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A chemical genetic approach reveals distinct EphB signaling mechanisms during brain development

Nature Neuroscience volume 15, pages 1645–1654 (2012)Cite this article

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Abstract

EphB receptor tyrosine kinases control multiple steps in nervous system development. However, it remains unclear whether EphBs regulate these different developmental processes directly or indirectly. In addition, given that EphBs signal through multiple mechanisms, it has been challenging to define which signaling functions of EphBs regulate particular developmental events. To address these issues, we engineered triple knock-in mice in which the kinase activity of three neuronally expressed EphBs can be rapidly, reversibly and specifically blocked. We found that the tyrosine kinase activity of EphBs was required for axon guidance in vivo. In contrast, EphB-mediated synaptogenesis occurred normally when the kinase activity of EphBs was inhibited, suggesting that EphBs mediate synapse development by an EphB tyrosine kinase–independent mechanism. Taken together, our data indicate that EphBs control axon guidance and synaptogenesis by distinct mechanisms and provide a new mouse model for dissecting EphB function in development and disease.

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Figure 1: A chemical genetic approach to studying EphB signaling.
Figure 2: Generation of AS-EphB TKI mice.
Figure 3: Selective inhibition of the kinase function of EphBs in AS-EphB TKI mice.
Figure 4: The kinase function of EphBs is required for growth cone collapse in ventrotemporal (VT) retinal ganglion cells.
Figure 5: The kinase function of EphBs is required for the formation of the ipsilateral retinal projection in vivo.
Figure 6: The kinase activity of EphBs is required for the formation of the corpus callosum in vivo.
Figure 7: The kinase activity of EphBs is dispensable for the formation of dendritic spines and functional excitatory synapses in culture.
Figure 8: The kinase activity of EphBs is dispensable for the formation of dendritic spines and functional excitatory synapses in hippocampal slices.

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References

  1. Klein, R. Eph/ephrin signaling in morphogenesis, neural development and plasticity. Curr. Opin. Cell Biol. 16, 580–589 (2004).

    Article  CAS  PubMed  Google Scholar 

  2. Lai, K.O. & Ip, N.Y. Synapse development and plasticity: roles of ephrin/Eph receptor signaling. Curr. Opin. Neurobiol. 19, 275–283 (2009).

    Article  CAS  PubMed  Google Scholar 

  3. Genander, M. & Frisen, J. Ephrins and Eph receptors in stem cells and cancer. Curr. Opin. Cell Biol. 22, 611–616 (2010).

    Article  CAS  PubMed  Google Scholar 

  4. Sanders, S.J. et al. De novo mutations revealed by whole-exome sequencing are strongly associated with autism. Nature 485, 237–241 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Merlos-Suárez, A. & Batlle, E. Eph-ephrin signaling in adult tissues and cancer. Curr. Opin. Cell Biol. 20, 194–200 (2008).

    Article  PubMed  CAS  Google Scholar 

  6. Pasquale, E.B. Eph receptors and ephrins in cancer: bidirectional signaling and beyond. Nat. Rev. Cancer 10, 165–180 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Cissé, M. et al. Reversing EphB2 depletion rescues cognitive functions in Alzheimer model. Nature 469, 47–52 (2011).

    Article  PubMed  CAS  Google Scholar 

  8. Sheffler-Collins, S.I. & Dalva, M.B. EphBs: an integral link between synaptic function and synaptopathies. Trends Neurosci. 35, 293–304 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Henkemeyer, M. et al. Nuk controls pathfinding of commissural axons in the mammalian central nervous system. Cell 86, 35–46 (1996).

    Article  CAS  PubMed  Google Scholar 

  10. Grunwald, I.C. et al. Kinase-independent requirement of EphB2 receptors in hippocampal synaptic plasticity. Neuron 32, 1027–1040 (2001).

    Article  CAS  PubMed  Google Scholar 

  11. Henderson, J.T. et al. The receptor tyrosine kinase EphB2 regulates NMDA-dependent synaptic function. Neuron 32, 1041–1056 (2001).

