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Astrocytes regulate adult hippocampal neurogenesis through ephrin-B signaling

Abstract

Neurogenesis in the adult hippocampus involves activation of quiescent neural stem cells (NSCs) to yield transiently amplifying NSCs, progenitors, and, ultimately, neurons that affect learning and memory. This process is tightly controlled by microenvironmental cues, although a few endogenous factors are known to regulate neuronal differentiation. Astrocytes have been implicated, but their role in juxtacrine (that is, cell-cell contact dependent) signaling in NSC niches has not been investigated. We found that ephrin-B2 presented from rodent hippocampal astrocytes regulated neurogenesis in vivo. Furthermore, clonal analysis in NSC fate-mapping studies revealed a previously unknown role for ephrin-B2 in instructing neuronal differentiation. In addition, ephrin-B2 signaling, transduced by EphB4 receptors on NSCs, activated β-catenin in vitro and in vivo independently of Wnt signaling and upregulated proneural transcription factors. Ephrin-B2+ astrocytes therefore promote neuronal differentiation of adult NSCs through juxtacrine signaling, findings that advance our understanding of adult neurogenesis and may have future regenerative medicine implications.

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Figure 1: In vivo, SGZ type 2a NSCs, type 2b neuronal precursors and type 3 neuroblasts express EphB4, and hippocampal astrocytes express ephrin-B2.
Figure 2: Fc–ephrin-B2 promoted the neuronal differentiation of NSCs in vitro.
Figure 3: Intrahippocampal injection of Fc–ephrin-B2 increases neurogenesis in the SGZ.
Figure 4: Efnb2 RNAi decreases the proneuronal effect of hippocampus-derived astrocytes in vitro.
Figure 5: Efnb2 RNAi decreases neuronal differentiation of BrdU+ cells in the SGZ.
Figure 6: Lineage tracing of ephrin-B2–induced NSC differentiation.
Figure 7: Ephrin-B2 instructs neuronal differentiation by activating β-catenin independent of Wnt signaling.

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Acknowledgements

We thank R. Fletcher for his guidance in mouse breeding and genotyping. This research was supported by US National Institutes of Health grant EB007295, Lawrence Berkeley National Lab Laboratory Directed Research and Development grant 3668DS and California Institute of Regenerative Medicine training grant T1-00007.

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Contributions

C.P. designed and executed the initial in vitro ephrin ligand studies. R.S.A. performed in vitro immunocytochemistry and A.C. performed in vivo immunohistochemistry. R.S.A. designed all of the ephrin-B2 in vivo gain-of-function and loss-of-function experiments, and A.C. and J.B. equally assisted in conducting the experiments. RNAi vectors were designed by R.S.A. and K.-I.L., cloned by K.-I.L. and P.S., and R.S.A. validated vectors experimentally and conducted in vitro RNAi and co-culture experiments. A.C. validated the expression of shRNA vectors in hippocampal astrocytes. R.S.A. conducted mouse breeding and designed and conducted fate mapping experiments with substantial input and assistance from A.C. A.C. conducted clonal analysis experiments, designed and conducted all of the experiments related to in vivo β-catenin activity studies, and analyzed tissue sample from both fate mapping and β-catenin activity studies. A.C. and C.P., and to a lesser extent R.S.A., executed the ephrin-B2, GSK3β S9A, β-catenin, Mash1 and NeuroD1 mechanistic studies. R.S.A. and A.C. performed statistical analysis. A.C. illustrated Supplementary Figure 8. R.S.A. and D.S. wrote the manuscript, with substantial input from A.C. D.V.S. supervised all aspects of this work and M.B. provided important scientific input and feedback.

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Correspondence to David V Schaffer.

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Ashton, R., Conway, A., Pangarkar, C. et al. Astrocytes regulate adult hippocampal neurogenesis through ephrin-B signaling. Nat Neurosci 15, 1399–1406 (2012). https://doi.org/10.1038/nn.3212

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