{"gene":"ROBO2","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":2004,"finding":"SLIT2/ROBO2 signaling in the nephrogenic mesenchyme restricts GDNF expression domain and thereby limits ureteric bud outgrowth to a single site; loss of either SLIT2 or ROBO2 causes anterior expansion of Gdnf expression and supernumerary ureteric buds.","method":"Mouse knockout (Slit2-/- and Robo2-/- mutants), in situ hybridization for Gdnf expression, histological analysis of kidney development","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 — reciprocal loss-of-function in two independent knockout lines with defined molecular phenotype (Gdnf domain expansion), replicated across Slit2 and Robo2 mutants","pmids":["15130495"],"is_preprint":false},{"year":2000,"finding":"Drosophila Robo2 and Robo1 have distinct roles in midline axon guidance; robo,robo2 double mutant phenocopies slit mutant (growth cones enter and fail to leave midline), demonstrating Robo2 is a second Slit receptor that together with Robo1 controls repulsive guidance at the midline.","method":"Drosophila genetics, double mutant analysis (robo;robo2 and slit), axon immunostaining","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis with double mutant phenocopying ligand loss, foundational paper with >200 citations","pmids":["11163264"],"is_preprint":false},{"year":2007,"finding":"ROBO2 loss-of-function (translocation disrupting ROBO2) produces dominant-negative ROBO2 proteins that abrogate SLIT-ROBO signaling in vitro, implicating the SLIT-ROBO2 pathway in vesicoureteral reflux and CAKUT pathogenesis.","method":"Translocation mapping, dominant-negative in vitro signaling assay, heterozygous mouse models, missense variant segregation analysis","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (translocation, in vitro dominant-negative assay, mouse models, family segregation)","pmids":["17357069"],"is_preprint":false},{"year":2015,"finding":"Slit2 acting through Robo1 and Robo2 promotes endothelial cell migration, Rac1 activation, and lamellipodia formation, driving retinal neovascularization; both receptors are required for Slit2- and VEGF-induced Rac1 activation.","method":"Conditional knockout mice (various Slit/Robo combinations), retinal angiogenesis assay, Rac1 activation assay, lamellipodia imaging, endothelial cell migration assay","journal":"Nature medicine","confidence":"High","confidence_rationale":"Tier 2 — conditional KO with defined cellular phenotype and biochemical readout (Rac1 activation), multiple receptor combinations tested","pmids":["25894826"],"is_preprint":false},{"year":2002,"finding":"Robo2 (astray) in zebrafish shapes retinal ganglion cell axon pathfinding by preventing and correcting pathfinding errors; robo2 mutant growth cones are larger and more complex, make midline and pre/post-midline errors that are rarely corrected, consistent with Slit2 and Slit3 acting as repulsive ligands via Robo2.","method":"Fixed-tissue and time-lapse imaging of zebrafish astray/robo2 mutant retinal axons, Slit expression analysis","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 — live imaging and genetic loss-of-function with specific morphological phenotype, >130 citations","pmids":["11804569"],"is_preprint":false},{"year":2012,"finding":"Robo2 is expressed on the basal surface of podocytes and forms a complex with nephrin through the adaptor protein Nck; Slit2-Robo2 signaling inhibits nephrin-induced actin polymerization and acts as a negative regulator of podocyte foot process architecture.","method":"Co-immunoprecipitation, co-localization studies, in vitro actin polymerization assay, Robo2 knockout mouse analysis, genetic interaction (Robo2 KO crossed to nephrin null)","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 — biochemical complex identification (Co-IP), in vitro activity assay, KO mouse with defined phenotype, genetic epistasis","pmids":["22840396"],"is_preprint":false},{"year":2016,"finding":"SLIT2/ROBO2 signaling activates a pathway through SRGAP1 and nonmuscle myosin IIA (NMIIA) in podocytes; MRLC (myosin II regulatory light chain) interacts directly with SRGAP1 and forms a ROBO2/SRGAP1/NMIIA complex in the presence of SLIT2; SLIT2 stimulation inhibits NMIIA activity, decreases focal adhesion formation, and reduces podocyte attachment to collagen.","method":"Co-immunoprecipitation, direct protein interaction assay, immunostaining, focal adhesion assay, cell adhesion assay, podocyte-specific Robo2 KO mouse, in vivo hypertension model","journal":"JCI insight","confidence":"High","confidence_rationale":"Tier 1-2 — direct interaction demonstrated, multiple downstream pathway components identified, in vivo validation with conditional KO","pmids":["27882344"],"is_preprint":false},{"year":2015,"finding":"Robo2 acts in trans (non-cell-autonomously) to inhibit Slit-Robo1 repulsion in pre-crossing commissural axons in Drosophila; Robo2's extracellular domains required for binding to Robo1 are also required for its ability to promote midline crossing, suggesting a receptor-receptor interaction mechanism.","method":"Drosophila genetics, gain-of-function and rescue assays, cell non-autonomous expression, extracellular domain deletion analysis","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 — multiple genetic assays (GOF, rescue, cell non-autonomous), domain-function mapping","pmids":["26186094"],"is_preprint":false},{"year":2008,"finding":"Robo2 receptor on ingressing placode cells interacts with its cognate ligand Slit1 expressed on early migrating cranial neural crest cells to mediate neural crest-placode interactions required for trigeminal ganglion assembly.","method":"Cell ablation experiments in chick, Robo2 function blocking antibodies, RNAi knockdown of Robo2 and Slit1, in situ hybridization for expression patterns","journal":"Nature neuroscience","confidence":"High","confidence_rationale":"Tier 2 — multiple independent loss-of-function approaches (antibody, RNAi for receptor and ligand) with defined cellular phenotype","pmids":["18278043"],"is_preprint":false},{"year":2009,"finding":"Slit1-Robo2 signaling modulates N-cadherin protein levels on the placodal cell surface (post-translationally, without changing total N-cadherin mRNA or protein) to regulate placodal cell adhesion during gangliogenesis; blocking Robo2 decreases surface N-cadherin, while increasing Slit-Robo signaling increases it.","method":"In vivo RNAi, dominant-negative Robo2 expression, N-cadherin manipulation, co-expression rescue experiments, in vivo chick trigeminal ganglion model","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 — bidirectional manipulation (gain and loss of function) of two pathways with defined molecular and cellular readouts","pmids":["19934013"],"is_preprint":false},{"year":2010,"finding":"Robo1 and Robo2 collaborate to prevent post-crossing commissural axons from reentering gray matter; Robo2 specifically is required for axons to project away from the floor plate into the lateral funiculus, while Robo1 prevents axonal stalling after crossing; epistatic analysis shows the double mutant is less severe than loss of all Slits, suggesting additional Slit receptor(s) exist.","method":"Mouse combination mutants (Robo1-/-, Robo2-/-, Robo3-/-, Slit1-3 triple mutant), commissural axon trajectory analysis","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 — systematic genetic epistasis across multiple mutant combinations revealing distinct receptor roles","pmids":["20631173"],"is_preprint":false},{"year":2007,"finding":"Robo-2 is required in a subset of olfactory sensory neurons (OSNs) for dorsoventral segregation of axons in the olfactory bulb; Slit-1 and Slit-3 expressed in the ventral OB act as repulsive ligands via Robo-2 to prevent dorsal OSN axons from projecting to the ventral OB.","method":"Robo-2 and Slit-1 knockout mice, olfactory axon tracing, expression analysis","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 — reciprocal receptor and ligand knockout phenotypes confirming ligand-receptor pair in specific circuit","pmids":["17715346"],"is_preprint":false},{"year":2009,"finding":"Robo2 is the major Slit receptor required for intraretinal axon guidance in mice; Robo1 knockout shows no intraretinal guidance defects, while Robo2 knockout phenocopies Slit1/Slit2 double mutant intraretinal pathfinding errors.","method":"Robo1 and Robo2 knockout mice, intraretinal axon tracing and pathfinding error quantification","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 2 — receptor-specific KO phenocopying ligand mutant, comparison with paralog KO establishing receptor specificity","pmids":["19782674"],"is_preprint":false},{"year":2007,"finding":"Slit1a inhibits retinal ganglion cell arborization and synaptogenesis via Robo2 in a cell-autonomous manner; increased arborization in astray/robo2 mutant is phenocopied by dominant-negative Robo2 in single RGCs and rescued by full-length Robo2; Slit1a acts both through Robo2-dependent and Robo2-independent mechanisms.","method":"Zebrafish astray/robo2 mutant, single-cell arbor imaging, dominant-negative and rescue constructs, morpholino knockdown, synaptic site labeling (YFP-Rab3)","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 — cell-autonomous rescue with full-length receptor, dominant-negative phenocopy, genetic pathway analysis","pmids":["17640525"],"is_preprint":false},{"year":2015,"finding":"ROBO2 restricts the nephrogenic field by limiting epithelial/mesenchymal interactions between the Wolffian duct/ureteric epithelium and mesenchyme; loss of Robo2 fails to separate the mesenchyme from Wolffian duct epithelium, exposing mesenchyme to abnormally high proliferative stimuli, expanding the nephrogenic cord cell number and the metanephric mesenchyme field.","method":"High-resolution 3D imaging of Robo2-null mouse embryos, ex vivo kidney explant experiments, cell proliferation analysis","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 2 — 3D imaging and ex vivo experiments defining cellular mechanism of field restriction","pmids":["26116176"],"is_preprint":false},{"year":2014,"finding":"SLIT/ROBO2 signaling in mammary basal cells restricts mammary stem cell (MaSC) renewal by negatively regulating WNT signaling; absence of SLIT/ROBO2 leads to increased nuclear β-catenin and repressed expression of p16(INK4a), delaying MaSC senescence.","method":"Robo2 conditional knockout mice, mammary stem cell functional assay, β-catenin localization, p16(INK4a) expression analysis","journal":"Stem cell reports","confidence":"Medium","confidence_rationale":"Tier 2 — conditional KO with molecular readouts, but single lab study","pmids":["25241737"],"is_preprint":false},{"year":2019,"finding":"In Drosophila tendons, Robo2 plays a non-signaling role by promoting Slit cleavage to produce the cleaved Slit N-terminal guidance signal; tendon-specific Robo2 expression (not muscle-specific) rescues muscle patterning defects, and membrane immobilization of Slit-N bypasses the requirement for tendon Robo2.","method":"Drosophila genetics, tissue-specific RNAi and rescue, membrane-tethered Slit constructs, muscle patterning analysis","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 — tissue-specific rescue and bypass experiments definitively placing Robo2 in non-signaling Slit processing role","pmids":["26400093"],"is_preprint":false},{"year":2011,"finding":"Pioneer myoblasts express Robo2 downstream of MyoD and Myf5; sclerotome-derived Slit1 acts through Robo2 to direct pioneer myoblast migration and fiber formation via RhoA; loss of Robo2 or sclerotome-derived Slit1 perturbs directional migration and fiber formation.","method":"Avian somite inversion experiments, Robo2/Slit1 loss-of-function in chick embryos, RhoA pathway analysis, fiber formation and migration assays","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 — mechanistic pathway (Slit1-Robo2-RhoA) established with tissue-specific loss-of-function","pmids":["21653616"],"is_preprint":false},{"year":2019,"finding":"Robo2 contains a cryptic binding site for NELL1 and NELL2 in its first fibronectin type III (FNIII) domain; this site is normally occluded in intact Robo2 