The Wnt/Wg pathway controls myriads of biological phenomena throughout the development and adult life of all organisms across the phyla. Thus, an aberrant Wnt signaling is associated with a wide range of pathologies in humans. Tight regulation of Wnt/Wg signaling is required for the maintenance of proper cellular homeostasis. Here we report a novel role of E3 ubiquitin ligase Deltex in Wg signaling regulation. Drosophila dx genetically interacts with wg and its pathway components. Further, Dx LOF results in a reduced spreading of Wg while its over-expression expands the diffusion gradient of the morphogen. We attribute this change in Wg gradient to the endocytosis of Wg through Dx which directly affects the short and long-range Wg targets. We also demonstrate the role of Dx in the regulation of Wg effector Armadillo where Dx down-regulates Arm through proteasomal degradation. We also showed the conservation of Dx function in the mammalian system where DTX1 is shown to bind with β-catenin and facilitates its proteolytic degradation, spotlighting a novel step that potentially modulates Wnt/Wg signaling cascade.
This is a useful study of the connection between the ubiquitin ligase protein deltex and the wingless signaling pathway. Two different links are inferred from genetic interactions in vivo between loss-of-function mutations and overexpression. While providing useful in vivo physiological context, the approach is necessarily incomplete in so far as it cannot distinguish between direct and indirect mechanisms.
Morphogens play a key role in cell fate determination in a concentration-dependent manner, and their functions are indispensable to tissue patterning during development. These molecules elicit long-range signaling by forming a concentration gradient and cells differentiate based on these positional cues. The roles of morphogens such as Wingless (Wg), Hedgehog (Hh), and Decapentaplegic (Dpp) during the development of Drosophila appendages have been extensively studied (Cadigan and Nusse, 1997; Lecuit et al., 1996; Nellen et al., 1996; Tabata, 2001)
Wg signaling is an evolutionarily conserved pathway and has multiple roles in the regulation of a variety of developmental processes, including cell-fate specification, cell proliferation, cell survival, and migration (Cadigan and Waterman, 2012; Logan and Nusse, 2004; MacDonald et al., 2009; Valenta et al., 2012). Any aberration in the expression of candidates in the Wg signaling cascade often leads to human diseases including cancer(Clevers, 2006; Coombs et al., 2008)
In the developing wing disc, Wg is secreted from the Dorsal-Ventral (D/V) boundary, and a gradient of Wg is formed to elicit long-range Wg signaling(Baena-Lopez et al., 2012; Diaz-Benjumea and Cohen, 1995). Secreted Wg, consecutively induces the expression of its target Senseless (Sens) and Distal-less (Dll). High threshold Wg target gene senseless is responsible for the formation of margin sensory bristles of adult wings(Couso et al., 1994), whereas the low threshold Wg target gene distal-less is required for proper wing growth and is expressed in a graded manner, declining towards the edges of the wing pouch (Couso et al., 1994; Neumann and Cohen, 1997). Wg also induces the expression of Cut by activating Notch in the D/V boundary(Bray, 1997).
Wg exercises its effect by regulating the transcription of the target genes in the responding cells. Armadillo (vertebrate homolog β-catenin) plays a pivotal role in the transduction of Wg signaling (Dierick and Bejsovec, 1998; Städeli et al., 2006). In the absence of the Wg ligand, a protein complex comprising of APC (Adenomatous polyposis coli), Axin, GSK3-β (Shaggy), and Casein Kinase-I (Cki) mediates Armadillo (Arm) phosphorylation, and ubiquitination, followed byits degradation via the proteasome pathway. On the contrary, binding of the ligand Wg to its receptor Frizzled (Fz), at the cell surface, initiates a signaling cascade that inhibits the function of the destruction complex via adaptor protein Dishevelled (Dsh), leading to the stabilization of Arm. Arm then translocates to the nucleus thereby activating the transcription of the downstream target genes (Mosimann et al., 2009). In canonical Wg signaling, Arm plays a dual role. The cytosolic Arm, as mentioned, is used for the transduction of the Wg signals,whereas the Arm localized at the adherens junctions (AJs) together with α-catenin and cadherin homologs is required for cell adhesion (Peifer et al., 1993).
We have earlier reported that deltex (dx), a known Notch signaling regulator, modulates Decapentaplegic morphogen gradient formation and affects Dpp signaling in Drosophila (Sharma et al., 2022). Here, in the present study, we report that deltex genetically interacts with the genes that encode Wg pathway components during wing development. Immuno-cytochemical analysis revealed that Dx facilitates the spreading of the morphogen, thereby directly affecting the expressionof the short and long-range target genes, Senseless and Vestigial. This change in the Wg gradient is attributed to the role of Dx in Wg trafficking. Here we speculate that Dx, like in the case of Dpp and Notch, aids in the vesicular trafficking of Wg to facilitate its gradient formation. Further, Dx was also seen to reduce the expression of the Wg effector Arm through proteasomal degradation. We also investigate the conservation of Dx function in β-catenin stability and degradation in cultured human cells. Here we show that human DTX1 binds with β-catenin and facilitates its degradation. This study thus presents a whole new avenue of Dx function in the regulation of Wg signaling.
