A gain-of-function suppressor screen for genes involved in dorsal-ventral boundary formation in the Drosophila wing

Abstract

The Drosophila wing primordium is subdivided into a dorsal (D) and a ventral (V) compartment by the activity of the LIM-homeodomain protein Apterous in D cells. Cell interactions between D and V cells induce the activation of Notch at the DV boundary. Notch is required for the maintenance of the compartment boundary and the growth of the wing primordium. Beadex, a gain-of-function allele of dLMO, results in increased levels of dLMO protein, which interferes with the activity of Apterous and results in defects in DV axis formation. We performed a gain-of-function enhancer-promoter (EP) screen to search for suppressors of Beadex when overexpressed in D cells. We identified 53 lines corresponding to 35 genes. Loci encoding for micro-RNAs and proteins involved in chromatin organization, transcriptional control, and vesicle trafficking were characterized in the context of dLMO activity and DV boundary formation. Our results indicate that a gain-of-function genetic screen in a sensitized background, as opposed to classical loss-of-function-based screenings, is a very efficient way to identify redundant genes involved in a developmental process.

Figure 1.—

Design of the genetic screen for suppressors of the Beadex wing phenotype. The wing primordium is subdivided into a dorsal (D) and a ventral (V) compartment by the restricted expression and activity of Apterous (Ap) in D cells. (A) ap-lacZ expression in a third instar wing disc visualized by histochemical staining for β-gal activity. (B) Early in development, Serrate (Ser) signals to V cells to activate Notch (N). Likewise, Delta (Dl) signals to D cells to activate Notch modified by Fringe (Fng) along the DV boundary. (C) Beadex¹/FM6; apterous^Gal4/CyO flies, which have a strong loss of wing-margin phenotype, were crossed with a large number of independent EP-containing lines. Gal4 expressed in D cells should bind to Gal4 binding sites within the target element enhancer and activate an adjacent endogenous gene X. Those lines that rescued the wing-margin phenotype were selected.

Figure 2.—

Increased Notch activity or Caps/Tartan expression rescues the Beadex wing phenotype. (A, C, and E) Cuticle preparations of Bx¹/Y; ap^gal4/+ (A), Bx¹/Y; ap^gal4/uas-caps (C) and Bx¹/Y; ap^gal4/uas-trn (E) adult wings. Note rescue of the wing-margin defects when Caps or Trn are expressed. Note also the blistered wing phenotype in C and E, probably due to defects in cell apposition between D and V wing surfaces in the presence of high levels of Caps or Trn expression. (B, D, and F) Cuticle preparations of Bx¹/+ (B), Bx¹/+, caps^65.2/+ (D), and Bx¹/+, trn^25/4/+ (F) adult wings. Note enhancement of the wing-margin defects when either caps or trn are removed. (G and H) Bx¹/Y; ap^gal4/+ (G) and Bx¹/Y; ap^gal4/uas-caps (H) wing discs labeled to visualize Gal4 (red) and Wingless (Wg, blue) protein expression. Dorsal (d) and ventral (v) compartments are marked. (I and J) Cuticle preparations of Bx¹/Ax^M1 (I) and Bx¹/+; H^E·31/+ (J) adult wings. Note rescue of the wing-margin defects when compared to the Bx¹/+ wing shown in B.

Figure 3.—

In situ hybridization to wild type (A, B, E, F, and H), ap^gal4/+; EP-732/+ (C), ap^gal4/+; EP-826/+ (D), and ap^gal4/+; EP-1199/+ (G) wing (A–G) or eye-antenna (H) imaginal discs with anti-sense (AS) or sense (SS) par 5 (A–C), CG8369 (D), and CG14073 (E–H) RNA probes. Note high levels of CG14073 expression at the morphogenetic furrow of the eye-antenna imaginal disc.

Figure 4.—

Wing phenotypes of the suppressor lines. Cuticle preparations of adult wings are from the following genotypes: (A) Bx¹/Y; ap^gal4/EP2-18 (Sly); (B) Bx¹/Y; ap^gal4/EP2-408 (cbt); (C) Bx¹/Y; ap^gal4/EP2-446 (nmd); (D) Bx¹/Y; ap^gal4/EP2-760 (CG5890); (E) Bx¹/Y; ap^gal4/EP2-1069 (chm); (F) Bx¹/Y; ap^gal4/EP2-1080 (CG4477); (G) Bx¹/Y; ap^gal4/EP2-1279 (shn); (H) Bx¹/Y; ap^gal4/EP2-1583 (ap); (I) Bx¹/Y; ap^gal4/EP3-26 (miR-282-RA); (J) Bx¹/Y; ap^gal4/EP3-364 (miR-279a); (K) Bx¹/Y; ap^gal4/EP3-378 (HLH-γ); (L) Bx¹/Y; ap^gal4/EP3-532 (skuld); (M) Bx¹/Y; ap^gal4/EP3-562 (Annex-IX); (N) Bx¹/Y; ap^gal4/EP3-1575 (tara); (O) Bx¹/+; shn⁴⁷³⁸/+; (P) Bx¹/+; NP0245/+; (Q) Bx¹/+; l(3)04708/+; (R) Bx¹/Y; skuld¹⁰¹⁹⁸/+; (S) Bx¹/+; l(3)neo54/+; and (T) Bx¹/+; tara¹/+.

