{"gene":"WNT5B","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":2003,"finding":"Wnt5b regulates the transition between different chondrocyte zones by differentially controlling cyclin D1 and p130 expression, as well as chondrocyte-specific Col2a1 expression, independently of the Ihh/PTHrP negative feedback loop.","method":"Genetic loss-of-function in mouse long bone development; expression analysis of cell cycle regulators","journal":"Development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined cellular phenotype with loss-of-function and multiple molecular readouts (cyclin D1, p130, Col2a1), single lab","pmids":["12538525"],"is_preprint":false},{"year":2005,"finding":"Wnt5b overexpression partially inhibits canonical Wnt/β-catenin signaling by reducing nuclear translocation of β-catenin, and promotes adipogenesis in 3T3-L1 preadipocytes, at least partly by antagonizing Wnt3a-induced canonical Wnt activation.","method":"Wnt5b overexpression in 3T3-L1 cells; microarray, RT-PCR, nuclear β-catenin localization assay, Wnt3a co-treatment rescue experiment","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (microarray, RT-PCR, β-catenin nuclear localization), single lab","pmids":["15796911"],"is_preprint":false},{"year":2009,"finding":"Wnt5b stimulates adipogenesis by activating PPARγ and aP2 expression and inhibits β-catenin-dependent Wnt signaling at the initiation of adipogenesis in 3T3-L1 preadipocytes.","method":"Wnt5b overexpression in 3T3-L1 cells; measurement of PPARγ, aP2, and β-catenin translocation","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — overexpression with multiple molecular readouts (PPARγ, aP2, β-catenin), single lab","pmids":["19577541"],"is_preprint":false},{"year":2010,"finding":"Wnt5b acts through the receptor Ryk (not solely Frizzled) to convey directional signals during zebrafish gastrulation; Ryk deficiency impairs Wnt5b-induced Ca2+ activity and directional cell movement, and Wnt5b-Ryk signaling promotes polarized cell protrusions. Fzd2, but not Ryk, recruits Dishevelled to the cell membrane upon Wnt5b stimulation, indicating separate downstream pathways.","method":"Zebrafish Ryk loss-of-function (morpholino); co-culture directional migration assay; Ca2+ imaging; cell membrane recruitment of Dishevelled assessed by imaging","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal assays (Ca2+ imaging, directional migration co-culture, Dvl membrane recruitment), loss-of-function in vivo, mechanistic pathway separation of Ryk vs Fzd2","pmids":["20660632"],"is_preprint":false},{"year":2010,"finding":"Rgs3 (Regulator of G protein Signaling 3) is required downstream of Wnt5b for appropriate frequency and amplitude of Ca2+ release during zebrafish somitogenesis; Rgs3 acts as a G protein GAP and its activity requires the ability to interact with Gα subunits.","method":"Zebrafish morpholino knockdown of rgs3 and wnt5b; in vivo Ca2+ imaging; rescue assays with Gα-interaction-deficient Rgs3 mutant","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo Ca2+ imaging, loss-of-function, mutagenesis-based epistasis rescue, replicated genetic interaction","pmids":["20628572"],"is_preprint":false},{"year":2011,"finding":"Wnt5b inhibits chondrocyte hypertrophy and regulates chondroprogenitor cell migration through JNK activation (planar cell polarity pathway) and disrupts mesenchymal condensation via Src-dependent β-catenin phosphorylation leading to increased cadherin receptor turnover.","method":"Wnt5b expression in chondrogenic cultures; JNK inhibition (SP600125); Src inhibition; cadherin turnover assay; migration assay","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple pathway inhibitors used (JNK, Src), multiple phenotypic readouts (hypertrophy, migration, cadherin turnover), single lab","pmids":["21413026"],"is_preprint":false},{"year":2011,"finding":"Def6, a novel guanine nucleotide exchange factor (GEF), functions downstream of Wnt5b signaling to regulate convergent extension cell movements during zebrafish gastrulation; overexpression of def6 rescues Wnt5b morphant phenotypes.","method":"Zebrafish morpholino knockdown; genetic epistasis rescue (def6 overexpression rescues wnt5b morphants); double knockdown of def6 and Wnt11","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis rescue in vivo, single lab, morpholino-based","pmids":["22039507"],"is_preprint":false},{"year":2013,"finding":"Wnt5b promotes cell motility (migration, invasion, filopodia formation) in oral squamous cell carcinoma cells through activation of Cdc42 and RhoA; siRNA knockdown of Wnt5b inhibits migration and filopodia, while recombinant Wnt5b stimulation increases active Cdc42 and RhoA levels.","method":"siRNA knockdown and recombinant protein stimulation; GTPase activity assays (active Cdc42, active RhoA); migration and invasion assays","journal":"International journal of oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal gain/loss-of-function with GTPase activity measurement, single lab","pmids":["24220306"],"is_preprint":false},{"year":2014,"finding":"Wnt5b forms a positive feedback loop with PKCα in arsenic-transformed cells: Wnt5b sustains phospho-PKC levels, and PKCα in turn maintains Wnt5b expression. This Wnt5b-PKCα axis activates Rac1, promoting actin cytoskeletal reorganization and cell migration. miR-200b suppresses this pathway by directly targeting PKCα.","method":"siRNA knockdown of Wnt5b and PKCα; forced expression of PKCα in miR-200b-overexpressing cells; Rac1 activation assay; 3'-UTR luciferase reporter for miR-200b targeting PKCα","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple reciprocal gain/loss-of-function experiments with mechanistic readouts (Rac1 activity, phospho-PKC), single lab","pmids":["24841200"],"is_preprint":false},{"year":2016,"finding":"Wnt5b is glycosylated at three asparagine residues and lipidated at one serine residue; these post-translational modifications are essential for Wnt5b secretion. Secreted Wnt5b phosphorylates Dvl2 and activates Rac1. In pancreatic cancer cells, ~55% of secreted endogenous Wnt5b is associated with exosomes, and Wnt5b-associated exosomes promote cancer cell migration and proliferation in a paracrine manner.","method":"Biochemical characterization of purified Wnt5b (glycosylation, lipidation); Wnt5b-KO PANC-1 cells (CRISPR); TSG101 knockdown; Dvl2 phosphorylation assay; Rac1 activation assay; centrifugation fractionation; immunoelectron microscopy; migration and proliferation assays","journal":"Cancer science","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro biochemical characterization of PTMs, KO validation, multiple orthogonal methods (immunoelectron microscopy, biochemical fractionation, functional assays)","pmids":["27762090"],"is_preprint":false},{"year":2016,"finding":"WNT5B induces partial endothelial-mesenchymal transition (EndoMT) in lymphatic endothelial cells (LECs), promoting tube formation via upregulation of Snail and Slug transcription factors; WNT5B-induced Snail/Slug expression is abolished by IWR-1-endo (Wnt/β-catenin inhibitor) and Rac1 inhibitors, implicating both canonical and non-canonical (PCP) Wnt pathways.","method":"Recombinant WNT5B treatment of LECs; WNT5B knockdown in OSCC cells; tube formation, permeability, migration assays; phalloidin staining; SNAIL/SLUG knockdown; pathway inhibitors","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple functional assays with pathway inhibitor dissection and downstream transcription factor knockdown, single lab","pmids":["27593938"],"is_preprint":false},{"year":2016,"finding":"WNT5B signals through the Frizzled-2 receptor and TAK1 to induce IL-6 and CXCL8 release in lung fibroblasts via activation of JNK, p38, and p65 NF-κB, without involving canonical β-catenin signaling; IL-6 and CXCL8 release are controlled through distinct downstream pathways (JNK/p38 for both; IKK for CXCL8 only).","method":"Recombinant WNT5B treatment; Frizzled-2 knockdown; TAK1 inhibition; JNK, p38, IKK inhibitors; cytokine secretion measurement (ELISA); β-catenin pathway assessment","journal":"American journal of physiology. Lung cellular and molecular physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — receptor identified by knockdown, multiple kinase inhibitors defining pathway branches, single lab","pmids":["27036869"],"is_preprint":false},{"year":2016,"finding":"WNT5B increases airway remodeling gene expression (fibronectin, MMP-2, MMP-9, Snail) in bronchial epithelial cells via TGF-β/Smad3 signaling.","method":"Exogenous WNT5B treatment of BEAS-2B and air-liquid interface PBECs; TGF-β/Smad3 pathway analysis","journal":"The European respiratory journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — exogenous protein treatment with pathway inhibitor validation (Smad3), multiple gene readouts, single lab","pmids":["27126693"],"is_preprint":false},{"year":2016,"finding":"WNT5B promotes colorectal cancer cell migration and invasion through activation of the JNK signaling pathway, leading to increased MMP-2 and MMP-9 expression; JNK knockdown reverses these effects.","method":"Stable WNT5B overexpression in COLO 205 cells; JNK siRNA knockdown; MTT, wound healing, Transwell assays; western blot for MMP2/9 and JNK pathway","journal":"Oncology reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain-of-function and loss-of-function (JNK RNAi) with multiple functional readouts, single