcriptional map of Biniou (a FoxF transcription factor) and Bagpipe (an Nkx factor) activity is presented as a first step to deciphering the developmental program regulating Drosophila visceral muscle development. A time course of chromatin immunoprecipitatation followed by microarray analysis (ChIP-on-chip) experiments and expression profiling of mutant embryos reveal a dynamic map of in vivo bound enhancers and direct target genes. While Biniou is broadly expressed, it regulates enhancers driving temporally and spatially restricted expression. In vivo reporter assays indicate that the timing of Biniou binding is a key trigger for the time span of enhancer activity. Although bagpipe and biniou mutants phenocopy each other, their regulatory potential is quite different. This network architecture was not apparent from genetic studies, and highlights Biniou as a universal regulator in all visceral muscle, regardless of its developmental origin or subsequent function. The regulatory connection of a number of Biniou target genes is conserved in mice, suggesting an ancient wiring of this developmental program (Jakobsen, 2007; full text of article).
The dynamic enhancer binding of Biniou suggested that the timing of Biniou occupancy is important for the timing of enhancer activity. To assess this in vivo, a number of regions from each of the three temporal clusters were linked to a GFP reporter. The timing of enhancer activity was assayed in vivo by in situ hybridization in transgenic embryos, to avoid time delays due to GFP protein folding and protein perdurance. All regions examined drive expression in a subset of Biniou-expressing cells and recapitulate all or part of the target genes' expression. This study focused on their temporal activity (Jakobsen, 2007).
The initiation of enhancer activity closely matches the first time point of Biniou binding for >90% of enhancers examined (10 of 11 CRMs). The early-bound enhancers (ttk, fd64a-e lmd, bap3) drive expression at stages 10-11, reflecting the binding of Biniou at these stages of development. Similarly, all four continuous-bound enhancers (HLH54F, otk, mib2, bap-FH) initiate expression at the first time period when Biniou binds. The two late-bound enhancers, in contrast, do not initiate expression at stages 10 or 11 of development, matching the lack of Biniou binding during these stages. Instead, the expression of the fd64a late enhancer initiates at stage 13, while the ken enhancer initiates VM expression at stage 14. This shift in the initiation of activity mirrors Biniou binding to these enhancers at stages 12-13 and 13-14, respectively. The only exception is the CG2330 enhancer, which initiates expression at stage 11, while Biniou enhancer binding was first detected at stage 13-14). As the expression of endogenous CG2330 does not initiate until stage 13, the apparent discrepancy in enhancer activity may simply reflect the exclusion of some regulatory motifs within the limits of the cloned region (Jakobsen, 2007).
Remarkably, the duration of enhancer activity is also tightly correlated with the time span of Biniou binding in 10 out of 11 CRMs examined. This is particularly striking in the early-bound enhancers: When Biniou ceases to bind to these CRMs (lmd, ttk, fd64a early, and bap3), their ability to regulate expression is lost. The converse is also true. Continuous Biniou binding correlates with continuous enhancer activity, specifically for bap-FH, HLH54F, and otk. The exception is the mib2 enhancer. In the context of this module Biniou binding it is not sufficient to maintain enhancer activity in the VM at late developmental time points (Jakobsen, 2007).
Taken together, these data indicate that the timing of Biniou enhancer binding is predictive for temporal enhancer activity in the large majority of cases (Jakobsen, 2007).
All 11 Biniou target enhancers examined in vivo regulate expression in more restricted patterns than Biniou itself. Since Biniou has broad temporal and spatial expression, additional regulatory inputs must refine Biniou's activity in a combinatorial manner. To identify other factors that may impinge on these enhancers overrepresented motifs were sought within the Biniou-bound CRMs. This analysis identified significant enrichment of a number of TF motifs. Of particular interest is the differential enrichment of motifs for Biniou, Mef2, and Nkx family proteins, Bap and Tin, between the three temporal classes of enhancers (Jakobsen, 2007).