Figure 5.—

master of thickveins as a suppressor of the the Beadex¹ phenotype. (A–C) Cuticle preparations of Bx¹/+; mtv⁶/+ (A), Bx¹/Y; ap^gal4/EP-473 (B), and Bx¹/Y; ap^gal4/uas-mtv (C) adult wings. (D) Bx¹/Y; ap^gal4/uas-mtv wing imaginal discs labeled to visualize Gal4 (green) and Wingless (Wg, red in the top and white in the bottom) protein expression. (E) Clones of cells mutant for master of thickveins (mtv⁶) and labeled by the absence of the GFP marker (green). Cut protein expression is shown in red (top) or white (bottom). Note loss of Cut expression in clones abutting the DV boundary. (F and G) Clones of cells mutant for master of thickveins (mtv⁶), labeled by the forked (f^36a) cuticle marker and marked by a red line. Note loss of wing-margin structures and ectopic vein tissue.

Figure 6.—

lilliputian as a suppressor of the the Beadex¹ phenotype. (A and B) Cuticle preparations of Bx¹/Y; ap^gal4/EP-27 (A) and Bx¹/+; lilli⁶³²/+ (B) adult wings. (C–F) Clones of cells mutant for lilliputian (lilli^4u5) and labeled by the absence of the GFP marker (green). Wingless (Wg) protein expression is shown in red (top) or white (bottom). Note reduced levels of Wg protein expression in clones abutting the DV boundary (white arrows) when compared to the endogenous level (red arrowhead) of Wg expression.

Figure 7.—

CG11399, the Drosophila phosphorylated C-terminal domain interacting factor, suppresses the Beadex¹ phenotype. (A) Genomic map of the CG11399 region. Exons are shown as boxes, and the ORF is marked in black. CG11399 was identified as a suppressor of the Beadex¹ phenotype by the EP-3-28 insertion (black arrowhead). Two other EP lines (GS11380 and EY11352, black arrowheads) are shown. (B–D) In situ hybridization to ap^gal4/+; EP-28/+ (B) and wild-type (C and D) wing imaginal discs with an anti-sense (B and C) and sense (D) CG11399 RNA probe. (E–L) Cuticle preparations of Bx¹/Y; ap^gal4/EP-28 (E), Bx¹/+; EY11352/+ (F), EY11352 (G), EY11352/Df(3L)ri-79c (H), N^ND1/Y (I), N^ND1/Y; EY11352/+ (J), dx^ENU/Y (K), and dx^ENU /Y; EY11352/+ (L) adult wings. Note ectopic vein tissue marked by a black arrow in G and H. (M–O) Cuticle preparations of wild-type (M), ap^gal4/+; EP-28/+ (N), and ap^gal4/+; GS11380/+ (O) adult nota. Note loss of macrochaetae in N and loss of both macro- and microchaetae in O.

Figure 8.—

miR-14 as a suppressor of the Beadex¹ phenotype: genomic map of the miR-14 region (blue box). miR-14 was identified as a suppressor of the Beadex¹ phenotype by four EP insertions (blue arrowheads). (B–E) Cuticle preparations of Bx¹/Y; ap^gal4/EP-235 (B), Bx¹/Y; ap^gal4/uas-p35 (C), Bx¹/Y; ap^gal4/uas-miR-14 (D), and Bx¹/Y; ap^gal4/EP-DIAP1 (E) adult wings. (F and G) Bx¹/Y; ap^gal4/+ (F) and Bx¹/Y; ap^gal4/ EP-235 (G) wing discs labeled to visualize Gal4 (red) and Wingless (Wg, blue) protein expression. (H) Cuticle preparation of a ap^gal4/uas-miR-14 adult wing. (I) ap^gal4/uas-miR-14 adult fly. Note the held-up wing phenotype. (J–L) ptc^gal4/uas-miR-14 (J and K) and wild-type (L) wing discs labeled to visualize GFP (red) and dLMO (green, top, or white, bottom) protein expression. Note reduced levels of dLMO protein in the GFP domain (white arrow). (M and N) Expression of the dLMO 3′-UTR sensor transgene (green or white) in ptc^gal4/uas-miR-14 (M) or wild-type wing discs (N). The sensor transgene was downregulated by miR-14 overexpression.