Twisted Activation Key Generator \/\/FREE\\\\

Twisted Activation Key Generator \/\/FREE\\\\

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The Twist 2 pattern generator begins as a familiar step-sequencer, and rapidly escalates into an unbelievable tool for sonic creation. In addition to pitch and velocity, the pattern generator provides additional tabs for adding precise measured-step control of crucial sound creation parameters. Portamento can be selected for each step, as can the filter frequency. LFO depth, rate, and sync can be defined per step. The harmonic width and content for each layer can also be specified. By automating even just one or two of these parameters, the pattern generator can infuse your sound with awe-inspiring depth and motion. Pattern presets can also be saved, recalled, and quickly modified on demand.

Symbol - twi FlyBase ID:FBgn0003900 Genetic map position - 2-[102] Classification - bHLH Cellular location - nuclear NCBI link: Entrez Gene twi orthologs: Biolitmine Recent literatureXie, S. and Martin, A. C. (2015) Intracellular signalling and intercellular coupling coordinate heterogeneous contractile events to facilitate tissue folding. Nat Commun 6: 7161. PubMed ID: 26006267 Summary: Cellular forces generated in the apical domain of epithelial cells reshape tissues. Recent studies highlighted an important role for dynamic actomyosin contractions, called pulses, that change cell and tissue shape. Net cell shape change depends on whether cell shape is stabilized, or ratcheted, between pulses. Whether there are different classes of contractile pulses in wild-type embryos and how pulses are spatiotemporally coordinated is unknown. This study developed a computational framework to identify and classify pulses and determine how pulses are coordinated during invagination of the Drosophila ventral furrow. Biased transitions in pulse behaviour were demonstrated, where weak or unratcheted pulses transition to ratcheted pulses. The transcription factor Twist directs this transition, with cells in Twist-depleted embryos exhibiting abnormal reversed transitions in pulse behaviour. Ratcheted pulses were shown to have higher probability of having neighbouring contractions, and that ratcheting of pulses prevents competition between neighbouring contractions, allowing collective behaviour.Lin, S., Ewen-Campen, B., Ni, X., Housden, B. E. and Perrimon, N. (2015). In vivo transcriptional activation using CRISPR-Cas9 in Drosophila. Genetics [Epub ahead of print]. PubMed ID: 26245833 Summary: A number of approaches for Cas9-mediated transcriptional activation have recently been developed, allowing target genes to be over-expressed from their endogenous genomic loci. However, these approaches have thus far been limited to cell culture, and this technique has not been demonstrated in vivo in any animal. The technique involving the fewest separate components, and therefore most amenable to in vivo applications, is the dCas9-VPR system, where a nuclease-dead Cas9 is fused to a highly active chimeric activator domain. This study characterized the dCas9-VPR system in Drosophila cells and in vivo. This system can be used in cell culture to upregulate a range of target genes, singly and in multiplex, and a single guide RNA upstream of the transcription start site can activate high levels of target transcription. Marked heterogeneity in guide RNA efficacy was observed for any given gene, and transcription was observed to be inhibited by guide RNAs binding downstream of the transcription start site. To demonstrate one application of this technique in cells, dCas9-VPR was used to identify target genes for Twist and Snail. In addition, both Twist and Snail were simultaneously activated to identify synergistic responses to this physiologically relevant combination. Finally, dCas9-VPR were shown to activate target genes and cause dominant phenotypes in vivo. Transcriptional activation using dCas9-VPR thus offers a simple and broadly applicable technique for a variety of over-expression studies.Crocker, J, Ilsley, G.R. and Stern, D.L. (2016). Quantitatively predictable control of Drosophila transcriptional enhancers in vivo with engineered transcription factors. Nat Genet [Epub ahead of print]. PubMed ID: 26854918 Summary: Genes are regulated by transcription factors that bind to regions of genomic DNA called enhancers. Considerable effort is focused on identifying transcription factor binding sites, with the goal of predicting gene expression from DNA sequence. Despite this effort, general, predictive models of enhancer function are currently lacking. This study combine quantitative models of enhancer function with manipulations using engineered transcription factors to examine the extent to which enhancer function can be controlled in a quantitatively predictable manner. These models, which incorporate few free parameters, can accurately predict the contributions of ectopic transcription factor inputs. The effect of individual transcription factors can be considered as independent submodules of activity that are combined in a linear manner to produce a sigmoidal output. Individual submodules can also encode more complex inputs. For example, a submodule is represented by the saturating cooperativity displayed by Dorsal and Twist, whose combined output has an upper limit. The models allow the predictable 'tuning' of enhancers, providing a framework for the quantitative control of enhancers with engineered transcription factors.

