Transcriptional dynamics elicited by a short pulse of notch activation involves feed-forward regulation by E(spl)/Hes genes.

Dynamic activity of signaling pathways, such as Notch, is vital to achieve correct development and homeostasis. However, most studies assess output many hours or days after initiation of signaling, once the outcome has been consolidated. Here we analyze genome-wide changes in transcript levels, binding of the Notch pathway transcription factor, CSL [Suppressor of Hairless, Su(H), in Drosophila], and RNA Polymerase II (Pol II) immediately following a short pulse of Notch stimulation. A total of 154 genes showed significant differential expression (DE) over time, and their expression profiles stratified into 14 clusters based on the timing, magnitude, and direction of DE. E(spl) genes were the most rapidly upregulated, with Su(H), Pol II, and transcript levels increasing within 5-10 minutes. Other genes had a more delayed response, the timing of which was largely unaffected by more prolonged Notch activation. Neither Su(H) binding nor poised Pol II could fully explain the differences between profiles. Instead, our data indicate that regulatory interactions, driven by the early-responding E(spl)bHLH genes, are required. Proposed cross-regulatory relationships were validated in vivo and in cell culture, supporting the view that feed-forward repression by E(spl)bHLH/Hes shapes the response of late-responding genes. Based on these data, we propose a model in which Hes genes are responsible for co-ordinating the Notch response of a wide spectrum of other targets, explaining the critical functions these key regulators play in many developmental and disease contexts.

Drosophila p53 controls Notch expression and balances apoptosis and proliferation.

A balance between cell proliferation and apoptosis is important for normal development and tissue homeostasis. Under stress conditions, the conserved tumor suppressor and transcription factor Dp53 induces apoptosis to contribute to the maintenance of homeostasis. However, in some cases Dp53-induced apoptosis results in the proliferation of surrounding non-apoptotic cells. To gain insight into the Dp53 function in the control of apoptosis and proliferation, we studied the interaction between the Drosophila Dp53 and Notch genes. We present evidence that simultaneous reduction of Dp53 and Notch function synergistically increases the wing phenotype of Notch heterozygous mutant flies. Further, we found that a Notch cis-regulatory element is responsive to loss and gain of Dp53 function and that over-expression of Dp53 up-regulates Notch mRNA and protein expression. These findings suggest not only that Dp53 and Notch act together to control wing development but also indicate that Dp53 transcriptionally regulates Notch expression. Moreover, using Notch  gain and loss of function mutations we examined the relevance of Dp53 and Notch interactions in the process of Dp53-apoptosis induced proliferation. Results show that proliferation induced by Dp53 over-expression is dependent on Notch, thus identifying Notch as a new player in Dp53-induced proliferation. Interestingly, we found that Dp53-induced Notch activation and proliferation occurs even under conditions where apoptosis was inhibited. Our findings highlight the conservation between flies and vertebrates of the Dp53 and Notch cross-talk and suggest that Dp53 has a dual role regulating cell death and proliferation gene networks to control the homeostatic balance between apoptosis and proliferation.

The cytolinker Pigs is a direct target and a negative regulator of Notch signalling.

Gas2-like proteins harbour putative binding sites for both the actin and the microtubule cytoskeleton and could thus mediate crosstalk between these cytoskeletal systems. Family members are highly conserved in all metazoans but their in vivo role is not clear. The sole Drosophila Gas2-like gene, CG3973 (pigs), was recently identified as a transcriptional target of Notch signalling and might therefore link cell fate decisions through Notch activation directly to morphogenetic changes. We have generated a null mutant in CG3973 (pigs): pigs(1) mutants are semi-viable but adult flies are flightless, showing indirect flight muscle degeneration, and females are sterile, showing disrupted oogenesis and severe defects in follicle cell differentiation, similar to phenotypes seen when levels of Notch/Delta signalling are perturbed in these tissues. Loss of Pigs leads to an increase in Notch signalling activity in several tissues. These results indicate that Gas2-like proteins are essential for development and suggest that Pigs acts downstream of Notch as a morphogenetic read-out, and also as part of a regulatory feedback loop to relay back information about the morphogenetic state of cells to restrict Notch activation to appropriate levels in certain target tissues.

Deadpan contributes to the robustness of the notch response.

Notch signaling regulates many fundamental events including lateral inhibition and boundary formation to generate very reproducible patterns in developing tissues. Its targets include genes of the bHLH hairy and Enhancer of split [E(spl)] family, which contribute to many of these developmental decisions. One member of this family in Drosophila, deadpan (dpn), was originally found to have functions independent of Notch in promoting neural development. Employing genome-wide chromatin-immunoprecipitation we have identified several Notch responsive enhancers in dpn, demonstrating its direct regulation by Notch in a range of contexts including the Drosophila wing and eye. dpn expression largely overlaps that of several E(spl) genes and the combined knock-down leads to more severe phenotypes than either alone. In addition, Dpn contributes to the establishment of Cut expression at the wing dorsal-ventral (D/V) boundary; in its absence Cut expression is delayed. Furthermore, over-expression of Dpn inhibits expression from E(spl) gene enhancers, but not vice versa, suggesting that dpn contributes to a feed-back mechanism that limits E(spl) gene expression following Notch activation. Thus the combined actions of dpn and E(spl) appear to provide a mechanism that confers an initial rapid output from Notch activity which becomes self-limited via feedback between the targets.

Drosophila Reporter Vectors Compatible with ΦC31 Integrase Transgenesis Techniques and Their Use to Generate New Notch Reporter Fly Lines.

Complex spatial and temporal regulation of gene activity is fundamental to development and homeostasis. The ability to decipher the DNA sequences that accurately coordinate gene expression is, therefore, of primary importance. One way to assess the functions of DNA elements entails their fusion to fluorescent reporter genes. This powerful approach makes it possible to visualize their regulatory capabilities when reintroduced into the developing animal. Transgenic studies in Drosophila have recently advanced with the introduction of site-specific, ΦC31 integrase-mediated approaches. However, most existing Drosophila reporter vectors are not compatible with this new approach and have become obsolete. Here we describe a new series of fluorescent reporter vectors optimized for use with ΦC31 transgenesis. By using these vectors to generate a set of Notch reporter fly lines, we demonstrate their efficacy in reporting the function of gene regulatory elements.

Direct response to Notch activation: signaling crosstalk and incoherent logic.

Notch is the receptor in one of a small group of conserved signaling pathways that are essential at multiple stages in development. Although the mechanism of transduction impinges directly on the nucleus to regulate transcription through the CSL [CBF-1/Su(H)/LAG-1] [corrected] DNA binding protein, there are few known direct target genes. Thus, relatively little is known about the immediate cellular consequences of Notch activation. We therefore set out to determine the genome-wide response to Notch activation by analyzing the changes in messenger RNA (mRNA) expression and the sites of CSL occupancy within 30 minutes of activating Notch in Drosophila cells. Through combining these data, we identify high-confidence direct targets of Notch that are implicated in the maintenance of adult muscle progenitors in vivo. These targets are enriched in cell morphogenesis genes and in components of other cell signaling pathways, especially the epidermal growth factor receptor (EGFR) pathway. Also evident are examples of incoherent network logic, where Notch stimulates the expression of both a gene and the repressor of that gene, which may result in a transient window of competence after Notch activation. Furthermore, because targets comprise both positive and negative regulators, cells become poised for both outcomes, suggesting one mechanism through which Notch activation can lead to opposite effects in different contexts.