BIOLOGICAL OVERVIEW

daughterless is so named because of its role in sex determination. It is required for the maturation of follicle cells during egg chamber morphogenesis. The dimerization partner of daughterless in the maturation of follicle cells is unknown (Gonzalez-Crespo, 1993 and Cummings, 1994). In other roles daughterless interacts with Dorsal to bring about the induction of twist and snail, genes required for gastrulation. daughterless is a cofactor for their activation.

However, it is the involvement of daughterless in neural differentiation that is considered of primary developmental importance. Although daughterless is not required for the formation and delamination of "nascent" neuronal precursors from the epidermal layer, it is required for expression of neuron specific genes. Mutation of da blocks transformation of presumptive precursors into true precursors. Since AS-C genes are required for cells to become neuronal precursors, this requirement is fulfilled in the absence of daughterless (Vassin, 1994). This result is paradoxical because it is presumed that DA is the dimerization partner of AS-C proteins. How can Achaete and Scute carry out their proneural function without DA?

The list of genes activated by Daughterless as a cofactor with achaete-scute complex genes will continue to grow. Known targets include prospero, cyclin A and calmodulin (Vaessin, 1994 and Kovalick, 1992).

Proneural gene products like Daughterless and Lethal of scute can bind to promoters of Enhancer of split and achaete genes, and by so doing, activate their transcription. Two proteins of the E(spl)-C (HLH-M5 and Enhancer of split) attenuate the transcriptional activation mediated by the proneural genes. This observation begins to untangle the complicated role of E(spl)-C genes in neurogenesis. Once neuroblasts have segregated, products of proneural genes become restricted to the neuroblasts. Products of the E(spl)-C genes are restricted to cells remaining in the epithelium. Therefore it appears that E(spl)-C functionally antagonizes the proneural proteins and thus silences expression of genes that are activated by the proneural genes (Oellers, 1994).

Daughterless couples the control of differentiation and cell cycle programs in in the developing sensory organ precursor (SOP). Although Daughterless is required for the proper expression of neuronal precursor genes and lineage identity genes in the peripheral nervous system (PNS) of Drosophila embryos, this requirement does not explain the failure of the nascent PNS precursors to undergo a normal cell cycle and divide in da mutants. Four genes whose products are required for various stages of the cell cycle are misexpressed in the PNS of da mutant embryos. Cyclin A, barren, disc proliferation abnormal and Histone H1 transcripts are significantly reduced or undetectable in the precursors of the PNS at stages 11 and 12. Precursors are still present at these stages in da mutants. This suggests that all aspects of PNS precursor differentiation examined so far are under the transcriptional control of da. Sensory organ precursors lacking Da may fail to express and/or accumulate other factors, such as critical differentiation genes, required for SOP entry into the cell cycle. It should be pointed out that these factors are unlikely to be the thus-far described neuronal precursor genes, as mutations in these genes do not result in any obvious cell cycle defects. Thus daughterless controls the expression of cell cycle genes in the PNS sensory organ precursors but nowhere else (Hassan, 1997).

The basic helix-loop-helix transcription factor Twist regulates a series of distinct cell fate decisions within the Drosophila mesodermal lineage. These twist functions are reflected in its dynamic pattern of expression, which is characterized by initial uniform expression during mesoderm induction, followed by modulated expression at high and low levels in each mesodermal segment, and finally restricted expression in adult muscle progenitors. Two distinct partner-dependent functions for Twist were found that are crucial for cell fate choice. Twist can form homodimers and heterodimers in vitro with the Drosophila E protein homolog, Daughterless. Using tethered dimers to assess directly the function of these two particular dimers in vivo, it has been shown that Twist homodimers specify mesoderm and the subsequent allocation of mesodermal cells to the somatic muscle fate. Misexpression of Twist-tethered homodimers in the ectoderm or mesoderm leads to ectopic somatic muscle formation overriding other developmental cell fates. In addition, expression of tethered Twist homodimers in embryos null for twist can rescue mesoderm induction as well as somatic muscle development. Loss of function analyses, misexpression and dosage experiments, and biochemical studies indicate that heterodimers of Twist and Daughterless repress genes required for somatic myogenesis. It is proposed that these two opposing roles explain how modulated Twist levels promote the allocation of cells to the somatic muscle fate during the subdivision of the mesoderm. Moreover, this work provides a paradigm for understanding how the same protein controls a sequence of events within a single lineage (Castanon, 2001).

