ABSTRACT Wnt/β-catenin signalling directs fundamental processes during metazoan development and can be aberrantly activated in cancer. Wnt stimulation induces the recruitment of the scaffold

protein Axin from an inhibitory destruction complex to a stimulatory signalosome. Here we analyse the early effects of Wnt on Axin and find that the ADP-ribose polymerase Tankyrase

(Tnks)—known to target Axin for proteolysis—regulates Axin’s rapid transition following Wnt stimulation. We demonstrate that the pool of ADP-ribosylated Axin, which is degraded under basal

conditions, increases immediately following Wnt stimulation in both _Drosophila_ and human cells. ADP-ribosylation of Axin enhances its interaction with the Wnt co-receptor LRP6, an

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PROMOTE WNT SIGNALING Article Open access 05 June 2025 INTRODUCTION The Wnt/β-catenin signal transduction pathway directs fundamental processes during metazoan development and tissue

homeostasis, whereas deregulation of Wnt signalling underlies numerous congenital disorders and carcinomas1,2. Two multimeric protein complexes with opposing functions—the cytoplasmic

destruction complex and the plasma membrane-associated signalosome—control the stability of the transcriptional co-factor β-catenin to coordinate the state of Wnt pathway activation. In the

absence of Wnt stimulation, β-catenin is targeted for proteasomal degradation by the destruction complex, which includes the two tumour suppressors: Axin and Adenomatous polyposis coli

(APC), and two kinases: casein kinase 1α (CK1α) and glycogen synthase kinase 3 (GSK3)3,4,5,6. Engagement of Wnt with its transmembrane receptors, Frizzled and low-density lipoprotein

receptor-related protein 5/6 (herein LRP6), induces rapid LRP6 phosphorylation, recruitment of Axin to phospho-LRP6, and assembly of the signalosome, which includes two other Axin-associated

components, GSK3 and Dishevelled (Dvl)7,8,9,10,11,12,13,14. Signalosome assembly results in the inhibition of β-catenin proteolysis; consequently stabilized β-catenin promotes the

transcriptional regulation of Wnt pathway target genes. As a key component in both the destruction complex and the signalosome, Axin is tightly regulated. Under basal conditions, Axin is

maintained at very low levels, and serves as the concentration-limiting scaffold for assembly of the destruction complex15,16. Following Wnt exposure, the rapid association of phospho-Axin

with phospho-LRP6 (refs 7, 12, 14) triggers Axin dephosphorylation, inducing a conformational change that inhibits Axin’s interaction with both the destruction and signalosome

complexes14,17,18. Axin is subsequently degraded7,18,19,20,21,22,23,24; however, Axin proteolysis occurs several hours after Wnt exposure, and thus does not regulate Axin’s essential role

during the initial activation of the Wnt pathway. The mechanisms that rapidly reprogram Axin from inhibitory to stimulatory roles following Wnt exposure remain uncertain. In current models,

Wnt stimulation induces Axin’s dissociation from the destruction complex, thereby promoting its interaction with the signalosome1,5,6,13,14,18,22,25,26,27. As Wnt stimulation induces Axin

dephosphorylation3,18,24, decreased phosphorylation was postulated to facilitate the dissociation of Axin from the destruction complex17; however, recent work revealed that the interaction

of Axin with LRP6 precedes Axin dephosphorylation, and that dephosphorylation serves to inhibit, rather than enhance this interaction14. Furthermore, some findings have challenged prevailing

models, providing evidence that Axin’s interaction with the destruction complex is not diminished upon Wnt stimulation2,19. Thus, whereas the rapid switch in Axin function following Wnt

stimulation is essential for the activation of signalling, the underlying mechanisms remain uncertain. During investigation of this critical process, we have discovered an unanticipated role

for the ADP-ribose polymerase Tankyrase (Tnks) in the reprogramming of Axin activity following Wnt exposure. As Tnks-mediated ADP-ribosylation is known to target Axin for proteolysis28,

small molecule Tnks inhibitors have become lead candidates for development in the therapeutic targeting of Wnt-driven cancers29,30,31. Here we identify a novel mechanism through which Tnks

regulates Axin: by promoting Axin’s central role in rapid Wnt pathway activation. We find that Wnt stimulation modulates Axin levels biphasically in both _Drosophila_ and human cells.

Unexpectedly, Axin is rapidly stabilized following Wnt stimulation, before its ultimate proteolysis hours later. In an evolutionarily conserved process, the ADP-ribosylated pool of Axin is

preferentially increased immediately following Wnt exposure. ADP-ribosylation enhances Axin’s association with phospho-LRP6, providing a mechanistic basis for the rapid switch in Axin

function following Wnt stimulation. Our results thus indicate that Tnks inhibition not only increases basal Axin levels, but also impedes the Wnt-dependent interaction between Axin and LRP6,

suggesting a basis for the potency of Tnks inhibitors in Wnt-driven cancers. Thus, Tnks not only targets Axin for proteolysis independently of Wnt stimulation, but also promotes Axin’s

central role in Wnt pathway activation, which may be relevant to the context-dependent activation of Wnt signalling and the treatment of Wnt-driven cancers with Tnks inhibitors. RESULTS AN

_IN VIVO_ ASSAY FOR AXIN REGULATION FOLLOWING WG EXPOSURE The precise onset of Wingless (Wg) /Wnt expression in fourteen segmental stripes during early _Drosophila_ embryogenesis has

provided a powerful _in vivo_ model for the signalling events triggered by Wg (Supplementary Fig. 1)32,33. However, previous studies reported contradictory conclusions regarding Axin

regulation, with the primary response to Wg exposure described as either Axin proteolysis23, or Axin recruitment from cytosol to plasma membrane34. Due to technical limitations in these

studies, the analysis of Axin was restricted to several hours after the onset of Wg exposure. In addition, Axin was overexpressed at levels high enough to abrogate Wg signalling, which

disrupts physiological regulation. We reasoned that the conflicting conclusions in previous reports might have resulted from these experimental barriers. Therefore, we sought to develop a

system that would permit an _in vivo_ analysis of Axin at near-physiological levels immediately following Wg stimulation. Because endogenous levels of Axin in _Drosophila_ embryos are too

low to be visualized with Axin antibodies, we generated transgenes encoding Axin tagged with a V5 epitope (_Axin-V5)_, and used site-specific integration35,36 coupled with the UAS/Gal4

system37 to direct their expression at near-physiological levels. Expression of _Axin-V5_ during early embryogenesis was induced with a maternal _α-tubulin_ enhancer (_mat-Gal4_) that drives

ubiquitous transcription38. In _Axin_ null mutant embryos, Wg signalling is aberrantly activated throughout the ectoderm39, as revealed by the ectopic expression of both _engrailed_ and

_wg_ (Supplementary Fig. 2a–h). Expression of _Axin-V5_ in _Axin_ null mutants completely prevented this constitutive activation of the Wg pathway, and critically, did not disrupt normal

signalling (Supplementary Fig. 2i–n). Thus, Axin-V5 replaced the function of endogenous Axin, and was expressed at levels that were regulated physiologically. Further, in wild-type embryos

expressing _Axin-V5_, the segmentally striped pattern of Wg, the resultant stabilization of Armadillo (Arm)/β-catenin in Wg-responding cells40, the Wg-dependent expression of

_engrailed_41,42, and the specification of Wg-dependent cell fates, as revealed by the patterning of the larval cuticle43, were normal (Supplementary Fig. 3a–h). To compare the relative

levels of Axin-V5 and endogenous Axin, we examined embryonic lysates. Detection of endogenous Axin by immunoblotting of cell extracts had not been possible with existing Axin antibodies18.

