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. 2011 Feb;187(2):485-99.
doi: 10.1534/genetics.110.125138. Epub 2010 Nov 23.

A Novel Interaction Between Hedgehog and Notch Promotes Proliferation at the Anterior-Posterior Organizer of the Drosophila Wing

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Free PMC article

A Novel Interaction Between Hedgehog and Notch Promotes Proliferation at the Anterior-Posterior Organizer of the Drosophila Wing

David J Casso et al. Genetics. .
Free PMC article

Abstract

Notch has multiple roles in the development of the Drosophila melanogaster wing imaginal disc. It helps specify the dorsal-ventral compartment border, and it is needed for the wing margin, veins, and sensory organs. Here we present evidence for a new role: stimulating growth in response to Hedgehog. We show that Notch signaling is activated in the cells of the anterior-posterior organizer that produce the region between wing veins 3 and 4, and we describe strong genetic interactions between the gene that encodes the Hedgehog pathway activator Smoothened and the Notch pathway genes Notch, presenilin, and Suppressor of Hairless and the Enhancer of split complex. This work thus reveals a novel collaboration by the Hedgehog and Notch pathways that regulates proliferation in the 3-4 intervein region independently of Decapentaplegic.

Figures

F<sc>igure</sc> 1.—
Figure 1.—
N function is required for Hh-dependent patterning of the wing. (A) Control (w). (B) FM6/+; ptcGAL4 smo RNAi. (C) Df(1)N-8/+; note wing-margin notching. (D) Df(1)N-8/+; ptcGAL4 smo RNAi; note vein 3–4 thickening and fusion. (E) N55e11/+ (N null); note notching at margin and veins 3 and 5 thickening. (F) N55e11/+; ptcGAL4 smo RNAi. (G) ptcGAL4 N RNAi (NIG.3936R-2). (H) ptcGAL4 N RNAi smo RNAi. (I) ptcGAL4 psn RNAi (P{GD4624}v43082). (J) ptcGAL4 psn RNAi smo RNAi. (K) ptcGAL4 Su(H) RNAi (NIG.3497R-3). (L) ptcGAL4 Su(H) RNAi smo RNAi. (M) ptcGAL4 smo RNAi; E(Spl)/+. (N) ptcGAL4 UAS-N. (O) ptcGAL4 UAS-N smo RNAi. (P) ptcGAL4 Su(dx) RNAi (NIG.4244R-1). (Q) ptcGAL4 Su(dx) RNAi smo RNAi. (R) ptcGAL4 UAS-HLH-mΔ. (S) ptcGAL4 UAS-HLH-mΔ smo RNAi. (T) ptcGAL4 UAS-E(spl)m8. (U) ptcGAL4 UAS-E(spl)m8 smo RNAi.
F<sc>igure</sc> 2.—
Figure 2.—
N signaling at the AP organizer. Three stages of wing development: (A–C) late third instar disc; (D–F) early pupal wing disc when the dorsal and ventral surfaces of the everting disc are coming into apposition (∼3 hr after pupariation); (G–I) early pupal wing (∼9–12 hr after pupariation). (A, D, and G) Su(H)lacZ expression (green) indicates N signaling both at the DV border (yellow arrowheads) and at the AP organizer (white arrowheads). The DV boundary is in the middle of the wing pouch (A), near the edge in a partially everted disc (D), and at the edge of the pupal wing (G). As development advances, Su(H)lacZ expression increases at the AP border (white arrowheads) and decreases at the DV border (yellow arrowheads). (B, E, and H) Ci expression (magenta) marks anterior cells; the posterior extent of Ci expression marks the AP border. (C, F, and I) Merged images show that Su(H)lacZ expression at the AP organizer is in Ci-expressing anterior cells; Ci staining is enhanced in enlarged views (C′ and F′) to show the position of the AP border and the coincidence of Ci and LacZ expression. (A–F) Orientation: anterior is left and ventral is up; (G–I) anterior is up and distal is right.
F<sc>igure</sc> 3.—
Figure 3.—
Expression of the N reporter E(spl)m-α-GFP at the AP organizer in pupal wings. (A and D) E(spl)m-α-GFP expression (green). (B and E) Ci expression (magenta). (C and F) A merge of GFP and Ci. (A–C) A control wing. (D–F) A fu1 wing. The position of the AP border where Ci expression ends is indicated by a white arrowhead.
F<sc>igure</sc> 4.—
Figure 4.—
N protein levels are elevated at the AP organizer. (A–C) Late third instar wing disc. (D–F) Pupal wing disc. (G–I) Early pupal wing. (A, D, and G) Expression of N protein (red) is high at both the AP (white arrows) and the DV (yellow arrows) border regions. (B, E, and H) Expression of Su(H)lacZ (green) indicates activation of N signaling at both DV and AP borders. (C, F, and I) Merged images show that Su(H)lacZ expression at the AP border is in cells with high levels of N protein.