At stage 10, in response to transcriptional regulators such as Sloppy paired and Even skipped, as well as signals from the overlying ectoderm such as Wingless, the uniform expression of Twist modulates into regions of high and low expression within each segment. Da is expressed uniformly in the mesoderm at this time. The region that maintains high Twist levels subsequently gives rise to somatic muscles whereas the region that has lower Twist levels gives rise to tissues such as visceral muscle, fat body, gonadal mesoderm and some glia cells. The heart is derived from the region that initially expresses high levels of Twist; however these cells lose Twist expression, an event necessary for the execution of heart fate. Expressing high Twist levels in cells destined to become visceral muscle, for example, blocks visceral muscle differentiation and promotes somatic muscle. Reduction of Twist levels in cells normally expressing high Twist levels blocks somatic myogenesis (Castanon, 2001 and references therein).

Several possible mechanisms are provided to explain these observations and illustrate the in vivo roles for the two opposing activities of Twist homodimers and Twist/Da heterodimers. Regions that normally express lower Twist levels do not form somatic muscles owing to higher concentrations of Twist/Da heterodimers as compared to Twist homodimers. These heterodimers repress transcription of pro-muscle genes, such as lísc as well as founder cell genes such as Kr, thereby prohibiting somatic muscle development. Other differentiation programs for visceral muscle or fat body development can proceed unaffected. No evidence is found that Twist/Da heterodimers promote visceral mesoderm or fat body fate through the direct activation of targets such as Fas III. Regions that normally express higher Twist levels do form somatic muscle owing to higher concentrations of Twist homodimers as compared to Twist/Da heterodimers. Dimer competition, then, restricts the developmental potential of mesodermal cells, by not allowing Twist homodimers to convert all mesodermal cells into somatic muscle (Castanon, 2001).

These conclusions are consistent with the observations that increasing Twist/Da levels, either by overexpression of Da or the tethered Twist-Da heterodimer, repress the earliest steps in somatic myogenesis. These are the same steps that are activated by Twist homodimers. For example, Lísc expression, which marks clusters of equipotential cells that segregate the muscle founder cells, is drastically reduced or absent upon an increase of Twist/Da heterodimers. This indicates an early failure in the somatic muscle program. Likewise failure in subsequent steps is seen; for example, few founder cells as well as few identifiable muscles are detected. These failures in muscle development are interpreted as an outcome of the initial block in the differentiation pathway. The possibility that overexpression of Da or of Twist-Da could directly repress these subsequent steps is not eliminated. Gal4 lines that drive expression at later stages of muscle development or in particular subsets of muscle cells (i.e., the S59-expressing founder cells) could provide insight into this alternative (Castanon, 2001).

Daughterless dictates Twist activity in a context-dependent manner during somatic myogenesis

Somatic myogenesis in Drosophila relies on the reiterative activity of the basic helix-loop-helix transcriptional regulator, Twist (Twi). How Twi directs multiple cell fate decisions over the course of mesoderm and muscle development is unclear. Previous work has shown that Twi is regulated by its dimerization partner: Twi homodimers activate genes necessary for somatic myogenesis, whereas Twi/Daughterless (Da) heterodimers lead to the repression of these genes. This study examined the nature of Twi/Da heterodimer repressive activity. Analysis of the Da protein structure revealed a Da repression (REP) domain, which is required for Twi/Da-mediated repression of myogenic genes, such as Dmef2, both in tissue culture and in vivo. This domain is crucial for the allocation of mesodermal cells to distinct fates, such as heart, gut and body wall muscle. By contrast, the REP domain is not required in vivo during later stages of myogenesis, even though Twi activity is required for muscles to achieve their final pattern and morphology. Taken together, evidence is presented that the repressive activity of the Twi/Da dimer is dependent on the Da REP domain and that the activity of the REP domain is sensitive to tissue context and developmental timing (Wong, 2008).

