{"gene":"PLXND1","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2009,"finding":"Sema3e expressed by selected motor neuron pools binds its high-affinity receptor PlexinD1 (Plxnd1) expressed on proprioceptive sensory neurons; this repellent signaling determines synaptic specificity in sensory-motor reflex circuits. Changing the profile of Sema3e-Plxnd1 signaling in sensory or motor neurons results in functional and anatomical rewiring of monosynaptic connections without altering motor pool specificity.","method":"Molecular genetic manipulation in mice (conditional gain/loss of Sema3e and Plxnd1 in defined neuronal populations), electrophysiology, anatomical tracing","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal genetic manipulation in vivo with functional (electrophysiological) and anatomical readouts, published in high-impact journal with clear mechanistic specificity","pmids":["19421194"],"is_preprint":false},{"year":2013,"finding":"Loss of PlexinD1 on double-positive thymocytes leads to aberrant migration and cortical retention after TCR-mediated positive selection. Loss of PlexinD1 on thymic endothelial cells causes ectopic medulla formation linked to dysregulation of thymic angiogenesis. Loss on thymic epithelium produces neither abnormality. These findings establish non-redundant, cell-type-specific roles for PlexinD1 in thymocyte migration and medullary topology.","method":"Conditional knockout mice (Plxnd1-flox crossed to pLck-Cre, pKeratin14-Cre, pTek-Cre); histology; immunofluorescence localization of thymocyte subsets","journal":"Frontiers in immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple cell-type-specific conditional knockouts with distinct phenotypic readouts, disambiguating cell-autonomous roles","pmids":["24312099"],"is_preprint":false},{"year":2015,"finding":"De novo loss-of-function mutations in PLXND1 cause Möbius syndrome by disrupting facial branchiomotor neuron migration in the hindbrain. Plxnd1 mutant mice show convergent defects at the facial branchiomotor nucleus, specifically affecting motoneuron migration.","method":"Whole-exome sequencing of MBS patients identifying de novo mutations; analysis of Plxnd1 mutant mice for facial branchiomotor nucleus defects","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — human genetics combined with mouse mutant validation, single lab, two orthogonal approaches","pmids":["26068067"],"is_preprint":false},{"year":2016,"finding":"In zebrafish, PlexinD1 mediates repulsive Sema3d signaling from mesenchymal cells to guide outgrowth of the common cardinal vein endothelial cells. Separately, autocrine Sema3d signals through Neuropilin1 (not PlexinD1) to regulate actin network organization and endothelial cell morphology via RhoA-Rock, stabilizing the endothelial sheet.","method":"Zebrafish genetic/pharmacological manipulation, live imaging of endothelial cell migration, epistasis between PlxnD1 and Nrp1 signaling branches","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis in zebrafish with multiple orthogonal readouts (migration, actin organization, morphology) clearly separating PlxnD1 and Nrp1 branches","pmids":["27799363"],"is_preprint":false},{"year":2022,"finding":"PLXND1 acts as a scaffold protein on endocardial endothelial cell membranes in atrial fibrillation; it binds ORAI1 and decreases ORAI1-mediated calcium influx. Reduced calcium influx decreases CAMK2 phosphorylation, which inhibits autophagic flux and results in endocardial endothelial cell dysfunction.","method":"Isolation of endocardial endothelial cells from AF model mice; co-immunoprecipitation/binding assay of PLXND1 with ORAI1; measurement of intracellular calcium and autophagic flux; CAMK2 phosphorylation assay","journal":"Frontiers in physiology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP binding of PLXND1-ORAI1 with downstream functional readouts (calcium, CAMK2 phosphorylation, autophagy), single lab","pmids":["36017337"],"is_preprint":false},{"year":2022,"finding":"PLXND1 promotes epithelial-mesenchymal transition (EMT) and cell invasion in colorectal cancer via activation of the PI3K/AKT pathway. The furin-cleaved form of SEMA3E (p61-SEMA3E) binds PLXND1 to enhance invasiveness and migration; furin inhibition blocks this binding and suppresses EMT. PLXND1 knockdown decreases cell migration and invasion in vitro and suppresses EMT in vivo.","method":"PLXND1 knockdown in CRC cells; cell migration/invasion assays; PI3K/AKT pathway activity measurements; furin inhibitor treatment; in vivo xenograft experiments","journal":"Annals of surgical oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined signaling pathway readout and in vivo validation, single lab","pmids":["35917012"],"is_preprint":false},{"year":2022,"finding":"Disease-associated PLXND1 rare variants in anomalous pulmonary venous return (APVR) patients convergently disrupt the GTPase-activating protein (GAP)-related domain of PLXND1. Plxnd1 knockout mice display abnormal migration and vascular formation of vascular endothelial cells consistent with APVR pathology.","method":"Whole-exome sequencing of APVR patients; functional domain mapping of variants to GAP-related domain; Plxnd1 knockout mouse analysis of endothelial cell migration and vascular formation","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — human genetic variant mapping to specific functional domain combined with mouse KO validation, single lab, two orthogonal approaches","pmids":["34791216"],"is_preprint":false},{"year":2024,"finding":"Plexin-D1 acts as a mechanosensor of blood flow in endothelial cells of the developing dorsal aorta; its mechanosensing activity positively regulates vessel caliber. Plexin-D1 mechanosensing activates the flow-responsive transcription factor KLF2, which acts as a downstream effector to enlarge endothelial cells and widen the vessel.","method":"Zebrafish and human endothelial microvascular network models; flow and genetic manipulations; loss-of-function of Plxnd1; KLF2 activity measurements; custom imaging software for vessel caliber quantification","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic and flow manipulation with defined transcriptional effector (KLF2) and cellular/structural readout; preprint, single lab","pmids":["38328196"],"is_preprint":true},{"year":2025,"finding":"SEMA3C inhibits cortical neuron dendrite outgrowth through PLXND1-dependent signaling; PLXND1 is required downstream of astrocyte-secreted SEMA3C for the dendritic inhibition observed in Rett syndrome model mice.","method":"Conditioned media from RTT astrocytes applied to cortical neurons; PLXND1-dependent epistasis tested by blocking PLXND1 signaling; rescue of dendritic arborization, synaptic activity, and behavior by normalizing SEMA3C levels in vivo","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis placing PLXND1 downstream of SEMA3C with multiple orthogonal functional readouts (dendrites, synaptic activity, behavior); preprint, single lab","pmids":["bio_10.1101_2025.10.10.681027"],"is_preprint":true}],"current_model":"PLXND1 (PlexinD1) is a transmembrane receptor for class 3 semaphorins (including Sema3E, Sema3C, Sema3D) that functions as a repulsive guidance and mechanosensing molecule in multiple contexts: it mediates repellent Sema3E signaling to establish synaptic specificity in spinal sensory-motor circuits, guides vascular endothelial cell migration and controls aortic caliber via mechanosensing and downstream KLF2 activation, directs thymocyte migration and thymic medullary organization in a cell-type-specific manner, and promotes epithelial-mesenchymal transition via PI3K/AKT signaling in cancer; its GAP-related domain is critical for cardiovascular morphogenesis, and it can act as a scaffold to bind ORAI1 and regulate calcium influx and downstream CAMK2-autophagy signaling in endocardial endothelial cells."},"narrative":{"mechanistic_narrative":"PLXND1 (PlexinD1) is a transmembrane class 3 semaphorin receptor that transduces repulsive guidance and mechanosensory cues to control cell migration and tissue patterning across neural, vascular, and immune systems [PMID:19421194, PMID:38328196]. In the nervous system it serves as the high-affinity receptor for Sema3E on proprioceptive sensory neurons, where repellent signaling establishes the specificity of monosynaptic sensory-motor reflex circuits, and it guides facial branchiomotor neuron migration in the hindbrain, where loss-of-function disrupts motoneuron positioning [PMID:19421194, PMID:26068067]. PLXND1 also acts downstream of SEMA3C to inhibit cortical dendrite outgrowth [PMID:bio_10.1101_2025.10.10.681027]. In the vasculature, PLXND1 mediates repulsive Sema3D signaling from mesenchyme to guide endothelial outgrowth and controls endothelial migration and vessel formation through a GTPase-activating protein (GAP)-related domain that is the convergent target of disease-associated variants [PMID:27799363, PMID:34791216]; it additionally operates as a mechanosensor of blood flow that enlarges endothelial cells and widens vessel caliber via the flow-responsive transcription factor KLF2 [PMID:38328196]. Beyond canonical semaphorin signaling, PLXND1 functions in a cell-type-specific manner to direct thymocyte migration and thymic medullary topology [PMID:24312099], promotes epithelial-mesenchymal transition and invasion through PI3K/AKT activation upon binding the furin-cleaved p61-SEMA3E in colorectal cancer [PMID:35917012], and acts as a membrane scaffold that binds ORAI1 to restrain calcium influx and downstream CAMK2-autophagy signaling in endocardial endothelial cells [PMID:36017337]. De novo loss-of-function mutations in PLXND1 cause Möbius syndrome [PMID:26068067], and rare variants disrupting its GAP-related domain are associated with anomalous pulmonary venous return [PMID:34791216].","teleology":[{"year":2009,"claim":"Established that PlexinD1 is the functional receptor whose ligand-defined expression pattern sets synaptic wiring specificity, showing the receptor's repulsive signaling has discrete circuit-level consequences rather than merely diffuse guidance.","evidence":"Reciprocal conditional gain/loss of Sema3e and Plxnd1 in defined mouse neuronal populations with electrophysiology and anatomical tracing","pmids":["19421194"],"confidence":"High","gaps":["Downstream cytoplasmic effectors of Plxnd1 in sensory neurons not defined","Does not address whether the same signaling logic operates outside reflex circuits"]},{"year":2013,"claim":"Dissected cell-autonomous versus non-cell-autonomous roles, demonstrating PlexinD1 acts non-redundantly in thymocytes for migration and in endothelium for medullary topology but is dispensable in epithelium.","evidence":"Cell-type-specific conditional knockout mice (Lck-Cre, Keratin14-Cre, Tek-Cre) with histology and immunofluorescence","pmids":["24312099"],"confidence":"High","gaps":["Ligand driving thymocyte migration not identified in this study","Molecular link between endothelial PlexinD1 loss and ectopic medulla unresolved"]},{"year":2015,"claim":"Linked PLXND1 loss-of-function to a human Mendelian disease, establishing facial branchiomotor neuron migration as a clinically relevant readout of receptor function.","evidence":"Whole-exome sequencing of Möbius syndrome patients identifying de novo mutations plus Plxnd1 mutant mouse facial branchiomotor nucleus analysis","pmids":["26068067"],"confidence":"Medium","gaps":["Single lab; specific signaling pathway downstream of the migration defect not mapped","Ligand responsible for branchiomotor guidance not pinned down"]},{"year":2016,"claim":"Separated parallel guidance branches in vascular development, showing PlexinD1 transduces repulsive Sema3D for endothelial outgrowth while a distinct Neuropilin1 branch controls actin/morphology, clarifying receptor-specific output.","evidence":"Zebrafish genetic/pharmacological epistasis with live imaging of cardinal vein endothelial migration and actin organization","pmids":["27799363"],"confidence":"High","gaps":["Cytoplasmic effectors coupling PlexinD1 to migration in this context not defined","Whether PlexinD1 and Nrp1 branches cross-regulate not addressed"]},{"year":2022,"claim":"Identified the GAP-related domain as the functional hotspot for disease-causing variants in anomalous pulmonary venous return, tying a specific structural module to endothelial migration and vascular morphogenesis.","evidence":"Whole-exome sequencing of APVR patients with variant domain mapping plus Plxnd1 knockout mouse endothelial analysis","pmids":["34791216"],"confidence":"Medium","gaps":["GAP catalytic targets (substrate GTPases) not directly assayed","Single lab; quantitative effect of variants on GAP activity not measured"]},{"year":2022,"claim":"Extended PLXND1 function into cancer, showing the furin-cleaved p61-SEMA3E ligand drives PLXND1-dependent EMT and invasion through PI3K/AKT, defining a pro-tumorigenic signaling axis.","evidence":"PLXND1 knockdown in colorectal cancer cells, migration/invasion assays, PI3K/AKT readouts, furin inhibition, and xenografts","pmids":["35917012"],"confidence":"Medium","gaps":["Direct biochemical demonstration of p61-SEMA3E/PLXND1 binding affinity not detailed","Single lab; how PLXND1 engages PI3K/AKT mechanistically unresolved"]},{"year":2022,"claim":"Revealed a non-canonical scaffolding role in which PLXND1 binds ORAI1 to restrain calcium influx and thereby tune CAMK2-autophagy signaling, expanding receptor function beyond guidance.","evidence":"Co-immunoprecipitation of PLXND1-ORAI1 in endocardial endothelial cells from atrial fibrillation model mice with calcium, CAMK2 phosphorylation, and autophagic flux readouts","pmids":["36017337"],"confidence":"Medium","gaps":["Single Co-IP without reciprocal structural validation of the interaction","Whether scaffolding depends on semaphorin ligand engagement unknown"]},{"year":2024,"claim":"Defined PlexinD1 as a flow mechanosensor that positively sets vessel caliber via the transcription factor KLF2, distinguishing mechanotransduction from ligand-driven guidance.","evidence":"Zebrafish and human endothelial microvascular models with flow and genetic manipulation, KLF2 activity, and caliber quantification (preprint)","pmids":["38328196"],"confidence":"Medium","gaps":["Preprint; molecular mechanism by which PlexinD1 senses force not resolved","Relationship between mechanosensing and semaphorin-ligand signaling unclear"]},{"year":2025,"claim":"Placed PLXND1 downstream of astrocyte-secreted SEMA3C in dendritic inhibition, implicating the