lab","pmids":["27121420"],"is_preprint":false},{"year":2015,"finding":"Wnt5b requires Wntless (Wls) for its secretion from pharyngeal tissue and regulates chondrogenic cell proliferation by fine-tuning fgf3 expression; fgf3 mRNA introduction rescues cartilage defects in Wnt5b- and Wls-deficient zebrafish larvae.","method":"Zebrafish wnt5b mutant and wls morphant analysis; fgf3 expression measurement; mRNA rescue experiment; cell proliferation and apoptosis assays","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo epistasis rescue, secretion mechanism identified by Wls dependence, single lab","pmids":["25934698"],"is_preprint":false},{"year":2015,"finding":"Glypican 4 (Gpc4) and Wnt5b cooperate to regulate chondrocyte stacking and intercalation during craniofacial cartilage morphogenesis in zebrafish; Gpc4 functions cell-autonomously in chondrocytes, and double heterozygous wnt5b;gpc4 embryos show enhanced phenotype, suggesting genetic interaction independent of core Wnt/PCP molecules.","method":"Zebrafish wnt5b and gpc4 mutant analysis; cell-autonomous rescue with chimeric transplants; double heterozygous epistasis","journal":"Mechanisms of development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in vivo (double heterozygotes, cell-autonomous transplants), single lab","pmids":["26459057"],"is_preprint":false},{"year":2015,"finding":"Wnt5b and Wnt5a act through the JNK signaling pathway to mediate mechanical tension-induced osteogenic differentiation of rat tendon-derived stem cells; shRNA knockdown of Wnt5a/Wnt5b reduces UMT-induced Runx2 and P-JNK, and JNK activation rescues Runx2 expression.","method":"shRNA knockdown of Wnt5a and Wnt5b in rTDSCs; JNK inhibitor (SP600125) and activator (anisomycin); JNK1-shRNA and JNK1-cDNA; Runx2 and P-JNK protein measurement","journal":"Cellular physiology and biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with rescue via JNK activation, multiple molecular readouts, single lab","pmids":["25966835"],"is_preprint":false},{"year":2018,"finding":"IMP3 stabilizes WNT5B mRNA indirectly by repressing miR-145-5p (which targets WNT5B), resulting in TAZ activation via alternative WNT signaling. WNT5B also facilitates SLUG transcription, which is required for TAZ nuclear localization.","method":"IMP3 knockdown/overexpression; miR-145-5p manipulation; WNT5B mRNA stability assay; TAZ nuclear localization; SLUG expression measurement","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple genetic manipulations with mechanistic readouts (mRNA stability, TAZ localization), single lab","pmids":["29847788"],"is_preprint":false},{"year":2019,"finding":"Wnt5b signals through canonical Wnt (β-catenin) pathway in zebrafish to direct Nkx2.5+ mesoderm into pacemaker cardiomyocytes, activating transcription factors Isl1 and Tbx18 while silencing Nkx2.5; this mechanism is evolutionarily conserved and can be applied to direct human pluripotent stem cells toward pacemaker cardiomyocyte fate.","method":"Zebrafish Wnt5b loss-of-function; cell lineage tracing; transcription factor expression (Isl1, Tbx18, Nkx2.5); hPSC directed differentiation applying Wnt5b pathway findings","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic loss-of-function combined with lineage tracing, multiple transcription factor readouts, cross-species (zebrafish to human PSC) validation","pmids":["31402282"],"is_preprint":false},{"year":2020,"finding":"Wnt5b/Ryk signaling promotes membrane trafficking of P2X3 receptors to the DRG neuron surface via CaMKII activation, leading to enhanced P2X3 currents and pain hypersensitivity; anti-Ryk antibody and CaMKII inhibitor (KN93) block these effects.","method":"Wnt5b application to cultured DRG neurons; anti-Ryk antibody; CaMKII inhibitor KN93; electrophysiological recording of P2X3 currents; membrane P2X3 protein quantification; in vivo intrathecal injection","journal":"Experimental neurology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — receptor identification by antibody blockade, CaMKII mechanistic link, electrophysiology and protein trafficking assays, single lab","pmids":["32979370"],"is_preprint":false},{"year":2020,"finding":"Wnt5b and Fak1a (focal adhesion kinase 1a) converge in regulating Rac1 and Cdc42 to mediate gastrulation cell movements in zebrafish; overexpression of fak1a rescues wnt5b morphant convergence defects and vice versa, and Rac1/Cdc42 activation synergistically rescues both morphants.","method":"Zebrafish morpholino knockdown; CRISPR/Cas9 fak1a mutants; cross-rescue overexpression; Rac1 and Cdc42 activation measurement","journal":"Open biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal genetic rescue and GTPase activity measurements, single lab","pmids":["32097584"],"is_preprint":false},{"year":2021,"finding":"WNT5B suppresses osteoblast differentiation and mineralization via ROR1/ROR2 receptors, which activate DVL2/3–RAC1–CDC42–JNK–SIN3A signaling and inhibit β-catenin activity; ERα and NFATc1 bind to an enhancer containing SNP rs2887571 to repress WNT5B expression, with the GG genotype showing greater ERα binding and greater WNT5B suppression than AA.","method":"ChIP-qPCR; CRISPR-Cas9 allele editing; ROR1/2 receptor manipulation; DVL2/3, RAC1, CDC42, JNK pathway analysis; alkaline phosphatase activity; mineralization assay; IL-6 measurement; correlation of genotype with WNT5B expression in 110 patient osteoblasts","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — CRISPR allele editing, ChIP-qPCR, multiple receptor/pathway components tested, functional osteoblast readouts, validated in 110 patient samples","pmids":["34906330"],"is_preprint":false},{"year":2021,"finding":"WNT5B promotes VSMC proliferation and migration via non-canonical Wnt signaling and induces mitochondrial fission; inhibition of mitochondrial fission (by mdivi-1) abolishes WNT5B-induced VSMC proliferation and migration, while secreted frizzled-related protein 2 (SFRP2, a Wnt scavenger) attenuates VSMC proliferation/migration by promoting mitochondrial fusion.","method":"Primary pulmonary artery SMC culture; WNT5B overexpression; mitochondrial division inhibitor 1 (mdivi-1); SFRP2 treatment; proliferation and migration assays; mitochondrial morphology analysis","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain-of-function and inhibitor-based pathway dissection with mechanistic readout (mitochondrial dynamics), single lab","pmids":["34368954"],"is_preprint":false},{"year":2023,"finding":"Wnt5b binds to Ror2 receptor in the producing cell; active Wnt5b-Ror2 complexes are loaded onto cytonemes (long cellular protrusions) and transferred to receiving cells, enabling initiation of Wnt-PCP signaling in recipient cells regardless of their own Ror2 status; this cytoneme-dependent spreading controls convergence and extension in zebrafish gastrula.","method":"Live imaging of Wnt5b-Ror2 on cytonemes in zebrafish; co-culture transfer assays; Ror2-deficient receiving cells; convergent extension phenotype analysis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — live imaging plus functional co-culture assays with Ror2-deficient recipients, multiple orthogonal approaches, high-impact journal","pmids":["38123680"],"is_preprint":false},{"year":2024,"finding":"WNT5B signals through FZD3 receptor to recruit DVL3 to the plasma membrane (requiring DVL3's DEP domain) in a WNT5B ligand-dependent manner, activating JNK signaling via RAC1 (WNT-PCP pathway) to promote malignant phenotype of NSCLC cells; deletion of the DEP domain of DVL3 abrogates these effects.","method":"Co-IP of WNT5B-FZD3-DVL3; DVL3 DEP domain deletion mutant; membrane recruitment assay; JNK and RAC1 activation; in vivo and in vitro tumor assays","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — Co-IP identifying the complex, domain deletion mutagenesis, in vivo validation, multiple pathway readouts","pmids":["39094673"],"is_preprint":false},{"year":2024,"finding":"Wnt5b signals through Ror1 (not Ror2) to promote PDAC cell proliferation in a cell-autonomous manner; knockdown of either Ror1 or Wnt5b in PANC-1 cells inhibits proliferation in vitro, and Ror1 knockout inhibits tumor growth in vivo.","method":"Ror1 and Wnt5b knockdown in PANC-1 cells; Ror1 CRISPR knockout; in vitro proliferation assays; in vivo xenograft tumor assay","journal":"Genes to cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — receptor identified by genetic KO with in vivo validation, single lab","pmids":["38531660"],"is_preprint":false},{"year":2024,"finding":"WNT5B drives osteosarcoma cancer stem cell expansion and promotes lung/liver metastasis in vivo; WNT5B signals via ROR1 to induce SOX2 expression, and also upregulates HYAL1 to degrade hyaluronic acid in the tumor microenvironment; ROR1 antibody-mediated inhibition of WNT5B/ROR1 signaling reduces stemness, sphere size, and SOX2 expression.","method":"WNT5B overexpression and knockdown in osteosarcoma spheres and PDX; in vivo metastasis assay; ROR1 antibody treatment; SOX2, HYAL1 measurement; hyaluronic acid quantification","journal":"Clinical and translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo metastasis model, receptor targeted by antibody, multiple molecular readouts, single lab","pmids":["38689429"],"is_preprint":false},{"year":2024,"finding":"Astrocyte-secreted WNT5B activates non-canonical Wnt signaling in brain microvascular endothelial cells via receptor ROR1, leading to JNK/c-JUN activation; c-JUN