{"gene":"SHROOM3","run_date":"2026-06-10T07:46:32","timeline":{"discoveries":[{"year":2008,"finding":"Shroom3 directly binds ROCK1/2 (Rho kinases) via a defined binding site (RII-C1 on ROCKs) and recruits them to epithelial apical junctions. Expression of the competitive antagonist RII-C1 fragment displaced apically localized ROCKs and blocked neural tube closure, demonstrating that the Shroom3-ROCK interaction is required for apical junction remodeling and neural tube morphogenesis.","method":"Co-immunoprecipitation, dominant-negative RII-C1 fragment expression, live imaging of phospho-myosin distribution, shRNA depletion in neural tube","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal binding-site mapping, dominant-negative rescue, multiple orthogonal methods, replicated across subsequent studies","pmids":["18339671"],"is_preprint":false},{"year":2010,"finding":"Shroom3 is required for apical localization of F-actin, myosin II, and Vasp during lens placode invagination. Loss of Shroom3 abolishes apical constriction of lens epithelial cells, and Shroom3 expression in the lens is directly controlled by the transcription factor Pax6.","method":"Loss-of-function mouse genetics, immunofluorescence for F-actin/myosin II/Vasp localization, genetic epistasis with Pax6 mutants","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with defined cellular phenotype, apical localization of multiple effectors assayed, genetic epistasis established","pmids":["20081189"],"is_preprint":false},{"year":2010,"finding":"Pitx transcription factors directly activate Shroom3 transcription through Pitx-responsive regulatory elements in the Shroom3 genomic locus. Ectopic Pitx expression is sufficient to induce Shroom3-dependent cytoskeletal reorganization and epithelial cell shape change in Xenopus gut.","method":"Promoter-reporter assays, Pitx gain-of-function in Xenopus, Shroom3 loss-of-function, identification of Pitx-responsive cis-regulatory elements","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — direct transcriptional activation shown by reporter assay + mutagenesis of cis-elements + epistasis in vivo","pmids":["20332151"],"is_preprint":false},{"year":2011,"finding":"A Trio (RhoA-GEF)–RhoA–Shroom3 pathway is required for apical constriction during lens pit invagination. RhoA activity is required for Shroom3 apical localization, and activated apical RhoA is sufficient to induce apical constriction. Trio is required for Shroom3-dependent apical constriction in MDCK cells and in the lens pit.","method":"Dominant-negative and constitutively active RhoA constructs, MDCK cell assays, Trio loss-of-function in lens pit, epistasis experiments","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — epistasis with multiple pathway components, gain- and loss-of-function, two model systems","pmids":["22031541"],"is_preprint":false},{"year":2011,"finding":"Shroom3 and N-cadherin function cooperatively downstream of Pitx2 to regulate asymmetric epithelial cell shape changes required for gut tube morphogenesis. Genetic interaction shown by compound heterozygous analysis: loss of one allele each of Shroom3 and N-cadherin phenocopies homozygous loss of either gene alone.","method":"Mouse genetics, compound heterozygous analysis, morphometric analysis of dorsal mesentery epithelial cells","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis via compound heterozygote analysis, single lab, two orthogonal approaches (genetics + cell morphometry)","pmids":["21726547"],"is_preprint":false},{"year":2012,"finding":"Shroom3 expression in the lateral line primordium is downstream of FGF signaling. Shroom3 drives apical constriction and rosette assembly by coordinating Rho kinase recruitment and non-muscle myosin activation. Gain- and loss-of-function experiments in zebrafish demonstrated Shroom3 is the main organizer of cell shape changes during proneuromast assembly.","method":"FGF pathway manipulation, shroom3 gain- and loss-of-function in zebrafish, quantitative rosette detection, myosin inhibition","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — gain- and loss-of-function in vivo with defined cellular phenotype, pathway placement downstream of FGF, multiple orthogonal methods","pmids":["23136387"],"is_preprint":false},{"year":2014,"finding":"A point mutation (R1838C) in mouse Shroom3, identified by ENU mutagenesis, abolishes ROCK binding without affecting Shroom3 expression or localization, rendering Shroom3 non-functional for cell morphology regulation and causing neural tube defects. This establishes ROCK as the major downstream effector of Shroom3 in neural tube morphogenesis.","method":"ENU mutagenesis, biochemical binding assays, site-directed mutagenesis, neural tube phenotype analysis","journal":"Biology open","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro binding assay with mutagenesis, clear loss-of-function phenotype, mechanistic link to ROCK established","pmids":["25171888"],"is_preprint":false},{"year":2014,"finding":"p120-catenin recruits Shroom3 to adherens junctions and is required for apical constriction during lens placode morphogenesis. Genetic interaction between p120-catenin and Shroom3 was established; p120-catenin loss phenocopies Shroom3 loss in lens AC.","method":"Mouse genetics, genetic interaction analysis (compound heterozygotes), immunolocalization of Shroom3 at junctions, lens cell morphometry","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis, localization rescue, multiple orthogonal methods in a single rigorous study","pmids":["25038041"],"is_preprint":false},{"year":2014,"finding":"SHROOM3 facilitates canonical TGF-β1 signaling (increasing COL1A1 expression) in renal tubular cells downstream of a β-catenin/TCF7L2-mediated pathway. The intronic SNP rs17319721 acts as a TCF7L2-dependent enhancer element increasing SHROOM3 transcription. Tubule-specific knockdown of Shroom3 abrogated interstitial fibrosis in a mouse model of ureteric obstruction.","method":"Luciferase reporter assay for enhancer function, siRNA knockdown in renal tubular cells, inducible tubular-specific Shroom3 knockdown mouse, unilateral ureteric obstruction model, collagen expression assays","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reporter assay for enhancer, in vitro signaling assays, in vivo conditional knockdown with fibrosis readout, multiple orthogonal methods","pmids":["25437874"],"is_preprint":false},{"year":2014,"finding":"Variants disrupting the actin-binding domain of SHROOM3 cause podocyte foot process effacement and impairment of the glomerular filtration barrier, as shown in zebrafish rescue experiments where the FHH rat Shroom3 allele (carrying an actin-binding domain mutation) failed to rescue glomerular defects.","method":"Zebrafish shroom3 knockdown, allele-specific rescue with wild-type vs. FHH mutant Shroom3, glomerular filtration assays, congenic rat mapping","journal":"Genome research","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — allele-specific rescue assay in zebrafish, domain-function mapping, complemented by rat congenic data","pmids":["25273069"],"is_preprint":false},{"year":2015,"finding":"Shroom3 physically interacts with Dishevelled 2 (a PCP pathway component) and co-distributes with it in cells. Shroom3 and PCP components (Vangl2, Wnt5a) genetically interact during neural tube closure; simultaneous depletion increases NTD liability and CE failure. Multiple Shroom3 pathway components are planar-polarized along mediolateral junctions in the neural plate in a Shroom3- and PCP-dependent manner.","method":"Co-immunoprecipitation (Shroom3–Dishevelled 2), compound heterozygous mouse genetics, immunofluorescence for planar polarity markers, neural tube defect scoring","journal":"Biology open","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, genetic epistasis with multiple PCP components, co-localization, multiple orthogonal methods","pmids":["25596276"],"is_preprint":false},{"year":2015,"finding":"A small molecule (CCG-17444) covalently targets Shroom3 Cys1816 to inhibit the Shroom3-ROCK protein-protein interaction in vitro. Inhibition of the Shroom3-ROCK interaction with CCG-17444 counteracts Nogo66-mediated inhibition and enhances neurite outgrowth, placing Shroom3 downstream of NogoA/Nogo66 in conveying axon outgrowth inhibitory signals.","method":"High-throughput screen, in vitro binding assay, covalent mechanism characterization, site-directed mutagenesis (Cys1816), neurite outgrowth assay","journal":"BMC neuroscience","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro biochemical assay with site identification, cell-based neurite outgrowth assay, single lab","pmids":["26077244"],"is_preprint":false},{"year":2016,"finding":"Shroom3 null mice exhibit glomerular abnormalities (cystic and collapsing glomeruli), disrupted podocyte arrangement, and altered Rho-kinase/myosin II signaling with loss of apically distributed actin. Heterozygous mice develop adult-onset glomerulosclerosis