{"gene":"SEMA3D","run_date":"2026-06-10T07:46:30","timeline":{"discoveries":[{"year":2016,"finding":"Sema3d regulates collective endothelial cell migration in zebrafish through two distinct mechanisms: (1) mesenchymal Sema3d guides outgrowth of the common cardinal vein via repulsion signaling through PlexinD1, and (2) autocrine Sema3d signaling within endothelial cells regulates actin network organization and cell morphology through Neuropilin1, which then signals via RhoA upstream of Rock to stabilize the endothelial sheet.","method":"Zebrafish genetic/knockdown experiments with receptor-specific dissection, live imaging of endothelial cell migration, and pathway manipulation (RhoA/Rock inhibition)","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal receptor-specific genetic dissection in vivo, two orthogonal signaling mechanisms identified with pathway validation, replicated across multiple experimental approaches","pmids":["27799363"],"is_preprint":false},{"year":2016,"finding":"Sema3d is stabilized by the Hapln1a-dependent extracellular matrix (ECM) during zebrafish fin regeneration; Hapln1a knockdown reduces hyaluronan and aggrecan, destabilizing Sema3d, and Sema3d overexpression rescues hapln1a knockdown phenotypes, placing Sema3d functionally downstream of the Hapln1a-ECM in Cx43-dependent skeletal patterning and growth.","method":"Double morpholino knockdown, rescue by Sema3d overexpression, epistasis analysis in zebrafish fin regeneration","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis via double knockdown and rescue experiment, single lab, clear functional phenotype","pmids":["26828861"],"is_preprint":false},{"year":2022,"finding":"Sema3d interacts with the actin-binding protein Filamin A (FLNA) as identified by co-immunoprecipitation combined with mass spectrometry, and this interaction inactivates the PI3K/Akt signaling pathway and remodels the cytoskeleton to suppress hepatocellular carcinoma proliferation, invasion, and metastasis.","method":"Co-IP combined with mass spectrometry, RNA sequencing with GSEA, gain- and loss-of-function experiments in HCC cell lines and mouse xenograft models","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP/MS identification of FLNA interaction plus functional in vitro and in vivo validation, single lab, multiple orthogonal methods","pmids":["35957887"],"is_preprint":false},{"year":2026,"finding":"Sema3d secreted by activated fibroblasts signals through the endothelial Plexin D1 receptor, leading to Arf6 activation and integrin β1 internalization, thereby suppressing focal adhesion formation, inducing cytoskeletal collapse, and preventing endothelial cell migration and angiogenesis, which promotes renal fibrosis in the AKI-CKD transition.","method":"Single-cell RNA sequencing, conditioned medium experiments on HUVECs, mechanistic pathway analysis (Arf6/integrin β1), and AAV9-shRNA knockdown of Sema3d in mouse uIRI model","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined receptor (PlxnD1) and downstream effectors (Arf6, integrin β1) with in vivo rescue, single lab, multiple orthogonal methods","pmids":["41608631"],"is_preprint":false},{"year":2025,"finding":"Citrullination of IGF2BP1 at R167 promotes the mRNA stability of SEMA3D by enhancing the interaction between IGF2BP1 and its cofactor ELAVL1, thereby upregulating SEMA3D expression and promoting proliferation, migration, and invasion of rheumatoid arthritis fibroblast-like synoviocytes.","method":"Transcriptomic sequencing, functional assays in RA-FLSs with citrullination site-specific mutagenesis (R167K knock-in mice), co-IP to detect IGF2BP1-ELAVL1 interaction, mRNA stability assays","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-specific mutagenesis in vivo (knock-in mice) plus Co-IP and mRNA stability assay, single lab, multiple orthogonal methods","pmids":["39809921"],"is_preprint":false},{"year":2014,"finding":"Disruption of SEMA3D (partial tandem duplication producing a truncated mRNA) in a patient with congenital heart defects suggests that Sema3d is required for correct migration of cardiac neural crest cells (CNCC) into the outflow tract; Sema3d-null mice present with congenital heart defects consistent with CNCC migration failure.","method":"Array comparative genomic hybridization, breakpoint sequencing, fiber FISH, expression analysis in patient lymphoblasts, reference to Sema3D knockout mouse phenotype","journal":"Human mutation","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single patient case with expression data and mouse KO reference, no direct mechanistic in vitro/in vivo experiment for CNCC migration in this study","pmids":["25230848"],"is_preprint":false},{"year":2017,"finding":"Genetic epistasis testing in mice showed that loss of Sema3d (null homozygotes) did not affect survival, myenteric plexus