Figure 8
Reelin and EphB/ephrin B expression in the hippocampus. (A) Expression of EphB1, EphB2 and their ligands ephrin B2 (Efnb2) and ephrin B3 (Efnb3) in the developing hippocampus. Using animals in which most of the intracellular domains of the EphB and ephrin B transmembrane proteins is substituted by the lacZ gene, we determined their expression patterns at E16.5 by β-galactosidase staining. EphB1 protein is almost exclusively expressed by migratory CA3 pyramidal cell precursors. EphB2-expressing cells were also located throughout the CA3 field (arrow). Both Efnb2 and Efnb3 are not expressed in this region prenatally. (B) Double immunofluorescence of β-galactosidase and NeuroD, a speficic marker for CA3 and DG neuronal precursors, in the developing EphB1lacZ brain demonstrated the neural expression of EphB1 in CA3 precursor cells. Arrowheads in the left panel showed β-galactosidase-positive cells throughout the CA3 subfield. Middle, higher magnification of the CA3 region (boxed area) showed robust NeuroD and β-galactosidase co-localization, confirming the expression of EphB1 (arrowheads) by CA3 migrating neurons. In contrast, NeuroD-positive cells in the dentate gyrus area (right, higher magnification of boxed area) showed reduced EphB1 expression (arrows). (C) β-galactosidase staining in adult EphB1lacZ hippocampus further confirmed that EphB1 is expressed almost exclusively in the CA3 region (arrows). (D) Reelin (green) is expressed medially to the developing CA3 region prenatally (left). The levels of Reelin expression (green, arrowheads) in both WT (top) and Ephb1−/−;Ephb2−/− mice (bottom) are comparable at both E16.5 (left) and E18.5 (right). Counterstain with DAPI (blue). (E) Normal Dab1 phosphorylation in primary neurons from mice lacking EphB1 (left) or both EphB1 and EphB2 (right) after treatment with Reelin for 15 min. β-tubulin served as a loading control. Scale bars, 100 μm (A), 100 μm (B, left), 20 μm (B, middle and right), 500 μm (C) and 200 μm (D, left) and 50 μm (D, middle and right).