and proteinuria, establishing Shroom3's role in maintaining podocyte architecture via actomyosin network modulation.","method":"Shroom3 gene trap knockout mouse, immunofluorescence for actin and myosin II, renal histology, proteinuria measurement","journal":"Journal of the American Society of Nephrology : JASN","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO and heterozygous mouse models with defined molecular and cellular phenotypes, multiple readouts","pmids":["26940091"],"is_preprint":false},{"year":2016,"finding":"SHROOM3 is required for apical constriction in the thyroid bud epithelium; its subcellular localization is disrupted in Cdc42-deficient embryos. In Shroom3 gene trap mutants, the thyroid bud epithelium lacks apical constriction, causing it to protrude into the foregut lumen rather than invaginating into the mesenchyme.","method":"Shroom3 gene trap mouse, Cdc42 conditional knockout, immunofluorescence for SHROOM3 localization, histological analysis of thyroid bud morphogenesis","journal":"Biology open","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gene trap KO with defined morphogenetic phenotype, localization study, single lab","pmids":["26772200"],"is_preprint":false},{"year":2018,"finding":"SHROOM3 interacts with FYN (a Src-family kinase) via a critical SH3-binding domain distinct from its ROCK-binding domain. The SHROOM3-FYN interaction is required for FYN kinase activation and downstream nephrin phosphorylation in podocytes. Glomerular-specific Shroom3 knockdown induces albuminuria and foot process effacement, while altering podocyte cytoskeleton, adhesion, and migration.","method":"Co-immunoprecipitation of endogenous SHROOM3 with FYN in human podocytes, inducible shRNA knockdown mouse (glomerular vs. tubular), electron microscopy, glomerular RNA-seq, in vitro kinase/nephrin phosphorylation assays","journal":"Journal of the American Society of Nephrology : JASN","confidence":"High","confidence_rationale":"Tier 2 / Strong — endogenous Co-IP, domain-mapping, in vivo conditional knockdown with molecular and ultrastructural phenotype, multiple orthogonal methods","pmids":["30341149"],"is_preprint":false},{"year":2018,"finding":"A SHROOM3 coding variant P1244L (associated with CKD) attenuates the interaction of SHROOM3 with 14-3-3 proteins, suggesting involvement of the Hippo pathway. A novel SHROOM3 isoform lacking the PDZ domain was identified, regulated by an alternative transcription start site controlled by the noncoding CKD-risk variant rs17319721, which disrupts TCF7L2 binding in podocyte nuclear extracts.","method":"Biophysical binding assays (SHROOM3-14-3-3 interaction), allele-specific transcription factor binding (TCF7L2 binding disruption), CRISPR/Cas9 editing, molecular cloning of novel isoform, nuclear extract pulldown","journal":"Journal of the American Society of Nephrology : JASN","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical binding assay for 14-3-3 interaction, allele-specific TF binding assay, CRISPR validation, single lab","pmids":["29476007"],"is_preprint":false},{"year":2020,"finding":"SHROOM3 physically and genetically interacts with Dishevelled 2 downstream of PCP signaling during cardiac development. Loss of Shroom3 in mice causes congenital heart defects (VSDs, DORV, thin LV myocardium) with disrupted actomyosin cytoskeleton, cardiomyocyte polarity, organization, proliferation, and morphology, phenocopying PCP disruption.","method":"Co-immunoprecipitation (SHROOM3-Dishevelled 2), Shroom3 gene trap KO mouse, cardiac histology, immunofluorescence for PCP/actomyosin components, proliferation assays","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, genetic epistasis with PCP components, in vivo KO with multiple defined molecular and cellular readouts","pmids":["32511952"],"is_preprint":false},{"year":2021,"finding":"Shroom3 is required for epithelial repair and redifferentiation after ischemic AKI. Shroom3 heterozygous null mice show increased mortality, worse kidney function, and impaired epithelial redifferentiation after ischemia, associated with disrupted Rho-kinase/myosin signaling and disorganized apical F-actin. MDCK cell experiments showed Shroom3 levels directly correlate with apical organization of actin and actomyosin regulators.","method":"Bilateral ischemia in Shroom3 heterozygous mice, renal histology, apoptosis/proliferation assays, immunofluorescence for actomyosin regulators, MDCK cell knockdown","journal":"Kidney360","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo heterozygous KO with defined molecular/cellular readout, complementary cell culture data, single lab","pmids":["35368578"],"is_preprint":false},{"year":2022,"finding":"Medial actin accumulation drives apical constriction non-cell-autonomously in neighborhoods of cells during neural tube closure. Mosaic crispant analysis of Shroom3 in Xenopus revealed both cell-autonomous and non-cell-autonomous effects on apical constriction.","method":"Tissue-scale time-lapse imaging, mosaic CRISPR crispant embryos in Xenopus, multivariate regression modeling of cell behavior","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live imaging with mosaic genetic perturbation and quantitative modeling, single lab","pmids":["36113571"],"is_preprint":false},{"year":2022,"finding":"Regional differences exist in Shroom3 function during cranial vs. spinal neural tube closure in Xenopus. Mosaic shroom3 mutations reveal distinct cell biological requirements in anterior versus posterior neural tube closure.","method":"CRISPR mosaic mutagenesis in Xenopus, high-resolution tissue-level time-lapse microscopy, regional analysis of actin and N-cadherin dynamics","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live tissue imaging with genetic perturbation, regional cell behavior analysis, single lab","pmids":["35244026"],"is_preprint":false},{"year":2023,"finding":"SHROOM3 knockout in a human brain organoid model causes expansion of the apical lumen and reduced F-actin polarization, with increased apical cell surface area due to reduced apical constriction. This phenocopies valproic acid treatment, establishing impaired apical constriction as a shared mechanism.","method":"SHROOM3 CRISPR/Cas9 knockout in self-organizing single-rosette cortical organoids, high-throughput imaging, F-actin immunostaining, lumen size quantification","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO with defined cellular phenotype in human organoid model, single lab, two orthogonal readouts","pmids":["37443734"],"is_preprint":false},{"year":2024,"finding":"Loss of SHROOM3 in cynomolgus monkey neuroepithelial organoids results in shorter cells with smaller nuclei due to insufficient apical recruitment of F-actin, myosin II, and phospho-myosin light chain. These defects are not rescued by folate supplementation.","method":"SHROOM3 knockout in cynomolgus monkey neuroepithelial organoids, immunofluorescence for F-actin/myosin II/pMLC, RNA sequencing, folate supplementation experiment","journal":"Zoological research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO in primate organoid model with defined molecular phenotype, multiple cytoskeletal markers assayed, single lab","pmids":["38287904"],"is_preprint":false},{"year":2024,"finding":"NRP1 physically interacts with SHROOM3 via protein-protein interaction, and this interaction depends on glycosaminoglycan (GAG) modification of NRP1 at Ser612. Shroom3 knockdown inhibits osteo/odontogenic differentiation of dental pulp stem cells, placing Shroom3 downstream of NRP1 in this pathway.","method":"Co-immunoprecipitation (NRP1-SHROOM3), protein docking, confocal co-localization, NRP1 S612A mutant (non-GAG-modified), shroom3 siRNA knockdown, ALP/mineralization assays","journal":"Biochimica et biophysica acta. Molecular cell research","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP confirmed interaction, domain mutagenesis, KD with functional readout, single lab","pmids":["39033931"],"is_preprint":false},{"year":2024,"finding":"The ASD2 domain of Shroom3 (ROCK-binding domain) is the specific profibrotic motif mediating tubulo-interstitial fibrosis. Transgenic mice overexpressing ASD2-deleted Shroom3 (ASD2Δ-Sh3) show reduced ROCK activation (phospho-MYPT1), reduced profibrotic/pro-inflammatory transcripts, and reduced fibrosis in UUO and aristolochic acid nephropathy models compared to WT-Sh3 overexpressors. A distinct FYN-binding motif of Shroom3, separate from ASD2, is required for its anti-albuminuric function.","method":"ASD2 domain deletion transgenic mice, in vitro ROCK activation assays, in vivo fibrosis models (UUO, AAN), transcriptome analysis, albuminuria measurement, Fyn-binding domain mutant","journal":"bioRxiv (preprint)","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — domain deletion transgenic mice with multiple fibrosis models, in vitro assays; preprint, not yet peer reviewed","pmids":["39605692"],"is_preprint":true},{"year":2025,"finding":"The ASD2-domain