Celebrating Discoveries in Wnt Signaling: How One Man Gave Wings to an Entire Field.

This year's Gairdner Foundation Award for Biomedical Research goes to Roel Nusse for his pioneering work on the Wnt signaling pathway and its many roles in development, cancer, and stem cells.

Transformed Drosophila cells evade diet-mediated insulin resistance through wingless signaling.

The risk of specific cancers increases in patients with metabolic dysfunction, including obesity and diabetes. Here, we use Drosophila as a model to explore the effects of diet on tumor progression. Feeding Drosophila a diet high in carbohydrates was previously demonstrated to direct metabolic dysfunction, including hyperglycemia, hyperinsulinemia, and insulin resistance. We demonstrate that high dietary sugar also converts Ras/Src-transformed tissue from localized growths to aggressive tumors with emergent metastases. Whereas most tissues displayed insulin resistance, Ras/Src tumors retained insulin pathway sensitivity, increased the ability to import glucose, and resisted apoptosis. High dietary sugar increased canonical Wingless/Wnt pathway activity, which upregulated insulin receptor gene expression to promote insulin sensitivity. The result is a feed-forward circuit that amplified diet-mediated malignant phenotypes within Ras/Src-transformed tumors. By targeting multiple steps in this circuit with rationally applied drug combinations, we demonstrate the potential of combinatorial drug intervention to treat diet-enhanced malignant tumors.

Control of fate specification within the dorsal head of Drosophila melanogaster.

During development, unique combinations of transcription factors and signaling pathways carve the nascent eye-antennal disc of the fruit fly Drosophila melanogaster into several territories that will eventually develop into the compound eye, ocelli, head epidermis, bristles, antenna and maxillary palpus of the adult head. Juxtaposed patterns of Hedgehog (Hh) and Decapentaplegic (Dpp) initiate compound eye development, while reciprocal domains of Dpp and Wingless (Wg) induce formation of the antennal and maxillary palp fields. Hh and Wg signaling, but not Dpp, contribute to the patterning of the dorsal head vertex. Here, we show that combinatorial reductions of the Pax6 transcription factor Twin of Eyeless and either the Wg pathway or the Mirror (Mirr) transcription factor trigger a transformation of the ocelli into a compound eye and the neighboring head epidermis into an antenna. These changes in fate are accompanied by the ectopic expression of Dpp, which might be expected to trigger these changes in fate. However, the transformation of the field cannot be replicated by increasing Dpp levels alone despite the recreation of adjacent Hh-Dpp and Wg-Dpp domains. As such, the emergence of these ectopic organs occurs through a unique regulatory path.

Wnt signaling modulates the response to DNA damage in the Drosophila wing imaginal disc by regulating the EGFR pathway.

Despite the deep conservation of the DNA damage response (DDR) pathway, cells in different contexts vary widely in their susceptibility to DNA damage and their propensity to undergo apoptosis as a result of genomic lesions. One of the cell signaling pathways implicated in modulating the DDR is the highly conserved Wnt pathway, which is known to promote resistance to DNA damage caused by ionizing radiation in a variety of human cancers. However, the mechanisms linking Wnt signal transduction to the DDR remain unclear. Here, we use a genetically encoded system in Drosophila to reliably induce consistent levels of DNA damage in vivo, and demonstrate that canonical Wnt signaling in the wing imaginal disc buffers cells against apoptosis in the face of DNA double-strand breaks. We show that Wg, the primary Wnt ligand in Drosophila, activates epidermal growth factor receptor (EGFR) signaling via the ligand-processing protease Rhomboid, which, in turn, modulates the DDR in a Chk2-, p53-, and E2F1-dependent manner. These studies provide mechanistic insight into the modulation of the DDR by the Wnt and EGFR pathways in vivo in a highly proliferative tissue. Furthermore, they reveal how the growth and patterning functions of Wnt signaling are coupled with prosurvival, antiapoptotic activities, thereby facilitating developmental robustness in the face of genomic damage.

