EPHB4 Mouse

What is EPHB4 and what is its significance in mouse models?

EPHB4 is a receptor tyrosine kinase that belongs to the Eph family, which binds ephrin ligands (particularly ephrin-B2) to initiate bidirectional signaling. In mouse models, EPHB4 plays critical roles in various biological processes including placental development, neurogenesis, and testicular function . The high conservation of the Eph/ephrin system between mice and humans makes mouse models particularly valuable for translational research on EPHB4 functions.

What methods are most effective for detecting EPHB4 expression in mouse tissues?

Multiple complementary approaches should be employed for robust EPHB4 detection:

• Immunohistochemistry/Immunofluorescence: Using specific antibodies like Goat Anti-Mouse EPHB4 Antigen Affinity-purified Polyclonal Antibody (as described in search result 3) allows visualization of spatial expression patterns . This method is particularly valuable for examining co-expression with cell-type specific markers.

• Western Blotting: Essential for quantifying protein levels and assessing phosphorylation status of EPHB4, which indicates active signaling .

• RT-PCR: For detecting EPHB4 mRNA expression, as demonstrated in testicular tissue analysis across different developmental stages .

Each method offers unique advantages, and researchers should select based on their specific experimental questions.

How does EPHB4 expression change during mouse development?

EPHB4 expression shows distinct temporal patterns depending on the tissue examined:

• In testicular macrophages, EPHB4 immunoreactivity is weak during the first 2 weeks of postnatal development, becomes strong at 3 weeks of age, and maintains this expression through 8 weeks (adulthood) .

• In placental tissues, EPHB4 expression is higher in early gestation and significantly reduces at term, with residual expression remaining at the apical side of the syncytiotrophoblast and in a subset of villous capillaries .

These dynamic expression patterns suggest important developmental roles that vary across different organ systems.

What role does EPHB4 play in placental development and function?

EPHB4 serves as a critical regulator of trophoblast behavior in placental development:

• It reduces trophoblast proliferation through mechanisms that remain incompletely characterized.

• It increases trophoblast apoptosis by upregulating caspase-3 activity.

• It inhibits trophoblast migration and invasion by decreasing MMP2 and MMP9 expression and suppressing the phosphatidylinositol 3-kinase (PI3K) signaling pathway .

EPHB4 is also expressed in decidual endothelial cells, where it impairs spiral artery remodeling and endothelial integrity by decreasing VEGF-A expression .

How does dysregulated EPHB4 contribute to placentation disorders in mouse models?

Upregulation of EPHB4 in mouse models leads to abnormal placentation through multiple mechanisms:

• Inhibition of trophoblast proliferation

• Increased trophoblast apoptosis via upregulation of caspase-3

• Inhibited trophoblast invasion through decreased expression of MMP2, MMP9, and PI3K

These effects collectively result in shallow spiral artery remodeling and defective uteroplacental circulation, which can trigger preeclampsia-like conditions. This is consistent with observations of EPHB4 upregulation in placentae of preeclamptic women .

How might EPHB4 signaling interact with hypoxic conditions in placental development?

The relationship between EPHB4 and hypoxia in placentation appears complex:

• Hypoxia can cause a significant but transient increase in EPHB4 expression.

• Preeclamptic placentae show downregulation of ephrin-B2 and upregulation of EPHB4 .

How does EPHB4 modulate neurogenesis following ischemic stroke in mice?

EPHB4 plays a dual role in post-stroke recovery in mouse models:

• Enhanced Neurogenesis: EPHB4 overexpression increases the number of proliferating cells (BrdU+, Ki67+) and differentiated neural cells (Nestin+, Sox2+, DCX+, and NeuN+) in the cerebral cortex following middle cerebral artery occlusion (MCAO) .

• Reduced Neuroinflammation: EPHB4 overexpression alleviates inflammatory injury in MCAO model mice, suggesting an anti-inflammatory effect alongside its pro-neurogenic function .

This dual action makes EPHB4 a particularly interesting target for potential stroke therapy development.

What signaling pathways mediate EPHB4's effects on post-stroke neurogenesis?

The ABL1/Cyclin D1 signaling pathway appears to be a key mediator of EPHB4's pro-neurogenic effects:

• EPHB4 overexpression significantly increases expression of proteins related to the ABL proto-oncogene 1, non-receptor tyrosine kinase (ABL1)/Cyclin D1 signaling pathway.

• This activation suggests that restoration or enhancement of EPHB4 levels promotes neurogenesis through ABL1/Cyclin D1 signaling .

Understanding these molecular mechanisms may inform therapeutic approaches targeting EPHB4 for stroke recovery.

What experimental approaches are optimal for studying EPHB4 function in neurogenesis?

