EFNB1 Human

What is EFNB1 and what are its primary functions in human development?

EFNB1 encodes EPHRIN-B1, a member of the Eph/ephrin family of membrane-linked signaling molecules that plays a crucial role in boundary formation during development . This process involves signaling between adjacent cells and often includes cell segregation, which is essential for proper tissue organization . The Eph/ephrin signaling pathway is particularly important in several developmental processes including vertebrate hindbrain development and limb bud formation .

EPHRIN-B1 functions through interaction with Eph receptors at the cell surface, creating bidirectional signaling that influences cell behavior. The signaling cascade affects various cellular processes including cell adhesion, migration, and proliferation, making it fundamental to embryonic development.

How do mutations in EFNB1 relate to craniofrontonasal syndrome?

Craniofrontonasal syndrome (CFNS) is characterized by craniosynostosis, hypertelorism, a broad nasal tip, and occasionally cleft lip and palate, caused by mutations in the ephrin-B1 gene (EFNB1) . Human genetic studies have indicated that mosaicism for EFNB1 mutation is central to CFNS pathology, a phenomenon termed cellular interference .

Specific mutations in EFNB1, such as the Ser118Ile missense mutation, have been identified in CFNS patients . This particular mutation occurs at the interface between EFNB1 and its receptor proteins. Ser118 is located in the G-H loop of the extracellular ephrin domain and is highly evolutionarily conserved across species, from rodents to fish . The mutation disrupts the dimerization interface with Eph receptors, potentially impeding the protrusion of the G-H loop and disturbing Eph-Ephrin signal transduction .

What methodologies are commonly used to detect EFNB1 mutations?

Researchers typically employ several complementary approaches to detect EFNB1 mutations:

• Direct Sequencing: Polymerase chain reaction (PCR) products are sequenced using autosequencers to identify heterozygous or homozygous mutations . This approach allows for the precise identification of specific nucleotide changes.

• Protein Structure Analysis: Since the structure of EFNB1 is not always directly available, researchers may utilize the protein structure of related proteins (like EFNB2) to characterize the spatial locality of mutations . This approach helps predict how specific mutations might affect protein function.

• Expression Analysis: Quantitative real-time PCR (qRT-PCR) is used to measure EFNB1 transcript levels in different cell types or developmental stages . This helps understand how mutations might affect gene expression patterns.

• Functional Assays: Cell segregation assays and co-culture experiments help determine how EFNB1 mutations affect cellular behaviors and interactions.

How can researchers effectively model CFNS using stem cell technologies?

Modeling CFNS using stem cell technologies involves several sophisticated approaches:

• hiPSC Derivation: Human induced pluripotent stem cells (hiPSCs) can be generated from CFNS patient-derived human dermal fibroblasts (HDFs) . Research has shown that EFNB1 expression is not necessary for reprogramming, as EFNB1 mutant HDFs demonstrate comparable reprogramming ability to control HDFs .

• Neural Differentiation: Control and CFNS hiPSCs can be differentiated into human neuroepithelial (hNE) cells, which express neural stem cell markers and several members of the Eph/ephrin family, including EFNB1 . Expression of EFNB1 varies between hNE lines and between independent differentiations of the same hiPSC line, indicating inherent variability in differentiations .

• Mosaicism Modeling: Since hiPSCs are clonally derived cell lines, CFNS heterozygous (CFNShet) hNE lines are not naturally mosaic for EFNB1 expression. Therefore, alternative approaches are necessary to model cellular interference effectively . Co-culture experiments with wild-type and EFNB1-null cells can simulate the mosaic environment.

• Expression Monitoring: EFNB1 expression is typically higher in hNE cells than in hiPSCs, suggesting that increased EFNB1 expression is characteristic of the hNE cell type . Additionally, EFNB1 expression decreases as hNE cells are maintained over time, indicating that higher EFNB1 expression may mark a progenitor stage in the differentiation program .

What computational approaches are being used to study EFNB1 structure and interactions?

Advanced computational techniques provide valuable insights into EFNB1 structure and interactions:

• Molecular Dynamics Simulations: Software such as YASARA Structure can be used to simulate EFNB1 with potential drug targets or binding partners . These simulations analyze properties such as the root mean square deviation (RMSD) of the α-carbons and binding energies of ligands .

• Stability Analysis: RMSD analysis conducted over extended simulation periods (e.g., 100 ns) provides insights into the structural stability of EFNB1 under different conditions . For instance, EFNB1 in its apo form (without ligand) typically stabilizes around 25 ns, with an average RMSD of approximately 11.79 Å and fluctuations below 2 Å, suggesting a biologically active conformation .