Wnt/Wingless signaling promotes lipid mobilization through signal-induced transcriptional repression.

The Wnt/Wingless signaling pathway plays critical roles in metazoan development and energy metabolism, but its role in regulating lipid homeostasis remains not fully understood. Here, we report that the activation of canonical Wnt/Wg signaling promotes lipolysis while concurrently inhibiting lipogenesis and fatty acid ß-oxidation in both larval and adult adipocytes, as well as cultured S2R+ cells, in Drosophila. Using RNA-sequencing and CUT&RUN (Cleavage Under Targets & Release Using Nuclease) assays, we identified a set of Wnt target genes responsible for intracellular lipid homeostasis. Notably, active Wnt signaling directly represses the transcription of these genes, resulting in decreased de novo lipogenesis and fatty acid ß-oxidation, but increased lipolysis. These changes lead to elevated free fatty acids and reduced triglyceride (TG) accumulation in adipocytes with active Wnt signaling. Conversely, downregulation of Wnt signaling in the fat body promotes TG accumulation in both larval and adult adipocytes. The attenuation of Wnt signaling also increases the expression of specific lipid metabolism-related genes in larval adipocytes, wing discs, and adult intestines. Taken together, these findings suggest that Wnt signaling-induced transcriptional repression plays an important role in regulating lipid homeostasis by enhancing lipolysis while simultaneously suppressing lipogenesis and fatty acid ß-oxidation.

Genetic mechanism regulating diversity in the placement of eyes on the head of animals.

Despite the conservation of genetic machinery involved in eye development, there is a strong diversity in the placement of eyes on the head of animals. Morphogen gradients of signaling molecules are vital to patterning cues. During Drosophila eye development, Wingless (Wg), a ligand of Wnt/Wg signaling, is expressed anterolaterally to form a morphogen gradient to determine the eye- versus head-specific cell fate. The underlying mechanisms that regulate this process are yet to be fully understood. We characterized defective proventriculus (dve) (Drosophila ortholog of human SATB1), a K50 homeodomain transcription factor, as a dorsal eye gene, which regulates Wg signaling to determine eye versus head fate. Across Drosophila species, Dve is expressed in the dorsal head vertex region where it regulates wg transcription. Second, Dve suppresses eye fate by down-regulating retinal determination genes. Third, the dve-expressing dorsal head vertex region is important for Wg-mediated inhibition of retinal cell fate, as eliminating the Dve-expressing cells or preventing Wg transport from these dve-expressing cells leads to a dramatic expansion of the eye field. Together, these findings suggest that Dve regulates Wg expression in the dorsal head vertex, which is critical for determining eye versus head fate. Gain-of-function of SATB1 exhibits an eye fate suppression phenotype similar to Dve. Our data demonstrate a conserved role for Dve/SATB1 in the positioning of eyes on the head and the interocular distance by regulating Wg. This study provides evidence that dysregulation of the Wg morphogen gradient results in developmental defects such as hypertelorism in humans where disproportionate interocular distance and facial anomalies are reported.

Dissociation of Drosophila Evi-Wg Complex Occurs Post Apical Internalization in the Maturing Acidic Endosomes.

Signaling pathways activated by secreted Wnt ligands play an essential role in tissue development and the progression of diseases, like cancer. Secretion of the lipid-modified Wnt proteins is tightly regulated by a repertoire of intracellular factors. For instance, a membrane protein, Evi, interacts with the Wnt ligand in the ER, and it is essential for its further trafficking and release in the extracellular space. After dissociating from the Wnt, the Wnt-unbound Evi is recycled back to the ER via Golgi. However, where in this trafficking path Wnt proteins dissociate from Evi remains unclear. Here, we have used the Drosophila wing epithelium to trace the route of the Evi-Wg (Wnt homolog) complex leading up to their separation. In these polarized cells, Wg is first trafficked to the apical surface; however, the secretion of Wg is believed to occurs post-internalization via recycling. Our results show that the Evi-Wg complex is internalized from the apical surface and transported to the retromer-positive endosomes. Furthermore, using antibodies that specifically label the Wnt-unbound Evi, we show that Evi and Wg separation occurs post-internalization in the acidic endosomes. These results refine our understanding of the polarized trafficking of Wg and highlight the importance of Wg endocytosis in its secondary secretion.