formation, or intestinal transcriptome, and there was no evidence of genetic interaction between Ret and Sema3d on these phenotypes — a negative finding for Sema3d's role in enteric nervous system development in this context.","method":"Double-null intercross mouse genetics, acetylcholinesterase staining for myenteric plexus, RNA-sequencing of embryonic intestine","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — rigorous genetic epistasis experiment with multiple phenotypic readouts and transcriptomics; result is explicitly negative for Sema3d-Ret interaction","pmids":["28334784"],"is_preprint":false},{"year":2009,"finding":"In the developing rat dorsal root ganglia, Sema3D expression is confined to proprioceptive neurons that co-express TrkC and Runx3, but not to nociceptive TrkA-expressing neurons, suggesting a selective role for Sema3D in axonal navigation of large-diameter proprioceptive DRG neurons.","method":"In situ hybridization and double immunohistochemical analysis of rat embryonic DRG with TrkA, TrkC, Runx3, and Neuropilin-1 markers","journal":"Neuroscience letters","confidence":"Low","confidence_rationale":"Tier 3 / Weak — co-localization by immunohistochemistry and ISH, single lab, no functional loss-of-function experiment performed","pmids":["19429098"],"is_preprint":false},{"year":2024,"finding":"SEMA3D protein is detected as extracellular granules in the tongue primordia at E11.5, while NRP1 is expressed in the hypoglossal nerve; SEMA3D granules are excluded from regions where hypoglossal nerve axons elongate, consistent with a repulsive guidance role for SEMA3D on NRP1-expressing hypoglossal axons during tongue development.","method":"Transcriptomic ROKU analysis, quantitative PCR, and immunohistochemistry on mouse embryonic tongue primordia","journal":"Acta histochemica et cytochemica","confidence":"Low","confidence_rationale":"Tier 3 / Weak — localization by immunohistochemistry without functional loss-of-function validation, single lab, single method for mechanistic inference","pmids":["38463205"],"is_preprint":false}],"current_model":"SEMA3D is a secreted class III semaphorin that functions as a repulsive guidance cue and signaling molecule: it signals through PlexinD1 to repel and guide endothelial and cardiac neural crest cell migration, signals through Neuropilin1 to organize the actin cytoskeleton via RhoA/Rock in an autocrine manner, is stabilized by the Hapln1a-dependent ECM, interacts with Filamin A (FLNA) to inactivate PI3K/Akt signaling, and its mRNA stability is regulated by citrullinated IGF2BP1 acting through ELAVL1."},"narrative":{"mechanistic_narrative":"SEMA3D is a secreted class III semaphorin that functions as a repulsive guidance cue and signaling molecule controlling collective cell migration, vascular patterning, and cytoskeletal organization [PMID:27799363]. In endothelial systems it acts through two receptor-defined routes: mesenchymal SEMA3D guides vein outgrowth by repulsive signaling through PlexinD1, while autocrine SEMA3D signals via Neuropilin1 to organize the actin network and stabilize the endothelial sheet through RhoA acting upstream of Rock [PMID:27799363]. SEMA3D-PlexinD1 signaling drives Arf6 activation and integrin β1 internalization, suppressing focal adhesion formation and inducing cytoskeletal collapse to block endothelial migration and angiogenesis [PMID:41608631]. SEMA3D also engages the actin-binding protein Filamin A, an interaction that inactivates PI3K/Akt signaling and remodels the cytoskeleton to suppress tumor cell proliferation, invasion, and metastasis [PMID:35957887]. Its extracellular activity depends on stabilization by a Hapln1a-dependent extracellular matrix [PMID:26828861], and its mRNA stability is controlled by citrullinated IGF2BP1 acting together with the cofactor ELAVL1 [PMID:39809921]. A SEMA3D-disrupting tandem duplication in a patient with congenital heart defects, together with the SEMA3D-null mouse phenotype, links the gene to cardiac neural crest cell migration into the outflow tract [PMID:25230848].","teleology":[{"year":2009,"claim":"An early question was whether SEMA3D acts selectively on defined neuronal populations; restricted expression in proprioceptive DRG neurons established a candidate role in axonal navigation of large-diameter sensory neurons.","evidence":"In situ hybridization and double immunohistochemistry in rat embryonic dorsal root ganglia with TrkA/TrkC/Runx3/Neuropilin-1 markers","pmids":["19429098"],"confidence":"Low","gaps":["No functional loss-of-function experiment performed","Co-localization does not demonstrate a guidance function","Receptor engagement not tested"]},{"year":2014,"claim":"It was unknown whether SEMA3D disruption causes human disease; a tandem duplication producing truncated SEMA3D in a congenital heart defect patient, alongside the null