• Ligand Interaction Studies: Various compounds have been studied for their interaction with EFNB1. For example, sotrastaurin (average RMSD = 11.59 Å) and bicuculline (average RMSD = 10.59 Å) stabilize EFNB1 around 20 ns, maintaining an RMSD slightly lower than the apo conformation, indicating possible stabilization of the protein structure . In contrast, tolvaptan (average RMSD = 12.95 Å) reaches stabilization later (approximately 45 ns) with a higher RMSD than the apo form, potentially indicating a conformational change .

• Steered Molecular Dynamics (SMD): This technique evaluates the residence time of ligands in the binding site under the application of an external force field, providing insights into the strength and stability of molecular interactions .

How does EFNB1 expression compare between human and non-human primates?

Comparative studies between human and chimpanzee neural progenitors offer insights into evolutionary differences:

• Cytoarchitectural Similarities: Research indicates that the cytoarchitecture, cell type composition, and neurogenic gene expression programs of humans and chimpanzees are remarkably similar regarding EFNB1 and related pathways . This suggests evolutionary conservation of these fundamental developmental mechanisms.

• Expression Patterns: When comparing neural progenitor cells, approximately 75% of genes with expression specific to apical progenitors (APs) or neurons in humans are also specific to each cell type in mice, suggesting that these gene expression programs were already established in the common ancestor of mouse, human, and chimpanzee some 90 million years ago .

• Primate-Specific Expression: About 12% of genes specific to apical progenitors or neurons in both human and chimpanzee are not specific to these cell types in mice, suggesting potential involvement in developmental processes unique to primate cerebral cortex .

What are the key methodological considerations for studying EFNB1 in neural stem cells?

Researchers working with EFNB1 in neural stem cells should consider several methodological aspects:

How can researchers effectively analyze EFNB1 mosaicism in clinical samples?

Studying EFNB1 mosaicism in clinical samples requires specialized approaches:

• Single-Cell Analysis: Single-cell RNA sequencing or single-cell genotyping can reveal the heterogeneous expression patterns characteristic of mosaicism.

• Spatial Transcriptomics: These techniques preserve spatial information while assessing gene expression, allowing visualization of EFNB1 expression boundaries in tissue samples.

• Immunohistochemistry Combined with Genetic Analysis: This approach can reveal the correlation between genetic mutation status and protein expression at the cellular level.

• Digital Droplet PCR: This highly sensitive technique can quantify the fraction of cells carrying specific EFNB1 mutations in a mixed population.

• Laser Capture Microdissection: This method allows isolation of specific cell populations from heterogeneous tissue samples for subsequent molecular analysis.

Beyond CFNS, what other human conditions are associated with EFNB1 dysregulation?

EFNB1 dysregulation has been implicated in several pathological conditions:

How does EphB1 expression correlate with clinical outcomes in acute myelogenous leukemia?

Analysis of EphB1 expression in acute myelogenous leukemia (AML) has revealed important clinical correlations:

What are the emerging technologies that may advance EFNB1 research?

Several cutting-edge technologies show promise for advancing EFNB1 research:

• Organoid Models: Brain organoids derived from patient-specific iPSCs offer three-dimensional models to study EFNB1 function in a complex tissue-like environment that better recapitulates in vivo development.

• CRISPR-Based Approaches: Beyond traditional knockout strategies, CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) allow for precise modulation of EFNB1 expression without altering the genomic sequence.

• Live Imaging Technologies: Advanced microscopy techniques that allow for real-time visualization of protein dynamics can help elucidate the spatial and temporal aspects of EPHRIN-B1 signaling.

• Computational Drug Design: Molecular dynamics simulations using software like YASARA Structure are being employed to study EFNB1 interactions with potential drug candidates . This approach includes analysis of protein stability (through RMSD measurements) and binding energy calculations.

• Multi-omics Integration: Combining transcriptomics, proteomics, and metabolomics data can provide a comprehensive view of how EFNB1 functions within broader cellular networks.

What are the key unresolved questions in EFNB1 research?

Despite significant advances, several important questions remain in EFNB1 research:

• Molecular Mechanisms of Cellular Interference: While mosaicism is known to be central to CFNS pathology, the precise molecular mechanisms by which EFNB1 mosaicism leads to cellular interference and subsequent developmental abnormalities remain incompletely understood.

• Non-developmental Functions: Most research has focused on EFNB1's role in development, but its functions in adult tissues, including potential roles in tissue homeostasis and repair, warrant further investigation.

• Therapeutic Targeting: Whether EFNB1 signaling can be therapeutically modulated in conditions like CFNS or cancer remains an open question. Current research is exploring potential drug candidates and their interactions with EFNB1, but no known inhibitors have been established for clinical use .

• Evolutionary Significance: While we know that about 12% of genes specific to neural progenitors in both humans and chimpanzees are not specific to these cell types in mice , the evolutionary significance of these differences in EFNB1 regulation and function across species requires further exploration.