Glypicans shield the Wnt lipid moiety to enable signalling at a distance.

A relatively small number of proteins have been suggested to act as morphogens-signalling molecules that spread within tissues to organize tissue repair and the specification of cell fate during development. Among them are Wnt proteins, which carry a palmitoleate moiety that is essential for signalling activity1-3. How a hydrophobic lipoprotein can spread in the aqueous extracellular space is unknown. Several mechanisms, such as those involving lipoprotein particles, exosomes or a specific chaperone, have been proposed to overcome this so-called Wnt solubility problem4-6. Here we provide evidence against these models and show that the Wnt lipid is shielded by the core domain of a subclass of glypicans defined by the Dally-like protein (Dlp). Structural analysis shows that, in the presence of palmitoleoylated peptides, these glypicans change conformation to create a hydrophobic space. Thus, glypicans of the Dlp family protect the lipid of Wnt proteins from the aqueous environment and serve as a reservoir from which Wnt proteins can be handed over to signalling receptors.

Founding the Wnt gene family: How wingless was found to be a positional signal and oncogene homolog.

The Wnt family of developmental regulators were named after the Drosophila segmentation gene wingless and the murine proto-oncogene int-1. Homology between these two genes connected oncogenesis to cell-cell signals in development. I review how wingless was initially characterized, and cloned, as part of the quest to identify developmental cell-to-cell signals, based on predictions of the Positional Information Model, and on the properties of homeotic and segmentation gene mutants. The requirements and cell-nonautonomy of wingless in patterning multiple embryonic and adult structures solidified its status as a candidate signaling molecule. The physical location of wingless mutations and transcription unit defined the gene and its developmental transcription pattern. When the Drosophila homolog of int-1 was then isolated, and predicted to encode a secreted proto-oncogene homolog, it's identity to the wingless gene confirmed that a developmental cell-cell signal had been identified and connected cancer to development.

Regulation of morphogen pathways by a Drosophila chondroitin sulfate proteoglycan Windpipe.

Morphogens provide quantitative and robust signaling systems to achieve stereotypic patterning and morphogenesis. Heparan sulfate (HS) proteoglycans (HSPGs) are key components of such regulatory feedback networks. In Drosophila, HSPGs serve as co-receptors for a number of morphogens, including Hedgehog (Hh), Wingless (Wg), Decapentaplegic (Dpp) and Unpaired (Upd, or Upd1). Recently, Windpipe (Wdp), a chondroitin sulfate (CS) proteoglycan (CSPG), was found to negatively regulate Upd and Hh signaling. However, the roles of Wdp, and CSPGs in general, in morphogen signaling networks are poorly understood. We found that Wdp is a major CSPG with 4-O-sulfated CS in Drosophila. Overexpression of wdp modulates Dpp and Wg signaling, showing that it is a general regulator of HS-dependent pathways. Although wdp mutant phenotypes are mild in the presence of morphogen signaling buffering systems, this mutant in the absence of Sulf1 or Dally, molecular hubs of the feedback networks, produces high levels of synthetic lethality and various severe morphological phenotypes. Our study indicates a close functional relationship between HS and CS, and identifies the CSPG Wdp as a novel component in morphogen feedback pathways.

Assessing the contribution of genes involved in monogenic bone disorders to the etiology of atypical femoral fractures.

