BIOLOGICAL OVERVIEW

twist and snail, genes whose transcription is directed by Dorsal, define the mid-ventral domain of the blastoderm. Cells from this domain, through the action of twist and snail are fated to become mesoderm, after they invaginate through the ventral furrow at gastrulation. The interface between mesoderm and ectoderm defines the mesectoderm, fated to become the ventral midline. Invagination forms an inner cell layer that gives rise to internal organs (including somatic and visceral muscles, the heart, and fat body). After invagination, mesodermal cells spread dorsally to form a monolayer of cells coating the inner face of the ectoderm.

Contact of mesodermal cells with the overlying ectoderm has major consequences for the fate of the mesoderm. This contact is central to the organization of mesoderm into somatic and visceral subdivisions, and the organization of mesoderm in terms of segmentation. Twist takes on a new role upon contact, both in dorsal/ventral subdivision and segmentation.

Mesodermal parasegments (embryonic segments) mirror the segmentation of the ectoderm. Each parasegment can be divided into an anterior region with weak twist expression and a posterior region with high twist expression. There is a gradual change, an increase in twist expression between anterior and posterior, then an abrupt decrease in expression at the next boundary between posterior and the next anterior parasegment. The sharp border of twist expression lies along a stripe of ectodermal engrailed expressing cells.

Visceral mesoderm arises from the mesodermal cells that are low twist expressors and presumably high bagpipe expressors. These cells move inward forming prominent clusters at segmental intervals. Within this migrating group of cells, the dorsal most cells give rise to the fat body. High twist expressors, remaining as the dorsal most mesodermal cells arrayed under the ectoderm, give rise to somatic muscles. These associate with the segmental borders by the invagination of the ectoderm to form a furrow. (See stripe site for additional information). There is an obvious physical gap between the precursors of dorsal muscles that develop in close association with the heart and the progenitors of the more ventral muscles. These two groups are physically separated by a landmark consisting of the longitudinal tracheal trunk. The dorsal crests of the mesodermal cells expressing twist give rise to progenitors for the heart, including the central tube of cardial cells and their flanking pericardial cells on either side. These latter cells express even-skipped (Dunin-Borkowski, 1995).

Thus the heart and somatic muscles, and even the visceral mesoderm are formed in intimate contact and regulatory feedback with the dorsal ectoderm, which itself is structured by segment polarity genes. It is apparent that the secreted ligands Wingless, DPP and Hedgehog have a central role in orchestrating not only the segmentation of the ectoderm, but the morphogenesis of the mesoderm as well.

The roles of Twist and Notch have been examined during adult indirect flight muscle development. The observations suggest that twist repression is a requirement for the initiation of muscle differentiation in some muscles of the fly. Persistent twist expression aborts the development of these muscles. Markers of differentiation, such as myosin, are greatly reduced. Erect wing, a transcription factor required for indirect flight muscle differentiation begins to be expressed as twist expression declines. Reduction in levels of Twist leads to abnormal myogenesis. It is thought that reduction of Twi levels causes premature differentiation and thus results in fewer myoblasts that are correctly positioned to contribute to muscle development. Notch reduction causes a similar mutant phenotype and reduces Twist levels. Conversely, persistent expression, in myoblasts, of activated Notch causes continued twist expression and failure of differentiation as assayed by myosin expression. The gain-of-function phenotype of Notch is very similar to that seen when twist is persistently expressed. Two models are proposed for Notch function:

(1) Notch function in muscle differentiation is proposed to be similar to the function of Notch in neurogenesis in that loss of Notch function would result in more founder cells and twist expression would decrease as a result of the onset of differentiation.
(2) Notch signaling could play a direct role in maintaining the un-differentiated state until myoblasts are correctly positioned to receive appropriate environmental signals to differentiate.

Until markers for founder cells in adult myogenesis are identified, it will be difficult to distinguish between these two possiblities. An intriguing result obtained in this study of persistent expression of activated Notch and twist is the significant difference in effects on very closely related muscles; the indirect flight muscles (which are sensitive to Notch and Twist levels), and the direct flight muscles (which are not). These two groups of muscles are clonally related and share progenitors at least until the late third larval instar, and then the progenitors differentiate into very different muscle types. Notch activity might function to delineate myoblast precursors of these two groups of muscles (Anant, 1998).

Notch signaling patterns Drosophila mesodermal segments by regulating Twist

One of the first steps in embryonic mesodermal differentiation is allocation of cells to particular tissue fates. In Drosophila, this process of mesodermal subdivision requires regulation of the bHLH transcription factor Twist. During subdivision, Twist expression is modulated into stripes of low and high levels within each mesodermal segment. High Twist levels direct cells to the body wall muscle fate, whereas low levels are permissive for gut muscle and fat body fate. Su(H)-mediated Notch signaling represses Twist expression during subdivision and thus plays a critical role in patterning mesodermal segments. This work demonstrates that Notch acts as a transcriptional switch on mesodermal target genes, and it suggests that Notch/Su(H) directly regulates twist, as well as indirectly regulating twist by activating proteins that repress Twist. It is proposed that Notch signaling targets two distinct 'Repressors of twist' - the proteins encoded by the Enhancer of split complex [E(spl)C] and the HLH gene extra machrochaetae (emc). Hence, the patterning of Drosophila mesodermal segments relies on Notch signaling changing the activities of a network of bHLH transcriptional regulators, which, in turn, control mesodermal cell fate. Since this same cassette of Notch, Su(H) and bHLH regulators is active during vertebrate mesodermal segmentation and/or subdivision, this work suggests a conserved mechanism for Notch in early mesodermal patterning (Tapanes-Castillo, 2004).