deletion of Shroom3 (ASD2Δ-Sh3) abolishes ROCK binding and prevents TGFβ1/Wnt/Ctnnb1 profibrotic signaling in tubular cells and fibroblasts. In vivo, tubular-specific (but not fibroblast-specific) ASD2Δ-Sh3 overexpression mitigates tubulo-interstitial fibrosis. A distinct Fyn-binding deficient mutant Shroom3 (FBDM-Sh3) induces albuminuria, dissociating the profibrotic (ROCK-binding ASD2) from the antiproteinuric (FYN-binding) function of Shroom3. Small molecule P2Is (including BT1137) targeting the Shroom3-ROCK interaction inhibit ROCK activation and mitigate fibrosis in WT-Sh3 mice.","method":"Transgenic overexpression (WT-Sh3, ASD2Δ-Sh3, FBDM-Sh3), cell-based ROCK activation assays, in vivo conditional (tubular/fibroblast-specific) overexpression, fibrosis models (UUO, AAN), small molecule P2I development and testing","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — domain-specific mutagenesis with in vitro and in vivo orthogonal validation, separation-of-function mutants, peer-reviewed","pmids":["41469391"],"is_preprint":false},{"year":2025,"finding":"Shroom3 is apically localized in neural retina and retinal pigmented epithelium. Shroom3 deficiency increases apical surface area of neural retina and RPE cells and disrupts optic fissure alignment. Neural retina-specific ablation showed that Shroom3 function in RPE is sufficient for tissue alignment but the fusion process fails due to inability of neural tissue to re-establish apical-basal polarity. Shroom3 deficiency also impairs other polarity-dependent epithelial fusion events (lens vesicle separation, eyelid formation, palate closure).","method":"Conditional/tissue-specific Shroom3 knockout mouse, immunofluorescence for apical localization, histological analysis of optic fissure and lens vesicle, quantification of apical cell areas","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — tissue-specific KO with defined localization and polarity phenotypes, multiple epithelial fusion processes examined, single lab","pmids":["40113025"],"is_preprint":false},{"year":2025,"finding":"VANGL2 and its downstream effector SHROOM3 control bilateral symmetry of the splanchnic mesoderm caudal to the venous pole during heart tube morphogenesis. Apically localized VANGL2 acts through SHROOM3 to regulate this process.","method":"Quantitative 3D heart geometry analysis, cell labeling in chick, genetic manipulation of Vangl2 and Shroom3 in mouse, immunolocalization","journal":"bioRxiv (preprint)","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, pathway placement of Shroom3 downstream of VANGL2 based on genetic analysis but limited direct mechanistic validation of SHROOM3's specific contribution","pmids":["bio_10.1101_2025.09.05.674213"],"is_preprint":true},{"year":2025,"finding":"Shroom3 is specifically expressed in cardiomyocytes of the developing and adult mouse heart. CM-specific deletion of Shroom3 (using Nkx2-5-Cre) causes increased cardiomyocyte hyperpolyploidization and left ventricular dilation with reduced ejection fraction. Functional characterization identified two SHROOM3 coding variants that disrupt SHROOM3-ACTIN interaction and alter expression of DNA replication genes.","method":"Genome-wide association mapping in Hybrid Rat Diversity Panel, CM-specific Cre-lox Shroom3 deletion, ploidy assays, echocardiography, functional variant characterization (SHROOM3-actin interaction assay), transcriptome analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — CM-specific KO with defined molecular and physiological phenotype, functional variant testing of actin interaction, GWAS plus in vivo validation","pmids":["42189988"],"is_preprint":false},{"year":2025,"finding":"SHROOM3 deficiency in podocytes exacerbates adriamycin-induced nephropathy. In vitro, SHROOM3 deficiency impairs podocyte size and adhesion with downregulation of focal adhesion molecules (talin1, vinculin, paxillin) and stress fiber regulators (synaptopodin and RhoA), as well as calpain activation and RhoA inactivation.","method":"Podocyte-specific SHROOM3 knockout mice, adriamycin nephropathy model, immunofluorescence for focal adhesion and stress fiber components, RhoA activity assay, calpain activity assay","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — podocyte-specific KO with molecular pathway readouts, in vitro corroboration, single lab","pmids":["40558522"],"is_preprint":false}],"current_model":"SHROOM3 is an F-actin-binding scaffold protein that drives apical constriction and epithelial cell shape change by recruiting Rho kinases (ROCK1/2) to apical junctions via its ASD2 domain, thereby activating actomyosin contractility; it operates downstream of Pax6 and Pitx transcription factors, the Trio-RhoA GEF pathway, and the planar cell polarity (PCP) component Dishevelled, while also engaging the Src-family kinase FYN via a distinct SH3-binding domain to phosphorylate nephrin in podocytes, and interacting with p120-catenin for junctional recruitment, collectively explaining its roles in neural tube closure, lens and gut morphogenesis, cardiac development, and glomerular/tubular kidney homeostasis."},"narrative":{"mechanistic_narrative":"SHROOM3 is an F-actin-binding scaffold protein that drives epithelial apical constriction and cell shape change by recruiting Rho kinases (ROCK1/2) to apical junctions and activating actomyosin contractility, thereby governing morphogenetic processes from neural tube closure to organ invagination [PMID:18339671, PMID:20081189, PMID:25171888]. The ROCK interaction is mediated by a defined binding site (ASD2 domain) on SHROOM3 and the RII-C1 region of ROCK; competitive disruption or a point mutation (R1838C) that abolishes ROCK binding renders SHROOM3 non-functional and causes neural tube defects, establishing ROCK as its principal downstream effector [PMID:18339671, PMID:25171888]. SHROOM3 concentrates F-actin, myosin II, and Vasp apically to power apical constriction, and this output operates downstream of an upstream regulatory network: it is transcriptionally activated by Pax6 in the lens and by Pitx factors in the gut, positioned downstream of FGF signaling and the Trio–RhoA GEF pathway (RhoA activity being required for SHROOM3 apical localization), and recruited to adherens junctions by p120-catenin [PMID:20081189, PMID:20332151, PMID:22031541, PMID:23136387, PMID:25038041]. SHROOM3 also acts within the planar cell polarity pathway, physically interacting with Dishevelled 2 and genetically with Vangl2/Wnt5a to coordinate planar-polarized constriction during neural tube and cardiac morphogenesis [PMID:25596276, PMID:32511952]. In the kidney, SHROOM3 serves two genetically separable functions: an ASD2/ROCK-binding arm that drives TGF-β1/Wnt–β-catenin profibrotic signaling in tubular cells, and a distinct FYN-binding (SH3) arm that activates FYN to phosphorylate nephrin and maintain podocyte foot process architecture and the glomerular filtration barrier [PMID:25437874, PMID:30341149, PMID:39605692, PMID:41469391]. Loss or actin-binding-domain variants of SHROOM3 cause podocyte foot process effacement, glomerulosclerosis, and proteinuria, while it is also required for epithelial repair after ischemic injury [PMID:25273069, PMID:26940091, PMID:35368578]. Coding and noncoding SHROOM3 variants are linked to chronic kidney disease, acting through altered TCF7L2-dependent transcription, 14-3-3/Hippo interaction, and disrupted actin binding [PMID:25437874, PMID:25273069, PMID:29476007].","teleology":[{"year":2008,"claim":"Established the core effector mechanism: how SHROOM3 converts its scaffold role into actomyosin contractility at apical junctions.","evidence":"Co-IP, dominant-negative RII-C1 fragment, live phospho-myosin imaging and shRNA in neural tube","pmids":["18339671"],"confidence":"High","gaps":["Does not resolve how SHROOM3 itself is recruited to apical junctions","Structural basis of the ROCK-binding interface not defined"]},{"year":2010,"claim":"Placed SHROOM3 downstream of tissue-specific transcription factors, explaining how its apical-constriction output is deployed in distinct organs.","evidence":"Mouse LOF with apical F-actin/myosin/Vasp localization and Pax6 epistasis (lens); Pitx promoter-reporter and Xenopus gain-of-function (gut)","pmids":["20081189","20332151"],"confidence":"High","gaps":["Whether Pax6 and Pitx act through shared cis-elements not addressed","Post-transcriptional control of SHROOM3 not examined"]},{"year":2011,"claim":"Defined the upstream GTPase and junctional inputs that localize and activate SHROOM3 for apical constriction.","evidence":"Dominant-negative/constitutively active RhoA, Trio LOF and MDCK assays (lens); compound heterozygote Shroom3/N-cadherin genetics (gut)","pmids":["22031541","21726547"],"confidence":"High","gaps":["Direct biochemical link between RhoA and SHROOM3 localization not established","N-cadherin/SHROOM3 cooperation mechanism inferred from genetics only"]},{"year":2012,"claim":"Generalized SHROOM3 as the central organizer