BACKGROUND: Recent studies suggested that genetic variants associated with monogenic bone disorders were involved in the pathogenesis of atypical femoral fractures (AFF). Here, we aim to identify rare genetic variants by whole exome sequencing in genes involved in monogenic rare skeletal diseases in 12 women with AFF and 4 controls without any fracture. RESULTS: Out of 33 genetic variants identified in women with AFF, eleven (33.3%) were found in genes belonging to the Wnt pathway (LRP5, LRP6, DAAM2, WNT1, and WNT3A). One of them was rated as pathogenic (p.Pro582His in DAAM2), while all others were rated as variants of uncertain significance according to ClinVar and ACMG criteria. CONCLUSIONS: Osteoporosis, rare bone diseases, and AFFs may share the same genes, thus making it even more difficult to identify unique risk factors.

High sugar diet promotes tumor progression paradoxically through aberrant upregulation of pepck1.

High dietary sugar (HDS), a contemporary dietary concern due to excessive intake of added sugars and carbohydrates, escalates the risk of metabolic disorders and concomitant cancers. However, the molecular mechanisms underlying HDS-induced cancer progression are not completely understood. We found that phosphoenolpyruvate carboxykinase 1 (PEPCK1), a pivotal enzyme in gluconeogenesis, is paradoxically upregulated in tumors by HDS, but not by normal dietary sugar (NDS), during tumor progression. Targeted knockdown of pepck1, but not pepck2, specifically in tumor tissue in Drosophila in vivo, not only attenuates HDS-induced tumor growth but also significantly improves the survival of Ras/Src tumor-bearing animals fed HDS. Interestingly, HP1a-mediated heterochromatin interacts directly with the pepck1 gene and downregulates pepck1 gene expression in wild-type Drosophila. Mechanistically, we demonstrated that, under HDS conditions, pepck1 knockdown reduces both wingless and TOR signaling, decreases evasion of apoptosis, reduces genome instability, and suppresses glucose uptake and trehalose levels in tumor cells in vivo. Moreover, rational pharmacological inhibition of PEPCK1, using hydrazinium sulfate, greatly improves the survival of tumor-bearing animals with pepck1 knockdown under HDS. This study is the first to show that elevated levels of dietary sugar induce aberrant upregulation of PEPCK1, which promotes tumor progression through altered cell signaling, evasion of apoptosis, genome instability, and reprogramming of carbohydrate metabolism. These findings contribute to our understanding of the complex relationship between diet and cancer at the molecular, cellular, and organismal levels and reveal PEPCK1 as a potential target for the prevention and treatment of cancers associated with metabolic disorders.

Ecdysone exerts biphasic control of regenerative signaling, coordinating the completion of regeneration with developmental progression.

In Drosophila melanogaster, loss of regenerative capacity in wing imaginal discs coincides with an increase in systemic levels of the steroid hormone ecdysone, a key coordinator of their developmental progression. Regenerating discs release the relaxin hormone Dilp8 (Drosophila insulin-like peptide 8) to limit ecdysone synthesis and extend the regenerative period. Here, we describe how regenerating tissues produce a biphasic response to ecdysone levels: lower concentrations of ecdysone promote local and systemic regenerative signaling, whereas higher concentrations suppress regeneration through the expression of broad splice isoforms. Ecdysone also promotes the expression of wingless during both regeneration and normal development through a distinct regulatory pathway. This dual role for ecdysone explains how regeneration can still be completed successfully in dilp8- mutant larvae: higher ecdysone levels increase the regenerative activity of tissues, allowing regeneration to reach completion in a shorter time. From these observations, we propose that ecdysone hormone signaling functions to coordinate regeneration with developmental progression.

Differential gene expression analysis identified determinants of cell fate plasticity during radiation-induced regeneration in Drosophila.