receptor in disease-relevant neuronal morphology beyond axon/circuit guidance.","evidence":"RTT astrocyte conditioned media on cortical neurons with PLXND1-blocking epistasis and in vivo rescue of dendrites, synaptic activity, and behavior (preprint)","pmids":["bio_10.1101_2025.10.10.681027"],"confidence":"Medium","gaps":["Preprint; intracellular signaling from PLXND1 to dendrite cytoskeleton not mapped","Whether SEMA3C binds PLXND1 directly versus via co-receptor not resolved"]},{"year":null,"claim":"The cytoplasmic GAP-related domain's direct substrate GTPases and the unifying mechanism connecting PLXND1's ligand-driven guidance, mechanosensing, and ORAI1-scaffolding outputs remain undefined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No direct identification of GTPase substrates of the GAP domain in the corpus","No structural model integrating mechanosensing and ligand-binding modes","Co-receptor requirements (e.g., neuropilins) across contexts not systematically resolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,3]},{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[7]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[4,6]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[4]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,3,5]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[0,2,3,6]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[2,5,6]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1]}],"complexes":[],"partners":["SEMA3E","SEMA3C","SEMA3D","ORAI1","NRP1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9Y4D7","full_name":"Plexin-D1","aliases":[],"length_aa":1925,"mass_kda":212.0,"function":"Cell surface receptor for SEMA4A and for class 3 semaphorins, such as SEMA3A, SEMA3C and SEMA3E. Plays an important role in cell-cell signaling, and in regulating the migration of a wide spectrum of cell types. Regulates the migration of thymocytes in the medulla. Regulates endothelial cell migration. Plays an important role in ensuring the specificity of synapse formation. Required for normal development of the heart and vasculature (By similarity). Mediates anti-angiogenic signaling in response to SEMA3E","subcellular_location":"Cell membrane; Cell projection, lamellipodium membrane","url":"https://www.uniprot.org/uniprotkb/Q9Y4D7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PLXND1","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/PLXND1","total_profiled":1310},"omim":[{"mim_id":"620997","title":"SEMAPHORIN 3G; SEMA3G","url":"https://www.omim.org/entry/620997"},{"mim_id":"620294","title":"CONGENITAL HEART DEFECTS, MULTIPLE TYPES, 9; CHTD9","url":"https://www.omim.org/entry/620294"},{"mim_id":"608166","title":"SEMAPHORIN 3E; SEMA3E","url":"https://www.omim.org/entry/608166"},{"mim_id":"604293","title":"PLEXIN B2; PLXNB2","url":"https://www.omim.org/entry/604293"},{"mim_id":"604282","title":"PLEXIN D1; PLXND1","url":"https://www.omim.org/entry/604282"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PLXND1"},"hgnc":{"alias_symbol":["KIAA0620"],"prev_symbol":[]},"alphafold":{"accession":"Q9Y4D7","domains":[{"cath_id":"-","chopping":"96-224_231-258","consensus_level":"medium","plddt":78.0178,"start":96,"end":258},{"cath_id":"2.130.10.10","chopping":"272-288_315-470","consensus_level":"medium","plddt":80.6723,"start":272,"end":470},{"cath_id":"2.60.40.10","chopping":"754-849","consensus_level":"medium","plddt":83.9953,"start":754,"end":849},{"cath_id":"2.60.40.10","chopping":"887-981","consensus_level":"medium","plddt":88.574,"start":887,"end":981},{"cath_id":"2.60.40.10","chopping":"987-1068","consensus_level":"medium","plddt":87.396,"start":987,"end":1068},{"cath_id":"2.60.40.10","chopping":"1079-1174","consensus_level":"medium","plddt":77.0254,"start":1079,"end":1174},{"cath_id":"2.60.40.10","chopping":"1179-1266","consensus_level":"medium","plddt":79.4886,"start":1179,"end":1266},{"cath_id":"3.10.20.90","chopping":"1553-1676","consensus_level":"medium","plddt":82.4514,"start":1553,"end":1676}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y4D7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y4D7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y4D7-F1-predicted_aligned_error_v6.png","plddt_mean":79.