In vivo study of gene expression with an enhanced dual-color fluorescent transcriptional timer.

Fluorescent transcriptional reporters are widely used as signaling reporters and biomarkers to monitor pathway activities and determine cell type identities. However, a large amount of dynamic information is lost due to the long half-life of the fluorescent proteins. To better detect dynamics, fluorescent transcriptional reporters can be destabilized to shorten their half-lives. However, applications of this approach in vivo are limited due to significant reduction of signal intensities. To overcome this limitation, we enhanced translation of a destabilized fluorescent protein and demonstrate the advantages of this approach by characterizing spatio-temporal changes of transcriptional activities in Drosophila. In addition, by combining a fast-folding destabilized fluorescent protein and a slow-folding long-lived fluorescent protein, we generated a dual-color transcriptional timer that provides spatio-temporal information about signaling pathway activities. Finally, we demonstrate the use of this transcriptional timer to identify new genes with dynamic expression patterns

Enhancer Priming Enables Fast and Sustained Transcriptional Responses to Notch Signaling.

Information from developmental signaling pathways must be accurately decoded to generate transcriptional outcomes. In the case of Notch, the intracellular domain (NICD) transduces the signal directly to the nucleus. How enhancers decipher NICD in the real time of developmental decisions is not known. Using the MS2-MCP system to visualize nascent transcripts in single cells in Drosophila embryos, we reveal how two target enhancers read Notch activity to produce synchronized and sustained profiles of transcription. By manipulating the levels of NICD and altering specific motifs within the enhancers, we uncover two key principles. First, increased NICD levels alter transcription by increasing duration rather than frequency of transcriptional bursts. Second, priming of enhancers by tissue-specific transcription factors is required for NICD to confer synchronized and sustained activity; in their absence, transcription is stochastic and bursty. The dynamic response of an individual enhancer to NICD thus differs depending on the cellular context.Wellcome Trus

Activation of the Notch Signaling Pathway In Vivo Elicits Changes in CSL Nuclear Dynamics.

A key feature of Notch signaling is that it directs immediate changes in transcription via the DNA-binding factor CSL, switching it from repression to activation. How Notch generates both a sensitive and accurate response-in the absence of any amplification step-remains to be elucidated. To address this question, we developed real-time analysis of CSL dynamics including single-molecule tracking in vivo. In Notch-OFF nuclei, a small proportion of CSL molecules transiently binds DNA, while in Notch-ON conditions CSL recruitment increases dramatically at target loci, where complexes have longer dwell times conferred by the Notch co-activator Mastermind. Surprisingly, recruitment of CSL-related corepressors also increases in Notch-ON conditions, revealing that Notch induces cooperative or "assisted" loading by promoting local increase in chromatin accessibility. Thus, in vivo Notch activity triggers changes in CSL dwell times and chromatin accessibility, which we propose confer sensitivity to small input changes and facilitate timely shut-down

Decoding the Notch signal: investigating dynamics of transcription directed by Notch responsive enhancers

Information from developmental signalling pathways must be accurately decoded to generate transcriptional outcomes. Notch is an evolutionarily conserved signalling pathway, which, despite its apparent simplicity, regulates very different processes across tissues and developmental contexts. After activation of the pathway via interaction of the Notch receptor with its ligands on adjacent cells, the intracellular domain (NICD) transduces the signal directly to the nucleus. How enhancers decipher NICD in the real time of developmental decisions is not known. In order to determine how enhancers respond to Notch activity in real time, I have used the MS2-MCP system to visualize nascent transcripts in Drosophila embryos. To do so, I used two well-characterized Notch-responsive enhancers that drive expression in a stripe of mesectoderm (MSE) cells and analyzed their transcription profile over time at the single cell level. Strikingly, all MSE cells initiated transcription within a few minutes of one another, and once active, each nucleus produced a sustained profile of transcription. By manipulating NICD levels and altering key motifs within the enhancers, I uncover two key principles. First, the ability of NICD to confer synchronized and sustained activity in MSE requires that the enhancers be primed by tissue-specific transcription factors Twist and Dorsal. In their absence, MSE enhancers confer stochastic and bursty transcription profiles, demonstrating that different response profiles can be generated from a single enhancer according to which other factors are present. Second, changing Notch levels modulate the transcription burst size but not the inter-burst periods, in contrast to most current examples of enhancer activation. Next, I performed a small knockdown screen of maternal factors that could be required to promote sustained transcription in response to Notch. I found that reduced activity of some of these factors decreased the mean levels of transcription in different ways. Unexpectedly, knockdown of transcriptional repressors reduced the levels of transcription, although it is unclear if they affect Notch dependent transcription directly or indirectly. Lastly, I investigated the relationship between morphogenetic processes occurring at this stage of embryogenesis and Notch dependent transcription. I found cellularization acts as a limiting factor in the onset of transcription, and that Notch-Delta signalling likely occurs at the lateral membranes before cellularization finishes. Notch responsive transcription presents a clear transition in levels at the time of gastrulation, which correlates with the start of mesoderm invagination in genetic perturbation experiments. I hypothesize that gastrulation influences transcription in a Notch sensitive but not specific manner, through an unknown mechanism that likely occurs downstream of pathway activation.Wellcome Trust 1+3 PhD studentship, Developmental Mechanisms, 109144/Z/15/