    Article  CAS  PubMed  Google Scholar 

  12. Kayser, M.S., Nolt, M.J. & Dalva, M.B. EphB receptors couple dendritic filopodia motility to synapse formation. Neuron 59, 56–69 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Kayser, M.S., McClelland, A.C., Hughes, E.G. & Dalva, M.B. Intracellular and trans-synaptic regulation of glutamatergic synaptogenesis by EphB receptors. J. Neurosci. 26, 12152–12164 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Himanen, J.P., Saha, N. & Nikolov, D.B. Cell-cell signaling via Eph receptors and ephrins. Curr. Opin. Cell Biol. 19, 534–542 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Dalva, M.B. et al. EphB receptors interact with NMDA receptors and regulate excitatory synapse formation. Cell 103, 945–956 (2000).

    Article  CAS  PubMed  Google Scholar 

  16. Nolt, M.J. et al. EphB controls NMDA receptor function and synaptic targeting in a subunit-specific manner. J. Neurosci. 31, 5353–5364 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Palmer, A. et al. EphrinB phosphorylation and reverse signaling: regulation by Src kinases and PTP-BL phosphatase. Mol. Cell 9, 725–737 (2002).

    Article  CAS  PubMed  Google Scholar 

  18. Henkemeyer, M., Itkis, O.S., Ngo, M., Hickmott, P.W. & Ethell, I.M. Multiple EphB receptor tyrosine kinases shape dendritic spines in the hippocampus. J. Cell Biol. 163, 1313–1326 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Orioli, D., Henkemeyer, M., Lemke, G., Klein, R. & Pawson, T. Sek4 and Nuk receptors cooperate in guidance of commissural axons and in palate formation. EMBO J. 15, 6035–6049 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. O'Donnell, M., Chance, R.K. & Bashaw, G.J. Axon growth and guidance: receptor regulation and signal transduction. Annu. Rev. Neurosci. 32, 383–412 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Cowan, C.A. et al. Ephrin-B2 reverse signaling is required for axon pathfinding and cardiac valve formation, but not early vascular development. Dev. Biol. 271, 263–271 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Mendes, S.W., Henkemeyer, M. & Liebl, D.J. Multiple Eph receptors and B-class ephrins regulate midline crossing of corpus callosum fibers in the developing mouse forebrain. J. Neurosci. 26, 882–892 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Petros, T.J., Shrestha, B.R. & Mason, C. Specificity and sufficiency of EphB1 in driving the ipsilateral retinal projection. J. Neurosci. 29, 3463–3474 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Chenaux, G. & Henkemeyer, M. Forward signaling by EphB1/EphB2 interacting with ephrin-B ligands at the optic chiasm is required to form the ipsilateral projection. Eur. J. Neurosci. 34, 1620–1633 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Ethell, I.M. et al. EphB/syndecan-2 signaling in dendritic spine morphogenesis. Neuron 31, 1001–1013 (2001).

    Article  CAS  PubMed  Google Scholar 

  26. Bishop, A.C. et al. A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature 407, 395–401 (2000).

    Article  CAS  PubMed  Google Scholar 

  27. Blethrow, J., Zhang, C., Shokat, K.M. & Weiss, E.L. Design and use of analog-sensitive protein kinases. Curr. Protoc. Mol. Biol. 18, 18.11 (2004).

    Google Scholar 

  28. Chen, X. et al. A chemical-genetic approach to studying neurotrophin signaling. Neuron 46, 13–21 (2005).

    Article  PubMed  CAS  Google Scholar 

  29. Bishop, A.C. et al. Design of allele-specific inhibitors to probe protein kinase signaling. Curr. Biol. 8, 257–266 (1998).

    Article  CAS  PubMed  Google Scholar 

  30. Alaimo, P.J., Shogren-Knaak, M.A. & Shokat, K.M. Chemical genetic approaches for the elucidation of signaling pathways. Curr. Opin. Chem. Biol. 5, 360–367 (2001).

