Fig. 5. Overall ephrin-B1 protein in transfection domains exhibits a graded distribution that parallels the endogenous ephrin-B1 gradient. (A) Schematic of a coronal section through an E10 tectum. Dividing cells are present in the neuroepithelium (ne) and stratum griseum et fibrosum superficiale (SGFS). RGC axons extend through the stratum opticum (SO) at the pial surface of the tectum. Boxes indicate areas shown in B and C. (B,C) E10 tectum incubated with EphB2-Fc reveals the distribution of ephrin-B1 along radially aligned processes (arrows) and in the SO (brackets). (B) Lateral tectum has low levels of ephrin-B in the SO. (C) Medial tectum has high levels of ephrin-B in the SO. The images in B and C are of the same section, taken sequentially using the same confocal settings and processed identically. (D-F) Coronal section through E7 lateral tectum after electroporation at E1.5 with RCAS-ephrin-B1-IRES-eGFP. Tectum was stained with EphB2-Fc to reveal the distribution of ephrin-B1. Infected cells and processes are in green and EphB2-Fc staining is red. Many infected cells are present in the ne as well as the SGFS. Lateral is to the left and medial is to the right of each panel. (E) EphB2-Fc reveals the presence of ephrin-B1 in the SO (bracket) and along radially aligned processes (arrow). Within the transfection domain (between arrowheads) a gradient of ectopic ephrin-B1 that parallels the endogenous ephrin-B1 gradient is apparent. (F) The eGFP reveals the extent of the transfection (between arrowheads). The level of eGFP is relatively consistent across most of the transfection domain. At these ages (E7-E10), only ephrin-B1 is expressed within the tectum; therefore the EphB2-Fc staining reveals the distribution of ephrin-B1 protein selectively (Braisted et al., 1997). Scale bar: 40 µm (B,C) and 50 µm (D-F).

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