Transcriptional Regulation

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).

Targets of Activity

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).

Hand is a direct target of the forkhead transcription factor Biniou during Drosophila visceral mesoderm differentiation

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).

Temporal ChIP-on-chip reveals Biniou as a universal regulator of the visceral muscle transcriptional network

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 Bagpipe’s 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, Bagpipe’s 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 Bagpipe’s 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).

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).

Differential regulation of mesodermal gene expression by Drosophila cell type-specific Forkhead transcription factors

A common theme in developmental biology is the repeated use of the same gene in diverse spatial and temporal domains, a process that generally involves transcriptional regulation mediated by multiple separate enhancers, each with its own arrangement of transcription factor (TF)-binding sites and associated activities. By contrast, the expression of the Drosophila Nidogen (Ndg) gene at different embryonic stages and in four mesodermal cell types is governed by the binding of multiple cell-specific Forkhead (Fkh) TFs [including Biniou (Bin), Checkpoint suppressor homologue (CHES-1-like) and Jumeau (Jumu)] to three functionally distinguishable Fkh-binding sites in the same enhancer. Whereas Bin activates the Ndg enhancer in the late visceral musculature, CHES-1-like cooperates with Jumu to repress this enhancer in the heart. CHES-1-like also represses the Ndg enhancer in a subset of somatic myoblasts prior to their fusion to form multinucleated myotubes. Moreover, different combinations of Fkh sites, corresponding to two different sequence specificities, mediate the particular functions of each TF. A genome-wide scan for the occurrence of both classes of Fkh domain recognition sites in association with binding sites for known cardiac TFs showed an enrichment of combinations containing the two Fkh motifs in putative enhancers found within the noncoding regions of genes having heart expression. Collectively, these results establish that different cell-specific members of a TF family regulate the activity of a single enhancer in distinct spatiotemporal domains, and demonstrate how individual binding motifs for a TF class can differentially influence gene expression (Zhu, 2012).

To drive expression in the visceral mesoderm (VM), the Fkh1 site in the Ndg enhancer is required in concert with either the Fkh2 or Fkh3 site. The trans-acting factor responsible for this activity of the Ndg enhancer is likely to be Bin because: (1) Bin binds to all three Fkh sites in vitro; (2) among the three candidate Fkh genes with appropriate VM expression, eliminating the function of only bin resulted in a significant reduction of Ndg expression in this tissue; and (3) Bin overexpression in the mesoderm is associated with Ndg enhancer activity in additional mesodermal cells. There are multiple precedents for Bin activating the expression of other VM genes. Moreover, chromatin immunoprecipitation assays show that Bin binds in vivo to the Ndg enhancer throughout embryonic stages 14 to 15, precisely when it would be expected to regulate this element in the visceral musculature (Zhu, 2012).

Somatic muscles in Drosophila are formed by the sequential fusion of individual muscle founder cells (FCs) with multiple fusion-competent myoblasts (FCMs). Both the endogenous Ndg gene and the reporter driven by the minimal enhancer used in this study are expressed in a subset of FCs, but not in any FCMs. Mutating all three Fkh-binding sites had no effect on Ndg expression in FCs, suggesting that Fkh factors do not play a role either in activating Ndg reporter expression in certain FCs, or in repressing it in other FCs. By contrast, binding of the FCM-expressed Fkh TF CHES-1-like to the Fkh2 or Fkh3 sites mediated repression of Ndg expression in FCMs. The design of the experiment prevented unambiguous determination of whether this repression also required CHES-1-like binding to the Fkh1 site. These results reveal a mechanism for regulating somatic myoblast gene expression that has not been previously recognized (Zhu, 2012).

Prior studies have focused on the contributions of signal-activated, tissue-specific and FC identity TFs in specifying the unique genetic programs of this class of myoblast. Similarly, TFs such as Lmd are known to be responsible for activating FCM-specific genes. However, this study has uncovered a novel mode of regulation in which FC genes are excluded from FCMs by an FCM-restricted repressor, in this case in the form of a Fkh domain protein. CHES-1-like is unlikely to be the only repressor playing such a role, as the de-repression in CHES-1-like mutants is limited to only a subset of the FCMs and is weaker than that seen for the Ndg enhancer with mutated Fkh sites. Although not verified functionally, it is possible that Lmd could play a similar repressive role in FCMs as a chromatin immunoprecipitation study found that this TF is bound extensively to FC genes (Cunha, 2010). However, given the widespread expression of CHES-1-like in FCMs, it is anticipated that many other FC genes will also be repressed by this Fkh domain TF (Zhu, 2012).