    Article  CAS  PubMed  Google Scholar 

  31. Rozakis-Adcock, M., Fernley, R., Wade, J., Pawson, T. & Bowtell, D. The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1. Nature 363, 83–85 (1993).

    Article  CAS  PubMed  Google Scholar 

  32. Moeller, M.L., Shi, Y., Reichardt, L.F. & Ethell, I.M. EphB receptors regulate dendritic spine morphogenesis through the recruitment/phosphorylation of focal adhesion kinase and RhoA activation. J. Biol. Chem. 281, 1587–1598 (2006).

    Article  CAS  PubMed  Google Scholar 

  33. Torres, R. et al. PDZ proteins bind, cluster, and synaptically colocalize with Eph receptors and their ephrin ligands. Neuron 21, 1453–1463 (1998).

    Article  CAS  PubMed  Google Scholar 

  34. Chumley, M.J., Catchpole, T., Silvany, R.E., Kernie, S.G. & Henkemeyer, M. EphB receptors regulate stem/progenitor cell proliferation, migration, and polarity during hippocampal neurogenesis. J. Neurosci. 27, 13481–13490 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Yucel, S., Dravis, C., Garcia, N., Henkemeyer, M. & Baker, L.A. Hypospadias and anorectal malformations mediated by Eph/ephrin signaling. J. Pediatr. Urol. 3, 354–363 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Risley, M., Garrod, D., Henkemeyer, M. & McLean, W. EphB2 and EphB3 forward signalling are required for palate development. Mech. Dev. 126, 230–239 (2009).

    Article  CAS  PubMed  Google Scholar 

  37. Choi, Y. et al. Discovery and structural analysis of Eph receptor tyrosine kinase inhibitors. Bioorg. Med. Chem. Lett. 19, 4467–4470 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Qiu, R. et al. Regulation of neural progenitor cell state by ephrin-B. J. Cell Biol. 181, 973–983 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Cowan, C.W. et al. Vav family GEFs link activated Ephs to endocytosis and axon guidance. Neuron 46, 205–217 (2005).

    Article  CAS  PubMed  Google Scholar 

  40. Williams, S.E. et al. Ephrin-B2 and EphB1 mediate retinal axon divergence at the optic chiasm. Neuron 39, 919–935 (2003).

    Article  CAS  PubMed  Google Scholar 

  41. Luria, V., Krawchuk, D., Jessell, T.M., Laufer, E. & Kania, A. Specification of motor axon trajectory by ephrin-B:EphB signaling: symmetrical control of axonal patterning in the developing limb. Neuron 60, 1039–1053 (2008).

    Article  CAS  PubMed  Google Scholar 

  42. Williams, S.E., Mason, C.A. & Herrera, E. The optic chiasm as a midline choice point. Curr. Opin. Neurobiol. 14, 51–60 (2004).

    Article  CAS  PubMed  Google Scholar 

  43. Bush, J.O. & Soriano, P. Ephrin-B1 regulates axon guidance by reverse signaling through a PDZ-dependent mechanism. Genes Dev. 23, 1586–1599 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Takasu, M.A., Dalva, M.B., Zigmond, R.E. & Greenberg, M.E. Modulation of NMDA receptor–dependent calcium influx and gene expression through EphB receptors. Science 295, 491–495 (2002).

    Article  CAS  PubMed  Google Scholar 

  45. McClelland, A.C., Sheffler-Collins, S.I., Kayser, M.S. & Dalva, M.B. Ephrin-B1 and ephrin-B2 mediate EphB-dependent presynaptic development via syntenin-1. Proc. Natl. Acad. Sci. USA 106, 20487–20492 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Holmberg, J. & Frisen, J. Ephrins are not only unattractive. Trends Neurosci. 25, 239–243 (2002).