Sandler, J. E. and Stathopoulos, A. (2016). Quantitative single-embryo profile of Drosophila genome activation and the dorsal-ventral patterning network. Genetics [Epub ahead of print]. PubMed ID: 26896327Summary: During embryonic development of Drosophila melanogaster, the Maternal to Zygotic Transition (MZT) marks a significant and rapid turning point when zygotic transcription begins and control of development is transferred from maternally deposited transcripts. Characterizing the sequential activation of the genome during the MZT requires precise timing and a sensitive assay to measure changes in expression. This study used the NanoString nCounter instrument, which directly counts mRNA transcripts without reverse transcription or amplification, to study over 70 genes expressed along the dorsal-ventral (DV) axis of early Drosophila embryos, dividing the MZT into 10 time points. Transcripts were quantified for every gene studied at all time points, providing the first data set of absolute numbers of transcripts during Drosophila development. Gene expression was found to change quickly during the MZT, with early Nuclear Cycle (NC) 14 the most dynamic time for the embryo. twist is one of the most abundant genes in the entire embryo and mutants were used to quantitatively demonstrate how it cooperates with Dorsal to activate transcription and is responsible for some of the rapid changes in transcription observed during early NC14. Elements within the gene regulatory network were uncovered that maintain precise transcript levels for sets of genes that are spatiotemporally co-transcribed within the presumptive mesoderm or dorsal ectoderm. Using this new data, it was shown that a fine-scale, quantitative analysis of temporal gene expression can provide new insights into developmental biology by uncovering trends in gene networks including coregulation of target genes and specific temporal input by transcription factors.Khoueiry, P., Girardot, C., Ciglar, L., Peng, P. C., Gustafson, E. H., Sinha, S. and Furlong, E. E. (2017). Uncoupling evolutionary changes in DNA sequence, transcription factor occupancy and enhancer activity. Elife 6. PubMed ID: 28792889 Summary: Sequence variation within enhancers plays a major role in both evolution and disease, yet its functional impact on transcription factor (TF) occupancy and enhancer activity remains poorly understood. This study assayed the binding of five essential TFs over multiple stages of embryogenesis in two distant Drosophila species (with 1.4 substitutions per neutral site), identifying thousands of orthologous enhancers with conserved or diverged combinatorial occupancy. The five factors examined, Twist, Mef2, Tinman (Tin), Bagpipe and Biniou, are the major drivers of the subdivision of the mesoderm into different muscle primordia and form part of a highly interconnected gene regulatory network. These binding signatures were used to dissect two properties of developmental enhancers: (1) potential TF cooperativity, using signatures of co-associations and co-divergence in TF occupancy. This revealed conserved combinatorial binding despite sequence divergence, suggesting protein-protein interactions sustain conserved collective occupancy. (2) Enhancer in-vivo activity, revealing orthologous enhancers with conserved activity despite divergence in TF occupancy. Taken together, this study has identified enhancers with diverged motifs yet conserved occupancy and others with diverged occupancy yet conserved activity, emphasising the need to functionally measure the effect of divergence on enhancer activity.Khuong, T. M., Wang, Q. P., Manion, J., Oyston, L. J., Lau, M. T., Towler, H., Lin, Y. Q. and Neely, G. G. (2019). Nerve injury drives a heightened state of vigilance and neuropathic sensitization in Drosophila. Sci Adv 5(7): eaaw4099. PubMed ID: 31309148Summary: Injury can lead to devastating and often untreatable chronic pain. While acute pain perception (nociception) evolved more than 500 million years ago, virtually nothing is known about the molecular origin of chronic pain. This study provides the first evidence that nerve injury leads to chronic neuropathic sensitization in insects. Mechanistically, peripheral nerve injury triggers a loss of central inhibition that drives escape circuit plasticity and neuropathic allodynia. At the molecular level, excitotoxic signaling within GABAergic (gamma-aminobutyric acid) neurons required the acetylcholine receptor nAChRalpha1 and led to caspase-dependent death of GABAergic neurons. Conversely, disruption of GABA signaling was sufficient to trigger allodynia without injury. Last, the conserved transcription factor Twist was identified as a critical downstream regulator driving GABAergic cell death and