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PLXND1","jax_strain_url":"https://www.jax.org/strain/search?query=PLXND1"},"sequence":{"accession":"Q9Y4D7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y4D7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y4D7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y4D7"}},"corpus_meta":[{"pmid":"19421194","id":"PMC_19421194","title":"Specificity of sensory-motor connections encoded by Sema3e-Plxnd1 recognition.","date":"2009","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/19421194","citation_count":150,"is_preprint":false},{"pmid":"26068067","id":"PMC_26068067","title":"De novo mutations in PLXND1 and REV3L cause Möbius syndrome.","date":"2015","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/26068067","citation_count":71,"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":"35917012","id":"PMC_35917012","title":"PLXND1/SEMA3E Promotes Epithelial-Mesenchymal Transition Partly via the PI3K/AKT-Signaling Pathway and Induces Heterogenity in Colorectal Cancer.","date":"2022","source":"Annals of surgical oncology","url":"https://pubmed.ncbi.nlm.nih.gov/35917012","citation_count":16,"is_preprint":false},{"pmid":"24312099","id":"PMC_24312099","title":"Plxnd1 expression in thymocytes regulates their intrathymic migration while that in thymic endothelium impacts medullary topology.","date":"2013","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/24312099","citation_count":11,"is_preprint":false},{"pmid":"36581828","id":"PMC_36581828","title":"Poland-Möbius syndrome: a case report implicating a novel mutation of the PLXND1 gene and literature review.","date":"2022","source":"BMC pediatrics","url":"https://pubmed.ncbi.nlm.nih.gov/36581828","citation_count":6,"is_preprint":false},{"pmid":"36017337","id":"PMC_36017337","title":"PLXND1-mediated calcium dyshomeostasis impairs endocardial endothelial autophagy in atrial fibrillation.","date":"2022","source":"Frontiers in physiology","url":"https://pubmed.ncbi.nlm.nih.gov/36017337","citation_count":5,"is_preprint":false},{"pmid":"37073425","id":"PMC_37073425","title":"Downregulation of circ_PLXND1 inhibits tumorigenesis of non-small cell lung carcinoma via miR-1287-5p/ERBB3 axis.","date":"2023","source":"Thoracic cancer","url":"https://pubmed.ncbi.nlm.nih.gov/37073425","citation_count":4,"is_preprint":false},{"pmid":"34791216","id":"PMC_34791216","title":"Association of PLXND1 with a novel subtype of anomalous pulmonary venous return.","date":"2022","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/34791216","citation_count":3,"is_preprint":false},{"pmid":"38328196","id":"PMC_38328196","title":"Plxnd1-mediated mechanosensing of blood flow controls the caliber of the Dorsal Aorta via the transcription factor Klf2.","date":"2024","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/38328196","citation_count":3,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.10.10.681027","title":"Upregulated SEMA3C in astrocytes contributes to Rett Syndrome phenotypes","date":"2025-10-12","source":"bioRxiv","url":"https://doi.org/10.1101/2025.10.10.681027","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7590,"output_tokens":2612,"usd":0.030975,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9777,"output_tokens":3680,"usd":0.070443,"stage2_stop_reason":"end_turn"},"total_usd":0.101418,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2009,\n      \"finding\": \"Sema3e expressed by selected motor neuron pools binds its high-affinity receptor PlexinD1 (Plxnd1) expressed on proprioceptive sensory neurons; this repellent signaling determines synaptic specificity in sensory-motor reflex circuits. Changing the profile of Sema3e-Plxnd1 signaling in sensory or motor neurons results in functional and anatomical rewiring of monosynaptic connections without altering motor pool specificity.\",\n      \"method\": \"Molecular genetic manipulation in mice (conditional gain/loss of Sema3e and Plxnd1 in defined neuronal populations), electrophysiology, anatomical tracing\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal genetic manipulation in vivo with functional (electrophysiological) and anatomical readouts, published in high-impact journal with clear mechanistic specificity\",\n      \"pmids\": [\"19421194\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Loss of PlexinD1 on double-positive thymocytes leads to aberrant migration and cortical retention after TCR-mediated positive selection. Loss of PlexinD1 on thymic endothelial cells causes ectopic medulla formation linked to dysregulation of thymic angiogenesis. Loss on thymic epithelium produces neither abnormality. These findings establish non-redundant, cell-type-specific roles for PlexinD1 in thymocyte migration and medullary topology.\",\n      \"method\": \"Conditional knockout mice (Plxnd1-flox crossed to pLck-Cre, pKeratin14-Cre, pTek-Cre); histology; immunofluorescence localization of thymocyte subsets\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple cell-type-specific conditional knockouts with distinct phenotypic readouts, disambiguating cell-autonomous roles\",\n      \"pmids\": [\"24312099\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"De novo loss-of-function mutations in PLXND1 cause Möbius syndrome by disrupting facial branchiomotor neuron migration in the hindbrain. Plxnd1 mutant mice show convergent defects at the facial branchiomotor nucleus, specifically affecting motoneuron migration.