Therefore, we generated a new Axin antibody that allowed sensitive detection of endogenous Axin, and confirmed its specificity using RNAi-mediated _Axin_ knockdown (Supplementary Fig. 3i).

We used this Axin antibody to immunoblot lysates from wild-type embryos expressing _Axin-V5_ (Supplementary Fig. 3j). Axin-V5 was distinguished from endogenous Axin by its slower migration

rate in electrophoresis, resulting from the V5 tag. Immunoblotting revealed that Axin-V5 was not overexpressed relative to the level of endogenous Axin; instead, Axin-V5 levels were lower

than the level of endogenous Axin both before and after the onset of Wg expression (Supplementary Fig. 3j–l). We conclude that Axin levels were increased by less than twofold following the

expression of Axin-V5. BIPHASIC REGULATION OF AXIN FOLLOWING WG AND WNT EXPOSURE Thus, expression of these _Axin_ transgenes in wild-type embryos enabled an _in vivo_ analysis of Axin at

near-physiological levels before, during, and after the onset of Wg stimulation. We found that during the first 3 h of embryogenesis, before and during the onset of Wg expression, Axin was

distributed uniformly throughout the ectoderm (Figs 1a–l and 2a–c). However, within 30 min after the onset of Wg expression in segmental stripes, a major change in Axin was observed,

starting in late stage 8 and persisting throughout stage 9. Axin was no longer distributed uniformly, but instead was present in wide segmental stripes of increased intensity (Fig. 1m–r;

Supplementary Fig. 4). The Axin fluorescence intensity in these stripes was two- to threefold higher than that in the interstripes (Supplementary Fig. 4). Moreover, the temporal appearance

of the Axin stripes was coincident with the stabilization of Arm/β-catenin in segmental stripes40 (Supplementary Figs 5d–f and 6a–c), suggesting that the Axin stripes were an early indicator

of the initial cellular response to Wg stimulation. To determine the relative position of the segmental stripes of Axin and Wg, we co-stained embryos expressing _Axin-V5_ with V5 and Wg

antibodies. Within 30 min after the onset of Wg expression in segmental stripes, all Axin stripes were centered over Wg stripes (Fig. 2d–f,m–r; Supplementary Fig. 5a–c). As Wg is not

required for embryonic segmentation43, we tested whether Wg was important for generating the segmental stripes of Axin. In contrast with wild-type embryos, no Axin stripes were observed in

_wg_ null mutants; instead, the Axin-V5 staining was uniform (Supplementary Fig. 6d–g). Thus, Wg is required for the differential regulation of Axin in the embryonic ectoderm. To rule out

the possibility that the segmental Axin stripes were caused artifactually by the V5 epitope tag, we generated transgenic flies expressing GFP-tagged Axin; _Axin-GFP_ was also expressed using

the _mat-Gal4_ driver. Before, during, and after the onset of Wg expression, the Axin-GFP and Axin-V5 patterns were identical, as were the kinetics of Wg-dependent changes in their

distribution (Supplementary Fig. 7). These data supported the hypothesis that within 30 min after Wg exposure, Axin is tightly regulated in Wg-responding cells, and that the rapid appearance

of Axin in stripes of increased intensity coincides with the initial activation of the pathway. To further test this hypothesis, we examined endogenous Axin levels in lysates from wild-type

embryos. Embryos were collected at developmental stages that just preceded (1.5 to 2.5 h of development) or followed (3.5 to 4.5 h of development) the onset of Wg expression. To monitor the

initial activation of the Wg pathway, we examined the accumulation of phosphorylated Arrow/LRP6 with an antibody directed against phosphothreonine1572 in human LRP6 (ref. 44), a residue

that is conserved in _Drosophila_ (Supplementary Fig. 8a). RNAi-mediated _arrow_ knockdown in S2R+ cells confirmed the specificity of this signal (Supplementary Fig. 8b,c). After the onset

of Wg stimulation, phospho-Arrow levels increased and remained elevated (Supplementary Fig. 9a). We found that Axin levels increased by approximately threefold after Wg stimulation

(Supplementary Fig. 9a,b; stage 9). The increase in Axin levels likely resulted from increased protein stability, as the _Axin_ gene is not a transcriptional target of the Wg pathway, and

instead _Axin_ transcripts are expressed ubiquitously39. These findings provided further evidence that Axin is unexpectedly stabilized following Wg stimulation, and supported the kinetics of

Axin stabilization observed in the embryos by immunostaining. In addition, we found that after the initial increase in Axin during the early response to Wg exposure, Axin levels

subsequently decreased (Supplementary Fig. 9a,b; stage 13, 9h 20 min to 10h 20 min of development), as reported previously for mammalian Axin18,19,22,23,24. To investigate the kinetics of

this process, we examined the _in vivo_ pattern of embryonic Axin stripes. We found that the Axin stripes persisted for 80 to 100 min following Wg exposure (Fig. 2d–f,m–r; Supplementary Figs

5a–c and 7m–o; late stage 8 to stage 9). After that time, Axin levels decreased, initially in cells exposed to the highest levels of Wg (Fig. 2g–i, 2 h following Wg exposure; mid-stage 10),

and subsequently, in their neighbours (Fig. 2j–l,s–x, 4 h following Wg exposure; Stage 11-13). Thus, when Axin was stabilized in the early phase following Wg exposure, Axin and Wg stripes

overlapped (Fig. 2d–f,m–r; Supplementary Figs 5a–c and 7m–o). By contrast, when Axin was destabilized in the delayed phase, Axin and Wg stripes were spatially juxtaposed (Fig. 2j–l,s–x), as

observed previously23. In summary, our unprecedented ability to visualize Axin before, during, and after the onset of Wg exposure revealed that Axin is regulated biphasically in response to