2.1. dx genetically interacts with Wg pathway components
Wg controls the growth and patterning of the Drosophila wing and aberrant Wg signaling interferes with normal wing development. In order to ascertain the role of dx in Wg signaling, first, we tried to check if dx genetically interacts with wg alleles. It is interesting to note that both the wg loss-of-function alleles tested (wgCX3, and wgCX4) showed lethality in homozygous condition; however, in heterozygous condition, they do not exhibit any phenotype (Fig. 1.B1, C1). The two mutant alleles of dx (a null allele dx152and a loss-of-function allele dx) show wing vein thickening phenotype in hemizygous condition, however, dx152 and dx heterozygote female flies exhibit normal wings. When the loss-of-function allele of wg (wgCX3 and wgCX4) was brought in heterozygous condition with either dx152 or dx mutant alleles, they showed wings that were indistinguishable from the wild type (data not shown). However, when the dosage of wg was reduced in dx152 and dx mutant alleles in the hemizygous background, an enhanced wing vein thickening phenotype was observed (n=100). Moreover, an appreciable number of flies show notching in the wing margin (Fig. 1.B1-C3), suggesting that a complete absence of dx creates a sensitized background, which is more responsive to a lower dosage of wg during wing development. Further, the expression of Cut, a downstream target of Wg signaling activity 23 at the D/V boundary of the wing disc, was found to be reduced in wing discs from these larvae. Third instar larval wing discs displayed an interrupted Cut expression at the intersection of anterior-posterior (A/P) and dorsal-ventral (D/V) boundary from individuals carrying heterozygous wg loss-of-function allele in dx hemizygous null background (Fig. 1.H1-H4). These results clearly indicate that dx may play a role in Drosophila Wg signaling activity.
Further, we tried to elucidate if the receptor of wg, frizzled (fz), does exhibit any interaction with dx. When the hypomorphic allele of fz (fz1), was brought in heterozygous condition with dx152 null, the male flies displayed wing notching phenotype in addition to wingvein thickening (n=100) (Fig. 1.D1-E3). senseless (sens), a short-range target of Wg signaling also displayed a strong genetic interaction with dx. A loss-of-function allele of sens (sensE58) is recessive lethal in homozygous condition; however, in heterozygous condition, they do not display any phenotype. On the contrary, on reducing the dosage of sens in dx null and dx loss-of-function allele in hemizygous condition, a marked serration in the wing margin was observed (n=100) (Fig. 1F1-F3). These results displayed a strong genetic interaction between dx and Wg pathway components, suggesting that Dx may have functional implications in Drosophila wing development.
2.2 Loss of dx reduces Wg signaling
To better understand the role of dx in Wg signaling regulation, we studied the expression of Wg and its target genes in dx null wing discs. It was found that in dx null third instar larval wing discs, the gradient of Wg at the D/V boundary was shortened. High-magnification pictures revealed a significant decrease in the amount of Wg emanating from the Wg-producing cells at the D/V boundary (Fig. 2.A2). Thus, Dx is required for the proper Wg gradient formation, as the range of Wg expression and the number of Wg puncta was significantly reduced in dx null condition. Similar interruption in Wg gradient was also observed by Hori et al, where dx24 allele showed a reduction in Wg expression 41, however, the study focuses on the indispensable role of dx in Notch signaling.
We also analyzed the expression of Wg target genes in dx null discs. The domain of Vestigial, the long-range target of Wg was found to be significantly broadened in dx null discs (Fig. 2.C2). This is in contrast to the reported partial suppression of Vg expression in ptc-GAL4 expressed Dx dominant negative discs(Hori et al., 2004). Moreover, in another set of experiments, we found that the expression of the short-range target Sens showed slight deformity in the expression pattern with a constricted expression at the D/V boundary (Fig. 2.B2).
Expression of the short-range target such as Sens needs a higher threshold of Wg morphogen gradient than the long-range target like Vestigial which often requires a less concentrated Wg gradient. Thus, our results show that a proper Wg gradient is not formed in the absence of Dx, and this supports that Dx may have a profound role in regulating Wg signaling through its gradient formation.
2.3 Dx down-regulates Wg signaling
Based on the loss-of-function genetic interaction studies between dx and wg mutant alleles that suggest the role of Dx in Wg signaling, we further tried to investigate the gain-of-function effect of Dx on Wg signaling activity. For this, UAS-GAL4 binary system was exploited. We over-expressed FLAG-tagged UAS-Dx, with engrailed-GAL4(en-GAL4) driver strain, that drives the expression of the transgene in the posterior compartment of the wing imaginal disc, and inturn in the adult wing. Over-expression of Dx with en-GAL4 shows pupal lethality at 25°C, however, at 18°C, the flies emerge with massive morphological defects. The wing displayed blisters along with vein loss (Supplementary Fig. 1.A2). Besides, Dx over-expression with apterous-GAL4 (ap-GAL4) also resulted in abnormal wings that failed to appose properly and show large blisters (Supplementary Fig. 1.A3), and since Wg signaling modulates the expression of cell adhesion molecules in Drosophila wing imaginal disc cells (Bienz, 2005; Wodarz et al., 2006) such phenotypes are often associated with impaired Wg signaling.
To check if Dx is involved in Wg pathway regulation in other developmental contexts, we over-expressed Dx in the primordia of the dorsal thorax using ap-GAL4 and pnr-GAL4. Dx over-expression in the thorax leads to reduced and disoriented macrochaetae and reduction in scutellar bristles (Supplementary Fig. 1.B2-B3). A reduced tarsal segment along the proximodistal axis and dorsoventral shift of the sex combs was observed when Dx was over-expressed with distal-less-GAL4 (dll-GAL4) in the leg (Supplementary Fig. 1.C2). All these phenotypes are characteristics of reduced Wg signaling as also shown earlier for ectopic expression of another gene required for epithelial planar polarity (EPP), nemo (Zeng and Verheyen, 2004). Dx shows a similar phenotype when overexpressed in flies suggesting that like nemo, Dx may antagonize the Wg signaling activity.