and becomes accessible upon acidic conditions or proteolytic digestion; specific amino acids in the FNIII domain critical for NELL1 binding to Robo2 (but not Robo1) were identified by mutagenesis.","method":"Binding assays (NELL1/2 vs Robo family members), deletion mutant analysis, single amino acid mutagenesis, acidic pH binding experiments","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 — binding assay with mutagenesis, but in vitro only and single lab","pmids":["30700556"],"is_preprint":false},{"year":2022,"finding":"Conformational change of the Robo2 ectodomain (hairpin-like structure loosening) at acidic pH unmasks the NELL1/2-binding site; the interaction between Ig-like and FNIII domains maintains the occluded conformation at neutral pH; alternative splicing isoforms affect this conformational equilibrium and NELL1/2-binding affinity.","method":"FRET-based conformational indicator, size exclusion chromatography, binding assays with isoforms, acidic pH experiments","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 1 — structural/biophysical experiments, but single lab and no in vivo validation","pmids":["35940226"],"is_preprint":false},{"year":2019,"finding":"Robo2 regulates synaptic oxytocin content by controlling local actin dynamics via Cdc42; robo2 mutant zebrafish show decreased synaptic OXT levels, reduced Lifeact-EGFP mobility in OXT synapses, and slower vesicle accumulation; dominant-negative Cdc42 (a downstream effector of Robo2) modulates OXT content, placing Slit3-Robo2-Cdc42 in a pathway controlling actin dynamics at OXT synapses.","method":"Zebrafish robo2 mutant, live imaging of OXT vesicles and actin probe, FRAP, dominant-negative Cdc42 expression, genetic epistasis","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (live imaging, FRAP, genetic tools) establishing signaling pathway","pmids":["31180321"],"is_preprint":false},{"year":2021,"finding":"Robo2 acts postsynaptically in hippocampal CA1 pyramidal neurons to promote formation of excitatory (but not inhibitory) synapses specifically in proximal dendritic compartments; this synaptogenic activity involves a trans-synaptic interaction with presynaptic Neurexins and also binding to its canonical ligand Slit.","method":"In vivo conditional KO, in vitro synaptogenesis assays, co-immunoprecipitation (Robo2-Neurexin interaction), 2-photon Ca2+ imaging of place cells in behaving mice","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 — reciprocal binding partner identification, in vivo KO with defined synaptic phenotype, in vivo circuit-level functional consequence","pmids":["34686348"],"is_preprint":false},{"year":2019,"finding":"Robo2 binds Baiap2 (IRSp53) through its IRSp53/MIM homology domain in renal epithelial cells; this interaction allows Robo2 to phosphorylate MDM2 at Ser166 via Baiap2, maintaining p53 homeostasis; disruption of Robo2-Baiap2 complex causes MDM2 dephosphorylation, elevated active p53, p53-mediated cellular senescence via p21, and decreased polarity/ciliary proteins, leading to cystic kidney disease.","method":"Co-immunoprecipitation, phosphorylation assays, Robo2 KO and double KO (Robo2/p53) mouse models, ciliogenesis and polarity analysis","journal":"JCI insight","confidence":"High","confidence_rationale":"Tier 2 — complex identification (Co-IP), pathway placement (MDM2 phosphorylation), double KO epistasis rescuing phenotype","pmids":["31534052"],"is_preprint":false},{"year":2018,"finding":"Epithelial Robo2 loss in the pancreas leads to activation of Robo1+ myofibroblasts and induction of TGF-β and Wnt pathways in a non-cell-autonomous manner; TGF-β inhibitor galunisertib suppresses the myofibroblast activation, collagen crosslinking, and immune infiltration caused by Robo2 loss.","method":"Pdx1Cre;Robo2F/F conditional KO mice, pancreatitis and PDAC mouse models, cell culture, TGF-β inhibitor treatment, collagen analysis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — conditional KO with pathway identification, pharmacological rescue confirming TGF-β mechanism","pmids":["30504844"],"is_preprint":false},{"year":2012,"finding":"Robo2 acts as the predominant receptor directing dorsal longitudinal axon tract formation in mouse spinal cord and also has a distinct function in repelling neuron cell bodies from the floor plate; Robo1 is the predominant receptor for ventral longitudinal tracts.","method":"Conditional reduction of Robo levels in mouse embryos, genetic sensitization strategy, longitudinal axon tract imaging","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 — genetic dissection of receptor isoform roles, but single lab","pmids":["21820427"],"is_preprint":false},{"year":2013,"finding":"Robo2 and Dcc coordinate zebrafish ADt neuron axonal projection choices; Robo2 responds to repellent Slit signals and suppresses attractive Netrin signals; knockdown of Robo2 abolishes the ipsilateral SOT projection from ADt neurons.","method":"Zebrafish morpholino knockdown of Robo2 and Dcc/Netrin1, single-cell labeling of ADt neurons, projection pattern analysis","journal":"The Journal of neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 — morpholino knockdown for multiple pathway components establishing epistatic relationship, but morpholino-only approach","pmids":["22956848"],"is_preprint":false},{"year":2019,"finding":"Mammalian ROBO1 and ROBO2 are subject to temporal regulation via alternative splicing at a conserved microexon; NOVA splicing factors regulate developmental expression of ROBO1 and ROBO2 variants with distinct guidance activities; early isoforms activate axonal repulsion to prevent premature crossing, later isoforms allow crossing, and postcrossing isoforms are disinhibited.","method":"Alternative splicing analysis, NOVA splicing factor mutant mice, commissural axon guidance assays, isoform-specific functional assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 — isoform-specific functional differences established in vivo with identified upstream regulators (NOVA factors)","pmids":["31392959"],"is_preprint":false},{"year":2014,"finding":"Robo3.1A promotes degradation of Robo2 protein by recruiting it into a late endosome- and lysosome-dependent pathway; cotransfection of Robo3.1A significantly reduces Robo2 protein levels in HEK293 cells and cerebellar granule cells; Robo2 and Robo3 colocalize in intracellular vesicles positive for late endosome/lysosome markers.","method":"Cotransfection experiments, cell surface binding assay, co-immunoprecipitation, siRNA knockdown, immunocytochemical colocalization with organelle markers","journal":"Journal of neuroscience research","confidence":"Medium","confidence_rationale":"Tier 3 — biochemical evidence of degradation mechanism, colocalization with endosomal markers, but single lab","pmids":["24936616"],"is_preprint":false},{"year":2021,"finding":"Robo2 function in regenerating peripheral nerve axons is required and sufficient for target-selective regeneration at nerve branch-points; Robo2 acts in response to locally positioned glia to prevent and correct axonal errors during regeneration.","method":"Zebrafish motor nerve regeneration model, cell-type-specific expression and knockdown of robo2, live imaging, glia ablation","journal":"The Journal of neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 — necessity and sufficiency shown with clean genetic tools, but single lab","pmids":["34916258"],"is_preprint":false},{"year":2011,"finding":"Robo2 is required for maintenance of an anti-reflux mechanism at the ureterovesical junction; Robo2-deficient mice develop high-grade VUR due to a dilated and incompetent ureterovesical junction rather than ureteral obstruction; Robo2 is expressed around the developing ureterovesical junction.","method":"High-resolution micro-ultrasonography, microbubble contrast agent, ultrasound-guided aspiration, expression analysis, Robo2 KO mouse model","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo imaging definitively distinguishing obstruction from reflux mechanism, expression localization","pmids":["21949750"],"is_preprint":false},{"year":2020,"finding":"Slit2-mediated growth cone collapse and axonal retraction are eliminated by Nox2 inhibition; slit2 increases growth cone hydrogen peroxide levels via Nox2 activation; astray/nox2 double heterozygotes show reduced retinotectal innervation, placing NADPH oxidase 2 downstream of slit2/Robo2 signaling.","method":"Zebrafish RGC culture, pharmacological Nox inhibition, Nox2 mutant fish, ROS biosensor imaging, growth cone collapse assay, astray/nox2 genetic interaction","journal":"Developmental neurobiology","confidence":"Medium","confidence_rationale":"Tier 2 — ROS imaging links Robo2 signaling to Nox2 activation, confirmed with genetic double heterozygote interaction","pmids":["33191581"],"is_preprint":false},{"year":2021,"finding":"Soluble TREM-1 (sTREM-1) is identified as a novel ligand for Robo2; sTREM-1 binds Robo2 on hepatic stellate cells and activates downstream Smad2/3 and PI3K/Akt signaling pathways, promoting HSC activation and liver fibrosis; HSC-specific knockdown of Robo2 inhibits sTREM-1-induced HSC activation.","method":"Pull-down assay with mass spectrometry (receptor identification), co-IP and immunofluorescence (interaction verification), Robo2 siRNA knockdown in LX-2 cells, AAV-mediated HSC-specific Robo2 knockdown in mouse fibrosis model","journal":"Journal of cellular and molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 — novel ligand identified by unbiased pulldown/MS and confirmed biochemically, with in vivo validation","pmids":["34750987"],"is_preprint":false},{"year":2021,"finding":"The Slit-binding Ig1 domain of Drosophila Robo2 is required for midline repulsion, lateral axon pathway formation, and proper subcellular localization in embryonic neurons; removal of Ig1 from Robo2 (but not Robo1) disrupts axonal localization, suggesting a unique role for this domain in Robo2.","method":"CRISPR/Cas9-based endogenous gene replacement (robo2ΔIg1), in vivo axon guidance analysis, protein localization imaging","journal":"Genesis","confidence":"Medium","confidence_rationale":"Tier 2 — endogenous CRISPR replacement with rigorous in vivo readouts, comparison with Robo1 controls","pmids":["34411419"],"is_preprint":false},{"year":2020,"finding":"ROBO2 expression in podocytes is upregulated after glomerular injury; loss of ROBO2 in podocytes (conditional KO) protects from foot process effacement and proteinuria after glomerular injury (protamine sulfate or nephrotoxic serum); overexpression of ROBO2 in cultured mouse podocytes compromises cell adhesion.","method":"Podocyte-specific Robo2 conditional KO, glomerular injury models (protamine sulfate, NTS), electron microscopy, proteinuria measurement, ROBO2 overexpression in cultured podocytes","journal":"The American journal of pathology","confidence":"High","confidence_rationale":"Tier 2 — conditional KO and overexpression in same cellular context, multiple injury models, ultrastructural and functional readouts","pmids":["32220420"],"is_preprint":false},{"year":2023,"finding":"Robo2 conditional deletion in adult β cells causes loss of pancreatic islet architecture without affecting β cell identity or maturation, demonstrating that Robo2 actively maintains adult islet architecture rather than solely setting it during development.","method":"Adult conditional Robo2 deletion in β cells (inducible Cre), islet architecture quantification, β cell identity/maturation markers","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 — adult conditional KO with defined architectural phenotype, but single lab study","pmids":["37972678"],"is_preprint":false},{"year":2020,"finding":"ROBO2 in the common nephric duct (CND) regulates CND migration and fusion with the primitive bladder via its novel binding partner RALDH2; delayed apoptosis due to failure of CND fusion in Robo2-/- embryos causes abnormal ureter connection; retinoic acid rescues ureter anomalies in Robo2-/- embryos.","method":"Robo2 knockout mouse, protein interaction studies (novel binding partner RALDH2), retinoic acid rescue experiment, apoptosis analysis","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 — novel binding partner identified, pharmacological rescue, but single lab","pmids":["32562756"],"is_preprint":false},{"year":2013,"finding":"Lhx2 transcription factor controls thalamocortical axon guidance and topographic sorting by regulating expression of Robo1 and Robo2 guidance receptors; augmenting Robo1 function restores normal axon guidance in Lhx2-overexpressing neurons, placing Lhx2 upstream of Robo1/Robo2 in thalamocortical circuit formation.","method":"Conditional deletion of Lhx2 in thalamus, Lhx2 overexpression, thalamocortical axon tracing, Robo1/Robo2 expression analysis, Robo1 rescue experiment","journal":"The Journal of neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 — genetic rescue establishing Lhx2→Robo1/2 transcriptional pathway, but not mechanistically dissecting Robo2-specific role","pmids":["22457488"],"is_preprint":false},{"year":2009,"finding":"Robo2 is required in Xenopus RGCs for dendrite branching but not guidance; Slit/Robo2 signaling stimulates dendrite branching via Robo2 (Robo3 has no effect on dendrites); in the same cells, both Robo2 and Robo3 function in concert in axons to mediate axonal guidance and respond to Slits, demonstrating distinct functions of the same receptor in axons versus dendrites.","method":"Antisense knockdown, dominant-negative Robo2/Robo3 constructs, in vitro Xenopus RGC cultures, dendrite and axon morphometry","journal":"Mechanisms of development","confidence":"Medium","confidence_rationale":"Tier 3 — functional dissection of compartment-specific roles with dominant-negative constructs and KD, single lab","pmids":["19961927"],"is_preprint":false},{"year":2022,"finding":"Robo2 and Gen1 coregulate ureteric budding by activating the GDNF/RET pathway and downstream MAPK/ERK signaling, promoting cell proliferation; double gene disruption (Robo2 + Gen1) significantly increases CAKUT phenotypes, and this is rescued by MAPK/ERK inhibitor U0126.","method":"Robo2/Gen1 double mutant mouse model (piggyBac transposon), MAPK/ERK pathway analysis, cell proliferation assay, U0126 pharmacological rescue","journal":"Frontiers in medicine","confidence":"Medium","confidence_rationale":"Tier 2 — genetic interaction with pharmacological rescue confirming pathway, but limited mechanistic dissection of Robo2-specific contribution","pmids":["35071283"],"is_preprint":false},{"year":2004,"finding":"Drosophila robo2 and robo3 are necessary for serotonergic neuron differentiation, functioning independently of their ligand Slit; loss of robo2 or robo3 causes loss of serotonin transporter (SerT) expression and loss of eagle (eg) transcription factor expression in serotonergic neurons; robo2 and eg interact genetically to regulate SerT expression.","method":"Drosophila genetics, loss-of-function mutants, slit mutant comparison, genetic interaction (robo2;eg double mutant), SerT and Eg expression analysis","journal":"Development (Cambridge, England)","confidence":"Medium","confidence_rationale":"Tier 2 — Slit-independent function established by slit mutant comparison, genetic epistasis with transcription factor","pmids":["14973268"],"is_preprint":false},{"year":2026,"finding":"CD47 stabilizes ROBO2 protein by sequestering the E3 ubiquitin ligase ITCH, thereby blocking ubiquitination and proteasomal degradation of ROBO2; loss of CD47 leads to ROBO2 degradation, reducing GBM cell proliferation and migration.","method":"Co-immunoprecipitation (CD47-ITCH-ROBO2 complex), ubiquitination assay, proteasome inhibitor experiments, CD47 and ROBO2 loss-of-function in GBM cells, in vivo tumor burden assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — biochemical complex identification, ubiquitination mechanism established, in vivo validation","pmids":["41871254"],"is_preprint":false},{"year":2025,"finding":"Slit1b/2-Robo2 repulsive signaling in the amacrine cell layer is essential to initiate apical horizontal cell migration during retinal lamination; disruption of this pathway causes basal retention of horizontal cells.","method":"Zebrafish transcriptomics, targeted CRISPR screening, live imaging of horizontal cell migration","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — CRISPR KO and live imaging establishing signaling requirement, but preprint","pmids":["bio_10.1101_2025.07.23.666134"],"is_preprint":true}],"current_model":"ROBO2 is a transmembrane receptor that binds Slit ligands (SLIT1, SLIT2, SLIT3) and other novel ligands (NELL1/2, sTREM-1) through distinct extracellular domains to mediate repulsive axon guidance, cell migration, and organogenesis across multiple tissues; its intracellular signaling activates downstream effectors including Rac1, RhoA, Cdc42, SRGAP1/NMIIA, and PI3K/Akt to regulate cytoskeletal dynamics, cell adhesion, and neuropeptide trafficking, while its protein stability is controlled by ITCH-mediated ubiquitination/proteasomal degradation (antagonized by CD47) and Robo3-mediated lysosomal degradation; in kidney development, Slit2-Robo2 restricts the GDNF expression domain and nephrogenic field to position ureteric bud outgrowth, while in mature podocytes ROBO2 inhibits nephrin-induced actin polymerization and destabilizes adhesion via SRGAP1/NMIIA, and in the nervous system Robo2 acts both cell-autonomously to guide axons and dendrites and non-cell-autonomously (in trans) to modulate Robo1 repulsion."},"narrative":{"teleology":[{"year":2000,"claim":"Establishing ROBO2 as a second Slit receptor required for midline repulsion resolved whether a single Robo receptor was sufficient for all Slit-dependent guidance, showing that Robo1 and Robo2 have non-redundant roles and together account for the full Slit mutant phenotype.","evidence":"Drosophila robo;robo2 double mutant phenocopying slit mutant, axon immunostaining","pmids":["11163264"],"confidence":"High","gaps":["Distinct downstream signaling cascades of Robo1 vs Robo2 not resolved","Vertebrate Robo2 function not yet tested"]},{"year":2002,"claim":"Live imaging of zebrafish robo2 (astray) mutants demonstrated that Robo2 prevents and corrects retinal axon pathfinding errors in vivo, establishing a vertebrate axon guidance function and showing that loss leads to larger, more complex growth cones.","evidence":"Fixed-tissue and time-lapse imaging of zebrafish astray/robo2 mutant retinal axons","pmids":["11804569"],"confidence":"High","gaps":["Whether growth cone morphology change is direct or secondary to guidance errors","Downstream cytoskeletal effectors unknown"]},{"year":2004,"claim":"Discovery that Slit2–Robo2 restricts the GDNF expression domain in nephrogenic mesenchyme to position ureteric bud outgrowth revealed a non-neuronal morphogenetic role, explaining how loss causes supernumerary ureteric buds and kidney malformations.","evidence":"Slit2−/− and Robo2−/− mouse knockouts with in situ hybridization for Gdnf","pmids":["15130495"],"confidence":"High","gaps":["Mechanism by which Robo2 signaling suppresses Gdnf transcription unknown","Whether Robo1 contributes to this restriction not tested"]},{"year":2004,"claim":"A Slit-independent role for Robo2 in serotonergic neuron differentiation was uncovered, showing that Robo2 regulates serotonin transporter expression via the eagle transcription factor independently of ligand binding.","evidence":"Drosophila loss-of-function mutants with slit mutant comparison and robo2;eg genetic epistasis","pmids":["14973268"],"confidence":"Medium","gaps":["How Robo2 signals without Slit in this context is mechanistically unclear","Not confirmed in vertebrate serotonergic neurons"]},{"year":2007,"claim":"Translocation disrupting ROBO2 in human families linked the Slit–ROBO2 pathway to vesicoureteral reflux and CAKUT, providing the first direct human genetic evidence for ROBO2 in urinary tract disease.","evidence":"Translocation mapping, dominant-negative in vitro assay, heterozygous mouse models, family segregation","pmids":["17357069"],"confidence":"High","gaps":["Frequency and penetrance of ROBO2 variants in CAKUT populations not established","Whether dominant-negative mechanism applies to all reported variants unknown"]},{"year":2007,"claim":"Cell-autonomous requirement of Robo2 for limiting retinal ganglion cell arborization and synaptogenesis established that Robo2 controls not only axon pathfinding but also terminal arbor size and synapse number, with Slit1a acting through both Robo2-dependent and -independent mechanisms.","evidence":"Zebrafish astray/robo2 mutant, single-cell arbor imaging, dominant-negative and full-length rescue","pmids":["17640525"],"confidence":"High","gaps":["Robo2-independent Slit1a receptor not identified","Downstream signaling controlling arbor size not mapped"]},{"year":2008,"claim":"Identification of Robo2 on ingressing placode cells interacting with Slit1 on cranial neural crest cells established a non-axonal cell–cell interaction mechanism for tissue assembly during gangliogenesis.","evidence":"Chick cell ablation, Robo2 blocking antibodies, RNAi for receptor and ligand","pmids":["18278043"],"confidence":"High","gaps":["Downstream intracellular effectors in placode cells not identified at this stage"]},{"year":2009,"claim":"Slit1–Robo2 signaling was shown to modulate surface N-cadherin levels post-translationally on placode cells, linking Robo2 to cell adhesion regulation rather than purely repulsive guidance.","evidence":"Bidirectional manipulation (RNAi, dominant-negative, gain-of-function) in chick trigeminal ganglion model","pmids":["19961927","19934013"],"confidence":"High","gaps":["Mechanism of post-translational N-cadherin surface regulation unknown","Whether this adhesion role generalizes beyond placode cells"]},{"year":2010,"claim":"Systematic epistasis across Robo1/2/3 and Slit1-3 mouse mutants distinguished Robo2 as the receptor directing post-crossing axons into the lateral funiculus, resolving the distinct contributions of each Robo family member to commissural axon guidance.","evidence":"Multiple mouse combination mutants with commissural axon trajectory analysis","pmids":["20631173"],"confidence":"High","gaps":["Identity of additional Slit receptor(s) suggested by epistasis not determined"]},{"year":2011,"claim":"Robo2 was placed downstream of MyoD/Myf5 in pioneer myoblasts where it transduces Slit1 signals via RhoA to direct directional migration and fiber formation, extending Robo2 function to myogenesis.","evidence":"Avian somite inversion, Robo2/Slit1 loss-of-function, RhoA pathway analysis","pmids":["21653616"],"confidence":"High","gaps":["Direct RhoA activation mechanism by Robo2 intracellular domain not characterized"]},{"year":2012,"claim":"Discovery that Robo2 forms a complex with nephrin via Nck and inhibits nephrin-induced actin polymerization established a podocyte-specific signaling axis explaining foot process regulation.","evidence":"Co-IP, in vitro actin polymerization assay, Robo2 KO mouse, genetic epistasis with nephrin null","pmids":["22840396"],"confidence":"High","gaps":["Structural basis of Robo2–Nck–nephrin complex not resolved","Whether Slit ligands are required for this interaction in vivo"]},{"year":2015,"claim":"Robo2 was shown to act in trans to inhibit Slit–Robo1 repulsion in pre-crossing commissural axons, revealing a non-cell-autonomous receptor–receptor interaction mechanism distinct from canonical signaling.","evidence":"Drosophila gain-of-function, rescue, cell non-autonomous expression, extracellular domain deletion mapping","pmids":["26186094"],"confidence":"High","gaps":["Biophysical basis of Robo2–Robo1 trans interaction not structurally defined","Whether this trans mechanism operates in vertebrates unknown"]},{"year":2015,"claim":"Slit2–Robo2 was found to drive retinal neovascularization by promoting endothelial migration and Rac1 activation, establishing a pro-angiogenic role that contrasts with its repulsive axon guidance function.","evidence":"Conditional KO mice with retinal angiogenesis, Rac1 activation, endothelial migration assays","pmids":["25894826"],"confidence":"High","gaps":["How the same receptor produces attraction in endothelial cells vs repulsion in neurons not mechanistically resolved"]},{"year":2016,"claim":"A