CD93 Mouse

What cell types express CD93 in the mouse central nervous system?

CD93 expression in the mouse CNS exhibits a specific cellular distribution pattern. It is predominantly expressed by neurons, vascular endothelial cells, and ramified microglia . Importantly, CD93 is not expressed in astrocytes or oligodendrocytes . This distinctive expression pattern suggests cell-type specific functions in the CNS environment. When analyzing CD93 expression in tissue samples, immunohistochemical staining reveals that CD93 localizes differently depending on the cell type - appearing at the neuronal cell membrane and in cytoplasmic vesicles in neurons, while showing exclusively membrane localization in endothelial cells .

What are the primary phenotypic changes observed in CD93 knockout mice?

CD93 knockout (CD93^-/-) mice exhibit multiple phenotypic alterations across different physiological systems. In the central nervous system, these mice display increased astrogenesis during neural development at the expense of neuron production . Behaviorally, CD93^-/- mice demonstrate autism-like behaviors, suggesting neurodevelopmental implications . From an immunological perspective, CD93-deficient mice show more robust brain and spinal cord inflammation in experimental autoimmune encephalomyelitis (EAE) models, characterized by increased numbers of infiltrating M1 macrophages and more activated microglial phenotypes . Additionally, CD93^-/- mice demonstrate defects in the clearance of apoptotic cells, although the direct engulfment capability of their macrophages appears unaffected in ex vivo studies .

How does CD93 expression change during mouse neural development?

During mouse neural development, CD93 expression follows a temporal pattern that correlates with the sequential differentiation of neural stem cells. Studies indicate that CD93 is expressed in neural stem cells and neurons but not in astrocytes . Importantly, CD93 expression declines as differentiation proceeds . This temporal regulation appears critical for proper timing of astrogenesis, as CD93 functions as a negative regulator of astrocyte production during the early embryonic period . The declining expression of CD93 over time corresponds with the transition from neurogenesis to astrogenesis during cortical development.

Through what molecular pathway does CD93 regulate astrogenesis in mice?

CD93 regulates astrogenesis through a complex molecular signaling cascade. When CD93 recognizes and binds to its ligand Multimerin 2 (MMRN2) in the extracellular environment, it initiates a signaling pathway that ultimately represses astrocyte differentiation . Mechanistically, CD93 delivers signals to β-Catenin through a series of phosphorylation cascades . β-Catenin then translocates to the nucleus where it activates the transcription of Zfp503 . This transcriptional repressor, ZFP503, subsequently inhibits the transcription of glial fibrillary acidic protein (Gfap) by binding to the Gfap promoter with the assistance of Grg5 . Since GFAP is a key marker and functional protein in astrocytes, this repression effectively inhibits astrogenesis during early developmental stages.

How does glycosylation affect CD93 function and stability?

Glycosylation plays a critical role in maintaining CD93 stability on the cell surface. Research demonstrates that inhibition of CD93 glycosylation (through treatment with benzyl 2-acetamido-2-deoxy-alpha-D-galactopyranoside or expression in glycosylation-defective cell lines) results in decreased CD93 expression on the cell surface with concurrent detection of CD93 in culture media . This indicates that O-glycosylation of CD93 is essential for its stable expression at the cell surface . Without proper glycosylation, CD93 is rapidly released into the extracellular environment, which could significantly alter its biological functions including its roles in angiogenesis, phagocytosis, and neural development.

What is the relationship between CD93 and blood-brain barrier integrity?

CD93 functions as a critical regulator of blood-brain barrier (BBB) integrity during inflammatory conditions. In experimental models of CNS inflammation, CD93-deficient mice exhibit more severe disruption of the BBB compared to wild-type counterparts . This is evidenced by increased fibrinogen transudation into the brain parenchyma, particularly pronounced in white matter regions but also present in gray matter . The compromised BBB integrity likely contributes to the elevated recruitment of immune cells into the brain parenchyma observed in CD93^-/- mice during neuroinflammation . This suggests that CD93 normally functions to maintain BBB integrity during inflammatory challenges, potentially through its expression on vascular endothelial cells that form the BBB.

How does CD93 deficiency affect experimental autoimmune encephalomyelitis (EAE) progression?

CD93 deficiency significantly exacerbates the inflammatory response in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. CD93^-/- mice exhibit more robust brain and spinal cord inflammation compared to wild-type mice with EAE . This enhanced inflammation is characterized by increased weight loss, greater fibrinogen deposition in brain parenchyma (indicating BBB disruption), and significantly more infiltrating immune cells expressing cyclooxygenase-2 (Cox2) . The enhanced inflammatory response in CD93-deficient mice suggests that CD93 normally functions as a negative regulator of neuroinflammation, working to control and limit the inflammatory processes in the CNS during autoimmune challenges.

What changes occur in microglial phenotype in CD93-deficient mice during inflammation?

In CD93-deficient mice, microglia demonstrate a more activated phenotype during neuroinflammatory conditions. During EAE, CD93^-/- mice show increased numbers of amoeboid microglia that express higher levels of activation markers compared to wild-type controls . Specifically, these microglia exhibit elevated expression of Tomato Lectin and cyclooxygenase-2 (Cox2), indicating a more pronounced pro-inflammatory state . This enhanced microglial activation likely contributes to the more severe neuroinflammation and neuronal injury observed in CD93-deficient mice. The data suggest that CD93 normally acts to restrain microglial activation during inflammatory challenges, serving as a neuro-immune regulator in the CNS.

How does CD93 influence macrophage polarization during CNS inflammation?

