Arf6 is necessary for senseless expression in response to wingless signalling during Drosophila wing development

In order to determine the level at which Arf6 is required in Wg signal transduction, we began by activating the Wg signalling pathway in an Arf6 mutant background. We suppressed the activity of the destruction complex by expressing a dominant-negative form of the Drosophila GSK3β homologue, shaggy (sggA81T) (Bourouis, 2002) or knocking-down axin. Both treatments induce high level Wg signalling and the formation of ectopic bristles in the wing blade (Fig. 3A,B). The number of ectopic bristles was dominantly suppressed in heterozygous Arf6 mutant backgrounds (Fig. 3A,A′,B,B′). These data indicate that the loss of bristles and Sens expression in the Arf6 mutants is not a result of the hyperactivation of the Arm destruction complex, and suggest that Arf6 acts downstream of Arm stabilisation.

We next confirmed that Arf6 acts downstream of the stabilisation of Arm by expressing two constitutively active forms of Arm: ArmS10 and ArmNDel (Pai et al., 1997). Importantly, these N-terminally truncated forms of Arm accumulate in the cytoplasm, triggering constitutive, high level Wg signalling in a ligand independent manner (Pai et al., 1997; Somorjai and Martinez-Arias, 2008). We expressed ArmS10 in a broad domain overlapping the D/V boundary with the C96-Gal4 driver, while ArmNDel expression is directly driven by the vestigial quadrant and margin enhancers (subsequently referred to as vgArmNDel). Both Arm variants induced ectopic bristles in the wing blade (Fig. 3C,C′,D,D′). Importantly, the bristles induced by vgArmNDel were not dependent on endogenous Wg signalling (Fig. S4A,A′,B,B′,B″) and vgArmNDel is active in canonical Wg signalling (Fig. S4C). The ectopic bristles induced by both constructs were dominantly suppressed in the Arf6 mutant background (Fig. 3C,C′,D,D′). Moreover, vgArmNDel or ArmS10 did not rescue the wing margin bristles lost in the wing margin of Arf6KO flies, and instead caused an enhancement of the Arf6 mutant phenotype (Fig. 3E,E′; Fig. S5A,A′). Over-expressing wild-type dsh also induced ectopic bristles that were suppressed in a heterozygous Arf6KO background (closed orange arrowhead, Fig. S5B,B′). dsh over-expression also enhanced of the heterozygous Arf6KO phenotype (compare Fig. S5B,C,C). This is unlikely to be due to a dominant negative effect of ArmS10 or Dsh overexpression as expressing either of these constructs in a wild-type background did not induce wing margin defects. Moreover, we did not observe a change in the levels of endogenous Arm and Cadherin at the adherens junctions in Arf61 mutant clones (Fig. S6A,A′), suggesting that Arf6 does not regulate Wg signalling through the sequestration of Arm to the adherens junction in Drosophila (Grossmann et al., 2013; Pellon-Cardenas et al., 2013). Altogether, these data demonstrate that Arf6 is required genetically downstream of Arm stabilisation in order to activate high level Wg signalling.

To test whether stabilised Arm had a generally reduced signalling activity in the Arf6 mutants, we stained for both Sens and Dll in wing imaginal discs expressing vgArmNDel in either a wild-type (Fig. 3F) or heterozygous Arf6KO background (Fig. 3F′). Clusters of ectopic Sens positive nuclei were apparent far from the D/V boundary in control wing discs expressing vgArmNDel (closed orange arrowheads, Fig. 3F) accompanied by an upregulation of Dll (open orange arrowheads, Fig. 3F). Removing a single copy of Arf6 led to an almost complete suppression of the ectopic Sens expression, including at the D/V boundary but both the ectopic and endogenous Dll remained (closed blue arrowheads, Fig. 3F′). These data indicate that although vgArmNDel is still able to activate low level signalling in the Arf6 mutant background, its ability to activate Sens expression is strongly attenuated. Importantly, although the Arf6 margin phenotype was mildly enhanced in a heterozygous arf1 (arf1182-1) mutant background, the signalling activity of ArmNDel was not suppressed in a heterozygous arf1182-1 background (Fig. S7A,A′,B,B′). This suggests that although Arf1 contributes to wing patterning, it likely does so in a distinct manner to Arf6 (Hemalatha et al., 2016).

Together, these results emphasise the specific requirement for Arf6 for the cell autonomous establishment of sens expression in response to high level Wg signalling. The loss of margin bristles in the Arf6 mutants is therefore likely to be due to a loss of the Sens-positive proneuronal clusters of the wing margin due to a suppression of high level Wg signalling.

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