Analysis of Notch mutant embryos revealed that Notch signaling is essential for Twist regulation at mesodermal subdivision. However, comparison of Notch and Su(H) mutant embryos indicated that Notch regulates Twist differently from Su(H). At stage 10, uniform high Twist expression was maintained in N^null mutants; by contrast, Su(H)^null mutants have a wild-type-like Twist pattern. Furthermore, while constitutive activation of Notch represses Twist expression at stage 10, constitutive expression of a transactivating form of Su(H) [Su(H)-VP16] increases Twist expression. Despite these differences, double mutant analysis and rescue experiments demonstrate that Notch requires Su(H) to repress Twist. Moreover, further rescue experiments show that Notch signaling acts as a transcriptional switch, which alleviates Su(H)-mediated repression and promotes transcription. In addition, genetics, combined with promoter analysis, suggest that Notch and Su(H) have multiple inputs into twist. Notch/Su(H) signaling both directly activates twist and indirectly represses twist expression by activating proteins that repress Twist. Finally, the data indicate that Notch targets two distinct 'Repressors of twist' - E(spl)-C genes and Emc. It is proposed that Notch signaling activates expression of E(spl)-C genes, which then act directly on the twist promoter to repress transcription. Since removing groucho enhances the phenotype of the E(spl)-C mutant embryos, it is suggested that the corepressor, Groucho, acts with E(spl)-C proteins and the Hairless/Su(H) repressive complex to mediate direct repression of twist. The second 'Repressor of twist', Emc, mediates repression of Twist in an alternative fashion. It is hypothesized Emc activity inhibits dimerization of Da with itself or another bHLH protein. This, in turn, prevents Da from binding DNA and activating twist transcription. Since Emc is expressed in the embryo prior to stage 10, it is likely that the transition from uniform high Twist expression to a modulated Twist pattern involves Emc inhibition of Da activity at stage 9. In conclusion, this work uncovers how Notch signaling impacts a network of mesodermal genes, and specifically Twist expression. Given that Notch signaling directs cell fate decisions in many Drosophila embryonic and adult tissues and that Notch regulates Twist in adult flight muscles, these data may suggest a more universal mode of Notch regulation (Tapanes-Castillo, 2004).

The distinct mesodermal phenotypes of Notch and Su(H) mutants can be explained by Notch acting as a transcriptional switch. This aspect of Notch signaling has been described in other systems, and the early Drosophila mesoderm appears no different in this regard. However, these data suggest that there is more to the phenotypes; that is, additional layers of Notch regulation in the transcriptional control of twist (Tapanes-Castillo, 2004).

Genetic experiments, as well as promoter analysis, raised the hypothesis that Notch signaling regulates twist directly, as well as indirectly by activating expression of a 'repressor of twist.' This indirect repression of twist concurs with the role of Notch in activating E(spl) transcriptional repressors. Moreover, a mechanism involving direct and indirect regulation is consistent with Su(H) mutant phenotypes. In Su(H)^null embryos, neither twist nor repressor of twist (for example, emc) are repressed. The de-repression of both genes at the same time results in Twist expression appearing 'wild-type-like'. When a constitutively activating form of Su(H) is expressed, both twist and repressor of twist are activated. In these embryos, high Twist domains are expanded, but uniform high Twist expression is not observed because repressor of twist is expressed (Tapanes-Castillo, 2004).

However, simple direct and indirect regulation [through emc and E(spl)-C genes] by Notch still does not fully explain the phenotypes of Notch mutants. Both twist and repressor of twist should be repressed in N^null embryos because Su(H) will remain in its repressor state. While the N^null phenotype was consistent with repressor of twist being repressed, twist was still strongly expressed. Additionally, constitutive Notch activation should cause both twist and repressor of twist to be expressed. Consequently, N^intra was expected to cause a phenotype similar to that caused by Su(H)-VP16. Contrary to these predictions, panmesodermal expression of N^intra represses Twist, consistent with only repressor of twist being strongly expressed. Taken together, these results suggested that at stage 10, the twist promoter is less receptive to Notch/Su(H) activation than to Notch/Su(H) repression. As a result, constitutive activation of Notch represses twist, while loss of Notch activates twist ectopically (Tapanes-Castillo, 2004).

While Notch signaling has the ability to activate twist, Notch/Su(H) signaling ultimately leads to repression of twist at stage 10. This predominance of repression can be explained in two ways: (1) direct Notch activation of the twist promoter is overpowered by Notch activated repressors of twist; and (2) a repressor of twist gene, such as E(spl), is more responsive to Notch/Su(H) activation than twist. These ideas are discussed below in light of the results (Tapanes-Castillo, 2004).

