Notch inhibits Yorkie activity in Drosophila wing discs

Abstract

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.

Figure 1. Notch signaling inhibits ex expression in the wing pouch.

A. In third instar larval wing discs, the expression of the Yki target expanded monitored using the ex⁶⁹⁷-lacZ 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 NRE-GFP reporter (green; A″) is highest at the D/V (yellow arrowhead). 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 dpp-Gal4 (along the line indicated by the yellow arrow), leads to the repression of ex-lacZ (red; B′; yellow arrowhead), while E-Cadherin levels are unaffected (E-Cad; green; B″). C. 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).

Figure 2. Notch acts at the level or downstream of yki.

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

Figure 3. E(spl) and Cut repressors do not mediate the effects of Nicd on ex-lacZ expression.

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

Figure 4. Vg mediates in part the effects of Nicd on ex-lacZ expression.

A–B. Effects of over-expressing the Notch target vestigial (vg; A) or RNAi constructs against vg (B) in the posterior compartment using hh-Gal4 (marked by GFP; green) on ex-lacZ expression (red; A′&B′). Vg over-expression and RNAi leads to the inhibition and up-regulation of ex-lacZ respectively (A′; B′; yellow arrowheads). C. 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 ex-lacZ expression is higher in vg depleted compartments. Standard error to the mean is shown; unpaired two-tailed student t-test was performed showing significance with p value = 0.0003 (***). D–E. 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 ex-lacZ (white; D), co-expressing an RNAi against vg suppresses this effect, and ex-lacZ expression is found throughout the ptc domain including the D/V boundary (white; E; yellow arrowhead).

Figure 5. Vg represses th/DIAP1 expression in the wing pouch.

A–B. Expression of the SWH pathway specific th/DIAP1 reporter IAP2B2C-lacZ (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). C–E. Quantification of the posterior compartment increase of IAP2B2C-lacZ expression after vg RNAi knock-down. C. Higher magnification of the image in B′ showing IAP2B2C-lacZ expression in hh-Gal4 vg-RNAi expressing discs. D. 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. E. 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 IAP2B2C-lacZ expression is 12% higher in the vg depleted compartment. Standard error to the mean is shown; unpaired two-tailed student t-test was performed showing significance with p value = 0.0006 (***).