neuropathic allodynia. Together, this study has defined how injury leads to allodynia in insects, and describe a primordial precursor to neuropathic pain may have been advantageous, protecting animals after serious injury. Irizarry, J., McGehee, J., Kim, G., Stein, D. and Stathopoulos, A. (2020). Twist-dependent ratchet functioning downstream from Dorsal revealed using a light-inducible degron. Genes Dev. PubMed ID: 32467225 Summary: Graded transcription factors are pivotal regulators of embryonic patterning, but whether their role changes over time is unclear. A light-regulated protein degradation system was used to assay temporal dependence of the transcription factor Dorsal in dorsal-ventral axis patterning of Drosophila embryos. Surprisingly, the high-threshold target gene snail only requires Dorsal input early but not late when Dorsal levels peak. Instead, late snail expression can be supported by action of the Twist transcription factor, specifically, through one enhancer, sna.distal. This study demonstrates that continuous input is not required for some Dorsal targets and downstream responses, such as twist, function as molecular ratchets.Howard, A. M., Milner, H., Hupp, M., Willett, C., Palermino-Rowland, K. and Nowak, S. J. (2020). Akirin is critical for early tinman induction and subsequent formation of the heart in Drosophila melanogaster. Dev Biol. PubMed ID: 32950464Summary: The regulation of formation of the Drosophila heart by the Nkx 2.5 homologue Tinman is a key event during embryonic development. This study identified the highly conserved transcription cofactor Akirin as a key factor in the earliest induction of tinman by the Twist transcription factor. akirin mutant embryos display a variety of morphological defects in the heart, including abnormal spacing between rows of aortic cells and abnormal patterning of the aortic outflow tract. akirin mutant embryos have a greatly reduced level of tinman transcripts, together with a reduction of Tinman protein in the earliest stages of cardiac patterning. Further, akirin mutants have reduced numbers of Tinman-positive cardiomyoblasts, concomitant with disrupted patterning and organization of the heart. Finally, despite the apparent formation of the heart in akirin mutants, these mutant hearts exhibit fewer coordinated contractions in akirin mutants compared with wild-type hearts. These results indicate that Akirin is crucial for the first induction of tinman by the Twist transcription factor, and that the success of the cardiac patterning program is highly dependent upon establishing the proper level of tinman at the earliest steps of the cardiac developmental pathway.Domsch, K., Schroder, J., Janeschik, M., Schaub, C. and Lohmann, I. (2021). The Hox Transcription Factor Ubx Ensures Somatic Myogenesis by Suppressing the Mesodermal Master Regulator Twist. Cell Rep 34(1): 108577. PubMed ID: 33406430Summary: Early lineage-specific master regulators are essential for the specification of cell types. However, once cells are committed to a specific fate, it is critical to restrict the activity of such factors to enable differentiation. To date, it remains unclear how these factors are silenced. Using the Drosophila mesoderm as a model and a comparative genomic approach, the Hox transcription factor Ultrabithorax (Ubx) was shown to be critical for the repression of the master regulator Twist. Mesoderm-specific Ubx loss-of-function experiments using CRISPR-Cas9 and overexpression studies demonstrate that Ubx majorly impacts twist transcription. A mechanistic analysis reveals that Ubx requires the NK-homeodomain protein Tinman to bind to the twist promoter. Furthermore, these factor interactions were found to be critical for silencing by recruiting the Polycomb DNA binding protein Pleiohomeotic. Altogether, these data reveal that Ubx is a critical player in mediating the silencing of Twist, which is crucial for coordinated muscle differentiation. Denk-Lobnig, M., Totz, J. F., Heer, N. C., Dunkel, J. and Martin, A. C. (2021). Combinatorial patterns of graded RhoA activation and uniform F-actin depletion promote tissue curvature. Development. PubMed ID: 34124762Summary: During development, gene expression regulates cell mechanics and shape to sculpt tissues. Epithelial folding proceeds through distinct cell shape changes that occur simultaneously in different regions of a tissue. Using quantitative imaging in Drosophila melanogaster, this study investigated how patterned cell shape changes promote tissue bending during early embryogenesis. The transcription factors Twist and Snail combinatorially regulate a multicellular pattern of lateral F-actin density that differs from the previously described myosin-2 gradient. This F-actin pattern