The first model proposes that while Notch signaling might directly promote both twist and repressor of twist activation, repressors of twist might suppress an increase in twist transcription. The data suggest that Notch regulates multiple repressors of twist, including E(spl)-C genes and Emc. On the twist promoter, these multiple repressors could overwhelm Su(H) activation. Hence, twist would be transcriptionally repressed rather than activated. In Su(H)-VP16 embryos, the constitutive activating ability of Su(H) on the twist promoter might inhibit some of this repression. Consequently, Twist is ectopically expressed at high levels (Tapanes-Castillo, 2004).

The data are also consistent with the second model, which proposes that twist and a repressor of twist gene, such as E(spl), respond differently to Notch activation. The reason for this differential response is provided by the concept of Notch instructive and permissive genes. Transcription of Notch instructive genes requires the intracellular domain of Notch (N^icd) first to alleviate Su(H)-mediated repression and then to serve as a coactivator for Su(H). Transcription of Notch permissive target genes requires N^icd solely to de-repress Su(H); Su(H) bound to other coactivators and/or other transcriptional activators is necessary for permissive gene activation. Since panmesodermal expression of N^intra does not activate twist, it is concluded that simple de-repression of Su(H) is insufficient to activate twist expression and that other factors are required. Hence, Notch acts permissively on the twist promoter. By contrast, panmesodermal expression of N^intra is sufficient to activate a repressor of twist, resulting in the strong Twist repression. Since E(spl)-C genes have been categorized as Notch instructive target genes, it is suggested that E(spl)-C genes are the Notch instructive repressor of twist genes in this system. Although Notch can upregulate Emc expression, the inability to see a change in Emc expression in N^null and Su(H)^null mutants suggests Emc is not a Notch instructive target gene. Thus, based on all of this work, the instructive and permissive target gene regulation model is currently favored (Tapanes-Castillo, 2004).

In Drosophila, Notch signaling is activated by the Delta (Dl) and Serrate ligands. Delta is expressed throughout the mesoderm at late stage 9 and stage 10, while Serrate is not embryonically expressed until stage 11. While the germline requirement for Delta prevents germline clone embryos from being produced by recombination, embryos lacking zygotically expressed Dl exhibit a wild-type-like Twist pattern. In addition, expression of a full-length Notch protein missing the two EGF repeats critical for Dl binding (EGF repeats 11 and 12) rescues Twist modulation in N^null mutant embryos. Thus Notch does not require EGF-like repeats 10-12 to repress Twist. These preliminary data suggest that Delta may use EGF-like repeats other than 10-12 to activate Notch. Alternatively, Notch may not be activated by canonical Delta signaling; a novel (non-DSL) ligand may activate Notch in the early mesoderm. Further experiments are required to evaluate whether the maternal component of Delta regulates Twist (Tapanes-Castillo, 2004).

While this work elucidates the molecular mechanism by which Notch represses Twist, how Notch signaling establishes a segmentally repeated pattern of low and high Twist domains -- that is, periodicity in Twist expression -- has yet to be understood. Two models, consistent with the data, are proposed to describe how Notch signaling contributes to a modulated Twist pattern. Model I proposes that during the transition from a uniform to a modulated Twist pattern, Notch signaling represses twist only in presumptive low Twist domains. Transcriptional activators, such as Da, maintain high Twist expression in presumptive high Twist domains. While Notch signaling components such as Notch, Su(H), and Delta are expressed throughout the mesoderm at late stage 9 and stage 10, this model predicts that Notch signaling is simply not activated in presumptive high Twist domains. Model II proposes that during the transition in Twist expression, Notch signaling represses twist throughout the mesoderm, but Notch independent transcriptional activators antagonize Notch repression in what will become high Twist domains, thereby promoting the formation of high Twist domains. For example, transcriptional effectors of Notch signaling [such as Su(H) and E(spl)] and an 'activator' that is only expressed in presumptive high Twist domains may converge and compete on the twist promoter (Tapanes-Castillo, 2004).

Consistent with model II, the segmentation gene sloppy-paired (slp) is a spatially regulated 'high Twist domain' activator. At stages 9-10, Slp is expressed in the mesoderm in transverse stripes that correspond to high Twist domains. Moreover, loss- and gain-of-function experiments indicate that Slp is required for high Twist expression. No change in Slp expression is found in Notch and Su(H) mutant embryos through mid-embryogenesis, indicating that slp is not regulated by Notch signaling at these stages. Mesodermal slp expression is activated by Wingless signaling; therefore, Wingless signaling is likely to alleviate Notch repression in high Twist domains. In the future, it will be important to establish the mechanism through which Notch signaling is antagonized in high Twist domains. Slp and Notch effectors may converge on the twist promoter to regulate expression. Additionally, Wingless signaling components may directly regulate and/or inhibit Notch (Tapanes-Castillo, 2004).

During vertebrate segmentation, mesodermal segments (called somites) are progressively segregated from a terminal undifferentiated growth zone called the presomitic mesoderm. Somites are then patterned though a process of subdivision, so that cells are allocated cells to distinct tissue fates. The first subdivision partitions each somite across the anterior-posterior axis into rostral and caudal halves. Later each somite is further subdivided across the dorsal-ventral axis into dermomyotome, which gives rise to dermis and skeletal muscle, and sclerotome, which develops into the axial skeleton. The Notch signal transduction pathway has been shown to play a central role in both somite segmentation and rostral/caudal subdivision (Tapanes-Castillo, 2004).

