Distribution of EphB receptors and ephrin-B1 in the developing vertebrate spinal cord

Abstract

Contact-dependent interactions between EphB receptors and ephrin-B ligands mediate a variety of cell-cell communication events in the developing and mature central nervous system (CNS). These predominantly repulsive interactions occur at the interface between what are considered to be mutually exclusive EphB and ephrin-B expression domains. We previously used receptor and ligand affinity probes to show that ephrin-B ligands are expressed in the floor plate and within a dorsal region of the embryonic mouse spinal cord, while EphB receptors are present on decussated segments of commissural axons that navigate between these ephrin-B domains. Here we present the generation and characterization of two new monoclonal antibodies, mAb EfB1-3, which recognizes EphB1, EphB2, and EphB3, and mAb efrnB1, which is specific for ephrin-B1. We use these reagents and polyclonal antibodies specific for EphB1, EphB2, EphB3, or ephrin-B1 to describe the spatiotemporal expression patterns of EphB receptors and ephrin-B1 in the vertebrate spinal cord. Consistent with affinity probe binding, we show that EphB1, EphB2, and EphB3 are each preferentially expressed on decussated segments of commissural axons in vivo and in vitro, and that ephrin-B1 is expressed in a dorsal domain of the spinal cord that includes the roof plate. In contrast to affinity probe binding profiles, we show here that EphB1, EphB2, and EphB3 are present on the ventral commissure, and that EphB1 and EphB3 are expressed on axons that compose the dorsal funiculus. In addition, we unexpectedly find that mesenchymal cells, which surround the spinal cord and dorsal root ganglion, express ephrin-B1.

Zic2 Regulates Retinal Ganglion Cell Axon Avoidance of ephrinB2 through Inducing Expression of the Guidance Receptor EphB1

“…To confirm the specificity of the EphB1 antibody, we used antibody preabsorption techniques, because this EphB1 antibody is known to have some cross-reactivity to EphB2 and EphB3 (Jevince et al, 2006). When the EphB1 antibody was preabsorbed with EphB1-Fc fusion protein, staining in VT growth cones completely disappeared (Fig.…”

“…To assess whether EphB1 protein is expressed in the growth cones of VT retinal axons, the only RGCs that express EphB1 mRNA, we used a monoclonal EphB1 antibody (Jevince et al, 2006) in both IHC and Western blot analysis of retinal explants plated on polyornithine/laminin without ephrinB2. By IHC, at E14.5-E15.5, the time when VT axons in mouse reach the chiasm midline, EphB1 protein is expressed exclusively on VT growth cones and not on the growth cones of axons emanating from explants from contralaterally projecting regions of the retina, here, DT retina ( Fig.…”

“…Immunohistochemistry using EphB1 antibodies (4A7) (Jevince et al, 2006), preabsorbed as above to eliminate cross-reactivity to EphB2 and B3, was used at 1:50 to stain RGC growth cones on neurites extending from retinal explants, taken from E14.5 animals. Anti-doublecortin and anti-GAP-43 antibody (Millipore Bioscience Research Reagents) were used at 1:1000 to stain retinal neurites.…”

“…To eliminate cross-reactivity from EphB2 and EphB3 (Jevince et al, 2006), the EphB1 antibody was preabsorbed with EphB2-Fc and EphB3-Fc fusion proteins (R&D Systems) (details of preabsorption protocol in the study by Bundesen et al, 2003). Anti-␤-tubulin antibody (monoclonal, T 5293, clone 2-28-33; Sigma) was used as a loading control.…”

“…To detect EphB1 protein, Western blot analysis was performed on lysates taken from E14.5 VT retinal explants cultured in vitro for 18 h, following standard protocols using an antibody to EphB1 (4A7 or mAb EfB1-EfB3, made against EphB1-Fc; gift from Zaven Kaprielian, Albert Einstein School of Medicine, Bronx, NY) (Jevince et al, 2006). Retinal cultures were homogenized with a sonicator in lysis buffer (50 mM Tris-HCl, pH 7.4, 5 mM DTT, 1% NP-40, and 5 g/ml leupeptin, aprotinin, and pepstatin).…”