    Article  CAS  PubMed  Google Scholar 

  47. Moon, M.S. & Gomez, T.M. Balanced Vav2 GEF activity regulates neurite outgrowth and branching in vitro and in vivo. Mol. Cell. Neurosci. 44, 118–128 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Banko, M.R. et al. Chemical genetic screen for AMPKα2 substrates uncovers a network of proteins involved in mitosis. Mol. Cell 44, 878–892 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Knight, Z.A. & Shokat, K.M. Chemical genetics: where genetics and pharmacology meet. Cell 128, 425–430 (2007).

    Article  CAS  PubMed  Google Scholar 

  50. Batlle, E. et al. EphB receptor activity suppresses colorectal cancer progression. Nature 435, 1126–1130 (2005).

    Article  CAS  PubMed  Google Scholar 

  51. Petros, T.J., Bryson, J.B. & Mason, C. Ephrin-B2 elicits differential growth cone collapse and axon retraction in retinal ganglion cells from distinct retinal regions. Dev. Neurobiol. 70, 781–794 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Plump, A.S. et al. Slit1 and Slit2 cooperate to prevent premature midline crossing of retinal axons in the mouse visual system. Neuron 33, 219–232 (2002).

    Article  CAS  PubMed  Google Scholar 

  53. Xu-Friedman, M.A. & Regehr, W.G. Presynaptic strontium dynamics and synaptic transmission. Biophys. J. 76, 2029–2042 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank M. Thompson, Y. Zhou and H. Ye of the Children's Hospital Boston Intellectual and Developmental Disabilities Research Center Mouse Gene Manipulation Core for ES cell work and blastocyst injection, T. Kuwajima and members of C. Mason's laboratory for advice on retinal explants and DiI labeling, M. Lopez for help selecting and generating PP1 analogs, Z. Wills and A. Mardinly for help with synapse analysis, S. Cohen for advice on electrophysiological recordings, P. Zhang for assistance with animal management, and L. Hu for antibody work. This research was funded by US National Institutes of Health grants RO1-NS-045500 (M.E.G.) and RO1-EY-018207 (C.W.C.). H.-Y.H.H. was supported by the Marion Abbe Fellowship of the Damon Runyon Cancer Research Foundation and US National Institutes of Health training grants in neurodegeneration and cancer biology. M.J.S. was supported by a National Science Foundation Graduate Research Fellowship. M.A.R. was supported by a training grant from the National Institute on Drug Abuse (T32 DA07290).

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  1. Chao Zhang & Christopher W Cowan

    Present address: Present address: Department of Chemistry, University of Southern California, Los Angeles, California, USA (C.Z.), Department of Psychiatry, McLean Hospital, Harvard Medical School, Belmont, Massachusetts, USA (C.W.C.).,

  2. Michael J Soskis and Hsin-Yi Henry Ho: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA

    Michael J Soskis, Hsin-Yi Henry Ho, Brenda L Bloodgood, Athar N Malik, Bulent Ataman, Alex A Rubin, Janine Zieg, Nikhil Sharma & Michael E Greenberg

  2. Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Michael A Robichaux & Christopher W Cowan

  3. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California, USA

    Chao Zhang & Kevan M Shokat

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Contributions

M.J.S., H.-Y.H.H. and M.E.G. conceived and designed the study. M.J.S. and H.-Y.H.H conducted all of the experiments unless otherwise noted. B.L.B. and N.S. performed electrophysiological recordings. J.Z. generated EphB1 and EphB3 targeting constructs. M.A.R. and C.W.C. contributed to the axon guidance experiments. A.N.M. and A.A.R. generated shRNAs for EphBs. B.A. performed qPCR experiments. C.Z. and K.M.S. designed and synthesized inhibitors. M.J.S., H.-Y.H.H. and M.E.G. wrote the manuscript.

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Correspondence to Michael E Greenberg.

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Soskis, M., Ho, HY., Bloodgood, B. et al. A chemical genetic approach reveals distinct EphB signaling mechanisms during brain development. Nat Neurosci 15, 1645–1654 (2012). https://doi.org/10.1038/nn.3249

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