Notch-dependent and -independent transcription are modulated by tissue movements at gastrulation.

Cells sense and integrate external information from diverse sources that include mechanical cues. Shaping of tissues during development may thus require coordination between mechanical forces from morphogenesis and cell-cell signalling to confer appropriate changes in gene expression. By live-imaging Notch-induced transcription in real time, we have discovered that morphogenetic movements during Drosophila gastrulation bring about an increase in activity-levels of a Notch-responsive enhancer. Mutations that disrupt the timing of gastrulation resulted in concomitant delays in transcription up-regulation that correlated with the start of mesoderm invagination. As a similar gastrulation-induced effect was detected when transcription was elicited by the intracellular domain NICD, it cannot be attributed to forces exerted on Notch receptor activation. A Notch-independent vnd enhancer also exhibited a modest gastrulation-induced activity increase in the same stripe of cells. Together, these observations argue that gastrulation-associated forces act on the nucleus to modulate transcription levels. This regulation was uncoupled when the complex linking the nucleoskeleton and cytoskeleton (LINC) was disrupted, indicating a likely conduit. We propose that the coupling between tissue-level mechanics, arising from gastrulation, and enhancer activity represents a general mechanism for ensuring correct tissue specification during development and that Notch-dependent enhancers are highly sensitive to this regulation

Additional Materials of the Thesis "Decoding the Notch signal: investigating dynamics of transcription directed by Notch responsive enhancers". Julia Falo Sanjuan, January 2020