directly binds to and represses the ZO-1 promoter, impairing tight junction integrity and disrupting the blood-brain barrier during meningitic E. coli infection.","method":"WNT5B treatment of BMECs; ROR1 pathway manipulation; dual luciferase reporter of ZO-1 promoter; ChIP for c-JUN binding; ZO-1 expression and tight junction measurement","journal":"Molecular neurobiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — receptor identified, dual luciferase and ChIP confirm c-JUN/ZO-1 mechanism, single lab","pmids":["38896157"],"is_preprint":false},{"year":2026,"finding":"Astrocytic WNT5B activates NFATc2 transcription factor via non-canonical signaling, inducing sustained MMP14 expression, which degrades extracellular matrix, damages medium spiny neurons, and increases mutant huntingtin aggregation in Huntington's disease models; ERα overexpression suppresses NFATc2 transcriptional activity, and genistein antagonizes NFATc2 to reduce MMP14 transcription.","method":"WNT5B gain-of-function in HD transgenic mice (N171-82Q); NFATc2 reporter assays; MMP14 measurement; ERα overexpression; genistein treatment; motor behavior and lifespan analysis","journal":"Signal transduction and targeted therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo gain-of-function, NFATc2-MMP14 pathway mechanistically defined, pharmacological rescue, single lab","pmids":["41549079"],"is_preprint":false},{"year":2026,"finding":"Wnt5b secreted by renal epithelial cells via exosomes activates canonical Wnt signaling in renal fibroblasts through cooperation of FZD1 and LRP6 receptors, triggering β-catenin cytoplasmic stabilization and nuclear translocation, driving fibroblast activation and renal fibrosis under hypoxia.","method":"Wnt5b knockdown in vivo (hypoxia mouse model); exosome isolation; FZD1 and LRP6 receptor identification; β-catenin nuclear translocation assay; fibroblast activation measurement","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo KD with exosome-mediated intercellular signaling mechanism, receptor cooperation identified, single lab","pmids":["42256303"],"is_preprint":false},{"year":2013,"finding":"CNBP transcription factor directly binds to the wnt5b gene locus and down-regulates wnt5b expression during zebrafish embryonic development, as confirmed by EMSA, ChIP, and loss-of-function experiments.","method":"Yeast one-hybrid; EMSA; ChIP; zebrafish CNBP loss-of-function with wnt5b expression measurement","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple chromatin/binding assays (EMSA, ChIP) plus in vivo functional validation, single lab","pmids":["23667590"],"is_preprint":false},{"year":2018,"finding":"Wnt5b is required for basal constriction at the zebrafish midbrain-hindbrain boundary (MHBC); focal adhesion kinase (Fak) acts downstream of Wnt5b in this process, functioning cell-autonomously within MHBC cells; dominant-negative Gsk3β expression overcomes wnt5b knockdown, placing Gsk3β in the pathway.","method":"Zebrafish wnt5b knockdown; Fak knockdown; tissue-specific Fak knockdown; dominant-negative Gsk3β rescue; Fak immunostaining; 3D reconstruction of MHBC cells","journal":"Biology open","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis (Gsk3β rescue, Fak downstream), cell-autonomous tissue-specific knockdown, single lab","pmids":["30305282"],"is_preprint":false}],"current_model":"WNT5B is a secreted, glycosylated, and lipid-modified ligand that primarily activates non-canonical (β-catenin-independent) Wnt signaling through receptors including Ryk, ROR1, ROR2, and Frizzled2/3, as well as canonical Wnt/β-catenin signaling via FZD1/LRP6 in certain contexts; it signals through downstream effectors DVL2/3, RAC1, CDC42, JNK, PKCα, CaMKII, NFATc2, and TAK1/NF-κB to regulate chondrocyte differentiation, adipogenesis, gastrulation cell movements, pacemaker cardiomyocyte specification, osteoblast differentiation, cancer cell migration and stemness, BBB integrity, and inflammatory gene expression, and is transported in active form via exosomes and cytonemes."},"narrative":{"mechanistic_narrative":"WNT5B is a secreted, post-translationally modified Wnt ligand that predominantly drives β-catenin-independent (non-canonical) signaling to control cell movement, differentiation, and migration across development and disease [PMID:20660632, PMID:27762090, PMID:34906330]. Its secretion depends on N-linked glycosylation at three asparagines and lipidation at one serine, and on the cargo receptor Wntless; secreted WNT5B is delivered to target cells in active form via exosomes and via Ror2-loaded cytonemes [PMID:27762090, PMID:25934698, PMID:38123680]. At the receptor level WNT5B engages Ryk, Frizzled-2/3, and ROR1/ROR2, channeling signal through Dishevelled (DVL2/3) recruitment to the membrane and activation of the small GTPases RAC1, CDC42, and RhoA, with downstream JNK, PKCα, CaMKII, and NFATc2 effectors [PMID:20660632, PMID:24220306, PMID:27036869, PMID:34906330, PMID:39094673]. Through this PCP/calcium machinery WNT5B directs convergent-extension and gastrulation cell movements—requiring the GEF Def6, focal adhesion kinase, and the RGS3/Gα calcium module—and shapes chondrocyte and craniofacial cartilage morphogenesis [PMID:20660632, PMID:20628572, PMID:22039507, PMID:32097584, PMID:26459057]. In several contexts WNT5B antagonizes canonical Wnt/β-catenin signaling, reducing nuclear β-catenin while promoting adipogenesis and suppressing osteoblast differentiation via the ROR1/ROR2–DVL–RAC1–CDC42–JNK–SIN3A axis [PMID:15796911, PMID:19577541, PMID:34906330]; conversely it can act through FZD1/LRP6 to stabilize β-catenin in renal fibrosis and through canonical signaling to specify pacemaker cardiomyocytes [PMID:42256303, PMID:31402282]. In cancer, WNT5B promotes migration, invasion, EndoMT, cancer stem-cell expansion, and metastasis through Cdc42/RhoA, JNK-driven MMP expression, ROR1–SOX2 stemness signaling, and a self-reinforcing PKCα–Rac1 loop [PMID:24220306, PMID:24841200, PMID:27121420, PMID:39094673, PMID:38689429]. WNT5B also drives inflammatory and remodeling programs, inducing IL-6/CXCL8 via FZD2–TAK1–NF-κB, blood-brain-barrier disruption via ROR1–JNK–c-JUN repression of ZO-1, and neurodegenerative ECM damage via NFATc2–MMP14 [PMID:27036869, PMID:38896157, PMID:41549079].","teleology":[{"year":2003,"claim":"Established that WNT5B has a developmental role distinct from the classic Ihh/PTHrP loop, controlling chondrocyte zone transitions through cell-cycle regulators.","evidence":"Mouse long-bone loss-of-function with cyclin D1, p130, and Col2a1 readouts","pmids":["12538525"],"confidence":"Medium","gaps":["No receptor or signaling pathway identified","Mechanism of cyclin D1/p130 regulation undefined"]},{"year":2005,"claim":"Showed WNT5B can antagonize canonical Wnt/β-catenin signaling while promoting adipogenic differentiation, defining its non-canonical, anti-canonical character.","evidence":"Wnt5b overexpression in 3T3-L1 cells with nuclear β-catenin and Wnt3a co-treatment assays","pmids":["15796911"],"confidence":"Medium","gaps":["Overexpression-based; endogenous role untested","Receptor mediating antagonism unknown"]},{"year":2009,"claim":"Refined the adipogenic mechanism to PPARγ/aP2 activation coupled with β-catenin suppression at adipogenesis initiation.","evidence":"Wnt5b overexpression in 3T3-L1 with PPARγ, aP2, and β-catenin readouts","pmids":["19577541"],"confidence":"Medium","gaps":["Direct link between WNT5B receptor and PPARγ unresolved","Single-lab overexpression model"]},{"year":2010,"claim":"Identified Ryk as a WNT5B receptor distinct from Frizzled and dissected parallel directional-signaling and Dishevelled-recruitment branches during gastrulation.","evidence":"Zebrafish Ryk morpholino, Ca2+ imaging, directional migration co-culture, Dvl membrane recruitment","pmids":["20660632"],"confidence":"High","gaps":["Biochemical WNT5B-Ryk binding not shown","How Ryk and Fzd2 outputs integrate unclear"]},{"year":2010,"claim":"Placed an RGS3/Gα calcium-regulating module downstream of WNT5B in somitogenesis, linking the ligand to G-protein-dependent Ca2+ dynamics.","evidence":"Zebrafish rgs3/wnt5b knockdown, Ca2+ imaging, Gα-interaction-deficient rescue","pmids":["20628572"],"confidence":"High","gaps":["Identity of the relevant Gα and receptor not defined","Connection to PCP effectors unclear"]},{"year":2011,"claim":"Connected WNT5B to JNK/PCP control of chondrocyte hypertrophy and to Src-dependent β-catenin phosphorylation regulating cadherin turnover and condensation.","evidence":"Chondrogenic cultures with JNK and Src inhibitors, cadherin turnover and migration assays","pmids":["21413026"],"confidence":"Medium","gaps":["Receptor not identified","Inhibitor-based pathway assignment, no genetic confirmation"]},{"year":2011,"claim":"Identified Def6 as a GEF acting downstream of WNT5B in convergent-extension movements, providing a molecular link to cytoskeletal regulation.","evidence":"Zebrafish morpholino with def6 epistasis rescue and Wnt11 double knockdown","pmids":["22039507"],"confidence":"Medium","gaps":["Direct biochemical link from WNT5B to Def6 not shown","Morpholino-based"]},{"year":2013,"claim":"Established CNBP as a direct transcriptional repressor of wnt5b, identifying upstream regulation of ligand levels in development.","evidence":"Yeast one-hybrid, EMSA, ChIP, and zebrafish CNBP loss-of-function","pmids":["23667590"],"confidence":"Medium","gaps":["Mammalian