Wg stimulation _in vivo_, which reconciles the contradictory conclusions in previous reports23,34. To determine whether the biphasic regulation of Axin in response to Wg stimulation is

evolutionarily conserved, we examined Axin1 levels in cultured human cells exposed to Wnt (Supplementary Fig. 9c–f). Treatment of two cell lines, HEK293T or SW480, with Wnt3A protein

resulted in rapid pathway activation, with the accumulation of phospho-LRP6 within 15 to 30 min (Supplementary Fig. 9c,e). In both cell lines, the levels of endogenous Axin1 protein

increased within 15 to 30 min following Wnt3A treatment (Supplementary Fig. 9c–f). This increase in Axin1 likely resulted from increased protein stability, as the _Axin1_ gene is not a

transcriptional target of the Wnt pathway45,46, and the increased Axin1 protein level was observed within 15 min of Wnt exposure. By 2 h after Wnt treatment, Axin1 levels decreased

(Supplementary Fig. 9c–f), as reported previously18,24. These findings suggested that the initial stabilization and subsequent destabilization of Axin in response to Wnt exposure, and the

kinetics of this response, are evolutionarily conserved. TNKS PROMOTES AXIN STABILIZATION FOLLOWING WG EXPOSURE As Tnks is known to target Axin for proteolysis28, we sought to determine

whether Tnks regulates Axin before and/or following Wg exposure. The Tnks-dependent proteolysis of Axin requires direct interaction of Tnks with a small region in the Axin amino terminus,

the Tnks-binding domain (TBD; refs 28, 47, 48). To determine whether the evolutionarily conserved TBD facilitates the degradation of _Drosophila_ Axin, we generated an _Axin-V5_ transgene

with a 21 amino-acid deletion that eliminates the TBD (AxinΔTBD; Supplementary Fig. 10a,b). To enable their direct comparison, the _Axin-V5_ and _AxinΔTBD-V5_ transgenes were integrated at

the same genomic site (_attP33)_, and were expressed using the same _mat-Gal4_ driver. By comparison with Axin-V5, the levels of AxinΔTBD were increased by three- to fourfold (Supplementary

Fig. 10c–g), even before the onset of Wg expression (0–2 h of embryogenesis, up to stage 4; Supplementary Fig. 10c–e). We conclude that the TBD is important to control the basal levels of

_Drosophila_ Axin independently of Wg exposure. To determine whether the TBD has additional roles in Axin regulation following Wg exposure, we analysed embryos expressing _AxinΔTBD_ after

the onset of Wg expression. Unexpectedly, and in contrast with Axin-V5 (Fig. 3a–c), AxinΔTBD-V5 was not present in segmental stripes 30 min after Wg exposure; instead, AxinΔTBD levels were

increased uniformly in all ectodermal cells (Fig. 3g–i; Supplementary Fig. 11a–c; late stage 8 and stage 9). Thus, the early stripes of Axin that form in response to Wg exposure were

dependent on the TBD; deletion of the TBD resulted in the aberrant Axin stabilization in all cells. To determine whether the TBD is also required for the subsequent degradation of Axin

induced by Wg, we examined embryos at later developmental stages (Fig. 3j–l). By comparison with their neighbours, the levels of AxinΔTBD were decreased in Wg-responding cells 2 h after Wg

exposure, with kinetics similar to those observed for wild-type Axin (Fig. 3d–f,j–l; Supplementary Fig. 12i–k; mid-stage 10). These findings indicated that the TBD is not required for the

Wg-dependent proteolysis of Axin. Therefore, we hypothesized that Tnks facilitates the stabilization of Axin following Wg exposure, but is dispensable for the subsequent Axin degradation. To

test this hypothesis, we examined _Tnks_ null mutant embryos expressing _Axin-V5_. In contrast with wild-type embryos, no early stripes of Axin were present in _Tnks_ mutants; instead, Axin

levels were uniformly high throughout the embryonic ectoderm (Fig. 3m–o; Supplementary Fig. 11d–f). Two hours after Wg exposure, the levels of Axin decreased in Wg-responding cells in

_Tnks_ mutants, as we had observed in wild-type embryos (Fig. 3d–f,p–r). Thus, the same phenotype was present in embryos expressing _AxinΔTBD_ or in _Tnks_ null mutant embryos: the early

Axin stripes were absent, whereas the late Axin stripes were present. These data indicated that in the absence of Tnks, the initial Wg-dependent regulation of Axin did not occur, and Axin

was aberrantly stabilized in all cells, but that the subsequent Wg-dependent Axin proteolysis occurred with normal kinetics. To determine whether the regulation of Axin at the earliest times

after Wg exposure promotes activation of the Wg pathway, we analysed the expression of the transcription factor Engrailed. During early embryogenesis, the initial transcription of

_engrailed_ is not dependent on Wg; however, Wg signalling is required subsequently to maintain _engrailed_ expression41,42. The expression of _engrailed_ is among the earliest readouts for

the activation of Wg signalling. Upon expression of _Axin-V5_ in either wild-type or _Axin_ null mutant embryos, both the onset and maintenance of _engrailed_ expression in segmental stripes

were normal (Fig. 4a,b and Supplementary Fig. 2m; compare with Supplementary Fig. 2b). In contrast, upon expression of _AxinΔTBD-V5_ in either wild-type or _Axin_ null mutant embryos, the

initiation of _engrailed_ expression was normal, but the maintenance of _engrailed_ expression was markedly diminished, as revealed by the decreased width of Engrailed stripes (Fig. 4c,d;

Supplementary Fig. 12m), indicating defects in Wg signalling. Similarly, examination of larval cuticles following expression of _Axin-GFP_ revealed normal epidermal patterning, in which

regions of naked cuticle alternated between ventral segmental denticle belts (Supplementary Fig. 13a). In contrast, a segment polarity phenotype was observed upon expression of

_AxinΔTBD-GFP_ (Supplementary Fig. 13b), in which the ventral epidermis formed denticles in place of naked cuticle43. These findings suggested that Tnks promotes not only the initial

Wg-dependent regulation of Axin, but also the activation of Wg signalling. We tested this hypothesis by examining Wg pathway activation in _Tnks_ null mutant embryos expressing _Axin-V5_. In

_Tnks_ mutant embryos expressing _Axin-V5_, the initiation of _engrailed_ expression was normal (Fig. 4e; stage 8), but its maintenance was markedly disrupted (Fig. 4f; stage 9). Therefore,

the same defects in Wg pathway activation were observed in _Tnks_ mutant embryos expressing _Axin-V5_ and in wild-type embryos expressing _AxinΔTBD-V5_. Nonetheless, no defects in

_engrailed_ expression were observed in _Tnks_ mutants (Supplementary Fig. 14), and these mutants were viable under standard laboratory conditions49,50,51. These findings revealed that a

less than twofold increase in Axin levels by the expression of _Axin-V5_ (Supplementary Fig. 3l) uncovers the importance of Tnks in promoting Wg signalling during embryogenesis, and

suggested that the regulation of Axin by Tnks following Wg exposure can be functionally compensated until Axin reaches a critical threshold level. Altogether, these findings indicated that