While Notch does not appear to be involved in fly segmentation, this work uncovers a previously uncharacterized role for Notch in the subdivision of Drosophila mesodermal segments. Notch repression is required to subdivide each mesodermal segment into a low and high Twist domain. Hence, Drosophila, like vertebrates, utilizes Notch and bHLH regulators to subdivide the mesoderm and transform uncommitted mesoderm into patterned segments. Since the homologs and/or family members of the bHLH regulators studied here -- Twist, Emc, Da and E(spl) -- are involved in vertebrate segmentation and/or somite subdivision, it will be interesting to determine whether these proteins are regulated in vertebrates in a manner similar to that governing their regulation in the fly (Tapanes-Castillo, 2004).

A core transcriptional network for early mesoderm development in Drosophila consists of Twist, Mef2, Tinman and Dorsal

Embryogenesis is controlled by large gene-regulatory networks, which generate spatially and temporally refined patterns of gene expression. This study reports the characteristics of the regulatory network orchestrating early mesodermal development in the fruitfly, where the transcription factor Twist is both necessary and sufficient to drive development. Through the integration of chromatin immunoprecipitation followed by microarray analysis (ChIP-on-chip) experiments during discrete time periods with computational approaches, >2000 Twist-bound cis-regulatory modules (CRMs) were identified and almost 500 direct target genes. Unexpectedly, Twist regulates an almost complete cassette of genes required for cell proliferation in addition to genes essential for morphogenesis and cell migration. Twist targets almost 25% of all annotated Drosophila transcription factors, which may represent the entire set of regulators necessary for the early development of this system. By combining in vivo binding data from Twist, Mef2, Tinman, and Dorsal an initial transcriptional network was constructed of early mesoderm development. The network topology reveals extensive combinatorial binding, feed-forward regulation, and complex logical outputs as prevalent features. In addition to binary activation and repression, it is suggested that Twist binds to almost all mesodermal CRMs to provide the competence to integrate inputs from more specialized transcription factors (Sandmann, 2007).

Twist-bound enhancers and direct Twist target genes

ChIP-on-chip was performed at two consecutive developmental time periods: 2-4 h (stages 5-7) and 4-6 h (stages 8-9), covering the stages of gastrulation, mesoderm expansion, migration, and early subdivision into different primordia. For each time period, four independent ChIPs were performed using two different anti-Twist antibodies to reduce possible off-target effects (Sandmann, 2007).

To systematically identify Twist-bound regions in an unbiased, global manner, a high-density microarray tiling across the Drosophila melanogaster genome was designed with ~380,000 60mer oligonucleotide probes. Twist binds to E-box motifs: As a degenerate E-box (CANNTG) is expected to occur every ~256 base pairs (bp) in the Drosophila genome, a 60mer oligonucleotide was designed for each E-box motif within the nonrepetitive, noncoding regions of the genome. This design made no assumptions about the specificity of the E-box bound by Twist, yet ensured all putative E-boxes were covered and that each Twist-bound sequence was detected by at least two neighboring 60mers (Sandmann, 2007).

These experiments identified 2096 nonoverlapping genomic regions significantly bound by Twist within one or both developmental time periods. This set includes all known Twist-bound enhancers tested, except the eve-cardiac enhancer that is regulated outside the period of development assayed. The majority of Twist-bound regions are found within introns of gene loci, rather than noncoding 5' and 3' regions. A similar positional bias was also observed for p53 and Krüppel, suggesting that introns close to the transcriptional start site represent hotspots for active CRMs. Intronic binding of Twist correlates significantly with the misregulation of these genes' expression in twist loss-of-function mutant embryos and their expression within the ventral blastoderm and mesoderm (Sandmann, 2007).

One of the major challenges for ChIP-on-chip studies is to accurately link the TF-bound enhancers to their appropriate target gene. Rather than simply taking the closest 5' or 3' gene, a more stringent approach was taken and a Twist-bound region was not assigned to a gene based on proximity alone. The results demonstrate that Twist binds more frequently to gene loci genetically downstream from the TF and/or expressed in the same cells as the TF. These criteria to systematically match all 2096 Twist-bound regions (intronic or intergenic) to their likely targets, leading to a high-confidence gene assignment for 854 Twist-bound sequences. This increased the number of Twist direct targets from the previously known 11 to 494 genes. All Twist-bound regions and surrounding genes can be visualized and searched at http://furlonglab.embl.de (Sandmann, 2007).

The RedFly database contains a comprehensive collection of previously described Drosophila enhancers, mainly characterized through single gene studies. Of the 2096 Twist-bound regions, 143 overlap with known enhancers for 62 genes, confirming that these regions have regulatory potential in vivo. Twist was not known to bind to many of these enhancers; this overlap therefore provides strong evidence for a regulatory link between Twist and the 62 target genes (e.g., Abd-A, Abd-B, aop, Brd, slp1, and bap). To further examine the regulatory potential of Twist-bound regions, reporter constructs of new putative enhancer sequences were tested in transgenic animals. Six Twist-bound regions within or close to the following gene loci were assayed: T48, trbl, retn, CG4221, CG8788, and CG32372. All regions proved sufficient to function as enhancers in vivo and could reproduce all or part of the endogenous spatio-temporal gene-expression pattern (Sandmann, 2007).

