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Ephrin-B3 reverse signaling through Grb4 and cytoskeletal regulators mediates axon pruning

Nature Neuroscience volume 12, pages 268–276 (2009)Cite this article

Abstract

It has been suggested that ephrin-B proteins have receptor-like roles in the control of axon pathfinding by repulsion, although it is largely unknown how the reverse signals are coupled to downstream intracellular molecules and how they induce cytoskeletal reorganization at the axon terminal. We found that ephrin-B3 (EB3) was able to function as a repulsive guidance receptor and mediate stereotyped pruning of murine hippocampal mossy fiber axons during postnatal development. Targeted intracellular point mutants showed that axon pruning requires tyrosine phosphorylation–dependent reverse signaling and coupling to the SH2/SH3 adaptor protein Grb4 (also known as Nckβ/Nck2). Furthermore, we found that the second SH3 domain of Grb4 is required and sufficient for axon pruning/retraction by mediating interactions with Dock180 and PAK to bring about guanine nucleotide exchange and signaling downstream of Rac, respectively. Our results reveal a previously unknown pathway that controls axon pruning and elucidate the biochemical mechanism by which ephrin-B reverse signals regulate actin dynamics to bring about the retraction of growth cones.

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Figure 1: Defective hippocampal mossy fiber pruning in Efnb3 mutants.
Figure 2: Defective hippocampal mossy fiber pruning in Ephb mutants.
Figure 3: Mossy fiber pruning requires tyrosine phosphorylation of the EB3 cytoplasmic tail.
Figure 4: Axon pruning of hippocampal granule cell neurons induced by Cos-1 coculture in vitro requires EB3 tyrosine phosphorylation.
Figure 5: Grb4 is a key mediator for transducing EB3 reverse signals involved in axon pruning.
Figure 6: EB3 reverse signaling requires PAK and Dock180.
Figure 7: Activation of EB3 reverse signaling leads to increased Rac and Cdc42 GTP levels.

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Acknowledgements

We thank K.S. Ravichandran and Jane Wu for Dock180 constructs; Toshio Ohshima for Pak1 dominant-negative expressing vector; Wei Zhang and Luis Parada for mCherry-Rac1 constructs; Jan La and Robert Silvany for technical help; Suya Sun for genotyping, histological assistance and tissue culture; and Michael Chumley for helpful comments on the study. This research was supported by the NIH (R01 MH066332 and R01 EY017434).

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  1. Department of Developmental Biology, Kent Waldrep Center for Basic Research on Nerve Growth and Regeneration, University of Texas Southwestern Medical Center, Dallas, 75390-9133, Texas, USA

    Nan-Jie Xu & Mark Henkemeyer

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Correspondence to Mark Henkemeyer.

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Xu, NJ., Henkemeyer, M. Ephrin-B3 reverse signaling through Grb4 and cytoskeletal regulators mediates axon pruning. Nat Neurosci 12, 268–276 (2009). https://doi.org/10.1038/nn.2254

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