of cell-shape change downstream of FGF signaling in a vertebrate sensory primordium.","evidence":"FGF pathway manipulation and shroom3 gain/loss-of-function with rosette quantification in zebrafish","pmids":["23136387"],"confidence":"High","gaps":["Direct transcriptional link between FGF and shroom3 not mapped"]},{"year":2014,"claim":"Proved ROCK binding is the essential output and identified the junctional recruitment receptor, cementing the constriction module.","evidence":"ENU R1838C mutant abolishing ROCK binding (neural tube); p120-catenin compound heterozygote genetics and SHROOM3 localization rescue (lens)","pmids":["25171888","25038041"],"confidence":"High","gaps":["Whether p120-catenin binds SHROOM3 directly not shown","Other recruitment partners at distinct junctions unexplored"]},{"year":2014,"claim":"Opened the kidney axis, distinguishing a tubular profibrotic transcriptional/signaling role from a podocyte actin-barrier role.","evidence":"TCF7L2-dependent enhancer reporter, tubule-specific knockdown UUO fibrosis model; zebrafish allele-specific rescue with actin-binding-domain mutant","pmids":["25437874","25273069"],"confidence":"High","gaps":["How a single gene's distinct cellular roles are coordinated not resolved at this stage","Direct SHROOM3 effectors in fibrosis vs. barrier maintenance not yet separated"]},{"year":2015,"claim":"Extended ROCK-interaction dependence to axon outgrowth and demonstrated druggability of the SHROOM3-ROCK interface.","evidence":"HTS, covalent inhibitor CCG-17444 targeting Cys1816, neurite outgrowth assay placing SHROOM3 downstream of NogoA","pmids":["26077244"],"confidence":"Medium","gaps":["In vivo relevance of the NogoA-SHROOM3 axis not tested","Single lab, biochemical and cell-based only"]},{"year":2015,"claim":"Connected SHROOM3 to PCP machinery, showing it acts within planar polarity signaling rather than as an isolated constriction effector.","evidence":"Reciprocal Co-IP with Dishevelled 2, compound heterozygote genetics with Vangl2/Wnt5a, planar polarity marker imaging in neural plate","pmids":["25596276"],"confidence":"High","gaps":["Domain on SHROOM3 binding Dishevelled 2 not mapped","Whether PCP input is upstream of or parallel to ROCK recruitment unclear"]},{"year":2016,"claim":"Demonstrated SHROOM3 maintains podocyte architecture and drives invagination in additional organs, defining adult kidney disease and developmental roles.","evidence":"Shroom3 KO/heterozygous mice with glomerulosclerosis and actomyosin readouts; gene-trap thyroid bud apical constriction with Cdc42 dependence","pmids":["26940091","26772200"],"confidence":"High","gaps":["Mechanism linking Cdc42 to SHROOM3 localization not defined","How heterozygosity produces adult-onset disease not resolved"]},{"year":2018,"claim":"Identified a second, ROCK-independent SHROOM3 signaling arm (FYN/nephrin) and a 14-3-3/Hippo-linked CKD variant, revealing functional modularity.","evidence":"Endogenous SHROOM3-FYN Co-IP, SH3-binding domain mapping, glomerular shRNA mouse with EM/RNA-seq; SHROOM3 P1244L 14-3-3 binding assay and novel PDZ-less isoform cloning","pmids":["30341149","29476007"],"confidence":"High","gaps":["Direct kinase mechanism of FYN activation by SHROOM3 not fully resolved","Functional role of the PDZ-less isoform untested in vivo"]},{"year":2020,"claim":"Showed the SHROOM3-Dishevelled2/PCP axis governs cardiomyocyte polarity and cardiac chamber morphogenesis.","evidence":"SHROOM3-Dishevelled 2 Co-IP, Shroom3 gene-trap KO with congenital heart defects and actomyosin/proliferation readouts","pmids":["32511952"],"confidence":"High","gaps":["Whether cardiac defects arise cell-autonomously not fully separated","Link between proliferation phenotype and actomyosin role unclear"]},{"year":2022,"claim":"Refined the cellular logic of constriction, showing SHROOM3 acts both cell-autonomously and non-cell-autonomously across cell neighborhoods.","evidence":"Mosaic CRISPR crispants and tissue-scale time-lapse imaging with regression modeling in Xenopus; regional cranial vs. spinal analysis","pmids":["36113571","35244026"],"confidence":"Medium","gaps":["Molecular basis of non-cell-autonomous coupling not identified","Regional functional differences not linked to specific partners"]},{"year":2023,"claim":"Validated SHROOM3's apical-constriction role in human and primate neuroepithelial models and tied it to folate-independent neural tube defect mechanisms.","evidence":"SHROOM3 CRISPR KO in human cortical organoids (lumen/F-actin); KO in cynomolgus monkey neuroepithelial organoids with folate supplementation test","pmids":["37443734","38287904"],"confidence":"Medium","gaps":["Single-lab organoid models","Does not establish SHROOM3 variants as human NTD causes"]},{"year":2025,"claim":"Achieved separation-of-function, dissociating the ROCK-binding (profibrotic) and FYN-binding (antiproteinuric) arms and demonstrating therapeutic targeting of the SHROOM3-ROCK interaction.","evidence":"Domain-deletion transgenic mice (ASD2Δ, FBDM-Sh3), cell-based ROCK activation, UUO/AAN fibrosis models, small-molecule P2Is (BT1137)","pmids":["41469391","39605692"],"confidence":"High","gaps":["Long-term efficacy and specificity of P2Is in vivo not established","How the two arms are differentially deployed across cell types not fully resolved"]},{"year":2025,"claim":"Expanded SHROOM3's roles to epithelial fusion, cardiomyocyte ploidy/contractile function, and podocyte focal adhesion regulation.","evidence":"Tissue-specific KO of optic fissure/retina; CM-specific Nkx2-5-Cre deletion with ploidy/echo and actin-variant testing; podocyte-specific KO with focal adhesion/RhoA/calpain readouts","pmids":["40113025","42189988","40558522"],"confidence":"High","gaps":["Mechanism linking SHROOM3 to DNA replication genes and ploidy unclear","Direct effectors at focal adhesions vs. apical actin not separated"]},{"year":null,"claim":"How SHROOM3's distinct molecular arms (ROCK/actomyosin, FYN/nephrin, 14-3-3/Hippo, focal adhesion regulation) are selectively engaged and coordinated within a single cell type remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model integrating the multiple binding interfaces","Upstream switch determining which arm is active in a given context unknown","Direct mechanism by which SHROOM3 alters DNA replication gene expression undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,6,7,14]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[1,9,27]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,14,24]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[1,12,17,21]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,7,13,25]}],"pathway":[{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[0,1,2,5,16]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,10,14,16]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[8,9,12,24]}],"complexes":[],"partners":["ROCK1","ROCK2","FYN","DVL2","CTNND1","NRP1","RHOA","YWHAE"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8TF72","full_name":"Protein Shroom3","aliases":["Shroom-related protein","hShrmL"],"length_aa":1996,"mass_kda":216.9,"function":"Controls cell shape changes in the neuroepithelium during neural tube closure. Induces apical constriction in epithelial cells by promoting the apical accumulation of F-actin and myosin II, and probably by bundling stress fibers (By similarity). Induces apicobasal cell elongation by redistributing gamma-tubulin and directing the assembly of robust apicobasal microtubule arrays (By similarity)","subcellular_location":"Cell junction, adherens junction; Cytoplasm, cytoskeleton; Apical cell membrane","url":"https://www.uniprot.org/uniprotkb/Q8TF72/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SHROOM3","classification":"Not Classified","n_dependent_lines":12,"n_total_lines":1208,"dependency_fraction":0.009933774834437087},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SHROOM3","total_profiled":1310},"omim":[{"mim_id":"611179","title":"SHROOM FAMILY MEMBER 1; SHROOM1","url":"https://www.omim.org/entry/611179"},{"mim_id":"604570","title":"SHROOM FAMILY MEMBER 3; SHROOM3","url":"https://www.omim.org/entry/604570"},{"mim_id":"300103","title":"SHROOM FAMILY MEMBER 2; SHROOM2","url":"https://www.omim.org/entry/300103"},{"mim_id":"182940","title":"NEURAL TUBE DEFECTS, SUSCEPTIBILITY TO; NTD","url":"https://www.omim.org/entry/182940"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/SHROOM3"},"hgnc":{"alias_symbol":["ShrmL","SHRM","KIAA1481","APXL3"],"prev_symbol":[]},"alphafold":{"accession":"Q8TF72","domains":[{"cath_id":"2.30.42.10","chopping":"26-110","consensus_level":"high","plddt":88.8135,"start":26,"end":110},{"cath_id":"-","chopping":"1664-1707_1778-1955","consensus_level":"high","plddt":89.5861,"start":1664,"end":1955}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TF72","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TF72-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TF72-F1-predicted_aligned_error_v6.png","plddt_mean":43.91},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SHROOM3","jax_strain_url":"https://www.jax.org/strain/search?query=SHROOM3"},"sequence":{"accession":"Q8TF72","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8TF72.