Tnks promotes not only the stabilization of Axin after Wg exposure, but also the activation of Wg signalling. TNKS PROMOTES WG SIGNALLING THROUGH A NOVEL MECHANISM We sought to elucidate the

mechanism through which Tnks regulates Axin and the activation of Wg signalling. In the current model, Tnks promotes Axin degradation, limiting the activity of the destruction complex, and

thereby facilitating the activation of Wg signalling28. Alternatively, Tnks might also regulate the rapid transition of Axin following Wg stimulation. To distinguish between these two

possibilities, we tested whether an increase in the basal levels of Axin, comparable to the higher levels present in AxinΔTBD or in _Tnks_ mutants, was sufficient to inhibit Axin regulation

or Wg pathway activation. We generated transgenic flies in which the _Axin-V5_ transgene was integrated at a different site in the genome (_attP40_), which is known to result in higher

expression levels than the original site (_attP33_)35. Indeed, immunoblotting of lysates revealed that the basal levels of Axin in embryos expressing _Axin-V5_ integrated at the _attP40_

site were significantly higher than the Axin levels in embryos expressing either _Axin-V5_ or _AxinΔTBD_ integrated at the _attP33_ site (Fig. 5a,b). Similarly, comparison of fluorescence

intensity revealed that levels of _attP40 Axin-V5_ were higher than _attP33 Axin-V5, attP33 AxinΔTBD-V5_, or _Tnks_ null mutant embryos expressing _attP33 Axin-V5_ (Supplementary Fig. 15).

However, despite the increased levels of Axin present in embryos expressing _attP40 Axin-V5_, the early regulation of Axin in Wg-responding cells, as revealed by the position, width and

timing of appearance of the Axin stripes, was the same in embryos expressing either _attP40 Axin-V5_ or _attP33 Axin-V5_ (Fig. 5d-i, compare with Fig. 2d–f and Supplementary Fig. 6a–c).

Further, both the initiation and the maintenance of _engrailed_ expression were similar in embryos expressing either _attP33 Axin-V5_ or _attP40 Axin-V5_, as was the embryonic hatch rate

(Fig. 5c,j,k, compare with Fig. 4a,b). These results were in sharp contrast with the absence of early Axin stripes, the disrupted _engrailed_ expression, and the decreased embryonic hatch

rate in embryos expressing _AxinΔTBD_ or in _Tnks_ mutants expressing _attP33 Axin-V5_ (Figs 3g–i,m–o and 4c–f; Supplementary Fig. 12l–n; Fig. 5c). Altogether, these findings demonstrate

that the level to which Axin is increased in _Tnks_ mutant embryos expressing _Axin-V5_ is not sufficient to disrupt Axin regulation or to inhibit Wg signalling. Instead, these results

suggested that in addition to its known role in Axin proteolysis, Tnks also acts through a distinct mechanism to promote the initial regulation of Axin and the activation of signalling

following Wg exposure. WNT STIMULATION INCREASES THE ADP-RIBOSYLATED AXIN LEVEL Our unanticipated findings suggested that Tnks-mediated ADP-ribosylation of Axin facilitates rapid Axin

regulation and the activation of signalling following Wnt exposure. To test this hypothesis, we examined whether Wnt stimulation modulates the levels of ADP-ribosylated Axin. Detection of

ADP-ribosylated Axin has been challenging, as the levels are very low under basal conditions52, and ADP-ribosylation does not markedly alter the electrophoretic mobility of Axin28,52.

Therefore, to detect ADP-ribosylated Axin, we utilized a previously developed pull down assay based on the Trp–Trp–Glu (WWE) domain of the RING-type E3 ubiquitin ligase RNF146/Iduna coupled

to glutathione _S_-transferase (GST)52. Tnks and RNF146 are known to form a stable complex that targets substrates for ADP-ribosylation and subsequent ADP-ribose-dependent

ubiquitination52,53,54,55. The WWE domain of RNF146 interacts directly with the poly(ADP-ribose) in these Tnks substrates to promote their ubiquitination. As a control for specificity of the

GST-WWE assay, pull downs were simultaneously performed using a GST-WWER164A mutant control, in which an arginine to alanine substitution in the RNF146 WWE domain abolishes interaction with

poly(ADP-ribose)52. As an additional control for specificity, we generated a construct that encodes mouse Axin1 with a nine amino-acid deletion in the TBD, _Axin1ΔTBD_, which is predicted

to prevent the Tnks-dependent ADP-ribosylation of Axin1 (refs 28, 47, 48). GST-WWE pull downs were performed with lysates from HEK293T cells expressing either _Flag-Axin1_ or

_Flag-Axin1ΔTBD_, followed by immunoblotting with Flag antibody (Fig. 6a). Although the levels of Axin1 and Axin1ΔTBD in these lysates were comparable, GST-WWE pulled down Axin1, but not

Axin1ΔTBD. Further, neither Axin1 nor Axin1ΔTBD was pulled down by the GST-WWER164A control. Tnks-mediated ADP-ribosylation does not markedly alter the electrophoretic mobility of Axin28,52;

nonetheless, we detected small mobility shifts in the ADP-ribosylated Axin isolated by GST-WWE pull down (Supplementary Fig. 16a), suggesting that Axin is pulled down through its

ADP-ribosylation, rather than through association with another ADP-ribosylated protein. These findings confirmed the specificity of the GST-WWE pull-down assay for the detection of

ADP-ribosylated Axin. To determine whether Wnt stimulation induces changes in the level of endogenous ADP-ribosylated Axin, we performed GST-WWE pull downs on lysates from HEK293T cells

treated with Wnt3A protein. The accumulation of phospho-LRP6 was used to monitor activation of the Wnt pathway (Fig. 6b, input). Unexpectedly, within 30 min of Wnt3A exposure, the level of

ADP-ribosylated Axin1 increased by four- to fivefold (Fig. 6b,c). These results demonstrate that the pool of ADP-ribosylated Axin increases rapidly following Wnt exposure. To determine

whether the increase in the pool of ADP-ribosylated Axin in response to Wnt exposure is an evolutionarily conserved process, we used GST-WWE pull downs to analyse lysates from _Drosophila_

S2R+ cells treated with Wg conditioned medium (Fig. 6d). We analysed two downstream signalling events to monitor the temporal response of these cells to Wg exposure: the phosphorylation of

Arrow/LRP6 (refs 8, 56), and the stabilization of Arm/β-catenin40,57 (Supplementary Fig. 8d). Treatment of S2R+ cells with Wg conditioned medium resulted in increased levels of phospho-Arrow

within 10 min, which peaked at 40 to 50 min and remained elevated for more than 2 h. In addition, Arm/β-catenin was stabilized within 20 min of Wg exposure and remained elevated for 2 h

(Supplementary Fig. 8d), as reported previously57. Our new Axin antibody allowed sensitive detection of endogenous Axin, and revealed a mobility shift following Wg stimulation (Figs 6d and