conservation of CNBP regulation untested"]},{"year":2013,"claim":"Demonstrated a pro-migratory role for WNT5B in carcinoma cells via activation of Cdc42 and RhoA GTPases.","evidence":"siRNA and recombinant protein in OSCC, GTPase activity and invasion assays","pmids":["24220306"],"confidence":"Medium","gaps":["Receptor mediating GTPase activation unidentified"]},{"year":2014,"claim":"Revealed a WNT5B–PKCα positive feedback loop activating Rac1 to drive cytoskeletal reorganization and migration in transformed cells, with miR-200b as a brake.","evidence":"siRNA, forced PKCα expression, Rac1 assay, and 3'-UTR luciferase reporter in arsenic-transformed cells","pmids":["24841200"],"confidence":"Medium","gaps":["Receptor link to PKCα not defined","Single transformation model"]},{"year":2015,"claim":"Defined WNT5B secretion as Wntless-dependent and linked it to fgf3 fine-tuning, and showed cooperation with Glypican 4 in cartilage morphogenesis.","evidence":"Zebrafish wnt5b/wls and gpc4 mutants, mRNA rescue, cell-autonomous transplants","pmids":["25934698","26459057"],"confidence":"Medium","gaps":["Biochemical Gpc4-WNT5B interaction not shown","Receptor for fgf3 regulation unknown"]},{"year":2016,"claim":"Established the biochemical basis of WNT5B secretion (glycosylation, lipidation) and its delivery via exosomes, with downstream Dvl2 phosphorylation and Rac1 activation in cancer.","evidence":"PTM characterization of purified protein, CRISPR KO, TSG101 knockdown, immunoelectron microscopy in PANC-1","pmids":["27762090"],"confidence":"High","gaps":["Receptor on recipient cells not defined here","Exosomal vs free ligand functional split unresolved"]},{"year":2016,"claim":"Expanded WNT5B's range to inflammatory and remodeling programs, defining FZD2-TAK1-NF-κB cytokine induction, EndoMT via Snail/Slug, JNK-MMP-driven CRC invasion, and TGF-β/Smad3 airway remodeling.","evidence":"Recombinant WNT5B with receptor/kinase knockdowns and inhibitors across fibroblasts, LECs, epithelial and CRC cells","pmids":["27036869","27593938","27121420","27126693"],"confidence":"Medium","gaps":["Whether one or multiple receptors mediate these contexts unclear","Mostly exogenous-ligand single-lab studies"]},{"year":2018,"claim":"Positioned WNT5B in an IMP3–miR-145-5p axis feeding TAZ activation and SLUG transcription, linking the ligand to Hippo-pathway effector output.","evidence":"IMP3/miR-145-5p manipulation, mRNA stability and TAZ localization 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fission driving VSMC proliferation/migration, antagonized by SFRP2-promoted fusion.","evidence":"Pulmonary artery SMC overexpression with mdivi-1 and SFRP2, mitochondrial morphology analysis","pmids":["34368954"],"confidence":"Medium","gaps":["Receptor and signaling intermediate to fission machinery undefined"]},{"year":2023,"claim":"Revealed cytoneme-based intercellular transport: Wnt5b-Ror2 complexes assemble in producing cells and transfer on cytonemes to initiate PCP signaling in recipients independent of their own Ror2.","evidence":"Zebrafish live imaging, co-culture transfer assays with Ror2-deficient recipients, CE phenotype analysis","pmids":["38123680"],"confidence":"High","gaps":["Recipient-cell receptor for transferred complex not defined","Relative contribution of cytonemes vs exosomes unresolved"]},{"year":2024,"claim":"Resolved receptor specificity across cancers—FZD3/DVL3-DEP/RAC1/JNK in NSCLC, Ror1 (not Ror2) in PDAC proliferation, and ROR1-SOX2/HYAL1 in osteosarcoma 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research","url":"https://pubmed.ncbi.nlm.nih.gov/37817257","citation_count":8,"is_preprint":false},{"pmid":"28078366","id":"PMC_28078366","title":"Genetic Screening of WNT4 and WNT5B in Two Populations with Deviating Bone Mineral Densities.","date":"2017","source":"Calcified tissue international","url":"https://pubmed.ncbi.nlm.nih.gov/28078366","citation_count":7,"is_preprint":false},{"pmid":"39102216","id":"PMC_39102216","title":"Targeting WNT5B and WNT10B in osteosarcoma.","date":"2024","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/39102216","citation_count":6,"is_preprint":false},{"pmid":"33666148","id":"PMC_33666148","title":"Understanding the binding affinities between SFRP1, SFRP1, Wnt and frizzled receptors 2, 3 and 7 using MD simulations.","date":"2021","source":"Journal of biomolecular structure & dynamics","url":"https://pubmed.ncbi.nlm.nih.gov/33666148","citation_count":5,"is_preprint":false},{"pmid":"31285641","id":"PMC_31285641","title":"Insights into Molecular Interactions of human Wnt5b and Frizzled proteins for their role in teratogenicity.","date":"2019","source":"Bioinformation","url":"https://pubmed.ncbi.nlm.nih.gov/31285641","citation_count":4,"is_preprint":false},{"pmid":"40612684","id":"PMC_40612684","title":"WNT5B regulates myogenesis and fiber type conversion by affecting mRNA stability.","date":"2025","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/40612684","citation_count":3,"is_preprint":false},{"pmid":"38531660","id":"PMC_38531660","title":"Role of Wnt5b-Ror1 signaling in the proliferation of pancreatic ductal adenocarcinoma cells.","date":"2024","source":"Genes to cells : devoted to molecular & cellular mechanisms","url":"https://pubmed.ncbi.nlm.nih.gov/38531660","citation_count":3,"is_preprint":false},{"pmid":"28801288","id":"PMC_28801288","title":"[Expression of Wnt5b in patients with HBV-related hepatocellular carcinoma and its clinical significance].","date":"2017","source":"Nan fang yi ke da xue xue bao = Journal of Southern Medical University","url":"https://pubmed.ncbi.nlm.nih.gov/28801288","citation_count":2,"is_preprint":false},{"pmid":"27329882","id":"PMC_27329882","title":"[The role of Wnt5b in the promotion of osteogenic differentiation by puerarin].","date":"2016","source":"Shanghai kou qiang yi xue = Shanghai journal of stomatology","url":"https://pubmed.ncbi.nlm.nih.gov/27329882","citation_count":1,"is_preprint":false},{"pmid":"41549079","id":"PMC_41549079","title":"Astrocytic noncanonical WNT5B signaling modulates extracellular matrix remodeling and neuropathology in Huntington's disease.","date":"2026","source":"Signal transduction and targeted therapy","url":"https://pubmed.ncbi.nlm.nih.gov/41549079","citation_count":0,"is_preprint":false},{"pmid":"41091903","id":"PMC_41091903","title":"ITLN1 Improves Endothelial Dysfunction in Hypertensive Mice via Wnt5b-JNK Signaling.","date":"2025","source":"Hypertension (Dallas, Tex. : 1979)","url":"https://pubmed.ncbi.nlm.nih.gov/41091903","citation_count":0,"is_preprint":false},{"pmid":"41463832","id":"PMC_41463832","title":"The SCD5 Gene Modulates Adipogenic Differentiation via the WNT5B Signaling Pathway in Xinjiang Brown Cattle.","date":"2025","source":"Animals : an open access journal from MDPI","url":"https://pubmed.ncbi.nlm.nih.gov/41463832","citation_count":0,"is_preprint":false},{"pmid":"41872383","id":"PMC_41872383","title":"Giant cell tumor of bone inhibits osteoblastogenesis via WNT5B.","date":"2026","source":"Journal of bone and mineral metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/41872383","citation_count":0,"is_preprint":false},{"pmid":"41681368","id":"PMC_41681368","title":"Genome-Wide Characterization of the wnt Gene Family Reveals a wnt5b-Mediated Regulatory Mechanism of Testicular Development in Cynoglossus semilaevis.","date":"2026","source":"Animals : an open access journal from MDPI","url":"https://pubmed.ncbi.nlm.nih.gov/41681368","citation_count":0,"is_preprint":false},{"pmid":"42256303","id":"PMC_42256303","title":"Wnt5b/FZD1/LRP6 signaling drives renal fibrosis by triggering cytoplasmic stabilization and nuclear translocation of β-catenin under hypoxia.","date":"2026","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/42256303","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.11.22.624939","title":"Key Regulatory Elements of the TGFβ-LRRC15 Axis Predict Disease Progression and Immunotherapy Resistance Across Cancer Types","date":"2024-11-25","source":"bioRxiv","url":"https://doi.org/10.1101/2024.11.22.624939","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":35009,"output_tokens":8306,"usd":0.114808,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":17992,"output_tokens":6507,"usd":0.126317,"stage2_stop_reason":"end_turn"},"total_usd":0.241125,"stage1_batch_id":"msgbatch_01UT1AahFPCAyn2uVweS7A9z","stage2_batch_id":"msgbatch_01HuKWtKNT4Pqj1bc8eEP5RV","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2003,\n      \"finding\": \"Wnt5b regulates the transition between different chondrocyte zones by differentially controlling cyclin D1 and p130 expression, as well as chondrocyte-specific Col2a1 expression, independently of the Ihh/PTHrP negative feedback loop.\",\n      \"method\": \"Genetic loss-of-function in mouse long bone development; expression analysis of cell cycle regulators\",\n      \"journal\": \"Development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined cellular phenotype with loss-of-function and multiple molecular readouts (cyclin D1, p130, Col2a1), single lab\",\n      \"pmids\": [\"12538525\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Wnt5b overexpression partially inhibits canonical Wnt/β-catenin signaling by reducing nuclear translocation of β-catenin, and promotes adipogenesis in 3T3-L1 preadipocytes, at least partly by antagonizing Wnt3a-induced canonical Wnt activation.