The formation of the DG and the molecules involved in this developmental process are beginning to be illuminated. Here we show that Eph-ephrin signaling contributes to the early development of this structure, as EphB2 tyrosine kinase-dependant forward signaling is required for the migration of neural progenitors into the dorsal region of the tertiary matrix to form the LSB. We demonstrate that ephrin-B1 acting as the ligand is also necessary for correct formation of the LSB, and that these molecules likely regulate the formation of the DG at least in part by influencing expression of Reelin in the marginal zone area directly above where the LSB will form.
Reelin is an extracellular matrix molecule expressed by Cajal-Retzius cells located in the marginal zone of the hippocampus (Del Rio et al., 1997
). Loss of Reelin has drastic effects on the structure of the DG as observed in Reeler
mutant mice, at least in part by controlling the formation of the radial glial scaffold of the developing DG (Forster et al., 2002
; Frotscher et al., 2003
). Reelin has also been shown to determine aspects of the migration of DG progenitors from the ventricle to the developing DG (Li et al., 2009
). Interpreting the phenotypes observed in the EphB2
mutants, Reelin is likely acting as an attractant to neural progenitors in the tertiary matrix, and loss of Reelin above the dorsal half of the developing DG leads to a failure of neural progenitors to migrate into this area. Interestingly, the effects on DG structure observed in EphB2
mutants compared to Reeler
mice are strikingly different. Granule neurons in Reeler
mice fail to coalesce into densely packed layers and are loosely distributed throughout the developing DG (Drakew et al., 2002
), whereas granule neurons in EphB2 forward signaling mutants are selectively absent in the LSB and remain tightly packed throughout the remainder of the DG. This discrepancy may be explained by the observation that Reelin expression is specifically lost above the LSB in EphB2
mutant mice, and is still expressed surrounding the other regions of the developing DG.
Although our data strongly suggests that Eph-ephrin signaling in DG development is of primary importance immediately surrounding the developing LSB, we cannot rule out a role for these signaling events at the ventricular proliferative zone. Ephrin-B1 is expressed at a high concentration in the ventricular zone where both cortical and DG neuron precursors arise (). In an early study involving thymidine labeling during embryonic development of the hippocampus, it was demonstrated that proliferating DG neuron progenitors arise in an area of the lateral ventricles termed the primary dentate neuroepithelium, otherwise known as the dentate notch (Altman and Bayer, 1990a
). This area transitions into a secondary matrix, which produces proliferative cells that migrate medially from this ventricular zone subregion to the site of DG formation, termed the tertiary matrix. Previously, it was demonstrated that ephrin-B1 expressed in progenitor cells in the cortical ventricular zone is involved with the maintenance of the progenitor pool in cortical neurogenesis (Qiu et al., 2008
). This function was linked to ephrin-B1 reverse signaling, which does not play a role in the DG phenotype described here, as we have analyzed ephrin-B1 reverse signaling mutants (Bush and Soriano, 2009
) and did not observe any abnormalities in LSB morphology (data not shown). As the DG neuron progenitors leave the secondary matrix and migrate in a medial direction towards the tertiary matrix, ephrin-B1 expression is lost and EphB2 expression is observed. These EphB2 positive cells then migrate to the tertiary matrix. DG granule layer development occurs in a gradient beginning at the crest and expanding in a lateral direction over time (Altman and Bayer, 1990b
). Our analysis of EphB2
mutant mice demonstrated that disruption of EphB2 forward signaling did not lead to a reduction in progenitor numbers in the ventral half of the tertiary matrix, indicating a role in the migration of progenitor cells from the ventral to the dorsal half of the tertiary matrix, and not in the migration from the ventricular zone to the tertiary matrix.