ROBO2/SRGAP1/NMIIA signaling complex was identified in podocytes where Slit2 stimulation inhibits myosin IIA activity and destabilizes focal adhesions, providing the downstream effector pathway for podocyte detachment.","evidence":"Co-IP, direct interaction assay, focal adhesion and cell adhesion assays, podocyte-specific Robo2 KO in hypertension model","pmids":["27882344"],"confidence":"High","gaps":["Whether SRGAP1/NMIIA pathway operates in non-podocyte ROBO2-expressing cells","Structural basis of SRGAP1–MRLC interaction"]},{"year":2018,"claim":"Epithelial Robo2 loss in the pancreas non-cell-autonomously activates myofibroblasts via TGF-β and Wnt signaling, rescued by TGF-β inhibition, revealing a tumor-suppressive stromal regulatory function.","evidence":"Pdx1Cre;Robo2F/F conditional KO, pancreatitis/PDAC models, galunisertib rescue","pmids":["30504844"],"confidence":"High","gaps":["Mechanism by which epithelial Robo2 loss triggers stromal TGF-β activation unclear","Whether Slit ligands are involved in this context not determined"]},{"year":2019,"claim":"NOVA-regulated alternative splicing at a conserved microexon was shown to produce ROBO2 isoforms with temporally distinct guidance activities — early isoforms promoting repulsion, later ones allowing crossing — resolving how the same gene achieves stage-specific guidance decisions.","evidence":"NOVA splicing factor mutant mice, isoform-specific functional assays, commissural axon guidance analysis","pmids":["31392959"],"confidence":"High","gaps":["Whether microexon splicing regulation is conserved across all Robo2-expressing tissues","Structural consequence of microexon inclusion not determined"]},{"year":2019,"claim":"A cryptic NELL1/2-binding site in the first FNIII domain of Robo2 was identified that is conformationally occluded at neutral pH and exposed at acidic pH, revealing a pH-regulated non-Slit ligand interaction and suggesting context-dependent ligand engagement.","evidence":"In vitro binding assays, deletion/point mutagenesis, FRET-based conformational analysis, isoform-dependent affinity measurements","pmids":["30700556","35940226"],"confidence":"Medium","gaps":["No in vivo validation of NELL1/2–Robo2 signaling","Physiological context where acidic pH unmasking occurs not identified"]},{"year":2019,"claim":"Robo2 was found to regulate synaptic oxytocin content by controlling local actin dynamics via Cdc42, establishing a Slit3–Robo2–Cdc42 neuropeptide trafficking pathway.","evidence":"Zebrafish robo2 mutant, live imaging of OXT vesicles, FRAP, dominant-negative Cdc42 epistasis","pmids":["31180321"],"confidence":"High","gaps":["Whether this mechanism generalizes to other neuropeptidergic synapses unknown","Direct link between Robo2 and Cdc42 activation not biochemically shown"]},{"year":2019,"claim":"A non-canonical Robo2 signaling axis through Baiap2/IRSp53 to MDM2 phosphorylation was discovered in renal epithelial cells, where Robo2 loss causes p53-dependent senescence and cystic kidney disease, rescued by p53 co-deletion.","evidence":"Co-IP, phosphorylation assays, Robo2 KO and Robo2/p53 double KO mice, polarity and ciliogenesis analysis","pmids":["31534052"],"confidence":"High","gaps":["Whether this MDM2-p53 axis operates in other Robo2-expressing tissues unknown","Kinase mediating MDM2 Ser166 phosphorylation downstream of Robo2–Baiap2 not identified"]},{"year":2019,"claim":"In Drosophila tendons, Robo2 was shown to have a non-signaling role promoting Slit cleavage to produce the Slit-N guidance signal, demonstrating a ligand-processing function distinct from canonical receptor signaling.","evidence":"Tissue-specific RNAi/rescue, membrane-tethered Slit constructs bypassing Robo2 requirement","pmids":["26400093"],"confidence":"High","gaps":["Mechanism by which Robo2 promotes Slit cleavage unknown","Whether this processing role exists in vertebrate tissues"]},{"year":2020,"claim":"Nox2-generated hydrogen peroxide was placed downstream of Slit2–Robo2 signaling in growth cone collapse, identifying reactive oxygen species as an effector of Robo2-mediated repulsion.","evidence":"Zebrafish RGC culture, pharmacological Nox inhibition, Nox2 mutant, ROS biosensor, astray/nox2 genetic interaction","pmids":["33191581"],"confidence":"Medium","gaps":["How Robo2 activates Nox2 is mechanistically unclear","Whether ROS pathway is specific to retinal neurons or general Robo2 effector"]},{"year":2021,"claim":"Robo2 was identified as a postsynaptic organizer of excitatory synapses in hippocampal CA1 neurons through trans-synaptic interaction with Neurexins, revealing a synaptogenic function beyond axon guidance.","evidence":"Conditional KO, synaptogenesis assays, Co-IP of Robo2–Neurexin, 2-photon calcium imaging in behaving mice","pmids":["34686348"],"confidence":"High","gaps":["Which Neurexin isoform(s) bind Robo2 and which Robo2 domains mediate this interaction","Whether Slit and Neurexin binding are competitive or cooperative"]},{"year":2021,"claim":"Soluble TREM-1 was identified as a non-Slit ligand for Robo2 on hepatic stellate cells, activating Smad2/3 and PI3K/Akt signaling to promote fibrosis, broadening the ligand repertoire beyond Slit family members.","evidence":"Unbiased pull-down/mass spectrometry, Co-IP, HSC-specific Robo2 knockdown in mouse fibrosis model","pmids":["34750987"],"confidence":"Medium","gaps":["Binding domain on Robo2 for sTREM-1 not mapped","Whether sTREM-1–Robo2 interaction is direct or through an intermediary not fully resolved"]},{"year":2025,"claim":"ROBO2 protein stability was shown to be controlled by the E3 ubiquitin ligase ITCH, which ubiquitinates ROBO2 for proteasomal degradation; CD47 stabilizes ROBO2 by sequestering ITCH, linking immune checkpoint signaling to guidance receptor turnover.","evidence":"Co-IP of CD47–ITCH–ROBO2 complex, ubiquitination assays, proteasome inhibitor experiments, in vivo GBM tumor model","pmids":["41871254"],"confidence":"High","gaps":["Specific ubiquitination sites on ROBO2 not mapped","Whether CD47-dependent stabilization of ROBO2 operates outside GBM context"]},{"year":null,"claim":"Major unresolved questions include: (1) how the same receptor produces repulsion in neurons but attraction/migration in endothelial cells and other cell types, (2) the structural basis for isoform- and pH-dependent ligand selectivity (Slit vs NELL1/2 vs sTREM-1 vs Neurexin), and (3) whether the ITCH-mediated degradation and Robo3-mediated lysosomal degradation pathways intersect or operate independently to control ROBO2 protein levels.","evidence":"","pmids":[],"confidence":"Low","gaps":["No unified structural model of full-length ROBO2 ectodomain with ligands","Context-dependent signaling polarity (attraction vs repulsion) mechanistically unexplained","Cross-talk between proteasomal (ITCH) and lysosomal (Robo3) degradation pathways untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,1,3,4,5,8,11,13,20,21]},{"term_id":"GO:0098631","term_label":"cell adhesion mediator activity","supporting_discovery_ids":[9,6,33]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[7,16]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[5,6,21,32,33]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[27]}],"pathway":[{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[0,2,14,34,35]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,6,15,22,31]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[1,4,10,11,13,20,21,26]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[2,23,29,38]}],"complexes":["ROBO2/nephrin/Nck","ROBO2/SRGAP1/NMIIA","ROBO2/Baiap2(IRSp53)"],"partners":["SLIT2","SLIT1","SRGAP1","NRXN1","NEPHRIN","BAIAP2","ITCH","ROBO1"],"other_free_text":[]},"mechanistic_narrative":"ROBO2 is a transmembrane receptor for Slit family ligands that functions as a central mediator of repulsive guidance, cell migration, tissue morphogenesis, and synaptic organization across multiple organ systems. In the nervous system, ROBO2 transduces Slit-dependent repulsive signals to guide axons at the midline, sort olfactory and retinal projections, regulate dendritic branching and synaptogenesis, and control neuropeptide vesicle trafficking via downstream effectors including Rac1, RhoA, Cdc42, and Nox2 [PMID:11163264, PMID:17640525, PMID:31180321, PMID:33191581]; it also acts postsynaptically to promote excitatory synapse formation through trans-synaptic interaction with Neurexins [PMID:34686348]. In kidney development, Slit2–ROBO2 signaling restricts the GDNF expression domain and nephrogenic field to position ureteric bud outgrowth, while in mature podocytes ROBO2 inhibits nephrin-induced actin polymerization and destabilizes focal adhesions through a SRGAP1/NMIIA complex, and loss-of-function mutations cause congenital anomalies of the kidney and urinary tract (CAKUT) including vesicoureteral reflux [PMID:15130495, PMID:22840396, PMID:27882344, PMID:17357069]. ROBO2 protein stability is regulated by ITCH-mediated ubiquitination antagonized by CD47, and by Robo3-dependent lysosomal degradation, while its temporal activity is tuned by NOVA-regulated alternative splicing that controls isoform-specific guidance functions [PMID:41871254, PMID:34916258, PMID:31392959]."},"prefetch_data":{"uniprot":{"accession":"Q9HCK4","full_name":"Roundabout homolog 2","aliases":[],"length_aa":1378,"mass_kda":151.2,"function":"Receptor for SLIT2, and probably SLIT1, which are thought to act as molecular guidance cue in cellular migration, including axonal navigation at the ventral midline of the neural tube and projection of axons to different regions during neuronal development","subcellular_location":"Membrane","url":"https://www.uniprot.org/uniprotkb/Q9HCK4/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ROBO2","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/ROBO2","total_profiled":1310},"omim":[{"mim_id":"620672","title":"LEUCINE-RICH REPEAT-CONTAINING PROTEIN 24; LRRC24","url":"https://www.omim.org/entry/620672"},{"mim_id":"616771","title":"MICRO RNA 218-2; MIR218-2","url":"https://www.omim.org/entry/616771"},{"mim_id":"616770","title":"MICRO RNA 218-1; MIR218-1","url":"https://www.omim.org/entry/616770"},{"mim_id":"610878","title":"VESICOURETERAL REFLUX 2; VUR2","url":"https://www.omim.org/entry/610878"},{"mim_id":"606523","title":"SLIT-ROBO RHO GTPase-ACTIVATING PROTEIN 1; SRGAP1","url":"https://www.omim.org/entry/606523"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Vesicles","reliability":"Approved"},{"location":"Plasma membrane","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"brain","ntpm":10.4},{"tissue":"ovary","ntpm":13.1}],"url":"https://www.proteinatlas.org/search/ROBO2"},"hgnc":{"alias_symbol":["KIAA1568"],"prev_symbol":[]},"alphafold":{"accession":"Q9HCK4","domains":[{"cath_id":"2.60.40.10","chopping":"33-129","consensus_level":"high","plddt":92.321,"start":33,"end":129},{"cath_id":"2.60.40.10","chopping":"132-222","consensus_level":"medium","plddt":92.588,"start":132,"end":222},{"cath_id":"2.60.40.10","chopping":"231-311","consensus_level":"medium","plddt":93.3736,"start":231,"end":311},{"cath_id":"2.60.40.10","chopping":"320-413","consensus_level":"medium","plddt":90.0687,"start":320,"end":413},{"cath_id":"2.60.40.10","chopping":"415-514","consensus_level":"medium","plddt":86.4589,"start":415,"end":514},{"cath_id":"2.60.40.10","chopping":"521-617","consensus_level":"medium","plddt":91.6296,"start":521,"end":617},{"cath_id":"2.60.40.10","chopping":"628-733","consensus_level":"medium","plddt":92.045,"start":628,"end":733},{"cath_id":"2.60.40.10","chopping":"742-834","consensus_level":"high","plddt":83.2137,"start":742,"end":834}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9HCK4","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9HCK4-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9HCK4-F1-predicted_aligned_error_v6.png","plddt_mean":67.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ROBO2","jax_strain_url":"https://www.jax.org/strain/search?query=ROBO2"},"sequence":{"accession":"Q9HCK4","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9HCK4.