The T48, tribbles, retained, CG4221, and CG8788 enhancers initiate expression within the early blastoderm. The T48 module mirrors the expression of the endogenous gene within the presumptive mesoderm. The zygotic expression of tribbles is highly dynamic, which is reflected by the assayed CRM. This enhancer drives expression very transiently in the ventral blastoderm and quickly becomes ubiquitously expressed. The relatively small enhancer region for retained is activated in the anterior and posterior ventral blastoderm, where it is coexpressed with Twist, and its expression extends into the dorsal blastoderm. The CRMs for CG4221 and CG8788 initiate expression in the presumptive mesoderm, and continue to drive expression throughout the trunk mesoderm at later stages. The expression of the CG32372 module initiates after gastrulation in the head mesoderm, a domain that overlaps with twist expression. It is interesting to note that Twist binds to multiple enhancer regions for many of these genes. This feature is also evident more globally: Almost 50% of Twist target genes have two or more Twist-bound enhancers, reflecting the complexity of their regulation (Sandmann, 2007).

In summary, these results demonstrate that ChIP-on-chip experiments provide a sensitive and accurate global map of Twist-bound regulatory regions during key stages of early mesoderm development (Sandmann, 2007).

Twist activity is essential for target gene expression

To assay the requirement of Twist function for target gene expression, the expression was examined of six novel direct targets in twist mutant embryos. These genes are expressed in the presumptive mesoderm prior to gastrultion, and therefore at stages when the role of twist function can be assessed. Mesodermal cells are absent in twist mutant embryos later in development due to a block in gastrulation. Triple-fluorescent in situ hybridization was performed using probes directed against twist (blue channel; while twist¹ is a protein-null allele, twist RNA is still expressed), inflated (red channel; this gene is dependent on twist for its expression and was used as a marker to distinguish homozygous mutant embryos from their siblings), and a probe directed against one of the six direct target genes (green channel). The spatial expression of all six targets overlaps with twist within the presumptive mesoderm (Sandmann, 2007).

Importantly, twist activity is essential for the expression of five out of six genes examined. Note, for CG32982 and CG9005, residual expression remains outside the twist expression domain in the dorsal and posterior blastoderm, respectively. These results, in combination with in vivo binding data, indicate that Twist binding to a CRM is a prerequisite to activate target gene expression for a large percentage of its targets. The role of Twist binding to the NetA enhancer remains unclear. Twist may act redundantly with other TFs, or alternatively may function in a more subtle manner to modulate the levels of expression (Sandmann, 2007).

Twist and Dorsal collaborate much more extensively than previously predicted

One of the earliest functions of Twist within the pregastrula embryo is the coregulation of D-V patterning with the NFkappaB ortholog Dorsal. Dorsal acts as a morphogen by regulating its target genes at (at least) three threshold concentrations along the D-V axis. Type I-regulated Dorsal enhancers receive high levels of Dorsal, contain low-affinity Dorsal sites and drive expression in ventral mesodermal domains (e.g., sna, htl, twi). Type II enhancers receive intermediate levels of Dorsal and drive expression in mediolateral domains of different sizes (e.g., sim, brk, vn), while Type III enhancers receive low levels of Dorsal, contain high-affinity Dorsal sites, and can be either activated (sog, ths) or repressed (dpp, tld, zen) by Dorsal. This system has been studied so intensively that the level of knowledge is sufficient for quantitative modeling of cis-regulatory interactions. It was therefore of interest to determine whether global analysis could reveal new insights into this process. The data identified in vivo binding of Twist to both Type I and II Dorsal enhancers, as expected. The boundaries of Twist binding are in remarkable agreement with the limits of characterized minimal enhancers (e.g., htl, rho, and ths). More importantly, new CRMs were identified for several of these well-characterized genes (Sandmann, 2007).

Seven novel enhancers for D-V patterning genes reveal the regulatory complexity of Twist-bound CRMs: The cactus, stumps, and wntD enhancers drive expression in a domain overlapping Twist within the ventral blastoderm and likely represent Type I enhancers. Cactus, an IkappaB ortholog, is expressed both maternally and zygotically and sequesters Dorsal within the cytoplasm. While the regulation of zygotic cactus expression was previously not understood, these data reveal a Twist-bound CRM that is sufficient to drive expression in the presumptive mesoderm. Twist also binds to a CRM of Toll. Although the function of cactus'and Toll's zygotic regulation remains unclear in Drosophila, positive feedback regulation of zygotic Toll-receptor expression is required to refine the Dorsal nuclear gradient in the flour beetle Tribolium castaneum (Sandmann, 2007).