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8TF72/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TF72"}},"corpus_meta":[{"pmid":"18339671","id":"PMC_18339671","title":"Shroom3-mediated 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Expression of the competitive antagonist RII-C1 fragment displaced apically localized ROCKs and blocked neural tube closure, demonstrating that the Shroom3-ROCK interaction is required for apical junction remodeling and neural tube morphogenesis.\",\n      \"method\": \"Co-immunoprecipitation, dominant-negative RII-C1 fragment expression, live imaging of phospho-myosin distribution, shRNA depletion in neural tube\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal binding-site mapping, dominant-negative rescue, multiple orthogonal methods, replicated across subsequent studies\",\n      \"pmids\": [\"18339671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Shroom3 is required for apical localization of F-actin, myosin II, and Vasp during lens placode invagination. Loss of Shroom3 abolishes apical constriction of lens epithelial cells, and Shroom3 expression in the lens is directly controlled by the transcription factor Pax6.\",\n      \"method\": \"Loss-of-function mouse genetics, immunofluorescence for F-actin/myosin II/Vasp localization, genetic epistasis with Pax6 mutants\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with defined cellular phenotype, apical localization of multiple effectors assayed, genetic epistasis established\",\n      \"pmids\": [\"20081189\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Pitx transcription factors directly activate Shroom3 transcription through Pitx-responsive regulatory elements in the Shroom3 genomic locus. Ectopic Pitx expression is sufficient to induce Shroom3-dependent cytoskeletal reorganization and epithelial cell shape change in Xenopus gut.\",\n      \"method\": \"Promoter-reporter assays, Pitx gain-of-function in Xenopus, Shroom3 loss-of-function, identification of Pitx-responsive cis-regulatory elements\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — direct transcriptional activation shown by reporter assay + mutagenesis of cis-elements + epistasis in vivo\",\n      \"pmids\": [\"20332151\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"A Trio (RhoA-GEF)–RhoA–Shroom3 pathway is required for apical constriction during lens pit invagination. RhoA activity is required for Shroom3 apical localization, and activated apical RhoA is sufficient to induce apical constriction. Trio is required for Shroom3-dependent apical constriction in MDCK cells and in the lens pit.\",\n      \"method\": \"Dominant-negative and constitutively active RhoA constructs, MDCK cell assays, Trio loss-of-function in lens pit, epistasis experiments\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — epistasis with multiple pathway components, gain- and loss-of-function, two model systems\",\n      \"pmids\": [\"22031541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Shroom3 and N-cadherin function cooperatively downstream of Pitx2 to regulate asymmetric epithelial cell shape changes required for gut tube morphogenesis. Genetic interaction shown by compound heterozygous analysis: loss of one allele each of Shroom3 and N-cadherin phenocopies homozygous loss of either gene alone.\",\n      \"method\": \"Mouse genetics, compound heterozygous analysis, morphometric analysis of dorsal mesentery epithelial cells\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via compound heterozygote analysis, single lab, two orthogonal approaches (genetics + cell morphometry)\",\n      \"pmids\": [\"21726547\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Shroom3 expression in the lateral line primordium is downstream of FGF signaling. Shroom3 drives apical constriction and rosette assembly by coordinating Rho kinase recruitment and non-muscle myosin activation. Gain- and loss-of-function experiments in zebrafish demonstrated Shroom3 is the main organizer of cell shape changes during proneuromast assembly.\",\n      \"method\": \"FGF pathway manipulation, shroom3 gain- and loss-of-function in zebrafish, quantitative rosette detection, myosin inhibition\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — gain- and loss-of-function in vivo with defined cellular phenotype, pathway placement downstream of FGF, multiple orthogonal methods\",\n      \"pmids\": [\"23136387\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"A point mutation (R1838C) in mouse Shroom3, identified by ENU mutagenesis, abolishes ROCK binding without affecting Shroom3 expression or localization, rendering Shroom3 non-functional for cell morphology regulation and causing neural tube defects. This establishes ROCK as the major downstream effector of Shroom3 in neural tube morphogenesis.\",\n      \"method\": \"ENU mutagenesis, biochemical binding assays, site-directed mutagenesis, neural tube phenotype analysis\",\n      \"journal\": \"Biology open\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro binding assay with mutagenesis, clear loss-of-function phenotype, mechanistic link to ROCK established\",\n      \"pmids\": [\"25171888\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"p120-catenin recruits Shroom3 to adherens junctions and is required for apical constriction during lens placode morphogenesis. Genetic interaction between p120-catenin and Shroom3 was established; p120-catenin loss phenocopies Shroom3 loss in lens AC.\",\n      \"method\": \"Mouse genetics, genetic interaction analysis (compound heterozygotes), immunolocalization of Shroom3 at junctions, lens cell morphometry\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis, localization rescue, multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"25038041\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SHROOM3 facilitates canonical TGF-β1 signaling (increasing COL1A1 expression) in renal tubular cells downstream of a β-catenin/TCF7L2-mediated pathway. The intronic SNP rs17319721 acts as a TCF7L2-dependent enhancer element increasing SHROOM3 transcription. Tubule-specific knockdown of Shroom3 abrogated interstitial fibrosis in a mouse model of ureteric obstruction.\",\n      \"method\": \"Luciferase reporter assay for enhancer function, siRNA knockdown in renal tubular cells, inducible tubular-specific Shroom3 knockdown mouse, unilateral ureteric obstruction model, collagen expression assays\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reporter assay for enhancer, in vitro signaling assays, in vivo conditional knockdown with fibrosis readout, multiple orthogonal methods\",\n      \"pmids\": [\"25437874\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Variants disrupting the actin-binding domain of SHROOM3 cause podocyte foot process effacement and impairment of the glomerular filtration barrier, as shown in zebrafish rescue experiments where the FHH rat Shroom3 allele (carrying an actin-binding domain mutation) failed to rescue glomerular defects.\",\n      \"method\": \"Zebrafish shroom3 knockdown, allele-specific rescue with wild-type vs. FHH mutant Shroom3, glomerular filtration assays, congenic rat mapping\",\n      \"journal\": \"Genome research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — allele-specific rescue assay in zebrafish, domain-function mapping, complemented by rat congenic data\",\n      \"pmids\": [\"25273069\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Shroom3 physically interacts with Dishevelled 2 (a PCP pathway component) and co-distributes with it in cells. Shroom3 and PCP components (Vangl2, Wnt5a) genetically interact during neural tube closure; simultaneous depletion increases NTD liability and CE failure. Multiple Shroom3 pathway components are planar-polarized along mediolateral junctions in the neural plate in a Shroom3- and PCP-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation (Shroom3–Dishevelled 2), compound heterozygous mouse genetics, immunofluorescence for planar polarity markers, neural tube defect scoring\",\n      \"journal\": \"Biology open\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, genetic epistasis with multiple PCP components, co-localization, multiple orthogonal methods\",\n      \"pmids\": [\"25596276\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"A small molecule (CCG-17444) covalently targets Shroom3 Cys1816 to inhibit the Shroom3-ROCK protein-protein interaction in vitro. Inhibition of the Shroom3-ROCK interaction with CCG-17444 counteracts Nogo66-mediated inhibition and enhances neurite outgrowth, placing Shroom3 downstream of NogoA/Nogo66 in conveying axon outgrowth inhibitory signals.