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

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

Comparative Analysis of Cas9 Activators Across Multiple Species

Several groups have generated programmable transcription factors based on the versatile Cas9 protein, yet their relative potency and effectiveness across various cell types and species remain unexplored. Here, we compare Cas9 activator systems and examine their ability to induce robust gene expression in several human, mouse, and fly cell lines. We also explore the potential for improved activation through the combination of the most potent activator systems and assess the role of cooperativity in maximizing gene expression

Deadpan contributes to the robustness of the notch response

This is an open-access article distributed under the terms of the Creative Commons Attribution License.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. © 2013 Babaoglan et al.Research supported by an MRC programme grant G0800034 to SJB (http://www.mrc.ac.uk/index.htm).Peer Reviewe

Several Notch-regulated enhancers associated with <i>dpn</i> gene.

<p>(<b>A</b>) Su(H) bound genomic regions obtained by chromatin immunoprecipitation (ChIP) using wing (pink) and DmD8 (blue) cells show strong overlap with Su(H) binding motifs (all motifs, light grey; conserved motifs, dark grey). Green a, b and c boxes represent peaks that are cloned into a reporter construct expressing GFP. (<b>B–C</b>) Thorax region of wing discs showing expression from <i>dpn[a]GFP</i> (B, green; B″ single channel) and <i>dpn[b]GFP</i> (C, green; C″ single channel) in relation to the adult muscle precursors (AMPs; red nuclei in B,C) which have similar characteristics to DmD8 cells. <i>dpn[b]GFP (C′′)</i> and endogenous Dpn (blue, single channel B′′′, C′′′) expression is detected in some of the AMPs. (<b>D–E)</b> Third instar wing discs immunostained with anti-Dpn (Blue, D, E; single channels D′, E′), anti-GFP (green D,E; single channel, D′′, E′′) and anti-Cut (red, D,E) antibodies. Both the <i>dpn</i> reporters <i>[a]/[b]</i> overlap with Dpn and Cut expression at the D/V boundary (purple arrows); <i>dpn[a]</i> also fully recapitulates Dpn expression in the interveins D-D″), whereas <i>dpn[b]</i> directs weak expression in those regions (e.g. red arrow; E-E″). (<b>F</b>) Expression of endogeneous Dpn (F′), and the <i>dpn[b]</i> reporter (F′′) overlap with <i>E(spl)mδ0.5LacZ</i> (F′′′), a Notch responsive enhancer, in third instar eye discs. Yellow and blue arrows mark the R4 and R7 cells, respectively.</p

<i>dpn</i> regulates <i>Cut</i> expression.

<p><i>dpn</i> downregulation, mediated by <i>en>RNAi</i>, in the posterior compartment of late (B-B′′) and early (C-C′′) third instar wing discs. <i>GFP-RNAi</i> was used as a control (A-A′). Discs were stained with Cut (red) and Dpn (green). Yellow brackets (B′-B′′ and C′-C′′) represent the Cut expression at the posterior D/V boundary.</p

<i>dpn</i> modifies <i>Notch</i> phenotypes and directly alters the expression of the Notch target <i>cut</i>.

<p>(<b>A</b>) Wild type wing showing the wing margin and the veins: anterior cross vein (acv), posterior cross vein (pcv), L2, L3, L4 and L5. (<b>B–F</b>) <i>Notch</i> (<i>N[55e11]/+</i>) heterozygous phenotype of mild distal wing notches (D) is genetically modified in flies also heterozygous for <i>dpn</i> alleles, <i>dpn</i>[1] (B,E) and <i>dpn</i>[6] (C, F). (<b>G–H</b>) <i>dpn-RNAi</i> driven by <i>engrailed-Gal4 TubGal80 (en>)</i> caused single nicks in 19% of the wings (H). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075632#pone.0075632.s005" target="_blank">Table S1</a>. (<b>I</b>) Quantification of nick occurrences in each phenotype, <i>dpn</i> alleles significantly enhance the wing notching of Notch heterozygotes (p<<0.01). (<b>J-J′</b>) <i>dpn</i>[6] homozygous mutant clones do not alter Wingless (Wg) expression at the D/V boundary.</p

<i>dpn</i> regulates <i>E(spl)</i> genes.

<p>Early (<b>A, B, E and F</b>) and late (<b>C, D, G and H</b>) third instar wing discs with <i>en</i>> driving <i>dpn</i> expression. Yellow brackets highlight the posterior compartment in which engrailed is expressed. <i>E(spl)mβ</i> (A′, C′) and <i>E(spl)m8-lacZ</i> (E′, G′) expression were reduced in both early and late instar discs, in comparison to GFP overexpression (B and D) and wild type <i>E(spl)m8-lacZ</i> (F and H) controls. (<b>I</b>) Misexpression of <i>E(spl)m8</i> fails to alter Dpn expression. (<b>J</b>) Misexpression of <i>dpn</i> has no effect on <i>NRE-GFP</i>.</p

<i>dpn</i> contributes to the robustness of the Notch response.

<p>Diagram summarizing regulatory interactions between the genes indicated. Involvement of additional gene, X, is inferred due to the fact that HES genes appear to function as dedicated repressors.</p

<i>dpn</i> and <i>E(spl)</i> genes may act redundantly.

<p>(<b>A–C</b>) The mild wing vein phenotype of <i>E(spl)mγ-mβ</i> (A) is dominantly modified by <i>dpn</i>[1] (B and C). (<b>B</b>) Arrows point to vein thickening at the tips of L4 and L5, observed in 74% of <i>dpn</i>[1]<i>/+ E(spl)mγ-mβ</i> wings (<b>C</b>) 26% of <i>dpn</i>[1]<i>/+ E(spl)mγ-mβ</i> wings also showed ectopic veins surrounding L5 (arrow). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075632#pone.0075632.s006" target="_blank">Table S2</a>. (<b>D–E</b>) <i>E(spl)</i>m<i>β</i> (E-E′) or both <i>dpn</i> and <i>E(spl)</i>m<i>β</i> (D-D′) levels were knocked down by RNAi at early third instar wing discs. Cut expression (green) was dramatically reduced in the double RNAi (D′) compared to <i>E(spl)</i>m<i>β</i>-<i>RNAi</i> alone (E′). (<b>F-F′</b>) <i>E(spl)-C</i> loss of function MARCM clones produce reduction in <i>Cut</i> expression (F′) when combined with <i>dpn RNAi</i>.</p