\",\n      \"method\": \"Whole-exome sequencing of MBS patients identifying de novo mutations; analysis of Plxnd1 mutant mice for facial branchiomotor nucleus defects\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — human genetics combined with mouse mutant validation, single lab, two orthogonal approaches\",\n      \"pmids\": [\"26068067\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In zebrafish, PlexinD1 mediates repulsive Sema3d signaling from mesenchymal cells to guide outgrowth of the common cardinal vein endothelial cells. Separately, autocrine Sema3d signals through Neuropilin1 (not PlexinD1) to regulate actin network organization and endothelial cell morphology via RhoA-Rock, stabilizing the endothelial sheet.\",\n      \"method\": \"Zebrafish genetic/pharmacological manipulation, live imaging of endothelial cell migration, epistasis between PlxnD1 and Nrp1 signaling branches\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis in zebrafish with multiple orthogonal readouts (migration, actin organization, morphology) clearly separating PlxnD1 and Nrp1 branches\",\n      \"pmids\": [\"27799363\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PLXND1 acts as a scaffold protein on endocardial endothelial cell membranes in atrial fibrillation; it binds ORAI1 and decreases ORAI1-mediated calcium influx. Reduced calcium influx decreases CAMK2 phosphorylation, which inhibits autophagic flux and results in endocardial endothelial cell dysfunction.\",\n      \"method\": \"Isolation of endocardial endothelial cells from AF model mice; co-immunoprecipitation/binding assay of PLXND1 with ORAI1; measurement of intracellular calcium and autophagic flux; CAMK2 phosphorylation assay\",\n      \"journal\": \"Frontiers in physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP binding of PLXND1-ORAI1 with downstream functional readouts (calcium, CAMK2 phosphorylation, autophagy), single lab\",\n      \"pmids\": [\"36017337\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PLXND1 promotes epithelial-mesenchymal transition (EMT) and cell invasion in colorectal cancer via activation of the PI3K/AKT pathway. The furin-cleaved form of SEMA3E (p61-SEMA3E) binds PLXND1 to enhance invasiveness and migration; furin inhibition blocks this binding and suppresses EMT. PLXND1 knockdown decreases cell migration and invasion in vitro and suppresses EMT in vivo.\",\n      \"method\": \"PLXND1 knockdown in CRC cells; cell migration/invasion assays; PI3K/AKT pathway activity measurements; furin inhibitor treatment; in vivo xenograft experiments\",\n      \"journal\": \"Annals of surgical oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined signaling pathway readout and in vivo validation, single lab\",\n      \"pmids\": [\"35917012\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Disease-associated PLXND1 rare variants in anomalous pulmonary venous return (APVR) patients convergently disrupt the GTPase-activating protein (GAP)-related domain of PLXND1. Plxnd1 knockout mice display abnormal migration and vascular formation of vascular endothelial cells consistent with APVR pathology.\",\n      \"method\": \"Whole-exome sequencing of APVR patients; functional domain mapping of variants to GAP-related domain; Plxnd1 knockout mouse analysis of endothelial cell migration and vascular formation\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — human genetic variant mapping to specific functional domain combined with mouse KO validation, single lab, two orthogonal approaches\",\n      \"pmids\": [\"34791216\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Plexin-D1 acts as a mechanosensor of blood flow in endothelial cells of the developing dorsal aorta; its mechanosensing activity positively regulates vessel caliber. Plexin-D1 mechanosensing activates the flow-responsive transcription factor KLF2, which acts as a downstream effector to enlarge endothelial cells and widen the vessel.\",\n      \"method\": \"Zebrafish and human endothelial microvascular network models; flow and genetic manipulations; loss-of-function of Plxnd1; KLF2 activity measurements; custom imaging software for vessel caliber quantification\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and flow manipulation with defined transcriptional effector (KLF2) and cellular/structural readout; preprint, single lab\",\n      \"pmids\": [\"38328196\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SEMA3C inhibits cortical neuron dendrite outgrowth through PLXND1-dependent signaling; PLXND1 is required downstream of astrocyte-secreted SEMA3C for the dendritic inhibition observed in Rett syndrome model mice.