Ionizing radiation (IR) is used to treat half of all cancer patients because of its ability to kill cells. IR, however, can induce stem cell-like properties in non-stem cancer cells, potentiating tumor regrowth and reduced therapeutic success. We identified previously a subpopulation of cells in Drosophila larval wing discs that exhibit IR-induced stem cell-like properties. These cells reside in the future wing hinge, are resistant to IR-induced apoptosis, and are capable of translocating, changing fate, and participating in regenerating the pouch that suffers more IR-induced apoptosis. We used here a combination of lineage tracing, FACS-sorting of cells that change fate, genome-wide RNAseq, and functional testing of 42 genes, to identify two key changes that are required cell-autonomously for IR-induced hinge-to-pouch fate change: (1) repression of hinge determinants Wg (Drosophila Wnt1) and conserved zinc-finger transcription factor Zfh2 and (2) upregulation of three ribosome biogenesis factors. Additional data indicate a role for Myc, a transcriptional activator of ribosome biogenesis genes, in the process. These results provide a molecular understanding of IR-induced cell fate plasticity that may be leveraged to improve radiation therapy.

The role of miR-128 and MDFI in cardiac hypertrophy and heart failure: Mechanistic.

Heart failure (HF) prognosis depends on various regulatory factors; microRNA-128 (miR-128) is identified as a regulator of cardiac fibrosis, contributing to HF. MyoD family inhibitor (MDFI), which is reported to be related with Wnt/ß-catenin pathway, is supposed to be regulated by miR-128. This study investigates the interaction between miR-128 and MDFI in cardiomyocyte development and elucidates its role in heart injury. Gene expression profiling assessed miR-128's effect on MDFI expression in HF using qPCR and Western blot analysis. Luciferase assays studied the direct interaction between miR-128 and MDFI. MTT, transwell, and immunohistochemistry evaluated the effects of miR-128 and MDFI on myocardial cells in mice HF. Genescan and luciferase assays validated the interaction between miR-128 and MDFI sequences. miR-128 mimics significantly reduced MDFI expression at mRNA and protein levels with decrease rate of 55%. Overexpression of miR-128 promoted apoptosis with the increase rate 65% and attenuated cardiomyocyte proliferation, while MDFI upregulation significantly enhanced proliferation. Elevated miR-128 levels upregulated Wnt1 and ß-catenin expression, whereas increased MDFI levels inhibited these expressions. Histological analysis with haematoxylin and eosin staining revealed that miR-128 absorption reduced MDFI expression, hindering cell proliferation and cardiac repair, with echocardiography showing corresponding improvements in cardiac function. Our findings suggest miR-128 interacts with MDFI, playing a crucial role in HF management by modulating the Wnt1/ß-catenin pathway. Suppression of miR-128 could promote cardiomyocyte proliferation, highlighting the potential value of the miR-128/MDFI interplay in HF treatment.

A unified mechanism for the control of Drosophila wing growth by the morphogens Decapentaplegic and Wingless.

Development of the Drosophila wing-a paradigm of organ development-is governed by 2 morphogens, Decapentaplegic (Dpp, a BMP) and Wingless (Wg, a Wnt). Both proteins are produced by defined subpopulations of cells and spread outwards, forming gradients that control gene expression and cell pattern as a function of concentration. They also control growth, but how is unknown. Most studies have focused on Dpp and yielded disparate models in which cells throughout the wing grow at similar rates in response to the grade or temporal change in Dpp concentration or to the different amounts of Dpp "equalized" by molecular or mechanical feedbacks. In contrast, a model for Wg posits that growth is governed by a progressive expansion in morphogen range, via a mechanism in which a minimum threshold of Wg sustains the growth of cells within the wing and recruits surrounding "pre-wing" cells to grow and enter the wing. This mechanism depends on the capacity of Wg to fuel the autoregulation of vestigial (vg)-the selector gene that specifies the wing state-both to sustain vg expression in wing cells and by a feed-forward (FF) circuit of Fat (Ft)/Dachsous (Ds) protocadherin signaling to induce vg expression in neighboring pre-wing cells. Here, we have subjected Dpp to the same experimental tests used to elucidate the Wg model and find that it behaves indistinguishably. Hence, we posit that both morphogens act together, via a common mechanism, to control wing growth as a function of morphogen range.

Exome sequencing identified mutations in the WNT1 and COL1A2 genes in osteogenesis imperfecta cases.