mouse, implicated SEMA3D in cardiac neural crest cell migration into the outflow tract.","evidence":"Array CGH, breakpoint sequencing, fiber FISH, lymphoblast expression analysis, and reference to the Sema3D-null mouse","pmids":["25230848"],"confidence":"Low","gaps":["Single patient case without direct mechanistic CNCC migration experiment in this study","Causality not established by independent families or functional rescue","Receptor and downstream pathway in CNCC not defined"]},{"year":2016,"claim":"The central mechanistic question of how SEMA3D directs collective migration was answered by showing two receptor-specific routes: PlexinD1-mediated repulsion guides vein outgrowth, while autocrine Neuropilin1 signaling organizes the actin cytoskeleton via RhoA/Rock.","evidence":"Zebrafish genetic/knockdown with receptor-specific dissection, live imaging of endothelial migration, and RhoA/Rock pathway manipulation","pmids":["27799363"],"confidence":"High","gaps":["Structural basis of differential PlexinD1 versus Neuropilin1 engagement not resolved","Whether the two routes operate in mammalian vasculature not tested here"]},{"year":2016,"claim":"How extracellular SEMA3D protein levels are maintained was addressed by placing SEMA3D downstream of a Hapln1a-dependent ECM that stabilizes the protein during regeneration.","evidence":"Double morpholino knockdown, SEMA3D-overexpression rescue, and epistasis analysis in zebrafish fin regeneration","pmids":["26828861"],"confidence":"Medium","gaps":["Molecular basis of ECM-mediated stabilization not defined","Direct biochemical interaction between SEMA3D and matrix components not shown"]},{"year":2017,"claim":"A possible role in enteric nervous system development was tested and excluded, narrowing SEMA3D's functional scope by showing no Ret-Sema3d genetic interaction in myenteric plexus formation.","evidence":"Double-null intercross mouse genetics, acetylcholinesterase staining, and embryonic intestine RNA-seq","pmids":["28334784"],"confidence":"Medium","gaps":["Negative result confined to enteric phenotypes","Does not address SEMA3D function in other neural crest derivatives"]},{"year":2022,"claim":"An intracellular/cytoskeletal mode of action was uncovered by identifying Filamin A as a SEMA3D partner whose binding inactivates PI3K/Akt and remodels the cytoskeleton to suppress tumor progression.","evidence":"Co-IP/mass spectrometry, RNA-seq with GSEA, and gain/loss-of-function in HCC cell lines and mouse xenografts","pmids":["35957887"],"confidence":"Medium","gaps":["Single Co-IP/MS identification without reciprocal structural validation","How a secreted semaphorin accesses cytoplasmic FLNA not resolved","Relationship to canonical PlexinD1/Neuropilin1 signaling unclear"]},{"year":2024,"claim":"The spatial logic of SEMA3D guidance in development was reinforced by showing SEMA3D granules are excluded from regions of NRP1-expressing hypoglossal axon elongation in tongue primordia.","evidence":"Transcriptomic ROKU analysis, qPCR, and immunohistochemistry on mouse embryonic tongue","pmids":["38463205"],"confidence":"Low","gaps":["Localization-based inference without functional loss-of-function validation","Repulsive effect on hypoglossal axons not directly demonstrated"]},{"year":2025,"claim":"Post-transcriptional control of SEMA3D was defined by showing that citrullination of IGF2BP1 at R167 enhances IGF2BP1-ELAVL1 interaction to stabilize SEMA3D mRNA and drive synoviocyte invasion.","evidence":"Transcriptomic sequencing, R167K knock-in mice, Co-IP, and mRNA stability assays in RA-FLSs","pmids":["39809921"],"confidence":"Medium","gaps":["Direct binding of IGF2BP1 to SEMA3D mRNA elements not mapped","Generality beyond rheumatoid arthritis synoviocytes untested"]},{"year":2026,"claim":"The downstream effector chain of SEMA3D-PlexinD1 signaling in disease was clarified by linking it to Arf6 activation and integrin β1 internalization that suppress focal adhesions and angiogenesis to promote renal fibrosis.","evidence":"scRNA-seq, conditioned-medium experiments on HUVECs, Arf6/integrin β1 pathway analysis, and AAV9-shRNA knockdown in a mouse uIRI model","pmids":["41608631"],"confidence":"Medium","gaps":["Direct biochemical link between PlexinD1 and Arf6 not reconstituted","Single lab; effector cascade not independently confirmed"]},{"year":null,"claim":"It remains unresolved how SEMA3D's secreted receptor-mediated repulsion and its intracellular Filamin A/PI3K-Akt activity are mechanistically reconciled within a single cell.