7a, input), consistent with the Wnt-dependent dephosphorylation of Axin observed previously in mammalian cells14,17,18. GST-WWE pull down followed by immunoblotting with the Axin antibody

revealed that the levels of ADP-ribosylated Axin increased within one hour of stimulation with Wg. In contrast, Axin was not pulled down by the GST-WWER164A control, confirming specificity

of the signal (Fig. 6d). The increase in ADP-ribosylated Axin levels ranged from four- to sevenfold, indicating that, as observed in human cells, ADP-ribosylated Axin accumulated rapidly

following Wg stimulation in _Drosophila_ cells (Fig. 6d,e). To further test this hypothesis _in vivo_, we quantitated changes in the levels of ADP-ribosylated Axin following Wg stimulation

in lysates from staged _Drosophila_ embryos expressing _Axin-V5_ using GST-WWE pull downs. These studies revealed that the total levels of Axin-V5 increased by approximately threefold in

stage 9 embryos (after Wg stimulation) by comparison with stage 4–5 embryos (before Wg stimulation; Fig. 6f,g). Importantly, the level of ADP-ribosylated Axin-V5 increased by ∼13-fold after

Wg stimulation (Fig. 6f,h). As described above, small mobility shifts were detected in ADP-ribosylated Axin (Supplementary Fig. 16b). These results indicated that the pool of ADP-ribosylated

Axin is preferentially increased following Wg exposure. Therefore, we conclude that the rapid increase in ADP-ribosylated Axin is an evolutionarily conserved response to Wnt/Wg stimulation.

ADP-RIBOSYLATION ENHANCES THE INTERACTION OF AXIN WITH LRP6 On the basis of the rapid accumulation of ADP-ribosylated Axin in both _Drosophila_ and human cells following Wnt exposure, we

speculated that ADP-ribosylation of Axin might facilitate Axin’s interaction with phospho-LRP6 and the subsequent activation of the Wnt pathway. Indeed, we observed that in lysates of human

cells exposed to Wnt, not only Axin, but also phospho-LRP6 was pulled down by GST-WWE (Fig. 6b). Analogously, in lysates of _Drosophila_ cultured cells or embryos exposed to Wg,

phospho-Arrow was pulled down by GST-WWE, but not the GST-WWER164A control, indicating a requirement for ADP-ribosylation (Fig. 6d,f). Thus, we hypothesized that both phospho-LRP6 and

phospho-Arrow were pulled down through their association with ADP-ribosylated Axin following Wnt exposure. To test this hypothesis, we repeated the GST-WWE pull downs after depleting

_Drosophila_ Axin using RNAi-mediated knockdown in cultured _Drosophila_ cells (Fig. 7a). Treatment of S2R+ cells with _Axin_ dsRNA resulted in a marked reduction in Axin levels, to the

extent that Arm/β-catenin was aberrantly stabilized in the absence of Wg stimulation (Supplementary Fig. 3i). In the presence or absence of _Axin_ knockdown, the level of phospho-Arrow was

comparable (Fig. 7a, input); however, the pull down of phospho-Arrow by GST-WWE was completely dependent on Axin (Fig. 7a). Although we cannot exclude the possibility that phospho-LRP6/Arrow

is ADP-ribosylated in an Axin-dependent manner, these findings support the hypothesis that following Wg exposure, phospho-Arrow interacts with ADP-ribosylated Axin. To further test the

hypothesis that the ADP-ribosylation of Axin promotes its interaction with phospho-LRP6 following Wnt stimulation, we treated HEK293T cells expressing Flag-Axin1 with either a dimethyl

sulfoxide (DMSO) control or with the small molecular inhibitor XAV939 (ref. 28), to inhibit the catalytic activity of the two human Tnks proteins. We immunoprecipitated from lysates of these

cells with Flag antibody, followed by immunoblotting with phospho-LRP6 antibodies. Treatment with XAV939, but not the DMSO control, markedly decreased the Wnt-dependent interaction between

Axin1 and phospho-LRP6 within 30 min of Wnt3A exposure (Fig. 7b). To further test this hypothesis, we immunoprecipitated lysates of HEK293T cells expressing _Flag-Axin1_ or _Flag-Axin1ΔTBD_

with Flag antibody, followed by immunoblotting with phospho-LRP6 antibodies directed against either phospho-serineS1490 [S1490 (ref. 8)] or phosphothreonine1572 [T1572 (ref. 44)]. By 30 min

after treatment with Wnt3A, Axin1 interacted with phospho-LRP6, as revealed by both phospho-LRP6 antibodies (Fig. 7c,d)8,12. In contrast, deletion of the TBD in Axin1 diminished the

interaction of Axin with phospho-LRP6 (Fig. 7c,d). Analogously, deletion of the TBD in _Drosophila_ Axin decreased the Wg-dependent interaction between Axin and phospho-Arrow (Supplementary

Fig. 17a). Deletion of the TBD in human Axin1 had no detectable effect on Axin1’s interaction with GSK3β, β-catenin or Dvl (Supplementary Fig. 17b,c). Altogether, these results indicated

that the Tnks-dependent ADP-ribosylation of Axin promotes Axin’s rapid interaction with phospho-LRP6 following Wnt stimulation. Finally, we tested whether the poly(ADP-ribosyl) polymerase

(PARP) activity of Tnks promotes the initial activation of Wnt signalling. We generated HA-tagged _Tnks_ transgenes for expression in _Drosophila_ embryos: one transgene encoded wild-type

Tnks, whereas the other transgene was identical, except for a methionine to valine substitution (M1064V) in the catalytic PARP domain (Fig. 8a). The analogous mutation (M1054V) in the highly

conserved PARP domain of human Tnks2 is known to abolish its catalytic activity58. Both _Tnks_ transgenes were integrated at the same genomic site, and expressed using the _mat-Gal4_

driver. As noted above, in _Tnks_ null mutant embryos expressing _Axin-V5_, the Wg-dependent expression of _engrailed_ was disrupted (Fig. 4f). In contrast, _engrailed_ expression was fully

restored in _Tnks_ mutant embryos expressing the wild-type _Tnks_ transgene (Fig. 8b), but not the _Tnks__M1064V_ transgene (Fig. 8c). Together, these findings support the conclusion that

the ADP-ribosylation of Axin by Tnks promotes not only the interaction between Axin and phospho-LRP6 following Wg exposure, but also the initial activation of Wg signalling. DISCUSSION We

demonstrate that Wnt exposure induces biphasic regulation in the level of Axin, and a large increase in the level of ADP-ribosylated Axin immediately after stimulation. ADP-ribosylation

enhances the interaction of Axin with phospho-LRP6, and promotes the activation of Wnt signalling. Our findings lead to three major revisions of the current model for the role of Tnks in the

activation of the Wnt pathway. First, Tnks serves bifunctional roles under basal conditions and after stimulation, revealing a remarkable economy and coordination of pathway components.