Finally, in the heart, it was shown that CHES-1-like and Jumu repress Ndg expression in Odd-PCs and in all Cardial cells (CCs) other than Tin- Lb-CCs. Repression in these cardiac cell types is mediated by binding of CHES-1-like to all three of the Fkh sites in the Ndg enhancer, and of Jumu to at least the Fkh2 site (Zhu, 2012).

A common occurrence in development is the repeated function of the same gene in multiple biological contexts and regulatory processes, requiring that the gene be expressed in distinct spatial and temporal domains. Such expression patterns are often generated by differential transcription mediated by multiple enhancers, each with its own arrangement of TF-binding sites and associated activities (Davidson, 2006). A notable exception is the case of genes regulated by Hox TFs, where different family members exhibit similar binding sequence specificity but exert differential effects on the same target genes (Zhu, 2012).

The results of the present study identify another class of TFs, the Fkh proteins, which exhibit a similar role in the Drosophila embryonic mesoderm. Specifically, it was shown that various cell-specific members of the Fkh TF family associate with the same binding sites within a single enhancer, thereby regulating the different spatiotemporal expression patterns of the associated target gene. Furthermore, it was shown that the distinct tissue-specific gene expression responses to these Fkh TFs are mediated by the TFs binding to different combinations of Fkh primary and secondary motifs that are represented by these sites. Thus, it was interesting to see that in several Drosophila species, where the Fkh1 site (which is the only site corresponding to the Fkh primary motif in D. melanogaster) is absent, its role may be compensated for by the overlapping Fkh2 and Fkh3 sites (which match only the secondary motif in D. melanogaster), which correspond to both Fkh primary and secondary motifs in these species. Similar evolutionary shuffling of motifs has been described previously (Zhu, 2012).

Finally, this study used a computational approach to attempt to generalize the potential involvement of the two classes of Fkh sites in cardiac gene regulation. Specifically, within putative enhancers in the noncoding regions of heart-expressed genes, a statistically significant overrepresentation of combinations of binding sites for known cardiogenic TFs was observed along with primary and secondary Fkh motifs. These observations are in agreement with previous studies that have documented an inability of a single consensus binding site to explain all aspects of in vivo TF binding. In addition, it has recently been shown that the regulatory specificity of a myoblast homeodomain TF is mediated by sequences preferentially bound by that particular homeodomain and not by other related family members (Busser et al., 2012b). In light of these findings, it will be interesting to determine whether other Fkh TFs and members of other TF families mediate differential gene expression responses by acting through distinct sequence motifs (Zhu, 2012).

HLH54F is required for the specification and migration of longitudinal gut muscle founders from the caudal mesoderm of Drosophila

HLH54F, the Drosophila ortholog of the vertebrate basic helix-loop-helix domain-encoding genes capsulin and musculin, is expressed in the founder cells and developing muscle fibers of the longitudinal midgut muscles. These cells descend from the posterior-most portion of the mesoderm, termed the caudal visceral mesoderm (CVM), and migrate onto the trunk visceral mesoderm prior to undergoing myoblast fusion and muscle fiber formation. HLH54F expression in the CVM is regulated by a combination of terminal patterning genes and snail. HLH54F mutations were generated and this gene was shown to be crucial for the specification, migration and survival of the CVM cells and the longitudinal midgut muscle founders. HLH54F mutant embryos, larvae, and adults lack all longitudinal midgut muscles, which causes defects in gut morphology and integrity. The function of HLH54F as a direct activator of gene expression is exemplified by analysis of a CVM-specific enhancer from the Dorsocross locus, which requires combined inputs from HLH54F and Biniou in a feed-forward fashion. It is concluded that HLH54F is the earliest specific regulator of CVM development and that it plays a pivotal role in all major aspects of development and differentiation of this largely twist-independent population of mesodermal cells (Ismat, 2010).

The Drosophila mesoderm forms from the ventral-most cells of the early embryo that invaginate during gastrulation. The expression and function of two transcription factors, the basic helix-loop-helix (bHLH) protein Twist (Twi) and the zinc-finger protein Snail (Sna), in ventral cells located between ~15% and 85% egg length are essential for their invagination and for subsequent mesodermal tissue development. Although the absence of either twi or sna activity results in similar phenotypes, the molecular roles of the two genes in this pathway differ. twi functions in activating a variety of mesoderm-specific target genes, including several that are known to regulate the invagination, patterning and differentiation of the mesoderm. By contrast, sna is thought to act largely, if not exclusively, as a repressor of a number of neuroectodermal targets, permitting mesoderm formation by restricting the expression of these genes to areas outside the presumptive mesoderm (Ismat, 2010).