correlates with whether cells apically constrict, stretch, or maintain their shape. The myosin-2 gradient and F-actin depletion do not depend on force transmission, suggesting that transcriptional activity is required to create these patterns. The myosin-2 gradient width results from a gradient in RhoA activation that is refined through the balance between RhoGEF2 and the RhoGAP C-GAP. These experimental results and simulations of a 3D elastic shell model show that tuning gradient width regulates tissue curvature.Rose, M., Domsch, K., Bartle-Schultheis, J., Reim, I. and Schaub, C. (2022). Twist regulates Yorkie activity to guide lineage reprogramming of syncytial alary muscles. Cell Rep 38(4): 110295. PubMed ID: 35081347Summary: Genesis of syncytial muscles is typically considered as a paradigm for an irreversible developmental process. Notably, transdifferentiation of syncytial muscles is naturally occurring during Drosophila development. The ventral longitudinal heart-associated musculature (VLM) arises by a unique mechanism that revokes differentiation states of so-called alary muscles and comprises at least two distinct steps: syncytial muscle cell fragmentation into single myoblasts and successive reprogramming into founder cells that orchestrate de novo fiber formation of the VLM lineage. This study provides evidence that the mesodermal master regulator twist plays a key role during this reprogramming process. Acting downstream of Drosophila Tbx1 (Org-1), Twist is regulating the activity of the Hippo pathway effector Yorkie and is required for the initiation of syncytial muscle dedifferentiation and fragmentation. Subsequently, fibroblast growth factor receptor (FGFR)-Ras-mitogen-activated protein kinase (MAPK) signaling in resulting mononucleated myoblasts maintains Twist expression, thereby stabilizing nuclear Yorkie activity and inducing their lineage switch into founder cells of the VLM.Rose, M., Domsch, K., Bartle-Schultheis, J., Reim, I. and Schaub, C. (2022). Twist regulates Yorkie activity to guide lineage reprogramming of syncytial alary muscles. Cell Rep 38(4): 110295. PubMed ID: 35081347Summary: Genesis of syncytial muscles is typically considered as a paradigm for an irreversible developmental process. Notably, transdifferentiation of syncytial muscles is naturally occurring during Drosophila development. The ventral longitudinal heart-associated musculature (VLM) arises by a unique mechanism that revokes differentiation states of so-called alary muscles and comprises at least two distinct steps: syncytial muscle cell fragmentation into single myoblasts and successive reprogramming into founder cells that orchestrate de novo fiber formation of the VLM lineage. This study provides evidence that the mesodermal master regulator twist plays a key role during this reprogramming process. Acting downstream of Drosophila Tbx1 (Org-1), Twist is regulating the activity of the Hippo pathway effector Yorkie and is required for the initiation of syncytial muscle dedifferentiation and fragmentation. Subsequently, fibroblast growth factor receptor (FGFR)-Ras-mitogen-activated protein kinase (MAPK) signaling in resulting mononucleated myoblasts maintains Twist expression, thereby stabilizing nuclear Yorkie activity and inducing their lineage switch into founder cells of the VLM that is critical for regulating myosin activity, leads to structural defects. It was further shown that Rbfox1 directly binds the 3'-UTR of target transcripts, regulates the expression level of myogenic transcription factors myocyte enhancer factor 2 and Salm, and both modulates expression of and genetically interacts with the CELF family RNA-binding protein Bruno1 (Bru1). Rbfox1 and Bru1 co-regulate fiber type-specific alternative splicing of structural genes, indicating that regulatory interactions between FOX and CELF family RNA-binding proteins are conserved in fly muscle. Rbfox1 thus affects muscle development by regulating fiber type-specific splicing and expression dynamics of identity genes and structural proteins.