The stumps CRM is expressed in a subset of Twist-expressing cells, yielding a salt and pepper pattern that may reflect the requirement for a second, partially redundant enhancer (e.g., the 'stumps_early' enhancer) to give robust expression. The wntD CRM is highly expressed at the anterior and posterior poles of the ventral blastoderm, but is very weakly expressed within the central region. This mirrors the transient expression of the endogenous gene at this stage of development. This single enhancer reflects the regulatory logic deduced from genetic studies: The inputs from Twist and Dorsal activate WntD, while Snail represses its transcription within the presumptive mesoderm. The CRM for crumbs reproduces the endogenous genes expression. This 480-bp region can function as an enhancer in the ectoderm while acting as a silencer within the ventral blastoderm. This ventral repression is most likely due to direct input from Snail on this CRM. Therefore, even at the same stage of development, these four Twist-bound CRMs drive expression in different spatial patterns within a small population of cells. This complexity is clearly mediated by context-dependent integration of additional inputs. Three additional CRMs for mir-1 (Type I), vn, and sim (Type II Dorsal targets) drive expression later in development, reproducing part of the endogenous gene’s expression (Sandmann, 2007).

Unexpectedly, Twist also binds to characterized Dorsal Type III enhancers known to regulate dpp, ind, and ths. Dorsal and its associated corepressors Cut, Retained, and Capicua recruit Groucho to repress dpp, confining its expression to the dorsal blastoderm. The cobinding of Twist and Dorsal to Type III CRMs suggests that these factors may also collaborate in transcriptional repression. Interestingly, Twist binds to regulatory regions of all three Dorsal corepressors, providing another level at which Twist may modulate Dorsal-mediated repression. Overall, this exhaustive map of new CRMs for D-V patterning genes greatly extends previous knowledge and will likely improve predictive models for this system (Sandmann, 2007).

Twist targets functional modules required for diverse aspects of mesoderm development

Twist is not only required for D-V patterning. The 494 direct target genes are significantly enriched in functional groups of genes involved in cell communication, signal transduction, cell motility, and cell adhesion. Genes in these categories are essential for multiple aspects of development, including gastrulation and directed migration of mesodermal cells. Genetic studies have demonstrated a requirement for twist in these processes; however, the molecular mechanism remained ill-defined. These data reveals Twist binding to CRMs for entire functional modules necessary for both gastrulation and migration (the FGF pathway) (Sandmann, 2007).

The present study highlights a new direct connection between Twist and many key components involved in cell cycle progression and cell growth. Members of both the Cdk2/CyclinA/B and Cdk2/CyclinE complexes are targeted, as well as modifiers of their activity and genes involved in cytokinesis and replication. In many cases, Twist binds to several CRMs of these genes (e.g., cyclinE and E2f), revealing the complexity of their regulation. This surprising link between Twist and the cell cycle is highly likely to be of regulatory significance; twist mutant embryos have proliferative defects that can be genetically separated from the block in mesoderm gastrulation (Sandmann, 2007).

These three functional groups of target genes (involved in morphogenesis, migration, and cell proliferation) have been defined as essential developmental network 'plug-ins.' Twist orchestrates early mesoderm development by binding to CRMs of virtually all genes within functional groups essential for gastrulation, mesoderm proliferation, migration, and specification. In contrast, few CRMs for genes involved in terminal differentiation (e.g., sarcomere structure) are targeted by Twist (Sandmann, 2007).

Twist is a highly connected hub targeting a large repertoire of TFs

This global map of Twist-bound CRMs provides a first glimpse of Twist’s connectivity to the rest of the regulatory genome. Remarkably, TFs represent the largest group of Twist targets: Twist binds to CRMs of a striking 25% (113/454) of all sequence-specific Drosophila TFs. Among these are TFs essential for mesoderm development, including gap (hb, hkb, kr, kni), pair rule (eve, slp, opa, odd, prd, run), and segmentation genes (en, hh, ptc, wg), as well as homeotic genes (pb, Scr, Antp, Abd-A, Abd-B, Ubx). These classes of target genes implicate a new role for Twist in the establishment or maintenance of anterior-posterior patterning within the mesoderm in addition to its known role in D-V axis formation. Although the function of many of the remaining TFs is unknown, this data links these regulators to mesoderm development. The sheer number of TFs regulated by Twist does not support a simple hierarchical network, where Twist regulates a small set of TFs, which in turn control another layer of regulators, and so forth. Rather, the data suggests a model for Twist contributing to the regulation of the majority of TFs involved in every aspect of early mesoderm development (Sandmann, 2007).

Temporal enhancer occupancy by Twist reveals stage-specific coregulators

Although Twist is expressed during both developmental time periods assayed, it binds to CRMs in a temporally regulated manner. Approximately half of the enhancer regions are detected at both time periods, indicating continuous binding of Twist throughout these developmental stages. In contrast, 23% of Twist-CRMs are only bound in early development (2-4 h), while 26% are specific to later time periods (4-6 h). This dynamic occupancy reveals that the ability of Twist to bind to CRMs is tightly controlled beyond the mere presence of a suitable binding site, and is likely regulated by other TFs that aid or inhibit binding. To identify additional regulators that could differentiate between temporally bound CRMs, a search was performed for overrepresented sequence motifs, using two complementary computational approaches: statistical enrichment of position weight matrices (PWMs) for characterized TFs, and the de novo detection of overrepresented motifs (Sandmann, 2007).

Twist and Snail consensus motifs are significantly overrepresented in all three groups of CRMs, indicating a potential for extensive cobinding between these two TFs. In contrast, Dorsal motifs are exclusively enriched in the early-bound CRMs, and not in the late group. While Tinman motifs are specifically overrepresented in the continuous and late-bound CRMs. A number of other motifs were also uncovered, including sites for potential Twist/Daughterless heterodimers, suggesting additional mechanisms to generate diverse outputs from Twist-CRMs (Sandmann, 2007).