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9HCK4/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9HCK4"}},"corpus_meta":[{"pmid":"15130495","id":"PMC_15130495","title":"SLIT2-mediated ROBO2 signaling restricts kidney induction to a single site.","date":"2004","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/15130495","citation_count":300,"is_preprint":false},{"pmid":"11163264","id":"PMC_11163264","title":"Short-range and long-range guidance by slit and its Robo receptors. 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loss of either SLIT2 or ROBO2 causes anterior expansion of Gdnf expression and supernumerary ureteric buds.\",\n      \"method\": \"Mouse knockout (Slit2-/- and Robo2-/- mutants), in situ hybridization for Gdnf expression, histological analysis of kidney development\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal loss-of-function in two independent knockout lines with defined molecular phenotype (Gdnf domain expansion), replicated across Slit2 and Robo2 mutants\",\n      \"pmids\": [\"15130495\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Drosophila Robo2 and Robo1 have distinct roles in midline axon guidance; robo,robo2 double mutant phenocopies slit mutant (growth cones enter and fail to leave midline), demonstrating Robo2 is a second Slit receptor that together with Robo1 controls repulsive guidance at the midline.\",\n      \"method\": \"Drosophila genetics, double mutant analysis (robo;robo2 and slit), axon immunostaining\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with double mutant phenocopying ligand loss, foundational paper with >200 citations\",\n      \"pmids\": [\"11163264\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"ROBO2 loss-of-function (translocation disrupting ROBO2) produces dominant-negative ROBO2 proteins that abrogate SLIT-ROBO signaling in vitro, implicating the SLIT-ROBO2 pathway in vesicoureteral reflux and CAKUT pathogenesis.\",\n      \"method\": \"Translocation mapping, dominant-negative in vitro signaling assay, heterozygous mouse models, missense variant segregation analysis\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (translocation, in vitro dominant-negative assay, mouse models, family segregation)\",\n      \"pmids\": [\"17357069\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Slit2 acting through Robo1 and Robo2 promotes endothelial cell migration, Rac1 activation, and lamellipodia formation, driving retinal neovascularization; both receptors are required for Slit2- and VEGF-induced Rac1 activation.\",\n      \"method\": \"Conditional knockout mice (various Slit/Robo combinations), retinal angiogenesis assay, Rac1 activation assay, lamellipodia imaging, endothelial cell migration assay\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with defined cellular phenotype and biochemical readout (Rac1 activation), multiple receptor combinations tested\",\n      \"pmids\": [\"25894826\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Robo2 (astray) in zebrafish shapes retinal ganglion cell axon pathfinding by preventing and correcting pathfinding errors; robo2 mutant growth cones are larger and more complex, make midline and pre/post-midline errors that are rarely corrected, consistent with Slit2 and Slit3 acting as repulsive ligands via Robo2.\",\n      \"method\": \"Fixed-tissue and time-lapse imaging of zebrafish astray/robo2 mutant retinal axons, Slit expression analysis\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — live imaging and genetic loss-of-function with specific morphological phenotype, >130 citations\",\n      \"pmids\": [\"11804569\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Robo2 is expressed on the basal surface of podocytes and forms a complex with nephrin through the adaptor protein Nck; Slit2-Robo2 signaling inhibits nephrin-induced actin polymerization and acts as a negative regulator of podocyte foot process architecture.\",\n      \"method\": \"Co-immunoprecipitation, co-localization studies, in vitro actin polymerization assay, Robo2 knockout mouse analysis, genetic interaction (Robo2 KO crossed to nephrin null)\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — biochemical complex identification (Co-IP), in vitro activity assay, KO mouse with defined phenotype, genetic epistasis\",\n      \"pmids\": [\"22840396\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SLIT2/ROBO2 signaling activates a pathway through SRGAP1 and nonmuscle myosin IIA (NMIIA) in podocytes; MRLC (myosin II regulatory light chain) interacts directly with SRGAP1 and forms a ROBO2/SRGAP1/NMIIA complex in the presence of SLIT2; SLIT2 stimulation inhibits NMIIA activity, decreases focal adhesion formation, and reduces podocyte attachment to collagen.\",\n      \"method\": \"Co-immunoprecipitation, direct protein interaction assay, immunostaining, focal adhesion assay, cell adhesion assay, podocyte-specific Robo2 KO mouse, in vivo hypertension model\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct interaction demonstrated, multiple downstream pathway components identified, in vivo validation with conditional KO\",\n      \"pmids\": [\"27882344\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Robo2 acts in trans (non-cell-autonomously) to inhibit Slit-Robo1 repulsion in pre-crossing commissural axons in Drosophila; Robo2's extracellular domains required for binding to Robo1 are also required for its ability to promote midline crossing, suggesting a receptor-receptor interaction mechanism.\",\n      \"method\": \"Drosophila genetics, gain-of-function and rescue assays, cell non-autonomous expression, extracellular domain deletion analysis\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple genetic assays (GOF, rescue, cell non-autonomous), domain-function mapping\",\n      \"pmids\": [\"26186094\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Robo2 receptor on ingressing placode cells interacts with its cognate ligand Slit1 expressed on early migrating cranial neural crest cells to mediate neural crest-placode interactions required for trigeminal ganglion assembly.\",\n      \"method\": \"Cell ablation experiments in chick, Robo2 function blocking antibodies, RNAi knockdown of Robo2 and Slit1, in situ hybridization for expression patterns\",\n      \"journal\": \"Nature neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple independent loss-of-function approaches (antibody, RNAi for receptor and ligand) with defined cellular phenotype\",\n      \"pmids\": [\"18278043\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Slit1-Robo2 signaling modulates N-cadherin protein levels on the placodal cell surface (post-translationally, without changing total N-cadherin mRNA or protein) to regulate placodal cell adhesion during gangliogenesis; blocking Robo2 decreases surface N-cadherin, while increasing Slit-Robo signaling increases it.\",\n      \"method\": \"In vivo RNAi, dominant-negative Robo2 expression, N-cadherin manipulation, co-expression rescue experiments, in vivo chick trigeminal ganglion model\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — bidirectional manipulation (gain and loss of function) of two pathways with defined molecular and cellular readouts\",\n      \"pmids\": [\"19934013\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Robo1 and Robo2 collaborate to prevent post-crossing commissural axons from reentering gray matter; Robo2 specifically is required for axons to project away from the floor plate into the lateral funiculus, while Robo1 prevents axonal stalling after crossing; epistatic analysis shows the double mutant is less severe than loss of all Slits, suggesting additional Slit receptor(s) exist.\",\n      \"method\": \"Mouse combination mutants (Robo1-/-, Robo2-/-, Robo3-/-, Slit1-3 triple mutant), commissural axon trajectory analysis\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — systematic genetic epistasis across multiple mutant combinations revealing distinct receptor roles\",\n      \"pmids\": [\"20631173\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Robo-2 is required in a subset of olfactory sensory neurons (OSNs) for dorsoventral segregation of axons in the olfactory bulb; Slit-1 and Slit-3 expressed in the ventral OB act as repulsive ligands via Robo-2 to prevent dorsal OSN axons from projecting to the ventral OB.\",\n      \"method\": \"Robo-2 and Slit-1 knockout mice, olfactory axon tracing, expression analysis\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal receptor and ligand knockout phenotypes confirming ligand-receptor pair in specific circuit\",\n      \"pmids\": [\"17715346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Robo2 is the major Slit receptor required for intraretinal axon guidance in mice; Robo1 knockout shows no intraretinal guidance defects, while Robo2 knockout phenocopies Slit1/Slit2 double mutant intraretinal pathfinding errors.\",\n      \"method\": \"Robo1 and Robo2 knockout mice, intraretinal axon tracing and pathfinding error quantification\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — receptor-specific KO phenocopying ligand mutant, comparison with paralog KO establishing receptor specificity\",\n      \"pmids\": [\"19782674\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Slit1a inhibits retinal ganglion cell arborization and synaptogenesis via Robo2 in a cell-autonomous manner; increased arborization in astray/robo2 mutant is phenocopied by dominant-negative Robo2 in single RGCs and rescued by full-length Robo2; Slit1a acts both through Robo2-dependent and Robo2-independent mechanisms.\",\n      \"method\": \"Zebrafish astray/robo2 mutant, single-cell arbor imaging, dominant-negative and rescue constructs, morpholino knockdown, synaptic site labeling (YFP-Rab3)\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cell-autonomous rescue with full-length receptor, dominant-negative phenocopy, genetic pathway analysis\",\n      \"pmids\": [\"17640525\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ROBO2 restricts the nephrogenic field by limiting epithelial/mesenchymal interactions between the Wolffian duct/ureteric epithelium and mesenchyme; loss of Robo2 fails to separate the mesenchyme from Wolffian duct epithelium, exposing mesenchyme to abnormally high proliferative stimuli, expanding the nephrogenic cord cell number and the metanephric mesenchyme field.\",\n      \"method\": \"High-resolution 3D imaging of Robo2-null mouse embryos, ex vivo kidney explant experiments, cell proliferation analysis\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — 3D imaging and ex vivo experiments defining cellular mechanism of field restriction\",\n      \"pmids\": [\"26116176\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SLIT/ROBO2 signaling in mammary basal cells restricts mammary stem cell (MaSC) renewal by negatively regulating WNT signaling; absence of SLIT/ROBO2 leads to increased nuclear β-catenin and repressed expression of p16(INK4a), delaying MaSC senescence.\",\n      \"method\": \"Robo2 conditional knockout mice, mammary stem cell functional assay, β-catenin localization, p16(INK4a) expression analysis\",\n      \"journal\": \"Stem cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with molecular readouts, but single lab study\",\n      \"pmids\": [\"25241737\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In Drosophila tendons, Robo2 plays a non-signaling role by promoting Slit cleavage to produce the cleaved Slit N-terminal guidance signal; tendon-specific Robo2 expression (not muscle-specific) rescues muscle patterning defects, and membrane immobilization of Slit-N bypasses the requirement for tendon Robo2.\",\n      \"method\": \"Drosophila genetics, tissue-specific RNAi and rescue, membrane-tethered Slit constructs, muscle patterning analysis\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — tissue-specific rescue and bypass experiments definitively placing Robo2 in non-signaling Slit processing role\",\n      \"pmids\": [\"26400093\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Pioneer myoblasts express Robo2 downstream of MyoD and Myf5; sclerotome-derived Slit1 acts through Robo2 to direct pioneer myoblast migration and fiber formation via RhoA; loss of Robo2 or sclerotome-derived Slit1 perturbs directional migration and fiber formation.