F<sc>igure</sc> 5.—
Figure 5.—
Disruption of N signaling does not alter Hh signaling. Ci (magenta) and Ptc (red) expression in third instar wing discs (A–L) and pupal wings (M–P). Clones are negatively marked by lack of GFP or lacZ expression (green). (A and B) w wing discs. (C and D) Wing discs from Nl1N-ts1 larvae shifted to 30° (restrictive temperature) for 24 hr. (E–H) Clone of null allele N55e11 within the AP organizer. (I–L) Ventral clone in the posterior compartment and a dorsal clone in the anterior compartment of null allele Su(H)del47. (H and L) Merged images showing the positions of these clones in relation to Ptc and Ci expression. (M–P) Expression of Ptc and Ci in w and temperature-shifted Nts pupal wings. (M and N) w pupal wings. Note that at this stage dorsal and ventral surfaces of the wing are not apposed to each other, resulting in the apparent splitting of the Pct and Ci stripes in the proximal region (left) of the wing. (O and P) Nl1N-ts1 pupal wings shifted to 30° for 24 hr.
F<sc>igure</sc> 6.—
Figure 6.—
Dl expression in the vein 4 primordium is in the posterior compartment. (A) A wild-type wing imaginal disc showing stripes of Dl expression in veins 3, 4, and 5 primordia. (B–D) High magnification of the AP organizer region in the dorsal wing pouch. Veins 3 and 4 stripes of Dl expression (red) flank the AP border indicated by arrowheads. Expression of Ci (green) marks the cells of the anterior compartment. The Dl vein 3 stripe is anterior of the border, while the vein 4 stripe is posterior and directly adjacent to the border. (A and B–D are from two different discs.)
F<sc>igure</sc> 7.—
Figure 7.—
N activation in the AP organizer depends on vn. N activation in a vn1/vnC221 mutant (A–D) late third instar wing disc. (E–H) Pupal disc. (I–L) Early pupal wing. Relative to wild type (Figure 4), expression of Su(H)lacZ (green) and N (red) is reduced at the AP organizer (white arrowheads), whose position is marked by Ci (magenta). DV boundary is indicated by yellow arrowheads.
F<sc>igure</sc> 8.—
Figure 8.—
N signaling and expression at the AP organizer depend on fu. Three stages of wing development: (A–D) late third instar disc; (E–H) early pupal wing disc when the dorsal and ventral surfaces of the everting disc are coming into apposition (∼3 hr after pupariation); and (I–L) early pupal wing (∼9–12 hr after pupariation). (A, E, and I) Su(H)lacZ expression (green) indicates N signaling both at the DV border (yellow arrowheads) and at the AP organizer (white arrowheads). The DV boundary is in the middle of the wing pouch (A), partially everted in E, and at the edge of the pupal wing (I). As development advances, Su(H)lacZ expression increases at the AP border (white arrowheads) and decreases at the DV border (yellow arrowheads). (B, F, and J) Ci expression (magenta) marks anterior cells; the posterior extent of Ci expression marks the AP border. (C, F, and I) Merged images show that Su(H)lacZ expression at the AP organizer is in Ci-expressing anterior cells. (A–F) Orientation: anterior is left and ventral is up. (I–L) Anterior is up and distal is right.
F<sc>igure</sc> 9.—
Figure 9.—
Expression of key genes at the AP organizer and a model for N activation by Hh. (A) Ovals represent the width of expression domains in cell diameters perpendicular to the AP axis, and oval sizes reflect expression levels. Expression domains were measured in late third instar wing discs in the ventral compartment intermediate between the edge of the wing pouch and the DV border. Antibodies were used except where lacZ insertions are indicated. (B) Hh-dependent activation of vn at the AP organizer is required for the expression of Dl in vein 3 and vein 4 stripes. N activation induces cell proliferation within the organizer at least in part through the activation of the E(spl) genes.

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