To further test whether Dx affects the Wg signaling output we tried to analyze the expression of the Wg target genes in the wing imaginal discs of the third instar larvae. Wg activates the expression of the proneural gene senseless (sens) in the sensory organ precursor, which later develops into bristles of the adult wing margin (Blair, 1996; Couso et al., 1994). The expression of Senseless, a canonical target normally activated by high Wg levels in two discrete stripes at the D/V boundary (Nolo et al., 2000) was found to be significantly reduced in the posterior compartment where Dx was over-expressed (Fig. 3.A2). It is known that Wg can also indirectly induce the expression of Cut. A positive feedback loop between Wg expressing cells along the D/V boundary and Serrate and Delta expressing cells in adjacent cells maintains the Notch signaling activity along the D/V boundary, which consecutively induces the expression of Cut (Bray, 1997). Similar to Sens, we found that the expression of Cut was also drastically abolished upon Dx over-expression (Fig. 3.B2). Moreover, to the contrary, the expression of Vestigial (Vg), which is activated in response to low ligand concentrations (Neumann and Cohen, 1997; Zecca et al., 1996) was found to be expanded in the posterior domain of the wing imaginal disc (Fig. 3.C2), indicating that Dx reduces the Wg gradient. These results strongly suggest that Dx modulates Wg signaling output.
Also, in our earlier reports, we demonstrated the role of Dx in inflicting apoptosis. Dx synergizes with JNK ligand Eiger and triggers caspase-dependent cell death (Dutta et al., 2018). Similarsynergistic action of Dx was also reported with TRAF6 where the association triggers JNK activation in an Eiger independent manner (Sharma et al., 2021). Thus, we tried to dissect if the loss of these markers (Sens and Cut) is due to cell death. We inhibited apoptosis with caspase inhibitor protein p35 in Dx over-expression background and no rescue in the level of Sens and Cut expression was observed in the posterior compartment of the wing disc (Fig. 3.D1-E4)suggesting the loss of Sens and Cut is independent of cell death.
It is worth mentioning here that similar to our findings, the expression of Sens was found to be down-regulated when Reggie-1, a component of the membrane micro-domainwas up-regulated. The reduction in Sens and Cut expression, in their studies, was attributed to an enhanced spreading of the morphogen, Wg (Katanaev et al., 2008).
Drosophila Dx interacts with the ankyrin-rich repeats of the Notch intracellular domain and regulates Notch signaling positively(Matsuno et al., 1995). Moreover, negative regulation of Dx in association with non-visual β-arrestin, Kurtz was reported earlier, where polyubiquitination mediated degradation of Notch by Dx was attributed to this loss (Mukherjee et al., 2005). A similar reduction of Notch and its targets was reported lately when Dx synergistically interacts with TRAF6, and Hrp48 (Dutta et al., 2017; Mishra et al., 2014). Human Deltex (DTX1) was also shown to regulate NOTCH1/HES1 signaling negatively in osteosarcoma cells (Zhang et al., 2010). It was reported earlier that in D/V boundary of the wing disc, the regulation of Wg is Notch-dependent (Neumann and Cohen, 1996), we tried to elucidate if the regulation of Wg via Dxis through Notch. Thus, we examined the status of Deadpan (Dpn) and NRE (Notch response element), a direct target of Notch(Zacharioudaki and Bray, 2014). In contrast to reduced levels of Sens and Cut, the expression of Dpn was found to remain unaltered in Dx over-expressed conditions (Supplementary Fig. 2. A1-A4), suggesting that Dx can possibly regulate the two signaling cascades, viz. Notch and Wg independent of each other. Further, we also checked the status of the Notch response element (NRE-GFP) in Dx over-expression background and found a punctate NRE expression in the posterior domain (Supplementary Figure 2. B1-B4). The role of Dx in Notch regulation is evident however if Dx regulates Wg through Notch or if the two events are independent is still a crucial aspect to investigate. Our results however indicate an independent role of Dx in Wg signaling regulation. We however do not rule out the alternate possibility of Wg regulation through Dx via Notch.
2.4 Over-expression of Dx expands the Wg diffusion gradient
In the third instar wild-type wing discs, Wg is secreted from the producing cells at the D/V boundary, which diffuses on either side to form a concentration gradient. Over-expression of Dx, by the A/P boundary-specific ptc-GAL4, results in an erosion of the Wg gradient (Fig. 3.A2 and B2). Wg expression can be visualized as spreading far into the disc from the site of production. A high magnification picture further revealed far-spreading punctate Wg expression more significantly in the ventral compartment. It is interesting to note that the number of Wg puncta and their apparent size is also significantly increased when Dx was over-expressed using ptc-GAL4 (Fig. 4.B2, Hori et al.,2004). Similardiffusion of Wg gradient was also observed when Dx was over-expressed with posterior domain-specific en-GAL4 driver (Fig. 4.C2).