\",\n      \"method\": \"Wnt5b overexpression in 3T3-L1 cells; microarray, RT-PCR, nuclear β-catenin localization assay, Wnt3a co-treatment rescue experiment\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (microarray, RT-PCR, β-catenin nuclear localization), single lab\",\n      \"pmids\": [\"15796911\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Wnt5b stimulates adipogenesis by activating PPARγ and aP2 expression and inhibits β-catenin-dependent Wnt signaling at the initiation of adipogenesis in 3T3-L1 preadipocytes.\",\n      \"method\": \"Wnt5b overexpression in 3T3-L1 cells; measurement of PPARγ, aP2, and β-catenin translocation\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — overexpression with multiple molecular readouts (PPARγ, aP2, β-catenin), single lab\",\n      \"pmids\": [\"19577541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Wnt5b acts through the receptor Ryk (not solely Frizzled) to convey directional signals during zebrafish gastrulation; Ryk deficiency impairs Wnt5b-induced Ca2+ activity and directional cell movement, and Wnt5b-Ryk signaling promotes polarized cell protrusions. Fzd2, but not Ryk, recruits Dishevelled to the cell membrane upon Wnt5b stimulation, indicating separate downstream pathways.\",\n      \"method\": \"Zebrafish Ryk loss-of-function (morpholino); co-culture directional migration assay; Ca2+ imaging; cell membrane recruitment of Dishevelled assessed by imaging\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal assays (Ca2+ imaging, directional migration co-culture, Dvl membrane recruitment), loss-of-function in vivo, mechanistic pathway separation of Ryk vs Fzd2\",\n      \"pmids\": [\"20660632\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Rgs3 (Regulator of G protein Signaling 3) is required downstream of Wnt5b for appropriate frequency and amplitude of Ca2+ release during zebrafish somitogenesis; Rgs3 acts as a G protein GAP and its activity requires the ability to interact with Gα subunits.\",\n      \"method\": \"Zebrafish morpholino knockdown of rgs3 and wnt5b; in vivo Ca2+ imaging; rescue assays with Gα-interaction-deficient Rgs3 mutant\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo Ca2+ imaging, loss-of-function, mutagenesis-based epistasis rescue, replicated genetic interaction\",\n      \"pmids\": [\"20628572\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Wnt5b inhibits chondrocyte hypertrophy and regulates chondroprogenitor cell migration through JNK activation (planar cell polarity pathway) and disrupts mesenchymal condensation via Src-dependent β-catenin phosphorylation leading to increased cadherin receptor turnover.\",\n      \"method\": \"Wnt5b expression in chondrogenic cultures; JNK inhibition (SP600125); Src inhibition; cadherin turnover assay; migration assay\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple pathway inhibitors used (JNK, Src), multiple phenotypic readouts (hypertrophy, migration, cadherin turnover), single lab\",\n      \"pmids\": [\"21413026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Def6, a novel guanine nucleotide exchange factor (GEF), functions downstream of Wnt5b signaling to regulate convergent extension cell movements during zebrafish gastrulation; overexpression of def6 rescues Wnt5b morphant phenotypes.\",\n      \"method\": \"Zebrafish morpholino knockdown; genetic epistasis rescue (def6 overexpression rescues wnt5b morphants); double knockdown of def6 and Wnt11\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis rescue in vivo, single lab, morpholino-based\",\n      \"pmids\": [\"22039507\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Wnt5b promotes cell motility (migration, invasion, filopodia formation) in oral squamous cell carcinoma cells through activation of Cdc42 and RhoA; siRNA knockdown of Wnt5b inhibits migration and filopodia, while recombinant Wnt5b stimulation increases active Cdc42 and RhoA levels.\",\n      \"method\": \"siRNA knockdown and recombinant protein stimulation; GTPase activity assays (active Cdc42, active RhoA); migration and invasion assays\",\n      \"journal\": \"International journal of oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal gain/loss-of-function with GTPase activity measurement, single lab\",\n      \"pmids\": [\"24220306\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Wnt5b forms a positive feedback loop with PKCα in arsenic-transformed cells: Wnt5b sustains phospho-PKC levels, and PKCα in turn maintains Wnt5b expression. This Wnt5b-PKCα axis activates Rac1, promoting actin cytoskeletal reorganization and cell migration. miR-200b suppresses this pathway by directly targeting PKCα.\",\n      \"method\": \"siRNA knockdown of Wnt5b and PKCα; forced expression of PKCα in miR-200b-overexpressing cells; Rac1 activation assay; 3'-UTR luciferase reporter for miR-200b targeting PKCα\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple reciprocal gain/loss-of-function experiments with mechanistic readouts (Rac1 activity, phospho-PKC), single lab\",\n      \"pmids\": [\"24841200\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Wnt5b is glycosylated at three asparagine residues and lipidated at one serine residue; these post-translational modifications are essential for Wnt5b secretion. Secreted Wnt5b phosphorylates Dvl2 and activates Rac1. In pancreatic cancer cells, ~55% of secreted endogenous Wnt5b is associated with exosomes, and Wnt5b-associated exosomes promote cancer cell migration and proliferation in a paracrine manner.\",\n      \"method\": \"Biochemical characterization of purified Wnt5b (glycosylation, lipidation); Wnt5b-KO PANC-1 cells (CRISPR); TSG101 knockdown; Dvl2 phosphorylation assay; Rac1 activation assay; centrifugation fractionation; immunoelectron microscopy; migration and proliferation assays\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro biochemical characterization of PTMs, KO validation, multiple orthogonal methods (immunoelectron microscopy, biochemical fractionation, functional assays)\",\n      \"pmids\": [\"27762090\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"WNT5B induces partial endothelial-mesenchymal transition (EndoMT) in lymphatic endothelial cells (LECs), promoting tube formation via upregulation of Snail and Slug transcription factors; WNT5B-induced Snail/Slug expression is abolished by IWR-1-endo (Wnt/β-catenin inhibitor) and Rac1 inhibitors, implicating both canonical and non-canonical (PCP) Wnt pathways.\",\n      \"method\": \"Recombinant WNT5B treatment of LECs; WNT5B knockdown in OSCC cells; tube formation, permeability, migration assays; phalloidin staining; SNAIL/SLUG knockdown; pathway inhibitors\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple functional assays with pathway inhibitor dissection and downstream transcription factor knockdown, single lab\",\n      \"pmids\": [\"27593938\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"WNT5B signals through the Frizzled-2 receptor and TAK1 to induce IL-6 and CXCL8 release in lung fibroblasts via activation of JNK, p38, and p65 NF-κB, without involving canonical β-catenin signaling; IL-6 and CXCL8 release are controlled through distinct downstream pathways (JNK/p38 for both; IKK for CXCL8 only).\",\n      \"method\": \"Recombinant WNT5B treatment; Frizzled-2 knockdown; TAK1 inhibition; JNK, p38, IKK inhibitors; cytokine secretion measurement (ELISA); β-catenin pathway assessment\",\n      \"journal\": \"American journal of physiology. Lung cellular and molecular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — receptor identified by knockdown, multiple kinase inhibitors defining pathway branches, single lab\",\n      \"pmids\": [\"27036869\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"WNT5B increases airway remodeling gene expression (fibronectin, MMP-2, MMP-9, Snail) in bronchial epithelial cells via TGF-β/Smad3 signaling.\",\n      \"method\": \"Exogenous WNT5B treatment of BEAS-2B and air-liquid interface PBECs; TGF-β/Smad3 pathway analysis\",\n      \"journal\": \"The European respiratory journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — exogenous protein treatment with pathway inhibitor validation (Smad3), multiple gene readouts, single lab\",\n      \"pmids\": [\"27126693\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"WNT5B promotes colorectal cancer cell migration and invasion through activation of the JNK signaling pathway, leading to increased MMP-2 and MMP-9 expression; JNK knockdown reverses these effects.\",\n      \"method\": \"Stable WNT5B overexpression in COLO 205 cells; JNK siRNA knockdown; MTT, wound healing, Transwell assays; western blot for MMP2/9 and JNK pathway\",\n      \"journal\": \"Oncology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain-of-function and loss-of-function (JNK RNAi) with multiple functional readouts, single lab\",\n      \"pmids\": [\"27121420\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Wnt5b requires Wntless (Wls) for its secretion from pharyngeal tissue and regulates chondrogenic cell proliferation by fine-tuning fgf3 expression; fgf3 mRNA introduction rescues cartilage defects in Wnt5b- and Wls-deficient zebrafish larvae.