Notably, however, there is at least one group of mesodermal cells that requires sna, but not twi, for its initial phase of development. This cell group is located ventrally within the domain of early twi and sna expression, but is restricted to the posterior tip of the mesoderm between ~7.5% and 15% egg length. Because it is fated to develop into the longitudinal muscles of the midgut, it has been termed the caudal visceral mesoderm (CVM) primordium. In light of the apparent lack of a requirement for twi for the initial development of the CVM, it is interesting that the cells of the CVM primordium are marked by the expression of another bHLH-encoding gene, HLH54F. This situation raises the possibility that, in the caudal-most portion of the mesoderm, HLH54F instead of twi cooperates with sna to control early CVM development. However, until now, specific mutants for HLH54F have not been available to test this possibility (Ismat, 2010).

The Drosophila midgut musculature consists of syncytial fibers that arise through myoblast fusion between gut muscle founder cells and fusion-competent myoblasts and forms a meshwork of circular and longitudinal muscles around the endodermal layer. The CVM appears to be the sole source of founder cells of the longitudinal midgut muscles. After their ingression, these cells migrate anteriorly and spread over the future midgut, where they fuse with resident fusion-competent cells to form the multinucleated longitudinal muscle fibers of the midgut. The fusion-competent cells for this event come from a different source, the so-called trunk visceral mesoderm (TVM), which provides a second (and major) contribution of precursors to the musculature of the midgut. The primordia of the TVM are arranged bilaterally as 11 metameric cell clusters within the dorsal mesoderm, which subsequently merge with each other into the contiguous band of the TVM. The TVM primordia are marked by the expression of the NK homeodomain gene bagpipe (bap) and the FoxF gene biniou (bin), both of which are essential for TVM formation. Within the TVM, the founder cells of the circular midgut muscles are induced by Jelly belly (Jeb) signals acting through the receptor tyrosine kinase Alk. The founder myoblasts from the TVM fuse one-to-one with adjacent fusion-competent myoblasts into binucleated syncytia that form the circular midgut muscles. Subsequently, after the migrating CVM-derived founder cells have arrived at their destinations, each fuses with multiple fusion-competent cells from the TVM left over from the TVM founder cell fusions. It is these multinucleated syncytia that will then differentiate into the longitudinal visceral muscles, which run perpendicularly to the circular muscles along the entire length of the midgut. The longitudinal gut muscle fibers from the outer layer of the developing visceral musculature are tightly interwoven with the circular gut muscle fibers from the inner layer (Ismat, 2010).

HLH54F is the earliest known marker of the CVM primordia and its expression is maintained throughout the development and differentiation of the longitudinal midgut muscles. To test whether HLH54F plays an important role in the development of the CVM and longitudinal midgut musculature, loss-of-function mutations for this gene were generated by imprecise P-excision and EMS mutagenesis screens. This study demonstrates that in the absence of HLH54F activity, no longitudinal gut muscle founder cells are formed. The absence of all tested CVM markers and the observed apoptotic death of the cells that would normally be destined to form CVM in HLH54F mutants, show that HLH54F has an essential role in determining the CVM and in specifying the founder cells of the longitudinal gut musculature. This function includes feed-forward regulation and direct binding to target enhancers (e.g., from the Dorsocross genes). It was also shown that ectopic expression of HLH54F can interfere with normal somatic muscle, cardiac and TVM development. Further, the known pathway of CVM development has been extended by showing that the initiation of HLH54F expression is largely independent of twi, but depends critically on the combined activities of sna and terminal patterning genes, particularly the synergistic activities of fork head (fkh) and brachyenteron (byn). Hence, the CVM primordia are determined at the intersection of the domains of these mesodermal and terminal regulators (Ismat, 2010).

This study has shown that HLH54F is a key regulator in the CVM, a population of cells in which the bHLH gene twi appears to have only minor functions. Although twi is initially co-expressed with HLH54F in these cells, it makes only a small contribution to activating HLH54F expression, and the expression of both bHLH genes rapidly becomes mutually exclusive. Instead of twi, the activation of HLH54F in the CVM primarily involves the combined activities of mesodermal sna and the terminal genes fkh and byn. As sna is generally thought to act as a ventral repressor of non-mesodermal genes in early mesoderm development, it will be interesting to determine whether the positive requirement for sna in the activation of HLH54F expression is direct, which would be unique to date. Alternatively, HLH54F might be activated by high levels of nuclear Dorsal and repressed by lateral genes that are repressed by sna ventrally. Along the anteroposterior axis, the posterior border of HLH54F expression is apparently defined by the posterior expression border of sna, which is delineated by the repressive action from hkb. It is proposed that the anterior border of HLH54F is determined by near-maximal threshold levels of tll, the expression of which declines steeply in the area anterior to the HLH54F domain. However, tll acts largely indirectly, through the combined activities of its downstream genes byn and fkh, in activating HLH54F. The low residual levels of HLH54F mRNA in fkh byn double mutants suggest the involvement of direct inputs from additional posterior activities, possibly tll or maternal torso. It appears that high-level expression of zfh1 in the CVM largely depends on tll and sna, whereas HLH54F and zfh1 do not depend on one another (Ismat, 2010).

