(A–L). Photomicrographs of transverse cryosections obtained from the forelimb level of E11, E12 or E13 rat embryos labeled with mAb EfB1-3, anti-EphB1, anti-EphB2 or anti-EphB3. At E11 (A–D), both mAb EfB1-3 (A) and anti-EphB2 (C) label the ventricular zone (vz) and longitudinally projecting spinal accessory nerves (SAN; long arrows), which lie adjacent to and alongside the lateral exit points positioned midway along the dorsoventral axis of the spinal cord. No specific labeling was obtained with either anti-EphB1 (B) or anti-EphB3 (D). In the E12 (E–H) and E13 (I–L) spinal cord, each of the antibodies labeled the marginal zone (short arrows), ventral commissure (vc), motor neurons (mn) and, to some extent, ventrally-located pre-crossing commissural axons (arrowheads). mAb EfB1-3 (E, I), anti-EphB1 (F, J) and anti-EphB3 (H, L), but not anti-EphB2 (G, K), also label the dorsal funiculi (df) at these embryonic ages. mAb EfB1-3 binding was detected with a fluorescein-conjugated goat anti-mouse IgG secondary antibody. Anti-EphB binding was detected with a Cy3-conjugated donkey anti-goat IgG secondary antibody. Scale bar in L = 100 µm (A–D); 120 µm (E–H); 140 µm (I–L).

(A–F). Open book preparations (see Materials and Methods) derived from E13 rat (A–C) and E12 mouse (D–F) spinal cord were labeled with mAb EfB1-3 (A, D), anti-L1 (B, E) and anti-TAG1 (C, F). In both the rat and mouse preparations, EphB receptors (A, D) are expressed on longitudinally projecting, decussated commissural axons contained within ventral funiculi (arrowheads) located immediately adjacent to the floor plate (fp). EphB receptors (A, D) are also expressed on decussated commissural axons that extend significant distances away from the floor plate and compose the lateral funiculi (arrows). In contrast, TAG1 (C, F) is expressed on pre-crossing commissural axons, which project in the transverse plane toward the floor plate. mAb EfB1-3 (A, D), anti-L1 (B, E) and anti-TAG1 (C, F) each label the dorsal funiculi (df). In the anti-L1 labeled E13 rat (B) and mAb EfB1-3 labeled E12 mouse (D) open-book preparations, intense labeling of the ventral funiculi obscures the unlabeled floor plate. Primary antibody binding was detected with AP-conjugated secondary antibodies. Omission of primary antibodies resulted in no specific labeling of the open-book preparations. Scale bar in F = 250 µm (A–F).

(A–C). Photomicrographs of spinal cord-containing transverse cryosections obtained from E12 mouse (A), E5 chick (B) and 36 hours post fertilization (hpf) zebrafish (C) labeled with mAb EfB1-3. In both the mouse and chick spinal cord, mAb EfB1-3 (A, B) labels the marginal zone (arrows), ventral commissure (vc), dorsal funiculus (df) and a subset of pre-crossing commissural axons (arrowheads). mAb EfB1-3 also labels the ventricular zone (vz) in the chick (B), but not the mouse (A) spinal cord. In the zebrafish spinal cord (C), mAb EfB1-3 prominently labels the neuropil (np), which contains longitudinally projecting axons that compose the ventral longitudinal fasciculus (VLF, arrowhead), medial longitudinal fasciculus (MLF, arrowhead), and the dorsal longitudinal fasciculus (DLF, arrowhead). mAb EfB1-3 binding was detected with a fluorescein-conjugated goat anti-mouse IgG secondary antibody. Scale bar in A = 100 µm (A, B) and 25 µm (C).

(A–J). Photomicrographs of transverse cryosections obtained from the forelimb level of E10 (A, B), E11 (C, D), E12 (E, F), E13 (G, H) or E14 (I, J) mouse embryos labeled with mAb efrnB1 (A, C, E, G, I) or anti-ephrin-B1 (B, D, F, H, J). As detected by both mAb efrnB1 (A, C, E, G) and anti-ephrin-B1 (B, D, F, H), ephrin-B1 is expressed in the roof plate (rp) from E10-E13. While mAb efrnB1 also labels a dorsal region of the spinal cord (*) at E12 (E), anti-ephrin-B1 dorsal spinal cord labeling (*) is evident as early as E10 (D) and persists until E13 (H). Between E12 and E14, bright mAb efrnB1 (E, G, I) and anti-ephrin-B1 (F, H, J) immunoreactivity (arrowheads) surrounds the spinal cord and dorsal root ganglia (drg). At E14, mAb efrnB1 (I) and anti-ephrin-B1 (J) labeling of the spinal cord proper is dramatically reduced. mAb efrnB1 and anti-ephrin-B1 binding was detected with a Cy2-conjugated goat anti-Armenian Hamster IgG and a Cy3-conjugated donkey anti-goat IgG secondary antibody, respectively. Scale bar in J = 100 µm (A–D); 130 µm (E–H); 160 µm (I, J).