\",\n      \"method\": \"Zebrafish wnt5b mutant and wls morphant analysis; fgf3 expression measurement; mRNA rescue experiment; cell proliferation and apoptosis assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo epistasis rescue, secretion mechanism identified by Wls dependence, single lab\",\n      \"pmids\": [\"25934698\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Glypican 4 (Gpc4) and Wnt5b cooperate to regulate chondrocyte stacking and intercalation during craniofacial cartilage morphogenesis in zebrafish; Gpc4 functions cell-autonomously in chondrocytes, and double heterozygous wnt5b;gpc4 embryos show enhanced phenotype, suggesting genetic interaction independent of core Wnt/PCP molecules.\",\n      \"method\": \"Zebrafish wnt5b and gpc4 mutant analysis; cell-autonomous rescue with chimeric transplants; double heterozygous epistasis\",\n      \"journal\": \"Mechanisms of development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in vivo (double heterozygotes, cell-autonomous transplants), single lab\",\n      \"pmids\": [\"26459057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Wnt5b and Wnt5a act through the JNK signaling pathway to mediate mechanical tension-induced osteogenic differentiation of rat tendon-derived stem cells; shRNA knockdown of Wnt5a/Wnt5b reduces UMT-induced Runx2 and P-JNK, and JNK activation rescues Runx2 expression.\",\n      \"method\": \"shRNA knockdown of Wnt5a and Wnt5b in rTDSCs; JNK inhibitor (SP600125) and activator (anisomycin); JNK1-shRNA and JNK1-cDNA; Runx2 and P-JNK protein measurement\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with rescue via JNK activation, multiple molecular readouts, single lab\",\n      \"pmids\": [\"25966835\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"IMP3 stabilizes WNT5B mRNA indirectly by repressing miR-145-5p (which targets WNT5B), resulting in TAZ activation via alternative WNT signaling. WNT5B also facilitates SLUG transcription, which is required for TAZ nuclear localization.\",\n      \"method\": \"IMP3 knockdown/overexpression; miR-145-5p manipulation; WNT5B mRNA stability assay; TAZ nuclear localization; SLUG expression measurement\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple genetic manipulations with mechanistic readouts (mRNA stability, TAZ localization), single lab\",\n      \"pmids\": [\"29847788\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Wnt5b signals through canonical Wnt (β-catenin) pathway in zebrafish to direct Nkx2.5+ mesoderm into pacemaker cardiomyocytes, activating transcription factors Isl1 and Tbx18 while silencing Nkx2.5; this mechanism is evolutionarily conserved and can be applied to direct human pluripotent stem cells toward pacemaker cardiomyocyte fate.\",\n      \"method\": \"Zebrafish Wnt5b loss-of-function; cell lineage tracing; transcription factor expression (Isl1, Tbx18, Nkx2.5); hPSC directed differentiation applying Wnt5b pathway findings\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic loss-of-function combined with lineage tracing, multiple transcription factor readouts, cross-species (zebrafish to human PSC) validation\",\n      \"pmids\": [\"31402282\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Wnt5b/Ryk signaling promotes membrane trafficking of P2X3 receptors to the DRG neuron surface via CaMKII activation, leading to enhanced P2X3 currents and pain hypersensitivity; anti-Ryk antibody and CaMKII inhibitor (KN93) block these effects.\",\n      \"method\": \"Wnt5b application to cultured DRG neurons; anti-Ryk antibody; CaMKII inhibitor KN93; electrophysiological recording of P2X3 currents; membrane P2X3 protein quantification; in vivo intrathecal injection\",\n      \"journal\": \"Experimental neurology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — receptor identification by antibody blockade, CaMKII mechanistic link, electrophysiology and protein trafficking assays, single lab\",\n      \"pmids\": [\"32979370\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Wnt5b and Fak1a (focal adhesion kinase 1a) converge in regulating Rac1 and Cdc42 to mediate gastrulation cell movements in zebrafish; overexpression of fak1a rescues wnt5b morphant convergence defects and vice versa, and Rac1/Cdc42 activation synergistically rescues both morphants.\",\n      \"method\": \"Zebrafish morpholino knockdown; CRISPR/Cas9 fak1a mutants; cross-rescue overexpression; Rac1 and Cdc42 activation measurement\",\n      \"journal\": \"Open biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal genetic rescue and GTPase activity measurements, single lab\",\n      \"pmids\": [\"32097584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"WNT5B suppresses osteoblast differentiation and mineralization via ROR1/ROR2 receptors, which activate DVL2/3–RAC1–CDC42–JNK–SIN3A signaling and inhibit β-catenin activity; ERα and NFATc1 bind to an enhancer containing SNP rs2887571 to repress WNT5B expression, with the GG genotype showing greater ERα binding and greater WNT5B suppression than AA.\",\n      \"method\": \"ChIP-qPCR; CRISPR-Cas9 allele editing; ROR1/2 receptor manipulation; DVL2/3, RAC1, CDC42, JNK pathway analysis; alkaline phosphatase activity; mineralization assay; IL-6 measurement; correlation of genotype with WNT5B expression in 110 patient osteoblasts\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — CRISPR allele editing, ChIP-qPCR, multiple receptor/pathway components tested, functional osteoblast readouts, validated in 110 patient samples\",\n      \"pmids\": [\"34906330\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"WNT5B promotes VSMC proliferation and migration via non-canonical Wnt signaling and induces mitochondrial fission; inhibition of mitochondrial fission (by mdivi-1) abolishes WNT5B-induced VSMC proliferation and migration, while secreted frizzled-related protein 2 (SFRP2, a Wnt scavenger) attenuates VSMC proliferation/migration by promoting mitochondrial fusion.\",\n      \"method\": \"Primary pulmonary artery SMC culture; WNT5B overexpression; mitochondrial division inhibitor 1 (mdivi-1); SFRP2 treatment; proliferation and migration assays; mitochondrial morphology analysis\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain-of-function and inhibitor-based pathway dissection with mechanistic readout (mitochondrial dynamics), single lab\",\n      \"pmids\": [\"34368954\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Wnt5b binds to Ror2 receptor in the producing cell; active Wnt5b-Ror2 complexes are loaded onto cytonemes (long cellular protrusions) and transferred to receiving cells, enabling initiation of Wnt-PCP signaling in recipient cells regardless of their own Ror2 status; this cytoneme-dependent spreading controls convergence and extension in zebrafish gastrula.\",\n      \"method\": \"Live imaging of Wnt5b-Ror2 on cytonemes in zebrafish; co-culture transfer assays; Ror2-deficient receiving cells; convergent extension phenotype analysis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — live imaging plus functional co-culture assays with Ror2-deficient recipients, multiple orthogonal approaches, high-impact journal\",\n      \"pmids\": [\"38123680\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"WNT5B signals through FZD3 receptor to recruit DVL3 to the plasma membrane (requiring DVL3's DEP domain) in a WNT5B ligand-dependent manner, activating JNK signaling via RAC1 (WNT-PCP pathway) to promote malignant phenotype of NSCLC cells; deletion of the DEP domain of DVL3 abrogates these effects.\",\n      \"method\": \"Co-IP of WNT5B-FZD3-DVL3; DVL3 DEP domain deletion mutant; membrane recruitment assay; JNK and RAC1 activation; in vivo and in vitro tumor assays\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — Co-IP identifying the complex, domain deletion mutagenesis, in vivo validation, multiple pathway readouts\",\n      \"pmids\": [\"39094673\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Wnt5b signals through Ror1 (not Ror2) to promote PDAC cell proliferation in a cell-autonomous manner; knockdown of either Ror1 or Wnt5b in PANC-1 cells inhibits proliferation in vitro, and Ror1 knockout inhibits tumor growth in vivo.\",\n      \"method\": \"Ror1 and Wnt5b knockdown in PANC-1 cells; Ror1 CRISPR knockout; in vitro proliferation assays; in vivo xenograft tumor assay\",\n      \"journal\": \"Genes to cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — receptor identified by genetic KO with in vivo validation, single lab\",\n      \"pmids\": [\"38531660\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"WNT5B drives osteosarcoma cancer stem cell expansion and promotes lung/liver metastasis in vivo; WNT5B signals via ROR1 to induce SOX2 expression, and also upregulates HYAL1 to degrade hyaluronic acid in the tumor microenvironment; ROR1 antibody-mediated inhibition of WNT5B/ROR1 signaling reduces stemness, sphere size, and SOX2 expression.\",\n      \"method\": \"WNT5B overexpression and knockdown in osteosarcoma spheres and PDX; in vivo metastasis assay; ROR1 antibody treatment; SOX2, HYAL1 measurement; hyaluronic acid quantification\",\n      \"journal\": \"Clinical and translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo metastasis model, receptor targeted by antibody, multiple molecular readouts, single lab\",\n      \"pmids\": [\"38689429\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Astrocyte-secreted WNT5B activates non-canonical Wnt signaling in brain microvascular endothelial cells via receptor ROR1, leading to JNK/c-JUN activation; c-JUN directly binds to and represses the ZO-1 promoter, impairing tight junction integrity and disrupting the blood-brain barrier during meningitic E. coli infection.