Second, our results provide a mechanistic basis for the rapid reprogramming of Axin function in response to Wnt stimulation, and thereby reveal an unanticipated role for Tnks in this process

(Fig. 8d). These findings suggest that Wnt exposure either rapidly increases the ADP-ribosylation of Axin or inhibits the targeting of ADP-ribosylated Axin for proteasomal degradation,

through mechanisms yet to be elucidated. Finally, we demonstrate that pharmacologic inactivation of Tnks diminishes the interaction of Axin with LRP6, revealing a previously unknown

mechanism through which small molecule Tnks inhibitors disrupt Wnt signalling, distinct from their known role in stabilizing the destruction complex. In the absence of Wnt stimulation, the

concentration-limiting levels of Axin regulate its scaffold function in the destruction complex15,16. As components of the destruction complex participate in other signalling pathways, the

low levels of Axin were proposed to maintain modularity of the Wnt pathway16. Our new findings indicate that Axin levels are not only regulated in the absence of Wnt, but also regulated

biphasically following Wnt stimulation. This sequential modulation of Axin divides activation of the pathway into an early, fast phase and a delayed long-term phase. During embryogenesis,

the earliest expression of Wg triggers the rapid appearance of Axin in segmental stripes, which is a novel hallmark for the initial activation of the pathway. Our findings reveal that Wnt

exposure induces a rapid increase in the total level of Axin, and importantly, a preferential increase in the level of the ADP-ribosylated Axin. The early Axin stripes are absent in _Tnks_

null mutant embryos and are also absent when the Tnks binding domain in Axin is deleted. Therefore, we propose that Axin ADP-ribosylation contributes to Axin stabilization and to the rapid

response to Wg stimulation. We postulate that the initial increase in levels of ADP-ribosylated Axin jump-starts the response to Wnt stimulation by enhancing the Axin-LRP6 interaction,

whereas the subsequent decrease in Axin levels prolongs the duration of signalling by reducing destruction complex assembly. Thus, Wnt stimulation induces rapid increases in the levels of

not only cytoplasmic β-catenin, but also ADP-ribosylated Axin. Previous work that coupled mathematical modelling with experimental analysis revealed that several Wnt signalling systems were

responsive to the relative change in β-catenin levels, rather than their absolute value59. This dependence was proposed to impart robustness and resistance to noise and cellular variation.

Our data raise the possibility that a similar principle applies to changes in Axin levels on the Axin-LRP6 interaction, as the marked increase in ADP-ribosylated Axin levels following Wnt

stimulation is evolutionarily conserved. Thus, the relative change in levels of ADP-ribosylated Axin may promote signalling following Wnt exposure by facilitating the fold change in

β-catenin levels. Our findings have relevance for the context-specific _in vivo_ roles of Tnks in Wnt signalling suggested in previous studies. Tnks inhibition disrupts Wnt signalling in a

number of cultured cell lines28, but _in vivo_ studies in several model organisms suggested that the requirement for Tnks in promoting Wnt signalling is restricted to specific cell types or

developmental stages. In mice, functional redundancy exists between the two Tnks homologues60, such that _Tnks_ single mutants are viable and fertile, whereas double mutants display

embryonic lethality without overt Wnt-related phenotypes60 (see Discussion in ref. 61). However, a missense mutation in the TBD of Axin2 that is predicted to disrupt ADP-ribosylation

resulted in either activating or inhibiting effects on Wnt signalling that were dependent on developmental stage. Tnks inhibitors resulted in the same paradoxical effects, suggesting complex

roles in mouse embryonic development61. Analogously, treatment of fish with Tnks inhibitors resulted in no observed defects in Wnt-mediated processes during development28; however, the

regeneration of injured fins in adults, a process that requires Wnt signalling62, was disrupted28,63. Similarly, the finding that _Drosophila Tnks_ null mutants are viable49,50,51 was

unexpected, as Tnks is highly evolutionarily conserved, and no other Tnks homologues exist in fly genomes. Nonetheless, our studies reveal that a less than twofold increase in Axin levels

uncovers the importance of Tnks in promoting Wg signalling during embryogenesis. Therefore, we postulate that Tnks loss can be compensated during development unless Axin levels are

increased, but that the inhibition of Wg signalling resulting from Tnks inactivation cannot be attributed solely to increased Axin levels (Supplementary Fig. 3, Figs 4f and 5). Furthermore,

_Drosophila_ Tnks is essential for Wg target gene activation in the adult intestine, and exclusively within regions of the gradient where Wg is present at relatively low concentration49,64.

Thus, the context-specific roles of Tnks observed in different model organisms may reflect the mechanisms described herein, which reveal that the Wnt-induced association of Axin with LRP6

occurs even in the absence of Axin ADP-ribosylation, but is markedly enhanced in its presence. We postulate that by enhancing this interaction, Tnks-dependent ADP-ribosylation of Axin serves

to amplify the initial response to Wnt stimulation, and thus is essential in a subset of _in vivo_ contexts. The recent discovery that Tnks enhances signalling in Wnt-driven cancers has

raised the possibility that Tnks inhibitors will offer a promising new therapeutic option28,63. Indeed, preclinical studies have supported this possibility30. Tnks inhibitors were thought

previously to disrupt Wnt signalling solely by increasing the basal levels of Axin, and thus by increasing destruction complex activity. However, our findings indicate that the degree to

which the basal level of Axin increases following Tnks inactivation is not sufficient to disrupt Wnt signalling in some _in vivo_ contexts. Instead, our results reveal that Tnks inhibition

simultaneously disrupts signalling at two critical and functionally distinct steps: by promoting activity of the destruction complex and by diminishing an important step in signalosome

assembly: the Wnt-induced interaction between LRP6 and Axin. On the basis of these findings, we propose that the efficacy of Tnks inhibitors results from their combined action at both of

these steps, providing a rationale for their use in the treatment of a broad range of Wnt-driven cancers. Therefore, our results suggest that in contrast with the current focus on tumours in

which attenuation of the destruction complex aberrantly activates Wnt signalling (such as those lacking APC), the preclinical testing of Tnks inhibitors could be expanded to include cancers

that are dependent on pathway activation by Wnt stimulation. These include the colorectal, gastric, ovarian and pancreatic cancers that harbour inactivating mutations in _RNF43_ (refs 65,

66, 67, 68, 69, 70), a negative Wnt feedback regulator that promotes degradation of the Wnt co-receptors Frizzled and LRP6 (refs 70, 71, 72). METHODS _DROSOPHILA_ STOCKS AND GENETICS To

generate the _pUASTattB Axin-V5_ transgene, we isolated a KpnI/HindIII fragment from _pAc5.1-Daxin-3xHA_ (ref. 28) and a HindIII/XbaI fragment encoding a triple V5 epitope from _pBlue

