Notch inhibits Yorkie activity in Drosophila wing discs.

During development, tissues and organs must coordinate growth and patterning so they reach the right size and shape. During larval stages, a dramatic increase in size and cell number of Drosophila wing imaginal discs is controlled by the action of several signaling pathways. Complex cross-talk between these pathways also pattern these discs to specify different regions with different fates and growth potentials. We show that the Notch signaling pathway is both required and sufficient to inhibit the activity of Yorkie (Yki), the Salvador/Warts/Hippo (SWH) pathway terminal transcription activator, but only in the central regions of the wing disc, where the TEAD factor and Yki partner Scalloped (Sd) is expressed. We show that this cross-talk between the Notch and SWH pathways is mediated, at least in part, by the Notch target and Sd partner Vestigial (Vg). We propose that, by altering the ratios between Yki, Sd and Vg, Notch pathway activation restricts the effects of Yki mediated transcription, therefore contributing to define a zone of low proliferation in the central wing discs

Vg represses <i>th/DIAP1</i> expression in the wing pouch.

<p><b>A–B.</b> Expression of the SWH pathway specific <i>th/DIAP1</i> reporter <i>IAP2B2C-lacZ</i> (red; A′&B′). While IAP2B2C-lacZ are uniform between the anterior and posterior of the pouch in control discs (A), a slight increase in the posterior compartment could be detected in discs where vg activity is lowered by RNAi only in the posterior compartment (B; marked by GFP; green; yellow arrowhead). <b>C–E.</b> Quantification of the posterior compartment increase of <i>IAP2B2C-lacZ</i> expression after vg RNAi knock-down. <b>C.</b> Higher magnification of the image in B′ showing <i>IAP2B2C-lacZ</i> expression in <i>hh-Gal4 vg-RNAi</i> expressing discs. <b>D.</b> Example of the profile of grey levels (reflecting IAP2B2C-lacZ levels), along the yellow box indicated in C. Levels are higher in the posterior compartment expressing the vg-RNAi than in anterior, except for effects at the boundary that are not understood. <b>E.</b> Quantification of the average IAP2B2C-lacZ intensity ratios between equivalent area picked in the posterior (green box in C) and anterior (white box in C) compartments in several discs. While the ratio is at 0.990 in control discs reflecting similar expression levels between the posterior and anterior compartments, this ratio rises to 1.122 in experimental discs showing that <i>IAP2B2C-lacZ</i> expression is 12% higher in the <i>vg</i> depleted compartment. Standard error to the mean is shown; unpaired two-tailed student t-test was performed showing significance with p value = 0.0006 (***).</p

Notch acts at the level or downstream of yki.

<p><b>A.</b> Over-expression of Yki along the A/P boundary of the wing disc using <i>dpp-Ga</i>l4 (along the line indicated by the yellow arrow), leads to the strong increase in expression of <i>ex-lacZ</i> in the pouch (red; A′), except at the D/V where Notch activity is highest (yellow arrowhead). <b>B.</b> When co-expressed with Yki (using dpp-Gal4; yellow arrow), Nicd imposes an inhibition of <i>ex-lacZ</i> expression in the pouch (red; B′; yellow arrowhead) but not at the periphery. E-Cadherin staining (green, A; B) outlines all cells.</p

Notch signaling inhibits <i>ex</i> expression in the wing pouch.

<p><b>A.</b> In third instar larval wing discs, the expression of the Yki target <i>expanded</i> monitored using the <i>ex⁶⁹⁷-lacZ</i> reporter line (ex-lacZ; red; A′) is strongest at the periphery and absent at the dorso-ventral boundary (D/V; yellow arrowhead). The activity of the Notch pathway monitored by the <i>NRE-GFP</i> reporter (green; A″) is highest at the D/V (yellow arrowhead). <b>B.</b> Over-expression of the active form of the Notch receptor (Nicd) in a stripe of cells along the antero-posterior (A/P) boundary of the wing disc using <i>dpp-Gal4</i> (along the line indicated by the yellow arrow), leads to the repression of <i>ex-lacZ</i> (red; B′; yellow arrowhead), while E-Cadherin levels are unaffected (E-Cad; green; B″). <b>C.</b> Inhibition of the Notch pathway in randomly generated clones overexpressing an RNAi against Notch (N-RNAi; positively marked by GFP; green; D″), leads to the upregulation of ex-lacZ in the pouch (red; D′; yellow arrowheads).</p

E(spl) and Cut repressors do not mediate the effects of Nicd on <i>ex-lacZ</i> expression.

<p><b>A–C.</b> When over-expressed in the posterior compartment using <i>hh-Gal4</i> (marked by GFP; green), the individual HES factors have different effects on <i>ex-lacZ</i> (red; A′, B′, C′). While E(spl)m<i>7</i> (and others see text; m7; A) has no effect on <i>ex-lacZ</i>, E(spl)m8 (m8; B) and Deadpan (dpn; C) induce a strong down-regulation of <i>ex-lacZ</i> (yellow arrowhead; B′, C′). <b>D.</b> MARCM clones, marked positively by GFP (green), which are homozygous mutant for all <i>E(spl)</i> genes (<i>Df(3R)E(spl)[Gro^b32.2</i>]) and express a strong RNAi against <i>dpn</i> show normal expression of <i>ex-lacZ</i> (red; D′). <b>E–F.</b> Early third instar larval wing discs showing <i>ex-lacZ</i> expression. Over-expression of Cut (ct) along the A/P boundary of the wing disc using <i>dpp-Gal4</i> (along the line indicated by the yellow arrow) inhibits <i>ex-lacZ</i> expression (F; yellow arrowhead) compared to controls (E). <b>G.</b> Over-expressing an RNAi construct against <i>ct</i> in the posterior compartment using <i>hh-Gal4</i> (marked by GFP; green) has no effect on <i>ex-lacZ</i> expression (red; G′).</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

Vg mediates in part the effects of Nicd on <i>ex-lacZ</i> expression.

<p><b>A–B.</b> Effects of over-expressing the Notch target <i>vestigial</i> (vg; A) or RNAi constructs against <i>vg</i> (B) in the posterior compartment using <i>hh-Gal4</i> (marked by GFP; green) on <i>ex-lacZ</i> expression (red; A′&B′). Vg over-expression and RNAi leads to the inhibition and up-regulation of <i>ex-lacZ</i> respectively (A′; B′; yellow arrowheads). <b>C.</b> Quantification of the average ex-lacZ intensity ratios between equivalent area picked in the posterior and anterior compartments in several discs. While the ratio is at 1.160 in control discs reflecting a slightly higher expression in the posterior compartment, this ratio rises to 1.620 in experimental discs showing that <i>ex-lacZ</i> expression is higher in <i>vg</i> depleted compartments. Standard error to the mean is shown; unpaired two-tailed student t-test was performed showing significance with p value = 0.0003 (***). <b>D–E.</b> While the overexpression of Nicd at the antero-posterior boundary (along the line indicated by the yellow arrow, using the ptc-Gal4 driver), leads to an inhibition of <i>ex-lacZ</i> (white; D), co-expressing an RNAi against <i>vg</i> suppresses this effect, and <i>ex-lacZ</i> expression is found throughout the ptc domain including the D/V boundary (white; E; yellow arrowhead).</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

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