\",\n      \"method\": \"Avian somite inversion experiments, Robo2/Slit1 loss-of-function in chick embryos, RhoA pathway analysis, fiber formation and migration assays\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic pathway (Slit1-Robo2-RhoA) established with tissue-specific loss-of-function\",\n      \"pmids\": [\"21653616\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Robo2 contains a cryptic binding site for NELL1 and NELL2 in its first fibronectin type III (FNIII) domain; this site is normally occluded in intact Robo2 and becomes accessible upon acidic conditions or proteolytic digestion; specific amino acids in the FNIII domain critical for NELL1 binding to Robo2 (but not Robo1) were identified by mutagenesis.\",\n      \"method\": \"Binding assays (NELL1/2 vs Robo family members), deletion mutant analysis, single amino acid mutagenesis, acidic pH binding experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — binding assay with mutagenesis, but in vitro only and single lab\",\n      \"pmids\": [\"30700556\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Conformational change of the Robo2 ectodomain (hairpin-like structure loosening) at acidic pH unmasks the NELL1/2-binding site; the interaction between Ig-like and FNIII domains maintains the occluded conformation at neutral pH; alternative splicing isoforms affect this conformational equilibrium and NELL1/2-binding affinity.\",\n      \"method\": \"FRET-based conformational indicator, size exclusion chromatography, binding assays with isoforms, acidic pH experiments\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — structural/biophysical experiments, but single lab and no in vivo validation\",\n      \"pmids\": [\"35940226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Robo2 regulates synaptic oxytocin content by controlling local actin dynamics via Cdc42; robo2 mutant zebrafish show decreased synaptic OXT levels, reduced Lifeact-EGFP mobility in OXT synapses, and slower vesicle accumulation; dominant-negative Cdc42 (a downstream effector of Robo2) modulates OXT content, placing Slit3-Robo2-Cdc42 in a pathway controlling actin dynamics at OXT synapses.\",\n      \"method\": \"Zebrafish robo2 mutant, live imaging of OXT vesicles and actin probe, FRAP, dominant-negative Cdc42 expression, genetic epistasis\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (live imaging, FRAP, genetic tools) establishing signaling pathway\",\n      \"pmids\": [\"31180321\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Robo2 acts postsynaptically in hippocampal CA1 pyramidal neurons to promote formation of excitatory (but not inhibitory) synapses specifically in proximal dendritic compartments; this synaptogenic activity involves a trans-synaptic interaction with presynaptic Neurexins and also binding to its canonical ligand Slit.\",\n      \"method\": \"In vivo conditional KO, in vitro synaptogenesis assays, co-immunoprecipitation (Robo2-Neurexin interaction), 2-photon Ca2+ imaging of place cells in behaving mice\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal binding partner identification, in vivo KO with defined synaptic phenotype, in vivo circuit-level functional consequence\",\n      \"pmids\": [\"34686348\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Robo2 binds Baiap2 (IRSp53) through its IRSp53/MIM homology domain in renal epithelial cells; this interaction allows Robo2 to phosphorylate MDM2 at Ser166 via Baiap2, maintaining p53 homeostasis; disruption of Robo2-Baiap2 complex causes MDM2 dephosphorylation, elevated active p53, p53-mediated cellular senescence via p21, and decreased polarity/ciliary proteins, leading to cystic kidney disease.\",\n      \"method\": \"Co-immunoprecipitation, phosphorylation assays, Robo2 KO and double KO (Robo2/p53) mouse models, ciliogenesis and polarity analysis\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — complex identification (Co-IP), pathway placement (MDM2 phosphorylation), double KO epistasis rescuing phenotype\",\n      \"pmids\": [\"31534052\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Epithelial Robo2 loss in the pancreas leads to activation of Robo1+ myofibroblasts and induction of TGF-β and Wnt pathways in a non-cell-autonomous manner; TGF-β inhibitor galunisertib suppresses the myofibroblast activation, collagen crosslinking, and immune infiltration caused by Robo2 loss.\",\n      \"method\": \"Pdx1Cre;Robo2F/F conditional KO mice, pancreatitis and PDAC mouse models, cell culture, TGF-β inhibitor treatment, collagen analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with pathway identification, pharmacological rescue confirming TGF-β mechanism\",\n      \"pmids\": [\"30504844\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Robo2 acts as the predominant receptor directing dorsal longitudinal axon tract formation in mouse spinal cord and also has a distinct function in repelling neuron cell bodies from the floor plate; Robo1 is the predominant receptor for ventral longitudinal tracts.\",\n      \"method\": \"Conditional reduction of Robo levels in mouse embryos, genetic sensitization strategy, longitudinal axon tract imaging\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic dissection of receptor isoform roles, but single lab\",\n      \"pmids\": [\"21820427\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Robo2 and Dcc coordinate zebrafish ADt neuron axonal projection choices; Robo2 responds to repellent Slit signals and suppresses attractive Netrin signals; knockdown of Robo2 abolishes the ipsilateral SOT projection from ADt neurons.\",\n      \"method\": \"Zebrafish morpholino knockdown of Robo2 and Dcc/Netrin1, single-cell labeling of ADt neurons, projection pattern analysis\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — morpholino knockdown for multiple pathway components establishing epistatic relationship, but morpholino-only approach\",\n      \"pmids\": [\"22956848\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Mammalian ROBO1 and ROBO2 are subject to temporal regulation via alternative splicing at a conserved microexon; NOVA splicing factors regulate developmental expression of ROBO1 and ROBO2 variants with distinct guidance activities; early isoforms activate axonal repulsion to prevent premature crossing, later isoforms allow crossing, and postcrossing isoforms are disinhibited.\",\n      \"method\": \"Alternative splicing analysis, NOVA splicing factor mutant mice, commissural axon guidance assays, isoform-specific functional assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — isoform-specific functional differences established in vivo with identified upstream regulators (NOVA factors)\",\n      \"pmids\": [\"31392959\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Robo3.1A promotes degradation of Robo2 protein by recruiting it into a late endosome- and lysosome-dependent pathway; cotransfection of Robo3.1A significantly reduces Robo2 protein levels in HEK293 cells and cerebellar granule cells; Robo2 and Robo3 colocalize in intracellular vesicles positive for late endosome/lysosome markers.\",\n      \"method\": \"Cotransfection experiments, cell surface binding assay, co-immunoprecipitation, siRNA knockdown, immunocytochemical colocalization with organelle markers\",\n      \"journal\": \"Journal of neuroscience research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — biochemical evidence of degradation mechanism, colocalization with endosomal markers, but single lab\",\n      \"pmids\": [\"24936616\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Robo2 function in regenerating peripheral nerve axons is required and sufficient for target-selective regeneration at nerve branch-points; Robo2 acts in response to locally positioned glia to prevent and correct axonal errors during regeneration.\",\n      \"method\": \"Zebrafish motor nerve regeneration model, cell-type-specific expression and knockdown of robo2, live imaging, glia ablation\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — necessity and sufficiency shown with clean genetic tools, but single lab\",\n      \"pmids\": [\"34916258\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Robo2 is required for maintenance of an anti-reflux mechanism at the ureterovesical junction; Robo2-deficient mice develop high-grade VUR due to a dilated and incompetent ureterovesical junction rather than ureteral obstruction; Robo2 is expressed around the developing ureterovesical junction.\",\n      \"method\": \"High-resolution micro-ultrasonography, microbubble contrast agent, ultrasound-guided aspiration, expression analysis, Robo2 KO mouse model\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo imaging definitively distinguishing obstruction from reflux mechanism, expression localization\",\n      \"pmids\": [\"21949750\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Slit2-mediated growth cone collapse and axonal retraction are eliminated by Nox2 inhibition; slit2 increases growth cone hydrogen peroxide levels via Nox2 activation; astray/nox2 double heterozygotes show reduced retinotectal innervation, placing NADPH oxidase 2 downstream of slit2/Robo2 signaling.\",\n      \"method\": \"Zebrafish RGC culture, pharmacological Nox inhibition, Nox2 mutant fish, ROS biosensor imaging, growth cone collapse assay, astray/nox2 genetic interaction\",\n      \"journal\": \"Developmental neurobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ROS imaging links Robo2 signaling to Nox2 activation, confirmed with genetic double heterozygote interaction\",\n      \"pmids\": [\"33191581\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Soluble TREM-1 (sTREM-1) is identified as a novel ligand for Robo2; sTREM-1 binds Robo2 on hepatic stellate cells and activates downstream Smad2/3 and PI3K/Akt signaling pathways, promoting HSC activation and liver fibrosis; HSC-specific knockdown of Robo2 inhibits sTREM-1-induced HSC activation.\",\n      \"method\": \"Pull-down assay with mass spectrometry (receptor identification), co-IP and immunofluorescence (interaction verification), Robo2 siRNA knockdown in LX-2 cells, AAV-mediated HSC-specific Robo2 knockdown in mouse fibrosis model\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — novel ligand identified by unbiased pulldown/MS and confirmed biochemically, with in vivo validation\",\n      \"pmids\": [\"34750987\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The Slit-binding Ig1 domain of Drosophila Robo2 is required for midline repulsion, lateral axon pathway formation, and proper subcellular localization in embryonic neurons; removal of Ig1 from Robo2 (but not Robo1) disrupts axonal localization, suggesting a unique role for this domain in Robo2.\",\n      \"method\": \"CRISPR/Cas9-based endogenous gene replacement (robo2ΔIg1), in vivo axon guidance analysis, protein localization imaging\",\n      \"journal\": \"Genesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — endogenous CRISPR replacement with rigorous in vivo readouts, comparison with Robo1 controls\",\n      \"pmids\": [\"34411419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ROBO2 expression in podocytes is upregulated after glomerular injury; loss of ROBO2 in podocytes (conditional KO) protects from foot process effacement and proteinuria after glomerular injury (protamine sulfate or nephrotoxic serum); overexpression of ROBO2 in cultured mouse podocytes compromises cell adhesion.\",\n      \"method\": \"Podocyte-specific Robo2 conditional KO, glomerular injury models (protamine sulfate, NTS), electron microscopy, proteinuria measurement, ROBO2 overexpression in cultured podocytes\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO and overexpression in same cellular context, multiple injury models, ultrastructural and functional readouts\",\n      \"pmids\": [\"32220420\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Robo2 conditional deletion in adult β cells causes loss of pancreatic islet architecture without affecting β cell identity or maturation, demonstrating that Robo2 actively maintains adult islet architecture rather than solely setting it during development.