Interestingly, the Bap motif is specifically enriched in the early-bound enhancers, and not in the continuous- or late-bound group. Tin motifs are also enriched in the early-bound group. This is in agreement with the transient expression of both TFs in the trunk VM during early stages development, and suggests that one or both of these TFs could impart some of the specificity for Biniou transient binding to these enhancers (Jakobsen, 2007).
The Mef2 motif is highly enriched in both early- and continuous-bound enhancers, but not in late VM enhancers. This was surprising since Mef2 regulates muscle differentiation genes and is therefore expected to coregulate late-bound enhancers. To substantiate this further, in vivo bound Mef2 enhancers were compared with the Biniou-bound enhancer regions at the same stages of development. In agreement with the motif enrichment, there is substantial combinatorial binding of Biniou and Mef2 on the early-bound and continuous-bound enhancers: 65.1% and 50.4%, respectively. In contrast, only 20.1% of the late Biniou-bound enhancers are cobound by Mef2. The same trend holds true in the other direction: There is no significant Biniou binding to many enhancers regulated by Mef2 at late developmental stages (e.g., the Mef2-bound enhancers for the contractile proteins Mhc, Mlc1, and Mlc2). This indicates that the VM may have two largely independent differentiation programs, one governed by Mef2 regulating more general muscle contractile proteins, and a second more VM-specific program driven by Biniou (Jakobsen, 2007).
Biniou consensus motifs are overrepresented in all three classes of temporal enhancers, providing global confirmation of the specificity of the ChIP-bound regions. Biniou motifs are particularly highly enriched in the continuous-bound and late-bound enhancers. This highlights a prominent role for Biniou in regulating enhancer activity at late stages of VM development. The inability of Biniou to bind to the late enhancers at early stages of development implies a mechanism that either blocks Biniou binding to these CRMs early in development or enhances Biniou's binding later in development. This could be mediated by many different mechanisms. Binding of the C. elegans FoxA TF, PHA-4, to early versus late pharyngeal muscle enhancers is primarily determined by the presence of high or low affinity binding sites, respectively (Gaudet, 2002). No apparent differences were detected in the Biniou motif between the early- and late-bound VM enhancers, and therefore a combinatorial model is favored with as-yet-unidentified cofactors. This is strongly supported by the restricted expression of all Biniou-bound CRMs examined, necessitating extensive combinatorial regulation to limit their activity (Jakobsen, 2007).
The specific enrichment of Bagpipe motifs in Biniou early-bound CRMs, in addition to the similarity of bagpipe and biniou mutant phenotypes, implies a potential for combinatorial regulation by these two TFs during the stages of VM specification. Since Biniou is downstream from Bagpipe, it has been very difficult to differentiate between a direct regulatory role by Bagpipe versus an indirect requirement via Biniou using genetic studies. To investigate the molecular function of bagpipe and its potential occupancy on Biniou-bound CRMs, ChIP-on-chip experiments were performed using anti-Bagpipe antibodies. This experiment identified 80 Bagpipe-bound genomic regions, using the same criteria as the Biniou experiments (Jakobsen, 2007).
A number of genomic regions are exclusively bound by Bagpipe, with no detectable Biniou binding at stages 10-11 of development. For example, the Bagpipe-bound region within the intron of CG8503: This enhancer is sufficient to drive transient expression in the trunk VM at stages 10-11, reflecting the transient expression of bagpipe in this tissue. Other Bagpipe-bound enhancer regions contain low levels of Biniou binding. The slp1 enhancer is within this class. This region drives expression in the foregut VM, recapitulating the endogenous gene's expression. Together these enhancers demonstrate that Bagpipe provides a direct regulatory role within the VM developmental program, independently of Biniou (Jakobsen, 2007).