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model integrating PlexinD1, Neuropilin1, and FLNA engagement","Cell-type-specific receptor usage not systematically mapped","Causal human disease mechanism beyond a single CHD case not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,3]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[2]}],"localization":[{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[0,3,8]},{"term_id":"GO:0031012","term_label":"extracellular matrix","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,3]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[0,5]}],"complexes":[],"partners":["PLXND1","NRP1","FLNA"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O95025","full_name":"Semaphorin-3D","aliases":[],"length_aa":777,"mass_kda":89.7,"function":"Induces the collapse and paralysis of neuronal growth cones. Could potentially act as repulsive cues toward specific neuronal populations. Binds to neuropilin (By similarity)","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/O95025/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SEMA3D","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SEMA3D","total_profiled":1310},"omim":[{"mim_id":"609907","title":"SEMAPHORIN 3D; SEMA3D","url":"https://www.omim.org/entry/609907"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Vesicles","reliability":"Approved"}],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"lymphoid tissue","ntpm":38.1},{"tissue":"thyroid gland","ntpm":33.1}],"url":"https://www.proteinatlas.org/search/SEMA3D"},"hgnc":{"alias_symbol":["coll-2","Sema-Z2"],"prev_symbol":[]},"alphafold":{"accession":"O95025","domains":[{"cath_id":"3.30.1680.10","chopping":"468-592","consensus_level":"medium","plddt":88.1978,"start":468,"end":592},{"cath_id":"2.60.40.10","chopping":"595-688","consensus_level":"high","plddt":89.0555,"start":595,"end":688}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O95025","model_url":"https://alphafold.ebi.ac.uk/files/AF-O95025-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O95025-F1-predicted_aligned_error_v6.png","plddt_mean":84.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SEMA3D","jax_strain_url":"https://www.jax.org/strain/search?query=SEMA3D"},"sequence":{"accession":"O95025","fasta_url":"https://rest.uniprot.org/uniprotkb/O95025.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O95025/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O95025"}},"corpus_meta":[{"pmid":"15727893","id":"PMC_15727893","title":"New serum biochemical markers (Coll 2-1 and Coll 2-1 NO2) for studying oxidative-related type II collagen network degradation in patients with osteoarthritis and rheumatoid arthritis.","date":"2005","source":"Osteoarthritis and cartilage","url":"https://pubmed.ncbi.nlm.nih.gov/15727893","citation_count":115,"is_preprint":false},{"pmid":"27799363","id":"PMC_27799363","title":"Sema3d controls collective endothelial cell migration by distinct mechanisms via Nrp1 and PlxnD1.","date":"2016","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/27799363","citation_count":69,"is_preprint":false},{"pmid":"27876815","id":"PMC_27876815","title":"Variable expressivity and genetic heterogeneity involving DPT and SEMA3D genes in autosomal dominant familial Meniere's disease.","date":"2016","source":"European journal of human genetics : EJHG","url":"https://pubmed.ncbi.nlm.nih.gov/27876815","citation_count":56,"is_preprint":false},{"pmid":"26073756","id":"PMC_26073756","title":"CTNNA3 and SEMA3D: Promising loci for asthma exacerbation identified through multiple genome-wide association studies.","date":"2015","source":"The Journal of allergy and clinical immunology","url":"https://pubmed.ncbi.nlm.nih.gov/26073756","citation_count":39,"is_preprint":false},{"pmid":"20684831","id":"PMC_20684831","title":"Possible association of the semaphorin 3D gene (SEMA3D) with schizophrenia.","date":"2010","source":"Journal of psychiatric research","url":"https://pubmed.ncbi.nlm.nih.gov/20684831","citation_count":24,"is_preprint":false},{"pmid":"16804892","id":"PMC_16804892","title":"Sema3D and Sema7A have distinct expression patterns in chick embryonic development.","date":"2006","source":"Developmental dynamics : an official publication of the American Association of Anatomists","url":"https://pubmed.ncbi.nlm.nih.gov/16804892","citation_count":23,"is_preprint":false},{"pmid":"25230848","id":"PMC_25230848","title":"Disruption of the SEMA3D gene in a patient with congenital heart defects.","date":"2014","source":"Human mutation","url":"https://pubmed.ncbi.nlm.nih.gov/25230848","citation_count":23,"is_preprint":false},{"pmid":"16261621","id":"PMC_16261621","title":"Sema3D, Sema3F, and Sema5A are expressed in overlapping and distinct patterns in chick embryonic heart.","date":"2006","source":"Developmental dynamics : an official publication of the American Association of Anatomists","url":"https://pubmed.ncbi.nlm.nih.gov/16261621","citation_count":15,"is_preprint":false},{"pmid":"26828861","id":"PMC_26828861","title":"Cx43-Dependent Skeletal Phenotypes Are Mediated by Interactions between the Hapln1a-ECM and Sema3d during Fin