We also observed a significant increase in the extracellular Wg expression upon Dx overexpression further corroborating our initial results (Fig. 4.D2). Earlier it was shown that Swim (Secreted Wg interacting molecule) promotes long-range Wg signaling by maintaining Wg solubility (Mulligan et al., 2012). The report postulates that Swim reduction results in an increase in extracellular Wg which consequently increases the expression of long range Wg target, Dll (Mulligan et al., 2012).We have observed a very similar situation with another long range Wg target, Vg in increased Dx expression condition (Fig. 3.C2). Here we do not rule out the possibility that Dx potentially aids in Wg mobilization from the adherens junction. Several models from studies in Drosophila embryonic epidermis and wing disc have been put forward to explain Wg movement across the cells (Cadigan, 2002; Tabata and Takei, 2004). Our result indicates that Dx facilitates Wg spreading which flattens its gradient. This, in turn, results in the reduction of the short-range Wg targets (Fig. 3.A1-A3), which often require high levels of Wg. On the contrary, the long-range Wg target showed an expansion in their expression domain (Fig. 3.C1-C3), suggesting that Dx affects the Wg gradient formation.
2.5 Dx-mediated Wg signaling is regulated by endocytosis
Extensive evidence suggests that signaling and endocytosis are intricately linked. Classically, endocytosis has been thought to reduce the number of accessible receptors, either through its sequestration in the endosomal vesicles or degradation in lysosomes, which consecutively downregulates the signaling. However, some reports support the notion that endocytosis can also positively regulate the signaling cascade, its initiation, and propagation (Hupalowska and Miaczynska, 2012; Seto and Bellen, 2006).
Different mechanisms have been suggested for the role of endocytosis in various signaling cascades. Notch is continuously internalized into early endosomes and followed by its internalization, Notch is sorted to other endocytic compartments including multivesicular bodies/late endosomes, and lysosomes, or recycling endosomes (Fortini and Bilder, 2009). Thus, this step helps to maintain a quantitative as well as a qualitative pool of Notch. Once endocytosed, Notch follows a vesicular trafficking pathway that determines its final fate. In the canonical pathway, Notch gets internalized after binding to its ligand. However, during the non-canonical pathway, Notch can be internalized in the absence of ligand interaction. Once internalized, the Notch protein faces almost the same pathway that begins with its incorporation into the early endosomal vesicles. From here, it is either recycled back to the cell membrane, or it becomes a part of late endosomal vesicles/mature endosomes (Fortini and Bilder, 2009; Vaccari et al., 2008; Wilkin et al., 2008; Yamada et al., 2011).
In Notch trafficking and endocytosis, Dx plays a key role. Dx facilitates the trafficking of Notch receptors from the cell membrane into the early and then to the late endosomal vesicles in the cytoplasm (Hori et al., 2004). It was observed that Dx, directly or indirectly, increased the half-life of these accumulated Notch proteins in the endosomal vesicles (Hori et al., 2004). Ifthe transport of Notch into the late endosome was blocked, it hampered Dx-mediated Notch signal activation (Hori et al., 2004). Further study, using a similar approach, showed that Dx genetically interacts with the components of HOPS and AP-3 endocytic complexes that, successively, have a significant influence on Notch signaling (Wilkin et al., 2008). Dx has also been shown to play a critical role in Dpp endocytosis and trafficking (Sharma et al., 2022) .
Like Notch and Dpp, evidence supporting Wg endocytosis has been reported earlier (Seto and Bellen, 2006). Wg is found to co-localize with Rab5 and Rab7 in the early and late endosomes respectively (Marois et al., 2006). We also observed a significant co-localization of Wg with Dx in Rab5-positive vesicles (Figure 5.A1-A4). Rab5 is a smallGTPase that is necessary for endosome fusion to form early endosomes (Clague and Urbé, 2001). Impaired endocytosis affects Wg trafficking and in turn the signaling. Expression of a Dominant-negative form of Rab5 (Rab5S43N) blocks the early steps of endocytosis, including internalization and early-endosome fusion (Entchev et al., 2000), and significantly affects the Wg signaling (Seto and Bellen, 2006). A more punctate Wg expression was observed when the endocytosis is blocked by over-expressing a GDP-bound Rab5 (Rab5DN or Rab5S43N) with a D/V boundary specific C96-GAL4 (Fig. 5.C1). A further reduction in Wg puncta was observed when Dx was over-expressed in the same background, suggesting the role of Dx in Wg trafficking (Fig. 5.C2).
Additionally, C96-GAL4 mediated over-expression of Rab5DN, results in a loss of wing tissue similar to the loss of Wg, implying the role of endocytosis in Wg signaling (Baker, 1988; Couso et al., 1994). This loss of wing tissue was further aggravated when Dx was over-expressed along with Rab5DN suggesting a more diffused Wg expression at the D/V boundary, which further limits the threshold required to form the adult wing margin (Fig. 5.B2). This synergism bringsabout 90% lethality, further implicating the role of Dx in Wg regulation via endocytosis. In addition to Rab5, the late endosome protein Rab7, when over-expressed with Dx, showed a punctate Wg expression (Fig. 5.F2), suggesting an important role of Dx in Wg trafficking. Rab7 targets the endocytic cargo from the early endosome to the late endosome and then to the lysosome for degradation. Over-expression of a wild type Rab7 increases trafficking towards the lysosome. This is due to enhanced vesicle fusion, resulting in enlarged late endosomal structures, prone to lysosomal degradation (Entchev et al., 2000). When UAS-Rab7 was co-expressed with Dx, a more punctate Wg expression was observed evincing that Dx might facilitate the functions of Rab7. Our study suggests the involvement of Dx in Wg endocytosis, however, further analysis is required to unravel the detailed mechanistic aspect in Wg endocytosis and trafficking via Dx.