\",\n      \"method\": \"Conditioned media from RTT astrocytes applied to cortical neurons; PLXND1-dependent epistasis tested by blocking PLXND1 signaling; rescue of dendritic arborization, synaptic activity, and behavior by normalizing SEMA3C levels in vivo\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis placing PLXND1 downstream of SEMA3C with multiple orthogonal functional readouts (dendrites, synaptic activity, behavior); preprint, single lab\",\n      \"pmids\": [\"bio_10.1101_2025.10.10.681027\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"PLXND1 (PlexinD1) is a transmembrane receptor for class 3 semaphorins (including Sema3E, Sema3C, Sema3D) that functions as a repulsive guidance and mechanosensing molecule in multiple contexts: it mediates repellent Sema3E signaling to establish synaptic specificity in spinal sensory-motor circuits, guides vascular endothelial cell migration and controls aortic caliber via mechanosensing and downstream KLF2 activation, directs thymocyte migration and thymic medullary organization in a cell-type-specific manner, and promotes epithelial-mesenchymal transition via PI3K/AKT signaling in cancer; its GAP-related domain is critical for cardiovascular morphogenesis, and it can act as a scaffold to bind ORAI1 and regulate calcium influx and downstream CAMK2-autophagy signaling in endocardial endothelial cells.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PLXND1 (PlexinD1) is a transmembrane class 3 semaphorin receptor that transduces repulsive guidance and mechanosensory cues to control cell migration and tissue patterning across neural, vascular, and immune systems [#0, #7]. In the nervous system it serves as the high-affinity receptor for Sema3E on proprioceptive sensory neurons, where repellent signaling establishes the specificity of monosynaptic sensory-motor reflex circuits, and it guides facial branchiomotor neuron migration in the hindbrain, where loss-of-function disrupts motoneuron positioning [#0, #2]. PLXND1 also acts downstream of SEMA3C to inhibit cortical dendrite outgrowth [#8]. In the vasculature, PLXND1 mediates repulsive Sema3D signaling from mesenchyme to guide endothelial outgrowth and controls endothelial migration and vessel formation through a GTPase-activating protein (GAP)-related domain that is the convergent target of disease-associated variants [#3, #6]; it additionally operates as a mechanosensor of blood flow that enlarges endothelial cells and widens vessel caliber via the flow-responsive transcription factor KLF2 [#7]. Beyond canonical semaphorin signaling, PLXND1 functions in a cell-type-specific manner to direct thymocyte migration and thymic medullary topology [#1], promotes epithelial-mesenchymal transition and invasion through PI3K/AKT activation upon binding the furin-cleaved p61-SEMA3E in colorectal cancer [#5], and acts as a membrane scaffold that binds ORAI1 to restrain calcium influx and downstream CAMK2-autophagy signaling in endocardial endothelial cells [#4]. De novo loss-of-function mutations in PLXND1 cause Möbius syndrome [#2], and rare variants disrupting its GAP-related domain are associated with anomalous pulmonary venous return [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Established that PlexinD1 is the functional receptor whose ligand-defined expression pattern sets synaptic wiring specificity, showing the receptor's repulsive signaling has discrete circuit-level consequences rather than merely diffuse guidance.\",\n      \"evidence\": \"Reciprocal conditional gain/loss of Sema3e and Plxnd1 in defined mouse neuronal populations with electrophysiology and anatomical tracing\",\n      \"pmids\": [\"19421194\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream cytoplasmic effectors of Plxnd1 in sensory neurons not defined\", \"Does not address whether the same signaling logic operates outside reflex circuits\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Dissected cell-autonomous versus non-cell-autonomous roles, demonstrating PlexinD1 acts non-redundantly in thymocytes for migration and in endothelium for medullary topology but is dispensable in epithelium.\",\n      \"evidence\": \"Cell-type-specific conditional knockout mice (Lck-Cre, Keratin14-Cre, Tek-Cre) with histology and immunofluorescence\",\n      \"pmids\": [\"24312099\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ligand driving thymocyte migration not identified in this study\", \"Molecular link between endothelial PlexinD1 loss and ectopic medulla unresolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Linked PLXND1 loss-of-function to a human Mendelian disease, establishing facial branchiomotor neuron migration as a clinically relevant readout of receptor function.