SK-3xV5_ (ref. 73), and ligated these fragments into _pUASTattB_ (refs 36, 37), at the KpnI and XbaI sites. To generate the _pUASTattB-AxinΔTBD-V5_ transgene, residues D-12 through K-32 were

deleted by PCR-based mutagenesis of _pUASTattB-Axin-V5_ using the oligonucleotide: 5′-GGTATCTGCTACCCCTTCGGTCATATGTTTCCGGATTCC-3′. The resulting _AxinΔTBD-V5_ fragment was digested with KpnI

and XbaI, and inserted into the _pUASTattB vector_ at the KpnI and XbaI sites. Transgenic flies were generated using phiC31-based integration36 at the _attP33_ or _attP40_ sites on

chromosome 2 (ref. 35). _pUASTattB Axin-GFP_ was generated using the Gateway LR Clonase system (Invitrogen), with an entry plasmid containing an _Axin_ cDNA amplified by PCR from an ovarian

library74 and the _pTWG_ destination vector containing sequences encoding _GFP_ (Drosophila Genomics Resource Center). _Axin-GFP_ was amplified by PCR using primers that introduced KpnI and

XbaI restriction sites at the 5′ and 3′ ends, respectively. Following KpnI and XbaI digestion, the amplified product was cloned into _pUASTattB_. Transgenic flies were generated using

phiC31-based site-specific integration at the _attP33 site_. _Axin-GFP_ has an 18 amino-acid linker sequence between the carboxy terminus of Axin and the amino terminus of GFP:

KPSD/kgradpaflykvvssat/(GFP) (‘/’ represents the junction between Axin and GFP, with additional amino acids shown in lowercase). The _pUASTattB-AxinΔTBD-GFP_ transgene was generated by

PCR-based mutagenesis of _pUASTattB-Axin-GFP_, in which residues D-12 through K-32 were deleted using the oligonucleotide: 5′-GGAATCCGGAAACATATGACCGAAGGGGTAGCAGATACC-3′. The resulting

_AxinΔTBD-GFP_ fragment was digested with KpnI and XbaI and inserted into the _pUASTattB_ vector at the KpnI and XbaI sites. To generate the _pUASTattB Tnks-HA_ transgene, a PCR fragment

encoding Tnks was amplified from the cDNA _LD22548_ (Drosophila Genomics Research Center), with the addition of an XhoI restriction site to 5′ end using the primer

5′-ACTCTCGAGATGGCCAACAGCAGCCGAAGT-3′. A fragment encoding a double-HA epitope followed by a KpnI restriction site was added to the 3′ end of the _Tnks_ cDNA using the PCR primer

5′-CATAGTCCGGGACGTCATAGGGATAGCCCGCATAGTCAGGAACATC-3′. The _Tnks-HA_ fragment was digested with XhoI and KpnI, and inserted into the _pUASTattB_ vector at the XhoI and KpnI sites. _pUASTattB

Tnks__M1064V__-HA_ was generated by replacing the methionine at position 1,064 with valine by PCR-based mutagenesis of _pUAST-Tnks-2_ × _HA_ using the oligonucleotide

5′-ATTGGCGGCGTGTTTGGGGCT-3′, and cloned into the XhoI and KpnI restriction sites of _pUASTattB._ Transgenic flies were generated using phiC31-based integration at the _attP40_ site. To

obtain embryos expressing _Axin-V5_ driven by _mat-Gal4_: _mat-Gal4_ females were crossed to _UAS-Axin-V5_ males. F1 female progeny of the genotype _UAS-Axin-V5 / mat-Gal4_ were crossed to

_UAS-Axin-V5_ males. To obtain _Tnks_ null mutant embryos expressing _Axin-V5_ driven by _mat-Gal4_: _UAS-Axin-V5; Tnks__19_ _mutant_ males were crossed to _mat-Gal4; Tnks__19_ females. F1

female progeny of the genotype _UAS-Axin-V5/mat-Gal4; Tnks__19_ were crossed to _UAS-Axin-V5_; _Tnks__19_ males. To obtain _Tnks_ null mutant embryos expressing _Tnks-HA_ and _Axin-V5_

driven by _mat-Gal4_: _UAS-Tnks-HA_, _UAS-Axin-V5_; _Tnks__503_ mutant males were crossed to _mat-Gal4; Tnks__503_ females. F1 female progeny of the genotype _UAS-Tnks-HA_,

_UAS-Axin-V5/mat-Gal4_; _Tnks__503_ were crossed to _UAS-Tnks-HA_, _UAS-Axin-V5_; _Tnks__503_ males. To obtain Tnks null mutant embryos expressing _Tnks__M1064V__-HA_ and _Axin-V5_ driven by

_mat-Gal4: UAS-Tnks__M1064V__-HA, UAS-Axin-V5; Tnks__503_ mutant males were crossed to _mat-Gal4; Tnks__503_ females. F1 female progeny of the genotype _UAS-Tnks__M1064V__-HA,

UAS-Axin-V5/mat-Gal4; Tnks__503_ were crossed to _UAS-Tnks__M1064V__-HA, UAS-Axin-V5; Tnks__503_ males. ANTIBODIES The primary antibodies used for immunostaining were rabbit anti-V5

(1:1,000, Abcam, ab9116), rabbit anti-GFP (1:200, Invitrogen, A11122), mouse anti-Engrailed (1:100, concentrated 4D9; Developmental Studies Hybridoma Bank, DSHB), mouse anti-Wg (1:200, 4D4

concentrated antibody, DSHB), mouse anti-Arm (1:20, N2 7A1, DSHB) and mouse anti-Neurotactin (1:20, BP106, DSHB). Secondary antibodies were goat or donkey Alexa Fluor 488 or 555 conjugates

(1:400, Invitrogen, A21202, A11008, A31572, A31570). The primary antibodies used for immunoblotting were mouse anti-V5 (1:5,000, Invitrogen, 46-1157), guinea pig anti-Axin (1:1,000 (ref.

49)), mouse anti-Wg (1:100, 4D4, DSHB), rabbit anti-Kinesin Heavy Chain (1:10,000, Cytoskeleton, AKIN01), mouse anti-Arm (1:100, N2 7A1, DSHB), mouse anti-α-Tubulin (1:10,000, DM1A, Sigma,

T6199), rabbit anti-α-Tubulin (1:10,000, Sigma, SAB3501071), mouse anti-Flag M2 (1:1000, Sigma, F3165), rabbit anti-GSK3β (1:1,000, Cell Signaling, 9315), mouse anti-β-catenin (1:4,000, BD

Biosciences, 610154), rabbit anti-Axin1 (1:1,000, Cell Signaling, #2074), rabbit anti-LRP6 (1:1,000, Cell Signaling, 2560), rabbit anti-c-Myc (1:1,000, Santa Cruz Biotechnology, sc-40),

rabbit anti-phospho-LRP6 [Ser1490] (1:1,000, Cell Signaling8, 2568), rabbit anti-phospho-LRP6 [Thr1572] (1:1,000, Millipore44, 07-2187), and guinea pig anti-Arrow (1:1,000 (ref. 75)).