\",\n      \"method\": \"High-throughput screen, in vitro binding assay, covalent mechanism characterization, site-directed mutagenesis (Cys1816), neurite outgrowth assay\",\n      \"journal\": \"BMC neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro biochemical assay with site identification, cell-based neurite outgrowth assay, single lab\",\n      \"pmids\": [\"26077244\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Shroom3 null mice exhibit glomerular abnormalities (cystic and collapsing glomeruli), disrupted podocyte arrangement, and altered Rho-kinase/myosin II signaling with loss of apically distributed actin. Heterozygous mice develop adult-onset glomerulosclerosis and proteinuria, establishing Shroom3's role in maintaining podocyte architecture via actomyosin network modulation.\",\n      \"method\": \"Shroom3 gene trap knockout mouse, immunofluorescence for actin and myosin II, renal histology, proteinuria measurement\",\n      \"journal\": \"Journal of the American Society of Nephrology : JASN\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO and heterozygous mouse models with defined molecular and cellular phenotypes, multiple readouts\",\n      \"pmids\": [\"26940091\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SHROOM3 is required for apical constriction in the thyroid bud epithelium; its subcellular localization is disrupted in Cdc42-deficient embryos. In Shroom3 gene trap mutants, the thyroid bud epithelium lacks apical constriction, causing it to protrude into the foregut lumen rather than invaginating into the mesenchyme.\",\n      \"method\": \"Shroom3 gene trap mouse, Cdc42 conditional knockout, immunofluorescence for SHROOM3 localization, histological analysis of thyroid bud morphogenesis\",\n      \"journal\": \"Biology open\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gene trap KO with defined morphogenetic phenotype, localization study, single lab\",\n      \"pmids\": [\"26772200\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SHROOM3 interacts with FYN (a Src-family kinase) via a critical SH3-binding domain distinct from its ROCK-binding domain. The SHROOM3-FYN interaction is required for FYN kinase activation and downstream nephrin phosphorylation in podocytes. Glomerular-specific Shroom3 knockdown induces albuminuria and foot process effacement, while altering podocyte cytoskeleton, adhesion, and migration.\",\n      \"method\": \"Co-immunoprecipitation of endogenous SHROOM3 with FYN in human podocytes, inducible shRNA knockdown mouse (glomerular vs. tubular), electron microscopy, glomerular RNA-seq, in vitro kinase/nephrin phosphorylation assays\",\n      \"journal\": \"Journal of the American Society of Nephrology : JASN\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — endogenous Co-IP, domain-mapping, in vivo conditional knockdown with molecular and ultrastructural phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"30341149\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"A SHROOM3 coding variant P1244L (associated with CKD) attenuates the interaction of SHROOM3 with 14-3-3 proteins, suggesting involvement of the Hippo pathway. A novel SHROOM3 isoform lacking the PDZ domain was identified, regulated by an alternative transcription start site controlled by the noncoding CKD-risk variant rs17319721, which disrupts TCF7L2 binding in podocyte nuclear extracts.\",\n      \"method\": \"Biophysical binding assays (SHROOM3-14-3-3 interaction), allele-specific transcription factor binding (TCF7L2 binding disruption), CRISPR/Cas9 editing, molecular cloning of novel isoform, nuclear extract pulldown\",\n      \"journal\": \"Journal of the American Society of Nephrology : JASN\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical binding assay for 14-3-3 interaction, allele-specific TF binding assay, CRISPR validation, single lab\",\n      \"pmids\": [\"29476007\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SHROOM3 physically and genetically interacts with Dishevelled 2 downstream of PCP signaling during cardiac development. Loss of Shroom3 in mice causes congenital heart defects (VSDs, DORV, thin LV myocardium) with disrupted actomyosin cytoskeleton, cardiomyocyte polarity, organization, proliferation, and morphology, phenocopying PCP disruption.\",\n      \"method\": \"Co-immunoprecipitation (SHROOM3-Dishevelled 2), Shroom3 gene trap KO mouse, cardiac histology, immunofluorescence for PCP/actomyosin components, proliferation assays\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, genetic epistasis with PCP components, in vivo KO with multiple defined molecular and cellular readouts\",\n      \"pmids\": [\"32511952\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Shroom3 is required for epithelial repair and redifferentiation after ischemic AKI. Shroom3 heterozygous null mice show increased mortality, worse kidney function, and impaired epithelial redifferentiation after ischemia, associated with disrupted Rho-kinase/myosin signaling and disorganized apical F-actin. MDCK cell experiments showed Shroom3 levels directly correlate with apical organization of actin and actomyosin regulators.\",\n      \"method\": \"Bilateral ischemia in Shroom3 heterozygous mice, renal histology, apoptosis/proliferation assays, immunofluorescence for actomyosin regulators, MDCK cell knockdown\",\n      \"journal\": \"Kidney360\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo heterozygous KO with defined molecular/cellular readout, complementary cell culture data, single lab\",\n      \"pmids\": [\"35368578\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Medial actin accumulation drives apical constriction non-cell-autonomously in neighborhoods of cells during neural tube closure. Mosaic crispant analysis of Shroom3 in Xenopus revealed both cell-autonomous and non-cell-autonomous effects on apical constriction.\",\n      \"method\": \"Tissue-scale time-lapse imaging, mosaic CRISPR crispant embryos in Xenopus, multivariate regression modeling of cell behavior\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live imaging with mosaic genetic perturbation and quantitative modeling, single lab\",\n      \"pmids\": [\"36113571\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Regional differences exist in Shroom3 function during cranial vs. spinal neural tube closure in Xenopus. Mosaic shroom3 mutations reveal distinct cell biological requirements in anterior versus posterior neural tube closure.\",\n      \"method\": \"CRISPR mosaic mutagenesis in Xenopus, high-resolution tissue-level time-lapse microscopy, regional analysis of actin and N-cadherin dynamics\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live tissue imaging with genetic perturbation, regional cell behavior analysis, single lab\",\n      \"pmids\": [\"35244026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SHROOM3 knockout in a human brain organoid model causes expansion of the apical lumen and reduced F-actin polarization, with increased apical cell surface area due to reduced apical constriction. This phenocopies valproic acid treatment, establishing impaired apical constriction as a shared mechanism.\",\n      \"method\": \"SHROOM3 CRISPR/Cas9 knockout in self-organizing single-rosette cortical organoids, high-throughput imaging, F-actin immunostaining, lumen size quantification\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO with defined cellular phenotype in human organoid model, single lab, two orthogonal readouts\",\n      \"pmids\": [\"37443734\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Loss of SHROOM3 in cynomolgus monkey neuroepithelial organoids results in shorter cells with smaller nuclei due to insufficient apical recruitment of F-actin, myosin II, and phospho-myosin light chain. These defects are not rescued by folate supplementation.\",\n      \"method\": \"SHROOM3 knockout in cynomolgus monkey neuroepithelial organoids, immunofluorescence for F-actin/myosin II/pMLC, RNA sequencing, folate supplementation experiment\",\n      \"journal\": \"Zoological research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO in primate organoid model with defined molecular phenotype, multiple cytoskeletal markers assayed, single lab\",\n      \"pmids\": [\"38287904\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NRP1 physically interacts with SHROOM3 via protein-protein interaction, and this interaction depends on glycosaminoglycan (GAG) modification of NRP1 at Ser612. Shroom3 knockdown inhibits osteo/odontogenic differentiation of dental pulp stem cells, placing Shroom3 downstream of NRP1 in this pathway.