Wing defects in Drosophila xenicid mutant clones are caused by C-terminal deletion of additional sex combs (Asx)

Abstract

Background: The coordinated action of genes that control patterning, cell fate determination, cell size, and cell adhesion is required for proper wing formation in Drosophila. Defects in any of these basic processes can lead to wing aberrations, including blisters. The xenicid mutation was originally identified in a screen designed to uncover regulators of adhesion between wing surfaces [1].

Principal findings: Here, we demonstrate that expression of the betaPS integrin or the patterning protein Engrailed are not affected in developing wing imaginal discs in xenicid mutants. Instead, expression of the homeotic protein Ultrabithorax (Ubx) is strongly increased in xenicid mutant cells.

Conclusion: Our results suggest that upregulation of Ubx transforms cells from a wing blade fate to a haltere fate, and that the presence of haltere cells within the wing blade is the primary defect leading to the adult wing phenotypes observed.

Figure 1. Identification of xenicid as an allele of Additional Sex Combs (Asx).

A) Maps of the genomic region from 50F-51B and nucleotides 10,400,000–10,500,000 on the right arm of the second chromosome, adapted from www.flybase.com. Genes (boxes), P-element insertions (ovals) and deficiencies (lines) that fail to complement Df(2R)03072/CyO are colored purple. P-element insertions or genes that complement are colored yellow. The breakpoints of Df(2R)03072 are shown in blue. B) Schematic diagram of WT and predicted mutant Asx proteins. Polyalanine (black box), nuclear localization sequences (red boxes), glutamine repeats (green boxes), and a cysteine-rich region predicted to form a double zinc finger (blue box) are indicated. The 14-base-pair deletion in exon 8 of Asx^xen is predicted to result in production of a truncated version of Asx (1235 aa vs. 1670 aa in the wild-type protein) that lacks the C-terminal nuclear localization sequence and Zinc finger domains.

Figure 2. Five phenotypes result from Asx mutation in clones of cells in the adult wing blade.

A) Wild-type wing with no defects. B) Wing bearing Asx^xen mutant cells with a Type A wing blister (see text). C) Crumpled wing (Type C blister) resulting from Asx^XF23 mutation. D) Bent wing bearing Asx^C849 mutant clones. E) Dual-lobed wing bearing Asx^C849 mutant clones. F) Crumpled wing with necrotic cells resulting from Asx^XF23 mutation. All five phenotypes were observed at similar penetrance in the three different Asx alleles analyzed. G) “Rippled” Ubx/+ wing bearing Asx^xen mutant cells (genotype is Flp; FRT^42D Asx^xen/FRT^42D GFP; Ubx/+). Arrowhead points to a ripple associated with a clone of small cells. Arrow indicates a ripple lacking associated small cells.

Figure 3. Asx mutation has no effect on βPS integrin or Engrailed expression.

Wing discs bearing Asx mutant clones (lacking GFP, green) were immunostained with anti-βPS (A) or anti-Engrailed (B) antibodies.

Figure 4. Asx mutant clone adult wings have patches of small cells containing more than one hair.

Boxed areas in wing blades are magnified at the right. Small cell clones are outlined in black. A) Wild type. B,C) Wings bearing Asx^xen mutant clones with small cells. Clone on edge of wing (B) and dual-lobed wing with small cells in the cleft between the lobes.

Figure 5. The primary defect in Asx mutants is upregulation of Ubx.

A) Larval wing discs bearing Asx^xen mutant clones (lacking GFP, green) were immunostained stained with anti-Hth (top) or anti-Ubx (bottom). Hth is expressed in the presumptive wing hinge in wild-type larval wing discs, and no effect is seen on Hth expression in Asx mutant clones. Ubx is expressed ubiquitously in haltere cells in imaginal discs, but is not normally expressed in wild-type wing blade cells. Ubx expression is strongly up-regulated within Asx mutant clones. B) Expression of UAS-Ubx throughout the wing blade using vestigial-Gal4 leads to wing defects that are indistinguishable from those seen in Asx mutant clones. C) Quantitation of wing phenotypes associated with Asx^xen mutant clones in flies that are wild-type at the Ubx locus (column 1, N = 383) or heterozygous (column 2, N = 46). Phenotypes are shown in order of increasing severity from bottom to top. Reduction of Ubx levels partially suppresses Asx^xen defects.