This study explores the regulation of Twi activity through mesoderm development and somatic myogenesis in Drosophila . Focus was placed on how Twi is modulated by its dimer partner, Da. The examination of Twi/Da dimers revealed that the activity of these dimers is acutely sensitive to their tissue environment: both between germ layers (the ectoderm versus the mesoderm), and within cell lineages (early mesoderm versus somatic muscle). This sensitivity is determined, in part, by the activity of the Da REP domain, which is critical for Twi/Da activity during mesodermal subdivision and FC specification, but is not required for the later activity of Twi/Da during muscle differentiation. This work provides insight to the mechanism of Twi/Da activity and calls attention to the effect of tissue context and developmental timing on bHLH protein regulation (Wong, 2008).

One of the most striking aspects of this study is the role of the Da REP domain in switching Twi/Da behaviour between a repressor and an activator function. This 'switchable' behaviour of Twi/Da activity was initially observed by its ability to inhibit myogenesis in the mesoderm, but activate myogenesis in the ectoderm. Notably, the deletion of the REP domain from Da has little effect on Da activity in the absence of Twi, as demonstrated by cell culture transcriptional assays. However, the activity of Twi-Da^Δ tethered dimers has a distinct effect on the mesoderm. Overexpression of these dimers had the greatest effect on somatic myogenesis during the process of mesodermal subdivision. The detection of increased numbers of founder cells (FCs), which appear to be specified normally, indicated an increased number of mesodermal cells being allocated to a somatic muscle fate at the expense of cardiac and visceral mesoderm (Wong, 2008).

An outstanding question is how the Da REP domain functions to modulate Twi/Da activity. Since Twi/Da dimers bind DNA and therefore may actively regulate the transcriptional state of a target gene, it was initially postulated that the REP domain must directly interact with transcriptional corepressors or factors that were expressed solely in the mesoderm and therefore were required for the repressive activity of Twi/Da in that tissue context. Exhaustive studies were conducted to identify these factors but no protein that satisfies all necessary criteria has been identified (Wong, 2008).

Deletion analysis of the E protein Rep domain suggested that this domain is required for the repression of the E protein activation domains, AD1 and AD2. Like the Da REP domain, the E protein Rep domain has specific activities depending on its dimer partner and tissue context (Markus, 2002). Informed by this work, the current data was interpreted to suggest that the Da REP domain is a cis-acting repressor, which functions to repress both Da AD1 and AD2 when Da is dimerized to Twi and bound to myogenic enhancers. Moreover, the effect of the Da REP domain is not restricted to the E protein/Da protein family. This work suggests that the Da REP domain also represses Twi's activation domains, Twi-AD1 and Twi-AD2, in Twi/Da dimers. It is proposed that the Da REP domain acts to mask the activation domains in both Twi and Da. Therefore, the net effect of the Da REP domain results in the recruitment of corepressors to myogenic enhancers by Twi/Da dimers. Alternatively, Twi/Da dimers may not actively repress target myogenic genes: instead, these dimers could compete for myogenic E boxes or transcriptional cofactors and machinery. In this model of passive repression, the Da REP domain could function to stabilize interactions with Twi or other factors that are required to properly mediate repression of myogenic target genes. These aspects of Da REP domain repression are currently being evaluated (Wong, 2008).

To date, various transcriptional regulators have been shown to have different activities and target genes in different tissues and be modulated by dimerization partners. Recently, ChIP-on-chip analyses have identified almost 500 direct Twi targets throughout mesodermal development. This study, however, is one of the first that focuses on how Twi activity is dynamically modulated through multiple developmental stages of a specific cell lineage, and how this regulation affects expression of Twi target genes (Wong, 2008).