2.6 Dx modulates Wg signal transduction
Arm, the Drosophila orthologue of mammalian β-catenin has a dual function. It acts as a nuclear transcription factor that transduces the Wg signaling on one hand and maintains the cell integrity on the other (Coombs et al., 2008). Within the wing disc, Arm concentrates apically where it binds with the transmembrane cadherins to build up the adherens junction that connects the actin filaments across polarized epithelial cells, thereby acting as a bridge between E-cadherin and the actin cytoskeleton. To investigate the functional aspect of Dx in Wg signaling regulation, we analyzed the status of this key effector of Wg signaling. Upon Dx over-expression with en-GAL4, the accumulation of Arm was strongly reduced in the whole posterior compartment (Fig. 6.A2), and most evidently at the D/V boundary. A similar reduction in Arm expression was observed in the eye disc in the GMR domain (Supplementary Fig. 3.B1.) indicating that Dx might disrupt the cell fate specification in the eye tissue. Moreover, confocal Z stack analysis revealed a reduction of apical Arm accumulation (Fig. 6.B2). The reduction of the apical Arm is consistent with the decline of cell adhesion, leading to the structural reorganization of the cytoskeleton (Wodarz et al., 2006). Highermagnification pictures further revealed a disorganized cell morphology upon Dx over-expression. Moreover, it was also observed that Dx co-localizes with endogenous Arm(marked by arrow) and is in the same vesicle upon Dx over-expression, suggesting that the two proteins might physically interact (Fig. 6.C3 and C4).
Further, upon over-expressing activated Arm (Arms10), with the wing margin specific C96-GAL4, ectopic hairs were observed throughout the wings, and this is more evident near the wing margin (Fig. 6.E1 and E2). This is a Wg gain-of-function phenotype (Vuong et al., 2018). The ectopic hair phenotype, however, was significantly reduced when Dx was over-expressed in the samebackground (Fig. 6.E5 and E6), corroborating with our immuno-staining results. A similar reduction in Arm gain-of-function phenotype was observed when the activated Arm was over-expressed together with Dx in the Drosophila eye. Over-expressing Arms10 with eye-specific GMR-GAL4 renders flies with reduced eye size (Freeman and Bienz, 2001) and severely diffused ommatidia (Fig. 6.F1 and F2). Moreover, upon over-expressing Dx in the same background, the Arm gain-of-function phenotype was found to be suppressed (Fig. 6.F5 and F6).
Interestingly, it was also observed that Dx co-localizes with endogenous Arm and is in the same vesicle upon Dx over-expression (Fig. 6.C3). Since Arm accumulation was reduced in the cytoplasm of Dx over-expressed cells in the disc, we also checked if there is any alteration of nuclear localization of Arm in these cells. To investigate this, we analyzed the nuclear versus cytoplasmic localization of Arm protein in the wing imaginal disc. High magnification pictures gave us a hint of Arm being exclusively in cytoplasm in Dx over-expressed condition. This inference was made since no localization of Arm in the nucleus (DAPI-stained area) was observed in the posterior compartment of the wing disc, though the anterior compartment showed significant localization of Arm with DAPI (marked by arrow) (Supplementary Fig. 4. A1-A10). The small size of cells in the wing disc made it difficult to draw a definite result and a more robust and facilitated assay system is required to visualize the changes i.e., cytoplasm versus nuclear localization.
Since Dx possesses an E3 ubiquitin ligase activity, so this down-regulation can be a consequence of ubiquitination-mediated degradation of Arm which needs to be determined.
2.7 Dx degrades Armadillo by the proteasome-mediated mechanism
The stabilization of Arm/β-catenin is critical in Wg/Wnt signaling regulation and the ubiquitination-dependent proteasomal degradation is considered to be a major mechanism for controlling its stability. β-TrCP, Jade1, casitas B-lineage lymphoma (c-Cbl), and SIAH1 [(the human homolog of Drosophila seven in absentia (sina)], are known E3 ligases that mediate β-catenin ubiquitination in the cytoplasm (Spiegelman et al., 2000). Moreover, β-catenin ubiquitination in the nucleus is mediated by c-Cbl and TRIM33 (tripartite-motif containing protein 33). C-Cbl is a RING finger E3 ubiquitin ligase that binds to the non-phosphorylated nuclear β-catenin via the armadillo repeat region (Chitalia et al., 2013). Though the ubiquitination of β-catenin is not always linked with its degradation and there are reports where E3 ubiquitin ligases enhanced the stability of β-catenin by ubiquitinating through Lys-29 or Lys-11 linked ubiquitin chains (Hay-Koren et al., 2011). Thus, to elucidate if Arm downregulation through Dx is a ubiquitination-dependent phenomenon, first, we tried to test if Dx physically interacts with Arm. Co-immunoprecipitation experiments using FLAG-tagged Dx revealed that FLAG-tagged Dx pulled down Arm when co-expressed in eye tissue (Fig. 7. A). Likewise, FLAG-tagged Dx was coimmunoprecipitated with Arm using an anti-Arm antibody when the two proteins were expressed together (Fig. 7. B).
Further, to investigate the role of Dx in Arm degradation we incubated the imaginal discs with MG312, a proteasome inhibitor, before performing Arm staining. A rescue in Arm loss post MG132 treatment was observed in the posterior domainof the wing disc suggesting it to be a ubiquitination-dependent phenomenon (Fig. 7.C1-E4). Moreover, Western blot reconfirmed our immunofluorescence studies where a decline in Arm expression was observed in Dx over-expression which was significantly rescued after MG132 treatment (Fig. 7. G, H). These results unravel a novel mechanistic aspect of Dx in Wg signaling regulation.