Secondary antibodies used for immunoblotting were goat anti-rabbit HRP conjugate (1:10,000, Biorad, 170-6515), goat anti-mouse HRP conjugate (1:10,000, Biorad, 170-6516) and goat anti-guinea

pig HRP conjugate (1:10,000, Jackson ImmunoResearch). GST-WWE PULL DOWNS For GST pull downs, GST-WWE and GST-WWER164A beads were generated as described previously52. Embryos, S2R+ or

HEK293T cells were treated as indicated, then washed once with cold 1 × PBS and lysed in lysis buffer (50 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1% NP-40, 10% glycerol, 1.5 mM EDTA [pH 8.0]) or

RIPA buffer (50 mM Tris-HCl (pH 8.5), 300 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS) supplemented with 1 μM of the poly(ADP-ribose) glycohydrolase inhibitor ADP-HDP (Enzo Life

Sciences), and protease and phosphatase inhibitor cocktail (1:100, Thermo Scientific). Lysates were incubated with GST-WWE or GST-WWER164A beads overnight at 4 °C. Following incubation,

beads were washed four times in wash buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% NP-40, 10% Glycerol, 1.5 mM EDTA [pH 8.0]) supplemented with 1 μM ADP-HPD and protease and phosphatase

inhibitor cocktail (1:100). Bound materials were eluted with 2 × sample buffer and resolved by SDS–PAGE, transferred to PVDF or nitrocellulose membranes and blotted with the indicated

antibodies. PLASMIDS Plasmids used for transfection of human cell lines were _pCS2+ Flag-mouse Axin1_ (ref. 8) and _pCS2+ Flag-mouse Axin1ΔTBD_. To generate the _pCS2+ Flag-mouse Axin1ΔTBD_

plasmid, residues P-26 through E-34 were deleted by PCR-based mutagenesis of _pCS2+ Flag-mouse Axin1_ using the oligonucleotide: 5′-GATGCCGGAGAACTGGTATCTACTGAT-3′. The resulting Flag-mouse

_AxinΔTBD_ fragment was digested with ClaI and BglII, and then inserted into the _pCS2+ vector_ at the ClaI and BglII sites. Plasmids used for transfection of _Drosophila_ cells were

_pAc5.1-Axin-V5_ and _pAc5.1-AxinΔTBD-V5_. To generate the _pAc5.1-Axin-V5_ and _pAc5.1-AxinΔTBD-V5_ plasmids: fragments encoding Axin-V5 and AxinΔTBD-V5 from _pUASTattB-Axin-V5_ and

_pUASTattB-AxinΔTBD-V5_, respectively, were digested using KpnI and XbaI. The resulting fragments were inserted into the _pAc5.1_ vector (Invitrogen) at the KpnI and XbaI sites. STUDY DESIGN

AND STATISTICAL ANAYSIS In all experiments reported in this study, Student’s _t_-test and ANOVA test were performed using Prism (GraphPad Software Inc., CA, USA) or the SAS version 9.4

(Statistical Analysis System Institute Inc., NC, USA). Student’s _t_-test was used to compare two groups for all data sets. ANOVA test was used for comparison of more than two groups. _P_

values and detailed test statistics are provided in the figure legends. No blinding was done and no particular randomization method was used. All experiments were performed at least three

times. Additional methods and fly stocks used are described in Supplementary Information. ADDITIONAL INFORMATION HOW TO CITE THIS ARTICLE: Yang, E. _et al_. Wnt pathway activation by

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the Wingless morphogen gradient. _Development_ 133, 307–317 (2006). Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank J. Cho, G. Deshpande, S. Ogden and E.

Wieschaus for comments on the manuscript, K. Beckett, J. Byun, F. Cong, Y. Han, S. Kim, A. Lavanway, B. MacDonald and S. Ogden for technical advice, the investigators listed in Methods for

generously sharing reagents, and V. Marlar for technical assistance. This work was funded by grants from the NIH (RO1CA105038 to Y.A., R01GM081635 and R01GM103926 to E.L., P40OD018537 to the

Bloomington Drosophila Stock Center), the Emerald Foundation (to Y.A.), the Norris Cotton Cancer Center (to Y.A. and S.F.), the Academic Research Fund (MOE2014-T2-2-039 to NT) and the

National Science Foundation (DBI-1039423 for purchase of a Nikon A1RSi confocal microscope). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Genetics and the Norris Cotton Cancer

Center, Geisel School of Medicine at Dartmouth College, Hanover, 03755, New Hampshire, USA Eungi Yang, Ofelia Tacchelly-Benites, Zhenghan Wang, Michael P. Randall, Ai Tian, Hassina

Benchabane, Claudio Pikielny & Yashi Ahmed * Department of Pharmacology and Toxicology, Geisel School of Medicine at Dartmouth College, Hanover, 03755, New Hampshire, USA Sarah

Freemantle * Department of Biological Sciences, Yale-NUS College, National University of Singapore, Singapore, 138615, Singapore Nicholas S. Tolwinski * Department of Cell and Developmental

Biology, Vanderbilt Ingram Cancer Center, and Vanderbilt Institute of Chemical Biology, Nashville, 37232, Tennessee, USA Ethan Lee Authors * Eungi Yang View author publications You can also

search for this author inPubMed Google Scholar * Ofelia Tacchelly-Benites View author publications You can also search for this author inPubMed Google Scholar * Zhenghan Wang View author

publications You can also search for this author inPubMed Google Scholar * Michael P. Randall View author publications You can also search for this author inPubMed Google Scholar * Ai Tian

View author publications You can also search for this author inPubMed Google Scholar * Hassina Benchabane View author publications You can also search for this author inPubMed Google Scholar

* Sarah Freemantle View author publications You can also search for this author inPubMed Google Scholar * Claudio Pikielny View author publications You can also search for this author

inPubMed Google Scholar * Nicholas S. Tolwinski View author publications You can also search for this author inPubMed Google Scholar * Ethan Lee View author publications You can also search

for this author inPubMed Google Scholar * Yashi Ahmed View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS E.Y., O.T.-B., Z.W., A.T., H.B.,

S.F., C.P., N.S.T., E.L. and Y.A. conceived the study. E.Y., O.T.-B., Z.W. and Y.A. designed and performed the experiments. M.P.R. assisted in the experiments. C.P. provided input. E.Y.,

O.T.-B. and Y.A.wrote the paper. CORRESPONDING AUTHOR Correspondence to Yashi Ahmed. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests.

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Wang, Z. _et al._ Wnt pathway activation by ADP-ribosylation. _Nat Commun_ 7, 11430 (2016). https://doi.org/10.1038/ncomms11430 Download citation * Received: 18 May 2015 * Accepted: 23 March

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