In contrast, 51% of Bagpipe enhancers are cobound by Biniou at the same stage of development. This extensive combinatorial binding provides the first evidence of global coregulation by these TFs during early stages of VM specification. These cobound enhancers suggests that transient Biniou occupancy on early group enhancers, may in part be due to cobinding with Bagpipe, which is transiently expressed at these stages. To investigate this, the temporal profile of Biniou binding to the 80 Bagpipe-bound CRMs was examined using K-means clustering. Two distinct classes of Biniou-Bagpipe-cobound CRMs were apparent: Group 1 enhancers are cobound at stages 10-11 and remain continuously bound by Biniou at later developmental time points. This indicates that Biniou does not require the presence of Bagpipe to bind to the trunk VM enhancers among this class. In contrast, Group 2 enhancers are cobound by Biniou and Bagpipe at stages 10-11 of development, but are largely not bound by Biniou later in development. In the context of these early enhancers, Bagpipe binding may be the temporal cue dictating transient Biniou binding. Many of these CRMs are likely to be cooperatively regulated by both TFs (Jakobsen, 2007).
In summary, this study used two complementary genomic approaches to systematically dissect the transcriptional program driving VM development in vivo: a time course of ChIP-on-chip experiments and expression profiling of mutant embryos performed during consecutive stages of embryogenesis. This global view revealed the following insights into the underlying cis-regulatory network (Jakobsen, 2007):
(1) Biniou binds to enhancers in a temporally regulated manner. Since Biniou is expressed from VM specification until the end of development, this demonstrates that additional regulatory inputs are necessary to restrict Biniou activity. For the early-bound enhancers, some temporal specificity likely stems from combinatorial binding with Bagpipe. However, other TFs are also likely to be involved (Jakobsen, 2007).
(2) Biniou-bound CRMs drive expression in diverse subtypes of VM. This restricted spatial expression again necessitates combinatorial regulation with additional factors. It is proposed that much of this spatial specificity is conferred through Biniou-mediated feed-forward regulation: Biniou regulates a large group of spatially restricted TFs and components of cell signaling pathways that likely target different subsets of these CRMs. Such feed-forward regulation is a prevalent feature in many developmental networks (Jakobsen, 2007).
(3) The timing of Biniou enhancer occupancy is tightly correlated with the time span of enhancer activity. This is surprising given the extensive combinatorial binding necessary to produce restricted spatio-temporal expression of Biniou CRMs and suggests Biniou recruitment is the key trigger for enhancer activity. Taken together, these data indicate that Biniou provides VM enhancers with the competence to be expressed within the VM at the appropriate stage, and that these modules integrate extensive inputs from additional factors to restrict Biniou activity (Jakobsen, 2007).
(4) Although bagpipe and biniou mutants phenocopy each other, their regulatory role within the underlying network is very different. The majority of Bagpipeís regulation occurs via combinatorial binding to Biniou-Bagpipe CRMs to regulate a shared set of target genes. From a limited number of enhancers assayed in vitro, Bagpipeís contribution to enhancer activity is mainly cooperative, with little regulatory potential by itself. In contrast, Biniou targets an additional large group of CRMs during VM specification, and can regulate their activity independently of Bagpipe. This underlying nature of Biniou and Bagpipeís regulatory potential was not apparent from genetic studies due to the severity of their mutant phenotypes (Jakobsen, 2007).
(5) Biniou provides regulatory input at all stages of VM development, not just specification. Moreover, the temporal regulation of target genes at either early or late stages reflects developmental progression. For example, 17% of target genes regulated late in development are involved in the formation or function of the neuromuscular junction, compared with 4% of continuously regulated targets and 0% of early targets. This reflects the requirement of neuronal stimulation for gut muscle contraction at the end of embryogenesis. These results also revealed a new role for Biniou as a direct regulator of the transcriptional program within the foregut and hindgut VM (Jakobsen, 2007).
(6) The underlying cis-regulatory circuitry between Biniou and its target genes is at least partially conserved from flies to mice. Four genes that are directly regulated by Biniou in flies require FoxF function for their expression in mice. Due to the limited number of characterized FoxF direct target genes in vertebrates, it is currently too early to determine if VM development represents an ancient trans-bilaterian kernel (Jacobsen, 2006).
Taken together, these data indicate that Biniou serves as a universal regulator of VM: The broad expression of Biniou in all VM subtypes and its extensive occupancy on VM enhancers strongly suggests that Biniou provides all VM cells, regardless of their origin or ultimate cell fate, with their VM identity (Jakobsen, 2007).
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