Movies and plasmid sequences described in the Thesis "Activity and specificity of Notch regulated enhancers". Julia Falo Sanjuan, January 2020 Time-lapse confocal microscopy of Drosophila embryos in which transcription from Notch responsive was imaged live. Movie 01 - Expression of m5/m8. Movie showing transcription from m5/m8-peve in the mesectoderm stripe during nc14. Also note earlier, Notch independent transcription in broad domains in nc10-13 and in few scattered cells during the first few minutes of nc14, followed by a long period (approximately 20 min) of inactivity before MSE stripe cells initiate transcription. Maximum intensity projection (19x1\textmu m stacks) of the MCP-GFP (grey in left panel, green in right panel) and His2Av-RFP (blue in right panel) channels. 0.36 um/px XY resolution and final time resolution of 10s/frame. Anterior to the left; embryo imaged from the ventral side. Movie 02 - Expression of sim. Movie showing transcription from sim-peve in the mesectoderm stripe and some mesodermal cells during nc14. Also note activity in scattered cells before nc14. Maximum intensity projection (29x1um stacks) of the MCP-GFP (grey in left panel, green in right panel) and His2Av-RFP (blue in right panel) channels. 0.36 um/px XY resolution and final time resolution of 15s/frame. Anterior to the left; embryo imaged from the ventral side. Movie 03 - Ectopic expression of m5/m8 with eve2-NICD. Movie showing ectopic transcription from m5/m8-peve in the eve2 domain during nc14. Maximum intensity projection (29x1\textmu m stacks) of the MCP-GFP (grey in left panel, green in right panel) and His2Av-RFP (blue in right panel) channels. 0.36 um/px XY resolution and final time resolution of 15s/frame. Anterior to the left; embryo imaged from the ventral side. Movie 04 - Ectopic expression of sim with eve2-NICD. Movie showing ectopic transcription from sim-peve in the eve2 domain during nc14. Maximum intensity projection (29x1um stacks) of the MCP-GFP (grey in left panel, green in right panel) and His2Av-RFP (blue in right panel) channels. 0.36 um/px XY resolution and final time resolution of 15s/frame. Anterior to the left; embryo imaged from the ventral side. Movie 05 - Regions of ectopic expression of m5/m8 and sim with eve2-NICD. Combined movie of m5/m8-peve (left) and sim-peve (right) showing ectopic transcription in the eve2 stripe. The maximum projection of the MCP-GFP signal is overlaid with tracked nuclei false colored with the maximum intensity pixel in each nucleus. Active nuclei in each of the analyzed regions is marked with a different color: red (mesoderm), purple (mesectoderm), blue (neuroectoderm) and green (dorsal ectoderm). Anterior to the left; embryo imaged from the ventral side. Movie 06 - Transcription of two m5/m8-peve MS2 reporters in the presence of ectopic NICD. Movie showing ectopic transcription from m5/m8-peve x2 in the eve2 domain during nc14. Maximum intensity projection (29x1um stacks) of the MCP-GFP (grey in left panel, green in right panel) and His2Av-RFP (blue in right panel) channels. 0.36 um/px XY resolution and final time resolution of 15s/frame. Anterior to the left; embryo imaged from the ventral side. Movie 07 - Changes in nuclear size and shape during nc14. Movie showing maximum projection of medial slices and orthogonal views of Nup107-GFP. 0.18 um/px XY resolution and final time resolution of 15s/frame. Anterior to the left; embryo imaged from the ventral side. Movie 08 - Expression of m5/m8 starts during cellularization. Movie showing cellularizing membranes using the marker Gap43-mCherry (maximum intensity projection of medial slices and orthogonal views, left) and transcription from m5/m8-peve (maximum intensity projection (29x1um stacks) of the MCP-GFP channel, right). 0.36 um/px XY resolution and final time resolution of 15s/frame. Anterior to the left; embryo imaged from the ventral side. Movie 09 - Localization of Notch and Delta during cellularization. Movies showing maximum projection of medial slices and orthogonal views of Ni-GFP (left) and Dl-mScarlet (right). 0.36 um/px XY resolution and final time resolution of 60s/frame. Anterior to the left; embryo imaged from the ventral side. Movie 10 - Expression of vnd-peve. Movie showing transcription from vnd-peve in the neuroectoderm. Maximum intensity projection (29x1um stacks) of the MCP-GFP (grey in left panel, green in right panel) and His2Av-RFP (blue in right panel) channels. 0.36 um/px XY resolution and final time resolution of 15s/frame. Anterior to the left; embryo imaged from the ventral side. Movie 11 - Transcription of m5/m8 MS2 in a fog mutant background. Mesoderm invagination is delayed and does not proceed properly, resulting in a folded gastrulation phenotype. Maximum intensity projection (29x1um stacks) of the MCP-GFP (grey in left panel, green in right panel) and His2Av-RFP (blue in right panel) channels. 0.36 um/px XY resolution and final time resolution of 15s/frame. Anterior to the left; embryo imaged from the ventral side. Plasmid sequences - annotated sequences of all generated plasmids: pattB-m5/m8-peve-MS2-LacZ pattB-m5/m8-hsp70-MS2-LacZ pattB-m5/m8-pm5-MS2-LacZ pattB-m5/m8-pm6-MS2-LacZ pattB-m5/m8-pm7-MS2-LacZ pattB-m5/m8-pm8-MS2-LacZ pattB-m5/m8-psimE-MS2-LacZ pattB-sim-peve-MS2-LacZ pattB-sim-psimE-MS2-LacZ pattB-m8NE-peve-MS2-LacZ pattB-m8NE-pm8-MS2-LacZ pattB-vnd-peve-MS2-LacZ pattB-pUbi-MS2-LacZ pattB-m5/m8_insSPS-peve-MS2-LacZ pattB-sim_SPS-peve-MS2-LacZ pattB-m5/m8_Dtwi-peve-MS2-LacZ pattB-m5/m8_Ddl-peve-MS2-LacZ pattB-m5/m8_DtwiDdl-peve-MS2-LacZ pCR4-E-m5m8 pCR4-NE-sim pCR4-NE-m8NE' pCR4-E-m5m8_insSPS pCR4-NE-sim_SPS pCR4-E-m5m8_Ddl pCR4-E-m5m8_Dtwi pCR4-E-m5m8_DtwiDdl pattB-eve2x2-peve-FRT-STOP-FRT-NICD-eve3'UT

Membrane architecture and adherens junctions contribute to strong Notch pathway activation.