A. Photomicrograph of a transverse cryosection obtained from the forelimb level of an E13 rat embryo labeled with mAb EfB1-3. In the ventral spinal cord, mAb EfB1-3 labels the ventral funiculi (vf) and lateral funiculi (lf), which contain longitudinally projecting commissural axons, as well as motor neurons (mn) and their ventral roots (vr). Weaker mAb EfB1-3 labeling is detected on the ventral commissure (vc), which is comprised of commissural axons that project in the transverse plane through the floor plate. mAb EfB1-3 also labels the dorsal funiculi (df), which are comprised of longitudinally projecting sensory axons, and to a lesser extent, the dorsal root ganglia (drg). mAb EfB1-3 binding was detected with a fluorescein-conjugated goat antimouse IgG secondary antibody. Scale bar = 100µm in A. B. In an immunoblot analysis performed under reducing conditions, mAb EfB1-3 labeled EphB1-Fc (EB1), EphB2-Fc (EB2) and EphB3-Fc (EB3), but not EphB4-Fc (EB4) and EphB6-Fc (EB6). Each of the recombinant EphB-Fc fusion proteins (1 µg loaded per lane) are approximately 100–117kD and represents the extracellular domain of the particular receptor linked to human Ig Fc. The weakly labeled lower molecular weight bands in the EB1 and EB3 lanes are likely to be degradation products since they are more prominent in samples that have been subjected to repeated freeze-thaw cycles. mAb EfB1-3 does not recognize Fc alone (data not shown). mAb EfB1-3 binding was detected with a HRP-conjugated goat anti-mouse IgG secondary antibody. Specific signals were not detected when mAb EfB1-3 was omitted from the analyses. C. In an immunoblot analysis performed under reducing conditions, anti-EphB1, anti-EphB2 and anti-EphB3 polyclonal antibodies selectively bind EphB1-Fc (EB1), EphB2-Fc (EB2) and EphB3-Fc (EB3), respectively. The anti-EphB-positive proteins migrate at the same molecular weights as the corresponding mAb EfB1-3-positive bands in B. None of the anti-EphB antibodies recognized EphB4-Fc, EphB6-Fc or Fc alone (data not shown). For these immunoblots, 250 ng of a given recombinant EphB-Fc fusion protein was loaded per lane. Anti-EphB binding was detected with a HRP-conjugated rabbit anti-goat IgG secondary antibody. (D–F). Photomicrographs of serial transverse cryosections obtained from the forelimb level of an E13 rat labeled with anti-EphB1 (D), anti-EphB2 (E) or anti-EphB3 (F). All three antibodies label the ventral and lateral funiculi contained within the marginal zone (arrows), the ventral commissure (vc), motor neurons (mn) and the ventral roots (arrowhead). Both anti-EphB1 and anti-EphB3, but not anti-EphB2, brightly label the dorsal funiculi (df), whereas only anti-EphB2 labels the ventricular zone (vz). Anti-EphB binding was detected with a Cy3-conjugated donkey anti-goat IgG secondary antibody. Specific signals were not detected in the absence of primary antibody. Scale bar in F = 100 µm in D–F.

(A–E). Photomicrographs of semi-serial transverse cryosections obtained from the forelimb/trunk level of an E12.5 rat embryo labeled with either mAb EfB1-3 (A) or several well-established commissural axon markers (B–E). mAb EfB1-3 (A), anti-DCC (C), anti-L1 (D), and anti-GAD65 (E) label the marginal zone (arrows), which contains longitudinally projecting, decussated commissural axons. While each of these antibodies also labels motor neurons (mn) and the ventral commissure (vc), mAb EfB1-3 (A) does not significantly label pre-crossing commissural axons. In contrast, anti-L1 (D) and anti-GAD65 (E) label ventrally located pre-crossing commissural axons (arrowheads), whereas anti-TAG1 (B) and anti-DCC (C) label pre-crossing commissural axons that emanate from more dorsal regions of the spinal cord (arrowheads). Anti-GAD65 (E) and, to some extent, anti-DCC (C), conspicuously label small clusters of cell bodies located in ventral and intermediate regions of the spinal cord (asterisks). Primary antibody binding was detected with biotinylated secondary antibodies. Scale bar in A = 100 µm (A–E).

