Ephrins stimulate neurite outgrowth during early cortical neurogenesis

The Eph receptor ligands, the ephrins, are membrane-bound molecules that play important roles in establishing intercellular communication after neurogenesis by regulating cell migration, axon pathfinding, and topographic mapping. In diverse systems, such as embryonic day 17.5 (E17.5) hippocampal and cortical neurons, repulsive/inhibitory mechanisms underlie these cellular effects. However, although ligand/receptor expression occurs far earlier, during brain neurogenesis, little is known about potential ephrin functions in initial process outgrowth. We have examined ligand/receptor expression in E13.5 cortex in vivo and in culture, using alkaline phosphatase (AP)-conjugated reagents and RNase protection assay. B ephrins are highly expressed, including B1, B2, and B3, whereas A ephrins exhibit low expression levels. In contrast, the Eph receptors demonstrate an opposite pattern, exhibiting high levels of Eph A3, A4, and A5 mRNA transcripts and low levels of the B-class receptors. To examine effects on neurite outgrowth, soluble ephrins were incubated with antihuman IgG antibody, producing oligomeric agonist complexes, and dried onto culture dishes. Unexpectedly, both ephrin A and B complexes increased process outgrowth: Seventy to eighty percent of neuronal precursors exhibited long neurites on ephrins, whereas only 5-10% of cells had neurites on IgG control substrates, indicating that ephrins stimulated neuritogenesis by early cortical neurons. These observations suggest that ephrin ligand/receptor systems play ontogenetic roles not previously considered, activating mechanisms other than cellular repulsion. Ephrin systems may induce initial process elaboration by early cortical neurons that is restricted at later stages by well-characterized repulsive signaling mechanisms.