BACKGROUND: Osteogenesis imperfecta (OI) is a heritable connective tissue disorder characterized by bone deformities, fractures and reduced bone mass. OI can be inherited as a dominant, recessive, or X-linked disorder. The mutational spectrum has shown that autosomal dominant mutations in the type I collagen-encoding genes are responsible for OI in 85% of the cases. Apart from collagen genes, mutations in more than 20 other genes, such as CRTAP, CREB3L1, MBTPS2, P4HB, SEC24D, SPARC, FKBP10, LEPRE1, PLOD2, PPIB, SERPINF1, SERPINH1, SP7, WNT1, BMP1, TMEM38B, and IFITM5 have been reported in OI. METHODS AND RESULTS: To understand the genetic cause of OI in four cases, we conducted whole exome sequencing, followed by Sanger sequencing. In case #1, we identified a novel c.506delG homozygous mutation in the WNT1 gene, resulting in a frameshift and early truncation of the protein at the 197th amino acid. In cases #2, 3 and 4, we identified a heterozygous c.838G > A mutation in the COL1A2 gene, resulting in a p.Gly280Ser substitution. The clinvar frequency of this mutation is 0.000008 (GnomAD-exomes). This mutation has been identified by other studies as well and appears to be a mutational hot spot. These pathogenic mutations were found to be absent in 96 control samples analyzed for these sites. The presence of these mutations in the cases, their absence in controls, their absence or very low frequency in general population, and their evaluation using various in silico prediction tools suggested their pathogenic nature. CONCLUSIONS: Mutations in the WNT1 and COL1A2 genes explain these cases of osteogenesis imperfecta.

Ferrying wingless across the synaptic cleft.

Secreted Wnt morphogens mediate cell-cell communication, but the mechanism of Wnt transfer between cells is unknown. Korkut et al. (2009) report that the transmembrane protein Evi is a versatile carrier that guides Wingless to presynaptic terminals of motor neurons and then escorts it across the synaptic cleft. In postsynaptic muscles, Evi promotes Frizzled-2 trafficking.

Interpretation of the wingless gradient requires signaling-induced self-inhibition.

In a classical view of development, a cell can acquire positional information by reading the local concentration of a morphogen independently of its neighbors. Accordingly, in Drosophila, the morphogen Wingless produced in the wing's prospective distal region activates target genes in a dose-dependent fashion to organize the proximodistal pattern. Here, we show that, in parallel, Wingless triggers two nonautonomous inhibitory programs that play an important role in the establishment of positional information. Cells flanking the source of Wingless produce a negative signal (encoded by notum) that inhibits Wingless signaling in nearby cells. Additionally, in response to Wingless, all prospective wing cells produce an unidentified signal that dampens target gene expression in surrounding cells. Thus, cells influence each other's response to Wingless through at least two modes of lateral inhibition. Without lateral inhibition, some cells acquire ectopic fates. Lateral inhibition may be a general mechanism behind the interpretation of morphogen gradients.

Trans-synaptic transmission of vesicular Wnt signals through Evi/Wntless.

Wnts play pivotal roles during development and in the mature nervous system. However, the mechanism by which Wnts traffic between cells has remained elusive. Here we demonstrate a mechanism of Wnt transmission through release of exosome-like vesicles containing the Wnt-binding protein Evenness Interrupted/Wntless/Sprinter (Evi/Wls/Srt). We show that at the Drosophila larval neuromuscular junction (NMJ), presynaptic vesicular release of Evi is required for the secretion of the Wnt, Wingless (Wg). We also show that Evi acts cell-autonomously in the postsynaptic Wnt-receiving cell to target dGRIP, a Wg-receptor-interacting protein, to postsynaptic sites. Upon Evi loss of function, dGRIP is not properly targeted to synaptic sites, interfering with postsynaptic Wnt signal transduction. These findings uncover a previously unknown cellular mechanism by which a secreted Wnt is transported across synapses by Evi-containing vesicles and reveal trafficking functions of Evi in both the Wnt-producing and the Wnt-receiving cells. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online.