Regeneration.","date":"2016","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/26828861","citation_count":14,"is_preprint":false},{"pmid":"28334784","id":"PMC_28334784","title":"Testing the Ret and Sema3d genetic interaction in mouse enteric nervous system development.","date":"2017","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/28334784","citation_count":7,"is_preprint":false},{"pmid":"32219083","id":"PMC_32219083","title":"Aberrant Expressions and Variant Screening of SEMA3D in Indonesian Hirschsprung Patients.","date":"2020","source":"Frontiers in pediatrics","url":"https://pubmed.ncbi.nlm.nih.gov/32219083","citation_count":7,"is_preprint":false},{"pmid":"19429098","id":"PMC_19429098","title":"Expression of Sema3D in subsets of neurons in the developing dorsal root ganglia of the rat.","date":"2009","source":"Neuroscience letters","url":"https://pubmed.ncbi.nlm.nih.gov/19429098","citation_count":7,"is_preprint":false},{"pmid":"35957887","id":"PMC_35957887","title":"Sema3d Restrained Hepatocellular Carcinoma Progression Through Inactivating Pi3k/Akt Signaling via Interaction With FLNA.","date":"2022","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/35957887","citation_count":6,"is_preprint":false},{"pmid":"35813868","id":"PMC_35813868","title":"Transmembrane Transporter Sema3D Serves as a Tumor Suppressor in Localized Clear Cell Renal Cell Carcinoma.","date":"2022","source":"Journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/35813868","citation_count":2,"is_preprint":false},{"pmid":"38463205","id":"PMC_38463205","title":"Tissue-Targeted Transcriptomics Reveals SEMA3D Control of Hypoglossal Nerve Projection to Mouse Tongue Primordia.","date":"2024","source":"Acta histochemica et cytochemica","url":"https://pubmed.ncbi.nlm.nih.gov/38463205","citation_count":1,"is_preprint":false},{"pmid":"35173464","id":"PMC_35173464","title":"SEMA3D Plays a Critical Role in Peptic Ulcer Disease-Related Carcinogenesis Induced by H. pylori Infection.","date":"2022","source":"International journal of general medicine","url":"https://pubmed.ncbi.nlm.nih.gov/35173464","citation_count":1,"is_preprint":false},{"pmid":"39809921","id":"PMC_39809921","title":"Citrullinated IGF2BP1 promotes rheumatoid synovial aggression via increasing the mRNA stability of SEMA3D.","date":"2025","source":"Communications biology","url":"https://pubmed.ncbi.nlm.nih.gov/39809921","citation_count":1,"is_preprint":false},{"pmid":"41608631","id":"PMC_41608631","title":"Sema3dhi Fibroblasts Promote Acute Kidney Injury Fibrotic Progression Through Confining Endothelial Cell Migration.","date":"2026","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/41608631","citation_count":0,"is_preprint":false},{"pmid":"37727374","id":"PMC_37727374","title":"Transcriptome profiling of intact bowel wall reveals that PDE1A and SEMA3D are possible markers with roles in enteric smooth muscle apoptosis, proliferative disorders, and dysautonomia in Crohn's disease.","date":"2023","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/37727374","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.08.18.670897","title":"Single Cell RNA sequencing reveals transitional states and signaling shifts in nephron progenitor cells of the late-gestation rhesus macaque kidney","date":"2025-08-22","source":"bioRxiv","url":"https://doi.org/10.1101/2025.08.18.670897","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12536,"output_tokens":2430,"usd":0.037029,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9581,"output_tokens":3417,"usd":0.066665,"stage2_stop_reason":"end_turn"},"total_usd":0.103694,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2016,\n      \"finding\": \"Sema3d regulates collective endothelial cell migration in zebrafish through two distinct mechanisms: (1) mesenchymal Sema3d guides outgrowth of the common cardinal vein via repulsion signaling through PlexinD1, and (2) autocrine Sema3d signaling within endothelial cells regulates actin network organization and cell morphology through Neuropilin1, which then signals via RhoA upstream of Rock to stabilize the endothelial sheet.\",\n      \"method\": \"Zebrafish genetic/knockdown experiments with receptor-specific dissection, live imaging of endothelial cell migration, and pathway manipulation (RhoA/Rock inhibition)\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal receptor-specific genetic dissection in vivo, two orthogonal signaling mechanisms identified with pathway validation, replicated across multiple experimental approaches\",\n      \"pmids\": [\"27799363\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Sema3d is stabilized by the Hapln1a-dependent extracellular matrix (ECM) during zebrafish fin regeneration; Hapln1a knockdown reduces hyaluronan and aggrecan, destabilizing Sema3d, and Sema3d overexpression rescues hapln1a knockdown phenotypes, placing Sema3d functionally downstream of the Hapln1a-ECM in Cx43-dependent skeletal patterning and growth.