The Notch pathway mediates cell-to-cell communication in a variety of tissues, developmental stages and organisms. Pathway activation relies on the interaction between transmembrane ligands and receptors on adjacent cells. As such, pathway activity could be influenced by the size, composition or dynamics of contacts between membranes. The initiation of Notch signalling in the Drosophila embryo occurs during cellularization, when lateral cell membranes and adherens junctions are first being deposited, allowing us to investigate the importance of membrane architecture and specific junctional domains for signalling. By measuring Notch-dependent transcription in live embryos, we established that it initiates while lateral membranes are growing and that signalling onset correlates with a specific phase in their formation. However, the length of the lateral membranes per se was not limiting. Rather, the adherens junctions, which assemble concurrently with membrane deposition, contributed to the high levels of signalling required for transcription, as indicated by the consequences of α-Catenin depletion. Together, these results demonstrate that the establishment of lateral membrane contacts can be limiting for Notch trans-activation and suggest that adherens junctions play an important role in modulating Notch activity

Mi-2/NuRD complex protects stem cell progeny from mitogenic Notch signaling.

To progress towards differentiation, progeny of stem cells need to extinguish expression of stem-cell maintenance genes. Failures in such mechanisms can drive tumorigenesis. In Drosophila neural stem cell (NSC) lineages, excessive Notch signalling results in supernumerary NSCs causing hyperplasia. However, onset of hyperplasia is considerably delayed implying there are mechanisms that resist the mitogenic signal. Monitoring the live expression of a Notch target gene, E(spl)mγ, revealed that normal attenuation is still initiated in the presence of excess Notch activity so that re-emergence of NSC properties occurs only in older progeny. Screening for factors responsible, we found that depletion of Mi-2/NuRD ATP remodeling complex dramatically enhanced Notch-induced hyperplasia. Under these conditions, E(spl)mγ was no longer extinguished in NSC progeny. We propose that Mi-2 is required for decommissioning stem-cell enhancers in their progeny, enabling the switch towards more differentiated fates and rendering them insensitive to mitogenic factors such as Notch

Ras2, the TC21/R-Ras2 Drosophila homologue, contributes to insulin signalling but is not required for organism viability

Ras1 (Ras85D) and Ras2 (Ras64B) are the Drosophila orthologs of human H-Ras/N-Ras/K-Ras and R-Ras1-3 genes, respectively. The function of Ras1 has been thoroughly characterised during Drosophila embryonic and imaginal development, and it is associated with coupling activated trans-membrane receptors with tyrosine kinase activity to their downstream effectors. In this capacity, Ras1 binds and is required for the activation of Raf. Ras1 can also interact with PI3K, and it is needed to achieve maximal levels of PI3K signalling in specific cellular settings. In contrast, the function of the unique Drosophila R-Ras member (Ras2/Ras64B), which is more closely related to vertebrate R-Ras2/TC21, has been only studied through the use of constitutively activated forms of the protein. This pioneering work identified a variety of phenotypes that were related to those displayed by Ras1, suggesting that Ras1 and Ras2 might have overlapping activities. Here we find that Ras2 can interact with PI3K and Raf and activate their downstream effectors Akt and Erk. However, and in contrast to mutants in Ras1, which are lethal, null alleles of Ras2 are viable in homozygosis and only show a phenotype of reduced wing size and extended life span that might be related to reduced Insulin receptor signalling.Secretaría de Estado de Investigación, Desarrollo e Innovación, Grant/Award Number: BFU2015-64220-P and BFU2018-094476. We would also like to acknowledge the support from the Drosophila transgenesis and confocal microscopy CBMSO scientific services. The CBMSO enjoys institutional support from the Ramón Areces and Santander Foundatio