One gene that is regulated by Twi dimers throughout somatic myogenesis is Dmef2. Dmef2 protein is expressed throughout and necessary for all stages of myogenesis. Dmef2 coordinates multiple processes necessary for proper somatic myogenesis. Moreover, it has been suggested that Dmef2 is required in combination with Twi to regulate the expression of a subset of Twi target genes in a feed-forward mechanism. The current data support these arguments, since mesodermal phenotypes were observed in Twi/Da^Δ (activation) or Twi/Da (repression) overexpressing embryos that mirror those of embryos overexpressing Dmef2 or in Dmef2 mutant embryos, respectively. For example, increased Dmef2 reporter gene expression and increased numbers of FCs were observed in embryos that overexpress Twi/Da^Δ panmesodermally. Consistent with these observations, Dmef2 has been shown to regulate components of the Ras/MAPK and Notch pathways, which are both required for the proper specification of FCs, and the expression of a subset of FC identity genes. Dmef2 has also been shown to regulate a subset of genes that are required for myoblast fusion and muscle attachment, processes required for proper muscle morphogenesis. This study found that Twi/Da and Twi/Da^Δ dimers disrupt myoblast fusion and muscle differentiation, which is likely due to these dimers repressing Dmef2 expression. In agreement with this observation, muscle analysis revealed that embryos overexpressing Twi-Da and Twi-Da^Δ dimers have muscle phenotypes that are similar to those observed in Dmef2⁴²⁴ hypomorph embryos and Dmef2^22.21 null embryos that have been partially rescued by UAS-Dmef2 transgenes. Taken together, these results supported our conclusions of the pivotal regulation of Dmef2 activity by Twi dimers throughout myogenesis (Wong, 2008).

Another notable question is how the Da REP domain is required for Twi/Da mediated transcriptional repression during mesodermal subdivision, but not during muscle morphogenesis. One possibility is that during somatic muscle differentiation, the repressive activity of Twi/Da relies on a different protein domain. Another possibility includes the changes in Twi/Da target genes through the course of somatic myogenesis. Studies conducted on chromatin remodeling have emphasized the specificity involved with the transcriptional regulation of a single gene. Therefore, it is likely that the regulation of multiple sets of genes through time would rely on the modular nature of transcriptional regulators. The Da REP domain may be required for the repression of a subset of Twi/Da target genes, whereas other target genes are unresponsive to this domain's repressive activity (Wong, 2008).

In summary, these results suggest that the regulation of Dmef2 by Twi/Da throughout myogenesis and the subsequent feed-forward mechanism by which Dmef2 and Twi regulate myogenic genes is critical for the coordination of the various disparate processes-mesodermal subdivision, FC specification, and muscle differentiation-necessary for somatic myogenesis (Wong, 2008).

Twi proteins are conserved across species [mouse, chicken, C. elegans, and jellyfish] and have been shown to dimerize with Da homologs, suggesting that REP domain regulation of Twi activity is conserved. Similarly to flies, Mouse Twi1 (MTwi1) heterodimerizes with E proteins to compete with MyoD/E proteins for binding sites on myogenic enhancers. In this manner, MTwi1/E protein heterodimers act like Twi/Da dimers to repress myogenesis. In other tissues, however, MTwi1/E protein heterodimers have been identified as an activator of targets, such as thrombospondin-1 during cranial suture formation. Therefore, like Twi/Da, MTwi1/E protein heterodimers are sensitive to tissue contexts. Of particular interest would be the examination of the E protein Rep domain in vivo. The function of this domain has been studied in mammalian cell culture, but not yet investigated in developmental processes. Moreover, the function of the E protein Rep domain has not been addressed in MTwi1/E protein dimers (Wong, 2008).

Notably, Twi proteins have also been implicated in a variety of tumourigenic processes, such as the inhibition of apoptosis and the coordination of metastasis. Mouse models and correlative data from human tumour samples suggest that MTwi1 and human Twi1 (HTwi1), respectively, direct epithelial-to-mesenchymal transitions (EMT) during breast cancer metastasis. The involvement of Twi1 in the complex process of cancer has many similarities to the developmental processes that Twi directs in the fly mesoderm, which include cell proliferation and cell migration, processes that have been recently revealed to be directly regulated by Twi. The role of the Da REP domain in directing Twi/Da transcriptional repression, and the tissue specificity of this domain's activity has illuminated various aspects of Twi regulation. It is anticipated that these findings will shed light on mammalian Twi1 activity and the Twi family of proteins in development and disease (Wong, 2008).

GENE STRUCTURE

Bases in 5' UTR - 229

Exons - two

Bases in 3' UTR - 992

PROTEIN STRUCTURE

Amino Acids - 710

Structural Domains

One-third of the way into the protein there is a tandem repeat of a histidine-rich region. This is followed by a PEST sequence, a "myc similarity region," (a basic HLH domain) and a C terminal lysine repeat region (Caudy, 1988 and Cronmiller, 1988).

date revised: 15 November 2001  

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