(A–E). Half-open book explants derived from E13 rat spinal cord were cultured for 48 h and then labeled as whole mount preparations with mAb EfB1-3 (B), anti-L1 (C), anti-β tubulin (D) or anti-TAG1 (E). mAb EfB1-3 (B), anti-L1 (C) and anti-β tubulin (D) label axons that have grown through the floor plate (fp) and extended out of the explants. Anti-L1 (C) also labels longitudinally oriented axons (perpendicular to those that extend out of the explants) within the explant that presumably correspond to ipsilaterally projecting axons in vivo. Anti-TAG1 (E) selectively labels pre-crossing segments (confined to the explant) of transversely projecting axons that extend to the floor plate. Primary antibody binding was detected with fluorescein-conjugated secondary antibodies. Omission of primary antibodies resulted in no specific labeling of the cultured explants. Scale bar in E = 100 µm (A–E).

A. Photomicrograph of a transverse cryosection obtained from the forelimb region of an E13 mouse embryo labeled with mAb efrnB1. mAb efrnB1 labels a dorsal region of the spinal cord that includes the roof plate (rp) and ventricular zone (vz). mAb efrnB1 immunoreactivity also surrounds the spinal cord and dorsal root ganglia (drg), where it is presumably associated with mesenchymal cells (arrowheads). mAb efrnB1 binding was detected with a Cy2-conjugated goat anti-Armenian Hamster IgG secondary antibody. Scale bar = 100 µm in A. B. In an immunoblot analysis performed under non-reducing conditions, mAb efrnB1 labeled ephrin-B1-Fc (eB1), but not ephrin-B2-Fc (eB2) or ephrin-B3-Fc (eB3). Each of the recombinant ephrin-B-Fc fusion proteins (250 µg loaded per lane) migrates at approximately 207 kD under non-reducing conditions and represents the extracellular domain of the particular ligand linked to human Ig Fc. The weakly labeled higher molecular weight bands in the eB1 lane are likely to be aggregates of ephrin-B1-Fc because the sample was not reduced or boiled. mAb efrnB1 does not recognize Fc alone (data not shown). The epitope recognized by mAb efrnB1 is destroyed in the presence of 0.2 M β-mercaptoethanol (reducing condition; data not shown). mAb efrnB1 binding was detected with a HRP-conjugated goat anti-Armenian Hamster IgG secondary antibody. No specific signals were detected when mAb efrnB1 was omitted from the immunohistochemical and immunoblot analyses. C. In an immunoblot analysis performed under reducing conditions, anti-ephrin-B1 polyclonal antibody binds ephrin-B1-Fc (eB1; migrates at approximately 60kD under reducing conditions), but not ephrin-B2-Fc (eB2), ephrin-B3-Fc (eB3) or Fc alone (data not shown). For this immunoblot analysis, 100 ng of a given recombinant ephrin-B-Fc fusion protein was loaded per lane. Anti-ephrin-B1 binding was detected with a HRP-conjugated rabbit anti-goat IgG secondary antibody. D. Photomicrograph of a serial transverse cryosection obtained from the forelimb level of an E13 mouse embryo labeled with anti-ephrin-B1. Just as mAb efrnB1, anti-ephrin-B1 labels a dorsal region of the spinal cord that includes the roof plate (rp) and ventricular zone (vz), as well as mesenchymal cells (arrowheads) that surround the spinal cord and dorsal root ganglia (drg). Anti-ephrin-B1 binding was detected with a Cy3-conjugated donkey anti-goat IgG secondary antibody. Scale bar = 100 µm in D. No specific signals were detected when the primary antibodies were omitted from the immunohistochemical and immunoblot analyses.

(A–C). Photomicrographs of a transverse cryosection derived from the forelimb level of an E12 mouse embryo double-labeled with anti-ephrin-B1 to visualize ephrin-B1 expression and mAbEfB1-3 to detect the collective expression of EphB1, EphB2 and EphB3. Ephrin-B1 is expressed in the dorsal spinal cord (*), and by mesenchymal cells (arrowheads) that surround the entire spinal cord and dorsal root ganglia (drg). EphB receptors are mainly expressed in the ventral spinal cord by axon tracts that compose the marginal zone (arrows) and those that form the ventral commissure (vc). Despite these complementary expression patterns, some overlap between mAb EfB1-3 and anti-ephrin-B1 labeling is apparent in dorsolateral regions of the spinal cord (C). Anti-ephrin-B1 and mAb EfB1-3 labeling was detected with a Cy3-conjugated donkey anti-goat IgG and fluorescein-conjugated goat anti-mouse IgG secondary antibody, respectively. df = dorsal funiculi. Scale bar in C = 100 µm (A–C).