2.8 Conserved mechanism of β-catenin degradation by human DTX1
In order to elucidate the conserved role of Dx in Wg signaling regulation across the phyla, we examined the status of β-catenin in human DTX1over-expressed HEK-293 cells. In the Drosophila system we have observed that Arm physically interacts with Dx and proteasome inhibitor, MG132, treatment resulted in stabilization of Arm in Dx-overexpressed wing imaginal discs. Thus, to determine the functional conservation of human homolog of Drosophila Dx, DTX1 in the β-catenin degradation, first we checked the binding of human DTX1 with β-catenin in HEK-293 cells through co-immunoprecipitation experiment. Consistent with the Drosophila studies, the results here undoubtedly reveal the fact that DTX1 binds to β-catenin (Figure 8.A). In addition, as in the case of Drosophila, β-catenin also gets stabilized in the presence of MG132 in human HEK-293 cells overexpressing DTX1. Post MG132 treatment, the HEK-293 cell lines transfected with HA-tagged DTX1 showed a rescue in β-catenin expression (Fig. 8.B) These results clearly indicate that there is aconserved mechanism through which DTX1 degrades β-catenin and in turn regulates Wnt signaling.
The diverse role of Wg in different developmental contexts depends on the Wg signaling activity. Endocytosis and intracellular trafficking regulate Wg signaling levels. Internalization and endosomal transport of Wg not only affect levels and distribution of Wg ligand but also regulates signal transduction through Arm stabilization. Regulation of Wg pathway is tightly monitored at multiple steps. Canonically, Wg exerts its effects by regulating the transcription of the target genes through the effector molecule, Arm (Dierick and Bejsovec, 1998; Städeli et al., 2006). In theabsence of the Wg signaling the multi-protein destruction complex (APC, Axin, GSK3-β, andCki) phosphorylates and degrades Arm. However, in the presence of the ligand Wg, the cell surface primes a signaling cascade with the aid of the scaffold protein, Axin, which in turn inhibits the destruction complex through the adaptor molecule Dishevelled leading to the stabilization of Arm. The stabilized Arm thereby enters the nucleus to activate the transcription of the target genes (Bilic et al., 2007; Mosimann et al., 2009). The stabilization of Arm is therefore a critical step where Wg regulation can take place. An activated Wg signaling also can relocate Axin from intracellular vesicles to the plasma membrane, thereby inactivating the destruction complex and stabilizing Arm (Cliffe et al., 2003). Relocation of Axin through intracellular trafficking strengthens the fact that Wg signaling is equally regulated through a non-canonical mechanism. Intracellular trafficking affects the efficiency of Wg signaling significantly. Knockdown of dynamin, a protein essential for clathrin-mediated internalization, significantly reduces Wg signaling. A Rab5 knockdown further down-regulates Wg signaling suggesting that an internalization and endosomal transport of Wg is critical for its regulation (Seto and Bellen, 2006).
Here we show that Dx plays an important role in the intracellular trafficking of Wg and consequently facilitates the spreading of the Wg gradient. Dx regulates Wg signaling activity both at the level of spreading of Wg ligand as well as directly at the level of Arm stabilization. We hypothesize that since Dx has an E3 ubiquitin ligase activity, it can directly degrade Arm through ubiquitination followed by proteasomal degradation and thus diminish Wg signaling.
Dx is a cytoplasmic protein and is best known for its regulation of Notch signaling (Hori et al., 2004; Matsuno et al., 1995). Dx regulates Notch signaling in a non-canonical manner and in an over-expressed condition Dx depletes Notch from the cell surface and accumulates it in the endocytic vesicles (Hori et al., 2004). Involvement of Dx in signaling cascade other than Notch has been reported from our earlier studies, where we show that Dx interacts with the TNF ligand Eiger,and regulates JNK signaling by facilitating the re-localization of Eiger from the cell membrane to the cytoplasm (Dutta et al., 2018). Moreover, we have also reported that Dx synergizes with TRAF6, the adaptor molecule in the JNK cascade, and activates JNK signaling at a level downstream of the ligand-receptor interaction (Sharma et al., 2021). A novel function of Dx in Toll pathway activation has also been reported lately, where we have reported a JNK-independent Toll pathway activation through Dx (Cliffe et al., 2003).
In this study, we present evidence for the first time that dx genetically interacts with wg and other components of the Wg signaling cascade in particular the transcription factor, arm. Our loss-of-function and gain-of-function studies show that Dx is involved in the regulation of Wg signaling. We show that over-expression of Dx plays an important role in promoting the spreading of the morphogen Wg. This leads to an erosion of the Wg gradient which consecutively results in a loss of the short-range targets, Sens and Cut. We also observed that the loss of Wg downstream targets upon Dx over-expression is independent of Notch. On the contrary, reducing the dosage of Dx narrows the gradient of Wg expression, supporting further the direct role of Dx in Wg spreading. Moreover, on knocking down Rab5, the small GTPase required for early endosome formation, along with Dx over-expression, a reduction in wing size was observed with a reduction in the Wg expression. A similar reduction in Wg expression was observed when Rab7 was over-expressed along with Dx. Rab7 facilitates the late endosome formation, and along with Dx it might facilitate Wg trafficking and in turn, maintains its proper gradient.
We propose a model of Wg regulation through Dx via down-regulation of Arm. Dx down-regulates Arm when it was over-expressed in the wing and eye tissue. Since Dx has an E3 ubiquitin ligase activity and therefore this down-regulation of Arm can be attributed to being a ubiquitination-dependent phenomenon. Indeed, our results support this hypothesis since post MG132 treatment rescues the loss of Arm when Dx is over-expressed in the background. In addition, we have also shown that human Deltex (DTX1) also displays a conserved function ofproteasomal degradation of β-catenin.