\",\n      \"method\": \"Adult conditional Robo2 deletion in β cells (inducible Cre), islet architecture quantification, β cell identity/maturation markers\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — adult conditional KO with defined architectural phenotype, but single lab study\",\n      \"pmids\": [\"37972678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ROBO2 in the common nephric duct (CND) regulates CND migration and fusion with the primitive bladder via its novel binding partner RALDH2; delayed apoptosis due to failure of CND fusion in Robo2-/- embryos causes abnormal ureter connection; retinoic acid rescues ureter anomalies in Robo2-/- embryos.\",\n      \"method\": \"Robo2 knockout mouse, protein interaction studies (novel binding partner RALDH2), retinoic acid rescue experiment, apoptosis analysis\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — novel binding partner identified, pharmacological rescue, but single lab\",\n      \"pmids\": [\"32562756\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Lhx2 transcription factor controls thalamocortical axon guidance and topographic sorting by regulating expression of Robo1 and Robo2 guidance receptors; augmenting Robo1 function restores normal axon guidance in Lhx2-overexpressing neurons, placing Lhx2 upstream of Robo1/Robo2 in thalamocortical circuit formation.\",\n      \"method\": \"Conditional deletion of Lhx2 in thalamus, Lhx2 overexpression, thalamocortical axon tracing, Robo1/Robo2 expression analysis, Robo1 rescue experiment\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic rescue establishing Lhx2→Robo1/2 transcriptional pathway, but not mechanistically dissecting Robo2-specific role\",\n      \"pmids\": [\"22457488\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Robo2 is required in Xenopus RGCs for dendrite branching but not guidance; Slit/Robo2 signaling stimulates dendrite branching via Robo2 (Robo3 has no effect on dendrites); in the same cells, both Robo2 and Robo3 function in concert in axons to mediate axonal guidance and respond to Slits, demonstrating distinct functions of the same receptor in axons versus dendrites.\",\n      \"method\": \"Antisense knockdown, dominant-negative Robo2/Robo3 constructs, in vitro Xenopus RGC cultures, dendrite and axon morphometry\",\n      \"journal\": \"Mechanisms of development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — functional dissection of compartment-specific roles with dominant-negative constructs and KD, single lab\",\n      \"pmids\": [\"19961927\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Robo2 and Gen1 coregulate ureteric budding by activating the GDNF/RET pathway and downstream MAPK/ERK signaling, promoting cell proliferation; double gene disruption (Robo2 + Gen1) significantly increases CAKUT phenotypes, and this is rescued by MAPK/ERK inhibitor U0126.\",\n      \"method\": \"Robo2/Gen1 double mutant mouse model (piggyBac transposon), MAPK/ERK pathway analysis, cell proliferation assay, U0126 pharmacological rescue\",\n      \"journal\": \"Frontiers in medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic interaction with pharmacological rescue confirming pathway, but limited mechanistic dissection of Robo2-specific contribution\",\n      \"pmids\": [\"35071283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Drosophila robo2 and robo3 are necessary for serotonergic neuron differentiation, functioning independently of their ligand Slit; loss of robo2 or robo3 causes loss of serotonin transporter (SerT) expression and loss of eagle (eg) transcription factor expression in serotonergic neurons; robo2 and eg interact genetically to regulate SerT expression.\",\n      \"method\": \"Drosophila genetics, loss-of-function mutants, slit mutant comparison, genetic interaction (robo2;eg double mutant), SerT and Eg expression analysis\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Slit-independent function established by slit mutant comparison, genetic epistasis with transcription factor\",\n      \"pmids\": [\"14973268\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"CD47 stabilizes ROBO2 protein by sequestering the E3 ubiquitin ligase ITCH, thereby blocking ubiquitination and proteasomal degradation of ROBO2; loss of CD47 leads to ROBO2 degradation, reducing GBM cell proliferation and migration.\",\n      \"method\": \"Co-immunoprecipitation (CD47-ITCH-ROBO2 complex), ubiquitination assay, proteasome inhibitor experiments, CD47 and ROBO2 loss-of-function in GBM cells, in vivo tumor burden assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — biochemical complex identification, ubiquitination mechanism established, in vivo validation\",\n      \"pmids\": [\"41871254\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Slit1b/2-Robo2 repulsive signaling in the amacrine cell layer is essential to initiate apical horizontal cell migration during retinal lamination; disruption of this pathway causes basal retention of horizontal cells.\",\n      \"method\": \"Zebrafish transcriptomics, targeted CRISPR screening, live imaging of horizontal cell migration\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — CRISPR KO and live imaging establishing signaling requirement, but preprint\",\n      \"pmids\": [\"bio_10.1101_2025.07.23.666134\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"ROBO2 is a transmembrane receptor that binds Slit ligands (SLIT1, SLIT2, SLIT3) and other novel ligands (NELL1/2, sTREM-1) through distinct extracellular domains to mediate repulsive axon guidance, cell migration, and organogenesis across multiple tissues; its intracellular signaling activates downstream effectors including Rac1, RhoA, Cdc42, SRGAP1/NMIIA, and PI3K/Akt to regulate cytoskeletal dynamics, cell adhesion, and neuropeptide trafficking, while its protein stability is controlled by ITCH-mediated ubiquitination/proteasomal degradation (antagonized by CD47) and Robo3-mediated lysosomal degradation; in kidney development, Slit2-Robo2 restricts the GDNF expression domain and nephrogenic field to position ureteric bud outgrowth, while in mature podocytes ROBO2 inhibits nephrin-induced actin polymerization and destabilizes adhesion via SRGAP1/NMIIA, and in the nervous system Robo2 acts both cell-autonomously to guide axons and dendrites and non-cell-autonomously (in trans) to modulate Robo1 repulsion.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"ROBO2 is a transmembrane receptor for Slit family ligands that functions as a central mediator of repulsive guidance, cell migration, tissue morphogenesis, and synaptic organization across multiple organ systems. In the nervous system, ROBO2 transduces Slit-dependent repulsive signals to guide axons at the midline, sort olfactory and retinal projections, regulate dendritic branching and synaptogenesis, and control neuropeptide vesicle trafficking via downstream effectors including Rac1, RhoA, Cdc42, and Nox2 [PMID:11163264, PMID:17640525, PMID:31180321, PMID:33191581]; it also acts postsynaptically to promote excitatory synapse formation through trans-synaptic interaction with Neurexins [PMID:34686348]. In kidney development, Slit2–ROBO2 signaling restricts the GDNF expression domain and nephrogenic field to position ureteric bud outgrowth, while in mature podocytes ROBO2 inhibits nephrin-induced actin polymerization and destabilizes focal adhesions through a SRGAP1/NMIIA complex, and loss-of-function mutations cause congenital anomalies of the kidney and urinary tract (CAKUT) including vesicoureteral reflux [PMID:15130495, PMID:22840396, PMID:27882344, PMID:17357069]. ROBO2 protein stability is regulated by ITCH-mediated ubiquitination antagonized by CD47, and by Robo3-dependent lysosomal degradation, while its temporal activity is tuned by NOVA-regulated alternative splicing that controls isoform-specific guidance functions [PMID:41871254, PMID:34916258, PMID:31392959].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Establishing ROBO2 as a second Slit receptor required for midline repulsion resolved whether a single Robo receptor was sufficient for all Slit-dependent guidance, showing that Robo1 and Robo2 have non-redundant roles and together account for the full Slit mutant phenotype.\",\n      \"evidence\": \"Drosophila robo;robo2 double mutant phenocopying slit mutant, axon immunostaining\",\n      \"pmids\": [\"11163264\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Distinct downstream signaling cascades of Robo1 vs Robo2 not resolved\", \"Vertebrate Robo2 function not yet tested\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Live imaging of zebrafish robo2 (astray) mutants demonstrated that Robo2 prevents and corrects retinal axon pathfinding errors in vivo, establishing a vertebrate axon guidance function and showing that loss leads to larger, more complex growth cones.\",\n      \"evidence\": \"Fixed-tissue and time-lapse imaging of zebrafish astray/robo2 mutant retinal axons\",\n      \"pmids\": [\"11804569\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether growth cone morphology change is direct or secondary to guidance errors\", \"Downstream cytoskeletal effectors unknown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Discovery that Slit2–Robo2 restricts the GDNF expression domain in nephrogenic mesenchyme to position ureteric bud outgrowth revealed a non-neuronal morphogenetic role, explaining how loss causes supernumerary ureteric buds and kidney malformations.\",\n      \"evidence\": \"Slit2−/− and Robo2−/− mouse knockouts with in situ hybridization for Gdnf\",\n      \"pmids\": [\"15130495\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which Robo2 signaling suppresses Gdnf transcription unknown\", \"Whether Robo1 contributes to this restriction not tested\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"A Slit-independent role for Robo2 in serotonergic neuron differentiation was uncovered, showing that Robo2 regulates serotonin transporter expression via the eagle transcription factor independently of ligand binding.\",\n      \"evidence\": \"Drosophila loss-of-function mutants with slit mutant comparison and robo2;eg genetic epistasis\",\n      \"pmids\": [\"14973268\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How Robo2 signals without Slit in this context is mechanistically unclear\", \"Not confirmed in vertebrate serotonergic neurons\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Translocation disrupting ROBO2 in human families linked the Slit–ROBO2 pathway to vesicoureteral reflux and CAKUT, providing the first direct human genetic evidence for ROBO2 in urinary tract disease.\",\n      \"evidence\": \"Translocation mapping, dominant-negative in vitro assay, heterozygous mouse models, family segregation\",\n      \"pmids\": [\"17357069\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Frequency and penetrance of ROBO2 variants in CAKUT populations not established\", \"Whether dominant-negative mechanism applies to all reported variants unknown\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Cell-autonomous requirement of Robo2 for limiting retinal ganglion cell arborization and synaptogenesis established that Robo2 controls not only axon pathfinding but also terminal arbor size and synapse number, with Slit1a acting through both Robo2-dependent and -independent mechanisms.\",\n      \"evidence\": \"Zebrafish astray/robo2 mutant, single-cell arbor imaging, dominant-negative and full-length rescue\",\n      \"pmids\": [\"17640525\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Robo2-independent Slit1a receptor not identified\", \"Downstream signaling controlling arbor size not mapped\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identification of Robo2 on ingressing placode cells interacting with Slit1 on cranial neural crest cells established a non-axonal cell–cell interaction mechanism for tissue assembly during gangliogenesis.