\",\n      \"method\": \"Double morpholino knockdown, rescue by Sema3d overexpression, epistasis analysis in zebrafish fin regeneration\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via double knockdown and rescue experiment, single lab, clear functional phenotype\",\n      \"pmids\": [\"26828861\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Sema3d interacts with the actin-binding protein Filamin A (FLNA) as identified by co-immunoprecipitation combined with mass spectrometry, and this interaction inactivates the PI3K/Akt signaling pathway and remodels the cytoskeleton to suppress hepatocellular carcinoma proliferation, invasion, and metastasis.\",\n      \"method\": \"Co-IP combined with mass spectrometry, RNA sequencing with GSEA, gain- and loss-of-function experiments in HCC cell lines and mouse xenograft models\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP/MS identification of FLNA interaction plus functional in vitro and in vivo validation, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"35957887\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Sema3d secreted by activated fibroblasts signals through the endothelial Plexin D1 receptor, leading to Arf6 activation and integrin β1 internalization, thereby suppressing focal adhesion formation, inducing cytoskeletal collapse, and preventing endothelial cell migration and angiogenesis, which promotes renal fibrosis in the AKI-CKD transition.\",\n      \"method\": \"Single-cell RNA sequencing, conditioned medium experiments on HUVECs, mechanistic pathway analysis (Arf6/integrin β1), and AAV9-shRNA knockdown of Sema3d in mouse uIRI model\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined receptor (PlxnD1) and downstream effectors (Arf6, integrin β1) with in vivo rescue, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"41608631\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Citrullination of IGF2BP1 at R167 promotes the mRNA stability of SEMA3D by enhancing the interaction between IGF2BP1 and its cofactor ELAVL1, thereby upregulating SEMA3D expression and promoting proliferation, migration, and invasion of rheumatoid arthritis fibroblast-like synoviocytes.\",\n      \"method\": \"Transcriptomic sequencing, functional assays in RA-FLSs with citrullination site-specific mutagenesis (R167K knock-in mice), co-IP to detect IGF2BP1-ELAVL1 interaction, mRNA stability assays\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific mutagenesis in vivo (knock-in mice) plus Co-IP and mRNA stability assay, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"39809921\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Disruption of SEMA3D (partial tandem duplication producing a truncated mRNA) in a patient with congenital heart defects suggests that Sema3d is required for correct migration of cardiac neural crest cells (CNCC) into the outflow tract; Sema3d-null mice present with congenital heart defects consistent with CNCC migration failure.\",\n      \"method\": \"Array comparative genomic hybridization, breakpoint sequencing, fiber FISH, expression analysis in patient lymphoblasts, reference to Sema3D knockout mouse phenotype\",\n      \"journal\": \"Human mutation\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single patient case with expression data and mouse KO reference, no direct mechanistic in vitro/in vivo experiment for CNCC migration in this study\",\n      \"pmids\": [\"25230848\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Genetic epistasis testing in mice showed that loss of Sema3d (null homozygotes) did not affect survival, myenteric plexus formation, or intestinal transcriptome, and there was no evidence of genetic interaction between Ret and Sema3d on these phenotypes — a negative finding for Sema3d's role in enteric nervous system development in this context.\",\n      \"method\": \"Double-null intercross mouse genetics, acetylcholinesterase staining for myenteric plexus, RNA-sequencing of embryonic intestine\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — rigorous genetic epistasis experiment with multiple phenotypic readouts and transcriptomics; result is explicitly negative for Sema3d-Ret interaction\",\n      \"pmids\": [\"28334784\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"In the developing rat dorsal root ganglia, Sema3D expression is confined to proprioceptive neurons that co-express TrkC and Runx3, but not to nociceptive TrkA-expressing neurons, suggesting a selective role for Sema3D in axonal navigation of large-diameter proprioceptive DRG neurons.