The importance of Wnt/Wg signaling is acknowledged by its implicit role in myriads of biological phenomena throughout development. Wnt/Wg signaling has been implicated in many physiological processes, including development, tissue homeostasis, and tissue regeneration 60. Thus, it is no surprise that mutations in the Wnt pathway components are associated with many hereditary disorders and cancers such as colorectal cancer, Wilms tumor, Familiar Adenomatous Polyposis (FAP), leukemia (Kinzler et al., 1991; Nishisho et al., 1991; Polakis, 2007; Zhan et al., 2017).Thus, understanding the mechanism of Wnt/Wg signal regulation is a potent area of investigation. Increased expression of DTX and its involvement in cell migration and invasiveness in different cancers such as follicular lymphomas, osteosarcoma, glioblastoma, melanoma have also been reported earlier (Gupta-Rossi et al., 2004; Huber et al., 2013). Here, we have uncovered an important functionof the cytoplasmic protein Dx in the gradient formation of the morphogen Wg. These results strengthen the role of Dx in the trafficking of morphogens, thereby allowing the proper activation of the short-range and long-range target genes. In addition, the E3 ubiquitin ligase activity of Dx further opens a new avenue of Wg signaling regulation by maintaining the critical concentration of the effector molecule Arm. A clear understanding of Dx-mediated regulation of Wg signaling will be very useful for future research in the field of therapeutic intervention of diseases involved in aberrant Wg signaling.
4. MATERIALS AND METHODS
3.1. Drosophila Genetics
All fly stocks were maintained on standard cornmeal/yeast/molasses/agar medium at 25°C as per standard procedures. UAS-NICD, UAS-FLAG-Dx, dx, and dx152 were kindly provided by Prof. Spyros Artavanis Tsakonas.. wgCX3(BL-2977), wgCX4(BL-6908), fz1 (BL-1676), fzMB07478 (BL-25554), SensE58(BL-5312), UAS-armS10 (BL-4782), UAS-Rab5DN (BL-9771), UAS-Rab7-GFP (BL-42706), UAS-Dx-RNAi (BL-35677), NRE-GFP (BL-30728), UAS-p35, UAS-NICD, vg-lacZ, en-GAL4, dpp-GAL4, C96-GAL4, ap-GAL4, pnr-GAL4, dll-GAL4, and GMR-GAL4 stocks were obtained from Bloomington Stock Center.
All crosses were performed at 25°C unless otherwise mentioned. To induce the expression of the gene in the specific domain GAL4-UAS binary system was used 20. Combination stocks were made with the help of appropriate genetic crosses.
4.1. Immunocytochemistry and Microscopy
Drosophila third instar larval wing discs were dissected out in ice-cold 1X-PBS, and tissues were fixed in a 1:1 mixture of 3% paraformaldehyde in PBS and at room temperature for 1 min, followed by a second fixation in 3% paraformaldehyde and 5% DMSO for 20 min. Immunostaining was done as described previously 22. For extra-cellular Wg staining permeabilization of tissue was omitted. Third instar larval wing discs were incubated in ice with thrice the antibody concentration before fixation. Conventional staining protocol was followed thereafter. To inhibit proteasomal degradation, imaginal discs were incubated with 10mM of MG132 (Sigma M7449) dissolved in Schneider Culture Media for 3 hours before proceeding with immunostaining.
The following primary antibodies were used in this study Mouse anti-Cut (1:00, DSHB), Mouse anti-Wg (1:100, DSHB), Mouse anti-Arm (1:50, DSHB), Rabbit anti-Flag (1:00, Sigma), Guinea pig anti-Sens (1:10,000, kindly gifted by Prof. Hugo Bellen) Rabbit anti-Dpn (1:200,a generous gift from Prof.Yuh Nung Jan), guinea pig anti-Rab5 (1:1000; a generous gift from Prof. Akira Nakamura), Alexa488-, or Alexa555-, or Alexa405-conjugated secondary antibodies (1:200, Molecular Probes) were used to detect the primary antibodies. Imaging was performed using a Carl Zeiss LSM 780 laser scanning confocal microscope and images were processed with Adobe Photoshop7.
4.2. Plasmid and Construct generation
DNA fragments coding for full-length human Deltex1 (1863bps) was inserted into the pcDNA3.1-HA (Addgene #128034) mammalian expression vector with an N-terminal HA tag. Forward primer 5‣CGCAGGGTACCATGTCACGGCCAGGCCACGGTG3‣ and reverse primer 5‣GCGAGTCTAGACTATCAAGCCTTGCCTGCAGCCTC 3‣, were used for PCR amplification of full-length Deltex1 cDNA and inserted into pcDNA3.1-HA vector utilizing KpnI and XbaI restriction sites.
4.3. Cell Culture and Transfection
HEK-293 (ATCC CRL-1573) was maintained in DMEM supplemented with 10% fetal bovine serum and penicillin/streptomycin (Invitrogen). Cells were cultured according to the manufacturer’s instructions (ATCC). Plasmids transfection was done by Lipofectamine 2000 (ThermoFisher Scientific) as described by the manufacturer.
