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).

GENE STRUCTURE

Exons - two

Bases in 3' UTR - 251

date revised:  3 July 97