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

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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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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).

Author information

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.

Correspondence to Michael E Greenberg.

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

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