\",\n      \"method\": \"In situ hybridization and double immunohistochemical analysis of rat embryonic DRG with TrkA, TrkC, Runx3, and Neuropilin-1 markers\",\n      \"journal\": \"Neuroscience letters\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — co-localization by immunohistochemistry and ISH, single lab, no functional loss-of-function experiment performed\",\n      \"pmids\": [\"19429098\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SEMA3D protein is detected as extracellular granules in the tongue primordia at E11.5, while NRP1 is expressed in the hypoglossal nerve; SEMA3D granules are excluded from regions where hypoglossal nerve axons elongate, consistent with a repulsive guidance role for SEMA3D on NRP1-expressing hypoglossal axons during tongue development.\",\n      \"method\": \"Transcriptomic ROKU analysis, quantitative PCR, and immunohistochemistry on mouse embryonic tongue primordia\",\n      \"journal\": \"Acta histochemica et cytochemica\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — localization by immunohistochemistry without functional loss-of-function validation, single lab, single method for mechanistic inference\",\n      \"pmids\": [\"38463205\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SEMA3D is a secreted class III semaphorin that functions as a repulsive guidance cue and signaling molecule: it signals through PlexinD1 to repel and guide endothelial and cardiac neural crest cell migration, signals through Neuropilin1 to organize the actin cytoskeleton via RhoA/Rock in an autocrine manner, is stabilized by the Hapln1a-dependent ECM, interacts with Filamin A (FLNA) to inactivate PI3K/Akt signaling, and its mRNA stability is regulated by citrullinated IGF2BP1 acting through ELAVL1.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SEMA3D is a secreted class III semaphorin that functions as a repulsive guidance cue and signaling molecule controlling collective cell migration, vascular patterning, and cytoskeletal organization [#0]. In endothelial systems it acts through two receptor-defined routes: mesenchymal SEMA3D guides vein outgrowth by repulsive signaling through PlexinD1, while autocrine SEMA3D signals via Neuropilin1 to organize the actin network and stabilize the endothelial sheet through RhoA acting upstream of Rock [#0]. SEMA3D-PlexinD1 signaling drives Arf6 activation and integrin \\u03b21 internalization, suppressing focal adhesion formation and inducing cytoskeletal collapse to block endothelial migration and angiogenesis [#3]. SEMA3D also engages the actin-binding protein Filamin A, an interaction that inactivates PI3K/Akt signaling and remodels the cytoskeleton to suppress tumor cell proliferation, invasion, and metastasis [#2]. Its extracellular activity depends on stabilization by a Hapln1a-dependent extracellular matrix [#1], and its mRNA stability is controlled by citrullinated IGF2BP1 acting together with the cofactor ELAVL1 [#4]. A SEMA3D-disrupting tandem duplication in a patient with congenital heart defects, together with the SEMA3D-null mouse phenotype, links the gene to cardiac neural crest cell migration into the outflow tract [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"An early question was whether SEMA3D acts selectively on defined neuronal populations; restricted expression in proprioceptive DRG neurons established a candidate role in axonal navigation of large-diameter sensory neurons.\",\n      \"evidence\": \"In situ hybridization and double immunohistochemistry in rat embryonic dorsal root ganglia with TrkA/TrkC/Runx3/Neuropilin-1 markers\",\n      \"pmids\": [\"19429098\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No functional loss-of-function experiment performed\",\n        \"Co-localization does not demonstrate a guidance function\",\n        \"Receptor engagement not tested\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"It was unknown whether SEMA3D disruption causes human disease; a tandem duplication producing truncated SEMA3D in a congenital heart defect patient, alongside the null mouse, implicated SEMA3D in cardiac neural crest cell migration into the outflow tract.\",\n      \"evidence\": \"Array CGH, breakpoint sequencing, fiber FISH, lymphoblast expression analysis, and reference to the Sema3D-null mouse\",\n      \"pmids\": [\"25230848\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Single patient case without direct mechanistic CNCC migration experiment in this study\",\n        \"Causality not established by independent families or functional rescue\",\n        \"Receptor and downstream pathway in CNCC not defined\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"The central mechanistic question of how SEMA3D directs collective migration was answered by showing two receptor-specific routes: PlexinD1-mediated repulsion guides vein outgrowth, while autocrine Neuropilin1 signaling organizes the actin cytoskeleton via RhoA/Rock.