CD93 plays a significant role in regulating macrophage polarization during CNS inflammation. In EAE models, CD93-deficient mice show increased numbers of infiltrating M1 macrophages (identified as CD11c+ CD206-) in the CNS . This suggests that CD93 normally functions to limit the infiltration of pro-inflammatory M1 macrophages or potentially influences the polarization state of these cells. The enhanced M1 macrophage presence in CD93^-/- mice likely contributes to the more severe inflammation and tissue damage observed in these animals. This finding positions CD93 as an important regulator of the innate immune response in the CNS, potentially through controlling the balance of pro-inflammatory versus anti-inflammatory macrophage populations.

What methods are available for detecting CD93 expression in mouse tissue samples?

Multiple methodological approaches can be employed to detect CD93 expression in mouse tissue samples. Immunohistochemistry using specific anti-CD93 antibodies is effective for visualizing CD93 localization in tissue sections, with rabbit anti-mouse CD93 against full-length fusion protein being one validated option . For multi-label immunofluorescence studies, researchers can combine CD93 staining with markers for specific cell types (e.g., NeuN for neurons, CD11b for microglia, CD34 for endothelial cells) to assess cell-specific expression patterns . Western blot analysis provides quantitative assessment of CD93 protein levels, typically using antibodies at a 1:200 dilution alongside appropriate loading controls . For gene expression analysis, PCR techniques can be employed to measure CD93 mRNA levels. When interpreting results, it's important to consider that CD93 expression varies significantly between cell types and can be altered during development or inflammatory conditions.

How can nanobodies against CD93 be generated and characterized?

Nanobodies (Nbs) against CD93 can be generated and characterized through a systematic process involving phage display technology. The process begins with solid-phase screening to identify CD93-specific Nbs . Following multiple rounds of panning, phage titer assays can confirm enrichment of antigen-bound clones, with successful enrichment typically showing a ~100-fold increase in bound clones in later rounds . Individual clones are then evaluated by monoclonal phage ELISA, with positive clones identified by target/negative (T/N) ratios exceeding 2.0 . Selected positive clones undergo DNA sequencing to determine their complementarity-determining regions (CDRs), particularly CDR3 which often exhibits the greatest variability . For protein expression, Nb genes can be inserted into appropriate vectors (e.g., PET24a) and expressed in E. coli BL21 (DE3) after IPTG induction, followed by purification using Ni-NTA affinity chromatography . Characterization of nanobody affinity involves indirect ELISA to calculate equilibrium association constants, while binding specificity can be assessed using flow cytometry with appropriate cell lines expressing CD93 .

What are the key considerations when designing experiments with CD93 knockout mice?

When designing experiments with CD93 knockout mice, several critical factors must be considered to ensure robust and interpretable results. First, appropriate backcrossing to establish the knockout on a pure genetic background (typically C57BL/6) is essential for minimizing confounding genetic effects . Age-, strain-, and sex-matched mice must be used as controls in each experiment to account for these variables . When investigating neuroinflammatory phenotypes, established models such as MOG-induced EAE or antibody-dependent EAE provide standardized frameworks for assessing CD93's role . Comprehensive phenotyping should include behavioral assessments (particularly for autism-like behaviors), histological analysis for cellular changes, immunostaining for inflammatory markers, and evaluation of BBB integrity through fibrinogen transudation assays . When studying developmental phenotypes, temporal considerations are crucial, as CD93's effects on neural differentiation are stage-specific . Finally, for mechanistic studies, both in vivo and ex vivo approaches may be necessary, as some CD93-dependent phenomena (like apoptotic cell clearance) show differences between these contexts .

How can molecular docking be used to study CD93 interactions with potential binding partners?

Molecular docking provides valuable insights into CD93 interactions with potential binding partners at the structural level. For CD93 research, the process begins with homology modeling of the protein structures using tools like I-TASSER, which generates tertiary structure models evaluated by confidence metrics including C-score (ideally between -5 and 2) and TM-score (optimally >0.5) . Once high-quality structural models are obtained, HADDOCK software can perform semiflexible docking between CD93 and potential binding partners, using solvent-exposed residues for interaction analysis . The quality of docking models is assessed by Z-scores (more negative values indicating better models) and RMSD values relative to the lowest energy structure . Post-docking analysis with tools like PDBePISA identifies specific interacting residues, categorizing them into interactions such as hydrogen bonds and salt bridges . This approach has successfully identified paratope-epitope interactions between CD93 and nanobodies, revealing that only select amino acids (e.g., ASN265) may be involved in multiple binding interactions . Molecular docking thus provides structural insights that complement experimental approaches and can guide the development of therapeutic agents targeting CD93.

What is known about the interaction between CD93 and MMRN2 in neural development?

The interaction between CD93 and its ligand Multimerin 2 (MMRN2) represents a critical extracellular signaling mechanism in neural development. Research has established that CD93 responds specifically to MMRN2 to trigger the repression of astrogenesis during early embryonic development . This ligand-receptor interaction initiates a downstream signaling cascade that ultimately regulates the timing of astrocyte production. The binding of MMRN2 to CD93 leads to signal transduction through β-Catenin, involving phosphorylation cascades that ultimately regulate gene expression . The specificity of this interaction suggests a precise extracellular control mechanism for neural stem cell fate determination. Understanding the structural aspects of CD93-MMRN2 binding could provide insights into potential therapeutic interventions for neurodevelopmental disorders, particularly those involving abnormal gliogenesis or the autism-like behaviors observed in CD93 knockout mice .