\",\n      \"method\": \"Co-immunoprecipitation (NRP1-SHROOM3), protein docking, confocal co-localization, NRP1 S612A mutant (non-GAG-modified), shroom3 siRNA knockdown, ALP/mineralization assays\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP confirmed interaction, domain mutagenesis, KD with functional readout, single lab\",\n      \"pmids\": [\"39033931\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The ASD2 domain of Shroom3 (ROCK-binding domain) is the specific profibrotic motif mediating tubulo-interstitial fibrosis. Transgenic mice overexpressing ASD2-deleted Shroom3 (ASD2Δ-Sh3) show reduced ROCK activation (phospho-MYPT1), reduced profibrotic/pro-inflammatory transcripts, and reduced fibrosis in UUO and aristolochic acid nephropathy models compared to WT-Sh3 overexpressors. A distinct FYN-binding motif of Shroom3, separate from ASD2, is required for its anti-albuminuric function.\",\n      \"method\": \"ASD2 domain deletion transgenic mice, in vitro ROCK activation assays, in vivo fibrosis models (UUO, AAN), transcriptome analysis, albuminuria measurement, Fyn-binding domain mutant\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — domain deletion transgenic mice with multiple fibrosis models, in vitro assays; preprint, not yet peer reviewed\",\n      \"pmids\": [\"39605692\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The ASD2-domain deletion of Shroom3 (ASD2Δ-Sh3) abolishes ROCK binding and prevents TGFβ1/Wnt/Ctnnb1 profibrotic signaling in tubular cells and fibroblasts. In vivo, tubular-specific (but not fibroblast-specific) ASD2Δ-Sh3 overexpression mitigates tubulo-interstitial fibrosis. A distinct Fyn-binding deficient mutant Shroom3 (FBDM-Sh3) induces albuminuria, dissociating the profibrotic (ROCK-binding ASD2) from the antiproteinuric (FYN-binding) function of Shroom3. Small molecule P2Is (including BT1137) targeting the Shroom3-ROCK interaction inhibit ROCK activation and mitigate fibrosis in WT-Sh3 mice.\",\n      \"method\": \"Transgenic overexpression (WT-Sh3, ASD2Δ-Sh3, FBDM-Sh3), cell-based ROCK activation assays, in vivo conditional (tubular/fibroblast-specific) overexpression, fibrosis models (UUO, AAN), small molecule P2I development and testing\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — domain-specific mutagenesis with in vitro and in vivo orthogonal validation, separation-of-function mutants, peer-reviewed\",\n      \"pmids\": [\"41469391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Shroom3 is apically localized in neural retina and retinal pigmented epithelium. Shroom3 deficiency increases apical surface area of neural retina and RPE cells and disrupts optic fissure alignment. Neural retina-specific ablation showed that Shroom3 function in RPE is sufficient for tissue alignment but the fusion process fails due to inability of neural tissue to re-establish apical-basal polarity. Shroom3 deficiency also impairs other polarity-dependent epithelial fusion events (lens vesicle separation, eyelid formation, palate closure).\",\n      \"method\": \"Conditional/tissue-specific Shroom3 knockout mouse, immunofluorescence for apical localization, histological analysis of optic fissure and lens vesicle, quantification of apical cell areas\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — tissue-specific KO with defined localization and polarity phenotypes, multiple epithelial fusion processes examined, single lab\",\n      \"pmids\": [\"40113025\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"VANGL2 and its downstream effector SHROOM3 control bilateral symmetry of the splanchnic mesoderm caudal to the venous pole during heart tube morphogenesis. Apically localized VANGL2 acts through SHROOM3 to regulate this process.\",\n      \"method\": \"Quantitative 3D heart geometry analysis, cell labeling in chick, genetic manipulation of Vangl2 and Shroom3 in mouse, immunolocalization\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, pathway placement of Shroom3 downstream of VANGL2 based on genetic analysis but limited direct mechanistic validation of SHROOM3's specific contribution\",\n      \"pmids\": [\"bio_10.1101_2025.09.05.674213\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Shroom3 is specifically expressed in cardiomyocytes of the developing and adult mouse heart. CM-specific deletion of Shroom3 (using Nkx2-5-Cre) causes increased cardiomyocyte hyperpolyploidization and left ventricular dilation with reduced ejection fraction. Functional characterization identified two SHROOM3 coding variants that disrupt SHROOM3-ACTIN interaction and alter expression of DNA replication genes.\",\n      \"method\": \"Genome-wide association mapping in Hybrid Rat Diversity Panel, CM-specific Cre-lox Shroom3 deletion, ploidy assays, echocardiography, functional variant characterization (SHROOM3-actin interaction assay), transcriptome analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CM-specific KO with defined molecular and physiological phenotype, functional variant testing of actin interaction, GWAS plus in vivo validation\",\n      \"pmids\": [\"42189988\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SHROOM3 deficiency in podocytes exacerbates adriamycin-induced nephropathy. In vitro, SHROOM3 deficiency impairs podocyte size and adhesion with downregulation of focal adhesion molecules (talin1, vinculin, paxillin) and stress fiber regulators (synaptopodin and RhoA), as well as calpain activation and RhoA inactivation.\",\n      \"method\": \"Podocyte-specific SHROOM3 knockout mice, adriamycin nephropathy model, immunofluorescence for focal adhesion and stress fiber components, RhoA activity assay, calpain activity assay\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — podocyte-specific KO with molecular pathway readouts, in vitro corroboration, single lab\",\n      \"pmids\": [\"40558522\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SHROOM3 is an F-actin-binding scaffold protein that drives apical constriction and epithelial cell shape change by recruiting Rho kinases (ROCK1/2) to apical junctions via its ASD2 domain, thereby activating actomyosin contractility; it operates downstream of Pax6 and Pitx transcription factors, the Trio-RhoA GEF pathway, and the planar cell polarity (PCP) component Dishevelled, while also engaging the Src-family kinase FYN via a distinct SH3-binding domain to phosphorylate nephrin in podocytes, and interacting with p120-catenin for junctional recruitment, collectively explaining its roles in neural tube closure, lens and gut morphogenesis, cardiac development, and glomerular/tubular kidney homeostasis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SHROOM3 is an F-actin-binding scaffold protein that drives epithelial apical constriction and cell shape change by recruiting Rho kinases (ROCK1/2) to apical junctions and activating actomyosin contractility, thereby governing morphogenetic processes from neural tube closure to organ invagination [#0, #1, #6]. The ROCK interaction is mediated by a defined binding site (ASD2 domain) on SHROOM3 and the RII-C1 region of ROCK; competitive disruption or a point mutation (R1838C) that abolishes ROCK binding renders SHROOM3 non-functional and causes neural tube defects, establishing ROCK as its principal downstream effector [#0, #6]. SHROOM3 concentrates F-actin, myosin II, and Vasp apically to power apical constriction, and this output operates downstream of an upstream regulatory network: it is transcriptionally activated by Pax6 in the lens and by Pitx factors in the gut, positioned downstream of FGF signaling and the Trio–RhoA GEF pathway (RhoA activity being required for SHROOM3 apical localization), and recruited to adherens junctions by p120-catenin [#1, #2, #3, #5, #7]. SHROOM3 also acts within the planar cell polarity pathway, physically interacting with Dishevelled 2 and genetically with Vangl2/Wnt5a to coordinate planar-polarized constriction during neural tube and cardiac morphogenesis [#10, #16]. In the kidney, SHROOM3 serves two genetically separable functions: an ASD2/ROCK-binding arm that drives TGF-β1/Wnt–β-catenin profibrotic signaling in tubular cells, and a distinct FYN-binding (SH3) arm that activates FYN to phosphorylate nephrin and maintain podocyte foot process architecture and the glomerular filtration barrier [#8, #14, #23, #24]. Loss or actin-binding-domain variants of SHROOM3 cause podocyte foot process effacement, glomerulosclerosis, and proteinuria, while it is also required for epithelial repair after ischemic injury [#9, #12, #17]. Coding and noncoding SHROOM3 variants are linked to chronic kidney disease, acting through altered TCF7L2-dependent transcription, 14-3-3/Hippo interaction, and disrupted actin binding [#8, #9, #15].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Established the core effector mechanism: how SHROOM3 converts its scaffold role into actomyosin contractility at apical junctions.\",\n      \"evidence\": \"Co-IP, dominant-negative RII-C1 fragment, live phospho-myosin imaging and shRNA in neural tube\",\n      \"pmids\": [\"18339671\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not resolve how SHROOM3 itself is recruited to apical junctions\", \"Structural basis of the ROCK-binding interface not defined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Placed SHROOM3 downstream of tissue-specific transcription factors, explaining how its apical-constriction output is deployed in distinct organs.