4.4. Co-immunoprecipitation and Western Blotting
FLAG-tagged Dx was overexpressed in the wing discs under the control of en-GAL4 driver. Wing discs were dissected and homogenized in 1X RIPA buffer (Cell Signaling Technology) containing PMSF1mM (Sigma), and 1X Protease Inhibitor (Roche). For control, en-GAL4/+ protein samples were used. To inhibit proteasomal degradation, discs were incubated with 10mM of MG132 (Sigma M7449) dissolved in Schneider Culture Media for 3 hours before proceeding with sample preparation. Protein concentration was quantified by Nanodrop. 1µg of protein was subjected to Western blotting using Immun-blot PVDF membrane (Bio-Rad). Mouse anti-Arm (1:1000), Mouse anti-β-tubulin (1:2000, DSHB), and anti-mouse IgG-AP conjugate (1:2000, Molecular probes) were used as primary and secondary antibodies to probe the blot. Colour was developed, after washing the membrane three times in TBST, using Sigma FAST BCIP/NBT (Sigma).
For immunoprecipitation of Dx and Arm, protein lysates were prepared in 1× RIPA buffer (Millipore, #20-188) from Drosophila head tissue expressing UAS-FLAG-Dx, UAS-arms10, or both UAS-arms10and UAS-FLAG-Dx using a GMR-GAL4 driver. Crude lysates containing 2 mg of total protein were mixed with 5 µl of rabbit anti-FLAG antibody or mouse anti-arm antibody and 30 µl of protein A/G beads before being incubated overnight with end-over-end rotation at 4°C. UAS-arms10 or UAS-FLAG-Dx lysates were used as control samples. Beads were collected after washing thrice with 1× RIPA buffer and separated on a 12% denaturing SDS polyacrylamide gel followed by transfer onto Immuno-blot PVDF membranes (Bio-Rad). Mouse anti-arm antibody at 1:1500 dilution or rabbit anti-FLAG antibody at 1:1000 dilution and goat anti-rabbit IgG–AP conjugate at 1:2000 or goat anti-mouse IgG-AP conjugate at 1:200 dilution (Molecular Probes) was used as primary and secondary antibodies to probe the blot. Colorimetric detection was performed using Sigma FAST BCIP/NBT.
For visualizing conservation across the phyla, human Deltex1 was expressed in HEK-293 cells by transient transfection as mentioned (See 2.4). Proteasomal degradation was inhibited by subjecting the transfected cells to 50 µM MG132 for 4 hr. Protein samples were quantified for Western blotting. Immunoprecipitation was carried out as described by abcam immunoprecipitation kit (#ab206996). After washing the samples with IP buffer, samples were denatured and run in 4-20% Mini-PROTEAN TGX precast protein gels (Biorad #4561098) followed by an overnight transfer onto a PVDF membrane. After blocking the membrane with 2.5% BSA in TBST for 1 hr, the blots were charged with primary antibody followed by an HRP-conjugated secondary antibody. Signal was developed using Chemiluminescence (ThermoFisher Scientific). Primary antibodies used for Immunoprecipitation and Immunoblotting were mouse monoclonal anti-HA (1:1000, Sigma), rabbit anti-β-catenin (1:1000, Cell Signaling), Rabbit anti-GAPDH (1:1000, Cell Signaling). Images were obtained with Chemidoc XRS+ (Biorad).
4.5. Imaging of adult tissues and Eye imprints
For adult eye imaging, the anesthetized flies of the desired genotype were kept on a bridge slide to maintain a proper angle. Documentation was done with the Leica MZ105 system. Eye imprints using nail polish were made for analyzing ommatidial defects and were examined under differential interference contrast (DIC) optics in a Nikon Eclipse Ni microscope. For wing and leg wholemount preparation, adult tissues from the desired genotype were removed with the help of a needle and scalpel and cleaned with isopropanol before mounting under coverslips in the mounting media. The slides were observed under a bright-field microscope and imaging was done at proper magnification.
4.7 Statistical analysis
For measuring wing and tarsal segment size, the area of the wing and the tarsal region were measured in arbitrary units using image J software. The size of the wild-type wing and leg was considered as 100% and the size of wing and legs from other genotypes were measured in percent ratio to the wild-type wings and legs. For Arm staining, intensity per unit area of the discs was measured along the anterior-posterior axis using Image J software, and the graphs were produced using GraphPad Prism 5 software. Each dataset was repeated at least three times. T-test and One-way analysis of variance (ANOVA) with Tukey’s multiple comparison post-test were employed to determine the significance of the level of difference among the different genotypes. p-value < 0.05 was accepted as statistically significant.
The authors extend sincere thanks to Prof. Spyros Artavanis-Tsakonas (Harvard Medical School), Prof. Florencio Serras (University of Barcelona), Prof. Hugo Bellen (Baylor College of Medicine), Prof. Yuh Nung Jan (University of California, San Francisco), Prof. Akira Nakamura (Institute of Molecular Embryology and Genetics, Kumamoto, Japan) and the Bloomington Stock Centre for fly stocks and antibody. Some of the antibodies used in the work were obtained from Developmental Studies Hybridoma Bank, University of Iowa. We also acknowledge the confocal facility of DBT-BHU-ISLS, Banaras Hindu University. Artwork is created with BioRender (BioRender.com).
6. AUTHOR CONTRIBUTIONS
AM and VS designed the experiments. VS, BS, and NS did all the experiments. AM, MM,and VS analyzed all the data. AM and VS wrote the manuscript.
This work was supported by grants from the Department of Science and Technology, Ministry of Science and Technology, India (CRG/2021/006975), and Institute of Eminence Scheme, Banaras Hindu University, India. Fellowship support to VS and BS was provided by the Council of Scientific and Industrial Research (CSIR), Government of India.
8. COMPETING INTERESTS
The authors declare no competing interest.
9. DATA AVAILABILITY
The datasets supporting the conclusions of the article are included within the article. Data available upon request.
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