\",\n      \"method\": \"WNT5B treatment of BMECs; ROR1 pathway manipulation; dual luciferase reporter of ZO-1 promoter; ChIP for c-JUN binding; ZO-1 expression and tight junction measurement\",\n      \"journal\": \"Molecular neurobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — receptor identified, dual luciferase and ChIP confirm c-JUN/ZO-1 mechanism, single lab\",\n      \"pmids\": [\"38896157\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Astrocytic WNT5B activates NFATc2 transcription factor via non-canonical signaling, inducing sustained MMP14 expression, which degrades extracellular matrix, damages medium spiny neurons, and increases mutant huntingtin aggregation in Huntington's disease models; ERα overexpression suppresses NFATc2 transcriptional activity, and genistein antagonizes NFATc2 to reduce MMP14 transcription.\",\n      \"method\": \"WNT5B gain-of-function in HD transgenic mice (N171-82Q); NFATc2 reporter assays; MMP14 measurement; ERα overexpression; genistein treatment; motor behavior and lifespan analysis\",\n      \"journal\": \"Signal transduction and targeted therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo gain-of-function, NFATc2-MMP14 pathway mechanistically defined, pharmacological rescue, single lab\",\n      \"pmids\": [\"41549079\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Wnt5b secreted by renal epithelial cells via exosomes activates canonical Wnt signaling in renal fibroblasts through cooperation of FZD1 and LRP6 receptors, triggering β-catenin cytoplasmic stabilization and nuclear translocation, driving fibroblast activation and renal fibrosis under hypoxia.\",\n      \"method\": \"Wnt5b knockdown in vivo (hypoxia mouse model); exosome isolation; FZD1 and LRP6 receptor identification; β-catenin nuclear translocation assay; fibroblast activation measurement\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo KD with exosome-mediated intercellular signaling mechanism, receptor cooperation identified, single lab\",\n      \"pmids\": [\"42256303\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CNBP transcription factor directly binds to the wnt5b gene locus and down-regulates wnt5b expression during zebrafish embryonic development, as confirmed by EMSA, ChIP, and loss-of-function experiments.\",\n      \"method\": \"Yeast one-hybrid; EMSA; ChIP; zebrafish CNBP loss-of-function with wnt5b expression measurement\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple chromatin/binding assays (EMSA, ChIP) plus in vivo functional validation, single lab\",\n      \"pmids\": [\"23667590\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Wnt5b is required for basal constriction at the zebrafish midbrain-hindbrain boundary (MHBC); focal adhesion kinase (Fak) acts downstream of Wnt5b in this process, functioning cell-autonomously within MHBC cells; dominant-negative Gsk3β expression overcomes wnt5b knockdown, placing Gsk3β in the pathway.\",\n      \"method\": \"Zebrafish wnt5b knockdown; Fak knockdown; tissue-specific Fak knockdown; dominant-negative Gsk3β rescue; Fak immunostaining; 3D reconstruction of MHBC cells\",\n      \"journal\": \"Biology open\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis (Gsk3β rescue, Fak downstream), cell-autonomous tissue-specific knockdown, single lab\",\n      \"pmids\": [\"30305282\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"WNT5B is a secreted, glycosylated, and lipid-modified ligand that primarily activates non-canonical (β-catenin-independent) Wnt signaling through receptors including Ryk, ROR1, ROR2, and Frizzled2/3, as well as canonical Wnt/β-catenin signaling via FZD1/LRP6 in certain contexts; it signals through downstream effectors DVL2/3, RAC1, CDC42, JNK, PKCα, CaMKII, NFATc2, and TAK1/NF-κB to regulate chondrocyte differentiation, adipogenesis, gastrulation cell movements, pacemaker cardiomyocyte specification, osteoblast differentiation, cancer cell migration and stemness, BBB integrity, and inflammatory gene expression, and is transported in active form via exosomes and cytonemes.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"WNT5B is a secreted, post-translationally modified Wnt ligand that predominantly drives β-catenin-independent (non-canonical) signaling to control cell movement, differentiation, and migration across development and disease [#3, #9, #21]. Its secretion depends on N-linked glycosylation at three asparagines and lipidation at one serine, and on the cargo receptor Wntless; secreted WNT5B is delivered to target cells in active form via exosomes and via Ror2-loaded cytonemes [#9, #14, #23]. At the receptor level WNT5B engages Ryk, Frizzled-2/3, and ROR1/ROR2, channeling signal through Dishevelled (DVL2/3) recruitment to the membrane and activation of the small GTPases RAC1, CDC42, and RhoA, with downstream JNK, PKCα, CaMKII, and NFATc2 effectors [#3, #7, #11, #21, #24]. Through this PCP/calcium machinery WNT5B directs convergent-extension and gastrulation cell movements—requiring the GEF Def6, focal adhesion kinase, and the RGS3/Gα calcium module—and shapes chondrocyte and craniofacial cartilage morphogenesis [#3, #4, #6, #20, #15]. In several contexts WNT5B antagonizes canonical Wnt/β-catenin signaling, reducing nuclear β-catenin while promoting adipogenesis and suppressing osteoblast differentiation via the ROR1/ROR2–DVL–RAC1–CDC42–JNK–SIN3A axis [#1, #2, #21]; conversely it can act through FZD1/LRP6 to stabilize β-catenin in renal fibrosis and through canonical signaling to specify pacemaker cardiomyocytes [#29, #18]. In cancer, WNT5B promotes migration, invasion, EndoMT, cancer stem-cell expansion, and metastasis through Cdc42/RhoA, JNK-driven MMP expression, ROR1–SOX2 stemness signaling, and a self-reinforcing PKCα–Rac1 loop [#7, #8, #13, #24, #26]. WNT5B also drives inflammatory and remodeling programs, inducing IL-6/CXCL8 via FZD2–TAK1–NF-κB, blood-brain-barrier disruption via ROR1–JNK–c-JUN repression of ZO-1, and neurodegenerative ECM damage via NFATc2–MMP14 [#11, #27, #28].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established that WNT5B has a developmental role distinct from the classic Ihh/PTHrP loop, controlling chondrocyte zone transitions through cell-cycle regulators.\",\n      \"evidence\": \"Mouse long-bone loss-of-function with cyclin D1, p130, and Col2a1 readouts\",\n      \"pmids\": [\"12538525\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No receptor or signaling pathway identified\", \"Mechanism of cyclin D1/p130 regulation undefined\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Showed WNT5B can antagonize canonical Wnt/β-catenin signaling while promoting adipogenic differentiation, defining its non-canonical, anti-canonical character.\",\n      \"evidence\": \"Wnt5b overexpression in 3T3-L1 cells with nuclear β-catenin and Wnt3a co-treatment assays\",\n      \"pmids\": [\"15796911\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Overexpression-based; endogenous role untested\", \"Receptor mediating antagonism unknown\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Refined the adipogenic mechanism to PPARγ/aP2 activation coupled with β-catenin suppression at adipogenesis initiation.\",\n      \"evidence\": \"Wnt5b overexpression in 3T3-L1 with PPARγ, aP2, and β-catenin readouts\",\n      \"pmids\": [\"19577541\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct link between WNT5B receptor and PPARγ unresolved\", \"Single-lab overexpression model\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identified Ryk as a WNT5B receptor distinct from Frizzled and dissected parallel directional-signaling and Dishevelled-recruitment branches during gastrulation.\",\n      \"evidence\": \"Zebrafish Ryk morpholino, Ca2+ imaging, directional migration co-culture, Dvl membrane recruitment\",\n      \"pmids\": [\"20660632\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Biochemical WNT5B-Ryk binding not shown\", \"How Ryk and Fzd2 outputs integrate unclear\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Placed an RGS3/Gα calcium-regulating module downstream of WNT5B in somitogenesis, linking the ligand to G-protein-dependent Ca2+ dynamics.\",\n      \"evidence\": \"Zebrafish rgs3/wnt5b knockdown, Ca2+ imaging, Gα-interaction-deficient rescue\",\n      \"pmids\": [\"20628572\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the relevant Gα and receptor not defined\", \"Connection to PCP effectors unclear\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Connected WNT5B to JNK/PCP control of chondrocyte hypertrophy and to Src-dependent β-catenin phosphorylation regulating cadherin turnover and condensation.\",\n      \"evidence\": \"Chondrogenic cultures with JNK and Src inhibitors, cadherin turnover and migration assays\",\n      \"pmids\": [\"21413026\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Receptor not identified\", \"Inhibitor-based pathway assignment, no genetic confirmation\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identified Def6 as a GEF acting downstream of WNT5B in convergent-extension movements, providing a molecular link to cytoskeletal regulation.