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 TwistOne 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 Nnull 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)-Cmutant 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. Thisaspect of Notch signaling has been described in other systems, and theearly 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 hypothesisthat Notch signaling regulates twist directly, as well as indirectlyby activating expression of a 'repressor of twist.' This indirectrepression of twist concurs with the role of Notch in activatingE(spl) transcriptional repressors. Moreover, a mechanism involvingdirect and indirect regulation is consistent with Su(H) mutantphenotypes. In Su(H)null embryos, neither twistnor repressor of twist (for example, emc) are repressed. Thede-repression of both genes at the same time results in Twist expressionappearing '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 Twistexpression is not observed because repressor of twist isexpressed (Tapanes-Castillo, 2004).However, simple direct and indirect regulation [through emc andE(spl)-C genes] by Notch still does not fully explain the phenotypes of Notch mutants. Both twistand repressor of twist should be repressed inNnull embryos because Su(H) will remain in its repressorstate. While the Nnull phenotype was consistent withrepressor of twist being repressed, twist was still stronglyexpressed. Additionally, constitutive Notch activation should cause both twist and repressor of twist to be expressed. Consequently,Nintra was expected to cause a phenotype similar to that caused by Su(H)-VP16. Contrary to these predictions, panmesodermal expression ofNintra represses Twist, consistent with only repressor oftwist 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 atstage 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 thattwist and a repressor of twist gene, such asE(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 (Nicd) first to alleviateSu(H)-mediated repression and then to serve as a coactivator for Su(H).Transcription of Notch permissive target genes requires Nicd solely to de-repress Su(H); Su(H) bound to other coactivators and/or othertranscriptional activators is necessary for permissive gene activation. Since panmesodermal expression of Nintra 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 permissivelyon the twist promoter. By contrast, panmesodermal expression ofNintra is sufficient to activate a repressor of twist,resulting in the strong Twist repression. Since E(spl)-Cgenes have been categorized as Notch instructive target genes, it issuggested that E(spl)-C genes are the Notch instructive repressor of twist genes in this system. Although Notch can upregulate Emc expression, the inability tosee a change in Emc expression in Nnull andSu(H)null mutants suggests Emc is not a Notch instructive target gene. Thus, based on all of this work, theinstructive 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 proteinmissing the two EGF repeats critical for Dl binding (EGFrepeats 11 and 12) rescues Twist modulation in Nnullmutant embryos. Thus Notch doesnot require EGF-like repeats 10-12 to repress Twist. These preliminary datasuggest that Delta may use EGF-like repeats other than 10-12 to activateNotch. Alternatively, Notch may not be activated by canonicalDelta signaling; a novel (non-DSL) ligand may activate Notch in the earlymesoderm. 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 segmentallyrepeated pattern of low and high Twist domains -- that is, periodicity in Twistexpression -- has yet to be understood. Two models, consistent with the data, are proposed to describe howNotch signaling contributes to a modulated Twist pattern. Model I proposesthat during the transition from a uniform to a modulated Twist pattern, Notchsignaling represses twist only in presumptive low Twist domains.Transcriptional activators, such as Da, maintain high Twist expression inpresumptive high Twist domains. While Notch signaling components such asNotch, 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 activatedin presumptive high Twist domains. Model II proposes that during thetransition in Twist expression, Notch signaling represses twistthroughout the mesoderm, but Notch independent transcriptional activatorsantagonize Notch repression in what will become high Twist domains, thereby promoting the formation of high Twist domains. For example, transcriptionaleffectors of Notch signaling [such as Su(H) and E(spl)] and an 'activator'that is only expressed in presumptive high Twist domains may converge andcompete 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. Atstages 9-10, Slp is expressed in the mesoderm in transverse stripes thatcorrespond to high Twist domains. Moreover, loss- and gain-of-functionexperiments 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 isrequired to subdivide each mesodermal segment into a low and high Twistdomain. 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 willbe interesting to determine whether these proteins are regulated invertebrates 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 DorsalEmbryogenesis 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 genesChIP-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 (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 expressionTo 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 predictedOne 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