Notably, neither twi nor HLH54F is required individually for the internalization of the CVM cells during gastrulation, although a redundant function cannot be excluded. The posterior portion of the mesoderm, which includes the CVM and portions of the presumptive HVM, bends around during gastrulation to form a second, internal mesodermal layer. It is conceivable that this movement is a passive process brought about by the invagination of the PMG rudiment. However, for subsequent migrations of CVM cells from these positions, the activity of HLH54F, but not twi, is crucial. In addition, byn, zfh1 and fkh are required for normal migration after stage 10. Whereas their respective functions are likely to be cell-autonomous, the observed requirement of twi for normal pathfinding of CVM cells is likely to be due to the absence of the migration substrate normally formed from the trunk mesoderm (Ismat, 2010).

The genetic data show that, after the caudal mesodermal cells have ingressed in this manner, they do not develop any further in the absence of HLH54F activity and undergo apoptosis. In the normal situation, HLH54F is needed for the activation of several transcription factor-encoding genes at this stage, including bin, croc and the Doc genes. Although the functions of these genes in CVM development have not been defined, it is likely that they regulate specific aspects of CVM development downstream of, and perhaps in combination with, HLH54F. The data from loss-of-function and ectopic expression analyses of HLH54F show that this gene is essential, but not sufficient, for specification of longitudinal gut muscle founders. Parallel inputs, albeit less pervasive, appear to come from high-level zfh1, which like HLH54F is required for croc/croc-lacZ expression. Altogether, it is proposed that HLH54F is necessary for activating the vast majority of early CVM-specific genes, with one known exception being high-level zfh1, and that zfh1, byn and fkh in various combinations act together with HLH54F to activate certain targets during the specification and early migration of CVM cells (Ismat, 2010).

The continuous expression of HLH54F in the CVM and longitudinal gut muscles suggests that this gene is not only required for specification, but is also directly involved in many other developmental processes, including the continued migration, myoblast fusion and differentiation of the CVM cells. Possible downstream targets of HLH54F in the promotion of proper cell migration include beat-IIa, which encodes an as yet uncharacterized membrane-anchored Ig domain protein, and the FGF receptor-encoding gene heartless (htl), which is known to be required for normal migration. In this context, it is interesting that the vertebrate orthologs of HLH54F are expressed prominently in specific migrating populations of mesodermal cells as well. For example, musculin is expressed in myoblasts at the myotomal lips that migrate into the developing limbs, and capsulin is expressed in the migrating pro-epicardial cells . Therefore, it is possible that parts of the regulatory circuit in the control of cell migration have been conserved, even though they occur in different mesodermal cell types. capsulin is also expressed prominently in the splanchnopleura and tissues derived from it, including the developing smooth muscles of the stomach and gut. Therefore, HLH54F and capsulin might share some functions in the terminal differentiation of the respective gut musculatures in the different systems. Both Capsulin and Musculin have been characterized largely as repressors. However, their activity (and likewise that of HLH54F) as repressors versus activators might well be context specific with respect to the particular enhancer, tissue or developmental stage in question and might depend on the relative concentrations of particular heterodimerization partners (Ismat, 2010).

Based on the phenotype of byn mutants, a role of the CVM in promoting midgut constrictions has also been proposed. The phenotype of HLH54F mutants confirms this effect, although it was found that partial constrictions can frequently occur and that the effect is variable. It is inferred that the physical interactions between developing longitudinal and circular muscle fibers are necessary to provide the full force required for the efficient constriction of the midgut endoderm at the well-defined signaling centers. In the fully developed midgut, scanning electron microscopy images have revealed that the longitudinal fibers are tightly interwoven with the web-shaped circular fibers, which may explain the mechanical strength of this meshwork. Indeed, it was found that the mechanical stability and integrity of the midgut, particularly in HLH54F mutant adults, are severely compromised (Ismat, 2010).

In summary, HLH54F appears to sit at the top of the regulatory hierarchy of CVM development and is likely to fulfill additional key roles during the course of development of the CVM and the longitudinal gut muscles. Future efforts need to be directed towards dissecting additional downstream events that regulate the different steps of cell migration, myoblast fusion, morphogenesis and terminal differentiation of the longitudinal midgut musculature (Ismat, 2010).

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

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.