\",\n      \"evidence\": \"Zebrafish morpholino with def6 epistasis rescue and Wnt11 double knockdown\",\n      \"pmids\": [\"22039507\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical link from WNT5B to Def6 not shown\", \"Morpholino-based\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Established CNBP as a direct transcriptional repressor of wnt5b, identifying upstream regulation of ligand levels in development.\",\n      \"evidence\": \"Yeast one-hybrid, EMSA, ChIP, and zebrafish CNBP loss-of-function\",\n      \"pmids\": [\"23667590\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mammalian conservation of CNBP regulation untested\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrated a pro-migratory role for WNT5B in carcinoma cells via activation of Cdc42 and RhoA GTPases.\",\n      \"evidence\": \"siRNA and recombinant protein in OSCC, GTPase activity and invasion assays\",\n      \"pmids\": [\"24220306\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Receptor mediating GTPase activation unidentified\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Revealed a WNT5B–PKCα positive feedback loop activating Rac1 to drive cytoskeletal reorganization and migration in transformed cells, with miR-200b as a brake.\",\n      \"evidence\": \"siRNA, forced PKCα expression, Rac1 assay, and 3'-UTR luciferase reporter in arsenic-transformed cells\",\n      \"pmids\": [\"24841200\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Receptor link to PKCα not defined\", \"Single transformation model\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined WNT5B secretion as Wntless-dependent and linked it to fgf3 fine-tuning, and showed cooperation with Glypican 4 in cartilage morphogenesis.\",\n      \"evidence\": \"Zebrafish wnt5b/wls and gpc4 mutants, mRNA rescue, cell-autonomous transplants\",\n      \"pmids\": [\"25934698\", \"26459057\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Biochemical Gpc4-WNT5B interaction not shown\", \"Receptor for fgf3 regulation unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Established the biochemical basis of WNT5B secretion (glycosylation, lipidation) and its delivery via exosomes, with downstream Dvl2 phosphorylation and Rac1 activation in cancer.\",\n      \"evidence\": \"PTM characterization of purified protein, CRISPR KO, TSG101 knockdown, immunoelectron microscopy in PANC-1\",\n      \"pmids\": [\"27762090\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Receptor on recipient cells not defined here\", \"Exosomal vs free ligand functional split unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Expanded WNT5B's range to inflammatory and remodeling programs, defining FZD2-TAK1-NF-κB cytokine induction, EndoMT via Snail/Slug, JNK-MMP-driven CRC invasion, and TGF-β/Smad3 airway remodeling.\",\n      \"evidence\": \"Recombinant WNT5B with receptor/kinase knockdowns and inhibitors across fibroblasts, LECs, epithelial and CRC cells\",\n      \"pmids\": [\"27036869\", \"27593938\", \"27121420\", \"27126693\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether one or multiple receptors mediate these contexts unclear\", \"Mostly exogenous-ligand single-lab studies\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Positioned WNT5B in an IMP3–miR-145-5p axis feeding TAZ activation and SLUG transcription, linking the ligand to Hippo-pathway effector output.\",\n      \"evidence\": \"IMP3/miR-145-5p manipulation, mRNA stability and TAZ localization assays\",\n      \"pmids\": [\"29847788\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct WNT5B-to-TAZ signaling mechanism not resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showed WNT5B drives basal constriction at the midbrain-hindbrain boundary with Fak downstream and Gsk3β in the pathway.\",\n      \"evidence\": \"Zebrafish wnt5b/Fak knockdown, dominant-negative Gsk3β rescue, 3D cell reconstruction\",\n      \"pmids\": [\"30305282\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Receptor not identified\", \"Canonical vs non-canonical engagement of Gsk3β ambiguous\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrated a context-specific canonical role: WNT5B/β-catenin directs Nkx2.5+ mesoderm to pacemaker cardiomyocyte fate, conserved to human PSCs.\",\n      \"evidence\": \"Zebrafish loss-of-function, lineage tracing, transcription factor readouts, hPSC directed differentiation\",\n      \"pmids\": [\"31402282\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Receptor/co-receptor for canonical engagement not specified\", \"Switch between canonical and non-canonical use unexplained\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Linked WNT5B/Ryk to CaMKII-dependent P2X3 receptor trafficking and pain hypersensitivity, and showed Fak1a convergence on Rac1/Cdc42 in gastrulation.\",\n      \"evidence\": \"DRG neuron electrophysiology with Ryk antibody and CaMKII inhibitor; zebrafish fak1a cross-rescue with GTPase assays\",\n      \"pmids\": [\"32979370\", \"32097584\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct WNT5B-Ryk binding affinity unquantified\", \"How CaMKII couples to P2X3 trafficking not detailed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined the ROR1/ROR2–DVL2/3–RAC1–CDC42–JNK–SIN3A axis by which WNT5B suppresses osteoblast differentiation, and identified ERα/NFATc1 enhancer (rs2887571) repression of WNT5B in patient osteoblasts.\",\n      \"evidence\": \"CRISPR allele editing, ChIP-qPCR, receptor/pathway manipulation, mineralization assays in 110 patient samples\",\n      \"pmids\": [\"34906330\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of ROR receptor engagement absent\", \"Causal disease mutation not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Connected WNT5B non-canonical signaling to mitochondrial fission driving VSMC proliferation/migration, antagonized by SFRP2-promoted fusion.\",\n      \"evidence\": \"Pulmonary artery SMC overexpression with mdivi-1 and SFRP2, mitochondrial morphology analysis\",\n      \"pmids\": [\"34368954\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Receptor and signaling intermediate to fission machinery undefined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealed cytoneme-based intercellular transport: Wnt5b-Ror2 complexes assemble in producing cells and transfer on cytonemes to initiate PCP signaling in recipients independent of their own Ror2.\",\n      \"evidence\": \"Zebrafish live imaging, co-culture transfer assays with Ror2-deficient recipients, CE phenotype analysis\",\n      \"pmids\": [\"38123680\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Recipient-cell receptor for transferred complex not defined\", \"Relative contribution of cytonemes vs exosomes unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Resolved receptor specificity across cancers—FZD3/DVL3-DEP/RAC1/JNK in NSCLC, Ror1 (not Ror2) in PDAC proliferation, and ROR1-SOX2/HYAL1 in osteosarcoma stemness and metastasis.\",\n      \"evidence\": \"Co-IP and DEP-domain deletion in NSCLC; CRISPR KO and xenografts in PANC-1; ROR1 antibody and PDX metastasis models\",\n      \"pmids\": [\"39094673\", \"38531660\", \"38689429\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why different ROR receptors dominate in different tissues unclear\", \"FZD3-ROR co-receptor relationship not integrated\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Implicated astrocyte-secreted WNT5B in blood-brain-barrier disruption via ROR1-JNK-c-JUN repression of the ZO-1 tight-junction promoter during infection.\",\n      \"evidence\": \"WNT5B treatment of BMECs, ROR1 manipulation, ZO-1 luciferase and c-JUN ChIP\",\n      \"pmids\": [\"38896157\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo BBB rescue by WNT5B blockade not shown\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Extended WNT5B's pathological reach to renal fibrosis (exosomal FZD1/LRP6/β-catenin) and Huntington's disease (NFATc2-MMP14 ECM damage), with ERα and genistein as suppressors.\",\n      \"evidence\": \"In vivo hypoxia kidney model with exosome isolation and receptor identification; HD transgenic mice with NFATc2 reporters and pharmacological antagonism\",\n      \"pmids\": [\"42256303\", \"41549079\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Receptor for canonical renal signaling and NFATc2 upstream receptor not jointly defined\", \"Single-lab in vivo models\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How WNT5B selects between canonical (β-catenin) and multiple non-canonical outputs in a given cell type, and the structural basis of its engagement with Ryk/ROR/Frizzled co-receptors, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of WNT5B-receptor complexes in the corpus\", \"Determinants of canonical vs non-canonical pathway choice unknown\", \"Relative in vivo roles of exosome vs cytoneme delivery undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [3, 9, 21, 23, 24]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 2, 11, 21]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [9, 14, 23, 29]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [9, 29]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3, 23, 24]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 11, 21, 24]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0, 3, 18, 23]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [13, 26, 27, 28, 29]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [11, 27]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"RYK\", \"ROR1\", \"ROR2\", \"FZD2\", \"FZD3\", \"DVL3\", \"LRP6\", \"FZD1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}