Based on published research methodologies:

• In vivo stroke models: Middle cerebral artery occlusion (MCAO) followed by reperfusion provides a clinically relevant model for studying EPHB4's role in post-stroke neurogenesis .

• Immunofluorescence analysis: Multi-marker approaches using combinations of proliferation markers (BrdU, Ki67) and stage-specific neural markers (Nestin, Sox2, DCX, NeuN) enable detailed characterization of EPHB4's effects on different stages of neurogenesis .

• Pathway analysis: Western blotting for signaling components, such as those in the ABL1/Cyclin D1 pathway, helps elucidate molecular mechanisms .

• EPHB4 overexpression: Genetic approaches to increase EPHB4 expression can reveal its sufficiency to promote neurogenesis and reduce inflammation .

What is known about EPHB4 expression in mouse testicular macrophages?

Intra-testicular-resident macrophages express both EPHB4 and ephrin-B1:

• F4/80-positive macrophages in the mouse testis show immunoreactivity for both EPHB4 and ephrin-B1.

• These macrophages are primarily located in intertubular spaces and more densely around the intra-testicular excurrent duct system.

• The number of these macrophages increases gradually during postnatal development until 5 weeks of age (puberty) and remains consistent thereafter .

How does EPHB4 expression in testicular macrophages change during development?

EPHB4 expression in testicular macrophages follows a distinct developmental pattern:

• Weak expression during early postnatal development (first 2 weeks)

• Strong expression beginning at 3 weeks of age

• Maintained strong expression through 8 weeks of age (adulthood)

Notably, ephrin-B1 expression follows a similar pattern but with consistently stronger intensity than EPHB4 at all examined ages .

What evidence supports active EPHB4 signaling in mouse testes?

Western blotting has demonstrated that EPHB4 is tyrosine-phosphorylated in mouse testis, providing direct evidence of active signaling . This phosphorylation indicates that EPHB4-expressing cells are interacting with ephrin-B-expressing cells within the testis, triggering forward signaling through the EPHB4 receptor.

How might EPHB4/ephrin-B1 signaling contribute to testicular function?

The co-expression of EPHB4 and ephrin-B1 in intra-testicular macrophages suggests potential roles in:

• Macrophage-Leydig cell interactions: Given the close physical relationship between testicular macrophages and Leydig cells, EPHB4/ephrin-B1 signaling may mediate communication important for Leydig cell development and function.

• Spermatogonial stem cell niche: The expression of these molecules may contribute to the organization of niche-like clusters that support spermatogenesis.

• Maintenance of male fertility: The tyrosine phosphorylation of EPHB4 in testis indicates active signaling that may participate in maintaining testicular function and fertility .

What controls are essential when studying EPHB4 phosphorylation and signaling?

Rigorous experimental design for EPHB4 phosphorylation studies should include:

• Mutation controls: Comparing wild-type EPHB4 with phosphorylation-deficient mutants (such as Y774F-EPHB4) to establish specificity of phosphorylation events .

• Ligand stimulation: Using soluble ephrin-B2/Fc fusion proteins to activate EPHB4 in a controlled manner, with appropriate time-course analyses (0-60 minutes after stimulation) .

• Functional readouts: Assessing cellular behaviors such as migration to correlate phosphorylation states with biological outcomes .

• Phospho-specific antibodies: Employing antibodies that specifically recognize phosphorylated tyrosine residues on EPHB4.

How should researchers address contradictory findings in EPHB4 research?

When confronted with contradictory findings regarding EPHB4, researchers should:

• Evaluate methodological differences between studies, including antibody specificity, detection methods, and experimental conditions.

• Consider tissue heterogeneity and potential differences in EPHB4 expression across cell types within the same organ.

• Examine temporal dynamics, as EPHB4 expression and function may change throughout development or disease progression.

• Investigate regulatory mechanisms, such as microRNAs (e.g., miR-20b, miR-17, miR-20a) that may differentially modulate EPHB4 expression under various conditions .

• Design targeted experiments that directly address the contradictions with appropriate controls.

What approaches are recommended for investigating bidirectional signaling between EPHB4 and ephrin-B ligands?

Effective investigation of bidirectional signaling requires:

• Distinguishing forward from reverse signaling: Using phosphorylation-specific antibodies to separately assess EPHB4 receptor activation (forward signaling) and ephrin-B ligand phosphorylation (reverse signaling).

• Soluble fusion proteins: Employing ephrin-B2/Fc chimeras to activate specifically forward signaling through EPHB4 without triggering reverse signaling .

• Mutant constructs: Utilizing phosphorylation-deficient mutants such as Y774F-EPHB4 to disrupt specific aspects of signaling and determine their functional consequences .

• Cell-specific genetic approaches: Manipulating EPHB4 or ephrin-B expression in specific cell populations to determine the cellular contexts in which each signaling direction operates.