\",\n      \"evidence\": \"Mouse LOF with apical F-actin/myosin/Vasp localization and Pax6 epistasis (lens); Pitx promoter-reporter and Xenopus gain-of-function (gut)\",\n      \"pmids\": [\"20081189\", \"20332151\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Pax6 and Pitx act through shared cis-elements not addressed\", \"Post-transcriptional control of SHROOM3 not examined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined the upstream GTPase and junctional inputs that localize and activate SHROOM3 for apical constriction.\",\n      \"evidence\": \"Dominant-negative/constitutively active RhoA, Trio LOF and MDCK assays (lens); compound heterozygote Shroom3/N-cadherin genetics (gut)\",\n      \"pmids\": [\"22031541\", \"21726547\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct biochemical link between RhoA and SHROOM3 localization not established\", \"N-cadherin/SHROOM3 cooperation mechanism inferred from genetics only\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Generalized SHROOM3 as the central organizer of cell-shape change downstream of FGF signaling in a vertebrate sensory primordium.\",\n      \"evidence\": \"FGF pathway manipulation and shroom3 gain/loss-of-function with rosette quantification in zebrafish\",\n      \"pmids\": [\"23136387\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct transcriptional link between FGF and shroom3 not mapped\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Proved ROCK binding is the essential output and identified the junctional recruitment receptor, cementing the constriction module.\",\n      \"evidence\": \"ENU R1838C mutant abolishing ROCK binding (neural tube); p120-catenin compound heterozygote genetics and SHROOM3 localization rescue (lens)\",\n      \"pmids\": [\"25171888\", \"25038041\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether p120-catenin binds SHROOM3 directly not shown\", \"Other recruitment partners at distinct junctions unexplored\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Opened the kidney axis, distinguishing a tubular profibrotic transcriptional/signaling role from a podocyte actin-barrier role.\",\n      \"evidence\": \"TCF7L2-dependent enhancer reporter, tubule-specific knockdown UUO fibrosis model; zebrafish allele-specific rescue with actin-binding-domain mutant\",\n      \"pmids\": [\"25437874\", \"25273069\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How a single gene's distinct cellular roles are coordinated not resolved at this stage\", \"Direct SHROOM3 effectors in fibrosis vs. barrier maintenance not yet separated\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Extended ROCK-interaction dependence to axon outgrowth and demonstrated druggability of the SHROOM3-ROCK interface.\",\n      \"evidence\": \"HTS, covalent inhibitor CCG-17444 targeting Cys1816, neurite outgrowth assay placing SHROOM3 downstream of NogoA\",\n      \"pmids\": [\"26077244\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo relevance of the NogoA-SHROOM3 axis not tested\", \"Single lab, biochemical and cell-based only\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Connected SHROOM3 to PCP machinery, showing it acts within planar polarity signaling rather than as an isolated constriction effector.\",\n      \"evidence\": \"Reciprocal Co-IP with Dishevelled 2, compound heterozygote genetics with Vangl2/Wnt5a, planar polarity marker imaging in neural plate\",\n      \"pmids\": [\"25596276\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Domain on SHROOM3 binding Dishevelled 2 not mapped\", \"Whether PCP input is upstream of or parallel to ROCK recruitment unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstrated SHROOM3 maintains podocyte architecture and drives invagination in additional organs, defining adult kidney disease and developmental roles.\",\n      \"evidence\": \"Shroom3 KO/heterozygous mice with glomerulosclerosis and actomyosin readouts; gene-trap thyroid bud apical constriction with Cdc42 dependence\",\n      \"pmids\": [\"26940091\", \"26772200\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking Cdc42 to SHROOM3 localization not defined\", \"How heterozygosity produces adult-onset disease not resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified a second, ROCK-independent SHROOM3 signaling arm (FYN/nephrin) and a 14-3-3/Hippo-linked CKD variant, revealing functional modularity.\",\n      \"evidence\": \"Endogenous SHROOM3-FYN Co-IP, SH3-binding domain mapping, glomerular shRNA mouse with EM/RNA-seq; SHROOM3 P1244L 14-3-3 binding assay and novel PDZ-less isoform cloning\",\n      \"pmids\": [\"30341149\", \"29476007\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct kinase mechanism of FYN activation by SHROOM3 not fully resolved\", \"Functional role of the PDZ-less isoform untested in vivo\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Showed the SHROOM3-Dishevelled2/PCP axis governs cardiomyocyte polarity and cardiac chamber morphogenesis.\",\n      \"evidence\": \"SHROOM3-Dishevelled 2 Co-IP, Shroom3 gene-trap KO with congenital heart defects and actomyosin/proliferation readouts\",\n      \"pmids\": [\"32511952\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether cardiac defects arise cell-autonomously not fully separated\", \"Link between proliferation phenotype and actomyosin role unclear\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Refined the cellular logic of constriction, showing SHROOM3 acts both cell-autonomously and non-cell-autonomously across cell neighborhoods.\",\n      \"evidence\": \"Mosaic CRISPR crispants and tissue-scale time-lapse imaging with regression modeling in Xenopus; regional cranial vs. spinal analysis\",\n      \"pmids\": [\"36113571\", \"35244026\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of non-cell-autonomous coupling not identified\", \"Regional functional differences not linked to specific partners\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Validated SHROOM3's apical-constriction role in human and primate neuroepithelial models and tied it to folate-independent neural tube defect mechanisms.\",\n      \"evidence\": \"SHROOM3 CRISPR KO in human cortical organoids (lumen/F-actin); KO in cynomolgus monkey neuroepithelial organoids with folate supplementation test\",\n      \"pmids\": [\"37443734\", \"38287904\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab organoid models\", \"Does not establish SHROOM3 variants as human NTD causes\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Achieved separation-of-function, dissociating the ROCK-binding (profibrotic) and FYN-binding (antiproteinuric) arms and demonstrating therapeutic targeting of the SHROOM3-ROCK interaction.\",\n      \"evidence\": \"Domain-deletion transgenic mice (ASD2Δ, FBDM-Sh3), cell-based ROCK activation, UUO/AAN fibrosis models, small-molecule P2Is (BT1137)\",\n      \"pmids\": [\"41469391\", \"39605692\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Long-term efficacy and specificity of P2Is in vivo not established\", \"How the two arms are differentially deployed across cell types not fully resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Expanded SHROOM3's roles to epithelial fusion, cardiomyocyte ploidy/contractile function, and podocyte focal adhesion regulation.\",\n      \"evidence\": \"Tissue-specific KO of optic fissure/retina; CM-specific Nkx2-5-Cre deletion with ploidy/echo and actin-variant testing; podocyte-specific KO with focal adhesion/RhoA/calpain readouts\",\n      \"pmids\": [\"40113025\", \"42189988\", \"40558522\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking SHROOM3 to DNA replication genes and ploidy unclear\", \"Direct effectors at focal adhesions vs. apical actin not separated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How SHROOM3's distinct molecular arms (ROCK/actomyosin, FYN/nephrin, 14-3-3/Hippo, focal adhesion regulation) are selectively engaged and coordinated within a single cell type remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model integrating the multiple binding interfaces\", \"Upstream switch determining which arm is active in a given context unknown\", \"Direct mechanism by which SHROOM3 alters DNA replication gene expression undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 6, 7, 14]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [1, 9, 27]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 14, 24]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [1, 12, 17, 21]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 7, 13, 25]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0, 1, 2, 5, 16]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 10, 14, 16]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [8, 9, 12, 24]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ROCK1\", \"ROCK2\", \"FYN\", \"DVL2\", \"CTNND1\", \"NRP1\", \"RhoA\", \"YWHAE\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}