This study has revealed a particularly interesting aspect of Eph-ephrin signaling in the hippocampus, as EphB1 and EphB2 appear to have both overlapping and separate roles in the DG. Both EphB1 and EphB2 control progenitor number throughout the SGZ in the adult brain, and we show that this activity is linked to the PDZ-binding ability of EphB2. However, only loss of the tyrosine kinase activity of EphB2 affected the number of mature granule cell neurons, a phenotype not observed in the EphB1−/−
mutant mice (Chumley et al., 2007
). EphB2 plays a major role in development of the DG as the formation of the LSB is disrupted in the embryonic brain. EphB1 does not appear important in the early development of the DG, as genetic deletion of EphB1 does not affect the DG volume. Interestingly, both EphB1 (Chumley et al., 2007
) and EphB2 (this study) are expressed on migrating neural progenitors as they migrate to the site of DG development. While we have shown that EphB1 and EphB2 are expressed in the embryonic DG neural progenitors, we have not established levels of expression at the cell membrane. EphB2 could be expressed at a much higher level than EphB1, making it more influential in the early development of the DG than EphB1. Alternatively, EphB1 and EphB2 may transduce somewhat different forward signals to bring about their distinct effects on the migration and proliferation of dentate precursors. This latter possibility has foundations in other studies that show the intracellular signaling domains of EphB1 and EphB2 have very different abilities to mediate the ipsilateral routing of retinal ganglion cell axons despite a high degree of sequence identity between the two receptors (Petros et al., 2009
The fact that such a specific part of the DG is affected while the remainder of the structure remains relatively normal could have specific effects on hippocampal function. A number of studies have shown a separation of function between the dorsal and the ventral DG. The dorsal DG has been linked to spatial learning and memory (Hunsaker and Kesner, 2008
), while the ventral DG has been linked to behavior correlated with anxiety (Eadie et al., 2009
). The structure of the adult DG in EphB2
mutants suggests that these mice should have spatial information processing deficits, and yet anxiety behavior should remain relatively unaffected.
As well as the demonstrated effects on Reelin expression, Eph-ephrin signaling may affect DG morphogenesis via other signaling pathways that have been shown to control the development of the DG (Li and Pleasure, 2007
). Elimination of the transcription factors Sox2 or neurogenin 2 in neural progenitors greatly decreases the size of the DG (Galichet et al., 2008
; Favaro et al., 2009
). Loss of Draxin, a protein linked to repulsive axon guidance functions, was also shown to cause a decrease in size of the entire DG (Zhang et al., 2010
). Mice lacking the chemokine receptor CXCR4 and the ligands SDF-1 and Cxcl12 were shown to greatly decrease the number of cells that populate the DG (Lu et al., 2002
; Li et al., 2009
). Likewise, sonic hedgehog (Shh) signaling is also required for formation of the dentate stem cell niche (Machold et al., 2003
). Other signaling molecules have been shown to specifically affect the radial glial scaffold, such as Wnts (Galceran et al., 2000
; Zhou et al., 2004
). Our analysis of ephrin-B1
knockout mutant DGs at E18 shows that the GFAP expression around the developing LSB is disrupted. However, unlike Wnt
signaling mutants, the majority of the DG is still able to form correctly in the EphB2
mutants with only the LSB affected, indicating that Eph-ephrin and Wnt signaling have separate roles in DG morphogenesis. Interestingly, these two signaling pathways have been linked in regulating the stem cell niche in the intestinal epithelium (Batlle et al., 2002
; Holmberg et al., 2006
), suggesting the possibility that Wnt and Eph-ephrin signals are common features that dictate stem cell migration and proliferation throughout the body.
It remains to be determined exactly how EphB2 kinase-dependant forward signaling affects Reelin expression in the MZ. We observed some co-expression of Reelin and EphB2 in Cajal-Retzius cells, suggesting that EpB2 forward signaling may play a cell-autonomous role in the expression and secretion of Reelin once activated by the ephrin-B1-positive cells surrounding the MZ. Alternatively, EphB2 activity may determine the migration of Cajal-Retzius cells into the MZ, and loss of forward signaling would result in fewer numbers of Cajal-Retzius cells above the LSB. Interestingly, we show that EphB2 and ephrin-B1 are expressed in opposing gradients surrounding Reelin, with EphB2 highly expressed below and ephrin-B1 highly above the observed Reelin expression in the MZ. Presumably, Eph-ephrin signaling is strongest at the point where these gradients overlap, immediately next to the cluster of Reelin above the developing LSB. This data suggests that Reelin expression may require a gradient of Eph-ephrin signaling strength.