These data reveals Twist binding to almost all previously characterized Dorsal enhancers. Twist and Dorsal are known to interact physically and to coregulate enhancers in the early, but not the late, time window of this experiment. It is therefore hypothesized that Dorsal may be coregulating many of the newly discovered Twist CRMs, in keeping with the specific enrichment of Dorsal consensus motifs within these enhancers. To experimentally test Dorsal's presence on predicted sites in vivo, ChIP experiments were performed at 2-4 h of development. Significant binding of Dorsal was detected by quantitative real-time PCR to all seven predicted sites tested. Similarly, since Tinman consensus sites were significantly enriched in 4-6-h CRMs, the in vivo occupancy of predicted sites by Tinman was tested at this stage of development. ChIP experiments detected significant binding of Tinman to 10 of 11 sites tested. Given the large number of early and late CRMs, the enrichment of these motifs highlights extensive combinatorial binding of Dorsal and Twist at 2-4 h, and Tinman and Twist at 4-6 h. A substantial part of Twist's temporal specificity likely stems from its association with these upstream and downstream coregulators (Sandmann, 2007).

A core transcriptional network for early mesoderm development

To delineate the combinatorial relationships between Twist and other TFs, an initial transcriptional network was generated for early mesoderm development. The temporal binding map for Twist was integrated with in vivo binding data for Mef2, Dorsal, and Tinman. A previous study of Mef2-bound enhancers offers the largest collection of regulatory regions bound at this stage of development to date. As it is difficult to visualize all 494 Twist target genes, focus was placed on TFs whose CRMs are cobound by two or more regulators during these stages of development. Therefore, all links in this network represent direct connections to the same CRM at the same stages of development (Sandmann, 2007).

The resulting core network of 51 TFs is already relatively complex, with nine genes [nau, E(spl), eve, bap, Ubx, lbe, odd, hth, and Ptx1] being targeted by three out of the four examined regulators. The topology of the network provides several insights into how Twist functions to regulate multiple aspects of early mesoderm development. Extensive combinatorial binding and feed-forward regulation are abundant features. Dorsal activates twist, which in turn coregulates the majority of known direct Dorsal targets. This network motif is even more prominent within the mesoderm: Twist regulates the expression of Mef2 and tinman, and cobinds with these TFs to many of their targets' enhancers. In fact, Twist co-occupies 42% of all Mef2-bound enhancers during early mesoderm development. Depending on the logical inputs from the two upstream regulators (transcriptional repression or activation), feed-forward loops can aid in cellular decision making by filtering out noisy regulatory inputs or control the timing of a transcriptional response. For example, early gene expression in the mesoderm (e.g., activation of tin) depends on Twist alone, while transcription of other genes initiated at a later stage may require the input from both Twist and Tinman proteins (Sandmann, 2007).

Twist-bound CRMs correspond to silencers as well as enhancers of transcription

Through the integration of ChIP-on-chip analysis with expression profiling data during early stages of Drosophila development, this study has identified >2000 Twist-bound regulatory regions and almost 500 direct target genes. This data, in combination with in vivo binding data for other TFs, lays the foundation of a transcriptional network describing early mesoderm development. The resulting network view reveals regulatory features that form the basis of Twist's functional versatility (Sandmann, 2007).

The data revealed extensive Twist binding to characterized Dorsal enhancers and also, surprisingly, to Dorsal-regulated silencers (e.g., dpp). Moreover, many of the new regulatory regions identified for D-V patterning genes can function either as enhancers or integrated enhancer-silencer modules (e.g., WntD and crumbs). This ability of Twist to act within the context of silencers, as well as enhancers, may partially explain the widespread recruitment of Twist to many regulatory regions and its ability to regulate diverse developmental processes (Sandmann, 2007).

An attractive molecular explanation for this bifunctionality is the potential of Twist to form both homodimers and heterodimers. Twist homodimers drive gene activation in Drosophila, while Twist-Daughterless heterodimers are associated with transcriptional repression. This model is supported by the significant overrepresentation of the Twist/Daughterless heterodimer consensus motif in both 2-4-h and 4-6-h CRMs. Direct protein-protein interactions between Twist and Dorsal is an alternative mechanism for Twist's incorporation into repressive complexes (Sandmann, 2007).

A network with unexpected topology governs early mesoderm development

Although the network generated in this study is far from complete, it represents the largest set of combinatorial-bound CRMs during these stages of development described to date, and therefore provides a comprehensive resource to decipher general regulatory principles. The resulting network topology was surprising. Instead of Twist regulating a restricted group of TFs, which in turn regulate a successive wave of transcription in a relay model, Twist directly impinges on CRMs for the vast majority of genes expressed in the early mesoderm (Sandmann, 2007).