\",\n      \"evidence\": \"Chick cell ablation, Robo2 blocking antibodies, RNAi for receptor and ligand\",\n      \"pmids\": [\"18278043\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream intracellular effectors in placode cells not identified at this stage\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Slit1–Robo2 signaling was shown to modulate surface N-cadherin levels post-translationally on placode cells, linking Robo2 to cell adhesion regulation rather than purely repulsive guidance.\",\n      \"evidence\": \"Bidirectional manipulation (RNAi, dominant-negative, gain-of-function) in chick trigeminal ganglion model\",\n      \"pmids\": [\"19961927\", \"19934013\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of post-translational N-cadherin surface regulation unknown\", \"Whether this adhesion role generalizes beyond placode cells\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Systematic epistasis across Robo1/2/3 and Slit1-3 mouse mutants distinguished Robo2 as the receptor directing post-crossing axons into the lateral funiculus, resolving the distinct contributions of each Robo family member to commissural axon guidance.\",\n      \"evidence\": \"Multiple mouse combination mutants with commissural axon trajectory analysis\",\n      \"pmids\": [\"20631173\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of additional Slit receptor(s) suggested by epistasis not determined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Robo2 was placed downstream of MyoD/Myf5 in pioneer myoblasts where it transduces Slit1 signals via RhoA to direct directional migration and fiber formation, extending Robo2 function to myogenesis.\",\n      \"evidence\": \"Avian somite inversion, Robo2/Slit1 loss-of-function, RhoA pathway analysis\",\n      \"pmids\": [\"21653616\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct RhoA activation mechanism by Robo2 intracellular domain not characterized\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Discovery that Robo2 forms a complex with nephrin via Nck and inhibits nephrin-induced actin polymerization established a podocyte-specific signaling axis explaining foot process regulation.\",\n      \"evidence\": \"Co-IP, in vitro actin polymerization assay, Robo2 KO mouse, genetic epistasis with nephrin null\",\n      \"pmids\": [\"22840396\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of Robo2–Nck–nephrin complex not resolved\", \"Whether Slit ligands are required for this interaction in vivo\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Robo2 was shown to act in trans to inhibit Slit–Robo1 repulsion in pre-crossing commissural axons, revealing a non-cell-autonomous receptor–receptor interaction mechanism distinct from canonical signaling.\",\n      \"evidence\": \"Drosophila gain-of-function, rescue, cell non-autonomous expression, extracellular domain deletion mapping\",\n      \"pmids\": [\"26186094\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Biophysical basis of Robo2–Robo1 trans interaction not structurally defined\", \"Whether this trans mechanism operates in vertebrates unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Slit2–Robo2 was found to drive retinal neovascularization by promoting endothelial migration and Rac1 activation, establishing a pro-angiogenic role that contrasts with its repulsive axon guidance function.\",\n      \"evidence\": \"Conditional KO mice with retinal angiogenesis, Rac1 activation, endothelial migration assays\",\n      \"pmids\": [\"25894826\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the same receptor produces attraction in endothelial cells vs repulsion in neurons not mechanistically resolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"A ROBO2/SRGAP1/NMIIA signaling complex was identified in podocytes where Slit2 stimulation inhibits myosin IIA activity and destabilizes focal adhesions, providing the downstream effector pathway for podocyte detachment.\",\n      \"evidence\": \"Co-IP, direct interaction assay, focal adhesion and cell adhesion assays, podocyte-specific Robo2 KO in hypertension model\",\n      \"pmids\": [\"27882344\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SRGAP1/NMIIA pathway operates in non-podocyte ROBO2-expressing cells\", \"Structural basis of SRGAP1–MRLC interaction\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Epithelial Robo2 loss in the pancreas non-cell-autonomously activates myofibroblasts via TGF-β and Wnt signaling, rescued by TGF-β inhibition, revealing a tumor-suppressive stromal regulatory function.\",\n      \"evidence\": \"Pdx1Cre;Robo2F/F conditional KO, pancreatitis/PDAC models, galunisertib rescue\",\n      \"pmids\": [\"30504844\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which epithelial Robo2 loss triggers stromal TGF-β activation unclear\", \"Whether Slit ligands are involved in this context not determined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"NOVA-regulated alternative splicing at a conserved microexon was shown to produce ROBO2 isoforms with temporally distinct guidance activities — early isoforms promoting repulsion, later ones allowing crossing — resolving how the same gene achieves stage-specific guidance decisions.\",\n      \"evidence\": \"NOVA splicing factor mutant mice, isoform-specific functional assays, commissural axon guidance analysis\",\n      \"pmids\": [\"31392959\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether microexon splicing regulation is conserved across all Robo2-expressing tissues\", \"Structural consequence of microexon inclusion not determined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"A cryptic NELL1/2-binding site in the first FNIII domain of Robo2 was identified that is conformationally occluded at neutral pH and exposed at acidic pH, revealing a pH-regulated non-Slit ligand interaction and suggesting context-dependent ligand engagement.\",\n      \"evidence\": \"In vitro binding assays, deletion/point mutagenesis, FRET-based conformational analysis, isoform-dependent affinity measurements\",\n      \"pmids\": [\"30700556\", \"35940226\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vivo validation of NELL1/2–Robo2 signaling\", \"Physiological context where acidic pH unmasking occurs not identified\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Robo2 was found to regulate synaptic oxytocin content by controlling local actin dynamics via Cdc42, establishing a Slit3–Robo2–Cdc42 neuropeptide trafficking pathway.\",\n      \"evidence\": \"Zebrafish robo2 mutant, live imaging of OXT vesicles, FRAP, dominant-negative Cdc42 epistasis\",\n      \"pmids\": [\"31180321\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this mechanism generalizes to other neuropeptidergic synapses unknown\", \"Direct link between Robo2 and Cdc42 activation not biochemically shown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"A non-canonical Robo2 signaling axis through Baiap2/IRSp53 to MDM2 phosphorylation was discovered in renal epithelial cells, where Robo2 loss causes p53-dependent senescence and cystic kidney disease, rescued by p53 co-deletion.\",\n      \"evidence\": \"Co-IP, phosphorylation assays, Robo2 KO and Robo2/p53 double KO mice, polarity and ciliogenesis analysis\",\n      \"pmids\": [\"31534052\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this MDM2-p53 axis operates in other Robo2-expressing tissues unknown\", \"Kinase mediating MDM2 Ser166 phosphorylation downstream of Robo2–Baiap2 not identified\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"In Drosophila tendons, Robo2 was shown to have a non-signaling role promoting Slit cleavage to produce the Slit-N guidance signal, demonstrating a ligand-processing function distinct from canonical receptor signaling.\",\n      \"evidence\": \"Tissue-specific RNAi/rescue, membrane-tethered Slit constructs bypassing Robo2 requirement\",\n      \"pmids\": [\"26400093\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which Robo2 promotes Slit cleavage unknown\", \"Whether this processing role exists in vertebrate tissues\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Nox2-generated hydrogen peroxide was placed downstream of Slit2–Robo2 signaling in growth cone collapse, identifying reactive oxygen species as an effector of Robo2-mediated repulsion.\",\n      \"evidence\": \"Zebrafish RGC culture, pharmacological Nox inhibition, Nox2 mutant, ROS biosensor, astray/nox2 genetic interaction\",\n      \"pmids\": [\"33191581\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How Robo2 activates Nox2 is mechanistically unclear\", \"Whether ROS pathway is specific to retinal neurons or general Robo2 effector\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Robo2 was identified as a postsynaptic organizer of excitatory synapses in hippocampal CA1 neurons through trans-synaptic interaction with Neurexins, revealing a synaptogenic function beyond axon guidance.\",\n      \"evidence\": \"Conditional KO, synaptogenesis assays, Co-IP of Robo2–Neurexin, 2-photon calcium imaging in behaving mice\",\n      \"pmids\": [\"34686348\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which Neurexin isoform(s) bind Robo2 and which Robo2 domains mediate this interaction\", \"Whether Slit and Neurexin binding are competitive or cooperative\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Soluble TREM-1 was identified as a non-Slit ligand for Robo2 on hepatic stellate cells, activating Smad2/3 and PI3K/Akt signaling to promote fibrosis, broadening the ligand repertoire beyond Slit family members.\",\n      \"evidence\": \"Unbiased pull-down/mass spectrometry, Co-IP, HSC-specific Robo2 knockdown in mouse fibrosis model\",\n      \"pmids\": [\"34750987\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Binding domain on Robo2 for sTREM-1 not mapped\", \"Whether sTREM-1–Robo2 interaction is direct or through an intermediary not fully resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"ROBO2 protein stability was shown to be controlled by the E3 ubiquitin ligase ITCH, which ubiquitinates ROBO2 for proteasomal degradation; CD47 stabilizes ROBO2 by sequestering ITCH, linking immune checkpoint signaling to guidance receptor turnover.\",\n      \"evidence\": \"Co-IP of CD47–ITCH–ROBO2 complex, ubiquitination assays, proteasome inhibitor experiments, in vivo GBM tumor model\",\n      \"pmids\": [\"41871254\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific ubiquitination sites on ROBO2 not mapped\", \"Whether CD47-dependent stabilization of ROBO2 operates outside GBM context\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Major unresolved questions include: (1) how the same receptor produces repulsion in neurons but attraction/migration in endothelial cells and other cell types, (2) the structural basis for isoform- and pH-dependent ligand selectivity (Slit vs NELL1/2 vs sTREM-1 vs Neurexin), and (3) whether the ITCH-mediated degradation and Robo3-mediated lysosomal degradation pathways intersect or operate independently to control ROBO2 protein levels.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unified structural model of full-length ROBO2 ectodomain with ligands\", \"Context-dependent signaling polarity (attraction vs repulsion) mechanistically unexplained\", \"Cross-talk between proteasomal (ITCH) and lysosomal (Robo3) degradation pathways untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 1, 3, 4, 5, 8, 11, 13, 20, 21]},\n      {\"term_id\": \"GO:0098631\", \"supporting_discovery_ids\": [9, 6, 33]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [7, 16]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [5, 6, 21, 32, 33]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [27]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0, 2, 14, 34, 35]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 6, 15, 22, 31]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [1, 4, 10, 11, 13, 20, 21, 26]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [2, 23, 29, 38]}\n    ],\n    \"complexes\": [\n      \"ROBO2/nephrin/Nck\",\n      \"ROBO2/SRGAP1/NMIIA\",\n      \"ROBO2/Baiap2(IRSp53)\"\n    ],\n    \"partners\": [\n      \"SLIT2\",\n      \"SLIT1\",\n      \"SRGAP1\",\n      \"NRXN1\",\n      \"NEPHRIN\",\n      \"BAIAP2\",\n      \"ITCH\",\n      \"ROBO1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}