\",\n      \"evidence\": \"Whole-exome sequencing of Möbius syndrome patients identifying de novo mutations plus Plxnd1 mutant mouse facial branchiomotor nucleus analysis\",\n      \"pmids\": [\"26068067\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; specific signaling pathway downstream of the migration defect not mapped\", \"Ligand responsible for branchiomotor guidance not pinned down\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Separated parallel guidance branches in vascular development, showing PlexinD1 transduces repulsive Sema3D for endothelial outgrowth while a distinct Neuropilin1 branch controls actin/morphology, clarifying receptor-specific output.\",\n      \"evidence\": \"Zebrafish genetic/pharmacological epistasis with live imaging of cardinal vein endothelial migration and actin organization\",\n      \"pmids\": [\"27799363\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cytoplasmic effectors coupling PlexinD1 to migration in this context not defined\", \"Whether PlexinD1 and Nrp1 branches cross-regulate not addressed\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified the GAP-related domain as the functional hotspot for disease-causing variants in anomalous pulmonary venous return, tying a specific structural module to endothelial migration and vascular morphogenesis.\",\n      \"evidence\": \"Whole-exome sequencing of APVR patients with variant domain mapping plus Plxnd1 knockout mouse endothelial analysis\",\n      \"pmids\": [\"34791216\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"GAP catalytic targets (substrate GTPases) not directly assayed\", \"Single lab; quantitative effect of variants on GAP activity not measured\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended PLXND1 function into cancer, showing the furin-cleaved p61-SEMA3E ligand drives PLXND1-dependent EMT and invasion through PI3K/AKT, defining a pro-tumorigenic signaling axis.\",\n      \"evidence\": \"PLXND1 knockdown in colorectal cancer cells, migration/invasion assays, PI3K/AKT readouts, furin inhibition, and xenografts\",\n      \"pmids\": [\"35917012\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical demonstration of p61-SEMA3E/PLXND1 binding affinity not detailed\", \"Single lab; how PLXND1 engages PI3K/AKT mechanistically unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Revealed a non-canonical scaffolding role in which PLXND1 binds ORAI1 to restrain calcium influx and thereby tune CAMK2-autophagy signaling, expanding receptor function beyond guidance.\",\n      \"evidence\": \"Co-immunoprecipitation of PLXND1-ORAI1 in endocardial endothelial cells from atrial fibrillation model mice with calcium, CAMK2 phosphorylation, and autophagic flux readouts\",\n      \"pmids\": [\"36017337\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single Co-IP without reciprocal structural validation of the interaction\", \"Whether scaffolding depends on semaphorin ligand engagement unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined PlexinD1 as a flow mechanosensor that positively sets vessel caliber via the transcription factor KLF2, distinguishing mechanotransduction from ligand-driven guidance.\",\n      \"evidence\": \"Zebrafish and human endothelial microvascular models with flow and genetic manipulation, KLF2 activity, and caliber quantification (preprint)\",\n      \"pmids\": [\"38328196\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint; molecular mechanism by which PlexinD1 senses force not resolved\", \"Relationship between mechanosensing and semaphorin-ligand signaling unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Placed PLXND1 downstream of astrocyte-secreted SEMA3C in dendritic inhibition, implicating the receptor in disease-relevant neuronal morphology beyond axon/circuit guidance.\",\n      \"evidence\": \"RTT astrocyte conditioned media on cortical neurons with PLXND1-blocking epistasis and in vivo rescue of dendrites, synaptic activity, and behavior (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.10.10.681027\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint; intracellular signaling from PLXND1 to dendrite cytoskeleton not mapped\", \"Whether SEMA3C binds PLXND1 directly versus via co-receptor not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The cytoplasmic GAP-related domain's direct substrate GTPases and the unifying mechanism connecting PLXND1's ligand-driven guidance, mechanosensing, and ORAI1-scaffolding outputs remain undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct identification of GTPase substrates of the GAP domain in the corpus\", \"No structural model integrating mechanosensing and ligand-binding modes\", \"Co-receptor requirements (e.g., neuropilins) across contexts not systematically resolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [4, 6]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 3, 5]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0, 2, 3, 6]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [2, 5, 6]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"SEMA3E\", \"SEMA3C\", \"SEMA3D\", \"ORAI1\", \"NRP1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}