\",\n      \"evidence\": \"Zebrafish genetic/knockdown with receptor-specific dissection, live imaging of endothelial migration, and RhoA/Rock pathway manipulation\",\n      \"pmids\": [\"27799363\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of differential PlexinD1 versus Neuropilin1 engagement not resolved\",\n        \"Whether the two routes operate in mammalian vasculature not tested here\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"How extracellular SEMA3D protein levels are maintained was addressed by placing SEMA3D downstream of a Hapln1a-dependent ECM that stabilizes the protein during regeneration.\",\n      \"evidence\": \"Double morpholino knockdown, SEMA3D-overexpression rescue, and epistasis analysis in zebrafish fin regeneration\",\n      \"pmids\": [\"26828861\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Molecular basis of ECM-mediated stabilization not defined\",\n        \"Direct biochemical interaction between SEMA3D and matrix components not shown\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"A possible role in enteric nervous system development was tested and excluded, narrowing SEMA3D's functional scope by showing no Ret-Sema3d genetic interaction in myenteric plexus formation.\",\n      \"evidence\": \"Double-null intercross mouse genetics, acetylcholinesterase staining, and embryonic intestine RNA-seq\",\n      \"pmids\": [\"28334784\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Negative result confined to enteric phenotypes\",\n        \"Does not address SEMA3D function in other neural crest derivatives\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"An intracellular/cytoskeletal mode of action was uncovered by identifying Filamin A as a SEMA3D partner whose binding inactivates PI3K/Akt and remodels the cytoskeleton to suppress tumor progression.\",\n      \"evidence\": \"Co-IP/mass spectrometry, RNA-seq with GSEA, and gain/loss-of-function in HCC cell lines and mouse xenografts\",\n      \"pmids\": [\"35957887\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single Co-IP/MS identification without reciprocal structural validation\",\n        \"How a secreted semaphorin accesses cytoplasmic FLNA not resolved\",\n        \"Relationship to canonical PlexinD1/Neuropilin1 signaling unclear\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"The spatial logic of SEMA3D guidance in development was reinforced by showing SEMA3D granules are excluded from regions of NRP1-expressing hypoglossal axon elongation in tongue primordia.\",\n      \"evidence\": \"Transcriptomic ROKU analysis, qPCR, and immunohistochemistry on mouse embryonic tongue\",\n      \"pmids\": [\"38463205\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Localization-based inference without functional loss-of-function validation\",\n        \"Repulsive effect on hypoglossal axons not directly demonstrated\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Post-transcriptional control of SEMA3D was defined by showing that citrullination of IGF2BP1 at R167 enhances IGF2BP1-ELAVL1 interaction to stabilize SEMA3D mRNA and drive synoviocyte invasion.\",\n      \"evidence\": \"Transcriptomic sequencing, R167K knock-in mice, Co-IP, and mRNA stability assays in RA-FLSs\",\n      \"pmids\": [\"39809921\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct binding of IGF2BP1 to SEMA3D mRNA elements not mapped\",\n        \"Generality beyond rheumatoid arthritis synoviocytes untested\"\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"The downstream effector chain of SEMA3D-PlexinD1 signaling in disease was clarified by linking it to Arf6 activation and integrin \\u03b21 internalization that suppress focal adhesions and angiogenesis to promote renal fibrosis.\",\n      \"evidence\": \"scRNA-seq, conditioned-medium experiments on HUVECs, Arf6/integrin \\u03b21 pathway analysis, and AAV9-shRNA knockdown in a mouse uIRI model\",\n      \"pmids\": [\"41608631\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct biochemical link between PlexinD1 and Arf6 not reconstituted\",\n        \"Single lab; effector cascade not independently confirmed\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how SEMA3D's secreted receptor-mediated repulsion and its intracellular Filamin A/PI3K-Akt activity are mechanistically reconciled within a single cell.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No structural model integrating PlexinD1, Neuropilin1, and FLNA engagement\",\n        \"Cell-type-specific receptor usage not systematically mapped\",\n        \"Causal human disease mechanism beyond a single CHD case not established\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [0, 3, 8]},\n      {\"term_id\": \"GO:0031012\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0, 5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"PLXND1\", \"NRP1\", \"FLNA\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}