The extent of combinatorial binding was also unanticipated. There is extensive cobinding of Twist and Dorsal to early 2-4-h CRMs. In fact, the presence of Dorsal binding may be a general prerequisite for Twist binding to enhancers specific for early development. The cooperative binding of Dorsal and Twist to the rho and sim CRMs supports this model. At 4-6 h of development, the composition of TFs impinging on Twist-bound CRMs changes. Although genome wide ChIP-on-chip data is currently not available for Tinman, the significant overrepresentation of Tinman motifs in Twist-bound CRMs and the ability of Tinman to bind to the majority of sites tested indicates prevalent combinatorial binding between these two TFs during 4-6 h of development. Comparing Twist-bound CRMs with a previously generated data set for Mef2 revealed extensive cobinding to enhancers early in development. Converging regulatory connections through combinatorial binding can produce diverse logical outputs, depending on the nature of the TFs. The cobinding of several pan-mesodermal TFs (Twist, Tinman, and Mef2) may ensure robust gene expression. While in other contexts (for example, the WntD-enhancer) the combined inputs of Twist and Snail allow for spatial fine-tuning of gene expression (Sandmann, 2007).

The core network also revealed an abundance of feed-forward loops, providing directionality during early mesoderm development. This is prevalent with both upstream regulators of Twist (Dorsal and Twist) and downstream regulators (Tinman and Twist and Mef2 and Twist). This network motif will likely become even more widespread as additional ChIP-on-chip data becomes available. Twist targets an astounding number of TFs, which may represent an almost complete repertoire of TFs required for early mesoderm development. It is tempting to speculate that Twist participates in feed-forward regulation, with many of these factors through combinatorial binding to different subsets of the ~2000 Twist-bound CRMs (Sandmann, 2007).

Temporal network dynamics reflect developmental progression

Both the composition and connectivity of regulatory networks describing developmental progression will naturally change over time. To capture dynamic changes within the early mesodermal network, these experiments were performed at consecutive time periods. The data reveals temporally regulated binding of Twist to three classes of CRMs: early, continuous, and late. Similar temporally restricted enhancer occupancy has also been observed for other regulators with broad temporal expression, suggesting that this may be a general feature of developmental networks—e.g., MyoD, PHA-4, and Mef2 (Sandmann, 2007).

The temporal occupancy of specific CRMs by Twist reflects the development of this tissue. At 2-4 h of development, Twist and Dorsal coregulate genes essential for D-V patterning. Twist also targets an almost complete set of genes essential for gastrulation and is required to progress to the next phase of development, mesoderm maturation. During this developmental window, the predominant target genes are part of functional modules essential for the cell migration, proliferation, patterning, and specification events occurring within the mesoderm at these stages. As expected for a TF essential for early aspects of mesoderm development, Twist does not bind to significant numbers of CRMs for genes involved in terminal differentiation. This is in sharp contrast to Mef2, which first co-occupies CRMs involved in early mesoderm development with Twist, and later selectively regulates an alternative group of CRMs driving genes involved in later aspects of differentiation; e.g., sarcomere structure or muscle attachment (Sandmann, 2007).

Conserved regulation of functional classes of genes by Twist

Integrating these data with genetic evidence from other species suggests that the regulation of several functional gene cassettes by Twist is conserved throughout evolution, from flies to man. These include (1) the FGF signaling pathway: Mutations in human FGF receptors phenocopy mutants in human twist (Htwist). (2) Genes implicated in epithelial-mesenchymal transitions (EMTs): In mice and humans, Twist facilitates tumor metastasis through the promotion of EMTs. (3) Cell proliferation and apoptosis: Htwist has been classified as a potential oncogene, since it maintains tissue culture cells in a proliferative state. Interestingly, ectopic expression of Htwist in Drosophila also induces proliferation and inhibits p53-dependent apoptosis, indicating that the ability to regulate these processes is conserved. However, for each process, only a few Twist-regulated genes have been known. Extrapolating from the current findings in flies points toward a role for Twist in the direct regulation of entire gene modules required for each process in vertebrates (Sandmann, 2007).

An emerging model for Twist as a global competence factor for mesoderm development

The results provide an initial global view of the transcriptional network describing early mesoderm development within the metazoan Drosophila. Twist resides at the top of this network and binds to CRMs for the vast majority of genes that need to be expressed during these stages. In many cases, Twist is essential and sufficient to drive expression of the target gene. In other cases, however, the contribution of Twist remains unclear (e.g., crumbs and NetA) . Rather than acting as a binary switch, Twist may act redundantly with other TFs. Alternatively, Twist may provide the competence for more specific TFs to bind to these CRMs; for example, by acting as a pioneer TF to facilitate chromatin remodeling (Sandmann, 2007).

In species as diverse as flies, jellyfish, and mice, Twist is only expressed in mesodermal cells when they are in an immature state, and loss of twist expression correlates with the initiation of differentiation. Moreover, overexpression of Twist-1 in mice is sufficient to block osteoblast differentiation. It is suggested that Twist provides the mesoderm with the competence to be pluripotent: first, by providing these cells with the components necessary to respond to inductive cues directing further specification; and second, by providing an almost universal repertoire of mesodermal CRMs with the competence to respond to other TFs. Once bound by Twist, these regulatory regions may be primed for activation by more specialized TFs, and thereby allow rapid developmental progression at the appropriate time (Sandmann, 2007).

GENE STRUCTURE

Exons - two

Bases in 3' UTR - 251

date revised:  22 May 2023